Synthesis and characterization of 5H-8-oxa-4b, 7-diaza …shodhganga.inflibnet.ac.in/bitstream/10603/2186/11/11... · · 2012-05-31Chapter-2 19 Scheme 2.1: Synthesis of 1 from 2

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2.1 INTRODUCTION

Non-steroidal anti-inflammatory drugs (NSAIDs) are amongst the

most widely used prescription and over the counter medications for the

treatment of pain and inflammation particularly arthritis. Pain, swelling

and tenderness at the joints are a few symptoms associated with the

arthritis. In the 1970’s, it was demonstrated that aspirin and other

NSAIDs block the formation of prostaglandins (PGs) produced from the

metabolism of arachidonic acid by the enzyme cyclooxygenase (COX),

familiarly known as prostaglandin synthase.32 The COX inhibitors are of

three kinds, namely COX-1, COX-2 and COX-3. Role of COX-3 in

prostaglandin mediated physiological activity is not clear and further

studies are under way. Until very recently, all commercially available

NSAIDs were inhibitors of both COX-1 and COX-2. Selective inhibitors of

COX-2 are now widely recognized as offering promising treatment for

inflammatory conditions without adverse side effects as associated with

consumption of nonselective inhibitors.

3,4-Diaryl substituted isoxazole derivative, valdecoxib 1 (figure 2.1) is

classified as NSAID. Valdecoxib 1 acts as a selective inhibitor of COX-2,

an enzyme that facilitates the formation of prostaglandins, which

mediate the process of inflammation.33 Increased risk of gastrointestinal

ulceration associated with blockade of COX-1 derived prostaglandins led

to the development of selective COX-2 inhibitors.34 Valdecoxib 1 has

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18

been recognized as an effective medication in the treatment of

rheumatoid arthritis, osteoarthritis, and dysmenorrhea.

Figure 2.1: Structure of valdecoxib 1

In view of the growing importance of anti-inflammatory drugs with

selective COX-2 inhibitory activity in day to day life, a systematic study

was taken up to develop an efficient, scalable and commercially viable

new synthetic route that would provide the title compound 1 with high

degree of quality. As this molecule is a drug substance intended for

human use, the quality should be in agreement with regulatory

requirements and ICH guidelines.

2.2 LITERATURE PRECEDENCE

Talley et al. disclosed the process for the preparation of 1 (scheme

2.1).35,36 Deoxybenzoin 2 was treated with hydroxylamine hydrochloride

to result in the oxime intermediate that was in situ deprotonated using

n-butyl lithium to form the oxy-anion that was trapped with ethyl acetate

to result in hydroxyisoxazoline intermediate 3. Compound 3 underwent

dehydration followed by chlorosulfonation upon treatment with

chlorosulfonic acid, which was finally converted to 1 by treating with

ammonium hydroxide.

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19

Scheme 2.1: Synthesis of 1 from 2

Alternatively, 1 was prepared in a stepwise manner (scheme 2.2).

Dehydration of 3 by treatment with sulfuric acid provided isoxazole

derivative 4. Chlorosulfonation with reagents such as chlorosulfonic

acid, SO3-Py complex/phosphorous oxychloride and sulfuric chloride,

provided the chlorosulfonic acid derivative, which in turn was subjected

to amidation with ammonium hydroxide to yield 1.35

Scheme 2.2: Sulfamidation with different reagents

An alternative approach for the synthesis of 1 was described in US

5859257 patent.37 In this process, 2 was first subjected to sulfamidation

by treatment with chlorosulfonic acid followed by ammonium hydroxide

to afford sulfonamide derivative 5. Having installed the required

functionality, build up of the isoxazole ring was carried out by different

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20

methods (scheme 2.3). First method involved the protection of the amine

group in 5 as cyclic disilylamine derivative by treating with 1,2-bis-

(chlorodimethylsilyl)ethane, which upon reaction with hydroxylamine

hydrochloride under basic conditions provided oxime 6. Treatment of 6

with ethyl acetate in presence of lithium diisopropylamide (LDA) followed

by dehydration and deprotection of silyl group in presence of aqueous

TFA afforded 1.

In the second method, masking of the amine functionality in 5 as a

pyrrole ring was obtained by reacting with acetonylacetone, which was

treated with hydroxylamine hydrochloride to yield the oxime 7. By

following the same methodology adopted above, the isoxazole 1 was built

up from 7 (scheme 2.3).

Scheme 2.3: Synthesis of 1 with protection and deprotection approach

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21

In another report, 1 was prepared by the [3+2] cycloaddition between

nitrile oxide and the corresponding alkyne.38 The precursors for the

cycloaddition reaction were prepared as follows.

Compound 8 was treated with ammonium hydroxide to result in

sulfonamide derivative 9. The carbonyl functionality of 9 was converted

to the alkyne group under modified Seyferth-Gilbert homologation

conditions, by treating with LDA, diethyl chlorophosphate followed by

methylation using hexamethylphospharamide (HMPA) and iodomethane

to obtain compound 10. Finally, compound 10 was subjected to [3+2]

cycloaddition with the nitrile oxide (generated in situ from benzaldoxime

11) to produce 1 (scheme 2.4).

Scheme 2.4: Synthesis of 1 utilizing [3+2] cycloaddition

Waldo et al. designed their strategy for the preparation of 1 utilizing

two key reactions namely halo-isoxazolation and Suzuki coupling

reaction.39 Exposure of the ynone 12 to methoxylamine hydrochloride

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22

generated the O-methyl oxime 13, which was subjected to ICl

(chloroiodide) cyclization conditions to yield iodoisoxazole 14. Treatment

of iodoisoxazole 14 under standard Suzuki-Miyaura coupling conditions

with compound 15 afforded 1 (scheme 2.5). It is important to note that

diastereomeric oximes 13 namely E & Z isomers were formed on

treatment of 12 with methoxylamine hydrochloride. These diastereomers

were separable by column chromatography. It had been observed that

only the Z-isomer underwent inter halogen mediated cyclization to

furnish the corresponding iodoisoxazole.

Scheme 2.5: Synthesis of 1 using halo-isoxazolation and Suzuki

coupling reactions

A simpler approach to the synthesis of 1 starting from readily

available raw materials was devised by Nunno and co-workers.40 Lithium

enolate 17 was generated from phenylacetone (16) by treatment with

LDA, which was trapped in situ with nitrile oxide 18 by a [3+2]

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23

cycloaddition to form 3. Dehydration followed by sulfamidation of 3

under known reaction conditions afforded 1 (scheme 2.6).

Scheme 2.6: Synthesis of 1 by [3+2] cycloaddition of 17 and 18

The above reported synthetic routes to 1 indicate the usage of

a) Moisture sensitive and highly flammable reagents like n-butyl

lithium and LDA.

b) Protection and deprotection procedures.

c) Carcinogenic chemicals like diethyl chlorophosphate, hexamethyl-

phospharamide and iodomethane.

d) Purification by column chromatography.

e) Expensive and commercially unavailable raw materials.

With so many hurdles, the above mentioned processes appeared not

to be commercially viable. In view of these drawbacks, a comprehensive

study was undertaken and attempts were made to develop a new, simple,

efficient and scalable synthetic procedure for 1.

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24

2.3 OUR APPROACH

Based on the mechanistic pathway of [3+2] cycloaddition as shown in

figure 2.2 and the reports on the application of enamine as an olefin

equivalent in such type of reactions,41,42 a novel synthetic route involving

an alternative dipolarophile other than those described in the literature

and the known nitrile oxide as 1, 3-dipole was designed to synthesize 1

efficiently.

Figure 2.2: In situ generated nitrile oxide and its [3+2] cycloaddition

with olefin

It was envisioned that the enamine 19 would serve as dipolarophile in

the [3+2] cycloaddition reaction with the nitrile oxide to generate the

pyrrolidinylisoxazoline 21 which could be converted into 1 after few

transformations (scheme 2.7). As the synthetic route is modified, the

formation of new impurities in the sequence can be expected. Hence, the

interest lay in the identification and characterization of the related

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25

compounds. In addition, a detailed study relating to the formation and

synthesis of these related substances is presented in this chapter.

Scheme 2.7: Novel approach to the synthesis of 1

2.4 RESULTS AND DISCUSSION

In our endeavor, the synthesis of 1 was planned from the readily

available starting material 16. The route for the synthesis of 1 was based

on the masking of the ketone functionality as an enamine in order to

localize the formation of the double bond which would be in conjugation

with the benzene ring in the form of a styrene moiety. In this regard, the

ketone group in 16 was transformed into the respective pyrrolidine

derived enamine 19 using a reported protocol.41 The [3+2] cycloaddition42

between 19 and benzonitrile oxide (generated in situ from 20 using

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26

triethylamine in DCM43) afforded 21, which was subjected to

aromatization in the presence of aqueous hydrochloric acid to result in

the isoxazole 4 (scheme 2.7). Spectral data of this compound was found

to be in agreement with the reported data.40 After synthesizing the core

isoxazole entity, the focus was shifted on the decoration of the aromatic

ring with the suitable functionalities. The isoxazole derivative 4 was

subjected to chlorosulfonation in presence of chlorosulfonic acid,

followed by amidation in presence of aqueous ammonia to afford 1 in 50

% yield and 99.0 % purity. Intensive process optimization study was

carried out to improve the yield and quality of final product by various

purification techniques that finally improved the yield to 80 % and

quality to

99.92 %. The synthesized material met the requirement specified by

regulatory authorities, hence qualifying the material for human

consumption. This process has been successfully implemented on Kg

scale in the plant.

Aforementioned synthesis had overcome the disadvantages in prior

art processes.

Having synthesized the target molecule, a systematic study was taken

up for the identification and characterization of the impurities that were

formed during the synthesis of drug substance. Five related substances,

23 (impurity-A), 24 (impurity-B), 25 (impurity-C), 26 (impurity-D) and

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27

27 (impurity-E) were observed by a simple high performance liquid

chromatographic (HPLC) method (figure 2.3). A

U

-0.020

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

IMP

UR

ITY

-C -

6.0

11

IMP

UR

ITY

-D -

6.5

67

IMP

UR

ITY

-A -

9.9

31

Vald

ecoxib

- 1

6.0

15

IMP

UR

ITY

-B -

16.8

11

IMP

UR

ITY

-E -

23.7

05

Figure 2.3: HPLC chromatogram of 1 and its related substances

In this regard LC–MS analysis was performed on the drug substance

to obtain the molecular weights of the related substances that were

present along with the desired product, which would provide insight to

the probable structures. The LC–MS data revealed the molecular weights

as 393, 314, 314, 315 and 611. Based on the LC–MS data and synthetic

pathway, the following tentative structures were predicted (figure 2.4).

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28

Figure 2.4: Chemical structures for the related substances of valdecoxib

To substantiate our claim, efforts were directed for the synthesis of

the respective compounds (figure 2.4). The presence of these related

substances was confirmed by performing the co-injection in HPLC

analysis (figure 2.5) with standards that were synthesized separately.

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

IMP

UR

ITY

-C -

6.0

07

IMP

UR

ITY

-D -

6.5

57

IMP

UR

ITY

-A -

9.9

37

Vald

ecoxib

- 1

6.0

22

IMP

UR

ITY

-B -

16.8

40

IMP

UR

ITY

-E -

23.6

96

Figure 2.5: HPLC chromatogram of 1 co-injected with impurities

Chapter-2

29

2.4.1 3-[5-Methyl-4-(4-sulfamoylphenyl)isoxazol-3-yl]benzene

sulfonamide (Impurity-A, 23)

Isoxazole 4 is a pseudo symmetrical molecule containing two

activated aromatic nucleus that are available for the sulfonation reaction.

As the drug substance contains only one sulfonyl group, this means

there exists a need for the controlled monosulfonation of the starting

material. The reaction was optimized in such a manner to maximize the

formation of the main product 1. Even under these circumstances there

is a possibility for the chlorosulfonation at more than one position, which

eventually would be transformed to sulfonamide derivatives by the

subsequent reaction with aqueous ammonia.

Isoxazole derivative 4 was subjected to chlorosulfonation with excess

chlorosulfonic acid followed by reaction with aqueous ammonia to obtain

23 (scheme 2.8, experimental section 2.6.9). The obtained product was

characterized by analytical tools as described.

Scheme 2.8: Synthesis of 23

Mass spectrum of 23 showed protonated molecular ion peak at m/z

394 (figure 2.6) and IR spectrum displayed characteristic absorptions at

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30

3322 & 3241 cm–1 corresponding to N–H stretching and 1346 & 1161

cm–1 corresponding to O═S═O asymmetric & symmetric stretching,

respectively (figure 2.7).

Figure 2.6: Mass (+Ve) spectrum of 23

Figure 2.7: IR spectrum of 23

In the 1H NMR spectrum, two doublets appeared at δ 7.45 and 7.86,

corresponding to two aromatic protons of para substituted phenyl ring.

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31

On the other hand, singlet at δ 8.05, doublet at δ 7.90 and a triplet at

δ 7.58 were attributed to four aromatic protons of meta substituted

phenyl ring (figure 2.8).

Figure 2.8: 1H NMR (DMSO–d6, 400 MHz) spectrum of 23

The carbon NMR spectrum showed chemical shifts at δ 11.3

corresponding to methyl group, δ 168.0, 159.6 and 114.2 corresponding

to quaternary carbons of isoxazole ring. The signals observed at δ 144.8

and 132.6 due to quaternary carbons of para substituted phenyl ring

and δ 143.3 and 131.2 due to quaternary carbons of meta substituted

phenyl ring. The remaining aromatic carbons observed at δ 125.1, 126.0,

126.7, 129.0, 129.4 and 129.9 (figure 2.9). Based on the above

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32

discussion and spectral data, the proposed structure was confirmed

as 23.

Figure 2.9: 13C NMR (DMSO–d6, 50 MHz) spectrum of 23

2.4.2 4-[(3-Phenylisoxazol-5-yl)methyl]benzenesulfonamide

(Impurity-B, 24)

Valdecoxib 1 was synthesized from the reaction of 16 with pyrrolidine

to produce the required enamine 19, which was converted to the product

after performing few transformations. The possibility for the formation of

the isomeric enamine 19a, a by product in the preparation of 19 was

also not ruled out. The isomeric 19a can also participate in the [3+2]

cycloaddition with benzonitrile oxide to result in the formation of 4a. This

Chapter-2

33

can further be transformed to 24 under the reaction conditions

employed.

Compound 24 was efficiently synthesized from 16 by reacting with

pyrrolidine to produce the mixture of 19 and 19a (isomer) followed by

[3+2] cycloaddition with benzonitrile oxide (in situ generated from 20) in

dichloromethane and elimination of pyrrolidine ring in presence of conc

hydrochloric acid to yield a mixture of 4 and 4a. Compound 4 was

filtered and the filtrate containing 4a was treated with chlorosulfonic acid

followed by aqueous ammonia to provide 24 (scheme 2.9, experimental

section 2.6.10.1–2.6.10.2).


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34

Compound 24 was characterized based on its mass, IR, 1H NMR and

13C NMR spectral data. The mass spectrum showed a peak at m/z 315

corresponding to protonated molecular ion (figure 2.10).


IR spectrum displayed characteristic bands at 3363 & 3271 due to

N–H stretching and 1341 & 1158 cm–1 due to O═S═O asymmetric &

symmetric stretching, respectively (figure 2.11).


Chapter-2

35

The PMR spectrum revealed a singlet at δ 6.33 corresponding to

isoxazole nucleus proton and a singlet of two proton integration at δ 4.27

due to benzylic protons (figure 2.12).

Figure 2.12: 1H NMR (CDCl3, 200 MHz) spectrum of 24

13C NMR spectrum showed signals at δ 31.8 for benzylic carbon,

δ 100.4 for tertiary carbon of isoxazole and δ 171.7 & 161.9

corresponding to quaternary carbons of isoxazole. Chemical shifts due to

quaternary and tertiary carbons of sulfonamide substituted phenyl ring

were observed at δ 142.7 & 140.2 and δ 130.0 & 128.5, respectively. The

remaining signals corresponding to unsubstituted phenyl ring were

observed at δ 129.2, 129.0, 126.4 and 126.0 (figure 2.13). The proposed

structure of 24 was confirmed based on the above discussion and

spectral data.

Chapter-2

36


2.4.3 3-(5-Methyl-3-phenylisoxazol-4-yl)benzenesulfonamide

(Impurity-C, 25)

During the chlorosulfonation of 4, probability for the formation of

meta isomer 22a also exists along with desired product 22. Compound

22a upon further reaction with aqueous ammonia would lead to the

formation of 25.

Compound 25 was independently prepared by performing the

chlorosulfonation followed by amidation on compound 4.

Chlorosulfonation on compound 4 resulted in a mixture of 22 and 22a.

Compound 22 was removed from the mixture by filtration. Thus,

obtained filtrate containing meta isomer 22a and minimum amount of 22

Chapter-2

37

was treated with aqueous ammonia resulting in the precipitation of solid

material 1 which was filtered. The filtrate was subjected to the isolation

of 25 (scheme 2.10, experimental section 2.6.11).


The meta sulfonamide 25 was characterized by mass and IR spectra,

in which a peak at m/z 315 corresponding to protonated molecular ion

was observed in the mass spectrum (figure 2.14) and IR spectrum

displayed characteristic absorptions at 3337 & 3247 cm–1 corresponding

to N–H stretching and 1334 & 1165 cm–1 corresponding to O═S═O

asymmetric & symmetric stretching respectively (figure 2.15).


Chapter-2

38


Careful observation of proton NMR of this compound indicated the

absence of symmetrical splitting of the aromatic protons, thereby

distinguishing the impurity-C from the drug substance. A singlet at δ

7.71 and triplet at δ 7.59 appeared in the 1H NMR spectrum (figure 2.16).


Chapter-2

39

CMR spectrum displayed characteristic δ values at 167.0, 160.4 and

115.0 for isoxazole ring and chemical shifts corresponding to

sulfonamide substituted phenyl ring at δ 148.7, 147.4, 129.1, 128.0,

126.4 and 126.0. The remaining signals corresponding to unsubstituted

phenyl ring were observed at δ 129.8, 128.9, 128.2 and 127.9 (figure

2.17). Based on the above spectral data observations, the proposed

structure of 25 was confirmed.


2.4.4 4-(5-Methyl-3-phenylisoxazol-4-yl)benzenesulfonic acid

(Impurity-D, 26)

The final step in the synthesis of 1 involves amidation of 22 with

aqueous ammonia. During this reaction, 22 can undergo hydrolysis in

the presence of water to form the sulfonic acid derivative 26. This

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40

compound was independently prepared by subjecting the

sulfonylchloride derivative 22 to hydrolysis in a mixture of

tetrahydrofuran and water under reflux conditions (scheme 2.11,

experimental section 2.6.12).

Scheme 2.11: Synthesis of sulfonic acid derivative 26

Mass spectrum of 26 displayed protonated molecular ion peak at m/z

316 (figure 2.18). Bands at 3395 cm–1 due to OH stretching and 1396 &

1177 cm–1 due to asymmetric & symmetric stretching of O═S═O,

respectively appeared in the IR spectrum (figure 2.19).


Chapter-2

41


The PMR spectrum showed a singlet at δ 2.44 corresponding to the

methyl group, doublets at δ 7.64 and 7.19 corresponding to protons of

sulfonic acid substituted benzene ring and multiplet at δ 7.45–7.35 due

to five protons of another benzene ring (figure 2.20).

Chapter-2

42


In the 13C NMR spectrum, methyl carbon at δ 11.3 and quaternary

carbons of isoxazole at δ 167.0, 160.5 and 114.8 were observed. Signals

at δ 147.4, 129.8, 128.6 and 126.0 represent the sulfonic acid

substituted phenyl ring carbons. Signals corresponding to carbons of

unsubstituted phenyl ring were appeared at δ 129.6, 128.9, 128.7 and

128.0 (figure 2.21). Above discussion and spectral data confirmed the

proposed structure of 26.

Chapter-2

43


2.4.5 4-(5-Methyl-3-phenylisoxazol-4-yl)-N-[4-(5-methyl-3-

phenylisoxazol-4-yl)phenylsulfonyl]benzenesulfonamide

(Impurity-E, 27)

The formation of 27 was explained on the basis that a reaction of 1

with staring material 22 could occur. To substantiate our claims on its

formation, a reaction was performed between 1 and compound 22 in

pyridine, which provided 27 (scheme 2.12, experimental section 2.6.13).

Compound 27 was characterized as described below.


Chapter-2

44

Protonated molecular ion peak appeared at m/z 612 in the mass

spectrum of the 27 (figure 2.22).


The IR spectrum displayed characteristic bands at 3339 cm–1

corresponding to N–H stretching and 1369 & 1165 cm–1 corresponding to

O═S═O asymmetric & symmetric stretching, respectively (figure 2.23).


A singlet at δ 2.50 corresponding to six protons of two methyl groups,

doublets at δ 7.95 and 7.32 corresponding to eight protons of

sulfonamide substituted benzene rings and multiplet at δ 7.45–7.36

Chapter-2

45

corresponding to ten protons of another two benzene rings were observed

in 1H NMR spectrum (figure 2.24).

Figure 2.24: 1H NMR (CDCl3 + DMSO–d6, 400 MHz) spectrum of 27

13C NMR spectrum showed methyl carbon at δ 11.3 and quaternary

carbons of isoxazole at δ 167.2, 160.6 and 114.5. Signals corresponding

to sulfonamide substituted phenyl ring carbons were observed at

δ 145.4, 131.5, 128.5 and 126.5. The signals of unsubstituted phenyl

ring carbons were appeared at δ 129.7, 129.0, 128.7 and 128.1 (figure

2.25).

Chapter-2

46


The proton NMR and 13C NMR spectra were similar to valdecoxib, but

the protonated molecular ion peak at m/z 612 in the mass spectrum and

presence of a single band at 3339 cm–1 corresponding to NH absorption

in the IR spectrum indicated the dimer structure for 27. The above

spectral data and discussion confirmed the proposed structure of 27.

2.5 CONCLUSION

Application of [3+2] cycloaddition in the development of an efficient,

scalable and commercially viable new synthetic route for the synthesis of

1 has been described. A detailed study on the formation, synthesis and

characterization of potential related substances (impurities) has been

presented.

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47

2.6 EXPERIMENTAL SECTION

The 1H NMR and 13C NMR spectra were measured in CDCl3,

DMSO–d6, CDCl3 + DMSO–d6 and CDCl3 + CD3CN using 200 MHz on a

Gemini–2000 (200 MHz) and 400 MHz on mercury plus (Varian 400 MHz)

FT–NMR spectrometer. The FT–IR spectra were recorded in the solid state

as KBr dispersion using Perkin–Elmer 1650 FT–IR spectrophotometer.

The mass spectrum (70 eV) was recorded on HP–5989A LC–MS

spectrometer. The solvents and reagents were used without further

purification.

2.6.1 High Performance Liquid Chromatography (HPLC)

An in-house liquid chromatographic gradient method was developed

for the separation of all possible related substances of valdecoxib. An

agilent 1100 series HPLC (auto sampler) equipped with binary pump,

static mixer and UV-VIS detector (Agilent Technologies, USA) was used.

Mobile phase-A is buffer (Dissolved 1.36 g of KH2PO4 and 0.22 g of 1-

octane sulfonic acid sodium salt in 1000 mL milli Q water and adjusted

the pH to 3.3 with dil.H3PO4) and mobile phase-B is acetonitrile and

water in 7:3 ratio (v/v). Zorbax CN 150 mm x 4.6 mm x 3.5 µm column

(Agilent Technologies, USA) was used. A timed gradient program of T

(min)/%B: 0/35, 5/35, 20/60, 25/95, 30/95, 35/35, 40/35 with 1.0

mL/min of flow rate, 27 °C of column oven temperature was used.

Column eluent was monitored by UV at 240 nm.

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48

2.6.2 Benzaldoxime 11

A solution of hydroxylamine hydrochloride (32.5 g, 0.467 mol) in

water (90.0 mL) was added portion wise to the mixture of sodium

hydroxide (46 g, 1.150 mol), water (68 mL) and benzaldehyde (45 g,

0.424 mol) at 25–35 °C. The resultant reaction mixture was stirred at

25–35 °C for 30–45 min. Thereafter the pH of reaction mixture was

adjusted to 6.5–7.5 with hydrochloric acid (~11 mL) and extracted with

dichloromethane (3 x 75 mL). The combined organic layers were

concentrated below 50 °C to afford 49 g (95 %) of 11 as a brown color

residue.

Purity by HPLC: 99.0 %.

IR (KBr, cm–1): 3334, 3064, 2923, 1634, 1578, 1494, 755, 691.

1H NMR (200 MHz, CDCl3): δ 9.22 (bs, 1H), 8.18 (s, 1H), 7.70–7.22 (m,

5H).

MS (m/z): 122 (M+ + H).

2.6.3 1-[(1-Methyl-2-phenyl)ethenyl]pyrrolidine (19)

Chapter-2

49

A solution of phenylacetone (16, 45 g, 0.133 mol), pyrrolidine (42.5 g,

0.597 mol) and cyclohexane (370 mL) was maintained under azeotropic

reflux (70–80 °C) conditions for 8 h. Thereafter, cyclohexane and excess

pyrrolidine were removed from the reaction mixture under reduced

pressure below 50 °C to provide the 19 [residue was directly used in next

step without further isolation (63.1 g; crude yield was considered as

100 %)].

IR (KBr, cm–1): 3016, 2965, 2871, 1610, 1566, 1436, 1353, 775, 741.

1H NMR (200 MHz, CDCl3): δ 7.40–6.91 (m, 5H), 3.69 (s, 1H), 3.27 (t, J

═ 6.6 Hz, 2H), 2.87 (t, J ═ 6.6 Hz, 2H), 2.13 (s, 3H), 1.92 (t, J ═ 6.6 Hz,

2H), 1.69 (t, J ═ 6.6 Hz, 2H).

MS (m/z): 187 (M+ + H).

2.6.4 Chlorobenzaldoxime 20

To a solution of N-chlorosuccinimide (63.4 g, 0.475 mol) in

N, N-dimethylformamide (240 mL) was added a solution of 11 (48 g,

0.396 mol) in N, N-dimethylformamide (48 mL) over 60 min at 25–35 °C.

The resulted reaction mixture was stirred for 1–1.5 h and poured into

water (720 mL) at 25–35 °C. Thereafter, reaction mixture was stirred for

30–45 min and extracted with dichloromethane (3 x 100 mL). The

combined organic layers were washed with a solution of sodium

Chapter-2

50

hydrosulphite (hydrose) (4.8 g) and water (95 mL) followed by water (2 x

190 mL). The organic layer was concentrated under atmospheric

pressure below 50 °C followed by under reduced pressure below 50 °C to

afford 20 [residue was directly used in next step without further

isolation (61.6 g; crude yield was considered as 100 %)].

IR (KBr, cm–1): 3273, 3063, 2900, 1629, 1492, 1450, 995, 936, 766,

691.

1H NMR (200 MHz, CDCl3): δ 8.53 (s, 1H), 7.83 (d, J ═ 7.0 Hz, 2H),

7.50–7.35 (m, 3H).

MS (m/z): 156 (M+ + H).

2.6.5 3,4-Diphenyl-5-methyl-5-pyrrolidinylisoxazoline (21)

To a stirred solution of 19 (63 g, 0.336 mol) and triethylamine (53 g,

0.523 mol) in dichloromethane (575 mL) was added a solution of 20

(61 g, 0.392 mol) in dichloromethane (95 mL) at 5–10 °C. The

temperature of reaction mixture was raised to 25–35 °C and stirred for

2–3 h. Subsequently, water (360 mL) was added to the reaction mixture

and stirred for 30 min. The organic layer was separated and

concentrated below 55 °C under atmospheric pressure to provide 21

Chapter-2

51

[residue was directly used in next step without further isolation (121.3 g;

crude yield was considered as 100 %)].

IR (KBr, cm–1): 2915, 1600, 1495, 1455, 1391, 1350, 1113, 1076, 756,

709.

1H NMR (200 MHz, CDCl3): δ 7.70–7.20 (m, 10H), 5.50 (s, 1H), 3.80–

2.90 (m, 4H), 2.40–1.60 (m, 4H), 1.45 (s, 3H).

MS (m/z): 307 (M+ + H).

2.6.6 3,4-Diphenyl-5-methylisoxazole (4)

To a stirred mixture of 21 (121 g, 0.395 mol) and water (500 mL) was

added conc hydrochloric acid (36 %, 225 mL, 2.250 mol) at 40–50 °C.

The resulted reaction mixture was heated to 98–102 °C and maintained

for 1.5–2.5 h at the same temperature. Thereafter, reaction mixture was

cooled to 25–35 °C and the product was extracted with dichloromethane

(2 x 200 mL). The combined organic layers were washed with water

(150 mL) and concentrated under atmospheric pressure below 50 °C

followed by under reduced pressure below 50 °C. Subsequently,

isopropyl alcohol (75 mL) was added and maintained for 15–30 min at

45–50 °C. The resulted reaction mixture was cooled to 0–5 °C and stirred

for 45–60 min. The precipitated solid was filtered and washed with

Chapter-2

52

chilled isopropyl alcohol (5 mL). The filtered compound was recrystallised

from isopropyl alcohol to obtain 24 g (26 %) of 4.


IR (KBr, cm–1): 3050, 2928, 1618, 1597, 1463, 1433, 1415, 1377, 1304,

1240, 1074, 916, 768, 697.

1H NMR (200 MHz, CDCl3 + CD3CN): δ 7.45–7.11 (m, 10H), 2.44 (s, 3H).

MS (m/z): 236 (M+ + H).

2.6.7 4-(5-Methyl-3-phenyl-4-isoxazolyl)benzenesulfonyl

chloride (22)

To a stirred solution of chlorosulfonic acid (98 g, 0.841 mol) in

dichloromethane (75 mL) was added a solution of 4 (25 g, 0.106 mol) in

dichloromethane (50 mL) at 0–10 °C. The reaction mixture was heated to

reflux and stirred for 9–11 h, then cooled to 25–35 °C and quenched into

chilled water (175 mL) below 10 °C. Thereafter, temperature of the

reaction mixture was raised to 25–35 °C and organic layer was

separated. The aqueous layer was extracted with dichloromethane

(2 x 60 mL). The combined organic layers were washed with water

(3 x 100 mL) and concentrated under atmospheric pressure below 60 °C.

Cyclohexane (250 mL) was added to reaction mixture and heated to

Chapter-2

53

reflux for 15–30 min. Water (75 mL) was added to the reaction mixture

and maintained for 15–30 min under reflux. The organic layer was

separated, cooled to 25–35 °C and stirred for 45 min. The precipitated

solid was filtered and washed with cyclohexane (22 mL). Recrystallization

from cyclohexane was repeated twice and dried under vacuum at 50–55

°C to obtain 21.3 g (60 %) of 22.


IR (KBr, cm–1): 3090, 3063, 1625, 1590, 1491, 1464, 1396, 1383, 1191,

782, 754.

1H NMR (200 MHz, CDCl3 + CD3CN): δ 8.02 (d, J ═ 8.6 Hz, 2H), 7.50–

7.28 (m, 7H), 2.53 (s, 3H).

13C NMR (50 MHz, DMSO–d6): 167.1, 160.6, 147.2, 129.9, 129.6, 129.0,

128.7, 128.6, 128.1, 126.0, 114.8, 11.3.

MS (m/z): 334 (M+ + H).

2.6.8 4-(5-Methyl-3-phenyl-4-isoxazolyl)benzenesulfonamide (1)

To a stirred solution of 22 (20 g, 0.060 mol) in dichloromethane (120

mL) was added charcoal (1 g) and stirred for 30–45 min. Thereafter,

charcoal was filtered and washed with dichloromethane (40 mL). To the

combined filtrate was added aqueous ammonia (15 %, 90 mL, 0.794 mol)

Chapter-2

54

at 20–30 °C for 1–1.5 h. Subsequently, dichloromethane was distilled off

from the reaction mixture below 45 °C under atmospheric pressure, then

cooled to 5–10 °C and stirred for 30–45 min. The precipitated solid was

filtered and washed with water (20 mL). The wet compound was charged

into water (100 mL), stirred for 45–60 min and filtered the solid. Washed

with water (20 mL) and dried under vacuum at 80–85 °C to afford 15 g

(80 %) of 1.


IR (KBr, cm–1): 3378, 3250, 2926, 1622, 1595, 1563, 1465, 1392, 1333,

1151, 784.

1H NMR (400 MHz, DMSO–d6): δ 7.84 (d, J ═ 8.2 Hz, 2H), 7.50–7.32 (m,

9H), 2.47 (s, 3H).

13C NMR (100 MHz, DMSO–d6): 167.5, 160.6, 143.3, 133.3, 130.0,

129.7, 128.7, 128.4, 128.1, 126.1, 114.2, 11.3.

MS (m/z): 315 (M+ + H).

Anal. for C16H14N2O3S: calcd: C, 61.13; H, 4.49; N, 8.92; S, 10.19.

Found: C, 60.89; H, 4.35; N, 8.98; S, 10.21.

Chapter-2

55

2.6.9 3-[5-Methyl-4-(4-sulfamoylphenyl)isoxazol-3-yl]-benzene

sulfonamide (Impurity-A, 23)

To a stirred solution of chlorosulfonic acid (347 g, 2.978 mol) in


dichloromethane (40 mL) at 0–10 °C. The resulted reaction mixture was

refluxed for 7–8 h followed by cooling to below 10 °C and quenched with

water (900 mL). The organic layer was separated and the aqueous layer

was extracted with dichloromethane (2 x 100 mL). Aqueous ammonia

(15 %, 260 mL, 2.294 mol) was added to the combined organic layer at

25–35 °C and stirred for 1 h. The precipitated solid was filtered and dried

at 80–85 °C to provide 23.5 g (70 %) of 23.


IR (KBr, cm–1): 3322, 3241, 1636, 1424, 1346, 1161, 786.

1H NMR (400 MHz, DMSO–d6): δ 8.05 (s, 1H), 7.90 (d, J ═ 8.0 Hz, 1H),

7.86 (d, J ═ 8.4 Hz, 2H), 7.58 (t, J ═ 8.0 Hz, 1H), 7.45 (d, J ═ 8.4 Hz, 2H),

7.42–7.35 (m, 5H), 2.48 (s, 3H).

13C NMR (50 MHz, DMSO–d6): 168.0, 159.6, 144.8, 143.3, 132.6, 131.2,

129.9, 129.4, 129.0, 126.7, 126.0, 125.1, 114.2, 11.3.

Chapter-2

56

MS (m/z): 394 (M+ + H).

2.6.10.1 5-Benzyl-3-phenylisoxazole (4a)

A solution of 16 (100 g, 0.745 mol) and pyrrolidine (88 g, 1.237 mol)

in cyclohexane (740 mL) was maintained under azeotropic reflux

conditions until water collection stops. The resulted reaction mass was

concentrated under reduced pressure below 70 °C followed by addition

dichloromethane (1000 mL) and triethylamine (116 g, 1.146 mol) at

25–35 °C. To the resulted reaction mixture was added a solution of 20

(125 g, 0.803 mol) in dichloromethane (200 mL) and stirred at 25–35 °C

for 3 h. Subsequently, the reaction mixture was quenched with water

(750 mL) and organic layer was separated. The aqueous layer was

extracted with dichloromethane (200 mL) and the combined organic

layers were concentrated under reduced pressure below 55 °C.

Thereafter, conc hydrochloric acid (350 mL) and water (700 mL) were

charged to the reaction mixture. The resulted reaction mixture was

refluxed for 3 h at 100 °C, cooled to 25–35 °C and charged

dichloromethane (450 mL). Organic layer was separated and aqueous

layer was extracted with dichloromethane (200 mL). The combined

organic layers were concentrated under reduced pressure below 50 °C.

Chapter-2

57

After cooling the reaction mixture to 25–35 °C, isopropyl alcohol (100 mL)

was charged and filtered the solid (4). The obtained mother liquor was

concentrated below 60 °C under reduced pressure, charged acetone (200

mL) at 25–35 °C and stirred for 1h. Filtered the solid and dried at

40–45 °C to obtain 26 g (15 %) of 4a.


IR (KBr, cm–1): 2918, 1597, 1578, 1408, 775.

1H NMR (200 MHz, CDCl3): δ 7.82–7.70 (m, 2H), 7.53–7.22 (m, 8H), 6.33

(s, 1H), 4.13 (s, 2H).

MS (m/z): 236 (M+ + H).

2.6.10.2 4-[(3-Phenylisoxazol-5-yl)methyl]benzenesulfonamide

(Impurity-B, 24)

To a solution of chlorosulfonic acid (79.2 g, 0.680 mol) in

dichloromethane (50 mL) was added a solution of 4a (20 g, 0.085 mol) in

dichloromethane (50 mL) at 0–5 °C. The reaction mixture was stirred at

40 °C for 3–5 h and quenched with water (140 mL) below 10 °C. The

layers were separated and the product was extracted with

dichloromethane (2 x 50 mL) from aqueous layer. Thereafter, aqueous

Chapter-2

58

ammonia solution (15 %, 130 mL 1.147 mol) was added to the combined

organic layer at 25–35 °C and stirred for 1–2 h. The precipitated solid

was filtered and washed with water (40 mL) and dichloromethane (20 mL)

successively. Wet solid was dried at 80–85 °C to afford 16 g (60 %) of 24.


IR (KBr, cm–1): 3363, 3271, 1609, 1341, 1158, 771.

1H NMR (200 MHz, CDCl3): δ 8.03 (d, J ═ 8.4 Hz, 2H), 7.91–7.65 (m,

2H), 7.56 (d, J ═ 8.4 Hz, 2H), 7.44–7.35 (m, 3H), 6.33 (s, 1H), 4.27 (s,

2H).

13C NMR (100 MHz, DMSO–d6): 171.7, 161.9, 142.7, 140.2, 130.0,

129.2, 129.0, 128.5, 126.4, 126.0, 100.4, 31.8.

MS (m/z): 315 (M+ + H).

2.6.11 3-(5-Methyl-3-phenylisoxazol-4-yl)benzenesulfonamide

(Impurity-C, 25)

To a solution of chlorosulfonic acid (40 g, 0.343 mol) in


dichloromethane (10 mL) at 0–10 °C. The reaction mixture was refluxed

for 7–8 h. The resulted reaction mixture was quenched with water

Chapter-2

59

(100 mL) below 10 °C. The organic layer was separated and the aqueous

layer was extracted with dichloromethane (2 x 50 mL). The combined

organic layers were concentrated below 45 °C under reduced pressure.

Thereafter, cyclohexane (100 mL) was added to the residue at 25–35 °C

and stirred for complete solid separation. The precipitated solid (22) was

filtered and filtrate was concentrated below 60 °C under reduced

pressure to obtain 22a (not isolated) along with some quantity of 22 (not

quantified). To a solution of 22a (crude) and 22 in dichloromethane

(100 mL) was added aqueous ammonia (15 %, 65 mL, 0.573 mol) at

25–35 °C. The resulted reaction mixture was stirred for 1 h at 25–35 °C.

The organic layer was separated and the aqueous layer was extracted

with dichloromethane (2 x 20 mL). The combined organic layers were

concentrated under reduced pressure below 45 °C, dichloromethane

(10 mL) was added at 25–35 °C and cooled to 10–15 °C. After stirring for

1 h, the precipitated solid (1) was filtered. The filtrate was concentrated

and repeated the crystallization twice using dichloromethane to remove 1

completely. The filtrate (free from 1) was concentrated below 45 °C under

reduced pressure. Ethyl acetate was added to the resulted residue and

refluxed for 30 min followed by cooling to 0–5 °C and stirred for 1 h. The

precipitated solid was filtered and dried at 80–85 °C to provide 1.3 g

(10 %) of 25.


IR (KBr, cm–1): 3337, 3247, 1334, 1165.

Chapter-2

60

1H NMR (400 MHz, DMSO–d6): δ 7.83 (d, J ═ 7.8 Hz, 1H), 7.71 (s, 1H),

7.59 (t, J ═ 7.8 Hz, 1H), 7.49–7.36 (m, 8H), 2.45 (s, 3H).

13C NMR (100 MHz, DMSO–d6): 167.0, 160.4, 148.7, 147.4, 129.8,

129.6, 129.1, 128.9, 128.7, 128.2, 128.0, 127.9, 126.4, 126.0, 115.0,

11.1.

MS (m/z): 315 (M+ + H).

2.6.12 4-(5-Methyl-3-phenylisoxazol-4-yl)benzenesulfonic acid

(Impurity-D, 26)

A mixture of 22 (20 g, 0.060 mol), water (65 mL) and tetrahydrofuran

(280 mL) was heated to reflux and maintained for 24 h. Solvent was

distilled completely from the reaction mixture below 80 °C under reduced

pressure. Reaction mixture was cooled to 25–35 °C, toluene (200 mL) was

added and stirred for 1 h. The solid was filtered and dried at 50–55 °C to

obtain 18.5 g (98.4 %) of 26.


IR (KBr, cm–1): 3395, 1622, 1396, 1177.

1H NMR (400 MHz, DMSO–d6): δ 7.64 (d, J ═ 8.0 Hz, 2H), 7.45–7.35 (m,

5H), 7.19 (d, J ═ 8.0 Hz, 2H), 2.44 (s, 3H).

13C NMR (100 MHz, DMSO–d6): 167.0, 160.5, 147.4, 129.8, 129.6,

Chapter-2

61

128.9, 128.7, 128.6, 128.0, 126.0, 114.8, 11.2.

MS (m/z): 316 (M+ + H).

2.6.13 4-(5-Methyl-3-phenylisoxazol-4-yl)-N-[4-(5-methyl-3-

phenylisoxazol-4-yl)phenylsulfonyl]benzenesulfonamide

(Impurity-E, 27)

A mixture of 1 (10 g, 0.032 mol), 22 (16 g, 0.048 mol) in pyridine (50

mL) was heated to 110–115 °C and stirred for 9 h at the same

temperature. The reaction mixture was concentrated under reduced

pressure at 95 °C. The resulted solid was recrystallized from isopropyl

alcohol (300 mL) and the wet compound was again recrystallized from

the mixture of water (100 mL) and acetone (100 mL). The wet solid was

dried at 80–85 °C to afford 12.5 g (64 %) of 27.


IR (KBr, cm–1): 3339, 1626, 1369, 1165, 871.

1H NMR (400 MHz, CDCl3 + DMSO–d6): δ 7.95 (d, J ═ 8.2 Hz, 4H), 7.45–

7.36 (m, 10H), 7.32 (d, J ═ 8.2 Hz, 4H), 2.50 (s, 6H).

13C NMR (100 MHz, DMSO–d6): 167.2, 160.6, 145.4, 131.5, 129.7,

129.0, 128.7, 128.5, 128.1, 126.5, 114.5, 11.3.

MS (m/z): 612 (M+ + H).

Documents

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