ANGIOTENSIN CONVERTING ENZYME INHIBITORS

Abstract: Inhibitors of angiotensin converting enzyme which have the formula: wherein R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentanecar­ bony, tert-butyloxycarbonyl, cyclopentanecarbonyl-L-lysyl, pyro-L-glutamyl-L-lysyl, L-lysyl, L-arginyl or pyro-L-­ glutamyl; A is L-phenylalanyl, D-phenylalanyl, D, L-alanyl, D-alanyl, D-I-tryptophyl, D-tryptophyl, D,L-tyrosyl, D-tyrosyl, D,L-isoleucyl, D-isoleucyl, D,L-leucyl, D-leucyl, D,L-histidyl, D-histidyl, D,L-valyl, or D-valyl, the α-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-dehy­ droproline, L-3-hydroxyproline, L-4-hydroxyproline, L-thiazoline-4-carboxylic acid, or L-5-oxo-proline, the imino group thereof being in imide linkage with the adjacent --; and n is O or 1, such that when n is O, R₁ is methyl are disclosed as useful anti-hypertensive agents.

Description: ANGIOIENSIN CONVERTING ENZYttE -INHIBITURS Angiotensin converting enzyme (peptidyldipeptide hydrolase,hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of converting the decapeptide angiotensin I, having the sequence AspArgValTyrIleHisProPheHisLeu to an octapeptide, angiotensin II by removal of the carboxyterminal HisLeu. The symbols. for various chemical entities have the meaning given in the following table unless otherwise indicated: Ala = L-alanine Arg = L-arginine Asp = L-aspartic acid Boc = t-butyloxycarbonyl Glu = glutamic acid 6 flu = pyro-L-glutamic acid (L-5-oxo-proline) Gly = glycine Hip = Hippuric acid (Benzoyl glycine) His = L-histidine Ile = L-isoleucine Leu - L-leucine Phe = L-phenylalanine Pro = L-proline A Pro = L-3,4-dehydroproline Ser = L-serine Trp = L-tryptophan Tyr = L-tyrosine Val = L-valine ACE = Angiotensin converting enzyme Hepes = 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 plasmaS acting on aserum a-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, brady kinin, a nonapeptide with the sequence ArgProProGlyPhe-Ser ProPheArg is a powerful depressor (blood pressure lowering) agent. In addition. to a direct pressor effect, angiotensin II stimul-ates release of aldosterone which tends ta elevate blood pressure by causing retention of extracellul-ar salt and fluids. Angiotensin 11 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 angiotensin II. However it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. ECavras, I., et al., New Engi. J. Med.- 291, 817 (1974)]. ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end of a variety of acylated tripeptides and larger polypeptides having an unblocked -carboxyl group. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the car-boxyl-terminal end yielding as reaction products a dipeptide and a remnant. The reactivity of the enzyme varies markedly depending on the substrate. At least one type of peptide bond, having the nitrogen supplied byproline, is not hydrolyzed at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. For general discussion of the kinetic parameters of enzyme catalyzed reactions, see Lehninger, A., Bisohemist-ry,- Worth Publishers, Inc., New York, 1970, pp. 153-157. Many peptides which are called inhibitors of the enzymatic conversion of angiotensin I to angiotensin II are in fact -substrates having a lower Km than angiotensin I. Such peptides are more properly termed competitive substrates. Examples of'competitive substrates include bradykinin-, and the peptide BPP5a (also called SQ20475) from snake venom, whose sequence is < Glu'LysTrpAlaPro. Numerous synthetic peptide derivatives have been shown to be ACE inhibitors by Ondetti, et al. in U.S. patent 3,832,337 issued August 27, 1974 The role' of ACE in the pathogenesis of hypertension has prompted a search for inhibitors of the enzyme that could act -as arrtihypertensive drugs. See for example U.S. patents 3,891,'616, 3,947,575, 4,052,511 and 4,053,651. A highly effective inhibitor, with high biological activity when orally administered, is D-3-mercapto-?-methyIpropanoyl- L-proline, designated SQ14225, disclosed in U.S. patent 4,046,889 to Ondetti et al., issued September 6, 1977, and in scientific articles by Cushman, D.W. et al., Biochemistry 16, 5484 (1977), and by Ondetti, M. et al., Science 196, 441 (1977). The inhibitor SQ14225 reportedly has an 150 value of 2.3 x 10-8M. The I50 value reported by Cushman, et -al., supra, is the concentration of inhibitor -required to produce 50 inhibition of the enzyme under a standard assay system containing substrate at a level substantially above Km It will be understood that 150 values are directly comparable when all potential factors affecting the reaction are kept constant. These factors include the source of enzyme, its purity, the substrate used and its concentration, and the composition of the assay buffer. All I50 data reported herein have been performed with the same assay system and same enzyme (human urinary ACE) and with an approximately 1/2Km level of substrate and are therefore internally consistent. Discrepancies with data obtained by other workers may be observed. Indeed such discrepancies do exist in the literature, for unknown reasons. See, for example, the 150 values for BPP 9a reported by Cushman, D.W., et al., Experientia 29, 1032 (1973) and by Dorer, F.E., et al., Biochim. Biophys. Acta 429, 220 (1976). The mode of action of SQ 14,225 has been based upon a model of the active site of ACE developed by analogy with the better known related enzyme, carboxypeptidase A. The active site was postulated to have a cationic site for binding the carboxyl end group of the substrate and a pocket or cleft capable of binding the side chain of the Terminal amino acid and providing especially tight binding for the heterocyclic ring of a terminal proline residue. A similar pocket for the penultimate amino acid residue was postulated, and the published data suggested a rather stringent sterie require ment, since the D-form of the inhibitor was substantially more potent than its stereoisomer or the 3-methyl and unsubstituted analogs. The -sulfhydryl group the inhibitor, postulated to be bound at the active site near the'catalytic 'center, was believed to play a central role' in inactivation of the enzyme by combining with the zinc moiety known to be essential f-or catalytic activity. Substituents on the sulfhydryl, such as a methyl group, and an S-acetyl derivative, substantially reduced potency of the inhibitor. See Cushman, D.W., et al., Biochemistry, supra. In vitro study of the mechanism by which SQ 14,225 and its analogs act to inhibit ACE has been somewhat hampered by the instability of these molecules under ambient conditions. For example, it has been observed tha't a f'resh aq'ueous solution of concentration-, -e.g., 1 mg per ml of SQ 14,225 at a pH of about 8 becomes substantially less ac'tive upon standing for as little as 30 minutes, and that activity continues to decrease as the solution stands for longer, periods. It is believed that this loss in activity is mainly the results of dimerization of SQ 14,225 occurring at the sulfhydryl end groups, whereby a disulfide- is formed which is largely inactive as an inhibitor. Since the free sulfhydryl group is highly reactive and may be readily oxidized to polar acidic moieties such as sulfone and sulfoxide groups, it may also be that the observed ion vitro loss of activity of aqueous solutions of SQ 14,225 on standing is in some part of a consequence of one or more such oxidation reactions, with formation of a sulfone or sulfoxide which does not function effectively as an inhibit'or for ACE. Such reports of SQ 14,225 clinical testing as are currently available, some of which refer to the compound under the name "Captopril", suggest that the product is sufficiently stable in the normal gastric and inte'stinal' 'en-vironments of most patients to be an effective 'inhibitor -T'or ACE when administered orally. It is not yet cle'ar, however, whether there may be a group of patients for which SQ 14,225 is substantially ineffective. Because of the hi'gh reactivity of the free sulfhydry'l group, SQ 1.4,225 could readily form mixed disulfides with serum cellular proteins, peptides or other free suihydryl group-containing substances in the gastric or intestinal environments, in addition to the. possibility for dimer formation or oxidative degradation reactions. A mixed disulfide with protein. may be antigenic and, indeed, occasional allergic reactions have been clinically observed. See Gavras, et al., New England J. Med. 29R, 991 -(1978). Disulfides and oxidative degradation products of SO 14,225, if formed, may at best be expected to be largely ineffective as inhibitors. It may be postulated accordingly that dose response to SO 14,225 may vary with conditions of administration and among individual patients. Moreover, in at least some patients, unwanted side effects may occur and maintenance of an effective concentration of the inhibitor 'in the body may be difficult to control. Thioester compounds generally are.thought to be highly reactive in that the thioester linkage is readily hydrolyzable to a sulfhydryl moiety and a carboxylic moiety. Thioesters are accordingly often used as active ester intermediates for acylation under mild conditions. See groups as, e.g., acetylthio have been used as blocking groups in the above cited Ondetti, et al.patents. Thioester intermediates are also postulated to occur in the biosynthesis of cyclic peptides such as tyrocidin or gramicidin S. See Lipmann, F. in Accounts Chem. Res. 6,361 (1973) Compounds related to SO 14,225 have been disclosed by Ondetti, et al., U.S. patents 4,046,889, 4,052,511, 4,053,651, 4,113,7-15 and 4,154,840. Of interest are disclosed analogs of SO 14,225 having the five-membered heterocyclic ring of proline replaced by a four- or a six-membered ring. The inhibitory potencies of such analogs relative to SO 14,225 are not disclosed. Substitution of D-proline for L-proline is reported to drastically reduce inhibitory potency of 3-mercaptopropanoyl amino acids (Cushman, D.W., et al., supra.) The substitution of L-3,4-dehydroproline for proline has been studied in several systems. Substitution of L-3,4-APro in the 7 position--of bradykinin y'iel-d's a bradykinin deriv- ative which has sig-nificantly reduced physiolbgical activity. See Fisher, G.H. et al., Arch. Biochem. Biophys. 189, 81 (1978). On the other hand, substitution of L-3;,4-A Pro at the 3, 5, 8-or 9 position in ACE'inhibitor BPP 9a enhances its inhibitory activity. See Fisher, C'.H. et al., FEBS tatters' 107, 273 (1979). In'copending appli'cation Serial No. 958,180, applicants fou'nd that the compounds having APro, which are disclosed in said application, have high inhibitory potency and antihypertansive effectiveness. However, at present, no rationale can be advanced to explain the diversity of observed results following substitution of A Pro for proline. Similarly, no-clear picture has emerged of the effects of other proline derivatives or analogs substituted at -various lo'ci on ACE inhibitors. To date, the effect of the amino acid to the left of the sulfur in the thioester compounds disclosed in our copending applications, 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. Applicants have found that various amino acids are effective and that the hydroxyprolines, proline, L-, and D,L-,3,4-dehydroproline, thiazolidine-4-carboxylic acid, and L-5-oxo-proline derivatives are all effective antihypertensive agents and have- high inhibitory potency for ACE. Our co-pending European application No. 79301854.0 discloses ACE inhibitors with amino acid moieties in the L-form. It has since been determined that the D- and D,Lforms of these amino acids can als'o be used to prepare com- pounds which are ACE inhibitors. Accordingly, the present invention relates to novel inhibitors of ACE which have the general 'formula EMI6.1 wherein R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentanecarbonyl, tert-butyloxycarbonyl, cyclo pentanecarboyl-L-lysyl, pyro-Lglutamyl-L-lysyl, L-lysyl, L-arginyl or pyro-L-glutamyl; A is L-phenylalanyl, D-phenylalanyl, D,L-alanyl, D-alanyl, D,L-tryptophyl, D-tryptophyl, DXL-tyrosyl, D-tyrosyl, D,L-isoleucyl, D-isoleucyl, D,L-leucyl, D-leucyl, D,L-histidyl, D-histidyl, D,L-valyl, or D-valyl, the -amino group therof 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, L-thiazolidine-4-carboxylic acid, or L-5-oxo-proline, the imino group thereof'being in imide linkage with the adjacent ,, ; and -C n is O or 1, such that when n is 0, R1 is methyl. All of the above compounds are inhibitors of ACE and are useful as orally effective anti-hypertensive agents. DESCRIPTION OF THE -PREFERRED EMBODIMENTS A more complete appreciation of the invention will be realized by reference to the following specific examples which describe the details of the synthesis and operational effectiveness of the foregoing compounds. The following examples are not intended to limit the invention disclosed herein except to the extent that limitations are specifically stated or to the extent to which limitations appear in the appended claims. 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 NaCl and 0.75 M Na2SO4. 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 of ± 3H]BenzoylClyHisLeu (25 Ci/mmole). Enzyme was diluted in the above buffer such that 40 l: buffered enzyme was capable of hydrolyzing 13% of substrate in a 15-minute incubation at 370C. To initiate the assay, 40 ij3 of enzyme and 10 l of buffer or inhibitor dissolved in buffer were preincubated for five minutes at 370C. Substr-ate, 50 l, was then added to initiate reaction and the solution was incubated for' 15 minutes at 37 C. 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 rot'ary mixer and centrifuged briefly to separate the phases. An aliquot, -500 pl-, of the' ethyl' 'acetate -layer was 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 aseries of different concentrations was compared to activity in the absence of inhibitor. A plot of inhibitor concentration 'versus percent inhibition yielded the I50 value. EXAMPLE 2 Preparation of N-[3-(benzoyl-l-phenylalanylthio)-2-D methyl-propanoyl3-L-proline a. Preparation of N-[3-(Boc-L-phenylalanylthio)-2-D-1, methyl-p'ropanoyll-L-proline A solution of 133 mg (o,.5 mmole) of Boc-L-Phe-OH in 0.5 ml of redistilled dimethylformamide (DMF) was cooled to -200C in an ice-dry ice-acetone bath. A cooled solution of 87 mg (0.54 mmole) of 1'-carbonyldiimidazole in 1 ml of DMF was added and the resulting solution was stirred at -10 C for 2 h. DMF, 1.0 ml, containing 119.5 mg (0.55 mmole) of 2-D-methyl3-mercaptopropanoyl-L-proline and 0.075 ml (0.55 mmole) of N-ethylmorpholine, all at -10 C, was added, and the final solution was stirred at -100C for -1 h. The reaction mixture was slowly warmed to room temperature. Solvent was removed with a rotary evaporator. Ethyl acetate (10 ml) was added, and the solution was cooled in an ice bath. The organic solution was washed twice with about 2 ml 'of 1 'N citric acid and then was washed twi'ce with a saturated NaCl 'sol'uttion. The methyl acetate phase was dried over anhydrous MgS04 and then filtered. The solvent of the filt'rate -was removed with a rotary evaporator. The residue was dissolved in a small volume of isopropanol/tetrahyd-rofuran (7:3 by vol) and was applied to a column (1;12 x 99 cm) of Sephadex LH-20 equilibrated with the same solvent. Each column eluent fraction contained 2.55 ml. Fractions 31-36 were pooled and solvent was removed by rotary evaporation. The residue was again dissolved in the isopropanoylXtetrahydrofuran solvent and was applied to a column (1.2 x 98 cm) of Sephadex G-10 equilibrated and developed with the same solvent. Fraction's, 2.55 ml each, were collected. Fractions 19-23 were pooled and yielded 165 mg of the desired product. Purification of side fractions from the LH-20 chromatography step yielded 40 mg of product. The product behaved as a pure substance on paper electrophoresis at pH 5.0 and on thin layer chromatography (silica gel plates) using three solvent systems. Amino acid analysis: Phe1.00, Pro1.01. b. Preparation of N0[-3-(HC1.H-L-phenylalanylthio)-2-Dmethyl-propanoyl]-L-proline. Anisole (0.2 ml) was added to 200 mg of N-F3-(Boc-L- phenylalanylthio)-2-D-methyl-propanoyl]-L-proline, and the mixture was stirred at OOC. Anhydrous trifluoroacetic acid (0.4 ml) was added, and the solution was stirred at room temperature (¯22 C) for 45 min. Trifluoroacetic acid was removed by rotary evaporation at 30 C and then 1 ml of ethyl acetate saturated with hydrogen chloride was added. Excess HCl and ethyl acetate were removed with a rotary evaporator to yield an oily residue. Anhydrous ethyl ether was added, and the mixture was allowed to stand at C for 1 h. A white solid was recovered by filtration and washed several times with anhydrous ether. The solid material was dried in a vacuum desiccator over NaOH and P205. c. Preparation of N-[3-(benzoyl-L-phenylalanylthio)-2-Dmethyl-propanoyl]-L-proline. A solution of 93 mg (0.23 mmole) of N-3-(HCi'H-L- phenylalanylthio)-2-D-methyl-propanoyl]-L-proline in 0.5 ml of dioxane (redistilled over NB) was mixed with 0.029 ml (0.25 mmole) of benzoylchloride in 0.099 ml (0.73 mmole) of N-ethyl morpholine. After 1 h of reaction at room temperature, an additional 0.029 ml of benzoylchloride and 0.034 ml of Nethyl morpholine were added. One h after the second addition, glacial acetic acid 0.2 ml) was added. Solvent was removed by rotary evaporation at 350C. Th'e -crude 'product was purified by 'partition- chromatography [1.2 x 97 cm column. of Sephadex C-25 equi-librated with N-butanoliacetic- acid$H2O (4:1:5)]. The sample was dissolved in a mixture-of 0.3 ml of lower -phase and 0.3 ml of upper phase. Elution was begun with upper phase. Fractions (2.75 ml' 'each)' were collected. Fractions 16'-i8 were pooled, and solvent was removed at 37 C with a rotary evaporator. The product was then applied to a column (1.2 x 98 cm) of silica gel equilibrated and developed with ethyl acetate. Fractions (5.15 ml each) were collected. Fractions 46-60 were pooled and yielded 52.5 mg of product. The product-behaved like a pure substance on thin layer chromatography using two solvent systems. EXAMPLE 3 Preparation of N-[3-(benzoyl-D-phenylalanylthio)-2-Dmethyl-propanoyl]-L-proline a. Preparation of N-[3-(HC1-H-D-phenylalanylthio)-2-Dmethyl-propanoyl]-L-proline A solution of 531 mg (2 mmoles) of Boc-D-phenylalanine in 3 ml of redistilled dimethylformamide (DMF) was cooled to -20 C and stirred vigorously. 1,1'-carbonyldiimidazole (341 mg; 2.1 mmole) in 3 ml of DMF (at -10 C) was added, and the resulting solution was stirred at -100C for 2 h. A solution of 2-D-methyl-3-mercaptopropanoyl-L-proline (456 mg; 2.1 mmole) in 2 ml of DMF and 0.285 ml of N-ethyl morpholine (2.1 mmole) was added, and the reaction mixture was stirred at -10 C for 1 h. The mixture was slowly warmed to room temperautre. Solvent was removed by rotary evaporation at 350C. The residue was dissolved in ethyl acetate (25 ml) and 3 ml of water was added. The mixture was cooled to 0 C and then acidified with 0.3 ml of concentrated HC1. The organic phase was washed once with cold dilute CH1, three times with water, and three times with saturated Nazi. The organic phase was dried over anhydrous MgSO4 and then -was filtered. The solvent was removed by rotary evaporation to yield an oily residue. The product was purified on a column (2.2 x 98 cm) of Sephadex LH-20 equilibrated and developed with isopropanol/ tetrahydrofuran (7.3 by vol). Fractions (5.6 ml each) were collected. Fractions 34-58 were pooled. Fractions, 33, 39, 40 and 41 were pooled, solvents removed and reapplied to the LH-20 column. Fractions 37-40 of the rechromatographed material and fractions 34-38 of the first chromatography were further purified on a column (1.2 x 98 cm) of silica gel equilibrated and dev-eloped with ethyl acetate. Fractions (5.0 ml each) were collected. Fractions 22-29 were pooled, and solvent was removed by rotary evaporation. The residue was dissolved in 1 ml of anhydrous trifluoroacetic acid containing 0.5 ml of anisole. Deprotection proceeded at room temperature for 0.5 h. Solvent was removed by rotary evaporation at 350C. The residue was dissolved in 1 ml of ethyl acetate saturated with hydrogen chloride. Anhydrous ether, 5 ml, was added at '00C, and a white precipitate formed. After 1 h at OOC, the white solid was collected by filtration and was washed five times with ether. The product was dried overnight in a vacuum desiccator over NaOH and P2 05. Yield: 381 mg. b. Preparation of N-[3-(benzoyl-D-phenylalanylthio)-2-D- methyl-propanoyl]-L-proline The product of stage a. above, N-[3-(HC1-H-D-phenyl- alanylthio)-2-D-methyl-propanoyl]-L-proline (93 mg; 0.23 mmole), was reacted with benzoylchloride (0.029 ml; 0.25 mmole) in 0.5 ml or redistilled anhydrous dioxane (0 C) and 0.099 ml (0.71 mmole) of N-ethyl morpholine. The reaction mixture was stirred vigorously at room temperature for 3 h. Benzoylchloride, 0.015 ml, and Methyl morpholine, 0.018 ml, were added and the mixture was stirred for 1 h. Solvent was removed by rotary evaporation at 3Q C. The residue was dissolved in a small amount of upper and lower phase of nbutanol/acetic acid/H20 (4:1:5) and was applied to a column (1.'2 x 98 cm) of Sephadex G-25 equilibrated for partition chromatography. The column was developed with upper phase as the moving phase (2.6 ml fractions). Fractions 15-24 were pooled and solvent removed by rotary evaporation. The residue was dissolved in a small amount of ethyl acetate and further purified on silica gel (1.2 x 95 cm column) equilibrated and developed with ethyl acetate (5 ml fractions). The fractions containing the major peak were pooled and solvent was removed. Th'e material was furthe'r purified by chromatography on Sephadex LH-20 (1.8 x 89 cm column) equi librated and developed with isoprop'anol9/tetrahydrofuran (7:3. by vol). Fractions (5.7 ml each) were 'collected, and the desired material was eluted in fractions 15-18 (yield 98 mg). The final product, behaved as a pure substance on paper electrophoresis at pH 5.0 and 2.0 and on thin layer chromatography. EXAMPLE 4 Preparation of N-[3-(benzoyl-D,L-phenylalanylthio)-2-Dmethyl-propanoyl]-L-proline -By using a ,SO:50 mixture of Boc-D-phenylalanine and Boc'--L-'pbenylala'ni'n'e in the 'pro'c"edu're'a of Example 2 or 3 above, N-[3-(benzoyl-D,L-phenylalanylthio)-2-D-methyl-propanoyl]-L proline is prepared. EXAMPLE 5 Preparation of N-(2-benzoylphenylalanylthiopropanoyl)-Lproline and N-(3-benzoylphenylalanylthiopropanoyl)-L-proline derivat-ives' The desired compound is prepared by substituting either 2-mercaptopropanoyl-L-proline or 3-mercaptopropanoyl-L-proline for 2-D-methyl-3-mercaptopropanoyl-L-proline in the procedures of Examples 2-4 above, and reacting the mercapto compound with either Boc-D-phenylalanine, Boc-L-phenylalanine, or a 50:50 mixture thereof. The mercapto compounds can be prepared using the procedures described in-any of the applications of which this application is a continuation-in-part, which are incorporated herein by reference. EXAMPtE' -'6' Preparation of L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline and L-thiazolidine-4-carboxlyic acid derivatives The desired compound is prepared by using the appropriate mercapto compound in which L-3,4-dehydroproline, D,L-3,4- dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine-4-carboxylic acid is substi'tuted for L-proline in the procedures of Example 2-5 above. The appropriate mercapto compounds can be prepared using the procedures described in Serial Nos. 64,897 to '6-4,903, incorporated herein by reference. EXAMPLE 7 Preparation of D-alanyl, D-tryptophyl, D-tyrosyl, D-isoleu-cyl, D-leucyl, D-h-istidyl and D-valyl derivatives The desired compound is prepared by substituting Boc-D alanine, Boc-D-tryptophan, Boc-D-tyrosine, Boc-D-isoleucine, Boc-D-leucine, Boe-D-histidine or Boc-D-valine for Boc-D phenylalanine in the procedures of Examples 2, 5 and 6 above. EXAMPLE 8 Preparati-on Of D,L-alanyl, D,L-tryptoph-yl, D,L-tyrosyl, D,L-isoleucyl, D,L-leucyl, D,L-histidyl and D,L-valyl derivatives A 50:50 mixture of Boc-D-A and Boc-L-A, wherein A is alanine, tryptophan, tyrosine, isoleucine, leucine, histidine or valine, is substituted for the mixture of Boc-D-phenyl alanine and Boc-L-phenylalanine in the procedures of Examples 4-6 above to obtain the desired compounds. EXAMPLE 9 By substituting the appropriate compound for benzoyl chloride in Examples 2, 3 and 5-8 above and following pro cedures well known in the art, such as those described in Serial Nos. 64,897 to 64,903, the formyl, acetyl, propanoyl butanoyl, phenylacetate, phenylpropanoyl, cyclopentanecarbonyl, cyclopentanecarbonyl-L-yyl, pyro-L-glutamyl-L lysyl, L-lysyl, L-a;rginyl and pyro-L-glutamyl derivatives are prepared. EXAMPLE 10 Preparation of N-[3-(benzoyl-L-phenylalanylthio)-2methyl-propanoyl]-L-5-oxo-proline L-glutamic acid, -10 mmoles is reacted with 11 'mmoles of the acid chloride of methacrylic acid in 35 ml of 1 N sodium hydroxide. After 60 min. at room temperature, the reaction is terminated by adding 2 N HC1 to a pH of¯2. The reaction product is extracted twice with an equal volume of ethyl acetate. The organic phase is reduced to a small volume on a rotary evaporator, and the product is crystalljzed by add ing ethyl ether. The soli'd product is collected by filtr ation. Six mmoles of the product are reacted with 6 mmoles of cyclohexylcarbodiimide in: 25 ml of anhydrous tetrahydro furan at 0 C for 1 h and then at 4 C overnight. The dicyclo bexylurea is removed by filtration. Th'e solvent of the fil trate is removed with a rotary' evaporator. The resulting anhydride (5 mmoles) is dissolved in 3 ml of anhydrous THF and 6 ml of anhydrous ethyl ether. To the latter'solution is -added dicyclohexylamine, 5 mmoles in 2 ml of ethyl ether, ta yield methacryloyl-L-5-oxo-proline. The salt is converted to the free acid by adding 2 N HC1 to pH 2.0. The product is extracted into ethyl acetate, The' or'g'anic phase is' evap-- orated to dryness and the product is crystallized from a mixture of hexane and ethyl acetate. The methacryloyl-L-5- oxo-proline, 3 mmoles-in 5 ml of toluene, is reacted with thiolacetic acid, 3 mmoles, by refluxing for 1 h to yield 3 acetylthio-2-methyl-propanoyl-L-5-oxo-proline. The product is crystallized in a mixture of ethyl acetate and hexane. The 3-acetylthio-2-methyl-propanoyl-L-5-oxo-proline is deprotected in liquid -NH3 in methanol in the presence of anisole. Solvent is removed with a rotary evaporator. The product is dissolved in ethyl acetate and the organic phase is washed with cold dil-ute HC1. The solvent of the organic phase is removed with a rotary evaporator. The residue is dissolved in dimethylformamide, 4 ml, 1-Hydroxybenzotriaz-ole, 2 mmoles, and the N-hydroxy-succinimide ester of benzoyl-L phenylalanine, 2 mmoles, are added. The reaction is allowed to proceed at room temperature for 48 h. The solvent is removed with a rotary evaporato-r. The residue is dissolved in a small volume of ethy'l acetate. Th'e organic phase is washed with cold dilute HCA 'and the'n -saturated N'aC1.' The organic phase is dried over MgS04. The (MgSO4 is removed by filtration and solvent of the filtrate is removed with a rotary evaporator. The residue is dissolved in 0.5 ml of THF. 'The- resulting solution is applied ta a column (2.5 x 100 cm) of LH'-20 equilibrated an-d developed with THF. The fractions containing the desired product (detectable by its absorption at 280 nm) are combined, and solvent is removed in a rotary evaporator under high vacuum. EXAMP-LE 11 The inhibitory potency of various of the above-synthesized 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 I50 value for various compounds. The I50 value is the concentration of inhibitor required to produce 50 Ó inhibition of the enzyme under a standard assay system containing substrate at a level substantially below Km Table 1 Compound. Example 2 1.6 x 10 8M Example 3 9 x 10-9M

Claims: CLAIMS 1. An angiotensin converting enzyme inhibitor having the formula EMI16.1 wherein R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentanecarbonyl, tert-butyloxycarbonyl, cyclopentanecarbonyl-Llysyl, pyro-L-glutamyl-L-lysyl, L-lysyl, L-arginyl or pyro L-glutamyl; A is L-phenylalanyl, D-phenylal-anyl, D,L-alanyl, D-alanyl, D,L-tryptophyl, D-tryptophyl, D,L-tyrosyl, D-tyrosyl, D,lisoleucyl, D-isoleucyl, D,l-leucyl, D-leucyl, D,L-histidyl, D-histidyl, D,L-valyl, or D-valyl, the 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, Lthiazolidine-4- carboxylic acid, or L-5-oxo-proline, the imino group thereof being in imide linkage with the adjacent 0 -C-; and, n is O or 1, such that when n is 0, R1 is methyl. 2. The inhibitor of claim 1 wherein R is benzoyl, A is D-phenylalanyl or L-phenylalanyl, -R1 is methyl, R2 is Lproline and n is 1. 3. The inhibitor of claim 1 wherein R is hydrogen, A is L-p,henylalanyl, R1 is methyl, R2 is L-proline and n is 1. 4. The inhibitor of claim 1 whe'rein R is hydrogen, A is D-p-henylalanyl, R1 is methyl, R2 is L-proline and n is 1. 5. An inhibitor as defined in any preceding claim for use in inhibiting angiotensin converting enzyme in vivo. 6. An inhibitor as defined in any of claims 1 to 4 for use in reducing blood pressure in vivo.

Inventors: CHUNG, ALFRED, RYAN, JAMES WALTER
Applicants: UNIVERSITY OF MIAMI
Application year: 1981
Main classification: C07C103
IPC codes: A61P9, A61P43, A61K38, A61K31, C12N9, C07K14, C07D207, C07D403, C07D277, C07K5