NOVEL ANTI-HYPERTENSIVE MERCAPTOACYLAMINO ACID DERIVATIVES, THEIR PREPARATION AND USE

Abstract: There are described novel angiotensin converting enzyme inhibitors having the general formula: wherein R represents hydrogen or a formyl, acetyl, prop­ anoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentylcarbonyl, tert-butyloxycarbonyl, cyclopentylcarbonyl-L-lysyl, pyro-L-glutamyl-L-lysyl, L-arginyl, L-lysyl or pyro-L-glutamyl group; A represents a L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl, L-leucyl, L-histidyl or L-valyl group, the α-amino group thereof being in amide linkage with R; F₁ represents hydrogen or methyl; R₂ represents L-proline, L-3,4-dehydroproline, D,L-3,4-­ dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine, the imino group thereof being in imide link­ age with the adjacent --; and n is 0 or 1, such that when n=O, R₁ represents methyl. The preparation of these compounds is also described. They are useful as orally effective anti-hypertensive agents.

Description: ANTI-HYPERTENSIVE AGENTS, A PROCESS FOR THEIR PREPARATION, AND THEIR USE. BACKGROUND or THE 'INVENTION; Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of convertingthe decapeptide anqiotensin I, havina the sequence AspArgValTyrIleHisProPheHisteu to an octapeptide, angiotensinII by removal of the carboxyterminal HisLeu. The symbols for various chemical entities are explained in the following table: Ala 5 L-alanine Arg = L-arginine Asp = L-aspartic acid ( Glu = pyro-L-glutamic acid Gly = glycine Hip = Hippuric acid (Benzoyl-glycine) His = L-histidine lie = t-isoleucine Leu = L-leucine Phe = L-phenylalanine Pro = L-proline APro - t-3,4-dehydroproline Ser = L-serine Trp = L-tryptophan Tyr = L-tyrosine Val = L-valine ACE = Angiotensin converting enzyme Heps = N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid Angiotensin I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues and plasma, acting on a serum sL-2 globulin. Blood pressure is affected by certain peptides found in the blood. One of these, angiotensin II, is a powerful pressor (blood pressure elevating) agent. Another, bradykinin, a nonapeptide with the sequence ArgProProGlyPheSer ProPheArg is a powerful depressor (blood pressure lowering) agent. In addition to a direct pressor. effect, angiotensin II stimulates release of aldosterone which tends to elevate blood pressure by causing retention of extracellular salt and fluids. Angiotensin II is found in measurable amount in the blood of normal humans. However, it is found at elevated concentrations in the blood of patients with renal hypertension. The level of ACE activity is ordinarily in excess, in both normal and hypertensive humans, of the amount needed to maintain observed levels of antiogensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. EGavras, I., et al., New Engl. 3. Med. 291. 817. (1974)3. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel inhibitors of ACE which have the general formula EMI2.1 wherein R is hydrogen, formal, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentylcarbonyl, tert-butyloxycarbonyl, cyclopentylcarbonyl-L-lysyl, pyro-L glutamyl-L-lysyl, L-lysyl, L-arginyl or pyro-la-glutamyl; A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl, L-leucyl, L-histidyl or L-valyl, the amino group thereof being in amide linkage with R; R1 is hydrogen or methyl; ; R2 is In-proline, L-3, 4-dehydroproline, D,L-3,4 dahydroproline, L-3-hydroxyproline, L-4-hydroxyproline or Lthiazolidine. the imino group thereof being in imide linkage with the adjacent EMI2.2 and, n is 0 or 1, such that&gamma; when n=0, R1 is methyl. All amino acids discussed herein are in the L-configuration unless otherwise noted. The disclosed compounds are inhibitors of ACE and are useful as orally effective antihypertensive agents. The discovery of ACE inhibitory potency in the compounds of the present invention provides a unique approach to the desinn of inhibitory compounds. Although many prior art inhibitors are proline derivatives, substitution of other amino acids for proline has also yielded potent inhibitors. Arginine, phenyl-alanine and alanine are all effective substitutes for proline, so that a trend is not discernible. The substitution of L-3,4-dehydroproline for proline has been studied in several systems. Substitution of L-3, 4-Pro in the 7 position of bradykinin yields a bradykinin derivative which has significantly reduced physiological activity. See Fisher, C.. et al., Arch.- Biochem. Biophys. 189, 81 (1978). On the other hand, substitution of L-3, 4-bPro at the 3, 5 or 9 position in ACE inhibitor BPP9a enhances its inhibitory activity. However, at present, no rationale can be advanced to explain the diversity of observed results following substitution of #Pro for proline. Similarly, no clear picture has emerged of the effects of other proline derivatives or analogs substituted at various loci on ACE inhibitors. To date, the effect of the amino acid to the left of the sulfur in the above-shown formula, has not been determined. It is thought that this amino acid functions as an additional recognition site for the enzyme. If this is true, it would be expected that a compound with an amino acid here would be a better inhibitor. It was not known which, if any, amino acids would be effective in this position and which if any would enhance the inhibitory activity of a given compound. Applicants have found that various amino acids are effective and that the hydroxyprolines, proline, L-, and D,L-,3,4-dehydroproline, and thiazolidine derivatives are all effective anti-hypertensive. agents and have high inhibitory potency for ACE. The present invention will be further described by the following examples. In these examples, the thin-layer chromatography (TLC) was performed using silica gel plates. The numerical solvent systems for use in the TLC procedures are as follows. (1) is methanol:chloroform, 1:1 (parts by volume). (2) is benzene:water:acetic acid, 9:1:9 (Parts by volume). (3) is acetic acid:water:n-butanol 26:24:150 (parts by volume). (4) is n-butanol:pyridine:acetic acid:water, 15:10:3:12 (parts by volume). (5) is chloroform:methanol: ammonium hydroxide, 60:45:20 (parts by volume). The buffers for paper electrophoresis were pH 1.9 - formic acid:acetic acid:water, 3:2:25 (parts by volume); pH 5.0 - diethylene glycol:acetic acid:pyridine:water, 100:6:8.5:885 (parts by volume). The tert-butyloxycarbonyl, benzoyl, acetyl, formyl, propanoyl, butanoyl, phenylacetyl and phenylpropanoyl derivatives of the amino acids are commercially available. Sephadex LH-20, Sephadex G-10 and Sephadex G-25 are trademarks of Pharmacia, Inc., Uppsala, Sweden. Example 1 ACE activity assay. For most experiments described herein, the enzyme was assayed in 0.05 M Hepes buffer, pH 8.0 containing 0.1 M NaC1 and 0.75 M Blal'SO . The substrate employed was Benzoyl-GlyHisLeu at a final concentration of 1 x 10 4M, (Km ¯ 2 x 10-4M). together with about 130,000 cpm 3 of [ H]BenzoylGlyHisLeu (25 Ci/mmole). Enzyme was diluted in the above buffer such that 40p1 buffered enzyme was capable of hydrolyzing 13% of substrate in a 15-minute incubation at 370C. To initiate the assay, 40p1 of enzyme and 10p1 of water or inhibitor dissolved in water were preincubated for five minutes at 370C. Substrate, 50p1, was then added to initiate reaction and the solution was incubated for 15 minutes at 370C. To terminate the reaction, 1 ml of 0.1 M HC1 was added, following which 1 ml of ethyl acetate was added. The mixture was agitated on a rotary mixer and centrifuged briefly to separate the phases. An aliquot, 500 l, of the ethyl acetate layer waçst transferred to a liquid scintillation vial containing 10 ml of Riafluor, trademark New England Nuclear Corporation, Boston, Massachusetts. For determination of i50 values, enzyme activity in the presence of inhibitor at a series of different concentrations was compared to activity in the absence of inhibitor. A plot of inhibitor concentration versus percent inhibition yielded the 150 value. Example 2 Synthesis of 3-acetylthiopropanoyl-1-proline-t-butyl ester. 3-acetylthiopropanoic acid, 0.865 g, was dissolved in 2 ml redistilled tetrahydrofuran (THF) and cooled to OOC. A cooled solution of dicyclohexylcarbodiimide, 1.2031 g in 2 ml of THF was added, following which a cooled solution of L-proline-t-butyl ester, 1 g, was added. The reaction mixture was stirred at 0 C for one hour, then at 40C overnight. The reaction mixture was then filtered and the precipitate was washed with ethyl acetate. Solvents of the filtrates were removed under reduced pressure in a rotary evaporator. The residue was dissolved in ethyl acetate v/ffi) which was then washed three times with cold 1 N citric acid, twice with saturated Nazi, twice with cold 1 N NaHC03 and three times with saturated NaC1. The solution was dried over anhydrous MgS04 and filtered. The solvent was removed under reduced pressure in a rotary evaporator at 300C yielding a clear colorless oily product in approximately 87% yield. The product migrated as a single spot in thin layer chromatography in five solvent systems. Example 3 Svnthesis of 3-mercaptopropanol-L-proline. The product from Example 2, 3-acetylthiopropanoyl-L proline-t-butyl ester, 0.5 g, was mixed with 4.5 ml of 5.5 N methanolic ammonia at room temperature under nitrogen for one hour to remove the acetyl group. The solvent was then removed at 250C with a rotary evaporator. After the product was taken up in methanol and reevaporated twice more in the rotary evaporator, the clear oily residue was dissolved in ethyl ether, washed twice with 5% potassium bisulphate and once with saturated NaCl, dried over tAgSO4 and filtered. Residual solvent was removed in vacuo to yield a clear oily product, migrating as a single spot on thin layer chromatography in three separate solvent systems. The t-butyl estet protecting group was removed by reaction with trifluoroacetic acid in anisole. Examples 4-6 By substituting 2-acetylthiopropanoic acid, 3-acetylthio-2-D-methylpropanoic acid, or 3-acetylthio-2-D,L-methyl propanoic acid for the )-acetylthiopropanoic acid in Example 2 and substantially following the procedures of Examples 2 and 3, the following compounds are obtained. By removing the t-butyl ester protecting group with trifluoroacetic acid in anisole as a first step, the- dicyclohexylamine salt can be formed to assist in the resolution of isomers. The acetyl protecting group can be removed in a second step using methanolic ammonia, as described in Example 3. Exanipi e Compound 4 2-mercaptopropanoyl-L-proline 5 3-mercapto-2-D-methylpropanoyl-L-proline 6 3-mercapto-2-D,L-methylpropanoyl-L-proline Example 7 Synthesis of 3-mercapto-2-methyl-propanoyl-L-3,4 dehydroproline. L-3,4-dehydroproline (Zi3Prn), 1 mmole, is dissolved in DMF and the solution is cooled to -150C. The solution is neutralized by adding 1 equivalent of N-ethyl morpholine. In a separate reaction vessel at -10 C, one equivalent of 3acetylthio-2-methyl-propanoic acid in an equal volume of DMF is mixed with 1.1 equivalent of 1,1'-carbonyidiimidazole, and the solution is stirred for one hour The first solution Jv containing 2E 3Pro is mixed with the second, containing 3-acetylthio-2-methyl propanoic acid while maintaining the temperature at -lO0C. The combined solution is stirred for 1 hour at -lO0C. The solution is then allowed to warm 5 slowly to room temperature. The solvent is removed on a rotary evaporator under reduced pressure at 40 C. Ethyl acetate (25 ml) is added and the solution is cooled to OOC. Two ml of 1 N citric acid is added, the two phases are mixed and then allowed to separate. The phases are separated with a separating funnel, and the organic phase is washed twice more with 2 ml 1 N citric acid, two times with saturated NaC1 and finally dried over anhydrous ttgS04. The MgS04 is removed by filtration, and the solvent is removed with a rotary evaporator. The residue is dissolved and re crystallized from a non-polar solvent such as benzene to yield 3-acetylthio-2-D,L-methylpropanoyl-L-3,4-dehydropro line. When the 2-D-methyl isomer is desired, the residue is dissolved in acetonitrile (approximately 3 ml) and the solution is warmed to 40 C. One equivalent of dicyclo hexylamine is added, and the solution is allowed to stand at room temperature overnight. The crystals are collected by filtration and are washed three times with acetonitrile. When further purification is required, the material can be recrystallized from isopropanol. The acetyl protecting group can be removed as in Example 3. Examples a-il By substituting D,L-3,4-dehydroproline, L-3-hydroxy proline, L-4-hydroxyproline, or L-thiazolidine for the L-3, 4-dehydroproline in Example 7 and substantially following the procedures of Example 7 the following compounds are obtained. Example Compound 3-mercapto-2-D-methylpropanoyl-D,L-3,4-dehydroproline 9 3-mercapto-2-D-methylpropanoyl-L-3-hydroxyproline 10 3-mercapto-2-D-methylpropanoyl-L-4-hydroxyproline 11 3-mercapto-2-D-methylpropanoyl-L-thiazolidine Example -12 Similarly, by substituting 3-acetylthiopropanoic acid or 2-acetylthiopropanoic acid for the 3-acetylthio-2-methyl propanoic acid of Examples 7-11, the L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline and L-thiazolidine derivatives are obtained, following substantially the described procedures. Example 13 Synthesis of N&alpha;-[3-(N&alpha;-acetyl-L-phenylananylthio)-2 -D-methylpropanoyl]-L-proline. A solution of 41.5 mg of N&alpha;-acetyl-L-phenylelanine in 0.5 ml redistilled dimethylformamide (DMF) was cooled in an ice-dry ice-acetone bath at -2O0C. To this solution was added a cold solution of 35 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10 C for two hours and then was added to a cold solution of 48 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DtiF which was neutralized with N-ethyl morpholine. The reaction mixture was stirred at =lO0C for an additional hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 400C and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and washed with 0.1 N HC1 and then three times with saturated NaC1 solution. The organic solvent was removed with a rotary evaporator after drying over anhydrous MgS04, The product was purified by Sephadex LH-20tm column chromatography using a 1.2 cm by 95 cm column and eluted with isopropanol. The peak fractions were pooled and the solvent was removed under reduced pressure yielding 35.5 mg of the named product. This product was found to be homogeneous using TLC with solvent systems 1, 2, 3 and 5. Examples 14-21 By substituting Nacetyl-glycine, N&alpha;-acetyl- alanine, N&alpha;-acetyl-tryptophan, N&alpha;-acetyl-tyrosine, N-acetyl-isoleucine, N-acetyl-leucine, Ni -acetyl- histidine, or N-acetylvaline for the N&alpha;-acetyl-phenyl- alanine in Example 13 and substantially following the procedure of Example 13, the following compounds are obtained. Example Compound 14 N&alpha;-[3-(N&alpha;-acetylglycylthio)-2-D-methylpropanoyl]- L-proline 15 N&alpha;-[3-(N&alpha;-acetyl-L-tryptophylthio)-2-D-methyl- propanoyl]-L-proline 16 Ni-[3-(N -acetyl-L-tyrosylthio)-2-D-methylpropanoyll- L-proline 17 N -[3-(N--acetyl-L-isoleucylthio)-2-D-methylpropanoyl]- L-proline 18 N"'-[3-(Nacetyl-L-leucylthio) 2-D-methylpropanoyl]- L-proline 19 Ng-[3-(NS-acetyl-L-histidylthio)-2-D-methylpropanoyl]- L-proline 20 N&alpha;-[3-(N&alpha; ;-acetyl-L-valylthio)-2-D-methylpropanoyl]- L-proline 21 NS-[3-(Ni-acetyl-L-alanylthio)-2-D-methylpropanoyla- L-proline Exemple 22 Similarly, the L-3,4-dehydroproline, D,L-3,4-dehydro proline, L-3-hydroxyproline, L-4-hydroxyproline and L thiazolidine derivatives are obtained by substituting the products of Examples 3-12 for the 3-mercapto-2-D-methyl propanoyl-L-proline in Examples 13-21 and substantially following the procedure of Example 13. Example 23 By substituting the N&alpha;-formyl, N-propanoyl, N - butanoyl, N-phenylacetyl or NoL-phenylpropanoyl deriva tices of L-Phe, Gly, L-Ala, L-Trp, L-Tyr, L-Ile, L-Leu, L-His and L-Val for the N&alpha;-acetyl derivatives in Examples 13-22 and substantially following the procedure of Example 13, the formyl, propanoyl, butanoyl, phenylacetyl, and phenylpropanoyl derivatives are obtained. Example 24 Synthesis of N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L phenylelenylthio)-2-D-methylpropanoyl]-L-proline. A solution of 133 mg of Ni2-tert-butyloxycarbonyl-L- phenylalanine (N&alpha;-Boc-L-Phe) in 0.5 ml redistilled DMF was cooled in an ice-dry ice-acetone bath at -200C. To this solution was added a cold solution of 87 mg of 1,1'carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10 C for two hours and then was added to a cold solution of 119.5 mg of 3-mercapto-2-D,methyIpropanoyl- L-proline in 1 ml of DMF which was neutralized with N-ethyl morpholine. The reaction mixture was neutralized with Nethyl morpholine. The reaction mixture was stirred at -100C for an additional hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 400C and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and washed with 0.1 N HC1 and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO4. the product was purified by liquid chromatography on Sephadex G-10tm using a 1.2 cm by 95 cm column and eluted with THF:isopropanol, 3:7 (parts by volume). The peak fractions were pooled and the solvent removed under reduced pressure yielding 165 mg of the named product. This product was found to be homogeneous using paper electrophoresis at pH 5.0 and using TLC with solvent systems 1, 2 and 31 Examples 25-32 By substituting N i-Boc-glucine, N L-Bo°-alanine, N 6- Boc-tryptophan, N-Boc-tyrosine, N-Boc-isoleucine, N&alpha;- Boc.-leucine, N&alpha;-Boc-histidine or N&alpha;-Boc-valine for the N&alpha;-Boc-Phe in Example 24 and substantially following the procedures of Example 24, the following compounds are obtained. Example Compound 25 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonylglycylthio)-2-D methylpropanoyl]-L-proline 26 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L-tryptophylthio)-2-D methylpropanoyl]-L-proline 27 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L-tyrosylthio)-2-D methylpropanoyl]-L-proline 28 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L-isoleucylthio)-2-D methylpropanoyl]-L-proline 29 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L-leucylthio)-2-D methylpropanoyl]-L-proline 30 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L-histidylthio)-2-D- methylpropanoyl]-L-proline 31 N&alpha;-[3-(N&alpha;-tert-butyloxycarbonyl-L-valylthio)-2-D- methylpropanoyl]-L-proline 32 N&alpha;-[3-(N&alpha; ;-tert-butyloxycarbonyl-L-alanylthio)-2-D- methylpropanoyl]-L-proline Example 33 Similarly, the L-3,4-dehydroproline, D,L-3,4-dehydro- proline, L-3-hydroxyproline, L-4-hydroxyproline and L-thiazolidine derivatives are obtained by substituting the products of Examples 3-12 for the 3-mercapto-2-D-methylpropanoyl- L-proline in Examples 24-32 and substantially following the procedure in Example 24. Example 34 Synthesis of N&alpha;-cyclopentylcarbonyl-L-phenylalanine. A cool solution of 2.06 gm of dicyclohexylcarbodiimide in 10 ml of dichloromethane was added to a solution of 1.4114 gm of cyclopentanecarboxylic -acid in 5 ml of dichloromethane at -5 C. It was followed by the addition of 4.28 gm of L-phenylalanin benzoyl ester toulenesulfonate salt in 10 ml of DMF which was neutralized with 1.36 ml of N-ethyl morpholine. The reaction mixture was stirred at OOC for one hour and then at room temperature for three hours. Dicyclohexylurea was removed by filtration and 50 ml of ethyl acetate was added to the filtrate. The organic phase was washed until neutral, dried over anhydrous MgS04 and filtered. The solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol and hexane yielding 2.35 gm of white crystals having a melting point of 88-890C. Elemental analysis of these crystals yielded the following. Calculated: C=75.19; H=7.17; N=3.9855 Found: C=74.96; H=7.17; N=4.09 The benzyl ester was removed by hydrogenolysis with 2 gm of 10% palladium on carbon in absolute alcohol. The catalyst was removed by filtration and the ethanol was removed by a rotary evaporator. The residue was crystallized from ether and hexane yielding 1.15 gm white crystals of the named product having a melting point of 107-108 C. Elemente1 analysis of these crystals yielded the following. Calculated: C=6'8.94; H=7.33; N=5.36 Found: C=68.90; H=7.32; N=5.34 The product was found to be homogeneous using paper electrophoresis at pH 1.9 and at pH 5.0 and using TLC with solvent systems, 1, 2 and 3. The named product may be abbreviated as N&alpha;-cpc-L-Phe. Example 35 Synthesis of N-[3-(N-cyclopentylcarbonyl-L phenyl-alanylthio)-2-D-methylpropanoyl]-L-proline. A solution of 52.5 mg of the compound from Example 34 in 0.5 ml redistilled DMF was cooled in an ice-dry iceacetone bath at -200C. To this solution was added a cold solution of 34 mg of 1,11-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10 C for two hours and then mixed with a cold solution of 45.6 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethyl morpholine. The reaction mixture was stirred at -100C for an additional hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 4O0C and ethyl acetate was added to the residue. The mixture was cooled and washed with 0.1 N HC1 and then three times with saturated NaCI solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO4. The product was purified bySephadex LH-20tm column chromatography using a 1.2 cm by 95 cm column and eluted with isopropanol. The peak fractions were pooled and the solvent was removed under reduced pressure yielding 60.5 mg of the named product. This product was found to be homogeneous using TLC in solvent systems 1, 2, 3 and 5. Examples 36-43 By substituting the benzoyl ester toluenesulfonate salts of glycine, L-alanine, L-tryptophan, L-tyrosine, Lisoleucine, L-leucine, L-histidine or L-valine for the L-Phe salt in Example 34 and substantially following the procedures of Examples 34 and 35, the following compounds are obtained. Example Comp o'u"nd 36 N&alpha;-[3-N&alpha;-cyclopentylcarbonylglycylthio)-2-D- methylropanoyl -L-proiine 37 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-tryptophylthio)-2-D- methylpropanoyl]-L-proline 38 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-tyrosylthio)-2-D- methylpropanoyl]-L-proline 39 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-isoleucylthio)-2-D- methylpropanoyl]-L-proline 40 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-leucylthio)-2-D- methylpropanoyl]-L-proline 41 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-histidylthio)-2- methylpropanoyl]-L-proline 42 N&alpha;-[3-(N&alpha; ;-cyclopentylcarbonyl-L-valylthio)-2-D- methylpropanoyll-L-proline 43 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-alanylthio)-2-D- methylpropanoyl]-L-proline Example 44 Similarly, the L-3,4-dehydroproline, D,L-3,4-dehydro- proline, L-3-hydroxyproline, L-4-hydroxyproline and L-thia zolidine derivatives are obtained by substituting the prod ucts of Examples 3-12 for the 3-mercapto-2-D-mihylpropanoyl- L-proline in Examples 35-43 and substantially following the procedure of Example 35. Example 45 Synthesis of N 6-(3-L-phenylalanylthio-2-D-methvl- prdpanoyl)-L-proline. The product from Example 24 was deprotected by stirring a mixture of 30 mg of the product, 50yl of anisole and 200p1 of anhydrous trifluoroacetic acid (TFA) at room temperature for one hour. Anisole and TFA were removed under reduced pressure at 350C and the residue was triturated with anhydrous ether. The white residue was purified by liquid chromhtography on Sephadex G-10tm using a 1.2 cm by 95 cm column and eluted with 5% acetic acid. The peak fractions were pooled and freeze-dried yielding 17.5 mg of the named product. This product was found to be homogeneous using paper electrophoresis at pH 1.9 and 5.0 and using TLC with solvent systems 4 and 5. Examples -46-53 Similarly, by substantially following the procedure of Example 45, the products from Examples 25-32 are deprotected and the following compounds are obtained. Example Compound 46 N&alpha;-(3-glycylthio-2-D-methylpropanoyl)-L-proline 47 N&alpha;-(3-L-tryptophylthio-2-D-methylpropanoyl)-L- proline 48 N&alpha;-(3-L-tyrosylthio-2-D-methylpropanoyl)-L-proline 49 N&alpha;-(3-L-isoleucylthio-2-D-methylpropanoyl)-L-proline 50 N&alpha;-(3-L-leucylthio-2-D-methylpropanoyl)-L-proline 51 N&alpha;-(3-L-histidylthio-2-D-methylpropanoyl)-L- proline 52 N&alpha;-(3-L-valylthio-2-D-methylpropanoyl)-L-proline 53 N&alpha; ;-(3-L-alanylthio-2-methylpropanoyl)-L-proline Example 54 Similarly, the L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline, and L-thiazolidine derivatives are obtained by deprotecting the compounds described in Example 33. The deprotection is done by substantially following the procedure of Example 45. Example 55 Synthesis of the N-hydr.oxysuccinimide ester of cyclo- pentane carboxylic acid. A cool solution of 11.4 gm of dicyclohexylcarbodiimide in DMF was added drop-wise to a mixture of 5.71 gm of cyclo pentanecarboxylic acid and 5.76 gm of N-hydroxysuccinimide in DMF at OOC. The reaction mixture was stirred at 0 C for 30 minutes and then at 4 C overnight. Crystalline dicyclo hexylurea was removed by filtration and the precipitate was washed with ethyl acetate. Solvents from the combined filtrate were removed under reduced pressure and the residue was crystallized from benzene and hexane yielding 5.77 gm of white crystals having a melting point of 72.50-730C. The infrared absorptsori spectrum in chloroform gave a typical spectrum of N-hydroxysuccinimide esters. Elemental analysis of the crystals yielded the following results. Calculated: C=56.86; H=6.20; N=6.63 Found: C=56.77; H=6.07; N=6.66 Example 56 Synthesis of N"L-cyclopentylcarbonyl-NE-tert-butyl oxyeabbonyl-L-lysine. A solution of 1.2316 gm of N±-tert-butyloxyearbonyl- L-lysine and 420 mg of NaHCO3 in 10 ml of water and 5 ml of THF was cooled in an ice bath with stirring. To this solution was added a cold solution of 1.19 gm of the product from Example 55 in 10 ml of THF. The THF was removed with a rotary evaporator at 350C after the reaction mixture had been stirred overnight at room temperature. About 20 ml of water was added to the reaction mixture and the pH was adhusted to 9 with solid NaHC03. The aqueous phase was extracted three times with ethyl acetate and the organic phase was discarded. The aqueous solution was cooled in an ice bath and then acidified to pH 2 with 1 N HC1 in the presence of ethyl acetate. The organic phase was washed twice with ice water and then twice with a solution of saturated NaC1. The organic solution was dried over anhydrous MgS04 and then filtered. The solvent was removed with a rotary evaporator and the residue was crystallized from ether and hexane yielding 1.135 gm of white crystals having a melting point of 104.50-i050C. Elemental analysis of the crystals yielded the following. Calculated: C=59.63; H=8.83; N=8.18 Found: C=59.74; H=8.85; N=8.24 The product was shown to be homogeneous using paper electrophoresis at pH 1.9 and pH 5.0 and using TLC with solvent systems 1, 2 and 3. Example 57 Synthesis of N 4-cycl-opentylcarbonyl-NE -tert-butyloxy-carbonyl-L-lysine-N-hydroxysuccinimide ester. A solution of 1.027 gm of the product from Example 56 and 346 mg of N-hydroxysuccinimide in 10 ml of CH2C12 was cooled to -50C. To this solution was added with stirring a cold solution of 680 mg of dicyclohexylcarbodiimide in 5 ml of CH2C12. The reaction mixture was stirred at 0 C for 30 minutes and then at 40C overnight. Crystalline dicyclohexylurea was removed by filtration and was washed with ethyl acetate. The combined filtrate was washed twice with 1 N NaHCO3, twice with water and finally with a solution of saturated NaC1. The organic phase was dried over anhydrous MgS04, filtered, and the solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol yielding 0.844 gm of white crystals having a melting point of 140.50C. The infrared adsorption spectrum in chloroform gave a typical spectrum of N-hydroxysuccinimide esters. Elemental analysis of the crystals yielded the following. Calculated : C=57.39; H=7.57; N=9.56 Found: C=57.10; H=7.57; N=9.61 Example 58 Synthesis of N&alpha;-cyclopentylcarbonyl-N#-tert-butyl oxycarbonyl-L-lysyl-L-phenylalanine. A solution of 220 mg of the product from Example 57 in 2 ml of dioxane was added drop-wise to a mixture of 99.1 mg of L-phenylalanine and 51 mg of NaHC03 in a mixture of 2 ml of water and I ml of DMF. The reaction mixture-was stirred at room temperature overnight and dioxane was removed with a rotary evaporator at 350C. Ethyl acetate (10 ml) was added to the reaction mixture, cooled, acidified at pH 2 with 0.1 N HC1 and the aqueous solution was discarded. The organic phase was washed with ice water, a solution of saturated Nat1, and dried over anhydrous MgSO4; and the solvent was removed with a rotary evaporator. The residue was crystallized from ether and petroleum ether (b.p.30600C) yielding 95 mg of white solid having a melting point of 90-920C. The product was shown to be homogeneous using paper electrophoresis at pH 1.9 and pH 5.0 and using TLC with solvent systems 1, 2, 3 and 4. Elemental analysis yielded, Calculated: G=63.78; H=8.03; N=8.58 Found: C=63.40; H=8.07; N=8.34 Example 59 Synthesis of Nw -[3-(NX -cyclopentylcarbonyl-N±- tert-butyloxycarbonyl-L-'lysyl-L-phenylala'nylthio)-2-D- mekhylpropanoyl ]-L-pr-oline. A solution of 73.5 mg of the product from Example 58 in 0.5 ml redistilled DHF was cooled in an ice-dry iceacetone bath at -200C. To this solution was added a cold solution of 26 mg of 1,1'-carbonyidiimidazole in 1.0 ml of DMF. The solution was stirred at -10 C for two hours and then mixed with a cold solution of 36 mg of 3-mercapto2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethyl morpholine. The reaction mixture was stirred at -10 C for an additional hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 400C and ethyl acetate was added to the residue. The mixture was cooled in an ice water bath and washed with 1 N citric acid and then three times with saturated NaC1 solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO4 The product was purified by Sephadex LH-20 m column chromatography using a 1.2 cm by 95 cm column and eluted with THF:isopropanol, 3:7 (parts by volume). The peak fractions were pooled and the solvent was removed under reduced pressure yielding the named product which may be abbreviated as N&alpha;-[3-(N&alpha;-cpc-N#-cpc-Boc-L-Lys-Phe-thio)-2-D methylpropanoyl ]-L-proline. Example 60 Synthesis of N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L lysyl-L-phenylalanylthio) -2-D-methylpropenoyl )--proline The N#-Boc group was removed from the lysine by stirring a mixture of 30 mg of the product from Example 59 with 5Dl anisole and 200p1 of anhydrous trifluoroacetic acid (TFA) at room temperature for one hour. Anisole and TFA were removed under reduced pressure at 35 C and the residue was triturated with anhydrous ether. The residue was tm purified by liquid chromatography on Sephadex G-10 using a 1.2 cm by 95 cm column and eluted with 5% acetic acid. The peak fractions were pooled and freeze-dried yielding the named product which can be abbreviated as N&alpha;-[3-(N&alpha;-cpc-L-Lys-L-Phe-thio)-2-D-dimethylpropanoyl]- L-proline. Examples 61-68 By substituting glycine, L-alanine, L-tryptophan, L-tyrosine, L-isoleucine, L-leucine, L-histidine, or L valine for the L-phenylalanine in Example 58 and sub stantially following the procedures of Examples 58, 59 and 60, the following compounds are obtained. Example Compound 61 N"L-C3-("'-cyclopentylcarbonyl-l-lysyL-glycyl- thio) -2-D-methylpropanoyl] -L-proline 62 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-lysil-L-tryptophyl- thio)-2-D-methylpropanoyl]-L-proline 63 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-lysil-L-tyrosyl- thio)-2-D-methylpropanoyl]-L-proline 64 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-lysil-L-isoleucyl- thio)-2-D-methylpropanoyl]-L-proline 65 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-lysil-L-leucyl- thio)-2-D-methylpropanoyl]-L-proline 66 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-lysil-L-histidyl- thio)-2-D-methylpropanoyl]-L-proline 67 N&alpha;-[3-(N&alpha; ;-cyclopentylcarbonyl-L-lysil-L-valyl- thio)-2-D-methylpropanoyl]-L-proline 68 N&alpha;-[3-(N&alpha;-cyclopentylcarbonyl-L-lysil-L-alanylthio) -2-D-methylpropanoyl]-L-proline Example 69 Similarly, the L-3,4-dehydrcproline, D,L-3,4-dehydro proline, L-3-hydroxyproline, L-4-hydroxyproline and L-thia zolidine derivatives are obtained by substituting the prod ucts of Examples 3-12 for the 3,-mercapto-2-D-methylpropanoyl L-proline in Examples 59 and 61-68 and substantially following the procedures of Examples 59 and 60. Example 70 By substituting pyro-L-glutamic acid for the cyclopentanecarboxylic acid in Example 55 and substantially following the procedures of Examples 55-57, N-pyro-L- glutamyl-N±-tert-butyloxyearbonyl-L-lysine-N-hydroxy- succinimide ester is obtained. By substituting this product for the N&alpha;-cpc-N#-Boc-L-Lys-N-hydroxysuccinimide ester in Example 58 and substantially following the procedures of Examples 58-69, the pyro-L-glutamyl derivatives are obtained. Example 71 Preparation of pyro-L-glutamyl-L-phenylalanine benzyl ester. A solution ° 0.52 g of pyro-L-glutamic acid, .1.72 g of L-phenylalanine benzyl ester toluenesulfonic acid and 0.55 ml of N-ethylmorpholine in 5 ml of DMF and 20 ml of dichloromethane was cooled in an ice bath with stirring. A solution of 0.826 g of dicyclohexylcarbodiimide in 2 ml dichloromethane was added to the above reaction mixture. The reaction mixture was stirred in an ice water bath for .1 hour and then at room temperature overnight. Dicyclohexylurea was removed by filtration and the product was washed in ethyl acetate. Solvents of the combined filtrates were removed under reduced pressure. with a rotary evaporator at 400C. Ethyl acetate (25 ml) was added to the residue and the organic solution was washed until neutral. The organic phase was dried over anhydrous MgSO4, filtered and then the solvent was removed with a rotary evaporator. The material was crystallized from isopropanol and ether, yield; 1.01 9 of white needles, m.p. 103-104.50C. The material was homogeneous on all TLC and electrophoresis systems. Elemental analysis yielded, Calculated: C=68.84; H=6.05; N=7.64 Found: C=68.58; H=6.05; N=7'.56 'Example 72 Preparation of pyro-L-glutemyl-L-phenylalanine. The benzoyl cster protecting group of the compound of ampÚ 71 (1.0g) was removed by catalytic hydrogenolysis @ @ @ rg of 10% (by weight) Pd on carbon in 0.15 ml @ glacial acetic acid and 15 ml of ethanol at 20 psi of H2 at room temperature overnight. The catalyst was removed by filtration. Solvent was removed with a rotary evaporator. The material was crystallized from isopropanol and benzene, to yield a total of 402 mg of white crystals, m.p. 147-1490C. The material was homogeneous on electrophoresis at pH 1.9 and pH 5.0 and on TLC in systems 1, 2 and 3. Elemental analysis yielded, Calculated: C=60.86; H=5.84; N'=10.14 Found: C=60.37; H=5.85; N=9.98 Example 73 Preparation of N&alpha;(-[3-Pyro-L-glutamyl-L-phenyl- ala-nylthio)-2-D-methylpropanoyl]-L-prol¯ine. - - A solution of 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml DMF was added to a solution of 139 mg of the product of Example 72 in 0.5 ml DMF at 150C. The reaction mixture was stirred at -100C for 1 hour, and then a mixture of 119.5 mg of 3-mercapto-2-D-methylpropanoyl-L-proline and 0.072 ml of N-ethyl morpholine in 1 ml. DMF was added. The reaction mixture was stirred at -100C for an additional hour and then was slowly warmed to room temperature. DMF was removed under reduced pressure with a rotary evaporator at 40 C and then 7 ml ethyl acetate and 2 ml I N citric acid were added. The organic phase was washed two times with 7 N citric acid and two times with saturated NaC1. The organic phase was dried with anhydrous MgSO4 and then filtered. Solvent was removed using a rotary evaporator. The residue was purified on Sephadex G-25 tm (1.2 x 99 cm) partition column chromatography with n-butanol: acetic acid: H20 (4:1:5 by volume). The product was homogeneous on TLC (systems 1, 2 and 3) and on electrophoresis at pH 5.0. Example 74 By substituting N4&alpha;, N#-bis-t-butyloxycarbonyl-L- lysine (hereinafter bis-Boc-L-Lys) or N&alpha;, N&gamma;, N#-triad- amantyloxycarbonyl-L-arginine (herinafter tri-Adoc-Larginine) for pyro-L-glutamic acid and by following substantially the procedure of Example 71, the corresponding bis-Boc-L-Lys or tri-Adoc-L-Arg derivatives of L-Phe-benzyl ester will be synthesized. Bis-Boc-L-Lys is commercially available. Tri-Adoc-L-Arg is prepared according to Jager, E and Geiger, R., Chem. Ber. 103, 1727 (1970). Example 75 By substituting the benzyl esters of Gly, Ala, Trp, Tyr, Tle, Leu, His or Val, in the procedures of Examples 71 and 74, the corresponding pyro-L-glutamyl, bis-Boc-L Lys and tri-Adoc-L-Arg derivatives are obtained. The foregoing benzyl esters are commercially available. Benzyl ester protecting groups are removed essentially as described in Example 72. Example 76 The compounds resulting from Examples 72, 74 and 75 are reacted with any of the compounds resulting from the procedures of Examples 3-12, following essentially the procedures of Example 73, to yield the corresponding bis Boc-L-Lys and tri-Adoc-L-Arg derivatives. The bis-Boc and tri-Adoc protecting groups are removed by treatment with trifluoroacetic acid in anisole. Following essentially the procedures and methods of Examples 2-12 and 71-76, a family of thiolester compounds is obtained which have the general formula EMI21.1 wherein, R is L-arginyl, L-lysyl or pyro-L-glutamyl; is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, -- l L-isoleucyl, L-leucyl, L-histidyl, or L-valyl - .. no group is in amide linkage with R; R1 is nydrogen or methyl; R2 is L-proline, L-3,4-dehydroproline, D,L-3,4-dehydro- proline, L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine whose imino group is in imide linkage with the 0 - C -; and n is O or 1 such as when n=O, R1 is methyl. Example 77 Synthesis of N2-[3-(N&alpha;-benzoyl-L-tryphtophylthio) 2-Dimethylpropanoyl]-L-proline. A solution of 62 mg of N L-benzoyl-L-tryptophan in 0.5 ml redistilled dimethylformamide (DMF) was cooled in a icedry ice-acetone bath at -200C. To this solution was added a cold solution of 35 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -100C for two hours and then added to a cold solution of 48 mg of 3-mercapto-2 D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethyl morpholine. The reaction mixture was stirred at -i00C for an additional hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 400C and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and washed with 0.1 N HC1 and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO4, The product was purified tm by liquid chromatography on Sephadex LH-2O using a 1.2 cm by 95 cm column and eluted with isoprppanol. The peak fractions were pooled and the solvent removed under reduced pressure yielding 60.5 mg of the named product, as a foamlike material. This product was found to be homogeneous using TLC with solvent systems 1, 2, 3 and 5. Examples 78-85 By substituting N&alpha;-benzoyl-L-tyrosine, N&alpha;-benzoyl L-histidine, N i-benzoyl-L-phenylalanine, N&alpha; -benzoylglycine, N&alpha;-benzoyl-L-alanine, N&alpha;-benzoyl-L-isoleucine, N &alpha; -benzoyl-L-leucine or N -benzoyl-L-valine for the N&alpha;- benzoyl-L-tryptophan in Example 77 and substantially following the procedures of Example 77, the following compounds are obtained: : Example Compound 78 N&alpha;-[3-(N&alpha;-benzoyl-L-tyrosylthio)-2-D-methyl- propanoyl]-L-proline 79 N&alpha;-[3-(N&alpha;-benzoyl-L-histidylthio)-2-D-methyl- propanoyl]-L-proline 80 N&alpha;-[3-(N&alpha;-benzoyl-L-phenylalanylthio)-2-D-methyl- propanoyl]-L-proline 81 N&alpha;-[3-(N&alpha;-benzoyl-L-glycylthio)-2-D-methyl- proaanoyl 1 -L-pro line 82 N&alpha;-[3-(N&alpha;-benzoyl-L-alanylthio)-2-D-methyl- propanoyl] -L-proline 83 N&alpha;-[3-(N&alpha;-benzoyl-L-isoleucylthio)-2-D-methyl- propanoyl]-L-proline 84 N&alpha;-[3-(N&alpha; ;-benzoyl-L-leucylthio)-2-D-methyl- propanoyl]-L-proline 85 N&alpha;-[3-(N&alpha;-benzoyl-L-valylthio)-2-D-methyl- propanoyl]-L-proline Example -8B; Similarly, the L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline, and L-thiazolidine derivatives are obtained by substituting the products of Examples 3-12 for the 3-mercapto-2-D-methylpropanoyl L-proline in Examples 77-85 and substantially following the procedures of Example 77. An alternative method of preparing N&alpha;-[3-(N&alpha;-benzoyl- -L-phenylalanylhio)-2-D-methylpropanoyl]-L-proline is described in the following two examples. Example 87 Preparation on N&alpha;-benzoyl-L-phenylalanine-N-hydroxy succinimide ester. 1.347 g of benzoyl-phenylalanine and 0.576 g of N hydroxy succinimide were mixed in a 1:1 (by volume) mixture The rid 1F The mixture was incubated at 40C overnight the presence of 1.133 g of dicyclohexylcarbodiimide. action mixture was filtered and the solvent was removed under reduced pressure at 300C. A white residue remained which was recrystallized from THF-isopropyl alcohol to yield 1.194 g (65.2%) of a white solid, m.p. 1560C157 C. The infrared absorption spectrum in chloroform showed bands at 3440 cm-1 indicating an NH group, at 1818 cm-1, 1790 cm-1, and 1744 cm-1, characteristic of the N-carboxy succinimide group and 1669 cm-1, characteristic of the N-benzoyl moiety. Example 88 Synthesis of N&alpha;-[3-(N&alpha;-benzoyl-L-phenylalanylthio)- 2-D-methylpropanoyl]-L-proline. A reaction mixture containing 93.3 mg of 2-D-methyl 3-mercaptopropanoyl-L-proline, 62 mg of 1-hydroxybenzotriazole JHOBt), 165 mg of N-benzoyl-phenylalanyl-N-hydroxy-succini mide ester, prepared as described in Example 87, was cooled in an ice bath at approximately 0 C, after which 0.0544 ml of N-ethyl morpholine was added. The reaction mixture was stirred in an ice bath for three hours, incubated at 4 C overnight, then at room temperature for twenty hours. The reaction was terminated by the addition-of 25p1 of N,N dimethyl-1,3-propanediamine, and stirred for an additional two hours. Ethyl acetate was added to the reaction mixture which was then washed by extraction with cold 0.1 N HC1, followed by two washes with water and three washes with saturated NaC1. The mixture was then dried over anhydrous MgSO4 and filtered. The solvent was removed under reduced pressure in a rotary evaporator, yielding a clear, oily product. The product was purified by chromatography on a column of Sephadex LH-20tm, 1.2 cm x 96 cm. The column was eluted with TEiF, and 2.5 ml fractions were collected. Fractions 30 and 31 were pooled and yielded, after solvent removal under high vacuum, 110 mg of product. Anhydrous ether was added and a white gum-like material was obtained. The ether was decanted and the white gum-like material was dissolved in THF and transferred to a vial, dried in a stream of nitrogen, then further dried over P205 overnight. A foam-like product, 82 mg, was obtained. Fractions 29, 32 and 33 were rechromatographed under identical conditions. Fractions 33 through 36 of the second chromatography were pooled, worked up as described and yielded an additional 51 mg of product. Total yield was 71L An alternative method of prepaying Nv -[2-(N&alpha;-benzoyl L-phenylalanylthio)-propanoyl )-L-3,4-dehydroproline and related derivatives is described in the following two examples. Example 89 Synthesis of 2-benzoylpheynlalanylt-hiopropan-oic acid. A solution of benzoyl-phenylalanine in 30 ml of redistilled dimethylformamide (Dt1F) is cooled to -200C. A solution of 1,1 1ecarbonyldiimidazole in 10 ml redistilled DMF is added drop-wise with vigorous stirring. Temperature is not allowed to exceed -140C. Following the addition, the solution is stirred at -10 C for two hours. DJL-Thio- lactic acid in redistilled DMF previously neutralized with redistilled N-ethylmorpholine is then added with continued stirring at -100C for one hour. The solution is then slowly warmed to room temperature. An approximately equal volume of ethyl acetate is added. The mixture is then cooled and neutralized with co-ncentrated HC1 in saturated NaC1. The organic phase is then washed three times with subsaturated NaC1, i.e., five volumes saturated NaC1 diluted with one volume water. In some cases a three-layer system is observed. In such cases, the middle layer is. saved and combined with the lower aqueous phase. The organic phase is dried over anhydrous MgS04, filtered and placed in the rotary evaporator to remove solvent. The combined aqueous phase and middle phase is acidified at 0 C with concentrated HC1 to.pH 2, and extracted three times with ethyl acetate. The organic phase is washed with saturated NaC1 and dried over anhydrous magnesium sulfate, filtered and rotary evaprated. A clear oil is recovered. Exemple 90 Synthesis of N&alpha;-[2-(N&alpha;-benzoyl-L-phenylalanylthio propanoyl)-L-3,4-dehydroproline. Boc-L-3,4-dehydroproline (213 mg; 1 mmole) was de protected with d ml of anhydrous trifluoroacetic acid for 1 hour. Anhydrous ethyl ether, 3 ml, was added. were removed using a rotary evaporator at 30 C. The residue was washed three times with ether and then was dried in a vacuum dessicator over NaOH. A solution of 358 mg (1 mmole) of the product from Example 89 in 3 ml of redistilled DMF was cooled to -200C in a dry ice-acetone bath. A solution of 1,1'-carbonyl- diimidazole, 171 mg (1.05 mmole) in 1 ml of redistilled DMF was added drop-wise, and the temperature of the reaction mixture was maintained at -100C to -15oC. The reaction mixture was stirred for two hours. A cold solution of L-3, 4-dehydroproline trifluoroacetate in 2 ml of DMF (eutra- lized with triethylamine) was added. The reaction mixture was warmed slowly to room temperature. Solvent was removed by rotary evaporation, using high vacuum, at 35 C. The residue was dissolved in 10 ml of ethyl acetate and 2 ml ofH20. The mixture was acidified with 1 N HC1 to pH 2 at 0 C and was mixed. The organic phase was separated and saved. The aqueous phase was extracted three more times with 5 ml of ethyl acetate. The combined organic phase was washed twice with H2C), three times with saturated NÅaC1 and then was decolorized with charcoal. The organic phase was dried with anhydrous MgSû4 and then filtered. The filtrate was evaporated to dryness on a rotary evaporator, and an oily product, 366 mg, was obtained. The product was purified by chromatography on Sephadex LH-20tm (1.2 x 98 cm) equilibrated and eluted with methanol. Fractions of 150 drops (2.9 ml) were collected. The desired compound was eluted in fractions 24-29. Solvent was removed by rotary evaporation. Additional purificstion was achieved by a second chromatography step on Sephadex LH-20tm The second column was equilibrated and eluted with isopropanol, peak fractionswere pooled and solvent was removed by rotary evaporation. The product was approximately 95% pure as judged by thin layer chromatography in solvent systems (2) and (3). Example 91 The inhibitory potency of various of the abovesynthesized compounds in vitro was measured in the assay system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). Table 1 shows the 150 value for various compounds. The 150 value is the concentration of inhibitor required to produce 500D inhibition of the enzyme under a standard assay system containing substrate at a level substantially below K m Table 1 Compound mi Example 13 2.6 x 10-8 M Example 24 3.4 x 10-8 M Example 35 7.5 x 10-9 M Example 45 8.8 x 10-9 M Example 59 2.5 x 10-8 M Example 60 1.5 x 10-8 M Example 77 9.4 x 10-9 M Example 80 2.5 x 10-8 M Example 81 7.5 x 10-9 M Example 90 (racemic) 3 x 10-9 M Example 92 Oral effectiveness of various compounds Rats (210-290 g body weight) were fasted overnight and then anesthetized with intraperitoneal pentobarbital, 50-60 mg/kg. Trachesostomy was performed and the animals were ventilated mechanically. A cannula was inserted into a femoral vein for injection of angiotensin I, and a second cannula was inserted into a common carotid artery for direct measurement of arterial blood pressure. Heparin, 1,000 units, as injected via the femoral vein to prevent regulation Blood pressure was measured with a pressure transducer connected to a polygraph. The rats were injected with 160-400 ng/kg of angiotensin I in 20p1 of 0.9 g 0 NaCl; an amount of angiotensin I sufficient to raise mean arterial blood pressure by 27-50 mm Hg. After the responsiveness of a given rat to angiotensin I was established, the respective compound at 5.5-23 pmole/kg (drug dissolved in 0.15 ml of H20 plus 10 l of 1 N NaXCO3), was given viaea stomach tube. At timed intervals, the effects of 160-400 ng/kg of angiotensin I on mean arterial blood pressure were tested. Results are shown in Table 2. The dose of the particular compound is shown in parenthesis behind the compound number. For example 13 (13.6) indicates that 13.6 pmole/kg of the compound from Example 13 was administered orally. The time, in minutes, after oral administration of the particular compound when the effects of angiotensin I were tested is indicated in brackets behind the "% of control" reading. Table 2 Blood Pressure Response to 160-400 ng/kg of Angiotensin I Expressed as % of Control [minutes after oral administration] Compound (dose) 13 24 35 45 60 73 (13.6) (20) (23) (20) (17) (20) 100 [-5] 100 [-5] 100 [-5] 100 [-5] 100 [-5] 100 [-5] 40 [+3] 100 [+3] 71 [+2] 77 [+6] 59 [+5] 53 [+1] 40 [7] 43 [11] 63 [6] 69 [10] 44 [10] 66 [5] 28 [13] 27 [22] 49 [16] 46 [15] 44 [15] 26 [15] 24 [18] 24 [29] 43 [23] 38 [25] 44 [31] 26 [18] 20 [23] 22 [34] 43 [37] 38 [29] 38 [37] 21 [21] 18 [31] 19 [51] 40 [48] 33 [38] 44 [45] 21 [25] 20 [24] 26 [64] 33 [50] 44 [55] 24 [29] 20 [42] 26 [71] 31 [57] 36 [81] 18 [39] 16 [48] 37 [84] 31 [65] 36 [93] 16 [44] 16 [58] 37 [96] 26 [74] 38 [104] 16 [49] 20 [67] 40 [109] 36 [88] 18 [54] 20 [80] 54 [124] 18 [60] 18 [90] 51 [140] 18 [74] 16 [100] 63 [157] 21 [86] 20 [112] 77 [171] 21 [110] 20 [125] 26 [120] 30 [137] 26 [132] 50 [167] 26 [142] Table 2 (cont) Blood Pressure Response to 160-400 ng/kg of Angiotensin I Expressed as % of Control [minutes after oral administration] (dose) 77 77 80 80 81 90 (20) (20) (cont) (11) (5.5) (7.6) (23) 100 [-5] 64 [212] 100 [-6] 100 [-6] 100 [-5] 100 [-5] 92 [+3] 64 [226] 67 [+6] 111 [+6] 95 [+1] 100 [+3] 64 [7] 80 [242] 57 [12] 93 [12] 95 [6] 81 [9] 32 [21] 80 [260] 43 [24] 81 [18] 95 [11] 74 [14] 40 [26] 84 [291] 37 [35] 85 [24] 92 [22] 74 [24] 40 [32] 17 [48] 56 [35] 68 [28] 74 [28] 20 [38] 17 [58] 56 [58] 61 [33] 63 [34] 8 [50] 20 [68] 52 [68] 58 [39] 56 [44] 8 [56] 17 [78] 30 [88] 47 [45] 44 [54] 8 [70] 13 [88] 37 [118] 47 [60] 56 [64] 20 [79] 27 [185] 60 [138] 45 [67] 37 [85] 12 [87] 33 [205] 37 [169] 47 [74] 44 [94] 12 [104] 33 [213] 37 [185] 47 [82] 74 [104] 20 [114] 43 [226] 44 [205] 47 [92] 56 [114] 40 [124] 55 [137] 100 [146] 40 [136] 55 [142] 36 [148] 47 [152] 48 [173] 63 [185] 48 [186] 79 [207] 68 [199] 74 [230] Example 93 Intravenous effectiveness of various compounds. Anesthetized rats were prepared as described in Example 92. After the responsiveness of a given rat to angiotensin I was established, the respective compound, at 2 mole/kg in a volume of 15 l of 0.01N sodium bicarbonate, was injected via a femoral vein. At timed intervals, the effects of angiotensin I, 40ûng/kg, on mean arterial blood pressure were tested. Results are shown in Table 3. Tab 3 follows the same format as Table 2. Table 3 Blood Pressure Response to 400 ng/kg of Angiotensin I as % of Control (minutes after intravenous administration] Compound (dose) 24 .60 77 (2) (2) (2) 100% [-5] 100% [-5] 100% [-5] 0 [+0.5] 8 [+3] 8 [+0.5] 19 [3] 15 [9] 18 [5] 19 [9] 23 [14] 21 [11] 22 [13] 23 [19] 24 [14] 31 [18] 33 [24] 29 [17] 31 [22] 41 [37] 34 [20] 50 [34] 46 [45] 42 [24] 63 [47] 46 [54] 47 [32] 78 [65] 46 [77] 53 [43] 75 [103] 51 [87] 58 [49] 94 [113] 61 [56] 71 [71] 78 [76] 76 [88] 76 [99] 74 [109] 84 [125] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this pplication is intended to cover any variations, uses, or drptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims: CLAIMS 1. An angiotensin converting enzyme inhibitor having the formula: EMI33.1 wherein R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentyl carbonyl, tert-butyloxycarbonyl, cyclopentylcarbonyl-Llysyl, -pyro-L-glutamyl-L-lysyl, L-arginyl, L-lysyl or pyro L-glutamyl; A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl, L-leucyl, L-histidyl or L-valyl, themL-amino group thereof being in amide linkage with R; R1 is hydrogen or methyl; R2 is L-proline, L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine, the imino group thereof being in imide linkage with the adjacent EMI33.2 and, n is O or 1, such that when n=0, -R1 is methyl. 2. The inhibitor of claim 1 wherein R is benzoyl. 3. The inhibitor of claim 1 wherein R is hydrogen. 4. The inhibitor of claim 1 wherein R is formyl acetyl, propanoyl, butanoyl, phenylacetyl Dr phenylpropanoyl. 5. The inhibitor of claim 1 wherein R is cyclopentylcarbonyl or tert-butyloxycarbonyl. 6. The inhibitor of claim 1 wherein R is cyclopentylcarbonyl-L-lysyl or pyro-L-glutamyl-L-lysyl. 7. The inhibitor of claim 1 wherein R is L-arginyl, L-lysyl or pyro-L-glutamyl. 8. The inhibitor of claim 1 wherein R is benzoyl, A is L-phenylalanyl, R1 is methyl, R2 is L-proline and n=l. 9. An inhibitor as defined in any of claims 1 - 8 for re in inhibiting angiotensin in vivo. 10. An inhibitor as defined in any of claims i-to 8 = use in reducing blood pressure. 11. process for preparing an angiotensin converting :zyme inhibitor useful for treating hypertension having true formula EMI34.1 wherein R is formyl, acetyl, propanoyl, butanoyl, phenyla-etyl, phenylpropanoyl, benzoyl, cyclopentylcarbonyl, tert-butyloxycarbonyl, cyclopentylcarbonyl-L-lysyl, pyro L-glutamyl-L-lysyl, L-arginyl, L-lysyl or pyro-L-glutamyl; A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl, L-leucyl, L-histidyl or L-valyl, the dL-amino group thereof being in amide linkage with R; R1 is hydrogen or methyl; ; R2 is L-proline, L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine, the imino group thereof being in imide linkage with the adjacent EMI34.2 and n is O or 1, such that when n=O, R1 is methyl, which comprises the following steps: (1) reacting R3-S-(CH2) EMI34.3 with R2 to give R3-S-(CH2) EMI34.4 wherein R3 is acetyl and R1, R2 and n are defined as above; (2) removing R3 from the product of step (1) to give HS-(CH2) EMI34.5 (3) reacting the product of step (2) with R-A to nive R-A-S-(CH2) EMI34.6 wherein R, A, R1, R2 and n are defined as above, 12. The process of claim 11 wherein the reaction described in step (1) is conducted either in the presence of 1,1 '-carbonyldiimidazole or dicyclohexylcarbodiimide and the reaction described in step (3) is conducted in the presence of 1,1'-carbonyldiimidazole. 13. The process of claim 12 wherein the R2 in step (1) also has a protecting group for the carboxyl which is removed in step (2). 14. The process of claim 12 wherein R is benzoyl. 15. The process of claim 12 wherein R is formyl, acetyl, propanoyl, butanoyl, phenylacetyl and phenylpropanoyl. 16. The process of claim 12 wherein R is cyclopentylcarbonyl or tert-butyloxycarbonyl. 17. The process of claim 12 wherein R is cyclopentylcarbonyl-L-lysyl or pyro-L-glutamyl-L-lysyl. 18. The process of claim 12 wherein R is L-arginyl, L-lysyl or pyro-L-glutamyl. 19. The process of claim 16 wherein A - S - (CH2) - EMI35.1 R2 is formed by deprotecting tert-butyloxycarbonyl-A-S-(CH2) EMI35.2 wherein A, R1, R2 and n are as defined above. 20. The process of claim 12 wherein R is benzoyl, A is L-phenylalanyl, R1 is methyl, R2 is lrproline and n=l.

Inventors: CHUNG, ALFRED, RYAN, JAMES WALTER
Applicants: UNIVERSITY OF MIAMI
Application year: 1979
Main classification: C07C103
IPC codes: C07D207, A61K38, C07K5