IE870109L - Substituted peptide compounds - Google Patents

Description

Substituted Peptide Compounds This invention is directed to compounds which may be useful in the preparation of substituted peptide compounds of formula 1 and salts thereof (I) O II R--CH-C- 3 I .

NH I C-O I R I O d ■CH-—s-CH — C-X CD X is an amino or Imino acid of the formula £ <*, 'ff- • \e r*» • V\ -N C-COOR -N—C-COOR ' _ l„v.a r. _ r, . low 10 R R v r* ' C*) » -N—C-COOR, -N—C-COOR, -N —C-COOK, ml) 6 i (&) 6 ml) 6 h a h or -H CH-COOR- I I R« *5 Rj is hydrogen, lower alkyl, halogen, fceto, O W / 19 hydroxy, -NH-C-lower alkyl, azido, amino, -H , *» ~£-p O -0 R1S ~ a 1- or 2-naphthyloxy of the formula -0- , -S-lower alkyl. 14}p -S- Qj_ or a 1- or 2-naphthylthio '"l3'p -4- 15 20 of the formula -S-(CH2)m, 0 H R_ is keto, halogen, | / 15 " -0-C-N ' ***""*15 10 -O-(CHj)/ -O-lower alkyl, a 1- or -S-lower alkyl, -S-(CH2J^-/qN V—^CRl3»p or a 1- or 2-naphthylthio of the formula fOTOj- i*14»p 25 is keto or - (CHj) 13 p -5 10 15 20 R10 is halogen or -Y-R16. *ll' *11 ' *12 *nd R'l2 are independently selected from hydrogen and lower alkyl or R'j^ r12 and R'12 are hydrogen and R^ is R13 is hydrogen, lower alkyl" of 1 to 4 carbons, lower alkoxy of .1 to 4 carbons, lower alkylthio of 1 to 4 carbons, chloro, bromo, fluoro, trifluororaethyl, hydroxy, phenyl, phenoxy, phenylthio, or phenylmethyl. ^ is hydrogen, lower alkyl of 1 to 4 carbons, lower alkoxy of 1 to 4 carbons, lower alkylthio of 1 to 4 carbons, chloro, bromo, fluoro, trifluoromethyl, or hydroxy. m is zero, one, two, three, or four. p is one, two-or three provided that p is more than one only if R^3 or Rj^ is hydrogen, methyl, methoxy, chloro, or fluoro.

R^s is hydrogen or lower alkyl of 1 to 4 carbons.

T is oxygen or sulfur. *16 ia lower alkyl of 1 to 4 carbons. <*13>p , or the H^g groups join to 30 complete an unsubstituted 5- or 6-membered-ring -6- 10 15 20 25 or said ring in which one or more of the carbons has a lower alkyl of 1 to 4 carbons or a di(lower alkyl of 1 to 4 carbons) substituent.

R, is hydrogen, lower alkyl, .—.

-(CH2'iT© -(CH^-cycloalkyl, , -(C^),^ .

-«CH2>ir{0) - * B H -(CH2)r-SH, -(CH2)r-s-lower alkyl, -(CH^-NH-C , or -(CH2)r-C—NHj 30 VNH2. -7- r is an integer from 1 to 4.

R19 is lower alkyl, benzyl, or phenethyl. R20 is-hydrogen, lower alkyl, benzyl or phenethyl.

R is hydrogen, lower alkyl, cycloalkyl, ' -tCH2)2-MH2, -(CH2)3-HH2 , -(ch2)4-nh2, -(ch2)2-oh, -(ch2)3-oh, -Cch2)4-oh, -(ch2)2-sh, -cch2)3-sh, or -{ch2)4-sh.

R^ is hydrogen, lower'alkyl,-halo substituted 15 lower alkyl, /-~K /—\ -(CB2,£-© ' 10 20 -(ch2)f^-°h ' "tCH2>rl~3@ oh | h ■(ch2)y i| n / *(chj), "(chj)^~sh| 25 I B -(CHj)r-OH, -(CB2)r-s-lower alkyl, O MB | 30 -CCB2)r-NB-C-^' , or -(CB2Jr-C-HB2 NHj -H- 10 IS 25 provide^ th#t R^ is' hydrogen only if R is other than hydrogen..

R2 is -«c"a,r®>IR , ' -•"j'.-O 14 p s -(ch2>5KE^3 'or -'"Vi-fQ) • R3 is hydrogen, lower alkyl, '"m'p "^s-* , halo substituted .20 lower alkyl, -(CH,)m-cycloalkyl, -1CH,)^^-0H , OH (CH2)?inS) ' -JCH2)r-OH, . 30 -9- -lCH2)r-HH2, -(CH2)r-SH, -(CH2)r-S-lowcr alkyl, mu o " Kg is hydrogen, lower alkyl, benzyl, benzhydryl, J , ?21 {? 10 -ch-o-c-r, „ -c c-o-r i 18 , 23 1*17 *22 IS -CH-(CH-OH) _ , or -CH-CH—CH 2 ■ I I ob ob R^ is hydrogen, lower alkyl, cycloalkyl, or phenyl.

R ' is hydrogen, lower alkyl, lower alkoxy, or phenyl or RJ7 and Rjg taken together are -(CH2>2-, 20 -(ch2)3-, -ch-ch- - 25 R2^ and Rjj are independently selected from hydrogen and lower alkyl.

Rjj is lower alkyl. - 10 - In accordance vith the invention, there is prQvided a compound of the foraula (II) O O R R O ii I in R--C-NH-CH-C-CH--N - CH-C-OH • I »3 wherein: R is hydrogen, Prot, lower alkyl, cycloalkyl, ' -(.CH2)2-8-Prot , -(CH2)3-3-Prot, -jch2)4-S-Prot, -|ch2)J-Q-Prot, -(chj)j-O-Prot, -(CH2)4-0-Prot , -(C»2)2-S-Prot, -(CHj)j-S-Prot, or -(CHj)^-S-Prot; ' R^ is hydrogen, lower alkyl, halo substituted lower alkyl, f -(CH^p^^b-Prot, (CH2)r@-°-prot' -(cVrirn[ol O-Prot N (CHj), -(CH2)r-S-P.rot -, -(CM2)r-S-lower alkyl I Prot ™ H Ar (CH2)r-0-Prot, -(CH2)r-N-Prot, - ^ Nll-Prot - 11 10 o II or -(CH2>r-C-NH2 provided that R^ is hydrogen only when R is other than hydrogen; Prot is an easily removable protecting group; R2 is >s®\, , 14 p ,ir"@ R3 is hydrogen, lower alkyl, -(ch2)^-/qN , <*4>P • ■|ch2,S~5~3 s (CH2), ;—pOl » halo substituted lower alkyl.

- (CH2) ^-eycloalkyl, - (CH2) qh , OH - 12 - - (CH2} r fi l'ol ' _lCH2)r"OH' I H -(CH,) 2)f-lj jft , -(CH2)r-NH2, -(CH2)r-SH, -(CHj) r~S-lower alkyl, O -{CH2)r-C-NH2 1 j 5 R14 is hydrogen, lower alkyl of 1 to 4 carbons, lower alkoxy of 1 to 4 carbons, lower alkylthio of 1 to 4 carbons, chloro, bromo, fluoro, trifluoromethyl, or hydroxy; m is zero, one, two, three, or four; 10 p is one, two or three provided that p is more than one only if is hydrogen, methyl, methoxy, chloro, or fluoro; and z is an ■ integer from 1 to 4'.

-(CH.) -NH-C « r « ,NH NH„ 13 The tern lower alkyl used in defining various symbols refers to straight or branched chain radicals having up to seven carbons. The preferred lower alkyl groups are up to four carbons 5 with methyl and ethyl most preferred. Similarly the terms lower alkoxy and lower alkylthio ° refer to such lower alkyl groups attached to an oxygen or sulfur.

The term cycloalkyl refers to saturated 10 rings of 3 to 7 carbon atoms with cyclopentyl and cyclohexyl being most preferred.

The term halogen refers to chloro, bromo and fluoro.

The term halo substituted lower alkyl refers IS to such lower alkyl groups described above in which one or more hydrogens have been replaced by chloro, bromo or fluoro groups such as trifluoromethyl, which is preferred, penta-fluoroethyl, 2,2,2-trichloroethyl, chloromethyl, 2C bromomethyl, etc.

The symbols -(CHj^ , -(CH^ and "(CH2'm"|OJ bridge is attached to represent that the alkylene an available carbon atom. - 14 - The compounds of formula I may be prepared by coupling a compound of the invention with the amino or imino acid ester of the formula (HI) 5 HX in the presence of a coupling agent such as dicyclo-hexylcarbodiimide wherein in the definition of X is an easily removable protecting group.

Removal of the Rfi protecting group yields the 10 products of fonnula X wherein Rg is hydrogen.

A compound of the invention can be prepared by reacting an aminofcetone of formula (IV) O 0 ? II 1 1 R -C-NH-CH-C-CH2-NH 2 I 15 with a haloacetic acid ester of the formula (V) *1 ° r « halo-CH— C-O-Prot (X.) wherein Prot is an easily removable ester protecting group such as t-butyl to yield the ester (VI) o o r r, o p i I i1 r r -c-nh-ch-c-ch -nh- ch - C-O-Prot I *3 A compound of the formula (IV) can converting the carboxyalkylamine of the formula (VII) r I ho-c-ch2-h-r40 p o wherein R^g is a protecting group such as benzyloxy-carbonyl, to its acid chloride and then reacting with an oxazolone of formula (VIII) to yield (IX) O II r2-c-nh compound of the be prepared by I I -ch-c-ch2-n-r40 - 16 - Removal of the R^g protecting group such as by hydrogcnation yields the reactant of foraula (IV).

The aminoketone of foraula (IV) wherein R is other than hydrogen can also be prepared by reacting the ketone of formula (X) O O II I "V R2-C-NH-CH-C-CH,-halo I *3 wherein halo is CI or Br with a substituted amine of the foraula (XI) R-NHj Compounds of formula (X) and their preparation are described and 10 claimed in our Patent Application No. ( ).

In the above reactions if any or all of R, Rj , Rj and are -(CH2 OB -(CH2)r-N82. -«Vrir"f| ' -CCH2)r-SH, I B ^N" 15 -(CBj)r~OB, 2 - J7 - then the hydroxyl, amino, imidazolyl, mercaptan or guanidinyl function should be protected during- the reaction. Suitable protecting groups include benzyloxycarbonyl,. t-but'oxycarbonyl, benzyl, benzhydryl, trityl, etc., and nitro in the case of guanidinyl. The protecting group is removed by hydrogenation, treatment with acid, or other known methods following completion of the reaction.

Preferred compounds of this Invention with respect to the peptide part of the structure are those.wherein: R is hydrogen, straight or branched chain lower alkyl of 1 to 4 carbons, or phenyl.

R^ is hydrogen, straight or branched chain lower alkyl of 1 to 4 carbons, -CFj, -(CH2)r-NH2 wherein r is an integer from 1 to 4, ' -B^OR OB "CH2X3@ ' "CH2T^jf ' ~CVSH' -NH -(cb2)2-s-ch3, "(chjjjnhc^ , -chj-oh, nb2 O O II I -CHj-C-NHj, or -(CHjlj-C-NBj provided that R^ 2.) is hydrogen only if S is other than hydrogen.

Most preferred compounds of this invention with respect to the peptide part of the structure are those wherein: R is hydrogen or methyl.

R^ is hydrogen, methyl, or 25 especially methyl, provided that R^ is hydrogen only if R is other than hydrogen. - 18 - Preferred compounds of this invention with respect to the keto portion of the structure are chose wherein: R2 is ■ -B^©L . « *14 M wherein a is" zero, .one,- or two, R^ is' hydrogen .methyl, methoxy, methylthio, CX, Br, F, or hydroxy, and r is an integer from 1 to 4, especially benzyl.

An asynnetric center is present in the keto portion of 15 the compounds of the invention vhen R^ is other than hydrogen. Thus, the compounds can exist in diastereolsomeric forms or in mixtures thereof. The above described processes can utilize racemates, enantiomers or diastereomers as 20 starting materials. ' When diastereomeric products are prepared, they can be separated by conventional chromatographic or fractional crystallization methods.

The compounds of formula X, and the pharma-25 ceutically acceptable salts thereof, are hypotensive agents. - 19 - The compounds of formula X wherein X is -NH-CH-COOR also possess enkephalinase I 6 *5 inhibition activity and are useful as analgesic agents. 5 Compounds of formula (I), as well as pharmaceutical compositions containing them and additional processes for their preparation, are further described and claimed in parent Patent Application No. >655/83.

The following examples are illustrative of the 10 preparation of compounds of the invention, as well as their use in the preparation of compounds of formula (I). Temperatures are given in degrees centigrade. LH-20 refers to a Sephadex chromatography gel commercially available from Pharmacia Fine Chemicals. - 20 - Example I N-[N-t3-(Benzoylamlno)-2-oxo-4-phenylbutvl1-N-i:iethvl-glycyll -N-cyclohexylglyclne, mononydrochloride a) 13- (Benzoyl amino) -*2-oxo-4-phenylbutyllmcthyl-carbamlc acid, phenylmethyl ester N-methyl-N- [ (phenylmethoxy) carbony1] glycine (2.23 g., 10 mmole) is dissolved in 30 ml. of tetrahydrofuran and cooled in an ice-bath. Oxalyl chloride (1- nil., 11.5 mmole) is added followed by 2 drops of dimethylformamide. After stirring for 30 minutes in' the ice-bath, the mixture is then stirred at room temperature .for an hour. To this 0.25 ml. of oxalyl chloride is added. The mixture is evaporated, redissolved in 15 ml. of tetrahydrofuran, and stirred in an ice bath. A solution of 2-pl»enyl-4-(phenylmethyl)-5(4H)-oxazolone (3.1 g., 12.4 mmole) dissolved in 15 ml. of tetrahydrofuran is added to the above solution stirring in the ice-bath. Triethylamine' (1.4 ml., 10 mmole) is added and the solution is stirred at room temperature overnight. The precipitated triethylamine hydrochloride salt is' filtered off. Tetrahydrofuran is removed from the residue and it is then redissolved in pyridine (5 ml.) and p-dimethylamino pyridine (20 mg.) is added. After stirring at room temperature for 3 hours, acetic acid (5 ml.) is added and the.reaction mixture is kept at 105" for 30 minutes. The reaction mixture is.then evaporated, the residue is dissolved in ethyl acetate, and washed with aqueous sodium bicarbonate and water. After trituration with ethyl acetate/ hexane, 2.2 g. of homogeneous [3-(benzoylamino)-2-oxo-4-phenylbutyl]methylcarbamic acid, phenylmethyl ester is obtained; m.p. 140-141*. - 21 - b) (1)-N-13-(Methvlamino)-2-oxo-l-(pheny liaethyl) -propyl Ibenzamidei' hydrochloride {3- (Benzoylamino) -2-oxo- 4-pheny lbuty 1 ] me thy 1-carbamic acid, phenylmethyl ester (0.5 g.) is 5 dissolved in ethanol (50 ml.) containing IS hydrochloric acid (2 ml.). Palladium carbon catalyst (lOt, 100 mg.) is added and hydrogenation is continued overnight. The reaction.mixture is then filtered, evaporated, dissolved in water, 10 and lyophilized to 300 mg. of (±)-N-[3-(methyl-amino)-2-oxo-l-(phenylmethyl)propylJbenzamide, hydrochloride as a homogeneous white powder. c) ft) -M- [3- (Benzoylamino) -2-oxo-4-phenylbutyll-N-methylqlycine,1,1-dimethylethyl ester 15 To a solution of (±) -N-[3- (methy laraino)-2-. oxo-1-(phenylmethyl)propyl]benzamide (5.0 g., 15 mmole) , prepared as set forth in (b) above, in dimethylformamide (20 ml.) is added bromoaeetlc acid, 1,1-dimethylethyl ester (13.8 g., 3.15 ml., 20 19.5 mmole) and diisopropylethylamine (2.5 g., 3.4 ml., 19.5 mmole). After 'stirring overnight at room temperature, the.reaction mixture is poured into water (100 ml.) and extracted with ethyl acetate (3x). The combined ethyl acetate 25 extracts are washed with saturated sodium bicarbonate (twice), 10% potassium bis'ulfate (twice)', and water (twice), dried' (Ha^SO^), and concentrated into a yellow oil, .which bccomas * dried up foam upon drying in high vacuum, to give 5.4 g. of 30 (t)-N-13-(benzoylamino)-2-oxo-4-phenylbutyl ] -N-methylglycine, 1,1-dimethylethyl ester. - 22 - d) (D-N-C3-(Benzoylamino)-2-oxo-4-phcnylbutyll-M-methylglycine. monohydrochlorlde The ester produdt from pact (c) (4.51 g., 11 mmole) is treated with 2N hydrochloric acid/ 5 acetic acid (20 ml.). After stirring for 2.5 hours at room temperature, the reaction mixture is concentrated uiider rcduced pressure and the oily residue is triturated with ether to give 3.3 g. of (±) -N- (3- (benzoylamino) -2-oxo-4-phenylbutylJ-10 N-methylglycine, monohydrpchloride as an off-white solid. el M-Cyclohexylqlycine,' 1.l'-dimethylethyl ester Cyclohexylamine (70.35 ml.) and sodium bicarbonate (12.9 g.) are suspended with stirring IS in 200 ml. of absolute ethanol while stirring in an ice-bath. To this is' added bromoacetic acid, 1,1-dimethylethyl ester (20.78 ml.) dropwise. The ice-bath is removed. After 24 hours at room temperature, the reaction mixture is concentrated 20 to dryness, taken into chloroform and washed with water. The crude product (42 g.) is chromato-graphed on silica gel eluting with ethyl acetate: hexane (2:1) to give 27.4 g. of N-cyclohexyl-glycine, 1,1-dimethylethyl ester. 25 f) W- [N- f 3- (Benzoylamino) -2-oxo-4-phenylbutyl)-H- methylqlycyll -N-cyclohexylglycine. 1,1-dimcthylethyl ester To a solution of (i)-N-l 3-(benzoylamino) -2-oxo-4-phenylbutyl] -N-methylglycine, hydrochloride 30 (1.0 g., 2.6 mmole) in distilled tetrahydrofuran (50 ml.) is added'N-cyclohexylglycine, 1,1-dimethylethyl ester (0.55 g., 2.6 mmole), prepared as set forth in (e) above, hydroxybenzotriazole hydrate (0.39 g., 2.6 mmole),arid dicyclohexyl-35 carbodiimide (0.S5 g., 2.6 mmole). The reaction mixture is stirred overnight, the precipitated - 23 - dicyclohexylurea is filtered off, and the filtrate is concentrated. The residue is dissolved in ethyl acetate (SO ml.) and washed with saturated sodium bicarbonate (twice), lot potassium bisulfate 5 (twice), and water (twice), dried (Na^SO^) and concentrated into an oily residue (1.5 g.). Flash chromatography (100 g., Merck silica gel 60) gives 0.49 g. of N-(N-[3-(benzoylamino)-2-oxo-4-phenyl-butyl) -N-methylglycyl] -R-cyclohexylglyclne ,1,1-10 dimethylethyl ester as a foam. a) N-'[N-t3- (Benzoylamino) -2-oxo-4-phenylbutvl)-N-methylqlycyl)-N-cyclohexylglyclne, monohydrochlorlde The ester product from part (f) (0.48 g., 0.87 15 mmole) is treated with 2N hydrochloric acid/acetic acid (10 ml.). After stirring for 2 hours at room temperature, the reaction mixture is concentrated under reduced pressure and the oily residue is triturated with ether pvernight to give 0.32 g. 20 of N-[H-13- (benzoylamino) -2-oxo-4-phenylbutylJ-N-methylglycyl]-N-cyclohexylglycine, monohydrochlorlde as an off-white solid; m.p. 131-145°. Kg 0.36 (silica gel, n-butanol/acetic acid/water> 4:1:1). 25 Anal, calc'd. for C28H35H3°5 " I*C1' * °-7 H^O: C, 61.95; R, 6.95; N, 7.74; CI, 6.53 Found: C, 61.95; H, 6.74; N, 7.71; CI, 6.23.

. Example 2 IS1 —7— 11 [ (±)-3-(Benzoylamino) -2-oxo-4~phcnylbutyl)-' methylamlnolacetyl) -1.4-dithia-7-azaspiro[4.4)-nonane-8-carboxyllc acid, methyl ester To a solution of (±)-N-[3-(behzoylamino)-2-oxo-4-phenylbutyl] -N-methylglycine, monohydrochloride (1.0 9.-, 2.5 mmole), prepared as set. forth in Example 1(d), in distilled tetrahydrofuran (SO ml.) ia added (S)-l,4-dithia-7-azaspiro(4.4]nonane-8-« carboxylic acid, methyl ester, monohydrochloride (0.66 g., 2.S mmole), dicyclohexylcarbodiimide (0.54 g., 2.5 mmole), hydroxybenzotriazole hydrate (0.39 9., 2.5 'mmole) and diisopropylethylam'ine (0.9 ml., 5- mmole). The reaction mixture is stirred overnight, the precipitated dicyclohexylurea is filtered off, and the filtrate is concentrated. The residue is dissolved in ethyl acetate (100 ml.) and washed with saturated sodium bicarbonate (twice) and water (twice), dried (N«2SO^), and concentrated into a yellow oily residue (1.3 g.). Flash chromatography- (Merck silica gel, 251 ethyl acetate/methylene chloride. It methanol/methylene chloride) affords 0.53 9. of (S)-7-((I(±1-3-(benzoylamino) -2-oxo-4-phenylbutyl]methylaaino]-acetyll -1, 4-dithia-7.-azaspirof 4 .4]nonane-8-carboxylie acid, methyl ester as a white foam; m.p. 60 -r 62". 0.52' (silica gel, 51 methanol/ methylene chloride).

Axial, calc'd. for c2bH33N3°5S2 * 0,33 H2°* - 25 - C, 59.87; H, 6.04; N, 7.48; S, 11.42 found: C, 59.87; H, 5.94; N, 7.56; S, 11.36.

Example 3 (S) —7— f 11 (1) -3- (Benzoylamino) -2-oxo-4-phenvlbutyll-5 methylamino] acetyl] -1.4-dithla-7-a»asplrot 4.41 nonane-8-carboxyllc acid, methyl ester, monohydrochloride The methyl ester product from Example 2 (0.26 g., 0.46 mmole) is treated with 2N hydrochloric acid/acetic acid until homogeneous 10 (2 minutes), concentrated under reduced pressure, and the oily residue is triturated with ether (twice) to give 0.26 g. of (S)-7-II((i}-3-(benzoylamino) -2-oxo-4-p.henylbutyl]methylamino) -acetyl] -l,4-dithia-7-azaspiro[4.4 ] nonane-8-15 carboxylic acid, methyl ester, monohydrochloride as a white solid; m.p. 79-85". R^ 0.53 (silica gel, 5t methanol/methylene chloride).

Anal, calc'd. for C28H33N3°5S2 * HC* * 0.56 H^O: C, 55.84; R, 5.88; N, 6.98; S, 10.64; Cl,5.88 20 Found: C, 55.84; H, 5.95; N, 6.78; S, 10.43; Cl.5.66.

Example 4 (4S) -1- rw-f3- (Benzoylamino) -2-oxo-4-phenylbutyl 1 -N-methvlqlvcvll-4-(4-fluoroohenoxy)-I.-orollng. methyl ester, monohydrochloride 25 a) C 4S) -4- Ifluorophanoxy) -L-proIine. methyl ester.' monohydrochloride TO a suspension of (4S)-4-(fluorophenoxy)-L-proline (2.5 'g., 11 mmole) in methanol at -3.0* under an argon atmosphere is' added thionyl 30 chloride' (8.09 ■!., 11 mmole). The reaction mixture / ■ - - 26 is stirred at -20" for 2 hours, then at room temperature for 16 hours. Solvent is removed at reduced pressure and the 'residue is redissolved in methylene chloride (ISO ml.) and washed with' IN sodium carbonate (twice) and water (twice).

After drying (MgSOj), excess hydrochloric acid/ methanol is added and solvent is removed at reduced pressure. Addition of ether gives a light brown solid (2.6 g.). Recrystallization from methanol/ ether gives 1.49 g. of (4S)-4.-(.fluorophenoxy)-L- proline, methyl ester, monohydrochloride as a 20 light brown solid; m.p. 147-148"; (a]Q » +6.96" (c ■ 1.5S, methanol). b) (4S)-l-[N-[3- (Benzoylamino) -2-oxo-4-phenylbutyl'] -N-methylqlycyl] -4- (4-f luorophenoxy) -L-proline. methyl ester To a solution of (±)-N-[3-(benzoylamino)-2-oxo-4-phenylbutyl] -N-methylglycine, monohydrochloride (1.17 g., 3 mmole), prepared as set forth in Example 1(d), in distilled tetrahydrofuran (20 ml.) is added (4S)-4-(fluorophenoxy)-L-proline, methyl ester, monohydrochloride (0.82 g., 3 mmole), hydroxybenzotriazole hydrate (0.46 g., 3 mmole) and di'cyclohexylcarbodiimide (0.62 g., 3 mmole). The reaction mixture is stirred overnight, the' precipitated dicyclohexylurea is filtered off, and the 'filtrate is concentrated. The residue is dis'solved in ethyl acetate (50 ml.) and washed with saturated sodium bicarbonate (twice) and water (twice) , dried (Na^SO^) ,' and concentrated into an - 27 - oily residue (l.lg.). Flash chromatography (200 g.f Herck silica gel 60} 3% methanol/chloroforra) gives 0.1S g. of (4S)-1-III-13-(benzoylamino)-2-oxo-4-phenylbutyl] -N-mcthylglycyl J-4-(4-f luoro-S phenoxy)-L-proline, methyl ester as a foam. c) (4 S)-1-tN-13- (Benzoylamino) -2-oxo-4-phenvl-butvll -N-methylqlvcyl)-4- (4-fluorophenoxv) -2-prollne. methyl ester, monohydrochloride The methyl ester product from part (b) 10 (0.15 g., 0.26 mmole) is treated with 2N hydrochloric acid/acetic acid until homogeneous (2 minutes), concentrated under reduced pressure, and the oily residue is triturated with ether (twice) to afford 0.14 g. of (4S)-l-[N-[3-15 (benzoylamino)-2-oxo-4-phenylbutylI-N-methyl-glycyl ] - 4- (.4-fluorophenoxy) -L-proline, methyl ester, monohydrochloride as an off-white solid; m.p. 105-125". R^ 0.27 (silica gel, 5% methanol/ chloroform). 20 Anal, cale'd. for • HCl C, 62.79; B, 5.76; M, 6.86; F, 3.10; Cl,5.79 Found: C, 62.78; R, 5.73; N, 6.87; F, 2.83; Cl,5.33.

■Example 5 MS)-1-INtIS)-3-(Benzoylamino)-2-oxo-4-phcnylbutyll-L-alanyll-4-(4-fluorophenoxy)-L-prollne, monohydrochloride a) (S)-N-[3-(Benzoylamino)-2-oxo-4-phenylbutyl1-L-alanine, 1.1-dimethylethyl ester To a stirring solution of (S)-H-[3-chloro-2-oxo-l-(phenylsiethyl)propyl]benzamide (10.0 g., 33.1 mmole) in dimethylformamide (80 ml.) is added L-alanine, 1,1-dimethylethyl ester, hydrochloride (6.0 g., 33.1 mmole), sodium bicarbonate (6.1 g., 72 molt) and sodium iodide (4.9 g., 33.1 mmole). The resulting solution is stirred overnight at room temperature, poured into ether and washed with water (twice) and 10% sodium bicarbonate. The ether solution is extracted with IN hydrochloric acid (3x), the combined extracts are made basic by the addition of solid sodium' bicarbonate and extracted with ethyl acetate (4x). The organic extracts are combined, dried (HgSO^) and concentrated to give 8.9 g. of pal* yellow solid. A portion of this material is recrystallixed from ethyl acetate to give (S)-N-(3-(benzoylamino) -2-oxo-4-phenylbuty 1J -L-alanine, 1,1-dimethylethyl ester as a white solid; m.p. 106.5 - 110". - 29 - b) (S)-H- [3- (Bcrizoylaiftlno)-2-oxo-4-phcnylbutyl)-L-alanine, monohydrochloride A solution of the ester product from partes) (2.95 g., 5.4 mmole) in 1.4 N hydrochloric acid in 5 acetic acid- (39 ml.) is stirred at roon temperature for 2 hours. The resulting white precipitate is collected, rinsed with ether and dried to give 2.27 g. of (S)-N-f3-(benzoylamino)-2-oxo-4.-phenylbutyl]-L-alanine, monohydrochloride; 10 m.p. 208-209* (dec.); [o]D = -7,1* (c - 0.38% in methanol). 0.51 (silica gel; chloroform/-methanol/acetic fccid; 4:1:1).' Anal, calc'd. for C20H22N2°4 * HC^S C, 61.64; H, 5.93;. N, 7.17; CI, 9.07 15 Found: C, *1.33; H, 5.97; K, 7.17; CI, 8.79. c) " (S) -M- [S- (Benzoylamino)-2-oxo-4-tShghvlbutylI -W- T (pheny Imethoxy) carbonyl | -L-alanine Triethylamine (2.1 ml., 15 mmole)' is added to a mixture of (S) -N-(.3- (benroylamino)-20 2-qxo-4-phenylbutyl]-L-a.lanine, monohydrochloride (2.0 g., 5.1 mmole), benzyl chlorofpzmate (730 jil., . 5.1 mmole)water' (7 ml .J and dioxane' (7 ml.) at 25*. The resulting mixture is stirred at 25* for 3 hours, after which it' is' poured into 25 5% aqueous- sodium bicarbonate solution and ' washed with ether. The aqueous layer is' acidified (HCl)and extracted.into ethyl acetate (3x). The extract is' dried' (MgSO^) and concentrated to give a colorless oil*. ' Trituration with ether ' 30 produces a white granular solid '(150 'mg.! which - 30 - is collected and discarded. The mother liquor is concentrated in vacuo to give 1.7.5 g. of (S)-N-[N-(benzoylamino)-2-oxo-4-phenylbutyl]-N-((phenylmethoxy) carbonyl] -L-alanine as a white glass. d) (4S)-l-[N-t(S)-3-(Benzoylamino)-2-oxo-4-phenylbutvll -N- [ (phenylmethoxy) carbonyl 1 -L-alanyll-4-(4-fluorophenoxy)-L-proline. phenylmethyl ester A mixture of (S)-N-I.N-(benzoylamino)-2-oxo-4-phenylbutyl]-N- ('(pheny lmethoxy)carbonyl]-L^ alanine (300 mg., 0.62 mmole), (4S)-4-(4-fluorophenoxy) -L-p.roline, phenylmethyl ester, p-toluenesulfonic acid salt (300 mg., 0.62 mmole), triethylamine (90 yl., 0.62 mmole), dicyclo-hexylcarbodiimide (130 mg., 0.62 mmole), and hydroxybenzotriazole hydrate (90 mg., 0.62 mmole) in tetrahydrofuran (7 ml.) is stirred at 25* £or 20 hours. The mixture is then filtered and diluted with ethyl acetate. The resulting solution is washed sequentially with IN hydrochloric acid and 10% aqueous sodium bicarbonate solution, dried (MgSO^), filtered, and concentrated to give 500 mg. of (4S)-l-(N-[(S)-3-(benzoylamino)-2-oxo-4-phenylbutyl] -N- [ (phenylmethoxy) carbonyl] -L-alanyl] -4- (4-fluorophenoxy) -L-proline, phenylmethyl ester as a pale yellow oil. - 31 - e) (.4S)-l-m-'[ (S)-3-(Benzoylamino)-2-oxo-4-phcnylbutyl)-L-alanyll-4-(4-fluorophenoxy)-L-prollne. monohydrochloride A mixture of the ester product from part(d) 5 (500 mg., 0.6 mmole), palladium on carbon catalyst (10%, 100 mg.), absolute ethanol (15 ml.), and 1.0 N aqueous hydrochloric acid (800 yl., 0.8 mmole) is hydrogenated at 1 atmosphere and 25* for 17 hours, after which it is filtered and concentrated. 10 The residue is chromatographed on HP-20 a linear gradient from (9:1, 0.01N aqueous hydrochloric acid:methanol] tp [1:1, 0.01N aqueous hydrochloric acid:methanol]. Fractions containing the desired product (TLC) are combined and concentrated. The 15 residue is dissolved in a minimum amount of methanol. Ether.is added, resulting in a white precipitate which is collected and dried in vacuo to give 200 mg. of (4S) -1- [N-[ (S) -3- (benzoylamino) -2-oxo-4-phenylbutyl] -L-alanyl]-4- (4-f luorophenoxy) -20 L-proline, monohydrochloride; m.p. 152-153° (dec.); [alj® ■ -50s (c » 0.5, methanol). Rg 0.75 (silica gel, chloroforra/methanol/acetic acid, 4:1:1).

Anal, calc'd. for C3iH32FN3°£ * ^ * 1-5 HjO: 25 C, 59.57; H, 5.80; N, 6.72; CI', 5.67 Found: C, 59.68; H, 5.56; N, 6.67; CI, 5.99. - 32 -Example 6 [1 (S) .-4R1-1- [N-[3- (Benzoylamino) -2-oxo-4-phenvlbutvll-L-alanyll-4-phenyl-L-prollne. monohydrochloride aj (Si-N-[N-(Benzoylamino)-2-oxo-4-phenvlbutylj -N- 5 f (phenylmethoxy).carbonyl)-L-alanlnc. succinimido ester A mixture of (S)-N-[N-(benzoylamino)-2-oxo-4-phenylbutyl]-N-((phenylmethoxy)carbonyl]-L-alanine .(800 mg., 1.6 mmole), prepared as set forth In Example 5 (c), dicyclohexylcarbodiimi.de 10 (340 mg., 1.6 mmole), and N-hydroxysuccinimide (190 mg., 1.6 mmole) in tetrahydrofuran (5 ml.) is stirred at 25? for 18 hours. After this tiiae it is filtered and concentrated'to give 950 mg. of (S)-N-(N-(benzoylamino)-2-oxo-4-phenylbutyl]-15 H- [(phenylmethoxy)carbonyl]-L-alanine, succinimido ester. b) fl(S).4R]-l-rN-f3-(Benzoylamino)-2-oxo-4-phenylbutyll-N-t(phenylmethoxy)carbonyl]-L-alanyl]-4-phenyl-L-prollne 20 To a solution of (S)-N-[N-(benzoylamino)-2- oxo-4-phenylbutyl]-N-((phenylmethoxy)carbonyl]-L-alanine, succinimido ester (950 mg., 1.6 nuaole) in dimethylformamide (5 ml.) is added (4R)-4-phcnyl-L-proline, hydrochloride (375 mg., 1.7 mmole) 25 and triethylamine (40 pi., 3.2 mmole). The resulting mixture is .stirred at 25* for 24 hours, after which it is poured into excess.IN hydrochloric acid and extracted with ethyl acetate (3x). The extracts are combined, dried (MgSOj), 30 filtered, and concentrated to give 1.1 g. of IX (S) , 4RJ—1—IN-[3-(benzoylamino)-2-oxo-4-phenyl-butyl] -N-[(phenylmethoxy)carbonyl]-L-alanyl]-4-phenyl-L-proline. e) H(S).4RJ-1-IN-I3-(Benzoylamino)-2-oxo-4-phenvlbutvl]-L-alanvl]-4-phenyl-L-proline. mono- . hydrochloride The product from part (b) (1.0 g., 1.5 mmole), ethanol (20 ml.)* water (5 ml.), 1.0 N hydrochloric acid (1.5 ml., 1.5 mmole), and palladium on carbon catalyst (10%, 100 mg.) is hydrogenated at one atmosphere and 25s for 18 hours, after which it is filtered and concentrated. The residue is chromato-graphed on HP-20 -using a linear gradient (0.01 N aqueous hydrochloric acid:methanol, 40:60 to 10:90]. Fractions containing the desired product (TLC) are combined and concentrated. The residue is dissolved in .a minimum amount of methanol. Ether is added and the resulting white precipitate is collected and dried to give 300 mg. of [ 1 (S) ,4R]-1-IN-(3- (benzoylamino) -2-oxo-4-phenyl-butyl]-L-alanyl}-4-phenyl-L-prollne, monohydrochloride; m.p. 160-162* (dec.); la]** - -57* (c « 1.5., methanol). Rg 0.8 (silica gel; chloroform/methanol/acetic acid; 4:1x1).

Anal, calc'd. for C3lH33 N3<>5 • HC1 - 1.35 HjO: C, 63.27; H, 6.29; N, 7.14; CI, 6.02* Found: C, 63.27; R, 6.17; N, 7.19; CI, 5.97.

Example 7.

III5I . 4S1-1-[N-[3-(Benzoylamino)-2-oxo-4-pheny1-butyll-L-alanyll-4-phenyl-L-proline. monohydrochlorlde Following the procedure of Example 6 but employing (4S)-4-phenyl-L-prolinej hydrochloride in part (b), one obtains (1(S),4S]-l-[N-j3-(benzoyl-amino) -2-oxo-4-phenylbutyll-L-alanyl-4-phenyl-L-proline, monohydrochloride; m.p. 138-143*; (aJD ■ -61* (c ■ 0.34 in methanol). R^ 0.84 (silica gel, chloroform/methanol/acetic acid, 4:1:1).

Anal, calc'd. fpr C31H33N305 • HC1 • 2-13 HjO: C, .61.80; B, 6.3S; S, 6.98; CI, 5.88 Found: C, 61.80; H, 6.06; ft, 7.05; Cl, 5.65.

Example 8 tl(S) ,4Rl-I-tN-t 3- (Benzoylamino) - 2-oxo-4-pheny 1-butyll -L-alanyll-4-cvclohexyl-L-prollne. monohydrochloride Following the procedure of Example 6 but employing (4K)-4-cyclohexyl-L-proline, hydrochloride in part (b) , one obtain* (1(S),4R]-1-[N-[3- (benzdylaminb-2-oxo-4-phenylbutyl] -L-alanyl] -4-cyclohexyl-L-proline, monohydrochloride; m.p. 140-154* (dec.); [o]D » -83* (c - 0.36% in methanol). R^ 0.84 (silica gel; chloroform/ methanol/acetic acid; 4:1:1). - :js - Anal., calc'd. for CjjHjjNjOj • HC1 - 0.79 HjO: C, 63.71; H, 7.17; N, 7.19; Cl, 6.06 Found: C, 63.71; H, 7.21; N, 7.05; Cl, 5.82.

Example 9 5 (1(S).4 S1-1-[M-[3-(Benzoylamino)-2-oxo-4-phenyl-•butyll-L-alanyll-4-cyclohexyl-L-proline. monohydrochloride Following the procedure of Example 6 but employing (4S)-4-cyclohexyl-L-proline, hydrochloride 10 in part (b), one obtains [1 (S) ,4S]-l.-[N-[.3- (benzoylamino) -2-oxo-4-phenyIbutyl ] -L-alanyl 1-4-cyclohexyl-L-proline, monohydrochloride; ro.p. 139-141* (dec.); [o)g ™ -80* (c = 0.2% in methanol). Rj 0.83 (silica gel; chloroforra/methanol/ 15 acetic acid; 4:1:1)..

Anal, calc'd. for C3iH39H3°s * HC* " 1-54 HjO: C, 62.28; K, 7.26; H, 7.03; Cl, 5.93 Found: C, 62.28: H, 7.01; ft, 7.02;.'C1, 6.16.

Example id 20 (S)i-7- [ (S) -2-t t3- (BenzOylamino) -2-oxo-4-phenylbutyll-amino 1 -1-oxopropy 11 -1.4-dithia-7-a«tspiro(4.41 nonane-8-carboxylle "acid; monohydrochloride Following the procedure of Example 6 . but employing (S) -1,4-dithia-7-azaspiro (4.4] nonane-8-25 carboxylic acid, hydrochloride in part (b) for the L-proline reactant, one obtains (S)-7-[ (S)-2-[ (3-(benzoylamino) - 2,-oxo-4-phehy Ibutyl] amino] -1-oxopropy 1 ] -1, 4-dithia-7.-az aspiro [ 4'. 4 ] nonane- 8-carboxylifc acid', monohydrochloride; a.p. 170-172*; 30 - 36 - (ajp® » -26* (c ™ 1.4% In methanol). 0.78 (silica gel; chloroform/methanol/acetic acid; 6:1:1).

Anal, calc'd. for • HC1 • 0.77H2<>s 5 C, 54.77; H, 5.71; N, 7.10; S, 10.83; Cl, 5.99 Found: C, 54.77} H, 5.70; N, 6.94; S, 10.82; Cl, 6.07.

Example 11 10 f1 IS).5S1-1-[N-13-(Benzoylamino)-2-oxo-4- ohenylbutyll -L-alanyl"1 -4.5-dihydro-3-Phenyl-lH-pyrazole-5-carboxylic" acid, monohydrochloridie Following the procedure of Example 5 but employing (S)-4,5-dihydro-3-phenyl-lH-pyrazole-5-15 carboxylic acid for the L-proline reactant in part (b), one - obtains the above named compound. Example 12 (S)-2-fH-f(SV-3-(Benzoylamino)-2-oxo-4-phenylbUtyl1-' L-alanyl] -1.2.3 f 4-tetrahydro-3-'lsoquinolinecarboxylic 20 acid', monohydrochloride Following the procedure of Example 6 . but employing (S)-l,2,3,4-tetrahydro-3-isoquinoline-carboxylie acid, hydrochloride in part (b) in place of the proline reactant, one obtains (S)-2-(N-[.(S)-25 3- (benzoylamino) -2-oxo-4-phehy Ibutyl] -L-alanyl] -1, 2,3,4-tetrahydro-3.-'i'soquinblinecarboxylic acid, -monohydrochloride.

Example 13 1- [N-[ (S) -3- (Benzoylamino) -2r-oxo-4-phenylbutyll -L-alanyll-2-(2-hydroxyphenyl)-4(R)-thiagolldinecar-boxyllc acid, monohydrochloride Following the procedure of Example 6 but employing 2-[(2-ptienylmethoxy)phenyl]-4(R)-thia-zolidinecarboxylic acid, hydrochloride in part (b) in place of the proline reactant, one obtains after removal of the hydroxy protecting group l-JN-[ (S) -3- (benzoylamino) -2.-OXO-4-phenyIbutyl]-L-alanyl]-2-(2-hydroxyphenyl)-4(R)-thiazolidinfecarboxylic acid, monohydrochloride. - 38 -

Claims (1)

1. CLAIMS I . A compound of the formula £3^ O O R R O II |l I I II R--C-SH-CH-C-CH--M - CH-C-OH , 2 R3 wherein: R is hydrogen, Prot, lower alkyl, cycloalkyl, h H -(ch2)^^^ ' -(.C82)2-»-Prot , -(CH2)3-N-PrOt, -(CH^-N-Prot, -(CHj)2-0-Prot, -(CH2)3-0-Prot, -(CHj)4-0-Prot , -(CH2)a-S-Prot, -(CH2)3-S-Prot, or -(CHjI^-S-Protj - 39 - R^ is hydrogen, lower alkyl, halo substituted lower alkyl, _ _ -(CH2 ]f(Q) ~{CH2)F\0)-0-Prot' • (CH2) ^-^V-O-Prot , -