IE850640L - Renin inhibitors containing statine - Google Patents

Abstract

Polypeptides of formula (I) and their salts are new. R-W-W'-NH-CHR2-CHR3-CH2-COR1 (I) W = Phe, His, Leu, Tyr, 1-naphthyl-Ala or 2-phenylmethyl 2-phenylmethylpropionylene; W' = Phe, His, Leu, Tyr or Nle; when W = Phe and W' = His, then the peptide link between W and W' may be replaced by -CH(1-4C)Alkyl-NH-; R = H, amino-protecting acyl of molecular wt. up to 500, or Pro or pGlu both amino protected; R2 = H, 1-6C alkyl, Ph, 4-7C cycloalkyl, Ph-(1-3C)alkyl or 5-10C cycloalkylalkyl; R3 = OH, NH2, NHR9, NHCOR9, OR9 or OCOR9; R9 = 1-4C alkyl; R1 = A-E-B; a gp. of formula (II); NH2; 1-4C alkoxy; 5-benzylipiperazin-1-yl; 1,2,3,4-tetrahydroquinolin-1-yl or -2-yl; 1,2,3,4-tetrahydro-3-aminocarbonyl isoquinolin-2-yl or the corresp. 3-methoxycarbonyl gp.; 1,2,3,4,5,6,7,8-decahydro-3-methoxycarbonyl isoquinolin-2-yl; 2-methoxycarbonylpyrrolidin -1-yl; 2-aminocarbonylpyrrolindin-1-yl; 4-phenylmethylpiperidin-1-yl; Pro-B; or Lys-B; -X-NH-CHZ-(CH2)n-Q-CHZ1-(CH2)m-Y (II) A = Lys or Pro or when R3 = NH2, Ile; E = Phe, Gly, Ala, Val, Leu, Ile, Lys, Orn, Arg, Asp opt. gamma-esterified or Glu opt. delta-esterified; B = OR4, NR4R5, glutamic acid (-OR4)2, glutamic acid (-OR4)(-NR4R5) or glutamic acid (-NR4R5)2; R4, R5 = H, 1-4C alkyl, Ph-(1-3C)alkyl or 5-10C cycloalkylalkyl; X = absent or Ala, Ile, Lys, Pro, Orn, Arg, N-(1-4C alkyl)- or N,N-(di-(1-4C) alkyl)-Lys or -Orn; Z, Z1 = H, 1-6C alkyl, 4-7C cycloalkyl, 5-10C cycloalkylalkyl or Ph-(1-3C)alkyl; n, m = 0-6; Q = CH2, CH=CH, O, NH, CH(OH) or CO; Y = Me, Ph, COOR6, CONR6R7, NH2, benzyloxycarbonylamino, CO-glutamic acid (-OR6)2, CO-glutamic acid (OR6)(NR6R7) or CO-glutamic acid (-NR6R7); R6, R7 = H, 1-4C alkyl, Ph-(1-3C)alkyl or 5-10C cycloalkylalkyl.

Description

2 The proteolytic enzyme renin, which has a molecular i weight of about 40,000, is produced in and secreted into the blood by the kidney. It is known to be active in vivo in cleaving the naturally-occurring plasma glyco-5 protein angiotensinogen, in the case of human angiotensinogen at the bond between the leucine (10th) and valine (11th) amino acid residues at the N-terminal end of the angiotensinogen: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-Glu-10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 The circulating N-terminal decapeptide (angiotensin I) formed by the above cleaving action of renin is subsequently broken down by the body to an octapeptide known as angiotensin II. Angiotensin II is known to be 15 a potent pressor substance, i.e. a substance that is capable of inducing a significant increase in blood pressure, and is believed to act by causing the constriction of blood vessels and the release of the sodium-retaining hormone aldosterone from the adrenal 20 gland. Thus, the renin-angiotensinogen system has been implicated as a causative factor in certain forms of hypertension.

One means of alleviating the adverse effects of the functioning of the renin-angiotensinogen system is the 2 5 administration of a substance capable of inhibiting the • angiotensinogen-cleaving action of renin. A number of such substances are known, including antirenin antibodies, pepstatin and naturally-occurring phospholipid compounds. Patent Specification No. 51471 discloses a series of 3 renin-inhibiting polypeptide derivatives of the formula X-Y-Pro-Phe-His-A-B-Z-W in which X may be hydrogen or an amino-protecting group, Y may be absent, B is a lipophilic amino acid 5 residue, Z is an aromatic amino acid residue, W may be hydroxy1 and A may be, inter alia, 1 2 R R 0 -NH-CH-CH-CH--CH-C- i * OH 1 2 with each of R and R being a lipophilic or aromatic side chain. According to the definitions set forth in 10 this patent Specification, it is not contemplated that either A or Z could be statine or that 6 could be lysine.

Patent Specification No,. :<■ U'- discloses a series cf renin-inhibiting polypeptide compounds having a non-terminal statine 15 or statine derivative residue. Included within this series are compounds having a phenylalanine-histidine-statine sequence. However, this published patent application does not disclose the placement of lysine immediately after the -Phe-His-Sta- sequence.

We have now discovered that certain novel compounds are useful as renin-inhibiting agents. This series of novel compounds consists of polypeptides and polypeptide derivatives of the formula R2 R-W-t^-NH-CH-CH-CH^-COR1 I •3 R and the pharmaceutically acceptable salts thereof, wherein 4 W is phenylalanine, histidine, leucine, tyrosine, 1-naphthylalanine or 2-phenylmethyl-propionylene; W* is phenylalanine, histidine, leucine, tyrosine or norleucine, with the peptide link between W and W* 5 optionally being replaced by -CH(alkyl of from 1 to 4 carbon atoms)-NH- when W is phenylalanine and'W* is histidine; R is hydrogen, an amino-protecting acyl moiety having a molecular weight of less than 500, proline, 10 amino-protected proline, pyroglutamic acid or amino-protected pyroglutamic acid; 2 R is hydrogen, alkyl of from 1 to 6 carbon atoms, phenyl, cycloalkyl of from 4 to 7 carbon atoms, phenylalkyl of from 7 to 9 carbon atoms or cyclo-15 alkyl (alkylene) of from 5 to 10 carbon atoms; R3 is hydroxy1, amino, -NHR9, -NHCOR9, 9 9 9 -OR or -OCOR , with R being alkyl of from 1 to 4 carbon atoms; and R^ is -A-E-B or -A-B, with A being lysine or proline, E being phenylalanine, glycine, alanine, valine, leucine, isoleucine, lysine, ornithine, arginine, aspartic acid, gamma-esterified aspartic acid, glutamic acid or 4 delta-esterifled glutamic acid, B being -OR , 4 5 4 -MR R , -glutamic acid(-OR )^t -glutamic acid(-OR4)(-NR4R^) or -glutamic acid(-NR4R^),, 4 5 and R and R each being hydrogen, alkyl of from 1 to 4 carbon atoms, phenylalkyl of from 7 to 9 carbon atoms or cycloalkyl(alkyl) of 30 from 5 to 10 carbon atoms.

Of particular interest are those compounds wherein R is an amino-protecting acyl moiety or amino-protected proline, W is phenylalanine, W^" is histidine bonded to W p in a peptide link, R is isobutyl or cyclohexyKmethylene), 5 R^ is hydroxy and A is lysine.

The compounds of this invention exhibit antihypertensive activity ija vivo in mammals, including humans. At least a substantial portion of this activity results from their ability to inhibit the cleavage of angioten-10 sinogen'by renin- Although we do not wish to be limited by the following theory of mechanism, it is likely that the mechanism of the renin-inhibiting activity of the compounds of the invention is their selective binding (as compared to angiotensinogen) to renin. Because 15 of their low molecular weights the compounds of the invention exhibit favorable solubility characteristics in aqueous media, thus making oral administration feasible, and can be synthesized at a commercially realistic cost. The compounds of the present invention 20 are also useful as diuretics.

By "pharmaceutically acceptable" salts is meant those salts which are non-toxic at the dosages administered. Since compounds of the invention may contain both basic and acidic groups, both acid addition and 25 base addition salts are possible. Pharmaceutically 6 acceptable acid addition salts include e.g. the hydrochloride, hydrobromide, hydriodide, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, maleate, mesylate, fumarate, citrate, acid citrate, 5 tartrate, bitartrate, succinate, gluconate and saccharate salts. Pharmaceutically acceptable base addition salts include e.g. the sodium, potassium, calcium and magnesium salts. Conventional methods of forming acid addition and base addition salts may 10 be employed.

The group R at the N-terminal end cf formula I, which is bonded to the alpha-nitroqen of the residue W, is selected from the group consisting cf hydrogen, amino-protecting ecyl moieties having a molecular weight, 15 c-f less than 500, proline, ami no-protected proline, pyroglutamic acid and amino-protected pyroglutamic acid. It is to be understood that the amino-protecting group of amino-protected proline or amino-protected pyroglutamic acid is also an amino-protecting acyl moiety having a 20 molecular weight of less than 500. The term "amino-protecting acyl moieties" refers to those acyl groups that are capable of substantially inhibiting reaction at the alpha-nitrogen of W (or proline or pyroglutamic acid) _in vivo after oral administration. R has a 25 molecular weight of less than 500 in order to prevent an excessive detrimental effect on solubility characteristics. Examples of suitable amino-protecting acyl moieties are well known to those skilled in the art, e.g. the t-butyloxycarbonyl, t-butylacetyl, benzyl-30 oxycarbonyl, t-butyluriedo, (tris-hydroxy)-(t-butyluriedo) and phenoxyacetyl moieties. Preferably, R is of the formula 0 0 n m R8-M-C- or R8-M-C-Pro-, 7 with M being -0-, -CH_-, -NH- or -SO-NH- 8 * and R being alkyl of from 1 to 6 carbon atoms, phenyl, phenylalkyl of from 7 to 9 carbon atoms or cycloalkyl- (alkylene) of from 5 to 10 carbon atoms.

As used herein, the expressions -glutamic acid- (—OR4)(—NR4R5) and -CO-glutamic acid(-OR6)(-NR6R7) refer both to C-terminal groups that are amidified at the delta-carbon of glutamic acid and C-terminal groups that are esterified at the delta-carbon of glutamic acid.

As stated earlier, one preferred group of compounds of the invention consists of those wherein R? is hydroxyl 2 and R is isobutyl or cyclohexyl(methylene), more preferably 2 the latter, so that R -NH-CH-CH-CH--CO- P becomes -statine- or -cyclostatine-, R is an amino-protecting acyl moiety having a molecular weight of less than 500 or amino-protected proline, W is 2o phenylalanine, W* is histidine bonded to W in a peptide link and R^" is -Lys-E-B or -Pro-E-B.

Placement of a lysine residue (instead of, for example, a leucine, isoleucine, valine or alanine residue) immediately after -phenylalanine-histidine-(statine 25 or cyclostatine)- surprisingly results in a dramatic increase in the duration of in vivo renin-inhibiting activity. 8 Particularly valuable are the following compounds and their pharmaceutically acceptable salts: [N-(t-butyloxycarbonyl)-proline]-phenylalanine-histidine-cyclostatine-lysine-phenylalanine; 5 [N-(t-butyloxycarbonyl)-phenylalanine]-histidine- cyclostatine-lysine-phenylalanine; IN-(t-butyloxycarbonyl)-phenylalanine]-histidine-statine-lysine-phenylalanine; [N-(t-butyloxycarbonyl)-phenylalanine]-histidine- cyclostatine-proline methyl ester; [N-(t-butyloxycarbony1)-phenylalanine]-histidine-cyclostatine-lysine-statine.

The compounds of the invention may be prepared by methods familiar to those skilled in the art. The 15 basic sub-unit of the preferred chemical synthesis is the acylation of the unprotected alpha-amino group of an amino acid residue with an amino acid having an activated (for acylation purposes) carboxylic function and a suitable protecting group bonded to its own alpha-20 nitrogen to form a peptide bond between the two amino acid residues, followed by the removal of said protecting group. This synthesis sub-unit of coupling-deblocking is performed repeatedly to build up the polypeptide, starting from the C-terminal end of 25 the molecular structure and working to the N-terminal end. The alpha-amino acids utilized to synthesize the compounds of the present invention are commercially available (as free acids, salts or esters, etc.) in both alpha-amino protected and alpha-amino unprotected 9 forms. Statine is commercially available as N-(t-butyloxycarbony1)-statine; additionally, statine may be prepared (as a free acid or ester) in both the gamma-amino protected and gamma-amino unprotected forms by 5 methods set forth in the literature (see e.g. U.S.

Patent 4,397,786 and Rich, D.H. et al., Jour. Org. Chem., 43, pp. 3624 et seq. (1978). When desired, an appropriate n-unprotected amino acid analogue (free acid, salt or ester, etc.) such as 4-aminobutyric acid, 4-amino-10 valeric acid or 4-amino-4-sec butyl-butyric acid is used as a reactant in the first coupling step. Appropriate derivatives of statine may be prepared by conventional synthetic methods. Thus, for example, the compound isobutyl h2n-ch-ch-ch2-cooh nh2 which is referred to herein as aminostatine, may be prepared in a form with both amino groups protected by reacting an amino-protected statine ester with a sulfonyl chloride to form a sulfonate ester, reacting the sulfonate ester with sodium azide to form 20 an amino-protected 4-isobutyl-2-butenoic acid ester and an amino-protected 4-isobutyl-3-azido-butyric acid ester, reacting the alkenoic compound with a primary amine and/or hydrogenating the azido compound and then reducing the resulting intermediate compound 25 with an alkali metal hydride in the presence of an aldehyde to form (in both cases) a 4-isobutyl-3-sec amino derivative of butyric acid, hydrogenating this 4-isobutyl-3-sec amino derivative of butyric acid and then reacting the resulting compound with an acyl 1 0 halide under basic conditions. N-protected compounds Cyclohexyl ch2 Protecting group-NH-CH-CH-CH_-COOH , I ^ oh which are referred to herein as N-protected cyclostatine 5 compounds, may be prepared by hydrogenating the corresponding N-protected-4-amino-4-phenylmethy1-3-hydroxy-butyric acid compound, which in turn may be prepared in the manner set forth by Rich, D.H. et al., Jour. Med. Chem., 23(1) , pp. 27-33 ( 1980).

The activity of the compounds of the present invention as inhibitors of the angiotensinogen-cleaving activity of renin may be determined by studying (1) their ability to inhibit the angiotensinogen-cleaving activity of; renin in vitro and (2) their 15 ability to antagonize the exogenous renin-induced pressor response in vivo.

The compounds of the present invention can be administered as antihypertensive agents by either the oral or parental routes of administration, with the former being preferred for reasons of patient convenience and comfort. In general, these antihypertensive compounds are normally administered orally in dosages ranging from about 0.1 mg. to about 10 mg. per kg. of body weight per day; variations will necessarily occur depending upon the condition of the subject being treated and the particular compound being administered. Typically, treatment is commenced at a low daily dosage and increased by the physician only if necessary. It is to be noted that these compounds may be administered in combination with pharmaceutically acceptable carriers by either of the routes previously indicated, and that such administration can be carried out in both single and multiple dosages.

The novel compounds of the invention can be orally administered in a wide variety of different dosage forms, i.e., they may be formulated with various pharmaceutically acceptable inert carriers in the form of tablets, capsules lozenges, troches, hand candies, powders, sprays, aqueous suspensions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, such oral pharmaceutical formulations can be suitably sweetened and/or flavored by means of various agents of the type commonly employed for such purposes. In general, the compounds of this invention are present in such oral dosage forms at concentration levels ranging from about 0.5% to about 90% by weight of the total composition, in amounts which are sufficient to provide the desired unit dosage. 1 2 For purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch and 5 preferably potato or tapioca starch, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very 10 useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection would also include lactose or milk sugar as well as high molecular weight polyethylene 15 glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dvss anc, if so desired, emulsifying and/or suspending agents as well, 20 together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

The following examples illustrate the invention but are not to be construed as limiting the same.

EXAMPLE 1 [N-(t-butyloxycarbonyl)-Phe]-His-Sta-Lys-Phe A. [N-Alpha-(t-butyloxycarbonyl)-N-epsilon-benzyloxycarbonyl-Lysine]-Phenylalanine Benzyl Ester N-hydroxybenzotriazole (162 mg, 1.2 mmoles), N-methy1-morpholine (101.2 mg., 1 rranole), L-phenylalanine 30 benzyl ester p-toluenesulfonate (428 mg., 1 mmole), N-alpha-(t-butyloxycarbonyl)-N-epsilon-benzyloxycar-bo:yl-L-lysine (456 mg., 1.2 mmoles) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene 1 3 sulfonate (635 mg., 80% pure, 1.2 mmoles) were sequentially dissolved in methylene chloride (50 ml.) at 0°C. and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was then washed consecutively with 5 75 ml. of 5.5% aqueous HC1, 75 ml. of saturated aqueous NaHC03 and 75 ml. of brine, and dried over anhydrous MgSO.. After filtration and evaporation, 669 mg. of 4 i crude product was obtained as a foam ( H NMR, CDCl^* 1.5 delta, 9 Hs [30C]). This crude product was used without further purification in the next step.

B. (N-Epsilor.-benzyloxycarbor.yl-Lysine) - Phenylalanine Benzyl Ester Hydrochloride [N-Alpha-(t-butyloxycarbonyl)-N-epsilon-benzyl- oxycarbonv1-lysine]-phenylalanine benzyl ester from step A (650 mg., 1 mmole) was dissoleved in 7 rrl. of 3.7 N HCl/dicxane and allowed to stand for one hour at 20°C.

The solution was then evaporated to dryness to afford 583 mg. of crude product as an oil which was used without purification in the next step (^"H NMR, CD^OD, 5.2 2C delta, 2H s[benzyl CHj]).

C. [N-(t-butyloxycarbony1)-Statine]-(N-epsilon- benzyloxycarbonyl-Lysine)-Phenylalanine Benzyl Ester (N-Epsilon-benzyloxycarbony1-lysine)-phenylalanine benzyl ester hydrochloride from step B (583 mg., 1 mmole), N-methyl-morpholine (101.2 mg., 1 mmole), N-(t-butyloxycarbony1)-statine (330 mg., 1.2 mmoles), N-hydroxybenzotriazole (162 mg., 1.2 mmoles) and l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho- p-toluene sulfonate (635 mg., 80% pure, 1.2 mmoles) were sequentially dissolved in 50 ml. of methylene chloride at 0°C. and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was worked up as in step A to yield 760 mg. of crude product which was used without further purification in the next step (*H NMR, CDC13, 1.5 delta, 9H s[BOC]). 1 4 D. Statine-(N-epsilon-benzyloxycarbonyl-Lysine)-Phenylalanine Benzyl Ester Hydrochloride [N-(t-butyloxycarbonyl)-statine]-(N-epsilon-benzyl oxycarbonyl-lysine)-phenylalanine benzyl ester from step C (760 mg., 1 mmole) was dissolved in 10 ml. of 3.7 N HCl/dioxane and allowed to stand for one hour at 20°C. This reaction mixture was worked up as in step B to afford 620 mg. of crude product as a foam (*H NMR, CD^OD, 5.2 delta, 2H s[benzyl CH2]), which was used without purification in the next step.

E. [N-Alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-Histidine]-Statine-(N-epsilon-benzyl-oxycarbonyl-Lysine)-Phenylalanine Benzyl Ester Statine-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester hydrochloride from step D (600 mg., 0.857 mmoles), N-methyl-mcrpholine (86.7 mg., 0.857 mmoles), N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbony1)-L-histidine (365 mg., 1.03 mmoles) N-hydroxybenzotriazole (135 mg., 1.03 mmoles) and l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho p-toluene sulfonate (545 mg., 1.03 mmoles) were sequentially dissolved in 50 ml. of methylene chloride at 0°C. and allowed to stir for 19 hours at 20°C. This reaction mixture was worked up as in step A to yield 770 mg. of crude product as a foam (^H NMR, CDCl^# 1-5 delta, 2H s[BOC] and 1.6 delta, 9H s[B0C]), which was used in the next step without further purification. 1 5 F. Histidine-Statine-(N-epsilon-benzyloxycarbonyl-Lysine)-Phenylalanine Benzyl Ester Dihydrochloride [N-Alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-statine-(N-epsilon-benzyloxycarbonyl-lysine)-5 phenylalanine benzyl ester from step E (770 mg., 0.85 mmole) was dissolved in 10 ml. of 3.7 N HC1/ dioxane and allowed to stand for 1.5 hours at 20°C. The reaction mixture was worked up as in step B to afford 602 mg. of crude product as a foam (*H NMR, CD-jOD, 5.2 delta, 2H s [benzyl CHj] )» which was used in the next step without purification.

G. [N-(t-butyloxycarbonyl)-Phenylalanine]-Histidine-Statine-(N-epsilon-benzyloxycarbony1-Lysine)-Phenylalanine Benzyl Ester Histidine-statine-(N-epsilon-benzyloxycarbonyl- lysine)-phenylalanine benzyl ester dihydrochloride from step F (602 mg., 0.689 mmoles), N-methvl-morpholine (139 mg., 1.38 mmoles), N-(t-butyloxycarbonyl)- L-phenylalanine (219 mg., 0.827 mmoles), N-hydroxy- benzotriazole (112 mg., 0.827 mmoles) and 1-cyclohexyl- 3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (438 mg., 0.827 mmoles) were sequentially dissolved in 50 ml. of methylene chloride at 0°C. and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was worked up as in step A to yield 555 mg. of a foam which was purified by chromatograpy (silica gel, 95:5 CH-Cl,/MeOH) to afford 136 mg. of 1 purified product as a foam ( H NMR, CDCl^, 1.5 delta, 9H s [BOC]). 1 6 H. Title Compound [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statine-(N-epsilon-benzyloxycarbony1-lysine)-phenylalanine benzyl ester (136 mg., 0.17 mmole) and 70 mg. of 20% 5 PdtOH^J/C catalyst were sequentially added to 15 ml. of methanol and the resulting mixture hydrogenated for four hours at 50 psi of and 20°C. The reaction mixture was therf filtered through Super-Cel and evaporated to dryness to yield 76 mg. of a glass, 10 which was triturated with ether to afford 63 mg. of purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statine-lysine-phenylalanine as a powder (1NMRf CD3OD, 1.5 delta, 9H s [BOC]).

EXAMPLE 2 [N-(t-butyloxycarbonyl)-Phe]-His-Cyclostatine-Lys-Phe A. N-(t-butyloxycarbonyl)-Cyclostatine Cyclohexyl CH~ » ^ (t-butyloxycarbonyl)-NH-CH-CH-CH.--COOH I ^ OH Glacial acetic acid (6.5 ml.) and 10% Rh/C catalyst (1.5 g.) were added to a solution of N-(t-20 butyloxycarbonyl)-4(S)-amino-3(S)-hydroxy-5-phenyl-pentanoic acid (10.02 g., 9.70 mmoles) in methanol (200 ml.), and the resulting mixture was hydrogenated for 8 hours at room temperature and 4 5 psi Hj. The reaction mixture was then filtered and the filtrate 1 7 concentrated to a white solid, which was triturated in hexane to afford a purified product [8.46 g.; 83% yield; m.p. 109-110°C.; Rf = 0.72 in CHC13: MeOH:Acetic acid, 18:2:1; XH NMR (CDC13): 1.45 (s,9H), 5 2.55 (d,J = 6, 2H)] .

B. Title Compound In like manner to that described in Example 1, but using N-(t-butyloxycarbonyl)-cyclostatine in place of N-(t-butyloxycarbonyl)-statine, the title compound 10 was prepared (^NMR, CD3OD, 1.50 delta, 9H s [BOC]).

EXAMPLE 3 [N-(t-butyloxycarbonyl)-Pro]-Phe-His-Cyclostatine-Lys-Phe In like manner to that described in steps A to F of Example,1, but using N-(t-butyloxycarbonyl)-cyclostatine 15 in place of N-(t-butyloxycarbonyl)-statine, the compound histidine-cyclostatine-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester dihydrochloride was prepared. In like manner to that described in steps G and H of Example 1, but using [N-(t-butyloxycarbonyl)-20 proline]-phenylalanine (which is commercially available) in place of N-(t-butyloxycarbonyl)-phenylalanine and using 95:5 CHCl3:MeOH as the silica gel chromatography eluant, the title compound was prepared as a purified solid product (1NMR, CD3OD, 1.49 delta, 9H s [BOC]).

EXAMPLE 4 [N-(t-butyloxycarbonyl)-Phe]-His-Cyclostatine-Lvs-Sta In like manner to that described in Example 1, but using statine benzyl ester hydrochloride in place of L-phenylalanine benzyl ester p-toluenesulfonate and N-(t-butyloxycarbonyl)-cyclostatine in place of N-(t-butyloxycarbonyl)-statine, the title compound was prepared (NMR, CD3OD, 1.5 delta, 9H s [BOC]) 1 8 EXAMPLE 5 [N-(t-butyloxycarbonyl)-Phe]-His-Cyclostatine- Pro Methyl Ester A. [N-(t-butyloxycarbonyl)-Cyclostatine]- Proline Methyl Ester In like manner to that described in step C of Example 1, but using N-(t-butyloxycarbonyl)-cyclostatine (3.15 g./ 10 mmoles) in place of N-(t-butyloxycarbonyl)-statine and proline methyl ester hydrochloride (1.65 g.f 10 mmoles) in place of (N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester hydrochloride, the compound (N-(t-butyloxycarbonyl)-cyclostatine]-proline methyl ester was prepared as a white solid (3.945 g.).

B. Title Compound In like manner to that described in steps D to H of Example 1, the compound [N-(t-butyloxycarbonyl)-phenylalanine)-histidine-cyclostatine-proline methyl ester was prepared. 1 9 EXAMPLE 6 Inhibition of the Angiotensinogen-Cleaving Activity of Renin In Vitro Blood plasma was obtained from healthy laboratory 5 personnel, pooled and stored frozen until required.

Before use, a quantity of this plasma was defrosted and centrifuged, and the supernatant mixed with protease inhibitors and buffered to pH 7.4. Renin inhibitors were added at different levels to different aliquots of the 10 plasma supernatant, and the resulting mixtures (310 lambda) incubated for three hours at 37°C. along with renin inhibitor-free control mixtures. After incubation, the mixtures were quenched in ice water and each assayed for angiotensin I using angiotensin I antibody. The produc-15 tion of angiotensin I in the presence of a renin inhibitor was compared to that produced in the absence of the inhibitor, and a percentage inhibition was calculated. Using data obtained from duplicate incubations at each of several different inhibitor concentra-20 tions, the inhibitor concentration in the incubation mixture required to produce a fifty percent inhibition of the angiotensinogen-cleaving activity of renin, i.e. the ICjjq of the inhibitor, was calculated for various different inhibitors.

The angiotensin I in the quenched incubation mixtures was assayed by means of a radioimmunoassay, using components of a renin radioimmunoassay kit supplied by Becton Dickinson and Co. (Orangeburg, N.Y.). This radioimmunoassay was based upon the one developed by 30 Haber et al., J. Clin. Endocrinol., 29, pp. 1349-1355 ( 1969) . no Using the foregoing procedure, renin inhibition coefficients were determined for the compounds prepared in each of the Examples. In each case the experimental IC.-n value was less than 5 micromoles/liter. 5U c EXAMPLE 7 Antagonization of Exogenous Renin-Induced Pressor Response In Vivo ___ Male Sprague-Dawley rats (230 to 300 g. body weight) are anesthetized with sodium pentobarbital 10 (65 mg.,/kg. body weight, intraperitoneal), after which femoral vein and carotid artery catheters are implanted in each animal. Following completion of surgery, the animals are placed in the prone position and rectal temperature monitored continuously. Mean 15 arterial blood pressure (MAP) is recorded via the carotid artery catheter using a Statham P23 ID pressure transducer and a physiograph. Subsequent to a stabilization period, control renin pressor responses (dP) of 20 to 30 mm Hg are obtained by administration of hog 20 renin (30 to 80 mO/kg. body weight, intravenous). After the MAP returns to the baseline, a renin inhibitor is administered (10 mg./kg. body weight, intravenous), the animals are rechallenged with hog renin (same dosage as for control response) at 5, 15 and 30 minutes after 25 renin inhibitor administration and the corresponding renin pressor responses (dP) measured. Percent antagonization is calculated as (control dP - experimental dP) x 100% , control dP where control dP and experimental dP are the pressor changes in MAP before and after renin inhibitor administration, respectively. Preferably, at least three animals are used in each, test, with results averaged. Using the foregoing procedure, percent renin-35 induced pressor response antagonizations may be determined for the compounds of the invention.

Claims (15)

CLAIMS:

1. A compound of the formula R2 R-W-W1-NH-CH-CH-CH--COR1 I R3 and the pharmaceutically acceptable salts thereof, wherein W is phenylalanine, histidine, leucine, tyrosine, 1-naphthylalanine or 2-phenylmethyl-propionylene; W is phenylalanine, histidine, leucine, tyrosine or norleucine, with the peptide link between W and W^" optionally being replaced by -CH(alkyl of from 1 to 4 carbon atoms)-NH- wMen W is phenylalanine and W* is histidine;;R is hydrogen, an amino-protecting acyl moiety having a molecular weight of less than 500, proline, amino-protected proline, pyroglutamic acid or amino-;protected pyroglutamic acid;;2;R is hydrogen, alkyl of from 1 to 6 carbon atoms, phenyl, cycloalkyl of from 4 to 7 carbon atoms, phenylalkyl of from 7 to 9 carbon atoms or cycloalkyl (alkylene) of from 5 to 10 carbon atoms;;3 9 9 R is hydroxy1, amino, -NHR , -NHCOR ,;-OR9 or -OCOR9, with R9 being alkyl of from 1;to 4 carbon atoms; and;R1 is - A-E-B or A-B,;with A being lysine or proline, E being phenylalanine,;glycine, alanine, valine, leucine, isoleucine, lysine,;ornithine, arginine, aspartic acid, aamma-esterified aspartic acid, glutamic acid or;4;delta-esterified glutamic acid, B being -OR ,;4 5 4 , . >;-MR R , -glutamic acid(-OR )2, -glutamic acid (-OR4 ) (-NR4R^ ) or -glutamic acid ( -NR4R~*) 2 / and R4 and R^ each being hydrogen, alkyl of from 1 to 4 carbon atoms, phenylalkyl of from 7 to 9 carbon atoms or cycloalkyl(alkyl) of from 5 to 10 carbon atoms.

2. A compound of claim 1 wherein R is an amino-protecting acyl moiety having a molecular weight of less than 500 or an amino-protected proline, W is phenylalanine-, is histidine and W and are bonded in a peptide link. 2

3. A compound of claim 2 wherein R is isobutyl 3 and R is hydroxyl. 2

4. A compound of claim 2 wherein R is cyclohexyl-(methylene) and R3 is hydroxyl.

5. A compound of claim 3 or 4 wherein RX is -lysine-E-B or proline-E-B.

6. A compound of claim 5 wherein E is phenylalanine and B is hydroxyl.

7. A compound of claim 6 wherein R is t-butyloxycarbonyl .

8. A compound of claim 6 wherein R is N-(t-butyloxycarbony1)-proline.

9. A compound of claim 2 wherein R is of the 0 0 8 " 8 n formula R -M-C- or R -M-C-Proline-, with M being -0-, -NH- or -SO^HH- and R® being alkyl of from 1 to 6 carbon atoms, phenyl, phenylalkyl of from 7 to 9 carbon atoms or cycloalkyl (alkyl) of from 5 to 10 carbon atoms.

10. a compound of claim 9 wherein R is N-(t-butyloxycarbonyl)-proline.

11. - A pharmaceutical composition containing a renin inhibiting-effective amount of a compound of claim 1 as the essential active ingredient in a pharmaceutical ly acceptable carrier.

12. A compound according to Claim 1, substantially as hereinbefore described and exemplified.

13. A process for preparing a compound according to Claim 1, substantially as hereinbefore described and exemplified.

14. A compound according to Claim 1, whenever prepared by a process claimed in Claim 13.

15. A pharmaceutical composition according to Claim 11, substantially as hereinbefore described. F. R. KELLY & CO. AGENTS FOR THE APPLICANTS.