L-proline derivatives having anti-hypertensive properties

L-PROLINE DERIVATIVES HAVING ANTI-HYPERTENSIVE PROPERTIES

62203/2*

The present invention relates to anti-hypertensive compounds and constitutes a modification of the invention described and claimed in Israel Specification 58223.

Israel Patent No. 58223 discloses and claims inhibitors of antiotehsin converting enzyme having the formula

R - A - S - (CH,)״ - CH - C׳ - R, c 1 ח c ^R1 wherein:

R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentanecarbonyl, tert-butyloxycarbonyl, cycl opentanecarboyl-L-lysyl, or pyro-L-glutamyl ־,

A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-toleucyl, L-leucyl or L-valyl, the a-amino group thereof being in amide linkage with R when R is other than hydrogen, with the proviso that phenylalanyl is racemic when R is benzoyl ‘, is hydrogen or methyl;

R₂ is L-proline, L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline or L-4-hydroxyproline, the imino group thereof being

Q in imide linkage with the adjacent - C - group, and n is 0 or 1, such that when ח is 0, R₁ is methyl.

It has now been discovered that the optically pure D-isomer of phenylalanine, and the optically pure D-isomers and D,L-racemic mixtures of alanine, tryptophane, tyrosine, isoleucine, leucine and valine, can be used in place of L-phenylalanine, D,L-phenylalanine and the L-forms of alanine, tryptophane, tyrosine, isoleucine, leucine,and valine to produce useful antihypertensive agents.

BACKGROUND OF 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 converting the decapeptide antiotensin I, having the sequence

AspArgVaTTyrl1eHisProPheHisLeu to an octapeptide, angiotensin II by removal of the carboxy terminal 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 < Glu = pyro-L-glutamic acid (L-5-oxo-proline)

Gly = glycine

Hip = Hippuric acid (Benzoyl glycine)

His = L-hystidine j He = L-isoleucine

Leu = L-leucine

Phe = L-phenylalanine

Δ Pro = L-proline

Pro = L-3,4-dehydroproline

Ser = L-serine

Trp = L-tryptophan

Tyr- L-tyrosine

Vai = L-valine

ACE = Angiotensin converting enzyme

Hepes = N-2-hydroxyethylpiperazihe-N^l-ethanesulfonic acid a Angiotensin I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues and plasma, acting on a serum o(-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 ArgProProGlyPheSerProPheArg 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 ip 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 j both normal and hypertensive humans, of the amount needed ן 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 I inhibitors. [Gavras, I., et al., New Engl, J. Med. 291, 817 (1974)].

ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the Cvterminal. end of a variety of acylated tripeptides and larger polypeptides having an unblocked a-carboxyl group. The aption of ACE results in hydrplytic cleavage of the penultimate peptide bond from the carboxyl-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 by proline, 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., Biochemistry, Worth Publishers, Inc., New York, 1970, pp. 153-157. Many peptides which are :. called inhibitors of the enzymatic conversion of angiotensin i I to angiotensin II are in fact substrates having a lower Km I than angiotensin I. Such peptides are more properly termed competitive substrates. Examples of competitive substrates include bradykinin, and the peptide BPP5_a (also called SQ20475) from snake venom, whose sequence is <GluLysTrpAlaPro.

Numerous synthetic peptide derivatives have been shown I j to be ACE inhibitors by Ondetti, et al. in U.S. patent i 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 antihypertensive drugs. See for example U.S. patents !! 3,891,616, 3,947,575, 4,052,511 and 4,053,651. A highly jl !I effective inhibitor, with high biological activity when orally .,' administered, is D-3־mercapto-2־methylpropanoyl-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 i (1977), and by Ondetti, M. et al., Science 196, 441 (1977).

The inhibitor SQ14225 reportedly has an I50 value of

2.3 x 10θΜ. The I5Q value reported by Cushman, et al.,

I I ίί 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

It will be understood that I50 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 I5Q data reported herein have been performed with the same assay system and . same enzyme (human urinary ACE) and with־an approximately 1/2 Kjh 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 I50 values for BPPg_a reported by Cushman, D.W., et al., Experientia 29, 1032 (1973) and by Dorer, F.E., et al., Biochim.Biophys.Acta 429, 220 (1976). i — —

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 jj better known related enzyme, carboxypeptidase A. The active 1׳ site was postulated to have a cationic site for binding jj the carboxyl enS group of the substrate and a pocket or jj cleft capable of binding the side chain of the C-terminal amino jj acid and providing especially tight binding for the heterocyclic • 1 ring of a terminal proline residue. A similar pocket for ΐ the penultimate amino acid residue was postulated, and the published data suggested a rather stringent steric requirement, since the D־form of the inhibitor was substantially more potent than its stereoisomer or the 3-methyl and unsubstituted analogs. The sulfhydryl group on 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 for 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.

'i For example, it has been observed that a fresh aqueous solution of concentration, e.g., 1 mg per ml of SQ 14,225 at a pH of about 8 becomes substantially less active 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 result of dimerization of SQ 14,225 occurring at ן 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 in vitro

I -loss of activity of aqueous solutions of SQ 14,225 on standing is in some part a consequence of one or more such oxidation

I reactions, with formation of a sulfone or sulfoxide which ί ! does not function effectively as an inhibitor 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 intestinal environments of most patients to be an effective inhibitor for ACE when administered orally. It is not yet clear, however, whether there may be a group of patients for which SQ 14,225 is substantially ineffective. Because of the high reactivity of the free sulfhydryl group, SQ 14,225 could readily form mixed disulfides with serum, cellular proteins, peptides or other free sulfhydryl group-containing substances in the gastric or intestinal environments, in addition to the possibility for dimer formation or oxidative degradation reactions. A nixed disulfide with protein may be antigenic and, indeed, occasional allergic reactions have been clinically observed. See Gavras, et al., New England J. Med. 298, 991 (1978). Disulfides and oxidative degradation products of SQ 14,225, if formed, may at best be expected to be largely ineffective as inhibitors. It may be postulated accordingly that dose response to SQ 14,255 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).

Thioester compounds having potent ACE inhibitory activity.and oral effectiveness as anti-hypertensive agents have been disclosed in Israel Specification 58223 and in tha oorroapoftding U»S. applioationa 06Ί/807 through 964,903 the 15 teachings of which specification^ axe^incorporated herein by reference.

Compounds related to SQ 14,225 have been disclosed by Ondetti, et al., U.S. patents 4,046,889, 4,052,511, 4,053,651, 4,113,715 and 4,154,840. Of interest are disclosed analogs of SQ 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 SQ 14,225 are not disclosed. Substitution of D-proline for L-proline is reported to drastically reduce inhibitory potency of ' I '

ו-

3-mercaptopropanoyl amino acids (Cushman, D.W., et al. , [ supra). I

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 j which has significantly reduced physiological activity. See ׳ Fisher, G.H. et al., Arch.Biochem.Biophys. 189, 81 (1978). On the other hand, substitution of L-3,4- ZSPro at the 3, 5, 8 or 9 position in ACE inhibitor BPPg_a enhances its inhibitory activity. See Fisher, G.H. et al., FEBS Letters 107, 273 j — μ (1979). In copending application Serial No. 958,180, appli!1 .

!, cants found that the compounds having APro, which are j: disclosed in said application, have high inhibitory potency !1 and antihypertensive effectiveness. However, at present, no .’. rationale can be advanced to explain the diversity of observed |' results following substitution of ΔΡγο 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 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 anti-hypertensive agepts and have high inhibitory potency for ACE.

SUMMARY OF THE INVENTION (which served as priority documents for Israel specification 58223) It was believed when Serial Nos. 941,289 and 958,180/ .

i were filed that N-bepzoyl-L-phenylalanine was prepared by the methods disclosed in Examples 4 and 2, respectively, of these applications. Subsequently, it was appreciated from reading Fischer & Mooneyrat, Ber, 33, 2383 (1900) that what had actually been prepared by the method described in these examples was the racemized D,L-form of the compound rather than the L- form. It has now been determined that all three optical isomers of the compound N-benzoyl-D,L-phenylalanine, N-benzoyl-L-phenylalanine, and N-benzoyl-D-phenylalanine can be used to prepare compounds which are inhibitors of angiotensin converting enzyme.

It was likewise believed when Serial Nos. 64,897 through (which also served as priority documents for Israel specification 5822$ 64,903, inclusive/, were filed that all of tne amino acids disclosed therein had to be in the L- form in order for the compounds that were prepared to be inhibitors of ACE. It has since been determined that the D- and D,L- forms of these amino acids can also be used to prepare compounds which are ACE inhibitors.

Accordingly, the present invention relates to novel inhibitors of ACE which have the general formula

O

II

R - A - S - (CH₂)_n - CH - C - R₂R!

/2 where!η

R is hydrogen, formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentanecarbonyl, tert-butyloxycarbonyl, cyclopentanecarboyl-L-lysyl, pyro-L-glutamyl-L-lysyl, L-lysyl, Larginyl or pyro-L-glutamyl;

A is D-phenylalanyl, D,L-alanyl, D-alanyl, D,L-tryptophyl, , D-tryptophyl, D,L-tyrosyl, D-tyrosyl, D,L-isoleucyl, D-isoleucyl, D,L-leucyl, D-leucyl, D,L-valyl, or D-valyl, the a-amino group thereof being in amide linkage with R ,when R is other than hydrogen, L-5-0X0-' . J ־,' it being understood that when R is benzoyl and R? is/L-proline-or~dehydroproliaeA may also be L-phenylalanyl;

R! is hydrogen or methyl;

R₂ is L-proli nd, L-3,4-dehydropropli ne, D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline or L-5-oxo-proline, the imino group thereof being in imide linkage with the adjacent - c - ., and, n is 0 or !, such that when n is O, R.j 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 effectictiveness 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 NaCI and 0.75 M NajSO. The substrate

62203/2 employed was Benzoyl-GlyHisLeu at a final concentration of 1 x 1O‘\ x 10 ⁴M), together with about 130,000 cpm of 3

[ H]Benzoy1G1yHisLeu (25 Ci/mmole). Enzyme was diluted in the above buffer such that 40 μΐ buffered enzyme was capable of hydrolyzing 13% of substrate in a 15-minute incubation at 37°C. To initiate the assay, 40 μΐ of enzyme and 10 μΐ of buffer or inhibitor dissolved in buffer were preincubated for five minutes at 37°C. Substrate, 50 μΐ, was then added to initiate reaction and the solution was incubated for 1.5 minutes at 37°C. To terminate the reaction, 1 ml of 0.1 M HCl 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 μΐ, 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 Ι^θ 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 I_c_ value, □u

- וו EXAMPLE k 2

Preparation of N-[3-(benzoyl-D-phenylalanylthi0)-2-Dmethyl-propanoyl-L-proline .-- ;

a. Preparation of N-[3-(HC1H-D-phenylalanylthio)-2-D-methyl- !

propanoyl-L-pro line

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 wqs stirred at -10° 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 temperature. Solvent was removed by rotary evaporation at 35°C. 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 HC1, three times with water, and three times with saturated KaCl. The organic phase was dried over anhydrous MgSO^ 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 34-38 were pooled. Fractions 33, 39, 40 and 41 were pooled, solvents removed and reapplied to the LH-20 column. Fractions 37-40 _rof the rechromtographed material and fractions 34-38 of the . I first chromatography were further purified on a column (1.2 x 98 cm) of silica gel equilibrated and developed with f ethyl acetate. Fractions (5.0ml each) were collected.

־-־14־

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 35°C. The residue was dissolved in 1 ml of ethyl acetate saturated with hydrogen chloride. Anhydrous ether, 5 ml, was added at 0°C, and a white precipitate formed. After 1 h at 0“0, 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 P₂O₅. 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-(HCl׳H-D-phenylalanylthio)-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 of 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 N-ethyl morpholine, 0.018 ml, were added and the mixture was stirred for 1 h. Solvent was removed by rotary evaporation at 30°C. The residue was dissolved in a small amount of upper and lower phase of n־butanol/acetic acid/H^O (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. The material was further purified by chromatography on Sephadex LH-20 (1.8 x

-Ϊ562203/2 cm column) equilibrated and developed with isopropanol/ 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

Preparati on of N-(2-benzo.yl phenyl al any! thi opropano.yl )-L-prOl i ne and N-(3-benzoylphenylal any!thi opropanoyl )-L-proline derivati ves

The desired compound is prepared by substituting either 2mercaptopropanoyl-L-proline or 3-mercaptopropanoyl-L-proline for 2-D-methyl-3-mercaptopropanoyl-L-proline in the procedure of Example 2 above, and reacting the mercapto compound with Boc-Dphenylalanine.

The mercapto compounds can be prepared using the procedures described in Israel Patent Specification No. 58 223.

EXAMPLE

Preparati on of L-3,4-dehydroprOli ne, D,L-3,4-dehydroprOline, L-3-hydroxyproli ne and L-4-hydroxyproline derivati ves

The desired compound is prepared by using the appropriate mercapto compound in which L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3hydroxyproline or L-4-hydroxyproline is substituted for L-proline in the procedures of Examples 2 and 3.

The appropriate mercapto compounds can be prepared using the procedures described in Israel Specification No. 58223. :

62203/2

EXAMPLE 5 .

Preparation of D-alanyl, D-tryptophyl, D-tyrosyl j D-isoleucyl, D-leiicyl, D-histidyl and D-valyl derivatives

The desired compound is prepared by substituting.Boc-D-alanine, Boc-D-tryptophan, Boc-D-tyrosine, Boc-D-.isoleucine, Boc-O-leucine, BocD-histidine or Boc-D-valine for Boc-D*phenylalanine in the procedures of Examples 3 and.4 above.

EXAMPLE

Preparation of D,L-alanyl , ^,L-tryptophyl, D,L-tyrosyl, D,Lisoleucyl, 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-phenylalanine and Boc-L-phenylalanine in the procedures of Examples 3 and 4 above to obtain the desired compounds.

EXAMPLE 7

By substituting the appropriate compound for benzoyl chloride in Examples 26־ above and following procedures will known in the art, such as those described in Israel Specification No. 58223, the formyl, acetyl, propanoyl, butanoyl, phenyl acetate, phenylpropanoyl, cyclopentanecarbonyl, cyclopentanecarbonyl-L-lysyl, pyro-L-glutamyl-L-lysyl, L-lysyl,,/!-arginyl and pyro-L-glutamyl derivatives are prepared.

EXAMPLE

Preparation of N-[3-(benzoyl-D-phenylal any!thi0)-2-methyl-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 crystallized by adding ethyl ether. The solid product is collected by filtration. Six mmoles of the product are reacted with 6 mmoles of cyclohexylcarbodiimide (in 25 ml of anhydrous tetrahydrofuran at 0°C for 1 h and then at 4°C overnight. The di cyclohexyl urea is removed by filtration. The solvent of the filtrate 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 di cyclohexyl amine, 5 mmoles in 2 ml of ethyl ether, to yield methacryloyl-L5־-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 organic phase is evaporated 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-acetylthio2-methyl-propanoyl-L-5-oxo-proline. The product is crystallized in a mixture of ethyl acetate and hexane. The 3-aCetylthio-2-methyl-propan31yl-L-5-oxoproline is deprotected in liquid NH^ 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 dilute HC1. The solvent of the organic phase is removed with a rotary evaporator. The residue is dissolved in dimethyl formamide, 4-ml, 1-Hydroxybenzotri azole, 2 mmoles, and the N-hydroxy-succinimide ester of benzoyl-D-phenylalanine, 2 mmoles are added. The reaction is allowed to proceed at room temperature for 48 h. The solvent is removed with a rotary evaporator. The residue is dissolved in a small volume of ethyl acetate. The organic phase is washed with cold dilute HC1 and then saturated NaCl. The organic phase is dried over MgSO. The MgSO. is removed by filtration and solvent of the filtrate *T Τ’ is removed with a rotary evaporator. The residue is dissolved in 0.5 ml of

62203/2 of THF. The resulting solution is applied to a column (2.5 x 100 cm) of LH-20 equilibrated and 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.

EXAMPLE 9

The inhibitory potency of the above synthesized compound of Example 2 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.¼., et al., Tissue and Cell 10, 555 (1978). The L.״ value obtained --tjy was 9 x 10 M. The Ι₅θ value is the concentration of inhibitor required to produce 50% inhibition of the enzyme under a standard assay system containing substrate at a level substantially below K_ffl.

62203/2

EXAMPLE ΙΟ

<td> 28.5.84'</td><td> ' ' Compounds according to the present invention were prepared and analyzed as set forth in Table I hereinafter.</td>

The thin layer chromatography solvent systems employed, using silica gel plates, when determining the Rf numbers listed in Table I were as follows:

System 1 [Rf(1)] = trichloromethane:methanol:acetic acid, 2:1:0.003 parts by volume.

System 2 [Rf(2)] = butanol:acetic acid:water, 4:1:1 parts by volume.

The NMR spectra obtained were run at 60 MHz.

Table I

<td> Compound No.</td><td> Compound</td><td> Property</td>

<td> 1-1</td><td> N*׳- [ XN*׳-benzoyl-D-phenylalanylthio)-2-D-methylpropanoyl]-L-proline</td><td> Mn = 25.1־ Rf(I) = 0.56 R_f(2j 0.74 ־ NMR a (in CDCI3+CD3OD) : 1.19 (3H, b.d., CH3), 1.5- 2.4 (4H, m, CH2), 2.5- 3.3 (5H, m, CH and CH2), 3.3-3.8 (2H, m, CH2). 4.0-4.4 (1H, m, CH), 4.8-5.2 (1H, m, CH), 7.23 (5H, s, aromatic H), 7.2-7.9 (5H, m, aromatic H).</td>

<td> 1-2</td><td> N¹*-[ XN^-benzoyl-D-alanylthio)-2-D-methylpropanoyl]L-proline</td><td> [«] £ = -10 3.0 Rf(l) = 0.58 Rf(2) 0.75 ־־ NMR ύ (in CDCI3): 1.19 (3H, b.d., CH3), 1.48 (3H, d, CH3), 1.7-2.4 (4H, m, CH2), 2.5-3.2 (3H, m, CH and CH2)&gt; 3.25-3.8 Ϊ2Η, m, CH2), 4.2-4.7 (1H, m, CH?, 4.89 (1H, q, CH), 7.06 (1H, d, NH), 7.3-8.0 (5H, m, aromatic H), 10.21 (1H, s, COOH).</td>

<td>³־¹</td><td> N3 ] -'״- (N*-benzoyl-D-leucylthio)-2-D-methylpropanoyl]L-proline</td><td> =-59.0 Rf(l) = 0.58 NMR £ (in CD3OD): 0.99</td>

/2

28.5.84

N*'- [3- (N^-acetyl-D-phenylalanylthio)-2-D-methylpropanoyl]-L-proline

N**'- [ 3- (N^׳-propanoyl-Dtryptophylthio)-2-D-methylpropanoyl]-L-proline (6H, b.d., CH₃), 1.22 (3H, b.d., CH₃), 1.52.4 (7H, m, CH and CH₂), 2.5-3.IT (3H, m, CH and CH₂), 3.4-3.9 (ΣΗ, m, CH2), 4.1-4.45 (1H, m, CH), 4.6-5.0 (1H, m, CH), 7.45-8.1 (5H, m, aromatic H).

W_D 51.9־ ־־

Rf(l) = 0.54

Rf(2) = 0.54

NMR J (in CDC1₃): 1.17 (3H, b.d., CH3), 1.752.35 (4H, m, CH₂), 1.91 (3H, s, CH3), Σ.553.25 (5H, m, CH and CH₂), 3.4-3.8 T2H, m, CH2), 4.1-4.6 (1H, m, CH), 4.6-5.0 (1H, m, CH), 6.63 (1H, d, NH), 6.82 (1H, s, C&amp;OH), 7.17 (5H, b.s., aromatic H).

[&lt;x]n = 28.6־

Rf(l) = 0.52

Rf(2) = 0.75

NMR / (in CD3OD): 1.02 (3H, t, CH₃), 1.08 (3H, b.d., CH₃y, 1.8-2.5 (4H, m, CH₂), 2.20 (2H, q, CH₂'), 2.5-3.1 (3H, m, CH and CH₂),

3.1- 3.9 (iH, m, CH₂),

4.1- 4.5 (1H, m, CH), 4.7-5.0 (1H, m, CH), 6.95-7.8 (5H, m, ~ aromatic H).

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28.5.84

<td colspan="3"></td>

<td> 1-6</td><td> NA3]-׳-(N^-benzoyl-Dtryptophylthio)-2-D-methylpropanoyl]-L-proline</td><td> Md = 33.1־ Rf(l) = 0.56 Rf(2) = 0.73 NMR Γ (in CD3OD): 1.10 (3H, b.d., CH3), 1.65-2.3 (4H, m, CH₂), 2.6-3.1 (3H, tn, CH and CH2), 3.2-3.7 (4H, m, CH2), 4.054.35 (1H, m, CH), 4.8-5.1 (1H, m, CH), 6.88-7.82 (10H, m, aromatic H).</td>

<td> 1-7</td><td> N*- [3- (N«׳-cyclopentanecarbonyl-D-alanylthio)-2D-methylpropanoyl-L-proline</td><td> MD = -47.2 Rf(l) = 0.62 Rf(2) 0.74 ־ NMR ό (in CDCI3+CD3OD) 1.20 (3H, b.d., CH₃), 1.38 (3H, d, CH3), 1.5-2.4 (12H, m, CH2), 2.4-3.2 (4H, m, CH and CH₂), 3.3-3.8 (2H, m, CH₂), 4.2-4.8 (2H, m, CH).</td>

<td> 1-8</td><td> N⁰3 ] -׳׳- (N*-tertbutyloxycarbonyl-D-alanylthio)-2D-methylpropanoyl]-L-proline</td><td> Md = -62.5 R_f(l) - 0.59 Rf(2) 0.73 ־־</td>

<td> 1-9</td><td> NA3 ] -׳- (NA'-acetyl-D- leucylthio)-2-D-methylpropanoyl]L-proline</td><td> Md = -29.5 Rf(l) = 0.55 Rf(2) = 0.59</td>

<td> 1-10</td><td> N&lt; [3-(NA׳-acetyl-D-alanylthio)-2-D-methylpropanoyl]L-proline</td><td> Mp » -72.3</td>

<td> 1-11</td><td> NA3] -׳- (NA׳-acetyl-D-valylthio)-2-D-methylpropanoyl]L-proline</td><td> Md - 73.8־</td>

Ν*Ή 3-(N^-propanoyl-D- Mp ⁼ 62.0־ alanylthio)-2-D-methylpropanoyl]-L-proline

N&lt;* [3-(N*butanoyl-D-alanylthio)-2-D-methylpropanoyl]L-proline

Mp = 57.9