GB2045771A - Mercaptoacyldipeptides - Google Patents

Abstract

Compounds having the formula <IMAGE> and alkyl esters and salts thereof. R1 is hydrogen, alkanoyl, benzoyl or <IMAGE> R2 is hydrogen, alkyl, or phenylalkyl; n is 0 or 1; and A1 and A2 each is an ???-amino or ???-imino acid residue joined through a peptide bond, have hypotensive activity.

Description

SPECIFICATION Mercaptoacyldipeptides This invention provides compounds having the formula

and alkyl esters and salts thereof: such compounds have hypotensive activity. In formula I, and throughout the specification, the symbols are as defined below.

R1 is hydrogen, alkanoyl, benzoyl or

R2 is hydrogen, alkyl, or phenylalkyl; n isOori; and A1 and A2 each is an a-amino or a-amino acid residue joined through a peptide bond.

The term "alkyl", as used throughout the specification, refers to groups having 1 to 7 carbon atoms.

The term "alkanoyl" as used throughout the specification, refers to alkanoyl groups having 2 to 7 carbon atoms. Acetyl is the preferred alkanoyl group.

The a-amino and a-imino acid residues represented by A1 and A2 can be either naturally occuring or synthetic. Exemplary groups are prol ine, 4-hydroxyproline, 4,4-ethylenedioxyproline, 4-methoxyproline, 4-thiazolidinecarboxylic acid, tryptophane, glycine, alanine, leucine, isoleucine and valine. As set forth above, the A1 and A2 groups are linked by "a peptide bond"; i.e., the linkage

between the a-carboxyl group of the residue A1 and the a-amino or a-imino group of the group A2.

The mercaptoacyldipeptides of formula I wherein R1 is alkanoyl or benzoyl can be prepared by acylation of a dipeptide having the formula II A1-A2 or an alkyl ester derivative thereof, with a thio acid having the formula

In formula Ill, and throughout the specification, the symbol R; is alkanoyl or benzoyl. The above acylation can be accomplished using any one of the numerous techniques well known in the art. For example, the acylation can be affected in the presence of a coupling agent such as a carbodiimide (of which dicylohexylcarbodiimide is the most often used). Alternatively, the thio acid of formula Ill can be activated by formation of its mixed anhydride, symmetrical anhydride, acid chloride or active ester or by the use of Woodward reagent K or N-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline.For a more detailed discussion of various acylation techniques, reference is made to Methoden der organischen Chemie (Houben-Weyl), Vo..

XV, part II, page 1 etseq. (1974).

An alternative synthesis for the compounds of formula I wherein R1 is alkanoyl or benzoyl comprises sequential acylation, as opposed to the previously described direct acylation, that is, the acylation of the amino acid "A1,,, with a mercaptoalkanoyl acid of formula III (using the procedure described above) to obtain a mercaptoalkanoyl amino acid having the formula

followed by acylation of the amino acid "A2" or alkyl ester thereof, with a mercaptoalkanoyl amino acid of formula IV. The acylation can be accomplished using any one of the well known techniques for coupling amino acids. For a review of these techniques reference should be made to Bodanszkyand Ondetti, Pep tide Synthesis, Interscience Publishers (1966).

Compounds of formula I wherein R1 is hydrogen can be prepared by ammonolysis or alkaline hydrolysis of the corresponding compounds of formula I wherein R1 is alkanoyl or benzoyl which can be obtained utilizing either of the procedures described above. Compounds of formula I wherein

can be prepared by oxidation of the corresponding free thiol of formula I with iodine.

Alternatives to the above synthesis will be apparent to the practitioner of this invention. For example, if an ester of A1-A2 or A2 is used in one of the above syntheses, the corresponding free acid can be obtained from the esterified product by acid or alkaline hydrolysis.

Mercaptoalkanoyl acids of formula ill and mercaptoalkanoyl amino acids of formula IV, and methods for their preparation, are described in the literature; see, for example, United States patents 4,046,889, 4,105,776 and 4,053,651.

The compounds of this invention form basic salts with various inorganic and organic bases which are also within the scope of the invention. Such salts include ammonium salts, alkali metal salts like sodium and potassium salts (which are preferred), alkaline earth metal salts like the calcium and magnesium salts, salts with organic bases e.g., dicyclohexylamine salt, benzathine, N-methyl-D-glucamine, hydrabamine salts, salts with amino acids like arginine, lysine and the like. The nontoxid, physiologically acceptable salts are preferred, although other salts are also useful, e,g., in isolating or purifying the product.

The compounds of formula I, and the alkyl esters and salts thereof, are hypotensive agents. They inhibit the conversion of the decapeptide angiotensin I to angiotensin II and, therefore, are useful in reducing or relieving angiotensin related hypertension. The action of the enzyme renin on angiontensinogen, a pseudo-globulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin converting enzyme (ACE) to angiotensin 11. The latter is an active pressor substance which has been implicated as the causative agent in various forms of hypertension in various mammalian species, ag., rats and dogs.The compounds of this invention intervene in the angiotensinogen~(renin) < angiotensin I(ACE)angiotensin ll sequence by inhibiting angiotensin converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin ll. Thus by the administration of a composition containing one or a combination of compounds offormula I, angiotensin dependent hypertension in the species of mammal suffering therefrom is alleviated. A single dose, or preferably two to four divided daily doses, provided on a basis of about 0.1 to 100 mg. per kilogram of body weight per day, preferably about 1 to 50 mg. per kilogram of body weight per day is appropriate to reduce blood pressure.The substance is preferably administered orally, but parenteral routes such as the subcutaneous, intramuscular, intravenous or intraperitoneal routes can also be employed.

The compounds of formula I can be formulated for use in the reduction of blood pressure in compositions such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About 10 to 500 mg. of a compound or mixture of compounds of formula I is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.

The invention thus extends to a pharmatceutical composition comprising a compound of the invention and a pharmaceutical carrier.

The following examples are specific embodiments of this invention.

EXAMPLE 1 1- IN- (A cetylthioacetyl)glycylj-L-proline Glycyl-L-proline (1.72 g) is taken into 10 ml of 1N sodium hydroxide with stirring in an ice bath. To this 5 ml of 2N sodium hydroxide is added followed by 1.13 g of chloroacetyl chloride and the bath is removed. After three hours at room temperature, thiolacetic acid (836 mg) and potassium carbonate (960 mg) in 10 ml of water is added and the reaction is stirred for about 16 hours at room temperature. The reaction is acidified with sulfonated polystyrene cation exchange resin, eluted with water and concentrated to dryness in vacuo.

This was taken into acetic acid and the insolubles (melting point over 320"C) are filtered. The filtrate is concentrated to dryness in vacuo and 7:3 chloroform acetic acid is added. The product is centrifuged and the supernate applied to a 60 g silica gel column and eluted with 7:3 chloroform: acetic acid yielding 1.0 g of the title compound.

EXAMPLE 2 l-[N-(Mercaptoacetyl)glycyl]-L-prollne 1 -[N-(Acetylthioacetyl)glycyl]-L-proline (1.0 gj is treated for thirty minutes under argon in a solution of 6 ml water and 6 ml concentrated sodium hydroxide. The solution is concentrated in vacuo, acidified with sulfonated polystyrene cation exchange resin, applied to a column of the same and eluted with water. The 800 mg of material yielded therein (plus 200 mg from a previous preparation) is chromatographed on a 30 g column of silica gel with 7:3 chloroform: acetic acid. This product (740 mg) is applied to an 85 ml column of diethylaminoethyl dextran anion exchange resin and eluted with a linear gradient of 0.005 M ammonium bicarbonate to 0.5 M ammonium bicarbonate (750 ml each).The product is converted to the free acid on sulfonated polystyrene cation exchan'ge resin, and then treated with 7 ml of acetic acid and 350 ml of zinc dust with stirring under an argon blanket for six hours. The suspension is centrifuged, and the supernate concentrated to dryness in vacuo, taken into water and lyophilized. This is acidified with sulfonated polystyrene resin, applied to a column of the same and eluted with water yielding 365 mg of the title compound.

Analysis Calc. for: CgH14N204S(o3)H2o Calc.for: C,42.95; H,5.68; N,11.13; S-12.72 Found: C,42.92; H,5.78; N,10.94; S-12.68 SH: Calc. -1.00; found -0.98 EXAMPLE 3 t-Butyl ester of (S)- I-[3-(acetylthio)-2-methyl 1-oxopropyll-L-propyl-L-proline To a solution of 7.8 g of 1-[S-3-(acetylthio)-2-methyl-1 -oxopropyl]-L-proline in 75 ml of dichloromethane is added 5.13 g of t-butyl-L-prolinate, and the solution is cooled to 5"C. To this solution is added (dropwise) a solution of dicyclohexylcarbodiimide in 50 ml of dichloromethane. The reaction is carried out at room temperature for about 16 hours.After the precipitate is removed, the resulting solution is successively washed with water, 5% sodium bicarbonate, water, 10% potassium bisulfate, and water, and then dried over sodium sulfate. The crude product (12 g) that is obtained after the solvent is removed, solidifies on standing.

Recrystallization from ethyl acetate: pentaneyieldsthetitlecompound, melting point 104-106"C.

EXAMPLE 4 l-(S-3-mercapto-2-meth yl- 1-oxoprop yl)-L-prolyl-L-proline t-Butyl ester of 1-[S-3-(acetylthio)-2-methyl-1-oxopropyi]-L-proline (12 g) is taken into 60 ml of redistilled trifluoroacetic acid and the resulting solution is allowed to stand at room temperature for 1 hour.

Trifluoroacetic acid is removed in vacuo and the residue is carefully dried in high vacuo for 24 hours. The heavy oil is dissolved in 30 ml of methanol and to this is added 20 ml of concentrated ammonium hydroxide.

The mixture is kept at room temperature for 1 hour, and the solvent is then removed in vacuo and the oil residue is dried and treated with 6 ml of dicyclohexylamine. The salt is treated with ether and an oil precipitates. The oil is taken into ethyl acetate and the solution is washed with 10% potassium bisulfate and then dried over sodium sulfate. After removal of the solvent, the residue is taken into methanol and the entire amount is adsorbed on silica gel. The solvent is removed and the silica gel containing the adsorbed product is applied to a silica gel column made up in chloroform. The column is eluted with chloroform and the mercapto positive fractions are combined. The solvent is removed and the residue is lyophilized from water to yield 1.05 g of the title compound as a hygroscopic solid.

Analysis Calc. for: C14H22N2O4S.1/2H2O Calc.for: C,51.99; H,7.17; N,8.66; S,9.92 Found: C,52.12; H,7.44; N,8.18; S,9.37 Neutralization equivalent: Calc., 323 found 341 EXAMPLE 5 N-6Acetylthioacetyl)-L-valyl-L-proline A solution of t-butyloxycarbonyl-L-valyl-L-proline (3.1 g) in trifluoracetic acid (20 ml) is kept at room temperature for 15 minutes, the solvent is removed in vacuo and the oily residue is dried in vacuo for several hours. The oil is taken into dimethylformamide (25 ml) and to this solution N-hydroxysuccinimide ester of acetylmercapto acetic acid (2.2 g) is added. The reaction is carried out at pH 7.5 (adjusted with triethylamine) and at room temperature for 17 hours.After removal of the solvent, the residue is taken into ethyl acetate and the solution is washed successively with water, 10% potassium bisulfate, water, saturated sodium bicarbonate, water and finally dried over sodium sulfate, yielding 4.8 g of the title compound as an oil.

EXAMPLE 6 N-(Mercaptoacetyl)-L-valyl-L-prollne N-(Acetylthioacetyl)-L-valyl-L-proline (4.8 g) is taken into a mixture of 12 ml of methanol N-(Acetylthioacetyl)-L-valyl-L-proline (4.8 g) is taken into a mixture of 12 ml of methanol and 7.5 ml of concentrated ammonium hydroxide and the resulting solution is kept at room temperature for 1 hour. The pH is adjusted to 7 and methanol is removed in vacuo. The remaining aqueous layer is acidified to pH 2 with 2N hydrochloric acid and the solution is saturated with sodium chloride.The product is extracted with ethyl acetate, the solution is dried over sodium sulfate and the solvent is removed in vacuo, yielding 2 g of material, melting point 158-162"C. Recrystallization from ethyl acetate (with the addition of a small amount of methanol) yields the product, melting point 162-165"C. After drying a sample at 55"C for 2 hours the melting point is 190-192"C.

EXAMPLE 7 I-(S-3-IAcetylthio)-2-methyl- I-oxopropyl]-L-prolyIglycine, ethyl ester Following the procedure of Example 3, but utilizing 10.4 g of 1-[S-3-(acetylthio)-2-methyl-1-oxopropyl]-Lproline, 100 ml of dichloromethane, 5.6 g of the ethyl ester of glycine (prepared from 5.58 g of the hydrochloride salt of the ethyl ester of glycine and 5.6 ml of triethylamine in 50 ml of dichloromethane) and 8.24 g of dicyclohexyl-carbodiimide, yields 11.2 g of the title compound as a viscous oil.

EXAMPLE 8 (S)- 1- (3-Mercap to-2-meth yl- l-oxopropylJ-L-prolylglycine, lithium salt 1 -[S-3-(Acetylthio)-2-methyl-l -oxopropyl]-L-prolylglycine, ethyl ester (11.2 g) is taken into methanol (200 ml) to which an aqueous solution of potassium hydroxide (9 g in 40 ml of water) is added. The hydrolysis is allowed to proceed at room temperature for two hours. The organic solvent is removed in vacuo and the aqueous residue is adjusted to pH 7.0. The product is extracted with ethyl acetate, the solution dried over sodium sulfate and the solvent is removed in vacuo. The residue (3.5 g) is taken into water and the pH is adjusted to pH 2.0. The free acid is again taken into ethyl acetate using sodium chloride to decrease the solubility in water.The residue after removal of the solvent, is lyophilized from water, yielding 2.16 g of the free base of the title compound. The entire amount was taken into water and the pH carefully adjusted to pH 7.0 using 0.05N lithium hydroxide. The solution is lyophilized to yield 1050 mg of the title compound as a hygroscopic solid.

Analysis Calc. for: C11 H17N2O4S.Li Calc: C,47.14; H,6.12; S,11.44; Li,2.48 Found: C,46.56; H,6.42; S,11.37; Li,2.43 EXAMPLE 9 (S)- l-(3-Mercapto-2-meth yl- 1-oxopropyl)-L-prolyl-L-alanine Following the procedure of Examples 3 & , but utilizing 7.8 g of 1-[S-3-(acetylthio)-2-methyl-1-oxopropyl] L-proline, 75 ml of dimethylformamide, 6.2 g of dicyclohexylcarbodiimide, 4.2 g of the hydrochloride salt of the methyl ester of L-alanine and 4.2 ml of triethylamine (pH 7.5-8.0), yields 8 g of material. This material is passed through a silica gel column using ethyl acetate as an eluant. Two fractions are obtained (Rf=0.76 and Rf=0.4, silica gel, ethyl acetate).

The slower moving fraction (2 g Rf=0.4) is taken into potassium hydroxide/aqueous methanol (1.5 g potassium hydroxide in 7 ml of water and 35 ml of methanol) and the solution is kept at room temperature for 2 hours. The organic solvent is removed in vacuo and the aqueous layer is acidified to pH 2.0. After the addition of sodium chloride, the product is extracted five times with ethyl acetate. The solution is dried and the solvent is removed in vacuo. The solid residue is crystallized from hot ethyl acetate yielding 1 g of product, melting point 128-130"C.

EXAMPLE 10 A cetylthioacetyl-valyl-tryptophan methyl ester A solution of 15g of the methyl ester of t-butyloxycarbonyl-valyl-tryptophan in 150 ml oftrifluoraceticacid and 15 ml of anisole is stirred for 1 hour under nitrogen. The solution is stripped in vacuo to an oily residue.

A solution of 4.83 g of acetylthioacetic acid and 6.18 g of hydroxybenzotriazole monohydrate in 120 ml of tetrahydrofuran is cooled to 0 C and treated with a solution of 8.16 9 of dicyclohexylcarbodiimide in 60 ml of tetrahydrofuran. The mixture is stirred at 00C for 0.5 hour and then at room temperature for 1 hour The precipitated urea is filtered and the filtrate is stripped in vacuo to a white solid. The active ester is dissolved in 90 ml of dimethylformamide after which a solution of the above dipeptide methyl ester trifluoroacetic acid salt in 60 ml of dimethylformamide is added along with sufficient N-methyl morpholine to maintain the pH at 7.5 and the resultant mixture is stirred for 16 hours at room temperature.The reaction mixture is stripped in vacuo to an oil which is partitioned between ethyl acetate and water. The ethyl acetate phase is washed with 2N citric acid, water, saturated sodium bicarbonate solution, and water, and dried over sodium sulfate and stripped to a solid. The material is crystallized from ethyl acetate: pentane to yield 10 g of the title compound, melting point 110-112"C.

EXAMPLE 11 N-6Mercaptoacetyl)-L-valyl-L-tryptophan, lithium salt Acetylthioacetyl-valyl-tryptophan methyl ester (5.0 g) is dissolved in 52 ml of methanol and a solution of 2.1 g of potassium hydroxide in 10.5 ml of water is added. The mixture is stirred under nitrogen at room temperature for 1.5 hours after which the methanol is stripped in vacuo. The aqueous remainder is diluted with water, extracted with ethyl acetate and acidified to pH 2.0 with 6N hydrochloric acid. The solid is filtered, washed with water and dried yielding 3.4 g of material. The solid is chromatographed on a Sephadex LH-20 column using 7:3 methanol: water as eluant to yield 1.5 g of an oil. The oil is dissolved in 1:1 ethanol: water and adjusted to pH 7.0 with 0.1 N lithium hydroxide.A small amount of insoluble material is removed by filtration and the filtrate is stripped in vacuo to an oil. The oil is dissolved in water and lyophilized to yield 1.5 g of a hygroscopic solid.

Analysis Calc. for: Cl8H22N304S.Li Calc.: C,51.54; H,6.25; S,7.65; Li,1.65 Found: C,51.94; H,6.03; S,7.28; Li,1.62 Neutralization Equivalent: Calc. 419; found 413 EXAMPLE 12 (S)- 1-f3-(A cetylthio)-2-meth yl- 1-oxopropyU-L-prolyl-D-alanine methyl ester To a solution of 1-[S-3-(acetylthio)-2-methyl-1-oxopropyl]-L-proline (7.8 g) in acetonitrile (150 ml) carbonyldiimidazole (4.9 g) is added at OOC. The mixture is stirred for 1 hour at this temperature after which the solution becomes almost clear and D-alanine methyl ester hydrochloride (4.2 g) is added. The pH-value is adjusted to 7.5-8.0 with triethylamine (4.2 ml) and the reaction mixture is stirred for 17 hours at room temperature.The precipitate and the solvent are removed and the oily residue is taken into ethyl acetate. The solution is successively washed with water, 0.1 N HCI, water, saturated NaHCO3, water and dried. After removal of the solvent the remaining oil shows one spot on tic (silica gel, EtOAc and CHC13: MeOH 9:1) at Rf=0.5 with a trace at Rf=0.75 (in ethyl acetate). Yield: 4 g.

EXAMPLE 13 (S)- -(3-Mercap to-2-methyl- 1-oxoprop yl)-L-prolyl-D-alanine (S)-[3-(Acetylthio)-2-methyl-1 -oxopropyli-L-prolyl-D-alanine methyl ester (4 g) is taken into potassium hydroxide/aqueous methanol (3 g of potassium hydroxide in 14 ml of water and 70 ml of methanol) and the solution is kept at room temperature for 2 hours. The product (2.1 g) is isolated using the procedure described in Example 9, m.p. 152-155"C. as a crystalline solid.

Analysis Calc. for: C12H20N204S Calc.for: C,49.98; H,6.99; S,11.12 Found: C,50.02; H,7.24; S,10.90 Neutralization Equivalent: Calc. 288; found 284 EXAMPLE 14 l-JN-(mercaptoacetyl)-L-alan ylJ-L-prollne To a solution of acetylthioacetic acid (1.3 g) and hydroxybenzotriazole (1.44 g) in tetrahydrofuran (39 ml), dicyclohexylcarbodiimide (2 g) is added and the mixture is stirred at OOC for thirty minutes and at room temperature for one hour. The precipitate is removed by filtration, and to the filtrate is added L-alanyl-L-proline as its trifluoracetate salt (prepared from t-butyloxycarbonyl-L-alanyl-L-proline, 2.8 g, and trifluoroacetic acid). The pH is adjusted to 7.5-8.0 with N-methylmorpholine and the reaction is allowed to proceed at room temperature for about 16 hours.The solvents are removed in vacuo and the residue is dissolved in a mixture of methanol (18 ml) and concentrated ammonium hydroxide (18 ml). After thirty minutes the mixture is concentrated in vacuo, the residue is dissolved in water and applied to a column of sulfonated polystyrene cation exchange resin eluting with water. This material is further purified by silica gel chromatography (acetic acid: chloroform, 7:3) and crystallized from ethyl acetate, 850 mg, melting point 152-155 C[a]D=-128 (c=1.5,50% aqueous MeOH).

EXAMPLE 15 1-[N-rMercaptoacetyl)-L-alanyll-L-thiazolidine-4carboxyllc acid Following the procedure of Example 14, but substituting the trifluoroacetic acid salt of L-alanyl-Lthiazolidine-4-carboxylic acid for the trifluoroacetic acid salt of L-alanyl-L-proline in the procedure of Example 14, yields the title compound.

EXAMPLE 16 1-[N-rMercaptoacetyl)-L-alanyll-4-methoxy-L-proline Following the procedure of Example 14, but substituting the trifluoroacetic acid salt of L-alanyl-4-methoxy- L-proline for the trifluoroacetic acid salt of L-alanyl-L-prnline, yields the title compound.

EXAMPLE 17 1-[N-{Mercaptoacetyl-L-alanyll-4-hydroxy-L-proline Following the procedure of Example 14, but substituting the trifluoroacetic acid salt of L-alanyl-4-hydroxy L-proline for the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 18 1-IN-(Mercap toacetyl)-L-alanylj-4,4-ethylenedioxy-L-prollne Following the procedure of Example 14, but substituting L-alanyl-4,4-ethylenedioxy-L-proline for the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 19 l-JN-(2-Mercap toprnpanoyl)-L-alanyl]-L-prnllne Following the procedure of Example 14, but substituting 2-acetylthiopropionic acid for the 2acetylthioacetic acid, yields the title compound. as a waxy solid, m.p. (47")54-65"C.(Dec.).

Analysis Calcd. for: C11H18N204S.1/2H2O Calcd: C,46.63; H,6.76; S,9.89 Found: C,46.50; H,6.58; S,9.78 SH Titration: 100% EXAMPLE 20 1-[N-63-Mercapto- 1-oxopropyl-L-alanyll-L-proline Following the procedure of Example 14, but substituting 3-acetylthio-propionic acid for 2-acetylthioacetic acid, yields the title compound as a hygroscopic solid, m.p. (61")75-84"C.

Analysis Calcd. for: C11H18N204.0.75H2O Calcd: C,45.89; H,6.83; S,11.14 Found: C,45.75; H,6.62; S,10.98 SH Titration: 99.6% EXAMPLE 21 1- IN- (2-Mercap topropano yl)-L-alan yl]-4-hydroxy-L-prollne Following the procedure of Example 14, but substituting 2-acetyithiopropionic acid for acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-hydroxy-L-proline for the tricluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 22 1-[N-r2-Mercaptopropanoyl)-L-alanyll-L-thiazolidine-4carboxylic acid Following the procedure of Example 14, but substituting 2-acetylthiopropionic acid for acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid salt for the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 23 i-IN-(2-Mercaptopropan oyl)-L-alanylj-4-methoxy--proline Following the procedure of Example 14, but substituting 2-acetylthiopropionic acid for acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline for the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 24 1-[N-(2-Mercaptopropanoyl)-L-alanyl]-4,4-ethylenedioxy-L-proline Following the procedure of Example 14, but substituting 2-acetyithiopropionic acid for acetylthioacetic acid and L-alanyl-4,4-ethylenedioxy-L-prnlinefor the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 25 l-[N-(2-Mercapto-3-phen ylpropano yl)-Lalan yl]-4-h ydroxy-L-proline Following the procedure of Example 14, but substituting 2-acetylthio-3-phenylpropionic acid for acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-hydroxy-L-proline for the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 26 l-[N- (2-Mercapto-3-phen ylpropanoyl)-L-alan ylPL-thiazolidine-carboxyllc acid Following the procedure of Example 14, but substituting 2-acetylthio-3-phenylpropionic acid for acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid for the trifluoroacetic acid salt of L-alanyl-L-proline yields the title compound.

EXAMPLE 27 l-[N-(2-Mercapto-3-phen ylpropanoyl)-L-alan yl]-4-methoxy-L-prollne Following the procedure of Example 14, but substituting 2-acetylthio-3-phenylpropionic acid for acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline for the trifluoroacetic acid salt of L-alanyl-L-proline, yields the title compound.

EXAMPLE 28 I-[N-12-Mercap to3-propan o y-L-alan yl]-4,4-ethylenedioxy-L-proline Following the procedure of Example 14, but substituting 2-acetylthio-3-phenylpropionic acid for acetylthioacetic acid and L-alanyl-4,4-ethylenedioxy-L-proline for the trifluoroacetic acid salt of L-alanyl-Lproline, yields the title compound.

EXAMPLE 29 1-[N-(Mercaptoacetyl)-L-proline]-L-proline Method A Benzyloxycarbonyl-L-prolyl-L-proline (3 g) is hydrogentated in a mixture of absolute ethanol (50 ml) and N-hydrochloric acid (7.5 ml) in the presence of 10% palladium on charcoal. After five hours the catalyst is filtered off and the solvent is removed in vacuo. The residue is dissolved in a mixture of dimethylformamide (15 ml) and triethylamine (1.05 ml) and acetylthioacetic acid N-hydroxysuccinimido ester (1.7 g) is added.

After storage at room temperature for about 16 hours the solvents are removed in vacuo and the residue is chromotographed on a column of silica gel (benzene: hexane, 7:1). This material is dissolved in trifluoroacetic acid (5 ml) and anisole (2 ml) and the solution is kept at room temperature for one hour. The reaction mixture is concentrated to dryness, and the residue is dissolved in 7.7 ml of N sodium hydroxide with stirring under argon. After fifteen minutes the mixture is neutralized with sulfonated polystyrene cation exchange resin, applied to a column of the same resin and eluted with water. The material obtained is crystallized from ethyl acetate and has a melting point 182-183"C.

Method B Dicyclohexylcarbodiimide (2.06 g) and 1-(acetylthioacetyl)-L-proline (2.3 g) are added to a solution of L-prolinet-butyl ester (1.7 g) and hydroxybenzotriazole (1.5g) in dichloromethane (15 ml) chilled in an ice bath. After stirring at 5"C for about 16 hours, the resulting precipitate is filtered off and the filtrate is washed until neutral. The organic layer is concentrated to dryness and the residue is purified by silica gel chromatography and crystallized from ether-hexane, yielding material with a melting point 95-98"C. This material is deprotected by sequential treatment with trifluoroacetic acid and sodium hydroxide as described in Method A.

EXAMPLE 30 Acetylthioacetic-L-alanyl-L-tryptophanmethyl ester L-alanyl-L-tryptophan-methyl ester acetate (2.5 grams) was dissolved in 20 ml of dimethylformamide and 2.0 grams of acetylthioacetic acid N-hydroxysuccinimide ester was added. The pH of the solution was adjusted to 7.5 with N-methyl morpholine and the mixture was stirred overnight at room temperature. The solvent was removed in vacuo and the residual oil taken into ethyl acetate, washed with water, dried and stripped in vacuo to a foamy residue (3 grams). The residue was dissolved in ethyl acetate and applied to a 1.5 cm x 45 cm column containing 60 grams of Woelm basic alumina (activity 1). Elution with ethyl acetate gave 1.6 grams of a foamy solid which gave one spot at Rf 0.69 on a silica gel thin-layer chromatograph, chloroform-methanol (3:1).

EXAMPLE 31 N-[N-6MercaptoacetylJ-L-alanyll-L-tryptophan lithium salt A solution of the product of Example 30 (1.2 grams in 36 ml of methanol) was stirred with 6 ml of 1 N sodium hydroxide solution under a nitrogen atmosphere for one-half hour at room temperature. The reaction mixture was adjusted to pH 6 and the solvent was removed in vacuo. The residue was partitioned between ethyl acetate and saturated sodium bicarbonate solution (1:1). The aqueous phase was washed with ethyl acetate, saturated with sodium chloride, acidified to pH 2.0 and extracted with ethyl acetate. The ethyl acetate extract was dried with sodium sulfate and stripped in vacuo to a foamy residue. The residue was dissolved in a 50% aqueous ethanol solution and the pH adjusted to 7 with 1 N lithium hydroxide solution.The solution was stripped to dryness and the residue taken into water and lyophilized to an off-white solid (390 mg).

Analysis Calcd. for: C16H18N304S.U.2H2O Calcd: C,49.11; H,5.67; N,10.73; S,8.18 Found: C,98.78; H,5.07; N,10.41; S,7.85 EXAMPLE 32 (S)- l-(3-Mercapto-2-methyl- 1-oxopropylJ-L-prolyl-B-alanine A. 3.06 grams of B-alanine-ethyl ester, hydrochloride and 2.82 ml of triethylamine were added to a solution of 5.2 grams of 1 -[S-3-acetylthio)-2-methyl-1 -oxopropyl]-L-proline and 3.24 grams of carbonyldiimi- dazole at 0 C. and the resultant mixture was stirred overnight at room temperature. The reaction mixture was stripped of solvent and the residue was extracted with ethyl acetate.The ethyl acetate extract was then washed with water, IN hydrochloric acid solution, water again, saturated sodium bicarbonate solution and water again and then dried over sodium sulfate and stripped to a clear oil (3.4) grams. This material was proven by spectral analysis to be (S)-1 -(3-acetylthio-2-methyl-1 -oxopropyi)-L-propyl-B-alanine and was used as such in the next step.

B. 3.4 grams of the material from step A was dissolved in a solution of 43 ml of methanol and 1.7 grams of potassium hydroxide in 8.6 ml of water was added. The mixture was stirred for one hour at room temperature, 50 ml of water was then added and the methanol was removed. The aqueous remainder was acidified to pH 2.0 with dilute hydrochloric acid. The solvent was then removed in vacuo and the residue was extracted with methanol. After drying, the methanol solvent was removed to leave an oil (3.2 grams) which was chromatographed on an LH-20 Sephadex column and eluted with a methanol-water solution (1:9) to give 1.1 grams of the titled compound as a clear oil. TLC one spot, Rf 0.65 silica gel, methanol.

Analysis Calcd. for: C12H20N204S Calcd. for: C,49.99; H,6.99; N,9.71 Found: C,49.81; H,7.29.; N,8.67 EXAMPLE 33 (R)-1 -[N-(2-mercapto-1 -oxo-3-phenyl propyl)-L-alanyl]-L-proline A. fR)-2-acetylthio-3-phenyl-propionic acid Sodium nitrate (32.75 grams) was added to a cooled solution of L-phenylalanine (51 grams) and potassium bromide (125 grams) in 620 ml of 25 N sulfuric acid over a period of one hour while maintaining the temperature of the reaction mixture at O"C. The reaction mixture was stirred for one additional hour at 0 and then for one hour at room temperature. The reaction mixture was then extracted with ether and the ether extract was dried over sodium sulfate.The ether was removed to leave (S)-2-bromo-3-phenyl-propionic acid as an oily residue (44.9 grams) which was distilled at 142-144"C (0.25 mm.Hg).

A mixture of thioacetic acid (8.7 ml) and potassium hydroxide (6.8 g) in 225 ml of acetonitrile was stirred for one hour and fifteen minutes at room temperature. The mixture was cooled in an ice bath and 25.3 grams of (S)-2-bromo-3-phenyl-propionic acid in 25 ml of acetonitrile was added over ten minutes. After stirring for five hours, the reaction mixture was filtered and the solvent removed in vacuo and the oily residue redissolved in ethyl acetate and dried, and the solvent removed to yield 24 grams of crude (R)-2-acetylthio-3 phenyl-propionic acid. The crude material was purified via formation of the dicyclohexylamine salt using ether as a crystallizing solvent. The free acid, [a]D5 = +62 (C=2.8, chloroform) was regenerated with hydrochloric acid and extraction with ethyl acetate.

B. R-(2-acetylthio-3-phenyl)propion yl-L-alanyl-L-prollne The above product is prepared by reacting the (R)-2-acetylthio-3-phenylpropionic acid from part (A) above with L-alanyl-L-proline according to the procedure of Example 14, m.p. (124")135-138"C, [a125 = -23 (C=1.4, chloroform).

C. R- 1-[N-f2-mercapto- 7-oxo-2-phenylprop yl)-L-alan yl-L-proline By continuing the procedure of Example 14 with the material from part (B) above, and using sodium hydroxide instead of ammonium hydroxide, the titled product is obtained as a foamy solid, [aJ25 = -78.4" (C-1.1, chloroform).

Analysis Calcd. for: C,7H22N204S2H2o Calcd. C,52.83; H,6.78; N,7.25; S,8.30 Found: C,52.54; H,6.26; N,7.01; S,8.16 EXAMPLE 34 (S)-1 [N-(2-mercapto-1 -oxo-3-phenyl propyl )-L-alanyl-L-proline A. (S)-2-acetylthio-3-phen ylpropionic acid Following the procedure of part (A) of Example 33 and substituting D-phenylalanine for L-phenylalanine, m.p. 1 46-1470C from ethyl acetate as the dicyclohexylamine salt.The free acid was regenerated with hydrochloric acid and extracted with ethyl acetate, [a]D5 = -70.1 (C=1.91, (C=1.91,chloroform) B. (S)-(2-acetylthio-3-phenyl)propionyl-L-alanyl-L-proline By reacting the material from part (A) above with L-alanyl-L-proline according to the procedure of Example 33(B), the above titled compound is obtained as oily residue which on trituration with ethyl ether and hexane solidifies, m.p. 161-1630C, [a]D5 = -109.6 (C=1.5, chloroform).

C. (S)- 1-[N-{2-mercapto- 1-oxo-3-phenylpropyl)-L-alanyl-L-proline The above titled product is obtained by treating the material from part (B) above according to the procedure of Example 33(C), as a foamy solid, [a]D5 = 82.7 (C=1.5, chloroform).

Analysis Calcd. for C17H22N204S.H20 Calcd: C,55.42; H,6.56; N,7.60; S,8.70 Found: C,55.25; H,6.18; N,7.36; S,8.43 SH titration: 99.1% EXAMPLE 35 N-[N-[2-(Mercaptomethyl)-1 -oxo-3-phenylpropyl]glycyl]-L-arginine A. N-[2-[gAcetylthio)methyll- l-oxo-2-phen ylprop yl]glycine A 13 gram (0.067 mole) sample of benzyl malonic acid was mixed with 7.6 grams (0.068 mole) of 40% aqueous dimethylamine and 5.4 grams (0.068 mole) of 37% formalin in 150 ml of water. The voluminous solid which formed in 15 minutes was filtered after 2 hours, washed with water and dried partially in air to give 20.8 grams (theory=16.8 grams).The solid was melted in a 170" oil bath and heated for 10 minutes, until amine evolution had stopped and bubbling had virtually ceased. The cooled product, a mobile liquid, was acidified with 10% KHSO4, extracted with hexane, dried (Na2SO4) and evaporated to give 6.3 grams of solid.

The aqueous filrates from the Mannich reaction were allowed to stand overnight and were then heated at 100"C on a steam cone until bubbling ceased (2 hours). Cooling, acidification and extraction as above gave another 1.2 grams of solid for a total of 7.5 grams (75%) of benzylacrylic acid.

A 6.2 gram (0.04 mole) sample of benzylacrylic acid in 350 ml of dichloromethane was treated with 5.4 grams (004 mole) of ethyl glycinate hydrochloride and 3.9 grams (0.04 mole) triethylamine. The mixture was cooled in ice and 7.9 grams (0.04 mole) dicyclohexyl carbodiimide was added. The mixture was stirred at 25"C overnight, then filtered. The filtrates were shaken with aqueous bicarbonate, 10% KHSO4, then water, and dried (Na2SO4) and evaporated to an oil. This was taken up in 200 ml of methanol and treated with excess 10% NaOH. The mixture was heated for 15 minutes on a steam cone, then evaporated to an aqueous slurry. This was filtered and the filtrates extracted with ether.The aqueous was acidified (10% HCI) and extracted with chloroform, the extracts dried (Na2SO4) and evaporated to an oil 7.3 grams (80%).

Thiolacetic acid (10 ml) was added to 3.2 grams (0.014 mole) of the glycine derivative and the resulting solution was evaporated the next day in vacuo. Trituration with isopropyl ether gave a near-white solid, 2.9 grams. Recrystallization from ethyl acetate-hexane gave 2.5 grams (60%), m.p. 97-1010C.

Anal. Calcd. for: C14H17N04S Calcd.for: C,56.93; H,5.80; N,4.74; S,10.85 Found: C,56.68; H,5.87; N,4.77; S,10.81 B. N-[N-[2-[IA cetylthiolmeth y- 1-oxo-3-phenylpropyliglycyl-L-arginine A 0.3 gram sample (1 mmole) of the product of part (A) was dissolved in 25 ml of dry tetrahydrofuran, 0.11 g (1.1 mmole) of triethylamine was added and the mixture cooled to 0 C. Then 0.11 g (1 mmole) of ethyl chloroformate was added, and after the mixture was stirred for 40 minutes at 0 , it was filtered into a solution of 0.17 g (1 mmole) of arginine in 10 ml of water. After stirring at 25"C overnight the mixture was evaporated to an aqueous mixture which was diluted to 30 ml with water and extracted gently (to avoid emulsions) with ethyl acetate.The aqueous was chromatographed on 50 g of Avicel in water, taking 25 ml fractions. The two Sakaguchi positive fractions were combined and filtered through a nuclepore polycarbonate filter and lyophilized to give 0.3 g (63%) white powder, m.p. 105-120", which, after drying at 95-100"C for 6 hours in vacuo analyzed correctly for the presence of 1.5moles of water.

Anal. Calcd. for: C20H29N5O5S.1 .5H2o; Calcd. for: C,50.19; H,6.74; N,14.63; S,6.70 Found: C,50.38; H,6.56; N,14.95; S,6.50 C. N-IN-12- (Mercap tom ethyl)- 1-oxo-3-phen ylprop yI]glycyl]-L-arginine A 0.5 g (1.1 mmole) sample of the product of Part (B) was cooled in ice and treated with 4 ml of conc.

NH40H under argon. After stirring for 1 hour, the sample was evaporated in vacuo to a glass. This was triturated with 25 ml of acetonitrile for 2 hours, then the solvent was decanted and replaced with fresh solvent and stirred for 2 1/2 days under argon. The resulting white solid was filtered and washed with acetonitrile and ether. Drying at 45" over KOH, P205 and paraflin at 0.01 mm Hg overnight to give 0.25 g (55%) of solid, m.p. 120-137".

Anal. Calcd. for: Ca8H27N504S.1.75 H20 Calcd.for: C,49.02; H,6.97; N,15.88; S,7.27 Found: C,48.89; H,6.67; N,16.17; S,7.28

Claims (42)

1. Acompound having the formula

or such a compound in alkyl ester or salt form, wherein R1 is hydrogen, alkanoyl, benzoyl or

R2 is hydrogen, alkyl or phenylalkyl; nisOor1;and A1 and A2 each is an a-amino or a-imino acid residue joined through a peptide bond.

2. A compound in accordance with claim 1 wherein R1 is hydrogen.

3. A compound in accordance with claim 1 wherein R1 is alkanoyl.

4. A compound in accordance with claim 1 wherein R1 is

5. A compound in accordance with any of claims 1 - 4 wherein R2 is hydrogen.

6. A compound in accordance with any of claims 1 - 4 wherein R2 is alkyl.

7. A compound in accordance with any of claims 1 - 4 wherein R2 is phenylalkyl.

8. A compound in accordance with any of claims 1 - 7 wherein n is0.

9. A compound in accordance with any of claims 1 - 7 wherein n is 1.

10. A compound in accordance with any of claims 1 - 9 wherein A1 is an a-amino acid residue and A2 is an a-imino acid residue.

11. A compound in accordance with claim 10 wherein A2 is L-proline.

12. A compound in accordance with any of claims 1 - 9 wherein A1 and A2 each is independently proline, 4-hydroxyproline, 4,4-ethylenedioxyproline, 4-methoxyproline, 4-thiazolidinecarboxylic acid, tryptophane, glycine, alanine, leucine, isoleucine orvaline.

13. The compound in accordance with claim 1,1-lN-(acetylthioacetyl)glycyl]-L-proline.

14. The compound in accordance with claim 1, 1-(N-(mercaptoacetyl)glycylj-L-proline.

15. The compound in accordance with claim 1, t-butyl ester of (S)-1.[3-(acetylthio)-2-methyl-1-oxo- propyl]-L-prolyl-L-proline.

16. The compound in accordance with claim 1, 1 -(S-3-mercapto-2-methyl-1 -oxopropyl)-L-prolyl-L- proline.

17. The compound in accordance with claim 1, N-(acetylthioacetyl)-L-valyl-L-proline.

18. The compound in accordance with claim 1, N-(mercaptoacetyl)-L-valyl-L-proline.

19. The compound in accordance with claim 1, 1 -[S-3-(acetylthio)-2-methyl-1 -oxopropylj-L-prolylglycine, ethyl ester.

20. The compound in accordance with claim 1, 1 -(S-3-mercapto-2-methyl-1 -oxopropyl)-L-prolyl-glycine, lithium salt.

21. The compound in accordance with claim 1, (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-L-prolyl-L- alanine.

22. The compound in accordance with claim 1, acetylthioacetyl-valyl-tryptophan methyl ester.

23. The compound in accordance with claim 1, N-(mercaptoacetyl)-L-valyl-L-tryptophan, lithium salt.

24. The compound in accordance with claim 1, (S)-[3-(acetylthio)-2-methyl-1-oxopropyl]-L-prolyl-D- alanine methyl ester.

25. The compound in accordance with claim 1, (S)-(3-mercapto-2-methyl-1 -oxopropyl)-L-propyl-D- alanine.

26. The compound in accordance with claim 1, 1-[N-(mercaptoacetyl)-L-alanylj-L-proline.

27. The compound in accordance with claim 1, 1-[N-(3-mercapto-1-oxopropyl]-L-alanyl]-L-proline.

28. The compound in accordance with claim 1, acetylthioacetic-L-alanyl-L-tryptophan.

29. The compound in accordance with claim 1, N-[N-(mercaptoacetyl)-L-alanyl-L-tryptophan.

30. The compound in accordance with claim 1, (S)-i -(3-acetylthio-2-methyl-1 -oxo-propyl)-L-prolyl-B- alanine.

31. The compound in accordance with claim 1, (S)-i -(3-mercapto-2-methyl-1 -oxopropyl)-L-prolyl-B- alanine.

32. The compound in accordance with claim 1, R-(2-acetylthio-3-phenyl)propionyl-L-alanyl-L-proline.

33. The compound in accordance with claim 1, R-1 -[N-(2-mercapto-1 -oxo-2-phenyl-propyl )-L-alanyl-Lproline.

34. The compound in accordance with claim 1, (S)-(2-acetylthio-3-phenyl)propionyl-L-alanyl-L-proline.

35. The compound in accordance with claim 1, (S)-1 -[N-(2-mercapto-1 -oxo-3-phenyl-propyl)-L-alanyl-Lproline.

36. A method for reducing blood pressure in mammals which comprises administering to a mammal in need thereof, an effective amount of a compound having the formula

or an alkyl ester or a physiologically acceptable salt thereof, wherein R1 is hydrogen, alkanoyl or

R2 is hydrogen, alkyl or phenylalkyl, n is0 or 1; and A1 and A2 each is an a-amino or a-imino acid residue joined through a peptide bond.

37.A process for preparing a compound having the formula

or an alkyl ester of-a salt thereof, wherein R1 is hydrogen, alkanoyl or

R2 is hydrogen, alkyl or phenylalkyl; n isO or 1; and A1 and A2 each is an a-amino or a-imino acid residue joined through a peptide bond, characterized by acylating, directly or sequentially, a dipeptide of the formula A1-A2 or an alkyl -ester derivative thereof with a thio acid having theformula

wherein R; is alkanoyl or benzoyl, to form products wherein R1 is alkanoyl or benzoyl, and subjecting said products to conventional hydrolysis to form products wherein R1 is hydrogen, and oxidizing said products wherein R1 is hydrogen to form products wherein R1 is

38. A compound in accordance with claim 1 when prepared by a process in accordance with claim 37.

39. A compound in accordance with claim 1 as named in any of the Examples.

40. A pharmaceutical composition comprising a compound in accordance with any of claims 1 - 35, 38 and 39 and a pharmaceutical carrier.

41. A composition in accordance with claim 40 in the form of a tablet, capsule, elixir or sterile injectable preparation.

42. A composition in accordance with claim 40 or41, including an excipient, binder, preservative, stabilizer or flavor. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~