LU82316A1 - MERCAPTOACYLDIPEPTITIES WITH ANTI-HYPERTENSIVE ACTION - Google Patents

Description

% - 1

Mercaptoacyldipeptides with anti-hypertensive action

The compounds of formula: R2 R2 5 ^ -S-ÎCH ^ -CH-C-A ^ A., As well as their alkyl esters and their salts, have an activity usable against hypertension. In formula I above and throughout the specification, the symbols 10 have the following definitions: is a hydrogen atom or an alkanoyl, benzoyl or -S- radical (ch2> n — CH — C — Ax —A2 is 15 1 1 2 is a hydrogen atom or an alkyl or phenylalkyl radical? N is 0 or 1; and A ^ and A2 are each a residue of a-amino acid or a-20 imino acid which are joined to each other by a peptide bond.

The term "alkyl", as used throughout the specification, denotes radicals having from 1 to 7 carbon atoms.

The term "alkanoyl", as used throughout the specification, refers to the radicals, alkanoyl having 2 to 7 carbon atoms. The acetyl radical is the preferred alkanoyl radical.

The residues of α-amino acids and α-imino acids which are represented by A. ^ and A2 can be of natural or synthetic origin. Examples of these α-amino acids and these α-imino acids are proline, 4-hvdroxyproline, 4,4-ethylenedioxyproline, 4-methoxyproline, 4-thiazolidine carboxylic acid, tryptophan, glycine , alanine, leucine, isoleucine and valine. As indicated above, the radicals Α ^^ and A2 are joined by a peptide bond, that is to say by the bond -C-NE * between the a-carboxyl group of the residue A.

* »" Ί ο L · * 2 and the a-amino or α-imino group of the remainder A ^ ·

The raercaptoacyldipeptides of formula I in which R is an alkanoyl or benzoyl radical can be prepared by acylation of a dipeptide of formula: 5 II Αχ - A2 or of an alkyl ester of this dipeptide, using a sea -capto-acid of formula: ^ 2

III I

i, _ R’-S- (CH_) -CH-C-OH ♦ 10 1 2 n | l

O

In formula III and throughout the specification, the symbol denotes an alkanoyl or benzoyl radical. The foregoing acylation can be accomplished using any of the many techniques well known in the art. For example, acylation can be carried out in the presence of a condensing agent such as a carbodiimide (of which dicyclohexylcarbodiimide is most often used). Alternatively, the mercapto acid of formula III can be activated by forming its mixed anhydride, its symmetrical anhydride, its acid chloride or an active ester, or alternatively by using the reagent K from Woodward or N-ethoxycarbonyl-2 -ethoxy-1,2-dihydroquinoline. For a better overview of the different acylation techniques, see 3 Methoden der 25 organischen Chemie (Kouben-Weyl), Vol. XV, part II, page 1 and seq. (1974).

Another mode of synthesis of the compounds of formula I in which R is an alkanoyl or benzoyl radical comprises a sequential acylation, as opposed to the direct acylation already described, that is to say the acylation of the amino- acid using a mercaptoalkanoylic acid of formula III (using the procedure described above) to obtain a mercaptoalkanoylic amino acid of formula: IV Ro i2. 35 |

Ri — S—, CH2) n — CH — C — Ai; ** then the acylation of the amino acid A2 or of its alkyl ester b ‘3 using a mercaptoalkanoylic amino acid of formula IV.

Acylation can be accomplished using any of the well known amino acid condensation techniques. To review these techniques, see Bodanszky 5 and Ondetti, Peptide Synthesis, Interscience Publishers (1966).

The compounds of formula I in which the hydrocone is can be prepared by aramoniolysis or alkaline hydrolysis of the corresponding compounds of formula I in which is an alkanoyl or benzovl radical, which can be obtained using either of the procedures described above. The compounds of formula I in which is R2 -S- (CH2) -CK-C-A ^ -Ag can be prepared by oxidation of the corresponding free thiol of formula I, using iodine.

Variants of the preceding modes of synthesis will be obvious to those who will implement the present invention. For example, if an ester of or A2 is used in one of the preceding syntheses, 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, as well as methods for preparing them, are described in the literature; 25 see for example U.S. Patent Nos. 4,046,889, 4,105,776 and 4,053,651.

The compounds according to the invention form, with various mineral and organic bases, basic salts which also fall within the scope of the invention. These 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 formed with organic bases, for example the salt of dicyclohexylamine, benzathine, N-methyl-. 35 D-glucamine, hydrabamine, salts formed with amino acids like arginine, lysine, etc. Non-toxic - and physiologically acceptable salts are preferable, but other * salts are also usable, for example to isolate or to * 4 purify the product.

The compounds of formula I, as well as their alkyl esters and their salts, are useful as antihypertensive agents. They inhibit the transformation of angiotensin 5 I, a decapeptide, into angiotensin II, and are therefore useful for reducing or reducing hypertension caused by angiotensin. The action of an enzyme, renin, on angiotensinogen, a blood plasma pseudoglobulin, produces angiotensin I. Angiotensin I is transformed by the angiotensin-converting enzyme (ETA) into angiotensin II. The latter is an active pressure product which has been made responsible for various forms of hypertension in various species of mammals, for example in rats and dogs. The compounds according to the invention intervene in the sequence angiotensinogen + 15 (renin) - * angiotensin I - * ETA - * angiotensin II by inhibiting the angiotensin transforming enzyme and by reducing or suppressing the formation of the pressure product, l 'angiotensin II. Thus, by administering a composition which contains at least one compound of formula I, hypertension due to angiotensin is alleviated in mammalian species which suffer therefrom. A single dose, or preferably two to four staggered daily doses, administered at a rate of about 0.1 to 100 mg per kilogram of body weight per day, preferably at a rate of about 1 to 50 mg per kilogram of weight body and 25 per day, suitable for reducing blood pressure. The substance is preferably administered orally, but parenteral routes such as the subcutaneous, intramuscular, intravenous or intraperitoneal routes may equally well be used.

The compounds of formula I may be packaged for use in reducing blood pressure in the form of compositions such as tablets, capsules or elixirs for oral administration, or in the form of sterile solutions or suspensions for parenteral administration. About 10 to 500 mg of a compound or a mixture of compounds of formula I is mixed with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. , under a

In * 5 unit dosage form as required by accepted pharmaceutical practice. The amount of active ingredient in these compositions or preparations is such that a suitable dosage is obtained within the indicated range.

The following examples are specific embodiments of the invention.

EXAMPLE 1 1- [N- (Acetylthioacetyl) clycyl] -L-proline

1.72 g of glycyl-L-proline are taken up in 10 ml of IN sodium hydroxide solution, with stirring in an ice bath. 5 ml of 2N sodium hydroxide are added to the mixture, then 1.13 g of chloroacetyl chloride, and. take off the bath. After 3 hours at room temperature, 836 mg of thiolacetic acid and 960 mg of potassium carbonate in 10 ml of water are added, and the reaction mixture is stirred for about 16 hours at room temperature. The reaction mixture is acidified with a cation exchange resin based on sulfonated polystyrene, eluted with water, and concentrated to dryness in vacuo. The residue is taken up in acetic acid and the insoluble materials are filtered (melting point above 320 ° C.). The filtrate is concentrated to dryness under vacuum and a 7: 3 mixture of chloroform and acetic acid is added. The product is centrifuged and the supernatant is introduced into a 60 g silica gel column, and eluted with a 7: 3 mixture of chloroform and acetic acid to give 1.0 g of the title compound .

EXAMPLE 2 1-fN- (Mercaptoacetyl) glycyl] -L-proline

1.0 g of 1- [N- (acetylthioacetyl) glycyl] -L-proline 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 a cation exchange resin based on sulfonated polystyrene, introduced into a column of the same resin, and eluted with water. At 8C0 mg of the substance thus obtained (plus 200 mg from a previous preparation), chromatography is carried out on a column of 30 g of silica gel with a 7: 3 mixture of chloroform and acetic acid. This product (740 me) is introduced into a column of 85 ml of anion exchange resin based on diethyl- * Μ * 6 aminoethyl dextran, and eluted with a linear gradient of 0.005 M ammonium bicarbonate to bicarbonate 0.5 m ammonium (750 ml each). The product is transformed into free acid using a cation exchange resin based on poly-5 styrene sulfone, then treated with 7 ml of acetic acid and 350 ml of zinc powder, stirring under a tree cover for 6 hours. The suspension is centrifuged, and the supernatant is concentrated to dryness in vacuo, then it is taken up in water and lyophilized. The resulting product is acidified with a sulfonated polystyrene resin, introduced into a column of the same resin and eluted with water, giving 365 rag of the title compound.

Analysis of: C ^ H ^^ O ^ S. 0.3

Calculated: C 42.95; K, 5.68; N, 11.13; S, 12.72.

Found: C 42.92; H, 5.78; N, 10.94; S, 12.68.

SH: calculated 1.00: found 0.98.

EXAMPLE 3

(S) -1- [3- (acetylthio) -2-methyl-l-oxopropyl] -L ~ prolyl-L-proline tert-butyl ester oo A 7.8 ç solution of 1- [S-3 - (acetylthio) -2-methyl-1-oxopropyl] -L-proline in 75 ml of dichloromethane is added 5.13 ç of tert-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 approximately 16 hours. After removing the precipitate, the resulting solution is washed successively with water, 5% sodium bicarbonate, water, 10% potassium bisulfate, and water, then dried over sulfate sodium. The crude product (12 g) which is obtained after removal of the solvent solidifies on standing. Recrystallization from a mixture of ethyl acetate and pentane gives the title compound, mp 104-106 ° C.

EXAMPLE 4 35 1- (S-3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-L-proline

12 g of tert-butyl ester of l- [S-3- (acetylthio) -2-methyl-l-oxopropyl] -L-proline are taken up in 60 ml of redistilled * Γ '7 * * trifluoroacetic acid, and the resulting solution is allowed to stand at room temperature for one hour. The 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 ammonia are added to the solution. The mixture is kept at room temperature for one hour, then the solvent is 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 precipitates. This oil is taken up in ethyl acetate and the solution is washed with 10% potassium bisulfate and then dried over sodium sulfate. After removing the solvent, the residue is taken up in methanol and the whole is adsorbed on silica gel. The solvent is removed and the silica gel containing the adsorbed product is introduced into a column of silica gel packed with chloroform. The column is eluted with chloroform, and the fractions are combined giving a positive mercaptan test. The solvent is removed and the residue is lyophilized in water to obtain 1.05 g of the title compound as a hygroscopic solid.

* VSHgO analysis:

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.

25 Neutralization equivalent: calculated 323, found, 341.

EXAMPLE 5 N- (Acetylthioacetyl) -L-valyl-L-proline

A solution of 3.1 g of tert-butyloxycarbonyl-L-valyl-L-proline in 20 ml of trifluoroacetic acid is kept at room temperature for 15 30 minutes, the solvent is removed in vacuo and the oily residue is dried under empty for several hours. The oil is taken up in 25 ml of dimethylformamide and 2.2 g of acetylmercaptoacetic acid N-hydroxysuccinimide ester 35 are added to this solution. The reaction is carried out at pH 7.5 (adjusted using triethylamine) and at room temperature for 17 hours. After removing the solvent, the residue is taken up in ethyl acetate and the solution is washed successively with water, 10% potassium bisulphate, water, sodium bicarbonate. saturated sodium, water, and finally dried over sodium sulfate, which gives 4.8 g of the title compound in the form of an oil.

EXAMPLE 6 N- (Mercaptoacétvl) -L-valyl-L-proline

4.8 g of N- (acetylthioacetyl) -L-valyl-L-proline are taken up in a mixture of 12 ml of methanol and 7.5 ml of concentrated ammonia, and the resulting solution is kept at room temperature during one hour. The pH is adjusted to 7 and the methanol is removed in vacuo. The remaining aqueous layer is acidified to pH 2 with 2N hydrochloric acid and the sodium chloride solution is saturated. The product is extracted with ethyl acetate, the solution is dried over sodium sulfate.

15 sodium and the solvent is removed in vacuo, which gives 2 g of product, melting point 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 two 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 of the ethyl ester of glycine and 5.6 ml of triethylamine in 50 ml of dichloromethane) and 8.24 g of dicyclohexylcarbo-30 diimide, 11.2 g of the title compound are obtained in the form of a viscous oil.

EXAMPLE 8

(S) -1- (3-Mercapto-2-methyl-1-oxopropyl) -L-prolylglycine lithium salt 35 11.2 g of 1- [S-3- (acetyl thio) ethyl ester are taken up ) -2-methyl-1-oxopropyl] -L-prolyglycine in 200 ml of methanol, to which an aqueous solution of 9 g of potassium hydroxide in 40 ml of water was added. The hydrolysis is allowed to take place 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 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 is adjusted to 2.0. The free acid is again taken up in ethyl acetate using sodium chloride to reduce the solubility in water.

After removing the solvent, the residue is lyophilized in water to give 2.16 g of the free base of the title compound. The whole is taken up in water and the pH is carefully adjusted to 7.0 with 0.05 N lithium hydroxide. The solution is lyophilized to obtain 1050 mg of the title compound, in the form of a hygroscopic solid.

Analysis of C ^ H ^ l ^ O ^ S. 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-1-oxopropyl) -L-prolyl-L-alanine

Following the procedure of Examples 3 and 4, but 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 of the methyl ester of L-alanine and 4.2 ml of triethylamine (pH 7.5-8.0), 8 g of product are obtained. This product is passed through a column of silica gel using ethyl acetate as the eluent. Two fractions are obtained (R ^ = 0.76 and R ^ = 0.4, silica gel, ethyl acetate).

The slow pltis fraction is taken up (2 g; R_p = 0.4) in a mixture of potassium hydroxide and aqueous methanol (1.5 g of potassium hydroxide in 7 ml of water and 35 ml of methanol), and maintained the solution at room temperature for two hours. The organic solvent is removed in vacuo and the aqueous layer is acidified to pH 2.0. After adding 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, which gives 1 g of product, melting point 128-130 ° C.

* EXAMPLE 10 * -% 10

Methyl ester of acetylthioacetyl-valyl-tryptophan

A solution of 15 g of tert-butyloxycarbonyl-valyl-tryptophan methyl ester in 150 ml of trifluoroacetic acid and 15 ml of anisole is stirred for one hour under nitrogen. The solution is concentrated 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 tetrahydrofuran. The mixture is stirred at 0 ° C for half an hour and then at room temperature for one hour. The precipitated urea is filtered and the filtrate is concentrated in vacuo to a white solid. The active ester is dissolved in 90 ml of dimethylformamide, after which a solution of the trifluoroacetic acid salt of the methyl ester of the preceding dipeptide is added to 60 ml of dimethylformamide, together with a sufficient amount of N-methylmorpholine for maintain the pH at 7.5, and the resulting mixture is stirred for 16 hours at room temperature. The reaction mixture is concentrated in vacuo to an oil which is partitioned between ethyl acetate and water. The ethyl acetate phase is washed with 2 N citric acid, water, saturated sodium bicarbonate solution, and water, and dried over sodium sulfate, then concentrated to solid. The substance is crystallized from a mixture of ethyl acetate and pentane to obtain 10 g of the title compound, melting point 110 ~ 112 ° C.

EXAMPLE 11

N- (mercaptoacetyl) -L-valyl-L-tryptophan lithium salt 5.0 g of acetylthioacetyl-valyl-tryptophan methyl ester are dissolved in 52 ml of methanol, and a solution of 2 is added. , 1 g of potash in 10.5 ml of water. The mixture is stirred under nitrogen at room temperature for an hour and a half, after which the methanol is removed in vacuo. 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, which gives 3.4 g of product. The solid is subjected to a chromatography in a column of "Sephadex LH-20" using as eluent a 7: 3 mixture of methanol and water, to obtain 1.5 g of an oil. This oil is dissolved in a 1: 1 mixture of ethanol and water and the solution is adjusted to pH 7.0 with 0.1 N lithium hydroxide. A small amount of insoluble product is filtered off. , and the filtrate is concentrated in vacuo to an oil. This oil is dissolved in water and lyophilized to obtain 1.5 g of a hygroscopic solid.

10 Analysis of .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 15 (S) -1-Γ3- (acetylthio) -2-methyl-l-oxopropyl] -L-prolyl-D-alanine methyl ester To a solution of 7.8 g of l- [S-3- (acetylthio) -2-methyl-1-oxopropyl] -L-proline in 150 ml of acetonitrile, 4.9 g of carbonyldiimidazole are added at 0 ° C. The mixture is stirred for one hour at this temperature, after which the solution becomes almost clear and 4.2 g of D-alanine methyl ester hydrochloride are added. The pH is adjusted to 7.5-8.0 with 4.2 ml of triethylamine 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 up in ethyl acetate. The solution is washed successively with water, 0.1 N hydrochloric acid, water, saturated sodium bicarbonate, water, and dried. After removal of the solvent, the remaining oil shows a spot on thin layer chromatography (silica gel, ethyl acetate and 9: 1 mixture of chloroform and methanol) at = 0.5 with a trace at R ^ = 0.75 (in ethyl acetate). Yield 4 g.

EXAMPLE 13 (S) -1- (3-Mercapto-2-methyl-1-oxopropyl) -L-prolyl-D-alanine 4 g of methyl ester of (S) - [3- (acetylthio) are taken up ) -2-methyl-l-oxopropyl] -L-prolyl-D -alanine in a mixture of potash and aqueous methanol (3 g of potassium hydroxide in 14 ml of water and 70 ml of methanol), and the solution is maintained at room temperature for 2 hours. The product is isolated (2.1 g) using the procedure described in Example 9, m.p. 152-155 ° C, in the form of a crystalline solid.

Analysis of ^ 2 ^ 20 ^ 2 ° ^: 5 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 10 To a solution of 1.3 g of acetylthioacetic acid and 1.44 g of hydroxybenzotriazole in 39 ml of tetrahydro-furan, we add 2 g of dicyclohexylcarbodiimide and the mixture is stirred at 0 ° C for 30 minutes and at room temperature for one hour. The precipitate is separated by filtration, and to the filtrate is added L-alany1-L-proline in the form of its trifluoroacetate (prepared from 2.8 g of tert-butyloxy-carbonyl-L-alanyl-L -proline 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 18 ml of methanol and 18 ml of concentrated ammonia. After 30 minutes, the mixture is concentrated in vacuo, the residue is dissolved in water and introduced into a column of sulfonated polystyrene cation exchange resin, eluting with water. This substance is further purified by chromatography on silica gel (7: 3 mixture of acetic acid and chloroform) and is crystallized from ethyl acetate, yield 850 mg, melting point 152-155 ° C, [ a] D - 128 ° (c, 1.5 in 50 S aqueous methanol).

EXAMPLE 15

1- [N-Mercaptoacetyl) -L-alanyl] -L-thiazolidine-4-carboxylic acid

Following the procedure of Example 14, but replacing the trifluoroacetic acid salt of L-alanyl-L-proline with the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4 -carboxylic in the procedure of Example 14, the title compound is obtained.

* 13 EXAMPLE 16 1- [N- (Mercaptoacetyl) -L-alanyl] -4-methoxy-L-proline

Following the procedure of Example 14, but replacing the trifluoroacetic acid salt of L-alanyl-5 L-proline with the trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline , we get the title compound.

EXAMPLE 17 1- [N- (Mercaptoacetyl-L-alanyl] -4-hydroxy-L-proline

Following the procedure of Example 14, but replacing the trifluoroacetic acid salt of L-alanyl-L-proline with the trifluoroacetic acid salt of L-alanyl-4-hydroxy-L-proline , we get the title compound.

EXAMPLE 18 1-fN- (Mercaptoacetyl) -L-alanyl] -4,4-ethylenedioxy-L-proline 15 Following the procedure of Example 14, but replacing the trifluoroacetic acid salt of L-alanyl -L-proline with L-alanyl-4,4-ethylenedioxy-L-proline, the title compound is obtained.

EXAMPLE 19 20 1- [N- (2-? 4ercaptopropanoyl) -L-alanyl 1-L-proline

Following the procedure of Example 14, but replacing 2-acetylthioacetic acid with 2-acetylthiopropionic acid, the title compound is obtained in the form of a waxy solid, m.p. (47 °) 54-65 ° C (decomposition).

25 Analysis of C ^ H ^ g ^ O ^ S. 1/2 ^ 0:

Calculated: C, 46.63; H, 6.76; S, 9.89.

Found: C, 46.50; H, 6.58; S, 9.78.

SH titration: 100%.

EXAMPLE 20 30 1- [N- (3-Mercapto-1-oxopropyl-L-alanyl] L-proline

Following the procedure of Example 14, but replacing 2-acetylthioacetic acid with 3-acetylthiopropionic acid, the title compound is obtained in the form of a hygroscopic solid, m.p. (61 °) 75-84 ° C.

• 35 Analysis of x ^ x8N2 ^ 4 ^^ '75 ^ 0:

Calculated: C, 45.89; K, 6.83; S, 11.14.

• Found: C, 45.75; H, 6.62; S, 10.98.

• SH titration: 99.6%, * “·% 14 EXAMPLE 21 1 ~ ΓN- (2-Mercaptopropanoyl) -L-alanyl] -4-hydroxy-L-proline

Following the procedure of Example 14, but replacing acetylthioacetic acid with 2-acetyl-5 thiopropionic acid and the trifluoroacetic acid salt of L-alanvl-L-proline with the acid salt trifluoroacetic of L-alanyl-4-hydroxy-L-proline, the title compound is obtained.

EXAMPLE 22

1- [N- (2-roe.rcaptopropanoyl) -L-alanyl] -L-thiazolidine-4-10 carboxylic acid

Following the procedure of Example 14, but replacing acetylthioacetic acid with 2-acetylthiopropionic acid and the trifluoroacetic acid salt of L-alanyl-L-proline with the trifluoroacetic acid salt from L-alanyl-L-thiazolidine-4-carboxylic acid, the title compound is obtained.

EXAMPLE 23 1-fN- (2-Mercaptopropanoyl) -L-alanyl] -4-methoxy-L-proline

Following the procedure of Example 14, but replacing acetylthioacetic acid with 2-acetylthiopropionic acid and the trifluoroacetic acid salt of L-alanyl-L-proline with the acid salt trifluoroacetic of L-alanyl-4-methoxy-L-proline, the title compound is obtained.

EXAMPLE 24 1-fN- (2-Mercaptopropanoyl) -L-alanyl] -4,4-ethvlenedioxy-L-proline

Following the procedure of Example 14, but replacing acetylthioacetic acid with 2-acetylthiopropionic acid and the trifluoroacetic acid salt of L-30 alanyl-L-proline with L-alanyl- 4,4-ethylenedioxy-L-proline, the title compound is obtained.

EXAMPLE 25 1- [N- (2-Mercapto-3-phenylpropanoyl) -L-alanyl] -4-hydroxy-L-proline 35 Following the procedure of Example 14, but replacing acetylthioacetic acid with 1 2-acetylthio-3-phenylpropionic acid and the trifluoroacetic acid salt of L-alanyl-L-proline by the trifluoroacetic acid salt of L- * * 15 \ alanyl-4-hydroxy-L-proline, the title compound is obtained.

EXAMPLE 26

1- [N- (2-mercapto-3-phenylpropanoyl) -L-alanyl] -L-thiazolidine-4-carboxylic acid 5 Following the procedure of Example 14, but replacing acetylthioacetic acid with 2-acetyl-thio-3-phenylpropionic acid and the trifluoroacetic acid salt of L-alanyl-L-proline by the trifluoroacetic acid salt of L-alanyl-L-thiazolidine-4-carboxylic acid, we obtains the title compound 10.

EXAMPLE 27 1- [N- (2-Mercapto-3-phenylpropanoyl) -L-alanyl] -4-methoxy-L-proline

Following the procedure of Example 14, but replacing the acetylthioacetic acid with 2-acetyl-thio-3-phenylpropionic acid and the trifluoroacetic acid salt of L-alanyl-L-proline with trifluoroacetic acid salt of L-alanyl-4-methoxy-L-proline, the title compound is obtained.

EXAMPLE 28 1- 1- [N- (2-Mercapto-3-propanoyl) -L-alanyl] -4,4-ethylenedioxy-L-proline

Following the procedure of Example 14, but replacing acetylthioacetic acid with 2-acetylthio-3-phenylpropionic acid and the trifluoroacetic acid salt of L-25 alanyl-L-proline with L- alanyl-4,4-ethylenedipxy-L-proline, the title compound is obtained.

EXAMPLE 29 1- [N- (Mercaptoacetyl) -L-prolyl] -L-proline

METHOD A

3 g of benzyloxycarbonyl-L-prolyl-L-proline are hydrogenated in a mixture of 50 ml of absolute ethanol and 7.5 ml of N hydrochloric acid, in the presence of 10% palladium on carbon. After 5 hours, the catalyst is filtered off and the solvent is removed in vacuo. The residue is dissolved in a mixture of 15 ml of dimethylformamide and 1.05 ml of triethylamine, and 1.7 g of N-hydroxy succinimide ester of acetylthioacetic acid are added. After standing at room temperature for about 16 hours, the solvents are removed in vacuo and the residue is subjected to chromatography on a column of silica gel (7: 1 mixture of benzene and hexane). This substance is dissolved in 5 ml of trifluoroacetic acid and 2 ml of anisole 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 15 minutes the mixture is neutralized with a cation exchange resin based on sulfonated polystyrene, it is introduced into a column of the same resin, and eluted with water. The substance obtained is crystallized from ethyl acetate and it has a melting point of 182-183 ° C.

METHOD B

2.06 g of dicyclohexylcarbodiimide and are added. 2.3 g of 1- (acetylthioacetyl) -L-proline to a solution of 1.7 g of tert-butyl ester of L-proline and 1.5 g of hydroxybenzotriazole in 15 ml of cooled dichloromethane 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 it is neutral. The organic layer is concentrated to dryness and the residue is purified by chromatography on silica gel and it is crystallized from a mixture of ether and hexane, which gives a substance having a melting point of 95-98 ° C. . The protective group is removed from this compound by treating it successively with trifluoroacetic acid and sodium hydroxide, as described in Method A.

EXAMPLE 30

Acetylthioacetyl-L-alanyl-L-tryptophan methyl ester 2.5 g of L-alanyl-L-tryptophan methyl ester acetate were dissolved in 20 ml of dimethylformamide and 2.0 g were added. of N-hydroxysuccinimide ester of acetylthioacetic acid. The pH of the solution was adjusted to 7.5 with N-methylmorpholine and the mixture was stirred overnight at room temperature. The solvent was removed in vacuo and the residual oil was taken up in ethyl acetate, washed with water, dried and concentrated in vacuo to sparkling residue (3 g). This residue was dissolved in ethyl acetate and passed through a 1.5 cm x 45 cm column containing 60 g of basic Woelm alumina (activity I). Elution with ethyl acetate gave 1.6 g of a foamy solid which gave a spot at = 0.69 on a thin layer chromatograph of silica gel, by elution with a 3: 1 mixture of chloroform and methanol.

EXAMPLE 31

Lithium salt of N- [N- (mercaptoacetyl) -L-alanyl] -L-tryptophan A solution of the product of Example 10 was stirred (1.2 g in 36 ml of methanol) with 6 ml of IN sodium hydroxide solution, under a nitrogen atmosphere, for half an 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 a 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 concentrated in vacuo to a frothy residue.

This residue was dissolved in a 50% aqueous ethanol solution and the pH was adjusted to 7 with an IN lithium hydroxide solution. This solution was concentrated to dryness and the residue was taken up in water and lyophilized to a whitish solid (390 µm).

25 Analysis of, Li.2H20:

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. EXAMPLE 32 (S) -1- (3-Mercapto-2-methyl-1-oxopropyl) -L-prolyl-B-alanine 30 A. 3.06 g of B-alanine ethyl ester hydrochloride was added and 2.82 ml of triethylamine to a solution of 5.2 g of l- [S-3-acetylthio) -2-methyl-l-oxopropyl] - L-proline and 3.24 g of carbonyldiimidazole at 0 ° C, and the resulting mixture was stirred overnight at room temperature. The solvent was removed from the reaction mixture and the residue was extracted with ethyl acetate. This extract was then washed with water, with a hydrochloric acid solution * ^ ^ IN, again with water, with a saturated solution of * # 18 sodium bicarbonate, and again with water, then dried over sodium sulfate and concentrated to a clear oil (3.4 g). Spectral analysis revealed that this substance was (S) -1- (3-acetylthio-2-methyl-1-oxopropyl) -L-prolyl-5 B-alanine, and was used as such in the 'next step.

B. 3.4 g of the substance 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 for one hour at room temperature, then 50 ml of water was added and the methanol was removed. The aqueous residue 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 methanolic solvent was removed to leave an oil (3.2 g) which was subjected to chromatography in a column of "Sephadex LH-20", and eluted with a 1: 9 mixture of methanol and water to obtain 1.1 g of the title compound, in the form of a clear oil. TLC: a spot at R ^ 0.65, silica gel, methanol.

20 Analysis of Cl2H20N2O4S:

Calculated: C, 49.99; H, 6.99; N, 9.71.

Found: C, 49.81; H, 7.29; N, 8.67.

EXAMPLE 33 (R) -1-fN- (2-mercapto-1-oxo-3-phenylpropyl) -L-alanyl] -L-proline 25 A. Acid (R) -2-acetylthio-3-phenylpropionic * 32.75 g of sodium nitrite was added to a cooled solution of 51 g of L-phenylalanine and 125 g of potassium bromide in 620 ml of 25N sulfuric acid, over one hour, while maintaining the temperature of the mixture. reaction 30 at 0 ° C. The reaction mixture was stirred for another 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 the (S) -2-bromo-3-phenyl-propionic acid in the form of an oily residue (44.9 g) which was distilled at 142-144 ° C (0.25 mm Hg).

We stirred for an hour and a quarter at temperature

** I

ambient a mixture of 8.7 ml of thioacetic acid and 6.8 g * 19% of potassium hydroxide in 225 ml of acetonitrile. The mixture was cooled in an ice bath and 25.3 g of (S) --2-bromo-3-phenyl ~ propionic acid in 25 ml of acetonitrile were added over 10 minutes. After stirring for 5 hours, the reaction mixture was filtered and the solvent was removed in vacuo, and the oily residue was redissolved in ethyl acetate and dried, and the solvent was removed to obtain 24 g of (R) -2-acetylthio-3-phenyl-propionic acid. This crude product was purified by formation of the dicyclohexylamine salt using ether as the crystallization solvent. The free acid was regenerated, [a] ^ + 62 ° (c = 2.8, chloroform) using hydrochloric acid, and extracted with ethyl acetate.

B. (R) - (2-acetylthio-3-phenyl) propionyl-L-alanyl-15 L-proline

The above product is prepared by reacting (R) -2-acetylthio-3-phenylpropionic acid from part (A) above with L-alanyl-L-proline according to the procedure of Example 14, pf (1240) 135-138 ° C, [a] ^ 5 - 23 ° (C = 1.4, 20 chloroform).

C. (R) -1- [N- (2-mercapto-1-oxo-2-phenylpropyl) -L-alanyî} -L-proline

Continuing the procedure of Example 14 with the product of part (B) above, and using sodium hydroxide in place of potassium hydroxide, the title compound is obtained in the form of a solid. sparkling, [a] ^ - 78.4 ° (c = 1.1, chloroform me).

Analysis of ci7H22N2 ° 4S * 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 34 (S) -1 [N- (2-mercapto-1-oxo-3-phenylpropyl) -L-alanyî} -L-proline A. Acid (S) -2-acetylthio-3-phenylpropionic Following the mode procedure of part (A) of Example 33 and by replacing L-phenylalanine by D-phenyl-alanine, the title compound is obtained in the form of its dicyclohexylamine salt, pf 146-147 ° C after recrystallization from ethyl acetate. The free acid is regenerated using 20% hydrochloric acid and extracted with ethyl acetate, [α] ρ ^ “70.1 ° (c, = 1.91, chloroform ).

B. (S) - (2-acetylthio-3-phenyl) propionyl-L-alanyl-L-proline By reacting the product of part (A) above with L-alanyl-L-proline according to the mode procedure of Example 33 (B), the title compound is obtained in the form of an oily residue which, by trituration with ethyl ether and hexane, solidifies, mp 161-163 ° C, 10-109.6 ° (c, = 1.5, chloroform).

C. (S) -1- [N- (2-iÎiercapto-l-oxo-3-phenylpropyl) -L- • alanyîj-L-proline

The title product is obtained by treating the substance of part (B) above according to the procedure of Example 25 33 (C), in the form of a frothy solid, [a] ^ - 82, 7 ° (c = 1.5, chloroform).

Analysis of C17H22N204S * H2 °:

Calculated: C, 55.42; K, 6.56; N, 7.60; S, 8.70. Found: C, 55.25; H, 6.18; N, 7.36; S, 8.43.

20 SH titration: 99.1%.

EXAMPLE 35 N- [N- [2- (Mercaptoroethyl) -1-oxo-3-phenylpropyl] glycyl] -L-arginine A. N- [2-f (Acetylthio) methyl] -1-oxo-2-phenylpropyl] - glycine 25 A sample of 13 g (0.067 mole) of benzylmalonic acid was mixed with 7.6 g (0.068 mole) of 40 I aqueous dimethylamine and 5.4 g (0.068 mole) of formalin. 37% in 150 ml of water. The bulky solid which had formed after 15 minutes was filtered after two hours, washed with water, and partially air dried to obtain 20.8 g (theory: 16.8 g). The solid was melted in an oil bath at 170 ° and heated for 10 minutes, until the amine evolution ceased and the boiling almost ceased. The cooled product, a mobile liquid, was acidified with 10% potassium acid sulfate, extracted with hexane, dried over sodium sulfate and concentrated to give 6 , 3 g of solid. The aqueous filtrates from the Mannich reaction (ft 21) were allowed to stand overnight and then heated to 100 ° C on a steam cone until boiling ceased (2 hours). Cooling, acidification and extraction as above gave a further 1.2 g of solid for a total of 7.5 g (75%) of benzylacrylic acid.

A sample of 6.2 g (0.04 mole) of benzylacrylic acid in 350 ml of dichloromethane was treated with 5.4 g (0.04 mole) of ethyl glycinate hydrochloride and 3.9 a (0 , 04 mole) of triethylamine. The mixture was cooled in ice and 7.9 g (0.04 mole) of dicyclohexylcarbo-diimide was added. The mixture was stirred at 25 ° C overnight and then filtered. The filtrates were shaken with aqueous bicarbonate, 10% potassium bisulfate, then with water, and dried over sodium sulfate and then concentrated to an oil. This oil was taken up in 200 ml of methanol and treated with an excess of 10% sodium hydroxide.

The mixture was heated for 15 minutes on a steam cone, then concentrated to an aqueous slurry. This slurry was filtered and the filtrates were extracted with ether. The aqueous layer was acidified with 10% hydrochloric acid and extracted with chloroform, the extracts dried over sodium sulfate and concentrated to an oil, 7.3 g (80% ).

10 ml of thio-acetic acid was added to 3.2 g (0.014 mole) of the glycine derivative, and the mixture was concentrated. next day the resulting solution under vacuum. Trituration with isopropyl ether gave an almost white solid weighing 2.9 g. Recrystallization from a mixture of ethyl acetate and hexane gave 2.5 g (60%), m.p. 97-101 ° C.

30 Analysis of Cl4H17N04S:

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-phenylpropyl] glycy ^ -L-arginine A sample of 0.3 g (1 mmol) of the product was dissolved of part (A) in 25 ml of dry tetrahydrofuran, 0.11 g (1.1 mmol) of triethylamine was added and the mixture was cooled to 0 ° C. Then 0.11 g (1 mmol) of ethyl chloroformate was added, and after having stirred the mixture for 40 minutes at 0 °, it was filtered in a solution of 0.17 g (1 mmol ) of arginine in 10 ml of water. After stirring at 25 ° C overnight, the mixture was concentrated to an aqueous mixture which was diluted to 30 ml with water and extracted gently (to avoid the formation of emulsions ) with ethyl acetate. The aqueous layer was chromatographed on 50 g of "Avicel" in water, taking 25 ml fractions. The two positive fractions were combined in the Sakaquchi test and filtered through a "nuclepore" polycarbonate filter, and lyophilized to obtain 0.3 g (63%) of a white powder, mp105 -120 °, which, after drying at 95-100 ° C for 6 hours under vacuum, confirmed on analysis the presence of 1.5 moles of water.

Analysis of C20H29N5 ° 5S. i f 51 ^ 0:

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) -1-oxo-3-phenylpropyl] -glycyl] -L-arginine A sample of 0.5 g (1.1 mmol) of product of part (B) and it was treated with 4 ml of concentrated ammonia under argon. After stirring for one hour, the sample was concentrated in vacuo to a glass.

This glass was triturated with 25 ml of acetonitrile for two hours, then the solvent was decanted and replaced with fresh solvent and stirred for two and a half days under argon. The resulting white solid was filtered and washed with acetonitrile and ether. Drying at 45 ° on potassium hydroxide, phosphoric anhydride and paraffin under 0.01 mm Hg overnight gave 0.25 g (55%) of solid, m.p. 120-137 °.

Analysis of C ^ gH ^ Nj-O ^ S.l ^ S H20:

Calculated: C, 49.02; H, 6.97; N, 15.88; S, 7.27,

Found: C, 48.89; K, 6.67; N, 16.17; S, 7.28, *

Claims (10)

1. Compounds of formula: R2 R1-S- (CH2) n — CH-C — A1-A2, 50 as well as their alkyl esters and their salts, in the formula of which: R ^ is a hydrogen atom or a alkanoyl radical, R0 I2 1Û benzoyl or -S- (CH2) n '- ^ - ^ 1 A2' O. R2 is a hydrogen atom or an alkyl or phenylalkyl radical; n is 0 or 1 7 and 15 and A2 are each a residue of α-amino acid or amino imino, joined together by a peptide bond. 2. Compounds according to claim 1, in which R2 is hydrogen. 3. Compounds according to claim 1, in which R ^ is the alkanoyl radical. 4. Compounds according to claim 1, in which Rj is the group: R2 -S- (CH2) n Cü — C — Αχ · Α2 _ 25 0 * 5. Compounds according to claim 1, in which R2 is hydrogen. 6. Compounds according to claim 1, in which R2 is the alkyl radical. 7. Compounds according to claim 1, in which R2 is a phenylalkyl radical. 8. Compounds according to claim 1, in which n is equal to 0. 9. Compounds according to claim 1, in which 35. is equal to 1. 10. Compounds according to claim 1, in which is a residue of α-amino acid and A2 is a residue of α-imino acid. 24k 11. Compounds according to claim 10, in which A2 is a residue of L-proline. 12. Compounds according to claim 1, in which and Ά2 are each independently one, proline residue, 5 4-hydroxyproline, 4,4-ethylenedioxyproline, 4-methoxy-proline, 4-thiazolidinecarboxylic acid, tryptophan , glycine, alanine, leucine, isoleucine or valine. 13. A compound according to claim 1, which is 1- [N- (acetylthioacetyl) glycyl] -L-proline. 14. A compound according to claim 1, which is 1- (N-mercaptoacetyl) glycyl] -L-proline. 15. The compound according to claim 1, which is the tert-butyl ester of (S) -l- [3- (acetylthio) -2-methyl-1-oxo-propyl] -L-prolyl-L-proline. 16. The compound according to claim 1, which is 1- (S-3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-L-proline. 17. A compound according to claim 1, which is N- (acetylthioacetyl) -L-valyl-L-proline. 18. A compound according to claim 1, which is N-20 (mercaptoacetyl) -L-valyl-L-proline. 19. The compound according to claim 1, which is the ethyl ester of 1- [S-3- (acetylthio) -2-methyl-1-oxopropyl] -L-prolylglycine. 20. The compound of claim 1 which is the lithium salt of 1- (S-3-mercapto-2-methyl-1-oxopropyl) -L-prolylglycine. 21. The compound according to claim 1, which is (S) -1- (3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-L-alanine. 22. The compound of claim 1, which is the methyl ester of acetylthioacetyl-valyl-tryptophan. 23. The compound according to claim 1, which is the lithium salt of N- (mercaptoacetyl) -L-valyl-L-tryptophan. 24. The compound according to claim 1, which is the (S) - [3- (acetylthio) -2-methyl-1-oxopropyl] -L-prolyl-D-alanine methyl ester. 25. The compound according to claim 1, which is (S) - (3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-D-alanine. if 26. The compound according to claim 1, which is 1- 25 »k [N- (mercaptoacetyl) -L-alanyl] -L-proline. 27. A compound according to claim 1, which is ΙΕ N- (3-mercapto-l-oxopropy1] -L-alanyl] -L-proline. 28. The compound of claim 1, which is acetylthioacetyl-L-alanyl-L-tryptophan. 29. A compound according to claim 1, which is N- [N- (mercaptoacetyl) -L-alany ^ -L-tryptophan. 30. The compound according to claim 1, which is (S) - 1- (3-acetylthio-2-methyl-1-oxopropyl) -L-prolyl-B-alanine. 31. A compound according to claim 1, which is (S) - 1- (3-mercapto-2-methyl-1-oxopropyl) -L-prolyl-B-alanine. 32. The compound according to claim 1, which is (R) - (2-acetylthio-3-phenyl) propionyl-L-alanyl-L-proline. 33. The compound of claim 1, which is (R) - 1- 1- [N- (2-mercapto-1-oxo-2-phenylpropyl) -L-alanyl] -L-proline. 34. A compound according to claim 1, which is (S) - (2-acetylthio-3-phenyl) propionyl-L-alanyl-L-proline. 35. A compound according to claim 1, which is (S) -1- [N- (2-mercapto-1-oxo-3-phenylpropyl) -L-alany ^ -L-proline. 36. Composition for reducing blood pressure in mammals, characterized in that its active principle is a compound of formula: R9 I 2 R, —S— (CH0) —CH — C — A, —A0, 1. n U lz 25 d or an alkyl ester or a physiologically acceptable salt of this compound, in the formula of which: R1 is a hydrogen atom or an alkanoyl, benzoyl or 30 I ^ -S- (CH ~) —CH — Ç — A. A9; 2 η ο i z O R2 is a hydrogen atom or an alkyl or phenylalkyl radical; 35. is 0 or 1; and and A2 are each a residue of α-amino acid or α-imino acid, joined to each other by a peptide bond. ** 37. Process for the preparation of a compound of formula: “2 6 12 R— S - (CH2) - CH — Ç — Α — Λ2, 0 5 or an alkyl ester or a salt thereof compound, in the formula of which R. ^ is a hydrogen atom or an alkanoyl, benzoyl or R2 radical I2 -S- <CH21 n-CH-fi-A1-A2 f O R2 is a hydrogen atom or an alkyl or phenylalkyl radical; n is 0 or 1; and and A2 are each a residue of α-amino acid or α-iroino acid, joined to each other by * a peptide bond, characterized in that one acylates, either directly or in several stages , a dipeptide of formula A ^ -A. · ,, or an alkyl ester derivative of this dipeptide, with a mercapto-acid of formula: ^ R 'S- (CH_) -CH-COOH 1. n 20 in which R ^ is an alkanoyl or benzoyl radical, so as to form products in which R1 is an alkanoyl or benzoyl radical, and said products are subjected to conventional hydrolysis so as to form products in which R1 is a hydrogen atom, and oxidizing said products in which 25 is a hydrogen atom so as to form products in which R1 is a group of formula: R0 O. I2 II -S- (CH „) -CH-C-Αί-A_. 2 n 12 ""