NL8001675A - MERCAPTOACYL DIPEPTIDES. - Google Patents

Description

/ * 1 Ύ

Mercaptoacyl dipeptides.

It has been found that compounds of general formula 1 R.2 R-S- (CH0) -CH-C-A.-A0 (1) O5 and the alkyl esters and salts thereof have useful hypotensive activity. In Formula 1 and in the description below, the symbols have the following meanings:

Rj represents hydrogen, alkanoyl, benzoyl or a group of the formula R ^ 10 -S-CCH ^ -CH-C-Aj-A ^ R2 represents 0 hydrogen, alkyl or phenylalkyl, n is 0 or 1 and Aj and A2 each represent an α-amino or α-imino acid residue linked together via a peptide bond.

In this description, "alkyl" is understood to mean alkyl groups having 1-7 carbon atoms.

In this specification, "alkanoyl" is understood to mean alkanoyl groups of 2-7 carbon atoms. Acetyl is the preferred alkanoyl group.

The α-amino and α-imino acid residues, represented by Aj and A2, may be residues of either naturally occurring or synthetic acids. Examples thereof are proline, 4-hydroxyproline, 4,4-ethylenedioxyproline, 4-methoxyproline, 4-thiazolidine carboxylic acid, tryptophan, glycine, alanine, leucine, isoleucine and valine. As mentioned above, the groups Aj and A2 are linked together by a "peptide bond", -C-NH-ff i.e. the bond 0 between the α-carboxyl group of 80 0 1 6 75 2 the residue Aj and the α-amino or a-imino group of the group A2.

The mercaptoacyl dipeptides of the formula 1, wherein R 1 represents alkanoyl or benzoyl, can be prepared by acylation of a dipeptide of the formula 2 Aj-A2 (2) or an alkyl ester derivative thereof with a thioic acid of the formula 3 R2

R 1 -S- (CH 2) -CH-C-OH (3) 1n n 0 0 In the formula 3 and the description below, R 1 * is alkanoyl or benzoyl. The above acylation can be carried out using any procedures known in the art. For example, the acylation can be carried out in the presence of a coupling agent, such as a carbodiimide, of which dicyclohexylcarbodiimide is most frequently used. Alternatively, the thioic acid of the formula III can be activated by forming the mixed anhydride, symmetrical anhydride, acid chloride or active ester thereof or by using Woodward reagent K or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline . For a more detailed discussion of the various acylation techniques, reference is made to Methods of Organic Chemistry (Houben-Weyl), Vol.

XV, Part II, p. 1 et seq. (1974).

Another synthesis for the compounds of formula 1, wherein R 1 represents alkanoyl or benzoyl, comprises the sequential acylation, as opposed to the direct acylation described above, ie the acylation of the amino acid "Aj" with a mercaptoalkanoylic acid of the formula 3 (using the procedure described above) to form a mercaptoalkanoylamino acid of the formula * 2 R. * -S- (CH0) -CH-CA.

1 L II 11 1 followed by 0 acylation of the amino acid "A2" or the alkyl ester thereof with a mercaptoalkanoylamino acid of the formula 4. The acylation can be effected by any technique known in the 80 0 1 6 75 v * 3 procedures for the coupling of amino acids.

For an overview of these techniques, reference is made to Bodanszky and Ondetti, Petide Synthesis, Interscience Publishers (1966).

Compounds of formula 1, wherein R 1 represents hydrogen, may be prepared by ammonolysis or alkaline hydrolysis of the corresponding compounds of formula I, wherein R 1 represents alkanoyl or benzoyl, which may be obtained using any of the above procedures.

The compounds of formula 1, wherein R 1 represents * 2 -S- (CHJ -CH-C-A.-A-2 n-1 2 0) can be prepared by oxidation of the corresponding free thiol of the formula 1 with iodine.

Alternatives to the above syntheses will be apparent to those skilled in the art. For example, if an ester of Aj or is used in any of the above syntheses, the corresponding free acid can be obtained from the esterified product by acid or alkaline hydrolysis.

Mercaptoalkanoyl acids of the formula 3 and mercaptoalkanoyl aranoic acids of the formula 4 and methods for their preparation have been described in the literature, for example, reference may be made to US patents 4,046,889, 4,105,776 and 4,053,651.

The compounds of the invention form basic salts with different inorganic and organic bases, all of which are also within the scope of the invention. Such salts include ammonium salts, alkali metal salts such as sodium and potassium salts (which are preferred), alkaline earth metal salts such as the calcium and magnesium salts, salts with organic bases such as the dicyclohexylamine salt, benzathine, N-methyl-D -glucamine, hydrabamine salts, salts with 35 amino acids, such as arginine, lysine and the like. The non-toxic, physiologically acceptable salts are preferred, although other salts are also useful, for example, in the isolation or purification of the product.

The compounds of formula I and the alkyl esters and salts thereof are useful as hypotensive agents.

They inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-related hypertension. The action of the enzyme renin on angiotensinogen, a pseudo-globulin in blood plasma, forms angiotensin I. Angiotensin I 10 is converted into angiotensin II by angiotensin converting enzyme (ACE). The latter is an active pressor substance, which is considered to be the causative agent of varying forms of hypertension in different mammalian species, for example, rats and dogs. The compounds of the invention break the conversion order of angiotensinogen -> (renin) - * angiotensin I + (ACE) - * angiotensin II by inhibiting (blocking) angiotensin converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin II. Thus, administration of a composition containing one or more compounds of the formula 1 reduces angiotensin-dependent hypertension in affected mammalian species. A single dose, or preferably 2-4 partial doses daily, administered on a basis of about 0.1-100 mg per kg of body weight per day, and preferably about 1-50 mg per kg of body weight per day, is suitable for lowering blood pressure. The substance is preferably administered orally, but parenteral routes of administration, such as subcutaneous, intramuscular, intravenous or intraperitoneal administration, can also be used.

The compounds of the formula 1 can be used for lowering blood pressure in the form of preparations, such as tablets, capsules or elixirs for oral administration or sterile solutions or suspensions for parenteral administration. About 10-500 mg of a compound or mixture of compounds of the formula 1 are mixed with a physiologically acceptable vehicle, carrier material, excipient, binder, preservative, stabilizing agent, flavoring, etc., in a 0 0 1 6 75 5 I * unit dosage form as required in accepted pharmaceutical practice. The amount of active substance in these preparations is such that an appropriate dosage in the indicated range is obtained.

The invention is further illustrated but not limited by the following examples.

Example I

1- / N- (acetylthioacetyl) glycyl / -L-proline.

Glycyl-L-proline (1.72 g) is taken up in 10 ml of IN. sodium hydroxide with stirring in an ice bath. 5 ml of 2N are added to this. sodium hydroxide added, followed by 1.13 g chloroacetyl chloride, and the bath is removed. After 3 hours at room temperature, thiol acetic acid (836 mg) and potassium carbonate (960 mg) in 10 ml of water are added and the reaction mixture is stirred at room temperature for about 16 hours.

The reaction mixture is acidified with sulfonated polystyrene cation exchange resin, eluted with water and evaporated to dryness in vacuo. The material was taken up in acetic acid and the insoluble material (melting point above 320 ° C) is filtered off.

The filtrate is evaporated in vacuo and chloroform-acetic acid (7: 3) is added. The product is centrifuged and the supernatant is applied to a 60 g silica gel column and eluted with chloroform acetic acid (7: 3) to give 1.0 g of the title compound.

Example II

1- / N- (mercaptoacetyl) glycyl / -L-proline.

1- / N- (acetylthioacetyl) glycyl / L-proline (1.0 g) is treated in an argon atmosphere in a solution of 6 ml of water and 6 ml of concentrated sodium hydroxide for 30 minutes. The solution is concentrated in vacuo, acidified with sulfonated polystyrene cation exchange resin, applied to a column thereof and eluted with water. The 800 mg of the resulting material (plus 200 mg from a previous preparation) are chromatographed on a column of 30 g of silica gel with chloroform acetic acid (7: 3). This product (740 mg) is applied to a column of 85 ml of diethylaminoethyldextran 80 0 1 6 75 6 anion exchanger resin and eluted with a linear gradient from 0.005 M ammonium hydrogen carbonate to 0.5 M ammonium hydrogen carbonate (750 ml each). The product is converted into the free acid on sulfonated polystyrene cation exchange resin and then treated with 7 ml acetic acid and 350 ml zinc dust while stirring in an argon atmosphere for 6 hours. The suspension is centrifuged and the supernatant evaporated to dryness in vacuo, taken up in water and lyophilized. The resulting material is acidified with sulfonated polystyrene resin, applied to a column thereof and eluted with water to yield 365 mg of the title compound.

Analysis for CgH ^^ O ^ SS ^ 0 Calculated: C 42.95, H 5.68, N 11.13, S 12.72 Found: G 42.92, H 5.78, N 10.94, S 12 68 15 SH: Ber. -1.00; found. -0.98

Example III

t-butyl ester of (S) -1- / 3- (acetylthio) -2-methyl-1-oxopropyl / -L-p-ly 1-L-pro 1 ine.

To a solution of 7.8 g of 1 / S-3- (acetylthio) -Ι-ΙΟ methyl-1-oxopropyl / -L-proline in 75 ml of dichloromethane are added 5.13 g of t-butyl-L-prolinate and the solution is cooled to 5 ° C. A solution of dicyclohexylcarbodixmide in 50 ml of dichloromethane is added dropwise to this solution.

The reaction is carried out at room temperature for about 16 hours. After the precipitate is removed, the resulting solution is washed successively with water, 5% sodium hydrogen carbonate, water, 10% potassium hydrogen sulfate and water and then dried over sodium sulfate. The crude product (12 g), which is obtained after the removal of the solvent, is solidified on standing. The title compound is obtained by recrystallization from ethyl acetate-pentane; mp -104-106 ° C.

Example IV

1- (S-3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-L-proline.

35 t-butyl ester of 1- / S-3- (acetylthio) -2-methyl-1-oxopropyl / -L-proline (12 g) is taken up in 60 ml of redistillate 800 1 6 75 ♦ * 7 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 a high vacuum for 24 hours. The heavy oil is dissolved in 30 ml of methanol and 20 ml of concentrated ammonium hydroxide are added to the solution. 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 up in ethyl acetate and the solution is washed with 10% potassium hydrogen sulfate and then dried over sodium sulfate. After the removal of the solvent, the residue is taken up in methanol and the entire amount is adsorbed on silica-15 gel. The solvent is removed and the silica gel containing the adsorbed product is applied to a silica gel column prepared 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.

Analyzes for C ^ 22 ^ 2 ^ 4 ^. 1/2 ^ 0 Calculated: 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. equivalent: ber. 323, found. 341.

Example V

N- (acetylthioacety1) -L-valy1-L-proline.

A solution of t-butyloxycarbonyl-L-valyl-L-proline (3.1 g) in trifluoroacetic acid (20 ml) is kept at room temperature for 15 minutes, the solvent is removed in vacuo and the oily residue is stirred for several hour dried in vacuo. The oil is taken up in dimethylformamide (25 ml) and N-hydroxy succinimide ester of acetyl mercaptoacetic acid (2.2 g) is added to this solution.

The reaction is carried out at a pH of 7.5 (adjusted with triethylamine) and at room temperature for 17 hours. After the removal of the solvent, the residue is taken up in ethyl acetate and the solution is washed successively with water, 10% potassium hydrogen sulfate, water, saturated sodium hydrogen carbonate, water and finally dried over sodium 5 sulfate to give 4.8 g of the title compound as an oil.

Example VI

N- (mercaptoacetyl) -L-valyl-L-proline.

N- (acetylthioacetyl) -L-valyl-L-proline (4.8 g) 10 is taken up in 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 a pH of 2 with 2N. chloro-15 hydrochloric acid and the solution is saturated with sodium chloride. The product is extracted with ethyl acetate, the solution dried over sodium sulfate and the solvent removed in vacuo to give 2 g of material having a melting point of 158-162 ° C. The product is obtained by recrystallization from ethyl acetate (adding a small amount of methanol); mp 162-165 ° G. After drying a sample at 55 ° C for 2 hours, the melting point is 190-192 ° C.

Example VII

1- / S-3- (ηοβίν1ίίιίο) -2-ιηβί1ιν1-1-οχορΓορν1 / -1> ρΓο1ν ^ 1νοίη6-25 ethyl ester

According to the procedure of Example III, but using 10.4 g of 1- / S-3- (acetylthio) -2-methyl-1-oxopropyl / -L-proline, 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 dicyclohexylcarbodiimide, 11.2 g of the title compound are obtained as a viscous oil.

Example VIII

(S) -1- (3-mercapto-2-methyl-1-oxopropyl) -L-prolylglycine-35 lithium salt 1 / S-3 (acetylthio) -2-methyl-1-oxopropyl / -L-80 0 1 6 75 * 9 prolylglycine ethyl ester (11.2 g) is taken up in 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 2 hours. The organic solvent 5 is removed in vacuo and the aqueous residue is adjusted to a pH of 7.0. The product is extracted with ethyl acetate, the solution dried over sodium sulfate and the solvent removed in vacuo. The residue (3.5 g) is taken up in water and the pH is adjusted to 2.0. The free acid is again taken up in ethyl acetate using sodium chloride to reduce water solubility. After the removal of the solvent, the residue is lyophilized from water to yield 2.16 g of the free base of the title compound. The entire amount was taken up in water and the pH was carefully adjusted to 7.0 using 0.05N. lithium hydroxide. The solution is lyophilized to yield 1050 mg of the title compound as a hygroscopic solid.

Analysis for C 16 H 20 S 4 Li 20 Calculated: 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 IX

(S) -1- (3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-L-alanine.

Following the procedure of Examples III, and IV, 25 but using 7.8 g of 1- / S-3- (acetylthio) -2-methyl-1-oxopropyl / -Lp roll in, 75 ml of dimethylformamide, 6.2 g of dicyclohexyl carbodiimide, 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), 8 g of material are obtained. This material is passed through a silica gel column using ethyl acetate as an eluent. Two fractions are obtained (R ^ = 0.76 and R ^ = 0.4, silica gel, ethyl acetate).

The slower moving fraction (2 g, R ^ = 0.4) is taken up in potassium hydroxide / aqueous methanol (1.5 g potassium-35 hydroxide in 7 ml water and 35 ml 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 a pH of 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 to give 1 g of product, mp 128-130 ° C.

Example X.

Acetylthioacetylvalyltryptophan methyl ester.

A solution of 15 g of the methyl ester of t-butyloxycarbonylvalyltryptophan in 150 ml of trifluoroacetic acid and 15 ml of anisole is stirred in a nitrogen atmosphere for 1 hour. 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 g of dicyclohexylcarbodiimide in 60 ml of tetrahydrofuran. The mixture is stirred at 0 ° C for 0.5 hours and then at room temperature for 1 hour. The precipitated urea is filtered off 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 dimethylform-25 amide is added together with sufficient N-methylmorpholine to maintain the pH at 7.5 and the resulting mixture is stirred at room temperature for 16 hours. 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 hydrogen carbonate solution and water, dried over sodium sulfate and stripped to a solid. The material is crystallized from ethyl acetate-pentane to give 10 g of the title compound; melting point = 110-112 ° C.

Example XI

N- (mercaptoacetyl) -L-valyl-L-tryptophan lithium salt.

800 1 6 75 π

Acetylthioacetylvalyltryptophan 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 at room temperature in a nitrogen atmosphere for 1.5 hours, after which the methanol is stripped in vacuo. The aqueous residue is diluted with water, extracted with ethyl acetate and acidified to a pH of 2.0 with 6N. hydrochloric acid. The solid is filtered off, washed with water and dried, yielding 3.4 g of material. The solid 10 is chromatographed on a Sephadex LH-20 column using methanol-water (7: 3) as an eluent to yield 1.5 g of an oil. The oil is dissolved in ethanol-water (1: 1) and adjusted to a pH of 7.0 with 0.1N. 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 for C18H22N3 ° 4S * Li Calculated: C 51.54, H 6.25, S 7.65, Li 1.65 20 Found: C 51.94, H 6.03, S 7.28, Li 1.62

Neutralization Equivalent: Ber. 419; found. 413.

Example XII

(S) -1- / 3- (acetylthio) -2-methyl-1-oxopropyl / -L-prolyl-D-alanine methyl ester, 25 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 0 ° G. The mixture is stirred at this temperature for 1 hour, after which the solution becomes substantially clear, and D-alanine methyl ester hydrochloride (4.2 g) is added. The pH is adjusted to 7.5-8.0 with triethylamine (4.2 ml) and the reaction mixture is stirred at room temperature for 17 hours. The precipitate and solvent are removed and the oily residue is taken up in ethyl acetate. The solution is washed successively with water, 0.1N. HCl, water, saturated NaHCO2, water, and dried. After removal of the solvent, the residual oil 80 0 1 6 75 12 shows one spot on a thin layer chromatogram (tic) (silica gel, EtOAc and CHCl ^ MeOH 9: 1) at R ^ = 0.5 with a trace at R ^ « 0.75 (in ethyl acetate); yield 4 g.

Example XIII

5 (S) -1- (3-mercapto-2-methyl-1-oxopropy1) -L-proly1-D-alanine.

(S) - / 3- (acetylthio) -2-methyl-1-oxopropyl / -L-prolyl-D-alanine methyl ester (4 g) is taken up in potassium hydroxide / aqueous methanol (3 g potassium hydroxide in 14 ml 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 IX; melting point = 152-155 ° C as a crystalline solid.

Analysis for c12H20N2 ° 4S Calculated: C 49.98, H 6.99, S 11.12. Found: C 50.02, H 7.24, S 10.90.

Neutralization Equivalent: Ber. 288; found. 284.

Example XIV

1- / N- (mercaptoacetyl) -L-alanyl / -L-prOline.

Dicyclohexylcarbodiimide (2 g) is added to a solution of acetylthioacetic acid (1.3 g) and hydroxybenzotriazole (1.44 g) in tetrahydrofuran (39 ml) and the mixture is added at 0 ° C for 30 minutes and at room temperature for 1 hour. stirred. The precipitate is removed by filtration and L-alanyl-L-proline as the trifluoroacetate salt thereof (prepared from t-butyloxycarbonyl-L-alanyl-L-proline, 2.8 g, and trifluoroacetic acid) is added to the filtrate. 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 30 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 80 0 1 6 75 • * 13 point = 152-155 ° C; / ct_7D = -128 ° (c = 1.5 in 50% aqueous MeOH).

Example XV

1- / N- (mercaptoacetyl) -L-alanyl / -L-thiazolidine-4-carboxylic acid.

According to the procedure of Example XIV but using the trifluoroacetic acid salt of L-alanyl-L-thiazo-lidin-4-carboxylic acid instead of the trifluoroacetic acid salt of L-alanyl-L-proline in the procedure of example XIV, the following is obtained title connection.

Example XVI

1- / N- (mercaptoacety1) -L-alanyl / -4-methoxy-L-proline.

Following the procedure of Example XIV, but using the trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example XVII

1- / N- (mercaptoacetyl-L-alanyl / -4-hydroxy-L-proline.

Following the procedure of Example XIV, but using the trifluoroacetic acid salt of L-alanyl-4-20 hydroxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example XVIII

1 / N- (mercaptoacetyl) -L-alanyl / -4,4-ethylenedioxy-L-proline.

According to the procedure of Example XIV, but using L-alanyl-4.4-ethylenedioxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example XIX

1- / N- (2-mercaptopropanoyl) -L-alanyl / -L-proline.

Following the procedure of Example XIV, but using 2-acetylthiopropionic acid instead of 2-acetylthioacetic acid, the title compound aLs is obtained as a waxy solid; melting point (47 ° C) 54-65 ° C (dec.).

Analysis for Cj jHjg ^ O ^ S. 1/21 ^ 0 35 Calculated: C 46.63, H 6.76, S 9.89 Found: C 46.50, H 6.58, S 9.78 800 1 6 75 14 SH titration: 100%.

Example XX

1- / N- (3-mercapto-1-oxopropyl-L-alanyl / -L-proline.

Following the procedure of Example XIV, but using 3-acetylthiopropionic acid instead of 2-acetylthioacetic acid, the title compound is obtained as a hygroscopic solid; mp. (61 ° C) 75-84 ° C.

Analysis for Cj 0.751 ^ 0

Calculated: C 45.89, H 6.83, S 11.14, 10 Found: C 45.75, H 6.62, S 10.98 SH titration: 99.6%

Example XXI

1- / N- (2-mercaptopropanoyl) -L-alanyl / -4-hydroxy-L-proline.

According to the procedure of Example XIV, but using 2-acetylthiopropionic acid instead of acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-hydroxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title connection.

Example XXII

20 1- / N- (2-mercaptopropanoyl) -L-alanyl / -L-thiazolidine-4-carboxylic acid

Following the procedure of Example XIV, but using 2-acetylthiopropionic acid instead of acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid salt instead of the trifluoroacetic acid salt of L-alanyl-L-proline one the title compound.

Example XXIII

1- / N- (2-mercaptopropanoyl) -L-alanyl / -4-methoxy-L-proline.

Following the procedure of Example XIV, but using 2-acetylthiopropionic acid instead of acetyl-30 thioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L- proline, the title compound is obtained.

Example XXIV

1- / N- (2-mercaptopropanoyl) -L-alanyl / -4,4-ethylenedioxy-L-proline. Following the procedure of Example XIV, but using 2-acetylthiopropionic acid instead of acetyl thioacetic acid and L-alanyl-4,4-ethylenedioxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl- L-proline, the title compound is obtained.

Example XXV

5 l- / N- (2-mercapto-3-phenylpropanoyl) -L-alanyl / -4-hydroxy-L-proline.

Following the procedure of Example XIV, but using 2-acetylthio-3-phenylpropionic acid in place of acetylthioacetic acid and the trifluoroacetic acid salt of L-ala-ny -4-4-hydroxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl- L-proline, the title compound is obtained. Example XXVI

1- / N- (2-mercapto-3-phenylpropanoyl) -L-alanyl / -L-thiazolidine-4-carboxylic acid.

Following the procedure of Example XIV, but using 2-acetylthio-3-phenylpropionic acid instead

the title compound is obtained from acetylthioacetic acid and the trifluoroacetic acid salt of L-alkyl-L-thiazolidine-4-carboxylic acid instead of the trifluoroacetic acid salt of L-alanyl-L-proline. Example XXVII

1- / N- (2-mercapto-3-phenylpropanoyl) -L-alanyl / -4-methoxy-L-proline.

Following the procedure of Example XIV, but using 2-acetylthio-3-phenylpropionic acid in place of acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl- L-proline, the title compound is obtained.

Example XXVIII

1- / N- (2-mercapto-3-propanoyl) -L-alanyl / -4,4-ethylenedioxy-L-proline.

Following the procedure of Example XIV, but using 2-acetylthio-3-phenylpropionic acid in place of acetylthioacetic acid and L-alanyl-4,4-ethylenedioxy-L-proline in place of the trifluoroacetic acid salt of. L-alanyl-L-proline, the title compound is obtained.

35 Example XXIX

1- / N- (mercaptoacetyl) -L-proline / -L-proline.

800 1 6 75 16

Method A

Benzyloxycarbonyl-L-prolyl-L-proline (3 g) is hydrogenated in a mixture of absolute ethanol (50 ml) and N-hydrochloric acid (7.5 ml) in the presence of 10 Z's of palladium on charcoal. After 5 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 triethyl amine (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 chromatographed 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 1 hour. The reaction mixture 15 is evaporated to dryness and the residue is dissolved in 7.7 ml of N.

sodium hydroxide with stirring in an argon atmosphere. After 15 minutes, the mixture is neutralized with sulfonated polystyrene cation exchange resin, applied to a column of the same resin and eluted with water. The resulting material is crystallized from ethyl acetate and has a melting point of 182-183 ° C.

Method B

Dicyclohexylcarbodiimide (2.06 g) and 1- (acetyl-thioacetyl) -L-proline (2.3 g) are added to a solution of L-proline t-butyl ester (1.7 g) and hydroxybenzotriazole (1.5 g) in dichloromethane (15 ml), cooled 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 evaporated to dryness and the residue purified by silica gel chromatography and crystallized from ether-hexane to obtain a material having a melting point of 95-98 ° C. This material is deprotected by successive treatment with trifluoroacetic acid and sodium hydroxide as described under method (A).

35 Example XXX

Acetylthioacetic acid-L-alanyl-L-tryptophan methyl ester.

800 1 6 75 η L-alanyl-L-tryptophan methyl ester acetate (2.5 g) was dissolved in 20 ml dimethylformamide and 2.0 g acetylthioacetic acid N-hydroxysuccinimide ester was added. The pH of the solution was adjusted to 7.5 with N-methylmorpholine 5 and the mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residual oil taken up in ethyl acetate, washed with water, dried and stripped in vacuo to give a foamy residue (3g). The residue was dissolved in ethyl acetate and applied to a 1.5 x 45 cm column containing 60 g of Volum basic alumina (activity I). Elution with ethyl acetate gave 1.6 g of a foamy solid, which stained at an R 2 of 0.69 on a silica gel thin layer chromatogram, chlorofom-methanol (3: 1).

Example XXXI

N- / N- (mercaptoacetyl) -L-alanyl / -L-tryptophan-lithium salt ♦

A solution of the product of Example XXX (1.2 g in 36 ml of methanol) was stirred with 6 ml of IN. sodium hydroxide solution in a nitrogen atmosphere for 0.5 hour at room temperature. The reaction mixture was adjusted to a pH of 6 and the solvent was removed in vacuo. The residue was partitioned between ethyl acetate and saturated sodium hydrogen carbonate solution (1: 1). The aqueous phase was washed with ethyl acetate, saturated with sodium chloride, acidified to a pH of 2.0 and extracted with ethyl acetate. The ethyl acetate extract was dried with sodium sulfate and stripped in vacuo to give a foamy residue. The residue was dissolved in a 50% aqueous ethanol solution and the pH was adjusted to 7 with 1N. lithium hydroxide solution. The solution was stripped dry and the residue was taken up in water and lyophilized to a dirt white solid (390 mg).

Analysis for CjgHjgNgO ^ S.Li ^^ O Calculated: 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.

35 80 0 1 6 75 18

Example XXXII

(S) -1- (3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-B-alanine.

(A) 3.06 g of B-alanine ethyl ester hydrochloride and 2.82 ml of triethylamine were added to a solution of 5.2 g of 1- / S-3-acetylthio) -2-methyl-1-oxopropyl / - L-proline and 3.24 g of carbonyldiimidazole at 0 ° C and the resulting mixture was stirred at room temperature overnight. 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 hydrogen carbonate solution and water again, then dried over sodium sulfate and stripped to a clear oil (3.4 g). The spectral analysis showed that this material was (S) -1- (3-acetylthio-2-methyl-1-oxopropyl) -L-prolyl-B-15 alanine and this material was used as such in the next step.

(B) 3.4 g of the material from step (A) were dissolved in a solution of 43 ml of methanol and 1.7 g of potassium hydroxide in 8.6 ml of water were added. The mixture was stirred at room temperature for 1 hour, then 50 ml of water were added and the methanol was removed. The aqueous residue was acidified to a pH of 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 g), which was chromatographed on an LH-20 Sephadex column and eluted with a methanol-water solution (1: 9) to give 1.1 g of the title compound as a clear oil. TLC one spot; 0.65, silica gel, methanol.

30 Analysis for Cj2 ^ 20 ^ 2 ^ 4 ^

Calculated: C 49.99, H 6.99, N 9.71 Found: C 49.81, H 7.29, N 8.67 Example XXXIII

(R) -1- / N- (2-mercapto-1-oxo-3-phenylpropyl) -L-alanyl / -L-proline.

35 (A) (R) -2-acetylthio-3-enylpropionic acid.

Sodium nitrite (32.75 g) was added to a 800 1 6 75 19 cooled solution of L-phenylalanine (51 g) and potassium bromide (125 g) in 620 ml of 25N sulfuric acid over a period of 1 hour while maintaining the temperature of the reaction mixture at 0 ° C. The reaction mixture was stirred at 0 ° C for an additional 1 hour and then at room temperature for 1 hour. The reaction mixture was then extracted with ether and the ether extract was dried over sodium sulfate. The ether was removed leaving (S) -2-bromo-3-phenylpropionic acid as an oily residue (44.9 g), which was distilled at 142-144 ° C (0.25 mm 10 Hg).

A mixture of thioacetic acid (8.7 ml) and potassium hydroxide (6.8 g) in 225 ml acetonitrile was stirred at room temperature for 1.25 hours. The mixture was cooled in ice baths. 25.3 g of (S) -2-bromo-3-phenylpropionic acid in 25 ml of aceto-15 nitrile were added over a 10 minute period. After stirring for 5 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, yielding 24 g of crude (R) -2-acetylthio-3-phenylpropion-20 acid were obtained. The crude material was purified via the formation of the dicyclohexylamine salt using--25 ether as a crystallization solvent. The free acid, α / = = + 62 ° (c = 2.8 in chloroform), was regenerated with hydrochloric acid and extracted with ethyl acetate.

(B) R- (2-acetylthio-3-phenyl) propionyl-L-alanyl-L-proline.

The above product is prepared by reacting (R) -2-acetylthio-3-phenylpropionic acid from part (A) with L-alanyl-L-proline according to the procedure of Example XIV; 30 mp. (124 °) 135-138 ° C, α7-7 23 23 ° (c = 1.4 in chloroform).

(C) R-1- / N- (2-mercapto-1-oxo-2-phenylpropyl) -L-alanyl-L-proline.

By continuing the procedure of Example XIV with the material from section (B) and using sodium hydroxide instead of ammonium hydroxide, the title product is obtained as a foamy solid / a = 800 1 6 75 20 -78.4 ° (c = 1.1 in chloroform).

Analysis for C ^ * 2H2O

Calculated: 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 XXXIV

(S) -1- / N- (2-mercapto-1-oxo-3-phenylpropyl) -L-alanyl-L-proline.

(A) (S) -2-acetylthio-3-phenylpropionic acid.

According to the procedure of section (A) of Example XXXIII and using D-enylalanine instead of L-phenylalanine; mp. 146-147 ° C from ethyl acetate as the dicyclohexylamine salt. The free acid was regenerated with -25 hydrochloric acid and extracted with ethyl acetate; / a_ / p = -70.1 ° (c = 1.91 in chloroform).

(B) (S) - (2-Acetylthio-3-phenyl) propionyl-L-alanyl-L-proline.

By reacting the material from part (A) with L-alanyl-L-proline according to the procedure of Example XXXIII

(B) the title compound is obtained as an oily residue, which solidifies on trituration with ethyl ether and hexane; 20 mp. 161-163 ° G / a = -109.6 ° (c = 1.5 in chloroform).

(C) (S) -1- / N- (2-mercapto-1-oxo-3-phenylpropyl) -L-alanyl-L-proline.

The above title product is obtained by treating the material from section (B) according to the procedure of Example XXXIII (C) as a foamy solid -25%; / α_ / β = -82.7 (c = 1.5 in chloroform).

Analysis for. ^ 0

Calculated: 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 XXXV

N- / N- / 2- (mercaptomethyl) -1-oxo-3-phenylpropyl / glycyl / -L-arginine. (A) N- / 2 - / (acetylthio) methyl / -1-oxo-2-phenylpropyl / glycine.

A 13 g (0.067 mole) sample of benzyl malonic acid was mixed with 7.6 g (0.068 mole) 40% aqueous dimethylamine 800 1 6 75 21 and 5.4 g (0.068 mole) 37% formalin in 150 ml water. The bulky solid, which formed in 15 minutes, was filtered after 2 hours, washed with water and partially air-dried to give 20.8 g (theory 16.8 g). The solid was melted in an oil bath at 170 ° C and heated for 10 minutes until no amine development occurred and the bubble formation had clearly ceased. The cooled product, a mobile liquid, was acidified with 10% KHSO 2, extracted with hexane, dried (Na 2 SO 2) and evaporated to give 6.3 g of solid. The aqueous filtrates of the

Mannich reaction was left overnight and then heated on a steam bath at 100 ° C until bubble formation ceased (2 hours). Cooling, acidification and extraction as described above yielded 1.2 g of solid, i.e. 15 g in total of 7.5 g (75%) of benzylacrylic acid.

A 6.2 g (0.04 mol) sample of benzyl acrylic acid in 350 ml of dichloromethane was treated with 5.4 g (0.04 mol) of ethyl glycinate hydrochloride and 3.9 g (0.04 mol) of triethylamine.

The mixture was cooled in ice and 7.9 g (0.04 mol) of dicyclohexylcarbodiimide were added. The mixture was stirred at 25 ° C overnight and then filtered. The filtrates were shaken with aqueous hydrogen carbonate, 10% KHSO 2, then water, and dried (Na 2 SO 4) and evaporated to an oil. This was taken up in 200 ml of methanol and treated with an excess of 10% NaOH. The mixture was heated on a steam bath for 15 minutes and then evaporated to an aqueous suspension. This was filtered off and the filtrates were extracted with ether. The aqueous material was acidified (10% HCl) and extracted with chloroform, the extracts were dried (11 SO 2) and evaporated to an oil, 7.3 g (80%).

Thiol acetic acid (10 ml) was added to 3.2 g (0.014 mol) of the glycine derivative and the resulting solution was evaporated in vacuo the following day. Trituration with isopropyl ether gave an almost white solid, 2.9 g. Recrystallization from ethyl acetate-hexane gave 2.5 g (60%); mp. Mp 97-10 ° C.

800 1 6 75 22

Analysis for Cj ^ Hj ^ NO ^ S

Calculated: 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 - / (acetylthio) methyl / -1-oxo-3-phenyl-propyl / glycyl-L-arginine.

A 0.3 g sample (1 mmol) of the product of portion (A) was dissolved in 25 ml dry tetrahydrofuran, 0.11 g (1.1 mmol) triethylamine was added and the mixture cooled to 0 ° C. Then 0.11 g (1 mmol) ethyl chloroformate 10 was added and after the mixture was stirred at 0 ° C for 40 minutes, it was filtered in a solution of 0.17 g (1 mmol) arginine in 10 ml water. After stirring at 25 ° C overnight, the mixture was evaporated to an aqueous mixture, which was diluted to 30 ml with water and gently extracted (to avoid emulsions) with ethyl acetate. The aqueous material 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 yield 0.3 g (63%) of a white powder (melting point = 105-120 ° C) which, after drying at 95-100 ° C for 6 hours in vacuo gave a correct analysis for the presence of 1.5 moles of water.

Analysis for. 1.5 ^ 0

Calculated: C 50.19, H 6.74, N 14.63, S 6.70 25 Found: C 50.38, H 6.56, N 14.95, S 6.50 (C) N- / N - / 2- (mercaptomethyl) -1-oxo-3-phenyl-propyl / glycyl / -L-arginine.

A 0.5 g (1.1 mmol) sample of the product from portion (B) was cooled in ice and treated with 4 ml of concentrated NH 2 OH 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 and then the solvent was decanted and replaced with fresh solvent and stirred in an argon atmosphere for 2.5 days. The resulting white solid was filtered off and washed with acetonitrile and ether. Drying at 45 ° C over KOH, ^ 2 ^ 5 and 80 paraffin at 0.01 mm Hg overnight gave 0.25 g (55%) of solid; melting point = 120-137 ° C.

Analysis for 0, - ^ - 11, -0.3.1.75 H-0 18 27 5 4 2

Calculated: 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.

800 1 6 75

Claims (28)

1. Compounds of the general formula * 2 R, -S- (CH0) -CH-C-Δ, -Α »1 fa ii ff 1 L · 5 0 or alkyl esters or salts thereof, wherein R 1 represents hydrogen, alkanoyl, benzoyl or a group of the formula? 2 -S- (CH.) -CH-CA, -A'L 11 ff 1 L 10 o R 2 represents hydrogen, alkyl or phenylalkyl, n is 0 or 1 and Aj and A ^ each represent an α-amino or α-imino acid residue linked together by a peptide bond. 2. Compounds according to claim 1, characterized in that R 1 represents hydrogen. Compounds according to claim 1, characterized in that R 1 represents alkanoyl. Compounds according to claim 1, characterized in that R 1 represents a group of the formula 20 R2 -S- (CHJ -CH-C-Α, -Α. Zn „1 z 0 Compounds according to claim 1, characterized in that R 2 represents hydrogen. Compounds according to claim 1, characterized in that R 2 represents alkyl. Compounds according to claim 1, characterized in that R 2 represents phenylalkyl. 8. Compounds according to claim 1, characterized in that n is 0. Compounds according to claim 1, characterized in that η is 1. 10. Compounds according to claim 1, characterized in that Aj represents an α-amino acid residue and A2 represents an α-imino acid residue. Compounds according to claim 10, characterized in that it is L-proline. Compounds according to claim 1, characterized in that Aj and each independently represent proline, 4-hydroxyproline, 4,4-ethylenedioxyproline, 4-methoxyproline, 4-thiazolidine carboxylic acid, tryptophan, glycine, alanine, leucine, isoleucine or valine. 13. 1- / N- (Acetylthioacetyl) glycyl / -L-proline. 14. 1- (N- (Mercaptoacetyl) glycyl / -L-proline. 15. t-Butyl ester of (S) -1- / 3- (acetylthio) -2-10 methyl-1-oxo-propyl / -L- prolyl-L-proline 16. 1- (S-3-Mercapto-2-methyl-1-oxopropyl) -L-proly1-L-proline. 17. N- (Ace ty1thioacety1) -L-valy1-L-proline. 18. N- (Mercaptoacetyl) -L-valyl-L-proline. 19- / S-3- (Acetylthio) -2-methyl-1-oxopropyl / -L-prolylglycine ethyl ester. 20. 1- (S-3-Mercapto-2-methyl-1-oxopropyl) -L-prolyl-glycine lithium salt. 21. (S) -1- (3-Mercapto-2-methyl-1-oxopropyl) -L-prolyl-L-alanine. 22. Acetylthioacetyl-valy1-tryptophan-methyl ester. 23. N- (Mercaptoacetyl) -L-valy1-L-tryptophan lithium salt. 24. (S) - / 3- (Acetylthio) -2-methyl-1-oxopropyl / - L-prolyl-D-alanine methyl ester. 25. (S) -O-Mercapto-2-methyl-1-oxopropyl) -L-propyl-D-alanine. 26. 1- / N- (Mercaptoacetyl) -L-alanyl / -L-proline. 27. I- / N- (3-Mercapto-1-oxopropyl / -L-alanyl - / - L-proline. 28. Acetylthioacetic acid-L-alanyl-L-tryptophan. 29. N- / N- (Mercaptoacetyl) -L-aLanyl-L-tryptophan. 30. (S) -1- (3-Acetythio-2-methyl-1-oxo-propyl) -35 L-prolyl-B-alanine. 31. (S) -1- (3-Mercapto-2-methyl-1-oxopropyl) -L- 800 1 6 75 prolyl-B-alanine. 32. R- (2-Acetylthio-3-phenyl) propionyl-L-alanyl-L-proline. 33. R-1- / N- (2-mercapto-1-oxo-2-phenylpropyl) -L-5anyl-L-proline. 34. (S) - (2-Acetylthio-3-phenyl) propionyl-L-alanyl 1-L-proline. 35. (S) -1- / N- (2-mercapto-1-oxo-3-enylpropyl) -L-alanyl-L-proline. 36. A method of decreasing blood pressure in mammals, characterized in that an effective amount of a compound of the formula r1-s- (ch2) ii-ch-c-a1 is administered to a mammal with high blood pressure. -a2, 0 or an alkyl ester or a physiologically acceptable salt thereof, wherein R 1 represents hydrogen, alkanoyl or a group of the formula 20 * 2 -S- (CH 1) -CH-CA.-A, 2 n · 12 0 R 2 hydrogen, alkyl or phenylalkyl, n is 0 or 1 and Aj and A2 each represent an α-amino or α-imino acid residue linked via a peptide bond. 37. Process for preparing compounds of the general formula * 2 R -S-CCHJ -CH-C-A.-A. 1 zn i 30 or alkyl esters or salts thereof, wherein R 1 represents hydrogen, alkanoyl or a group of the formula * 2 -S- (CHJ -CH-C-A.-A. Z Π ff 1 z o R2 represents hydrogen, alkyl or phenylalkyl, n is 0 or 1 and Aj and A2 each represent an α-amino or α-imino acid residue linked 800 1 6 75 by a peptide bond, characterized in that one is directly or sequentially a dipeptide of the formula Aj-A2 acylates its alkyl ester derivative with a thioic acid of the formula * 2 5 R'-S- (CRJ -CH-COOH 1 n wherein R 1 'represents alkanoyl or benzoyl, to form products wherein R 1 represents alkanoyl or benzoyl, and subject these products to a conventional hydrolysis to form 10 products in which R 1 represents hydrogen and the products in which R 1 represents hydrogen oxidize to form products in which R 1 represents a group of the formula R 2 O -S- (CH 2) n-CH-CA-A2 Pharmaceutical compositions containing one or more of the compounds defined in claim 1. Pharmaceutical preparations according to claim 38 in the form of pharmaceutical articles, such as tablets or the like. 40. Compounds, preparations and methods as described in the description and / or examples. 25 800 1 6 75