NO150360B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE BRAND CAPACYLDIPEPTIDES - Google Patents

Abstract

Analogous process for the preparation of therapeutically active, especially hypotensive, compounds of the formula. and alkyl esters and salts thereof. R 1 is hydrogen, alkanoyl, benzoyl or. hydrogen, alkyl or phenylalkyl ,. n is 0 or 1, and. A. 2 and Aer are each an α-amino or α-amino acid residue linked together through a peptide bond.

Description

Connections with the formula

and alkyl esters and salts thereof have useful hypotensive activity. In formula I and in the description, the symbols have the meanings given below.

is hydrogen, alkanoyl, benzoyl or

1*2 is hydrogen, alkyl or phenylalkyl,

n is 0 or 1, and

and A2 is each an α-amino or α-imino acid residue selected from proline, tryptophan, glycine, phenylglycine, alanine, phenylalanine, leucine, isoleucine, valine and arginine, linked together through a peptide bond.

The term "alkyl" as used herein denotes groups with

1 to 7 carbon atoms.

The term "alkanoyl" as used herein denotes alkanoyl groups of 2 to 7 carbon atoms. Acetyl is the preferred alkanoyl group.

As stated above, the groups A^ and A2 are linked together by means of "a peptide bond", i.e. the link -CO-NH- between the α-carboxyl group in the residue A^ and the α-amino or α-imino group in the group A2.

The mercaptoacyl dipeptides of formula I where R., is alkanoyl or benzoyl can be prepared by acylation of a dipeptide of the formula

or an alkyl ester derivative thereof, with a thioacid of the formula

In formula III and further in the description, the symbol ' is alkanoyl or benzoyl. The acylation described above can be carried out using any of the methods known in the art. E.g. the acylation can be carried out in the presence of a coupling agent such as a carbodiimide (of which dicyclohexylcarbodiimide is the most commonly used). Alternatively, the thioacid of formula III can be activated by forming its mixed anhydride, symmetrical anhydride, acid chloride or active ester, or by using Woodward's reagent K or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. For a more detailed description of different acylation methods, reference is made to Methoden der organischen Chemie (Houben-Weyl), Vol. XV, part II, page 1 et seq. (1974).

An alternative synthesis for the compounds of formula I where R 1 is alkanoyl or benzoyl involves stepwise acylation as opposed to the previously described direct acylation, i.e. acylation of the amino acid "A 2 " with a mercaptoalkanoyl acid of formula III (using the above described procedure) to form a mercaptoalkanoyl amino acid of the formula

followed by acylation of the amino acid "A^" or an alkyl ester thereof with a mercaptoalkanoyl amino acid of formula IV. The acylation can be carried out by any of the well-known methods for linking amino acids. For an overview of these methods, reference can be made to Bodanszky and Ondetti, Peptide Synthesis, Interscience Publishers (1966). Compounds of formula I where is hydrogen can be prepared by ammonolysis or alkaline hydrolysis of the corresponding compounds of formula I where R 1 is alkanoyl or benzoyl, which can be prepared using any of the methods described above. Compounds of formula I where R 1 is

can be produced by oxidation

of the corresponding free thiol of formula I with iodine.

Alternatives to the syntheses described above will be apparent to a person skilled in the art. If e.g. an ester of A^- A^ or A^ is used in one of the syntheses described above, the corresponding free acid can be obtained from the esterified product by acid or alkaline hydrolysis.

Mercaptoalkanoyl acids of formula III and mercaptoalkanoyl amino acids of formula IV and methods for their preparation are described in the literature, see e.g. US Patents 4,046,889, 4,105,776 and 4,053,651.

The compounds produced according to the invention form basic salts with various inorganic and organic bases which can also be produced according to 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 calcium and magnesium salts, salts with organic bases, e.g. dicyclohexylamine salts, benzathine, N-methyl-D-glucamine, hydrabamine salts, salts with amino acids such as arginine, lysine and the like. The non-toxic, physiologically acceptable salts are preferred, although other salts are also useful, e.g. for isolation or purification of the product.

The compounds of formula I and the alkyl esters and salts

hence are useful as hypoteric agents. They inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-associated hypertension. The effect of the enzyme renin on angiotensinogen,

a pseudoglobulin in blood plasma, forms angiotensin I.

Angiotensin I is converted using angiotensin-converting enzyme

enzyme (ACE) to angiotensin II. The latter is an active pressor substance that has been proposed as the substance that causes different forms of hypertension in different mammalian species, e.g. rats and dogs. The compounds produced according to the invention intervene in the angiotensinogen -*-(renin) -*■ angiotensin I -> (ACE) angiotensin II course by inhibiting the angiotensin-converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin II. By administering a composition containing one or a combination of compounds of formula I, angiotensin-associated hypertension is alleviated in mammals suffering from it. A single dose or preferably two or more divided daily doses, given on the basis of

about. 0.1 to 100 mg per kg body weight per day, preferably approx. 1 to 50 mg per kg body weight per day, is appropriate to

reduce blood pressure. The compound is preferably administered orally, but parenteral routes can also be used such as subcutaneous, intramuscular, intravenous or intraperitoneal administration.

The compounds of formula I may be prepared for use in lowering blood pressure, in such preparations 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 mixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc., in a unit dosage form according to accepted pharmaceutical practice. The amount of active substance in these preparations is such that a suitable dosage in the specified range is achieved.

The following examples illustrate the invention.

Example 1

1-[N-(acetylthioacetyl)glycyl]-L-proline

Glycyl-L-proline (1.72 g) is introduced into 10 ml of 1N sodium hydroxide with stirring in an ice bath. To this mixture is added 5 ml of 2N sodium hydroxide followed by 1.13 g of chloroacetyl chloride, and the bath is removed. After 3 hours at room temperature, thiolacetic acid (836 mg) and potassium carbonate (960 mg) are added in 10 ml of water,

and the reaction mixture is stirred for approx. 16 hours at room temperature. The reaction mixture is acidified with sulphonated polystyrene cation exchange resin, eluted with water and concentrated to dryness in vacuo. The residue is taken up in acetic acid, and the insoluble components (melting point above 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 supernatant is applied to a 60 g silica gel column and eluted with 7:3 chloroform:acetic acid to give 1.0 g of the title compound.

Example 2

1-[ N-(mercaptoacetyl)glycyl]-L-proline

1-[N-(acetylthioacetyl)glycyl]-L-proline (1.0 g) is treated

for 30 minutes under argon in a solution of 6 ml of water and 6 ml of concentrated sodium hydroxide. The solution is concentrated in vacuo, acidified with sulfonated polystyrene cation exchange resin, fed to a column of the same and eluted with water. 800 mg of material which is thereby obtained (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 fed to an 85 mL column of diethylaminoethyl-dextran anion exchange resin and eluted with a linear gradient from 0.005 M ammonium bicarbonate to 0.5 M ammonium bicarbonate (750 mL of each). The product is converted to the free acid on sulfonated polystyrene cation exchange resin and then treated with 7 ml of acetic acid and 350 ml of zinc dust with stirring under an argon blanket for 6 hours. The suspension is centrifuged, the liquid on top is concentrated to dryness in vacuo, taken up in water and lyophilized. The product is acidified with sulfonated polystyrene resin, fed to a column of the same and eluted with water to give 365 mg of the title compound.

Analysis: CgH^N^S (0, 3) H2O:

Calculated 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: calculated -i.00, found -0.9 8

Example 3

t-butyl ester of (S)-1-[3-(acetylthio)-2-methyl-1-oxopropyl]-L-prolyl-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 cool 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 approx. 16 hours. After the precipitate is removed, the resulting solution is washed successively with water, 5% sodium bicarbonate, water, 10% sodium bisulfate, and water, and then dried over sodium sulfate. The crude product (12 g) obtained after the solvent has been removed solidifies on standing. Recrystallization from ethyl acetate:pentane gives the title compound, m.p. 104-106°C.

Example 4

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

t-butyl ester of L-[S-3-(acetylthio)-2-methyl-1-oxopropyl]-L-proline (12 g) is taken up in 60 ml of redistilled trifluoroacetic acid, and the resulting solution is allowed to stand at room temperature for 1 hour. Trifluoroacetic acid is removed under vacuum, and the residue is carefully dried under high vacuum for 24 hours. The heavy oil is dissolved in 30 ml of methanol, and 20 ml of concentrated ammonium hydroxide is added to this solution. The mixture is kept at room temperature for 1 hour, and the solvent is then removed in vacuo, and the oily residue is dried and treated with 6 ml of dicyclohexylamine. The salt is treated with ether, and an oil is precipitated. The oil is taken up in ethyl acetate, and the solution is washed with 10% potassium bisulphate and then dried over sodium sulphate. After removal of the solvent, the residue is taken up in methanol, and the entire amount is absorbed on silica gel. The solvent is removed, and the silica gel containing the absorbed product is fed to a silica gel column prepared in chloroform. The column is eluted with chloroform, and the mercapto-positive fractions are collected. The solvent is removed and the residue lyophilized from water to give 1.05 g of the title compound as a hygroscopic solid.

Analysis: <C>14H22N2°4S'1/2 H2°

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. Neutralization equivalent: calculated 323, found 341.

Example 5

N-(acetylthioacetyl)-L-valyl-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 dried in vacuo for several hours. The oil is introduced

in dimethylformamide (25 ml), and to this solution is added N-hydroxy-succinimide ester of acetyl mercaptoacetic acid (2.2 g).• The reaction is carried out at pH 7.5 (regulated with triethylamine) and at room temperature for 17 hours. After removal of the solvent, the residue is taken up in ethyl acetate, and the solution is washed successively with water, 10% potassium bisulfate, water, saturated sodium bicarbonate and water, and finally dried over sodium sulfate to give 4.8 g of the title compound as an oil.

Example 6

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

N-(acetylthioacetyl)-L-valyl-L-proline (4.8 g) is introduced 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 value 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 to give 2 g of material, mp 158-162°C. Recrystallization from ethyl acetate (with the addition of a small amount of methanol) gives 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

1-( S- 3-( acetylthio)- 2- methyl- l- oxopropyl]- L- prolylglycine ethyl ester

Following the procedure of Example 3, 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 gives 11.2 g of the title compound as a viscous oil.

Example 8

( S )- 1-( 3- mercapto- 2- methyl- l- oxopropyl)- L- prolylglycine-

lithium salt

1-[S-3-(acetylthio)-2-methyl-1-oxopropyl]-L-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 set. The hydrolysis is allowed to take place at room temperature for 2 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 is dried over sodium sulfate, and the solvent is removed in vacuo. The residue (3.5 g) is taken up in water, and the pH value is adjusted to pH 2.0. The free acid is taken up again in ethyl acetate using sodium chloride to reduce the soluble

the heat in water. After removal of the solvent, the residue is lyophilized from water to give 2.16 g of the free base of the title compound. The entire amount is taken up in water and the pH value is carefully adjusted to pH 7.0 using 0.05N lithium hydroxide. The solution is lyophilized to give 1050 mg of the title compound as a hygroscopic solid.

Analysis : 1]_H17N2°4S *Li

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 9

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

By following the procedure according to examples 3 and 4, however

by using 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), you get 8 g of material. This material is passed through a silica gel column using ethyl acetate as eluent. You get two factions

(R^ = 0.76 and R^ = 0.4, silica gel, ethyl acetate).

The slowest migrating fraction (2 g R^ = 0.4) is taken up in potassium hydroxide/aqueous methanol (1.5 g potassium 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 pH 2.0. After 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, melting point 128-130°C.

Example 10

Acetylthioacetyl- valyl- tryptophan- methyl ester

A solution of 15 g of the methyl ester of t-butyloxycarbonyl-valyl-tryptophan in 150 ml of trifluoroacetic acid and 15 ml of anisole is stirred for 1 hour under nitrogen. The solution is subjected to vacuum stripping 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 hour and then at room temperature for 1 hour. The precipitated urea is filtered off, and the filtrate

subjected to stripping 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 together with sufficient N-methylmorpholine to maintain the pH at 7.5, and the resulting mixture is stirred for 16 hours at room temperature. The reaction mixture is subjected to stripping in vacuum to an oil which is distributed 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 subjected to stripping to a solid. The material is crystallized from ethyl acetate:pentane to give 10 g of the title compound, mp 110-112°C.

Example 11

N-( mercaptoacetyl)- 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

in

is driven off in a vacuum. The aqueous residue 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 to give 3.4 g of material. The solid is chromatographed on a "Sephadex LH-20" column using 7:3 methanol:water as eluent to give 1.5 g of an oil. The oil is dissolved in 1:1 ethanol:water and adjusted to pH 7.0 with 0.1N lithium hydroxide. A small amount of insoluble material is removed by filtration, and the filtrate is subjected to vacuum stripping to an oil. The oil is dissolved in water and lyophilized to give 1.5 g of a hygroscopic solid.

Analysis for <c>i8<H>22<N>3°4<S>"Li

Calculated: 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: Calculated 419, found 413

Example 12

( S)- 1-[ 3-( acetylthio)- 2- methyl- l- oxopropyl]- L- propyl- 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) is added carbonyldiimidazole (4.9 g) at 0° C. 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 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 NaHCO 3 and water and dried. After removal of the solvent, the remaining oil shows a spot on TLC (silica gel, EtOAc and CHCl3:MeOH 9:1) at Rf = 0.5 with a trace at Rf = 0.75 (in ethyl acetate). Yield: 4 g.

Example 13

( S )- 1-( 3- mercapto- 2- methyl- l-oxopropyl)- L- prolyl- D- alariin (S)-[3-(acetylthio)-2-methyl-l-oxopropyl]-L-prolyl -D-alanine methyl ester (4 g) is taken up in 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 method described in Example 9, m.p. 152-155°C as a crystalline solid. Analysis for C12<H>20<N>2°4<S><:>

Calculated: C 49.98, H 6.99, S 11.12

Found: C 50.02, H 7.24, S 10.90.

Neutralization equivalent: Calculated 288, found 284.

Example 14

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 stirred at 0°C for 30 minutes and at room temperature for 1 hour. The precipitate is removed by filtration, and to the filtrate is added L-alanyl-L-proline as the trifluoroacetate salt (prepared from t-butyloxycarbonyl-L-alanyl-L-proline, 2.8 g and trifluoroacetic acid). The pH value is adjusted to 7.5-8.0 with N-methylmorpholine, and the reaction is allowed to take place at room temperature for approx. 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 fed to a column of sulphonated polystyrene cation exchange resin and eluted 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

[α]D = -128° (c = 1.5, 50% aqueous MeOH).

Example 15

1-[ N-( mercaptoacetyl)- L- alanyT] - L- thiazolidiri- 4- carboxylic acid

By following the method according to example 14, but using the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid instead of the trifluoroacetic acid salt of L-alanyl-L-proline in the method according to example 14, the title compound is obtained.

Example 16

1-[ N-( mercaptoacetyl)- L- alanyl]- 4- methoxy- L- proline

By following the procedure according to example 14, 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 17

1-[ N-(mercaptoacetyl-L-alanyl]-4-hydroxy-L-proline

By following the procedure according to example 14, but using the trifluoroacetic acid salt of L-alanyl-4-hydroxy-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example 18

1-[ N-( mercaptoacetyl)- L- alanyl]- 4, 4- ethylenedioxy- L- proline

By following the procedure according to example 14, 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 19

1-[ N-(2-mercaptopropanoyl)-L-alanyl]-L-proline

By following the procedure according to example 14, by using 2-acetylthiopropionic acid instead of 2-acetylthioacetic acid, the title compound is obtained as a waxy solid, m.p. (47°) 54-65°C (split.)

Analysis for <C>11<H>18<N>2°4<S*H>2°

Calculated: C 46 , 63, H 6 .76 , S 9.89

Found: C 46.50, H 6.58, S 9.78

SH titration: 100%

in

Example 20

1-[ N -( 3- mercaptol- l- oxopropyl- L- alanyl]- L- proline

By following the procedure according to example 14, but by using 3-acetylthio-propionic acid instead of 2-acetylthioacetic acid, the title compound is obtained as a hygroscopic solid, m.p. (61°) 75-84°C.

Analysis for cnHi8N2°4 *0' 75 H2°

Calculated: 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-[ N-( 2- mercaptopropanoyl)- L- alanyl]- 4- hydroxy- L- proline

By following the procedure according to example 14, 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 compound is obtained.

Example 22

1-[ N-(2- mercaptopropanoyl)- L- alanyl]- L- thiazolidine- 4- carboxylic acid

By following the procedure according to example 14, but using 2-acetylthiopropionic acid instead of acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example 2 3

1-[ N-(2- mercaptopropanoyl)- L- alanyl]- 4- methoxy- L- proline

By following the procedure according to example 14, but using 2-acetylthiopropionic acid instead 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 24

1-[ N-(2- mercaptopropanoyl)- L- alanyl]- 4, 4- ethylenedioxy- L- proline

By following the procedure according to example 14, but by

using 2-acetylthiopropionic acid instead of acetylthioacetic acid and L-alanyl-4,4-ethylenedioxide-L-proline instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example 25

1-[ N-( 2- mercapto- 3- phenylpropanoyl)- L- alanyl]- 4- hydroxy- L- proline

By following the procedure according to example 14, but by

using 2-acetylthio-3-phenylpropionic 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 gives the title compound.

Example 26

1-[ N-(2- mercapto-3- phenylpropanoyl)- L- alanyl]- L- thiazolidine-4- carboxylic acid

By following the procedure according to example 14, but by using 2-acetylthio-3-phenylpropionic acid instead of acetylthioacetic acid and the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid instead of the trifluoroacetic acid salt of L-alanyl-L-proline, the title compound is obtained.

Example 2 7

1-[ N -( 2- mercapto- 3- phenylpropanoyl)- L- alanyl]- 4- methoxy- L- proline

By following the procedure according to example 14, but using 2-acetylthio-3-phenylpropionic acid instead 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, you get the title connection.

Example 2 8

I-[ N-(2- mercapto-3- propanoyl)- L- alanyl]- 4, 4- ethylenedioxy-L- proline

By following the procedure according to example 14, but using 2-acetylthio-3-phenylpropionic acid instead of acetylthioacetic 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 29

1-[ N-(mercaptoacetyl)-L-proline]-L-proline

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% 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 triethylamine (1.05 ml), and acetylthioacetic acid N-hydroxy-succinimido ester (1.7 g) is added. After storage at room temperature for approx. After 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 is concentrated to dryness, and the residue is dissolved in 7.7 ml of N sodium hydroxide with stirring under argon. After 15 minutes, the mixture is neutralized with sulphonated polystyrene cation exchange resin, fed to a column of the same resin and eluted with water. The material obtained is crystallized from ethyl acetate and has a melting point of 182-183°C.

Method B

Dicyclohexylcarbodiimide (2.06 g) and 1-(acetylthioacetyl)-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 approx.

After 16 hours, the resulting precipitate is filtered off, and the filtrate is washed until it is neutral. The organic layer is concentrated to dryness and the residue is purified by silica gel chromatography and crystallized from ether-hexane to give a material with a melting point of 95-98°C. This material is freed from protective groups by stepwise treatment with trifluoroacetic acid and sodium hydroxide as described in method A.

Example 30

Acetylthioacetyl- L- alanyl- L- tryptophan methyl ester

L-alanyl-L-tryptophan methyl ester acetate (2.5 g) is dissolved

in 20 ml of dimethylformamide, and 2.0 g of acetylthioacetic acid-N-hydroxy-succinimide ester are added. The solution's pH value is adjusted to 7.5 with N-methylmorpholine, and the mixture is stirred overnight at room temperature. The solvent is removed in vacuo, and the remaining oil is taken up in ethyl acetate, washed with water, dried and subjected to stripping in vacuo to a foamy residue

(3g). The residue is dissolved in ethyl acetate and added to a

1.5 cm x 45 cm column containing 60 g Woelm basic aluminum oxide (activity I). Elution with ethyl acetate gives 1.6 g of a foamy solid which gives a spot with Rf 0.69 on a silica gel thin layer chromatograph, chloroform-methanol (3:1).

Example 31

N-[ N-( mercaptoacetyl)- L- alanyl]- L- tryptophan- lithium salt

A solution of the product from Example 30 (1.2 g in

36 ml of methanol) is stirred with 6 ml of IN sodium hydroxide solution under a nitrogen atmosphere for 1/2 hour at room temperature. The reaction mixture is adjusted to pH 6, and the solvent is removed in vacuo. The residue is distributed between ethyl acetate and saturated sodium bicarbonate solution (1:1). The aqueous phase is washed with ethyl acetate, saturated with sodium chloride, acidified to pH 2.0 and extracted with ethyl acetate. The ethyl acetate extract is dried with sodium sulfate and subjected to vacuum stripping to a foamy residue. The residue is dissolved in a 50% aqueous ethanol solution, and the pH value is adjusted to 7 with IN lithium hydroxide solution. The solution is evaporated to dryness, and the residue is taken up in water and lyophilized to a whitish solid (390 mg).

in

Analysis for ci6H18N3°4S-Li • 2H20:

Calculated: C 49.11, H 5.67, N 10.73, S 8.18

C 48.78, H 5.07, N 10.41, S 7.85.

Example 32

( R) - 1-[ N-( 2- mercaptol- l- oxo- 3- phenylpropyl)- L- alanyl]- L- proline A. ( R)- 2- acetylthio- 3- phenyl- propionic acid

Sodium nitrite (32.75 g) is added to a 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 the temperature of the reaction mixture is maintained at 0°C. The reaction mixture is stirred for a further 1 hour at 0°C and then for 1 hour at room temperature. The reaction mixture is then extracted with ether, and the ether extract is dried over sodium sulfate. The ether is removed to leave (S)-2-bromo-3-phenyl-propionic acid as an oily residue (44.9 g) which is 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 is stirred for 1 hour and 15 minutes at room temperature. The mixture is cooled in an ice bath, and 25.3 g of (S)-2-bromo-3-phenyl-propionic acid in 25 ml of acetonitrile are added over 10 minutes. After stirring for 5 hours, the reaction mixture is filtered, and the solvent is removed in vacuo, and the oily residue is redissolved in ethyl acetate and dried, and the solvent is removed to give 24 g of impure (R)-2-acetylthio-3-phenyl-propionic acid. The impure material is purified via formation of the dicyclohexylamine salt using ether as a crystallization solvent. The free acid, [a]D =6 2°

(c = 2.8, chloroform) 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 the (R)-2-acetylthio-3-phenylpropionic acid from part (A) above with L-alanyl-L-proline by the method according to example 14,

sm.p. (124°) 135-138°C, [α]<25> = -23° (c = 1.4, chloroform).

C. R-l-[N-(2-mercaptol-l-oxo-2-phenylpropyl)-L-alanyl-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 title product is obtained as a foamy solid, [a]25 = -78.4° (c = 1.1, chloroform ).

Analysis for <c>i7<H>22<N>2°4<S>'2H2°

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 33

( S)- 1-[ N-( 2- mercaptol- l- oxo- 3- phenylpropyl)- L- alanyl- L- proline

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

By following the procedure from part (A) in example 32 and using D-phenylalanine instead of L-phenylalanine, the product with m.p. 146-147°C from ethyl acetate in the form of the dicyclohexylamine salt. The free acid is regenerated with hydrochloric acid and extracted with ethyl acetate, [a]p = -70.1° (c = 1.91, chloroform)

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

When reacting the material from part (A) above with L-alanyl-L-proline by the method according to example 32(B)

the above title compound is obtained as an oily residue which solidifies on trituration with ethyl ether and hexane, m.p. 161-163°C, [α]<25> = -109.6° (c = 1.5, chloroform).

C. (S)-1-[N-(2-mercaptol-l-oxo-3--phenylpropyl)-L-alanyl-L-proline

The above title compound is obtained by treating the material from part (B) above by the method according to example 32(C), as a foamy solid,

[α]<25> = -82.7° (c = 1.5, chloroform).

Analysis for <C>17<H>22N2<0>4<S*>H20:

Calculated: C 55.42, H 6.56, N 7.50, S 8.70

Found: C 55.25, H 6.18, N 7.36, S 8.43

SH titration: 99.1%

Example 3 4

N-[ N-[ 2-(mercaptomethyl)-l-oxo-3-phenylpropylglycyl]-L-arginine

A. N-[2-[(acetylthio)methyl]-l-oxo-2-phenylpropyl]glycine

A sample of 13 g (0.06 7 mol) of benzylmalonic acid is mixed with

7.6 g (0.068 mol) 40% aqueous dimethylamine and 5.4 g (0.068 mol) 37% formalin in 150 ml water. The voluminous, solid substance that forms within 15 minutes is filtered off after 2 hours,

washed with water and partially dried in air to give 20.8 g (theoretical = 16.8 g). The solid substance is melted in a 170° oil bath

and heated for 10 minutes until amine evolution has stopped and bubbling has effectively ceased. The cooled product,

a volatile liquid, acidified with 10% KHSO 4 , extracted with hexane, dried (Na 2 SO 4 ) and evaporated to give 6.3 g of solid. The aqueous filtrates from the Mannich reaction are allowed to stand overnight and are then heated at 100°C on a steam cone until bubbling ceases (2 hours). Cooling, acidification and extraction as above gives an additional 1.2 g of solid, to give a total of 7.5 g (75%) of benzyl acrylic acid.

A sample of 6.2 g (0.04 mol) of benzyl acrylic acid in 350 ml of dichloromethane is treated with 5.4 g (0.04 mol) of ethyl glycinate hydrochloride and 3.8 g (0.04 mol) of triethylamine. The mixture is cooled in ice, and 7.9 g (0.04 mol) of dicyclohexylcarbodiimide is added. The mixture is stirred at 25°C overnight and then filtered. The filtrates are shaken with aqueous bicarbonate, 10% KHSO 4 , then water, and dried (Na 2 SO 4 ) and evaporated to an oil. This is taken up in 200 ml of methanol and treated with an excess of 10% NaOH. The mixture is heated for 15 minutes on a steam cone and then evaporated to an aqueous slurry. This is filtered, and the filtrates are extracted with ether. The aqueous phase is acidified (10% HCl) and extracted with chloroform, the extracts are dried (Na2SO4) and evaporated to an oil, 7.3 g (80%).

Thiolacetic acid (10 ml) is added to 3.2 g (0.014 mol) of the glycine derivative, and the resulting solution is evaporated the next day in vacuo. Trituration with isopropyl ether gives an off-white solid, 2.9 g. Recrystallization from ethyl acetate-hexane gives 2.5 g (60%), m.p. 97-10l°e.

Analysis for C,.H,-N0.S:

J 14 17 4

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]- l- oxo- 3- phenylpropyl] glycyl-L- arginine

A 0.3 g sample (1 mmol) of the product from part (A) is dissolved

in 25 ml of dry tetrahydrofuran, 0.11 g (1.1 mmol) of triethylamine is added, and the mixture is cooled to 0°C. 0.11 g (1 mmol) of ethyl chloroformate is then added, and after the mixture has been stirred for 40 minutes at 0°C, it is filtered into a solution of 0.17 g (1 mmol) of arginine in 10 ml of water.nn. After stirring at 25°C overnight, the mixture is evaporated to an aqueous mixture which is diluted to 30 ml with water and extracted carefully (to avoid emulsions) with ethyl acetate. The aqueous phase is chromatographed on 50 g of "Avicel" in water, collecting 25 ml fractions.

The two Sakaguchi-positive fractions are pooled and filtered through a so-called "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 a vacuum gives the correct analysis for the presence of 1.5 mol of water.

Analysis for C20H2gN505S•1.5 H20:

Calculated: 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-[ N-[ 2-( mercaptomethyl)- l- oxo- 3- phenylpropyl] glycyl]- L- arginine

A 0.5 g (1.1 mmol) sample of the product from part (B) is cooled in ice and treated with 4 ml conc. NH^OH under argon. After stirring for 1 hour, the sample is evaporated: vacuum into a glass. This is triturated with 25 ml of acetonitrile for 2 hours, and then the solvent is decanted and replaced with fresh solvent and stirred for 2 1/2 days under argon. The resulting white solid is filtered and washed with acetonitrile and ether.

Drying at 45° over KOH, P2°5°^ paraffin at 0.01 mm Hg

overnight yields 0.25 g (55%) solid, m.p. 120-137°C.

Analysis for C gH^N^S ■ 1.75 H2.0

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

Claims (1)

Analogous method for the preparation of a therapeutically active compound of the formula or an alkyl ester or a salt thereof, where is hydrogen, alkanoyl or R2 is hydrogen, alkyl or phenylalkyl, n is 0 or 1, and A₂ and A₂ are each an α-amino or α-imino acid residue selected from proline, tryptophan, glycine, phenylglycine, alanine, phenylalanine, leucine, isoleucine, valine and arginine, linked together through a peptide bond, characterized in that a dipeptide with the formula A-^-A,, or an alkyl ester derivative thereof, is acylated, directly or stepwise, with enHthio acid of the formula where R^' is alkanoyl or benzoyl, to form products where R^ is alkanoyl or benzoyl, and the products are subjected to conventional hydrolysis to form products where R^ is hydrogen, and the products where R^ is hydrogen are oxidized to form products where R ^ is