Profiles of Drug Substances Vol 05

Analytical Profiles of

Volume 5

Edited b y

Klaus Florey The Squibb Institute for Medical Research

New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Glenn A. Brewer, Jr. Lester Chafetz Bernard 2. Senkowski

Boen T. Kho Gerald J. Papariello

Compiled under the auspices of the Pharmaceutical Analysis and Control Section

Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London 1976 A Subsidiary of Harcourt Brace Jovanovich, Publishers

EDITORIAL BOARD

Norman W. Atwater Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari

Erik H. Jensen Boen T. Kho Arthur F. Michaelis Gerald .J. Papariello Bruce C. Rudy Bernard Z. Senkowski Frederick Tishler

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1976, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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Main entry under title:

Analytical profiles of drug substances.

Includes bibliographical references. Compiled under the auspices of the Pharmaceutical

Analysis and Control Section, Academy of Pharmaceutical Sciences.

Medical and pharmaceutical-Collected works. I. Florey, Klaus, ed. 11. Brewer, Glenn A. 111. Academy of Pharmaceutical Sciences. Pharmaceutical Analysis and Control Section. [DNLM: 1. Drugs-Analysis-Yearbooks. QV740 AA1 A551

1. Drugs-Analysis-Collected works. 2. Chemistry,

RM300.A56 616l.1 70-187259 ISBN 0-12-260805-4 (v. 5)

PRINTED IN THE UNITED STATES OF AMERICA

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

H. Y. Aboul-Enein, USV Pharmaceuticals, Tuckahoe, New York, now: Riyadh University, Riyadh, Saudi Arabia

G. D. Anthony, Searle and Company, Chicago, Illinois

N. W. Atwuter, Searle and Company, Chicago, Illinois

J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey

G. A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey

D. E. Cudwulluder, School of Pharmacy, University of Georgia, Athens, Georgia

L. Chufefz, Warner-Lambert Research Institute, Morris Plains, New Jersey

A. R. Chamberlin, Stanford Research Institute, Menlo Park, California

2. L. Chung, Searle and Company, Chicago, Illinois

A. P. K . Chatng, Stanford Research Institute, Menlo Park, California

E. M. Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania

J. P. Comer, Eli Ully and Company, Indianapolis, Indiana

R. D. Duley, Ayerst Laboratories, Rouses Point, New York

K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey

S. A. Fusuri, Parke, Davis and Company, Detroit, Michigan

vii

AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

G. G. Gallo, Gruppo LePetit, Milan, Italy

R. Gomez, Hoffmann-LaRoche, Inc., Nutley, New Jersey

R. B. Hagel, Hoffmann-LaRoche, Inc., Nutley, New Jersey

D. D. Hong, The Sterling-Winthrop Research Institute, Rensselaer, New York

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan

J , H. Johnson, Hoffmann-LaRoche, Inc., Nutley, New Jersey

H. W. Jun, School of Pharmacy, University of Georgia, Athens, Georgia

B. T. Kho, Ayerst Laboratories, Rouses Point, New York

P. Lim, Stanford Research Institute, Menlo Park, California

J. P. McDonnell, Salsbury Laboratories, Charles City, Iowa

E. A. MacMuZlan, Hoffmann-LaRoche, Inc., Nutley, New Jersey

A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

S. M. Miller, Wyeth Laboratories, Philadelphia, Pennsylvania

N. G. Nash, Ayerst Laboratories, Rouses Point, New York

C E. Onech, Ayerst Laboratories, Rouses Point, New York

G. J. Papanello, Wyeth Laboratories, Philadelphia, Pennsylvania

A. Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

P. Radaelli, Gruppo LePetit, Milan, Italy

J , A. Raihle, Abbott Laboratories, North Chicago, Illinois

R, J. Rucki, Hoffmann-LaRoche, Inc., Nutley, New Jersey

B. C. Rudy, Schering-Plough, Corp., Bloomfield, New Jersey

viii

AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

F. Russo-Alesi, The Squibb Institute for Medical Research, New Brunswick, New Jersey

B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey

C. M. Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania

F. Tishler, Ciba-Geigy, Summit, New Jersey

R J. Warren, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

E. H. Waysek, Hoffmann-LaRoche, Inc., Nutley, New Jersey

L. L . Wearley, Searle and Company, Chicago, Illinois

ix

PREFACE

Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degredation and metabolism. For drug substances important enough to be accorded monographs in the official compendia such supple- mental information should also be made readily available. To this end the Phar- maceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the fifth.

The concept of Analytical Profiles is taking hold not only for cornpendial drugs but, increasingly, in the industrial research laboratories. Analytical Profiles are being prepared and periodically updated to provide physico-chemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an Analytical Profile will require a minimum of effort whenever a new drug substance is selected for compendial status.

The cooperative spirit of our contributors had made this venture possible. All those who have found the profiles useful are earnestly requested to contrib- ute a monograph of their own. The editors stand ready to receive such contribu- tions.

Klaus Florey

xi

BENDROFLUMETHIAZIDE

Klaus FIorey and Frank M. Russo-Alesi

KLAUS FLOREY AND FRANK M . RUSSO-ALES)

1.

2.

3.

4.

5.

6.

7.

8.

9.

CONTENTS

Description 1.1 History 1.2 Name, Formula, Molecuiar Weight 1.3 Appearance, Color,Odor

Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Solubility 2.8 Crystal Properties 2.9 pKa

Synthesis

Stability and Degradation

Drug Metabolic Products - Pharmacokinetics

Methods of Analysis 6.1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Nonaqueous Titration 6.5 Chromatographic Analysis

6.51 Paper 6.52 Thin-Layer

Identification and Determination in Biological Fluids

Miscellaneous

References

2

BENDROFLUMETH IAZIDE

1. Description 1.1 Histor

d l u m e t h i a z i d e belongs to the class of thiazide diuretics. Its synthesis was first reported by Holdrege, Babel and Cheneyl in 1959 and its diuretic activity was first described in 960 almost simultaneously by Lund and Kobing and by Kennedy, Buchanan and Cunningham s .

1.2 Name, Formula, Molecular Weight Bendroflumethiazide (also bendrofluazide,

benydroflumethiazide, and benzylhydroflumethiazide) is 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3,4- dihydro-3-(phenyl methyl)-6-trifluoromethyl-l,l- dioxide or 3-Benzyl-3,4-dihydro-6-(trifluoromethyl)-2H-1,2,4-benzothiad~az~ne-7-sulfonam~de- 1,l dioxide [73-48-31,

0 0 NH SO '@('>H a

HC-CH2 CF 3

H

'1 SH1 qF 3N304 2 Mol. Wt. 421.41

1.3 Appearance, Color, Odor White or sliqhtly off-white, uniform free

flowing crystalline powder i slight floral odor.

2 . Physical Properties 2.1 Infrared Spectrum

The infrared spectrum f bendroflumethiazide is given in Figure 1 P .

2.2 Nuclear Magnetic Resonance The 60 MHz proton magnetic resonance

spectrum in d4-methanol containing tetramethylsilane as an intern93 reference (Figure 2) is assigned as follows :

3

.. h

m

rl

=w=

c u

cl fd

al a

fdk

k

C\

wrn

G

G

HH

rl

al

4 P

Figure 1. Infrared Spectrum of Bendroflumethiazide (batch #15); KBr, MeOH. Instrument: Perkin-Elmer 621.

m

Figure 2. NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated methanol (Instrument: Perkin Elmer R12B)

KLAUS FLOREY AND FRANK M. RUSSO-ALES1

H ~1.67(s)

not assigned Q T2.7

I n d6-DMSO (Figure 3 1 , t h e NH proton resonances were observed near ~ 1 . 7 5 ( s i n g l e t , superimposed with a r y l p ro ton ) , ~2 (b road) , 2 .45 ( s i n g l e t ) and 2.57 ( s i n g l e t ) (Figure 3) i n a d d i t i o n t o t h e o t h e r protons assigned from t h e dq-methanol spectrum. The higher f i e l d s i n g l e t s a t ~ 2 . 4 5 and 2.57 are t e n t a t i v e l y assigned t o t h e sulfonamide -NH2 protons. N o p reference of assignment i s made of t h e o t h e r two -NH protons. On a d d i t i o n of D 2 0 , t h e -NH protons a r e r a p i d l y exchanged f o r deuterium, r e s u l t i n g i n t h e disappearance of t h e NH protons and sharpening of t h e proton resonance near ~5 which now appears l i k e t h a t of t h e dq-methanol spectrum .

2.3 U l t r a v i o l e t Spectrum Squibb House Standard (batch #15) i n

methanol fxh ib i t ed t h r e e maxima a t 208 mu (El 745) ,

This ag i s with measurements by P i l sbu and JacksonfF and by Kracmar and Lastovkovafi who d i scuss t h e U . V . spectrophotometry of benzothia- d i a z i n e s and p resen t spec t r a .

273 mp (E 565) and 326 mu (Ei 9 6 ) 1 3 . 1

2 .4 Mass Spectrum The l o w r e s o l u t i o n mass spectrum

(Figure 4 ) shows only a very weak molecular ion (M+) of m / e 4 2 1 and a base peak of m / e 319 t h a t could arise by a double proton rearrangement, which i s not a common fragmentat ion pathway. The assignment of some of t h e fragment ions i s shown below:

6

0 L I 4 c 7 r S i n 0 L I 4 c 7 r S i n

Figure 3. NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated DMSO (Instrument: Perkin-Elmer R12B)

3753 SO15497 BFI#15

12-FEB-76 MR0587

I I 0 0 I

90..

80.-

70.-

60.-

5 0.-

40-

ie MRSS/CHRRGE

INTENSITY SUM = 1 8 3 3 9 BRSE PERK Z = 8 . 3 2

Figure 4. Low Resolution Mass Spectrum of Bendroflumethiazide (Instrument: AEI MS9)

BENDROFLUMETHIAZIDE

319 H

2"H2

330191 H 77

m/e 402 6 M+ m/e 4 2 1

m/e 239 <-\ m/e 255 4

m/e 303 -so2NH2 m/e 302 (-HI

m/e 319- m/e 302 -so2NH2, m/e 222 -so2

H

m/e 174 m/e 158 m/e 175 (+HI m/e 159 (+HI m/e 176 (+2H)

Although these appear t o be reasonable assignments, they should be considered t e n t a t i v e s i n c e t h e a s s i nments are not confirmed by h igh- reso lu t ion d a t a s 3.

2.5 Melting Range The melt ing p o i n t does no t occur

sharp ly and depends on t h e r a t e of hea t ing . The melt ing p o i n t of t h e Squibb House Standard (ba tch 15) w a s repor ted a t 223.20 - 226.20 C. L i t e r a t u r e va lues range from 220' - 228'.

2.6 D i f f e r e n t i a l Thermal Analysis Melting endotherm: 2210 C. (17).

2.7 S o l u b i l i t d i n water and chloroform.

9


Soluble i n a l k a l i , ace tone and a lcohol . Soluble i n one equiva len t of 0 . 1 N NaOH. S l i g h t l y so lub le i n e t h e r . Inso luble i n a c i d , benzene, l i g r o i n and petroleum e t h e r l 7 . S t a b l e concentrated s o l u t i o n i n polyethylene g l y c o l , water and dimethyl- a c e t a n i l i d e o r N-methyl-2-pyrolidinone have been claimed (18).

2.8 Crys ta l P r o p e r t i e s

presented i n Table I and Figure 517. The powder x-ray d i f f r a c t i o n p a t t e r n i s

TABLE I 0

d (A) * I/I0**

17.23 0.528 13.27 0.356 11.63 0.103 8.94 8.11 0*246 *d = i n t e r p l a n a r d i s t a n c e 0.148 7.64 0.118 - 1

7.36 6.70 6.34 5.91 5.48 5.31 4.82 4.67 4.53 4.46 4.31 4.19 3.96 3.84 3.75 3.69 3.63 3.54 3.44 3.40 3.19 3.11 2.99

0.100 0.199 0.124 0.341

11 n Z@

X = 1.539 A

0*141 Radiation: K a l and 0.194 0.162 Ka2 Copper 0.545

l'ooo ** Rela t ive i n t e n s i t y 0.206 0.175 0.516 0.213 0.357 0.185 0.248 0.289 0.205 0.140 0.145 0.166 0.145 0.157

based on h ighes t i n t e n s i t y of 1 . 0 0

10

Figure 5. Powder X-Ray Diffraction Pattern of Bendroflumethiazide (Instrument: Norelco)

I I


2.9 pKa A pKa of 8.5320.05 was determined b

using the solubility variation with pH method4<

3. Synthesis

by cyclization of 4-amino-6-trifluoromethyl-m- benzenedisulfonamide (11) with phenylacetaldehydel (111) was e c 1 e by Holdrege, Babel and Cheney . and others 4~ '~r ; ' t6 . Alternate approaches are condensation of 4-amino-6-trifluoromethyl-m-

8,9 benzenedisulfonyl chloride (IV) with phenyl- acetaldehyde (111) in the presence of ammonia , condensation of I11 with phenylacetimine (V) 10, reaction of 4-amino-6-trifluoromethyl-m-benzenedisulfonamide (11) with cetoxystyrene (VI) in the presence of acetic acid" and by hydrogenation of 3-benzyl-6-trifl~oromethyl-7-sulfamoyl-l,2~4- benzothiadiazine 1,l-dioxide (VII) with lithium aluminum hydridel2.

(see Figure 6) The synthesis of bendroflumethiazide (I)

4. Stability and Degradation Bendroflumethiazide, as a solid, appears to be

very stable. The solid, exposed to. 606-C. for a period of two weeks showed no decomposition as measured by I.R. and modified Bratton-Marshal reaction. In ethanol (1 mg/ml), it showed significant changes (22% decomposition) at the end of two weeks at 60 C. In aqueous suspension, almost complete breakdown to the disulfonamide (11, Figurfg6) occurred at the end of one week at 6OOC. .

In alkaline solution (pH 12) bendrof lu-

2@2the methiazide undergoes complete hydro1 sis 21 disulfonamide (11) in one hour at 351: C.

It is also unstable under certain acid conditions . 5 . Drug Metabolic Products - Pharmacokinetics

No drug metabolites have been identified so far. the rat24 and in human beings . In human beings, orally or intravenously administered doses of S35-labeled bendrof lumethiazide a excreted almost quantitatively in 24 hourss5. stability of the trifluoromethyl group was

The Pharmacokinetics ha55 been studied in

The

12

N

x-

V I

l-l

w 0 k a c a,

0

4J

In m

H

H

3

13

H2NSOZ

+

I1 @H2-CH0 H=CHOCOCH

CF3

J"/' I11

@ 0 0

0 0 v1 Y +S4

- C H 2 a 3

H 2 N S 0 L i A l H 4 ' NH2 "'mC! -CH2 -0 H

4

0

C F 3'

V I I NaO/ I k 4 0 H

Q c H -cH=NH SO'C1 +

B NH

I11 v

CF 3 IV

Figure 6 . S y n t h e t i c Pathways t o B e n d r o f l u m e t h i a z i d e


studied in ratsz6. fluoride uptake in the teeth of rats on a carious diet. Bendrof umethiazide was found to be 94%

There was no detectable

protein bound4 i! . 6. Methods of Analysis

6.1 Elemental Analysis

Found : Calc. % (Squibb House Standard Batch 15)

C 42.75 H 3.35 F 13.53 N 9.97 0 15.21 S 15.22

42.63 3.27 13.83 9.92

15.60 -

6.2 Spectrophotometric Analysis The ultraviolet absorption maximum at

273 mp (Ei 565) can be used for the quantitation of bendro lumethiazide in dosage forms23 I 27.

6.3 Colorimetric Analysis Bendroflumethiazide can be quantitatively

assayed in dosage forms by alkaline hydrolysis to- the disulfonamide (11, Figure 6) diazotization, coupling with N-(1-naphthyl) ethylenediamine and determ'nation of the absorption maximum at 515 nm28r3'. used29r31. determine the presence2pS2&he disulfonamide (11) in bendroflumethiazide

Other coupling agents have been This assay can also be used to

6.4 Non-Aqueous Titration An assay based on titration with sodium

methoxide in dimethylf ormamide using p-nitro- benzene-azo-resorcinol as indicator has been developed32. as indicator can also be used2*.

Pyridine as solvent and azo-violet

6.5 Chromatographic Analysis 6.51 Paper Chromatographic

Bendroflumethiazide (Rf 0.76) can be separated from the disulfonamide (11, Figure 6) using methylisobutylketone saturated with formamide as mobile phase and 30% formamide

14

in methanol as stationary phase. For quantitation, the spots are eluted with methanol and the concentration is determined spectrophotometrically (see 6.2)33.

Identification and separation from other thiazide diuretics by paper chromatography has also been reportedl5.

6.52 Thin-Layer Chromatography Thin layer chromatographic systems are compiled in the

following table.

Absorbent

250 Silica Gel G

G 250 Silica Gel G G

" G G

'I G " G " G

n 11

11 II

11 II

11 n

11 I1

I1 11

Silica Gel

Silica Gel Alumina GF-254 (Two Dimensional) Silica Gel Alumina G

Ref. - Solvent System

Benzene-Ethyl Acetate (2:8) and 34

Propanol-2/12N Ammonia (8:2) 34 Propanol-l/12N Ammonia (8 : 2) 34 Butanol-l/12N Ammonia (8 : 2) 34 Pentanol-l/12N Ammonia (8:2) 34 Ethyl Acetate/l2N Ammonia (8:2) 34 Chloroformflethanol (8 : 2) 34 Cyclohexane/Glacial Acetic Acid/ 34

Ethyl Acetate-Methanol Ammonium Hydroxide (85 : 10 : 5)

Acetone (4:1:5) Toluene/xylene/l-4-Dioxane/Isopropanol/ 35 25% Ammonia (50 : 10 : 30 : 10) Ethanol/Chloroform/Heptane (1:l:l) 36

Bu tanol

Ethanol 38

(1st) Ethanol: (2nd) Chloroform/ 37

Ethanol containing 1.5% Water 37

Rf

0.91 -

0.88 0.93 0.70 0.54 0.98 0.52 0.98

0.98 - 0.71 -

Absorbent

Silica Gel G Silica Gel

It I1

I1 I1

Silica Gel

Silica Gel

Silica Gel

Silica Gel G " G I1

11 G

Ref. - Solvent System

Ethanol/Benzene (80:20) 38 Ethyl Acetate/Benzene (8:2) 39 Benzene/Ethyl Acetate/25% Ammonia/ 39

Ethyl Acetate/Benzene/25% Ammonia/ 39

Ethyl Acetate/Benzene/Ammonia/ 39

Methanol (20 : 80 : 1 : 10)

Methanol (60 : 40 : 20)

25% Methanol (50:40:2:10) n-Hexane/Acetone/Ethylamine (60:30:10) 39 n-Hexane/Acetone/Diethylamine (40:40:20) 39

(80:15:5) Benzene/Ethyl Acetate (2:8) 40 Ethyl Acetate/Methanol/Ammonium 40 Hydroxide (8 5 : 10 : 5 Ethyl Acetate/Benzene (8:2) 41

Chloroform/Methanol/Diethylamine 39

Rf - - 0.70 0.75

0.40

0.68

0.10 0.46 0.75

0.82 0.82

0.98

Identification of oral hypoglycemic and diuretic drugs by TLC using metal ions has been described42.

7. Identification and Determination in Biological Fluids

In plasma: Colorimetrically12

In urine: Calorimetrically 24 Radioactivity 25 TLC 39 r 40 t 41

8. Miscellaneous 43,44 Pharmaceutical preparation of bendroflumethiazide have been patented

BEN DROFLUMETH IAZIDE

9.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

References

C. T. Holdrege, R. B. Babel and L. C. Cheney, J. Am. Chem. SOC. 81, 4807 (1959) A. C. Kennedy, K. b. Buchanan and C. Cunningham, Lancet 1960-1, 1267. F. Lund, W. 0. Godtfredsen, Brit. Pat. 863,474 (1961); C.A. 55, 19,971d (1961) also U . S . Pat. 3,392,168 n968). J. G. Topliss, M. H. Sherlock, F. H. Clarke, M. C. Daly, B. W. Pettersen, J. Lipski and N. Sperber, J. Org. Chem. 26, 3842 (1961). J. Klosa and H. Voigt, J. =act. Chem. 16, 264 (1962), also Ger. (East) Pat. 31,48r (1964); C.A. 63, 14,887g (1965), Brit. Pat. 1,049,322 (1960) C.A. 66, 65,5343 (1967) and Belg. Pat. 631,232 (1963); C.A. - 60, 14,528b (1964). K. Abildgaard, Fr. Pat. 1,586,602 (1970) C.A. 74, 100,112J (1971). E. Schoenfeldt and H. Thorsteinson, Brit. Pat. 879,592 (1961) ; C.A. - 57, 844f (1962). L. C. Cheney and C. T. Holdrege, Fr. Pat. 1,368,708 (1964); C . A . 62, 9157c (1965). J. Klosa, Brit. Pat. 1,063,102 (1967); C.A. 67, - 11,514e (1967). Brit. Pat. 898,109 (1962); C.A. - 57 12,516a (1962). Fr, Pat. 1,388,607 (1965); C.A. - 63, 7,024b (1965). Gl Hurka, Austrian Pat. 253,513 (1967); C.A. 67, 11,512~ (1967). J. Duxam, The Squibb Institute, Personal Communjcation. V V

14. J. Kracmar and M. Lastovkovi, Pharmazie - 25, 464 (1970); Cesk. Farm. 20, 287 (1971); C.A. 76, 144,8783 (1972).

15. V. B.Pilsbury and J. V. Jackson, J. Pharm. Pharmac. 18, 713 (1966).

16. F. J. Lunrand W. Kobinger, Acta Pharmacol. et Toxicol. - 16, 297 (1960).

17. H. Jacobson, The Squibb Institute, Personal Communication.

18. J. Ueda, Japan Pat. 25,692 (1963); C.A. - 60, 9,107f (1964).

19. M. Everhard, The Squibb Institute, Private Communication.

17


20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

30.

31.

32. 33.

34.

35.

36.

37.

38.

39.

40.

41. 42.

K. Matsushima and K. Kiyota, Iryo - 23, 1561 (1969); C.A. 73, 112,897m (1970). J. C. Turner, A. W. Nichols and J. E. Sloman, The Pharmaceutical J. 1970, 622. J. Dobrecky and B. G. Gonzalez, Rev. Farm. - 114, 34 (1972); C.A. 78, 7,794f (1973). A. I. Cohen, The Squis Institute, Personal Communication. J. J. Piala, J. W. Poutsiaka, C. J. Smith, J. C. Burke and B. N. Graves, J. Pharmacol. Expt. Therapeutics - 134, 273 (1961). H. R. Brettell, J. G. Smith and J, K. Aikawa, Arch. Internal. Med. 113, 373 (1964). G. Hasselmann and K. m o l d , J. Pharm. Pharmacol. 15, 339 (1963). A. M. Leal and M. B. S. Ramos Lopez, Rev. Port. Farm. 2, 48(1963); C.A. - 59, 11,188e (1964). National Formulary XIV, p. 61ff (1975). J. Bermejo, Galenica Acta - 14, 255 (1961); C.A. 56, 10,286e (1962). M. Ghxardoni and M. Fedi, Boll. Chim. Farm. - 101, 26 (1962); C.A. 57, 9582 (1962). J. F. Magalhaes and M.G. Piros, Rev. Farm. Bioquh. Univ. Sao Paulo - 8, 273 (1971); C.A. 75, 121,466~ (1971). H. C.Chiang, J. Pharm. Sci. 5 0 , 885 (1961). H. Roberts, The Squibb Institute, Personal Communication. P. J. Smith and T. S. Herman, Anal. Biochem. - 22, 134 (1968). K. C. Guven and S. Cobanlar, Ecsacilik Bul. - 9, 98 (1967); C.A. 67, 102,839f (1967). S. Gecgil, Ecsacmik Bul. - 7, 100 (1965); C.A. 64, 14,032d (1966). M. Duzene and L. Lapiere, J. Pharm. Belg. - 20, 275 (1965); C.A. 64, 7,970d (1966). R. Adam and C. L. Lapsre, J. Pharm. Belg. - 19, 79 (1964); C.A. 61, 8,134 (1964). R. Maes, M. Gijbxs and L. Larvelle, J. Chromatogr. 53, 408 (1970). D. Sohn, J. Simon, H. Moheeb, G. Shali and R. Tolba, J. Chromatogr. 87, 570 (1973). B. G. Osborne, J. ChromatGr. 70, 190 (1972). S. Agarwal, M. Walash, and M. I. Blake, Indian J. Pharm. - 35, 181 (1973).

18

BENDROFLUMETHIZAIDE

4 3 . M. G o l d b e r g , U.S. P a t . 3 , 2 6 5 , 5 7 3 ( 1 9 6 0 ) ; C.A. 6 5 , 1 3 , 4 5 9 ~ ( 1 9 6 6 ) .

4 4 . C . RifSirin and M. G o l d b e r g , U . S . P a t . 3 , 4 2 6 , 1 3 0 ( 1 9 6 9 ) ; C.A. 7 1 , 33,41513 ( 1 9 6 9 ) .

4 5 . A. xgren and T. Bzck, A z a . Pharm. S u e c i n a - 10, 2 2 3 ( 1 9 7 3 ) .

L i t e r a t u r e surveyed through June 1 9 7 4 .

19

CEPHRADINE

Klaus Florey

KLAUS FLOREY

1.

2.

3. 4.

5. 6.

7. 8. 9.

TABLE OF CONTENTS Description 1.1 Name 1.2 Definition 1.3 Formula and Molecular Weight 1.4 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectra 2.2 NMR Spectra 2.3 Mass Spectrum 2.4 Ultraviolet Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Thermogravimetric Analysis 2.9 Ionization Constant, pK 2.10 Solubility 2.11 Crystal Properties S yn the s i s Stabili ty-Degradation 4.1 Bulk Stability 4.2 Solution Stability Drug Metabolism, Pharmacokinetics Methods of Analysis 6.1 Elemental Analysis 6.2 Microbiological Analysis 6.3 Iodometric Analysis 6.4 Spectrophotometric Analysis 6.5 Fluorometric Analysis 6.6 Colorimetric Analysis 6.7 Chromatographic Analysis

6.71 Paper 6.72 Thin-layer 6.73 Column

Determination in Body Fluids and Tissues Determination in Pharmaceutical Preparations References

22

CEPHRADINE

1. Description 1.1 Names

Cephradine is 7-,@ ( - ) -2-amino- (1,4 cyclohexadien-l-yl)acetamido-3-rnethyl-8-0~0-5- thia-l-azabicyclo-oct-2-ene-2-carboxylic acid; also 7-p-amino-2-(1,4 cyclohexadienyl) acetamido7-desacetyl-cephalosporanic acid.

1.2 -Definition Cephradine, unless specified otherwise,

is defined as a hydrated form containing 3-6% of water (for further discussion see Section 2.11).

1.3 Formula,Molecular Weiqht

/+ U C H I - 8 - ! jT~++-cH3

NH2

0 CO2H

C16H19N304S Molecular Weights: 349.41 anhydrous

367.43 monohydra te 385.45 dihydrate

1.4 Appearance,Color,Odor White crystalline powder, odorless to

slightly sulphurous.

2. Physical Properties 2.1 Infrared Spectra

Spectra of cephradine (Batch #NN005NC) (Figure l), cephradine dihydrate(house standard batch #NNOO5NB) (Figure 2), cephradine monohydrate recrystallized from acetonitrile-water (sample MSA 38719) (Figure 3 ) and cephradine monohydrate recrystallized from methanol (sample MSA 38680) (Figure 4) are presented'.

23

FREQUENCY (a')

WAVELENGTH (MICRONS)

Figure 1. Infrared Spectrum of Cephradine Batch #NN005NC, KBr Pel let , Instrument, Perkin E l m e r 21.

Figure 2. I n f r a r e d Spectrum of Cephradine Dihydrate(House Standard Batch # NN005NB) KBr P e l l e t , I n s t rumen t , Perkin E l m e r 621.

Figure 3. Infrared Spectrum of Cephradine Monohydrate Recrystallized From Acetonitrile (Water Sample MSA 38719) KBr Pellet, Instrument, Perkin Elmer 621.

Figure 4. Infrared Spectrum of Cephradine Monohydrate, Recrystallized From Methanol (Sample MSA 38680) KBr Pellet, Instrument, Perkin Elmer 621.

KLAUS FLOREY

D 2 0

2 . 2 NMR S p e c t r a NMR s p e c t r a i n CF3COOH (F igure 5 ) and

( F i g u r e 6 ) a r e presented2 . I n t r i f l u o r o a c e t i c a c i d (TFA), t h e

compound ex i s t s i n p r o t o n a t e d form w i t h the NMR s h i f t o f the NH+ a t Z2.44 w i t h r e f e r e n c e t o in - t e r n a l t e t r a m e t h y l s i lane(TMS). The imide NH p ro ton appea r ing a s a d o u b l e t (J = 9.0 H z ) a t Z 2 . 0 0 i s coupled t o one o f B-lactam r i n g protons, which appea r a s a q u a r t e t (J = 9.0 ,4 .0 Hz) a t C4 .20 . The second @-lactam p r o t o n resonance i s a d o u b l e t (J = 4.0 H z ) a t % = 4.74. The S-CH2 group p r o t o n s appear a s AB q u a r t e t (J = 18.0 Hz) a t C 6 . 3 6 and 6.54 w h i l e t h e methyl group appea r s a t C7.61. I n t h e d ihydrophenyl r i n g , t h e o l e f i n i c p r o t o n s appear a t f 3.62 (1 H) and 4 .21 ( 2 H ) . T h e resonance a t Z 7.13 i s a s s i g n e d t o f o u r p ro tons of t h e d ihydro r i n g . F i n a l l y the methine p ro ton of the CHNHf group appea r s a s a m u l t i p l e t a t f 5 . 0 0 . The spectrum o b t a i n e d i n deuter ium o x i d e c o n t a i n i n g a drop of sodium deuter ium oxide was r eco rded us ing 3 - ( t r i m e t h y l s i l y1 ) -1 -p ropansu l - f o n i c a c i d sodium s a l t a s an i n t e r n a l r e f e r e n c e . The amine and NH group p r o t o n s are exchanged w i t h D 2 0 . I n t h e dihydrophenyl r i n g , t h e chemical s h i f t s a r e t w o o l e f i n i c hydrogens a t C 4 . 2 5 , one o l e f i n i c p r o t o n a t X 4 . 1 5 and t h e o t h e r f o u r hydrogens a t Z 7 . 3 1 . The @-lactam r i n g p r o t o n s a r e a p a i r of d o u b l e t s (J = 4.0 H z ) a t t 4.42 and 4 . 9 4 The S - m 2 group p r o t o n s appear a s a AB q u a r t e t (J = 18 .0 ) a t C 6 . 8 1 and 6.43. The methyl resonance is a d o u b l e t a t C 8 . 2 1 and may be a r e s u l t of p a r t i a l i s o m e r i z a t i o n of the double bond i n t h e b a s i c s o l u t i o n . F i n a l l y , t h e methine (CHNHd p ro ton h a s a chemical s h i f t o f 6.00C . amount of r e s i d u a l cepha lex in by comparing t h e a r e a under t h e phenyl ro ons ( .C2.50) and o l e f i n i c p r o t o n s ( z 3. E4) 5 .

2

NMR can a l s o be used t o de te rmine the

28

F i g u r e 5. NMR Spectrum of Cephradine Batch NN005NC i n CF3COOH Ins t rumen t : v a r i a n XL-100.

., , , - n I :

F i g u r e 6. NMR Spectrum of Cephradine Batch NNO05NC i n D20 Ins t rumen t : Var ian XL-100.

CEPHRADINE

2.3 Mass Spectrum Because of t h e low v o l a t i l i t y of ceph-

r a d i n e , a t r i m e t h y l s i l y l d e r i v a t i v e was made us ing t h e r e a g e n t BSA (N,O-Bis- (Tr imethyl s i l y l ) a c e t a m i d e ) . The low r e s o l u t i o n mass spectrum ( F i g u r e 7 ) i n d i c a t e s t h a t t w o and t h r e e t r i m e t h y l s i l y l g roups were added. The compound having t w o t r i m e t h y l s i l y l g roups was t h e predominant p roduc t w i t h molecular i o n a t m / e 493. The loss of CH3 from t h e molecu la r i o n ( t y p i c a l o f t r i- methyl s i l y l g roups) y i e l d e d an i o n a t m / e 4783. The d a t a s u p p o r t s t r u c t u r e I.

U An i n t e n s e i o n a t m/e 180 (see spectrum) cor responds t o 11,

I1 a n o t h e r i n t e r v a l i o n a t m / e 230 cor responds t o 111.

H

C - 0 Si(CH3I3 II

O I11 which i s a t y p i c a l f ragment o b t a i n e d from 8-lactam r i n g f r agmen ta t ions3 .

31

1 FIB

96l

815 >

f.n Z lL 68 k- Z - 56l Id

t. 715

2 915

j 315 $ 3 ;

K

zh:

161

/

MASS/CHARGE

F i g u r e 7. Low Resolu t ion Mass Spectrum of Tr imethyl S i l y l D e r i v a t i v e of Cephradine. I n s t r u m e n t : A E I MS-902.

2.4 U l t r a v i o l e t Spectrum Cephradine e x h i b i t s a s i n g l e absorbance peak a t 262 nm

= 220 f o r b a t c h ~ N 0 0 5 N c ) ~ . 2 .5 O p t i c a l R o t a t i o n

The s p e c i f i c r o t a t i o n of t h e house s t a n d a r d ( d i h y d r a t e , b a t c h #NN005NB) i n a pH 8 b u f f e r was found t o be + 88.3O(as i s ) 5. s p e c i f i c r o t a t i o n of seven b a t c h e s of cephrad ine a t a c o n c e n t r a t i o n of 0.5% i n 0.1M a c e t a t e b u f f e r (pH 4.6) and c a l c u l a t e d on an anhydrous b a s i s was found t o be + 91.6O w i t h a range from + 89.22O t o 92.90°. The e f f e c t o f pH on r o t a t i o n was found t o be s l i g h t , a s can be seen from Table 15.

The ave rage

Tab le 1 27O L 3 -7 conc. Approx. 1%

D

pH I n i t i a l 1 Hour 3 Hours 5 H o u r s 24 Hours 4 .01 + 77.450 + 79.45O + 76.58O + 78.45O + 75.380 5.00 + 78.250 + 78.75O + 77.550 + 77.65O + 75.86O

7.00 + 80.47O + 78.37O + 77.78O + 76.68O + 76.95O 8 .20 + 87.24O + 86.04O + 85.05O + 85.74O + 84.32O

6.03 -t 78.32O + 79.10° + 78. loo + 77.83O + 77.770

KLAUS FLOREY

2.6 M e l t i n q Range Cephradine melts with decomposition.

The melting range (USP) of cephradine d ihydra te house s tandard (Batch NN005NB) was 183-185O. The melting range of t y p i c a l batches of cephradine has va r i ed from 175-192O.

2 .7 D i f f e r e n t i a l Thermal Analysis The thermogram of cephradine e x h i b i t s a

s i n g l e exotherm a t approximately 200-203O, depending on hea t ing r a t e . This exotherm i n d i c a t e s oxida t ive decomposition accompanied by melting6. On the o t h e r hand t h e thermogram of cephradine d ihydra te e x h i b i t s two endothermic peaks a t about 90° and a t about 102O which a r e r e l a t e d t o the hydrat ion of t h e compound. A decomposition exotherm i s observed a t about 20OoC. The d i f f e rence i n temperature between t h e two endothermic peaks i s a measure of t he stress (gr inding, hea t ing) leading t o dehydration, to which the sample i s subjected6.

Thermograms of cephradine dihydrate , cephradine, cephalexin 7 , and cephradine monohydrate ( r e c r y s t a l l i z e d from ace toni t r i le -water ) ' a r e presented i n Figure a6. endotherm f o r cephradine suggests t h a t cephradine i s no t a t r u e hydrate and t h e water is n o t s t r u c t u r a l l y bound i n t h e c r y s t a l - l a t t i c e ( see a l s o s e c t i o n 2 .11) . On t h e o t h e r hand t h e d i - hydrate , t h e monohydrates obtained by r e c r y s t a l l i z a t i o n from ace ton i t r i l e -wa te r7 ( sharp endotherm a t 150-152O) and methanol8 (Endotherm a t 152O)as we l l a s cephalexin7 a r e t r u e hydra tes?

DTA has a l s o been used a s a screening technique t o s tudy the i n t e r a c t i o n of cephradine with p o t e n t i a l adjuvants t o a pa ren te ra l formula t ion9 .

The absence Of an

34

CEPHR AD I N E

Figure 8. D i f f e ren t i a l The rmog rams

35

KLAUS FLOREY

2 . 8 Thermogravimetr ic A n a l y s i s By thermogravimet r ic a n a l y s i s (TGA) of

t y p i c a l batches o f cephrad ine from 3 t o 6% v o l a t i l e s ( w a t e r ) , w e r e found ( t h e o r y f o r monohydrate w a t e r 4.93%). The d i h y d r a t e ( b a t c h B49988D)by TGA gave 9.8% v o l a t i l e s ( t h e o r y f o r d i h y d r a t e w a t e r 9.35%). VPC was used t o confirm the v o l a t i l e compound a s water48. The 13% loss shown by TGA a t abou t 200-210°(decomposition p o i n t ) i s assumed t o be due t o deca rboxy la t ion6 .

2.9 I o n i z a t i o n Cons tan t , pK

pK1 of 2.63 and pK2 o f 7 .27 w a s found6. By t i t r a t i o n w i t h sodium hydroxide a

2.10 S o l u b i l i t y There i s no d i f f e r e n c e i n s o l u b i l i t y of

the v a r i o u s c r y s t a l forms. The s o l u b i l i t y of cephrad ine i n b u f f e r s a t d i f f e r e n t pH v a l u e s i s r e p o r t e d i n Tab le 2 .

Table 2 S o l u b i l i t y o f Cephradine a t D i f f e r e n t pH Values

pH of S a t u r a t e d S o l u b i l i t y p H o f b u f f e r S o l u t i o n mq/ml

4.00 4.02 35.8 water 4 . 9 1 21.3

60% s u c r o s e 5 . 0 1 7 . 1 5 .00 5.04 2 1 . 1 6 .03 5.90 20.5 7 .20 6.12 2 8 . 2 8. 20 7 .09 36.7 9. i a 7.41 49 .6

Cephradine i s p r a c t i c a l l y i n s o l u b l e i n e t h y l e t h e r , chloroform, benzene and hexane. The a n t i b i o t i c i s v e r y s l i g h t l y s o l u b l e i n ace tone and a b s o l u t e e t h a n o l . I t i s f r e e l y so luble i n propylene g l y c o l . T h e s o l u b i l i t y te rminology used i s taken from U S P X V I I I .

c ephrad ine and i t s d i h y d r a t e as w e l l as The i n t r i n s i c d i s s o l u t i o n r a t e s of

36

CEPHRADINE

cephalexin were found i d e n t i c a l a t an a g i t a t i o n The observed i n t r i n s i c

d i s s o l u t i o n r a t e s (mg/ml/min/cm2) a r e a s follows : i n t e n s i t y of 100 rpm 10 .

Compound Temperature Cephradine 2 2oc

37oc

d ihydra te 2 2oc 37OC

Cephradine

Cephalexin 2 2% 3 7%

2 . 1 1 Crys ta l P rope r t i e s

I n t r i n s i c Disso lu t ion Rate

0.08 0 . 1

0 . 0 8 0.09

0.08 0.09

There i s considerable evidence f o r polymorphism and four polymorphs o r r a t h e r pseudopolymorphs have been charac te r ized so f a r .

1. ) Cephradine, hydrated. Although the water content v a r i e s from 3-6% i t i s no t a s to i ch iomet r i c hydrate s i n c e t h e water apparent ly moves f r e e l y i n the c r y s t a l l a t t i c e ( s e e s e c t i o n 2 . 7 ) . I t i s obtained from aqueous so lu t ion . Anhydrous cephradine has been prepared and was found t o be very s t a b l e and r e s i s t a n t t o oxida t ion t o cephalexin, when kept anhydrous(see sec t ion 4 . 1 ) . However, i t cannot be properly charac te r ized , s i n c e i t immediately hydrates on exposure t o the atmosphere.

compound which c r y s t a l l i z e s from aqueous s o l u t i o n under con t ro l l ed c o n d i t i o n s l l , i s a t r u e dihy- d r a t e ( see sec t ion 2 . 7 ) . I t i s very s t a b l e and r e s i s t a n t t o oxidat ion t o cephalexin. However, on dehydration ( loss of c r y s t a l s t r u c t u r e ) t h e d ihydra te becomes very uns tab le ( see s e c t i o n 4.1).

The s t r u c t u r e of cephradine d ihydra te was determined by s i n g l e c r y s t a l x-ray d i f f r a c - t i o n i n order t o i n v e s t i g a t e t h e pos i t i on of t he water molecules i n the c r y s t a l and the hydrogen

2. ) Cephradine dihydrate . This

37

KLAUS FLOREY

bonding of the water t o var ious s i tes i n the cephradine molecule50. The hydrogen bonding p a t t e r n i s ind ica t ed i n f i g u r e 9 . There are t w o molecules i n the u n i t cel l . The p ro jec t ion of one is shown. The second i s symmetry r e l a t e d by 180° r o t a t i o n i n t he plane of p ro jec t ion , and t r a n s l a t i o n of 1 /2 of t he b r epea t , Atoms of t h e second molecule involved i n hydrogen bonding a r e ind ica t ed with a prime nota t ion .

follows: Water oxygens a r e numbered 69 and 71. Water oxygen #69 bonds with t h e @-lactam carbonyl of molecule 1, the amide n i t rogen 13”, of molecule 2 , and wi th t h e second water molecule # 71. Water oxygen #71 Ponds with one of t h e carboxyl oxygens, 64 , of molecule 2 , and with water molecule #69. The amino n i t rogen , N5’ of molecule 2 bonds with both carboxyl oxygens.

from a c e t o n i t r i l e water8. This i s a t r u e hydra te (see sec t ion 2 . 7 ) .

hydrous methanol8 a l s o appears t o be a t r u e monohydrate , al though another one ha l f mole of unbound water was a l s o present . N o r e s i d u a l methanol was found.

1 2 a tendency t o form s o l v a t e s s i n c e a c e t o n i t r i l e and ethylene g lyco l so lva te s have been observed . Powder x-ray p a t t e r n s of the four c r y s t a l forms a r e presented i n Tables 3-613.

and ce l l conten t determinat ions of t he four c r y s t a l forms w e r e made by s i n g l e x-ray c r y s t a l l - ographyl and a r e presented i n Table 7.

c r y s t a l forms, see s e c t i o n s on I R ( 2 . 1 ) , DTA(2.7) and Bulk S t a b i l i t y ( 4 . 1 ) .

The bonding may be summarized a s

3 . ) Cephradine monohydrate r e c r y s t a l l i z e d

4 . ) Cephradine r e c r y s t a l l i z e d from an-

Cephradine, genera l ly , seems t o have

The u n i t c e l l dimension, space group

For f u r t h e r d i scuss ion on these

38

Ia oc

0 W

F i g u r e 9. Dis tr ibut ion of water molecules i n cephradine dihydrate.

KLAUS FLOREY

Table 3 Powder X-Ray P a t t e r n of Cephradine,Hydrated

Batch NN005NC d

15.80 11.90 8.04 5.98 5.61 5.34 4.92 4.66 4.48 4.32 4.20 4.00 3.93

1/10 14.1 47.4 15.4 19.2 24.4 100.0 21.8 11.5 21.8 57.7 26.9 30.8 14.1

Table 4 Powder X-Ray Pat t e r n

Batch NN005NB d 11.60 10.50 8.75 7.05 6.20 6.00 5.80 5.61 5.23 5.10 4.68 4.55 4.45 4.25 3.94 3.87 3.80

1/10 35.4 15,6 10.4 19.8 37.5 17.7 24.0 56.3 20.8 37.5 7.3 9.4

26.0 17.7 9.4 12.5 28.1

d 3.76 3.61 3.47 3.33 3.24 3.18 3.08 2.90 2.80 2.74 2.67 2.57 2.43

1/10 14.1 35.9 12.8 17.9 12.8 11.5 25.6 12.8 11.5 10.3 14.1 9.0 11.5

of Cephradine Dihydrate (House Standard)

d 1/10

3.76 22.9 3.67 26.0 3.55 100.0 3.45 43.8 3.42 46.9 3.19 28.1 3.08 30.2 2.92 54.2 2.80 10.4 2.68 15.6 2.61 15.6 2.57 13.5 2.49 14.6 2.46 17.7 2.39 7.3 2.31 12.5

CEPHRADINE

Table 5 Powder X-Ray Pattern of Cephradine Monohydrate

Recrystallized from Acetonitrile-Water

d 9.92 8.83 6.96 6.45 5.64 5.43 4.92 4.84 4.74 4.39 4.23 4.00 3.91 3.64 3.41 3.36 3.22 3.10

Sample #38720 1/10 d

100.0 3.05 94.0 3.02 82.0 2.96 24.0 2.90 80.0 2.80 35.0 2.77 15.0 2.75 22.0 2.70 91.0 2.65 45.0 2.60

100.0 2.47 45.0 2.42 35.0 2.38 21.0 2.35 32.0 2.24 29.0 2.21 24.0 2.14 61.0

1/10 17.0 18.0 25.0 14.0 17.0 35.0 14.0 13.0 18.0 26.0 11.0 26.0 23.0 31.0 25.0 17.0 25.0

41

KLAUS FLOREY

Table 6 Powder X-Ray Pattern of Cephradine Monohydrate

Recrystallized from Methanol Sample # 38708

d 11.20 8.83 8.18 7.83 6.80 6.23 5.82 5.64 5.21 4.86 4.74 4.59 4.34 4.19 4.13 4.00 3.89

1/10 100.0 17.0 19.0 10.0 18.0 19.0 25.0 13.0 14.0 37.0 45.0 43.0 32.0 34.0 25.0 26.0 32.0

d 3.78 3.63 3.51 3.30 3.17 3.08 2.94 2.82 2.74 2.65 2.62 2.54 2.45 2.42 2.36 2.30

1/10 14.0 26.0 33.0 24.0 15.0 16.0 26.0 18.0 21.0 17.0 18.0 16.0 14.0 14.0 17.0 14.0

42

Table 7

- CELL coNsTAms-> SPACE MEASURED C E L L COJTI'ENTS

12 cephradine

P 0

4 cephradine Acetonitrile

Recrystallized from 17.58 9.4 21.6 90 3568 P212121 1.33 8 cephradine Me th an o 1 8 water

Monohydra t'e I

3. Synthesis

toxycephalosporanic acid (7-ADCA) (11) with a protected derivative of dihydrophenylglycine (I),

Cephradine (111, Figure 10) is synthesized by coupling 7-aminodesace-

Figure 10 Synthetic Pathway to Cephradine

CH - COOH Intermediate + H2N 0 I hz COOH

0:" - CO - mJ-<J

/ CH3 m2

0 COOH

I11 such as the tert.-butoxylcarbonyl derivative which can be converted to a mixed anhydride with ethylchloroformate and reacted with 7-ADCA14. Cephradine can also be made by forming the methyl acetoacetic ester enamine derivative of dihydrophenylglycine-which is converted to a mixed

CEPHRADINE

anhydr ide w i t h benzoyl c h l o r i d e p r i o r t o coupl ing w i t h 7-ADCAI2.

I t a l s o can be c r y s t a l l i z e d from wa te r a l o n e , a s w e l l a s from o t h e r s o l v e n t s ( s e e s e c t i o n 2 . 1 1 ) .

Cephradine is then cr s t a l l i z e d from a b i p h a s i c MIBK-aqueous s o l u t i o n Y2 .

4. S t a b i 1 i ty-Degrada t i o n 4 . 1 Bulk S t a b i l i t y

Cephradinqwhen k e p t under d r y and c o o l s t o r a g e c o n d i t i o n s , h a s shown reasonab le b u l k s t a b i l i t y 1 2 . Like o t h e r 1 ,4 cyc lohexadienes such a s 2,5 d i h y d r ~ p h e n y l a l a n i n e ~ ~ , cephradine i s prone t o a slow r a t e of o x i d a t i o n of t h e cyc lo- hexadiene r i n g t o t h e benzenoid r i n g . The e x a c t mechanism of t h i s r e a c t i o n i s n o t known, b u t i n a d d i t i o n t o oxygen and wa te r , t race me ta l s such a s i r o n seem t o have an a c c e l e r a t i n g e f f e c t .

The o x i d a t i o n t o cepha lex in a s w e l l as d e g r a d a t i o n (loss of b iopotency) can be p reven ted o r s i g n i f i c a n t l y reduced by s t o r a g e a t low tempera ture and exc lus ion of oxygen, a s w e l l a s removal of water . Th i s l a s t method, however, i s i m p r a c t i c a l due t o t h e ex t remely hygroscopic n a t u r e of anhydrous cephradine12.

On t h e o t h e r hand cephrad ine d i h y d r a t e be ing a t r u e s o l v a t e (see s e c t i o n 2 , l l ) w h e r e water cannot move f r e e l y i n t h e c r y s t a l l a t t i c e e x h i b i t s remarkable r e s i s t e n c e t o cepha lex in format ion , loss of b iopotency and c o l o r develop- m e n t d u r i n g extended s t o r a g e under a i r l 2 . d i f f e r e n c e be tween cephradine and i t s d i h y d r a t e i s i l l u s t r a t e d i n Table 812.

of cephradine d i h y d r a t e under a v a r i e t y of c o n d i t i o n s , d r y i n g a t h i g h e r t empera tu res o r c e r t a i n k i n d s of m i l l i n g t h i s e x c e l l e n t s t a b i l i t y i s n o t ma in ta ined lz .

11

The

However upon p a r t i a l o r f u l l dehydra t ion

45

KLAUS FLOREY

Table 8 Average Bulk S t a b i l i t y Data for Three Batches of

Cephradine and f o r Three Batches of Cephradine Dihydrate Af t e r 9 Months of Storage Under A i r

Cephradine ("as i s " )

I n i t i a l 5Oc RT 4OoC 5OoC Bioassay, mcg/ml 949 947 942 924 849 Cephalexin, % 2 . 7 3.0 3.5 4 .6 7.0

Bioassay, mcg/ml 909 870 870 897 906 Cepha lex in , % 2.3 2.3 2.3 2 . 2 2.4

Cephradine Dihydra t e ("as i s")

Cephradine i s moderately l i g h t s e n s i t i v e . When exposed t o U.V. l i g h t , t he s o l i d turns yellow on t h su r face b u t no loss of bio- a c t i v i t y w a s noted . The s e n s i t i v i t y of cephalosporin C t o l i g h t has been previously no tedl6.

products of s o l i d cephradine have been i d e n t i f i e d TLC examination of degraded (loss of biopotency) samples revealed a m u l t i p l i c i t y of U.V. absorbing and f luo rescen t spots17.

f 2

Other than cephalexin, no degradation

4 . 2 S t a b i l i t y i n Solu t ion I n aqueous so lu t ion cephradine tends t o

be q u i t e s t a b l e a t pH 4.0 and below and much l e s s s t a b l e a t h igher pH u n i t s . A pH-s tab i l i ty p r o f i l e

6 is presented i n Table 9 . Cephradine was found to be f u l l y poten t for at l e a s t e i g h t hours i n a v a r i e t y of pa ren te ra l in fus ion solut ions23.

Although cephradine, l i k e o t h e r cephal- osporins is much more r e s i s t a n t t o opening of t he B-lactam r ing by a l k a l i than p e n i c i l l i n s , t h e r i n g does open up with subsequent f u r t h e r degradation, Ring opening a l s o leads the loss of U.V. absorbance a t 262 nm.

46

Table 9 S t a b i l i t y of Cephradinein Phosphate Buffer

a t Room Temperature

Percent of Remaining B i o a c t i v i t y * p H of Solu t ion 2 days 4 days 7 days 10 days 14 days

4.0 6.0 8.0 10.0

95.9 105.1 99.3 99.3 95.2 73.0 86.9 69.5 30.2 18. 5 67 .1 43 .1 24.5 13.4 10.9 64.0 41 .1 25.2 13. 3 11.7

* ph of samples a t s t a r t of study. The pH of t h e high pH samples d r i f t e d

toward lower pH va lues a s t h e s tudy progressed. 5

One of t h e a l k a l i n e degradat ion products p r e c i p i t a t e s from the s o l u t i o n and has been i d e n t i f i e d a s 2-[6-(1,4 cyclohexadien-l-yl)-2, 5-dioxo-3-piperazinyl~-5,6-dihydro-5-methyl-2H-l,3-thiazine-4-carboxylic ac id , sodium s a l tI8.

KLAUS FLOREY

The B-lactam r i n g of cephradine i s q u i t e re- s i s t a n t t o p e n i c i l l i n a s e , b u t opens r e a d i l y w i t h cepha lospor inase l9 .

s t u d i e s w i t h a 2% s o l u t i o n a t pH 1 .6 , h e l d a t 60°C, demonstrated t h a t t h e r e i s no 0-lactam r i n g opening. However p o s s i b l e s h i f t i n g of t h e double bond from carbons 2-3 t o 3-4 was observed by NMR and confirmed by loss of U.V. absorbancy a t 262 nm. A f t e r 93 hours NMR i n d i c a t e d a 50% double bond i somer i za t ion2 . Very v igorous t r ea tmen t w i t h s t r o n g a c i d w i l l l e a d a t l e a s t i n p a r t t o a s p l i t o f t h e amide l i nkage t o form 7-ADCA and dihydrophenylglycine12. This c leavage can a l s o be achieved enzymat i ca l ly w i t h p e n i c i l l i n acylase19 . pH 6 v igorous a e r a t i o n o r exposure t o room l i g h t d i d n o t a c c e l e r a t e degradation2'. hours of exposure t o a Hanovia U.V. lamp of an aqueous s o l u t i o n (pH 5 ) of cephradine more than doubled t h e cephalex in c o n t e n t w i t h l i t t l e change i n biopotency12. be s t a b l e f o r a t l e a s t t h i r t y days i n f rozen ( -S0C) human s e r u m and u r i n e . l o s s was d e t e c t e d by r e p e a t e d thawing and re f reez ing2 ' . w i th serum a t 37OC f o r 6 hours t h e r e was a 20% loss of a c t i v i t y . I n c u b a t i o n w i t h whole b lood under t h e same c o n d i t i o n s caused l i t t l e o r no loss of b iopotency22.

of cepha lospor ins c o n t a i n i n g a phenylg lyc ine moiety ( cepha log lyc in and t o a l e s s e r degree cephalex in and cephradine) i s p r o g r e s s i v e l y 10s t i n t h e presence of c u p r i c i on . Th i s degrading e f f e c t of c u p r i c i on can be i n h i b i t e d by d-penic i l lamine .

On t h e a c i d s i d e NMR s t a b i l i t y

I n a phosphate b u f f e r a t

However 10

Cephradine was found t o

N o s i g n i f i c a n t

On t h e o t h e r hand when incubated

I t was found5I, t h a t t h e a c t i v i t y

40

CEPHRADINE

5. Druq Metabolism

a d m i n i s t e r e d t o e i g h t normal male s u b j e c t s a s a s i n g l e o r a l dose i n a b i o a v a i l a b i l i t y s tudy24. Serum and u r i n e samples were a s sayed i n a double b l i n d f a s h i o n f o r b i o l o g i c a l a c t i v i t y and i n an open f a s h i o n f o r r ad iochemica l a c t i v i t y .

(7.0 2 1.0 kgm/ml - SE by b i o a s s a y and 7.8 f 0.9 pgm/ml - SE by r a d i o a s s a y ) o c c u r r e d 55 minutes a f t e r dos ing and dec reased w i t h a b i o l o g - i c a l h a l f - t i m e o f 40 minutes . The cumula t ive a r e a s under the cu rves f o r cephrad ine were 161.5f 29 and 177.3 f 34.7 min. pgm/ml f o r b i o a s s a y and r a d i o a s s a y cu rves r e s p e c t i v e l y . Cephradine was r a p i d l y e x c r e t e d . Approximately 77% of cephrad ine was e x c r e t e d w i t h i n t h e t h r e e hour p e r i o d fo l lowing drug a d m i n i s t r a t i o n . A f t e r 24 hour s approximate ly 87% of t h e a d m i n i s t e r e d dose of cephrad ine was recovered i n t h e u r i n e as

25 measured by both bio and r ad iochemica l a s s a y . Four h e a l t h y female subjects r e c e i v e d a s i n g l e i n t r a m u s c u l a r i n j e c t i o n of 1 gram of ~ e p h r a d i n e - ~ H ~ ~ . 10 f 1 .7 ug/ml - SE i n plasma w a s reached a t 2 hours a f t e r a d m i n i s t r a t i o n and then dec reased w i t h a b i o l o g i c a l h a l f - l i f e of 2 hours . Binding of cephrad ine t o plasma p r o t e i n was found t o be 6% ove r t h e r ange of c o n c e n t r a t i o n s found i n plasma d u r i n g the a b s o r p t i v e and e x c r e t o r y phases.

Absorp t ion , based on e x c r e t i o n of r a d i o - a c t i v i t y i n u r i n e ove r t h e 24 hour p e r i o d + + fo l lowing a d m i n i s t r a t i o n , w a s 92 - 6.6% - SE f o r t h e f o u r s u b j e c t s . Another 1% was e x c r e t e d i n f e c e s w i t h i n t h e 72-hr p e r i o d of t h e experiment . There w a s no s i g n i f i c a n t d i f f e r e n c e i n e x c r e t i o n based on t h e r ecove ry of r a d i o a c t i v i t y o r a n t i m i c r o b i a l a c t i v i t y i n u r i n e . Exc re t ion was 66 f 6.9% f SE a t t h e end of 6 h r and 8 5 f 6.5%

Cephradine-3H 250 mg d r y f i l l e d c a p s u l e s w e r e

The mean peak cephrad ine s e r u m c o n c e n t r a t i o n

The mean peak c o n c e n t r a t i o n o f

49

5 SE a t t h e end of 1 2 hours. Recovery of antimicrobial a c t i v i t y i n u r i n e and t h e a rea under t h e curve f o r concent ra t ion of a n t i b i o t i c i n plasma were i n e x c e l l e n t agreement wi th those found when l a b e l l e d cephradine w a s adminis tered o r a l l y .

Cephradine appears t o be slowly r e l eased from t h e s i t e of i n j e c t i o n t o g ive l e v e l s of a n t i b i o t i c i n plasma which, though reaching a maximum a t 1/3 those found a f t e r o r a l adminis t ra t ion , provide t h e same amount of b ioava i l ab le a n t i b i o t i c over a longer per iod of t i m e 2 4 -

N o canine o r human drug metabol i tes w e r e found so f a r except t r a c e amounts of cephalexin, a s i d e n t i f i e d by m a s s spectrometry26.

The above information was publ ished ( r e fe rences 27-31).

6. Methods of Analysis 6 .1 Elemental Analysis % C H N S

Calc. f o r anhydrous 55.01 5.48 12.03 9.16 Calc. f o r monohydrate 52.30 5.76 11.44 8.73 Found f o r cephradine batch NN057NC 51.96 5.83 11.16 9.00

Calc. f o r d ihydra te 49.85 6.01 10.90 8.31 Found f o r cephradine

d ihydra te ba tch NNOO5NB 49.77 6.06 10.95 8.39

CEPHRADINE

6.2 Mic rob io log ica l Assay For b u l k and formula ted p roduc t s a

t u r b i d i m e t r i c method us ing S t r ep tococcus f a e c a l i s A.T. C. C. 10,541 i s convenient . A l t e r n a t i v e l y a g a r p l a t e methods u s i n g S a r c i n a l u t e a A . T . C. C. 9341, B a c i l l u s pumilus A.T. C. C. 14,884 o r S taphylococcus au reus , A.T.C. C. 6538P = FDA 209P, a r e a l s o employed. For b lood and body f l u i d samples an aga r p l a t e method i s used employing S a r c i n a l u t e a a s t e s t o r anism because of i t s g r e a t s e n s i t i v i t y 32,46,47,

The minimum i n h i b i t o r y c o n c e n t r a t i o n ( M I C ) of cephradine f o r t h e f o u r t e s t c u l t u r e s i s a s fo l lows:

mcq/ml S a r c i n a l u t e a 0.04 S taphylococcus au reus 0 .40 B a c i l l u s p u m i l u s 0.40 S t r ep tococcus f a e c a l i s 50 .0

Cephradine i s s l i g h t l y more b i o a c t i v e a g a i n s t S t r ep tococcus f a e c a l i s and S a r c i n a l u t e a than cepha lex in , wh i l e t h e r e v e r s e h o l d t r u e f o r S taphylococcus a ~ r e u s ~ ~ .

6 . 3 Iodomet r i c A n a l y s i s Cephradine can be de termined by t h e

iodomet r i c a s say . The B-lactam r i n g i s opened wi th a l k a l i o r cepha lospor inase fo l lowed by

34 i o d i n a t i o n a t an a c i d pH (pH 4 .5 phosphate b u f f e r ) . About 4-5 moles o f i o d i n e a r e consumed, I t i s i n t e r e s t i n g t o n o t e t h a t p e n i c i l l i n s under t h e same c o n d i t i o n consume 8-9 moles o f i o d i n e . The p r e c i s i o n of t h e iodomet r i c a s s a y f o r cephradine i s n o t a s good a s t h a t f o r p e n i c i l l i n s when a l k a l i i s used f o r i n a c t i v a t i o n . The p r e c i s i o n can be c o n s i d e r a b l y improved by u s i n g cephalos o r i n a s e i n s t e a d o f a l k a l i f o r r i n g opening . Also , w i t h t h e l a t t e r method much bet ter agreement was o b t a i n e d w i t h the microbio-

16

51

logical assay (see Table 10) even with severely degraded bulk samples. It therefore can be considered stability indicating.

Table 10 Comparison of the cephradinase-iodometric, alkali-iodometric *

and bioassay methods for the determination of cephradine in bulk powders

Cephradine potency = mcg/mg

Sample

NN054ND NNO 5 9ND NN061ND NN054N.D NNO 5 9ND NNO61ND

Bioassay Cephalosporinase- Alkali-iodometric iodometric assay assay

812 8 54 7 78 578 424 405

813 04 3 84 6 578 46 6 464

97 9 991 980 744 643 63 9

* Results are the means of determinations on two consecutive days. The powders were stored at 5OoC two years in bottles with varying volumes of head space.

CEPHRADINE

The presence of 7-ADCA does n o t i n t e r - f e r e w i t h t h e iodometr ic a s s a y .

It was found t h a t when i o d i n a t i o n was c a r r i e d o u t a t an a l k a l i n e r a t h e r t han a c i d pH, 1 3 e q u i v a l e n t s of i o d i n e were consumed .

19

34

6 .4 Spec t ropho tomet r i c Ana lys i s The u l t r a v i o l e t absorbance peak of

cephradine a t 262 nm (see s e c t i o n 2 .4) can be used a s an i d e n t i f y and homogeneity a s s a y i n formula t ions35 .

Opening of t h e B-lactam r i n g w i t h a l k a l i o r p r e f e r a b l y , w i th cepha lospor inase a b o l i s h e s t h e U.V. absorbance a t 260nm. T h i s h a s been made t h e p r i n c i p a e of a q u a n t i t a t i v e a s s a y which appea r s t o be s t a b i l i t y i n d i c a t i n g i n t h e " p r a c t i c a l " range ( less than 20% loss of b i o a c t i v i t y ) 19.

6 .5 F luo romet r i c A n a l y s i s Cephradine can be as sayed q u a n t i t a t i v e -

l y i n a l k a l i n e medium by f l u o r i m e t r y , ( e x c i t a t i o n wave l e n g t h 350 nm, emiss ion wave l e n g t h 495 nm). Th i s method h a s been used f o r b lood l e v e l s t u d i e s b u t i s n o t recommended f o r t h e d e t e r m i n a t i o n i n u r i n e because of e r r a t i c r e s u l t s .

6 .6 C o l o r i m e t r i c A n a l y s i s The w e l l known hydroxylamine method

f o r p e n i c i l l i n has been adap ted by FDA t o cephradine a s a b a t c h i n g a s say . I t i s n o t s t a b i l i t y i n d i c a t i n g . S t r o n g l y a l k a l i n e r e a c t i o n c o n d i t i o n s and f e r r i c n i t r a t e w e r e used . When f e r r i c ammonium s u l f a t e was used a t pH 7 o n l y 15% of t h e response normal f o r p e n i c i l l i n s was ob ta ined3 7.

d i t h i o b i s (2 -n i t robenzo ic a c i d ) a t pH 9.2 produces a ye l low c o l o r which can be q u a n t i t a t e d by measuring t h e peak absorbance a t 412 nm38.

5

A c o l o r i m e t r i c method u s i n g 5 ,5 ' -

53

KLAUS FLOREY

6.7 Chromatoqraphic A n a l y s i s 6 . 7 1 Paper

Cephalexin (slower moving component) can be s e p a r a t e d from cephrad ine w i t h a n-butanol-t-amyl alcohol-~ater(7:l:4)system and q u a n t i t a t e d by b ioautography us ing S t r ep tococcus au reus 209P a s t h e a s s a y organisn?? Dihydrophenlyglycine(faster moving component) can be s e p a r a t e d from cephrad ine w i t h a t e r t . - amyl a lcohol -sec . -butanol-water (4:4: 1) system and q u a n t i t a t e d w i t h a ninhydrin-copper complex 40 .

6.72 Thin-Layer There a r e two TLC methods by

which cepha lex in and o t h e r i m p u r i t i e s can be s e p a r a t e d from cephrad ine a n d b o t h cepha lex in and

40 cephrad ine can be q u a n t i t a t e d . Both methods g i v e comparable r e s u l t s . I n t h e f i r s t method , s i l i c a g e l p l a t e s a r e impregnated w i t h s i l i c o n e f l u i d and developed f o r 2-1/2 hours i n a pH 4 . 1 McIlvaine b u f f e r and a c e t o n e ( 1 0 0 : l . S ) . The zones a r e l o c a t e d w i t h U.V. l i g h t , e l u t e d and q u a n t i - t a t e d s p e c t r o p h o t o m e t r i c a l l y a t 260 nm. The s e p a r a t i o n scheme of the known components is as fo l lows :

compound Rf Cephradine Dihydrocephradine 0 .31 0.72

A t 22OC

Cephradine 0 .43 1.00 Cephalexin 0 .51 1 .20 7-ADCA 0.65 1 . 5 1 7 -ACA 0.65 1 . 5 1 Dihydrophenylglycine 0.74 1.72 Phenylg lyc ine 0.80 1.88

q u a n t i t a t i v e l y , i t i s advantageous t o change t o a s o l v e n t system o f pH 6.5 McI lva ines b u f f e r - a c e t o n e ( 5 0 : l ) . The 7-ADCA zone i s e l u t e d w i t h 0.24 M sodium b i c a r b o n a t e and the absorbance a t

To de te rmine r e s i d u a l 7-ADCA

54

CEPHRADINE

260 nm i s measured41. The second method42, a modi f ica-

t i o n o f t h e f i r s t i s p r e f e r r e d because of g r e a t e r e a s e of performance. The p l a t e s a r e impregnated w i t h t e t r a d e c a n e i n s t e a d of s i l i c o n e and s i n c e t h i s i n t e r f e r e s w i t h t h e u. v. absorbance, q u a n t i t a t i o n i s c a r r i e d o u t w i t h n inhydr in .

T h i s method can a l s o b e used t o de termine d ihydrophenlyglyc ine and 7-ADCA semi- q u a n t i t a t i v e l y . Approximate Rf v a l u e s a r e a s f o l l o w s :

C e phra d i n e 0.2 Cephalexin 0 . 3 7 -ADCA 0.6 Dihydrophenyl- 0 .7

g l y c i n e 6.73 Column Chromatoqraphy

High p r e s s u r e l i q u i d chromoto- graphy (HPLC) h a s been used t o q u a n t i t a t e cephra- d i n e and r e s i d u a l cepha lex in i n cephradine . The mobile phase i s a pH 4 . 3 g l a c i a l acetic- anhydrous sodium s u l f a t e system, a Dupont s t r o n g c a t i o n exchange r e s i n , a t ambient t empera tu re and a p r e s s u r e o f 1000 p s i g . Sul famethaz ine is used as i n t e r n a l s t a n d a r d and the o r d e r of elu- t i o n i s su l f ame thaz ine , cepha lex in and ~ e p h r a d i n e ~ ~ . A m o d i f i c a t i o n , u s e s an a c e t a t e - 0.17M sodium s u l f a t e b u f f e r pH 4.7, Dupont Zipax c a t i o n exchange r e s i n , n- (4-methoxy-methyl- 6-methyl-2-pyrimidinyl s u l f a n i l a m i d e as i n t e r n a l s t a n d a r d and a p r e s s u r e o f 300 p s i g . The o r d e r o f e l u t i o n is cepha lex in , cephrad ine , i n t e r n a l s t a n d a r d . 7-ADCA, when p r e s e n t , w i l l appear i n t h e vo id volume44. the mobile phase t o 3.70 and r a i s i n g t h e p r e s s u r e t o 1800 p s i g , t h e system was able t o s e p a r a t e cephrad ine (d- isomer) from t h e synthe- t i c a l l y p repa red l-isomer which peaks between cepha lex in and cephradine . N o l-isomer was

When a d j u s t i n g t h e pH o f

55

KLAUS FLOREY

de tec t ed i n r egu la r cephradine samples4’. r eve r se phase system, use fu l for q u a n t i t a t i o n i n formulation has also been descr ibed49. A 1 mm x 2.1 mm i .d . column, ODS-Sil-X-I1 packing and 7 % methanol, 93% 0.05M ammonium carbonate as mobile phase were used. Pressure , 1 0 0 0 p i g ; f l o w 0 .6 ml/min; d e t e c t o r W (254 nm); s e n s i t i v i t y , 0.08 AUFS.

7 . Determination i n Body F l u i d s and Tissues Cephradine has been determined microbio-

l o g i c a l l y (see s e c t i o n 6.2) i n human serum, i n human u r ine , human lung t i s s u e , human eye t i s s u e and i n s p i n a l f l u i d .

I t has been determined f luo romet r i ca l ly (see sec t ion 6.5) i n dog serum.

A

8. Determination i n Pharmaceutical Prepara t ion I n pharmaceutical p repa ra t ions (capsules ,

oral suspensions and i n j e c t a b l e s ) i n f r a r e d has been used for i d e n t i t y tests, t h e hydroxylamine and iodometric assay f o r batching, t h e micro- b i o l o g i c a l and cephalosporinase-iodometric assays f o r s t a b i l i t y and chromatography f o r d e t e c t i o n of impur i t i e s .

56

CEPHRADI N E

9.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12 .

13.

14.

15.

16. 17.

Re fe rences

B. T o e p l i t z , The Squibb I n s t i t u t e , P e r s o n a l commun i ca t ion . M. S. Puar , The Squibb I n s t i t u t e , P e r s o n a l communication. P. T. Funke, The Squibb I n s t i t u t e , P e r s o n a l communication. J. Dunham, The Squibb I n s t i t u t e , P e r s o n a l communication. F. M. Russo-Alesi , The Squibb I n s t i t u t e , P e r s o n a l communication. H. Jacobson, The Squibb I n s t i t u t e , P e r s o n a l communication. L. P. M a r e l l i , A n a l y t i c a l P r o f i l e s of Drug Subs tances , Vol. 4, p 21, A c a d e m i c Press, 1974. M. S. Atwal, The Squibb I n s t i t u t e , P e r s o n a l communication. H. Jacobson and I. G i b b s , J. Pharm. S c i . 62, 1543 (1973) . D. E. Aus lander , The Squibb I n s t i t u t e , P e r s o n a l communication, F. Dursch, The Squibb I n s t i t u t e , U . S . P a t e n t 3 ,829,620, June 25, 1 9 7 4 . F. Dursch, The Squibb I n s t i t u t e , P e r s o n a l communication. Q. Ochs, The Squibb I n s t i t u t e , P e r s o n a l communication. J. E. D o l f i n i , H. E. Applega te , G. Bach, H. Basch, J. B e r n s t e i n , J. Schwartz and F. L. Weisenborn, J. Med. Chem. 14, 117 (1971) : U.S.Patent 3,485,819(1969).

M. L. Snow, C. Lauinger and C. Ressler, J. Org. Chem. 33, 1774(1968) . L. Demain, Nature 210,426 (1966) . H. R. Roberts, The Squibb I n s t i t u t e , Pe r sona l communication.

57

KLAUS FLOREY

18.

19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

A. I. Cohen, P. T. Funke and M. S. Puar, J. Pharm. S c i , 62, 1559 (1973). B. M. F ran tz , The Squibb I n s t i t u t e , Pe r sona l communication; J. Pharm. S c i . , i n p r e s s , R. V a l e n t i and H. Jacobson, The Squibb I n s t i t u t e , Pe r sona l communication. M. A. L e i t z , The Squibb I n s t i t u t e , P e r s o n a l commun i ca ti on, K. J. K r i p a l a n i and A. V. Dean, The Squibb I n s t i t u t e , Pe r sona l communication. D. Adam, Munch. Med. WSchr. 116, 1945(1974) . I. Weliky and R. Vukovich, The Squibb I n s t i t u t e , Pe r sona l communication. A. Vahid i , R. Vukovich, E. S. N e i s s and E. S c h r e i b e r , The Squibb I n s t i t u t e , Pe r sona l communication. P. T. Funke, The Squibb I n s t i t u t e , Pe r sona l communication. Weliky, I . , and Zaki, A . , S e l e c t e d Proceedings from t h e 8 t h I n t e r n a t i o n a l Congress of Chemotherapy, Sept . 8-14,1973, Athens, Greece, pp. 1-5. Vukovich, R . , Mar t inez , M . , and N e i s s , E .S . , S e l e c t e d Proceedings from t h e 8 t h I n t e r n a - t i o n a l Congress o f Chemotherapy, Sep t . 8-14, 1973, Athens, Greece, pp. 30-34. Zaki, A. , S c h r e i b e r , E .C. , Weliky, I . , K n i l l , J . R . , and Hubsher, J . A . , J. Cl in . Pharmacol, New Drugs 14: 118-126 (1974). I. Weliky, H. H. Gadebusch, K. K r i p i l a n i , P. Arnow and E. C. S c h r e i b e r , A n t i m i c r o b i a l Agents and Chemotherapy 2, 49(1974) . G. Renzin i , G. Ravagnan, B. O l iva , E. S a l v e t t i , and R. A u r i t i , Quaderni di A n t i b i o t i c a ; 1972, 17. T. B. P l a t t , The Squibb I n s t i t u t e , P e r s o n a l communication. S. Wind, The Squibb I n s t i t u t e , P e r s o n a l c o m u n i c a t i o n .

58

CEPHRADINE

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

J. Alicino, The Squibb Institute, Personal communication. H. Lerner and S. Willis, The Squibb Institute, Personal communication. A. F. Heald, C. E. Ita and E. Solney, The Squibb Institute, Personal communication. C. Sherman, The Squibb Institute, Personal communication. J. Kirschbaum, J. Pharm. Sci., 63 923 (1974). A. Vahidi and H. R. Roberts, The Squibb Institute, Personal communication. H. R. Roberts, The Squibb Institute, Personal communication. F. P. Targos, The Squibb Institute,Personal communication. I. R. Salmon, The Squibb Institute,Personal commun i ca t ion. A. Peterson and D. Guttman, Smith Kline & French Laboratories, Personal communication. J. Kirschbaum, The Squibb Institute, Personal communication. H. H. Pu and R. B. Poet, The Squibb Institute, Personal communication. D. M. Isaacson, The Squibb Institute, Personal communication. J. R. Ster, H. Weisblatt and J. D. Levin, The Squibb Institute, Personal communication. A. N. Niedermayer, The Squibb Institute, Persona 1 communication. E. R. White, M. A. Carroll, J.E. Zarembo and A. D. Bender, J. Antibiotics - 28, 205 (1975). J. Z. Gougoutas and B. K. Toeplitz, Univ.of Minnesota and Squibb Institute, Personal communication. J. V. Uri, P. Actor, L. Phillips and J. A. Weisbach, Experientia &54(1975).

59

CHMROQUINE PHOSPHATE

Donald D, Hong

DONALD D. HONG

CONTENTS

Analytical Profile - Chloroquine Phosphate

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2. Physical Properties 2.1 Ultraviolet Spectrum

rescence Spectrum ear Magnetic Resonance Spectrum

- 2.2 2.3 NA 2.4 Ma&% 2.5 ~ p t i 2.6 2.7 Dissociation Constant 2.8 DH

Spectrum cal Rotation morphism and Melting Range

2.9 seezing Point Depression 2.10 Solubility 2.11 Differential Scanning Calorimetry

3. Synthesis

4. Drug Metabolic Products 4.1 Biotransformation Products 4.2 Distribution in Human Tissues

5. Methods of Analysis 5.1 Phase Solubility Analysis 5.2 Identification by Spot Tests 5.3 Non-Aqueous Titration 5.4 Spectrophotometric Analysis 5.5 Fluorometric Analysis 5.6 Gravimetric Analysis 5.7 Chromatographic Analysis

5.71 Paper 5.72 Thin-Layer

5.8 Miscellaneous Methods 5.73 Gas

6. Ref erencea

62

CHLOROQUINE PHOSPHATE

1. Description

1.1 Name, Formula, Molecular Weight

The chemical name of chloroquine phosphate i n Chemical Abstracts is found under the heading Quinoline and designated as 7-Chloro- [4- (4-diethylamino-1-methylbutyl- amino) Iquinoline diphosphate. e a l t e are a l s o avai lable .

The hydrochloride and s u l f a t e

2H2PO4-

C@&CN3 2H3Po4 Molecular Weight: 515.87

1 .2 Appearance, Color, Odor

Chloroquine phosphate is a white , odorless , c r y s t a l l i n e powder having a b i t t e r taste: it discolore gradually on exposure t o l i g h t .

2. Physical Propert ies

2 .1 Ul t r av io l e t Spectrum

A 10 y/ml solut ion of chloroquine phosphate i n 0.01 N H C 1 when scanned between 360 and 210 nm exh ib i t s t h ree maxima, three minima and seve ra l shoulders i n the region from 270 t o 225 nm, as shown i n Figure 1. are located a t 343 nm ( a = 36.1), 328 nm (a = 32.6) and 222 nm ( a = 59.9). The r a t i o of /%% i s 1.11. Minima were observed a t 335 nm, 280 nm a342h3 nm.

The maxima

2.2 Fluorescence Spectrum

Figure 2 shows the fluorescence spectrum of chloroquine phosphate obtained on a solut ion of 0.2 mg/ml pH 7.9 phosphate buffer using an Aminco-Bowman spectro- photofluorometer. produced emission spectra with a maximum a t 400 nm, the

Excitation a t e i t h e r 320 nm o r 370 nm

63

- . .- ...~

... - ...

... ...

-

..

..

-

si

.........

......... .

..

..

..

.

..

..

.

..

..

..

..

..

..

..

... !

' . 1..

..

.

.. , .

I

..

.

j I ....

1

, i(

..

I

+

3-

I

"I i ,.

.

!.

i i ! I

,

64

CH LOR OQUlN E PHOSPHATE

Fluorescence

Figure2. F luorescence Spectrum of Chloroquine Phosphate in pH 7.9 Phosphote Buffer (Sterling-Winthrop House Reference Standard Lot N-087- JF)

70

WAVE L ENGTH-MILL IMICRONS

65

DONALD D. HONG

la t ter exc i t a t ion wavelength providing a higher emission response.

2.3 Nuclear Magnetic Resonance Spectrum (NMRl The spectrum i n Figure 3 was obtained w i t h a

Varian A60 NMR Spectrometer using a 20$ solut ion i n D20 containing TMS as an e x t e r n a l standard. assignments are summarized below (1).

The s p e c t r a l

c1

Protons

CH3 -CH2 CH -CH Cg- CH2 CH2-N CH-N NH

c b a C d

2.4

No. Protons Derived from a Integrat ion Chemical Shi

6 1-68-1 -80 3 1.90-1.9 4 2.35 6 3 -55-3 a 9 1 1 4.38-4.65

exchanged 5 -39

1 7.15-7 - 28 1 7 - 58-7 -75 1 7.75 1 8-33 -8.40 1 8 - 48-8-55

Mass Spectrum

; Mult ipl ic

doublet doublet broad s i n g l e t q u i n t e t broad s i n g l e t sharp s i n g l e t

doublet mult iplet s i n g l e t doublet doublet

The mass spectrum is shown i n Figure 4 and was obtained using a J o e l JMS-01SC mass spectrometer with an ionizing energy of 75 eV. m/e 319 i s a thermal breakdown product where two phosphoric acid moieties were l o s t from t h e parent compound. The base

The highest mass observed a t

peak at m/e 86 is due t o t h e N,N,N-diethYlmethylene fragment (1).

66

L

L

A/-+-

I . . . . , . . . . I . , . I I 1 I I I I . , . . I . . I

, . I . ' . . I . , . I , ' ' I I 1 I I I I U 7 1 00 ( 0 Y I 4 I 4 0 a n 1 0 $ 8 8

Figure 3. NMR Spectrum of Chloroquine Phosphate. Instrument: Varian A60

100

> v) 80 z W F 5 60

t

W

+ 40

A W

L a

a 20

0

DONALD D. HONG

Intensity

319

BACKGROUND

. . 20 40 60 ' 80 100 I20 140160 I80 250 300

m/e Fig.4 Mass Spectrum of Chloroquine Phosphate (Sterling-Winthrop

House Reference Standard Lot N-087-JF)

60

CHLOROQU IN E PHOSPHATE

2.5 Optical Rotation

Chloroquine phosphate exhibi ts e s s e n t i a l l y no o p t i c a l a c t i v i t y , ex i s t ing as a racemic mixture. Riegel and Sherwood (2) have shown t h a t ne i the r of t h e o p t i c a l l y ac t ive enantiomorphs showed any s i g n i f i c a n t differences i n an t ima la r i a l a c t i v i t y i n birds and f o r t o x i c i t y i n dogs.

2.6 Polymorphism and Melting Range

Chloroquine phosphate e x i s t s i n two polymorphic forms giving rise t o two melting ranges. one melting between 193" and 195" and the other between 210" and 215". Mixtures of t h e forms melt between 193" and 215". It is possible t o obtain one form se l ec t ive ly by regulat ing t h e rate of c r y s t a l l i z a t i o n (4) .

USP xn ( 3 ) r epor t s

2.7 Dissociation Constant

The pKa 's f o r chloroquine phosphate by t h e t i t r i m e t r i c method were found t o be 8.10 and 9.94 (5 ) .

2.8 @

A 18 aqueous solut ion has a pH of about 4.2.

2.9 Freezing Point Depression

Cryoscopic measurements were made on lo$ and 2o$ (w/v) solutions of t he drug.

Freezing Point Depression Calculated

Chloroquine phosphate * * 0.73 " Isotonic 7.oe

69

DONALD 0. HONG

The value f o r "calculated isotonic" solut ion was obtained by graphic in t e rpo la t ion t o FPD of 0.550", representing 0 . d sodium chloride solut ion (5 ) .

2.10 So lub i l i t y

Chloroquine phosphate i s f r e e l y soluble i n water: p rac t i ca l ly insoluble i n alcohol, i n chloroform and i n ether (3).

2.11 Di f f e ren t i a l ScanninR Calorimetry (DSC)

Two polymorphic forms of chloroquine phosphate A mixture of t h e two c r y s t a l forms a re exhibited by DSC.

may be demonstrated a l s o by the t r a n s i t i o n temperatures (6) . The DSC thermogram of a chloroquine phosphate s tandard shown i n Figure 5 was obtained on a Perkin-Elmer DSC-lB d i f f e ren - t i a l scanning calorimeter a t a heating rate of 10°C per minute under nitrogen. This i s an example of t h e low melting form. Figure 6 shows another sample of chloroquine phosphate containing a mixture of the low and high melting forms.

Both the low and high melting polymorphs may be obtained from t h e same aqueous solut ion of chloroquine phosphate by se l ec t ive c r y s t a l l i z a t i o n . form usually occurs as small c r y s t a l s while the low melting polymorph c r y s t a l l i z e s as s ign i f i can t ly l a rge r c r y s t a l s . The two forms exh ib i t s l i g h t differences i n t h e i r I R curves from a KBr matrix.

The high melting

3 . Synthesis

Elderf ie ld (7) and Kenyon is collaboration w i t h Wiesner and K w a r t l e r (8) and more r ecen t ly Basu, e t a1 ( 9 ) have summarized the American e f f o r t t o develop an a n t i - malarial drug necessitated by World W a r 11. This need was compounded when Japan seized control of t he East Indies, e f f ec t ive ly cu t t i ng off t h e na tu ra l sources of quinine, which was the drug of choice f o r malaria a t t he time. Chloroquine was one of t h e f r u i t s of t h i s concerted e f f o r t .

The synthesis of chloroquine was first reported by the German chemists Andersag, Breitner and Jung (10, 11). Since the patent l i terature lacked the de t a i l information required t o prepare the necessary intermediates, a research program w a s s t a r t e d a t Winthrop Chemical Company r e s u l t i n g i n a method of synthesis for chloroquine by Surrey and

70


Figure 5. OSC Thermogram of Chloroquine Phosphate (Sterling-Winthrop House Reference Standard Lot N-087-JF) Low Melting Form

189-202.5-207.5 O C (Corr.)

Ic -4

0 0 0 0 0 0 0 m * 0 (D r- OD Q,

t t t 0 t * t t

0 N

1 I I I I I I I

TEMPERATURE O K

71

DONALD D. HONG

Figure 6 DSC Thermogram of Chloroquine Phosphate (Lot An-K-67) Mixture of Low and High Melt ing Forms

1- 189-202-1 -207.5 OC (Corrl

21 5-223- 2 2 6 O C (Corr)

ENDO

t 1

0 0 0 0 a 00 Q, 0

e * Q d * In

0 m

1 I I I I J

O P c

E X 0

TEMPERATURE O K

72

CH LOROQUI NE PHOSPHATE

Kammer (12).

The two key intermediates required i n t h e synt hes is a r e 4 7-d i c h l o r oquino li ne and 4 -d ie thy lami no - 1 - methylbutylamine ( “novol diamine”). The synthet ic scheme i s shown i n Figure 7.

4. Drug Metabolic Products

4 .1 Biotransformation Products

Several metabolites in addi t ion t o t h e unchanged drug have been isolated from human t i s s u e s and urine. -- e t a1 (13) used counter-current d i s t r i b u t i o n t o i s o l a t e t h e desethyl compound (A ) from the ur ine of human volunteers. Kuroda (14) isolated four metabolites of chloroquine from human t i s s u e s using a combination of paper chromatography and W spectroscopy. ( A ) of Ti tus , they were the bisdesethyl (B), t h e carbinol (C) and the ~-amino-7-chloroquinoline ( D ) der ivat ives .

Titus,

I n addi t ion t o the desethyl compound

Similar observations were seen from urine of human subjects but no t r a c e of t h e 4-hydroxy-7-chloroquinoline w a s found. The unchanged drug was always found t o be t h e major compound. a fluorescence technique confirmed t h e above findings and i n addi t ion found t r a c e s of t h e hl-aldehyde (E) and t h e b’-carboxy (F) de r iva t ives . determinable excretory products as TO$ chloroquine, 23s desethylchloroquine, 1-2$ bisdesethylchloroquine and t h e others as t r a c e degradation products. I n addi t ion they reported t h a t about one-third of t h e administered chloroquine was unaccounted and remains obscure i n t h i s very complex blotransformation of t he drug.

McChesney, e t a1 (15, 16) using

They l i s t e d t h e amount of

73

DONALD D. HONG

Figure 7: Synthesis of Chloroquine Phosphate

- m-Chloroaniline Ethyl oxalacetate Ethyl(m-chloropheny1imino)- succinate

and isomer separation

OH (1) NaOH k I

co0c.$l5

heat

(2) HC1 c1 COOH

7-Chloro-4-hydroxyquinoline-2-carboxylic

acid

Ethyl-7-chloro-4-hydroxyquinoline-2-carboxylate

-COOH

1 CH3CHCH2CH2CH9 (CpHg )2

.- a> I!H2

Cl c1 N “novol diamine” .1,

7 -Chlor o - 4- 4,7-Dichloroquinoline hydroxyquinoline

H3P04 Chloroquine diphosphate <-

CL

Chloroquine. base

74

CHLOROQUIN E PHOSPHATE

Compound

Chloroquine

A

B

C

D

E

F

R

-CHCH$H2CH$I (C2H5 )2

I CH3

I CH3

I CH3

-CHCH2CH$H$IHC2Hrj

-CHCH$H$H$H2

-CHCH2CH2CH20H

4

-FHO -rHC82CH2CwH

H

CH3

CH3

4.2 Di s t r ibu t ion in Human Tissues

Chloroquine i s predominantly loca l ized i n t h e l i v e r and t o lesser degrees i n t h e spleen, hea r t , kidney, lung, b ra in , leucocytes and sk in . Chloroquine was o r i g i n a l l y used t o treat malaria. Subsequently it was found t o be e f f e c t i v e aga ins t o the r p a r a s i t i c d i so rde r s . Rubin, e t a1 (17) t r a c e s of t h e drug and its metabol i tes were found i n t h e blood and u r i n e of sub jec t s up t o f i v e yea r s after d iscont inuing long-term therapy.

According t o

75

DONALD D. HONG

5 . Methods of Analysis

5 . 1 Phase S o l u b i l i t y Analysis (PSA)

PSA i s probably not promising f o r chloroquine phosphate because t h e mul t ip l e pKa's would allow substan- t i a l d i sp ropor t iona t ion (18).

5.2 I d e n t i f i c a t i o n of Chloroquine Phosphate by Spot Tests

I n t h e approximate period of two decades fo l lowing World War I1 where t h e r e was inc reas ing use of chloroquine phosphate f o r mass prophylaxis of malaria it became necessary t o have a r e l a t i v e l y s i m p l e method t o in su re t h a t t h e d rug was taken r e g u l a r l y . T e s t s of t h i s type usua l ly measure t h e drug i n u r i n e . Other tests u t i l i z e var ious r eagen t s t o r e a c t e i t h e r w i th t h e pure drug o r t h e drug i n dosage form. Some of these tes ts are summarized i n Table 1.

5.3 Non-Aqueous T i t r a t i o n

Chloroquine phosphate can be t i t r a t e d wi th acetous 0.1 N perch lo r i c a c i d . The t i t r a t i o n may be c a r r i e d o u t manually wi th c r y s t a l v i o l e t as i n d i c a t o r o r determined po ten t lome t r i ca l ly . The t i t r a t i o n is r ap id al though non-se lec t ive . This nonspec i f i c i ty is no drawback, however, so long as good i d e n t i f i c a t i o n tests are a l s o adopted ( 3 0 ) . Wu, e t a1 (31) have repor ted t h e determin- a t i o n of e leven a n t i m a l a r i a l drugs us ing t h i s t i t r imet r ic procedure.

Each ml of 0.1 N HClO4 is equ iva len t t o 25.79 mg of chloroquine phosphate.

5.4 Spectrophotometr ic Assay

The W s p e c t r a of chloroquine base and phosphate sa l t are s i m i l a r i n 0.01 N HC1. Absorption maxima are observed a t 343, 328, 256 and 222 nm. most favorably made a t 343 nm where absorp t ion is most i n t e n s e and least a f f e c t e d by i n t e r f e r i n g subs tances i n t h e b i o l o g i c a l sample.

Measurements are

The chloroquine base is obtained by e t h e r o r chloroform e x t r a c t i o n of an a l k a l i n e homogenate of t h e b i o l o g i c a l sample. After sepa ra t ion of i n t e r f e r i n g

76

T e s t

CH LOROQUIN E PHOSPHATE

Table 1 - Spot Tes ts

1. Complex wi th Copper

2. Complex wi th Cobalt

3 . Dimethylaminobenz- aldehyde ( E h r l i c h ' s reagent )

4. Styphnic acid

5. Nitroprusside and Piperazine (Lewin's reagent )*

6. Eosin yellowish ( D i l l & Glazko)

7. Mercuric iodide/KI ( W e r -Tanre t ' s reagent )

8. Methyl orange

9. Complex with HClO 4/AuClj

10. Aconitic a c i d / a c e t i c anhydride/ e thylene d i c h l o r i d e

11. H2SO4/KClOj

12. H C ~ / K C ~ O ~

Form Color S e n s i t i v i t y Ref.

Tablet Pa le green NA

Tablet Vio le t NA

Table t Yellow NA

Pure drug Roset tes 0 . 5 ~ of p l a t e s

Pure drug NA SOY

Urine Yellow t o NA v i o l e t -red

Urine ** 2.5-9- 5~ per r n l u r ine

Urine Yellow 2 mg/liter

Pure drug Rose t tes 0 . 4 ~ or b i o l . and dendr i tes

e x t r a c t

Pure drug Red 57

Biol . Red - v i o l e t 5 Y e x t r a c t

Biol . Yellow 1 0 Y e x t r a c t

15. 25$ H2SOl+/Chlorinated Biol . Yellow 10Y l i n e e x t r a c t

1 4 . BPB/boric ac id Free base Blue-violet 0.8 mg$ to blue -green

19

1.9

20

21

22

23

24

25

26

27

20

28

28

29

* Speci f ic f o r N-ethyl group ** Turbidi ty i s measured

77

DONALD D. HONG

materials, the base i s i n tu rn extracted i n t o a solut ion of 0.1 or 0.01 N H C 1 and quan t i t a t ive ly determined by measuring i ts W absorption. Alternat ively t h e acid solut ion can be made a lka l ine and t h e base extracted w i t h e the r o r chloroform. The organic phase i s evaporated t o dryness and t h e residue further examined by I R and paper o r TLC.

A b r i e f summary of t h e spectrophotometric procedures f o r t h e quan t i t a t ive determination of chloroquine and metabolites is summarized i n Table 2.

Table 2 - Spectrophotometric Methods

Method of Analysis I so l a t ion from Human Reference

uv Tissues uv Blood UV, IR , TLC, GLC Tissues uv, I R , PC Tissues+ uv Urine

32 33 34 14 35

* Includes i d e n t i f i c a t i o n of chloroquine metabolites a l s o

5.5 Fluorometric Analysis

Fluorometric procedures have been extensively u t i l i z e d f o r t h e quan t i t a t ive determination of chloroquine i n b io log ica l materials. I n t h e e a r l y days of fluorescence when instrumentation was not s u f f i c i e n t l y advanced, an add i t iona l i r r a d i a t i o n s t e p was required t o convert t h e chloroquine t o a more intense fluorophore ( 3 6 ) . With t h e advent during t h e mid 1950's of t h e highly s e n s i t i v e spectrophotofluorometers u t i l i z i n g t h e xenon a r c source and monochromators, it is now possible t o measure t h e chloroquine fluorescence d i r e c t l y (15, 16, 37, 3 8 ) .

Brodie, e t a1 (36) found t h a t t he fluorescence of chloroquine i n pH 9.5 borate buffer after i s o l a t i o n from b io log ica l specimen may be s t ab i l i zed by t h e addi t ion of cysteine. and t h e measurement made using a s u i t a b l e fluorometer. The s e n s i t i v i t y of the procedure is about 0.lmcg.

The sample i s then irradiated with UV l i g h t

5.6 Gravimetric Analysis

Parikh and Mukherji (39) reported t h e quant i ta- t i v e formation of chloroquine-silicatungatate CSi02-

78

CH LOROQUI N E PHOSPHATE

1803 -2(C18H2@3Cl). 2H20] precipi ta te from chloroquine phosphate and s i l i c a t u n g s t i c a c i d . By use of t h e appro- p r i a t e grav imet r ic f a c t o r t h e var ious salts o f chloroquine could be determined. grav imet r ic method whereby t h e chloroquine base is measured.

USP XVI (10) a l s o contained a

5.7 Chromatographic Analysis

5.71 Paper Chromatosaphic Analysis

A number of paper chromatographic systems for chloroquine phosphate and i t s base are summarized i n Table 3. Goldbaum and Kazyak ( 4 1 ) use iodop la t ina t e reagent t o v i s u a l i z e t h e chloroquine which i n t u r n may be e lu t ed o f f t h e paper and t h e spo t quan t i t a t ed by o t h e r means.

5.72 Thin-Layer Chromatographic Analysis

The fo l lowing TU: systems (Table 4 ) are u s e f u l as an i d e n t i t y tes t and i n t he eva lua t ion of t he p u r i t y of t h e drug substance. The na tu re of t he impur i t i e s present is a l s o h e l p f u l i n t h a t it t e l l s i n d i r e c t l y , f o r example, s i m i l a r i t y o r d i e s i m i l a r i t y of t h e manufacturing process .

A l l of t h e systems u t i l i z e precoatcd s i l i c a g e l conta in ing a f l u o r e s c e n t i n d i c a t o r .

5.73 Gas-Liquid Chromatographic Analysis (GLC)

The fo l lowing procedures have been demonstrated t o be app l i cab le t o t h e GLC examination of chloroquine base. Vanden Heuvel, -- e t a1 ( 4 6 ) Viala, e t a1 (47) and Kazyak and Knoblock (48) have repor ted t h e d e t e c t i o n of t h e drug i n microgram amounts from b i o l o g i c a l samples after so lven t e x t r a c t i o n . Holtzman (49) has shown t h a t a8 l i t t l e as 5 nanogram q u a n t i t i e s of chloroquine base could be de tec ted us ing e l e c t r o n capture . The condi t ions repor ted , however, are f o r pure drug and m a y be of p o t e n t i a l value i n t h e a n a l y s i s of t h e substance i n b i o l o g i c a l ma te r i a l e .

The fol lowing condi t ions have been used f o r t he

Column: 3.8s S i l i c o n e Gum SE 30, 4 ft, g la se Support : Diatopor t S Detect ion: FID

CIA: de te rmina t ion of chloroquine base (50) .

79

Solvent System

1. n-Butanol s a t ' d w i t h b u f f e r

2. n-Butanol sat 'd wi th buffer

3 . n-Butanol sa t 'd wi th buffer

4. n-Butanol sat 'd W 0 w i t h b u f f e r

5. Ethanol-Water-Conc ammonia (35-63-2)

6. AS NO. 5 (45-53-2)

7 - AS NO. 5 (55-43-2)

8. AS NO. 5 (65-33-2)

9. AS NO. 5 (75-23-2)

10. As No. 5 (85-13-2)

11. As No. 5 (95-3-2)

Table 3 - Paper Chromatographic Systems

Species

base

base

base

base

base

base

base

base

base

base

base

Paper

Whatman No. 2 s a t ' d w i t h pH 3 .O MacIlvaine 's buffer

Whatman No. 2 sat 'd w i t h pH 5 .O MacIlvaine ' s buffer

Whatman No. 2 sat 'd w i t h pH 6.5 Sorensen's buffer

Whatman No. 2 sat 'd w i t h pH 7.5 Sorensen 's buffer

Whatman N o . 1 s a t ' d w i t h petroleum (195-220' f r a c t i o n )

as above

as above

as above

as above

as above

as above

Detect ion

uv

uv

W

uv

w

w w uv W

W

uv

R f

0.15

-

0.16

0.26

0.89

0.36

0.60

0.79

0.87

0.88

0.89

0.89

Reference

4 1

41

41

41

42

42

42

42

42

42

42


Table 4 - TLC Systems

System Spot t ing Soln 1 Methanolwater-conc Chloroform

a m o n i a (72:25:3)

Benzene-methanol- Chloroform' ieopropylamine (87:10:3)

Chlorof orm-aethanol- MeOH-H 0 isopropylamine (94:3 :3) (737

Chloroform-isopropylamine Chloroform2 (97:3 1

Ether -hexane - i sopropyl- m i n e (90:~:~) i sopropylaaine

n-Butanol-conc mmonia- Water water (85:k:ll)

Ethylacetate-conc ammonia- Water ebs . a lcohol (5:2:2)

25$ ammonia-benzene-dioxane- Water e thanol

Chloroform-cyclohexane- Water diethylamine ( 5 :4: 1 )

H20/MeOH/CHC13 /

(1 : 10:8 : 1)

25% ammonia-methanol (3:200)

Water

Acetone -water -ammonia abs. a lcoholS (90 : 40 : 1 )

Rf x 100

2%

41

40

15

28

59

60

28

40

20

15

Reference

43

43

43

43

43

43

44

44

44

44

45

Spot t ing s o l u t i o n 1. 2. Similar t o above b u t from human plasma. 3 . Spotted as t h e base .

The drug i s extracted i n t o chloroform a f t e r bas i fy ing with 10% Na2C03.

Detect ion A - W-254 B - Iodine vapor/20$ H2S04 C - Dragendorff's reagent D - w-360 E - Iodopla t ina te reagent

81

DONALD D. HONG

Temperature : I n j . port 275 "C Column 240 O C Detector 250 "C

Flaw Rate: 30 ml/min helium Retention Time: 7.0 min

5.8 Miscellaneous Methods of Analysis

Roushdi and Shafik (51) reported t h e determina- t i o n of chloroquine phosphate by t i t r a t i o n with an anionic surfactant , d i o c t y l sodium sulfosuccinate (Aerosol O.T. ), using dlmethyl yellow as indicator .

A complexometric method using bismuth complexonate t o p rec ip i t a t e t he chloroquine base and t i t r a t i n g t h e liberated EWIA w i t h zinc s u l f a t e was also reported by these authors (52).

Various quinine salts and chloroquine phosphate have been determined using ammonium reinckate (53). chloroquine phosphate t h e insoluble re inckate salt I s formed a t pH 1, removed, and t h e amount of excess reagent In t h e f i l t ra te is measured colorirnetrlcally. The difference i n absorbance between the sample and blank and t h e standard and blank represent t h e absorbances of t h e sample and standard solut ions, respect ively.

For

82

CH LOROQU IN E PHOSPHATE

6. References

1. S. Clemans, Sterling-Winthrop Research I n s t i t u t e , Personal Communication.

2.

3- 4.

5.

6.

7. 8.

9.

10.

11.

12. 13

14. 15.

16.

17 *

18.

19 20. 21.

22.

23 *

24. 25

B. Riegel and L. T. Sherwood , Jr. , JACS, 71, 1129 (1949)- United S ta t e s Pharmacopeia X M , p. 82 (1975). R. L. Kenyon, J. A. Wiesner and C. E. K w a r t l e r , Ind. and Ehg. Chem. , 41, 659 (1949). P. Dederick, Sterling-Winthrop Research I n s t i t u t e , Unpublished Data. W. Houghtaling, Sterling-Winthrop Research I n s t i t u t e , Personal Communication. R . C. Elderf ie ld , Chem. Eng. N e w s , 6, 2598 (1946). R . L. Kenyon, J. A. Wiesner and C. E. K w a r t l e r , Ind. and Eng. Chem. , 2, 654 (1949). U. P. Basu, A. Raychaudhuri and R. De, Res. and Ind.,

H. Andersag, S. Brei tner and II. Jung, German Patent 683,692 (1939). - Ibid . , U . S. Patent 2,233,970 ( t o Winthrop Chemical Company) (1941). A. R. Surrey and H. F. Hammer, JACS, 68, 113 (1946). E. 0. Titus , L. C. Craig, C. Golumbic, H. R. Mighton, I. M. Wampen and R. C. Elderf ie ld , J. Org. Chem.,

10, 165 (1965).

UJ 39 (194a)* K. Kuroda, J. Pharmacol. Exp. Ther., u, 156 (1962). E. W . McChesney, W. D. Conway, W . F. Banks, Jr. , J. E. Rogers, and J. M. Shekosky, fbid., 151, 482 ( 1966 . E. W . McChesney, M. J. Fasco and W. F. Banks, Jr., - Ibid. , 158, 323-(1967). M. Rubin. H. N . Bernstein and N. J. Zvaif ler , Arch. Ophthalmil. , 70, 474 (1963 ) . L. T. Grady, Drug Standards Laboratory, Personal Communication. P. Cooper, Pharmaceutical J., 177, 53 (1956)- - I b i d . , p. 495. E. G. C. Clarke, J. Pharm. and Pharmacol., l0, 194 (1958 *

E. Si lva, Anal. Chem., Proc. I n t . Symp. i n Honor of F r i t z Feigl , Birmingham, Engl., Apri l 1962. Elsevier, 1963 , p. 78. J. Lel i jveld and H. Kortmann, Bull. WHO, 42, 477

G. Fuhrmann, w., 22, 663 (1960). W. T. Haskins, Am. J. Trop. Med. Hyg., 7, 199 (1958).

( 1970 -

83

DONALD D. HONG

26

27. 28.

29

32.

33

34.

37

38-

39

40. 41.

42.

43

44.

45

46.

47

48.

49 50

A. F. Fartushnyi, Farmatsiya, l8, 77 (1969); CA 71: 47882 v (1969). R. Strufe , Clin. Chlm. Acta, 2, 753 (1960). A. F. Fartusknyl, Sudebro-Med. Ekspertiza. Mln. Zdravokhl SSR, lo, 45 (1967); CA 66:114281 k (1967). 0. Fuhrmann and K. Werrbach, 2. Tropenmed. Parasi tol . , l.6, 269 (1965): CA 65:12725 b (1966). M. E. Auerbach, Sterling-Winthrop Research Ins t i t u t e , Unpublished Data. T. S. Wu, T. 0. Sun and T. H. Tang, Yao Hsueh Hsueh Pao, 6, 253 (1958); CA 53~20691 d (1959). R . W. Prouty and K. Kuroda, J. Lab. Clin. Med., 2, 477 (1958)- V. Waaret, Archiv. For Pharmacl. Og. Cheml., 71, 116 (1964) A. E. Robinson, A. I. Coffer and F. E. Camps, J. Pharm. and Pharmacol., 22, 700 (1970). J. Stepan and B. Kakac, Med. Exp., ll, 352 (1964). B. B. Brodie, S. Udenfrlend, W. D i l l and T. Chenkin, J. Biol. Chem., x, 319 (1947). E. M. Ensor, Trans. Roy. SOC. Trop. Med. Hyg., 60,

E. W. McChesney, W . F. Banks and J. P. McAullff, Ant ibiot ics and Chemotherapy, 12, 583 (1962). P. M. Parlkh and S. P. Mukherji, Ind. J. Pharm., 5, 269 (1.963). United S ta t e s Pharmacopeia XVI, p. 151 (1960). L. R. Goldbaum and L. Kazyak, Anal. Chem., 28, 1289 (1956). J. Vecerkova, J. Solc and K. Kacl, J. Chromat., l0, 479 ( l963) . R. Crain, Sterling-Winthrop Research I n s t i t u t e , Unpublished Data. L. T. Grady, Drug Standards Laboratory, APhA, Unpublished Data. R. Castagnou and M. Sylvestre, Bull . SOC. Pharm. Bordeaux, 105, 121 (1966); CA 67~62821 k. W. J. A. VandenHeuvel, B. 0. A. Kaahti and E. C. Horning, Cl tn . Chem., g, 351 (1962). A. Viala, J. P. C a n 0 and A. Durand, J. Chromatog.,

L. KaWsk and B. C. Knoblock, A n a l . Chem., s, 1448 (1963) J. L. Holtzman, Anal. Biochem., u, 66 (1965). J. Grego, Sterling-Wlnthrop Research I n s t i t u t e , Unpublished Data.

75 (1966).

111, 299 (1975).

84

CHLOROOU IN E PHOSPHATE

51. I. M. Roushdi and R . M. Shafik, J. Pharm. Sci.

52. - Ibid. , p. 57. 53. S-K. Sheng, C - I . Hsieh and S-H. Peng, Yao Hsueh

U.A.R., 2, 65 (1968).

Hsueh Pao, l2, 662 (1965); CA 64:7967 c (1966).

Acknowledgments

The writer wishes t o thank Mr. E. L. P r a t t and D r . R . S. Browning f o r reviewing the mnuacript , D r . S. D. Clemans for the NMR and M.S. data, Mr. W . W . Houghtaling f o r the DSC data, Dr. L. T. Grady (USP Laboratories) f o r t h e TLC data, Mrs. E. V. Miller f o r the drawings and Miss S. F. Casey f o r typing t h e prof i le .

85

DAFSONE

Chester E. Orzech, Norris G. Nash, and Raymond D. Daley

CHESTER E. ORZECH ma/.

CONTENTS

1. DESCRIPTION 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2. PHYSICAL PROPERTIES 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectra 2.4 Mass Spectrum 2.5 Differential Thermal Analysis (DTA) 2.6 Differential Scanning Calorimetry (DSC) 2.7 Crystal Properties 2.8 Solubility 2.9 Fluorescence Spectra 2.10 Melting Point

3. SYNTHESIS

4. STABILITY-DEGRADATION

5. DRUG METABOLIC PRODUCTS

6. METHODS OF ANALYSIS 6.1 Identification Tests 6.2 Elemental Analysis 6.3 Colorimetric Methods 6.4 Titration Methods 6.5 Fluorometric Methods 6.6 Paper Chromatography 6.7 High Pressure Liquid Chromatography 6.8 Gas Chromatography 6.9 Thin Layer Chromatography

7. ACKNOWLEDGMENTS

8. REFERENCES

88

DAPSON E

1. DESCRIPTION

1.1 Name, Formula, Molecular Weight Dapsone i s 4,4'-diaminodiphenyl sulfone, a l s o

known as p i p '-sulf onyldianil ine and b i s (4-aminophenyl) sulfone. Chemical Abstracts indexes dapsone a s benzen- amine, 4,4'-sulfonylbis-, s t a r t i n g with Volume 76; previously the index name was an i l i ne , 4,4'-sulfonyldi-. The CAS Registry Number is [80-08-0].

0

H2N c $*H2 0

Mol. W t . : 248.31

1.2 Appearance, Color, Odor Dapsone is a white or creamy white odorless crys-

t a l l i n e powder.

2. PHYSICAL PROPERTIES

2.1 Infrared Spectra Infrared spectra of two c r y s t a l forms of dapsone

(designated Form 1-and Form 11) are shown i n Figures 1 and 2. The samples were prepared a s mineral o i l m u l l s between potassium bromide plates . A Beckman Model I R - 1 2 spectrophotometer was used. The spectrum of Form I i s similar t o those published by Pouchert (1) and by Hayden e t a 1 (2). been observed; s ince i t is d i f f e r e n t from the spectra of the anhydrous forms, samples should be dried a t 105OC before obtaining spectra. Some of the absorption bands may be assigned a s follaws (3):

The spectrum of a hydrate of dapsone has a l s o

89

(D 0

Figure 1. Infrared Spectrum of Dapsone (Form I ) , Mineral Oil Mull

Figure 2. Infrared Spectrum of Dapsone (Form II), Mineral O i l Mull

CHESTER E. ORZECH Hal .

Band frequency, cm -’ Assignment

3200-3500 N-H S t r e t ch

3000-3 100 Aromatic C-H S t r e t ch

2800-3000 Mineral O i l

1635 N-H Deformation

1590, 1500, 1440 Aromatic C=C S t r e t ch

1460, 1380 Mineral O i l

1300 Region Asymmetric -SO2- S t r e t ch

1150 Synunetric -SO2- S t r e t ch

830-840 2 Adjacent H on aromatic r ing

550 -SOz- Scissoring

2.2 Nuclear Magnetic Resonance Spectrum The NMR spectrum shown i n Figure 3 was obtained

by dissolving USP reference standard dapsone i n acetone- d containing tetramethylsilane as i n t e r n a l reference. Tke spectrum was produced using a Varian EM-360 NMR spectrometer. The s e r i e s of peaks centered a t 7.2 ppm is an A2Bp pa t t e rn t h a t is typical of para s u b s t i t u t i o n and the broad s i n g l e t a t 5.4 ppm is due t o the amine protons. The peaks a t 2 ppm and 3 ppm a r e due t o the solvent. These values co r re l a t e well with those previously reported ( 4 )

2.3 Ultraviolet Spectra Figure 4 is the u l t r a v i o l e t absorption spectrum

of dapsone i n methanol solut ion, run on a Cary Model 14 spectrophotometer. dapsone per l i t e r of methanol, and was run against methanol ( 1 cm c e l l s ) . The spectrum exh ib i t s peaks a t 295 nm and 260 nmwith abso rp t iv i t i e s of 30,100 and 18,300 l./mole c m respectively. The u l t r a v i o l e t i d e n t i t y t e s t of the B r i t i s h Pharmacopoeia (5) spec i f i e s peaks a t

The solut ion contained 8.0 mg of

92

I--- .

I I I I I I I I I I I 1

10 9 8 7 6 5 4 3 2 I 0 6

Figure 3. Nuclear Magnetic Resonance Spectrum of Dapsone

0

In

m

..

.

IL

z

I

I- 0

z

W

-

94 (D P

w v z 2 % a

m a

210 250 300 350 WAVELENGTH IN NANOMETERS

Figure 4 . Ultraviolet Spectrum of Dapsone, 8 mg/l., in Methanol, 1 cm Cells

CHESTER E. ORZECH eta/.

295 and 260 nm with a b s o r p t i v i t i e s of about 30,000 and 18,100 l./mole cm respectively.

A s imi l a r spectrum is obtained i n 95% ethanol so lu t ion , except t h a t the a b s o r p t i v i t i e s a r e apparently lower. Baliah and Ramakrishnan (6) r epor t peaks a t 295 and 261 nm wi th a b s o r p t i v i t i e s of 27,000 and 17,800 1./ mole cm.

dapsone a t various hydrogen ion concentrations; the da ta a re a s follows:

Hayden e t a 1 (2) r epor t peaks a t 295 and 260 nm. Maschka e t a 1 (7) reported u l t r a v i o l e t spec t ra of

Wave length Molar Absorptivity, Condition of Maxima, nm l./mole cm

1. pH 11.0 291.0 19,820 258.5 12,190

2. pH 6.8 291.0 259.0

17,950 10,810

3. pH 1.8 289.5 13,490

4. 22 H C l 273.5 264.5 234.5

1,850

10,790 2,000

2.4 Mass Spectrum Figure 5 shows the low re so lu t ion mass spectrum

of dapsone. The da ta w a s obtained wi th an LKB 9000s mass spectrometer, with an ion iza t ion voltage of 70 eV, source temperature 250'C. follows (8):

Some of the peaks may be assigned a s

- m/e Assignment

248 M+

140

108

92

95

0) a

v1

96

50

MASS/ CHARGE R AT10

Figure 5. Mass Spectrum of Dapsone

DAPSONE

2.5 Di f fe ren t ia l Thermal Analysis (DTA) The DTA curve in Figure 6 was obtained with a

DuPont Model 900 instrument. The curve shows a sharp endothermic sol id-sol id phase t r a n s i t i o n a t 84OC and a melting endotherm a t 178OC.

2.6 Di f fe ren t ia l Scanning Calorimetry (DSC) The DSC melting thermogram of dapsone c rys ta l l ized

from methanol and water i s shown i n Figure 7 , 'The thermogram was obtained with a Perkin-Elmer DSC-1B d i f f e r e n t i a l scanning calorimeter a t a heating r a t e of 1.25°C/minute i n a nitrogen atmosphere. The puri ty of samples of t h i s compound can be determined by analysis of the melting thermogram (9).

2.7 Crystal Properties B u t t (10) reported t h a t dapsone can be obtained i n

a t l e a s t two c r y s t a l forms, with d i f fe ren t melting points. Infrared spectra (see Section 2.1) and x-ray powder d i f - f rac t ion pat terns a l s o indicate tha t dapsone occurs in a t l e a s t two c r y s t a l forms, end t h a t it forms a c r y s t a l l i n e hydrate. Table 1. These pat terns were obtained with a Norelco d i f - fractometer, using n icke l - f i l t e red copper Kcr radiat ion.

The c r y s t a l and molecular s t ruc ture of dapsone has been determined by Alleaume and Decap (ll), and by Dickinson e t a 1 (12, 13). Both groups report 4 molecules i n an orthorhombic u n i t c e l l , symmetry P212121, with s imilar dimensions (I):

The powder d i f f r a c t i o n pat terns a re given i n

a=25.57 + - 0.01, b=8.07 + - 0.01, c=5.77 5 0.01 (11)

a=8.065 + - 0.005, b=25.57 + c 0.02, c=5.760 2 0.001 ( 1 2 )

97

Figure 6. Differential Thermal Analysis Curve of Dapsone

I I-

to

20

25

00

Irz 0

a, T

f II

i

a

30

\

0

99 (0 (0

6

21

OAPSONE x hHF ~4.90 KCAL.

6 PURITY = 99.99 %

Figure 7 . Differential Scanning Calorimeter Curve of Dapsone

CHESTER E. ORZECH eta / .

TABLE 1 DAPSONE X-RAY POWDER DIFFRACTION PATTERNS

d

12.79 7 . 7 4 6.86 6.42 5.88 5.66 5.28 5.03 4.71 4.64 4.42 4.30 4.12 4.00 3.84 3.78 3.65 3.46 3.42 3.32 3.21 3.16 3.09 2.98 2.94 2.88 2.80 2.78 2.74 2.64 2.56 2.51 2.44 2.40 2.35 2.31 2.25 2.20 2.17 2.13

c 1/10

4 8

32 53 4 9

6 0 3 4 17

100 4 9 62 1

4 8 22 44

2 1

10 2

10 4

46 2 9 4 6 4 4 7 3 2 5 5 3 2 5 3 4 8

- d

13.07 10.78

9.21 7.41 6.85 6.66 6.57 6.40 5.80 5.68 5.45 5.35 5.01 4.66 4.57 4.37 4.27 3.99 3.86 3.82 3.52 3.50 3.42 3.37 3.33 3.28 3.20 3.13 3.09 3.02 2.89 2.86 2.73 2.68 2.52 2.43 2.40 2.34 2.28 2.21

- F o r m I1

100

1/10 - 5 2 1 7 8

8 9 18 18

3 3

27 5 4 23 97 2 0 96

9 8

2 1 1 0 0

23 25

8 9

17 2 8

22 14 26

9 17

3 3 6 2 2 5 2 2

Hydrate

d

12.55 12.25 11.13 8.54 7.73 7.62 7.54 7.45 6 -82 6.39 6.25 5.98 5.74 5.68 5.62 5.53 5.45 5.32 5.12 5.00 4.93 4.86 4.79 4.68 4.59 4.55 4.45 4.39 4.31 4.27 4.22 4.18 4.09 4.05 4.01 3.95 3.86 3.83 3.77 3.72

- 1/10 - 1 4 3

16 2 9 13 47 28 1 7 7 4

28 35 1 9 17 11 4 3

2 1 58

9 2

1 0 0 39 75

6 27 13 5 4 3 1 12 8 7 1 9 13 4

15 1 9 4 3

DAPSONE

2.08 5 2.17 3 2.06 1 2.10 7 1.96 2

3.65 3.58 3.46 3.42 3.37 3.35 3.30 3.28 3.23 3.20 3.14 3.09 3.02 2.96 2.87 2.84 2.82 2.79 2.72 2.69 2.65 2.62 2.58 2.50 2.45 2 -42 2.27 2.15 2 .11 2.10 2.07 1.96

1 14 11

9 13 6

15 8

30 10 2 1 14 8 6 5 3 3 6 6 5 7 7 1 3 3 2 6 5 2 2 3 3

2.8 S o l u b i l i t y The s o l u b i l i t y a t room temperature is a s fol lows:

Solvent

Methanol Ethanol (95%) 2-Propanol Ethyl Acetate Ethyl Ether Chloroform Benzene Water 2,2,4-trimethylpentane

Approximate S o l u b i l i t y , mg/ml

52 28

6 34

0.9 3 0.5 0.2 (14)

c0.2

101


Dapsone is very soluble i n acetone and a c e t o n i t r i l e ; one gram of dapsone dissolves i n 1.8 m l of acetone or 5 m l of ace toni t r i le . It is a l s o soluble i n d i l u t e mineral acids; one gram dissolves i n about 10 m l of 1E hydrochloric acid.

2.9 Fluorescence Spectra Peters e t a 1 (15) found the exci ta t ion maximum a t

285 nm and the fluorescence maximum a t 350 nm, i n ethylene dichloride solut ion, Ellard and Gammon (16) reported an exci ta t ion maximum a t 298 nm and the fluorescence maximum a t 345 nm, i n e t h y l acetate . Glazko e t a 1 (73) and Cucinell e t a 1 (17) reported the exc i ta t ion m a x i m u m a t 297 nm and the fluorescence maximum a t 340 nm, i n e thyl ace ta te ,

2.10 Melting Point A range of melting points has been reported for

dapsone :

172'C 172-173OC 172 - 174'C 174OC 174- 176'C 175OC 175-176'C 176OC 176.3-177.5OC 178-179OC 1 7 9- 1 8 O o C

Bu t (10) reported tha t dapsone can -e obtained ,n two forms, one melting a t about 178.5OC, the other a t about 180.5'C.

3. SYNTHESIS

Dapsone has been prepared by various procedures, s t a r t - ing with 4,4'-dinitrodiphenyl sulf ide ( 2 1 , 2 2 , 31, 32) prepared from p-chloronitrobenzene (33), or by oxidation of 4,4'-diacetylaminodiphenyl sulf ide (19, 23, 25) prepared from 4-nitro-4'-aminodiphenyl su l f ide (19, 23, 24) or 4,4'- diaminodiphenyl sulf ide (25). It has a l s o been made from a su l f ina te and a halonitrobenzene (24, 26, 29 , 35) ; from 4- acetylaminobenzenesulfonyl chloride and acetani l ide (20, 36, 37 , 38); from acetani l ide and thionyl chloride ( 2 7 , 39, 40); from 4,4'-dichlorodiphenyl sulfone (18, 28, 30, 41, 42, 43); from the diphthaloyl derivative of 4,4'-diaminodi-

102

0" fn

A

U

m

x

d

4

U

9 Xm

a

U

.r

z

DU

rnx F 0" cn

2

cn

X"

8 r 0"

0 0

0 x 2" z

V

=O

0

$1 rnx

cu 0

-

rn 9)

G

0

m

i?

d

a

"

I 4

U

0

rn

rfl 0"

X

xm

I4 0

cw . co aJ I4

'4 ii a

103

CHESTER E. ORZECH era/.

phenyl sulfide (44, 45); and from 4,4'-dimethyldiphenyl sulfone by oxidation to the dicarboxylic acid and Hofmann degradation of the diamide (46). The 4,4'-diacetylaminodiphenyl sulfone obtained by any of these methods can easily be hydrolyzed to the diamino compound (23). Sanghavi (47) has reviewed the various methods of synthesis. these procedures are outlined in Figure 8.

4.

A few of

S TAB1 LITY -DEGRADAT I ON

No reports of stability studies or degradation of dapsone were found.

5, DRUG METABOLIC PRODUCTS

Dapsone metabolic products have been reported as follows:

Me t a bo 1 i te

Dapsone glucuronide

Dapsone sulfamate

Dapsone monohydroxylamine

Monoace tyl dapsone

Dapsone monohydroxylamine su 1 f ama te

Dapsone monohydroxylamine glucuronide

Monoace tyl dapsone sulfamate

Species

Man Monkey Rabbit

Man Rat

Man Dog

Man

Monkey Rabbit

Man

Man

Man Monkey

References

(48,49,50)

(48,51,53, (51,521

54,55)

(48,50 1 (51,521

(56 ,581 (56,57,58)

(15,51,57, 59,60,61) (51,52,62) (51,61,62)

(58)

(58 1

(50) (51,52)

104

p: ot;;:

z II

xx

0

0

n

Y .. a a

u rl

M

al

5 cd ru 1

cn

82

2-

hl

X

(I 8

ocv)+

o

z, X

X z

o=

u X

V 0

rl x

Xm

V

o=u

5:

105

Dapsone sulfamate (DDS-S) Dapsone glucuronide (DDS-G)

Dap s one (DDS )

I Dapsone monohydroxylamine

(DDS-NOH) Az ox y -d aps one (AZ my-DDS )

Monoace t y l dapsone Monoace t y l dapsone hydroxylamine Diace t y l azoxy-dapsone (MADDS (MADDS-NOH)

Figure 9. Some Dapsone Metabolites

CHESTER E. ORZECH era/.

Monoacetyl dapsone glucuronide Man (50,511 Monkey (52) Rabbit (51) Rat (51)

Monoace t y l dapsone hydroxylamine Man (57)

Az my-da ps one Man (571 R a t (57)

Diace t y l azoxy-dapsone Man (57)

The s t ruc tu res of some of these metabolites, a r e shown i n Figure 9.

6. METHODS OF ANALYSIS

6.1 Iden t i f i ca t ion Tests Dapsone is e a s i l y iden t i f i ed by the physical prop-

e r t i e s described i n sec t ion 2 above. Where iden t i f i ca t ion of dapsone i n formulations i s necessary, it can be extracted by various organic solvents such as methanol, chloroform, ethylene dichlor ide, e t c . I f the i d e n t i f i c a t i o n of very small amounts of dapsone i s necessary, the colorimetric method of Peters e t a1 (63), the color imetr ic t e s t s described i n sec t ion 6.3, or the fluorometric t e s t s described i n sec t ion 6.5 may be useful.

6.2 Elemental Analysis The elemental composition of dapsone is as follows:

Element %Theory

Carbon Hydrogen Nitrogen Su If u r Oxygen

58.04 4.87 11.28 12.91 12.89

6.3 Colorimetric Methods There a r e two basic colorimetric methods f o r dap-

sone. The f i r s t and most widely used is the method of Bratton and Marshall (64). Dapsone is diazotized, then coupled with N-(1-naphthy1)-ethylenediamine t o form an 820

dye. Other invest igators have used t h i s basic method but have used d i f f e r e n t coupling reagents: N i t t i e t a 1 (651, dimethyl- 0 -naphthylamine; Rose and Bevan (66), N-@-sulfa-

106

DAPSON E

toethyl-m-toluidine; Schoog (67), l-sulfomethylaminonaph- thalene-8-sulfonic acid; Merland (68), N-naphthyl-N' ,N'-di- e thy 1 pro py lene d iamine . Higgins (69), is based on the formation of a Schiff base between dapsone and 4-dimethylaminobenzaldehyde. This method is reported t o be 2.4 times more sens i t i ve than the Bratton-Marshall technique and, i n addi t ion, it i s reported t o be s p e c i f i c f o r dapsone i n the presence of i ts metabo- li tes.

The second type of method, reported by Levy and

6.4 T i t r a t i o n Methods Tomicek (70) t i t r a t e d dapsone potentiometrically

with perchloric acid i n g l a c i a l a c e t i c acid. Wojahn bro- minated dapsone with bromide-bromate and back-t i t ra ted the excess bromate with sodium th iosu l f a t e so lu t ion (71) . The USP method of assay i s t i t r a t i o n of the cooled, s t rongly acid sample so lu t ion with sodium n i t r i t e so lu t ion to an electrometric endpoint (72).

6.5 Fluorometric Methods Glazko (73) reported a fluorometric method fo r

dapsone i n plasma and ur ine samples. Complete d e t a i l s of sample preparation a r e given, The fluorescence of the e thy l ace t a t e ex t r ac t is determined by ac t iva t ing a t 297 nm and measuring the emission a t 340 nm. Ellard and Gammon (16) developed an ex t r ac t ion scheme t o determine dapsone and two possible metabolites, MADDS and DADDS, fluorometr ical ly i n plasma and urine samples. Peters e t a1 (15) modified and expanded the above procedure. They extracted with ethylene dichloride instead of e t h y l ace t a t e and thus eliminated the problem of quenching caused by small quan- t i t i e s of water i n the e thy l ace t a t e extract .

6.6 Paper Chromatography Longenecker (74) reported procedures fo r p a r t i t i o n

chromatography of dapsone by both ascending and descending chromatography on paper and s t r ing . Bushby and Woiwood (54, 55) u s e d paper chromatography t o i s o l a t e and i d e n t i f y dapsone and some diazot izable metabolites by paper chromatography and electrophoresis. Wadia e t a 1 (75) reported Rf values fo r dapsone and twenty f ive other diaminodiphenyl su l f ides , sulfoxides, and sulfones using four d i f f e r e n t solvent systems. Jardin and S t o l l (76), Khosla e t a 1 (77), and Tsu t sumi (48) a l l used paper chromatography i n t h e i r s tud ie s on the metabolism of dapsone.

107


6.7 High Pressure Liquid Chromatography Gordon and Peters (78) separated dapsone, mono-

ace ty l dapsone (MADDS), and d iace ty l dapsone (DADDS) a t the 1 t o 20 ,,g l eve l on a s i l ica ge l column with e t h y l acetate as solvent. The e f f luen t was monitored a t 280 nm. Murray e t a1 (79) used a fluorometric detector and increased the s e n s i t i v i t y of the determination t o the 10 ng level. This procedure was modified by Murray e t a 1 (80) t o increase the s e n s i t i v i t y t o 0.1 ng i n 0.5 m l . pressure accelerated chromatography through microparticulate s i l ica g e l columns, packed by centr i fugat ion, t o separate dapsone, MADDS, and DADDS, with chloroform-methanol (97:3) as solvent and u l t r a v i o l e t absorption a t 254 nm fo r detection.

chloroform-carbon te t rachlor ide (7:3) solvent t o examine dapsone f o r impurit ies. the absorption a t 280 nm. Fractions were col lected and evaporated, and the residues were dissolved i n ethylene d i - chloride. The fluorescence of the re-dissolved f r ac t ions was measured. Impurities determined were 2,4'-diaminodiphenyl sulfone and 4-aminodiphenyl sulfone .

Krol and Mannan (83) developed a method fo r the analysis af dapsone using a commercially avai lable s i l i c a ge l column 6 - P o r a s i P ) , a solvent containing isopropyl a l - cohol, a c e t o n i t r i l e , e thy l ace t a t e , and pentane (1:1:1:7), and detect ion a t 254 mo su i t ab le e i t h e r fo r assay f o r dapsone i n t a b l e t s or for analyzing fo r impurit ies i n dapsone. The following r e l a t ed compounds can be separated from dapsone: (a) 2,4'-diaminodiphenyl sulfone; (b) 4-aminodiphenyl sulfone; (c) 4-amino- 4'-chlorod iphenyl sulf one ; (d ) 4,4'-d iace tamidod iphenyl sulfone (DADDS) ; (e) 4-amino-4I-ace tamidodiphenyl sulf one (MADDS); ( f ) 4-amino-4'-hydroxydiphenyl sulfone; (g) 4,4'- diace tamidodiphenyl sulfoxide; 4,4'-diaminodiphenyl su l f ide (90).

Ribi e t a 1 (81) used

Gordon e t a1 (82) used a s i l i c a ge l column with

Column e f f l u e n t was monitored by

A 30 cm by 4 mm I.D. column was

6.8 Gas Chromatography Burchfield e t a 1 (84, 85) reported tha t dapsone can

be chromatographed d i r ec t ly . Because t h e i r l a t e r work required greater s e n s i t i v i t y , and, i n addi t ion, the determination of MADDS and DADDS, the iodo der ivat ives were prepared by diazot izat ion and an e l ec t ron capture detector w a s used. Using a 4 foot by 0.25 inch O.D. g las s U-tube packed with 3% Poly-A-103 on 100-120 mesh Gas Chrom Q a t 285'C with nitrogen, 280 pg of iodo der ivat ive was e a s i l y detectable.

108

DAPSONE

Chang e t a 1 (52) investigated the metabolism of dapsone by chromatographing t r ime thy l s i ly l de r iva t ives of the metabolic products on 3% OV-17 (Gas Chrom Q) a t 275OC.

6.9 Thin Layer Chromatography Thin layer chromatography systems have been com-

piled i n Table 2 .

7. ACKNOWLEDGMENTS

The w r i t e r s wish t o thank D r . B. T. Kho f o r h i s review of the manuscript, D r . G. Schi l l ing of Ayerst Research Laboratories f o r h i s mass s p e c t r a l data and in t e rp re t a t ion , the l i b r a r y s t a f f fo r t h e i r l i t e r a t u r e search, and Mrs. B. Juneau fo r typing the prof i le .

109

TABLE 2

Adsorbent

Silica gel

Silica gel DF-5

Silica gel G 11

11

11

11

11

11

I 1

I1

11

11

11

So Lvent S ys tem

Chloroform-Butanol-Pe troleum Ether (1 : 1 : 1)

Toluene-Ethyl acetate (1 : 1)

Chlorof om-Hep tane-E thanol (1: 1: 1)

Ace tone-Chloroform (1:9)

Chloroform-Ethyl Ether (85: 15)

Toluene-Ethyl acetate (1:l)

Chloroform-Methanol (95:5)

Chloroform-Methanol (9: 1)

Chloroform-Ethanol (9:l)

Chlor of orm-Ace tone -Die thanolamine ( 5 : 4 : 1 )

Me thano 1-Ace tone -Die thanolamine ( 50 : 50 : 1.5 )

Ace tone -Chlorof o m (9 : 1 )

Isopropyl alcohol-cyclohexane-259. Axunonia

Dime thy1 f ormamide-Die thylamine-Ethanol-

(65:25:10)

Ethyl acetate (1:1:6:12)

!% 0.62

0.30

0.60

0.20

0.11

0.27

0.47

0.48

0.65

0.69

0.78

0.84

0.85

0.88

Ref.

86

87

88

89

-

11

11

11

11

11

11

11

11

11

11

DAPSONE

8. REFERENCES

1.

2.

3.

4.

5.

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21. 22.

C. J. Pouchert, "The Aldrich Library of Infrared Spectra", Aldrich Chemical Company, Inc., Milwaukee, Wisconsin, 1970, spectrum 843F. A. L. Hayden, 0. R. Samul, G. B. Selzer, and J. Carol, J. Ass. Off. Agr. Chem. 45, 797-900 (19451, spectrum 29. L. G. Bellamy, "The Infra-red Spectra of Complex Molecules", 3rd ed., John Wiley and Sons, Inc., New York, N. Y., 1975. J. G. Grasse l l i (ed.), "Atlas of Spectral Data and Physical Constants f o r Organic Compounds", Chemical Rubber Company, Cleveland, Ohio, 1973, p. B-918. "Bri t ish Pharmacopoeia 1973", Her Majesty's Stat ionery Office, London, England, 1973, p. 140. V. Baliah and V. Ramakrishnan, J. Indian Chem. SOC.

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H. Dorn and G o Hilgetag, Ger. (East) Pa ten t 33,094; C.A. 64, PC65Og. F renchPa ten t , 829,926; C.A. 33, 1760. H. G. Biswas, S c i . Cul t . (Calcu t ta ) 28, 586-8 (1962); C.A. 2, 3806e. Yu-Li Chi and Jo-Yung Moh, K'e Hsueh Tung Pao 2, 50-1 (1957); C.A. 53, 18896i. Nietzki and Bothof, Ber. 27, 3261 (1894). Hodgson and Rosenberg, J. Chem. SOC. 180 (1930). A. M. Morales, Anales fac. farm. y bioquim., Univ. nacl . mayor San Marcos (Lima, Peru) 2, 607-14 (1950); C.A. 49, 210b. K e r e s z y and Wolf, Hung. Patent 120,021; C.A. 33, 4600. Indian Pa ten t 47,585; C.A. 49, 1101c. S w i s s Pa ten t 243,601; C.A. 43, 7960e. S. P. Raman and S. C. Niyogy, Ann. Biochem. and Exp t l . Med. ( Ind ia ) 2, 207-10 (1955); C.A. 50, 12880~ . W. Braun, Ger. Pa ten t 964,593; C.A. 53, P12240g. Brit. Pa ten t 506,227; C.A. 33, 9328. A. Girard, French Pa ten t 844,220; C.A. 34, 7543. Ya. Ya. Makarov-Zemiyanskii, Nauch. T r u d y Moskov. Tekhnol. In s t . Legkoi Prom., Sbornik 1957, No. 8 ,

V. A. Zasosov, E. I. Metel'kova, and M. I. Galchenko,

22-4 (1940); C.A. 34, 3704.

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Med. Prom. S.S.S.R. - 13, No. 2 , 18-20 (1959); C.A. 53, 19944a. S. Radulova and E. Topalova, Vet. Med. Nauki (Sof ia ) - 2 , 873-7 (1965); C.A. 64, 17460g. P.P.T. Sah, S. A. PeopGs, S. T. Kwan, and H. J. Sah, Arzneim.-Forsh. 17, 425-31 (1967); C.A. 67 , 424772. N. M. Sanghavi, Indian Chem. J. 6 (11, 179-81 (1971). S. T s u t s u m i , Chem. and Pharm. B u i l . (Tokyo) 2, 432-6 (1961); C.A. 56, 2850h.

112

DAPSONE

49.

50.

51.

52

53. 54 . 55.

56. 57.

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62

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64.

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67.

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70 .

J. O'D. Alexander, E. Young, T. McFadyen, N. G. Fraser, W. P. Duguid, and E. M. Meredith, B r i t . J. Dermatol. - 83, 620-31 (1970). J. H. Peters, G. R. Gordon, D. C. Ghoul, J. G. Tolentino, G. P. Walsh, and L. Levy, Am. J. Trop. Med. Hyg. 2, 450-7 (1972). A. J. Glazko, T. Chang, J. Baukema, S. F. Chang, A. Savory, and W. A. D i l l , Int. J. Leprosy 37, 462-3 (1969). T. Chang, S. F. Chang, J. Baukema, A. Savory, W. A. D i l l , and A. J. Glazko, Fed. Proc. 28, 289 (1969). G. A. Ellard, B r i t . J. Pharmacol. 26, 212-7 (1966). S. R. M. Bushby and A. J. Woiwood, Biochem. J. 63, 406-8 (1956). S. R. M. Bushby and A. J. Woiwood, Amer. Rev. Tuberc. - 72, 123-5 (1955). S. Tabare111 and H. Uehleke, Xenobiotica 1971, 501-2. 2. H. I s r a i l i , S. A. Cucinell, J. Vaught, E. Davis, J. M, Lesser, and P. G. Dayton, J. Pharmacol. Exp. Ther. 187, 138-51 (1973). H. U e h m e and S. Tabarel l i , Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 55-68 (1973). J. H. Peters, G. R. Gordon, L. Levy, M. A. Storkan, R. R. Jacobson, C. D. Enna, and W. F. Kirchheimer, Am. J. 'hop. Med. Hyg. 2, 222-30 (1974). G. A. Ellard and P, T. Gammon, In t . J. Leprosy 37, 398-405 (1969). J. H. Peters, G. R. Gordon, R. Gelber , and L. Levy, Int . J. Leprosy 37, 463 (1969). 0. R. Gordon, J. H. Peters, R. Gelber , and L. Levy, Proc. West. Pharmacol. SOC. 13, 17-24 (1970). J. H. Peters, S. C. Lin, and L. Levy, Int . J. Leprosy 36, 46-51 (1969). A. C. Bratton and E. K. Marshall, J. Biol. Chem. 128, 537-50 (193 9), F. N i t t i , D. Bovet, and V. Hamon, Compt. rend. soc. biol . 128, 26-8 (1938); C.A. 32, 6332. F. L. Rose and H. G. L. Bevan, Biochem. J. 38, 116 (1944).

M. Schoog, Arzneim.-Forsch. - 2, 512-4 (1952); C.A. 47, 3392b. R. Merland, Ann. biol . cl in. (Par is) 13, 21-32 (1955); C.A. 2, 8359c. L. Levy and L. J. Higgins, In t . J. Leprosy 34, 411-4 (1966). 0. Tomicek, Col l . Czech. Chem. Comuns. 2, 116-30 (1948); C.A. 42, 8108g.

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71.

72.

73.

74. 75.

76.

77.

78.

79.

80.

81.

82.

83 . 84.

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90.

H. Wojahn, Suddeut. Apoth.-Ztg. - 88 , 395-6 ( 1 9 4 8 ) ; C.A. 43, 1908f . T n i t e d S ta tes Pharmacopeia XIX", Mack Publishing Co. , Easton, Pa., 1975, pp. 118-19 and 626. A. J. Glazko, W. A. D i l l , R. G. Montalbo, and E. L. Holmes, Amer. J. Trop. Med. Hyg. 17, 465-73 (1968). W. H. Longenecker, Anal. Chem. 3, 1402-5 (1949) . P. S. Wadia, M. C. Khosla, and N. Anand, J. Sci . Ind. Research (India) E, 148-52 ( 1 9 5 5 ) ; C.A. 2, 12195d. C. Jardin and A. Stoll, Semaine hop. Therap. 34, 611- 16 ( 1 9 5 8 ) ; C.A. 52, 20627d. M. C. Khosla, J. D. Kohli, and N. Anand, J. Sci. Ind. Research (India) E, 51-6 (1959) ; C.A. 53, 19131d. G. R. Gordon and J. H. Peters, J. Chromatogr. 47, 269- 71 (1970). J. F. Murray, G. R. Gordon, and J. H. Peters, J. Lab. Clin. Med. 78, 464-71 (1971). J. F. MurraE G. R. Gordon, C. C. Gulledge, and J. H. Peters, J. Chromatogr. 107, 67-72 (1975). E. Ribi, S. C. Harris, J. Matsumoto, R. Parker, R. F. Smith, and S. M. S t ra in , J. Chrom. Sci . l.0, 708-11 (1972). G. R. Gordon, D. C. Ghoul, and J. H. Peters , J. Pharm. Sci. 64 , 1205-7 (1975). a. J.?rol and C. V. Mannan, Ayeret Laboratories, Inc., Rouses Point, N. Y., private communication. H. P. Burchfield, R. J. Wheeler, and E. E. Storrs , Int. J. Leprosy 37, 462 (1969). H. P. Burchfield, E. E. Storrs , R. J. Wheeler, V. K. Bhat, and L. L. Green, Anal. Chem. 45, 916-20 (1973). E. Sawicki, €I. Johnson, and K. K o s i z k i , Microchem. J. - 10 ( 1 - 4 ) , 72-102 (1966) . L. Fishbein and J. Fawkes, J. Chromatogr. 22, 323-9 (1966). T. S. Gloria, Rev. Fac. Farm. Bioquim. Univ. Sao Paulo - 4 , 391-400 (1966) ; C.A. 67, 8 4 9 1 7 ~ . L. M. S. Armasescu and M. Manda, Farmacia (Bucharest) - 22 ( 3 ) , 165-70 ( 1 9 7 4 ) ; C.A. 8 l , 158721t. Compounds (a) , (b), and (c) were kindly provided by G. R. Gordon, Stanford Research I n s t i t u t e , Menlo Park, Ca.

Li terature surveyed through June, 1975.

114

FLUCY TOSINE

Edward H. Waysek and James H. Johnson

EDWARD H. WAYSEK AND JAMES H. JOHNSON

INDEX

A n a l y t i c a l P r o f i l e - F l u c y t o s i n e

1 . D e s c r i p t i o n

1 . 1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2. Phys ica l P r o p e r t i e s

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12

I n f r a r e d Spectrum Nuclear Magnet ic Resonance Spectrum U 1 t r a v i o l e t Spectrum Fluorescence Spectrum Mass Spectrum Opt i ca 1 Rota t i o n M e l t i n g Range D i f f e r e n t i a l Scanni ng Ca lor imet ry Thermogravirnetr ic Ana lys is S o l u b i l i t y X-Ray C r y s t a l P r o p e r t i e s D i ssoc i a t i on Constant

3 . Synthesis

4. S t a b i l i t y

5. Drug Metabo l ic Products

5.1 Tox ico logy

6. Methods o f Ana lys is

6.1 Elemental Ana lys is 6.2 Phase S o l u b i l i t y Ana lys is 6.3 6.4 Non-Aqueous T i t r a t i o n 6.5 F luoromet r ic Ana lys is 6.6 6.7 F l u o r i n e Ana lys is

Th i n-Layer Chromatograph i c Ana 1 ys i s

D i r e c t Spect rophotornetr i c Ana lys is

6.71 Free F l u o r i d e Ana lys is 6.72 O r g a n i c a l l y Bound F l u o r i n e Ana lys is

116

FLUCYTOSIN E

6.8 Biological Assays

7 . Acknowledgements

8. References

117


1. Descr ip t ion


F 1 ucytos i ne i s 4-ami no-5-f 1 uoro-2 ( 1 H) -pyr i m i done.

C4H4FN30 Molecular Weight: 129.1

1.2 Appearance, Color, Odor

F lucytos ine i s a white, odorless, c r y s t a l l i n e powder.

2. Physical Proper t ies

2.1 I n f r a r e d Spectrum ( I R )

The i n f r a r e d spectrum of f l ucy tos ine i s presented i n F igure 1. The spectrum was recorded w i t h a Perkin-Elmer Model 621 Grat ing In f ra red Spectrophotometer as a Fluorolube suspension from 4000 t o 1350 cm’l and as a minera l o i l suspension from 1350 t o 400 cm”. ments f o r the c h a r a c t e r i s t i c bands i n the I R spectrum are l i s t e d i n Table 1 ( I ) .

The assign-

118

FIGURE 1

I n f r a r e d Spectrum of Flucytosine

W AVEL E NGTH MICROMETERS

100 100 7 8 9 10 12 14 1822 35 50 2.5 3 4 5 6

80 80 8

260 60

W

I-

5 40 40

20 20

0 0

a CtT t-

4000 3500 3000 2500 2000 1700 1400 1100 800 500 200

FREQUENCY (CM-')


Table I

I n f rared Ass i gnmen t s For F I ucytos i ne

Frequency (cm-I) Charac te r i s t i c O f

3374, 3138 Bonded NH 2800-2200 (broad) Enol i c OH (associated) 1684, 1672 C=O s t r e t c h 1647 N-C=H (amidine) 1554 C=C (extended conjugation)

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The NMR spectrum o f f l u c y t o s i n e i n F igure 2 was determined on a JEOL C-60 HL Spectrometer a t ambient temperature (ca. 25"). The sample was d isso lved i n 0.1N deuterium ch lo r i de conta in ing sodium 2,2-dimethyl-2-silapentane su l fonate as an i n te rna l reference (MeSi = 0 ppm). The C-6 proton exh ib i t ed a doublet (J 8.03 ppm.

= 5.5 Hz) a t h- f

2.3 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum o f f l ucy tos ine i n 0.1N

3 hydroch lo r ic ac id i n the reg ion 340 t o 210 nm e x h i b i t s a maximum a t 283-287 nm (E = 9.2 x 10 ) and a minimum a t 243-247 nm. The spectrum presented i n F igure 3 was obtained from a reference standard s o l u t i o n o f f l ucy tos ine a t a concen- t r a t i o n o f 0.85 mg per 100 m l o f 0.1N H C I ( 2 ) .

2.4 Fluorescence Spectrum

Figure 4 shows the e x c i t a t i o n and emission spectra o f a so lu t i on o f reference standard f l ucy tos ine from 250 t o 650 nm. measured i n a methanol so lu t i on o f f l u c y t o s i n e (0.025 mg/ml us ing a Farrand MK-1 spectro- f luorometer, showed one e x c i t a t i o n maximum a t 290 nm and one emission maximum a t 366 nm.

The spect ra

120

FLUCYTOSINE

FI GURE 2

NMR Spectrum of Flucytosine

121

.6 -

.5 -

w . 4 - 0

f 9.3

8 v) -

*‘t .I - I


FIGURE 3 Ultraviolet Spectrum of Flucytosine

.7

n 210 -I 250 _ _ 300 I - L 35 NANOMETERS

122

FIGURE 4: Fluorescence Spectra of Flucytosine

I I

Excitation Spectrum

\ Spectrum

250 300 350 400 450 500 550 600 650 NANOMETERS


2.5 Mass Spectrum

The mass spectrum o f f l u c y t o s i n e was obta ined us ing a CEC 21-110 mass spectrometer w i t h an i o n i z i n g energy o f 70 eV. mass spectrometer was analyzed and presented i n the form o f the bar graph, shown i n F igure 5, by a Varian 100 MS dedicated computer system

The output from the

( 3 )

The spectrum shows a s t rong molecular ion peak a t m/e 129. The peak a t m/e 101 i s due t o the loss o f CO from the parent mass and the f ragment o f m/e 86 i s due t o the loss o f HNCO, a l s o from the molecular ion.

2.6 Opt ica l Rotat ion

F lucy tos ine e x h i b i t s no o p t i c a l a c t i v i t y .

2.7 Me l t inq Range

A sharp mel t ing po in t i s no t observed w i t h f l u - cytos ine. The mel t ing range depends on the r a t e o f heat ing. When the USP Class l a procedure (4) i s used, the me l t i ng p o i n t l i e s between 292" and 298°C. Me l t ing i s accompanied by mel t decomposition.

2.8 D i f f e r e n t i a l Scanning Calor imetry (DSC)

When scanned a t a programmed r a t e o f 10°C per minute using n i t rogen as the i n e r t gas, decom- p o s i t i o n occurs before any observed t r a n s i t i o n , there fore , DSC i s not use fu l f o r character iza- t i o n o f t h i s compound ( 5 ) .

2.9 Thermogravimetric Analys is (TGA)

No weight loss was observed by TGA between ambient temperature and 210°C a t a heat ing r a t e o f 10°C per minute. A weight loss began a t about 210°C and amounted t o about 64% o f the sample weight a t 5 O O 0 C (6) .

124

.. v\

125

FIGURE 5: Mass Spectrum of Flucytosine


2.10 Solubi 1 i t y

The s o l u b i l i t y data f o r f l ucy tos ine obtained a t 25'C i s g iven i n Table I1 ( 7 ) . The s o l u b i l i t i e s were measured a f t e r an e q u i l i b r a t i o n pe r iod of three hours.

Table I 1

Flucytos ine S o l u b i l i t y

So 1 ven t

3A a lcohol Absolute ethanol Ace tone Benzene Chloroform D ie thy l e ther E thy l ace ta te Hexane 2 - p ropa no 1 Met hano 1 USP a lcohol Water

Solubi 1 i t y (mg/ml)

1.3 0.5

<o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1

1 . 1 5.0 1.7

14.2

2.11 X-Ray Crys ta l Proper t ies

The x-ray powder d i f f r a c t i o n pa t te rn o f f l u c y - tos ine i s presented i n Table I l l (8) . The instrumental cond i t ions are g iven below,

Instrumental Condit ions

General E l e c t r i c Model XRD-6 Spectrogoniometer

Genera t o r Tube Target Radiat ion Opt ics

50 KV, 12.5 mA Copper 0

Cu (Cu Ka = 1.5418 A) 0.1' Detector s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007" N i f i l t e r 4' take o f f angle

126

FLUCYTOSINE

Goniometer Scan a t 0.2"/minute 20 A m p l i f i e r g a i n - 16 coarse

Sealed p r o p o r t i o n a l counter tube and D.C. v o l t a g e a t p la teau, pu lse h e i g h t s e l e c t o r EL; 8.0 v o l t s , Eu; o u t

Rate meter T.C. 4, 2000 c/s f u l l s c a l e

Chart speed 1" per 5 min. Prepared by g r i n d i n g a t room

8.17 f i n e

temperature

Recorder Sample

Table I l l

X-Ray Powder D i f f r a c t i o n P a t t e r n of F l u c y t o s i n e

20

13.74 14.76 15.14 17.26 18.76

21.81 22.75 23.94 25.94 26.44 29.24 29.94 30.65;: 32.01* 33.48 37.57 39.98 40.70 42.74 51.75

-

20.18

*Broad Peaks

d ( i )

6.45 6 .OO 5.85 5.14 4.73 4.40 4.08 3.91 3.72 3.44 3.37 3.06 2.99

2.80 2.68 2.39 2.26 2.22 2.12 1.77

2.92

0.4 * 39 .76 .09 .22 .81 .98 .16

1 .oo .29 .67 .72 .14 .15 .55 .22 .22 .07 .09 .15 .15

127


(1) d - ( i n te rp lana r d is tance) nA 2 s i n 0

(2) [ / I 1 = r e l a t i v e i n t e n s i t y

2.12 D issoc ia t ion Constant

The apparent pKa values f o r f l u c y t o s i n e have been determined spect rophotometr ica l ly t o be 2.90 2 0.05 and 10.71 2 0.05 ( 9 ) . The apparent d i s s o c i a t i o n constants were ca lcu la ted from the UV spec t ra l data i n water a t var ious pH values. pKal (2.90) represents the pro tonat ion o f N-3 and pKa (10.71) represents deprotonat ion a t the ami8e moiety. These values a re i n good agreement w i t h the pKa's reported by Ueda and Fox f o r 3-methylcytosine (10).

3. Synthesis

F lucy tos ine may be prepared by the reac t ion scheme shown i n F igure 6. 5 -F luorourac i l i s re f l uxed w i t h d ime thy lan l l i ne and phosphorous oxychlor ide. The 2,4-di ch l o ro -5 - f 1 uoro-pyr i m i d ine formed i s reacted wi th ammonia and ethanol t o g i ve 2-chloro-4-amino- 5-f luoropyr imid ine which y i e l d s f lucy tos ine hydro- c h l o r i d e upon being t rea ted w i t h concentrated hydro- c h l o r i c acid. F lucy tos ine hydroch lo r ide i s neutra- l i z e d w i t h ammonium hydroxide forming 5- f luorocy tos ine ( 1 1 ) .


F lucytos ine s to red a t room temperature f o r f i v e years was s tab le when tes ted by u l t r a v i o l e t absorpt ion and th in - l aye r chromatography us ing e t h y l acetate: methano1:conc. NHhOH (50:50:2) as the so lvent system (12). The s t a b i l i t y o f 0.14; bu f fe red so lu t i ons i s shown i n Table I V (13).

1 28

A

LL =o

z -4

0 I

Lt

1 29

FIGURE 6: Synthesis of Flucytosine

0 CI

H 5-Fluorouracil 2.4-Dichloro-

5-f luoropyrimidine

y 2

NH40H

H Flucytosine

2-Chloro-4-amino- 5-f luoropyrimidine

fi HCI

H Flucytosine hydrochloride


~~

Before Heat i ng A f t e r 1 Hour a t 100°C

APHA APHA pH Color C l a r i t y pH Color C l a r i t y % Recover:

3.10 0-10 Clear 3.15 0-10 Clear 92.9 5.60 0-10 95.9 5.50 0-10 I1

6.90 0-10 99.0 6.90 0-10 I 1

9.40 0-10 I 1 9.40 0-10 98.5

I1

I1

I1

Table I V

Stabi 1 i t y o f 0.1% Buf fered So lu t i on

I H z T n g 1 A f t e r 1 Hour a t 100°C

APHA APHA pH Color C l a r i t y pH Color C l a r i t y % Recover:

3.10 0-10 Clear 3.15 0-10 Clear 92.9 5.60 0-10 95.9 5.50 0-10 I1

6.90 0-10 99.0 6.90 0-10 I 1

9.40 0-10 I 1 9.40 0-10 98.5

I1

I1

I1

Method Used: U l t r a v i o l e t absorpt ion and th in - l aye r chromatography.

The s t a b i l i t y o f f l ucy tos ine has a l so been s tud ied i n i t s dosage forms (14). I n ampuls f l ucy tos ine was s tab le t o the ex ten t o f 2% o r less breakdown a t room temperature f o r 36 months. No degradation products, urea, u rac i 1, and 5 - f luorourac i 1 , were detected i n capsules a f t e r s i x months a t 37°C and twelve months a t 25°C.

5. Drug Metabol ic Products

I t has been repor ted t h a t f l u c y t o s i n e d i d no t undergo s i g n i f i c a n t b io t rans format ion i n humans a f t e r o r a l dosage and 90% o f the f l u c y t o s i n e administered was excreted unchanged i n the u r i n e (15). Koechl in , e t a l . have s tud ied the metabolism o f f l u c y t o s i n e i n the r a t and i n man us ing drug labeled i n the 2- p o s i t i o n w i t h carbon-14. Rats given a parentera l dosage d i d not metabolize f l u c y t o s i n e and the dosage was recovered q u a n t i t a t i v e l y , mostly i n the u r ine . F lucy tos ine was deaminated t o 5 - f l uo rou rac i l t o the ex ten t o f 20-30% a f t e r o r a l admin i s t ra t i on t o r a t s , which was i n t u r n metabolized f u r t h e r t o a- f luoro- 6-ure ido-propion ic ac id , urea, and carbon d iox ide.

No metabol ic degradation o f f l ucy tos ine i n humans was

130

F LUCY TOSl N E

found a f t e r a s i n g l e o r a l 2 g dose (16). Polak and Scholer (17, 18) using rad io labe led f l u c y t o s i n e i n Candida a lb icans, determined t h a t f l u c y t o s i n e en ters the c e l l by means o f the enzyme cy tos ine permease and i s deaminated t o 5 - f luorourac i 1 by cy tos ine deaminase. The 5 - f luorourac i 1 thus formed i s incorporated i n t o the RNA i n place o f u r a c i l by means of the f o l l o w i n g pathway (19) .

5- f 1 uorocy tos i n e 5 - f 1 uorourac i l+5- f 1 uorour i dine monophospha t e

t r iphosphate

5 - f luorourac i 1 )

-+5-f1uorouridine diphosphate+5-fluorouridine

+RNA (up t o 50% o f u rac i 1 replaced by

5.1 Toxicology

The fo l l ow ing excerpt on the tox ico logy o f f l u - cy tos ine was taken from Drug Evaluat ion Data (15):

"The LD f o r f l u c y t o s i n e has been s tud ied i n r a t s adomice. The LD i n r a t s i s repor ted as 8 g/kg o r a l l y and 100 $&kg in t ravenously . The LD f o r mice i s repor ted t o be 2 g/kg o r a l l y anaOsubcutaneously, 1.2 g/kg i n t r a p e r i toneal ly and 500 mg/kg i n t ravenous 1 y . I '

6 . Methods o f Analys is

6.1 Elemental Analys is

The r e s u l t s from the elemental ana lys is are l i s t e d i n Table V (20).

131

EDWARD H. WAYSEK A N D JAMES H. JOHNSON

6.2

6.3

Table V

Elemental Analys is o f F lucy tos ine

E 1 emen t % Theory % Found

C 37.22 37.24 H 3.12 3.13 N 32.55 32.40 F 14.72 14.60

Phase S o l u b i l i t y

Phase s o l u b i l i t y ana lys is o f f l u c y t o s i n e may be c a r r i e d o u t us ing methanol as the so lvent . F igure 7 shows a t y p i c a l example and l i s t s the cond i t ions o f the ana lys is (21).

Thin-Layer Chromatographic Analys is (TLC)

The th in - l aye r chromatography o f f lucy tos ine and o ther f luoropyr im id ines has been ex tens ive ly s tud ied by Hawrylyshyn, Senkowski, and Wol l i sh (22). obta ined us ing var ious so lvent systems w i t h 10 c1g o f sample spot ted on a s i l i c a ge l p l a t e and developed f o r a t l eas t 10 cm. The p la tes were a i r d r i ed and viewed under shortwave u l t r a - v i o l e t rad ia t i on .

The R f values presented i n Table V I were

One and two-dimensional TLC systems are g iven by Koechlin, e t a l . (16) f o r the separat ion o f f lucy tos ine , a-fluoro-8-ureido-propionic acid, 5 - f l u o r o u r a c i l , and urea i n u r ine . The so lvent systems used were e t h y l acetate: formic ac id : water (60:5:35), 2-propanol :ammonia (20: l ) , and methanol :water (85: 15).

o f f a c i d GF P

The fo l l ow ing TLC system i s used f o r the detec- t i o n o f f l u o r o u r a c i l i n f l u c y t o s i n e drug substance (23). Twenty p1 o f a 25 mg/ml s o l u t i o n

ucytos ine i n a mix tu re o f g l a c i a l a c e t i c and water (8:2) i s spot ted on a s i 1 i ca gel a te and developed by ascending chromato-

132

FIGURE 7

PHASE SOLUBILITY ANALYSIS SAMPLE 8 FLUCYTOSINE SOLVENT METHANOL SLOPE: O.57t0.2% EQUILIBRATION: 20 HRS at 25. EXTRAPOLATED SOLUBILITY’ 7.01t0.1 mq wr q of SOLVENT

I I

25 50 75 SYSTEM COMPOSITION rng of SAMPLE per g of SOLVENT


graphy i n a m i x t u r e o f ch1oro form:g lac ia l a c e t i c a c i d (65:35). A f t e r development for a t l e a s t 14 cm, the a i r - d r i e d p l a t e i s viewed under shortwave u l t r a v i o l e t r a d i a t i o n . F l u c y t o s i n e has an approximate R va lue o f 0.3 w i t h t h i s system and f l u o r o u r a c i I 0.45 (24).

f

Table V 1

R f Values for F l u c y t o s i n e i n Var ious So lvent Systems (22)

Solvent System

1.

2.

3. 4. 5. 6 . 7. 8. 9.

10. 1 1 . 12.

6.4

6.5

E t h y 1 ace ta te : acetone: water

E t h y l acetate:methanol:conc.

Acetone Ether E t h y l a c e t a t e Methanol 2-propanol E t h y 1 a c e t a t e : methano 1 (80 : 20) E t h y l acetate:methanol (75:25) E t h y l acetate:water (100: I ) E t h y l acetate:water (100:3) E thy 1 acetate:me thanol : acet i c

(70:40: 10)

ammonium hydrox ide (75:25: 1 )

a c i d (75:25: 1)

R Value f

0.1

0.3 0.02 0.0 0.0 0.6 0.1 0.2 0.2 0.0 0.0

0.4

Non-Aqueous T i t r a t i o n

F l u c y t o s i n e may be t i t r a t e d i n a m i x t u r e of g l a c i a l a c e t i c a c i d : a c e t i c anhydr ide (2: 1) us ing acetous p e r c h l o r i c a c i d w i t h a g lass / calomel e l e c t r o d e system (23) .

F l u o r m e t r i c Ana lys is

A s p e c t r o f l u o r o m e t r i c method o f assay ing f l u c y - t o s i n e i n b i o l o g i c a l f l u i d s has been r e p o r t e d by Wade and Sudlow (25). F l u c y t o s i n e i s iso- l a t e d from p r o t e i n - f r e e b i o o g i c a l f l u i d s by TLC and e l u t e d from t h e s i l ca g e l w i t h water. A f t e r making the s o l u t i o n a k a l i n e , t h e

134

FLUCYTOSINE

6.6

6.7

6.8

f luorescence i s measured. E x c i t a t i o n was car- r i e d ou t a t a wavelength o f 300 nm and the emission was measured a t 365 nm.

D i r e c t Spect rophotomet r i c Ana lys i s

D i r e c t spectrophotometric ana lys is can be used t o ob ta in a q u a n t i t a t i v e assay o f f l u c y t o s i n e i n capsules (23). I n d i l u t e hydroch lo r ic ac id ( 1 i n 100) the repor ted maximum i s 283-287 w i t h

= 9.2 x 103.

F luo r ine Analys is

6.71 Free F luor ide Analys is

The determinat ion o f f r e e f l u o r i d e i n f l u c y t o s i n e drug substance can be c a r r i e d ou t by d i r e c t measurement w i t h a f l u o r i d e - s p e c i f i c ion e lec t rode (23). E lect rode p o t e n t i a l measurements a re made i n a c i t r a t e conta in ing acetate b u f f e r s o l u t i o n (pH between 5.0 and 5 . 5 ) . measurements a re converted t o f r e e f l u - o r i d e concent ra t ion by reference t o a c a l i b r a t i o n curve.

The p o t e n t i a l

6.72 Organ ica l l y Bound F luor ine Analys is

Bound f l u o r i n e may be determined by SchHniger combustion fo l lowed by measurement w i t h a f l u o r i d e - s p e c i f i c i on e lec- t rode (26).

B io loq i ca l Assays

Shadomy, e t a l . (27) ind ica ted t h a t the a c t i v i t y o f f l ucy tos ine should be s tud ied i n a completely syn the t i c medium. I n l a t e r s tud ies (28) Shadomy described a c y l i n d e r p l a t e bioassay f o r the determinat ion o f f l u c y t o s i n e i n sera, u r ines ,

ssue homogena tes . was & cerev is iae base agar supple-

cerebrosp i na The ind i ca to ATCC 9763 on

f l u i d s , and t organ i sm used

yeas t n i t rogen

135-


mented w i t h L-asparagine and dext rose. For sera and o t h e r b i o l o g i c a l f l u i d s the lower s e n s i t i v i t y 1 i m i t was 0.4 t o 0.5 pg f l u c y t o s i n e / m l . B lock and Bennett (29) repor ted a c y l i n d e r p l a t e b i o - assay t h a t p e r m i t t e d t h e de terminat ion o f f l u - c y t o s i n e i n b i o l o g i c a l f l u i d s i n the presence o f amphoter ic in B which o therw ise would i n t e r - f e r e . B laker and Dout t (30) descr ibed a d i s c d i f f u s i o n method f o r assay ing f l u c y t o s i n e i n serum and o t h e r body f l u i d s us ing a s t r a i n o f Saccharomyces c e r e v i s i a e as the t e s t organism on yeast n i t r a t e base agar. The method was use- f u l i n t h e c o n c e n t r a t i o n range 2 t o 6 pg/ml. Holt and Newman (31) a l s o repor ted a method fo r t h e r o u t i n e assay o f f l u c y t o s i n e i n b i o l o g i c a l f l u i d s us ing C . a l b i c a n s (Carshal ton 2606) as t h e i n d i c a t o r o r g a n i s m . The method de tec ted 0.1 pg f l u c y t o s i n e / m l . Ma E i c k h o f f (32) s t u d i e d the a n t i f u n g a o f f l u c y t o s i n e us ing b r o t h - d i l u t i o n t i t e r d i l u t i o n , agar-d i l u t i o n , and s u s c e p t i b i l i t y t e s t s . T h e i r i n v e s t i n d i c a t e d t h a t the d i s c s u s c e p t i b i l

read i l y ks and

a c t i v i t y micro-

d i s c g a t i o n t y t e s t

may be a p p l i c a b l e i n d i a g n o s t i c mic rob io logy l a b o r a t o r i e s f o r yeasts such as Candida and Toru lops i s .

7. Acknow 1 edgemen t s

The authors w ish to acknowledge M.V. Go, D r . K. Blesse l , E . Kohler, and the S c i e n t i f i c L i t e r a t u r e Department o f Hoffmann-La Roche Inc. for t h e i r ass is tance i n the p r e p a r a t i o n o f t h i s a n a l y t i c a l p r o f i le .

1 36

FLUCYTOSINE

8. References

1.

2 .

3 .

4.

5.

6.

7.

8.

9.

10.

1 1 .

12.

13.

14.

15.

16.

17.

18.

19.

20.

S . Traiman, Hoffmann-La Roche Inc., Personal Communication. R. Gomez, Hoffmann-La Roche Inc., Personal Communication. W. Benz, Hoffmann-La Roche Inc., Personal Comnun i cat i on. Pharmacopeia of t he h i t e d S ta t e s X I X , 652 (1975). R.J. Rucki, Hoffmann-La Roche Inc., Personal Communication. R.J. Rucki, Hoffmann-La Roche Inc., Personal Commun i ca t i on. E . Bass, Hoffmann-La Roche Inc., Personal Communi ca t ion . A.M. Chiu and W. McSharry, Hoffmann-La Roche Inc. , Personal Communication. V . Toome, Hoffmann-La Roche lnc. , Unpublished Data. T. Ueda and J.J. Fox, J . Am. Chem. Soc., 85, 4024 (1963). L.A. Dolan, Hoffmann-La Roche Inc., Unpublished Data . R. Gomez, Hoffmann-La Roche Inc., Unpublished Data. J. Guastel la, Hoffmann-La Roche Inc., Unpub- l i shed Data. J.B. Johnson, Hoffrnann-La Roche Inc., Unpub- l i shed Data. Drug In t e l l i gence and ClinicaZ Pharmacy, 1, 75 (1973). B. Koechlin, F. Rubio, S. Palmer, T. Gabr ie l , and R. Duschinsky, Biochemical PhamcoZogy, - 15, 435 (1966). A . Polak and H. Scholer, Path. Microbiol., 2, 148-159 (1973). A. Polak and H. Scholer, Path. MicrobioZ., 39, 334-347 ( 1973) . A . Polak and H. Scholer, Hoffmann-La Roche Inc., Basle, Switzerland, Unpublished Data. F. Scheid l , Hoffmann-La Roche Inc. , Unpublished Data .

137


21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

E. Bass, Hoffmann-La Roche Inc. , Personal Commun i c a t ion. M. Hawrylyshyn, B.Z. Senkowski, and E.G. W o l l i s h MicrochemicaZ Journal, 8, 15-22 (1 964) . Pharmcopeia of the UniTed States, X I X , 199 (1975). B.C. Rudy and B.Z. Senkowski, AnaZyticaZ Profiles of Drug Substances, Vol. 2, 1973, Academic Press, p . 239. D. Wade and H. Sudlow, J . Pharm. Sci., 62, 828 (1973). B . C . Jones, J.E. Heveran, and B.Z. Senkowski, J . Pharm. Sci . , &, I036 (1971). S. Shadomy, H.J. Shadomy, J.A. McCay, and J . P . U t z , Antimicrobial Agents and Chemotherapy, 452 (1968). S . Shadomy, AppZied Microbiology, 17, (6) , 871-877 (1969). . . . . . . E .R . B lock and J.E. Bennett , AntimkrobiaZ Agents and Chemotherapy, 1, (61, 476-482 (1 972) . R.G. B laker and B.J. Dout t , AntimicrobiaZ Agents and Chemotherapy, 2, (6), 502-503 (1972). R.J. H o l t and R.L. Newman, J . Clin. Path., - 26, 167-174 (1973). M . I . Marks and T.C. E i c k h o f f , AntimicrobiaZ Agents and Chemotherapy, 49 1 (1 970) .

138

GLUTETHIMIDE

Hassan Y. Aboul-Enein

HASSAN Y. ABOUL-ENEIN

CONTENTS Analytical Profile - Glutethimide

1. Description 1.1 Nomenc la ture

1.11 Chemical Names 1.12 Generic Name 1.13 Trade Names

1.21 Empirical 1.22 Structural

1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor

2.1 Crystal Properties 2.11 Crystallinity 2.12 X-Ray Diffraction 2.13 Melting Range 2.14 Differential Scanning Calorimetry

1.2 Formulae

2. Physical Properties

2.2 Solubility 2.3 Optical Activity 2.4 Molecular Orbital Calculations & Dipole

2.5 Acid Dissociation Constant 2.6 Identification 2.7 Spectral Properties

2.71 Ultraviolet 2.72 Infrared 2.73 Nuclear Magnetic Resonance 2.74 Mass Spectrum

Moment

3. Synthesis 4. Stability and Decomposition Products 5. Metabolism 6. Method of Analysis

6.1 Titrimetric Methods 6.11 Aqueous 6.12 Non-Aqueous

6.21 Qualitative 6.22 Quantitative

6.3 Polarographic Analysis 6.4 Ultraviolet Spectrophotometric 6.5 Fluorometric Analysis

6.2 Colorimetric

140

GLUTETHIMIDE

CONTENTS (cont'd)

6.6 Chromatographic Analysis 6.61 Paper 6.62 Thin Layer 6.63 Gas Chromatography

6.7 High Voltage Electrophoresis 6.8 Biological Assay 6.9 Nuclear Magnetic Resonance

141

HASSAN Y, ABOUL-ENEIN

1. Description

1.1 Nomenclature

1.11 Chemical Names: a. (+) - 2-Ethyl-2-phenylglutari-

mide . b. O(-ethyl-O(-phenylglutarimide. c. 3-ethyl-3-phenyl-2,6-piperi-

d. 3-ethyl-3-phenyl-2,6-dioxo-

e. 3-ethyl-3-phenyl-2,6-diketo-

dinedione.

piperidine.

piperidine.

1.12 Generic Name: Glutethimide

1.13 Trade Names: Doriden, Noxyron, Elrodorm.

1.2 Formulae:

1.21 Empirical :

C13H15N02

1.22 Structural :

H

1.3 Molecular Weight:

1.4 Elemental Composition:

217.26

C, 71.86; H, 6.96; N, 6.45; 0, 14.73

142

GLUTETH lMl DE

1.5 Appearance, Color, Odor: Glutethimide is a colorless crystalline white solid, odorless, has a bitter taste .

2. Physical Properties:

2.1 Crystal Properties:

2.11 Crystallinity Glutethimide is a crystalline

solid. A typical photomicrograph of glutethimide obtained by sublimation, recrystallization from 50% aqueous ethanol and recrystallization from concentrated ammonia is shown in Fig. (1). These crystalline structures can be used as a microscopic means of identification of glutethimide (1). The behavior of glutethimide crystals extracted from the tablet formulations was discussed by Penprose and Biles (1).

2.12 X-Ray Diffraction: Analytical x-ray diffraction data

for glutethimide, its anhydrous, hydrous forms and optical active antipodes were studied in detail by Bonamico et al. (2) along with other analogs. The elementarcrystal structure for these forms are shown in Table I.

Further information regarding conformation of the chemical structure of glutethimide through analysis of x-ray diffraction patterns are given by Bonamico et &. (2).

The optical crystallographic properties for glutethimide crystals are as follows ( 3 ) :

q1.572; p 1.585, x 1 . 5 9 0 Optic Sign: Negative 2V. large Extinction: parallel and inclined Elongation: positive System: 6-sided rods and plates

143

f

F A . 1 - - Photomicrographs showing variation in crystal structure of glutethimide (1). A-sublimation, B-from 50% EtOH, C-from conc. NH40H.

Table I X-Ray D i f f r a c t i o n Data f o r Glute th imide

0 0 0

B S p e c i a l G r o z Form a,A b,A _. C ,A - z v - Glutethimide hydrous 7 .5320 .02 30.50+0.09 10.83+0.03 8 2487E3 D;5 - ?bca

8 Glutethimide anhydrous 20.40+0.06 11 .3920 .03 20.6020.06 16 4791A3 92 40 220 P - c&

rhombic prism 0 0 . - A

monoc l in ic prisms 2’COr Pc - c; -

0

Glutethimide (+)or ( -1 6.8720.02 25.7550.07 6 .9020 .02 4 1221A3 ant ipode rhombic prisms


2.12 X-ray D i f f r a c t i o n (continued)

The c h a r a c t e r i s t i c d i f f r a c t i o n p a t t e r n s were obtained by sub jec t ing g l u t e t h i - mide powder t o CuK)I-radiation from x-ray spectrometer and record ing t h e d i f f r a c t e d r a d i a t i o n on a c h a r t , us ing a modified GM-tube wi th a recording potent iometer (1). The D-distances were ca l cu la t ed as shown i n Table 11.

Table I1

10.3 7.23 6 .22a 6 . 0 1 5. lla 4.59 3.78a 3.52 3.27 3.20

a - Strong

2.13 Melting Range The Nat ional Formulary X I V ( 4 )

s p e c i f i e s a melt ing range f o r g lu te th imide between 860 and 89O as a cri teria of a c c e p t a b i l i t y . Furthermore, it w a s repor ted t h a t g lu te th imide showed a m.p. range of 85O-880 with r e s i d u a l c r y s t a l s growing s lugg i sh ly only below 80° i n t o g r a i n s and prisms ( 5 ) . The m e l t s o l i d i f i e s t o a g l a s s . The g l a s sy powder showed nD 1.5403.

The e u t e c t i c temperature of glute th imide with azobenzene and with b e n z i l w a s repor ted t o be 53O and 61°, r e spec t ive ly .

Table I11 shows t h e melt ing range of glu te th imide r epor t ed i n t h e l i t e r a t u r e .

146

F*

Glutethirnide h y d r o u s rhombic prism

Glutethimide a n h y d r o u s m o n o c l i n i c prisms

Glutethimide (+)or ( - ) antipode rhombic prisms

T a b l e I X - R a y D i f f r a c t i o n Data f o r G l u t e t h i m i d e

0 0 0

C ,A - 2 !! - B S p e c i a l Group

7 . 5 3 2 0 . 0 2 3 0 . 5 0 f p . 0 9 1 0 . 8 3 + 0 . 0 3 8 2 4 8 7 ~ ~ D215 - ’bca

- a , A b,A - 0

0 o * - 2 0 . 4 0 + _ 0 . 0 6 1 1 . 3 9 + _ 0 . 0 3 2 0 . 6 0 + 0 . 0 6 16 4 7 9 1 ~ ~ 9 2 40 5 2 0 P - C j h

2’COr Pc - c;

6 . 8 7 2 0 . 0 2 2 5 . 7 5 5 0 . 0 7 6 . 9 0 2 0 . 0 2 4 1 2 d 3 P212121

HASSAN Y . ABOUL-ENEIN

Table I11

m.p., CO Reference 1 9 1-9 2 0

85-87, bo 3168 6 82-8 3 ( is6propanal) 7 86-88 anhydrous glutethimide 8 80-85 (EtOAc-pet. ether) 9 83-85 10 68.5.70.5 (ail EtOH) 11

2.14 Differential Scanning Calorimetry Reubke and Mollica (12) reported

the application of the differential scanning calorimetry in the quantitative estimation of purity of glutethimide and the detection of polymorphism. They reportedAH value for glutethimide to be 28.7+1 - cal/gm. at 86.80 - 87O.

2.2 Solubilit : d i n water (1.0 mg/ml) , soluble

1 in 5 of ethanol, 1 in 12 of ether and 1 in less than 1 of chloroform, dichloromethane. in acetone, ethyl acetate. A saturated solution in water is acidic to litmus.

Soluble

Recently, it was reported that the tetramethyl-substituted amides of pimelamide, suberamide, azelamide and sebacamide markedly enhance the solubility of glutethimide in aqueous solution (13). The solubility of glutethimide was increased significantly above the critical concentrations and from the nature of the solubility curves, a micellar type of solubili- zation appears to be dominant.

2.3 Optical Activity Glutethimide is marketed as the racemic

mixture of 2-ethyl-2-phenylglutarimide. Keberle et al. (14) describes a procedure for resolution

the racemic mixture to the pure optical antipodes as shown in Scheme 1. The sedative- hypnotic activity of the (+) isomer is 2-3 times more potent than the ( - 1 isomer (15).

148

GLUTETH IMI DE

H ( +,

Pi"" I t

c 6 H 5 -: -co 2 H (CH2) 2C02H

(2) -2 -e thy l-2 -pheny lg lu t a r i c ac id

reso lved wi th (+) and ( -1 d -phenethylamine I

J y 3

H (-)-isomer

Scheme 1

0 phno H

(+)-isomer

149

m

rl

* rl W

rl

I-

rl

W

rl

\D

rl

Pl 0

rl I ui

* 0 rl I Pl 0

rl

* 0 rl

I m

0

rl

Pl 0

rl I N

0

4

* 0 rl

* 0 rl - 0 rl

N

0

rl

- X 0

:

- X 0

2 h

8 +,

w

- X

w s v

rl

X

W

s Y

N

+I

W

W

rl

I

ui N

O

T

u

rl II V -

N

+I

W

I-

rl

-k

N

+I rl

m 4

I

I1 V Y

* W

rl

I

0

NO

T

ui N

O 0

ND

T

m

NO

n

8

- U

II u

U

Ll a,

8 Ll a,

d .r(

I + h

Y

1 50

GLUTETHIMIDE

2.3 Optical Activity (continued)

The specific rotations, melting points for the (+) and ( - 1 isomers are given in Table IV as recorded' in the literature.

2.4 Molecular Orbital Calculations and Dipole Moment The molecular orbital calculations of

glutethimide was discussed by Andrews (19) using two methods, extended Huckel theory (EHT) and complete neglect of differential overlap CND0/2. Calculated dipole moment (D) of glutethimide was found to be 8.67 using EHT, while the experimental dipole moment (D) was reported by Lee and Kumler (20) to be 2.83. Andrews showed that CNDO/2 method is more suitable for assessing net atomic charges than the EHT.

H

2.83D

Furthermore, Lee and Kumler (20) concluded from their study that of the dipole moments of several six-membered cyclic imides the imide groups in these compounds are in a cis-cis conformation.

2.5 Acid Dissociation Constant Doornbos and De-Zeeuw (21) measured the

"proton lost" dissociation constant K H and the "proton gained" K3H dissociation conszant for glutethimide by a previously described potentiometric titration method at 20°. for glutethimide at ionic strength of p=O.?O was found to be 4.518.

K2H and K

151


2 .6 I d e n t i f i c a t i o n The f o l l o w i n a i d e n t i f i c a t i o n tes ts are

pub l i shed i n B.P. 1973 ( 2 2 ) as a par t of t h e i d e n t i f i c a t i o n of g l u t e t h i m i d e .

1. The l i g h t a b s o r p t i o n , i n t h e r ange 230 t o 350 nm, of a 2-cm l a y e r of a 0.03 p e r c e n t w/v s o l u t i o n i n dehydra ted a l c o h o l e x h i b i t s t h r e e maxima, a t 252 nm, 258 nm, and 264 nm; e x t i n c t i o n a t 252 nm, abou t 1.0, a t 258 nm, a b o u t 1.1, and a t 2 6 4 nm, about 0.86.

2 . Heat 1 g w i t h 5 ml of sodium hydrox- i d e s o l u t i o n and 1 5 m l of water on a water -ba th f o r t h i r t y minu tes , cool and a c i d i f y t o l i t m u s paper w i t h d i l u t e h y d r o c h l o r i c acid; f i l t e r ; m e l t i n g p o i n t o f t h e p r e c i p i t a t e , a f t e r washing w i t h water and d r y i n g a t l o o o , abou t 159'.

Fur thermore , Imaoka and Ogura (23) pub- l i s h e d a procedure f o r i d e n t i f i c a t i o n of g lu - t e t h i m i d e i n t a b l e t s by s p o t t es t . A powdered sample (2-3 mg) on Whatman's T e s t Paper i s w e t t e d w i t h 2 d rops a c e t o n e , 1 d r o p 1% copper acetate or 1% c o b a l t acetate s o l u t i o n , k e p t f o r 30 seconds then 1 d r o p 1 0 % i sopropylamine s o l u t i o n i n ace tone w a s added, g l u t e t h i m i d e g i v e s a clear v i o l e t c o l o r e d s p o t .

Most bu lk ing a g e n t s , e .g . lactose, s t a r c h , s u c r o s e , gun a r a b i c , g lucose o r t a l c w i t h t h e e x c e p t i o n of a l g i n i c a c i d does n o t i n t e r f e r e w i t h t h e test.

2 .7 S p e c t r a l P r o p e r t i e s

2 .71 U l t r a v i o l e t G lu te th imide i n s o l u t i o n a b s o r b s

u l t r a v i o l e t r a d i a t i o n over a broad range t o produce a spec t rum w i t h maximum a t 257 251, 263 nm ( t y p i c a l spectrum, F i g . 2 1 , w i t h A !&, = 19 for t h e wave l e n g t h 257 nm (24-26) .

152

g0.40 OL

0

U 0 20

000

I L

I I I

220 260 300 340 WAVE L € N GT t 4, rni i 1 i m ic r ons

Fig. 2 -

U l t r a v i o l e t spectrum of g lu te th imide i n methanol ( 2 4 ) .

153

HASSAN Y . AEOUL-ENEIN

2.72 I n f r a r e d Spectrum The i n f r a r e d spec t rum of g l u t e t h i -

mide i s shown i n F i g . 3. The spectrum w a s ob- t a i n e d on a Perkin-Elmer 621 spec t rophotometer from a K B r p e l l e t .

The s t r u c t u r a l ass ignments have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e - quenc ie s :

Frequency ( c m - l ) Assignment 319 0-3100 NH s t r e t c h i n g imide 1710 C=O imide ca rbony l 1680 C=O imide ca rbony l n e x t

1200 C - 0 s t r e t c h i n g 760-700 Monosubs t i tu ted phenyl

t o t h e d - p h e n y l group

F u r t h e r i n fo rma t ion w i t h r e g a r d t o t h e i n f r a r e d s p e c t r a of g l u t e t h i m i d e i s g iven by s e v e r a l a u t h o r s (27-29) .

2.73 Nuclear Magnetic Resonance Spectrum A t y p i c a l NMR spectrum of g l u t e t h i -

mide is shown i n F i g . ( 4 ) . The sample w a s d i s s o l v e d i n carbon t e t r a c h l o r i d e . The spec t rum w a s de te rmined on a Var ian T-60 NMR Spec t rometer w i t h TMS as the i n t e r n a l s t a n d a r d .

The fo l lowing s t r u c t u r a l a s s i g n - ments have been made f o r F i g . ( 4 ) .

Chemical S h i f t (dl Assignment T r i o l e t 0.80 -CH 9 -CH 7

M u l k p l e t 1 .8

S i n g l e t 6.9 7 Broad s i n g l e t

--J t o 1.97 -CH2-CH3 and t w o -C>- o f g l u t a r i m i d e r i n g A r o m a t i c phenyl p r o t o n s

w i t h D 2 0 . 8.27 NH imide , exchangeable

F u r t h e r i n fo rma t ion c o n c e r n i n s t h e i n t e r p r e t a t i o n of t h e NMR spec t rum of g l u t e t h i - m i d e and r e l a t e d c y c l i c imides can be o b t a i n e d

154

FA. - 3 - I n f r a r e d spectrum of g lu te th imide , K B r p e l l e t .

Glutithimide ( 1 ) C

Fig . - - 4 - NMR spectrum of g lu te th imide i n CC14 conta in ing TMS as i n t e r n a l s tandard .

G L U T E T H IMlDE

from C a s i n i and S a l v i ' s ( 2 9 ) s t u d y of t h e NMR o f a series of c y c l i c imides and a l s o by Defay and Dor l e t ( 3 0 ) .

2 . 7 4 Mass S p e c t r a The mass spectrum of g l u t e t h i m i d e

o b t a i n e d by c o n v e n t i o n a l e l e c t r o n impact i o n i - z a t i o n shows a molecular i o n M+ a t m/e 2 1 7 . M+ i o n peak is o n l y 5% (F ig . 5 ) . The b a s e peak i s a t m / e 1 1 7 . The mass s p e c t r a l f r a g m e n t a t i o n mechanism w a s d i s c u s s e d by Rucker (31) as shown i n Scheme 2 . Mass spec t romet ry w a s a l s o used as a n a n a l y t i c a l t o o l f o r r a p i d , a c c u r a t e i d e n t i - f i c a t i o n o f and d i a g n o s i s of g l u t e t h i m i d e over - doses and po i son ing cases (32-33) .

The

The chemical i o n i z a t i o n ( C I ) mass spec t romet ry of g l u t e t h i m i d e w a s r e c e n t l y re- p o r t e d by S a f e r s t e i n and Chao ( 3 4 ) . The i o n i - z a t i o n w a s accomplished by t h e r e a c t i o n of t h e drug w i t h e i t h e r i o n i z e d gaseous methane or iso- bu tane . The r e s u l t i n g spec t rum w a s less complex t h a n t h a t produced by c o n v e n t i o n a l E I i o n i z a t i o n and u s u a l l y r e s u l t s i n t h e fo rma t ion of a molecular i o n p l u s one (M+ + 1) i .e . , 218 f o r g l u t e t h i m i d e .

3 . S y n t h e s i s

X(CH2) 2-C-CN

@ CH2CN N a N H 2 C2H5C:

H2S04 roac basic c a t a l y s t

X=-CN or E t

Scheme 3

157

w 0

5 k c, 0

a, a

(I)

'001 '0R

'09 '0

h

'032 '0

YH

3d 35HB

XI 3EltllN

3Xf3d

158

SRMPLE NUMBER 73Z

G L u T E T H 1 M ID E hi m

C D IG I T I2 E D 3

I

i 1 i I

115

5.m. M / E

I a m .

117

132

1 GO

I !in.

Fig. 5 - Mass Spectrum of Glutethimide (EI). - -

GLUTETHIMIDE

\ rnlr = 189 m/c . 146

m/e = 137 m/e = 117

Scheme - 2 - Mass spectral fragmentation mechanism of glutethimide (31).

159


Glutethimide is synthesized as shown in Scheme 3. Starting with benzylcyanide which is treated with ethyl chloride or bromide in the presence of sodium amide to yieldd-ethyl benzylcyanide. This is then allowed to react with methyl acrylate or acrylonitrile (Michael condensation) under the catalytic influence of piperidine or another suitable basic catalyst, thus, forming methyl 4-cyano-4-phenylhexanoate or its corresponding dinitrile derivative, respectively. The latter intermediate was purified by distillation and cyclized in the presence of acetic acid and 90% sulfuric acid to yield glutethimide (6, 35-37).

Another synthetic route for glutethimide is described by Iwase -- et al. ( 9 ) shown in Scheme 4. A mixture of l-phenyl-l- ethylpropane-1,3-dicarboxylic acid and p-nitro- soaniline was treated at 180-185O under nitrogen atmosphere for 5 hrs., refluxed with 2% sodium hydroxide for 3 hrs., acidified, washed with ether, neutralized with sodium carbonate, extracted with chloroform to yield glutethimide.

NO Et

1

NH2 H

Scheme 4

1 60

GLUTETHIMIDE 13.

Other r o u t e (38) f o r g l u t e t h i - mide s y n t h e s i s i s shown i n Scheme 5.

HC 1 i s o p r o p a n o l

CH =CHCN -9 C1CH2CH2C02CH (CH3) 2

i s o p r o p y l /3-chlarnproDionate

C gHg$HCN C2H5 1 dimethy l p i p e r i d i n e

s u l f a t e

‘gH5

H

Scheme 5

161


4. Stability & Decomposition Products Several reports showed that glutethimide is

hydrolyzed to h-ethyl-4-phenylglutaramic acid in alkaline solution (36, 39). Yamaha and Mitzukami (40) reported that this decomposition occurs in sodium hydroxide and sodium carbonate solutions by second order reaction but not in sodium bicarbonate solution. The degradation rate constants at 20°, 30° and 400 in 0.01N sodium hydroxide were 7.66 x 1.67 x 10-1 and 3.56 x 10-1 those in 0.01M sodium carbonate were 3.56 x 10- , respectively. The activation energies in 0.01N sodium hydroxide and 0.01M sodium carbonate were 14.3 and 14.6 K cal., respectively. Furthermore, Wesolowski et al. (41) studied the kinetics of d e g r a d a t i o n o f l u t e t h i m i d e in buffered aqueous solutions in the pH range 1.5-8.0. They supported the previous reports that the degradation of glutethimide is a base-catalyzed reaction since the contribution of ionized species to the reaction rate in the pH range studied is very small. The rate is first order with respect to the concentration of glutethimide. The apparent energy of activation Ea, for the degradation of glutethimide at pH8 is found to be in the order of 25.86 K cal./mole. However, the energy of activation corrected for heat of ionization of water at SOo, 60°, and 650 was found to be 13.47 K cal., 13.92 K cal., and 14.15 K cal./mole, respectively.

The mechanism proposed for the glutethimide degradation is shown in Fig. 6 and involves direct attack by a hydroxyl ion on the unhindered carbonyl of the glutarimide ring followed by the cleavage of the ring to 4-ethyl-4-phenyl-glu- taramic acid.

a?d

162

GLUTETHIMIDE

8 Ph OH,

0 0 Slow 7

N H

r 1

c =o HO

H

Fig. 6 The Mechanism of Glutethimide

Degradation ( 4 1 ) .

A p l o t of l og t+ versus pH (Fig. 7) shows t h a t below pH5, g lu te th imide is very s t a b l e s i n c e a t pH 1.0 and 5.0 t h e ra te i s independent of hydrogen ion concent ra t ion .

163

2 4 t

-J

0.6

04-

0.2

0.0

2.2 - 20 - 1.8 - 1.6 -

CI v) I 4 5 C r” 1.2

-

- 0

i? Q8C

k \

4 0 5.0 6 .O 7.0 t3c 9

PH

Fig. 7 - - -

Log t4, the half-life of the reaction in months against pH at 25’ (41).

164

GLUTETH l MI DE

5. Metabolism The distribution and metabolism of glutethi-

mide in the dog and rat were extensively studied and summarized by Keberle et al. (42). The metabolic fate in man was studied by B’itikofer et al. (43). Both of these studies indicated that the (+)-isomers are metabolized via two different Eiochemical routes. The (+)-isomer was hydroxylated on the glutarimide ring at the 4- position to yield 4-hydroxy-2-ethyl-2-phenylglutarimide, a small percent of which undergoes dehydration to give 2-ethyl-2-phenylglutaconimide. The (-)-isomer predominantly hydroxylated on the ethyl side chain to form 2-(l-hydroxyethyl)-2- phenylglutarimide, of which a small percent lose a moleculae of acetaldehyde to form 2-phenyl--. glutarimide, Fig. ( 8 ) . In 1969, Post and Schutz (44) identified a phenolic metabolite which they assumed to be 2-(4-hydroxyphenyl) glutarimide.

--

--

Recently Stillwell -- et al. (45) identified more polar metabolites present in the urine of rats and guinea pigs, (Fig. 9). They characterized these metabolites by combined GC/MS as follows:

a. b.

d. e. f.

C.

g. h.

4-hydroxy-2-ethyl-2-phenylglutarimide 3-hydroxy-2-ethyl-2-phenylglutarimide 2-(l-hydroxyethyl)-2-phenylglutarimide 2-ethyl-2-(4-hydroxyphenyl)glutarimide 2-ethyl-2-(3,4-dihydroxyphenyl)glutarimide 2-ethyl-2-(3,4-dihydroxy-l,5-~yclohexa- dien- l-yl) glutarimide 2-ethyl-2-phenylglutaconimide 2-phenylglutarimide

They demonstrated that the hydroxylation of the aromatic ring in the rat and guinea pig occurs V J an epoxide pathway. These authors also supported the fact that the rat and guinea pig, like the dog and man, metabolize the (-)-isomer of glutethimide by hydroxylation of the ethyl side chain to form 2- (1-hydroxyethyl) -2-phenylglutarimide. However, the metabolism of the ( + I -

165

. O C ~ H S G 1 u - o ~ o C 6 H 5

0 0 0

H H 2 % H 4 5 %

glucuronidation dog do9 man man rat rat guinea pig guinea pig

Glu = glucuronic acid

i

0 0

(+)-isomer Dextrorotatory

0: 0

h-

T

-CH3CHO, 0 C6H5

0 0

H (+) Glutethimide

(-)-isomer Levorotatory

- N - n 45% H

4 % man rat guinea pig and dog

do9 man r a t and guinea pig

L 1

Fig. 8 Metabolism of Glutethimide

H man

OH

H r a t guinea p ig

H Man (minor)

OH

N H H

0 N u n

r a t guinea p ig

r a t r a t guinea p ig (minor) guinea p ig

Fig. 9 Polar Metabol i tes of Glutethimide


isomer seems to be species dependent. In con- trast to man and dog, the rat apparently metabolized the (+)-isomer of glutethimide by hydroxy- lating the aromatic as opposed to the heterocyclic ring to form 2-ethyl-2-(4-hydroxyphenyl)- glutarimide, however, some hydroxylation of the heterocyclic ring occurs. The guinea pig hy- droxylates both the aromatic and the heterocyclic rings and these metabolites are presumably formed from the (+)-isomer. Further studies on the metabolism of the ( - ) and (+) isomers by the rat and guinea pig are needed to validate these conclusions.

Ambre and Fischer (46) showed that monohy- droxylated metabolite of glutethimide accumulated in the plasma of humans intoxicated with glutethimide overdose. This metabolite was subsequently isolated from urine of dogs given large doses of glutethimide and was chemically identified as 4-hydroxyglutethimide (47). This metabolite was found to possess twice the sedative-hypnotic activity of glutethimide (47).

Recently, it was shown that the concentration of 4-hydroxyglutethimide is highest when the respiratory status of the patient was near its lowest (48, 49). This led to the conclusion that 4-hydroxyglutethimide accumulation plays an important role in acute glutethimide poisoning. 4-Hydroxyglutethimide and other potential glutethimide metabolites were chemically synthesized and pharmacologically tested by Aboul-Enein et al. ( 5 0 ) .

--

Among other metabolites previously identified in animals and man, Andreson et al. (51) recently reported the identification and chemical characterization of 2-ethyl-2-(4-hydroxyphenyl) glutarimide in human urine from patients over- dosed with glutethimide as a major metabolite. They also characterized 2-ethyl-2-(3-methoxy-4- hydroxypheny1)-glutarimide as a new metabolite of glutethimide.

_ -

168

GLUTETHIMIDE

The toxic effects possibly caused by the metabolites of glutethimide has interested many research laboratories to continue investigating this problem.

6. Methods of Analysis

6.1 Titrimetric Methods

6.11 Aqueous A titrimetric method was developed

for analysis of glutethimide. volves the alkaline hydrolysis of glutethimide with standard alcoholic potassium hydroxide and subsequent back titratation of the unconsumed alkali with standard hydrochloric acid using phenolphthalein as indicator (521, a method adapted by the British Pharmacopeia (22).

The procedure in-

6.12 Non-Aqueous Ellert et al. (53) described a . . --

method for determination of glutethimide by nonaqueous titration with 0.1N sodium methoxide in methanol-benzene in a solution of ethylenedi- amines (against 0-nitroaniline) or in pyridine (against Azo Violet). Glutethimide can also be quantitated in tablet formulation by non-aqueous titration in dimethyl formamide and titrating with propanol-benzene 0.1N potassium hydroxide (using metanil yellow as indicator) (54).

6.2 Colorimetric

6.21 Qualitative The following color reaction is

published in the B.P. 1973 (22) as part of an identification scheme.

Shake 10 mg with 2 ml of water, 0.1 g of hydroxylammonium chloride and 1 ml of sodium hydroxide solution, allow to stand for ten minutes and add 2 ml of dilute hydrochloric acid and 1 ml of ferric chloride test-solution; a deep brownish-red color is produced.

169


6 . 2 2 Quan t i t a t ive Sheppard ( 3 9 ) and a s s o c i a t e s

u t i l i z e d a method based on t h e formation of t h e colored complex between f e r r i c ion and t h e hydroxamate r e s u l t i n g from t h e r e a c t i o n of glute thimide with a l k a l i n e hydroxylamine f o r d e t e r - mination of t h e drug i n u r ine . The c o l o r w a s read wi th in f i v e minutes a t 510 nm. The presence of l a c t o s e and anions which complex wi th f e r r i c ion o r s a l t s which form p r e c i p i t a t e s wi th f e r r i c ion i n t e r f e r e with t h i s method. Phang e t a l . ( 5 5 ) int roduced a modif icat ion t o Shep- pa rd ' s procedure so t h e f a t t y impur i t i e s do not i n t e r f e r e with the q u a n t i t a t i o n of g lu te th imide i n vomitus m a t e r i a l .

--

Belova and Zinakova (56) repor ted a s i m i l a r c o l o r i m e t r i c method i n which the co lo r developed was measured a t 490 nm with a no. 5 l i g h t f i l t e r and claimed t h a t t h e presence of l a c t o s e does not i n t e r f e r e with t h e r e s u l t of g lu te th imide determinat ion.

This procedure, however, i s not very s e n s i t i v e o r app l i cab le t o blood specimens and i s r e l a t i v e l y non-specif ic ( 5 7 ) .

6 . 3 Polarographic Analysis Glutethimide f a i l e d t o have polaro-

graphic wave on a .c . and d.c . polarograms us ing 0.1M l i th ium ch lo r ide , o r tetramethylammonium ch lo r ide as support ing e l e c t r o l y t e (58).

Recently, a detailed s tudy of an in - d i rec t polarographic determinat ion of g l u t e t h i - mide a f t e r n i t r a t i o n was repor ted by Lauermann ( 5 9 ) . The method can be used t o q u a n t i t a t e g lu te th imide i n post-mortum t i s s u e s and bio- l o g i c a l f l u i d s , wi th s e n s i t i v i t y of 0 .8 mg/100g of ma te r i a l . The conversion of t h e phenyl group t o n i t rophenyl group a f t e r n i t r a t i o n makes it poss ib l e t o u t i l i z e t h e polarographic technique i n a n a l y s i s of g lu te th imide .

1 70

GLUTETHlMl DE

6.4 Ultraviolet Spectrophotometric The ultraviolet absorption of glutethi-

mide in methanol at 257 nm (60) is-used as a sensitive criteria for its analysis. This method is sensitive to a concentration range of 50-500 pg/ml (61).

Ultraviolet spectroscopy has been extensively applied to glutethimide determination in biological fluids such as (62-64) serum and urine and pharmaceutical preparations (54, 65) with high degree of sensitivity.

6.5 Fluorometric Analysis A fluorometric procedure was described

for the quantitative determination of glutethimide in table formulations (66). The method is based on the fluorogen resulting from the reaction of glutethimide with conc. sulfuric acid containing formaldehyde. The fluorescent intensity is a straight line function of concentration over a wide range. The excitation maxima were observed at 280 and 365 nm and the corresponding maximum fluorescent emission occurred at 450 nm. The procedure is applicable for the determination of glutethimide in the presence of its degradation product 4-ethyl-4- phenylglutaramic acid, since the latter yields only 1/10 the fluorescence of glutethimide. The fluorescent reaction does not occur with compounds lacking a phenyl group or containing a substituted phenyl ring and there is no interference with the tablet excipients.


6.61 Paper The chromatographic behavior of

qlutethimide and related analogs was established 6y Davies and Nicholls (6 7) without interference from the chemically related barbiturates by ascending paper chromatography. Whatman No. 1 paper was used and some paper was impregnated with liquid paraffin ( 4 % in hexane), olive oil

171


(20% in acetone) or tributyrin (10% in acetone). All compounds were applied in amounts of 100 )Ig by the window technique.

Several reagents were used to detect glutethimide after chromatography. These include the hypochlorite reagent by Creig and Leaback (681, alkaline, hydroxylamine spray of Sheppard -- et al. (39), and 1% HgN03 solution (69). Some of the most useful solvent systems are shown in Table V.

Table V

Solvent System Reference P hMe -HOAC - H 0 10:5:4 67 CC1 HOACmH20 1:2:1 67 aq. 4iaC1 1 0 % w/v 67 0.066M Na3P04(pH 7.3 on tri- 67 butyrin paper

A method for rapid detection of glutethimide and other hypnotic was described (70) using pigment impregnated paper, and a combination of 2 dimensional chromatographs. Solvent system used in this technique were piperidine-petroleum ether (1:9) followed by cyclohexane, CHC13 (1:3) or solvent system piperidine-petroleum ether (1:9) followed by benzene: CHC13 (1 : 5 ) .

impregnated paper for the resolution of hypnotics in cadaveric material was also discussed.

The applicability of pigment

Kloecking (69) described the use of wedge-strip procedure to achieve better separation of glutethimide and other barbiturates in chemical-toxicological analysis than the usual ascending descending chromatograms. A good separation was obtained by using ascending chromatograms with dioxane-xylene-toulene-isopropanol-258 NH (1:2:1:4:2) upper phase on Schleicher and Zchnell 2045 Bgl paper for 24

1 72

GLUTETHlMl DE

hours a t 20°. Other p e r t i n e n t i n fo rma t ion i n t h i s r e g a r d i s t o be found i n r e f e r e n c e s ( 7 1 - 7 4 ) .

6.62 Thin Layer S e v e r a l t h i n layer chromato-

g r a p h i c sys tems have been developed t o s t u d y v a r i o u s a s p e c t s of g l u t e t h i m i d e . S e v e r a l pro- c e d u r e s w e r e d e s c r i b e d f o r i t s d e t e c t i o n , i d e n t i f i c a t i o n i n b i o l o g i c a l f l u i d s , f o r e n s i c and t o x i c o l o g i c a l a n a l y s i s , (82-90). S e v e r a l s o l v e n t systems used f o r i t s i d e n t i f i c a t i o n and s e m i q u a n t i t a t i o n of g l u t e t h i m i d e are shown i n Tab le V I .

6.63 G a s Chromatography Many i n v e s t i g a t o r s have used

g a s chromatography t o a n a l y z e g l u t e t h i m i d e . A l - though most of t h e pub l i shed work i s concerned almost e n t i r e l y w i t h a n a l y s i s of g l u t e t h i m i d e and i t s m e t a b o l i t e s i n b i o l o g i c a l f l u i d s and t i s s u e s , many of t h e methods could be modi f ied e a s i l y f o r a n a l y s i s of g l u t e t h i m i d e i n pharma- c e u t i c a l dosage forms.

A number of t h e sys tems used f o r GC are l i s t e d i n Tab le VII. Berry ( 9 1 ) conducted in-depth s t u d y f o r t h e g a s chromatographic be- h a v i o r of g l u t e t h i m i d e i n p re sence of s e v e r a l b a r b i t u r a t e s on 1 2 GC columns. OV-225 and CDMS were t h e most u s e f u l of t h e columns t e s t e d f o r a n a l y s i s of t h e b a r b i t u r a t e s , g l u t e t h i m i d e and i n t e r f e r i n g d rugs a t t h e r a p e u t i c and overdose l e v e l s i n plasma. The method w a s s e n s i t i v e , r a p i d and s u i t a b l e f o r emergency t o x i c o l o g i c a l cases.

B l o o m e r e t a l . (92) developed a r a p i d method f o r d i a g n o s i s of s e v e r a l a c i d i c and n e u t r a l s e d a t i v e - h y p n o t i c s u s i n g GC i n i n t o x i - c a t e d p a t i e n t s . The procedure p e r m i t s s imul- taneous i d e n t i f i c a t i o n and measurements of a

--

1 73

Table VI

Solvent Svstem CHC13:Et20 85:15

CHC13 :EtOH 90 :10

EtOAcaeOH:NH385:5:2.5

CHCI3 :Me2CO 9: 1

2

-4 EtOAc:MeOH:NH40H 85:lO :5 P

Cyc10hexane:CgHg :Et2NH 15 :3:2

CHC13 :Me2C0 9 : 1

I soPr OH : CHCl : 2 5 %NH4 OH 45: 45:lO

Developer KI-benzidine

KI-o-tolidine HC1 (after treatment with gaseous C12)

0.1% KMnO4 and 1% A g NO3

1% HgS04

0.2% aq. solution CuSO with pyridine (50 : 4, -grey color developed.

Rf 0.66

0.96

Reference 75

75

0.89 76

Rf were reptd. 77 on various commerc. avail. 77 silica gel tlc, plates, sheets, 77 films .

0.84

78

79

Table VI (cont'd)

Solvent System Et0Ac:cyclohexane-dioxane: MeOH:H20:NH40H 50:50:10:10: 1.5:0.5 50:50:10:10:0.5:1.5

EtOAc-cyclohexane :MeOH :NH40H 56:40:0.8:0.4 70:15:10:5

EtOAc:cyclohexane:NH~0H 50: 40 :O .1

E tOAc : cyclohexane : NH40H : MeOH:H20 70:15;2:8:0.5

CHC13:BuOH:HC02H 70:40:3.5 (chamber saturated with NH40H)

EtOAc (red is ti1 led) Dioxane: CH2C1 :H20 l:2:1 CHC13 :Me2C0 9 : 1

Developer 3

0.1% diphenyl carba- 0.89 zone (orange pink- spot) : 1% AgOAc (blue- purple spot: HgS04 (purple fades away).

1% diphenyl carba- zone; Hg (NO3)

W at 254 nm C12 and starch KI solution

0.68 0.96 0.55

Reference 79

79

79

79

80

81

Table VII Gas Chromatographic Systems Used for Glutethimide Analysis

All systems listed used flame ionization detectors.

Column Carrier Gas Column Temp, Co 3% OV-17 on Supelcoport 55 ml/min He 160-2200 at 7"/min (100-120 mesh) (derivatized with dimethylformanide dimethyl acetal) . 3% OV-1 on 100-120 mesh Gas 30 ml/min He Chrom Q

120-2200 at 1o0/min

15 A 4% OV-17 on 80-100 mesh o, Chromosorb WHPAW-DMSC psi N2

3.8% SE-30 on 80-100 mesh 25 Chromosorb WHPAW-DMSC psi N2

10% Dexsil 300 on 800-100 mesh Chromosorb WHP

N2

N2 17% Dexsil 300 on 80-100 mesh Chromosorb WHP

1% Carbowax 20M* 50-60 ml/min N2

* on 80-100 mesh Chromosorb W, HP.

183

183

220

220

205

Ref. 95

96

97

97

98

98

91

T a b l e V I I (cont 'd)

Co lumn 3% P o l y A - 1 0 3 *

3% NGA+0.07% t r i m e r acid*

1% SP-1000*

3% PPE-20*

1 0 % A p i e z o n L*

4 % CDMS*

3% OV-l*

2

-I -J

5% OV-17*

3% OV-25*

4 % ov-210*

4 % OV-225*

3% OV-225 o n 80-100 mesh Chromosorb W

C a r r i e r G a s 50-60 m l / m i n N2

50-60 m l / m i n N2

50-60 m l / m i n N2

50-60 m l / m i n N2

50-60 m l / m i n N2

50-60 m l / m i n N2

50-60 m l / m i n N 2

50-60 ml /min N 2

50-60 m l / m i n N 2

50-60 ml /min N 2

50-60 m l / m i n N 2

40 ml /min N2

Column Temp, Co 2 15

230

2 3 0

200

1 9 0

220-240

155

200

1 6 5

1 6 0

2 0 5

200

R e f . 9 1

9 1

9 1

9 1

9 1

9 1

9 1

9 1

9 1

9 1

9 1

9 9

Table VII (cont'd)

Column 8% X F 1112 on 60-80 mesh

Carrier Gas 25 ml/min N.,

4 Chromosorb WHMDS

7% DC-200 on G a s Chrom Q 50 ml/min He (80-100 mesh) derivatized as N methyl derivative

5% SE-30 on 70-80 mesh AW Chromosorb W

60 ml/min N2

4

-l Q) * on 80-100 mesh chromosorb, W, HP.

Column Temp, Co Ref. 19 5 10 0

19 0 10 1

19 5 10 2

GLUTETHIMIDE

variety of sedative agents. By virtue of acid- base extraction with chloroform, the acidic and neutral agents are separated. The chloroform extracts were dried, evaporated and the residue dissolved in isopropanol. Column used was 3% SE-30 on 80-100 mesh Chromosorb W.

Gel filteration using Sephadex LH-20 was used to remove impurities from chloroform extracts of acidified blood, urine and homogenized tissue sample. Quantitative determination was made by GC using 15% SE-30 in Chromosorb W at column temperature of 15Oo-25O0 ( 9 3 ) .

Fischer and Ambre ( 9 4 ) showed that analysis of urine from patients intoxicated with glutethimide on columns containing SE-30, OV-1 and OV-17 lead to an overestimation of the unchanged drug in urine. These columns were considered non-selective. However, 3% OV- 225 on 80-100 mesh Supelcoport and 2% Carbowax 20M on 100-120 mesh Supelcoport were selective to separate the drug from its potentially interfering metabolites and thus, eliminates the possible overestimation of glutethimide in biological samples.

Several gas chromatographic methods for measurement of glutethimide in biological fluids are reported employing a variety of extraction procedures and gas chromatographic conditions (103-123).

6.7 High Voltage Electrophoresis Hoffman et al. (124) d escribed a method --

for the separation, detection and semiquanti- tation of glutethimide and other barbiturates from urine and other biological fluids using high voltage electrophoresis. It was reported that glutethimide migrates at the cathode side at a distance of 0.22 relative to crotylbarbital.

1 79


6 . 8 Biological Assay Glu te th imide can be d e t e c t e d by hemag-

g l u t i n a t i o n i n h i b i t i o n method d e s c r i b e d by Valentour e t a l . ( 1 2 5 ) . A n t i s e r a from r a b b i t s i n j e c t e d w i t h g l u t e t h i m i d e - b o v i n e serum albumin ( 1 0 mg/ml) w e r e s e n s i t i v e enough t o d e t e c t 50 ng g l u t e t h i m i d e /0.1 m l plasma or u r i n e .

The method w a s used t o a n a l y z e u r i n e and plasma p a t i e n t s suspec ted of g l u t e t h i m i d e i n t o x i c a t i o n .

6 . 9 Nuclear Magnetic Resonance Aboul-Enein (126) h a s r e p o r t e d a method

f o r q u a n t i t a t i v e d e t e r m i n a t i o n of- g l u t e t h i m i d e by NMR bo th i n powder and t a b l e t f o r m u l a t i o n s . The method o f f e r s a r a p i d , a c c u r a t e procedure f o r a n a l y s i s of t h e drug . Fur thermore , i t p r o v i d e s a conf i rma to ry i d e n t i f i c a t i o n f o r g l u t e t h i m i d e .

1 80

GLUTETHIMIDE

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

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- -

-

-

1 84

GLUTETHIMI DE

7 3

74 .

75.

76 . 7 7

78. 79.

80.

81.

82.

83.

84.

85. 86.

87. 88.

89 . 90 .

9 1 92 .

93. 94 .

95.

96 .

97 .

W. Mohrschulz, Arch. Pharm., 289, 508 ( 1 9 5 6 ) C h e m . Abstr. 51, 6 7 5 3 a (1957). J. Schmidlin-Me'szbros, C h i m i a , 1 2 , 275 ( 1 9 5 8 ) ; C h e m . A b s t r . 53-a (1959) .

R . L i n d f o r s , A n n . M e d T E x p r l . B i o l . Fenniae ; C h e m . A b s t r . 6 1 , 1 2 3 4 6 b ( 1 9 6 4 ) . S.J. Mule, J .Chromatogr . , 55, 255 ( 1 9 7 1 ) . G. K a n a n e n , I. Sunshine, J. Montorte, J. - C h r o m a t o g r . , 52, 2 9 1 ( 1 9 7 0 ) . 0. H o r a k o v a , E s k . Farm., 21, 23 ( 1 9 7 2 ) . K . K . K a i s t h a , J . H . J a f f e , P h a r m . S c i . , - 61, 679 ( 1 9 7 2 ) . W.A. R o s e n t h a l , M.M. Kaser, K . N . M i l e w s k i , C l i n . C h i m . A c t a , 33, 5 1 ( 1 9 7 1 ) . P . E . H a y w o o d , M.W.Horner, and H . J . R y l a n c e , Ana lys t , 92, 7 1 1 ( 1 9 6 7 ) . I . Popa, T . S t a n , L . A r m a s e s c u , E . U r s u , Farmacia ( B u c h a r e s t ) , 20, 1 5 ( 1 9 7 2 ) . R.H. D r o s t , J . F . R e i t h T P h a r m . Weekbl., 1 0 5 , 1 1 2 9 ( 1 9 7 0 ) . - C . S : F r i n g s , C.A. Q u e e n , C l i n . C h e m . , 18, 1 4 4 0 ( 1 9 7 2 ) . E . V i d i c , A r c h . T o x i k o l . , 2 7 , 1 9 ( 1 9 7 0 ) . L . B . H e t l a n d , D.A. K n o w l t o c D . C o u r i , C l i n . C h i m . A c t a , 36, 473 ( 1 9 7 2 ) . W . Pau lus , A r c h . T z i k o l . , - 20, 1 9 1 ( 1 9 6 3 ) . R.B. H e r m a n s and P . E . K a m p , Pharm. Weekbl., - 102, 1 1 2 3 ( 1 9 6 7 ) ; C h e m . A b s t r . 161672 (1968). P. Schweda, A n a l . C h e m . , 39, 1 0 1 9 ( 1 9 6 7 ) . M . R i e c h e r t , Med. L a b . , 2 2 , 1 1 0 ( 1 9 6 9 ) ; C h e m . Abstr. 7 1 , 5 3 6 1 1 ~ (1969) .

H.A. B l o o m e r , R . K . , Maddock, J . B . Sheehe, E . J . A d a m s , A n n . I n t e rn . Med., 72, 2 3 3 ( 1 9 7 0 ) .

Y . Maeba, J . Forens i c S c i . , 1 5 , 92 ( 1 9 7 0 ) . L . F . F i s c h e r and J .J . A m b r e , J . C h r o m a t o g r . , 87, 379 ( 1 9 7 3 ) . K S . V e n t u r e l l a , V.M. G u a l a r i o , R .E. Lang, J. Pharm. S c i . , 62, 662 ( 1 9 7 3 ) . R . F . S k i n n e r , E . G . G a l l a h e r , B.D. Predmore, Anal. C h e m . , 45, 5 7 4 ( 1 9 7 3 ) . D . K a d a r , W. E l o w , J . Chromatogr., - 72, 2 1 ( 1 9 7 2 ) .

J. C h r o m a t o g r . , 86, 8 9 ( 1 9 7 3 ) . -

-

-

185


98.

99.

100.

101.

102. 103.

F.L. Fr icke , J. A s s o c . O f f . Anal. C h e m . , 55, 1162 (1972). KL. C o h e n , R . T . Koda, J . C h e m . E d u c . , 51, 133 (1974).

104.

105.

106.

107.

108. 109.

110 *

111.

E. B r o c h m a n n - H a n s s e n and A. B a e r h e i m Svendsen, J . Pharm. S c i . , 51, 318 (1962). B . P . K o r z u n , S.M. B r o d y , P X . Keegan, R.C. Luders, and C.R. R e h m , J. Lab . C l i n . Med., - 68, 333 (1966). I. Sunsh ine , R. Maes and R. Feracci, C l i n . C h e m . , 2, 595 (1968). P. G r i e v e s o n and J .S . G o r d o n , J . C h r o m a - t og r . , 44, 279 (1969). J. M a c G G , C l i n . C h e m . , 17, 587 (1971). H . E . S i n e , M . J . McKenna, T.A. R e l e n t and M.H. Murray, C l i n . C h e m . , 16, 587 (1970). L.R. G o l d b a u m and J.J. D o m G s k i , J. - Forens ic , S c i . , 11, 233 (1966).

A. Wins ten , and D. B r o d y , C l i n . C h e m . , 14, 589 (1967).

112. K.D. P a r k e r , J . A . Wright , A . F . H a l p e r n , and C.H. H i n e , J . Forensic. S c i . S O C . , 125, (1968).

113. W.C. G r i f f i t h s , S .K. O l e k s y k , I . D i a m o n d ,

114.

115. R . J . Flagagan , G . Withers , J. C l i n . P a t h o l ,

116.

C l i n . B i o c h e m . , 6, 124 (1973). J . H . Kaufman, Am, J. Med. Technol . , - 39, 338 (1973).

25, 899 (1972). A. P r e m e l - C a b i c , P . A l l a i n , T h e r a p i e , 28, 951 (1973).

117. R.G. C o o p e r , M . S . G r e a v e s , G . O w e n , C l i n . - 118. E . Watson, S . M . K a l m a n , C l i n . C h i m . A c t a ,

119. K M . Solon, Anal. I n s t r u m . , 10, 185 (1972). 120. C . C a r d i n i , V. Quercia, A. C a l o , B o l l .

C h e m . 18, 1343 (1972).

38, 33 (1972).

- -

186

GLUTETHIMIOE

Chim. Farm., - 107, 300 (1968).

19, 158 (1973).

Clin. Chem., 20, 195 (1974).

54, 19 (1974).

Pharmazie 29, 208 (1974).

121. M. Gold, E. Tassoni, M. Etzl, Clin. Chem.,

122. M. Gold, E. Tassoni, M. Etzl and G. Mathew,

123. K. hvy, T. Schwartz, Clin. Chim. Acta,

124. F. Hoffmann, M. Buchner , and W. Hebecker , 125. J.C. Valentour, W.W. Harold, A.B. Stavit-

sky, G. Kananen, I. Sunshine, Clin. Chem. Acta 43, 65 (1973).

399 (1974).

-1 - 126. H.Y. Aboul-Enein, Anal. Chim. Acta, - 73,

Acknowledgment

The author expresses appreciation to Miss Marie Belmonte for typing the manuscript and Mr. S. Kobrin for his technical assistance in photographing the figures in this profile.

187

LEVODOPA

Ralph Gomez, Robert B. Hagei, and Edward A . MacMulian

RALPH GOMEZ eta/.

INDEX

A n a l y t i c a l P r o f i l e - Levodopa

1. D e s c r i p t i o n 1.1 Name, Formula, Mol ecul a r Weight 1.2 Appearance, Color, Odor

2. Phys ica l Proper t ies 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 O p t i c a l Rota t ion 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a l Scanning Ca lor imet ry 2.9 Thermogravi m e t r i c Ana lys is 2.10 S o l u b i l i t y 2.11 C r y s t a l Proper t ies

2.12 D i s s o c i a t i o n Constant

2.111 X-Ray D i f f r a c t i o n 2.1 12 C r y s t a l S t r u c t u r e

3. Synthes is

4. Separat ion o f Racemates

5. Stabi 1 i ty and Degradation

6. Drug Metabo l ic Products

7. Methods o f Ana lys is 7.1 7.2 7.3

7.4 7.5 7.6 7.7

E 1 erne n t a 1 - An a 1 y s i s Phase S o l u b i l i t y Ana lys is Chromatographic Methods 7.31 Thin-Layer Chromatography 7.32 Paper Chromatography 7.33 Gas-Liquid Chromatography 7.34 Column Chromatography D i r e c t Spect rophotometr ic Analys is Col o r i me t ri c Ana 1 y s i s Non-Aqueous T i t r a t i on Determinat ion o f D-Dopa

190

LEVODOPA

8. Acknow 1 edgements

9 . References

191

RALPH GOMEZ et al.

1. Descri p t i on

1.1 Name, Formula, Molecular Wei qh t

Levodopa i s (-)-3-(3,4-Dihydroxyphenyl)-L- alanine.

HO b C H 2 C H C O O H I

NH2


Levodopa i s an odor less w h i t e t o o f f - w h i t e c r y s t a l 1 i ne powder.


2.1 I n f r a r e d Spectrum

The i n f r a r e d spectrum o f levodopa is shown i n F igure 1 ( 1 ) . Elmer Model 621 G r a t i n g I n f r a r e d Spectrophotometer and was measured i n a K B r p e l l e t which conta ined 1 mg o f levodopa i n 300 mg o f KBr.

f o r F igure 1 :

The spectrum was recorded on a Perk in-

The f o l 1 owi ng absorp t i ons have been assigned

a. OH s t r e t c h i n g (bonded): 3375 cm'l, 3210 cm-l b. NH +: 3070 cm-1, 2700-2300 cm-1 (broad) c. CO&: 1656 cm-1, 1569 cm-1 d. Aromatic CH o u t o f plane bending o f two ad jacent

f r e e H ' s : 821 cm-1, 816 cm-1


The NMR spectrum o f levodopa, recorded on a JEOL C60HL spectrometer, i s shown i n F igure 2 ( 2 ) . The spectrum was recorded us ing a s o l u t i o n o f 60 mg o f levodopa/0.4 m l D20 t 0.1 m l DCL. The s p e c t r a l assignments a r e l i s t e d i n Table I .

192

Id P

0

-0

0

>

W

-1

cc 0

5 L c,

V

Q) P

Ln

-0

W L

Id L

+ ET

U

t-

lo-

rD \,

I 1

I

8 0

d- 0

(0

0

“8 co

33

NV

lllWS

NV

Hl Yo

193

FIGURE 2

NMR Spectrum o f Levodopa

LEVODOPA

TABLE I

NMR Spectral Assignments f o r Levodopa

Proton Chemical S h i f t (6) Spectral S t ruc ture (J i n H z l

3.22 ppm Mu1 t i p l e t (7.5) 4.44 ppm T r i p l e t 6.80 ppm 6.92 ppm Doublet 7.05 ppm Doublet

Doublet (2 sets; 2.0, 8.0)

The remaining protons exchange i n the deuterated solvent used.

2.3 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum o f levodopa ( 4 mg o f levodopa/100 m l o f 0.1N hydroch lo r ic ac id ) i n the reg ion o f 230 t o 370 nm e x h i b i t s one maximum a t 280 nm ( E = 2.8 x 103) and one minimum a t 250 nm. shown i n Figure 3 ( 3 ) .

2.4 F1 uorescence Spectrum

The spectrum i s

The e x c i t a t i o n and emission spectra o f levodopa are presented i n Figure 4 (4 ) . The sample was dissolved i n methanol a t a concentrat ion o f 1.43 mg o f levodopa/ml and the spectra were recorded using a Farrand MK-1 recording spectrof luorometer. Levodopa e x h i b i t s e x c i t a t i o n maxima a t 236 nm and 286 nm and emission peaks a t 320 nm and 620 nm.

2.5 Mass Spectrum

The low r e s o l u t i o n mass spectrum o f levodopa i s shown i n Figure 5 (5 ) . The spectrum was obtained using a

195

0.6

0.f

0.4

w u O.!

2 8 a

m 0.2

0. I

C

RALPH GOMEZ etal.

FIGURE 3

U1 t r a v i ol e t Spectrum o f Levodopa

I I I 250 300 350

NANOMETERS

196

a P

0

-0

0

>

a

-I

cc 0

5 L c,

V a

P

Ln

a

V c a

V

v)

a

L 0 3

LL

F

-8

-8

-8

W

0

In

0

-8, K

U

I- O

U

a

02

-s.,

-$

-8

-8

-s: z

m

m

(u

197

AlIS

N3

1N

I 3h

llWl3

tl

198

100

90

80

70 5 3 60 I-

5 50 w 2 40 I- a -I 30 W a

20

10

0

FIGURE 5

Mass Spectrum o f Levodopa

50 100 150 200 m/e

LEVOOOPA

Varian MAT spectrometer w i t h an ionizing voltage of 70 eV, which was interfaced w i t h a Varian data system 6201. data system accepts the output of the spectrometer, calculates the masses, compares the i n t e n s i t i e s t o the base peak and plots this information as a se r ies of l i n e s whose heights are proportional t o the i n t e n s i t i e s .

The molecular ion f o r levodopa was measured a t m/e = 197. Other charac te r i s t ic masses were observed a t m/e = 179 corresponding t o the loss of H20 from the molecular ion, m/e = 152 which corresponds t o the loss of COOH from the parent mass, and m/e = 123, the base peak, which i s the 3,4 dihydrovphenyl moiety. A h i g h resolution scan confirmed the r e s u l t s o f the low resolution spectrum. Table I1 l i s t s the elemental compositions f o r the ions as determined by h i g h resolution mass spectrometry.

The

TABLE I 1

High Resolution Mass Spectrum of Levodopa N

197.0678 197.0689 9 11 1 179.0540 1 79.0583 9 9 1 177.01 37 1 77.01 89 9 5 0 175.9964 1 75.9984 8 2 1 165.0149 165.01 88 8 5 0 161.0472 161.0477 9 7 1 152.0722 152.071 3 8 10 1 151.0633 151.0634 8 9 1 139.0207 139.0270 6 5 1 136.0537 136.0525 8 8 0 134.0612 1 34.0580 5 10 0 1 32.0446 132.0423 5 a 0 1 30.0032 1 30.0055 8 2 0 127.01 62 1 27.01 84 9 3 0 123.0415 123.0447 7 7 0

- H - C Found Mass Calcd. Mass - 0

4

-

3 4

2

2.6 Optical Rotation The spec i f ic rotat ion of an aluminum complex

of levodopa i n an acetate buffer a t 25OC is plot ted versus

199

RALPH GOMEZ et a/.

wavelength i n Figure 6 ( 6 ) . observed a t 589 nm was approximately -41" while the rotat ion a t 365 nm was approximately -122". ch lor ic acid the spec i f ic rotat ion of levodopa a t 589 nm has been reported t o be -11" ( 7 ) .

spec i f ic rotat ion of an acidif ied solution of levodopa containing methenamine t o be approximately -165" a t 589 nm.

The spec i f ic rotat ion

In 0.1N hydro-

Chafetz and Chen (8) have reported the

2.7 Melting Range Levodopa me1 t s w i t h decomposition above 270°C

( 9 ) .

2.8 Differential Scanning Calorimetry An endotherm was obtained i n the 290"-300°C

region where me1 ti ng , accompanied by sample decomposi t i on, occurred. The temperatures observed f o r the decomposition t rans i t ions a re dependent on instrumental conditions as well as sample size and cannot be considered c h a r a c t e r i s t i c f o r the compound (10) .

2.9 Thermogravimetric Analysis ( T G A ) A TGA scan showed no loss o f weight as the

temperature increased from 30" t o 270°C a t a r a t e of 1 O " C / minute (10).

2.10 Solubi l i ty The approximate s o l u b i l i t y data obtained f o r a

sample of levodopa a t 25°C i s l i s t e d i n Table I11 (11). The equi l ibrat ion time was 20 hours f o r each system.

TABLE I11

Sol ubi 1 i t y Data f o r Levodopa Sol vent

Water 95% Ethanol 3A Alcohol

Sol ubi 1 i t y (mg/ml )

3.0 0.3 0.15

200

NO

tlVlO

tl 3M133d

S

201

FIGURE 6

Specific Rotati on Versus Wavelength for Levodopa

- -

-50" - z I-

0

- 2

a 0

- 2

2 -100" -

- -

LL

a v) -

- - -

-150" 300 400 500 600

WAVELENGTH IN NANOMETERS

RALPH GOMEZ eta/.

TABLE I11 (cont . )

Sol ubi 1 i t y Data f o r Levodopa

So 1 vent

Me t h a no1 2-Propanol Chloroform D i ethyl Ether Petroleum Ether (3O"-6O0C) Benzene Dimethylacetami de Propylene Glycol Benzyl A1 coho1 Acetone Acetoni tri 1 e

Sol ubi 1 i t y (mg/ml )

0.1 <0.01 0.1

<o. 01 <o * 01 <o. 01 <0.01 0.2

<0.01 0.03 0.07

2.11 Crystal Properties 2.111 X-Ray Diffraction

The X-ray powder d i f f rac t ion data f o r levodopa are presented i n Table IV (12); instrumental conditions are gi ven be1 ow. Instrument and Operating Conditions I n s t ru me n t G E Model XRD-6 Spectrogoniometer

Goni ometer Detector

Generator 50 KV, 12.5 mA Tube Target Optics 0.1" Detector s l i t

0

Copper ( C u Ka = 1.5418 A)

M.R. So l le r s l i t 3" Beam s l i t 0.0007" Ni F i l t e r 4" Take of f angle Scan a t 0.2" 20 per minute Amplifier gain-16 coarse; 8.7 f ine . Sealed proportional counter tube and DC voltage a t plateau. Pulse height select ion El 5 vol t s , Eu out. Rate meter T.C. 4, 2000 CIS f u l l sca le .

202

LEVODOPA

Recorder

Samples

20

6.54 13.08 14.75 15.31 16.85 17.91 18.45 19.75 21.21 22.38 22.73 23.05 23.85 24.91 25.86 26.35 26.92 28.57 29.61 31.25 33.19 33.64 34.31 35.43 36.15 36.28 37.25 37.74 38.34 39.35 40.04

-

Chart speed 1 "/5 minutes.

Prepared by gr ind ing a t room temperature.

TABLE I V

Levodopa Powder D i f f r a c t i o n Data

d (!)*

13.52 6.77 6.01 5.79 5.26 4.95 4.81 4.50 4.19 3.97 3.91 3.86 3.73 3.58 3.45 3.38 3.31 3.13 3.02 2.86 2.70 2.66 2.61 2.53 2.49 2.48 2.41 2.38 2.35 2.29 2.25

I/Io**

4 2 4 2

20 4

53 14

100 21 42 38

3 60 55

6 28 10 9

21 8

12 6 5 6

21 9

25 8 5

10

203

RALPH GOMEZ er a/.

TABLE IV (cont . )

20

40.80 41.24 41.82

*d

-

- -

**I/Io =

Levodopa Powder Diffraction Data

0 d ( A ) *

2.21 2.19 2.16

I/Io** 6 8 8

needles and low tempera 1 i ke crysta c rys ta l l ine

( interplanar distance) n h rn percent re la t ive intensi ty (based on maximum intensi ty o f 1 .OO)

112 Crystal Structure

plates. ures w i t h o u t agi ta t ion produced the needle-

forms (13) .

Levodopa has been observed t o e x i s t as Rapid recrystal l izat ion from water a t

habit . The needles were found t o exist i n two

Mostad, e t a l . (14) have determined the s t ructures of two monoclinic crystal forms of levodopa. A s tab le form was produced by the slow diffusion of absolute alcohol i n t o a half-saturated solution of levodopa in fgrmic acid, The ce l l dimensi ns are as follows: a = 13.629 A; b = 5.308 8; c = 6.049 1 ; and B = 97.53'.

A l ess s tab le form of levodopa was obtained as t h i n needles by using ethyl e ther as a precipi ta t ing agent. This form i s n o t s tab le when separated from the p t h e r liquo . I t s cel l dimensions are: a = 16.9 A; b = 5.88 8; c = 9.0 8; and B = 99'.

Additional data on crystal s t ructures have been reported (15, 16).

2.12 Dissociation Constants The dissociation constants for levodopa have been

determined t i t r imetr i cal ly and are reported as fol lows (1 7) :

204

LEVODOPA

i H 2 NHZ

3. Synthesis

start ing with three different compounds: 3-(3,4-methylene- dioxypheny1)-L-alanine ( I ) ; N-acetyl-3-(3,4-methylenedioxy- pheny1)-L-alanine (11 ) ; or N-acetyl-3-(3,4-methylenedioxy- pheny1)-L-alanine Z-menthyl ester (111). The s tar t ing materi a1 , red phosphorous and a mixture of HI and acetic anhydride are refluxed for several hours t o give (-)-3-(3,4 di hydroxyphenyl )-L-a1 ani ne. in Figure 7.

4. Separation of Racemates Methods described in the l i t e ra ture for the separation

of DL-3- ( 3,4-dihydroxyphenyl )alanine into i t s opti cal isomers general ly i nvol ve the resol uti on of an i ntermedi a te in the synthesis. The optically pure intermediate i s then reacted further t o give pure levodopa or D-Dopa (19-24).

Yamada, e t a l . (18) report the synthesis of levodopa

The reaction scheme i s shown

A procedure has been described which was used t o resolve DL-3-(3,4-dihydroxyphenyl)alanine into i t s optically active antipodes (25) . A supersaturated solution of the racemic mixture was seeded with crystals of one pure enantiomorph which crystallized pure enantiomorph. was transferred t o a separatory vessel, heated and treated with fresh finely ground racemic mixture; the amount added was approximately twice the amount of pure enantiomorph obtained in the f i r s t recrystallized stage. A preferential solution of the deficient enantiomorph occurred which l e f t the antipode behind as a crystall ine product. The method

The mother liquor

205

Iu 0 UJ

FIGURE 7

Synthesis of Levodopa

CH2- CH-COOH G ! ?HZ

t I

CH2 -CH-COOH I

NH- COCH3

II L EVO DO PA

CH2 - CH -COO d n I ‘0

m

LEVODOPA

i s applicable only when the opt ical ly act ive antipodes form a racemic mixture and. not a compound.

5. Stabi 1 i ty and Degradati on Levodopa, i n the presence of moisture, is rapidly

oxidized by atmospheric oxygen and turns green (9). The oxidation of levodopa i n basic solution resu l t s i n the formati on of me1 ani n and re1 ated intermediates (26). The s t a b i l i t y of levodopa i n the bulk form was studied under conditions of elevated temperature by s tor ing samples a t 105°C for varying periods of time. The samples were examined for evidence of degradation by measuring the color o f a 10% w / v solution i n 10% hydrochloric acid and by t h i n - layer chromatography. some darkening a f t e r 24 hours, and increased w i t h increasing heating time. these solutions by thin-layer chromatography (27).

6. Drug Metabolic Products The major metabolites of levodopa i n humans have been

reported t o be 3-methoyy-4-hydroxyphenylacetic acid, 3,4- d i hydroxyphenylacetic acid, dopamine, and 3-methoxytyrosine (28-33). levodopa show an increase i n the excretion of methylated derivatives such as o-methyldopa, 3-methoxytyramine and 5- hydroxyi ndol eaceti c acid. Wada and Fel lman (35) have presented evidence t h a t 2,4,5-trihydroxyphenylacetic acid i s a metabol i c product of 3,4-di hydroxyphenylpyruvate, a levodopa metabolite. Gjessing and Borud (36) and O'Gorman, e t a l . (30) have also studied the metabolic f a t e o f levodopa i n humans, Figure 8 shows the metabolism of levodopa as described by them. Figure 8 are l i s t e d i n Table V .

The color of the solutions showed

However, no degradation was detected i n

Barbeau (34) has reported tha t pat ients given

The chemical names o f the compounds i n

TABLE V

Metabolites of Levodopa Referred t o i n Figure 8

I , 3,4-dihydroxyphenylalanine 11. 3,4-dihydroxyphenylethylarnine

111. 3,4-dihydroxyphenylpyruvic acid IV. 3,4-di hydroyyphenyll a c t i c acid

V. 3,4-dihydroxyphenylacetic acid

207

RALPH GOMEZ eta / .

FIGURE 8

Metabolism o f Levodopa

208

LEVODOPA

TABLE V (con t . )

Metabo l i tes o f Levodopa Refer red t o i n F igure 8

VI. 3,4-dihydroxyphenylethanol 3,4-di hydroyyphenyl a c e t a l dehyde 3-methoxy-4-hydroxyphenyl a1 an i ne

IX. 3-methoxy-4-hydroxyphenyl p y r u v i c a c i d X. 3-methoxy-4-hydroxyphenyll a c t i c a c i d

XI. 3-methoxy-4-hydroxyphenyl a c e t i c a c i d

XIII. 3-methoxy-4-hydroxyphenylacetaldehyde

VI I. VI I I .

XI I. 3-methoxy-4-hydroxyphenylethanol

XI V . 3-methoxy- 4- hyd roxy pheny 1 e t hy 1 ami ne

7. Methods o f Ana lys is

7.1 Elemental Ana lys is

A t y p i c a l e lemental a n a l y s i s o f a sample o f levodopa i s presented i n Table V I (37) .

TABLE VI

Elemental Ana lys is o f Levodopa

Element % Theory % Found

C 54.82 54.82 H 5.62 5.74 N 7.10 7.10 0 32.46 32.34 (by d i f f e r e n c e )

7.2 Phase S o l u b i l i t y Ana lys is

Phase s o l u b i 1 i ty a n a l y s i s has been c a r r i e d o u t f o r levodopa u s i n g 1 :1 methanol :water as t h e s o l v e n t . example i s presented i n F igure 9 a long w i t h t h e c o n d i t i o n s under which t h e a n a l y s i s was performed ( 3 8 ) .

An

7.3 Chromatographic Methods

7.31 Thin-Layer Chromatography (TLC)

The f o l l o w i n g TLC procedure appears i n USP X I X and i s u s e f u l f o r s e p a r a t i n g levodopa f rom 3- methoxytyros ine and 3,4,6-trihydroxyphenylalanine ( 6 ) . An A v i c e l p l a t e , p r e v i o u s l y predeveloped i n t h e deve lop ing

209

Y 0

2.5

2.0

1.5:

-

-

- - - - - - - - - - - - - LEVOWPA PHASE SOLUBILITY ANALYSIS

3

LEVODOPA

solvent , is spotted w i t h 0.1 mg of levodopa from 9:l acetone:4% hydrochloric acid. ascending chromatography i n n-butanol : gl aci a1 a c e t i c acid: double d i s t i l l ed water:methanol (1 50: 75:75:15). After development of a t l e a s t 15 cm, the p l a t e i s a i r dried and sprayed w i t h 2:l 10% w/v f e r r i c chloride:5% w/v potassium ferricyanide. The approximate Rf values a re summarized i n Table VII.

The p la te i s subjected t o

TABLE VII

Summary of TLC Data Compound Approximate Rf -

3,4,6 Trihydroyyphenylalanine 0.25 Levodopa 0.4 3-Methoxytyrosi ne 0.5

Additional TLC separations of levodopa from related compounds o r metabolites have been reported (39-42). A summary of these methods i s found i n Table VIII.

TABLE VIII

Thin-Layer Chromatographic Systems f o r Levodopa

Adsorbent Solvent of Levodopa Reference Rf Value

Cellulose M N 300 Awl Alcoho1:Formic 0.50 39 Acid: Water (40 :40 : 20 1

Cellulose MN 300 n-Propano (65:25)

Cellulose M N 300 n-Heptane

:Water 0.39 39

Carbon 0.02 39 Tetrachloride: Methanol ( 70 : 40 : 30)

Polyami de Isobutanol :G1 aci a1 0.18 40 Acetic Acid:Cyclo- hexane (80:7:10)

21 1

RALPH GOMEZ et a/.

TABLE V I I I ( c o n t . )

Thin-Layer Chromatographi C Systems f o r Levodopa

Adsorbent

C e l l u l o s e

C e l l u l ose

C e l l u l o s e

C e l l u lose

7.32

Sol vent

n-Butanol : 61 a c i a1 Acet i c Ac id :Water ( 5 : l :3)

E t h y l Acetate: G1 a c i a1 A c e t i c Ac id :Water (5:1.5:3)

E t h y l Acetate:n- Butanol : G1 a c i a1 A c e t i c Ac id: Water (3:2:1:3)

Methyl E t h y l Ketone :Acetone: 2.5N G l a c i a l A c e t i c A c i d (40:20:20)

Rf Value of Levodopa Reference

0.28 41

0.23 41

0.33

0.16

41

Paper Chromatography

PaDer chromatowaohi c s e o a r a t i ons o f

42

levodopa have been used f o r i d e n t i f i c a t i o n as w e l l as f o r i t s separa t ion f rom r e l a t e d compounds (41, 43, 44, 4 5 ) . Table I X i s a summary o f some o f t h e paper chromatographic methods employed f o r levodopa. Whatman number 1 paper was used i n each case.

TABLE I X

Paper Chromatographic Systems f o r Levodopa

Rf Value Sol vent Development o f Levodopa Reference

n-Butanol :Gl a c i a1 Descending 0.27 41 A c e t i c Acid:Water (5:1:3)

21 2

LEVODOPA

TABLE IX (cont.)


Rf Value Sol vent Development o f Levodopa Reference

Ethyl Acetate: Des cendi n g 0.21 41 Glacial Acetic Acid: Water (5:1.5:3)

Methanol :Water: Des cendi n g 0.42 Quinoline (160: 40 :8)

n-Butanol :Pyridine: Descending 0.47 0.2N Sodium Acetate ( 1 : l : l )

n-Butanol :Pyridine: Descending 0.68 1M Sodium Acetate (1 :1:2)

n-Butanol :Pyridine Descending 0.40 (1 : 1 ) saturated w i t h 1 M Sodium Acetate

n-Butanol :Pyridine: Descending 0.54 Water (1 :1:1)

Benzene :Methanol : Descending 0.36 n-Butanol :Pyridine: Water (1 :2: 1 :1:1)

Methanol :Water: Descend i n g 0.46 Pyridine (20:5:1)

Methanol :Water: Ascending 0.37 Pyri d i ne (20:5: 1 )

43

44

44

44

44

44

44

44

21 3

RALPH GOMEZ etal.

TABLE IX (cont.)

Paper Chromatographic Systems for Levodopa

Sol vent

To1uene:Ethyl Acetate : Py ri d i ne : Water :Methanol ( 1 : l : l : l : l )

To1uene:Ethyl Acetate:Methanol : Water (1 :1:1 : l ) - Aqueous Phase

Water saturated w i t h methyl ethyl ketone

Water saturated w i t h methyl ethyl ketone

n-Butanol :Ethanol : Water ( 2 : l : l )

Development

Descending

Descending

Descending

Ascending

Descend

Methanol :n-Butanol : Descend Benzene: Water ( 2 : 1 : 1 : 1 )

Methanol :n-Butanol : Descending Benzene:Water (4:3:

Methanol :n-Butanol : Ascending Benzene:Water (4: 3:

2: l )

2 : l )

To1 uene: Ethyl Descending Acetate: Methanol : 0.1N H C 1 ( 1 : l : l : l )

Rf Value o f Levodopa Reference

0.59 44

0.62 44

0.05 44

0.02

0.23

0.3

0.2

0.20

0.75

44

44

44

44

44

44

21 4

LEVODOPA

TABLE IX (cont . )

Paper Chromatographic Systems for Levodopa

Sol vent Development

Methanol :Awl Descending A1 coho1 :Benzene : 2N HC1 (37:17.5: 35: 12.5)

n-Butanol saturated Descending w i t h 1N HC1

n-Butanol saturated Ascending w i t h 1N HC1

$ert . -Butanol : Descending Acetone: Formic Acid: Water (1 60 : 160: 1 : 39)

t er t . -8utanol: Ascending Acetone: Formic Acid: Water (160:160:1:39)

Ch1oroform:Glacial Descending Acet i c Acid: Water ( 2 : l : l )

n-Butanol : G 1 aci a1 Acetic Acid:Water ( 4 : l : l )

Descending

t e r t . -Butanol : Descending Acetone:Propionic Acid:Water (160: 160: 1 :39)

Benzene: Propionic Descending Acid:Water ( 2 : l : l )


0.51 44

0.19 44

0.18 44

0.08 44

0.06 44

0.80 44

0.21 44

0.06 44

0.83 44

21 5

RALPH GOMEZ et al.

TABLE I X (con t . )


Sol vent

t e r t . -Butanol : Methyl E thy l Ketone : Formi c Aci d :Water (40:30: 15: 15)

n-Butanol : G1 ac i a1 Acet ic Acid: Water (50: 25 : 25)

Phenol-Water (Lower Phase)

Water:sec. - Butanol : t e r t . - Butanol (48.4:43: 8.6 ) - Up per Phase

2-propanol :Water: Concentrated HC1 (65:18.4: 16.6)

see. -Butanol : Water-Upper Phase

Ethy l Acetate: Formi c Ac id :Water ( 70 : 20 : 10)

E thy l Acetate: Water:Formic Ac id (60: 35 :5)-Upper Phase

Rf Value Development o f Levodopa Reference

Ascending 0.42 45

Ascending 0.44

Ascending 0.38

Ascending 0.33

Ascending 0.40

Ascending 0.17

Ascending 0.30

Ascending 0.00

45

45

45

45

45

45

45

21 6

LEVODOPA

Sol vent

t e r t . -Butanol : Methyl Ethyl Ketone: Water: Formic Acid: (44: 44: 11 : 0.26)

TABLE IX (cont.)



0.10 45

Development

As cendi n g

7.33 - G s-Liquid Chrom tography A gas-1 i q u i d chromatographic procedure

f o r the determination of levodopa purity and detection of possible impurities has been developed (46) .

Gas-1 i q u i d chromatography permits the qua l i ta t ive ident i f icat ion of the impurities by re la t ing the retention times re la t ive t o an added internal standard. Deri vati za t i on was carried out by the reaction o f 1 evodopa and/or proposed impurities w i t h bis-trimethylsilyl- acetamide reagent (BSA). insoluble i n most non-acidic o r non-aqueous solvents, the conversion t o the trimethylsi lyl (TMS) derivative was accomplished without the use o f a solvent. addition of the BSA reagent t o the compound followed by moderate heating was suf f ic ien t for complete derivative formati on.

Because levodopa is v i r tua l ly

Direct

The TMS deri vati ves were successful ly chromatographed on a column packed w i t h 20% SE-30 on Gas Chrom Q under isothermal conditions and detected using a thermal conductivity detector. In order t o compensate for column charac te r i s t ics , instrumental var ia t i ons , and sample introduction technique, an internal standard, docosane, was employed for re la t ive retention time data and response data. t ion yielded a precision o f f 0.5% f o r f ive degrees of freedom.

Assay values calculated by area normaliza-

21 7

RALPH GOMEZ et al.

An absolute determination u s i n g an internal standard yielded a precision of 2 1.2% f o r f i v e degrees of freedom.

quant i ta t ive gas-liquid chromatographic separation of the N-trifluoroacetyl-n-butyl e s t e r of levodopa from 14 other non-protei n amino acids . The temperature programmed chromatography was carr ied out on columns of 60/80 mesh acid-washed Chromosorb W coated w i t h 5% w/w DC-550. Yields of 98 - + 3% were obtained.

the t r imethyls i lyl derivatives of levodopa, tyrosine, phenylalanine, N-acetyl tyramine, tyramine, N-acetyldopamine and dopamine isothermally on various columns. By using the column i n conjunction w i t h a flame ionization detector , levodopa was detected a t levels as low as 1-2 pg.

7.34 Col u r n Chromatography

Gehrke and S t a l l i n g (47) reported the

Atkinson, Brown and Gelby (48) separated

As part of a study of the biosynthesis and metabolism of catecholamines, Masuoka, e t a l . (49) developed a separation of the consti tuents of tissue extracts by column chromatography. The ex t rac ts were separated in to three fract ions on an alumina column by elut ing successively w i t h 0.5M (pH 6.1) ammonium acetate buffer, 0.01M (pH 4.0) ammonium acetate buffer and 2N ace t ic acid. Dowex 50 column which separated levodopa from the other consti tuents. the absorbance a t 279 nm.

The t h i r d f ract ion was then passed through a

The fract ions were monitored by measuring

Spiegel and Tonchen (50) described a method f o r the separation of levodopa from catecholamines found i n plasma. The sample was adsorbed on alumina, eluted w i t h 0.1N hydrochloric ac id , then adsorbed on and eluted from an AG50W-X4 cation-exchange column.

A method for the separation of catechol der ivat ives , including levodopa, from guinea p i g brains by column chromatography w i t h Duo1 i t e C-25 was described by Nakajima (51).

21 8

LEVODOPA

Rolland, Lasry and Lissitzky (52) separated levodopa from L-tyrosine and other amino acids contained in protein or natural extracts on Dowex 50. The l imit of detection of levodopa was reported t o be 50-200~.

7.4 Direct Spectrophotometric Analysis Levodopa, i n tablets or capsules, can be

assayed directly by an ultraviolet absorption procedure, The tablets are finely ground or the contents of the capsules are mixed. quantitatively diluted w i t h 0.4N hydrochloric acid, The contents are mixed, f i l tered and the absorbance of an appropriately diluted solution i s measured a t 280 nm. The amount of levodopa i n tablets or capsules i s determined by comparing the absorptivity of the sample a t 280 nm with the absorptivity of a solution of levodopa reference sample simi larly prepared and measured ( 6 ) .

A portion of the powder i s weighed and

7.5 Colorimetric Analysis An automated method uti l izing the Doty reaction

has been successfully applied t o the uantitative determination o f levodopa in tablets 753) . The method i s specific for the catechol confi gurati on and wi 11 indicate any decomposition due t o the oxidation of this moiety. The tablets are dissolved i n 1N sulfuric acid, homogenized w i t h d i s t i l l ed water, and quickly combined w i t h sodium bisulf i te solution t o prevent oxidation. c i t ra te solution i s introduced into the system followed by a strongly basic buffer which produces a stable purple color measureable a t 545 nm. The amount of levodopa i s calculated by comparison with a cal ibration curve prepared from pure levodopa, similarly treated (54).

A ferrous

7.6 Non-Aqueous Titration A potenti ometri c t i t r a t i on w i t h perch1 oric acid

i n glacial acetic acid is the method o f choice t o assay levodopa. acetic acid i s added, and the t i t ra t ion i s carried o u t with 0.1N perchloric acid i n glacial acetic acid. 0.100N perchloric acid i s equivalent t o 19.72 mg of levodopa (6 ) .

The sample i s dissolved in formic acid, glacial

Each ml of

21 9

RALPH GOMEZ et a/.

7.7 Determi n a t i on o f G-Dopa

Coppi, V id i and Bonardi (55) described a method f o r the determinat ion o f D-Dopa i n levodopa. The method i s based on the q u a n t i t a t i v e reac t ion o f a levodopa decarboxyl ase, present i n a Streptococcus frecaZis suspension, which converts 1 evodopa t o dopami ne w i thou t a f f e c t i n g D-Dopa. e l u t i n g a s o l u t i o n o f t he mix tu re w i t h 0.05M pH 6.0 phosphate b u f f e r on an Amber1 i t e I RC50 i on-exchange co l urn. The e lua te was assayed f l uo r ime t r i ca l l y f o r D-Dopa according t o Anton and Sayre (56).

D-Dopa was separated from dopamine by

8. Acknowledgements

the S c i e n t i f i c L i t e r a t u r e Department and t h e Research Records O f f i c e o f Hoffmann-La Roche Inc. f o r t h e i r 1 i t e r a t u r e searches.

The authors wish t o acknowledge the assistance o f

220

LEVODOPA

9. References

1.

2.

3.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Waysek, E., Hoffmann-La Roche Inc . , Personal Communication. Johnson, J., Hoffmann-La Roche Inc . , Personal Communi c a t i o n . Corrade, C., Hoffmann-La Roche, Inc., Personal Communi c a t i o n . Boatman, J., Hoffmann-La Roche, Inc. , Personal Communication. Benz, W., Hoffmann-La Roche Inc. , Personal Communication. "The Un i ted States Pharmacopei a X I X " , pp. 280, 281 (1975). Osborn, S., Hoffmann-La Roche Inc. , Personal Commun i c a t i on. Chafetz, L. and Chen, T.M., J . Pharm. S c i . , 63, 807 (1974). T e r c k Index", E igh th Ed., p. 397. Rucki, R., Hoffmann-La Roche Inc., Personal Communication. MacMull an, E. , Hoffmann-La Roche Inc . , Personal Communication. Chiu, A. M., Hoffmann-La Roche Inc . , Personal Communication. Sc i are1 1 0, J . and Sheridan, J . , Hoffmann-La Roche I n c . , Personal Communi c a t i o n . Mostad, A,, Ottersen, T. and Romming, Chr., Acta Chem. Scand. , 24, 1864 (1970). Jandacek, R. J . and Ear le , K. M., Acta Crys t . , - B27, 841 (1971). Becker, J. W., Thathachari , Y. T. and Simpson, P. G., Biochem. Biophys. Res. C o m n . , - 41, 444 (1970). Gorton, J. E . and Jameson, R. F., J . Chem. SOC. ( A ) , 2615 (1968). Yamada, S., F u j i i , T. and S h i o i r i , T., Gem. Pharm. BuZZ., 10, 693 (1962). Har r ing ton , C . T . and Randall , S. S., Biochem. J . , - 25, 1028 (1931). Vogler, K. and Baumgartner, H., HeZv. a i m . Acta, 35, 1776 (1952). Emada, S., F u j i i , T. and S h i o r i , T., Chern. Pham. BUZZ., 10, 680 (1962).

221

RALPH GOMEZ er a/.

22 *

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39 40.

Berenyi, E . , Budar, Z . , Pallos, L . , Magdanyi, L. and Benko, P . , Hung. Teljes 858, 28 Aug. 1970, Appl. 28 Oct. 1969; CA:74:64389u. Berenyi, E. Budai, Z. Pallos, L . , Benko P. and Magdanyi, L . , Hung. Teljes 859, 28 Aug. 1970, Appl . 28 Oct . 1969; CA: 64390n. Hever, I . and Villanyi, E . , Hung. Teljes 383, 6 June 1970, Appl. 16 Nov. 1968; CA: 64391~. Merck and Co., Inc., Neth. Appl. 6,514, 950, May 18, 1966. Lerner, A . 6. and Fi tzpatr ick, T. B . , Physical Rev,, 30, 91 (1950). Burke, C. M . , Sokoloff, H . K. and Heveran, J . E . , Hoffmann-La Roche Inc., Personal Communication. Abrams, W . B . , Spiegel, H . E . , Leon, A. S . , Pocelinko, R. M . , and Solomon, H. M . , Am. Med. Assoc. 119th Ann. Conv., Chicago, I l l i n o i s , June 21-25, 1970. Bronaugh, R . L . , McMurtry, R. J . , Hoehn, M. M . , and Rutledge, C. O . , Pharmacologist, - 15, 247 (1973). O'Gorman, L. P . , Borud, 0. Khan, I . A. and Gjessing, L. R., CZin. Ch im. Acta, 29, 111 (1 970) . Routh, J . I . , Bannow, R. E . , Fincham, R. W . , and S t o l l , J . L . , CZin. Chim., 1, 867 (1971). S i i r t o l a , T. and Hirviaho, L . , Scand. J. CZin. Lab. Invest., 3, (Supp l . 130), 14 (1973). Tyce, G. M . , Muentner, M . D . and Owen, C. A . , J r . , Mayo CZin. Proc., 45, 645 (1970). Barbeau, A , , and Mzowell, F. H . , "L-Dopa and Parkinsonism," F. A. Davis Co., Phi la . , Pa.,

Wada, G . H . and Fel lman, J . H . , Biochemistry, - 12, 5212 (1973). Gjessing, L. R . and Borud, O . , Scmd . J . CZin. Lab. Invest . , 17, 80 (1965). Scheidl, F . , Hoffmann-La Roche Inc. , Personal Communication. Bass, E . , Hoffmann-La Roche Inc., Personal Communication. O i ttmann J., J . Chromatogr., 32, 764 (1968). Sapi ra , J . D. , J , hromatogr. ,TL, 134 ( 1969).

pp. 360-362, 1970.

222

LEVODOPA

41.

42.

43. 44 *

45.

46.

47.

48.

49.

50.

51.

52.

53. 54.

55.

56.

Vahidi, A . and Siva Sankar, D. V., J . Chromatogr., 43, 135 (1969). Baumann, P. , Scherer, B . , Kramer, W . and Matussek, N . , J . Chromatogr., 59, 463 (1971). Mattok, G. L. , J . Chromatogr.,T~, 254 (1964). McGeer, E. G. and Clark, W. H. , J . Chromatogr., 14, 107 (1964). F i n k , K . , Cline, R. E . and F i n k , R . M., Anal. Cham., 35, 389 (1963). Mahn, F. P . , a n i u s , G . , Venturella, V . S . and Senkowski, B. Z . , Hoffmann-La Roche Inc. , Personal Communication. Gehrke, C. W . and S t a l l i n g , D. L . , S e p a r . Sci., - 2, 101 (1967). Atkinson, P . W . , Brown, W . V. and Gilby, A. R . , AnaZ. Biochem., 40, 236 (1971). Matsuoka, D. T . , Drell , W . , Schat t , H . F. , Alcaraz, A. F. and James, E . C . , AnaZ. Biochem., - 5, 426 (1963). Spiegel, H . E . and Tonchen, A. E . , CZin. Chem., 16, 763 (1970). Nakajima, K . , Osaka f/iagaku Igaku Zasshi, 72, 897 (1960). Rolland, M. Lasry, S . and Lissitzky, S . , BUZZ. SOC. Chim. BioZ., 42, 1065 (1960). Doty, J . R . , Anal. &em., 20, 1166 (1948). Billmeyer, A . , Geller, M., Venturella, V. and Senkowski , B . , Hoffmann-La Roche Inc. , Personal Communication. Coppi, G . , Vidi , A . and Bonardi, G . , J . Pharm. Sci. , 61, 1460 (1972). Anton,T. H . and Sayre, D. F. , J . PharmacoZ. Exp. Ther., 145, 326 (1964).

--r

223

SODIUM LEVOTHYROXINE

Alex Post and Richard J. Warren

ALEX POST AND RICHARD J. WARREN

CONTENTS

I . D e s c r i p t i o n

1 . 1 Nomenclature

1 . 1 1 Chemical Names

1.12 Gener ic Names

I . 13 Trade Names

I .2 Formula, Mo lecu la r Weight, S t r u c t u r e

I .21 Empi r i ca l Formula, Mo lecu la r Weight

1.22 S t r u c t u r e

I .3 Appearance, Color , Odor

2. Phys i ca l P r o p e r t i e s

2 . I Spect ra I P r o p e r t i e s

2.11 U l t r a v i o l e t Absorp t i on Spectra

2.12 I n f r a r e d Spectrum

2.13 Nuclear Magnet ic Resonance Spectra

2.131 Pro ton NMR Spectrum

2. I 3 2 Carbon- I 3 NMR Spectrum

2.14 Mass Spectrum

2. I5 S p e c i f i c O p t i c a l R o t a t i o n

2.2 S o l u b i l i t y

2.3 C r y s t a l P r o p e r t i e s

2.31 C r y s t a l l i n i t y

2.32 X-Ray D i f f r a c t i o n

226

SODIUM LEVOTHY ROX IN E

CONTENTS (continued)

2.4 pKa, Ionization Constant

2.5 Melting Range

2.6 Differential Thermal Analysis

2.7 Thermogravimetric Analysis

3. Synthesis

3.1 Chemical Synthesis

3.11 L-Thyroxine, Monosodium Salt

3.12 D-Thyroxine, Monosodium Salt

3.2 Nonenzymic Synthesis of L-Thyroxine

3 . 3 Synthesis of Radiolabeled L-Thyroxine

4. Stability

5. Drug Metabol ism

5.1 Metabolic Products

5.2 Biological Half-Life

6. Elemental Analysis

6.1 Determination of Organically Bound Iodine

6.11 Oxygen Flask Combustion t lodometric Titration

6.12 Ashing t N-Bromosuccinimide Titration

6.13 Oxygen Flask Combustion + Coulometric Titration

6.14 Specific Ion Electrode

6.2 Determination of Water

6.21 USP X I X

227


CONTENTS (con t inued)

6.22 Thermograv imetr ic A n a l y s i s

6.3 Chromatographic Ana lys i s

6.3 I Paper Chromatography

6.32 Th in Layer Chromatography

6.33 Column Chromatography

6.331 Ion Exchange Chromatography

6.332 Gel F i l t r a t i o n Chromatography

6.333 Gas L i q u i d Chromatography

6.334 High Performance L i q u i d Ckomatography

6.4 Neutron A c t i v a t i o n A n a l y s i s

6.5 Po I arograph i c Ana I y s i s

6.6 K i n e t i c Methods o f A n a l y s i s

6.7 Double-Isotope D i l u t i o n A n a l y s i s

6.8 De te rm ina t ion of S te reo isomer i c P u r i t y

6.9 E q u i l i b r i u m D i a l y s i s

Methods o f Ana lys i s - A Comp i la t i on 7.

8. References

228


I . D e s c r i p t i o n

Sodium l e v o t h y r o x i n e i s a p h y s i o l o g i c a l l y a c t i v e m a t e r l a l b e i n g t h e levo- isomer o f t h y r o x i n e . The da ta presented i n t h l s a n a l y t i c a l p r o f i l e , un less o t h e r w i s e s ta ted , w i l l r e f e r t o t h e levo- isomer. References t o data o b t a i n e d f o r t h e dextro- isomer, sodium d e x t r o t h y r o x i n e , t h e DL-form, or f o r t h e f r e e amino a c i d of e i t h e r s tereoisomer w i l l be so des i gnated.

I . I Nomenclature

1 . 1 1 Chemical Names

Several chemical names have been used t o desc r ibe sodium l e v o t h y r o x i n e and sodium d e x t r o t h y r o x i n e :

d i iodophenoxy)-3,5-di iodophenyl l -L-a l a n i n e l ( a ) Sodium d e r i v a t i v e o f 3-[4-(4-hydroxy-3,5-

(b 1 L-3,3', 5,5 ' -Tetra i odo thy ron 1 ne, sa I t, pentahydrate2

( c ) !3-[(3,5-D i iodo-4-hydroxyphenoxy d i iodophenyl l -a lan ine, sodium s a l t , pentahydratez

3,5-d

3,5-d

sod i um

-3,5-

( d ) D-Tyrosine, 0-(4-hydroxy-3,5-di odophenyl l - i odo-, monosod i um sa I t, hydra ted

( e l L-Tyrosine, 0-(4-hydroxy-3,5-di i odopheny l l - iodo-, monosodium s a l t , hyd ra te4

I . 12 Gener ic Names

Sodium d e x t r o t h y r o x i n e (D-T4, Na); sodium l e v o t h y r o x i n e (L-T4, Na); and L - thy rox ine , sodium (L-T4, Na).

I . 13 Trade Names

Cholox in , Syn th ro id , L e t t e r , Cyto len, Levoid, E l t r o x i n , Laevoxin, Levaxin, Oroxine, and S y n t h r o i d Sodium.

1.2 Formula, Mo lecu la r Weight, S t r u c t u r e

229


I .21 E m p i r i c a l Formula, Mo lecu la r Weight

( a ) Sodium S a l t , Pentahydrate: C15H1014NNa04 5H20

( b ) Anhydrous Sodium S a l t : cl S H l 01qNNa04

( c ) Free Acid: 1 5 1 1 * 4

888.96

798.86

776.93

I .22 S t r u c t u r e

I I

N a' -0 O--@-CH 2- YH-COOH 5H20

NH2 I' I

I . 3 Appearance, Color , Odor

The sodium s a l t , pentahydrate, i an odor less , w h i t e t o p a l e b u f f powder o r c r y s t a l l i n e powder s . The anhydrous powder I s l i g h t ye1 low t o b u f f c o l o r e d and hygroscopic3.

2. Phys i ca l P r o p e r t i e s

2. I Spectra I P r o p e r t i es

2.11 U l t r a v i o l e t Absorp t i on Spectra

Gemml I I s r e p o r t e d X max 325 ( E = 6207) I n 0.4 N KOH and A max 295 ( E = 4160) i n 0.4 N HCI. Evidence has been- presented t h a t t h e s h l f t I n t h e maxima I s due t o t h e d i s s o c i - a t i o n o f t h e p h e n o l i c hyd roxy l group. Edelhoch6 a l s o r e p o r t e d X max 325 ( E = 6180) i n 0.1 N NaOH. -

The u l t r a v i o l e t spectrum o f t h y r o x i n e i n a c i d i - f i e d ethanol (pH 2.017 i s shown i n F i g u r e I . 300 nm ( E = 4600) and a shou lde r a t 290 nm a r e bands cha rac te r - i s t i c o f t h e conjugated a romat i c system.

A maximum a t

F i u r e 2 i s t h e u l t r a v i o l e t spectrum i n a l k a l i n e e thano l (pH 13.0) 9 which produces t h e sodium s a l t o f t h e

230

ru Y

0.8 :$ 0.7

0.6 - 0.5 - 0.4 - 0.3 - 0.2 - 0.1 -

?

CONC: 2.004 x W5Y

SOLVENT: Acidifird Ethrnd (pH 2.0)

CELL: 5 an

I I I I

260 300 350 400

Figure 1

0

-0

d

0

-u

)

m

0

-0

0

0

-to

(v

N

Q)

232

13.0)

260 300 350 400

Figure 2

SOD I UM LEVOTHY ROXl N E

p h e n o l i c hyd roxy l on t h e a romat i c system. The spect rum shows t h e expected s h i f t i n maximum t o 328 nm ( E = 6580) w l t h co r resoond ing i nc rease i n i n t e n s i t y .

2.12 I n f r a r e d SDectrum

F i g u r e 3 i s t h e i n f r a r e d spectrum of t h y r o x i n e , USP, taken as a m ine ra l o i l d i s p e r s i o n from 4000-625 c m - l on a Perkin-Elmer Model 457 i n f r a r e d s p e c t r o p h o t m e t e r . The f o l lowing a b s o r p t i o n bands a r e a ~ s i g n e d : ~

Tab le I I n f r a r e d Spec t ra l Assignments

Wavelength (cm-l) Assignment

3600 OH 3500-3200 broad, NH2 + H20

1610 1415) coo- I245 R-0-R

The i n f r a r e d spectrum o f L - t h y r o x i ne has been r e p o r t e d by Hagen, e t a1.8

2.13 Nuclear Magnet ic Resonance Spectra

2.131 Pro ton NMR Spectrum

The p r o t o n NMR spectrum ( F i g u r e 4 ) was ob ta ined I n a DMs0-d~ s o l u t i o n which con ta ined about 100 mg thy rox ine /m l and t e t r a m e t h y l s i l a n e as t h e i n t e r n a l re fe rence . The spectrum was ob ta ined on a Perk in-Elmer Model R32 90 MHz spect rometer . The assignments a r e as f o l l o w s :

Table 2

P ro ton NMR Spec t ra l Assignments

233

Figure 3

235

MATERIAL:

INSTRUMENT: PrLRElmr R32.90 MHz

RUN IN: DMSO-dg

I

k

L I I I I I I I 1 I

m 9 B 1 6 6 4 3 2 1 0

Figure 4


Table 2 ( con t inued)

P ro ton P o s i t i o n S igna l Appearance Chemical S h i f t (ppm)

a broad m u l t i p l e t 3.00 b broad m u l t i p l e t 3.51 c broad, NH: t H20 5.30 d s i n g l e t 7.09 e s i n g l e t 7.83

2.132 Carbon-13 NMR Spectrum

The carbon-13 NMR spectrum o f t h y r o x i n e was taken i n DMSO-d, s o l u t i o n on a Var ian CFT-20 spect rometer . The spectrum i s shown i n F i g u r e 5. The assignments a r e as f o l lows:?

Table 3

Carbon-13 NMR Spec t ra l Assignments

I COOH I

Carbon-13 P o s i t i o n

a b

d e f and k 9 h i j

c

2.14 Mass Spectrum

Chemical S h i f t (ppm)

169.30 151.27 149.80 140.90 139.06 125.00 91.50 87.59 54.78

over lapped by s o l v e n t

The f i e l d deso rp t i on mass spectrum o f

236

MATERIAL Thyroxine

INSTRUMENT: Varian CFT 20

RUN IN: DMSOd6

N

Y

4000 3000 2000 1000 0

Figure 5


9 L-thyroxine was obta ined on a Varian MAT CH-5 spectrometer. The r e s u l t s a re presented as a bar graph I n F igure 6. The presence o f a t r a c e o f tr l iodothyronlne i s evidenced by f rag- ments a t m/e 577, 607 and 651. Mass spect rometr ic s tud ies o f thy rox ine t r l m e t h y l s i l y l de r i va t i ves have been reported.10

2.15 Spec i f i c Opt ica l Rotat ion

As both t h e lev0 and dex t ro isomers a re pharmacological ly important substances, t h e i r respec t ive s p e c i f i c r o t a t i o n values have been l i s t e d i n several compen- d i a as c r i t e r i a o f a c c e p t a b i l i t y . The fo l l ow ing are severa I repor ted va I ues, together w I t h references ( R I ) :

I somer

Lev&

L e v 4

Lev&

Levoa

Levoa

Levoa

Levoa

Lev&

Lev&

Lev&

Levoa

Dextro'

Dextrob

Concentrat ion

0.66 g

0.66 g

3%

2.2%

3.28%

3.25%

5%

3%

2%

3%

2%

2%

3%

*C X max [ a ] - - - Solvent

6.07 g 0.5 N NaOH

6.07 g 0.5 N NaOH 25 + 13.03 g CTH50H 0.13 N NaOH i n 2o 70% CFH5OH

+ 13.03 g CZH50H 21

I N NaOH : C2H50H

I N NaOH : C2H5OH

2o

2 ,

(1-72)

( 172) 24 g 0.5 N NaOH t 56 g C2A5OH

17,5

I N HCI : C2H50H 25 (172)

20 0.2 N NaOH I n 70% & H ~ O H I N HCIC: C ~ H S O H 20 ( 1-74] I N NaOH : C2HsOH 20

I N HCI : C2H50H 2o ( 174) I N HCI : C2H50H 2o ( 1741

( 1-72)

25 I N NaOH i n C2HsOH

aAnhydrous f ree amino ac i d bAnhydrous sod i urn

546

546

D

D

D

D

D

D

D

D

D

D

D

s a l t

-3.2 I 1

-3.2 2

-4.4 12

-5.7 1 2

-5.4 1 3

-4.6 1 3

+ I5 1 4

-4.5 14

+ I6 t o +20

4 -5 t o

-6

t i 9 . 1 25

-19.2 15

t5 t o 3 +6

238

A

Q

a L

0

I t

El 'r

HI +- +- E +' -

El 3

239

Matsrial: Thyroxine EmitbrWireCumnt: 23mA win dippad m DMSO sdution InmUm*n: Vrirn MAT CH-5 O f Soura Tan~emu re: 50°C P.akr>m

10 :il 0

Figure 6


S p e c i f i c r o t a t i o n s o f L - thy rox ine (C = 29, 0.2 N HCI-ethanol ) a t severa l wave1 engths and temperatures have been repor ted:15

Temp Cal 436 nm 364 nm ( O C ) 589 nm 578 nm 546 nm -

10 +19.5 t20.3 +23.3 +42.2 +7 I .O 20 t 1 9 . I +20. I t23 .2 +42.6 +70. I

40 +18.4 +19.3 +22. I +41.5 +62.7 30 +18.9 +19.7 +22.8 +42.0 +64.5

Equ iva len t s p e c i f i c r o t a t i o n va lues, b u t o f o p p o s i t e s ign , were a I so r e p o r t e d for D- thy rox i ne. I5

Us ing t h e above data, Felder , e t a l l 5 ob ta ined t h e o p t i c a l r o t a t o r y d i s p e r s i o n curves and e s t a b l ished t h e L- conf igura- t ion o f t h e samp l e o f L- thyrox ine.

2.2 Sol u b i I i t v

Table 4

L-Thyrox ine Solubi I i t y

Ref #

H20 0. 14 3 95% ethanol 0.3, 0.4 1

a1 k a l i hydrox ides so I ub 1 e 1

So I v e n t g/lOO ml -

c h I o r o f o m a lmos t l n s o l u b l e 1 e t h y l e t h e r a lmost i n s o l u b l e 1

pH 7.4 b u f f e r 0.022-0.044 1 6

Ever t16 used a phosphate b u f f e r a t pH 7.2-7.8 a t 38OC and a range o f i o n i c s t r e n g t h f rom 0.032-0.162. He p o s t u l a t e d t h a t t h e i nc rease i n i o n i c s t r e n g t h by t h e a d d i t i o n o f sodium c h l o r i d e produced a change i n t h e i o n i c atmosphere o f t h e c e n t r a l ion r e s u l t i n g i n a ' s a l t i n g - i n and s a l t i n g - o u t ' e f f e c t . U l t r a v i o l e t a b s o r p t i o n a n a l y s i s was used t o e s t a b l i s h t h e s o l u b i l i t i e s . E v e r t has presented s o l u b i l i t y curves which show t h e e f f e c t o f temperature (25' and 38OC) and o f i o n i c s t r e n g t h s on t h e s o l u b i l i t y o f L - t h y r o x i n e sodium. S o l u b i l i t y i s s i g n i f i c a n t l y increased above pH 7.4.

240


2.3 Crys ta I P r o p e r t i e s

2.31 C r y s t a l l i n i t y

C r y s t a l data on L - thy rox ine , sodium s a l t , pentahydrate, have been r e p o r t e d by Cody, e t a l .I7

2.32 X-Ray D i f f r a c t i o n

The c r y s t a l and mo lecu la r s t r u c t u r e s o f L - t h y r o x i n e hyd roch lo r i de , monohydrate have been determined by X-ray c r y s t a l lography and r e p o r t e d i n t h e I i t e r a t u r e . 2 8 The c r y s t a l system i s monoc l i n i c , and t h e sample c r y s t a l l i z e d i n space group C 2 w i t h a = 17.23, b = 5.14, C = 25. 15 R; $ = 90.47'; Z = 4.

2.4 pKa, I o n i z a t i o n Constant

The apparent pKa o f t h e phenol i c hydroxy l , ca rboxy l and amino f u n c t i o n s has been repo r ted :

Funct i o n Ref

pKa Ref

#

ca rboxy I 2.2 19 3.832 20 p h e n o l i c hydroxy l 6.7 5 8.085 20

amino 10. I 19 9.141 20

a l n 75% d l m e t h y l s u l f o x i d e - w a t e r and 0.1 M KNO3 T i t r a n t : p o t e n t i o m e t r i c w i t h sodium hyd rox ide

2.5 M e l t i n g Range

The fol lowing m e l t i n g ranges have been r e p o r t e d f o r t h y r o x i n e :

Reference # I somer M e l t i n g Range ( O C ) - L-T4 233-235 (decmp 1 1 2 L-T4 235-236 (decomp 1 2 D-T4 237 ( d e c m p ) 2 L-T4 236 ( c o r r ) 8

241


The m e l t i n g range o f L - t h y r o x l ne (SKBF r e f e r e n c e 88-2746-227) employing t h e USP X I X , C lass I procedure, was 235-236OC (decomp 1. 21

2.6 D i f f e r e n t i a l Thermal A n a l y s i s

A d i f f e r e n t i a l thermal a n a l y s i s c u r v e o f L - t h y r o x i n e (SK&F r e f e r e n c e 88-2746-2271, o b t a i n e d on a DuPont Model 900 D i f f e r e n t i a l Thermal Analyzer from r o o m temperature t o 25OOC a t a hea t ing r a t e o f 10°C p e r m inu te under n i t r o g e n sweep, i s shown i n F i g u r e 7. A sharp exothermic change occu r red from 230-235OC, i n d l c a t l n decomposi t ion w i t h no obse rvab le p r i o r m e l t i n g (endotherm). Be

2.7 Thermograv lmetr lc Ana lys i s

The the rmograv lme t r l c a n a l y s i s o f L - thy rox ine sodium, pentahydrate, was ob ta ined on a DuPont Thermograv lmetr ic Analyzer (see F i g u r e 8).z1 o f 2OoC pe r m inu te under n i t r o g e n sweep t o 475OC. loss of ~ 9 % was observed. As expected, i n c e p t i o n o f r a p i d decomposi t ion appeared t o occu r a t approx imate ly 20OoC. r e s u l t ob ta ined by t h e loss on d r y i n g procedure o f t h e USP X I X 4 was 8.75%.

The compound was heated a t a r a t e A we igh t

The

3. Synthes is

3.1 Chemical Syn thes i s

3. I I L-Thyroxl ne, Monosodi um Sa I t

The s y n t h e t i c r o u t e t o L - thy rox ine , and subse- q u e n t l y t o I t s monosodium s a l t , pentahydrate, was desc r ibed by Chalmers, e t a l l 2 and i s presented i n F i g u r e 9. was a l s o presented showing t h e s t e r e o s p e c i f i c i t y o f t h i s syn thes i s . The o v e r a l l y i e l d was 26%.

Evidence

To a coo led suspension o f L - t y r o s i n e ( 1 ) i n s u l f u r l c a c i d was added n i t r i c a c i d which on n e u t r a l i z a t i o n y i e l d e d 3 ,5 -d in i t ro -L - t y ros ine ( I I ) . An a l k a l i n e s o l u t i o n o f ( I I ) a c e t y l a t e d w i t h a c e t i c anhydr ide y i e l d e d t h e amide ( I l l ) . E s t e r i f i c a t i o n o f ( I l l ) t o ( I V ) was e f f e c t e d u s i n g e t h y l a l c o h o l and p - t o l u e n e s u l f o n i c a c i d and then azeo t rop ing t h e wa te r w i t h ch lo ro fo rm. The d lpheny l e t h e r ( V ) was prepared from ( I V ) by t r e a t m e n t w i t h p - to luenesu l fony lch lo r ide and

242

h) P 0

0 50 100 150 200 250

T, %(CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

Figure 7

N P P

1 1 I I 1 I I I I I I 100 150 200 250 300 350 400 450 500 0 50

T. 'C(CORRECTE0 FOR CHROM€l AlUMEl THERMOCOUPLES)

Figure 8

I

0

0 0

IN

I I 11

0-2

N I

0

I 0 I

\ ." I m 0

z I

I

0

8 IN

11

V -2

I

t

81

1;

Nr

n

II

00

0 v=o

II

0--z

I N

I 0

m

I

I0

0

0 v=o

11

0 --z

01

I1

f

C 0

Y-

O I

245

Q,

m I

o cv

m

IZ

00

O

I

8 o=o

II

I1

0-2

I, I

u

0, I

o

t I

0

0

0,

6 \

W I m

I

o N

m

I1

00

o o=o

II

0 --z

81

11

I N

I

Y

l;9 \'

0, I

0

a, U

L 0

.-

246

h

c

u

0

0

IN

r

x

I 0--z

iu I

0

0 I

IN

I I1

0--z

cu I

H O

H

0 I

H Jp

H 0 I

I I cu I

0

H

HY

0

I

H q\-

0

+ (0 z

247


p-methoxyphenol. Reduct ion o f ( V ) t o t h e d iamine ( V I ) was r e a d i l y ob ta ined by t r e a t i n g an a l coho l s o l u t i o n o f ( V ) w i t h Raney N i .

Replacement o f t h e amino groups was b rough t about v i a t e t r a z o t i s a t i o n and Sandmeyer procedures. T e t r a - z o t i s a t i o n was c a r r i e d o u t by t h e s low a d d i t i o n of a s o l u t i o n of ( V I ) i n a m i x t u r e o f a c e t i c and s u l f u r i c a c i d s t o one of sodium n i t r i t e i n t h e m i x t u r e o f t h e same ac ids . The d lazo- nium groups were removed by t h e a d d i t i o n of a s o l u t i o n o f i o d i n e i n aqueous sodium iod ide .

A l I t h e p r o t e c t i v e groups p resen t i n ( V I I ) were removed by t r e a t m e n t w i t h a m i x t u r e o f h y d r o i o d i c a c i d and g l a c i a l a c e t i c a c i d . l o d i n a t i o n o f ( V I I I ) w l t h a s o l u t i o n o f I o d i n e i n e thy lam lne y i e l d e d L - t h y r o x i n e ( I X ) . The a d d i t i o n o f L - t h y r o x i n e t o a b o i l i n g 2 2 sodium ca rbona te s o l u t i o n produced t h e d e s i r e d p roduc t ( X I .

3.12 D-Thyroxine, Monosodium S a l t

E lks and Wal lerZ3 prepared t h e D-isomer by f i r s t i n v e r t i n g t h e 3 , 5 - d i n i t r o - L - t y r o s i n e ( I I - f rom F i g u r e 9 ) t o t h e D-form ( X I I - F i g u r e 10) by t r e a t m e n t of ( I 1 ) w l t h n i t r o s y l b r o m l d e which, a f t e r ammonolysis o f (X I -F igu re l o ) , y i e l d e d t h e 3 ,5 -d in i t ro -D- t y ros ine (XI I - F i g u r e 10). The D- thy rox ine was t h e n prepared I n a s e r i e s of r e a c t i o n s e s s e n t i a l l y e q u i v a l e n t t o t h a t used by Chalmers, e t a l l 2 [see ( I I I ) - ( X I i n F i g u r e 91.

3.2 Nonenzymic Syn thes i s of - L-Thyroxine

I n an a t tempt t o e x p l a i n t h e b i o s y n t h e s i s o f t h y r o x i n e from 3,5-di iodo-L- tyros lne, Shi ba, e t a I z 4 proposed a nonenzymic model for t h i s pathway ( F i g u r e I I ) . A t n e u t r a l pH and a t room temperature and i n t h e presence o f sodium g l y o x y l a t e ( I l l , c u p r i c acetate, and oxygen, t h e r e a c t i o n proceeded r a p i d l y through a t ransamina t ion v i a a meta l c h e l a t e o f t h e S c h i f f base o f d l i o d o t y r o s i n e and g l y - o x y l i c a c i d ( I I I ) . d i l odopheny lpy ruv i c a c i d ( I V ) w i t h t y r o s i n e y i e l d e d t h e L- t h y r o x i n e ( V ) . The a n a l y t i c a l da ta presented e s t a b l i s h e d t h e s t e r e o s p e c i f i c i t y of t h i s s e r i e s o f r e a c f i o n s . The o v e r a l I y i e l d was 8%.

O x i d a t i v e coup1 ing o f 4-hydroxy-3,5-

240

NO2

KBr, NaN02 H O V CH2 -CH - COOH L-HO-I=)...-CH-COOH \ / NH2 I -> H2S04 B r I

NO2 n02

( I 1 - from F igure 9) ( X I 1

- aqueous NH3 H O O C H 2 - CH-COOH 1

NH2 \ I

NO2

( X I I )

F i g u r e 10

N

3 +

+

3

0 -0

--0

/+

I

"'Y"Z,,," -O

I2

Y 4 Y ?

z

I N

+

0-0

I

%Y& P +

+ N r:

250


3.3 Synthesis o f Radiolabeled L-Thyroxine

Weeke and O r s k o ~ ~ ~ have developed a ra the r f a c i l e rocedure f o r t h e synthes is and p u r i f i c a t i o n o f monolabeled

P251-LT4 w i t h a very h igh s p e c i f i c a c t i v i t y f o r use i n radiolmnune assay.

F i ve mCI o f 1251 ( s p e c i f i c a c t i v i t y > 14 mCi/ug) were added t o 50 p1 o f 50 mmol / l phosphate bu f fe r , pH 7 . 5 , f o r bu f fe r ing t h e a l k a l i n e 1251 so lu t i on . (20 pI 1 o f 3 ,5,3 ' - t r l iodo-L- thyronlne and 90 pg (25 1.11) o f chloramine-T, mix ing f o r 15 seconds and h a l t i n g t h e reac t i on w i t h 240 pg ( I 0 0 p l ) o f sodium metab isu l f l t e , y ie lded a 1251-LT4 o f very h igh s p e c i f i c a c t i v i t y (~3000 mCl/mg T4). The 1251 label i s presumably i n t h e 5' pos i t i on .

Add i t ion o f 2 pg

A more general and simple procedure f o r t h e synthe- s i s o f rad ioac t i ve L-thyroxine, c a r r y i n g t h e label I3lI or 14C e i t h e r i n t h e phenol i c r i n g o r i n the non-phenol i c r i n g and t h e s ide chain, was repor ted by Shiba and Cahnmann.26 I t i s based on t h e coupl ing o f 4-hydroxy-3,5-diiodophenyI- py ruv i c ac id and d i iodoty ros ine . d l lodophenylpyruv ic a c i d was prepared by the condensation of iod inated p-hydroxybenzaldehyde w i t h ace ty lg l yc ine and t h e hyd ro l ys i s o f t h e azlactone. labeled compound prepared, y i e l d s ranged from 12-209.

Labeled 4-hydroxy-3,S-

Depending on the s p e c i f i c


The USP,4 NF,3 and B P I s t a t e t h a t levothyrox dex t ro thyrox ine sodium a re t o be pro tec ted from exposure to l i g h t these compounds may assume a p In add i t ion , t h e USP4 s ta tes t h a t Ievothyrox ine dry a i r .

ne and ight . On nk co lo r . s s tab le i n

Several i nves t i ga to rs have s tud ied t h e s t a b i l i t y o f t hy rox ine as a func t i on o f t h e de lod ina t ion process which y i e l d s f ree iod ine and tri iodothyronine. reviewed the pathway and mechanism o f t h i s process by both i n v i t r o and I n v i vo experimentation. He ind ica ted t h a t t h e major f a c t o r I n t h e de iod ina t ion process i s invo lved w i t h t h e fact l e ion lzab l I i t y o f t h e hydroxyl group. Because o f t h i s , one can expect t h a t t h e lodlnes i n t h e 3' and 5 ' pos i t i ons are more l a b i l e than those i n t h e 3 and 5 p o s i t i o n s

StanburyaP has

251


( r e f e r t o § I ,221. The re leased i o d i d e i s t hen o x i d i z e d t o f r e e i od ine . That t h i s appears t o be t h e i n v i v o mechanism has subsequent ly been borne o u t by o t h e r i n v e s t i g a t o r s .

High energy g a m a r a d i a t i o n has been shown t o cause r a p i d d e i o d l n a t i o n and t h e for rnat lon o f o t h e r i o d l n a t e d o r g a n i c molecu 1 es .28

d e i o d i n a t i o n o f 1311 labe led t h y r o x i n e Is s t i I I o f concern. TaurogBg has shown t h a t t h e s p o t t i n g o f d i l u t e aqueous b u f f e r s o l u t i o n s on f i l t e r paper and d r y i n g for 10-20 minutes l ed t o a s i g n i f i c a n t and v a r i a b l e loss o f 1311 from t h e 1311- thyrox ine. paper and t h i n l a y e r s i I i c a g e l were used. Reduct ion i n t h e amount o f d e i o d i n a t i o n occu r red when ethanol or p ropy lene g l y c o l was added t o t h e sample. Shortwave u l t r a v i o l e t l i g h t g r e a t l y enhanced t h e r a t e o f d e i o d i n a t i o n .

The r a t i o n a l e and e x p l a n a t i o n f o r t h e spontaneous

T h i s e f f e c t was a l s o observed when g l a s s

These spontaneous d e i o d i n a t i o n e f f e c t s were a l s o s t u d i e d by J o l i n , e t a l . 30 They i n d i c a t e d t h a t t h i s e f f e c t a r i s e s when one o f t h e components o f t h e s o l u t i o n under s tudy becomes an a c t i v e d e i o d i n a t i o n agent d u r i n g t h e a n a l y t i c a l procedure. Thus, a s t r i c t adherence t o procedure must be f o l lowed.

Reviczky and Nagy3l found t h a t under u l t r a v i o l e t r a d i - a t i o n d e i o d i n a t i o n occu r red t o a g r e a t e r e x t e n t and more r a p i d l y i n aqueous s o l u t i o n s than i n butanol , t h e r e l e a s e o f I o d i d e i s p r o p o r t i o n a l t o t h e decrease i n pH, and chromatography showed t h a t i n a d d i t i o n t o t h e presence o f f r e e i o d i d e t r i i o d o t h y r o n i n e was t h e pr ime decomposi t ion p roduc t .

5 . Drug Metabol ism

5. I B i o l o g i c a l H a l f - L i f e

I n a study s i m i l a r t o t h a t of S t e r l i n g , e t a1,32 Demeester-Mi r k i ne showed t h a t t h e ha I f - I i f e o f 1311- thyrox i ne i s 7 days i n serum and 0.4 days i n t i ssues .33

5.2 Metabol i c Products

Thy rox ine i s conver ted t o t r i i o d o t h y r o n i n e i n hypo- t h y r o i d human s u b j e c t s ma in ta ined on s y n t h e t i c sodium L-

252


t h y r o x i n e admin i s te red o r a l ly.34 S t e r l i n g , e t a l ,32 who i n j e c t e d p u r i f i e d t h y r o x i n e l a b e l e d w i t h 14C i n r i n g A and i n t h e a l a n i n e s i d e cha in . Methods f o r de te rm in ing t h i s convers ion have been reviewed by Surks and Oppenheimer. 35

6. Elemental A n a l v s i s

T h i s was con f Inned by

The elemental composi t ion o f L - t h y r o x i n e as t h e monosodium s a l t pentahydrate, t h e anhydrous monosodium s a l t , and t h e anhydrous f r e e a c i d i s as f o l l o w s :

% (Theory)

Monosodium Monosodium Amino Ac id (Pentahydra t e ) (Anhydrous) (An hy d r o u s 1

Carbon 20.27 Hyd rog en 2.27 N it rogen I .58 Oxygen 16.20 Sod i urn 2.59 I o d i n e 57.10

22.55 23.19 I .26 I .43 1.75 I .80 8.01 8.24 2.88 -

63.54 65.34

The t h e o r e t i c a l percentage o f water i n t h e monosodium penta- h y d r a t e form i s 10.13%.

6.1 De te rm ina t ion of O r g a n i c a l l y Bound l o d l n e

As t h e c r i t e r i o n o f acceptabi I i t y i n bo th t h e USPd and t h e NF3 i s t h e assay v a l u e f o r i o d i n e , t h e methods o f a n a l y s i s employed t o determine t h i s element have been care- f u l l y i nves t i ga ted .

6 . I I Oxygen F l a s k Combustion36 + lodomet r i c T i t r a t ion37

This method i s desc r ibed i n bo th t h e USP X I X and NF X I V monographs f o r sodium l e v o t h y r o x i n e and sodium d e x t r o t h y r o x i ne.

Apparatus - The apparatus c o n s i s t s o f a heavy- w a l l e d c o n i c a l , deeply l i p p e d o r cupped 500-ml f l a s k (un less a l a r g e r f l a s k Is s p e c i f i e d ) , f i t t e d w i t h a ground-glass s topper t o which i s fused a sample c a r r i e r c o n s i s t i n g o f

253


heavy-gauge p l a t i n u m w i r e and a p i e c e o f welded p l a t i n u m gauze measuring about 1.5 x 2 cm.

samp 4 cm samp p l a t cons s o l u

Procedure - Weigh a c c u r a t e l y about 25 mg o f e on a p i e c e o f h a l i d e - f r e e f i l t e r paper measuring about square, and f o l d t h e paper t o enc lose it. P l a c e t h e e, t o g e t h e r w i t h a f i l t e r paper f u s e - s t r i p , i n t h e num gauze sample ho lde r . P lace t h e absorb ing l i q u i d , s t i n q of a m i x t u r e of 10 m i of sodium hyd rox ide i on 71 i n 1001, i n t h e f l a s k and mo is ten t h e j o i n t o f

t h e stoDoer w i t h water . F lush t h e a i r from t h e f l a s k w i t h a I ,

stream o f r a p l d l y f l o w i n g oxygen, s w i r l i n g t h e I i q u i d t o favo r i t s t a k i n g up oxygen. [NOTE--saturation o f t h e l i q u i d w i t h oxygen i s e s s e n t i a l f o r t h e success fu l performance o f t h e combustion procedure. ] I g n i t e t h e f u s e - s t r i p by s u i t a b l e means. I f t h e s t r i p i s i g n i t e d o u t s i d e t h e f l a s k , i n v e r t t h e f l a s k so t h a t t h e a b s o r p t l o n s o l u t i o n makes a seal around t h e stopper, and h o l d t h e s topper f i r m l y i n p lace. When t h e combustion i s complete, p l a c e a few m l o f water around t h e s topper and a l l o w t h e f l a s k t o s tand f o r about 15 minutes. Loosen t h e s topper , and r i n s e t h e s topper , sample h o l d e r , and s i d e s o f t h e f l a s k w i t h about 20 m l o f water, added i n smal l p o r t i o n s . Add I m l o f an o x i d i z i n g , s o l u t i o n prepared by adding 5 m l of bromide t o 100 ml of a I i n 10 s o l u t i o n o f sodium a c e t a t e i n g l a c i a l a c e t i c a c i d . I n s e r t t h e s topper I n t h e f l a s k , and shake v i g o r o u s l y f o r I minute. Add 0.5 m l o f f o rm ic a c i d , r e p l a c e t h e s topper , and shake v i g o r o u s l y f o r I minute. Remove t h e s topper , and r i n s e t h e s topper , t h e sample ho lde r , and t h e s ides o f t h e f l a s k w i t h severa l smal l p o r t i o n s o f water. remove t h e oxygen and excess bromine, add 500 mg o f potass ium iod ide, s w i r l t o d i s s o l v e , add 3 ml o f d i l u t e d s u l f u r i c ac id , s w i r l t o mix, and a l l o w t o s tand f o r 2 minutes. T i t r a t e w i t h 0.02 N sodium t h i o s u l f a t e , adding 3 m l o f s t a r c h TS as t h e endpoTnt i s approached. Each ml o f 0.02 - N sodium t h i o s u l f a t e i s e q u i v a l e n t t o 0.1057 mg o f i od ine .

Bubble n i t r o g e n through t h e f l a s k t o

6.12 Ash i ng t N-Bromosucc i n i m i de T i t r a t i o n

Bakarat, e t al,38 ashed t h e sample i n t h e presence o f potassium carbonate and, a f t e r d i l u t i o n w i t h water, t i t r a t e d t h e s o l u t i o n w i t h 0.02 N N-bromosuccinimide. Accuracy o f g r e a t e r than 99% was ob ta inzd f o r samples con- t a i n i n g 5-10 mg o f t h y r o x i n e .

254


6.13 Oxygen F lask Combustion + Cou lomet r i c T i t r a t i on

A modif i c a t i o n d g o f t h e oxygen f l a s k combustion method has been used p r i o r t o t h e c o u l o m e t r i c t i t r a t i o n 4 0 o f t h e i od ide :

To a 500-ml oxygen f l a s k f i l l e d w i t h oxygen I s added 10.00 m i o f 0.4 N potassium hyd rox ide and seve ra l m i l l i g r a m s o f hyd raz ine s u l f a t e . i g n i t e d , and a f t e r combust ion i s complete t h e f l a s k i s coo led f o r a few seconds i n a stream o f c o l d water and s e t a s i d e f o r a t l e a s t 30 minutes f o r complete a b s o r p t i o n of t h e combustion products . The abso rb ing s o l u t i o n i s a c i d i f i e d w i t h 10.00 m l o f a s o l u t i o n o f 0.6 N n i t r i c a c i d i n 20% g l a c i a l a c e t i c a c i d . The f l a s k i s s w i r l e d t o remove some o f t h e carbon d i o x i d e evolved, stoppered and v i g o r o u s l y shaken. An a l i q u o t of 4.00 m l i s t i t r a t e d c o u l o m e t r i c a l l y on a s u i t - a b l e c o u l o m e t r i c t i t r a t o r , such as t h e Aminco-Cotlove Auto- mat i c T i t r a t o r . 42j 42

The f i l t e r paper t a b i s

6. I4 S p e c i f i c Ion E l e c t r o d e

The s p e c i f i c i on e l e c t r o d e s e n s i t i v e t o i o d i d e has been used by P a l e t t a and Pantenbeck43 fo r samples of L-T4. The i o d i n e i s s t r i p p e d from t h e compound w i t h a c t i v a t e d a l u - minum f o i I a t pH I I a t 6OoC i n 10 minutes. A f t e r n e u t r a l l z - a t i o n w i t h d i l u t e h y d r o c h l o r i c ac id , t h e p o t e n t i a l is measured us ing t h e s p e c i f i c i on e lec t rode44 versus t h e calomel r e f e r e n c e e l e c t r o d e . The amount of i o d i d e p r e s e n t i s d e t e r - mined by comparing t h e p o t e n t i a l read ing w i t h t h o s e o b t a i n e d f o r a s e r i e s o f s tandard s o l u t i o n s c o n t a i n i n g t o lo-’ grams o f i o d i d e p e r m l . The assay r e s u l t s compared f a v o r a b l y w i t h those ob ta ined by t h e conven t iona l t i t r i m e t r i c procedure us ing a sodium t h i o s u l f a t e t i t r a n t .

6.2 De te rm ina t ion o f Water

L - t h y r o x i n e monosodium i s g e n e r a l l y prepared as a hydrate. Thus, i n o r d e r t o compare samples on an anhydrous bas is , t h e m o i s t u r e c o n t e n t i s determined.

6.21 G r a v i m e t r i c Method (USP X I X , Method I l l , p.668)

From t h e monograph f o r l e v o t h y r o x i n e sodium

255


t h e de te rm ina t ion i s as f o l l o w s :

'Dry about 500 mg, a c c u r a t e l y weighed, o v e r phosporous pen tox ide a t 6OoC and a t p ressu re n o t exceeding 10 mm of mercury f o r 4 hours.

6.22 Thermogravi m e t r i c Ana I ys i s (TGA)

The procedure desc r ibed i n 2.7 has been found t o y i e l d e q u i v a l e n t r e s u l t s t o t h e USP X I X procedure. sample (Batch #27) analyzed by t h e USP X I X procedure showed t h e presence y f 8.75% moisture, and by thermograv imetry I t showed 8.85%.

A

6.3 Chromatographic A n a l y s i s

As t h y r o x i n e i s o f t e n contaminated w i t h t r i i o d o - thy ron lne , and i n t h r y o i d powders w i t h a s e r i e s o f i o d i n a t e d t h y r o n i n e s and/or t y r o s i n e s , and i n se ra w i t h s i m i l a r compounds, it i s impor tan t t h a t t h e chromatographic system employed be capable o f s e p a r a t i n g these r e l a t e d compounds. Thus, i n t h e severa l chromatographic techniques used t o e v a l u a t e t h r y o x i n e and t h r y o x i n e - c o n t a i n i n g m a t e r i a l s , t h e R f , RT, e t c . of these r e l a t e d compounds a r e a l s o r e p o r t e d when avai l a b l e .

The f o l l o w i n g a b b r e v i a t i o n s o f t h r y o x i n e and r e l a t e d compounds w i l l be used:

T4 T3 T2 TI T I-

MIT DIT

6.31

TY

t h y r o x i n e 3,3', 5 - t r i i odothyron i ne 3,5-d i i odothyron i ne 3- iodothyron i n e t h y r o n i ne i norgan i c i od i de t y r o s i ne 3 - i o d o t y r o s i n e 3 ,5 -d i i odo ty ros ine

Paper Chromatography (PC)

A s i g n i f i c a n t amount of l i t e r a t u r e i s a v a i l a b l e concern i ng t h e app I i c a t i o n o f paper chromatography t o t h e separa t i on o f t h e iodoamino-acids. A few o f t h e r e l e v a n t pub1 i c a t i o n s have been c i ted.45-53

256

I & L c i R f Values o f Thyroxine, Analogs, and Related Compounds (Paper Chromatography)

M o b i l e Phase 2 3 4 5 6 7 8 9 10 I I 12

ComDound I R f

T4 T3 T2 T I T I' T MIT DIT

0.89 0.47 0.45 0.45 0.51 0.43 0.86 0.70 0.28 0.43 0.22 0.07 0.91 0.70 0.63 0.65 0.63 0.58 0.86 0.85 0.36 0.78 0.33 0.25 0.69 0.06 0.11 0.11 0.68 0.62 0.82 0.85 0.47 0.42

0.87 0.62 0.35 0.26

0.26 0.40 0.90 0.89 0.46 0.26 0.25 0.17 0.17 0.58 0.39 0.18 0.28 0.72

0.67 0.59

Mobile Phase: Reference #

I . n-Butanol:2N acetic acid ( I : I ) 46

3. 48 4 . C o l l i d i n e (conc. NHt,OH vapor) 46

2. n-Butanol :&hano1 :0.5N NH40H ( 5 : 1 :2) 47

5 . n-5utanol :ethanol :2N NH40H (5: I :2) 4 9 6. n-Butanol :2N NH40H T5:2) 49

n-Butano I : d I oxane :2& m40H ( 4 : I : 5 1

7. n-Butanol :Zv formic a c i d 50 8 . n-Butanol :6F NH40H 50 9. lsoamyl alcohol : ter t -amyl a lcoho l :6N NH40H ( 1 : I :2-upper phase) 4 5

52 12. 3% sodium c h l o r i d e 53

10. ter t -amyl a lcohol :2N NH40H:hexane ( 5 : 6 : I ) 53 I I . I , I-dimethy I propanol- I satura ted w i t h 6N - NH40H


Table 6

Detect 1 on Met hods Used i n Paper Chromatography o f Thyroxine

A comprehensive desc r ip t i on o f t h e f o l l o w i n g de tec t i on methods i s l i s t e d i n Reference 46 - they are on ly b r i e f l y descr i bed here :

' I . Cer ic -arsen i te reagent:

Th is method o f de tec t i on depends upon the l i b e r a t i o n o f i od ine from t h e compounds by ox ida t i on w i t h c e r l c su l fa te . The iod ine-conta in ing compounds appear as wh i te spots on a ye l low background. As l i t t l e as 0.1 pg o f L-T4 and L-T3 and 0.01 pg o f I- could be detected. o f t h i s method can be found i n Reference 139.

A d e t a i l e d account of the a p p l i c a t i o n

2. Fe r r i ch l o r 1 de- fer r icyan ide-arsenious a c i d reagent:

&el I n and Virtanen"' demonstrated t h a t t h e c a t a l y t i c acce le ra t ion by I' o f the simultaneous reduc t ion o f f e r r l c h l o r l d e and f e r r i c y a n t d e t o y i e l d a mix tu re of Tu rnbu l l ' s b lue and Prusslan b lue was a s e n s i t i v e de tec t ion method f o r iodothyronines and iodotyros ines (0.002 pg f o r each o f t h e amino ac ids and 0.001 pg f o r I-.)

3. Starch:

Datta, e t a I .49 showed t h e use o f a s ta rch spray, an overspray w i t h KI03, and exposure t o UV l i g h t t o be a s e n s l t i v e de tec t ion reagent. A t r a n s i e n t b lue c o l o r from as l i t t l e as 0.05 pg o f an iodoamino- ac id can be detected.

Less s e n s i t i v e de tec t ion reagents are:

4 . Dla to t i zed s u l f a n i l l c acid:

10-20 pg o f t hy rox ine can be detected. A range o f co lo rs from reddish purp le (L-T4) t o orange (L-TI ) i s obtained.

258

SOD1 UM LEVOTHY ROX I N E

Table 6 ( con t inued)

5 .

6.

7.

D i a z o t i zed N,N-di e thy I su I fan i lami de:

T h i s reagen t i s o n l y s l i g h t l y more s e n s i t i v e t h a n t h e above, and t h e c o l o r s a r e i n t h e p u r p l e range.

N i nhydr i n :

A l ess s p e c i f i c reagent, s i n c e it r e a c t s w i t h amino a c i d s . r a t h e r t h a n t h e t y p i c a l p u r p l e . The l i m i t o f d e t e c t i o n o f L-T4, L-T3, and L-T2 i s I ~ 9 . ~ ~

U l t r a v i o l e t l i g h t :

These compounds absorb s t r o n g l y i n UV l i g h t and can be de tec ted i f 20-30 pg a r e p resen t .

L-T4 appears as a p u r p l i s h brown c o l o r

A uni ue reagent (Emerson's)'41 was used by E i s d o r f e r and

presence and amount o f L-T2 and L-T3 i n L-T4. The reagen t i s prepared by d i s s o l v i n g 4 -aminoan t ipy r ine h y d r o c h l o r i d e i n aqueous sodium ca rbona te s o l u t i o n . The reagent i s s e n s i t i v e i n d e t e c t i n g I Pg o f each o f t h e iodoamino a c i d s fo rm lng a range o f c o l o r s : L-T2 (orange), L-T3 ( r e d ) , and L-T4 ( p u r p l e ) .

Post4 2 t o qua1 i t a t i v e l y and q u a n t i t a t i v e l y determine t h e

D e t e c t i o n of l abe led iodoarnino a c i d s can be accompl ished by:

I . Neutron a c t i v a t i o n a n a l y s i s

2. Radioautography (X-ray f i Im)

3. Automat ic chromatographfc scanners f i t t e d w i t h a Geiger- Mu I I e r d e t e c t o r

259

T a b l e 7

1-

MIT DIT

R f Values o f Thyrox ine, Analogs, and R e l a t e d Compounds

( T h i n Layer Chromatography)

( see n o t e s ) M o b i l e Phase I 2 3 4 5 6 7 8 9 10

R f

0.55 0.25 0.38 0.48 1.28 0.89 0.14 0.18 0.24 0.58 0.73 0.36 0.46 0.59 1.76 1.13 0.34 0.50 0.30 0.49

0.50 0.66 0.48 0.58 0.27 0.38 0.27

0.87 0.75 0.70 0.53 0.90 0.62 0.31 0.12

0.18 0.29 0.23 0.67 0.39 0.83 0.07 0.14 0.18 0.73 0.24 0.19 0.15 0.17 0.72 0.04 0.09 0.30

Mob; I e Phase

NOTES

Adsorbent D e t e c t i on Ref #

I. n-Butanol : c h l o r o f o r m (4:7) s a t u r a t e d K i e s e l g u h r G Rad i oscan 55 w i t h NH40H atmosphere

2. 30% NHhOH (w/v):methanol : c h l o r o f o r m S i l i c a Gel H C e r i c s u l f a t e : 56 (0. I :2:2, v / v ) sodium a r s e n i t e :

57 methy I ene b I ue

N

-

N

- D

.. a, u

u

LO

U

OD

(0

.- .. .- - .-

0

-0 a 0

D

n

.- V

m D

V

a, .- N

-

4

C

L

-0 r

L c

z

.- .-

C 0

Ln

ul m

._ .- .- t V a, + a, n

tc

o

mw

N

+-

(0

--0

.-

3 D

n

ulna

a, E

m v,

a, c3

I

a, V 3

0 a

ul a, ul 0

-

t

Q) a, L

cn

c3 c3

a, c3

- W a,

cn

- - 0

0

M

z z

- a, L3

+I

a,

I: h

P

ul m

:

n

L 0

ul D

a

C

m E

a, u

m 0

.- (0

V

.- a, ul a,

Y

.- 0

W

0

Lo

*

a,

Cn 5 .- +

t

ul m w

- a, u

c 0

V

I I

!-I H

k

c

E '8

P

I

01

02

T;I

V

(0

.- I

0 t I

Z

.. - 0 c 00

V

N

a,

- V

.- € 0 +

.. a,

t

a, u

s !?

C

2-0

Et

ma,

1Y

-

.. ..

rub

-

>x

u-3 ..

-

I-

22

I

IU

Z

L:

.. - t-

L>

a,L

tf

(0 r

t

a, E E e 0

Y-

0

0

L 0

V.- -

> --

nJ\ >

.I-..

h

>

\

>

In

M

0

- .. - .. c

a, ul m r

n

a, - .-

GO

-

v I ..

- ..

%'F

- 0

LW

V LL

Mob i l e Phase

3.

4.

5.

6 . h)

9

7 .

8.

9.

Tert-amy ( 2 5 : 8 : 7 ,

Table 7 (cont inued)

Adsorbent Detect ion Ref #

alcohol:acetone:NH40H Kieselge l G v/v)

Ter t -but -no l : ter t -amyl a lcoho l : NH40H :met hy I e t hy I ketone : H20

Eastman Sheets #6060 ( S i l i c a Gel)

(Rf &ven as reZat ive Rf of I-, Rf of I- not repor ted) Tert-amyl alcoho1:dioxane:iN NH40H 20% S i I i ca Gel G+ (2:2: I ) 80% Ce l lu lose

MN 300

(Rf given as reZative Rf of I-, Rf of I- not repor ted) Ethano I :methy I e thy I ketone:ZN - NH40H ( I :4: I )

-

Same as 5

Formic acid:H20 ( I :5)

Tert-butanol :2N - NH40H:ch lo ro fo rm (376:70:60)

Ethy1acetate:methanoI:ZN - NH40H (100:40:60, V / V )

10. Ch1oroform:methanol : fo rmic a c i d ( 7 0 : 15: 15, v/v)

Cel I u lose Powder

Same as 7

S i l i c a Gel G

Ninhydr in , PdCI2

Ninhydr in , PdC12

D iazo t ized s u l f a n i l i c ac id , PdC I 2

Same as 5

F e r r i c c h l o r i d e : f e r r i cyan i de: arsenious a c i d

Same as 7

Cer ic s u l f a t e : sod i um arseni t e

S i l i c a Gel G (not repor ted)

58

59

60

60

61

61

62

66


Table 5 l i s t s t h e R f o f severa l o f t h e iodoaminoacids i n d i f f e r e n t systems. D e t e c t i o n methods a r e l i s t e d i n T a b l e 6. Several Whatman papers have been used: Whatman I, 3, 4, 3MM be ing t h e most popular . I n a d d i t i o n , t h e procedure has been c a r r i e d o u t i n t h e ascending, descending and c i r c u l a r modes. I n each case, it has been t h e exper ience o f t h e au tho rs t h a t , under t h e p roper cond i - t i o n s o f temperature, equi I i b r a t i o n t ime, and l e n g t h o f run, t h e Rfs ob ta ined w i t h any o f t hese modes gave e s s e n t i a l l y e q u i v a l e n t r e s o l u t i o n from day t o day and l a b o r a t o r y t o I a bo ra to ry . have been eva lua ted and described I n Section 4.

Dei od I n a t I on e f f e c t s d u r i ng paper chromatog rap hy

Paper chromatography, us ing f o u r d i f f e r e n t s o l v e n t systems on Whatman # I paper, was used t o e s t a b l i s h t h e chromatographic p u r i t y o f t h e L - thy rox ine sodium r e f e r e n c e substance p r i o r t o i t s a d d i t i o n t o t h e B r i t i s h .Pharmacopoeia.54 e fhy rox ine va lues a r e r e p o r t e d fo r t h e compounds I i s t e d i n Tab le 7.

6.32 T h i n Layer Chromatography (TLC)

T h i n l a y e r chromatography o f f e r s some advantages t o paper chromatography i n t h a t b e t t e r s e p a r a t i o n s a r e general l y ob ta ined w i t h h i g h e r loadings. However, r e p r o d u c i b i l i t y o f R f va lues i s o f t e n t i m e s d i f f i c u l t t o o b t a i n because o f t h e b a t c h t o b a t c h d i f f e r e n c e s i n adsorbents, temperature v a r i a t i o n s w i t h i n a l a b o r a t o r y , and e q u i l i b r a t i o n t i m e s used by d i f f e r e n t i n v e s t i g a t o r s . Thus, t h e data presented i n Tab le 7 should be considered i n l i g h t o f t h e s e v a r i a b l e s ; and t h e a n a l y s t should be expected t o a l t e r t h e m o b i l e phase, a l t hough s l i g h t l y , t o e f f e c t a s u i t a b l e separa t i on .

The re fe rences c i ted55-62 i n d i c a t e t h e v a r i e t y o f systems a v a i l a b l e f o r t h e s e p a r a t i o n o f t hese compounds. Chapters f rom severa l t e x t ~ 4 6 , ~ 3 > @ c o n t a i n a d d i t i o n a l i n format ion.

A c r i t i c a l t h i n l a y e r chromatographic a n a l y s i s o f L - t h y r o x i n e sodium was made by a j o i n t commit tee o f t h e Pharmaceut ical Soc ie ty o f Great B r i t a i n and t h e B r i t i s h Pharmacopoeia p r i o r t o e s t a b l i s h i n g t h e sample as a r e f e r e n c e s ~ b s t a n c e . 5 ~ adsorbants were used t o e s t a b l i s h i t s p u r i t y .

F i v e d i f f e r e n t mob i l e phases on f i v e d i f f e r e n t

262

SODIUM LEVOTHYAOXINE

Schorn and Wink lerS5 sys temat i ca l gated more than two dozen s o l v e n t systems on S i I p l a t e s i n t h e The r e s u l t s c t o t h e separa

6.33

s p e c i f i c t e rm

separa t i on o f L-T4, L-T3, L-T2, L- e a r l y showed t h e v i a b i l i t y o f t h i s i on o f t h e iodoaminoacids.

y i n v e s t i - ca g e l G I and I-. technicrue

Col umn Chromatography

As column chromatography i s a r a t h e r non- t o desc r ibe a seDarat ion orocedure. we have

d e l i neated t h i s t echn ique i n t o ' f o u r spec i f i c t y p e s : exchange, ge l f i l t r a t i o n , gas l i q u i d , and h i g h performance I i q u i d chromatography. T h e i r i n d i v i d u a l appl i c a t i o n s a r e desc r ibed i n f o l lowing sec t i ons .

I on

6.331 Ion Exchange Chromatography ( IEC)

Resin column chromatography has been eva lua ted and employed by many i n v e s t i g a t o r s i n t h e separa- t i o n and q u a n t i t a i t o n o f iodoamino a c i d s and iodo thy ron ines i s o l a t e d from b i o l o g i c a I mater i a I ~ . 6 ~ - 7 8 procedures a r e amenable t o assaying t h y r o x i n e and t h y r o i d powders, t h e new separa t i on techn iques desc r ibed i n Sec t i ons 6.332, 6.333, and 6.334, have, i n genera l , supplanted i o n exchange chromatographic separa t i ons .

A I though these

A n i o n i c r e s i n s , Dowex I-X2 and 50-X4 (Dow Chemical Co., Midland, Mich.) , u s i n g ammonium ace ta te , sodium a c e t a t e or ammonium formate b u f f e r s a t pH rnages from 3.2-5.6, w i t h o u t or c o n t a i n i n g up t o 30% e thano l , have been used t o separa te severa l o f t h e i odo thy ron ines . Automated procedures f o r d e t e c t i n g t h e components i n e f f l u e n t s have a l s o been reported.68J73J74J77J7&? The conven t iona l c e r i c - a r s e n i t e r e a c t i o n d e t e r m i n a t i o n f o r i o d i n e i s t h e most f r e q u e n t l y used d e t e c t i o n method.

r e s i n s t o separa te t h e i odo thy ron ines has been repor ted. 71J 7 7 J 7 8 The c i t e d re fe rences deal a lmost e x c l u s i v e l y w i t h two iodoaminoacids and two iodothyronines, MIT, DIT, T3 and T4, r e s p e c t i v e l y . s e p a r a t i o n o f e i g h t d i f f e r e n t i odo thy ron ines on (1.0 x 15 cm) c a t i o n exchange r e s i n , AG 50W-X4 (30-35 pm), e q u i l i b r a t e d w i t h 0.04 M ammonium a c e t a t e b u f f e r , pH 4.7, c o n t a i n i n g 30% ( v / v ) e thanol a t 5OoC and a g r a d i e n t o f I nc reas ing pH. The

263

The a p p l i c a t i o n o f c a t i o n exchange

Recent ly , Sor imachi and U i79 r e p o r t e d t h e


g r a d i e n t c o n s i s t e d o f s t a r t i n g b u f f e r and 0.65 N NaOH. D e t e c t i o n was by t h e i o d i n e c a t a l y z e d c e r i c - a r s s n l t e r e a c t i o n . Tab le 8 l i s t s t h e e l u t i o n volumes o f a s e r i e s of lodoamino- a c i d s and i o d i d e ob ta ined by t h i s procedure.

Tab le 8

E l u t i o n Volumes o f lodoam noac ds

Approximate E l u t on Volumes ( m l 1 Com po u n d

I o d i d e MIT DIT T I T2 T3 T4

3'-T I 3,3'-T2 3 ' ,5 ' -T2

3,3 ' ,5' -T3

6 37 50 80 80

I 0 6 91 96

I 1 3 88 96

6.332 Gel F i l t r a t i o n Chromatography (GFC)

The appl i c a t i o n o f ge l f i l t r a t i o n separa t i on o f t h e i odo thy ron ines has been p r i m a r i l y used t o determine t h e i r i n d i v i d u a l con ten ts I n b i o l o g i c a l samples. The i n f o r m a t i o n p rov ided i n Table 9 i n d i c a t e s t h e chromato- g raph ic systems used t o separa te t h e iodoaminoacids i s o l a t e d from these samples. Each o f t h e c i t e d r e f e r e n c e s desc r ibes t h e impor tan t s teps r e q u i r e d t o p repare these columns (e.g., t h e i r dimensions, mesh s i z e of t h e g e l p a r t i c l e s , e t c . ) , t h e d e t e c t i o n systems used ( g e n e r a l l y t h e c e r i c - a r s e n i t e r e a c t i o n , u l t r a v i o l e t abso rp t i on , l i q u i d s c i n t i l l a t i o n c o u n t i n g o f tagged i s o l a t e s , e t c . ) , and t h e p r e c i s i o n and accuracy o f t h e p a r t i c u I a r v a r i a t i o n employed by t h e r e s p e c t i v e i nvesi t g a t o r .

B l a s i and DeMasiB0 have l i s t e d t h e p a r t i t i o n c o e f f i c i e n t s , Kd, o f severa l t y r o s i n e s and t h y r o - n i n e s as ob ta ined from t h e Sephadex G-25 column and t h e i r e l u t i o n c o n d i t i o n s . From these data, r e l a t i o n s h i p s between t h e s t r u c t u r e o f t h e compound and t h e e l u t i o n volume can be es tab l i shed . The Kd va lues a r e l i s t e d I n T a b l e 10.

264

S O D I UM LEVOTHY ROX INE

Tab le 9

Gel F i l t r a t i o n Chromatographic Separa t i on Systems

Co I umn Mater i a I

(a ) Sephadex G-25

Sephadex G-25

Sephadex G-25

Sephadex LH-20(a)

Sephadex G-25

Sephadex G- I 5 fa)

E 1 u e n t

0.01 ,N NaOH

te r t -amy l a l coho l s a t u r a t e d w i t h

0.02 N NaOH

e t h y I a c e t a t e : methanol : 2 N NH40H ( l00:25: 10, T/v) 0.1 N NaOH 0.007 - N NaCl

0.02 N NaOH

2 - N NH40H

-

-

Compounds Ref Separated

DIT, T3, T4 81

T3, T4 82

# -

Ty, MIT, DIT, T, 80 T I , ( b ) T2, T3, T4

MIT, DIT, T3, T4 83

I-, T3, T4 84

T3, T4 85

fa)Pharmac i a F i ne Chemi ca I s, I nc. , P i scataway , NJ

fb)3- i odo thy ron i ne. I n a d d i t i o n 3 ' ,5'-d i i odo thy ron i ne and 3,3' ,5'- t r i i odothyron i ne were a I so separated.

Tab le 10

Kd Values of l odo thy ron ines and Related Compounds

(a ) Comoou nd Kd

TY 0.32 MIT 0.36 D I T 0.52 T 0.52 3- l odo thy ron i ne 0.93 T2 1.13 3 , 5 ' -d i i odothyron i ne I .95 T3 2.35 3 , 3 ' , 5 ' - t r i i odo thy ron i n e 4.40 T4 5.20

(a) l .5 mg i n 0.5 ml 0.02 - N NaOH

265

Compound Separated

T4, T3 MIT, DIT, T

T4, T3, T2 MIT, D IT

,,, T4, T3, 8 MIT, DIT

T4, T3, MIT, DIT

T4, T3, MIT, DIT

T4, T3, T2, MIT, DIT, T

T4, T3, T2

T4, T3, T2, MIT, DIT, T

T a b l e I I Gas L i q u i d Chromatographic Separa t i on Systems

D e r i v a t i v e Co I umn

N,O-d met hy

N,O-b ace ty e s t e r

met hy

methy

N,O-d

N, 0-d

p i v a l y l 0.5% SE-30 e s t e r on Gas Chrom Q

s t r i f I uoro 3.8% SE-30 methy I on D i a t o p o r t S

p i v a l y I 1 % p o l y s u l f o n e e s t e r on Gas Chrom Q

p i v a l y l 3% OV-17 on e s t e r Gas Chrom P

TMS (CJ 3% O V - l on

TMS 0.5% SE-30 on

Gas Chrom Q

DMSC-treated f d ) Chromosorb G

N,O-d ip iva ly l 5% OV-17 on methyl e s t e r Gas Chrom Q

N,O-d ip iva ly l 5% OV-17 on methyl e s t e r Gas Chrom Q

Co I umn Temperature

Program

I O o / m i n

25OoC

I30-305OC

232OC

282OC

285OC

75-250°C 8 4.6'/rnin

225-235OC @ 5O/min

285OC

D e t e c t o r

F I D ( ~ )

FID

ECD f b )

ECD

ECD

FIC

F ID

ECD

Amount I n j ec ted

ug

0.01 umol

ng

P9

50-150 ng

20 ng

< I ug

3 ng

Ref #

66

-

86

87

87

88

89

90

90

T a b l e I I ( c o n t i n u e d )

Der i v a t i ve Compound Separated

T4, T3, T2 TMS DIT, Ty

T4, T3, T TMS DIT, MIT, Ty

T4, T3, T2, T, TMS MlT DIT, Ty

T4, T3, T2, T, TMS ru 3 MIT, DIT, Ty

T4, T3, T2 TMS

T4, (f) T3, N,O-dimethyl T2, DIT methy l e s t e r

T4, T3, T2 TFAA(9) methy l e s t e r s

T4, T3, T2 N,O-d i p i v a l y I methy I e s t e r s

T4, T3, TMS

Co I umn

2% SE-33 on Gas Chrom Q

3% OV-17 on Gas Chrom Q

1 % OV-1 on C h romosor b W HP

I % OV- I on Chromosorb WHP

3% O V - 1 7 on D i a t o m i t e CQ

3% OV-1 on Gas C h r m Q

2.3% O V - l on Gas Chrom Q

2.3% OV-1 on Gas Chrom Q

1 % O V - l on Gas Chrom Q

Co I umn D e t e c t o r TemDerature

I 50-280'C FID @ 1o0/min

165'C @ 3 m i n FID t o 265' @ 10 m i n

135-255'C FID @ 5'/min

180-225OC ECD 8 25'/min

(e l FID

25OoC F I D

f e ) EC D

29OoC ECD

165-285'C FID 8 1o0/min

Amount Ref I n j e c t e d -#-

50 ng 91

3-15 ug 92

'L4 llg 93

0.3-1.5 ng 93

5-20 ng 94

1.4-2.5 ng 96

6-8 ng 96

4-16 97

Tab I e I I (continued 1

Der i va t i ve Com po u n d Separated

Col umn Co I umn Amount Ref Temper a t u r e Detector In jec ted - #

0.25-3 ng 98 T4, T3 N,O-dipivalyl 3% DEXS 1 L 300 GC 305’C ECD methyl e s t e r on Chromosorb WHP

fa’FID = Flame I o n i z a t i o n Detector

ECD = E l ec t ron Capture Detector

fC’Trimethylsl l y l d e r i v a t i v e

OD ’ ‘d’Dimethylchlorosi lane

fe’Variable, depending on which compounds are t o be separated

ff’As t h e sod i um sa It, hydrate


6.333 Gas L i q u i d Chromatography (GLC)

Since t h e iodoaminoacids a r e n o t v o l a t i l e and t h u s n o t amenable t o a gas chromatographic a n a l y s i s , t h e p r e p a r a t i o n o f s u i t a b l e s t a b l e v o l a t i l e d e r i v a t i v e s p r i o r t o a n a l y s i s i s a p r e r e q u i s i t e .

The f i r s t success fu l gas chromato- g raph ic s e p a r a t i o n of T4, T3, DIT, MIT, and Ty, as t h e i r N,O-d ip iva ly l methy l e s t e r d e r i v a t i v e s , was r e p o r t e d by S t o u f f e r , e t S ince then t h i s techn iques has been c r i t i c a l l y eva lua ted because of i t s i n h e r e n t s e n s i t i v i t y , speed o f a n a l y s i s , and a p p l i c a b i l i t y t o t h e q u a n t i t a t i o n o f t hese compounds i n b i o l o g i c a l p r e p a r a t i o n s . As it i s beyond t h e scope o f t h i s monograph t o p r o v i d e p r e c i s e d e t a i l s o f t h e gas chromatographic methods, a t a b u l a t i o n o f t h e v a r i o u s r e p o r t s i s l i s t e d i n Tab le 1 1 . I t i s recommended t h a t t h e c i t e d re fe rences be r e f e r r e d t o f o r t h e p r e p a r a t i o n o f t h e v o l a t i l e d e r i v a t i v e s , t h e use o f i n t e r n a l s tandards f o r q u a n t i t a t i v e a n a l y s i s , and f o r t h e p r e p a r a t i o n o f t h e column subs t ra tes .

apparent d i p i va I y I former i s t h e y requ prepared

From t h e i n f o r m a t i o n I n Tab le I1 i t i s h a t t h e two favored d e r i v a t i v e s a r e t h e N,O- methyl e s t e r and t h e TMS. The advantage o f t h e t h a t t h e e s t e r s have g r e a t e r s t a b i l i t y . r e a two-step syn thes i s , whereas t h e l a t t e r can be n a s i n g l e s tep b u t a r e s e n s i t i v e t o m o i s t u r e .

However,

6.334 High Performance L i q u i d Chromatography

High performance I i q u i d chromatography (HPLC) has been used by Karger, e t a l . 99 t o separa te T4, T3 and T2 i n about two minutes i n a m o b i l e phase o f bu tano l and methy lene c h l o r i d e on a s i l i c a g e l column coated w i t h a m i x t u r e o f p e r c h l o r l c a c i d and sodium p e r c h l o r a t e . On a s i m i l a r system T4, MIT and DIT separated i n l ess t h a n e i g h t minutes. T4 and T3 on a s t r o n g c a t i o n exchange (SCX) column. Both of these methods showed e x c e l l e n t s e n s i t i v i t y , i n t h a t nanogram q u a n t i t i e s c o u l d be r e a d i l y de tec ted u s i n g h i g h l y s e n s i t i v e UV d e t e c t o r s a t 254 nm.

DuPont I ns t rumen ts loo r e p o r t e d t h e s e p a r a t i o n o f

Thy rox ine and t r i i o d o t h y r o n i n e have a l s o been separated i n l e s s than 12 minutes on a Micropak

269


C-18 ( r e v e r s e phase) column u s l n g a methanol-ammonium c l t r a t e mobi l e phase.101 separa t i on on a CI&oras i I column u s i n g a mob i l e phase con- s i s t i n g o f a c e t o n i t r i le-n-butanol :0.005 M sodium p e r c h l o r a t e . A c l e a r s e p a r a t i o n was e f f e c t e d i n l e s s than 20 minutes.

Waters Associates102 repo r ted a s lm i l a r

6.4 Neutron Act i v a t i on Ana I ys i s

Neutron a c t I v a t i o n ana I ys 1 s was used by A I soszo3 t o determine t h e L - t h y r o x i n e sodium c o n t e n t i n t a b l e t s . I n t h i s procedure, t h e t a b l e t was i r r a d i a t e d fo r 5 minutes I n a neutron f l u x of = 2.5 x 1012n/cm2/sec and q u i c k l y t r a n s f e r r e d t o a coun t ing t u b e fo r measurement o f 1281. standards o f potassium i o d i d e I r r a d i a t e d fo r t h e same t i m e y i e l d e d t h e c o n t e n t o f L - t h y r o x i n e sodium. I n t e r f e r i n g nuc lea r r e a c t i o n s were n e g l l g l b l e , and t h e e r r o r was l e s s than 2%.

Comparison w i t h

Neutron a c t i v a t i on ana I ys I s was a I so used by G I obe I, e t a I . 104 t o determl ne t h e re1 a t i v e amounts o f L - t h y r o x i ne (T4) and 3 ,5 ,3 ' - t r I i o d o t h y r o n i n e (T3) i n serum. These hormones were removed from t h e p r o t e l ns by pass i ng 20 m I of serum ( a t pH I I ) t h rough a Dowex IX-2 i on exchange column i n t h e a c e t a t e form. The e l u a t e was concen t ra ted and chromatographed on a c e l l u l o s e t h i n l a y e r p l a t e t o separa te t h e T4 and T3 from t h e i n o r g a n i c i od ide . The i s o l a t e d T4 and T3 f r a c t i o n s were i r r a d i a t e d I n a thermal neutron f l u x o f 5 x 1O2n/cm2/sec u s i n g a T r i g a Mark I r e a c t o r , f o l l o w e d by i d e n t i f i c a t i o n and measurement of induced 1281 a c t i v i t y w i t h a germanium ( L i ) sol i d s t a t e d e t e c t o r . The I i m i t s o f d e t e c t i o n were 5 ng.

Schmoelzer and Muel Ier1O5 used a s l m l l a r approach b u t separated t h e T4 and T3 on a QAE Sephadex A-25 column p r i o r t o a c t i v a t i o n a n a l y s i s .

6.5 Po I a rograp h i c Ana I y s i s

P o l a r o raphy was employed by Wacholz and P f e i f e r t o assay I h y r o x i n e ~ O 6 and t o determine t h e t h y r o x i n e c o n t e n t of t h y r o l d powders and t a b I e t s .

I n t h e assay o f t h y r o x i n e , 5-50 pg of sample i n I m i of 2 N n i t r i c a c i d i s heated a t 6OoC f o r I hour. A f t e r c o o l - i n g aKd t h e a d d i t i o n o f 5 m l o f 0.06 - N sodium hydrox ide, t h e

270

SOD1 UM LEVOTH Y R OX IN E

s o l u t i o n I s deoxygenated w i t h n i t rogen. The thy rox ine content I s determined by comparison o f wave he igh t of t he sample a t E& -0.6 t o -0.7V w i t h t h a t o f standards. The method i s s u f f l c l e n t l y s e n s i t i v e t o determine the thy rox ine content o f spots i so la ted by t h i n layer chromatography.

I n t h e assay f o r thy rox ine I n t a b l e t s and powders, p r i o r e x t r a c t i o n procedures are requ i red t o remove t h e thyrox ine and o the r iod lnated amino acids. A f t e r separat ion by t h i n layer chromatography, e l u t i o n o f t h e th ryox ine spot, concentrat ion and n l t ra t i on ,106 t h e wave he igh t a t t h e prev ious ly spec i f i ed E+ i s obtained.

6.6 K i n e t i c Methods o f Analys is

The a p p l i c a t i o n of k i n e t i c measurements of t h e iod ine ca ta lyzed c e r i c arseni t e reaction108,109 has been u t i I lzed f o r t h e determinat ion o f t hy rox ine iod ine i n chromatograph I c e luates. 76~1*0,111 rapid, have h igh p rec i s ion and a re sens i t i ve , genera l l y de tec t ing less than I ng o f T4.

The repor ted methods a re

6.7 Double-losotope D i l u t i o n Analys is

A double-isotope d e r i v a t i v e assay f o r serum iodothyron I n e s l l 2 (L-T4 and L-T3) has been mod i f l e d and improved upon by Hagen, e t a1.8 thyrox ine i s labeled by formation o f an ace ty l d e r i v a t l v e l l 3 us ing t r i t i u m - l a b e l e d a c e t i c anhydride. a c t i v i t y o f t h e t r i t i a t e d d e r i v a t i v e i s known, t h e thy rox ine content of t he sample can be ca lcu la ted . Losses I n t h e complex p u r i f i c a t i o n steps are accounted f o r by t h e a d d i t i o n of a h igh s p e c i f i c a c t i v i t y 1311-labeled thyrox ine .

I n t h i s procedure, t h e unknown

As the s p e c i f i c

6.8 Determination o f Stereoisomeric P u r i t y

In format ion presented by the J o i n t Committee of t h e Pharmaceutical Society of Great B r i t a i n and t h e B r i t i s h Pharmaceutical Committee54 ind ica tes t h a t a l I D-thyroxine conta ins t r a c e amounts o f t h e L-isomer. A method f o r determining t h e amount o f L-T3, an in termediate i n t h e synthes is o f D-T4, has been reported.45 An adaptat ion o f t h i s method has been appl led t o D-T4 conta in ing less than I % o f t he L- i somer. 114

271


6.9 Equi I i b r i u m D i a l y s i s

Several i n v e s t r g a t o r s 115-117 have used equ i I i b r i urn d i a l y s i s t o determine t h e f r e e t h r y o x i n e i n serum. A c r i t i - c a l s tudy o t h i s procedure was made by Lee and P i leggT.115 The e f f e c t s of pH, i n c u b a t i o n t i m e and temperature, b u f f e r composi t ion and concen t ra t i on , p r o t e i n concen t ra t i on , and specimen d l u t i o n were s t u d i e d . Using 1311-L- thyrox ine and a reusab le p l a s t l c d i a l y s i s c e l I , r e c o v e r i e s of 92-96% were ob ta ined when t h e d i a l y s i s was r u n a g a i n s t 0.05 M phosphate pH 7.6 b u f f e r a t 37OC fo r 18 hours. r u n p r e c i s i o n s were 10.6% and 14.2%, r e s p e c t i v e l y .

bag and 1251-L-thyroxine, determined t h e f r e e t h y r o x i n e con- t e n t a f t e r d i a l y s i s a t 4OC, a g a l n s t pH 7.4 phosphate b u f f e r f o r 18-24 hours.

W i th in - run and between-

Fang and Selenkow,'" us ing t h e conven t iona l d i a l y s i s

B i r d and Ab iodun lZ7 employed e q u i l i b r i u m d i a l y s i s and ion exchange chromatography t o determine free t h y r o x i n e . I on exchange chromatography was used t o separate t h e 1251 i o d i d e from t h e 1251-L-thyroxine p r i o r t o de te rm in ing t h e f r e e t h y r o x i n e con ten t .

The l i t e r a t u r e i s r e p l e t e w i t h a s i g n i f i c a n t number of p u b l i c a t i o n s d e s c r i b i n g m o d i f i c a t i o n s o f e q u i l i b r i u m d y a l y s i s or a combinat ion of t h i s procedure w i t h o t h e r s e p a r a t i o n technlques. l18-Z22 b a s i c background t o t h e a p p l i c a t i o n of t h i s method t o t h e s tudy o f t h e t h r y o x i n e - p r o t e i n b i n d i n g phenomenon.

The c i t e d papers o f f e r a good

7 . Methods of Ana lys i s - A Comp i la t i on

The f o l l o w i n g t a b l e s (12, 13, 14 and 15) i n c l u d e t h e references t o a n a l y t i c a l procedures for t h e a n a l y s i s of chemicals, t a b l e t s , powders, and serum and t i s s u e . Severa l a d d i t i o n a l re ferences which were n o t c i t e d i n t h e s p e c i f i c s e c t i o n s a r e i nc luded i n t h e fo l lowing compi ' la t ions Two rev iew a r t i c l e s , by CahnmannlZ3 and by Ral I , e t a l .i24, a r e a l s o noted.

272


Table 12

Analysis o f Thyroxine Chemicals

Ana lys i s :

I dent I f i c a t ion

T I t r I metry : I odornet r i c N-Bromosuccinimide Coulometric Spec i f i c Ion Elect rode

Cer i rnetry

Chromatography: PC T LC

I EC G FC GLC

HP LC

Neutron A c t i v a t i o n

Po I a rog r a p h y

K i n e t i c

Spect rophotornet r y

Automation

Elect rophores is

Phase S o l u b i l i t y

Ana I ys i s

T i t r ime t ry : I odometr i c

See Reference # :

3, 3, 4, 54, 125, 126

1, 3, 37, 107 38 39, 40 43

129

45, 54, 130, 131, 132, 1 3 3 54, 55, 58, 59, 61, 62, 107, 130, 131, 134 68, 69, 77, 79, 136 120, 127, 137 86, 87, 89, 90, 91, 92, 93, 94, 97, 98, 138 101, 142, 143

103

97, 106, 107

76, 78, 111

5, 6, 7, 107, 144

68, 77, 78, 110, 136, 145, 146, 147

54

54

Table 13

Analys ls o f Thyroxine Tablets

See Reference #

1, 3, 4

273


Table 13 (continued

Chromatog rap hy : PC TLC

Pol arography

Spectrop hotometry

Ana I ys i s

148 107, 144, 149

107

107, 144, 148, 149, 150, 151, 152, 153

Table 14

Analys is of Thyroxine Powders

See Reference #

Cer i metry 61

Chroma t o g rap h y PC T LC I EC GLC

154 59, 61, 149, 155, 156 154 94, 97, 157

S pectrophotometry 149, 150, 156

Titrlmetry 1

Table 15

Analysis o f Sera and/or Tissues f o r Thyroxine and Analogs

Anal ys 1 s

Cer i met r y

Chromatography : I EC

GFC G FC GLC

Neutron Act i va t ion

See Reference #

68, 69, 71, 73, 74, 75, 76, 77, 78, 79, 120, 133, 145, 146, 158, 159, 160, 161

67, 69, 70, 72, 72, 73, 74, 76, 77, 120, 117, 136, 145, 146, 147, 159, 163, 164 85, 110, 137, 165, 166, 167 87, 90, 98, 138

104, 105

274


T a b l e 15 ( con t inued)

K i n e t i c

D ia i ys i s

Spectrometry

Au toma t i on

Radloimmunoassay

Compet i t i ve P r o t e i n B i n d i n g

Nephelometry

E I e c t r o p hores i s

F I uoromet r y

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174, 175, 176

105, 128, 170, 171, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186; 187-

188

70, 135, 172

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157. R. B l l o u s , e t a l . , J . Pharm. Sc i . , 60, 1270 (1971). 158. E. Makowetz, e t a l . , Mlcrochem. J., 10, 194 (19661.

-

280


159. K. Horn, e t a t . , Z . K I i n . Chem. u k i n . B

160. N. E. Kontax is and D. E. P i cke r inc l . J. CI 99 (1972).

ochem., 2,

n. Endocr ino l . 161. 162. 163.

.,I Metab., 18, 774 (1958). J . D. AcEnd, Biochem., g , 177 (1957). E. E. Gussakovsk i i , e t a l . , Mol. B i o l . , 7, 598 (1974 J . A. Hathaway, e t a t . , Am. J . CI i n . Path. , - 53, 635 (1970).

164. V. V . Row, e t a l . , C I i n . Chim. Acta, 31, 473 (1971). 165. C. H. G. I r v i n e , Proc. As ia Oceania C z g r . Endocr ino

I , 401 (1974). .,

166. 167. P. J . Davis and R. I . Gregerman, J. CI i n . Endoc r ino l .

168.

R; Liewendahl, e t a l . , Acta Endoc r ino l . , 67, 793 (1971) .

Metab., 30, 236 (1970). R. R. C a E l i e r i and S. H. Ingbar, Methods Enzyrnol ., - 36, 126 (1975).

169. R. A. Pages, e t a l . , Biochem., 12, 2773 (1973). 170. W. C. G r i f f i t h s , e t a l . , C I i n . Biochem., 5, 13 (1972 171. N . M. Alexander and J . F. Jenni ngs, CI in.-Chem., - 20,

553 ( 1974). 172. S . Harnada, e t a l . , J. C I i n . Endoc r ino l . Metab., 2,

( I 970). 173. I . Posner, J . Lab. C I i n . Med., 57, 314 (1961). 174. A . Bercy, J . BeIge Rad io l . , 5 7 , 1 8 5 (1974) . 175. M. I . Surks, e t a l . , J. C l i n T l n v e s t . , 52, 805 (1973) . 176. I . J . Chopra, e t a l . , J . C l i n . Endoc r ino l . Metab., - 36,

66

31 I ( 1 9 7 3 ) . 177. F. W. S p i e r t o , e t a t . , C l i n . Chim. Acta, 5 178. B. Murphy and C. Patee, J . C I i n . Endocr ino

179. €3. Murphy and C. Patee, J . C I i n . Endocr ino (1966) *

( I 964 1 * 180. R. Ekins, C I i n . Chim. Acta, 5, 453 ( 1 9 6 0 ) - 181. R. Ekins. e t a l . , C I l n . BiocFem., 2, 253 (

I., C l i n . Ch%. Acta 182. F. J . L. Crombag, e t

183. D. E. Dal rymple and R

184. S . Barbadoro, Can. J . 185. H. Sel igson and D. Se

( 1 973) .

75, 325 (1970) . -

(1972) *

, 281 (1974) . 26, 247

24, 187

* ’ - .’ -

969) *

46, 345 - D. U t i g e r , J . Lab. C l i n . Ped.,

Med. Technol . , 35, 5 (1973) . igson, C I i n . C h x . Acta, - 38, 199

186. 287. S. Nobel and F. Barnhar t , C I i n . Chem., 15, 509 (1969) . 188. G. Hocman and L. Hegedus, Endok r ino log ie , - 55, 194

S. C. Thorson, e t a l . , Acta Endocr ino l . , 64, 630 (1970) .

(1969) .

261

METHOTREXATE

Arthur R. Chamberlin, Andrew P.K. Cheung, and Peter Lim

1.

2.

3 .

4 .

5 .

6. 6.1 6.2

6.3

6.4 6.5

6.6

6.7

ARTHUR R. CHAMBERLIN etal.

Contents --

Description 1.1 Name, Formulae, Molecular Weight 1.2 Isomeric Forms 1.3 Appearance, Color, Odor

Physical Properties 2.1 Infrared Spectrum 2.2 Proton Magnetic SpeCtrUm 2.3 Carbon-13 Magnetic Spectrum 2.4 Ultraviolet spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Dissociation Constants 2.8 Solubi l i ty

Synthesis

S t a b i l i t y 4 .1 Bulk 4.2 Solution

M e t a bol i s m

Methods of A n a l y s i s Elemental Analysis Equivalent Weight Determination 6.21 Nonaqueous tit r a t ion 6.22 Complex-formation t i t r a t ion Biological Assay 6.31 Microbiological Assay 6.32 Enzymic Assay Polarographic Assay Spectrophotometric Analysis 6.51 Fluoromet r ic 6.52 Ultraviolet /vis ible Chromatography 6.61 Paper 6.62 Thin-Layer

6.64 High Speed Liquid Proton Magnetic Resonance

6.63 column

284

METHOTREXATE

7. Acknowledgment

8. References

285

ARTHUR R. CHAMBERLIN eta/.

1 . Description

1.1 Name, S t ruc tu ra l and Empirical Formulae, Molecular Weight Methotrexate is N-[4-{[(2,4-diamino-6-pteridinyl)-

met hy 1 ] met hy 1 amine } benz oy 1 ] glut ami c acid . Frequent 1 y , t he name i s abbreviated t o MTX. Methotrexate a l s o is known as ~-amino-l0-methylfolic acid and amethopterin and i s iden t i f i ed by the National Cancer I n s t i t u t e code number NSC-740.

' 6' 7 0 Y FooH

COOH

CZ0Hz2N8O5 Mol. w t . 454.46

1.2 Isomeric Forms The presence of an asymmetric carbon i n the

glutamic acid moiety provides f o r op t i ca l isomerism. Unless sp2cif ied, commercially avai lable methotrexate i s prepared from L-glutamic ac id , Recently, Lee and co-workers' prepared methotrexate s t a r t i n g with D-glutamic ac id . The D-enantioxer of methotrexate was ac t ive against L-1210 i n the mouse and had less tox ic e f f e c t s than methotrexate it s e l f , the L-enant iomer .

1.3 Appearance, Color, Odor Methotrexate i s a bright yellow-orange, odorless It generally is hydrated t o the extent of 8 t o lo$ powder.

water. I t a l s o has been prepared as the hydrochloride ( 1: 0.3) and hydrate (1: 2.5)*.

li. Private communication from D r . H.B. Wood, Jr., of the National Cancer I n s t i t u t e and M r . D.F. Worth of Parke, Davis and Co.

286

METHOTREXATE

2 . Physical Properties

2 .1 In f r a red Spectrum -- -- Recorded as a suspension i n mineral o i l , t h e

spectrum i n Figure 1 shows r e l a t i v e l y broad s t r u c t u r e s , i n d i c a t i n g t h e complexity of t h e molecule and sugges t ing t h e lack of c r y s t a l l i n i t y of t h e sample. Table I g ives t h e assignments t o t h e major bands.

TABLE 1

In f ra red Assignments f o r Methotrexate

I n t e r p r e t a t ion -I----- I R Absorption B a n d b ) ---

2.90-3.10 H20, -W2) 3 .O0-4.03

5.90-6.10

-COOH

-C-( COOH, -C-N-)

- 9 R B

6.20, 6.50-6.60 Aryl systems

6.50-6.60 Amide I1

11.9 H

H ElH H

2.2 Proton Magnetic Resonance Spzctrum (pmr) The pmr spectrum i n Figure 2 was reco rded on a

Varian A&-A spectrometer wi th t h e sample a s a s o l u t i o n i n DMSO-de. The chemical s h i f t s are i n ppm r e l a t i v e t o TMS des igna ted a s 0.00.

Table I1 g ives t h e s t r u c t u r a l assignments t o t h e resonances i n Figure 2 .

287

N a, a,

0.10 O r w V z 0.20

K 0.30 0 0.40

Q

2

-

-I

2 3 4 5 6 7 8 9 10 11 12 13 14 15 WAVELENGTH - microns

FIGURE 1 INFRARED SPECTRUM OF METHOTREXATE

0

w

L-

m

d

h

X

w a

L

U 0

f N

Lu

289

TABLE I1

pnr Assignments for Methotrexate

Assignments s i c a l S h i f t s (ppm) M u l t i p l i c i t y J ( H z ) ___-I_

H2N-2 4 H-7 H-9

HSC-11 ~ - 2 ~ , 6 ~ , 3 ' ,5'

H-8' H u

H-B9y H(COOH, HOH)

7.42 8.64 4.81 3.21

6.81,7.78 8.21 4.43

1.70-2.60 6.21

s( broad) --

s( broad) -- -- S

-- S

d 8.2 d 8 .O q 7 .o m s( broad) --

--

The values agree reasonable w e l l w i t h those reported by Pastore2 who studied the p m spectra of methotrexate i n solutions a t pH 7.5.

METHOTREXATE

2.3 - Carbon-13 magnetic spectrum (cmr)

The "cmr spectrum i n Figure 3 w a s recorded on a Varian XL-100 spectrometer with t h e sample as a so lu t ion i n IMSO. The assignments given i n Table 111 f o r Figure 3 are i n general agreement with those reported by Ewers and co-workers,' but minor d i f fe rences e x i s t .

TABLE I11

13C Assignment For Methotrexate (TMS 0 .OO ppm)

Chemical S h i f t ( ppm)

162.67 3 160.01 148.31 148.96 150.94 121.66

rv 40 (amid DMSO) 51.96

121.24 128.94 111.12 150.85 166.41

54.82 26.17

1

39.55 173.96 3 174 .ll

Assignment

c-2 c-4 C-6 c-7 c-8a C-4a c-9 c-11 c-1 ~ - 2 1 , C-6' c-31, c-5' c-4 ' c-7' c a c-B C Y U-CoOH Y -COOH

2.1; U l t r av io l e t s p e c t r u ~ Figure =the UV spectrum of methotrexate i n 0.1 N

NaOH. The longest wavelength maximum appears a t 372 nm and is ascr ibable t o t h e diaminopteridine moiety. The i n t e r - mediate wavelength maximum occurs a t 303 nm and is due primarily t o the aminobenzoyl group. The shor t wavelength maximum is a t 258 nm and is a t t r i b u t e d t o both chromophores. In 0.1 N HC1 methotrexate expsriences a hypsochromic s h i f t r e s u l t i n g i n a UV spzctrum (Figure 5 ) t h a t e x h i b i t s

---

UV spec t ra were recorded on a Cary Model. 14 and the molar absorpt i v i t i e s a re based on anhydrous methotrexate .

*

291

i

3

W

I- a

X

W

a

I- 0 r

z 0

H

9

a

I- 0

W

n

v)

L LL

E' (? F

u

m

W

a 3

U

(3

292

I 1

I 1

I I

I 1

I 0

In d

0 0

d

&I t; a

ma

5

0

z

Z

W

0 0 0

0

In

hl

I

P 0 I- z w

2 2 w

I- a

X

a

I- 0

I

I- w

z 0

H 3

a

I- u

w L

v)

w

LL

t; 0

a

3

J

>

E

3

t

w a 3

12 LL

293

ARTHUR R. CHAMBERLIN eta:.

250 300 350 400 NANOMETERS

FIGURE 5

294

METHOTREXATE

maxima a t 307 and 243 nm. Table I V and i n genera l are i n agreement wi th those repor ted by Seeger and co-workers4,

The W d a t a are summarized i n

TABLE I V

Absorption Spac t ra of Methotrexate

Solvent

0.1 N HC1 0 .1 M ~ ~ 6 . 7 T r i s bu f fe r

0.1 N NaOH

-I

2.5 Mass Spectrum Methotrexat e it self

mass spectrum because of does not y i e l d a s a t i s f a c t o r y non-volat i l i t y . HoNever, t rea tment

of methotrexate wi th a TMS reagent a f f o r d s a mixture of tri-, tetra-, and psnta-TMS d e r i v a t i v e s t h a t does g ive use fu l mass spzctral d a t a . That s eve ra l TMS-containing d e r i v a t i v e s are formed is not su rp r i s ing , cons ider ing t h e number and v a r i e t y of func t iona l groups involved.

The mass f o r t h e tri-, summarized as

RZ-NH c

s p s c t r a l fragmentat ion p a t t e r n obtained tetra-, and psnta-TMS d e r i v a t i v e s is follows:

I

I

' COOTMS I

R1=R,=TMS: m / e 319 I 1 Rl,R2=1TMS, 1H; m/e 247

I Rl=R2=TMS m / e 452 Rl,R,=lTMS, 1 H m / e 380

I M1bR1=R2=R3=TMS m / e 814 M-, R1=R2=TMS, R,=H m / e 742 Mf-R1,R2=1TMS, l H , R3=H m / e 670

M-CH, m / e 727 M-HOTMS m / e 652 M-HOTMS-CH, m/e 637

295

ARTHUR R . CHAMBERLIN et al.

2.6 Optical Rotation 46

21 = 20.4 2 0.6' (c 1, N/10 NaOH) 589 546 = 26.9 0.8'

2.7 Dissociation Constants ~---- Neither t he a c i d i c nor the basic pKa f o r methotrex-

ate has been reported. reported t h a t t h e basic pKa values f o r 2,bdiaminopteridine w e r e < 0.5 and 5.32. Kallen and Jencks' reported the a c i d i c pKa values f o r p-aminobenzoylglutamic acid a s 4.83 and 3.76. By spectrometry, w e found a pKa of 5.60 2 0.03, which i s assignable t o the diaminopteridinyl moiety.

However, Albert and co-workers5

2.8 Solubi l i ty7 ___-- Methotrexate is prac t i ca l ly insoluble i n water,

alcohol, chloroform, and e t h e r . I t is f r e e l y soluble i n d i l u t e solut ions of a lkal ine and carbonates; it i s s l i g h t l y soluble i n d i l u t e hydrochloric acid (1 i n 2 ) .

3. Synthesis

Seeger and co-workers' is as follows: The first reported synthesis of methotrexate, t ha t by

CH2Br I I CHO

+ CHBr +H-N C-NH-

COOH

- ------- - --- +C These s p e c i f i c r o t a t i o n values a r e based on anhydrous

methotrexate.

296

METHOTREXATE

This r e a c t i o n o f t e n is r e f e r r e d t o as t h e Waller reac t ion , because Waller i n i t i a l l y prepared p te roylg lu tamic ac id by an analogous method.

Theore t ica l ly , the s u b s t i t u t i o n on t h e pyrazine r i n g can be at t h e C-6 o r t h e C-7 pos i t i on . is based on t h e p t e r i n e carboxyl ic a c i d obtained from an a l k a l i n e p3rmanganat.e ox ida t ion of methotrexate . Of t h e two a c i d s poss ib le , on ly t h e C-6 has been found. t i n c t i o n between t h e two poss ib le a c i d s has been based on comparative paper chromatography, a l though pmr or c m r a l s o could be used.

Proof of s t r u c t u r e

D i s -

Because t h e Waller r e a c t i o n gene ra l ly y i e l d s impure products t h a t are very d i f f i c u l t t o p i r i f y, many r e sea rche r s have attempted t o improve t h e syn thes i s of methotrexate . Among t h e more successfu l a t tempts are those by Taylor’ and by Pip2r and Montgomery”. are on t l ined as fol lows:

The r e spec t ive methods

Taylor

NC N CHZC1

0

0

0 I DMF

R=N-( glutamyl)

297

ARTHUR R. CHAMEERLIN eta/.

Piper and Montgomery

NH2 NH2

PMABG = N-( p-methylaminobenzoy1)glutamic acid DMAC = N,N-dimethylacetamide

These improved procedures not only y i e l d products t h a t are easier t o purify, but the subs t i t u t ion on the pyrazine is unambiguous.


4 .1 Bulk

----

When stored i n a cappsd, brown b o t t l e a t room temperature, samplings removed over a twelve-month period yielded indis t inguishable UV and papsr chromatographic d a t a , These r e s u l t s indicate that methotrexate is stable f o r a t least one year under these conditions.

4.2 Solution As d i l u t e solut ions (0 .5 mg and 0.05 mg/ml) i n pH

7 .O aqueous sodium bicarbonate maintained a t room tempera- t u r e i n darkness and under laboratory illumination, a l iquo t s removed over a 24-hour period y ie lded iden t i ca l papergrams, within experimental e r r o r s . On th i s basis, these solut ions were considered stable under these conditions.

298

METHOTREXATE

Under strongly a c i d i c aqueous conditions, t he amide is

Under highly alkaline subject t o hydrolysis, yielding N10-methyl-4-amino-4- deoxypteroic acid and glutarnic acid. aqueous conditions, e s p x i a l l y a t elevated temperatures, t he pr incipal decomposition products a re N1'-methylfolic acid, N1'-methylpteroic acid, and glutamic acid--all as the carboxylate ions.

Photodecompwition of c e r t a i n p t e r ines is well documented .I1 photochemistry of methotrexate has been found i n the l i t e r a t u r e .

However, no s p e c i f i c report on the

5. Metabolism

administered is excreted unchanged, 'la indicat ing t h a t l i t t l e metabolism of t h i s drug occurs.

In man, a very l a rge portion of the methotrexate

Johns and Loo" reported t h a t methotrexate is metabolized rapidly by rabbi t l i v e r aldehyde oxidase, and the metabolite w a s i den t i f i ed as 4-amino-k-deoxy-7- hydroxy-N1O-methylpteroylglut amic acid (7-hydroxy-MTX) .

Johns and Valerino13 have observed tha t , when methotrexate i s incubated with cecal contents from t h e mouse i n v i t ro , ~-amino-~-deoxy-N1O-methylpteroic acid is produced. Levy and Goldman'* have demonstrated t h a t t h i s metabolite can be produced from methotrexate by ac t ion of carboxypiptidases from s t r a i n s of Psudoxona_t.

6. Methods of Analysis - ---

6.1 Elemental Analysis ---- Table I V presents t h e r e s u l t s from a representative

elemental analysis of methotrexate ( reference standard) .

* Adjusted

TABLE V

Elemental Analysis of Methotrexate

Element -Theory -- $ Found 9

C 52.85 52.72 H 4.88 4.85 N 24.66 24.51

f o r the found water

299

ARTHUR R . CHAMEERLIN eta / .

6.2 Equivalent Weight Determinations __l_l --I_---_

6.21 Nonaqueous T i t r a t i o n

D e Carnevale e t a1.l’ descr ibed an equiva len t weight determinat ion based on a sodium methoxide t i t r a t i o n i n pyr id ine t o an azo-v io le t end p o i n t . Because t h e bas i s of t h e t i t r a t i o n is an ac id lbase n e u t r a l i z a t i o n , it lacks s p e c i f i c i t y . Consequently, t h e method has not been employed widely as an assay f o r methotrexate .

6.22 Complex-Formation T i t r a t i o n - ----- Guerel lo has described’’ a second t i t ra t ion

by which equiva len t weights of methotrexate samples can be obtain??. The aethod i s basad on the complexation between t h e C a and t h e glutamyl moiety and thereby a f f o r d s higher s p e c i f i c i t y than an ac id lbase n e u t r a l i z a t i o n . Because glutamic a c i d conta in ing impur i t i e s a r e commonly found i n methotrexate samples, r e s u l t s from t h i s t i t r a t i o n must be i n t e r p r e t e d c a r e f u l l y .

6.3 Bio logica l Assay ---------

6.31 Microbiological Assay Severa l microbio logica l a s says are descr ibed

i n t h e 1 i t e r a t ~ r e . l ~ very time consuming and thus have l i t t l e use fu lness f o r rou t ine ana lyses .

A l l are r e l a t i v e l y nonspec i f ic and

6.32 Enzymic Assay - - - - ~ - Werkheiser and co-workers” have developed

an enzymatic a s say f o r methotrexate us ing f o l i c acid reductase . Methotrexate binds very tenac ious ly and s t o i c h i o n e t r i c a l l y t o t h i s enzyme and is determined co lorometr ica l ly by t i t r a t i o n of t h e drug w i t h t h e enzyme.

6.4 Polarographic Assay

Asahi” has repor ted polarographic reduct ion of methotrexate . H e attribJtes t h e f i r s t wave as being the reduct ion t o dihydromethotrexate, the second wave a s being t h e r educ t ive cleavage of t h e CH2-N bond, and t h e t h i r d wave a s being t h e reduct ion t o 2,4, -diamino-6-methyl-5,6,7, 8-tetrahydropteridine ,

300

METHOTREXATE

6.5 Spectrophotometr ic Analys is ------- 6.51 Fluorometr ic Analysis ---

Fluorometr ic methods2' have been used widely i n t h e a n a l y s i s of methotrexate . The methods a r e based on a n ox ida t ive t ransformat ion of methotrexate t o the presumed p t e r i n e carboxyl ic a c i d which f l u o r e s c e s in t ense ly . Because many of t h e impur i t i e s commonly found i n methotrexate a l s o undergo t h e same reac t ion , t h e s e methods lack s p c i f i c i t y un le s s t h e ox ida t ion is preceded by a sepa ra t ion scheme i n which methotrexate is i s o l a t e d .

6 .52 -- Ul t rav io l e t /V i s ib l e Analysis Because commercial methotrexate sanples a r e

impure, and because t h e organic impu- i t i e s have W absorp- t i o n c h a r a c t e r i s t i c s t h a t a r e similar t o those of methotrexate, d i r e c t uv /v is spsc t ropho tone t r i c a n a l y s i s is e n t i r e l y t o o nonspec i f ic t o be of any value i n t h e q u a n t i t a t i v e a n a l y s i s of methotrexate . P r a c t i c a l l y a l l t h e contaminants l i k e l y t o be present i n a sample of met hot r exa t e- -N1' -me thy1 p t e r o i c acid, and so f o r t h - - w m l d i n t e r f e r e wi th a direct W o r v i s i b l e method.

6.61 Paper -- Nichol and co-workers21 have repor ted t h e

sepa ra t ions of methotrexate from related compounds on Whatman No. 1 wi th pH 7.0, 0.1 M sodium phosphate and wi th pH 5.0, 0.1 M sodium acetate. I n our labora tory , w e have used 0.5% NaHC03 and 0.5% Na2C03 w i th t h e sane papzr.

Balazs and co-workers22 repor ted a rap id assay f o r methotrexate based on ps.per chromatographic sepa ra t ion followed by W measurement of t h e i s o l a t e d methotrexate component. The l i m i t a t i o n s of t h i s assay a r e t h e accuracy of t h e re ference UV va lue and the recovery of methotrexate from t h e papergram. The molar a b s o r p t i v i t y a t 303 nm f o r methotrexate i n N / 1 0 NaOH reported by Balazs and co-workers i s i n e r r o r ; it should be 24,830.

The assay procedure f o r methotrexate cited i n USP XVIII is s i m i l a r t o t h e assay repor ted by Balazs et a1 except t h a t t h e re ference s tandard i s USP methotrexate

301

ARTHUR R. CHAMEERLIN etal.

which itself is impure.

6.62 Thin-layer Copenhaver and O'Brienz3 have used ion-

exchange thin-layer chromatography t o separa te a number of f o l i c ac id analogs including methotrexate . The cation- exchange r e s i n was AG50W-X4, and the developing solvent was 154 Na2HF04'12 H20, p H 8.5 buf fer containing 0.1 M mercaptoethanol .

6.63 Column Heinrich and c o - ~ o r k e r s ~ ~ reported the

use of Dowex 1-chloride w i t h very d i l u t e HC1 or N@ t o separate f o l i c acid and r e l a t ed compounds. Noble described a pu r i f i ca t ion procedure based on the use of a Dowex 1-acetate with pH 3.2, M ace t a t e buf fer . cited the use of DEAE ce l lu lose and pH 8 phosphate buf fer gradient t o separa te fo l i c acid analogs. Gal le l l i and YokoyamaZ7 developed a methotrexate assay procedure e n t a i l i n g a DEAE ce l lu lose separa t ion followed by a spectrophotometric measurement of the i so l a t ed component.

Oliverio2'

The use of DEAE cellulose t o separate fol ic ac id analogs is w e l l e s t ab l i shed . The pr inc ipa l l imi t a t ions of t h i s method are the t i m e required t o pack t h e column and the t i m e needed t o develop the chromatogram.

6.64 High Speed Liquid - In our work w i t h other f o l i c ac id antagon-

ists, cat ion exchange high speed l i q u i d chromatography (HSLC) has been very e f f e c t i v e i n separating c lose ly r e l a t ed p t e r id ines . Taking advantage of t h i s s e l e c t i v i t y , we have developed a HSLC method of ana lys i s for methotrexate that is specific, f a s t , and s e n s i t i v e . We use it t o assay bu lk and formulated methotrexate. The procedure r equ i r e s a reference methotrexate sample of known purity, which is used a s an ex te rna l reference o r i n conjunction wi th a n i n t e r n a l reference that, i n our laboratory, has been 2-amino-bmethylpyridine . The column, l m x 3mm 0 .D. Vydac Cation Exchange Packing, is held a t 55' during the developnent w i t h pH 4.30, 0 .1 M KH2P04 a t a f l o w rate of 1.0 ml/min. a t 254 nm. so lu t ions a s d i lu t ed a s 1 pg methotrexate/ml can be in j ec t ed

The e f f luen t is monitored by a UV-detector set With the de t ec to r s e t a t i t s highest s e n s i t i v i t y ,

302

METHOTREXATE

d i r e c t l y and de tec ted .

A second procedure employing a l m x 3mm O.D. column packed with reverse-phase phenyl and a mobile phase of 5% MeOH i n 0.05 M KH2P04, pH 7.0, buffer a l s o has been developed and found use fu l , The reverse-phase system is less sens i t i ve t o minor pH va r i a t ions r e s u l t i n g from sample impur i t ies . For t h i s reason, t h i s column produces high prec is ion more r ead i ly .

6.7 Proton Magnetic Resonance Assay K t h o t r e x a t e has been assayed by quan t i t a t ive

Accurately weighed pmr on a Varian A-&A spectrometer. portions of methotrexate and of an i n t e r n a l standard (2,4-dimethoxy-5-methylpyrimidine) were dissolved i n DMSO, and t h e spectrum was recorded. The psrcentage of anhydrous methotrexate i n t h e sample then was ca lcu la ted according t o t h e formula

$ MTX = Wr x A s x 454.4 x P - - W s A r 154.2

where W r = weight of i n t e rna l standard used

W s = weight of methotrexate sample

A r = in tegra ted a rea of i n t e r n a l standard He s ing le t (8.02 6 )

As = in tegra ted a rea of methotrexate H7 proton (8.64 6 )

154.2 = molecular weight of i n t e r n a l standard

454.4 = molecular weight of anhydrous MTX

P = pur i ty of t h e i n t e r n a l standard

Although the pmr method may lack s e n s i t i v i t y and possibly s p e c i f i c i t y i n complex mixtures, it i s one method of assay t h a t does not requi re a methotrexate reference sample of known pur i ty . Thus, it may be usefu l i n cases i n which no reference material is ava i l ab le .

303


7 , Acknowledgments ----

Supported by Cont rac t N01-CM-33723 from t h e d i v i s i o n of Cancer Treatment, Nat iona l Cancer I n s t i t u t e , Nat iona l I n s t i t u t e s of Health, Department of Health, Education, and Welfare. The opin ions expressed are t h o s e of t h e a u t h o r s and not n e c e s s a r i l y t h o s e of t h e Nat iona l Cancer I n s t i t u t e .

The a u t h o r s wish t o acknowledge t h e t e c h n i c a l a s s i s t a n c e rendered by Ms. Leslie Pont, M s . Barbara Senuta, Ms. Florence Yoshikawa, M r . Martin S t r u b l e and I r . John Jee of S tanford Research I n s t i t u t e .

304

METHOTREXATE

8. Reference?

1.

2. 3.

4 .

5.

6 .

7.

8.

9 .

10 *

11.

l l a . 12.

W.W. Lee, A.P. Martinez, and L . Goodman, J . Med. Chem. lJ, 326 (1974) . E . J . Pastore, Ann. N.Y. Acad. S c i . -9 185 43 (1971). U . Ewers, H. Gunther, and L . Jaenicke, Chem. B e r .

D.R. Seeger, D.B. Coslllich, J . M . Smith, and M.E. Hultquist, J . Am. Chein. SOC. 2, 1753 (1943). A . A l b e r t , D.J . Brawn, and G . Chessman, J . Chem. SOC., 4219 (1952). R.G. Kallen and W.P. Jencks, J . Biol. Chem. 24, 531.~5 (1956) . The United S ta tes Pharlnacopzia, The XVIII Revision (1970), p. 418. D . R . Seeger, J .M. Smith, and M.E. Hulquist, J . Am. Chem. S O C . 69, 2567 (1947) . E .C. Taylor,'Chernistry and Biology of Pteridines, Proceedings of the Fourth Internat ional SympDsium on Pteridines, Toba, 19s9, Internat ional Academic Print ing Co., Ltd., Tokyo, 1970, p. 79. J . R . Pipzr and J . A . Montgoaery, J . H e t . Chem. 11,

R.L. Blakely, The Biochemistry of Fol ic Acid and Related Pter idine s , Nort h-Holland Pub1 ishing Compnny, London, 1959, p. 77. Ibid p.495. D.G. Johns and T.L. Loo, J. Pharm. Sc i . 56, 356

- 106, 3351 (1973) *

I

273 (1974) *

- (1957) *

13. D.G. Johns and D.M. Valerino, Ann. N.Y. Acad. S c i . - 186, 384 (1971). C.C. Levy and P. Goldman, J . Biol. Chem. -- 242, 2933 14. (1967) *

15 . R.C.D. D e Carnevale, J . Dobrecky, and L.O. Guerello,

16.

17. J . H . Burchenal, G.B. Waring, R.R. Ell ison, and

Rev. F a m . (Buenos Aires) - 113, 15 (1971). L.O. Guerello, Rev. Asoc. Bioquim. Argent. - 34, 33 (1969) *

H.D. Reil ly, Proc. SOC. Exp. Biol. N.Y. i'8, 603 ( 1951) ; A . Z . Smolyanskaya and N.N. Agadzhanova, vop. Onkol . 13, 58 (1957) , Chem. Abst ., 46, 26036; D.E. Hunt anrR.F . P i t t i l l o , Cancer R e s . - 28, 1095 (1958) .

305


18.

19.

20.

21.

22.

23.

24.

25. 26. 27.

W.C. Werkheiser, S . F . Zakrzewski, and C.A. Nichol, J . Plannacol . Exp. Therap. m, 162 (19.52) . Y, Asahi, Yakugaku Zasshi E, 1570 (1959), Chea. Abst . 54, 10593d. S.G. CcGkrabarti and I . Bernstein, C l i n . Chem. 9, 1157 (1959); S . F . Zakrzewski and C.A. Nichol, J . Biol Chem. 205, 364 (1953); M.V. Freeman, J . Pharmacol. E X ~ . Therap. - 120, -- 1, (1957) and 121,

C.A. Nichol, S . F . Zakrewski, and A.D. Welch, Proc. So. Exp. Biol. Med. 5, 272 (1953); S.F. Zakrrewski and C.A. Nichol, J . Biol. Chea. 205, 352 (1953). M.K. Balazs, C.A. Anderson, and K-Lim, J . Pharm. sci. z, 2002 (1968). J.H. Copmhaver and K.L. O'Brien, Anal. Biochem.

M.K. Heinrich, V.C. Dewey, and G.W. Kidder, J . Chromatog. - 2, 296 (1959). E.P. Noble, Biochem. Prep. 8, 20 (1961). V.T. Oliverio, Anal. Chem. 3, 263, (1951). J . F . G a l l e l l i and G. Yokoyama, J . Pharm. Sc i . 55,

- -- 154 (1958) *

s, 454 (1969) '

357 (1957)

306

METHYCLOTHIAZLDE

James A . Raihle

JAMES A. RAIHLE

Contents

1. Description 1.1 Nomenclature

1.11 Chemical Names 1.12 Generic Name 1.13 Trade Names

1.21 Empirical 1.22 Structural

1.3 Molecular Weight 1.4 Elemental Composition 1.5 General

1.2 Formulae

2. Physical Properties 2.1 Infrared Spectrum 2.2 Raman Spectrum 2.3 Nuclear Magnetic Resonance Spectrum 2.4 Ultraviolet Spectrum 2.5 Mass Spectrum 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Dissociation Constant 2.9 Solubility 2.10 Crystal Properties

3. Synthesis

4. Stability-Degradation


6. Methods of Analysis 6.1 Titrimetric Methods

6.11 Argentimetric Titration 6.12 Potentiometric Titration

6.21 Column Chromatography 6.22 Paper and Thin-Layer Chromatography


6.3 Spectrophotometric Methods 6.4 Polarographic Method

7. References

308

METHYCLOTH lAZl DE

Methyclothiazide

1. Description

1.1 Nomenclature

1.11 Chemical Name

Methyclothiazide is 6-chloro-3-(chloromethyl) -3,4-dihydro-2-methy1-2H-1,2,4-benzothiadiazine-7-su1fon- amide 1,l-dioxide. (1) It is also known as 6-chloro-3- ch1oromethy1-3,4-dihydro-2-methy1-7-su1famoy1-1,2,4-benzothiadiazine lyl-dioxide; 6-chloro-3-chloromethyl-2-methyl- 7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine 1,l-dioxide (2) and by many slight variations of the particular nomenclature. The GAS Registry No. is [135-07-91.

1.12 Generic Name

Methyclothiazide

1.13 Trade Names

Enduro@ and Aquatensen@

1.2 Formulae

1.21 Empirical

‘gHl 1C12N304S 2

1.22 Structural

309

JAMES A. RAIHLE

1.3 Molecular Weight

360.23

1.4 Elemental Composition

C - 30.00; H - 3.08; C1 - 19.68; N - 11.66; 0 - 17.77; S - 17.80.

1.5 General

Methyclothiazide occurs as a white to practically white crystalline powder which is principally odorless.

2. Physi'cal Properties

2.1 Infrared Spectrum (IR)

The infrared spectrum of methyclothiazide (NF Ref- erence Standard, Lot No. 69196) is presented in Figure 1. The spectrum of a KBr pellet is taken on a Perkin-Elmer Model 521 Spectrophotometer. The assignments for the characteristic bands in the IR spectrum are listed in Table I. (3)

Table I

Characteristic Bands in the IR Spectrum of Methyclothiazide

Wavelength (em-l) Characteristic of

3270 and 3360 NH stretching vibration of sulfonamide group

1595 and 1502 C = C stretch of aromatics

1155 and 1330 (doublet)

S = 0 stretching vibrations of sulfonamide groups

This spectrum is consistent with that published by Fazzari and co-workers. (4)

31 0

2.5 WAVELENGTH (MICRONS)

3 4 5 6 7 8 9 10 12 15

FIGURE 1 - INFRARED SPECTRUM 0 F METH Y CLOTH IAZIDE

JAMES A. RAIHLE

2.2 Raman Spectrum

The raman spectrum of the methyclothiazide reference standard was determined in the solid phase on a Cary Model 83 Spectrophotometer. The assignments of the characteristic bands as shown in Figure 2 are listed in Table 11. (3)

Table I1

Characteristic Bands in the Raman Spectrum of Methyclothiazide

Wavelength (cm-l) Characteristic of

3270 and 3363 NH stretching vibration of sulfonamide group

1600 C = C stretch of aromatics

1160 s = 0 stretching vibrations of sulfonamide groups

2.3 Nuclear Magnetic Resonance Spectrum ("El)

The 60 MHz NMR spectrum of methyclothiazide is presented in Figure 3. The spectrum was determined in deuterated acetone (d6) on a Varian T-60 Spectrometer. Spectral assignments are given in Table 111. (5)

31 2

AlIS

N31N

I 0

0

0

- 0

0

0

v

el 0

0

Qo

9

31 3

FIGURE 3 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF METHYCLOTHIAZIDE

d

r

8.0 7.0 6.0 5.0 4.0 3.0 PPM (6)

2.0 1 .o

METHYCLOTH IAZIDE

Table 111

NMR Assignments for Methyclothiazide

Number of Chemical Assignment Protons Shift (ppm) Multiplicity

Aromatic proton 1 8.23 S at carbon 8

Aromatic proton 1 7.22 S at carbon 5

Exchangeable 3 6.87 M(b) protons: NH, NH2

Methyne proton at carbon 3

1 5.53 T

Methylene protons 2 4.07 D of chloromethyl group at carbon 3

Methyl protons 3 2.77 S of 2-methyl group

2.4 Ultraviolet Spectrum (W)

The UV spectrum of methyclothiazide prepared as a 1 in 100,000 solution in methanol is shown in Figure 4 . The spectrum exhibits three maxima and two minima characteristic of substituted benzothiadiazines. The maxima are at 226 nm (Em = 39,300), 267 nm (Em = 21,250) and ca 311 nm (Em = 3,300). The spectrum is consistent with previously published reports by Furman ( 6 ) and Fazzari, et. al. (4)

Minima were observed at 240 nm and 290 nm.

2.5 Mass Spectrum

The mass spectrum shown in Figure 5 was obtained using an Associated Electrical Industries Model 902 Mass Spectrometer with an ionizing energy of 50 eV and a temperature of 150°C. Methyclothiazide yields a spectrum with a base peak at m/e 359. Subsequent fragments, Table IV,

31 5

JAMES A. RAIHLE

FIGURE 4 - ULTRAVIOLET SPECTRUM OF METHYCLOTHIAZIDE

Ly

v 2

m = VI m

a

0

a

200 250 300 3 5 0

WAVE LENGTH (nm)

31 6

31 7

FIGURE 5, MASS 5PEClRUM OF MElHYCLOlHIAZIDE

I ' I ' L ' I ' I I,, .I, I .#I ,II ; I , , ,

I

250 300 350

JAMES A. RAIHLE

reflect the loss of the chloromethyl and sulfonamide groups as well as the fragmentation of the ring system. (7)

Table IV

High Resolution Mass Spectrum of Methyclothiazide

Composition Mass Relative Error Found Intensity (mu) - c L! ! 0 s c135

358.9565 3.06 -0.32 9 1 1 3 4 2 2

309.9711 100.00 -1.21 8 9 3 4 2 1

291.9629 0.56 1.18 8 7 3 3 2 1

229.9914 6.02 -0.26 8 7 2 2 1 1

166.0280 3.52 -1.78 8 7 2 0 0 1

131.0615 5.42 0.57 8 7 2 0 0 0

123.9952 7.89 -0.22 6 3 1 0 0 1

96.9847 5.95 0.16 5 2 0 0 0 1

90.0113 6.77 0.25 3 5 1 0 0 1

42.0345 63.94 0.13 2 4 1 0 0 0

2.6 Melting Range

Methyclothiazide melts with rapid decomposition between 225°C and 227°C. Slight discoloration of the solid may be observed at about 215°C.

2.7 Differential Thermal Analysis

The thermogram depicted in Figure 6 shows a large endothermic melt between 224°C and 231°C. The thermogram shows a subsequent exothermic response confirming the visual observation of rapid decomposition.

31 8

FIGURE 6 - DIFFERENTIAL THERMAL ANALYSIS CURVE OF METHYCLOTHIAZIDE

0 20 40 40 80 100 n o 140 160 in0 200 220 240 1, OC (CORRECTED FOR CHROME1 ALVMEL THERMOCOUPLES)

JAMES A. RAIHLE

2.8 Dissociation Constant

The pKa of methyclothiazide was determined by the titrimetric method by extrapolation of data from acetone- water mixed solvents to 100% water. The pKa is 9.4 (proton lost).

2.9 Solubility

The following solubilities have been determined for methyclothiazide at room temperature.

National Formulary Solvent Solubility Descriptive Term( 1)

Water Chloroform Benzene n-Butanol

E thano 1 Acetonitrile Methanol Acetone Pyridine

PEG- 600

0.06 mg/ml 0.03 mg/ml

< 1 mg/ml 1 mg/ml 1 mg/ml 11 mg/ml 5 mg/ml

> 10 mg/ml N - 200 mg/ml - - 200 mg/ml

Very Slightly Soluble Very Slightly Soluble Very Slightly Soluble ---- ---- Slightly Soluble

Sparingly Soluble Freely Soluble Freely Soluble

----

2.10 Crystal Properties

The X-ray powder diffraction pattern of methyclo-

An thiazide was determined by visual observation of a film obtained with a 143.2 mm Debye-Scherrer Powder Camera. Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA with nicker filtered copper radiation; ), = 1.5418 was employed. A listing of d-spacings and intensities is presented in Table V. (8)

320

METHYCLOTH IAZIDE

Table V

X-Ray Powder Diffraction Pattern d-Spacings and Intensities

dA - - dA -1 111

9.8 7.75 7.5 7.2 6.25 5.75 5.3 5.1 4.85 4.56 4.42 4.3 4.11 4.00 3.90 3.75B 3.6 3.52 3.44 3.32 3.30 3.12 3.07 3.00

5 50 30 5 20 50 5 10 100 80 20 15 20 2 2 20 3 20 5 5 8 5 8 5

2.95 2.90 2.81 2.72 2.68 2.62 2.51 2.42 2.27 2.24 2.15 2.11 1.99 1.88 1.85 1.81 1.77 1.75 1.73 1.69 1.64 1.5 B 1.35B 1.3 B

I/I1

5 LO 60 3 5 15 10 3 15 10 3 5 8 3 5 8 2 3 3 2 4 4 3 3

3. Synthesis

Methyclothiazide is synthesized by the reaction sequence shown in Figure 7. Sprague (9) described the reaction of 4-amino-6-chloro-1,3-benzenedisulfonamide with urea to form the 3-keto derivative. Close, et. al. (10) have preferentially alkylated the more acidic cyclic sulfonamide with methyl iodide to form 6-chloro-2-methyl-3- oxo-7-sulfamyl-3,4-dihydro-l,2,4-benzothiadiazine 1,l- dioxide. This intermediate is readily ring opened by alkaline hydrolysis and then re-cyclized with chloro- acetaldehyde to form the desired product.

321

0

N

I z

N

I

z

0=

0 z N

0

I

0 I

z

N

I6

6

-6

*

%

I

z

0

I

0

t

N

I

2

322

Figure 7

Synthesis of Methyclothiazide

CH31 0 II

+HpNCNHz +

METHYCLOTH IAZIDE

4 . Stability-Degradation

Methyclothiazide is stable in the solid state and under ordinary ambient conditions. It is rapidly decomposed in boiling acidic solutions to 2-methylsulfamyl-4-sulfarnyl-5- chloroaniline. In alkaline solution it rapidly loses one chlorine atom to form the 3-hydroxymethyl analog. This product has been shown to degrade further under severe conditions, however none of the alkaline degradation products contain primary aromatic amine centers. Solutions buffered at pH 4.0 show 22% hydrolysis after 7 days at 60"C, 10% after 28 days at 40"C, but only 2% after 28 days at 25°C. Solutions buffered at pH 6.0 gave 1.6% hydrolysis after 7 days at 6OoC, and less than 1% after 28 days at 40°C or 25°C.


No report of metabolic products related to methyclothiazide has been published.


6.1 Titrimetric Methods

6.11 Argentimetric Titration

The compendia1 procedure for the purity determination of the drug substance is based upon the argentimetric titration of the chloride liberated after reflux in methanolic potassium hydroxide. (1) The equivalent weight is 360.23 since only the chlorine from the 3-chloromethyl group is liberated during reflux. specific since this group is added during the final step of the synthesis and a limit of 0.02% free chloride is imposed on the drug substance.

The method is

6.12 Potentiometric Titration

Methyclothiazide can be titrated as an acid using either tetra-butylamonium hydroxide in chlorobenzene (11) or potassium hydroxide in isopropanol (12) as the titrant. The solvents are pyridine and acetone, respectively. Methyclothiazide consumes two equivalents of base per

323

JAMES A. RAIHLE

mole of drug substance. The first equivalent is from the neutralization of the free sulfamyl group. The exact lo- cation for the reaction of the second equivalent has not been determined, however, it may result from rapid hydrolysis of the 3-chloromethyl function.



Fazzari (13) has published a collaborative study on a column chromatographic method for the analysis of methyclothiazide from tablets. The drug is eluted from a 0.1 NaHC03-Celite column with chloroform and measured directly at 267 nm by W spectrophotometry. The precision of the method was 99.8 4 1.64% on a commercial preparation of 2.5 mg tablets.

6.22 Paper and Thin-Layer Chromatography

Paper chromatography has been applied by Pilsbury and Jackson (14) for the rapid detection and identification of thiazide diuretics in tablets, gastric fluid, and urine. Identification is accomplished by ascending reverse phase chromatography using tributyn treated paper and developing for 20 minutes at 90°C with a phosphate buffer (pH 7.4). The thiazides are located by ultraviolet light (254 nm) and confirmed by the red color given by alkaline sodium 1,2-naphthaquinone-4-sulfonate spray reagent. Paper chromatography can also monitor the extent of manufacturing by-products. Ascending chromatography using butanol saturated with 3% ammonium hydroxide and descending chromatography using butano1:acetic acid:water (50:15:60) have been employed. Visualization is by ultraviolet light.

Thin-layer chromatography using the system ch1oroform:methanol: ammonium hydroxide (170: 30: 2) on 0.25 mm silica gel GF254 plates and ultraviolet detection rapidly isolates and identifies the common manufacturing intermediate s ,

324

METHYCLOTH IAZIDE

6.3 Spectrophotometric Assays

Methyclothiazide can be determined after acid hydrolysis to 2-methylsulfamyl-4-sulfa~l-5-chloroaniline by a modified Bratton-Marshall procedure. This procedure without prior acid hydrolysis also monitors diazotizable substances in the drug substance. (1) Ultraviolet absorption at 267 nm is seldom directly employed since the intermediates and degradation products have similar absorption spectra. The ultraviolet procedure has been utilized after prior separation by chromatography (13) or for non-specific content uniformity measurements. (1)

6.4 Polarographic Method

The current compendia1 assay for methyclothiazide tablets is a polarographic assay in an aqueous system containing 6% v/v dimethylformamide and 0 . 1 _M tetra-n-butyl- ammonium chloride as the supporting electrolyte. A mercury anode is used in conjunction with the DME since the halfwave potential of methyclothiazide occurs in the region where potassium ions from classical agar electrodes would interfere.

7. References

1. The National Formulary, 14th Ed., Mack Publishing Go. , Easton, PA (1975).

2. The Merck Index, 8th Ed. , Merck and Go. , Inc., Rahway, NJ (1968).

3. Washburn, W., Abbott Laboratories, Personal Comunication.

4. Fazzari, F. R., Sharkey, M. F. , Yaciw, C. A. and Brannon, W. L., J. Ass. Offic. Anal. Chem., 2, 1154, (1968).

5. Egan, R. S., Abbott Laboratories, Personal Communication.

325

JAMES A. RAIHLE

6.

7.

8.

9.

10.

11.

12.

13.

14.

Furman, W. B., J. Ass. Offic. Anal. Chem., 51, 1111, (1968).

Mueller, S . , Abbott Laboratories, Personal Communication.

Quick, J. E. , Abbott Laboratories, Personal Communication.

Sprague, J. M., Ann. N. Y. Acad. Sci. , 71, 328, (1958).

Close, W. J., Swett, L. R., Brady, L. E., Short, J. H. , and Versten, M. , J. Am. Chem. SOC. , 82, 1132 (1960).

Luebke, D. 'R., Abbott Laboratories, Personal Communication.

Chang, S . L., Acta. Phann Sinica, 7, (1959). (Anal. Abstr., 1, 1909, (1960)).

Fazzari, R. , J. Ass. Offic. Anal. Chem., 56, 677, (1973).

Pilsbury, V. B., and Jackson, J. V., J. Pharm. Pharmacol., J-8, 713, (1966).

326

M ETRONIDAZOLE

Lorraine L. Wearley and Gaylord D. Anthony

LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

Cantents

1. Description

1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor


2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Scanning Calorimetry 2.8 Thermogravimetric Analysis 2.9 Solubility

3. Metabolism

4. Pharmacokinetics


5.1 Titrimetric Analysis 5.2 Spectrophotometric Analysis 5.3 Colorimetric Analysis 5.4 Chromatographic Analysis

5.41 Thin Layer Chromatography 5.42 Gas Chromatography

5.5 Polarographic Analysis

6. Synthesis

7. References

8. Acknowledgments

328

METRON IDAZOLE

1. Description


Metronidazole is 1- (2-hydroxyethyl) -2-methyl- 5- n i troimidazole

2.

CH2 CHZOH

N02aH3 l4olecular Weight: 171.16 'SHSN303

1 . 2 ADDearance. Color. Odor

Metronidazole is a white t o pale yellow, odorless crystal l ine powder.

Physical Properties

2.1 Infrared S p e c t m

The infrared absorption spectrum of a metronidazole reference standard compressed in a KBr pel le t is shown i n Figure 1. been made for absorption bands in Figure 1.

The following assignmenfs have

Band (an-1) Assignment

32 30 OH st re tch

3105 C=CH; C-H s t re tch

1538 6 1375 NO2; N-0 s t re tch

1078 C-OH; C-0 s t re tch

830 C-N02; C-N s t re tch

329

W

Figure 1

Infrared Spectrum of Metronidazole

METRON I DAZO LE

2.2 Nuclear Magnetic Resonance Spectrum

The M I spectrum of metronidazole in deuterated acetic acid is shown in Figure 2. Below are the assignments of the major signals. Positions of absorption bands are reported as shifts downfield from the signal of the protons in te ramethylsilane which was used as internal standard. I

Assignment \\ ,C - CH3 -

u

274 (triplet) $2 - c F r 2 - o H

155 singlet

241 (triplet) CH2 --ez - OH

a - 481 (singlet) ,C - H

2 . 3 Ultraviolet Spectrum

Metronidazole exhibits an absorption maxima at about 274 run. methanol as solvent. this solvent is 6333. spectrum is shown in Figure 3.

using 0.1 N sulfuric acid in The molar absorptivity in The ult2aviolet absorption

2.4 Mass Spectrum

The low resolution mass spectrum of metronidazole is shown h3Figure 4. as follows:

Structure assignments are

!YE Assignment % Relative Intensity

171 M**(molecular ion) 12.5

154 M-OH 4.0

125 M - NO2

125 M - N O Z - H

22.1

25.7

41 M - CH3CN 100

331

0 0 1u

. . . . 5.0 . . . m . . , . ( T ) 6.0 . . . . . . . . . . . . . . . . . . . . . . . 7.0 8.0 91) . . . . , . . . . ? . . . l ' " - I ' ' ~ ' . '

00 PD tm tm **,

m o m

I . . . 1

I 1 . . . . . . . I . . . I . . . . ' . . . . . . . L A r . . . . l . . . . ,

I . I . . . . , . . . . l I . . . . ,

I

ko 74 4.a

I . . . . I I . . I I 1 . - 1 . . .

Figure 2

Nuclear Magnetic Resonance Spectrum of Metronidazole

u1

Figure 3

traviolet Spectrum of Metronidazole in 0.1 N Sulfuric

I

-

Acid in Methanol

E

J

1

NBMINFIL- MRSSi-

Figure 4

Mass Spectnnn of Metronidazole

METRON IDAZOLE

2.5

2.6

2.7

2.8

2.9

Optical Rotation

Metronidszole exhibits no optical activity.

Melting Rang e

Theomelting range given in the USP XIX is 159' to 163 C.

Differential Scanning Calorimetry

The DSC thermogramoof metronidazole obtained at a heating rate of 20 C/minute is shown in Figuse 5, The endothermic change observed at about 163 C corresponds to the melting of the compound.

Thermogravimetric Analysis

The TGAof metronidazole obtained a t a heat inq

r a t e o f 10°C/minute i s shown i n f i g u r e 6. 4

Solubility

Solubilities in various solveps at 2S0C are given in the following table.

Solvent Solubility, mg/ml

Water 10.5

Methanol 32.5

Ethanol 15.4

Chlorof o m 3.8

Heptane co.01

3. Metabolism

Stambaugh et a1.6 found that the major urinary excretioA products of metronidazole in man and 0 - 1 mice were unchanged metronidazole, 1- (2-hyroxyethyl) -2-hydroxymethyl- 5-nitroimidazole and their ether glucuronides .

335

- OaN

3 O

X3 -

336

Figure 5

DSC Thennogram of Metronidazole

2- 2-

.c-+m -------- E W E R A T W E OC

Figure 6

TGA of Metronidazole


These products represented 70-90% of the total urinary nitro fraction. Minor metabolites were reported to be 1- (2 -hydroxyethyl) - 2 -carboxylic acid- 5-nitraimidazole and 1-acetic acid-2-methyl-5-nitroimidazole (see Fig. 7). Oxidation of the methyl group of metronidazole appears to occur more facilely than the hydroxyethyl group. Nitro reduction products have not been found in animal or human urine.

The major vaginal products in females given oral doses of drug were found by Manthei et a1 to be unchanged drug, and 1- (2 - hydroxyethyl) - 2 - hydroxymethyl - 5-ni troimidazole. These products were also present in the urine. thought to be a cyclized lactone was found.

In addition a fluorescent lipophilic product

4. Phannacokinetics

In a study on healthy females receiving single and multiple doses of 200 mg metronidazole tablets, Welling and Monro reported the biological half-life to be 6.2 hr. ment open model. Steady state serum concentrations of metronidazole on a regimen of 200 mg twice daily averaged 7.07 ug/ml maximum and 2.47 ug/ml m i n h .

Ings et al,’ studied the distribution of p4C]jmetroni- dazole in rats. rapidly absorbed from the gastrointestinal tract; and rapidly equilibrated between blood and most tissues. in the liver, kidney, gastro-intestinal tract and vaginal secretions. The half life of clearance was longest for the gastro-intestinal tract. showed similar distribution.

Serum concentration data fit a one compart-

They found that oral doses were

Radioactivity was found to concentrate

I.V. doses


5.1 Titrimetric Analysis

The titration with perchloric acid is the method of choice to assay metronidazole. The sample is dissolved in acetic anhydride, and warmed slightly to effect solution. After cooling, one drop of mlachite green T.S. is added, and the titration with 0.1 N perchloric acid to a yellow-green endpoint is carried out. A blank determination is

338

METRON IDAZOLE

Figure 7: Pathways proposed by Stambaugh et al. for the metabolism of metronidazole in man. (1) metronidazole glucuronide (R = glucuronide) (3) 1 - (2-hydroxyethy1)- 2-hydroxymethyl- 5-nitroimidazole (4) its corresponding glucuronide (5) 1-acetic acid-2-methyl- S-nitroimidazole (6) 1- (2 - hydroxyethyl ) - 2 -carboxyl ic acid- 5 -ni troimidazole .

(2) the corresponding ether

339

LORRAINE L. WEARLEY A N D GAYLORD D. ANTHONY

?ae * performed and any necessary correction is One equivalent of the compound is t i t ra ted .

5.2 Spectrophotometric Analysis

Spectrophotometric analysis of metronidazole m y be carried out using 0 .1 N sulfuric acid in methanol as the solvent. is a t about 274 nm.

The ultraviolet absorption maxima

5.3 Colorimetric Analysis

5.31 Metronidazole can be analyzed colorimetrically by reducing the n i t ro group to the corresponding mine , which is subsequently determined by diazotization and coupling w i t h N- (l-naphthyl) - ethylenediamine dihydrochlor ide (Brat ton- Marshall Reagent) .11

5.32 A variation of the above method involves the alkaline hydrolysis of the n i t ro group of metronidazole. The nitrous acid produced diazotizes sulfanilamide in acidic m e d i u m t o form a diazonium s a l t . After coupling with Bratton-Marshall reagent the concentra- t ion is detenined spectrophotmetrically by canparison t o n i t r i t e standards which have been arried through the colorimetric procedure 15


5.41 Thin Layer Chromatography - Several TLC systems and corre onding Rf values are smnarized below. Pi

340

METRONIDAZOLE

E€ Solvent System Absorbent Detect ion

Acetone Silica Gel 1 0.65

Ch1orofonn:Methanol: Silica Gel 1 0.76 Water:Acetic Acid 74:20:4:2

Benzene:methanol: Silica Gel 1 0.36 ammonium hydroxide 79:20:1

Ch1orofonn:methanol: Silica Gel 1, 2 0.66 water:acetic acid 70: 24 :4: 2

1. Spray Vith 1% aqueous titanium trichloride; heat at 130 C for 3 minutes. Spray with 1% Dimethyl- aminobenzaldehyde in 2 N HC1.

Saturate plate with t-butyl hypochlorite vapors. Spray with aqueous 1% starch/l% potassium iodide solution. (This spray has been found to be much more sensitive than #1.)

5.42 Gas-Liquid Chromatography - Metronidazole

2.

can be chranatographed as the trimethyl silyl derivative. The silyl derivative is prepared by dissolving metronidazole in a 1:l mixture of dimethylfomide and bis (trimethylsilyl) - trifluoroacetamide. The silylation reaction is compete in 30 minutes at room temperature.1

Instrumental Conditions

Column:

Column Temp. : 160' C

Carrier: Nitrogen at 70 ml/min

Detector: Hydrogen Flame Ionization

Retention Time: 4 . 1 minutes

6 ft. glass column packed with 3% OV-1 on Gas Chrom Q

341


-0.6 V

Figure 8

Polarograph of Metronidazole, 2% Solution in pH 3.8f 0.2 Buffer

342

METRONIDAZOLE

5.5 Polarographic Analysis

A suitable polarographic analysis of metronidazole may be carried out at a concentration of approximately 5 ug/ml in a 2% solution of pH 3 . 8 + 0.2 buffer. on such a solution, using differential pulse mode, 3 electrode system. solutions addition of a maxima suppressant may be necessary. -0.23 volts vs saturated calomel ele~trode.~

The scan shown in figure 8 was ohained

For less concentrated

Peak maximuni value is approximately

6.0 Synthesis

2-methyl-imidazole (I) is nitrated by reacting with nitric acid in the presence of sulfuric acid catalyst. The resulting 5-nitro product (11) can then be reacted with either chloroethanol or ethylene oxide to produce 1-(2-hydroxyethy1)-2- methyl-5-nitroimidazole (111). See figure 9.

CH2CH20H NwH3 CICH2CH20H or H 2 C ~ F H 2 ’

0 m

Figure 9

Synthesis o f Metronidazole

343


7 . References

1.

2.

3.

4.

5.

6.

7.

8.

9.

10. 11.

12.

13.

14.

Damascus, J., Searle Laboratories, personal conmumication. Aranda, E., Searle Laboratories, personal cammica t ion. Hribar, J., Searle Laboratories, personal conmumicat ion. Marshall, S., Searle Laboratories, personal cammication. Aranda, E., Searle Laboratories, personal camnrnicat ion. Stambaugh, J., Feo, L., Manthei, R., J, Phaxm. and Fxp. Therapeutics, Vol. 161, No. 2, 1968. Manthei, R., Feo, L., Stambaugh, J., Wiadmosci Parazytologiczne T. XV, Nr. 3-4, 1969. Welling, P., Monro, A., Arzneim - Forsch (drug Research) 22 , Nr. 1 2 (1972). Ings, R., McFadzian J., Onnerod, W., Xenobio- tica, 1975, Vol. 5, No. 4, 223-235. USP X I X , p. 327. Bratton, A., Marshall, E., Babbitt, D., Herdrick- son, A., J. Biol. Chm., 128, 537, (1939). Lau, E., Yao, C., Lewis, M., Senkwski, B., J. Phaxm. Sci., 58, No. 1, 55, (1969). Quirk, P., Chow,T., Searle Laboratories, personal cammication. Wood, N., Chow, R., Searle Laboratories, personal commmication.

8. Acknowledgments

The authors wish t o express special thanks t o Dr. J. W i t t for the information on synthesis; t o Dr. J. Opperman for his assistance i n preparing the sections on metabolism and pharmacokinetics; to Mr. S. Marshall and Mrs. M. Gerry for generating and interpreting the data on TGA, DSC and Polaro- graphy; to Mr, J. Damascus and Dr. J. Hribar for the information on I R , W, and Mass Spectra; and t o Ms. J. Janik for her secretarial assistance in preparing th i s manuscript.

344

NITROFURANTOIN

Donald E. Cadwallader and Hung Won Jun

DONALD E. CADWALLADER AND HUNG WON JUN

Table of Contents

1. Description 1. 1 1. 2

Name, F o r m u l a , and Molecular Weight Appearance, Color , Odor and T a s t e

2. Physical P r o p e r t i e s 2.1 Ultraviolet Spec t ra 2 . 2 Infrared Spec t rum 2 . 3 Nuclear Magnetic Resonance Spec t rum 2.4 Dissociation Constant 2 . 5 Melting Range 2.6 C r y s t a l P r o p e r t i e s

2.61 C r y s t a l Shape 2.62 C r y s t a l Size 2.63 Hydrates

2 .7 Sa l t s 2.8 Solubility

2.81 Solubility in Aqueous Media 2.82 Solubility in Organic Solvents

3 . Synthesis

4. Stability 4.1 Stability to Light and Metal 4 .2 Shelf-Life and Storage Conditions

5 . Metabol ism

6 . Methods of Analysis 6.1 Identification 6 . 2 Color Reaction T e s t 6. 3 Elemental Analysis 6 . 4 Chromatographic Sys tems

6.41 Thin L a y e r Chromatography 6.42 P a p e r Chromatography 6 .43 Column Chromatography

346

NITROFURANTOIN

Table of Contents, continued.

6 . 5 Quantitative Analysis 6.51 A s s a y of Dosage F o r m s 6.52 Quantitative Determinat ion in

Biological Samples

7. Biopharmaceut ic s and Pharmacokinet ic s 7 .1 Absorption 7 . 2 Distribution 7 . 3 Elimination 7.4 Bioavailability 7.5 Pharmacokinet ics

8. References

347


I. Description

1. 1 N a m e , F o r m u l a , and Molecular Weight

Nitrofurantoin is N-(5 -Nitro-2-furfurylidene) -1-

smino) hydantoin. Furadant in 8 and Macrodantin aminohydantoin; I - (5 -Ni ro-2-furfuryliden

m o s t commonly u s e d t rade m a r k s ; 11 additional are listed in the M e r c k Index (1).

are the

I / CH2- C, ‘ 0

‘gHgN4O5 Mol. wt . : 238.16

1.2 Appearance, Color , Odor and T a s t e

Nitrofurantoin is descr ibed as lemon-yellow, odor less c r y s t a l s o r fine powder, having a b i t te r tas te (1,2).


2.1 Ultraviolet Spec t ra

Values of E (170, lcm) in w a t e r a t 367.5 and 265 n m are found to be 760 and 540, respect ively (3). The m o l a r extinction coefficients, e at 367 and 265 n m are 13,100 and 17, 300, respect ively (4) .

340

NITROFURANTOIN

When the U V s p e c t r u m of nitrofurantoin in 270 dimethylformamide (DMF) was scanned f r o m 260 to 400 n m , two maxima o c c u r r e d a t 265 and 367 n m . T h e s p e c t r u m shown in F i g u r e 1 w a s obtained f r o m a solution of 10.00 mg of n i t rofuranto in / l i t e r of 270 DMF in w a t e r (3) .

F i g u r e 2 shows the effect of pH on maximal wavelength and E (170, I c m ) values of ni t rofurantoin ( 3 ) . KH2P04 - NaOH and bor ic acid - NaOH buffer systems w e r e employed. hydantoin r ing - opening in the alkaline solution s ince the shift w a s r e v e r s i b l e upon reacidification ( 3 ) .

The s p e c t r a l shift w a s not due to

2 .2 Inf ra red Spec t rum

T h e inf ra red s p e c t r u m of nitrofurantoin (Norwich P h a r m a c a l Reference Standard Pur i ty) a s a m i n e r a l oil mul l is shown in F i g u r e 3. Interpretat ion of the s p e c t r u m was made by Michels (3) using C r o s s , Stevens and Watts ( 5 ) a s a re ference .

Table I

IR Band Assignments for Nitrofurantoin

Wavelength ( m i c r o n s ) Assignment

3 .05 NH 5 . 6 - 5 . 7 5 hydantoin C = 0 6 . 6 - 7 . 4 5 d, -ni t rofuran

10.4 2,5-disubst i tuted furan 8 . 0 5 a s y m m e t r i c a l C - 0 - C 9. a s y m m e t r i c a l C-0-C

349

W 0 z Q

K 0 m

m

m a


1.c

. e

6

4

2

0

NANOMETERS

F i g u r e 1. Ultraviolet Spec t rum of Nitrofurantoin (Coleman 124)

350

NITROFURANTOIN

a z

390 r 3 7 0

380 -

3 7 0 -

360 I I I

5 6 7 8 9 10 360

5 6 7 8 9 10

F i g u r e 2. U l t r a v i o l e t Absorp t ion of N i t r o f u r a n t o i n as a Func t ion of pH

351

k

5

rcc 0

k

U

.rl

a m 0

E 5

k

V

a, a

cn U

k

(d

k

M

a, k

5

M

4000 3000 2000 1500 1000 900 800 700

100

h 80 FR v

Lu

60

2

2

k v) 40 Z

20 fi

0

Figure 3 . Infrared Spectrum of Nitrofurantoin (Perkin Elmer 467)

NITROFURANTOIN

2. 3 Nuclear Magnetic Resonance Spec t rum

T h e nuc lear magnetic resonance s p e c t r u m f o r nit r ofurantoin ( N o rwic h P h a r m a c a l Reference Standard Pur i ty) in DMSO - d6 containing a t e t r a - methyls i lane as the in te rna l re ference i s shown in F i g u r e 4 ( 3 ) . The s p e c t r a l ass ignments a r e presented in Table I1 (3) .

Table I1

NMR Spect ra l Assignments f r Nitr furantoin

Band ( p p m , d ) No. of Pro tons Assignment

Singlet 4.40 2 hydantoin CH2 Doublet 7. 15 ( J=4) 1 furan 3H Doublet 7 .62 ( J = 4 ) 1 furan 4H

Broad Singlet 11.4 1 hydantoin NH Singlet 7 .8 3 1 -CH=N-

(exchangeable)

2 . 5 - solvent

2 .4 Dissociation Constant

Michels (3) determined the pKa of the f r e e acid to be 7 .0 using the method descr ibed by Stockton and Johnson (6 ) . furantoin ( 1).

A pKa of 7 . 2 i s a l s o repor ted f o r n i t ro-

2 .5 Melting Range

Nitrofurantoin m e l t s a t 270-272' C. (1,7).

353

2.0 3.0 4.0 5.0 PPM(TJ 6.0 7.0 8.0 9.0 i o ' . ' ' ' , ' ' , ! . ' ' , ' ' ' ' . ' ! ' ' ' . ' ' ' ' , ' ! ' , ' ' ' ' ' ' ' ' ! ' . ' ' ' I ' ; ' , ' I ' ' ' ' ' . ' ' , ' I ' , ' '

I " I , ' ' : ' , ' '; ! '

++ a0 ,m m lm o cn

I

I . . _ . , . . . . I . . . . , . . . _ I . _ . . , . . . . I . _ . . , _ . . _ I . . . . I . . . . I . _ . . I . . . . I _ . . . I . . . . I . . . . , . . . . l . . . 1 . . . . I . . . . I . . . . I . . . . 1 . . . . 1 . . . . 1 . . . . 1 , . . . I

a.0 7.0 6.0 5.0 PPM( 6 ) 4.0 3.0 2.0 1 .o 0

Figure 4. NMR Spectrum of Nitrofurantoin (Varian A60A)

NITROFURANTOIN

2.6 C r y s t a l P r o p e r t i e s

2.61 C r y s t a l Shape

Crystal l izat ion f r o m diluted ace t ic acid yields needle- l ike c r y s t a l s (1) .

2.62 C r y s t a l Size

T h e c r y s t a l s i z e of ni t rofurantoin has been found to affect the d e g r e e of emesis and the r a t e s of gastrointest inal absorpt ion and u r i n a r y excretion following o r a l adminis t ra t ion (8). The dif- f e r e n t c r y s t a l s i z e s of ni t rofurantoin w e r e p r e - p a r e d by recrys ta l l iza t ion f r o m ni t romethane.

2 .63 Hydrates

It h a s been found that ni t rofurantoin can ex is t in anhydrous and monohydrate f o r m ( 9 ) . Anhydrous ni t rofurantoin and previously dried n i t r ofurantoin monohydrate become hydrated only a t v e r y high humidity (>92% R. H. ). Nitrofuran- toin monohydrate does not lose o r gain m o i s t u r e upon s t o r a g e a t var ious re la t ive humidities in the range of 31-9270 R.H. Monohydrate f o r m is v e r y s table in terms of retaining w a t e r a t 5OoC.

2. 7 Sal t s

T h e sodium s a l t of nitrofurantoin is available and is used to p r e p a r e p a r e n t e r a l formulat ions (10) . Aqueous solutions of the s a l t are v e r y unstable .

355


2 . 8 Solubility

2 . 8 1 Solubility in Aqueous Media

Solubilities of nitrofurantoin in aqueous media have been repor ted f o r var ious tempera- t u r e and pH conditions. These solubility values a re presented in Table 111.

Table I11

Solubilities of Nitrofurantoin in Aqueous Media

T e m p e r a t u r e , OC @ Solubility ( m g / l ) References.

24 24 24 25 30 37 37 37 37 37

45

5 7

7 d. H 2 0

1 .12 4 . 8 5

d. H 2 0 7 7 . 2

d. H 2 0

d. H 2 0

76. 3 131 .1 7 9 . 5

190 113 .4 154 125 167.8 174. 1 312. 1 374 251.2

2 . 8 2 Solubility in Organic Solvents

11 11 11

1 , 1 2 11 1 3 14 11 11 11 13 11

The solubili t ies of nitrofurantoin in var ious organic solvents a r e presented in Table IV.

356

NlTR 0 FUR ANT0 IN

Table IV

Solubilities of Nitrofurantoin in Organic Solvents:%

Solvent Solubility ( m g / l )

Acetone 5,100

Dime thy lfo r tnamide 80 , 000

Ethanol 47.570 189

70 % 712

9 570 5 10

Glycerin 600

Peanut Oil 20.7

Polyethylene glycol 300 15,100

Propylene glycol 2070 1,560

;::The solubility values w e r e repor ted in References (1) and (15). The tempera ture w a s not indicated.

357


3 . Synthesis

Nitrofurantoin can b e prepared by the react ion of 1- aminohydantoin a s the sulfate (16) and as the hydrochlo- r i d e (17) with 5-ni t rofurfural diacetate in isopropyl- alcohol-water media. furantoin w e r e descr ibed by Sanders e t at. ( 1 8 ) . react ion scheme is shown in F igure 5. w a s a l s o prepared by the condensation of 1-aminohydantoin with 5-nitro-2-furaldehyde (19). Another method f o r the synthesis of nitrofurantoin w a s descr ibed by J a c k (20).

Details of the production of n i t ro- The --

Nitrofurantoin

4. Stability

4. 1 Stability to Light and Metal

Nitrofurantoin c r y s t a l s and its solutions a re d i s - colored by alkali and by exposure to light, and are decomposed upon contact with meta ls other than s ta in less s tee l and aluminum ( 2 ) . Since ni t rofurantoin solutions are photosensitive, all analytical opera- tions must be conducted under subdued light. Nitro- furantoin solutions are a l so ex t remely sensi t ive to alkali ; therefore , a l l g lassware m u s t be analytically clean and d r y for a s s a y procedures .

4 .2 Shelf-Life and Storage Conditions

Storage conditions of nitrofurantoin and o r a l suspensions a r e recommended to be in tight, light- res i s tan t containers (2) . Recommended shelf-l ife of tablets and suspensions is f ive y e a r s when s tored at room tempera ture and in regular g lass containers (21).

358

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0

a, c 0

t

a,

L

.- z I

In

I

0

t

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z- 0

C

0

t

I=

z

i

3

II Y

- ..

I-u

t

.- I

2

\ Z

(v

.to: CU

I

I T

L

02

0

\\/ \4

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I 0

0

z- I I z

=

cu I

m

.r(

m

Q

8

ul . m Q

M

$ .r(

Fr

359


When the products a r e packaged in l ight-resis tant containers and s tored a t r o o m tempera ture , the drug showed negligible loss of potency over a f ive-year period of t ime. The nitrofurantoin products should not be s t o r e d where tempera tures a r e expected to exceed 86'F.

5. Metabolism

Reckendorf -- e t a l . ( 2 2 ) repor ted that la rge amounts of nitrofurantoin (30 -50%) of a n oral ly and intravenously adminis te red dose w e r e recovered intact in the ur ine of r a t , dog and man. Beutner _ - et at. (23) a l so found a s i m i l a r recovery in ur ine a f te r o r a l adminis t ra t ion. T h e s e s tudies suggest that nitrofurantoin undergoes metabolic t ransfor - mation in the body to a significant extent. The possible metabolic pathways of nitrofurantoin a r e not completely elucidated i n the l i t e ra ture . However, nitrofurantoin would follow somewhat s i m i l a r pathways in metabol ism to that for ni t rofurazone which undergoes reduction in the n i t r o group and hydrolysis in the aaomethane linkage.


6. 1 Identification

Nitrofurantoin may be identified by i t s melting point (270-272OC) and by means of i t s charac te r i s t ic in f ra red s p e c t r a (see Figure 3 ) .

6. 2 Color Reaction T e s t

A number of color react ion tes t s f o r the identification of nitrofurantoin has been presented in a publication (24) .

360

NITROFURANTOIN

6. 3 Elemental Analysis

The r e s u l t s of a n e lementa l analysis of nitrofurantoin (Norwich P h a r m a c a l C o . , Lot. No. E3769) a r e presented in Table V (25) .

Table V

Elementa l Analysis of Nitrofurantoin

Element yo Theory yo Found

C 40. 34 40.22

H 2.54 2 .57

N 23.53 23 .53

0 33.59

6.4 Chromatographic S y s t e m s

6 .41 Thin Layer Chromatography

The following T L C s y s t e m s have been found suitable f o r detection of possible postulated impur i t ies (26) .

Solvent Sys tem I

Acetone: Glacial Acetic Acid: Methanol 90 5 5 ( v / v )

361


Rf = 0 . 8 1 on Brinkman Silica Gel plate without f luorescent indicator as visualized by shor t - wave ul t raviolet light. l inear dynamic range w a s 0.25-1.10 p g / s p o t .

It w a s found that the

Solvent Sys tem I1

Acetone: Benzene: Glacial acet ic ac id 80 20 1 (v /v)

Rf = 0.95 on Brinkman Silica Gel plate without f luorescent indicator as visualized by short-wave ultraviolet light and H2SO4 char r ing .

Other T L C procedures f o r the identification and separat ion of nitrofurantoin have been des - cr ibed (27) .

6.42 P a p e r Chromatography

The use of paper chromatography in the analysis of nitrofurantoin and other ni t rofuran compounds has been repor ted by Breinl ich (28) , who examined s e v e r a l solvent s y s t e m s and methods for identification of the spots .


Bender -- e t al. (29) u s e d column chromatographic procedures to separa te nitrofurantoin f r o m other components of ur ine samples and subsequently determined the drug concentrations by spectrophotometr ic as say.

362

NITROFURANTOIN

6.5 Quantitative Analysis

6.51 Assay of Dosage Forms

The methods of analysis that a r e used € o r the determination of nitrofurantoin depend on i ts ultraviolet absorption characterist ics. The U. S. P. XIX (30) describes a spectrophotometric determination of nitrofurantoin in an acidic 27'0 dimethylformamide -wate r solution. method i s applicable to nitrofurantoin in tablet, capsule and suspension dosage forms where the excipients a r e U V non-absorbing. Under conditions where separations a r e not made, any drug degradation products a r e reasonably expected to be reflected in the value of the UV ratio; con- versely, constancy of the U V ratio a s compared to the initial formulation value can be taken a s evidence f o r stability (31). A polarographic ( 32) and gravimetric ( 3 3 ) determination of nitrofurantoin in tablets have been described.

This general

6 . 5 2 Quantitative Determination in Biological Samples

Paul -- et al. (34) utilized the ultraviolet absorption of nitrofurantoin to determine drug concentrations in r a t urine followed by extraction with ether, but the absorption maximum was pH dependent as indicated by Stoll and co-workers (35).

363


In 1965 Conklin and Hollifield (36) in t roduced the nitromethane-Hyamine procedure for the determinat ion of nitrofurantoin i n ur ine , and this method has been established a s the p r e - f e r r e d a s s a y for nitrofurantoin in biological samples . modified to a s s a y the drug in whole blood o r p lasma (37). 2 r g / m l and is specific f o r nitrofurantoin. However, this method w a s inadequate f o r quantita- ting nitrofurantoin blood levels in subjects who r e - ceived n o r m a l therapeutic doses . Mattock, McGilveray and Charet te (38) improved the n i t romethane-Hyamine method for the determination of nitrofurantoin in blood samples s o that s m a l l e r volumes are required (0 .8 ml) and the sensit ivity is g r e a t e r ( 0 . 2 u g / m l ) . A modified nitromethane- Hyamine method was descr ibed by Hollifield and Conklin (39) for the a s s a y of nitrofurantoin in ur ine i n the presence of phenazopyridine and i t s metabolites .

The original procedure has been

This procedure has a sensit ivity of

Buzard -- e t at. (40) developed a co lor imet r ic method f o r the analysis of nitrofurantoin in plasma o r s e r u m of the r a t a f te r var ious o r a l doses .

Jones e t at. (41) descr ibed a polarographic method f o r the determinat ion of nitrofurantoin in ur ine. The sensit ivity l imit of a s s a y is l p g / m l . S e v e r a l au thors (42, 43) have repor ted polarographic procedures for the a s s a y of the drug.

364

NITROFURANTOIN

A microbiological procedure for the a s s a y of nitrofurantoin in ur ine w a s a l so descr ibed by Jones -- e t a l . (41) . Gang and Shaikh (44) used an indicator organism in a turbidimetr ic method for the a s s a y of nitrofurantoin and other ni t rofuran der ivat ives in s e r u m and ur ine samples .

A column chromatographic method f o r the determinat ion of nitrofurantoin in ur ine w a s r e - ported by Bender e t a l . (29) .

Stone (45) and Pugl is i (46) have m e a s u r e d a t r a c e of nitrofurantoin in milk by the co lor imet r ic and spec t r o pho t ome t r i c methods , re spec tive 1 y . Both procedures a r e based on the conversion of nitrofurantoin to 5 -nitrofurfuraldehyde phenylhy- drazone and a r e followed by the extract ion and concentration on a chromatographic column. F ina l determinat ions depend on the development of a blue color by the addition of Hyamine base.

7. Biopharmaceutics and Pharmacokinet ics

7 .1 Absorption

Nitrofurantoin is efficiently and rapidly absorbed Limited drug absorp- a f te r o r a l adminis t ra t ion (47) .

tion o c c u r s when nitrofurantoin is adminis te red rectal ly (48).

The f i r s t evidence that absorpt ion and excret ion

This paper repor ted w e r e affected by differences in par t ic le s i z e of the drug was published in 1967 (8). on the relat ionship of par t ic le s ize of nitrofurantoin to emesis in dogs and to absorption and ur inary

365


excretion in man and rats . It was found that larger crystals of the drug caused less emesis in dogs and slower absorption in man and rats. Thus, i t was concluded that the use of large crystals of nitrofurantoin (Macrodant ine could minimize adverse effects of this drug such as nausea and vomiting by slowing the rate of absorption in the gastrointestinal tract.

Nitrofurantoin occasionally causes nausea, vomiting, drowsiness, headache and skin rashes (49). Incidence of these reactions may be reduced to some degree by administration of the drug with food (50,51).

Bates and co-workers (52) studied the effect of food on the absorption of nitrofurantoin from commercial dosage forms. They found considerably increased absorption in nonfasting a s compared to fasting subjects.

7 . 2 Distribution

After absorption into the blood circulation, nitrofurantoin is rapidly distributed into most body fluids (5 3) . During normal oral therapeutic regimen, blood o r plasma levels of the drug a r e usually very low, in the neighborhood of 1 pg/ml. However, nitrofurantoin levels about 3-5 t imes greater than this a r e usually found in these fluids when drug i s adminis- ter ed intravenous Iy o r intramuscularly. Detailed studies of the absorption and distribution of nitrofurantoin have been carr ied out by Buzard e t al. (54) in small animals.

366

NITROFURANTOIN

7. 3 Elimination

The biological half-life of nitrofurantoin in man appears to be 30 min. o r less (55, 56, 57, 22). In clinical studies in human subjects, urinary drug concentration of 200 to 400 mg/l i ter have been reported (50, 58). Renal excretion involves glomerular f i l t ra - tion and active tubular secretion. Schirmeister et at . (59) found that the clearance of nitrofurantoin in human subjects was lower in acid urine than in alkaline urine. During renal impairment, nitrofurantoin urinary excretion was significantly reduced (60).

--

Conklin and Wagner (61) and Conklin -- e t al. (62) reported that a s much a s 20% of the intravenous dose of nitrofurantoin sodium was excreted in hepatic bile in the dog.

7.4 Bioavailability

Bioavailability of nitrofurantoin has received a considerable attention in recent years . (63) presented a monograph on the bioavailability of nitrofurantoin in the special APhA Bioavailability pilot project. The general character is t ics and experimental c r i te r ia f o r bioavailability testing of nitrofurantoin were discussed. and co-workers (64) evaluated the bioavailabilities of 14 commercially available nitrofurantoin preparations by in vivo and in vitro procedures. They found that some products that met the official Compendia1 requirement, were less bioavailable than other products tested. Earl ier studies by various authors

Cadwallader

More recently, Meyer

--

367


(65, 66, 67) a l so repor ted bioavailability problems associated with the use of c o m m e r c i a l ni t rofurantoin tablets. of nitrofurantoin w a s recent ly presented by Cadwallader (68).

An updated monograph on the bioavailability

7. 5 Pharmacokinet ics

In m a n , p lasma levels of nitrofurantoin appear to decline exponentially with a half-l ife value of about 20 to 30 minutes (56, 57, 22). U r i n a r y excret ion and biotransformation appear to b e mainly and equally responsible f o r the elimination of nitrofurantoin. one-compartment model a p p e a r s to be adequate for descr ibing the kinet ics involved in nitrofurantoin absorption and elimination.

The

8. References

1.

2.

3.

4.

5.

6.

7.

8 .

The M e r c k Index, 8th e d . , M e r c k and C o . , Inc . , Rahway, N . J . , 1968, p. 738.

The United S ta tes Pharmacopeia , 19th e d . , Mack Publishing Co. , Easton, Pennsylvania 1975, p. 341. J. G. Michels , Norwich P h a r m a c a l C o . , P e r s o n a l Communication. V. E g e r t s , J. S t rad ins and M. Shimanska, "Analysis of 5-ni t rofuran Derivat ives , t r a n s - la ted by J. Schmarak , Ann Arbor Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 83. A. H. J. C r o s s , S. G. E. Stevens, and T . H. E. Watts , J . Appl. C h e m . , London, 1, 562 (1957). J. Stockton and C. R. Johnson, J. A m , P h a r m . A s s o c . , Sci. E d . , 33, 383 (1944). T h e Nitrofurans, Vol. 1, "Introduction to the Ni t rofurans , Eaton Labora tor ies , Norwich, N. Y. 1958, p. 16. H. E. Paul , K . J . Hayes, M. F. Paul and A . R. Borgmann, J. P h a r m . S c i . , 56, 882 (1967).

368

NITROFURANTOIN

9.

10.

1 1.

12.

13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

23.

B. G. P a t e l , Norwich P h a r m a c a l Co. , P e r s o n a l Communication. F u r a d a n t i n a Sodium P a r e n t e r a l , Eaton Labora- t o r i e s , Norwich, N. Y. L. K. Chen, M.S. T h e s i s , Universi ty of Georgia , 1975. H. E. P a u l and M. F. P a u l in Exper imenta l Chemotherapy, vol. 11. R. J. Schni tzer and F. Hawking, e d s . , Academic P r e s s , New York, N . Y . , 1964, p. 334. T . R. Bates , J. M. Young, C. M. Wu and H. A. Rosenberg, J. P h a r m . S c i . , 63, 643 (1974); T . R. Bates , SUNY a t Buffalo, P e r s o n a l Communication. M. F. Paul , R . C. Bender , and E. G. Nohle, A m e r . J. Phys io l . , 197, 580 (1959). The Ni t rofurans , vol. 1, "Introduction to the Nitrofurans, I ' Eaton Labora tor ies , Norwich,

A. Swirska , J. Lange, and Z . Buczkowski, P r z e m y s l . C h e m . , 11 (34) , 306-308 (1965). Through C. A . , 52, 14079 b (1958). C. J. O'Keefe, Norwich P h a r m a c a l G o . , P e r s o n a 1 C ommunication , H. J. Sanders , R. T . Edmunds, and W. B. Sti l lman, Ind. Enp. C h e m . , 47, 358 (1955). K. Hayes, U. S. Patent , 2,610, 181 (1952). D. Jack , J. P h a r m . P h a r m a c o l . , 11, Suppl. , 108 T (1959). J. F. S t a r k , Norwich P h a r m a c a l C o . , P e r s o n a l C ommuni ca ti on. H. K. Reckendorf, R. Cas t r ing ius , a n d H . Spingler, Med. Welt, 15, 816 (1963). E. H. Beutner , J. J . Pe t ronio , H. E. Lind, H. M. Traf ton , and M. C o r r e i a - B r a n c o , Antibiotics Ann., 1954-1955, 988 (1955).

N. Y . , 1958, pp. 14-15.

369


24.

25. 26.

27.

28. 29.

30.

31.

32.

33. 34.

35.

36.

V. E g e r t s , J. Stradins , and M. Shimanska, I'Analysis of 5-Nitrofuran Derivat ives , I' t r a n s - lated by J. Schmarak, Ann Arbor Science Publ i shers , Ann Arbor , Michigan, 1970, p. 18. Atlantic Microlab, Inc. , Atlanta, Georgia. M. J. Cardone, Norwich P h a r m a c a l Co., P e r s o n a l Communication. V. E g e r t s , J. Stradins , and M. Shimanska, "Analysis of 5-Nitrofuran Derivat ives , t r a n s - lated by J . Schmarak, Ann Arbor Science Publ i shers , Ann Arbor , Michigan, 1970, pp. 126- 127. J. Breinlich, Dtsch. Apotheker. Z tg . , 104, 535(1964). R . C. Bender , E . G. Nohle, and M. F . P a u l , Clin. Chem. , 2, 420 (1956). The United States Pharmacopeia , 19th ed . , Mack Publishing Co. , Easton, Pennsylvania, 1975, p. 342. R. A. Boice, Norwich P h a r m a c a l Co. , P e r s o n a l Communication. V. E g e r t s , J. Stradins , and M. Shimanska, "Analysis of 5-Nitrofuran Derivat ives , 'I t r a n s - lated by J. Schmarak, Ann Arbor Science Publ i shers , Ann Arbor , Michigan, 1970, pp. 73- 74. Ibid. , pp. 39-40. H. E. Paul , F. L. Austin, M. F. Paul and V. R . E l l s , J. Biol. Chem. , 180, 345 (1949). R . G. Stoll , T. R . Bates and J. Swarbrick, J. P h a r m . Sc i . , 62, 65 (1973). J. D. Conklin and R . D. Hollifield, Clin. Chem. , 11, 925 (1965). -

370

NITROFURANTOIN

37. J. D. Conklin and R. D. Hollifield, w., 3, 690 (1966).

38. G. L. Mattock, I. J. McGilveray, and C. Chare t te , ibid. , 16, 820 (1970).

39. R. D. Hollifield and J. D. Conklin, ibid. , 16, 335 (1970).

40. J. A . Buzard, D. M. Vrabl ic , and M. F. Paul , Antibiotics and Chemotherapy, 6, 702 (1958).

41. B. M. Jones , R. J . M. Ratcliffe and S. G. E. Stevens, J. P h a r m . Pharmacol . , 17, 525 (1965).

42. T . Sasaki , P h a r m . Bull. (Tokyo), 2, 104 (1954). 43. H. P. Moore and C. R. Guertal , J. Assoc. Offic.

44. D. M. Gang and K. Z. Shaikh, J. P h a r m . S c i . ,

45.

46. E. Pugl is i , J. Assoc. Offic. Agr. Chemis ts , 44,

47. J. D. Conklin, R. J. Sobers , and D. L. Wagner,

48. J. D. Conklin, Pharmacology, S, 178 (1972). 49.

50. P. F. MacLeod, G. S. Rogers , and B. R .

Agr. C h e m i s t s . , 43, 308 (1960).

- 61, 462 (1972). L. R. Stone, J. Assoc. Offic. Agr. Chemis ts , - 44, 2 (1961).

30 (1961).

1. P h a r m . Sc i . , 58, 1365 (1969).

G. C a r r o l l and R. V. Brennan, J. U r o l . , 71, 650 ( 1954).

Anylowar, Intern. Record Med. and Gen. P r a c t . Cl in . , 169, 561 (1956).

(1966). T . R. Bates , J. A. Sequeira , and A. V. Tembo, Clin. Pharmacol . T h e r a p . , 16, 63 (1974).

-- 51. A. G. Smith, J . Am. Med. ASSOC., 195, 1061

52.

371


5 3.

54.

55.

56. 57. 58.

59.

60.

61.

62.

63.

64.

65.

M. F. Paul , H. E. Paul , R . C. Bender , F. Kopko, C. M. Harr ington, V. R . E l l s , and J. A. Buzard , Antibiotics and Chemotherapy, 10, 287 (1960). J . A. Buzard , J . D. Conklin, E. O'Keefe and M. F. Paul , J. Pharmacol . Exptl. Therap . - 131, 38 (1961). W. M. Bennett, I. Singer , and C. H. Coggins, J. Am. Med. A s s o c . , 214, 1468 (1970). V. D. Kobvletzki. Med. Welt . . 19. 2010 (19681. . . I . . I - , - - I

C. M. Kunin, Ann. Internal Med. , 67, 151 (1967). W. A. Richard , E. R i s s , E. H. K a s s a n d M . Finland, A.M.A. Arch. Internal Med. , 96, 437 (1955). J. S c h i r m e i s t e r , F. Stefani, H. Willmann, and W. Hal louer , Antimicrobiol. Agents and Chemo- therapy, 1965, p. 223. J. Sachs , T . G e e r , P. Noell, and C. M. Kunin, New Engl. J. M e d . , 9, 1032 (1968). J. D. Conklin and D. L. Wagner, J. P h a r m a c o l . , - 43, 140 (1971). J . D. Conklin and R . J. Sobers and D . L. Wagner , Br . J. Pharmacology, 48, 273 (1973). D. E. Cadwallader , "Nitrofurantoin, I f The APhA Bioavailability Pi lot P r o j e c t , Amer ican P h a r m a - ceut ica l Association, Washington, D. C. Ju ly 1973 M. C. M e y e r , G. W . A. Slywka, R. E. Dann and P. L. Whyatt, J. P h a r m . S c i . , 63, 1693 (1974). I. J. McGilveray, G. L. Mattok, and R . D. Hossie , J. P h a r m . P h a r m a c o l . , 23, 246 S (1971) .

372

NITROFUR ANT0 IN

66. G. L. Mattok, R . D. Hossie , and I. J . McGilveray

67. I. J. McGilveray, G. L. Mattok, and R . D. Hossie Can. J. P h a r m . S c i . , 1, 84 (1972).

Rev. Can. Biol. 32, 99 (1973). 68. D. E. Cadwallader, J. Am. Pharm. ASSOC.,

NS15, 413 (1975).

The au thors wish to thank Dr. John Stark and other personnel at the Norwich P h a r m a c a l Company for the i r valuable ass i s tance and support . secretarial support of M r s . Kay W. Oliver is acknowledged.

T h e excellent

373

PIPERAZINE ESTRONE SULFATE

Zui L. Chang

ZUI L. CHANG

Contents

Analytical Profile - Piperazine Estrone Sulfate 1. Description

1.1 Name, Formula, Molecular Weight 1.2 Appearance, color, Odor 1.3 Elemental Composition


2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Raman Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Thermal Analysis 2.9 Solubility 2.10 Crystal Properties 2.11 Dissociation Constant 2.12 Fluorescence 2.13 Hygroscopic Behavior 2.14 Sublimation

3. Synthesis

4. Stability - Degradation 5. Drug Metabolic Products and Pharmacokinetics

6. Method of Analysis

6.1 Identification 6.2 Chromatographic Analysis

6.21 Thin-Layer Chromatography 6.22 Gas-Liquid Chromatography

6.3 Spectrophotometric Analysis 6.4 Colorimetric Analysis 6.5 Nitrogen Analysis 6.6 Titration

376

PlPERAZlNE ESTRONE SULFATE

7. Acknowledgements

8. References

377

ZUI L. CHANG

1. Description

1.1 Name, Formula, Molecular Weight Piperazine estrone sulfate is estra-1,3,5 (10)-

trien-l7-one, 3-(su1fooxy)-, compound with piperazine (1: 1). y 2

0

HO-SO' II 0

Piperazine Estrone Sulfate

c1 8H2205 s' C4H10N2 Molecular Weight 436.56

1.2 Appearance, Color, Odor Piperazine estrone sulfate is a white to yellow-

ish white, fine crystalline powder. It is odorless.

1.3 Elemental Composition C-60.53; H-7.39; N-6.42; 0-18.32; S-7.34.


2.1 Infrared Spectrum The infrared spectrum of piperazine estrone sul-

fate is presented in Figure 1. The spectrum was measured in the solid state as a potassium bromide dispersion. The following bands (cm-1) have been assigned for Figure 1.3

a. 3400-2300 cm-l broad complex of bands due mainly to N-H stretching vibrations of the amine salt.

378

F I G U R E 1 - I N F R A R E D S P E C T R U M O F P IPERAZINE E S T R O N E SULFATE

WAVELENGTH (MICRONS)

8 9 10 12 15 2.5 3 4 5 6 7

0 -4 (D

4000 3500 3000 2 500 2000 1500 1000 700

FREQUENCY (CM-1)

ZUI L. CHANG

b. 1730 cm-l characteristic C=O stretching vibration of the 17-keto group.

c. 1600 and 1490 cm-I characteristic skeletal stretching vibrations of the aromatic ring.

d. 1040 cm-l due to S-0 linkage

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The nuclear magnetic resonance spectrum of

piperazine estrone sulfate as shown in Figure 2 was obtained on a Varian HA-100 NMR Spectrometer in deuterated dimethylsulfoxide (d6) containing tetramethylsilane as the internal standard. presented in Table I.

The spectral peak assignments4 are

2.3 Ultraviolet Spectrum (W) When the W spectrum of 0.1% solution of pipera-

zine estrone sulfate in 0.04% sodium hydroxide solution was scanned from 400 to 210 nm, two maxima and two minima were observed (Figure 3).l ( c = 838) and 268 nm ( c = 851). The minima occur at 272 nm and 239 nm. Model 14 Recording Spectrophotometer.

The maxima are at 275 nm

The spectrum was obtained with a Cary

2.4 Mass Spectrum The mass spectrum shown in Figure 4 was obtained

using an Associated Electrical Industries Model MS-902 Mass Spectrometer with an ionizing energy of 70 ‘eV. mass spectrum of piperazine estrone sulfate indicates the presence of estrone, piperazine, and a sulfur-oxygen constituent, but it does not yield a molecular ion fo r the complete chemical entity. This is attributed to the following behavior:

The

(a) Dissociation of the amine salt into the free amine (piperazine) and the free acid (estrone hydrogen sulfate).

(b) Possible thermal decomposition of estrone hydrogen sulfate to estrone, S02, OH’, etc.

380

FIGURE 2 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF PIPERAZINE ESTRONE SULFATE

ZUI L. CHANG

Table I

NMR Spectral Assignments for Piperazine Estrone Sulfate

Proton Chemical Assignment Shift (ppm) Mu 1 tip 1 i c i ty

H

fl 0

QIH 0

H

7.17

6.92

5.5

2.89

doublet J=9. 5Hz

Mu1 t ipl e t

Broad

Singlet

382


Table I Cont.

Proton C hemi c a 1 Assignment Shift (ppm) Mu1 tip 1 ic i ty

C -CH,

2 - 3.0

0.84

Comp 1 ex

Singlet

383

ZUI L. CHANG

0.8 - 0.7 -

0.6 =

0.5

0.4

0.3

0.2

0.1

0

RUM ATE

I FIGURE 3 - ULTRAVIOLET SPECT OF PIPERAZINE ESTRONE SULF

FIGURE 3 - ULTRAVIOLET SPECTRUM OF PIPERAZINE ESTRONE SULFATE

\

2 00 250 300 350

WAVELENGTH (nm)

384

FIGURE 4 - MASS SPECTRUM OF PIPERAZINE ESTRONE SULFATE

I'

ZUI L. CHANG

The mass spectrum assignments of the prominent ions and subsequent fragments are shown in Table I1 and Figure 5.5

2.5 Raman Spectrum The Raman spectrum of piperazine estrone sulfate

as shown in Figure 6; was obtained in the solid state on a Cary Model 83 Spectrometer. have been assigned for Figure 6.

a.

The following bands (cm-l) 3

1733 ane1 due to the C=O stretching of the 17-keto group.

b. 1608 cm-l due to skeletal stretching mode of the aromatic ring.

c . 1050 cm-l due to s-0 linkage

2.6 Optical Rotation A 1% solution of piperazine estrone sulfate in

0.4% sodium hydroxide solution exhibited a rotation of [cu]~~ + 87.8" when determined on a Perkin-Elmer Model 141 Po 1 ar ime t er . 6

2.7 Melting Range Piperazine estrone sulfate melts at about 190°C

to a light brown, viscous liquid which re-solidifies, on further heatin and finally melts at about 245°C with de compo s i t ion. f 3

2.8 Differential Thermal Analysis (DTA) The DTA curve obtained on a Dupont Model 900

Analyzer as shown in Figure 7 confirms the observed melting characteristics described in section 2.7.

2.9 Solubility Approximate solubility data obtained at room

temperature are given in the following table: 's8

386


Table I1

High Resolution Mass Spectrum of Piperazine Estrone Sulfate

Found Mass

270.1620

213.1287

185.0960

172.0887

146.0725

86.0843

85.0771

63.9618

47.9669

Calculated Mass

270.1620

213.1279

185.0966

172.0888

146.0732

86.0844

85.0766

63.9619

47.9670

- C - H - - N O

18 22 0 2

15 17 0 1

13 13 0 1

12 12 0 1

10 LO 0 1

4 10 2 0

4 9 2 0

0 0 0 2

0 0 0 1

5

0

0

0

0

0

0

0

1

1

-

387

ZUI L. CHANG

FIGURE 5 - FRAGMENTATION PATHWAYS OF PIPERAZINE ESTRONE SULFATE

H I y

H I

bN>1+*

HO-S-0 O W II

'& II 0

0 II

HO-S-0 m / a 350 not observed

L J

m/a 64

m/a 270 [ so]+' ...I- -- [ H O W 1

I

[ ..m]+' m/a 172

I [ H o r n ] " m/a 146

388

FIGURE 6 - RAMAN SPECTRUM OF PIPERAZINE ESTRONE SULFATE

2000 1800 1600 1400 1200 loo0 800 600 400 200 0 I I I I I I I I I

80 - -

60 - -

-

-

0 - I 1 I 1 I I I 1 I

2000 1800 1600 1400 1200 lo00 800 600 400 200 0

RAMAN SHIFT ACM-’

-

80

60

40

20

0

w (D 0

0 X Ly

A -

I-

d '

v -

t 0 n

FIGURE 7 - DIFFERENTIAL THERMAL ANALYSIS CURVE OF PIPERAZINE ESTRONE SULFATE

-

-

s

1 I I I I I I I I I

0 5 0 100 150 200 2 5 0 300 3 5 0 400 4 5 0

T " C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)


Solvent Water

Solubility (mg/ml) 8

95% Ethanol 7 . 4

Chloroform 1

Ether Practically insoluble

Acetone 0.2

Benzene Practically insoluble

Methylene dichloride 1.6

Isopropanol Practically insoluble

0.1 Sodium hydroxide 57

Propylene glycol 3 5

Mineral oil 2

Sesame oil 2

2.10 Crystal Properties The X-ray powder diffraction pattern of pipera-

zine estrone sulfate was determined by visual observation of a film obtained with a 143 .2 mm Debye-Scherrer Powder Camera (Table 111). An Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA with nickel filtered copper radiation; ), = 1 .5418 , were employed. 9

2.11 Dissociation Constant The apparent pKa value of the unprotonated

piperazine nitrogen (proton gained) was found to be 3 . 6 , by titration in acetonitrile-water (80120, v/v) with aqueous sodium hydroxide. Attempts to find systems to extrapolate the pKa to 100% water were unsuccessful.

The pKa value of the protonated piperazine nitrogen (proton lost) was found to be 9.7 by titration in pyridine-water mixtures with methanolic KOH, and extrapolation to 100% water. 8

391

ZUI L. CHANG

Table 111

dA

16.5

7.7

7.4

6.6

6.0

5.67

5.23

4.55B

4.38

4.22

4.05

3.86

3.77

3.61

3.54

3.4013

3.22

3.05

X-Ray Powder Diffraction Pattern d-Spacings and Intensities

I/I

10

10

40

40

20

30

40

20

80

60

5

30

100

5

2

20

5

1

dA

2.98

2.92

2.86

2.73

2.53

2.46

2.37

2.32

2. 28

2.21

2.16B

2.11

2.07B

1.95

1.89

1.85B

1.75B

1.70

- if1

2

2

5

10

5

8

__.

1

5

5

1

5

1

8

2

2

3

5

3

392


2.12 Fluorescence Piperazine estrone sulfate does not exhibit

fluorescent properties in either methanol or in an alkaline aqueous solution. However, it does exhibit fluorescence at 488 nmwhen excited at 465 nm in 65% sulfuric acid solution.6 verts the piperazine estrone sulfate to estrone which reacts with sulfuric acid to yield the fluorescent species.

The strong sulfuric acid con-

2.13 Hygroscopic Behavior Piperazine estrone sulfate was not hygroscopic

when e posed to a relative humidity of 40-50% for three weeks. 3

moisture slight 1 y

2.14

Piperazine estrone sulfate does not absorb at 79% relative humidity, and is only very hygroscopic (0.89%) at 100% relative humidity. 7

Sublimation Piperazine estrone sulfate did not sublime when

it was stored at 105'C for one month.8 sublimation was noted when it was heated with a hot stage to 204°c.7

No evidence of

3. Synthesis

Piperazipg estrone sulfate was first prepared by Hasbrouck in 1951.

The compound can also be prepared from estrone by a fast, complete conversion reaction using a dimethylformamide/ sulfur troxide complex as the sulfating reactant. Excess dimethylformamide is the solvent. Th reaction is com- pleted by the addition of piperazine. '11

4. Stability-Degradation Piperazine estrone sulfate was found to be stable

when refluxed in water for 3 hours. But it degrades completely after 3 hours in refluxing 1 3 hydrochloric acid to yield estrone and piperazine sulfate. slightly in refluxing 1 sodium hydroxide after 3 hours to yield less than 10% free estrone.

It degrades

12

393

ZUI L. CHANG

Piperazine estrone sulfate yields about 10% free estrone when heated at 105OC for one month.

The rate of hydrolysis of piperazine estrone sulfate to estrone at 90°C was studied over the pH range of 2.5-9.1. spectrophotometric measurement in 0.1 1 sodium hydroxide solution. 13

The extent of degradation was determined by a

The hydrolysis of piperazine estrone sulfate to estrone follows first order kinetics with respect to the piperazine estrone sulfate concentration remaining. Futh rmore, the degradation is first order with respect to t' 2 hydrogen ion concentration, resulting in a 10-fold rate increase with each pH unit decrease.13 cons ants at different pH and temperature values are shown in I? oles IV and V. The activation energy of the degra- datit n reaction, Ea (obtained from the slope of the plot of 11 K, as a function of 1/T where T is the absolute temp rature), for three pH levels is shown in Table V.

Some rate

5. Drug Metabolic Products and Pharmacokinetics

media. The known metabolic intercon ersions of the The drug substance is hydrolyzed to estrone in acidic

estrone are summarized in Figure 8. 1Z

Purdy's15 work suggests that estrone, as the sulfate conjugate, is an important transport form of estrone in human plasma.

Urinary excretion studies of sodium estrone sulfate have been performed by Twombly and Levitz16, and Brown. 17

Biliary excretion was also studied by Twombly and Levitz. 16

A very exhaustive study of urinary and biliary meta- 18 bolites was described by Jirku and Levitz.

Quantitative determination of plasma estrogens by radioimmmunoassay has been developed by Vega. l9

394


Table IV

First Order Rate Constants of Piperazine Estrone Sulfate as a Function of pH at 90°C

Initial kl 95% Conf. Lim. - Concentration No. of -1 pH mg. /ml. Samp 1 e s X lo4, hour

2.50 0.3 8 1,830 + - 250 3.03 0.3 8 480 + 25 -

396 + 16

201 + 14

130 + 16

- 3.07 1.5 12

3 . 4 0 1.5 13

3.57 0.3 7

-

- 3.88 1.5 15 59.l 2.9

4.26 1.5

4.86 1.5

22.4 + 1.5

7.51 + 1.03 - 15

15 - 3.63 + .68

1.85 + .77 - 5.16 1.5 15

5.98 1.5 13 - 6.11 1.5 14 1.12 - + .44

.68 + .30

.72 + .17

.42 + .12

- 6.48 1.5 15

6.88 1.5 14

7.50 1.5 13

-

-

7.90 1.5 15 .14 + .11 -

8.43 1.5 13 22 + .08 -

.18 + .11 - 9.10 1.5 12

395

ZUI L. CHANG

Table V

F i r s t Order Rate Constants and Act iva t ion Energies of P iperaz ine Estrone S u l f a t e a t Three pH l e v e l s

pH 2.50 pH 3.03 pH 3.57

k l x l o 4 , hr.-’ 90°C. 1,830 + 250 480 +_ 25 130 + 16 8 O O C . 695 z 31 150 + 4.6 44.8 4 .0

262 + 15 52.2 7 2.2 14.6 f 2.3 7 O O C . - -

Arrhenius Rela t ionship

Linear Cor re l a t ion Coeff. 1.000 .999 1.000 Act iva t ion Energy, Ea 24,100 27,500 27,100

396

I

0

Ly c

4

Y 4

3

v)

Ly

z

0

2! c v) L

y

Ly

z

4 3

- A c &

4

Q

E

2 - U P) L

X

0

f Ef F(

P

397

W (0 -J

FIGURE 8 - METABOLIC PATHWAYS OF PIPERAZINE ESTERONE SULFATE

HO

HO \ HO

178 -errradio1 2 -h yd rox yertrone

OH

7

HO 2-methoxyestrone estriol

HO HO M a -h y drox y estrone

(also 168-) 15a-hydroxyestradiol 16-epiestriol

ZUI L. CHANG


6.1 Identification The presence of piperazine may be identified with

quinone T. S. or thin-layer chromatography (Section 6.21).

identified by thin-layer chromatography (Section 6.21). The presence of estrone hydrogen sulfate may be

Idied The presence of free estrone may be ident with a 2.5% solution of @-naphthol in sulfuric acid or thin-layer chromatography (Section 6.21).


6.21 Thin-Layer Chromatography A number of thin-layer chromatographic

systems on silica gel have been described for the separation of hydrolysis products of i erazine estrone sulfate from the parent substance. 1 9 6 9 2f 9 $2 piper azine es trone sulfate chromatographs as the piperazine and estrone sulfate moieties. Various systems,methods of detection and Rf valves are summarized in Table VI.

6.22 Gas-Liquid Chromatography Piperazine estrone sulfate can not be di-

rectly chromatographed. tion product, estrone, may be silylated with BSA and chromatographed using 3% QF-1 on Gas Chrom Qf2, or it may be silylated with a silylating mixture containing N-trimethylsilylimidazole, BSA and trimethylchorosilane in the ratio 1:30:bf, and chromatographed using 10% SE-30 on C hr om0 sor b W - AW .

However, the principal degrada-

6.3 Spectrophotometric Analysis Direct spectrophotometric analysis of piperazine

estrone sulfate is applicable provided significant quan- tities of interferring contaminents are not present. The drug substance may be examined directly in a methanol solution at 269 nm ( 6 = 860) or in 0.1 - N sodium hydroxide solution. 6

398

Table VI

Solvent System

Chloroform: Methanol (5:4)

n-Propanol: Ethanol : Conc. Ammonia

0 (D (2:1:2) (0

Chlorof o m : Methanol (3: 1)

n-Butanol: Water: Conc. Ammonia (1: 1: 1)

Chloroform: Methanol: Stronger Ammonia TS (85: 15: 1 )

TLC Systems f o r P ipe raz ine Estrone S u l f a t e

Refe- Detect ion P ipe raz ine S u l f a t e Estrone Pence

A r senomolybda te Or ig in 0.64 0.82 6

R f Rf Estrone

R f

spray

Short wave W 0.42 0.78 0.92 21 o r 10% S u l f u r i c Acid i n Ethanol

Short wave W 0.03 0.35 0.72 21 o r 10% S u l f u r i c Acid i n Ethanol

s h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol

Iodine Vapor

ZUI L. CHANG

The estrone content of piperazine estrone sulfate can be determined with a suitable spectrophotometer at 238 nm after the conversion of piperazine estrone sulfate to estrone with the use of hydrochloric acid.l

The degradation product, estrone,may be quantitated in the drug substance by a liquid-liquid extraction into chloroform and comparison to an estrone reference standard at 280 nm. Alternately, the chloroform extract may be evaporated and the residue dissolved in 65% suifuric acid solution. The estrone is dehydrated to a species which fluorescence at 488 nm with excitation at 465 nm.6

6.4 Colorimetric Analysis Piperazine estrone sulfate may be determined as

dehydrated estrone using a phenol-sulfuric acid mixture as the col r development reagent, The chromophore absorbs at 522 nm. 40

Piperazine estrone sulfate may also be determined

6 using 65% sulfuric acid as the reagent. The reaction product with sulfuric acid has a yellow chromophore at 450 nm.

These colorimetric methods may be used for the analysis of piperazine estrone sulfate in tablets or creams. The primary degradation product, estrone, can be removed by prior chloroform extraction.

6.5 Nitrogen Analysis The amount of nitrogen present in piperazine

estrone sulfate may be determined by converting the nitroqgn to ammonia and titrating with 0.05 acid.

sulfuric

6.6 Titration Piperazine estrone sulfate may be potentiometri-

cally titrated in pyridine-water mixtures using methanolic potassium hydroxide and glass-calomel electrodes. 8

7. Acknowledgements

The author wishes to thank Mr. V. E. Papendick and Mr. J. A. Raihle for their review of the manuscript, and Miss Sandra Hudson for typing and drawing of the manuscript.

400

ZUI L. CHANG

8. References

1. The National Formulary, 14th Revision, Mack Publishing Co., Easton, PA (1974).

2. Chemical Abstracts Service Registry No. 7280-37-7.

3. Washburn, W., Abbott Laboratories, Personal Commun ic at ion.

4. Cirovic, M., Abbott Laboratories, Personal C omuni c a t ion.

5. Mueller, S., Abbott Laboratories, Personal Communication.

6. Chang, 2. L., Abbott Laboratories, unpublished work.

7. Yunker, M., Abbott Laboratories, Personal Communication.

8. Wimer, D. C., Abbott Laboratories, Personal C ommu n i c a t ion.

9. Quick, J., Abbott Laboratories, Personal Commun i c a t ion.

10. Hasbrouck, R. B., Assignor to Abbott Laboratories, U. S. Patent 2,642,427.

11. Lex, C. G., Assignor to Abbott Laboratories, U. S. Patent 3,525,738.

12. Williamson, D. E., Abbott Laboratories, Personal communication.

13. Borodkin, S., Abbott Laboratories, Personal C ommun i c a t ion.

14. Kohn, F. E . , Abbott Laboratories, Personal Communication.

15. Purdy, R. H., Engel, L. L., and Oncley, J. L., J. Biol. Chem., 236, 1043, (1961).

401

ZUI L. CHANG

16. Twombly, G H., and Levitz, M., Am. J. Obstet. and Gynec., 80, 889, (1960).

17. Brown, J. B., J. Obstet, and Gynec. Brit. Emp., - 66, 795, (1969).

18. Jirku, H. and Levitz, M., J. Clin. Endocr., 2, 615, (1969).

19. Vega., S. M., Abbott Laboratories, Personal Communication.

20. Abbott Laboratories, J. Am. Med. Assoc., 149 (5), 443, (1952).

21. Birch, R. A., Dale, B. J. and Earl, J. M., Abbott Laboratories, Personal Communication.

22. Luebke, D. R., Abbott Laboratories, Personal Communication.

402

PROCARBAZINE HYDROCHLORIDE

Richard J. Rucki

RICHARD J. RUCK1

INDEX

1 .

2.

3 .

4.

5.

6 .

7.

a.

9.

A n a l y t i c a l Pro f i l e - Procarbazine Hydrochlor ide

Descr ip t ion 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

Phys i ca 1 Proper t i es 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F1 uorescence Spectrum 2.5 Mass Spectrum 2.6 Op t i ca l Rota t ion 2.7 Me l t i ng Range 2.8 D i f fe ren t i a1 Scanning Calor imetry 2.9 2.10 Solubi 1 i t y 2.11 Crys ta l Proper t ies 2.12 D issoc ia t ion Constant

Thermogravi met r i c Anal ys i s

Synthesis

S t ab i 1 i t y and Degradation

Drug Metabolic Products

Toxi c i t y

Methods o f Analysis 7.1 Elemental Analysis 7.2 Thin-Layer Chromatographic Analysis 7.3 D i r e c t Spectrophotometric Analysis 7.4 Coulometric Analysis 7.5 7.6 T i t r i r n e t r i c Analysis

Pol arog raph i c Ana 1 ys i s

Acknowledgements

References

404


1 . Descr ip t ion

1 . 1 Name, Formula, Molecular Weight

P roca r baz i ne hyd roch 1 o r i de i s N- i sop ropy 1 -a- (2-methylhydrazino)-~-toluamid~ hydroch lo r ide .

Procarbazi ne Hydrochlor ide

C12H19N30-HCl Molecular Weight: 257.76


White t o pa le ye1 low c r y s t a l 1 i ne powder w i t h a s l i g h t c h a r a c t e r i s t i c odor.

2. Phys i ca l Proper t ies

2.1 I n f r a r e d Spectrum ( 1 R )

The i n f r a r e d spectrum of procarbazine hydro- c h l o r i d e i s presented i n F igure 1 ( 1 ) . The instrument used was a Perkin-Elmer Model 621 Grat ing Spectrophotometer. The sample was dispersed i n FluorolubeR t o record the spectrum i n the reg ion o f 4000-1340 cm’l and i n minera l o i l f o r the reg ion o f 1340-400 cm-l. The f o l l o w i n g assignments have been made for the bands i n F igure 1 (1) .

Band (cm-’) Assignment

3277 and 3200 NH s t r e t c h 3035 Aromatic CH s t r e t c h 2961 and 2853 A 1 i p h a t i c CH s t r e t c h 2760-2300, main

band a t 2725 N H:

405

33NW

lllWS

NV

W

%

406

FIGURE 1

I n f r a r e d Spectrum of Procarbazine Hydrochloride

WAVELENGTH (MICRONS) 2.5 3 4 5 6 7 8 9 10 12 14 18 22 3550

4000 3500 3000 2500 2000 1700 1400 I loo 800 500 200

FREQUENCY (CM-’1


1660 and 1636 C=O stretch (Amide I ) 1556 Amide I t 1299 Amide I l l 1361 and 1351

85 7 Aromatic CH out-of- p 1 ane bend i ng


The NMR spectrum shown in Figure 2 was obtained by dissolving 50.4 mg o f procarbazine hydrochloride in 0.5 ml of DMSO-d6 containing tetramethylsilane as internal reference. The spectral assignments are shown in Table 1 (2).

a

Table 1

Chemi ca 1 Coup 1 i ng Shift Constant ,

Protons 6 (ppm) Mu1 tip1 ici ty J (in Hz)

1.20 Doub 1 et 2.72 Singlet 4.18 Mu1 ti p let 4.23 Sing 1 et 7.55 Doub 1 e t 7.99 Doublet 8.36 Doublet 6.33- Singlet 10.33 (broad)

6.0

6.0

8.5 8.5 7.0

--

--

--

407

408

P 0 0)

FIGURE 2

NMR Spectrum of Procarbazine Hydrochloride

1 1 1 1 1 1

6 5 4 3 2 1 0 PPM (8)


2.3 U I t r a v i o l e t Spectrum (UV)

The u l t r a v i o l e t spectrum o f procarbazine hydro- c h l o r i d e i n the reg ion o f 350 t o 200 nm i s shown i n F igure 3. (3 ) . one maximum a t 232 nm (E = 1.3 x 10 ) and a m i n i - mum a t 213 nm. The s o l u t i o n concent ra t ion was 0.01 mg/ml i n 0 . 1 N hydroch lo r i c a c i d and the quar t z c e l l width-was 1 cm.

The spect ruq e x h i b i t s

2.4 FI uorescence Spectrum

E x c i t a t i o n and emission scans were c a r r i e d o u t f o r procarbazine hydroch lo r ide i n s o l u t i o n s of 0 . 1 N hydroch lo r i c ac id , 0.1N sodium hydrox- i d e an7 water. observed under these cond i t ions (2).

No f luorescence, however, was

2.5 Mass Spectrum

The low r e s o l u t i o n mass spectrum shown i n F igure 4 was obta ined us ing a Varian MAT CH5 mass spectrometer, i n te r faced w i t h a Var ian data system, w i t h an i o n i z i n g energy o f 70 eV (4) . The data system accepted the o u t - pu t o f the spectrometer, ca l cu la ted the masses, compared t h e i r i n t e n s i t i e s t o the base peak and p l o t t e d t h i s in fo rmat ion as a se r ies o f l i n e s whose he igh ts were p ropor t i ona l t o the i n t e n s i t i e s .

The molecular i o n o f the f r e e base was observed a t m/e 221. The H C l moiety was observed a t m/e 36. The ions a t m/e 191, 177 and 163 co r -

3’ respond t o a loss from the f r e e base of NHCH NHNHCH , and NHCH(CH ) respec t ive ly . The ions a? m/e 149 and 3j$’correspond t o the loss o f C H I91 an9 177, respec t ive ly . m/e 118 r e s u l t s from the loss o f NHNHCH f rom m/e 163.

by McLaf fe r ty rearrangement from m/e The base peak a t

3

A h igh r e s o l u t i o n scan confirmed the r e s u l t s of the low r e s o l u t i o n spectrum. Table I I l i s t s the elemental compositions f o r the ions as

409

RICHARD J. RUCK1

FIGURE 3

U l t r a v i o l e t Spectrum o f Procarbazi ne Hydrochlor i de

0.5 -

0.4 -

w V z a

m a

8 0.3 - $

1 I I I 200 250 300 35c

NANOMETERS

41 0

L

All S N 3

1N

I 3 A I 1V

13 8

41 1

FIGURE 4

Mass Spectrum of Procarbazine Hydrochloride

100

90

80

70

60

50

40

30

20

10

0 1 1 I I I I I I I I I I I I I I I I I I I 50 100 I i o 200 2

m/e 0

RICHARD J. RUCK1

determined by h igh r e s o l u t i o n mass spectroscopy (4).

Table I I

High Resolut ion Mass Spectrum o f Procarbazine Hydrochlor ide

Found Mass Calcd. Mass - C H N - 11 8.0428 118.0419 8 6 0 135.0698 135.0684 8 9 1 149.0753 1 49.07 1 5 8 9 2 163.0870 163.0872 9 1 1 2 177.1170 177.11 54 11 15 1 191.1288 191. I 3 1 1 12 17 1 221.1533

2.6

2.7

2.8

2.9

221.1529 12 19 3

Op t i ca l Rota t ion

Procarbazine hydroch lo r ide e x h i b i t s no o p t i c a l a c t i v i t y (3 ) .

Me1 t i ng Range

According t o USP X I X , procarbazine hydro- c h l o r i d e mel ts a t about 223"C, w i t h decorn- p o s i t i o n (5).

D i f f e r e n t i a l Scanning Calor imetry (DSC)

DSC spect ra f o r procarbazine hydroch lo r ide a t a scan r a t e o f 10°C/min. e x h i b i t e d an endotherm a t about 23OoC, where me l t i ng was accompanied by sample decomposition. The endotherm was fo l lowed immediately by a slow exotherm (con t i nu ing decompos i t ion). observed temperature o f the melting/decomposi- t i o n endotherm i s dependent upon inst rumenta l cond i t ions and, there fore , i s no t c h a r a c t e r i s t i c o f the compound.

The

Thermogravimetric Analys is (TGA)

A thermogravimetric ana lys is performed on

41 2


procarbazine hydroch lo r ide e x h i b i t e d no l oss o f weight from 30-150"C. A t about 150"C, decomposition weight losses began and con- t inued t o 500°C (upper l i m i t o f ins t rument ) .

2.10 Solubi 1 i t y

Approximate s o l u b i l i t y data obta ined a f t e r 3 hours a t 25°C are g iven i n Table I l l (6,7).

Table I l l

S o l u b i l i t y o f Procarbazine Hydrochlor ide

Solvent

Water 95% Ethanol Absolute Ethanol Methanol Ace tone D ie thy l Ether Petroleum Ether (30-60") C h 1 oroform Benzene 3A Alcohol lsopropyl Alcohol Cyclohexane E thy l Acetate D i oxane A c e t o n i t r i l e Carbon Tet rach l o r ide Methylene Ch lor ide

Solubi 1 i t y (mg/ml)

200 27 8

59 4 . 5 <o. 1 <0.1 2

12 1

<O. 5 0.7

<O. 5 <0.5 <O. 5

<0.5

<0.5

2.11 Crys ta l Proper t ies

The x-ray powder d i f f r a c t i o n p a t t e r n of p rocarbazine hydroch lo r ide i s presented i n Table I V (8).

Instrument Condi t ions

Instrument

Genera t o r

GE Mode meter

50 K V ,

XRD-6 Spect rogon io -

2.5 m~

41 3

RICHARD J. RUCK1

Tube Target Opt ics

Goniometer Detec tor

Recorder Samp 1 es

0

Copper (Cu Ka = 1.5418 A) 0.1" Detector s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007'' N i F i l t e r 4" Take o f f ang le Scan a t 0.2" 2Wminute A m p l i f i e r gain-16 coarse 8.7 f i n e Sealed p r o p o r t i o n a l counter

tube and DC v o l t a g e a t . p la teau.

Pulse h e i g h t s e l e c t i o n El 5 v o l t s , Eu out .

Rate meter T.C. 4, 2000 c /s f u l l sca le.

Chart speed 1"/5 minutes Prepared by g r i nd i ng a t room

temperature.

Table I V

Procarbazine Hydroch lo r ide 0

- 20 d (A)+: - ' / ' Oft

11.240 7.8719 0.24 12.400 7.1380 0.46 18.020 4.9225 0.48 18.640 4.7601 0.23 19.800 4.4838 0.17 20.370 4.3596 0.40 21.550 4.1235 0.61 22.550 3.9428 0.35 23.930 3.7185 0.30 25.190 3.5352 1 .oo 25.800 3.4530 0.10 28.800 3.2090 0.14 28.410 3.1415 0.04 29.550 3.0228 0.22 29.650 3.0128 0.23 33.740 2.6564 0.07

;fd ( i n t e r p l a n a r d is tance) = nX/2 s in01 * * l / l o = r e l a t i v e i n t e n s i t y ( h i g h e s t i n t e n s i t y = 1.00)

41 4


2.12 D issoc ia t i on Constant

The d i s s o c i a t i o n constant f o r procarbazine hydroch lo r ide was determined t i t r i m e t r i c a l l y us ing 0.1N sodium hydroxide and a s o l u t i o n i o n i c s t rength o f 0.1. determined by t h i s method was 6.8 ( 9 ) .

The va lue f o r the pKa

3. Synthesis

Procarbazine hydroch lo r ide may be prepared by the reac t i on scheme shown i n F igure 5. 4-Formylben- z o i c a c i d isopropylamide ( I ) i s combined w i t h methy l - hydraz ine i n dimethylformamide t o prepare the cor responding methylhydrazone ( I I ) , which i s then reduced, us ing pal lad ium charcoal c a t a l y s t , t o N- isopropyl - a-(Z-methylhydrazino) -p-toluamide ( I IT). Hydrogen c h l o r i d e i s added t o t h e reac t i on mix tu re o f I l l con- v e r t i n g i t t o the hydroch lo r ide o f N-isopropyl-a- (2-methyl hydraz i no) -e-toluami de ( I VT.

Stab i 1 i t y and Degradation 4.

Procarbazine hydroch lo r ide , i n the presence o f mois ture o r i n aqueous s o l u t i o n , undergoes o x i d a t i o n by atmospheric oxygen. Th is au tox ida t i on has beeg repor t$$ t o be cata lyzed by metal ions such as Mn and Cu ( 1 0 , l l ) . The major products o f t h i s o x i - da t i on are N- i sopropyl -a- (2-methylazo) -p- to1 uami de (I I ) and N-Tsopropyl-a-(2-methyl hydrazof;o)-p-toluamide ( I 1 IT. To a much lesser ex ten t , t o l u i c a c i d isopropylamide ( I V ) and 4-formylbenzoic a c i d i so - propylamide (V) can a l so be formed (12,13). The o x i d a t i v e degradation scheme i s shown i n F igure 6. I n add i t i on , procarbazine hydroch lo r ide i s very s e n s i t i v e t o u l t r a v i o l e t l i g h t (14), necess i ta t i ng the use o f l ow-ac t i n i c glassware dur ing analyses. I n closed, amber b o t t l e s a t room temperature, degrada t i on o f procarbazine hydroch lo r ide i n the s o l i d s t a t e i s slow, and the compound remains s u i t a b l y s t a b l e f o r a t l eas t th ree years (15). A n i t r o g e n atmosphere has been found t o r e t a r d degradation.

U t i l i z i n g spectrophotometric (16) and po la rograph ic t ime stud ies, i t was determined t h a t procarbazine

2

41 5

FIGURE 5

Synthesis of Procarbazine Hydrochloride

CH3 H 0 H H CH3 H 0 H H HC-N-C I I II o A = O + CH~N-NHZ I ---+ H;- i - ! !OC=N-NCH3 I I

I I CH3 (I) CH3

CH3 H 0 H H CH3 H 0 H H

I I

Hh- l ! l - ! oCH2-N-N- -CH3 I I +- HCI HC-l!l-: I O C H z - N - N C H 3 I I

(nI) -HCI CH3 (Im CH3

+ 0

II r--0

LI

Y

H

- 0 I m

I r I I I

0

2

I

2

N

I

6 0

=0

I

z I

A

I

F

I Y

I

c)l I" 0

-0

-0

I

i 2

I z

II r

0

I 4 I"

0-0

-0

r

m I

0

I 2

II 2

I

L

66 Y

0=0

I r

I

mf

I" 0

-0

-0

r

QR cc

o=o I I

I ni

I" 0

-0

-0

I

m r

0 I

z

I

+ B

I z r

I

mI I"

0-0

-0

r

41 7

FIGURE 6

Deg rada t ion o f Procarbaz i ne Hydroch l o r i de

y 3 0

H C - N H - l e C H 2 - N H - N H - C H 3 HCI (I) I

CH3

(-2H) 7 \2HI

I CH3 0 CH3 0

I CH3

I

I H C - N H - ! o CH2-N=N-CH3 HC - NH- : o C H = N - N H - C H 3

(If) CH3 tm,

RICHARD J. RUCK1

hydroch lo r ide degrades r a p i d l y i n a l c o h o l i c media ( i nc lud ing a c i d i f i e d and deaerated a lcoho l ) and more s lowly i n aqueous media. S t a b i l i t y was best i n aqueous a c i d and decreased w i t h inc reas ing pH (13,16).

5. Drug Metabol ic Products

The metabolism o f the tumor - i nh ib i t i ng procarbazine hydroch lo r ide proceeds according t o a s i m i l a r p a t t e r n i n man, dog and r a t (17). The major metabo l i tes o f procarbazine hydroch lo r ide are N - i sopropyl -a- (2- methyl azo) - to1 uami de (AZO) , NTi sopropy 1 terep’htha 1 - amic a c i d $AC), methylhydrazin: and carbon d iox ide (17-24). A la rge p o r t i o n of the drug i s excreted i n u r i n e as the i n a c t i v e TAC, w h i l e both TAC and the a c t i v e metabo l i te A20 have been detected i n b lood (17, 20, 21).

A photometr ic method f o r the q u a n t i t a t i v e determina- t i o n o f procarbazine hydroch lo r ide i n blood plasma, u t i l i z i n g e x t r a c t i o n w i t h ethanol /ch loroform and o x i d a t i o n o f the hydrazine group w i t h f e r r i c y a n i d e , i s described by Raaflaub (17, 25).

The major metabol ic pathways cons is t o f rap id o x i - da t i on (microsomal and non-microsomal) of procarbazine t o A20 fo l lowed by N2-C cleavage o f A20 t o 4- formy 1 ben zo i c ac i d i sop ropy 1 am i de and me thy 1 - hydrazine. While several authors (17, 20, 26) suggest t h a t cleavage occurs a f t e r AZO i s isomerized t o 1- i sop ropy 1 -a- (2-methy 1 hyd razono-2- to1 uami de (HYDRAZONE), ’Baggio l i n i , e t a l . (18) present data which suggests t h a t the major pathway i s d i r e c t cleavage o f AZO. The metabol ic scheme proposed by the l a t t e r i s presented i n F igure 7. The chemical names o f the compounds i n F igure 7 are l i s t e d i n Table V.

Although the mode o f c y t o t o x i c a c t i o n o f procarba- z ine hydroch lo r ide has not y e t been c l e a r l y de f ined, there i s evidence t h a t the drug ac ts by i n h i b i t i o n of p ro te in , RNA and DNA synthes is (27-29) The N-methyl group o f procarbazine hydrochlor de and i t s azo metabo l i te , poss ib ly i n the form o f a methyl f r e e r a d i c a l (30), has been found t o be essent a1 f o r

41 8

FIGURE 7

Metabolic Products of Procarbazine Hydrochloride

CH3-NH-NH2 + HC-NH-C C=O + HN=N-CH3 I %3-y

I (Ip) cm1 CH3 I

1

( Y ) Oxidation by rnr,rosomol hydroxy lase

INMI = Oxidat ion 0th.r than by ( M I

IDH) * O l i d o t i o n by N A D - l inked d.hydroprnora

+ = Yost important atmpa

RICHARD J. RUCK1

b i o l o g i c a l a c t i v i t y as a tumor i n h i b i t o r (30-32). I t has a l s o been suggested tha t hydrogen peroxide, produced du r ing procarbazine ox ida t i on t o the azo compound, may be responsib le f o r the c y t o t o x i c e f f e c t (33, 3 4 ) .

Table V

Metabol i tes o f Procarbazine Hydrochlor ide Referred t o i n F igure 7

I .

I I .

I V . 4-formylbenzoic a c i d isopropylamide

1- i sopropyl -a- (2-methy 1 hydraz i no) -e-toluamide hydroch l o r i de (procarbazine hydrochlor ide) - N- i sopropy l -a- (2-methy lazo) -p - - to1 uamide

I I I . methylhydrazine

V . - N- i sopropy 1 tereph tha lami c ac i d

6 . T o x i c i t y

The major drug t o x i c i t i e s i n acute and chron ic animal s tud ies were hematologic w i t h granulocyte depression, thrombocyte depression and anemia. Reticuloendothe- l i a l system lymphocytic dep le t ion , marrow c e l l depres- s ion, t e s t i c u l a r atrophy and mucous membrane u l ce ra - t i o n were f u r t h e r evidence of i n v i v o c y t o t o x i t y (35) . Leukemia and pulmonary tumors i n mice, and mammary adenocarcinomas i n r a t s , have been observed subsequent to procarbazine hydrochlor ide admin i s t ra t i on i n h igh doses. The o r a l LD i n mice and r a t s was de te r - mined t o be 1320 + 66 mg%g and 785 2 3 4 mg/kg, res- p e c t i v e l y . I n rabb i t s the o r a l LD was 147 2 11.5

50 mg/kg (35) *

7. Methods o f Analys is

7.1 Elemental Analys is

A t y p i c a l elemental ana lys is o f a sample o f p rocarbazine hydroch lo r ide i s presented i n Table V I ( 3 6 ) .

420


Table V I

E lementa 1 Ana l ys i s o f Procarbazi ne Hydrochlor ide

Element % Theory 2 Found

C H N c 1

55.91 7.82

16.30 13.75

56.17 7.90

16.29 14.00

7.2 Thin-Layer Chromatographic Analys is

The f o l l o w i n g TLC procedure i s use fu l fo r sepa r a t i ng N- i sop ropy 1 -a- (2-methy 1 azo) -p- to1 ua- m i de and E-Ts op ropy 1 -a- (2- met h y 1 h yd razono) - p - toluamide from procarbazine hydroch lo r ide (77). The procedure i s used t o evaluate the s t a b i l i t y o f the dosage form. Using s i l i c a g e l GF p l a t e s (about 300 p t h i c k ) and e t h y l ace ta te as the developing so lvent , the equ iva len t o f 1 mg of procarbazine hydroch lo r ide i n 2.5% (w/v) c y s t e i n e hydroch lo r ide i n methanol i s spo t ted on the p l a t e and subjected t o ascending chromatography. A f t e r the so lvent f r o n t has ascended about 15 cm, t h e p l a t e i s a i r d r i ed and observed under shortwave u l t r a v i o l e t r a d i a t i o n . The p l a t e i s then sprayed e i t h e r w i t h mod i f ied E h r l i c h ' s reagent or w i t h g l a c i a l a c e t i c ac id fo l lowed by 10% aqueous f e r - r i c c h l o r i de: 5% aqueous potass i um f e r r i cyanide ( 1 : l ) . The approximate R values are as fo l l ows : f

Procarbazine hydroch lo r ide 0.0

2- to luamide 0.6

to1 uami de 0.7

- N- i sopropyl-a- (2-methyl hydrazono) -

- N- i sopropy 1-a- (2-methyl azo) -E-

7.3 D i r e c t Spectrophotometric Analys is

The procarbazine hydroch lo r ide content (as base equ iva len t ) i n capsules may be determined spec- t ropho tomet r i ca l l y by us ing the f o l l o w i n g procedure. An amount o f capsule f i l l i s mixed and a p o r t i o n o f the powder equ iva len t t o approx i -

421

RICHARD J. RUCK1

7.4

mately 25 mg o f procarbazine base i s weighed. The powder i s mixed w i t h 100 m l o f 0.1N hydro- c h l o r i c a c i d and the mix tu re i s f i l t e r F d through dry f i l t e r paper, d iscard ing the f i r s t 15 m l o f f i l t r a t e . A s u i t a b l e a l i q u o t o f the remaining f i l t r a t e i s d i l u t e d w i t h 0.1N hydroch lo r i c a c i d to g i v e a s o l u t i o n The absorbance o f the s o l u t i o n i s measured a t 232 2 2 nm us ing 0.1N hyd roch lo r i c a c i d as r e f - erence. The amount o f procarbazine base i s c a l - cu la ted by comparison t o the absorbance o f .a s o l u t i o n o f procarbazi ne hydroch lo r ide reference standard s i m i l a r l y prepared and measured ( 3 8 ) .

conta in iLg about 12.5 ug/ml.

Cou 1 ome t r i c Ana 1 y s i s

Procarbazine hydroch lo r ide can be assayed bo th as the drug substance and i n the dosage form by cou lomet r ic t i t r a t i o n . Using a p la t inum gener- a t i n g system and a po la r i zed p la t inum i n d i c a t i n g system, a constant cu r ren t of 96.5 mA i s a p p l i e d t o the sample i n s o l u t i o n w i t h O.5M potassium iod ide so lu t i on , bu f fe red a t pH 8.x 2 0.1 .

The t i t r a t i o n r e s u l t s i n the q u a n t i t a t i v e ox ida- t i o n o f the hydrazo moiety o f procarbazine hydro- c h l o r i d e t o the azo group, making the method q u i t e s p e c i f i c . Each coulomb o f e l e c t r i c i t y i s equ iva len t t o 1335 pg of procarbazine hydro- c h l o r i d e (39).

A cou lomet r ic t i t r a t i o n w i t h e l e c t r o l y t i c a l l y generated bromine has been used t o determine procarbazine hydroch lo r ide i n capsules (40). Th i s method i s no t a s s p e c i f i c as the above method o f O l iver i -V igh , e t a l . , s i nce e l e c t r o - chemical ly generated bromine no t on ly ox id i zes the hydrazine moiety bu t also subs t i t u tes i n t o the phenyl r i n g as f o l l ows (39):

422


(CH3)2-CH-NH-CD 0 CH2NH-NH-CH 3 +4 Br2+2H20

(CH ) - C H - N H - C U 3 2

7.5 Pol arograph i c Ana 1 ys i s

Polarographic ana lys is o f procarbazine hydro- c h l o r i d e i s u t i l i z e d as both an i d e n t i t y and an assay method f o r the dosage form (5), an i d e n t i t y f o r the drug substance (41), and a s t a b i l i t y t e s t i n g procedure f o r the dosage form (42, 43) and drug substance. I n pH 12 Br i t ton-Robinson b u f f e r , the halfwave p o t e n t i a l f o r the o x i d a t i o n o f procarbazine hydrochlor ide occurs a t about -0.16V versus a saturated calomel reference e lec t rode and the d i f f u s i o n cur ren t i s propor- t i o n a l t o concentrat ion. The polarographic wave i s a t t r i b u t e d t o the ox ida t i on o f the hydrazine moiety (-NH-NH-) t o the azo moiety (-N=N-) and i s , therefore, s p e c i f i c f o r the i n t a c t drug substance. e l e c t r o l y t e of aqueous sodium acetate:absolute ethanol (2 : l v/v) has a l s o been reported (13, 44).

A polarographic procedure u t i l i z i n g an

7.6 T i t r i m e t r i c Analysis

Procarbazine hydroch lo r ide i s assayed by d is - so l v ing the sample i n water and t i t r a t i n g w i t h 0.1N sodium hydroxide. The endpoint i s de ter - minzd po ten t iomet r ica l l y , using a glass-calomel

423

RICHARD J. RUCK1

e lec t rode system. x i d e i s equ iva len t t o 25.78 mg of-C H N O s H C I (5). A l t e r n a t i v e l y , the endpoint m& ae ter - mined v i s u a l l y using phenolphthalein T.S. as i n d i c a t o r (41, 45). T i t r i m e t r i c assay o f procarbazine hydrochlor ide has a l s o been reported us ing t i t r a n t s of aqueous s i l v e r n i t r a t e s o l u t i o n and p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d (13, 46). Bromide-bromate, potassium f e r r i c y - anide, and n i t r i t e t i t r a t i o n s have been attempted w i t h l i m i t e d success (13). T i t r a t i o n w i t h 0.1N iod ine led t o unfavorable stoichiometry, causez by l o c a l i z a t i o n o f excess t i t r a n t dur ing the ana lys is (13, 39).

Each m l o f 0.1N sodium hydro-

8. Acknowledgements

The author wishes t o acknowledge the assistance of the Research Records Of f i ce and the S c i e n t i f i c L i t e r a t u r e Department o f Hoffmann-La Roche Inc.

424

PROCARBAZINE HY DROCH LORlDE

9. References

1.

2.

3 .

4.

5. 6.

7.

8.

9.

10.

1 1 .

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Waysek, E. , Hoffmann-La Roche Inc., Personal Communication. Johnson, J.H., Hoffmann-La Roche Inc . , Per- sona 1 Communication. Corrado, C . , Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc. , Personal Commun i cat ion. United States Pharmacopeia XIX, p. 410 (1974). Bass, E . , Hoffmann-La Roche Inc., Personal Commun i cat i on. Uy Bar re ta , E. , Hoffmann-La Roche Inc., Per- sonal Communication. Munroe, M. , Hoffmann-La Roche Inc., Personal Commun i ca t ion. P h i l i p , C . , Hoffmann-La Roche Inc., Personal Commun i cat ion . Aebi, H., Dewald, B. and Suter, H., Helv. Chim. Acta, 48, 656 (1965). Erlenmeyer, H., e t a l . , HeZv. Chim. Acta, - 47, 876 (1964). Carstensen, J.T., Hoffmann-La Roche Inc. Unpub- l i shed Data. Weber, S. and Rigass i , L., Hoffmann-La Roche Inc. , Unpublished Data. G a l l e l l i , J.F., Uni ted States Department of Health, Education and Welfare, Unpublished Data. Boehler t , J., Hoffmann-La Roche Inc. , Unpublished Data. B i l lmeyer , A., Hoffmann-La Roche Inc., Personal Cornmun i cat i on. Raaflaub, J. and Schwartz, D.E., Experientia, 21, 44 (1965). B a g g i o l i n i , M., Dewald, B. and Aebi, H., Biochem. PharmacoZ., 18, 2187 (1969). Reed, D . , Wittkop, J. and Prough, R. , Fed. Proc., - 29, 346 (1970). 0 1 i v e r io , V.T. , e t a 1. , Cancer Cltemother. Rep., - 42, 1 (1964). Schwartz, D.E. , ExpeAentia, 3, 212 (1966).

425

RICHARD J. RUCK1

22.

23.

24.

25.

26.

27.

28.

29.

30.

33. 34.

35.

36.

37.

38.

39.

40.

41.

42.

Car te r , S.K. (ed.), Proc. Chemother. Conf. on Procarbazi ne (Ma t u l ane:NSC-772 13) : Development and App l i ca t i on , Bethesda, Md., 1970, p . 24. Bagg io l i n i , M. and B icke l , M.H. , L i f e Sc i . , 5, 795 (1966). Adamson, R.H., Ann. N.Y. Acad. Sci. , 179, 432 (1971). Raaflaub, J., Hoffmann-La Roche Inc . , Unpublished Data. Berneis, K., e t a l . , HeZv. Chim. Acta, 46, 2157 (1963). Rutishauser, A. and Bol lag, \-I. , Ezperient&, 23, 222 (1967). Wei tze l , G., e t a l . , 2. PhysioZ. Chem., 348, 443 ( 1967) . S a r t o r e l l i, A.C. and Tsunamura, S. , MoZec. Phar- macoz., 2, 275 (1966). Dost, F.N. and Reed, D.J., Biochem. PharmacoZ., - 16, 1741 (1967). Schwartz, D.E., Bo l lag, W. and Obrecht, P., Arzneim.-Forsch., 17, 1389 (1967). Kre is , W., Proc. Am. Assoc. Cancer Res., 2, 39 ( 1966) . Berneis, K., e t a l . , Expeuientia, 19, 132 (1963). J e l l i f f e , A.M. and Marks, J. ( e d s . r N a t u l a n (Ibenzmethyzin), B r i s t o l : John Wr ight and Sons Ltd., 1965, p. 4. Data on F i l e , Hoffmann-La Roche Inc., Nut ley, New Jersey. Scheid l , F., Hoffmann-La Roche Inc., Personal Communication. Boehler t , J . , Hoffmann-La Roche Inc . , Unpub- l i s h e d Data. Houghton, R.E., Hoffmann-La Roche Inc., Un- Dub1 ished Data. O l i ve r i -V igh , S., e t a l . , J . Pharm. Sci., 60, 1851 (1971). Bera l , H. and Stoicescu, V., Pharm. ZentraZh., - 108, 469 (1969). Van Dyk, M.A., Hoffmann-La Roche Inc . , Unpub- l i s h e d Data. Colarusso, R.J. and Scerbo, A., Hoffmann-La Roche Inc., Unpublished Data.

426


43. Johnson, J. B. and Venture1 l a , V .S . , BUZZ.

44. Levin, M., Hoffmann-La R z h e Inc. , Unpubl ished

45. Levin, M. and Mahn, F., Hoffmann-La Roche Inc. ,

46. Kragh, Hoffmann-La Roche Inc . , Unpublished Data.

Parentera2 Drug ASSOC., 25, 239 (1971).

Data.

Unpub 1 i shed Data.

427

PROMETHAZINE HYDROCHLORIDE

Charles M. Shearer and Susan M. Miller

CHARLES M. SHEARER AND SUSAN M. MILLER

CONTENTS

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2.1 Infrared Spectrum 2.2 Ul t r av io l e t Spectra 2.3 Nuclear Magnetic Resonance Spectrum 2.4 Mass Spectra 2.5 Melting Range 2.6 Di f f e ren t i a l Scanning Calorimetry 2.7 So lub i l i t y 2.8 Crystal Propert ies

2.81 X-Ray Diffract ion 2.82 Optical

2.9 Micelle Formation 2.10 Ionizat ion Constant 2.11 Metal Complex and Charge Transfer Complex Formation 2.12 Adsorption and Protein Binding Properties 2.13 Optical Act ivi ty 2.14 Dipole Moment 2.15 P a r t i t i o n Coefficients


3. Synthesis 4. S t a b i l i t y and Degradation 5. Metabolism and Pharmokinetics 6. Iden t i f i ca t ion 7. Methods of Analysis

7.1 Elemental Analysis 7.2 Phase So lub i l i t y Analysis 7.3 Direct Spectrophotometric Analysis 7.4 Colorimetric Analysis 7.5 Electrochemical Analysis 7.6 T i t r ime t r i c Analysis 7.7 Gravimetric Analysis 7.8 Fluorometric Analysis 7.9 Microbiological Analysis 7-10 Separation Methods of Analysis

7.101 Paper Chromatography 7.102 Thin Layer Chromatography 7.103 Gas Chromatography 7.104 Column Chromatography 7.105 Electrophoresis 7.106 Counter Current P a r t i t i o n

8. References

430


1. Description

1.1 Name, Formula, Molecular Weight Promethazine hydrochloride is 10H-phenothiazine-10-

ethanamine, N,N,d-trimethyl-, monohydrochloride , or 10- t2- (dimethy1mino)propya phenothiazine, monohydrochloride (1). Additional names are 10-(2-dimethylamino-2-methylethyl) phenothiazine hydrochloride and N-(2'-dimethylamino-2'- methy1)ethylphenothiazine hydrochloride. The most commonly used trade name is Phenergan. The empirical formula is C1,H20N2S-HC1 with a molecular weight of 320.88.

I CH, CH(C5 )N(Ch *HC1

The CAS Registry number is 60-87-7 for 10H-phenothiazine-10- ethanamine, N,N,o(-trimethyl and 58-33-3 for thehydrochloride ealt of this compound.


White to faint yellow, practically odorless, crye- talline powder. Slowly oxidizes, and acquires a blue color, on prolonged exposure to air (1).


2.1 Infrared Spectrum An infrared spectrum of a mineral oil dispersion of

promethazine hydrochloride (USP Reference Standard Material) was obtained on a Perkin Elmer Model 467 grating infrared spectrophotometer ( 3 ) . This spectrum is shown in Figure 1. The Spectral band assignments are listed in Table I. This spectrum compares favorably with that presented by Sunshine and Gerber (4) in which the promethazine (presumably the hydrochloride salt) is dispersed in a potassium bromide pellet. for this material because the possibility exists for salt interchange between the bromide and chloride if potassium bromide is used.

A mineral oil dispersion is the preferable technique

2.2 Ultraviolet Spectra The ultraviolet spectra of USP Reference Standard

promethazine hydrochloride in water is presented (7) as Fig- ure 2. Table I1 gives ultraviolet spectral data obtained on the USP Reference Standard in various solvents. The Merck Index (2) describes the ratio of absorbance by lo(A249/A298> = 8.0-8.8.

431

WAVELENGTH MICRONS

8 h)

FREQUENCY (CM-' )

Figure 1 - Infrared Spectrum of Promethazine Hydrochloride (USP Reference Standard), Mineral O i l D i spers ion

0

Fc V

h

X

2

w 0

433

WAVE LENGTH (nm)

Figure 2 - Ultraviolet Spectrum of Promethazine Hydrochloride (USP Reference Standard) Solvent - water


Table I Infrared Spectral A s s i gnment s f o r Promethazine Hydrochloride

(5, 6) Wave Number (ern-')

28QO -3000 2 200-2480

1591 1430-1470

1378 1334

850-860, 757 and 731

Vibration Mode CH s t r e t ch ing (Nujol) “H+ s t r e t ch ing aromaticCrC Stretching CH, and % bending CH, bending C-N s t r e t ch ing of

t e r t i a r y m i n e out of plane CH bending

of disubst i tuted aromatic

Table I1 Ultraviolet Spectral Character is t ics f o r Promethazine

Hydrochloride (7) Solvent

water 249 297

0.1 N HC1 249 297

USP alcohol 253 302

a - E - 89.9 28,770 10.6 3,400 89.4 28,690 10.6 3,400 95.5 30,640 11.4 3,660

2.3 Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum of prometha-

zine hydrochloride dissolved i n deuterochloroform containing tetramethylsi lane as the in t e rna l standard is presented (8) as Figure 3. The spec t r a l peak assignments a r e l i s t e d i n Table 111.

Table I11 NMR Spectral Assignments of Promethazine Hydrochloride

Chemical S h i f t (d) Protons S p l i t t i n g 1.50 doublet 2.85 E 2 I* sing 1 e t

3.5-4.3 N-C&-CH mu1 t i p l e t 4.6-5 0 N-% -2 mu1 ti p l e t 6.8-7.5 --- - - broad

2.4 Mass Spectra The mass spectrum of promethazine hydrochloride (USP

The Reference Standard Material) was obtained with a MS-902 double focusing, high resolut ion mass spectrometer (9).

434

P w 01

9 8 7 6 5 4 3 2 I t Figure 3 - Nuclear Magnetic Resonance Spectrum of Promethazine Hydrochloride (USP Reference

Standard) Solvent - deuterochloroform

n

P

S3U

/SN

3f N/ 3

A/N

73

&

a, P

L)

Q a

v1

m m

2

436

Figure 4 - Mass Spectrum of Promethazine Hydrochloride (USP Reference Standard)


probe temperature w a s 1 5 O o C and the ionizat ion electron beam energy was a t 70 eV. High resolut ion data were compiled and tabulated with the aid of the PDP-8 Digi ta l computer. These d a t a showed the molecular ion (formula C l,H20N2S) t o have a measured mass of 284.1359 compared to a calculated mass of 284.1347. Figure 4 i s a bar graph of the mass spectrum, with the molecular ion a t d z 284. (C H N) a r i s e s from the s ide chain by cleavage of the C-C bond which i s p t o both the s ide chain nitrogen and the heterocyclic nitrogen. Loss of t h i s fragment c rea t e s the peak a t nJ"2 212, and a proton t r a n s f e r before cleavage gen- e r a t e s mJ" 213. Loss of the complete s ide chain r e s u l t s i n n~/z 198, and elimination of su l fu r from the 212 fragment gives the o the r predominant peak a t d z 180. This spectrum and route of fragmentation i s i n agreement with t h a t presented by Gilber t (10) and Audier (11) and Fales (12). A chem- i c a l ionizat ion spectrum showed the parent peak a t M + 1, t h a t i s , dz 285 (9).

The base peak a t d~ 72

4 10

2.5 Melting RanPe The observed melting range (13) f o r the USP Ref-

erence Standard promethazine hydrochloride i s 219.5°C-220.50C with decomposition using the USP Class I a procedure. The melting range i s increased with increased heating rates . The Merck Index (2) reports a value of 23OOC with decomposition.

2.6 D i f f e ren t i a l Scanning Calorimetry (DSC) The DSC thermogram (14) of promethazine hydro-

chlor ide (USP Reference Standard) i s shown as Figure 5. The thermogram w a s obtained a t a heating r a t e of 10°C/minute i n a nitrogen atmosphere u t i l i z i n g a Perkin-Elmer DSC-2. The thermogram exhibi ts no endotherms o r exotherms o the r than t h a t associated with the decomposition melt.

2.7 S o l u b i l i t y The following s o l u b i l i t y data were obtained a t un-

control led room temperature (7).

Solvent e thyl ace t a t e ab s o 1 u t e e t h ano 1 i sopro panol USP ethanol me thano 1 chloroform w a t e r

85 9

150 320 335 5 00

437

I 1 n I I L 1 I I

I0 220 200 180 160 O C

Figure 5 - Differential Scanning Calorimetry Curve of Promethazine Hydrochloride (USP Reference Standard)


2.8 C r y s t a l Proper t ies

2.81 X-Ray Diffract ion - The X-ray powder d i f f r a c t i o n pa t t e rn of pro-

methazine hydrochloride (USP Reference Standard), obtained with a P h i l l i p s diffractometer using C u K a r ad ia t ion i s presented (14) i n Figure 6. The calculated Itd" spacings (Table I V ) a r e i n good agreement with those obtained by Rajeswaran and Kirk (15).

Table I V

X-Ray Diffract ion Pat tern of Promethazine Hydrochloride d(Ao * - d(Ao I/Io - 7.85 0.28 3.40 0.12 7.11 0.59 3.36 0.10 6.93 0.32 3.25 0.62 6.61 0.63 3.18 0.30 5.72 0.47 3.10 0.38 5.60 0.54 3.06 0.20 5.23 0.61 3.03 0.08 4.89 0.94 2.96 0.06 4.39 1.00 2.85 0.12 4.23 0.25 2.80 0.12 3.99 0.17 2.73 0.12 3.86 0.52 2.57 0.15 3.80 0.16 2.51 0.16 3.65 0.47 2.35 0.07 3.53 0.46 2.27 0.09 3.45 0.10

2.82 Optical Crys ta l Properties Promethazine hydrochloride has been described

as color less rods and i r r e g u l a r fragments. polarized l i g h t the ext inct ion i s paral le l and the s ign of the elongation i s posit ive. The r e f r a c t i v e indices are: o(= 1.617, p = 1.691, f = 1.733 a l l ? 0.002 (16). Escobar (17) describes the c r y s t a l l i n e s t ruc tu re of promethazine hydrochloride. The c r y s t a l l i n e parameters a re given. The space group i s P2(1)-C with four molecules per u n i t c e l l . Projections of t he s t ruc tu re along the OZ and OY axis a r e i l l u s t r a t e d .

I n p a r a l l e l

2.9 Micelle Formation Florence and P a r f i t t (18, 19) have determined

the c r i t i c a l micelle concentration of promethazine hydrochloride by the use of nuclear magnetic resonance and by pH measurements. Ionic s t rength e f f ec t s are also discussed. Stevens (20) discusses the

439

80-

70-

k

k! $ 50-

% 40-

30 - 20

1 1

"6 8 I0 I2 1'4 16 I8 20 22 24 26 28 30 32 34 k k 4 213

Figure 6 - X-Ray Dif f r ac t ion P a t t e r n f o r F'romethazine Hydrochloride (USP Reference Standard)


e f f e c t s of pH and temperature upon the c r i t i c a l micel le concentration. Zografi (21) and Vilallonga (22) discuss the surface a c t i v i t y of promethazine hydrochloride a t the air- solut ion interface.

2.10 Ionization Constant The ionizat ion constant f o r promethazine hydro-

chlor ide has been reported as 9.1 using the solubi1i;y method i n water (23) and as 9.1 using a potentiometric t i t r a t i o n with varying amounts of methanol i n water as t he solvent and extrapolating t h e values obtained t o zero percent methanol (24).

2.11 Metal Complex and Charge Transfer Complex Formation Promethazine hydrochloride has been shownto complex

with Fe(II1) , Co(II1) and Mn(II1) t o produce rose-red colored solut ions (25). The u l t r a v i o l e t and v i s i b l e spec t r a of the cobal t complex has been published (26). Pd(I1) has been used as a method of analysis (27). cobaltous thiocyanate complex with promethazine has been iso- l a t ed and characterized (28).

In the presence of promethazine the polarographic wave corresponding t o the reduction of oxygen disappears, and a new wave appears a t a more negative potent ia l . The l a t t e r apparently represents the reduction of the phenothiazine oxygen complex (29).

with promethazine which form i n a c e t o n i t r i l e have been detected by the use of wnductometric t i t r a t i o n s (30). band a t 1700-1650 cm-' (30). Association constants of promethazine with 1 , 4 dinitrobenzene chloroform were measured by nuclear magnetic resonance (31). Charge t r ans fe r spec t r a l s tud ie s of promethazine i n chloroform, a c e t o n i t r i l e and ethanol-acetone, with bromanil, chlor- a n i l , p-benzoquinone, m-dinitrobenzene and tetracyanoethylene have been reported (32,331. a lso been used a s spray reagents f o r detect ing promethazine on thin-layer chromatography p la t e s (34).

The complex with The

Charge t r ans fe r complexes of bromine and iodine

Infrared spect a of t he iodine complex showed a new

i n carbon t e t r ach lo r ide or

These electron acceptors have

2.12 Adsorption and Protein Binding Properties Promethazine hydrochloride i s adsorbed from aqueous

solut ion onto talc, kaolin, and act ivated charcoal (35,361. I t i s bound by carrageenan, furcel laren, carboxymethyl cel lu- lose, sodium a lg ina te , pectin, gum tragacanth, gum arabic , locust bean gum, gum agar and quince seed mucilage (37). It can also bond t o surface ac t ive agents such as polysorbate 80 (38) and sodium polyethylenesulfonate (39). The bonding of

441


promethazine hydrochloride t o bovine serum albumin has been studied (40, 41).

2.13 Optical Ac t iv i ty Promethazine as normally synthesized i s a r a c m i c - -

mixture. dibenzoyl-D-tartaric acid (42). obtained inDthie manner decompose a t 220-221OC. The o p t i c a l a c t i v i t y p ] of (+) promethazine i s + 7 . 6 O : t h a t of the (-1 form is -7.6! cen t r a l nervous act ion of t he separated op t i ca l isomers and the racemic promethazine a r e t h e same (42).

The op t i ca l isomers have been separated by use of Both the (+> and (-1 forme

The tox ic i ty , antihistaminic a c t i v i t y and

2.14 D i o l e Moment -ports the dipole moment fo r prometha-

z ine base a t 25OC as p(D) = 2.05 f 0;Ol. A second paper by t h e same author (44) discusses the conformation of the s i d e chain with respect t o t h e r ing portion of t he molecule.

2.15 P a r t i t i o n Coefficients Burger (45) has studied the d i s t r i b u t i o n of pro-

methazine hydrochloride between 0.5 N HC1 and various organic phases. Doyle ( 4 6 ) discusses the e f f e c t of solvent composition on the par- t i t i o n of amines i n general.

Table V P a r t i t i o n Coefficients of Promethazine Hydrochloride between

The r e s u l t s he obtained a r e given i n Table V.

0.5 N HCL and Vartous Organic Phases (45)

conc. i n 0.5 N HC1 conc. i n organic phase K =

organic phase bu tan0 1 chloroform amyl acetate e thyl acetate benzene ethyl e ther hexane

K 0.37 0.15 1.14 2.70 5.25

15.20 18.20

3. Synthesis

react ing of phenothiazine with 2-chloro-l-dimethylaminopropane i n the presence of various bases such as sodium hydroxide (47, 48, 491, eodamide (47, 50, 511, potassium hydroxide (48) , phenyl l i thium (52) and calcium carbonate (53). This synthet ic route i s i l l u s t r a t e d i n Figure 7. The s a l t can then be prepared by t r e a t i n g the promethazine base with hydrogen chloride.

The most d i r e c t synthesis of promethazine involves the

442


I

H

Figure 7

Synthesis of Promethazine

443


A second synthesis is via the Grignard reaction ( 5 4 , 55). Phenothiazine is ref luxed with methyl iodide and magnesium. To this is then added 2-chloro-1-dimethylaminopropane to give promethazine.

1-Phenothiazine-2-hydroxypropane p-toluenesulfonate when heated with dimethylamine gave promethazine (52).

Promethazine can also be synthesized by first reacting 2-bromo-2'-amino-diphenylsulfide with l-dimethylamino-2- chloropropane in xylene solution with sodamide present, to give 2-bromo-2 I - ( 2"-dimethylaminopropyl) -amino-diphenylsulfide. To bring about cyclization, this product is heated with potassium carbonate and copper powder in dimethylformamide (56).

4. Stability and Degradation

photolysis. complex with promethazine ( 5 7 , 29).

Promethazine is decomposed primarily by oxidation and/or Oxygen has been shown by polarography to form a

Promethazine hydrochloride when refluxed in an aqueous solution produced 10-methyl phenothiazine, acetaldehyde and dimethylamine ( 5 8 ) . It was concluded that the cleavage of the promethazine was probably due to a free radical mechanism and oxygen in some way caused an activation of the molecule. The same products were formed when a promethazine hydrochloride (pH 5 ) solution was placed in an ampule, sealed under nitrogen and irradiated with a fluorescent light (59). However, Yamamoto found no degradation in a similar solution of promethazine under nitrogen when exposed to a light intensity of 5000 luxes at 3 5 O (60). Aqueous solutions of promethazine hydrochloride stored at room temperature in dif- fused daylight for 2 days to six months decomposed to promethazine sulfoxide, 9, 9 dioxopromethazine, N-demethyl- promethazine and several more unidentified compounds (61, 62) . Phenothiazine was identified as a major degradation product when a solution of promethazine hydrochloride was exposed to sunlight (63).

A kinetic study was reported by Stevens ( 6 4 ) . The pH was controlled with Sorenson citrate buffer containing 0.1% EDTA and adjusted to constant ionic strength. The sample flask was held at constant temperature in the dark and the solution was bubbled with 4 at a flow rate of 10 ml/min. The first order rate constants are given for the pH range 1.2 to 5.0. Within this range promethazine is most unstable

444


a t a pH around 4.3. A t pH's between 5.5 and 7 t he degrada- t i o n no longer followed f i r s t order kinet ics .

The s t a b i l i t y of promethazine hydrochloride i n solut ion i s improved by the addition of ascorbic acid (65, 66, 67, 681, cyeteine (651, sodium metabisulf i te (69) o r rongol i te (68, 69). t he s t a b i l i t y of t h e mater ia l was discussed.

In several of these publications the e f f e c t of pH upon

5. Metabolism and Pharmokinetics

and metabolism of S35 labeled promethazine i n rats. excretion takes place during the f i r s t 24 hours. w a s r ead i ly metabolized, primarily t o the sulfoxide. Two other unidentified metabolites were also found. The sulfoxide w a s found as the only metabolite i n the b ra in and l i ve r . Rueiecki and Wysocka-Paruszewska (71) i n another study on rats found similar excretion pat terns - most intensiveduring the f i r s t 72 hours a f t e r administration and l a s t i n g no more than 5 days. S ix o r seven, depending upon the dose, meta- b o l i t e s were found i n the urine. These metabolites were not i den t i f i ed chemically, but characterized by Rf on t h i n layer chromatography. I n the organs (kidney, spleen, lungs and stomach) 24 hours a f t e r the drug w a s administered, only t r aces of promethazine and i t s metabolites were found. These metabolites were the same as found i n the urine. I n the blood 24 hours a f t e r administration, no unchanged prometha- z ine and only t r a c e s of two metabolites were found. Metabo- l i t e s of promethazine i n r a t s were characterized as beingpro- duced by sulfoxylation, aromatic hydroxylation and N-demethyk ation. I n human volunteers promethazine w a s found t o be excreted primarily as the glucuronide conjugate (72). Robinson (73, 74) has shown t h a t hydroxylation (two d i f f e r e n t products), dealkylation and demethylation of promethazine occur i n r a t l i v e r homogenate.

Hansson and Schmiterlow (70) investigated the excretion Maximum

The drug

6. Iden t i f i ca t ion

methazine are given i n Table V I . Kirk (75) describes the preparation of many of t he l e s s e r known reagents. (76) presents a systematic procedure f o r t he i s o l a t i o n of promethazine from biological material and dosage forms. The i d e n t i f i c a t i o n of t he i so l a t ed material i s by u l t r a v i o l e t spectrophotometry. combining preparative gas-liquid chromatography with micro- infrared spectroscopy f o r the i d e n t i f i c a t i o n of drugs. Infra- red spectroscopy can be used d i r e c t l y on the raw mater ia l f o r

Several color react ions f o r the i d e n t i f i c a t i o n of pro-

Bradford

DeLeenheer (77) describes a system f o r

445


Table V I

I d e n t i f i c a t i o n Color Tests f o r Promethazine Hydrochloride

Reagent

Palladium c h l o r i d e F e r r i c ch lo r ide Conc. $ SO,, Conc. H N 4 Mandelin Reagent Marquis Reagent Buckingham's Reagent Frahde 's Reagent Chloropla t in ic acid Bromine water 1% F'hos phomol ybda t e acid 10% K,,Fe(CNl6 10% QFe(CN)6

Mecke I s Reagent Reickard 's Reagent F luekkiger ' s Reagent V i t a l i s Reagent Schneider ' s Reagent 0.3 M Per iodic ac id i n

1 N %SO,, Ammonium cerr ic n i t r a t e

i n 5% n i t r i c acid Conc. $ SO,, , then 0.1%

E h r l i c h ' s Reagent 10% Chloramine plus

Fuming H N 4 V a n i l l i n wi th $SO,,

SO, -HN$ (1: 1 )

N a N 4

CHC 1,

Response Reference

red-purple p r e c i p i t a t e red c o l o r fusch ia c o l o r magenta t o yellow-orange pink c o l o r magenta c o l o r b r i l l i a n t red t o magenta ptnk c o l o r f l a t purple c r y s t a l s red fading t o c o l o r l e s s v i o l e t p r e c i p i t a t e yellow p r e c i p i t a t e yellow p r e c i p i t a t e pink tu rn ing yellow purple co lo r pink c o l o r red c o l o r yellow-pink c o l o r brownish-green co lo r

142 142

14, 143 14, 143 14, 143 14, 143

14, 75

14, 143

14

14

143 143 143 143

75 75 75 75 75

red co lo r 144

brownish-red c o l o r 145

red c o l o r orange co lo r

146 147

v i o l e t c o l o r 147 red c o l o r 143, 147 pink, red co lo r 147

446


its identification (1). identification of promethazine and isopromethazine. identification tests are described by Clarke (79). gives a microchemical test to distinguish promethazine hydrochloride from promethazine 8-chlorotheophyllinate.

tests for prornethazine. Andres (80) gives a description of the crystals and also photomicrographs of the products of promethazine reacted with platinum bromide, ammonium reineckate and gold chloride. Promethazine from tabletswas identified by these tests. An amorphous precipitate was obtained with picric acid. Bogs (81) obtained a crystalline product (mp 15 9- 1 60° C) by treating promethaz ine hydrochloride with sodium 4,4'-dichlorodiphenyldisulfimide.


Edge and Wragg (78) discuss the Several

He also

Several authors describe microcrystalline identification

7.1 Elemental Analysis The elemental analysis of promethazine hydrochloride

USP Reference Standard is presented below.

E 1 emen t C H N c1 S

% Calculated % Reported (8) 63.59 63.61 6.59 6.59 8.73 8.68 11.05 10.76 9.99 9.90

7.2 Phase Solubility Analysis Phase solubility analysis was carried out using ace-

tone as the solvent (13). Experience in this laboratory has shown that 24 hours with vibration at 31.2OC is suffi- cient for equilibration to be attained. A purity of 99.7 ? 0.5% w s obiained.

7.9 Direct Spectrophotometric Analysis Promethazine may be assayed by ultraviolet spectro-

photometry at 249 nm in water or at 256 nm in ether (82). Ultraviolet spectrophotometric methods are used (1) for the analysis of promethazine hydrochloride syrups, injections and tablets after separation from the inert ingredients by a bi- phase extraction or a partition column. describes a method which involves first, forming ion pairs of promethazine with lauryl sulfate or dioctylsulfosuccinate, second, extracting these from an aqueous into a non-miscible organic solvent and third, determining the concentration of promethazine by ultraviolet spectrophotometry. Separation prior to spec t ro pho tom et r i c determination by partition column chromatography (63, 841, ion exchange chromatography (85, 86) and reverse phase partition column

Pellerin (83)

447


chromatography (87) have been reported. describes a quan t i t a t ive in f r a red spectrophotometric determination of pr ome thaz i ne .

Salvesen (88)

7.4 Colorimetric Analysis Cavatora (89) describes methods f o r determining pro-

methazine i n the presence of promazine and chlorpromazine by react ing i t with mercuric s u l f a t e t o form a colored species.

Promethazine hydrochloride can be determined by forming a colored product with sodium 1 , 2 naphthoquinone-4- eulfonate i n 50% s u l f u r i c acid (90). ingredients i n pharmaceutical preparations do not i n t e r f e r e with the assay.

Many common i n e r t

Promethazine hydrochloride can be oxidized by FeN€&,(S0,,)2 and n i t r i c acid t o form a product which can be estimated spectrophotometrically (91).

Ryan (27) describes a colorimetric assay f o r promethazine by forming a colored complex of it with palladium chloride. The method i s s t a b i l i t y indicat ing with respect t o the oxidation of promethazine to the sulfoxide. A s imi l a r method i s discussed by Cavorta (89). Mercaldo and Gallo (92) have automated t h i s method of analysis using Technicon AutoAnalyzer equipment.

Hetzel (93) describes a colorimetric assay f o r compounds having a phenothiazine moiety by forming a yellow colored n i t r a t i o n product. The method w a s used f o r assaysof phenothiazine drugs i n biological samples.

Oxidation of promethazine hydrochloride by heating it with ammonium pe r su l f a t e gave a colored product with absorption maxima a t 520 nm (94). physiological s a l i n e and t o syrups by extract ing the promethazine base from a bas i c solut ion i n t o ether and t8gh extract ing the hydrochloride sal t i n t o 0.1 N HC1.

The method w a s applied t o

Sodium c h l o r i t e r eac t s with promethazine hydrochlor ide t o produce a rose-violet color which can bemeasured a t 533 run (95).

The react ion between promethazine hydrochloride and

The method was used f o r biological samples (96). p-benzoquinone can be applied t o the determination of promethazine.

448


Beer's law is followed in the range 2.0-3.2 mg/50 ml for promethazine hydrochloride reacted with 2-nitro-1,3- indandione in acetic acid, when measured at 540 run (97).

The reaction between antimony pentachloride and promethazine in dichloroethane can be used for the quantitative determination of the drug in solutions, or in biological media (98).

Eriochrome Black T with promethazine forms salts which can be extracted into chloroform and measured at 510- 520 nm (99).

Promethazine hydrochloride can be determined photo-

An automated application of the method is presented. metrically after extraction as ion-pairs with methyl orange (100).

Promethazine hydrochloride has been analyzed by the formation of the photooxidation products which are free radicals characterized by their strong absorption in the visible region. An automated system for the generation and determination of the free radicals is described (101).

Promethazine can be quantitatively precipitated as the reineckate salt and redissolved in acetone and determined spectrophotometrically (102). Promethazine can be determined by precipitating it with molybdophosphoric acid (103) or tungstophosphoric acid (104) and then redissolving it in dimethylformamide-methanol solution and measuring it- spectro- photometrically.

7.5 Electrochemical Analysis Kabaskalian and McGlotten (105) studied the polaro-

graphic oxidation of promethazine and other phenothiazine tranquilizers. A gold micro-electrode was used. The effects of pH, concentrations and temperature on the position and mag nitude of the anodic wave were investigated.

Meckle and Discher (106) found that controlled poterr tial electrolysis is suitable for the coulometric determination of promethazine. In 14 N $SO, promethazine is quantitatively oxidized to the free radical at + 0 . N vs SCE and to the sulfoxide at + 0.98V. Controlled potential electrolysis at + 0.70V was suitable for the analysis of the raw material.

Hynie (107) and Dusinsky (108) investigated the use of oscillographic polarography for analysis of promethazine.

449


Promethazine can be n i t r a t e d with conc. n i t r i c acid and t h e r e s u l t i n g product determined by polarography (109).

7.6 T i t r h n e t r i c Analysis Promethazine hydrochlor ide can be t i t r a t e d by the

Pifer-Wollish method using c r y s t a l v i o l e t as t h e endpoint i n d i c a t o r (1). Mainvi l le and Chatten (110) desc r ibe a s i m i - l a r t i t r a t i o n us ing a c e t o n i t r i l e a s the so lven t , 0.1 N per- c h l o r i c acid i n dioxane a s the t i t r a n t and endpoint determin- a t i o n by e i t h e r the co lo r change of c r y s t a l v i o l e t o r by potentiometry using a glass-calomel e l ec t rode combination. This procedure was s a t i s f a c t o r y f o r t h e drug substance bu t no t f o r promethazine hydrochlor ide t a b l e t s . uaed acetone a s a so lvent i n a Pifer-Wollish type t i t r a t i o n .

Kerney (111)

Promethazine hydrochlor ide can be determined by a two phase (chloroform and ac id) t i t r a t i o n using sodium l a u r y l s u l f a t e a s t h e t i t r a n t and methyl yellow as t h e i n d i c a t o r (112, 113, 114). This method has been appl ied t o prometha- z ine hydrochloride t a b l e t s (115). c i n a t e has a l s o been used as the t i t r a n t (116).

Sodium dioc ty lsu l fosuc-

Oxidimetric ti t r a t i o n s of promethazine hydrochlor ide wi th c e r r i c s u l f a t e and wi th potassium bromate (potassium bromide added) have been s tudied (117, 118, 119, 120). The endpoint can be determined by potentiometry, v i s u a l l y o r by t h e dead s t o p method. cerr ic ion were descr ibed by P a t r i a r c h e (121, 122). t e t r a a c e t a t e has been used a s the oxida t ive t i t r a n t f o r promethazine hydrochlor ide wi th endpoint determinat ion employing platinum f o i l as i n d i c a t o r and a calomel e l ec t rode a s t h e re ference (123).

S imi l a r methods using e lec t rogenera ted Lead

Promethazine hydrochlor ide can be t i t r a t e d wi th e i l i c o t u n g s t i c acid determining the endpoint e i t h e r conduc- tome t r i ca l ly (124) o r by t h e c o l o r change of congo red (125). The method has been appl ied t o drug dosage forms.

Promethazine base a f t e r ex t r ac t ion from a sodium b i - carbonate s o l u t i o n i n t o chloroform has been t i t r a t e d wi th an a ry l su l fon ic ac id i n dioxane using endpoint i nd ica to r (126).

has been determined by t h e use of a ch lo r ide i o n s e l e c t i v e e l ec t rode (127). Promethazine hydrochlor ide has been t i t ra - t e d with 0.1 N sodium hydroxide f o r t h e proton o f t h e a c i d i c por t ion of t h e s a l t by d i s so lv ing it i n dimethylformamide and

dimethyl yellow as t h e

The ch lo r ide po r t ion of promethazine hydrochlor ide

450


using thymol blue for endpoint determination (128).

Danek (129) precipitated promethazine reineckate, redissolved it in acetone-water and determined the amount of the reineckate present by titrating it with silver nitrate. Promethazine can be quantitatively precipitated with mercuric chloride and the excess mercury determined by a complexometric titration (130). Dilute solutions of promethazine hydrochloride in water, when treated with a known equal volume of 0.15% reineckate salt solution gives a precipitate which is removed by filtering and the excess reineckate determined by potassium bromate titrations (131).

Promethazine hydrochloride in dosage forms was determined by using an anion exchange resin and titration of the eluted promethazine free base (132, 133).

7.7 Gravimetric Analysis Gravimetric analysis of promethazine hydrochloride

can be carried out by precipitating it as the styphnate (1341 reineckate (135) , molybdophosphone complex (103) , or silico- tungstate (136). A different type of gravimetric determination of promethazine involves the oxidation and subsequent bromination of it. The resulting compound was extracted from base with chloroform, the organic layer evaporated and the residue weighed (137).

7.8 Fluorometric Analysis Promethazine hydrochloride can be oxidized in 50%

glacial acetic acid with hydrogen peroxide and heat to give a stable product which can be quantitatively determined by its fluorescence (138). mine promethazine in blood samples (139). ric procedure utilizes the fluorescent product produced by diluting into dimethylsulfoxide a solution of promethazinc in sulfuric acid. The procedure has also been used for analysis of promethazine hydrochloride in biological samples.

This procedure can be used to deter- Another fluoromet-

7.9 Microbiological Analysis Solution of greater than 0.5 mg/ml promethazine

hydrochloride can be assayed by a microbiological method using J. anthracin (141).

7.10 Separation Methods of Analysis

7.101 Paper Chromatography Promethazine has been chromatographed on

451

Table V I I

Eluent

Paper Chromatography of Promethazine

Paper

Butyl alcohol: amyl alcohol: acetic acid: water, 25: 75: 12: 1%

Butano1:water:citric acid, 50:50: 1

Acetone:l M sodium acetate:l M acetic acid, 10: 20: 5

Butanol: acetic acid:water, 4: 1: 2

Butano1:acetic acid:water, 4: 1:5

n-Butanol: acetic acid:water, 60: 15: 25

Phosphate buffer (pH 6.5)

0.1 M NaCl:methanol, 5:l

0.1 M NaCl:formamide, 5:l

Dichloroethane: acetic acidswater, 20: 8: 2

5% aqueous (NH,, I2 SOc :isobutanol, 1: 1

S & S 2045b

Whatman No. 1

Whatman No. 1

S & S 2043

Whatman No. 1

Munktell OB

CM2 paper CM-82, ion exchange

CM-82

CM-82

S & S 2043 or 2045

Whatman No. 1

0.66

0.7

0.83

0.85

0.31

0.40

0.51

0.69

0.62

Reference

148

149

150

151

70

152

153

153

153

154

155

Table IX

TLC Systems Using Silica Gel as Adsorbent

Eluent

Ethano1:water: acetic acid; 20: 20: 1 0.3% 4 in chloroform Cyclohexane: acetone; 50: 50 Cyclohexane: diethylamine; 9: 1 Methano1:ammonia; 100: 1.5 Ch1oroform:methanol; 50: 50 Benzene: ethanol: monia; 95: 15: 1 Methanol: acetone; 12: 88 Water: ethanol; 4: 96

P 1sopropanol:isopropyl ether; 16:84 0 Methano1:methyl acetate:cyclohexane; 18: 49: 33

Ethyl acetate: isopropanol: monia; 70: 25: 5 Ethyl acetate:dichloroethane: ammonia; 80: 20: 5 Benzene:dioxane: annnonia; 10:80: 10 Acetonermethanol: ammonia; 50: 50: 1 95% Ethanol’ Methanol’ Cyc1ohexane:benzene:methanol; 75: 15: 10 Methanol2 Ac e tone 2

m

2

‘Support prepared with 0.1 M NaXSOC or 0.1 M KHSq

2Support prepared with 0.1 N KOH or 0.1 N NaOH

,Rf- 0.41 0.29 0.15 0.62 0.61 0.44 0.41 0.26 0.21 0.16 0.47 0.73 0.56 0.77 0.53 0.23 0.45 0.46 0.47 0.37

Reference

159 164 165 166 166 167 168 169 169 169 169 170 170 171 172

173, 174 173, 174 173, 174 173, 174 173, 174

CHARLES M. SHEARER AND SUSAN M MILLER

paper under var ious condi t ions. Solvent systems and o the r pe r t inen t da t a are given i n Table V I I . Methods of de t ec t ion a re given i n Table V I I I .

Several of these systems (148, 152, 154) have been used i n analyzing u r ine and o the r b i o l o g i c a l mater- i a l s f o r metabolic products of promethazine.

Spray Reagents f o r Detect ion of Promethazine on Paper Table V I I I

Chromatography

Reagent Dragendorff 's Reagent s u l f u r i c acid-ethanol s u l f u r i c acid n i t r i c acid palladium ch lo r ide f e r r i c ch lor ide c e r r i c s u l f a t e E h r l i c h ' s reagent

Color orange red red rose orange rose rose rose

- Reference 148 148 156 156 156 156 156 156

7.102 Thin Layer Chromatography The var ious e luen t systems f o r t h i n l a y e r

chromatography of promethazine using S i l i c a Gel a s adsorbent a r e given i n Table IX. Systems using o the r adsorbants a r e included i n Table X. Table X I l i s t s spray reagents and o the r de t ec t ion methods success fu l ly used i n analyzing promethazine. D i rec t conversion of promethazine t o t h e su l foxide on t h e p l a t e , and subsequent ana lys i s by u l t r a v i o l e t spectroscopy has a l so been descr ibed (157, 158). Separat ion of pheno- t h i a z i n e was achieved using a pH gradien t from 7.0 t o 8.5 on S i l i c a Gel l aye r s (1591, and on alumina (160).

Table X TLC Systems Using Adsorbents Other Than S i l i c a

Eluent Adsorbent EL Reference

benzene: e thanol ; 98: 2 .34 161 benzene: e thanol ; 95: 5 .66 161 benzene: e thanol ; 90$10 * 73 161 ch1oroform:ethanol; 98: 2 .74 161 chloroform: ethanol ; 95: 5 .80 161 chloroform: n-propanol: 25%

anunonia; 98: 1: 1 .88 161 acetone:chloroform; 15: 85 .70 161 ether:petroleum e t h e r ; 1: 1 .55 161 cyclohexane: e thanol ; 85: 15 67 161

- alumina

454


i sobu ty l a lcohol s a t u r a t e d

chloroform: acetone: 25%

chloroform: e t h y l acetate: 25%

wi th 5% (NH,, I2 SO, .54 162

ammonia; 50: 50: 1 .96 163

ammonia; 50: 50: 1 .90 163

7.1013 Gas Chromatopraphy Gas chromatography has been used t o analyze

promethazine and t o sepa ra t e i t from o t h e r phenothiazines . The column cond i t ions and necessary d a t a f o r t h e var ious methods a r e given i n Table X I I . The var ious phenothiazines have been cha rac t e r i zed by gas chromatography of t h e i r py ro lys i s products (180).

Gas chromatography of promethazine has been used i n combination wi th micro- infrared spectroscopy (181).

Reagent Ani l ine vapors, then

bromine vapors Iodop la t i n a t e spray Iodine vapors 1.0% w/v se len ious ac id i n

conc. & SO, Concentrated Q SO, F o l i n ' s reagent Ceric s u l f a t e i n s u l f u r i c acid Marquis reagent Palladium c h l o r i d e Dragendorff ' s reagent Dime thy1 aminob enz a l d ehyd e Fur fu ra l i n s u l f u r i c ac id KMnO,, S u l f u r i c acid: e thanol ; 1: 9 Pe rch lo r i c ac id Phos phomol ybdic ac id

Table X I Detect ion Methods Used i n TLC of Promethazine

Reference 175

- Color brown changing -

t o green v i o l e t brown

red-purple fusch ia r ed -v io l e t red yellow-red red orange rose r o s e yellow rose red r o s e

176 176, 162

168 170 170 170 177

172, 178 176, 178

176 176 176 179 179 179

7.104 Column Chromatography Promethazine can be separa ted from o t h e r

dosage form components and drugs by p a r t i t i o n chromatrography (63, 841, ion exchange chromatography (85, 86, 132, 133) and r eve r se phase chromatography (87).

455

Table X I 1

G a s Chromatography of Promethazine Hydrochloride

Column

6 f t x 3 m i.d.; 0.07% SE-30 on 120-170 mesh g l a s s beads

6 f t x 3 mm i.d., 0.08 PDEAS on 120-170 mesh g l a s s beads

6 f t x 3 mm i.d., 1.1% XF-1150 on 100-120 mesh Chrmosorb W-HMDS

h 6

6 f t x 3 mm i.d.; 1.08% CW-2OM on 100-120 mesh G a s Chrom P

Q)

4 f t x 4 mm i.d.; 5% Apiezon L on 100-110 mesh Anachrom ABS

1.8 m e t e r x 4 m i.d.; 3.8% SE-30 on 80-100 mesh Diatoport S

6 f t x 5 nun i.d.; 2% SE-30 on 80-100 mesh G a s Chrom S

Temperature Retent ion T i m e

175' 7.2 min.

175O 21.7 min.

175O 27.9 min.

175O 17.1 min.

240"

2200

205 "

Reference

182

182

182

182

183

184

185


Pound and Sears (186) discuss the use of high pressure liquid chromatography for the analysis of promethazine hydrochloride.

7.105 Electrophoresis Promethazine was separated from other pheno-

Visualization was thiazines by paper electrophoresis (Whatman No. 2 paper) using a glycol-HC1 electrolyte (pH 3 . 3 4 ) . by Dragendorff, iodoplatinate, palladium chloride or cerric sulfate spray (187).

7.106 Counter Current Partition Promethazine was separated from promazine and

chlorpromazine using counter-current partition between chloroform and hydrochloric acid (188).

457


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CA:5EP1092b.

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CA: 78: 7784c.

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CA:61:14668b. L

465

RIFAMPIN

Gian G. Gallo and Pietro Radaelli

GlAN G. G A L L 0 AND PIETRO RADAELLI

TABLE OF CONTENTS

DESCRIPTION 1.1 Name 1.2 Formula and Molecular Weight 1.3 Potent ia l Stereoisomerism 1.4 Appearance, Color and Odor PHYSICAL PROPERTIES 2.1 Spectra

2.11 Ultraviolet Spectra 2.12 Infrared Spectra 2.13 Nuclear Magnetic Resonance Spectra

2.131 Proton Magnetic Resonance

2.132 Carbon Magnetic Resonance Spectra

Spectra

2.2 Ionization Constants 2.3 Polarography 2.4 Optical Rotation 2.5 Crystal Properties

2.14 Mass Spectra

2.51 X-ray Diffraction 2.52 Thermal Analysis

2.521 Melting Range 2.522 Different ia l Scanning Colorime-

t r y 2.6 Distribution Properties

2.61 Solubili ty 2.62 Lipid-Water Par t i t ion

2.621 Organic Solventda ter Par t i t ion 2.622 Silicon O i l d a t e r Par t i t ion 2.623 Surface Activity

STABILITY 3.1 S t a b i l i t y a s Powder 3.2 S t a b i l i t y i n Solution

4. SYNTHESIS

RlFAMPlN

5. METHODS OF ANALYSIS 5.1 5.2 5.3

5.4 5.5 5.6 5.7

5.8

Elemental Analysis I d e n t i f i c a t i o n Tests Spectrophotometric Methods 5.31 Spectrophotometric Assay on B u l k

5.32 Spectrophotometric Assay on Pharma-

5.33 Spectrophotometric Assay on Biological

F luorome t r i c De termina t ion Analysis by Complex F ormation Volumetric Methods Chromatographic Methods 5.71 T h i n Layer Chromatography 5.72 Column Chromatography 5.73 Paper Chromatography 5.74 High Pressure Liquid Chromatography Microbiological Methods 5.81 Diffusion P l a t e Assay Methods 5.82 S e r i a l Tube Dilut ion Methods 5.83 Turbidime t r i c Methods

Product

c e u t i c a l Preparat ions

F lu ids

6. PROTEIN BINDING 7. PHARMACOKINETICS AND METABOLISM I N MAN

8. REFERENCES

469

GlAN G. GALL0 AND PIETRO RADAELLI

1. DESCRIPTION

1.1 NAME Rifampin (1) i s designated by IUPAC r u l e s as 2,7-

(Epoxypentadeca[l ,11,13]trienimino)naphtho~2,1 -b l furan= 1 , 1 1 (2H) -dione, 5,6,9,17,19,21 -hexahydroxy-23-methoxy- 2,4,12,16,18,20,22-heptamethyl=8-CN-(4-methyl-l-pipe= raz iny l ) formimidoyl l -21 -acetate. However, i n the li-. terature, r i f amp in has been p r e f e r e n t i a l l y known as 3- [[( 4-methyl-1 -p iperaz iny l ) iminolmethyl] r i f amyc in SV, according t o t h e o r i g i n a l nomenclature o f r i famy- c i n s (2-4).

Rifampin (5,6) i s the USAN name f o r the compound, r i f a m p i c i n i s the i n t e r n a t i o n a l nonpropr ietary name (7) and other t r i v i a l names used are r i f amyc in AMP and r i- fa ldaz ine (8,9).

1.2 FORWIA AND MOLECULAR WEIGHT

36 35

C43H58N401 2 Mol.Wt. = 822.95

470

R I F AMP IN

This numbering system, according t o the spec ia l nomenclature o f rifamycins, i s adopted i n the present p r o f i l e .

The numbering system according t o IUPAC r u l e s i s :

The molecule o f r i f amp in i s usua l ly described as cons is t ing o f a naphthohydroquinone chrornophoric p a r t spanned by an a l i p h a t i c chain c a l l e d the ansa w i t h a piperazine s ide chain attached.

1.3 POTENTIAL STEREOISOMERISM Rifarnpin, a semi-synthetic a n t i b i o t i c (8) , even

though i t contains 9 asymmetric carbon atoms and 3 double bonds, i s found only as one isomer because a l l the isomerogenic centers belong t o the na tu ra l p a r t o f the molecule and only one isomer i s s p e c i f i c a l l y produced by the microorganism ( 4 , I O ) .

471


1.4 APPEARANCE, COLOR AND ODOR

powder. Rifampin i s a red-orange, odorless, c r y s t a l l i n e

2. PHYSICAL PROPERTIES

2.1 SPECTRA

2.1 1 U l t r a v i o l e t Spectra UV-VIS spectrophotometry has been used f o r

s t r u c t u r a l determinat ion o f var ious r i famycins t o ob ta in s p e c i f i c in fo rmat ion on the chromophoric p a r t o f t h e molecule. I n par t i cu la r , the V I S maximum, which undergoes a hypochromic e f f e c t and a smal l hypsochromic s h i f t w i t h s t rong acids, i s c h a r a c t e r i s t i c o f the naphthohydrg quinone form ca r ry ing the ac id i c ion izab le func t ion (1 1 - 13), and the auxochromic e f f e c t on the same V I S maximum depends on the nature o f t h e subst i tuents i n pos i t i on 3 ( 1 4-1 7).

The UV spectrum o f r i fampin, recorded on a Perk in Elmer model 40004 spectrophotometer i n aqueous phosphate b u f f e r pH 7.38 (8), e x h i b i t s the absorpt ion maxima given i n Table I.

Table I U l t r a v i o l e t absorpt ion o f r i f amp in

E X,ax' nm

237 33 , 200 255 32,100 334 27,000 475 15,400

The v a r i a t i o n o f the UV-VIS spectrum o f r i fampin w i t h pH ( f i g u r e 1) ind ica tes the presence o f an ion izab le funct ion, a t t r i b u t e d t o the ac id i c 8-hydroxyl group i n p e r i pos i t i on t o the hydroquinonic hydroxyl (13,18,19).

2.12 I n f r a r e d Spectra The i n f r a r e d spectra o f var ious r i famycins

usua l ly have been reported i n the l i t e r a t u r e bu t on ly

472

ELP

I-*

cc1 C

-%

N

01 . 5

.

0

01

I 4

.-. . 0

W I

0

m . -. I P

310 r*ILLIMICRONS

215 210 230 1.0 y y I lbo 270 PBO 2vo 1rm WAVUENGIH

Figure 1 - W spectra of rifampin i n methanol-water (2:3) solution at different pH's

pH 0.5; -.-.-.-.-.= pH 1.8;

........... pH 2.5; ---------_ 3.5-1 1 . o


occasionally have been discussed (1 9/20). spectral assignments have only recently been made (21).

form solution was reported by Maggi e t al. (8). Figures 2 and 3 are the spectra of rifampin recorded as mineral o i l m u l l and i n deuterochloroform solution, respectivelg w i t h a Perkin-Elmer mod.421 spectrophotometer. Inter- pretation of the spectrum o f rifampin (21) is given i n Table 11. I n CDCl solution, rifampin does not ex i s t as the zwitterion, while i n solid s t a t e it seems to.

Table I1

Exhaustive

The infrared spectrum of rifampin i n chloro-

3

Infrared spectra of rifampin

IR absorption band, cm-1 Interpretation lineral o i l m u l l

35003300 * n n

3200-2300

1715 1735

1670 and 1610 1570

1540 and 1520 1255,1050 and 1020

Non-transparen

CDCl solution

3480 3

2970, 2930 and 2880

2820 2800

3300-2300

1715 1 640

1 620 1570

1540 and 520 1255,1040 and 1020

region of the m

u0H-bonded uCH3

uCH30 uCH3-N uNH and vNH-bonded and vOH-bonded vC0 acetyl uCO furanone ( i n so. lution in t r a H- bonded) amide I vc=c amide I1 vC-0-C acetyl

neral o i l

2.13 Nuclear Maanetic Resonance Spectra 2.131 Proton Magnetic Resonance SDectra

'H-NMR spectroscopy has been widely used as a fundamental tool for s t ructural determination of various rifamycins (18,19,22-24).

474

WAVELENGTH [MICRONS)

2 5 3 4 5 6 7 8 9 10 12 15 20 25

3500 2500 Moo 1900 1800 17$XJOJE@&~ 1400 1300 1200 1100 loo0 900 800 700 600 500 400

Figure 2 - IR spectrum o f rifampin i n mineral o i l m u l l .

2.5 3 4 5

3500 3000 2500 zoo0 1900

WAVELENGTH 'MICRONS)

6 7 8 9 10 12 15 20 25

1400 1300 1200 900 8 b 700 603 5'20 AX 1100 lo00

F i g u r e 3 - IR s p e c t r u m of r i f a m p i n i n CDCl s o l u t i o n . 3

R I FAMP IN

1 The H-NMR spectrum o f r i f a m p i n i n

a t 60 MHz has been repor ted and some assignments

recorded a t 100 MHz w i t h a Var ian )(L-l00-15 NMR

C D C l 3

g iven (8). C D C l specqrometer. has been made (25) and i s g iven i n Table 111.

F i g u r e 4 i s the spectrum o f r i f a m p i n i n

Complete i n t e r p r e t a t i o n o f t h e spectrum

2.132 Carbon Magnetic Resonance Spectra %NMR spectroscopy has been re-

c e n t l y used f o r s t r u c t u r a l determinat ion i n t h e f i e l d o f r i f a m y c i n s (24,26-28).

decoupled NMR spectrum o f r i f a m p i n i n CDCl3 recorded a t 25.2 MHz w i t h a Var ian XL-100-15 NMR spectrometer, equipped w i t h an FT accessory. I n t e r p r e t a t i o n o f t h e spectrum has been made (25) and i s g iven i n Table I V .

2.14 Mass Spectra

13 F igure 5 i s the FT C proton-noise-

The mass spect ra o f some r i f a m y c i n s have been s tud ied and fragmentat ion pa t te rns have been i n - t e r p r e t e d (29). peaks correspond t o M?, CMICH30Hlt and t o the chromo- phor ic ions.

t a i n e d under 70eV e l e c t r o n impact i n DIS a t 200OC w i t h a Perk in Elmer 270 spectrometer. I n t e r p r e t a t i o n o f t h i s spectrum has been publ ished (30). The spectrum was o n l y p a r t i a l l y c h a r a c t e r i s t i c as the compound decomposes i n t h e i o n source and M'f and CMLH30HIf were absent, w h i l e t h e peak a t m/e 398 corresponded t o the chromophoric ion.

The mass spectrum o f r i f a m p i n was a l s o ob- t a i n e d under f i e l d desorp t ion i o n i z a t i o n c o n d i t i o n s a t w i r e c u r r e n t 12 mA w i t h a Var ian MAT 731 spectrometer

(31). the [M+1 I peak corresponded t o t h e i s o t o p i c c o n t r i b u t i o n . No f ragmentat ion peaks were observed.

2.2 IONIZATION CONSTANTS The i o n i z a t i o n p r o p e r t i e s o f r i f a m y c i n s have been

used i n con j u n c t i o n w i t h UV-VIS spectrophotometry (see Sect ion 2.1 1) t o o b t a i n i n f o r m a t i o n on the chromophoric

I n p a r t i c u l a r , the most c h a r a c t e r i s t i c

F i g u r e 6 i s t h e spectrum o f r i f a m p i n ob-

I t e x h i b i t e d t h e molecular i o n a t m/e 822 and

477

PPU 1%

I I 1 I I I I 1 ' I ' I I I I I I I I I 1

Figure 4 - ' H 4 M R spectrum at 100 MHz of rifampin i n C K l , solution.

RlFAMPlN

6

T a b l e I11

'H-NMR d a t a o f r i f a m p i n i n C K l n s o l u t i o n [ M u l t i p l i c i t y , chemica l s h i f t s (6, ppm) and v i c i n a l i n t e r p r o t o n coup- l i n g c o n s t a n t s (J,Hz) l .

J I

I

M u 1 t i p l . a ) P ro ton

NI-i C H 4 l

CH2-2' ,6' CH2-3 ' ,5 '

N-CH3

OH-8 , OH-4

CH3-1 3 : CH~-14

H-19 H-20 H-21

OH-23 H-22 H-23 H-24 H-25 H-26 H-27 H-28 H-29

CH3-30 CH3-31 CH3-32 CH3-33 CH3-34 CH3-36 CH3-37

S

S

m m S

bs

S

S

S

m

dd ddq

dd

bs

ddq dd

ddq dd

dds dd dd

d

d d d d

S

S

S

I 1

8.22 2.9-3.3 2.4-2.8 2.34

11.4-14.0 b)

13.16 b) 1.82 2.22

6.3-6.8

5.92 2.26 3.78

3.2-4.2 b)

1.70 3.04 1.52 4.96 1.22 3.58 5.00 6.20 2.10 0.88 1.01 0.58

-0.33 2.06 3.05

I I c )

c )

- -

- -

I - -

I - c )

~ 15 and 5

I 9 and 1 5 and 9 and 7

- I

1 and 1.5 and 71 1.5 and 10 I

10 and 1 and 7 1 1 and 10 ,

10 and 1.5 and 7j

7 and 13 ' 1.5 and 7

13

7 7 7 7

I

-

- -

a ) s z s i n g l e t ; d = d o u b l e t ; dd=doub le t of d o u b l e t s ; ddq=double t of d o u b l e t s o f q u a r t e t s ; m=mul t ip l e t ; bs=broad s i g n a l ;

b) it exchanges w i t h D 0 c ) not de t e rmined 2

479

A

S

.rl

C

.rl

Q

E

0

4-

.rl I4

4- 0

N

S

z

hl

UJ hl

c,

0

E 3

I4 c, 0

Q,

P

u)

480

T

13

3

F i g u r e 5 - FT C p r o t o n - n o i s e - d e c o u p l e d NMR s p e c t r u m a t 25.2 MHz of r i f a m p i n i n C D C l s o l u t i o n .

RlFAMPlN

1

Table I V 'IC-NMR data o f rifampin i n CDC13 solution. [Chemical sh i f t s 16, ppm) and one-bond coupling constants ( I J , H a .

I 1 2 3 4 5 6

I 7 I 8

9 I 10 I 11 I 12

13 14 15

I 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

CH=N- C-2'- C-3'- C-5'- C-6'-

I

I CH,-N-

138.6 105.9"' 110.8') 147.8 112.8a) 174.3 1 20.3a) 169.3 104.4') 117.8") 195.3 108.7 21.5 7.6

169.6 129.4 135.0 123.2 142.6 38.6 70.7 33.4 76.7 37.6 74.4 39.5 76.7

118.7 142.6 20.7 17.8 10.9 8.5 8.8

171.9 20.7 57.0

134.4 50.2 53.9 53.9 50.2 45.8 I

- - -

I - / - I -

- I - I - I

- - -

130 130 - -

1 50 150 1 50 125 140 125 140 125 140 125 140 I 155 190 , 130 130 130 I

130 130 I

130 1 40 , 1 70 130 I

130 130 130 130

1

- I

I I

I

1 I J

a) These assignments may be interchanged

481

I I

I I

I I

AlIS

N31N

I 3AIlVl3H

o=

ss

"=

o

L

cc 0

482

Figure 6 - MS spectrum of rifampin a t 70 eV i n DIS a t 200%.

R I FAMPlN

pro ton l o s t

p a r t o f the molecule and as a q u a n t i t a t i v e method o f ana lys is ( 1 1 - 1 3 , 18).

The pK values f o r r i f a m p i n have been determined spec t rophotomet r ica l l y (see f i g . 1 ) and po ten t iomet r i - c a l l y i n s o l u t i o n i n water and i n methy lce l loso lve- water ( 4 : l ) and are repor ted i n Table V. e x i s t s i n water s o l u t i o n as t h e z w i t t e r i o n w i t h i so- e l e c t r i c po in equal t o 4.8 (8,13) .

Rifampin

Table V I o n i z a t i o n constants o f r i f a m p i n

A t t r i b u t i o n IReference pKa *KS

1.7 3 .6 hydroxy l a t C-8 I ,13,18,19 proton gained 17.9 6.7

Rifampin i o n i z e s i n non-aqueous solvents, i.e., i n g l a c i a l a c e t i c a c i d the bas ic p iperaz ine n i t r o g e n can be t i t r a t e d w i t h p e r c h l o r i c a c i d (32) .

2 .3 POLAROGRAPHY The polarographic behaviour o f r i f a m y c i n s has been

descr ibed and the presence o f an o x i d a t i o n o r r e d u c t i o n wave a t about 0 v o l t s =. X E i n d i c a t e s t h e hydroquinone o r quinone ( 1 4/33-35) system, r e s p e c t i v e l y . These polarographic p r o p e r t i e s permi t amperometric t i t r a t i o n o f r i f amyc i n s (36) .

Rifampin i n methanol-acetate b u f f e r so lu t ion , pH 5.9, shows an o x i d a t i o n wave w i t h El12 = 4 . 1 0 v o l t s. SCE, a t t r i b u t e d t o t h e hydroquinone system ( 8 ) , and i n aqueous phosphate b f f e r , pH 6.88, t h e r e i s a l s o a re -

t r i b u t e d (37). p o t e n t i a l o f E1/2 = 4 . 0 6 v o l t d e t a i l s .

2.4 OPTICAL ROTATION

Sano e t a l . (38) : C a l k +10.60 ( G O . 5% i n CDC13).

d u c t i o n wave w i t h E y/ 2 -1.66 v o l t s s. SCE, no t a t -

Sano e t al . (38) repor ted an o x i d a t i o n E. SCE, w i t h o u t o ther

The o p t i c a l r o t t i o n f o r r i f a m p i n was repor ted by

p iperaz ine N-4 113

483


2.5 CRYSTAL PROPERTIES 2.51 X-ray Dif f rac t ion

Single-crystal X-ray d i f f r a c t i o n was used t o deduce the s t r u c t u r e of the p-iodoanilides of rifamycin B and rifamycin Y (10,39-42). T h e s ing le-crys ta l X-ray d i f f r ac t ion method was applied t o rifampin c r y s t a l l i z e d w i t h 5 water molecules i n the orthorombic system (43).

The X-ray powder d i f f r a c t i o n pa t te rn of rifampin, is reported i n Figure 7 (44) and Table V I g ives the values according t o the ASTM rules. The grinding causes the c r y s t a l l i n i t y of rifampin t o disappear and an amorphous form t o or ig ina te , a s shown i n Figure 8.

2.52 Thermal Analysis 2.521 Melting Range

Rifampin melts w i t h decomposition a t 183-1 88% (open cap i l l a ry g l a s s tube),

2.522 D i f f e r e n t i a l Scanning Calorimetry U n t i l 1 now, DSC has not been applied

T h e heating curve of rifampin has t o the f i e l d of rifamycins.

been obtained on a Du Pont D i f f e r e n t i a l Thermal Analyzer mod.990 w i t h a temperature r i s e of 100C/min and is reported i n f i g .9 (45). corresponding t o the melting, immediately followed by an exotherm corresponding t o the r e c r y s t a l l i z a t i o n of the melt, which t h e n decomposes exothermically a t about 240%.

2.6 DISTRIBUTION PROPERTIES 2.61 S o l u b i l i t y

I t shows an endotherm a t 193OC

The approximate s o l u b i l i t i e s of rifampin i n

The r e s u l t s a r e reported i n Table VII, various solvents have been determined a t room tempera- t u r e (32). according t o the de f in i t i on used by the US Pharmacopeia

X I X , Rifampin is soluble i n a c id i c and a lka l ine water (8).

were reported, as shown i n Table VIII. Quan t i t a t ive s o l u b i l i t y data fo r rifampin

484

Figure 7 - X-ray powder di f fract ion pattern of rifampin before grinding.

T a b l e VI X-rav powder d i f f r a c t i o n p a t t e r n of r i f a m p i n before q r i n d i n q , a c c o r d i n q to ASTM r u l e s

d ( i )

5.18 4.90 4.63 4.43 4.12 4.05 3.95 3.82 3.67 3.44 3.38 2.96

0 Rad. Cuka x = 1.5418 A

F i l t e r N i

1/11 h k l

13 17 2

loo 5 2 3 6 3 8 8 5

I/I Spectrometer 1

d ( i )

17.16 12.43 11.11 10.76 9.40 8.80 7.86 7.51 6.91 6.51 6.21 5.60

Rifampin ( sample before g r i n d i n g )

1/11 h k l

2 8 3 1 1 23 35 2 14 4 2 44

3a 20 10 3" 2w -_- -

_L, d - d Y - - I .a Figure 8 - X-ray powder diffraction pattern of rifampin after grinding

Figure 9 - Heating curve of rifampin

RI F AMP IN

Solvent

Table VII Approximate s o l u b i l i t i e s of rifampin

P a r t s of so lvent required f o r 1 p a r t of rifampin

Descr ip t ive term

chloroform methanol dimethylformamide dimethylsulphoxide e thanol 95 per cent acetone benzene c o r bon t e t r ac h l o r i d e n-hexane cyclohexane n-butanal propyleneglycol g l y c e r o l carbowax 400

from 1 t o 10 from 10 t o 30 from 10 t o 30 from 10 t o 30 from 100 t o 1,000 from 100 t o 1,000 from to00 t o 10,000

f r e e l y so luble so luble s o l u b l e s o l u b l e s l i g h t l y so luble s l i g h t l y so luble very s l i g h t l y so luble p r a c t i c a l l y i n s o l u b l e p r a c t i c a l l y inso luble p r a c t i c a l l y inso luble p r a c t i c a l l y i n s o l u b l e p r a c t i c a l l y inso luble p r a c t i c a l l y inso luble p r a c t i c a l l y inso luble

I I

Table VIII

-- S o l u b i l i t i e s of rifampin

I I I chloroform 1 349 I ' dichloromethane I 216 , e t h y l a c e t a t e

dioxane methanol

I acetone l n-hexane j petroleum e t h e r

water pH 7.3

108 I

39 25oc I 38 i 16 ,

14 I 0.43 I I 0.33 2.5

water pH 4.3 1.3 water pH 7.5 water pH 2.0 1 99.5 O.tN Hcl

2.8 'room j 46

!2O0.O ;37oc 1 47 ' 1 phosphate buffer pH 7.4 , 9.9 d ,

489


0.029

-0.293

2.62 Lipid-water p a r t i t i o n 2.621 Organic solvent-water P a r t i t i o n

The study of the p a r t i t i o n between water and organic so lvents a s an i n d i r e c t measure of t h e l ipid-water p a r t i t i o n has not been general ly applied t o t h e f i e l d of rifamycins. the system n-octanol/aqueous phosphate buffer, pH 7.4 was determined by Seydel (47) t o be K=15.6, w h i l e Curc i e t al. (48) have measured it i n the system benzene/ aqueous buf fers i n the range pH 5.5-7.0, andKequaled 9.7; a t pH 7.5, K=9.0; a t pH 8.0, K=4.6.

T h e p a r t i t i o n of rifampin i n

2.622 Si l i con oil-water p a r t i t i o n The l ipid-water p a r t i t i o n has been

obtained f o r var ious rifamycins from Rf values i n par- t i t i o n reverse-phase t h i n l ayer chromatography on S i l i c a g e l p l a t e s impregnated w i t h s i l i c o n o i l as sta- t ionary phase and water-acetone a s mobile phase (49-51 ).

f o r quan t i t a t ive co r re l a t ions between s t r u c t u r e and a n t i b a c t e r i a l (49,50) and a n t i v i r a l (51) a c t i v i t i e s .

and a r e reported i n Table IX.

The f r e e energy parameter RM=log(- 1 -1) (52), was used R

RM values were obtained f o r rifampin

30% acetone i n s i l i c o n o i l 49 water (v/v)

40% acetone i n s i l i c o n o i l water (v/v) I 50

Table I X RM values f o r rifampin

I RM 1 mobile phase ls ta t ionary phase I Ref. I

2.623 Surface a c t i v i t y Rifamycins have been shown t o have

sur face a c t i v i t y , from the va r i a t ion of the surface tension of buffer water so lu t ions w i t h concentration (53 I 54).

490

RlFAMPlN

The sur face proper t ies of rifampin depend on t h e pH of t h e medium. I n the a lka l ine t o neu t r a l range, rifampin i s a weak su r fac t an t and no assoc ia t ions a r e observed; i n the ac id i c range (pH 4-0) a pronounced lowering of the sur face tension w i t h concen t r a t ion is observed, and micel le formation is apparent a t a concentrat ion of about 10-5 mole/ l i ter (55)

3. STABILITY

3.1 STABILITY AS POWDER

sealed conta iners a t room temperature, a s described i n Table X (56). a l s o a t temperatures u p t o 70OC, as reported by Sano e t a l . (38). 3.2 STABILITY IN SOLUTION

been widely invest igated and t h e condi t ions and the transformation products a r e reported i n Table XI. other rifamycins, rifampin undergoes desace ty la t ion on a lka l ine t rea tment ,y ie ld ing the corresponding 25-des- a c e t y l de r iva t ive without s u b s t a n t i a l l o s s of an t i -

b a c t e r i a l a c t i v i t y (57).

presence of atmospheric oxygen a t room temperature, rifampin transforms i n t o rifampin quinone; t h i s oxidation can be prevented by sodium ascorbate. the same condi t ions and a t 60-70OC, rifampin y i e lds three main transformation products, which were iden- t i f i e d by Maggi e t a1.(22) a s 25-desacetyl-rifampin, 25-desacetyl-23-acetyl-rifampin and 25-desacetyl- 21 -acetyl-r i f ampin,

rifampin a t 250 i n aqueous so lu t ions a t d i f f e r e n t pH's: it decomposes rap id ly i n a c id i c o r a lka l ine condi t ions, but slowly i n neu t r a l ones. the main decomposition product of rifampin i n aqueous

Rifampin is very s t a b l e i n the s o l i d s t a t e i n

Rifampin i n t h e s o l i d s t a t e i s s t a b l e

The s t a b i l i t y of rifampin i n aqueous so lu t ion has

L i k e

I n mildly a lka l ine aqueous so lu t ions and i n the

Under

Sano e t a l . (38) have s tudied t h e s t a b i l i t y o f

3-Formylrifamycin SV is

491

Table X

s t o r t ing 12 months 21 months 30 months

' UV Microbig UV Microbig UV Microbig UV Microbig kssay l o g i c a l Assay l o g i c a l Assay l o g i c a l Assay l o g i c a l

% Assay % % Assay % % Assay % % Assay % c

96.4 99.0 100.0 100.2 95.4

P (D N

41 months

UV Microbig Assay l o g i c a l

% Assay %

I 97.3 95.9

S t a b i l i t y of rifampin i n t h e s o l i d s t a t e a t room temperature (56) -

I

I I TLC

t ~ f o r r n v l r i f a - i

TLC 1 I I I TLC TLC j

TLC ,

I I -

i 1 1.5-2% I 2 . 5 4 % rifampin 1 absent 1-1.5% 1 1.5-2%

: rifampin N- ! ! t r a c e s

oxide ! 1 1-1.5% 1 1-1 .!j% I 1-1.5%

1 1-1.5% I

25-desacetyl 1 1.5 - 2% 1 1 - 1.5% 1 1-1.5% rifampin 1 I i i , 0.5 - 1% j 1.5 - 2%

acetyl-rifampin I I I

R I F AMP IN

Tab le X I

S t a b i l i t y o f r i f a m p i n i n aqueous s o l u t i o n s

3-formyl-r i famycin SV r i f ampin-quinone + + pH 2.3, 20-22OC (8) tic1 0,1N, 37% (47)

pH 8.2, 20-22% (8)

r i f ampin

pH 8 .2 (22) 60-70% -i NaOH 5% i n e thanol

water ( 1 : l ) 20- 22% (57)

t 25-desacetyl-21-acetylrifampin

I 1

bl 25-desace ty l -23 -acety l r i f am p i n I

493


Time ’ Concentra- tion( mg/ml) Conditions

acidic medium (8). composition r a t e of rifampin i n 0.1N tC1 vs. temperature and, from the Arrhenius plot, calculated the activation energy, &la=l9.2 Kcal/mole/degree.

Rifampin does not lower its microbiological activ- i t y i n the various conditions reported i n Table XII.

Seydel (47) has determined the de-

Table XI1 S t a b i l i t y of the an t ibac ter ia l ac t iv i tv of rifamPin i n solution.

Ref . water-dimethyl- f ormamide ( 95 : 5) a t 5%

d ime t hyl - sulphoxide a t 150

1 5% days 58

10 8 months 59

t I I I I I I i

water-ethanol (8:2) a t 4% or 20%

1 8 weeks 6O,6l

4. SYNTHESIS

by condensation of 1 -amino-4-methylpiperazine w i t h 3- formyl-rifamycin SV i n peroxide-free tetrahydrofuran a t 100-150C. The 3-formyl-rifamycin SV (14) was obtained from rifamycin B, the fermentation product (62-64) , by the chemical procedure reported i n table XIII. tonio e t al . (65) have patented the production of rifampin by reacting rifamycin S d i r e c t l y w i t h aldehyde, tert-butylamine and MnO and condensing w i t h 1 -amino-4-methylpiperazine (see table XIII).

Rifampin is a semi-synthetic rifamycin, obtained

Gianan-

form- 2

494

R I F AMP IN

Table X l l l

Synthesis of r if a mpin STREPTOMYCES MEDITERRANEI

1 w H3C*

oxidation

reduct ion 0 0 I!

CH2-COOH '0

\ RlFAMYClN B(62-64) (fermentation product)

in aerated aqueous

solution

RlFAMYClN 0 ( 66)

Acidic hydrolyais

\ OH 0 oxidation

reduction

OH OH

Mannich reaction and reduction

OH OH

H3cf& CH2-N /RI

OH 'R2

RlFAMYClN SV(67)

tart-but lamine, dn02 in

tetrahydrofuran

2) l-amino- 4 -methyl-

piperazine (65) MANNICH DERIVATIVE OF RlFAMYClN SV (15)

Oxidation in acidjc / conditions

OH OH

"3c@cH>-ami;;~::thyl

OH i-NnN-CH3 OH v

RIFAMPIN ( 8 ) 3-FORMYL RlFAMYClN SV (14,681

495


5. METHODS OF ANALYSIS

5.1 ELEMENTAL ANALYSIS

Element C H N 0

T h e o r e t i c a l % 62.76 7.10 6.81

23.33

5.2 IDENTIFICATION TESTS Ri fampin i s i d e n t i f i e d and d i f f e r e n t i a t e d from

t h e o the r r i f a m y c i n s by i n f r a r e d and nuc lear magnetic resonance spectroscopy, f i e l d deso rp t i on mass s p e c t r o metry, po ten t i omet r i c t i t r a t i o n , d i f f e r e n t i a l scanning c a l o r i m e t r y and e lementa l ana lys i s , I n o rde r t o d e f i n e t h e homogeneity o f t h e r i f a m p i n sample, chromatographic procedures (paper and t h i n l a y e r ) and the rma l a n a l y s i s are used.

C o l o r i m e t r i c t e s t s based on o x i d a t i o n were descr ibed, 1 mg/ml), I m l o f 10% (w/v) ammonium persulphate i n phosphate b u f f e r , pH 7.38, i s added: t h e c o l o r t u r n s from orange-yellow t o v i o l e t - r e d (69). thod, employing f e r r i c n i t r a t e as o x i d i z i n g agent, was descr ibed by Eidus e t a l . (70,71), who used i t t o iden- t i f y the presence o f r i f a m p i n and desace ty l r i f a m p i n i n b i o l o g i c a l f l u i d s .

5.3 SPECTROPHOTOMETRIC METHODS

To 5 m l o f aqueous r i f a m p i n s o l u t i o n (about

A s i m i l a r me-

5.31 Spectrophotometr ic assay on b u l k product The V I S maximum a t 475 nm i n aqueous phos-

phate b u f f e r s o l u t i o n pH 7.38 w i t h an a b s o r p t i v i t y (g/a) value o f 18.7 (see Sec t ion 2.11) enables r i f a m p i n t o be q u a n t i t a t i v e l y assayed.

f o r m y l r i f a m y c i n SV and r i fampin-quinone. The two products can be spec t ropho tomet r i ca l l y quan t i t a ted : t h e

f i r s t one by an e x t r a c t i o n method us ing n-hexane:ethylacetate ( 2 : l ) (13) and the second one by a d i f f e r e n t i a l spectrophotometr ic method which takes advantage o f t he r e d u c t i o n by sodium ascorbate o f t h e quinone ( 1 3) .

The main i m p u r i t i e s o f r i f a m p i n a re 3-

496

RlFAMPlN

5.32 Spectrophotometric assay on pharmaceut ical p repara t ions Ri fampin can be determined i n pharmaceuti-

c a l preparat ions by us ing t h e d i f f e r e n t i a l spectropho- tomet r ic method (72), modi f ied by Pasqualucci e t a l .

(73) 5.33 Spectrophotometric assay i n b i o l o n i c a l

f l u i d s Numerous spectrophotometr ic methods f o r de-

te rmin ing r i f a m p i n i n b i o l o g i c a l f l u i d s have been desc r ibed i n t h e l i t e r a t u r e , bu t owing t o t h e i r low sen- s i t i v i t y they were s a t i s f a c t o r i l y app l ied o n l y t o t h e determinat ion o f r e l a t i v e l y h i g h l e v e l s (more than 5pg/ml). The methods were based on the e x t r a c t i o n o f r i f a m p i n and i t s metabol i tes w i t h organic so lvents and on the de terminat ion o f the V I S absorbance o f t h e or - ganic e x t r a c t . marized i n Table XIV.

5 . 4 FLUORQMETRIC DETERMINATION Rifamycins do n o t e x h i b i t n a t u r a l f luorescence. A

q u a n t i t a t i v e method was developed f o r r i f a m y c i n B (82), based on i t s t rans format ion i n t o the t r i a c e t y l der iva- t i v e . forming i t w i t h hydrogen peroxide (83) i n t o a f l u o r e - scent product. The maximum f luorescence develops i n an aqueous carbonate-bicarbonate bu f fe r , pH 9.2 a t 4-80 nm, when t h e e x c i t a t i o n wavelength i s 370 nm. The r e l a t i v e f luorescence i n t e n s i t y i s l i n e a r w i t h concentrat ions o f r i f a m p i n i n t h e range 0.1 t o 10 pg/ml.

5.5 ANALYSIS BY COMPLEX FORMATION

f o r r i f a m y c i n L (34) and f o r r i f a m y c i n S (84) .

t r e a t e d w i t h an aqueous s o l u t i o n o f A l C l 2:l (85); t h e i n t a b i l i t y constant i n waqer-methanol ( 1 : l ) i s 3.4.10 . Complex format ion was demonstrated t o take p lace a l s o w i t h Hg*, Cu", Ag' and Fe- (86) .

The exper imenta l c o n d i t i o n s are sum-

Ri fampin was determined f l u o r e m e t r i c a l l y by t rans-

Ri famycins complex w i t h m e t a l l i c ions, as repor ted

Rifampin i n methanol s o l u t i o n g ives a complex when i n t h e r a t i o

8

497


b i l e , u r i n e

Table X I V

Spectrophotometric methods f o r t he de te rm ina t ion o f r i f a m p i n and i t s me tabo l i t es i n body f l u i d s .

benzene RtDA

1 Compound

475nm and 335nm ( c a l i - b r a t i o n curve)

335nm ( c a l i - b r a t i o n curve)

478nm ( c a l i - b r a t i o n curve)

Assayed ar s t a t e d i n t h e r e f 2

Body 1 E x t r a c t i o n f l u i d so l ven t

75

76

I rence I

478nm (19.6) 77

u r ine , serum

482nm (15.6)

iso-amyl a l c o h o l

79

I R+DA

aerum

u r i n e

e t h y l a l c o h o l - e t h y l a c e t a t e R (1 :1)

I R I$;;ne-hexane

475nm (15.5)

u r i n e , serum, b i l e and t i ssues

RtDA butanol-hexane (4 : l )

80

u r i n e

~~ ~

u r i n e

1 I

cyclohexane- b u t y l ace ta te j R

I R 1 :;;p;ol-hexane

I(i :i)

t e e t h

' t i o n curve) I

butanol-hexane I (4 : l )

I R / I

serum 1;;;;;oLheptane I R

A n a l y t i c a l

e ference wavelength :abrorpt i v i t y , 9/11 *-

478nm ( C a l i - t i o n curve)

b lue l i g h t f i

498

RlFAMPlN

The format ion o f the complex w i t h A1C13 was employed fo r quan t i t a t i ve determinat ion o f r i f amp in i n bulk, i n pharmaceutical formulat ions and i n u r i ne (87), by measuring the cherry-red co lo r a t 507nm i n methanol- water (49:l) o r i n methanol-water-butanol-hexane (1 9: 1 :4:1),

5.6 VOLUMETRIC METHODS Rifampin i n capsules was determined by a volu-

met r ic method (88): the a n t i b i o t i c was ox id ized w i t h an excess o f f e r r i c chlor ide, which was then t i t r a t e d iodome tr i c a l l y .

5.7 CHROWTOGRAPHIC METHODS 5.71 Thin l aye r chromatography

TLC has been widely used i n the f i e l d o f r i famycins (89,90) and t h e co lored spots are d i r e c t l y located.

analys is o f r i fampin and i t s metabol i tes i n body f l u ids . The Rf values were shown t o be dependent on the concen- t r a t i o n s (91). The spots were quant i ta ted by using the bioautographic technique (92) o r the densi tometr ic one (93) o r by v i s u a l est imat ion (94) or, f i n a l l y , by the e l u t i o n method fol lowed by m ic rob io log i ca l assay (95). A reverse,,phase p a r t i t i o n TLC procedure has been descr ibed f o r the determinat ion o f r i f amp in (96) : s i l a n i z - ed S i l i c a g e l p la tes were used as s ta t i ona ry phase, phosphate buf fer , pH 7 conta in ing 0.1 % sodium ascorbate as mobile phase (other mobile phases are reported), The colored spot was e lu ted and measured spectrophotome- t r i c a l l y . Reverse-phase p a r t i t i o n TLC has been used f o r s t ruc tu re -ac t i v i t y co r re la t i ons (see Sect ion 2.622).

Many TLC procedures were developed f o r the

w i t h

5.72 Column chromatography The content o f r i f amp in and i t s metabol i tes

i n urine, b i l e and serum, was determined by ex t rac t i on w i t h chloroform and by column chromatography, fo l lowed by spectrophotometry (97). The l i q u i d - s o l i d chromatography was c a r r i e d out us ing a glass column 7 cm long, 4 mm I.D., packed w i t h S i l i c a g e l G, buf fered a t pH 6, and chloroform and chloroform-methanol i n progress ive ly

499


i nc reas ing amounts (5%, 10% and 16.6%) as so lvent , a t a f l o w r a t e o f 0.15 ml/min. Ri fampin and i t s me tabo l i t es were then q u a n t i t a t e d by read ing the absorbance o f t h e e l u a t e s a t 475 nm.

5.73 Paper chromatoqraphy A paper chromatographic technique f o r de-

t e r m i n a t i o n o f r i f a m p i n and 25-desacety l r i fampin was used f o r u r i n e and b i l e (74). was c a r r i e d o u t on Whatman 3MM paper us ing methanol: n- o c t a n o l (4 : l ) as s t a t i o n a r y phase and aqueous buf fer , pl-l 6 , as mobi le phase f o r 6 hr. The i n t e n s i t y o f t h e red-orange spots was measured by an A n a l y t r o l photoden s i t omete r (mod.Rl3-450 nm f i l t e r ) o r t he m a t e r i a l s determined b ioau tog raph ica l l y .

Akimoto e t a l . (98) repo r ted a paper chromatographic method f o r t h e separa t i on o f r i f a m p i n and i t s metabol i tes, us ing e t h y l acetate-water-dimethylformamide ( 1 O : l O : l ) .

5.74 H igh pressure l i q u i d chromatography

Descending chromatography

Ri fampin has been f r e q u e n t l y c i t e d as an emample o f t h e usefulness o f HPLC (99-102).

r i f a m y c i n SV were separated (99) under the f o l l o w i n g cond i t i ons : DuPont 820 Chromatograph equipped w i t h UV detector , ODS Permaphase column a t 50% and 1,000 ps i , water t o methanol w i t h l i n e a r g r a d i e n t 8%/min as mobi le phase and lml/min f l ow r a t e .

5 . 8 MICROBIOLOGICAL METHODS Micro b i o l o g i c a l met hods have been w i d e l y descr ibed

f o r t he de te rm ina t ion o f r i f a m p i n potency i n bu l k products, i n pharmaceut ical f o rmu la t i ons and i n body f l u i d s , as l i s t e d i n the review by Binda e t a l . (103). methods can be c l a s s i f i e d as a) d i f f u s i o n p l a t e methods, b) s e r i a l tube d i l u t i o n methods and methods,

Rifampin, 25desacetylr i fampi11, r i f a m p i n quinone and 3 - fo rmy l

These

c) t u r b i d i m e t r i c

5.81 D i f f u s i o n p l a t e assay methods These methods were used t o determine r i-

fampin i n serum, b i l e and u r i n e as w e l l as i n o the r body f l u i d s and organs (fragments o f organs and t i s s u e s were

500

RlFAMPlN

a p p r o p r i a t e l y t r e a t e d f o r e x t r a c t i n g r i f amp in ) . can be d i v i d e d i n d i f f e r e n t c lasses according t o tech- n i c a l aspects as repo r ted i n Table XV, where o t h e r ex- pe r imen ta l d e t a i l s a re summarized, The c y l i n d e r - p l a t e technique descr ibed by Grove e t a l . (104) has been adopted i n L e p e t i t S.p.A. (105)

They

5.82 S e r i a l tube d i l u t i o n methods T h i s method, f i r s t app l i ed by C l a r k e t a l .

(115) t o r i f a m i d e and r i f amp in , has been used f o r t he de te rm ina t ion o f r i f a m p i n i n serum by va r ious labs. Some d e t a i l s a re repo r ted i n Table XVI .

A t u r b i d i m e t r i c m i c r o b i o l o g i c a l assay o f 5.83 T u r b i d i m e t r i c met hods

r i f a m p i n us ing an automat ic analyzer was developed by Simoncin i e t a l . (120). The method i s based on the measurement o f t h e o p t i c a l d e n s i t y o f t h e b a c t e r i a l suspension ( K l e b s i e l l a Pneumoniae ATCC 10031 o r Esher i ch ia c o l i ATCC 10536 as t e s t microorganism) i n a D i f c o c u l t u r e medium c o n t a i n i n g r i fampin, a f t e r incu- b a t i o n a t 370 f o r 3.5 hr.

6 PROTEIN BINDING The b i n d i n g o f r i f a m p i n t o blood serum and plasma

p r o t e i n s i n man and o the r an imal species was w i d e l y studied. range, probably because o f t h e d i f f e r e n t methodologies used and o f t h e many parameters t h a t can i n f l u e n c e t h e phenomenon (1 21 ) . a l . w i t h a d i a l y s i s technique, and they r e p o r t e d t h a t a t t h e concen t ra t i ons reached i n v ivo, about 7545% o f r i f a m p i n b inds w i t h serum p r o t e i n s (48,122-125). I n - t e r e s t i n g l y , t hey found t h a t PAS ( para-amino-sal icylate) i n concen t ra t i ons from 100 t o 200 pg/ml decreases t h e b i n d i n g o f t h e a n t i b i o t i c (122). f u g a t i o n technique, Seydel (47) obta ined a percentage o f r i f a m p i n bound t o bovine albumin i n v i t r o i n t h e range from 50 t o 70%. From these data, Keber le e t a l . (126) de r i ved t h a t each albumin molecule has two b i n d i n g s i t e s f o r r i f amp in .

The d a t a obta ined cover a r e l a t i v e l y l a r g e

I n v i t r o experiments were c a r r i e d o u t by C u r c i e t

By an u l t r a c e n t r i -

Mashimo (127) has found t h e p r o t e i n

501

Table XV

Microbio loqica l ass= of r i fampin by d i f f u s i o n p l a t e assay methods

Medium Microorganism

Agar (Difco 0263-02) ISarcina l u t e a (ATCC 9341) ,

Technica l a s p e c t s Reference

1 05 metal c y l i n d e r s containing - t h e l i q u i d t o be assayed wells ( l o m m ) con ta in ing t h e l i q u i d t o be assayed

wells (9mm)

d i s k s (6mm) soaked i n the l i q u i d t o be assayed

d i s k s (9mm) d i s k s

d i s k s

: d i s k s (9mm) Salt-glucose-peptone k o r c i n a l u t e a (IP 5345) broth (Pas t eu r Inst. 1 Codex) I

112

1 ~ d i s k s (9mm) ' Casein peptone-soya !Staphylococcus coagulase 113

'Sarc ina l u t e o (ATCC 9341) j 114 I 1 d i s k s ( 6 m m )

1 peptone-agar lnega t ive I

' Agar (Difco 0263-02) 1 I-. i

RlFAMPlN

Microorganism

Sarcina l u t e a

Staphylococcus aureus (ATCC 6538P) Staphylococcus aureus (Oxford, s e n s i t i v e t o O.O2ug/ml) Mycobacterium t u b e r c u l o s i s

(H37RV)

Table X V I

M i c r o b i o l o q i c a l assays o f r i f a m p i n by s e r i a l tube d i l u t i o n methods.

Inoculum

0.031111 o f a 1 :250 d i l u t i o n 115 o f 18h c u l t u r e 0.05ml o f a 1 : lo0 d i l u t i o n 116 o f 18h c u l t u r e 0.05ml o f a 1 :200 d i l u t i o n 117 o f 24h c u l t u r e

5-1 OO*1O4 v i a b l e u n i t s 118

i e f e r e nc e Medium

Mycobacterium t u b e r c u l o s i s (H37Rv o r F u hrmann

Loc kemann -

non-spec i f i e d b r o t h

119 j

D i f c o sucrose b r o t h w i t h phenol r e d mannite and phenol red s e l e c t i v e b r o t h Youmans

b ind ing o f r i f a m p i n by d i a l y s i s , u l t r a f i l t r a t i o n and u l t r a c e n t r i f u g a t i o n , a t a concent ra t ion o f 2Opg/ml, t o be lo%, 90% and 70%, respec t ive ly . t a t i o n was g iven f o r the d i f f e r i n g data,

I n v i v o experiments o f p r o t e i n b ind ing were c a r r i e d ou t us ing ' ' k - r i fampin (128) and about 75% o f r i f a m p i n was found t o be bound t o human serum pro te in , P i l h e u e t a l . (129) found r a t i o s o f 15 t o 40% between cerebrosp ina l f l u i d (considered as a p r o t e i n - f r e e f l u i d ) and plasma l e v e l s . b ind ing o f r i f a m p i n t o the serum p r o t e i n s o f var ious animals: i n the horse 40-46%, i n t h e r a b b i t 13-17% and i n man 17-38%. r i f a m p i n (131) showed t h a t 86.1% and 88.9% o f r i f a m p i n were bound t o t h e plasmas o f tubercu la r p a t i e n t s and

No c l e a r i n t e r p r e -

Aoyagi has repor ted (130) t h e

A recent study by d i a l y s i s us ing

503


healthy volunteers, respect ively. have suggested, on t h e basis of e lectrophoresis and microbiological assay, t h a t rifampin assoc ia tes w i t h a1-t 012- and P-globulins of human serum.

Yokosawa e t a l . (1 32)

7. PHARM4COKINETICS AND METABOLISM I N

s t u d i e d and the serum leve l s of rifampin a f t e r s ing le and repeated administration of d i f f e r e n t doses were determined by various inves t iga tors and have been reviewed (103). rifampin is well-absorbed w i t h the maximum plasma lev- e l s a t 1.5-3 h r over a wide range of s ing le dose, i .e . , 0.1 t o 1.2 g. The l eve l s a r e s t i l l appreciable (5-10% of the peak l eve l ) a f t e r 12 h r only a t the high doses (103).

i n the various body f l u i d s (105), as indicated i n Table X V I I . fampin (47,413).

Rifampin el iminat ion, which m u s t be considered slow, occurs mainly through the b i l e b u t a l s o through the ur ine (103,128,133). inated i n the b i l e i s not proportional t o t h e dose administered (48,116,134) while t h e urinary el iminat ion increases w i t h t he dose (77,105,109,116,133-136). I n an inves t iga t ion car r ied out over 6 days (105), about 22% was eliminated i n the faeces and about 26% i n the u r i n e , I n another study, however, Riess(128) found t h a t 96 hrs a f t e r administration of 300 mg of labeled rifampin, 94% of the t o t a l r ad ioac t iv i ty had been recovered i n equal percentages i n the faeces and ur ine,

The main metabolite of rifampin i n man was iden- t i f i e d , a f t e r i so l a t ion from b i l e and urine, a s 25- desacetylrifampin (74,92,94,137). less l ipoph i l i c than rifampin and it is e a s i l y excreted i n urine (46) and not reabsorbed (138,139). (93) reported t h a t i n addi t ion t o 25 -de~ace ty l r i f ampin~ a second metabolite i s 3-formyl rifamycin SV.

The pharmacokinetics of rifampin has been widely

To summarize, a f t e r o r a l administration,

Rifampin is widely d i f f u s i b l e i n the t i s s u e s and

T h i s i s re la ted t o the h i g h lipotropism of ri-

The t o t a l amount of rifampin elim-

T h i s compound is much

Sano e t a l ,

504

R I F AMP IN

Table XVII Distribution of rifampin i n human tissues and f lu ids a f t er oral administration of a s ingle 450 mg dose (105)

16 spleen l 2 f lung

adm i n i- stration

6 I-\ D l i v e r 36

13 I ] gallbladder wall

colon wall

12 s k i n muscle

12 kidney

15 mammary gland

ovarian cyst wall b

505


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51 3

SULF ASALAZINE

J. Patrick McDonnell

J. PATRICK McDONNELL

CONTENTS

1.

2.

3 .

4.

5 .

6.

7 .

8 .

9.

10.

11.

Analy t i ca l P r o f i l e - Sul fa sa l az ine

Descr ip t ion 1.1 Name Formula, Molecular Weight 1.2 Appearance, Color Odor

Phys ica l P r o p e r t i e s 2 . 1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2 . 3 U l t r a v i o l e t - v i s i b l e Spectrum 2.4 Mass Spectrum 2.5 Melt ing Range 2.6 Di s soc ia t ion Constants 2 . 7 S o l u b i l i t y 2.8 D i s t r i b u t i o n Coef f i c i en t

Synthes is

Impur i t i e s - S t a b i l i t y

Drug Metabolic Products

Methods of Analysis 6 .1 U l t r a v i o l e t Spectrophotometry 6.2 Q u a n t i t a t i v e Thin-Layer Chromatography 6 .3 Column Chromatography 6.4 Polarography 6.5 T i t r i m e t r i c Analysis 6 .6 High Speed Liquid Chromatography

D i s t r i b u t i o n i n F lu ids and Tissues

P ha rma c okine t i c s

Determination i n Body F lu ids and Tissues 9 . 1 Spectrophotome t r y 9 .2 Polarography 9 . 3 Paper Chromatography

Acknowledgments

References

51 6

SULF ASALAZIN E

SULFASALAZINE

1. Descr ip t ion

1.1 Name, Formula , Molecular Weight Su l f a sa l az ine i s known chemical ly a s benzolc a c i d ,

2 -hyd;oxy-5 -L 14- / (2 -pyr i d inylamino) sul fonyi lphenyi la 227- ; 5-1 b- (2-pyr i ~ y l ~ u l famoyl) phenyi/azg/ sa l i c y l i c a c i d (1) o r 4-Tpyr idyl-2-amidosul f onyl) -3 -carboxy-& -hydroxya zobenzene (2 ) . Common names f o r t h i s drug substance a r e s a l a z o s u l f a - py r id ine and s a l i c y l a z o s u l f a p y r i d i n e (2).

.. COOH

Molecular Weight 398.39

1 .2 Appearance, Color , Odor The compound i s a b r i g h t yellow t o brownish-

yellow, o d o r l e s s , f i n e powder.

2 . Phys i ca l P r o p e r t i e s

2 . 1 I n f r a r e d Spectrum The i n f r a r e d abso rp t ion spectrum of s u l f a s a l a z i n e

(Salsbury Reference Standard XP-2) i s presented i n F igure 1. The spectrum was taken i n a potassium bromide p e l l e t w i th a Perkin-Elmer, Model 735B I n f r a r e d Spectrophotometer. The assignments a r e a s fol lows: 2500 - 3400 OH s t r e t c h (bonded) 1670 - 1680 C = 0 v i b r a t i o n 1580 - 1630 C = C o r N = N v i b r a t i o n s 1360 C02NH v i b r a t i o n 1280 - 1290 SO2 asymmetrical 1260 - 1278 C - 0 s t r e t c h i n g v i b r a t i o n o r OH deformation 1170 - 1195 S02NH 1135 SO2 group - symmetrical 770, 790, 800 CH deformation (aromatic r i n g )

51 7

H a 8 - N

8 9 0

s 8 w ? N 8 N 0

0

w 0

f 0

0

0

N

NO

lSS

lWS

NV

Ml lN

33M3d

tn m

W

rl 7

cn I

C

H

51 8

SULFASALAZINE

2.2 Nuclear Magnetic Resonance Spectrum The nuc lea r magnetic resonance spectrum of s u l f a -

s a l a z i n e (U.S.P. LN231547, Lot F) i s shown i n F igure 2 . The spectrum was run on a Varian, Model HA-100 a s 100 MHZ pro ton spectrum us ing t e t r ame thy l s i l ane a s the r e fe rence lock s igna l . Sweepwidth was 1000 cyc le s i n the f i e l d sweep mode. Deuterated N , N-dimethylformamide (d7 - DMF) was the so lven t . The s p e c t r a l p a t t e r n a r i s e s from aromatic pro tons on d i f f e r e n t r i ngs . These a r e found i n the r eg ion of 6 .8 ppm t o 8.8 ppm. Each of the smal l s epa ra t ed groups co r re - sponds t o one proton. Three groups l i e u p f i e l d of the r eg ion of mixed absorp t ions and one l i e s downfield ( 3 ) .

2.3 U l t r a v i o l e t - v i s i b l e Spectrum The u l t r a v i o l e t - v i s i b l e sDectrum of s u l f a s a l a z i n e

(U.S.P. LN231547, Lot F) i s shown i n Figure 3. us ing a Cary, Model 15 Spectrophotometer on a 1.9 x 10-%I s o l u t i o n of the compound. The maximum and minimum absorp- t i o n s a r e s i m i l a r t o those repor ted i n the l i t e r a t u r e (4, 5). I n the pH range of 1 t o 10 a ueous s o l u t i o n s of s u l f a - salazi i le (concent ra t ion 1.9 x 10-$4) exh ib i t ed s p e c t r a l abso rp t ion maxima a t 235-240 nm and 350-360 nm. Absorpt ion minimum was shown a t 285-290 nm. A t pH above 11.6 absorp- t i o n maxima were exh ib i t ed a t 450 nm, 235-240 nm and about

It was made '

290 nm (4).

2.4 Mass Spectrum The low r e s o l u t i o n mass spectrum of s u l f a s a l a z i n e

(U.S.P. LN231547, Lot F) was obtained us ing a Varian/MAT CHq Mass Spectrometer. The sample temperature was 3600 C (See Figure 4) .

i n a t i o n of S02H (m/e = 65) from the molecule (M - 65 = 333) followed by e l imina t ion of C 0 2 ( m / e = 45) from the molecule (m - 109 = 289). This may i n d i c a t e an o r i g i n a l molecular s t r u c t u r e involv ing c y c l i z a t i o n of the sulfonamide , pyr id ine n i t rogen and the 2 p o s i t i o n of t he neighboring phenyl group (6) .

The primary fragmentat ion p a t t e r n i n d i c a t e s e l i m -

2.5 Mel t ing Range It m e l t s a t about 255O C w i t h decomposition (1).

51 9

I I A I " " " ' ' ' ' ' ' I ' ' ' ' '

-3 I W 600 400 . 2c4

m

LO 9 8 7 6 5 . , , 0

~ . : . : , : . ~ . ~ . : . ~ , ~ . . . ~ . . . ~ , . . . . I . . . . 1 . . . . I . . . . t . . . . I . . . . 1 . . . . I . . . I . . . . j . . . . I . . 1 PPM I I

Figure 2 Nuclear Magnetic Resonance Spectrum - Sulfasalazine

1 (

O !

0.t

0.;

0.f QJ z 2 0.E

0 0 ’ 0.4

0.3

0.2

0.1

o.a

SU LFASALAZI NE

Figure 3 Ul travio let -v is ib le Spectrum

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1

I pH 12.3

Wavelength, MI

521

al c *rl N

m

rl m

rn m

w

rl 7

v)

rn rn

al &

1

M

522

1 0 0

9 0

80

7 0

6 0

5 0

Y O

3 0

2 6

1 0

5 0 1110 1 5 0

Figure 4 Mass Spectrum - S u l f a s a l a z i n e

SU LF ASALAZI N E

2.6 pK Values (Di s soc ia t ion Constants) The four d i s s o c i a t i o n cons t an t s of s u l f a s a l a z i n e

determined spec t rophotometr ica l ly a r e a s follows: 0 .6 ; pK2 = 2.4; pK3 = 9.7 and pQ = 11.8 (4) .

pK1 =

2.7 S o l u b i l i t y The s o l u b i l i t y of s u l f a s a l a z i n e i s a s fol lows (1) :

P r a c t i c a l l y in so lub le i n water P r a c t i c a l l y in so lub le i n e t h e r P r a c t i c a l l y in so lub le i n chloroform P r a c t i c a l l y in so lub le i n benzene Very s l i g h l y so lub le i n a l coho l Soluble i n aqueous s o l u t i o n s of a l k a l i hydroxides

3. Synthes is

and r e a c t i n g t h i s w i th an a l k a l i n e s o l u t i o n of s a l i c y l i c ac id ( 7 , 8). Other r epor t ed methods of p repa ra t ion a re : - a ) r e a c t i n g 5 - n i t r o s o s a l i c y l i c ac id wi th su l f apyr id ine , and - b) r e a c t i n g n i t roso - su l f apyr id ine wi th 5-aminosa l icy l ic a c i d (8) (See F igure 5) .

Su l f a sa l az ine is prepared by d iazo t i z i n g s u l fapyr id ine

4 . Impur i t i e s - S t a b i l i t y P a r t i a l c h a r a c t e r i z a t i o n of impur i t i e s i n commercial

s u l f a s a l a z i n e was repor ted by Stone e t a 1 (9). Of the t h r e e impur i t i e s s t u d i e d , one was proposed t o be a pos i - t i o n a l isomer of s u l f a s a l a z i n e ; the second t o be polymeric r e s u l t i n g from the formation of a benzyne in t e rmed ia t e ; and the t h i r d an undiazot ized sulfamide (See F igure 6) . The compound d id no t degrade when d isso lved i n dimethylformamide and was sub jec t ed t o thermal stress a t 80° C f o r 196 hours (10) .

5. Drug Metabol ic Products I n man, s u l f a s a l a z i n e i s metabolized by r educ t ive

c leavage , presumably by the gu t f l o r a , t o s u l f a p y r i d i n e and 5-aminosalicy i c ac id . The s u l f a p y r i d i n e p o r t i o n is then s u b j e c t t o &-ace ty l a t ion o r py r id ine r i n g hydroxyl- a t i o n , followed by conjugat ion t o glucuronic a c i d , o r both. The 5-aminosa l icy l ic ac id moiety i s N-acetylated (11) (See Figure 7 ) . In r a t s , s u l f a s a l a z i n e i s metabolized s i m i l a r t o t h a t i n man (12, 13).

523


Figure 5 Synthesis of S u l f a s a l a z i n e

Example 1: +

Example 2:

kOOH

Example 3:

+Q =O

I NH

Base

OH

Pcoon CH coon - 3 9 / 9

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Figure 6 Sulfasalazine Impurities

Benzyne Intermediate

Undiazot ized Sul famide

525


Figure 7 Su l fasa laz ine Metabolic Pathway

Sul fapvridfae 5 - A m i n o s a l i c ~ l i c Acid

COOH &-Acetyl su1f.D-

I N-Acetyl-5-aminosa l i c y l i c Acid

5 ' -HydKoxysulfaD.rr id i

&-Ace t y 1- 5 ' -hydroxmul f a m + r i d k

5 ' -Hydroxysul fapyridine -o-Rlucuranide

N4-Acetvl-5 ' -hvdroxvsul fapyridine-a-glucuronide

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6 .1 U l t r a v i o l e t Spectrophotometry Berggren e t a 1 (5) developed a spectrophotometr ic

procedure f o r s u l f a s a l a z i n e which cons i s t ed of d i s s o l v i n g the sample i n 0.1 E sodium hydroxide, a d j u s t i n g the pH t o between 4 and 5 wi th 0 .1 E a c e t i c a c i d and determining the absorbance i n a 1 cm qua r t z c e l l a t 359 mu. This method was a l s o r epor t ed i n the 1953 E d i t i o n of Tes t s and Standards f o r New and Nonof f i c i a l Remedies (14).

6.2 Q u a n t i t a t i v e Thin-Layer Chromatography (TLC) Two methods of q u a n t i t a t i v e TLC a n a l y s i s of s u l f a -

s a l a z i n e have been publ ished (1 , 15). Both use the same developing so lven t repor ted by Kiger e t a 1 (16) and a r e s i m i l a r i n the method of prepar ing and cond i t ion ing the developing tank. The Powell e t a 1 method (15) c o n s i s t s of d i s s o l v i n g the sample and r e fe rence s tandards i n dimethylformamide/methanol s o l u t i o n , s p o t t i n g the s o l u t i o n s on a TLC p l a t e , developing the p l a t e , c u t t i n g the s u l f a s a l a z i n e s p o t (Rf. 0.6-0.7) from the p l a t e and e l u t i n g i t i n a 0.016 N sodium hydroxide s o l u t i o n . The s o l u t i o n i s f i l - t e r ed and an a l i q u o t combined w i t h a 0 .1 so lu t ion . The absorbance of t he r e fe rence s tandard and sample s o l u t i o n s a r e compared a t 360 nm us ing water a s a blank.

The method provided i n the Four teenth E d i t i o n o f t he Nat iona l Formulary (1) involves d i s s o l v i n g the sample and r e fe rence s tandard i n dimethylformamide, s p o t t i n g the s o l u t i o n on a TLC p l a t e , developing the p l a t e , s c rap ing the s u l f a s a l a z i n e s p o t ( R f . about 0 .6) and e l u t i n g i t wi th dimethylformamide. s o l u t i o n absorbancies a r e compared a t 406 nm.

a c e t i c ac id

The r e s u l t a n t s tandard and sample

6 .3 Column Chromatography Stone e t a 1 (9) r epor t ed a n a s say procedure f o r

s u l f a s a l a z i n e us ing column chromatography. The s u l f a - s a l a z i n e sample i s d isso lved i n py r id ine then sepa ra t ed on a s i l i c i c a c i d column. The developing s o l v e n t system i s methyl e t h y l ketone/acetone/water (16: 16: 1) and the flow r a t e is 1.2 m l per minute. The second of the t h r e e bands t o e l u t e is s u l f a s a l a z i n e which is c o l l e c t e d and the so lven t removed by evapora t ion . The r e s u l t a n t sample i s taken up w i t h 0.1 E sodium hydroxide and t o t h i s is added

527


0.1 a c e t i c ac id and d i l u t e d t o volume wi th water . The absorbance of t h i s s o l u t i o n is determined a t 359 tun us ing water a s a blank.

6 . 4 Polarography A polarographic method of a n a l y s i s f o r s u l f a -

s a l a z i n e has been p r e l i m i n a r i l y o u t l i n e d by Nygard (17) and independent ly a l s o by Lastovkove e t a 1 (18). Based on the work of these i n v e s t i g a t o r s , Nygard et (19) developed a polarographic assay method which they used t o determine s u l f a s a l a z i n e p u r i t y .

6.5 T i t r i m e t r i c Analysis Berggren e t a 1 (5) a l s o developed a t i t r i m e t r i c

method of a n a l y s i s which was adopted i n the 1953 New and Nonoff ic ia l Remedies (14). The sample is d isso lved i n 2 ammonium hydroxide and d i l u t e d t o volume wi th water . The s o l u t i o n i s heated t o 70' C and carbon dioxide is passed through i t . Hydrochlor ic a c i d and 0.1 t i t an ium tri- c h l o r i d e a r e added and the mixture i s maintained a t 70° C u n t i l a l l t he p r e c i p i t a t e has gone i n t o s o l u t i o n . It i s cooled t o 150 C and the excess t i t an ium t r i c h l o r i d e i s t i t r a t e d wi th 0.1 f e r r i s ch lo r ide us ing 2 m l of 10% ammonium th iocyanate a s the ind ica to r .

6.6 High Speed Liquid Chromatography High speed l i q u i d chromatography methods of a n a l -

y s i s have been used t o determine the p u r i t y of s u l f a - s a l a z i n e bulk powder and t o q u a n t i f y the l e v e l of s u l f a s a l a z i n e i n t a b l e t e d dosage forms (10, 2 0 ) . Bighley -- e t a 1 (10)used a r eve r se phase C o r a s i l c18 column and 10% 2-propanol i n pH 7 . 7 phosphate b u f f e r a s the mobile phase t o accomplish ana lys i s . The s u l f a s a l a z i n e bulk drug o r t a b l e t was d isso lved i n dimethylformamide and propyl para- ben added as the i n t e r n a l s tandard . Quan t i t a t ion was e f f e c t e d by peak he igh t o r peak a rea us ing a UV photo- met r ic d e t e c t o r (254 nm r a d i a t i o n us ing a low p res su re mercury source) a s the d e t e c t i o n sys t e m .

- a 1 (20). Thei r mobile phase was 13% a c e t o n i t r i l e i n pH 7 . 3 phosphate bu f fe r and the i n t e r n a l s tandard was methyl meta-ni t robenzoate . The s u l f a s a l a z i n e was d i s so lved i n dimethylformamide. he igh t measurement and the d e t e c t i o n made a t 254 nm us ing

A C o r a s i l C 1 8 column packing was used by Frahm et

Q u a n t i t a t i o n was e f f e c t e d by peak

528

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a low p res su re mercury source.

7. D i s t r i b u t i o n i n F lu ids and Tissues Following intravenous i n j e c t i o n i n mice, s u l f a s a l a z i n e

a t tached t o connect ive t i s s u e and was p re sen t a l s o i n high concent ra t ions i n p e r i t o n e a l , p l e u r a l and synovia l f l u i d s , i n the l i v e r , and i n the i n t e s t i n a l lumen (21, 22) .

8, Pharmacokine t i c s

s u l f a s a l a z i n e and i ts me tabo l i t e s ob ta ined by Day 5 (dose 4 grams d a i l y ) were a s fol lows:

The s t e a d y - s t a t e serum concent ra t ions i n humans f o r

Sul fa s a l a z i ne Range Median

4 .7 t o 45 u d m l 12 u d m l To ta l Sul fapyr id ine Metabol i tes 37 t o 92 ug'jml To ta l 5-Aminosalicylic Acid Less than 2 ug/ml -

50 ug/ml

The u r ina ry e x c r e t i o n of unmetabolized s u l f a s a l a z i n e ranged from 1.7% t o 10% of the dose. Approximately 80% of the dose was excre ted i n the u r ine a s me tabo l i t e s of s u l f a - py r id ine and one- th i rd of the dose was excre ted a s the me tabo l i t e of 5-aminosa l icy l ic a c i d . Feces d i d n o t con ta in any s u l f a s a l a z i n e , bu t about 5% of the dose was p r e s e n t a s metabol i tes of su l fapyr id ine , and a n unknown amount a s metabol i tes of 5-aminosa l icy l ic ac id (11).

9 . Determinat ion i n Body F lu ids and Tissues

9 . 1 Spectrophotometry von Pora t (23) published two co lo r ime t r i c methods . . -

f o r the de te rmina t ion of s u l f a s a l a z i n e i n serum. One was a d i r e c t measurement of t he drug c o l o r i n a l k a l i n e serum. The serum blank, however, was h igh , corresponding t o 14 ug/ml of s u l f a s a l a z i n e . s e r a i n t e r f e r r e d cons iderably wi th the measurement. The o t h e r method involved a combined p r e c i p i t a t i o n and e x t r a c - t i o n method. The serum blank decreased t o 5 ug/ml and the i n t e r f e r e n c e from i c t e r i c s e r a diminished.

method s i m i l a r t o von P o r a t ' s . He used an a c i d i f i e d p o r t i o n of t he serum sample as a r e fe rence . The e f f e c t of s l i g h t hemolysis and i c t e r i c s e r a were thus , t o a l a rge e x t e n t , e l imina ted .

I n a d d i t i o n , hemolyzed and i c t e r i c

Bo t t ige r (24) repor ted a d i r e c t c o l o r i m e t r i c

529


Sandberg (25) presented a spectrophotometr ic method f o r determining s u l f a s a l a z i n e i n serum and u r ine by e x t r a c t i n g the compound i n t a c t and measuring spectrophoto- m e t r i c a l l y a t 455 run. I n b i l e and feces the drug was reduced t o su l f apyr id ine which was ex t r ac t ed and then determined wi th a s l i g h t l y modified Brat ton-Marshal l procedure. A f t e r t he e x t r a c t i o n s t e p , serum, u r ine , b i l e and feces gave low b lank va lues corresponding t o 0.3 and 1 ug/ml s u l f a s a l a z i n e i n serum and u r ine and less than 1 ug/ml i n b i l e and feces .

9.2 Polarography Nygard (17) descr ibed a polarographic method f o r

ana lyz ing s u l f a s a l a z i n e i n serum. Due t o the formation of a polarographic , non-reducible adduct between serum p r o t e i n s and s u l f a s a l a z i n e , 1,2-diphenyl-3,5-dioxo-4-n- b u t y l pyrazol id ine (Butazol idinR) was added t o d i sp l ace s u l f a s a l a z i n e from i t s molecular adduct wi th albumin.

9.3 Paper Chromatography Hanngren e t a 1 (21, 22) used paper chromatography

i n conjunct ion w i t h autoradiograms of the chromatograms t o semiquan t i t a t ive ly determine s u l f a s a l a z i n e and i ts meta- b o l i c by-products i n u r i n e , l i v e r e x t r a c t s and i n t e s t i n a l e x t r a c t s . iso-any1 a lcohol /water (35:35:30). Whatman No. 1 f i l t e r paper was used and the chromatograms were developed by descending chroma tograp hy .

The developing system was a mixture of p y r i d i n d

10. Acknowledgments The au thor wishes t o acknowledge the a s s i s t a n c e of D r .

Lyle D. Bighley i n reviewing t h i s a n a l y t i c a l p r o f i l e , Mary Lou Bullard f o r typing the manuscript and Harold E . Haus f o r prepar ing the a r t work.

11. References

(1) Nat iona l Formulary, Fourteenth E d i t i o n , Mack Publ i sh ing Co. , Easton , Pa. 667 (1975)

(2) The Merck Index, Eighth Ed i t ion , Merck & Co. , Inc. Rahway, N . J . 930 (1968)

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(3) James J. Downs, Personal Communication, Midwest Research I n s t i t u t e , Kansas C i t y , Mo. 64110

(4) Nygard, B. , Olofsson, J . & Sandberg, Acta Pharma- c e u t i c a Suecica 3, 313 (1966)

(5) Berggren, A. & Hansen, S . , Farm Rev. 34, 537 (1952)

(6) James J. Downs, Personal Communication, Midwest Research I n s t i t u t e , Kansas C i t y , Mo. 64110

(7) Doroswamy, K. R. & Guha, P. C. , Journal of Ind ian Chemical Soc ie ty 23, 278 (1946)

(8) U. S. Pa t en t 2,396,145 (Patented March 5 , 1946)

(9) Stone, J. C . & Gorby, R. , Journal of Pharma- c e u t i c a l Sciences 63, 1296 (1974)

(10) Bighley, L. D. & McDonnell, J. P. , Journa l of Pharmaceut ical Sciences 64, 1549 (1975)

(11) Schroder , H. & Campbell, D. E. S . , C l i n i c a l Pharmacolopy & Therapeut ics 12, 539 (1972)

(12) Peppercorn, M. A . & Goldman, P. , Gast roentero logy - 64, 240 (1973)

(13) Peppercorn, M. A. & Goldman, P. , Journa l of Pharmacology & Experimental Therapeut ics 181, 555 (1972)

(14) Tes t s & Standards €or New & Nonof f i c i a l Remedies, L ipp inco t t , Phi lade lphia , Pa. , 256-258 (1953)

(15) Powell , D. R. & Burton, B. A. , Journa l of Pharma- c e u t i c a l Sciences 63, 1290 (1974)

(16) Kiger , J. L. & Kiger , J. G. , Annales Pharma- ceu t iques Franca ises 24, 593 (1966)

(17) Nygard, B . , Svenska Kem. T i d s k r . , 333 (1958)

531


(18) Lastovkova, N. & Vackova, A . , Cesk Farm 1, 570 (1958)

(19) Nygard, B . , Olofsson , Pha rma ceu t i c a Sue c i c a

(20) Frahm, L. J . , Scherb, S a l s b u r y L a b o r a t o r i e s

J. & Sandberg, M. , Acts - 3 , 343 (1966)

Y. & McDonnell, J. P. , (unpubl ished d a t a )

(21) Hanngren, A . , Hansson, E. , S v a r t y , N. & U l l b e r g , S. , Acta Medica Scandinavica 173, 6 1 (1963)

(22) Hanngren, A. , Hansson, E . , S v a r t z , N. & U l l b e r g , S . , Acta Medica Scandinavica 173, 391 (1963)

von P o r a t , B. , Nord Med. 24, 2070 (1944) (23)

(24) B o t t i g e r , L. E . , Scand. J . Chem. Lab Inv. lo, 108 (1958)

(25) Sandberg, M. & Hanson, K. A . , Acta Pharmaceutica Suecica l0, 107 (1973)

I n t h e p r e p a r a t i o n of t h i s A n a l y t i c a l P r o f i l e , the l i t e r a t u r e was reviewed through September, 1975.

532

TESTOLACTONE

Klaus Florey

KLAUS FLOREY

1.

2.

3. 4. 5. 6.

7.

CONTENTS

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, odor Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Crystal Properties Synthesis Stability, Degradation Drug Metabolism Methods of Analysis 6.1 Elemental Analysis 6.2 Phase solubility Analysis 6.3 Colorimetric Analysis 6.4 Fluorometric Analysis 6.5 Non-aqueous Titration 6.6 Chromatographic Analysis

6.61 Paper 6.62 Thin Layer 6.63 column 6.64 Vapor Phase

Determination in Pharmaceutical Preparations 8. References

534

TESTOLACTONE

1. Description 1.1 Name, Formula, Molecular Weiqht

At -testololactone, l-dehydro-testololactone, 13,17 secoandrosta-1,4-dien-l7-oic acid, 13- hydroxy-3-oxo-~-lactone and D-homo-17a-oxaandros- ta-1,4-diene-3,17-dione. SQ 9538.

Testolactone is also known as

'1 gH2403 M.W. 300.38

1.2 Appearance, Color, Odor Testolactone is a white to off-white,

odorless crystalline powder.

2 . Physical Properties 2.1 Infrared Spectrum

The infrared spectrum (KBr pellet) of testolactone (batch 36B) is presented in Figure 1. It supports the presence of a lactone with a carbonyl stretching frequency of 1703 cm'l and an A ring dienone with a carbonyl stretching frequency at 1650 cm'l and C=C sf.r$iching frequencies of 1620 and 1595 cm- . This

and the spectrum given by Gual, Corfman and Rosenkrantz25. The same authors also reported frequencies in the fingerprint region 26 .

agrees with data presented by Fried et.al. 1,12

535

Figure 1. Infra R e d Spectrum of Testolactone (batch 36B). KBr Pellet. Instrument: Perkin-Elmer 621.

TESTOLACTONE

2.2 Nuclear Magnet ic Resonance Spectrum The 60 MHz pro ton spectrum i s shown i n

F i g u r e 2. There a r e 24 p r o t o n s p r e s e n t . The C - 1 8 and C-19 p r o t o n s occur a s 3-proton s i n g l e t s , t h e C - 1 proton i s a d o u b l e t , t h e C-2 p ro ton i s a q u a r t e t and the C-4 p ro ton appea r s a s a b road m u l t i p l e t (see Table 1) 24.

2 .3 U l t r a v i o l e t Spectrum F r i e d e t . a l . r e p o r t e d max 242 nm

( 6 = 1 5 , 8 0 0 ) .

l e n g t h was r e p o r t e d f o r Squibb House S t a n d a r d ( L o t # 41040-102) 27.

An Elcrn o f 545 a t t h e same wave- 1%

* T a b l e I 60 MHz NMR Data i n CDC13

Group

c- 1

c- 2

c-4 C- 18 c-19

SQ 9,538 Batch #36B

= 10 1 9 2

294

7 .05 d; J

6 .28 q; J = 1 . 5

J 1 , 2 = 10 6 .13 m 1 .25 s 1.40 s

* The chemical s h i f t s are i n d e l t a , w i t h i n t e r n a l r e f e r e n c e TMS.

s = s i n g l e t ; d = d o u b l e t ; m = m u l t i p l e t ; q = q u a r t e t J = coup l ing c o n s t a n t i n Hz.

537

. . . .. . . . -.

Figure 2. NMR Spectrum of Testolactone (batch 36B) in CDC13. Instrument: Perkin-Elmer R12B (60 MHz)

TESTOLACTONE

2.4 Mass Spectrum The low- reso lu t ion mass spectrum of

t e s t o l a c t o n e ( F i g u r e 3 ) was o b t a i n e d by direct i n s e r t i o n of t h e sample i n t o a 18OoC s o u r c e of an AEIMS-902 spectrometer. The M+ of m / e 300 co r re sponds t o t h e formula of ClgH2403. f ragment i o n s shown below a r e d i a g n o s t i c f o r t h i s s t r u c t u r e 2 8 .

The t w o

539

Figure 3. Low-resolution mass spectrum of Testolactone (Batch 36B). Instrument: AEI-MS-902.

TESTOLACTONE

23 0 2 .5 Optical R o t a t i o n F r i e d e t . a l . 1 r e p o r t e d [ a y -44

(c = 1.29 i n CHC13).[alD - 49.2 w a s f e c o r d e d f o r Squibb House S tanda rd Lot 41040-10227.

2 . 6 Mel t ing Range A m e l t i n g range of 218-219O w a s

r e p o r t e d by F r i e d e t . a l . ’ on t h e Kof l e r h o t

The m e l t i n g behav io r s t a g e h a s been descr ibed35.

2.7 D i f f e r e n t i a l Thermal Ana lys i s D . T . A . of house s t a n d a r d Lot 4 40-102 $9 showed a l a r g e s i n g l e endotherm a t 219OC ,

2 . 8 S o l u b i l i t y T e s t o l a c t o n e i s s l i g h t l y s o l u b l e i n

wa te r and i n benzy la l coho l , i s s o l u b l e i n a l c o h o l and i n chloroform and i s i n s o l u b l e i n e t h e r and i n s o l v e n t h e ~ a n e ~ ~ .

2 .9 C r y s t a l P r o p e r t i e s N o polymorph of t e s t o l a c t o n e have been

d e s c r i b e d so f a r excep t t h a t , t w o m o d i f i c a t i o n s were observed on t h e K o f l e r h o t stage35.

The powder x-ray d i f f r a c t i o n p a t t e r n of t e s t o l a c t o n e i s p r e s e n t e d i n Table 2 , cor responding t o t h e p a t t e r n i n F igu re 4.

541

KLAUS FLOREY

** Tab le 2 1 dl * R e l a t i v e I n t e n s i t y

7 . 7 5 6.52 6 .20 5.60 5 . 30 4 .85 4.62 4.34 4.05 3.95 3.80 3 .85 3 . 7 1 3 .45 3.29 3.22 3.16 3.10 2.94 2.86 2.67 2.57 2.44 2.32 * 0

d i n A = i n t e r p l a n a r d i s t a n c e based on h i g h e s t i n t e n s i t y of **

R a d i a t i o n : Kcl l and Ka2 Copper I n s t r u m e n t : P h i 11 i p s

0.74 0.40 0 . 1 1 0.11 1.00 0.72 0 .09 0.06 0 .15 0.14 0.32 0.17 0.57 0.43 0 .15 0.14 0 . 4 1 0.16 0.17 0.06 0.04 0.09 0.05 0.04

-

1.00

542

c 0 JJ

u m 4

'0

U

d

a,

d

543

Figure 4. Powder X-ray diffraction pattern of testolactone (batch 41040-102) Instrument: Phillips

KLAUS FLOREY

3. Synthesis

identified as a bio-oxidative product of the fermentation of progesterone (I1), with Cylindrocarpon radicola by Fried, Thoma and Klingsbergl, (see Figure 5). Peterson, Thoma, Perlman and Fried2 were able to show that l-dehydro-testosterone (111) and A1,4-androstadiene-3,17-dione (Iv) are intermediates in this conversion. Conversion of testololactone to testolactone ('1 by the same microorganism has been observed5. been used for the production of testo- lactone6'11j 19. issued12-17. tone starting with epiandrosterone acetate via epiandrololactone and dihydrotestololactone has been reported i8 .

Testolactone") was first isolated and

(V)

other microorganisms have also

Patents also have been A chemical synthesis of testolac-

544

fH3 c=o 0

0 & I1 -lz2??:# I11

F i g u r e 5 . S y n t h e s i s and Degradat ion .

KLAUS FLOREY

4. Stability, Deqradation

strongly alkaline solution the lactone ring will open to A' -testolic acid2'(VI). be accomplished by microbiological degradation. Other microbiological transformation products are 15-keto-testo-lactone (VII) l-methyl-cyclo- hexan-l-ol-4-keto 2,3-diproprionic acid lactone (VIII) and surprisingly 7a-methylhexahydrindan- 1,5-dione-4a- (3-prapionic acid) (IX) 22 (see Table 4).

and prednisolone when exposed to ultraviolet radiation or ordinary fluorescent laboratory lighting in alcoholic solutions, undergo photolytic degradation of the A-ring. Since testolactone has the same A-ring as prednisolone it probably also is labile under these conditions.

Testolactone is very stable as a solid. In

This can also

It has been reported23 that hydrocortisone

5. Drug Metabolism In human subjects the urinary metabolites

found were unchanged testolactone and 3a, 13a- dihydroxy-13,17-seco-5~-and~O~ta-l-ene-l7-oic acid lactone the latter both in the free form and as the glucuronide.

Bovine blood albumin contains a A4-5f3- hydrogenase system which also reduces testolac tone4.

546

TESTOLACTONE

6. Methods of A n a l y s i s 6 . 1 Elementa l A n a l y s i s

C a l c f o r C1gH2403 Found 1 27 F r i e d , e t . a 1 House S td .

C 75.97 76.29 75.89 H 8 .05 7 .81 8 .23

6.2 Phase S o l u b i l i t y Ana lys i s The p u r i t y of Squibb House S tanda rd l o t

41040-102 was found t o be 99.6% pure by phase s o l u b i l i t y ana lys i s27 . was n-propanol-methanol ( 2 : l ) . E q u i l i b r a t i o n was c a r r i e d o u t over 24 hour s a t 25OC. The e x t r a p o l a t e d s o l u b i l i t y was determined a t 25.5 mg/g of s o l v e n t .

The s o l v e n t system used

6 . 3 C o l o r i m e t r i c Ana lys i s The r e a c t i o n of t h e 3-ket0-A’’~-diene

system of t e s t o l a c t o n e w i t h i s o n i c o t i n i c a c i d hydraz ide t o form hydrazone wi th an a b s o r p t i o n maximum a t 415 nm3’ has been made t h e b a s i s f o r t h e cornpendial a s s a y of t e s t o l a c t o n e i t s e l f and i t s dosage forms29.

A s o t h e r s t e r o i d l a c t o n e s , tes t o l a c t o n e forms a p ink chromogen when s u b j e c t e d t o t h e .

a c t i o n of potassium hydroxide, hydroxylamine and f e r r i c ~ h l o r i d e ~ ~ j ~ ~ . cornpendial i d e n t i t y tes t29.

T h i s h a s been used f o r a

6 .4 F luo romet r i c Ana lys i s When t e s t o l a c t o n e i s h e a t e d i n 85%

phosphor ic a c i d a t 100°C f o r 30 minutes a f luo rogen i s formed which on d i l u t i o n w i t h methanol e x h i b i t s e x c i t a t i o n and f l u o r e s c e n t s p e c t r a w i t h maxima a t 275 nm and 375 nm, r e s p e c t i v e l y . T h i s can be used f o r t h e d e t e r - mina t ion of t e s t o l a c t o n e i n f e rmen ta t ion b r o t h .

547

KLAUS FLOREY

Progesterone and 1-dehydro-progesterone do no t i n t e r f e re31 .

6 .5 Nonaqueous T i t r a t i o n I t has been reported32 t h a t t e s t o -

lac tone a f t e r p r e c i p i t a t i o n with sodium phenylborate can be t i t r a t e d with c e t y l pyridinium ch lo r ide and thymol b lue i n d i c a t o r with acceptab le accuracy.

6 .6 Chromatographic Analysis 6.61 Paper Chromatoqraphic Analysis

The following paper chromatographic so lven t systems have b e n reported: 1. ) Methycyclohexane-carbitol . 2. ) Toluene-propylene g lycol2 .

4.) 5. ) Impregnation with formamide-rnethanol(20:80)

s 3 . ) Propylene glycol-cyclohexane-chloroform 7 .

Toluene s a t u r a t e d with propylene glyco133.

then methyl i sobu ty l ketone-formamide (20 : 1) 33, - -

6. )

from progesterone , A1, $-andros ta-diene-dione and t e s to l ac tone . I n system 4 progesterone and t e s t o l o l a c t o n e have g r e a t e r m o b i l i t i e s than t e s to l ac tone . t e s t o l a c t o n e (both wi th an R f of 0.68) can be separa ted from t e s to l ac tone (Rf 0 .60) . I n system 6 the o rde r of s epa ra t ion (with inc reas i rq Rf) i s a s follows: t e s to l ac tone (Rf 0.23) t e s t o l o l a c t o n e , t e s t o s t e r o n e , and progesterone. System 4, 5 and 6 have a l s o been used f o r q u a n t i t a t i o n following the genera l procedure of Roberts and F 1 0 r e y ~ ~ . I n systems 4, 5 and 6 e l u t i o n with an a c i d i f i e d methanolic s o l u t i o n of i s o n i c o t i n i c a c i d hydrazide was used,followed by q u a n t i t a t i o n (see sec t ion 6 .3 ) . A l t e r n a t e l y i n system 6 e l u t i o n with 95% e thanol and measure-

Methylcyclohexane-chloroform (4: 1) ”. A l l systems sepa ra t e t e s t o l a c t o n e

I n system 5 A I 4 - and A I 5 -

ment of absorption at 242 nm can also be used33. 6.62 Thin Layer Chromatographic Analysis

The following thin-layer chromatographic solvent systems have been reported: 1.) The method of Belic and Socic19 is summarized in Table 3:

Table 3. Rf-values and color reaction of steroids with 50% sulphuric acid on silica gel developed with cyclohexane/ethylacetate (1:2).

Steroid

P ogesterone A -Androstene-3,17-dione Testosterone A’, 4-Androstadiene-3, 17-dione l-Dehydro tes tos terone Te s to lo lac tone l-Dehydrotestololactone l-Dehydroprogesterone A’, 4-Andros tadien-3, 17-dionea Testosteronea

2 2

Rf

0.63 0.52 0.44 0.45 0. 36 0.24 0.18 0.53 0.45 0.40

Daylight Re la tive Color In tens i ty ye 1 low weak blue-green strong blue-green strong bright-red intense ochre-red strong blue-green medium ochre medium pink weak bright-red intense blue-green strong

Fluores- cence

greenish pale-blue blue orange orange green orange ochre orange blue

developed with benzene/ethylacetate(l:l). a

KLAUS FLOREY

2 . ) Ethyl-acetate-hexane(5:S) o r chloroform- methanol(97:3) on s i l i c a g e l HF Foated g l a s s p l a t e s . De tec t ion by U . V . l i g h t . 3 . ) Chloroform-acetone (94:6) on s i l i c a g e l SF. Detec t ion by U.V. l i g h t o r s u l f u r i c a c i d spray . The fo l lowing Rf va lues were found: t e s t o l a c t o n e 0.22; A1- tes tos te rone , 0.27; andros t ad iened ione 0 .49; A1-progesterone, 0 .58; p roges t e rone , 0 . 6 1 li .

4 . ) Butylacetate-acetone(4:l) on s i l i c a g e l . De tec t ion by U.V. l i g h t 2 9 .

6.63 Column Chromatographic Ana lys i s Column hromatography on

'?" h a s been used t o alumina o r s i l i c a g e l s e p a r a t e t e s t o l a c t o n e from o t h e r s t e r o i d s and i m p u r i t i e s i n f e rmen ta t ion e x t r a c t s . Petroleum e ther -ch loroform (1: 1) been used a s e l u t i o n s o l v e n t s .

1

and e t h y l a c e t a t e 5 have

6.64 Vapor Phase Chromatoqraphic Ana lys i s T e s t o l a c t o n e has been q u a n t i t a -

t e d t o g e t h e r w i t h i t s b ioconvers ion p r e c u r s o r s on a 6 - f t g l a s s column c o n t a i n i n g 3% SE-30 on 80-100 mesh D i a t o p o r t . Column and flame- i o n i z a t i o n d e t e c t o r tempera tures were 25OoC and t h e c a r r i e r g a s was helium a t 50 rnl/minll.

7. Determination i n Pharmaceut ica l P r e p a r a t i o n s

a c i d hydraz ide assay2' (see s e c t i o n 6 . 3 ) , t h e paper chromatographic systems (see s e c t i o n 6.61) 2 and 6 have been used a s a s t a b i l i t y i n d i c a t i n g a s s a y f o r t e s t o l a c t o n e i n suspens ion and t a b l e t s 3 3 fo l lowin t h e g e n e r a l procedure of

I n a d d i t i o n t o t h e compendia1 i s o n i c o t i n i c

Rober t s and F lo rey 34 .

550

TESTOLACTONE

8.

1.

2.

3.

4.

5.

6.

7 .

8.

9.

10.

11.

12.

13.

14.

15.

16.

1 7 .

References

J. F r i e d , R. W. Thoma and A . Kl ingsberg , J. Am. Chem. SOC. 75,5764 (1953) . G. E. Pe t e r son , R. W. Thoma, D. Perlman and J. F r i e d , J. B a c t e r i o l . 74 ,684(1957) . E. L. Rongone, A. S e g a l o f f , J. F r i e d and E. Sabo, J. B i o l . Chem. *,2624(1961). E . L. Rongone, Arch.Biochm.Biophys. 98,292 (1962) .

E. J. K u s n e r and R. D. G a r r e t t , S t e r o i d s l7, 521 (1971). M. Nishikawa, S. Noguchi, T. Hasegawa and I. Banno, J. Pharm. SOC. Japan 76,383 (1956) ; Pharm. B u l l . ( Japan) 3 ,322 (1955) , C. A. 50 , 13165d( 1956) . S. A . S z p i f o g e l , M. S . de W i n t e r and W. J. Alsche, R e c . t r a v . chim 75 ,402(1956) . A . Capek and 0. Hanc, F o l i a mic rob io l . - 5 251 (1960) C . A . 55, 3729a (1961) . E. Kondo, Shionogi Kenkyusho Numpo l O , 9 1 (1960) C.A. 54, 25026(1960) K. Singh and S. Rahki t , Biochim. Biophys. Acta 144 ,139 (1967). T. L. M i l l e r , Biochim. Biophys. Acta, - 270,167 (1972) . U.S. P a t e n t 2,744,120, May 1,1956;C.A. 50 14812 (1956) . U.S. P a t e n t 2,823,171, February 11,1958; C . A . 52, 9235d(1958). U.S .Pa ten t 2,868,694,January 13,1959;C.A.53, 9295e (1959) . B r i t . P a t e n t 792 ,803 ,Apr i l 2,1958;C.A. 53, 2293a (1959) . Gen. P a t e n t 1,021,845, J anua ry 2, 1958; C . A . 54, 4686(1960). Japan P a t e n t 31 (158) Janua ry 10; C.A. 52 , 17629 (1958).

551

KLAUS FLOREY

18.

19.

20.

21 .

22 .

23.

24.

25.

26.

27.

2 8 .

29. 30.

31.

32.

L. N. V o l o v e l s k i i , G. V. Knorozova and M. Y. Yakovleva, Zh. Obshch. Khim. - 37,1252 (1967)C.A. 68, 3078w (1968) . J. B e l i c , E. P e r t o t , H. Soc ic , J . S t e r o i d Biochem I, 105 (1970) C. A . 73,425859 (1970) ; a l s o H. S o c i c and J. B e l i c , F r e s e n i u s J. Anal. Chem. 243,291 (1968) . C. H. Holmlund, R. H. Blank, K. J. Sax and R. H. Evans, Jr., Arch. Biochem. Biophys. - 103, 105 (1963) . S. L. Neidleman, P. A. D i a s s i , B. J u n t a , R. M. Palmere and S. C. Pan, Te t rahedron L e t t e r s 44,5337 (1966). K. Schuber t , K. H. Bahme, F. R i t t e r and C. Hbrhold, J. S t e r o i d . Biochem. 2, 245 (1971). W. E. Hamlin, T. Chulsk i , R. H. Johnson and J. G. Wagner, J. Am. Pharm. Assoc. S c i . Ed. 49, 253(1963) and D. R. Barton and W. C. Taylor , J. Am. Chem. SOC. 80,244 (1958) , J. Chem. SOC. 1958, 2500. M. S. Puar, The Squibb I n s t i t u t e , p e r s o n a l comm un i c a t i o n . C. Gual, R. I. Dorfman and H. Rosenkrantz, Spectrochim. Acta 13 ,248 (1958) . H. Rosenkrantz and C. Gual, Spectrochim. Acta 1 3 , 2 9 1 (1959). B. N. Kabadi, E. R. Squibb and Sons, p e r s o n a l communication, P. T. Funke, The Squibb I n s t i t u t e , pe r sona l communication. N.F. X I V , 1975 p.683. C. A. Lendzian, The Squibb I n s t i t u t e , pe r sona l communication. E. I vashk iv , The Squibb I n s t i t u t e , p e r s o n a l communication. R.C.D. D e Carnevale Bonino, J. Dobrecky, L. 0. Guere l lo , Rev. Farm. 113, 15(1971) C.A. 75,67518s (1971).

552

TESTOLACTON E

33. H. R. R o b e r t s , The Squ ibb I n s t i t u t e ,

34. H. R. Roberts and K. F l o r e y , J. Pharm. S c i . ,

35. K u h n e r t - B r a n d s t a t t e r , P. G a s s e r , P. D. Lark ,

p e r s o n a l communication.

51,794 (1962) .

P. L i n d e r and G. K r a m e r , Microchem J. 2 , 7 1 9 (1972) .

p e r s o n a l communication. 3 6 . H. J acobson , The Squ ibb I n s t i t u t e ,

L i t e r a t u r e su rveyed th rough 1973.

553

ADDENDA AND ERRATA

D i a t r i z o i c Acid Volume 4 , p.

ADDENDA AND ERRATA

151

Under 5.2, Free Iodine and Free Halide, rep lace sentence s t a r t i n g wi th , "An a l t e r n a t e procedure . . . . . * I with the following:

For d e t e c t i o n of f r e e iod ine and iod ide , t h e f i l t r a t e i s a c i d i f i e d , chloroform and sodium n i t r i t e a r e added, and t h e reddish co lo r i n t he chloroform l aye r i s compared t o a s tandard so lu t ion ( 4 , 1 2 1 .

1sosorbi.de D i n i t r a t e V o l u m e 4 , p. 225

The correct name of t h e f i r s t author i s S i v i e r i , no t S i l v i e r i .

Phenformin Hydrochloride V o l u m e 4 , p. 319

To t h e embarrassment and r e g r e t of t h e e d i t o r , t h e f u l l l i s t of au thors was inadver ten t ly no t presented by him for t h i s P r o f i l e . The correct t i t l e is:

Phenformin Hydrochloride

Michael J. O'Hare, Hridaya Bhargava, Ruth Wasserman and Joseph E. Moody

5.

6.5

Chemical i o n i z a t i o n m a s s spectrometry was used f o r t h e determinat ion of Phenformin Hydrochloride i n b i o l o g i c a l f l u i d s by S. B. Matin, J. K. Karam, P. H. Forsham and J. B. Knight, Biomedical Mass Spectrometry - 1, 320 ( 1 9 7 4 ) .

An HPLC method f o r t he determinat ion of Phenformin Hydrochloride has been developed by R. K. Gilp in , J. A. Korpi and C . A. J a n i c k i , J. of Chromat. 1 0 7 , 115 (1975).

-

556

ADDENDA AND ERRATA

*- 4, p. 386

It was pointed out by Prof. H. Vanderhaeghe of Leuven, Belgium that in the structural formula on p. 386 the substituent on C15 should be H (see formula on p. 400). Also, since the absolute configuration is known (see Klyne and Buckingham, "Atlas of Stereochemistry , I' Chapman and Hall (1974) 1 , the structure given is that of the inactive enantiomer.

The correct structural formula is:

bCH3 OCH3

Tolbutamide Volume 3, p. 513

5. Solid probe c..emical ion-zation mass spectrometry and gas chromatography has been used for the determination of Tolbutamide and its metabolites in human plasma by S . B. Matin and J. B. Knight, Biomedical Mass Spectrometry L, 323 (1974).

Triflupromazine Hydrochloride Volume 2, p. 523

2.5 pKa

The pKa of 6.5 as listed in Volume 2, p. 533 is in error. Reexamination by Dr. H. Jacobson (The Squibb Institute) gave a pKa value of 9.45, in reasonable agreement with values determined by

557

ADDENDA AND ERRATA

references 8 and 9 .

2.10 The c r y s t a l structure of Triflumpromazine Hydrochloride has been determined by x-ray d i f f r a c t i o n by D . W . Phelps and A. W; Cordes, Acta Cryst. g, 2812 ( 1 9 7 4 ) .

558

CUMULATIVE INDEX Italic numerals refer to Volume numbers.

Acetaminophen, 3 , l Acetohexamide, 1.1; 2,573 Alpha-Tocopheryl Acetate, 3.11 1 Amitriptyline Hydrochloride, 3,127 Ampicillin.2, 1;4,517 Bendroflumethiazide 5, 1 Cefazolin, 4 , l Cephalexin, 4 , 2 1 Cephalothin Sodium, 1 , 3 19 Cephradine, 5, 21 Chloral Hydrate, 2,85 Chloramphenicol, 4,47 ,5 17 Chlordiazepoxide, 1, 15 Chlordiazepoxide Hydrochloride, 1. 39; 4,

Chloroquine Phosphate, 5,61 Chlorprothixene, 2.63 Clidinium Bromide, 2,145 Clorazepate Dipotassium, 4,91 Cloxacillin Sodium, 4.113 Cycloserine, 1, 5 3 Cyclothiizide, 1,66 Dapsone, 5. 87 Dexamethazone, 2, 163; 4, 5 18 Diatrizoic Acid,4, 137,5 556 Diazepam, 1,79;4,517 Digitoxin, 3,149 Dioctyl Sodium Sulfosuccinate, 2,199 Diphenhydramine Hydrochloride, 3,173 DisuKiam, 4,168 Echothiophate Iodide, 3,233 Erthromycin Estolate, 1,101;2,573 Estradiol Valerate, 4, 192 Ethynodiol Diacetate, 3,253 Fiucytosine, 5, 115 Fludrocortisone Acetate, 3,281 Fluorouracil, 2,22 1 Fluphenazine Enanthate, 2,245; 4,523 Fluphenazine Hydrochloride, 2, 263; 4,

517

5 18

Gluthethimide, 5, 139 Halothane.1, 119;2,573 Hydroxyprogesterone Caproate, 4,209 Iodipamide, 3,333 Isocarboxazid, 2,295 Isopropamide, 2 , 3 15 Isosorbide Dinitrate, 4,225;5, 556 Levartereno1 Bitartrate, 1,49; 2,573 Levallorphan Tartrate, 2,339 h o d o p a , 5, 189 Levothyroxine Sodium, 5. 225 Meperidine Hydrochloride, 1,175 Meprobamate, 1,209; 4,s 19 Methadone Hydrochloride, 3, 365; 4, 5 19 Methaqualone, 4, 245,519 Methotrexate, 5, 283 Methyclothiazide, 5. 307 Methyprylon, 2,363 Metronidazole, 5, 327 Nitrofurantoin, 5, 345 Norethindrone, 4,268 Norgestrel, 4, 294 Nortriptyline Hydrochloride, 1,233; 2,

Oxazepam, 3,441 Phenazopyridine Hydrochloride, 3,465 Phenelzine Sulfate, 2, 383 Phenformin Hydrochloride, 4, 319;5. 429 Phenoxymethyl Penidllin Potassium, 1.249 Phenylephriie Hydrochloride, 3,483 Piperazine Estrone Sulfate, 5, 375 Rimidone, 2,409 Procainamide Hydrochloride, 4, 333 Rocarbazine Hydrochloride, 5,403 Promethazine Hydrochloride, 5. 429 Propiomazine Hydrochloride, 2,439 Ropoxyphene Hydrochloride, 1,301; 4,

Reserpine, 4,384;5, 557 Rifampin, 5,467

573

5 19

559

CUMULATIVE INDEX

Secobarbital Sodium, I , 343 Spironolactone, 4 .43 1 Sulfamethoxazole, 2, 467;4, 520 Sulfasalazhe, 5, 5 15 Sulfisoxazole, 2, 487 Testolactone, 5. 533 Testosterone Enanthate, 4, 452 Theophylline, 4,466 Tolbutamide, 3, 513;5, 557 Triamcinolone, I . 367;2, 571;4, 520,523 Triamcinolone Acetonide, I , 397; 2, 571;

4 ,520

Triamcinolone Diacetate, 1, 423 Triclobisonium Chloride, 2, 507 Triflupromazine Hydrochloride, 2, 5 23;

Trimethaphan Camsylate, 3,545 Trimethobenzamide Hydrochloride, 2,

Tropicamide, 3, 565 Tybamate, 4, 494 Vinblastine Sulfate, I , 443 Vincristine Sulfate, I , 463

4, 520;5, 557

55 1

A 6 8 7 C 8 D 9 € 0 F 1 C 2 H 3 1 4 1 5

560

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