2444 Med Chem 1991 34 24442452

was resuspended in times the original volume of buffer and Registry No 134133-69-0 134233-45-7 134233-46-8

incubated at 37 for 15 mm centrifuged again and resuspended 134233-47-9 134133-70-3 S.HC1 134233-67-3 134233-48-0

in the original volume of buffer binding assay using 9.HC1 134308-16-0 th-10 52079-30-8 5R-11 58879-35-9

bremazocine 0.5 aM was carried out as described above 5S-11 58879-36-0 12 134133-71-4 13 134233-49-1 14

134133-72-5 15 134233-50-4 16 isomer 134133-73-6 16 isomer

Acknowledgment We greatly appreciate receiving134233-51-5 18 isomer 134133-74-7 18 isomer

samples of benzomorphan standards its enantiomer23134233 5592413423356-02513423357 130 134233-58-2

R- and S-3 and the enantiorner of S-3 from Dr31 134233-59-3 32 isomer 134133-76-9 32 isomer

Herbert Merz Boehringer Ingelheim KG Germany We 134233 60-6 33 isomer 134133 77 33 isomer 134233 61

acknowledge the support of this work by the National 33 isomer 1-HC1 134233-62-8 33 isomer 2.HC1 134308-15-9Institute on Drug Abuse through research grants DA-03933 34 isomer 134233-63-9 34 isomer 134233-64-0 35 isomer

and DA-06675 134233-65-1 35 isomer 134233-66-2

Preparation and Anticonvulsant Activity of Series of Functionalized

a-Heteroatoin-S ubstituted Amino Acids

Harold Kohnt Kailash Sawhney Philippe LeGall David Robertsonh and David Leander

Department of Chemistry Uniuer8ity of Houston Houston Texas 77204-5641 and Lilly Research Laboratories Eli Lilly

Company Indianapolis Indiana 46285 Receiued December 1990

Potent anticonvulsant activity has been reported for RS-2-acetamido-N-benzyl-2-methylacetamide 2a Select

a-heteroatom substituted derivatives of 2a have been prepared 26 examples in which the a-methyl group has been

replaced by nitrogen 3aq oxygen 3ru and sulfur 3vz containing moieties The functionalized amino acid

derivatives were evaluated in the maximal electroshock seizure MES and horizontal screen tox tests in mice

The most active compounds were RS-2-acetaniido-N-benzyl-2-methoxyaminoacetamide 31 and RS-2-acetamido-N-benzyl-2-methoxymethylaminoacetaniide 3n After ip administration the MES ED values for

316.2 mg/kg and 3n 6.7 mg/kg compared favorably with phenytoin 9.50 mg/kg

Nonnaturally occurring amino acids have become in- amino acid derivatives 26 examples are presented In

creasingly important in the design of pharmacologically eluded in this survey are selected oxygen nitrogen and

active peptides and peptidomimetics.1 Recently we re- sulfur-functionalized amino acids Analysis of the comported the excellent anticonvulsant activity of certain posite data set disclosed trends that further define the

functionalized amino acid derivatives 1.2 Potent pro- structureactivity relationships for this class of amino acid

tection against maximal electroshock seizures MES in derived anticonvulsant agentsmice was observed for functionalized amino acid racemates Selection of Compoundsontaining both an

Ns ai RS Acetamido benzyl methylacetamide3 2astituent revealed that stringent steric and electronic re-

represented the parent compound in this study wherein

quirements must be met for optimal activity The medianthe methyl group was replaced by select functionalized

effective dose ED for the a-methyl 2a 76.5 mg/kgnitrogen oxygen and sulfur substituents Table In

and a-phenyl 2b 20.3 mg/kg derivatives4 comparedall cases the racemates were prepared and tested No

favorably with that observed for phenobarbital7 21attempts were made at this stage to resolve the enan

mg/kg while those of the a-pyrrolyl 2c 16.1 mg/kgtiomeric mixtures The a-nitrogen-substituted adducts

and -furan 2d 10 /k adducts6 rivaled thconsisted of the parent ammo 3a the monoalkylammo

reported for phenytoin7 950mg/kg Furthermore3bc the dialkylamino 3de and the trialkylammonium 3f

parison of the two individual enantiomers of 2abd re-derivatives as well as the corresponding monoaryl ana

vealed that in each case the anticonvulsant activity residedlogues 3g and 3h Included in our a-nitrogen subset of

primarily in the stereoisomer 256 compounds were three classes of functionalized amino

derivatives These were the monoacyl derivatives 31 and

3j the N-hydroxyamino adducts 3ko and the N-

hydrazino compounds 3p and 3q The second set of

RlCNH.NHR3 CH3CNH-f-NHCH structurally modified amino acid derivatives were the

For leading references see Shunian Ornstein25 CH3 Paschal Gesellchen Org Chem 199055 738

Ph Ojima Komata Qiu Am Chem Soc 1990 112

2-pyrrolyl770 and references therein

A2 2-fKohn Conley Chem Br 1988 24 231

uranyCortes Liao Z.-K Watson Kohn Med Chem

In the present study the synthesis and anticonvulsant 1985 28 601

Conley Kohn Med Chern 1987 30 567properties of novel series of a-heteroatom-substituted Kohn Conley Leander Brain Res 1988 457

371

Kohn Sawhney LeGall Conley Robert-

Author to whom correspondence should be addressed to at son Leander Med Chem 1990 33 919the University of Houston Porter Cereghino Gladding Hessie

tUniversity of Houston Kupferberg Scoville White Cleveland Clin

Lilly Research Laboratories 1984 51 293

0022-2623/91 1834-2444$02.50/0 1991 American Chemical Society

MYLAN - EXHIBIT 1012

Functionalized a-Heteroatom-Substituted Amino Acids Journal of Medicinal Chemistry 1991 Vol 34 No 2445

Table Selected Physical and Pharmacological Data in Mice for a-Heteroatom-Substituted Functionalized Amino Acid Derivatives 3s

II II

CH3CNH CH CNHCH2Ph

MESCno mpb EDs TD

3a NH2 131133 65.1

56.275.33b NHCH3 115117 44.5

37.052.43c NHCH2CH3 123125 42.4

37.247.83d NCH32 104106 45.3NO 171172 30 100

31 NCH33 BF4 171173 dec 1003g NHPh 183185 300

3hNN

135137 100

NH31 NHCOCH3 265267 dec 100 3003j NHCOCF3 228-230 3003k NHOH 144146 dec -10031 NHOCH3 9597 6.2 46.0

5.47.2 38.056.03m NCH3OH 159161 303n NCH3OCH3 165167 6.7 50.5

5.77.7 40.459.9

30 P\ 149151 31.4

26.737.8

3p NHNHPh 132134 -100

3q NHNHCO2CH2Ph 152154 55.6

49.363.93r OH 136138 80.1

70.691.03sf OCH3 145146 98.3 100 300

84.4114.03t1 OCH2CH3 153155 62.0 112

51.178.43u OPh 125-128 1003v SCH3 155157 1003w SCH2CH3 140142 30 1003x SPh 165167 3003y1 SOCH2CH3 135137 1003y-mix SOCH2CH3 135137 1003z SO2CH2CH3 161163 1002a CH3 138139 76.5 4539

66.689.0 416.6501.02b Ph 202203 20.3 96.9

16.924.5 79.8118.42c 2-pyrrolyl 174175 16.1 30 100

13.219.92d 2-furanyl 178179 10.3 -40

9.111.6phenytoin 9.5

8.110.4 52.572.1phenobarbital 21.8 69.0

15.022.5 62.872.9

valproate 272 426

247338 369450

The compounds were administered intraperitoneally ED and TD values are in milligrams per kilogram Number in parentheses are

95% confidence intervals Time of peak effects in hours as determined in the Experimental Section is denoted in brackets 5Melting points

are uncorrected MRS maximal electroshock seizure test Compound was suspended in 30% PEG unless otherwise noted Tox

neurologic toxicity determined from horizontal screen unless otherwise noted e0 determined tReference Compound 3u was sus

pended in acacia Reference Neurologic toxicity determined using the rotorod test Reference Reference Reference

a-oxygen-substituted compounds 3ru This group was similar battery of a-sulfur-substituted compounds i.ecomprised of the a-hydroxy adduct 3r the two a-alkoxy 3vx8 was selected for evaluation Attempts to synthesize

derivatives 3s and 3t8 and the a-phenoxy compound 3u the parent a-thiol derivative in the series were unsuc

________________________________________________cessful however In addition to 3vx both the sulfoxide

LeGall Sawhney Conley Kohn mt 3y and the sulfone 3z derivatives of the ethylthio adduct

Peptide Protein Res 1988 32 279 3w were prepared

2446 Journal of Medicinal Chemistry 1991 Vol 34 No Kohn et al

Scheme Preparation of a-Heteroatom-Substituted characterized due to the sensitivity of to moisture andFunctionalized Amino Acid Derivatives its poor solubility in nonhydroxylic solvents Accordingly

Method either the addition of an excess of the heteroatom speciesBrO

_______or the sequential addition of triethylamine and the nu

Br

CH3CNH-CH2-COCH2CH3 CH3CNH-CH-COCH2CH2cci4 cleophilic heteroatom-containing reagent to THF mixture

/7 containing furnished in moderate amounts 138%yields from 3t This method was employed for the

preparation of compounds 3d 3k 3p and 3uw Includedexcess xH THF -78

in the product mixture for 3k were the diastereomeric

adducts 8a and 8b in which equiv of reacted withX0PhCH2NH2 NH2OH Implementation of this procedure for the syn

cH3NH-cH-cNHCH2Ph CH3CNH-CH-COCH2CH3

thesis of 31o was not successful due to the difficulty en-

CH3OH countered in obtaining solutions of the free hydroxyaminesin nonhydroxylic solvents Use of methanolic solutions of

Met/rod excess XH the hydroxyamines furnished only 3sBr THF-78

BBr3II II

21 CH3CNH-CH-CNHCH2Ph II II

CH2CI2 or CH3CNH1Et3N

..CNHCH2..Q2XH NOH

THF -78

_____CH3CNH.CCNHCH2_QMethodC

II ii

HOCH330

BF

BF NCH33excess xti

CH3CNH-CH-CNHCH2Ph

Employment of the third protocol method outlined

in Scheme provided convenient procedure to circum

Method vent this obstacle Methylation of the dimethylamino

adduct 3d with trimethyloxonium tetrafluoroborate in

nitromethane furnished the quaternary ammonium deQEt BFEt2O

E120 rivative 3f in high yields Subsequent treatment of 3f withCH3CNH-CH-CNCH2Ph methanolic solution containing the requisite hydroxy

excess XHamine led to production of 31o in good yields 4282%

Synthesis of the a-hydroxy 3r a-ethylthio 3w and

a-thiophenoxy8 3x amino acid derivatives was accomChemistry

pushed using the last technique depicted in Scheme in

Four different synthetic approaches Scheme methods which 3t was treated with BF3.Et20 in the presence of H20AD were employed for the preparation of most of the EtSH and PhSH respectively Scheme methoda-heteroatom-substituted functionalized amino acid de- similar protocol was utilized by us for the preparation ofrivatives In the first route method the a-bromo a-heteroaromatic functionalized amino acid derivatives.68

ester was prepared in near quantitative yield using the This procedure proved superior than that of method for

protocol of Kober and Steglich9 from commercially the preparation of 3wavailable0 ethyl acetamidoacetate Treatment of with Two of the remaining compounds listed in Table 3i

an excess of the nucleophilic heteroatom species furnished and 3j were obtained by treatment of 3a with acetic an-the corresponding ethyl 2-substituted-2-acetamidoacetate

hydride and trifluoroacetic anhydride respectively The6192% yields Formation of is presumed to proceed final compounds 3y and 3z were prepared directly from

through the intermediacy of ethyl 2-acetyliminoacetate the a-ethylthio adduct 3w Interestingly treatment of 3wIn most cases was isolated as thick oil and used directly with m-chloroperbenzoic acid in CH2C12 led to the stein the subsequent step without extensive purification reoselective production of the a-sulfoxide 3y-l 3C NMRTreatment of with benzylamine and catalytic amount

analysis of the initial reaction mixture indicated theof NaCN gave the desired product in moderate yields

presence of only single diastereomeric enantiomeric pair2974% This method was used to synthesize compounds compound The precise stereochemical identity of this

3ac 3e 3g and 3h Attempts to utilize this protocol to adduct has not been established Correspondingly use of

prepare 3d 3k 3m and 3n were unsatisfactory In these stoichiometric amounts of Na104 at room temperature in

cases the final benzylamine-mediated step did not proceed an aqueous methanolic solution yielded 21 diastereo

cleanly meric mixture 3C NMR analysis of 3y-1 and 3y-2 in

The difficulty encountered in converting several which the major compound present corresponded to thefunctionalized esters to the corresponding benzylamides product generated in the m-chloroperbenzoic acid reaction

led to the development of the second procedure method 3y-l Attempts to completely separate these diastereo

depicted in Scheme In this route the benzylamide mers by either TLC or recrystallization proved unsucmoiety was incorporated within the framework of the am- cessful Accordingly 23 mixture of 3y-l and 3y-2 obmo acid derivative prior to the introduction of the tamed after fractional recrystallization was analyzed for

heteroatom substituent Treatment of 3t with BBr3 inanticonvulsant activity and is identified as 3y-mix We

CH2CI2 led to the formation of the presumed a-bromo have tentatively attributed the diastereoselectivity of thederivative This adduct could not be fully purified or

m-chloroperbenzoic acid mediated process to the preor

_________________________________________ ganization of the oxidant with 3w in CH2CI2 Employment

of excess Na104 with 3w at elevated temperatures 5060Kober Steglich Liebigs Ann Chem 1983 599

gave the ce-sulfone 3z in 32% yield10 Aldrich Chemical Co11 Hogberg Strom Ebner Ramaby Org Chern Of note all 17 aa-diamino acid derivatives 3aq in-

1987 52 2033 cluding the trimethylammonium adduct 3f were well

Functionalized a-Heteroatom-Substituted Amino Acids Journal of Medicinal Chemistry 1991 Vol 34 No 2447

defined stable compounds.2 Only 3f 3i and 3k melted Conclusions

with decompositionStraightforward procedures have been employed for the

Pharmacological Evaluation preparation of a-heteroatom-substituted amino acid de

The a-heteroatom substituted amino acid derivativesrivatives Despite the fact that these compounds have

were tested for anticonvulsant activity by using thegeminal heteroatoms to the carbonyl they are chemically

procedures described by Krall et aL3 All compounds werewell-defined and are exrected to serve as useful substrates

administered intraperitoneally ip to mice Table listsin future chemical and pharmacological studies The

the median effective dose ED values required to preventpharmacological data obtained in this investigation pro-

seizures in the MES test by racemic Included in thisvided additional information concerning the structure

table are the median neurotoxic dose TD values de- activity profile of functionalized amino acid anticonvul

termined for select compounds using the horizontal screensanta The biological activities for reinforced our notions

test.4that stringent steric and electtonic requirements exist for

Evaluation of the results listed in Table revealedmaximal anticonvulsant activity in this class of compounds

several important observations First the a-amino 3a The potencies of 31 and 3n in the MES test were compa

a-alkylamino 3be and a-trimethylainmonium 3f de-rable to those of phenytoin and 2d Additional studies in

rivatives all displayed anticonvulsant activities comparableprogress are aimed at investigating the generality of this

class of compounds as well as their mode of actionto that observed for the a-methyl analogue 2a.34 Secondthe a-arylamino derivatives 3g and 3h were devoid of Experimental Section

activity at doses below 100 mg/kg comparable reduc-Chemistry General Methods Melting points were deter

tion in activity has been observed in proceeding from the mined with Thomas-Hoover melting point apparatus and are

a-methyl derivative 2a to the corresponding a-benzyl uncorrected Infrared spectra IR were run on Perkin-Elmer 1330adduct4 and has been attributed in part to the stringent and 283 spectrometers and were calibrated against the 1601-cm

steric requirements that exist for maximal anticonvulsant band of polystyrene Absorption values are expressed in wave-

activity in this class of compounds Third conversion of numbers cm Proton NMR and carbon 3C NMR nuclear

the a-amino derivative 3a to the corresponding a-acyl- magnetic resonance spectra were taken on Nicolet NT-300 and

amino adducts 3i and 3j led to decrease in activity of theGeneral Electric QE-300 NMR instruments Chemical shifts

test compound Fourth incorporation of an a-N-alkoxy-are in parts per million ppm relative to Me4Si and couplingconstants values are in hertz Low-resolution mass spectra

amino moiety i.e 31 3n 3o within the backbone of the MS were recorded at an ionizing voltage of 70 eV with Variancompound led to pronounced improvement of the po- MAT CH-5 spectrometer at the Lilly Research Laboratoriestency of the compound in the MES test compared to either Microanalyses were provided by the Physical Chemistry De2a or 3a corresponding enhancement in activity was partment of the Lilly Research Laboratories Benzyl carbazate

not observed for the two N-hydroxyamino adducts 3k and was obtained from Lancaster Synthesis Ltd Windham NH3m The anticonvulsant activities of racemic 31 EDJ Thin-layer chromatography were run on precoated silica gel GHLF6.2 mg/kg and 3n EDse 6.7 mg/kg were comparable microscope slides 2.5 10 cm Analtech No 21521

to that of the RS-2-furanyl derivative 2d6 EDse 10.3 Preparation of a-Heteroatom-Substituted Amino Acids

mg/kg and phenytoin7 ED 9.5 mg/kg ImportantlyMethod Synthesis of Ethyl 2-Acetamido-2-substi-

tuted-acetates General Procedure cooled -78 solutionin the most potent analogues 2d 31 and 3n func-

of 59 equiv in THF mmol/10 mL was added slowly totionalized oxygen atom existed two atoms removed from THF mmol/4 mL solution of the nitrogen nucleophile 510the a-carbon atom This pattern suggests that substi-

equiv at 78 The reaction was stirred at this temperaturetuted /3-heteroatom may be necessary for maximal activity 0.5 and then at room temperature The insoluble ma-Fifth the a-hydrazine derivatives 3p and 3q did not dis- terials were filtered and washed with THF The filtrate was

play significant anticonvulsant activity Once again this concentrated in vacuo and the residue was purified by flash

property has been attributed in part to the steric size of chromatography on Si02 gel using the indicated solvent as the

these substituents Sixth the a-hydroxy 3r and the two eluent to give the desired product

a-alkoxy adducts 3s 3t displayed activity comparableBy use of this procedure the following compounds were pre

to that reported for 2a The potency of the a-oxygen seriespared

was somewhat diminished from that observed for theSynthesis of Ethyl 2-Acetamido-2-aminoacetate 6a

Compound 2.00 8.93 mmol and liquid NH3 56 equivcorresponding a-amino derivatives 3ac In agreement

yielded light brown residue which on purification by flash

with previous findings4 the a-phenoxy adduct 3u displayed column chromatography on Si02 gel 5% MeOH/CHC13 gaveno activity at doses of 100 mg/kg or less Seventh within 1.00 70% of 6a as yellow oil 0.21 5% MeOH/CHC13the a-sulfur series only the a-ethylthio adduct 3w ex- NMR CDC13 61.31 7.1 IIz 2.03 2.61

hibited anticonvulsant activity at doses less than 100 br 4.24 7.1 Hz 5.21 7.1 Hzmg/kg Eighth no enhancement of activity was noted for 7.50 7.1 Hz 3C NMR CDC13 13.72 22.68 59.70

the three sulfur-oxygenated derivatives 3y-1 3y-mix and 61.73 170.40 170.68 ppm3z versus 3w This observation is consistent with the Synthesis of Ethyl 2-Acetamido-2-methylaminoacetate

results obtained for the two N-hydroxyamino adducts 3k6b Use of 2.00 8.93 mmol and MeNH2 2.50 80.6 mmol

and 3m versus 3a gave an oily residue 1.50 The residue was purified by flash

column chromatography on 8102 gel 3% MeOH/CHC13 to yield

_______________________________________________1.00 65% of Gb as an oil R1 0.303% MeOH/CHCI3 NMR

12 For recent reports on the preparation of aa-diamino acid de-CDC13 1.32 7.1 Hz 2.07 2.36

rivatives and related compounds see Katritzky4.26 7.1 Hz 5.20 7.4 Hz 6.60 br

Urogdi Mayence Chem Soc Chem Commun 1989 the remaining amino proton was not detected 3C NMR337 Katritzky Urogdi Mayence Org CDC13 14.02 23.06 30.84 62.04 65.72 170.09 170.40 ppmChem 1990 55 2206 Bock DiPardo Synthesis of Ethyl 2-Acetanaido.2-ethylaminoacetate

Freidinger Ibid 198651 3718 Fischer Hassner 6c Employing 2.10 9.38 mmol and EtNH2 1.40 31.04

Ibid 1990 55 5225 and references therein mmol gave brown residue The residue was purified by flash

13 Krall Penry White Kupferberg column chromatography on 8102 gel 3% MeOH/CHC13 to yield

Swmyard Epilepsia 1978 19 409 0.90 51% of Ge as light yellow oil R1 0.36 4% MeOH/14 Coughenour McLean Parker Pharmacol CHC13 NMR CDC13 0.93 6.7 Hz 1.12

Bjochem Behau 1977 351 6.8 Hz 1.87 2.48 6.7 Hz 4.05

2448 Journal of Medicinal Chemistry 1991 Vol 34 No Kohn et at

6.8 Hz 5.05 7.1 Hz 7.09 7.1 Hz MeOH/CHC13 to give 0.60 66% of Sn as an oil R1 0.53 2%the remaining amino proton was not detected 3C NMR MeOH/CHC1 NMR CDC1 1.35 7.0 Hz 2.12

CDC1 13.64 14.55 22.53 39.06 61.3864.14 170.09 170.20 ppm 2.62 3.48 4.30 7.0 Hz 5.36

Synthesis of Ethyl 2-Acetamido-2-dinsethylaminoacetate 8.9 Hz 6.66 8.9 Hz NMR CDC13Sd Compound 2.00 8.93 mmol and MeNH 56 equiv 14.06 22.89 40.30 60.01 61.89 70.16 168.14 170.53 ppmgave 6d 1.50 89% as yellow oil NMR CDC13 1.25 Synthesis of 2-Acetamido-N-benzyl-2-substituted-acet-

7.1 Hz 2.02 2.23 4.104.25 amides General Procedure mixture of equiv5.24 8.3 Hz 6.59 8.3 Hz NMR benzylamine 1.2 equiv and NaCN 0.1 equiv in MeOH

CDC1 14.05 23.00 40.28 61.84 69.24 169.38 170.57 ppm mmol/25 mL was stirred at 4550 18 The solvent was

Synthesis of Ethyl 2-Acetamido-2-4-morpholinoacetate removed in vacuo and the residue was purified with either

Se Use of morpholine 1.71 19.64 mmol and 2.00 8.93 trituration with EtOAc or flash column chromatography on Si02

mmol gave an oily residue which was purified by flash column gel using the indicated solvent as the eluent

chromatography on SiO gel 2% MeOH/CHC1 to give 1.90 By use of this procedure the following compounds were pre93% of Se as thick oil R0.29 3% MeOH/CHC1 NMR

paredCDC1 1.32 6.8 Ffz 2.07 2.432.72

Synthesis of 2-Acetamido-N-benzyl-2-aminoacetamide3.583.78 4.26 6.8 Hz 5.27 3a Compound Sa 1.00 6.25 mmolbenzylamine 0.80 7.5

7.9 Hz 6.39 7.9 Hz NMR CDCI 14.21 mmol and NaCN 0.03 0.61 mmol gave residue that solidified23.25 48.47 62.06 66.71 69.22 169.00 170.46 ppm on standing 18 The reaction mixture was triturated with

Synthesis of Ethyl 2-Acetanildo-2-N-anilinoacetate 6g EtOAc 20 mL The white solid 1.00 72% that remainedUse of aniline 1.83 19.6 mmol and 2.00 8.93 mmol

was filtered and then further purified by recrystallization fromprovided brown residue which was purified by flash column EtOAC mp 131133 dec R1 0.21 5% MeOH/CHC1 IRchromatography on SiO gel CHC12% MeOH/CHC1 gradient KBr 3300 1650 br 1530 br 1450 740 cm1 NMRto yield 1.80 85% of 6g mp 8789 recrystallized from

DMSO-d6 61.83 2.35 hr 4.28 4.4 Hzethyl acetate/petroleum ether R0.52 4% MeOH/CHC1 IR

4.91 7.0 Hz 7.207.32 8.31 hrKBr 3340 1720 1635 1590 1490 730 710 cm NMR8.51 hr 13C NMR DMSO-d6 22.66 42.05 60.29

CDC1 61.29 7.1 Hz 1.84 4.27 7.1126.67 127.10 128.18 139.23 169.24 170.67 ppm mass

Hz 5.89 8.2 Hz 6.43 8.2 Hz6.686.71 6.806.83 7.177.22 in the

spectrum m/e relative intensity 222 100 221

remaining amino proton was not detected NMR CDC129 133 Anal CHNO

13.96 22.98 60.19 62.41 113.87 119.29 129.37 144.09Synthesis of 2-Acetamido-N-benzyl-2-methylainino-

169.77 170.14 ppm mass spectrum FD 237 Md Anal acetamide 3b Compound Sb 1.50 8.63 mmol benzylamine

CH6NO 1.11 10.35 mmoland NaCN 0.04 0.82 mmol gave brown

Synthesis of Ethyl 2-Acetamido-2-3-pyrazolylamino-residue that was purified by flash column chromatography on Si02

acetate 6h Use of 2.00 8.93 mmol and 3-aminopyrazolegel 2% MeOH/CHC1 to yield 1.00 49% of 3b mp 115117

1.85 22.32 mmol and purification of the reaction product by recrystallized from ethyl acetate/petroleum ether R1 0.33

chromatography on SiO gel 2% MeOH/CHC1 gave 1.80 MeOH/CHC1 IR KBr 3240 1610 br 1500 br 1430

89% of 6h as yellow oil R0.35 8% MeOH/CHC1 725670 cm NMR DMSO-d6 61.87 2.18

CDC1 61.21 7.1 Hz 1.89 4.20 7.14.20-4.29 in 4.87 7.9 Hz 7.247.35

Hz 5.64 1.8 Hz 5.71 br 5.738.14 7.9 Hz 8.55 hr the remaining amino

7.1 Hz 7.29 1.8 Hz 7.98 7.1 Hz proton was not detected NIvIR DMSO-d6 22.52 31.3742.04

the remaining amino proton was not detected NMR 65.99 126.68 127.12 128.18 139.28 169.51 169.83

CDCI 13.73 22.49 61.41 62.02 91.79 130.53 153.02 169.96 ppm AnaL CH7NO170.93 ppm Synthesis of 2.Acetamido-N-benzyl-2-ethylaminoacet-

Synthesis of Ethyl 2-Acetamido-2-hydroxyaminoacetate ainide 3c Use of Sc 0.90 4.79 mmolbenzylamine 0.62

6k Use of 2.10 9.38 mmol and anhydrous NHOH5 0.93 5.75 mmol and NaCN 0.03 0.51 mmol gave an oily residue

28.00 mmol gave an oily residue The residue was purifiedthat was purified by flash column chromatography on SiO gel

by flash column chromatography on SiO gel 5% MeOH/CHC1 3% MeOH/CHCI to give 0.35 29% of 3c as white solid

to give 1.00 61% of 6k The product was recrystallized from in 123125 recrystallized from ethyl acetate/hexane R1 0.34

EtOH to give white flaky solid mp 119121 R1 0.24 5% 4% MeOH/CHC1 IR KBr 3250 1620 br 1510 br 1450

MeOH/CHC1 IR KBr 3300 1750 1660 1540 1390 610 cm br 740 680 cm NMR DMSO-d6 0.93 6.8 HzI-I NMR DMSO-d8 1.19 6.9 Hz 1.87 1.81 2.08 br 2.402.48 in 4.22

4.10 6.9 Hz 5.09 dd 4.0 8.0 Hz 6.06 br 5.5 Hz 4.90 7.8 Hz 7.207.27 in7.63 8.50 8.0 Hz NMR 8.08 7.8 Hz 8.48 5.5 Hz NMR

DMSO-d6 14.05 22.46 60.82 67.37 169.19 169.48 ppm mass CDC1 15.14 22.97 37.65 43.53 65.68 127.44 127.50 128.64

spectrum FD 177 M4 Anal C8HN204 137.73 169.75 171.20 ppm Anal CH9NOSynthesis of Ethyl 2-Acetamido-2-methylhydroxy- Synthesis of 2-Acetamido-N-benzyl-2-4-morpholino-

aminoacetate Sm MeNHOH 17.39 mmol prepared from acetamide 3e Use of Se 0.59 2.56 mmolbenzylamine 0.28

MeNHOH.HC1 2.00 23.95 mmol and NaOMe 0.94 17.39 2.82 mmol and NaCN 0.01 0.26 mmol gave thick oily

minol and 1.00 4.46 mmol gave an oily residue The residue residue The residue was triturated with EtOAc mL and the

was triturated with EtOAc mL and the solid 0.70 82% white solid 0.35 that remained was collected by filtration to

that remained was filtered and recrystallized from EtOH to give give 3e The filtrate was concentrated and the residue was

Sm as white solid mp 148150 R1 0.34 5% MeOH/CHC1 purified by flash column chromatography on SiO gel 2%IR KBr 3320 3200 br 1760 1660 1530 1470 720 640 cm MeOH/CHC1 The initial fractions furnished trace amount

NMR DMSO-d6 1.17 7.0 Hz 1.89 0.09 of 2-acetamido-N-benzyl-2-benzylaminoacetamide2.37 4.004.20 5.04 9.2 Hz 8.17 Continued elution gave additional amounts 0.20 of 3e

8.43 9.2 Hz NMR DMSO-d6 14.04 2-Acetainido-N-benzyl-2-benzylaminoacetamide yield

22.28 43.78 60.7971.48 168.29 170.23 ppm mass spectrum FD 0.09 11% mp 135138 R1 0.52 4% MeOH/CHC1 JR

192 Anal C7H4N204.0.25HO KBr 3250 br 1630 br 1500 br 1425 750 700 cm NMRSynthesis of Ethyl 2-Acetamido-2-methoxymethyl- DMSO-d6 1.83 3.52 hr 3.56 13.6 Hz

aminoacetate Sn MeNHOMe 17.40 mmol prepared from 3.66 13.6 Hz 4.23 5.4 Hz 4.89

MeNHOMe.HCI 2.18 22.32 mmol and NaOMe 0.94 17.40 8.0 Hz 7.057.38 10 8.20 8.0 Hzmmol and 1.00 4.46 mmol gave residue which was 8.51 5.4 Hz NMR DMSO-d6 22.63 42.11

purified by flash column chromatography on SiO gel 1% 48.5764.41 126.70 127.13 128.002 128.13 128.22

128.30 139.29 140.12 169.61 169.90 ppm mass spectrum

m/e relative intensity 312 12 311 178 1115 Hurd Inorg Synth 1939 87 177 100 Anal C8HNO-0.25HO

Functionalized a-Heteroatom-Substjtuted Amino Acids Journal of Medicinal Chemistry 1991 Vol 34 No 2449

2-Acetaniido.N-benzyl-2-4-morpholinoacetamide 3e 170.31 ppm mass spectrum FD 250 M4 Anal C3-yield 0.55 71% mp 171172 recrystallized from EtOAc H9N302R1 0.35 4% MeOH/CHC13 lB KBr 3250 1620 1515 1440 740 Synthesis of 2.Acetansido-N.benzyl-2-hydroxyamino-690 cm NMR DMSO.d6 51.87 2.292.45 acetamide 3k Use of 3t 2.00 8.0 mmol BBr3 in

3.51 4.1 Hz 4.23 dd 6.1 151 Hz 4.28 CHC12 8.8 mL 8.8 mmol and anhydrous NH2OH5 56 equiv

dd 6.1 15.1 Hz 5.08 9.1 Hz 7.127.30 gave an oily residue The residue was separated into three corn

8.24 9.1 Hz 8.58 6.1 Hz 13C ponents by flash chromatography on Si02 gel 7.5% MeOH/DMSO-d6 22.39 42.20 48.43 66.03 69.24 126.76 CHCI127.13 128.21 139.42 168.02 170.20 ppm mass 2-Acetamido-N-benzyl-2-hydroxyaminoacetamide 3kspectrum m/e relative intensity 292 233 158 yield 0.14 7% mp 144146 dec recrystallized from EtOH19 157 100 116 26 115 100 106 29 9172 Anal R10.30 8% MeOH/CHC13 IR KBr 3320 br 1660 br 1540

C5H21N303 br 1460 750 700 cm NMR DMSO-d6 1.88 4.31

Synthesis of 2-Acetamido-N-benzyl-2-N-anilinoacet- 5.7 Hz 5.08 dd 4.4 8.1 Hz 5.94 ddamide 3g fig 2.00 8.47 mmol benzylamine 1.09 10.00 2.8 4.4 Hz 7.197.35 7.52 2.8 Hzmmol and NaCN 0.04 0.84 mmol gave white solid that 8.26 8.1 Hz 8.42 5.7 Hz 3C NMRseparated during the course of the reaction The precipitate was DMSO-d6 22.69 42.25 67.86 126.69 127.14 128.18

filtered and purified by recrystallization from absolute EtOH to 139.08 168.53 169.67 ppm mass spectrum FD 238

give 1.10 44% of 3g mp 183185 IR KBr 3270 1630 Anal C1H5N3031520 1490 1430 740 690 cm1 NMR DMSO-d6 51.84 Compound 8a yield 0.05 3% mp 177179 recrys

4.31 5.8 Hz 5.67 8.1 Hz 6.04 tallized from EtOH R1 0.27 8% MeOH/CHC13 IR KBr 3240

8.1 Hz 6.596.64 6.706.72 7.067.11 br 1640 br 1510 br 1450 690 cm NMR DMSO-d67.207.33 8.41 8.1 Hz 8.72 1.82 4.254.34 5.21 9.3 Hz

5.8 Hz 3C NMR DMSO-d6 22.46 42.25 60.42 113.21 7.207.33 10 8.16 9.3 Hz 8.26 5.8 Hz117.22 126.72 127.16 128.18 128.77 138.99 8.51 3C NMR DMSO.d6 22.54 42.30

145.88 168.65 169.70 ppm mass spectrum m/e relative intensity 67.55 126.63 127.13 128.11 139.02

297 239 164 28 163 100 122 20 121 100 106 168.24 169.33 ppm mass spectrum FD 442

47 104 65 93 63 91 77 Anal C17H19N302 Anal C22H27N505Synthesis of 2.Acetamido-N-benzyl-2-3-pyrazolyl- Compound 8b yield 0.10 6% mp 184186 recrys

aminoacetamide 3h solution of 6h 1.60 7.1 rnmol in talllzed from MeOH R1 0.18 8% MeOH/CHC13 IR KBr 3300

MeOH 40 mL containing benzylamine 0.83 7.8 mmol and br 1660 br 1530 br 1450 740 700 cm NMR DMSO-d6NaCN 50 mg mmol was stirred at 4555 18 TLC 1.87 4.20 dd 5.3 15.3 Hz 4.44 dd 6.2

analysis 8% MeOH/CHC13 of the reaction mixture indicated 15.3 Hz 5.28 9.0 Hz 7.157.31 10 8.00

the presence of only minor amount of product second lot 9.0 Hz 8.39 dd 5.3 6.2 Hz 8.51

of NaCN 50 mg mmol was then added and the reaction was 3C NMR DMSO-d6 22.50 42.58 69.98 126.73

allowed to proceed at 4555C and then at room temperature 127.23 128.22 139.08 167.60 169.57

48 The solvent was removed in vacuo and the residue was ppm mass spectrum FD 442 Anal CnHvN5O5triturated with EtOAc 15 mL The insoluble solid that remained

was filtered and purified by flash column chromatography on Si02 Improved Synthesis of 2-Acetamido-N-benzyl-2-hy-gel 7% MeOH/CHCI3 to give 0.90 44% of 3h mp 135137 droxyaminoacetamide 3k Compound prepared from 3t

R1 0.35 8% MeOH/CHCI3 IR KBr 3230 br 1620 br 3.00 12.0 mmol and BBr3 in CH2C12 17.2 mL 17.2 mmol1500 br 1430 730 690 cm NMR DMSO-d6 11.82 was dissolved in THF 250 mL cooled 10 and then added

4.29 5.9 Hz 5.515.55 7.187.40 dropwise 30 mm to suspension of NHOH 56 equiv in THF8.36 br 8.53 br 11.66 br 3C NMR 50 mL at 10 The reaction mixture was stirred 30 mm

DMSO-d6 22.59 42.29 61.79 90.68 126.67 127.07 128.17 at this temperature and then allowed to warm to room temper-

129.10 139.41 153.53 169.19 169.67 ppm mass spectrum ature The insoluble materials were filtered and the filtrate

m/e relative intensity 288 64 287 230 28 was concentrated in vacuo The residue was separated into two

229 100 153 46 Anal C4H7N50.0.5H20 components by flash column chromatography on SiO gel 7.5%Preparation of Functionalized a-Heteroatom-Substituted MeOH/CHC1

Amino Acids Method General Procedure BBr3 2-Acetamido-N-benzyl-2-hydroxyaminoacetamide 3ksolution in CH2C12 1.1 equiv was added to solution of yield 0.66 23% mp 144146C dec recrystallized from EtOH3t68 equiv in CHCl 10 mmol/125 mL The mixture was Compound 8b yield 0.10 5% mp 184186 recrysstirred at room temperature and then concentrated to tallized from MeOHdryness in vacuo to give as pale yellow crystalline material Compound 8a was not observed under these conditions

The bromo adduct was then dissolved in THF 10 mmol/250 Synthesis of 2-Acetamido-N-benzyl-2-N.phenyl-mL cooled 78 and then added over 15-mm interval to hydrazinoacetamide 3p Use of 3t 2.00 8.0 mmol BBr

cooled 78 solution of the heteroatom nucleophile in THF in CHC12 10.0 mL 10.0 mmol and phenylhydrazine 2.60

mmol/ mL The reaction mixture was stirred at this tern- 24.0 mmol gave pale yellow oily residue which was purified

perature 30 mm and then at room temperature 90 mm The by flash column chromatography on SiO gel 2% MeOH/CHC1insoluble salts were filtered and the filtrate was concentrated in to give 0.75 29% of 3p The product was recrystallized from

vacuo The residue was then purified by flash column chroma- chloroform/hexane as light yellow solid mp 132134

tography on SiO gel using the indicated solvent as the eluent 0.26 2% MeOH/CHC1 IR KBr 3300 1640 br 1610 1520

By using this procedure the following compounds were pre br 1460 760 700 cnf NMR DMSO-d6 11.89 4.28

pared 5.8 Hz 4.89 5.2 Hz 5.09 dd 5.2

Synthesis of 2-Acetamido.N-benzyl-2-dlmethylainino- 7.4 Hz 6.61 7.4 Hz 6.707.28 10 8.29

acetamide 3d By making use of 3t 3.00 12.0 mmol BBr 7.4 Hz 8.60 5.8 Hz Nvffi DMSO-d6in CHC12 13.2 mL 13.2 mmol and Me2NH 56 equiv 22.88 42.2266.22 112.66 117.57 126.65 127.08 128.15

brown residue was obtained that was purified by flash column 128.53 139.12 149.90 168.66 170.04 ppm mass

chromatography on Si02 gel 2.5% MeOH/CHCI to give 1.20 spectrum FD 313 Anal C7HN4O40% of 3d The product was recrystallized from ethyl ace- Synthesis of 2-Acetamido-N-benzyl-2-N2-benzyloxy-

tate/hexane to give light yellow cubic crystals mp 104106 carbonylhydrazinoacetamide 3q By employing 3t 3.00

R1 0.39 5% MeOH/CHCI IR KBr 3280 1670 br 1500 br 12.0 mmol BBr3 in CHCl 15.0 mL 15.0 mmol and

1460 760 700 cm NMR DMSO-d5 1.91 2.11 benzyl carbazate 4.58 27.6 mmol 0.95 21% of 3q was

4.22 dd 5.2 14.7 Hz 4.34 dd 6.1 14.7 Hz obtained The product was recrystallized from chloroform/hexane5.11 8.3 Hz 7.237.31 8.18 to give white amorphous solid mp 152154 R1 0.32 2%

8.3 Hz 8.55 br NMR DMSO-d6 22.43 40.33 MeOH/CHC13 IR KBr 3325 1620 br 1500 br 1440 74042.28 69.42 126.73 127.27 128.21 139.49 168.49 680 cm1 1H NMR DMSO-d6 1.85 4.27 4.4

2450 Journal of Medicinal Chemistry 1991 Vol 34 No Kohn et al

Hz 5.00 5.14 dd 3.1 8.0 Hz 5.23 br 3C NMR DMSO-d8 22.64 42.28 61.42 66.25 126.743.1 Hz 7.257.35 10 8.26 8.0 Hz 127.19 128.19 139.11 167.95 169.66 ppm mass

8.56 bra 8.66 br NMR DMSO-d6 22.71 42.28 spectrum FD 252 Anal C12H17N30365.56 6597 126.69 127.16 127.61 127.77 128.13 Synthesis of 2-Acetamido-N-benzyl-2-niethylhydroxy-

128.27 136.74 138.87 168.04 169.95 ppm mass spectrum aminoacetamide 3m MeOH solution 30 mL of MeNHOHFD 371 Anal C19H22N404 21.74 mmol prepared from MeNHOH.HCI 2.36 28.26 mmol

Synthesis of 2-Acetamido-N-benzyl-2-phenoxyacetamide and NaOMe 1.17 21.74 mmol and 3f 2.20 6.25 minol gave3u Use of 3t 3.00 12.0 mmol BBr3 in CH2CI2 15.0 residue that was purified by flash column chromatography on

mL 15.0 mmoland NaOPh 4.18 30 somol gave brown oily Si02 gel 6% MeOH/CHC13 to give 0.95 61% of 3m as white

residue which was purified by flash column chromatography on solid The product was then purified by recrystallization from

Si02 gel using first CHC13 and then 2% MeOH/CHCI3 as the EtOH mp 159161 R1 0.32 8% MeOH/CHC19 IR KBreluents to give 0.80 22% of 3u The compound was recrys- 3440 br 3300 1640 1530 1460 750 700 cm 1H NMRtailized from chioroform/hexane mp 125128 softens at 122 DMSO-d8 1.95 2.43 4.26 dd 5.7 15.1

0.58 3% MeOH/CHC13 JR KBr 3300 1650 br 1600 Hz 4.35 dd 5.7 15.1 Hz 5.09 9.1 Hz1530 br 1490 1450 760 700 cm1 1H NMR DMSO-d6 1.83 7.217.29 8.05 8.18 9.1 Hz

4.35 5.7 Hz 6.18 9.4 Hz 8.23 5.7 Hz 3C NMR DMSO-d6 22.40 42.34 43.92

6.946.99 7.027.33 in 8.98 5.7 Hz 71.49 126.62 127.12 128.12 139.14 167.82 170.28

9.10 9.4 Hz 3C NMR DMSO-d8 22.54 42.24 76.44 ppm mass spectrum FD 252 Anal C2H7N3O3116.09 121.78 126.84 127.26 128.25 128.44

138.84 155.97 166.63 170.73 ppm mass spectrum FD 299 Synthesis of 2-Acetamido-N-benzyl-2-methoxymethyl-Anal C7H8N203.0.5H20 aminoacetamide 3n MeOH solution 20 mL of MeN-

Synthesis of 2-Acetamido-N-benzyl-2-methyltbioacet- HOMe 17.39 mmol prepared from MeNHOMe.HC1 2.20amlde 3v cooled 78 solution of Et3N 4.85 48.0 mmol 23.02 mmol and NaOMe 0.94 17.39 mmol and 3f 2.10

in THF 20 mL was added to cooled 78 solution of 5.97 mmol gave solid residue Flash column chromatography

prepared from 3t 4.00 16.0 mmol and BBr in CH2C12 of the solid on Si02 gel 2% MeOH/CHC13 yielded pure 3n 1.30

20.0 mL 20.0 mmol in THF 275 mL cooled 78 82% The product was recrystallized from EtOH mp 165167solution of excess MeSH 56 equiv in THF 55 mL was then R1 0.39 2% MeOH/CHC13 IR KBr 3300 1640 br 1540

added The reaction mixture was stirred at this temperature 30 hi 1460 750 700 cm NMR DMSO-d6 1.93 s3 2.43

mm and then at room temperature The insoluble materials 3.32 4.25 dd 5.9 14.9 Hz 4.37 ddwere filtered and the filtrate was evaporated to dryness in vacuo 5.9 14.9 Hz 5.19 9.4 Hz 7.217.35

The oily residue obtained was purified by flash column chro- 8.31 9.4 Hz 8.56 5.9 Hz 18 NMRmatography on Si02 gel 2% MeOH/CHC13 to give 1.10 27% DMSO-d6 22.36 39.68 42.34 59.16 70.33 126.74 127.41

of 3v as yellow orange oil The product was purified by second 128.21 139.30 167.38 170.30 ppm mass spectrum FD 266

flash column chromatography on Si02 gel 2% MeOH/CHCI3 Anal C3H9N303to give 0.72 of the pure product as white solid mp 155157 Synthesis of 2-Acetamido-N-benzyl-2-N-isoxazolidi-

R10.65 3% MeOH/CHC13 JR KBr 3320 1650 br 1520 noacetamide 3o 3f 1.60 4.55 mmol isoxazolidine prebr 1460 750 cnc NMR CD3NO2 61.98 2.08 pared from isoxazolidine hydrobromide 2.41 15.65 mmol and

4.39 dd 6.1 15.2 Hz 4.49 dd 6.1 15.2 Hz NaOMe 0.70 13.04 mmcl gave 0.80 64% of 3o Compound5.51 7.8 Hz 7.15 7.8 Hz 7.177.41 3o was recrystallized from chloroform/hexane to give white

8C NMR CD3NO2 12.28 22.94 44.26 56.03 128.46 amorphous solid mp 149151 R0.29 4% MeOH/CHC13128.60 129.77 140.17 169.19 171.06 ppm mass JR KBr 3400 br 3300 1650 1530 1470 740 700 610 cmspectrum FD 253 Anal C2H6N202S NMR DMSO-d6 61.91 2.052.20 2.452.89

Synthesis of 2-Acetamido-N-benzyl-2-ethylthioacet- 2.983.07 3.743.90 4.25 ddamide 3w By using the procedure described for the synthesis 6.1 15.3 Hz 4.35 dd 6.1 15.3 Hz 5.23

of 3v 3t 2.00 8.0 mmol and EtSH 0.65 10.4 mmol were 9.2 Hz 7.157.35 8.49 9.2 Hz 8.56

converted to 0.80 38% of 3w The compound was further br 3C NMR DMSO-d6 22.26 28.26 42.15 48.9466.19

purified by recrystallization from chloroform/hexane to give 68.77 126.64 127.02 128.13 139.22 167.43 170.27

beige solid mp 146148 11 0.604% MeOH/CHC1 JR KBr ppm mass spectrum FD 278 Anal C4H9N3033240 1620 hr 1510 br 1415 680 640 cm1 NMRDMSO-d6 61.56 7.4 Hz 1.88 2.492.67 Preparation of Functionalized a-Heteroatom-Substituted

4.23 dd 5.9 15.2 Hz 4.32 dd 5.9 15.2 AmIno Acids Method General Procedure 2-Acet-

Hz 5.55 9.1 Hz 7.207.35 8.59 amido-N-benzyl-2-ethoxyacetamide 3t equiv was suspended

9.1 Hz 8.75 5.9 Hz 3C NMR DMSO-d6 in Et20 100 mL/10 mmol and then BF3.Et20 1.62.4 equiv

14.73 22.43 23.73 42.10 53.70 126.87 127.14 128.32 was rapidly added and the resulting solution was stirred 10 mm139.01 167.89 169.02 ppm mass spectrum FD 267 The nucleophile H20 or EtSH 1.64.0 equiv was then addedAnal C3H5N202S.0.25H20 and the reaction mixture was stirred at room temperature 1848

Preparation of Functionalized a-Heteroatom-Substituted The reaction was then quenched by the addition of an aqueous

Amino Acids Method General Procedure mixture NaHCO 100 mL/10 mmol/ice mixture The experimental

of 3f equiv and the nitrogen nucleophile 45 equiv in MeOH workup varied slightly for 3r and 3w and is described in the

mmol/ mL was stirred at 5560 The solvent was following sections along with the observed spectral propertiesremoved in vacuo and the residue was purified by flash column Synthesis of 2-Acetaniido-N-benzyl-2-hydroxyacetamide

chromatography on SiO2 gel by using the indicated solvents as 3r Reacting 3t 1.00 4.0 mmol BF3.Et20 0.91 6.4 mmolthe eluent and H20 0.12 6.7 mmol followed by aqueous NaHCO5 workup

By using this procedure the following compounds were pre- gave an aqueous reaction mixture The solution was then expared tracted with EtOAc 50 mL and the combined EtOAc ex

Synthesis of 2-Acetamido-N-benzyl-2-niethoxyamino- tracts were dried Na2SO4 and concentrated in vacuo The residue

acetamlde 31 Use of MeOH solution of MeONH prepared was purified by flash column chromatography on Si02 gel 3%from MeONH2.HCI 2.83 33.9 mmol and NaOMe 1.41 26.1 MeOH/CHC13 to give 0.30 34% of 3r as white solid mpmmol and 3f 2.70 7.67 mmol gave an oily residue that was 136138 R1 0.14 3% MeOH/CHC13 JR KBr 3300 1620

purified by flash column chromatography on SiO gel 2% 1530 br 1430 hi 730 690 cm 1H NMR DMSO-d6 1.85

MeOH/CHC13 to give 0.80 42% of 31 The product was 4.29 5.9 Hz 5.48 dd 5.5 8.6 Hzrecrystallized from chlorofonn/hexane xnp 9597 R1

0.23 2% 6.47 5.5 Hz 7.217.35 8.52 5.9 HzMeOH/CHCI3 JR KBr 3300 1650 1620 1510 br 1440 750 8.59 8.6 Hz 3C NMR DMSO-d6 22.66 41.99

680 cm1 NMR DMSO-d6 1.88 3.38

4.224.41 in 5.18 dd 4.9 7.8 Hz 6.78 ____________________________________________________

4.9 Hz 7.217.32 8.33 7.8 Hz 8.56 16 Hine Evangelists Am Chem Soc 1980 102 1649

Functionalized cg-Heteroatom-Substjtuted Amino Acids Journal of Medicinal Chemistry 1991 Vol 34 No 2451

71.42 126.66 127.22 128.13 139.20 169.47 169.62 126.89 127.27 128.24 138.55 164.66 170.18 ppm mass

ppm mass spectrum m/e relative intensity 223 spectrum FD 283 Anal C3H8N203S163 11 134 106 46 91 100 77 22 65 38 Anal Synthesis of 2-Acetamido-N-benzyl-2-ethylthioacet-

C11H4N203 amide S-Oxide 3y-l and 3y-2 solution of Na104 1.77

Synthesis of 2-Acetamido-N-benzyl-2-ethylthioacet- 8.27 mmol in H20 20 mL was added dropwise into stirred

amide 3w Use of 3t 2.00 8.0 mmol BF3.Et20 2.72 19.2 solution of 3w 2.00 7.52 mmol in MeOH 25 mL premmol and EtSH 2.38 38.4 mmol gave an aqueous reaction cipitate appeared rapidly H20 30 mL was added to the

mixture The solution was extracted with CHC13 104 mL mixture to dissolve most of the suspension and the reaction

The combined CHC13 layers were dried Na2SO4 and then con- mixture was stirred at room temperature The reaction was

centrated in vacuo to give 1.90 89% of 3w as white solid concentrated in vacuo and the remaining aqueous mixture was

mp 148149 mixed melting point with an authentic sample extracted with CHC13 100 mL The combined CHC13 ex

was undepressed R1 0.60 4% MeOH/CHC13 tracts were dried Na2SO4 and the solvent was removed in vacuo

Synthesis of 22-Diacetaxnido-N-benzylaoetamide 3i The oily residue 1.95 92% solidified on drying in vacuo NMRAc20 mL was added to solution of 3a 1.10 4.98 mmol analysis DMSO-d6 of the product showed that it was 21

in dry pyridine 10 mL and then CH2C12 20 mL was added mixture of the two diastereomers 3y-l and 3y-2 respectively The

The mixture was stirred at room temperature and then the reaction was recrystallized from EtOAc to give nearly pure dia

volatile materials were removed in vacuo The residue was then stereomer 3y-1 1.20 The EtOAc mother liquor was concentreated with saturated aqueous NaHCO3 solution 50 mL The trated and the remaining residue 0.75 was recrystallized from

white solid 1.20 92% that remained was filtered dried ethyl acetate/hexane to give diastereomeric mixture 0.41

Na2SO4 and recrystallized from MeOH mp 265267 dec of 3y-1 and 3y-2 in 23 ratio respectively mp 135137IR KBr 3260 1530 1500 740 690 cm NMR DMSO-d6 softens at 117 R1 0.60 4% MeOH/CHC1 IR KBr 3300

1.84 4.26 5.8 Hz 5.71 7.6 Hz br 1640 br 1510 br 1370 1230 1100 1020 900 cm mass

7.207.31 8.44 7.6 Hz 8.48 5.8 spectrum FD 283 Anal C3H5N203S The

Hz 3C NMR DMSO-d6 22.44 42.26 56.99 126.62 following NMR spectral properties have been assigned to corn-

127.02 128.12 139.15 168.19 169.39 ppm mass pounds 3y-1 and 3y-2

spectrum FD 264 Anal C3H17N30 Diastereomer 3y-l NMR DMSO-d6 1.16 7.5

Synthesis of 2-Acetamido-N-benzyl-2-trlfluoroacet- Hz 2.00 2.492.72 4.284.39

amldoacetamide 3j Ice-cold trifluoroacetic anhydride mL 5.56 9.7 Hz 7.217.34 8.718.77 rnwas added in one portion to ice-cold 3a 1.00 4.53 mmol The 3C NMR DMSO-d6 7.10 22.43 42.48 42.57 67.23 126.98 127.36

reaction was accompanied by the evolution of heat After stirring 128.33 138.63 164.73 170.25 ppmmmthe volatile materials were removed in vacuo The residue Diastereomer 3y-2 NMR DMSO-d6 1.13 7.6

was treated with saturated aqueous NaHCO3 solution 20 mL Hz 1.94 2.492.72 4.284.39

and the solid that remained was filtered and washed with H20 5.71 9.9 Hz 7.217.34 8.83 9.9 Hzto give 3j 1.00 70% The product was recrystallized from 8.98 5.6 Hz 3C NMR DMSO-d6 6.47 22.43EtOH mp 228-230 R1 0.34 8% MeOH/CHC13 JR KBr 41.53 42.55 67.90 126.98 127.36 128.33 138.39 164.43

3300 1720 1660 1520 1380 760 700 cm1 NMR DMSO-d6 169.82 ppm1.90 4.30 5.1 Hz 5.85 8.0 Hz Synthesis of 2-Acetamido-N-benzyl-2-ethanesulfonyl-

7.217.35 8.64 8.0 Hz 8.75 5.1 acetamide 3z An aqueous solution 20 mL of Na104 3.00

Hz 10.04 18C NMR DMSO-d6 22.52 42.52 57.42 14.02 mmol was added to MeOH solution 20 mL of 3w 0.95

117.4 288.3 Hz 126.80 127.16 128.21 138.93 3.57 mmol The initial homogeneous solution rapidly became

156.14 35.3 Hz 166.39 169.88 ppm mass spectrum FD turbid HO -10 mL was then added dropwise until the system

318 Anal C13H4N303F3 became homogeneous The solution was stirred 18 at 5060

Synthesis of 2-Acetamido-N-benzyl-2.trimethyl- MeOH 50 mL was added to the reaction solution and the

ananaonloacetamide Tetrafluoroborate 3f solution of 3d precipitated salt was filtered and washed with MeOH 10 mL1.93 7.76 mmol in nitromethane mL was added slowly to The filtrate was concentrated and the remaining solution was

an ice-cold solution of trimethyloxonium tetrafluoroborate 1.26 extracted with CHC13 50 mL The combined CHC13 extracts

8.54 mmol in nitromethane mL The reaction mixture was were dried Na2SO4 and concentrated in vacuo The residue was

stirred at this temperature 15 mm and then at room temperature purified by flash chromatography on Si02 gel 1% MeOH/CHC13Anhydrous Et20 -50 mL was added to the reaction to give 0.34 32% of the desired product Compound 3z was

mixture and the white solid that separated was filtered washed further purified by recrystallization from EtOH mp 161-163

with Et20 and dried in vacuo yield 1.95 72% rep 171173 R10.34 3% MeOH/CHC13 JR KBr 33002940 1660 1520 1310

dee JR KBr 3300 1680 br 1530 1490 710 cm NMR 1230 1120 900 crn NMR DMSO-d6 1.22 7.4 HzCD3NO2 2.14 3.18 4.50 5.8 Hz 1.99 3.043.24 rn 431 dd 5.7 15.3 Hz5.70 9.3 Hz 7.307.41 7.57 9.3 Hz 4.41 dd 5.7 15.3 Hz 5.93 9.8 Hz

7.70 br mass spectrum FD 264 Anal 7.227.35 9.13 5.7 Hz 9.17 9.8 HzC4HN3O2BF4 NMR DMSO-d6 5.72 22.27 42.63 45.43 69.14 127.02

Synthesis of 2-Acetamido-N-benzyl-2-ethylthioacet- 127.28 128.33 138.16 161.88 169.83 ppm mass

amide S-Oxide 3y-1 solution of m.chloroperbenzoic acid spectrum FD 298 M4 Anal CH8N204S1.00 65% 3.76 mmol in CH2C12 10 mL was added Pharmacology Initial anticonvulsant evaluation of was

dropwise into stirred cooled 10 to 15 CHCl solution conducted with at least three dose levels 30 100 and 300 mg/kg125 mL of 3w 1.00 3.76 mmol under N2 The reaction was administered intraperitoneally All tests were performed with

stirred 30 mm at this temperature and then the m-chlorobenzoic male CF-i mice from Charles River Breeding Laboratories

acid was precipitated as its animonium salt by passing NH3 gas Portage MI Test solutions of all compounds were dissolved

over the surface of the reaction solution The excess NH3 was in 30% polyethylene glycol 400 Four mice at each dose level

removed by passing N2 gas through the solution 20 mm at room were tested at 0.5 and after administration to determine

temperature The ammoniurn salt was filtered and the filtrate if there was protection against MES seizures

was concentrated in vacuo The residue was purified by flash MES seizures were elicited by electrical current ac 60 cps 50

chromatography on Si02 gel 2% MeOH/CHC1 to give 0.55 mA 0.2 applied via corneal electrodes drop of 0.9% saline

52% of the desired product Compound 3y-l was recrystallized was instilled on the eye prior to application of the electrodes to

from chloroforin/hexane as white granular solid mp 135137 ensure electrical contact Abolition of the hind limb tonic ex

R1 0.23 2% MeOH/CHC1 IR KBr 3300 br 1640 br tension component of the seizure was defined as protection in

1510 br 1370 1230 1100 1020 900 cm NMR DMSO-d6 the MES test

11.15 7.5 Hz 1.99 s3 2.492.56 Afterthetimeofpeakactivityandtheapproximatedoserange2.652.72 4.34 5.7 Hz 5.55 9.5 Hz were determined doseresponse curve was generated with at

7.237.34 8.74 9.5 Hz 8.77 least three or four doses and 1012 mice per dose The MES EDn5.7 Hz 8C NMR DMSO-d6 7.03 22.34 42.40 42.47 67.15 is the estimated dose from the doseresponse data to protect 50%

2452 Med Chem 1991 34 24522463

of the mice in the MES test Neurological impairment was 3d 133873-20-8 3e 133873-27-5 3f 133873-22-0 3g 133873-28-6measured in mice using the horizontal screen tox text.4 Pre- 3h 133873-29-7 31 133873-30-0 3j 133873-31-1 3k 133873-32-2viously trained mice were dosed with the compound and then 31 133873-33-3 3m 133873-34-4 3n 133873-35-5 3o 133873-36-6

placed individually on top of square 13 13 cm wire screen 3p 133873-37-7 3q 133873-38-8 3r 133873-39-9 3s 133873-23-1no mesh which was mounted on vertical rod The rod was 3t 117422-80-7 3u 133873-40-2 3v 133873-41-3 3w 133873-24-2then rotated 180 and the number of mice that returned to the 3x 133887-00-0 3y diastereomer 133873-42-4 3y diastereomer

top of the screen within mm was determined To determine 133886-68-7 3z 133873-43-5 124421-42-7 6a 133873-08-2

EDes values doseresponse curve was determined at the time Gb 133873-09-3 Ge 133873-10-6 Gd 133873-11-7 Ge 133873-12-8of peak anticonvulsant activity with at least three to four doses Gg 133873-13-9 Gh 133873-14-0 Bk 133873-15-1 Gm 133873-16-2and 12 mice per dose The TDes is the estimated dose that 6n 133873-17-3 133873-18-4 diastereomer 133873-45-7

impaired 50% of the mice diastereomer 133873-46-8 PhNH2 62-53-3 PhCH2NH2

Acknowledgment We thank Beedle Dennis 100-46-9 PhNHNH2 100-63-0 H2NNHCOOCH2Ph 5331-43-1

NaOPh 139-02-6 MeSH 74-93-1 EtS 75-08-1 108-98-Thompson and Lawson Lilly Research Laboratories -AcNHCHNHCH2PhCONHCH2Ph 133873-44-6 morphofor their expert technical assistance

line 110-91-8 3-aminopyrazole 1820-80-0 isoxazolidine hydroRegistry No 3a 133873-19-5 3b 133873-25-3 3c 133873-26-4 bromide 111780-15-5

Arocalciferols Synthesis and Biological Evaluation of Aromatic Side-Chain

Analogues of la25-Dlhydroxyvitamin D31a

Bruno Figadereib Anthony NormanQd Helen Henry Phillip Koeffler Jian-Yuan Zhouie and

William OkamuraTh

Department of Chemistry the Division of Biomedical Sciences and the Department of Biochemistry at the University of

California Riverside California 92521 and the Department of Medicine Division of Hematology/Oncology Cedars-SinaiMedical Center University of California Los Angeles California 90024 Received September 21 1990

Aromatic side-chain analogues arocalciferols 69 of the steroid hormone la25-dihydroxyvitamin D3 were

synthesized and biologically evaluated The analogues were prepared by coupling the vitamin A-ring enyne 14

with the appropriate enol triflate of modified CD steroid fragment of the type 22 The resulting dienyne 23 wasthen transformed in three steps to the vitamin analogues 6-9 Biological evaluation of these analogues have providedinformation concerning side-chain topographical effects on in vivo and in vitro activity

Introduction has accordingly been an increased interest in the further

The steroid hormone la25-dihydroxyvitamin D3development of 125OH2D3 analogues for these new

125OH2D3 the physiologically active form of vitamin therapeutic purposes

calciferol is considered to be one of the most potentThe clinical utility of 125OH2D3 is limited because

stimulators of calcitropic effects intestinal calcium ab- therapeutically effective doses induce hypercalcemia

sorption ICA and bone calcium mobilization BCM.2 This has led investigators toward the development of an

Besides its traditional role as hormone in calcium ho- analogue with more useful therapeutic index specifically

meostasis3 125OH2D3 induces differentiation and ff directed toward analogues with high cell differentiating

cellular proliferation indicating its possible use in the ability and low calcitropic action series of remarkably

treatment of certain cancers and skin disordersfr Therediverse side-chain analogues of 125OH2D3 exhibiting

promising therapeutic indices have in fact been reported

Paper 39 in the series Studies of Vitamin Calciferol and Differentiation of leukemia cells Honma HozumiIts Analogues For paper 38 see Enas Shen G.-Y Abe Konno Fukushima Hats NishiiOkamura Am Chem Soc 1991 113 3873 De- DeLuca I-I Suda Proc Nati Acad Sci U.S.A 198380partment of Chemistry University of California Riverside 201 Koeffler Amatruda Ikekawa KobayashiUCR Division of Biomedical Sciences UCR De- DeLuca Cancer Res 1984 5624 Munkerpartment of Biochemistry UCR Department of Medicine Norman Koeffler Clinical Invest 1986 78Cedars-Sinai Medical Center University of California Los 424 Differentiation of skin cells psoriasis treatmentAngeles MacLaughlin Gange Taylor Smith Holick

Norman Vitamin the Calcium Homeostatic Proc NatI Acad Sci U.S.A 1985 82 5409 MoriSteroid Hormone Academic Press New York 1979 For moto Onishi Irnanaka Yukawa Kosuka

description of recent advances in the field including leading Kitano Yoshikawa Calcif Tissue mt 1986 38 119

references see Norman Schaeffer Grigoleit H.-G Abe Miyaura Sakagami Takeda KonnoHerrath Eds Vitamin Molecular Cellular and Yamazaki Yoshiki Suda Proc Natl Acad SciClinical Endocrinology Walter de Gruyter and Co Berlin U.S.A i981 78 4990 Coiston Colston Feld1988 Pardo Santelli Bull Chim Soc Fr 1985 98 man Endocrinology 1981 108 1083

Jones guest editor Steroids 1987 49 1196 Shiina Abe Miyaura Tanaka YamadaIkekawa Med Res Rev 1987 333 Ohnori Nakayama Takayama Matsuyama

Minghetti Norman FASEB 1988 3043 Nishii DeLuca Suda Arch Biochern BiophysCarson-Jurica Schrader OMalley 1983 220 9094 Shiina Miyaura Tanaka Abe

Endocrine Rev 1990 ii 201 The amino acid sequence of Yamada Yamamoto moe Takayarna Mat-the human 125OH2D3 receptor has been determined Baker sumaya Nishii Suda Med Chem 1985 28

MacDonnell Hughes Crisp Man- 11531158gelsdorf Haussler Pike Shine OMal- Koeffler Hirji Itri The Southern California

ley Proc Nati Acad Sci U.S.A 1988 85 3294 Leukemia Group Cancer Treat Rep 1985 69 1399

0022-2623/91/1834-2452$02.50/0 1991 American Chemical Society