HU181965B - Process for preparing new substituted proline derivatives - Google Patents

Description

The present invention relates to novel substituted proline derivatives of formula I, their isomers and their salts with bases, and to pharmaceutical compositions containing these compounds.

In the general formula I

R is hydroxy or C 1-4 alkoxy,

R 1 and R ₄ independently of one another are hydrogen, C 1 -C 7 alkyl or phenyl (C 1 -C 7) alkyl, but R 4 may also be phenyl,

R ₂ is hydrogen, C 1-4 alkyl, phenyl (C 1-4) alkyl, triphenyl (C 1-2) alkyl, R ₅ -C- or (a)

O group of formula, and the above groups _R5 is C1-4 alkyl or phenyl and R is the (a) group of the formula, R, R ₃ and R ₄ are as defined above and at the same time the same in R, R 'R ₃ and R _{4 in} the formula I,

R ₃ is hydrogen, hydroxy or C 1-7 alkyl and n is 0, 1 or 2.

When R _{2 is} (a), the compound of formula I forms a symmetric disulfide.

The compounds of Formula I have valuable pharmacological properties, namely, their antihypertensive activity by virtue of their activity as an inhibitor of the action of the enzyme angio2 tensin.

According to its angiotensin converting enzyme inhibitory activity, the first hypotensive agent, Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro nonapeptide, is non-orally active (Biochemistry 10, 4033 (1971), Science 196). 441-444 (1977)].

The carbon atoms having the substituents R ₁ , R ₃ and R ₄ are each asymmetric carbon when the substituent is other than hydrogen; it allows the formation of asymmetric carbon atoms. Stereoisomers in which proline is in the L-configuration are preferred.

: 5 In the formula I, any alkyl group is straight or branched and if its carbon number is

Up to 4 - methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl; when its carbon number is 7, it may also be pentyl, isopentyl, hexyl or heptyl, or the corresponding isomer thereof; C 1-4 alkyl is preferred, especially methyl and ethyl. The alkoxy group may be said C 1 -C 4 linear or branched alkyl group attached to an oxygen atom such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy or tert-butoxy. Preferred are methoxy and ethoxy. A preferred phenylalkyl group is benzyl.

The novel proline derivatives of Formula I can be prepared by various methods, direct or indirect acylation.

a) a formula II proline compound - R and R ₃ are as defined above - a III general merkaptoalkánsavval formula - R _t, R ₄ and n as defined above, R ₂ is the same as _R2 defined above or represents protecting sulfur moiety - or reaction, and then deprotecting the R ₂ , if any.

The desired product can be prepared not only by the direct acylation method described above, but also indirectly by:. 10

b) the proline compound of the formula II - R and R ₃ as defined above - with an acid derivative of the formula IIa - R ₍ R ₄ and na above) X represents a halogen atom, preferably a chlorine, bromine or iodine atom or reactive 15 derivatives or, if n is only 1, with a dehydrohalogenated derivative of the general formula IIIb,

R 1 and R ₄ are reacted with the above - or a reactive derivative thereof - and the resulting compound is substituted or added with a R ₂ S- sulfide of the general formula R ₂ -SH from thiol or thiol acid 2C - R ₂ reaction and remove any R a protecting group which may be present.

c) In the formula I compounds having 25 wherein n is 1 or 2 and R ₂ is hydrogen may also be prepared by the 11 general prolinvegyületet formula - R and R ₃ have the above meanings - a general thiolactone formula IV - R _(and R _{4 is} as defined above, n is 1 or 2

When the acylation of process a) is carried out with a protected mercaptoalkanoic acid or a reactive derivative thereof to produce a compound wherein R _{2 is} hydrogen, the protected compound is represented by the formula wherein Y is a sulfur protecting group such as ρ-methoxybenzyl, 2-tetrahydro-2H-pyranyl, acetamidomethyl or R-C-CH- (CH) _n -S-

II I I

OR [R ₄ 40 or another sulfur protecting group.

Deprotection can be accomplished by conventional techniques such as reaction with hot trifluoroacetic acid, cold trifluoromethanesulfonic acid, mercury (II) acetate, sodium ammonium, zinc and hydrochloric acid in liquid ammonia or the like. For a summary of these methods, see Methoden dér Organischen Chemie (Houben-Weyl), XV. Volume,

Part I, page 736 (1974).

When an acid of formula III is used as an acylating agent, the acylation is conveniently carried out in the presence of a coupling reagent such as dicyclohexylcarbodiimide or the like, or the acid to form a mixed anhydride, symmetric anhydride, acid chloride or ester, or Woodward-K reagent, N- ( ethoxycarbonyl) -2-55-ethoxy-1,2-dihydroquinoline or the like. For methods of acylation, see Methoden der Organischen Chemie (Houben-Weyl), XV. Volume II. part, page 1 (1974).

Compounds of formula II include, for example, proline, hydroxyproline, 4-methylproline, their C 1-4 alkyl esters, etc. Most of the acylation of such compounds is described below.

According to one preferred method for the preparation of compounds of formula I, especially when R ₂ 65 is R ₅ -CO-, an acid or ester of formula II with a haloalkanoic acid of formula IIa, X is halogen, preferably chloro or bromine - react reactively. This can be accomplished by any of the known methods in which the acid of formula IIa is reacted with an acid of formula II before forming a mixed anhydride, symmetric anhydride, acid chloride, forming an active ester or Woodward-K reagent, EEDQ [N- (ethoxycarbonyl) -2-ethoxy]. -1,2-dihydroxyquinoline] or the like.

The reaction product is a compound of formula (V). Reaction of the compound of formula V in a substitution reaction with a sulfide of formula R ₂ S- from thio acid R ₂ -SH provides a product of formula I. If R _{5 is} -CO-, this product is then converted to the product of formula Ia by ammonolysis. When R ₂ protecting group, a compound of formula Ia is obtained by removing the protective group in the manner described above. When R is an ester group (i.e., R is a C 1-4 alkoxy group), the ester group may be removed, for example, by treating the ester of formula I or Ia with trifluoroacetic acid and anisole to give the corresponding free acid. When other alkoxy groups are present, the corresponding acid is obtained by alkaline hydrolysis.

In variant (b) of the process, an acrylic acid derivative of formula IIIb may also be used as starting material. The acrylic acid derivative is suitably first converted to the acid halide and then reacted with a compound of formula II to give a compound of formula Va, which is reacted with R (S) sulfide in the addition reaction as described above.

A tosyl-oxyalkanoic acid of the formula Iile may also be used to acylate the acid of formula II, followed by the substitution reaction, etc., as described above.

The acrylic acid of formula IHb can be first reacted with R ₂ S sulfide to give a product of formula III, 1, which is then converted to an acid halide, such as thionyl chloride, and then coupled to the compound of formula II, as described above. .

The acid or ester or reactive derivative of Formula II can also be acylated with a "protected" form of a lile of formula ω-mercapto-alkanoic acid, wherein R _{s is} a "protecting group". Such "protecting groups" are described above.

Following acylation, the product is deprotected by one of the known methods described above.

Another type of acylating agent may be a thiolactone, such as β-propiothiolactone, z-methyl-p-propiothiolactone or the like.

Further details of the preferred methods for preparing the compounds of the invention are provided by way of example, without limitation. "

In a particularly preferred embodiment, the acid or ester of formula II is acylated with a (haloalkanoyl) halide of formula IIIf wherein X is independently halogen, preferably chlorine or bromine;

R 1 is hydrogen, C 1 -C 7 alkyl or phenyl-C 1 -C 7 alkyl; and n is 0, 1 or 2,

R ₄ is as defined above.

The reaction is usually carried out in an alkaline medium such as dilute alkaline hydroxide solution, alkaline bicarbonate solution or alkaline carbonate solution at low temperatures, for example about 0 ° C to 15 ° C. The reaction product is also reacted with said R5S-sulfate in an alkaline medium, preferably an alkali carbonate solution, and then worked up in the usual manner. The product of the reaction, when R ₂ in formula I is a group R ₅ -CO-, can be converted to the product by ammonolysis such as an alcoholic ammonia solution or concentrated ammonium hydroxide solution, or by alkaline hydrolysis such as an aqueous alkali metal hydroxide solution. wherein R ₂ is hydrogen.

When an acid of formula 11 is used as a starting material, the final product obtained as the free carboxylic acid can be converted into an ester, for example, with a diazoalkane such as diazomethane, a 1-alkyl 1 -3- (p-tolyl) triazine, e.g. -butyl) -3- (p-tolyl) triazine or the like.

Alternatively, an ester of Formula II, preferably tert-butyl ester, is reacted in anhydrous medium, such as dichloromethane, tetrahydrofuran, dioxane, or the like with a thiolalkanoic acid of Formula III, R ₄ hydrogen, to form a cyclohexylcarbodiimide, N, In the presence of N'-carbonyldiimidazole, ethoxyacetylene, (diphenylphosphoryl) azide or the like, at a temperature in the range of from about 0 ° C to about 10 ° C. The ester group R can be removed, for example, by reaction with trifluoroacetic acid and anisole at about room temperature.

When an ester of formula II, wherein R is C 1-4 alkoxy, especially tert-butoxy, is acylated with a thiolactone, such as β-propiothiolactone, α-methyl · β-propiothiolactone or the like, the reaction is carried out in an anhydrous solvent. such as tetrahydrofuran, dioxane, dichloromethane or the like, at a temperature from about 0 ° C to about room temperature. The ester group can be removed with anisole and trifluoroacetic acid as described above.

When R ₂ is a group of formula (R), R, R ₍ , R ₃ and R _{4) are} as defined for formula I, the symmetric disulfides can be prepared by direct oxidation of a compound of formula I, R _{2, with} hydrogen.

The compounds of formula I have one or more asymmetric carbon atoms. If R,. R ₃ or R ₄ other than hydrogen, then the carbon atom to which the substituent is attached is asymmetric. Accordingly, the compounds exist in stereoisomeric forms or in racemic mixtures thereof. The preparation of all these is within the scope of the invention. The synthesis described above may use the racemate or one of the enantiomers as starting materials. When the racemic starting material is used in the synthesis, the resulting stereoisomers may be separated by conventional chromatography or fractional crystallization techniques. Generally, the L-isomer of the amino acid carbon is the preferred isomeric form. The D-muscle is also preferred for the carbon at the α-position (i.e., R, substituted on the acyl side chain) of the acyl side chain.

81 965

The compounds of the present invention form salts with a variety of inorganic and organic bases 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 calcium and magnesium salts, with organic bases such as dicyclohexylamine, N, N'-dibenzyl (ethylene). salts with N-methyl-D-glucosamine, N, N'-bis (dehydroabiethyl) ethylenediamine, salts with amino acids such as ar10 ginine, lysine and the like. Non-toxic pharmaceutically acceptable salts are preferred, although other salts are also used, for example in the isolation or purification of the product, as shown in the examples for the dicyclohexylammonium salt.

Salts are conventionally formed by reacting the free acid form of the product with one or more equivalents of the appropriate base to provide the desired cation in a solvent or medium in which the salt is insoluble or in water and freeze-drying the water. The salt is neutralized with an insoluble acid such as a cation exchange resin in the hydrogen cycle (Dowex 50 or similar polystyrene sulfonic acid resin) or aqueous acid and extracted with an organic solvent such as ethyl acetate, dichloromethane or the like to give the desired free acid form. in the case of other salts.

Further experimental details can be found in the Examples which are preferred embodiments of the invention and serve as a model for the production of other members of the group. The compounds of the invention inhibit the conversion of angiotensin I decapeptide to angiotensin ΙΙ and are therefore useful in reducing and eliminating angiotensin-induced hypertension. The effect of renin on angiotensinogen, a pseudoglo35 bulb in the blood plasma, produces angiotensin I. Angiotensin T is converted by angiotensin converting enzyme (ACE) to angiotensin ΙΙ. The latter is an active pressor substance that has been shown to cause various forms of hypertension in various mammalian strains, such as rats and dogs. By inhibiting the angiotensin converting enzyme, the compounds of the present invention interfere with the azangiotensin (renin) * angiotensin I - angiotensin II chain and reduce or eliminate the formation of the angiotensin II pressor. Thus, the administration of a compound of formula I, or a composition comprising one of its pharmaceutically acceptable salts, or combinations thereof, may alleviate angiotensin-induced hypertension in mammalian species. A single dose providing about 0.1 to 100 mg / kg / day, preferably about 1 to 50 mg / kg / day, or preferably daily

Doses in 2 to 4 doses are appropriate to reduce blood pressure, as indicated by results from model animal studies. [S. L. Engel, T. R. Schaeffer, Μ. H. Waugh and B. Rubin, Proc. Soc Exp Bioi. Med., 143, 483 (1973)]. The substance is preferably administered orally but may also be administered parenterally, for example subcutaneously, intramuscularly, intravenously or intraperitoneally.

The compounds of the present invention may be used as compositions for oral administration, such as tablets, capsules or elixirs, or as sterile solutions or suspensions for parenteral administration, for the reduction of blood pressure. In conventional pharmaceutical practice, about 10 to about 500 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof in unit dosage form are recommended.

-3181965, or mixtures thereof, with a pharmaceutically acceptable excipient, carrier, saturator, binder, preservative, stabilizer, flavor, and the like. material. The amount of active ingredient in these formulations is within the appropriate dosage range indicated above.

Examples of excipients which may be used in tablets, capsules and the like are: tragacanth, acacia, corn starch or gelatin; the saturating agent may be dicalcium phosphate; cornstarch, potato starch, alginic acid and the like; the lubricant may be magnesium stearate; a sweetening agent may be sucrose, lactose or saccharin; flavoring agents may be peppermint, juniper or cherry oil. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. There may also be various other materials which modify the physical form of the dosage unit as a wrapper or otherwise. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir contains the active ingredient, sucrose as a sweetening agent, methyl or propyl p-hydroxybenzoate as a preservative, a dye and cherry or orange as a flavoring.

Sterile injectable preparations according to standard pharmaceutical practice include the active ingredient in a carrier such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fatty carrier such as ethyl. by dissolving or suspending it. It may also contain buffering agents, preservatives, antioxidants and the like.

The invention is illustrated by the following examples and particularly preferred embodiments thereof. All temperature data are in degrees Celsius.

Example 1 1- [2- (Benzoylthio) acetyl] -L-proline

5.75 g of L-proline are dissolved in 50 ml of 1 N sodium hydroxide solution and the solution is cooled in an ice-water bath. 26 ml of 2N sodium hydroxide solution are added and

Chloroacetyl chloride (5.65 g) was added and the mixture was stirred vigorously at room temperature for three hours. add

A suspension of 7.5 g of thiobenzoic acid and 4.8 g of potassium carbonate in 50 ml of water. After stirring at room temperature for 18 hours, the reaction mixture was acidified and extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo. The residue (14.6 g) was dissolved in ethyl acetate (150 mL) and dicyclohexylamine (11 mL) was added. The crystals were filtered off and recrystallized from ethyl acetate to give 5.7 g of m.p. 151-152 °. The crystals were dissolved by shaking with 100 ml of 5% aqueous potassium bisulfate solution and 300 ml of ethyl acetate to convert the salt to acid. The organic layer was washed once with water, then dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo to give 3.45 g.

Example 2 1- (2-Mercaploacetyl) -L-proline

3.4 g of 1- [2- (benzoylthio) acetyl] -L-proline are dissolved in a mixture of 10.5 ml of water and 6.4 ml of concentrated ammonium hydroxide solution. After 1 hour, the reaction mixture was diluted with water and filtered. The filtrate was extracted with ethyl acetate, then acidified with concentrated hydrochloric acid, saturated with sodium chloride, and extracted twice with ethyl acetate. The ethyl acetate extracts were washed with saturated sodium chloride solution and evaporated to dryness to give 1.5 g. The product, 1- (2-mercaptoacetyl) -L-proline, is crystallized from ethyl acetate. 133-135 ° C.

Example 3 1- [2- (Benzoylthio) -propionyl] -L-proline

5.75 g of L-proline are dissolved in 50 ml of 1 N sodium hydroxide solution and the solution is cooled in an ice-water bath with stirring. 25 ml of 2N sodium hydroxide solution and 8.57 g of 2-bromopropionyl chloride were then added and the reaction mixture was removed from the ice-cooling bath and stirred for one hour at room temperature. A mixture of 7.5 g of thiobenzoic acid, 4.8 g of potassium carbonate and 50 ml of water is added and the mixture is stirred overnight at room temperature. After acidification with concentrated hydrochloric acid, the aqueous phase is extracted with ethyl acetate and the organic phase is washed with water, dried and evaporated to dryness. The residue (14.7 g) was chromatographed on a column of silica gel (440 g) with benzene-acetic acid (7: 1). The fractions containing the desired material were combined, evaporated to dryness and the residue was precipitated twice from ether-hexane and converted into dicyclohexylammonium salt in ether-hexane. Yield: 9.4 g; mp (142) -148-156 °. The dicyclohexylammonium salt is converted to the acid according to the procedure described in Example 1. Yield: 5.7 g.

Example 4 1- (2-Mercaptopropionyl) -L-proline

5.7 g of 1- [2- (benzoylthio) propionyl] -L-proline are dissolved in 12 ml of water and 9 ml of concentrated ammonium hydroxide with stirring. After 1 hour, the mixture was diluted with water (10 mL) and filtered. The filtrate was washed twice with ethyl acetate, evaporated to one third of its original volume, acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried and evaporated to dryness in vacuo. Crystallization of the remaining 1- (2-mercaptopropionyl) -L-proline from ethyl acetate-hexane gave 3 g of melting point (105) -116-120 °.

Example 5 - 1- [3- (Benzoylthio) -propionyl] -L-proline

5.75 g of L-proline are dissolved in 50 ml of 1N sodium hydroxide solution and the solution is cooled in an ice bath. 8.5 g

3-Bromopropionyl chloride and 27 ml of 2N sodium hydroxide solution were added and the mixture was stirred for 10 minutes in an ice bath and then for three hours at room temperature. A suspension of 7.5 g of thiobenzoic acid and 4.5 g of potassium carbonate in 50 ml of water is added and the mixture is stirred for 18 hours at room temperature. After acidification with concentrated hydrochloric acid, the aqueous phase is extracted twice with ethyl acetate. The organic layers were dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo to give 7.1 g of 1- [3- (benzoylthio) propionyl] -L-proline, which was recrystallized from ethyl acetate-hexane at 101-102 ° C. melted.

Example 6

L-Proline tert-butyl ester

230 g of L-proline are dissolved in 1 liter of water and 400 ml of 5N sodium hydroxide solution. The solution was cooled in an ice bath and 460 mL of 5N sodium hydroxide solution and 340 mL of benzyloxycarbonyl chloride were added in 5 equal portions over half an hour with vigorous stirring. After stirring for one hour at room temperature, the mixture was extracted twice with ether and acidified with concentrated hydrochloric acid. The precipitate was filtered off and dried. Yield 442 g; mp 78-80 °.

180 g of the benzyloxycarbonyl-L-proline thus obtained are dissolved in a mixture of 300 ml of dichloromethane, 800 ml of liquid isobutylene and 7.2 ml of concentrated sulfuric acid. The solution was shaken in a pressure flask for 72 hours. The pressure was released, the isobutylene was allowed to distill and the solution was washed with 5% sodium carbonate solution, water, dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo to give 205 g of (benzyloxycarbonyl) -L-proline tert-butyl. ester.

205 g of (benzyloxycarbonyl) -L-proline tert-butyl ester are dissolved in 1.2 liters of anhydrous ethanol and hydrogenated under normal pressure with 10 g of 10% palladium on carbon catalyst until only a trace of carbon dioxide can be detected in the effluent hydrogen ( 24 hours). The catalyst was filtered off and the filtrate was evaporated at 30 mm Hg. The residue was distilled in vacuo to give L-proline tert-butyl ester. Boiling point 50-5Γ / 1 mmHg.

Example 7

1- [3- (acetyl-lio) propionyl] -L-proline t-butyl phenylmethyl ester

5.13 g of L-proline tert-butyl ester are dissolved in 40 ml of dichloromethane and the solution is cooled in an ice bath. A solution of 6.18 g of dicyclohexylcarbodiimide in 20 ml of dichloromethane is added, followed immediately by 4.45 g of 3- (acetylthio) propionic acid. After stirring in an ice-water bath for 15 minutes, then at room temperature for 16 hours, the precipitate was filtered off and the filtrate was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate and washed to neutral. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo to give 9.8 g of 1- [3- (acetylthio) propionyl] -L-proline tert-butyl ester.

Example 8 1- [3- (Acetylthio) propionyl] -L-proline

4.7 g of 1- [3- (acetylthio) propionyl] -L-proline tert-butyl ester were dissolved in a mixture of 34 ml of anisole and 68 ml of trifluoroacetic acid and kept at room temperature for 1 hour. The solvent was evaporated in vacuo and the residue was stirred several times with ether-hexane.

The residue (3.5 g) was dissolved in acetonitrile (25 mL) and dicyclohexylamine (2.8 mL) was added. The crystalline salt is filtered off and recrystallized from isopropanol. Yield: 3.8 g; mp 176-177 °. The salt was converted to 1- [3- (acetylthio) propionyl] -L-proline as described in Example 1;

yield 1.25 g; m.p. 89-90 ° (crystallized from ethyl acetate-hexane).

Example 9 1- (3-Mercaptopropionyl) -L-proline tert-butyl ester

To a solution of 3.42 g of L-proline tert-butyl ester in 10 ml of tetrahydrofuran is added 1.76 g of propothiolactone in an ice bath. After 5 minutes in an ice bath, then for 3 hours at room temperature, the reaction mixture was diluted with ethyl acetate (200 mL) and washed with 5% potassium bisulfate solution and water. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo. The remaining 1- (3-mercaptopropionyl) -L-proline tert-butyl ester was crystallized from ether-hexane; yield 3.7 g; mp 57-58 °.

Example 10

- (3-Mercapto-propionyl) -L-proline

Procedure A)

4.9 g of 1- [3- (benzoylthio) propionyl] -L-proline are dissolved in a mixture of 8 ml of water and 5.6 ml of concentrated ammonium hydroxide solution and stirred for one hour under argon. The reaction mixture was diluted with water, filtered and the filtrate was extracted with ethyl acetate. The aqueous phase was acidified with concentrated hydrochloric acid, saturated with sodium chloride, and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo. The remaining 1- (3-mercaptopropionyl) -L-proline was crystallized from ethyl acetate-hexane; yield 2.5 g; mp 68-70 °.

Procedure B

0.8 g of 1- [3- (acetylthio) propionyl] -L-proline is dissolved in 5 ml of 5.5N methanolic ammonia solution and the solution is kept under argon at room temperature. After 2 hours, the solvent was removed in vacuo, the residue was dissolved in water and applied to a column of analytical grade Dowex 50 ion exchange resin in hydrogen and eluted with water. The fractions giving the positive thiol reaction were combined and evaporated to dryness to give 0.6 g. The product was prepared as described in process A)

-5181965 was crystallized from ethyl acetate-hexane to give 1- (3-mercaptopropionyl) -L-proline.

Procedure C

2.3 g of 1- (3-mercaptopropionyl) -L-proline tert-butyl ester are dissolved in a mixture of 20 ml of anisole and 45 ml of trifluoroacetic acid. After one hour at room temperature under argon, the reaction mixture was evaporated to dryness in vacuo and the residue was repeatedly mixed several times with ethyl acetate-hexane. The residue (1.9 g) was dissolved in ethyl acetate (30 ml) and dicyclohexylamine (1.85 ml) was added. The crystalline salt is filtered off and recrystallized from isopropanol. Yield: 2 g; mp 187-188 °.

The salt was converted to the acid in the same manner as in Example 1; Yield 1.3 g. The product is crystallized from ethyl acetate-hexane as in Process A.

Many:

sodium:

Dissolve 500 mg of 1- (3-mercaptopropionyl) -L-proline in a mixture of 2.5 ml of water and 2.5 ml of 1N sodium hydroxide solution. The solution was freeze-dried to obtain the sodium salt.

magnesium:

500 mg of 1- (3-mercaptopropionyl) -L-proline, 49.5 mg of magnesium oxide and 10 ml of water were stirred under gentle heating until a perfect solution was obtained. The solvent was then removed by freeze-drying to give the magnesium salt.

calcium:

500 mg of 1- (3-mercaptopropionyl) -L-proline are dissolved in a mixture of 91 mg of calcium hydroxide and 10 ml of water and freeze-dried to give the calcium salt.

potassium salt:

1- (3-mercaptopropionyl) -L-proline (500 mg) was dissolved in a mixture of potassium bicarbonate (246 mg) and water (10 ml) and freeze-dried to give the potassium salt.

N-Methyl-D-glucosamine salt;

500 mg of 1- (3-mercaptopropionyl) -L-proline and 480 mg of N-methyl-D-glucosamine are dissolved in 10 ml of water and freeze-dried to give the N-methyl-D-glucosamine salt .

Example 11 1- (3-Mercaptopropionyl) -L-hydroxyproline

In the procedure of Example 3, L-proline was replaced by L-hydroxyproline and then reacted according to the procedure of Example 10 (A) with 1- [3- (benzoylthio) propionyl] -L-hydroxyproline, and 1- (3-mercaptopropionyl) -L-hydroxyproline. The latter has a melting point of 193.5-195 ° as the dicyclohexylamine salt.

Example 12

- (3-Mercapto-propionyl) -D-proline

In the procedure of Example 13, L-proline was replaced by D-proline and then reacted according to the procedure of Example 18, A- [3- (benzoylthio) -propionyl] -D-proline and 1- (3-mercapto). -propionyl) -D-proline, m.p. 68-70 °.

Example 13

A) A mixture of 3- (acetylthio) -2-methylpropionic acid (g) thioacetic acid and 40.7 g methacrylic acid was heated in a hot water bath for one hour and then allowed to stand at room temperature for 18 hours. After the MMR spectroscopy confirmed that the methacrylic acid had completely reacted, the reaction mixture was distilled under vacuum. The desired 3- (acetylthio) -2-methylpropionic acid is obtained in a fraction of 128.5-131 ° C at 2.6 mmHg; yield 64 g.

B) 1- [3- (Acetylthio) -2-methylpropionyl] -L-proline tert-butyl ester

Dissolve 5.1 g of L-proline tert-butyl ester in 40 ml of dichloromethane and cool with stirring in an ice bath. A solution of dicyclohexylcarbodiimide (6.2 g) in dichloromethane (15 ml) was added, followed immediately by a solution of 3- (acetylthio) -2-methylpropionic acid (4.9 g) in dichloromethane (5 ml). Stir in an ice bath for 15 minutes and then at room temperature for 16 hours. The precipitate was filtered off and the filtrate was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate and washed to neutral. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo. The remaining 1- [3- (acetylthio) -2-methylpropionyl] -L-proline tert-butyl ester was purified by column chromatography (silica gel-chloroform); yield 7.9 g. Oil.

Example 14 1- [3- (Acetylthio) -2-D- and -DL-methylpropionyl] -L-proline

Procedure A)

7.8 g of 1- [3- (acetylthio) -2-methylpropionyl] -L-proline tert-butyl ester obtained in Example 13 are dissolved in a mixture of 55 ml of anisole and 110 ml of trifluoroacetic acid. After standing for one hour at room temperature, the solvent was distilled off in vacuo and the residue was repeatedly mixed several times with ether-hexane. The residue (6.8 g) was dissolved in acetonitrile (40 mL) and dicyclohexylamine (4.5 mL) was added. The crystalline salt was refluxed with 100 ml of fresh acetonitrile, cooled to room temperature and filtered. yield

3.8 g; mp (165) -187-188 °. The material was recrystallized from isopropanol; [α] _D = -67 ° (c = 1.4, ethanol). The crystalline dicyclohexylammonium salt is suspended in a mixture of 5% potassium bisulfate solution and ethyl acetate. The organic phase is washed with water and evaporated to dryness. The residue was crystallized from ethyl acetate-hexane to give 1- [3- (acetylthio) -2-D-methylpropionyl] -L-proline; mp 83-85 °; [α] D = -162 ° (c = 1.7, ethanol).

Procedure B

8.1 g of 3- (acetylthio) -2-methylpropionic acid and 7 g of thionyl chloride are added and the suspension is stirred for 16 hours at room temperature. The reaction mixture was evaporated to dryness and distilled under vacuum (boiling: 80 °). 5.4 g of 3- (acetylthio-2-methylpropionyl chloride) and 15 ml of 2N sodium hydroxide solution are added to a solution of 3.45 g of L-proline in 30 ml of 1N sodium hydroxide in an ice bath. After stirring for 3 hours at room temperature, the mixture was extracted with ether, the aqueous phase was acidified and extracted with ethyl acetate, the organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness with 1- [3- (acetylthio) -2-DL-methyl]. -propionyl] -L-proline is obtained.

Procedure C

Methacryloyl chloride (4.16 g) was added to a solution of 3.45 g of L-proline in a mixture of 100 ml of water and 12 g of sodium bicarbonate under ice-water cooling and vigorous stirring. When the addition was complete, the mixture was stirred at room temperature for two hours and then extracted with ether. The aqueous phase was acidified with 1N hydrochloric acid and extracted with ethyl acetate. The organic phase is evaporated to dryness in vacuo, the residue is mixed with 3.5 g of thioacetic acid, a few crystals of azo-bis-isobutyronitrile are added and the mixture is heated in a hot water bath for 2 hours. The reaction mixture was dissolved in benzene: acetic acid (75:25) and chromatographed on a silica gel column. Elution with the above solvent mixture gave 1- [3- (acetylthio) -2-DL-methylpropionyl] -L-proline.

Example 15 1- (3-Mercapto-2-D-methylpropionyl) -L-proline 1- (3-Mercapto-2-D-methylpropionyl) -L-proline from the product of Example 14 A is obtained by the following reaction:

0.85 g of the thioester is dissolved in 5.5 N methanolic ammonia solution and the solution is kept at room temperature for 2 hours. The solvent was evaporated in vacuo, the residue was dissolved in water and chromatographed on a column of Dowex 50 ion exchange resin in analytical grade, eluting with water. The fractions giving the positive thiol reaction were collected and freeze-dried. The residue was crystallized from ethyl acetate-hexane; yield 0.3 g. 1- (3-mercapto-2-D-methylpropionyl) -L-proline has a melting point of 103-104 °; [ _α ] _D = -131 ° (c = 2, ethanol).

Example 16

A) 3- (Acetylthio) -2-benzylpropionic acid

In the procedure of Example 13, methacrylic acid is replaced by 2-benzylacrylic acid to give 3- (acetylthio) -2-benzylpropionic acid.

B) 1- [3- (Acetylthio) -2-benzylpropionyl] -L-proline tert-butyl ester

In the procedure of Examples 5 and 10, 3- (acetylthio) -2-methylpropionic acid is replaced by 3- (acetylthio) -2-benzylpropionic acid, 1- [3- (acetylthio) -2- benzylpropionyl] -L-proline tert-butyl ester is obtained.

C) 1- [3- (Acetylthio) -2-benzylpropionyl] -L-proline

In Example 14, Method A, 1- [3- (acetylthio) -2-methylpropionyl] -L-proline tert-butyl ester is replaced by product B) 1- [3- (acetylthio) -2-benzylpropionyl] -L-proline is obtained.

D) 1- (3-Mercapto-2-benzylpropionyl) -L-proline 1- [3- (Acetylthio) -2-benzylpropionyl] -L-proline is reacted with methanolic ammonia according to the procedure of Example 15. - (3-Mercapto-2-benzylpropionyl) -L-proline was obtained as an oil. _Rf = 0.47 (silica gel: benzene-acetic acid = 75: 25).

Example 17 1- (3-Mercapto-2-methylpropionyl) -L-hydroxyproline

Example 17 1- (3-Mercapto-2-methylpropionyl) -L-hydroxyproline

In the procedure of Example 13 using L-hydroxy-proline tert-butyl ester tert-butyl ether, the product was then reacted according to the procedure of Example 14A and then the procedure of Example 15 to give 1- [ 3- (acetylthio) -2-methylpropionyl] -L-hydroxyproline tert-butyl ester tert-butyl ether followed by 1- [3- (acetylthio) -2-methylpropionyl ] -L-hydroxyproline and finally 1- (3-mercapto-2-methylpropionyl) -L-hydroxyproline are obtained as the amorphous material. [.Alpha.] D @ 20 = -61.9 DEG (c = 2 in ethanol).

Example 18 1- [4- (Benzoylthio) butyryl] -L-proline

To a solution of 2.88 g of L-proline in 25 ml of 1N sodium hydroxide solution in an ice bath are added 12.5 ml of 2N sodium hydroxide solution and 3.5 g of 4-chlorobutyryl chloride. The reaction mixture was stirred for 3.5 hours at room temperature and a suspension of 3.75 g of thiobenzoic acid and 2.4 g of potassium carbonate in 25 ml of water was added. After stirring overnight at room temperature, the reaction mixture was acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness in vacuo. The residue was chromatographed on a silica gel column with benzene-acetic acid (7: 1). The fractions containing the desired material were combined and evaporated to dryness to give 1.35 g. A small sample of the material was dissolved in ethyl acetate and dicyclohexylamine was added to pH 8-10 (measured with wet pH paper). The dicyclohexylammonium salt crystallizes immediately. Melting point: 159-161 °.

Example 19 1- (4-Mercaptobutyryl) -L-proline

1.08 g of 1- [4- (benzoylthio) butyryl] -L-proline is dissolved in a mixture of 4 ml of water and 2.7 ml of concentrated ammonium hydroxide solution. After stirring for one hour at room temperature, the mixture was diluted with water, filtered, extracted with ethyl acetate, and the aqueous phase was concentrated in vacuo. 1- (4-mer7)

-7181965 Capio-butyryl -) - L-proline ammonium salt was purified by column chromatography on DEAE-Sephadex by ion exchange chromatography with ammonium bicarbonate gradient to give 0.7 g of product. The ammonium salt was dissolved in 2 mL of water and loaded onto a column of analytical grade hydrogen Dowex 50 sulfonic acid resin in the hydrogen cycle and the free acid eluted with water. Fractions containing the desired material (positive sulfhydryl and carboxylic acid reaction) were combined and freeze-dried to give 1- (4-mercaptobutyryl) -L-proline. The dicyclohexylammonium IC salt was prepared according to the procedure of Example 18; mp 157-158 °.

Example 20 1

A) 1- [3- (Acetylthio) butyryl] -L-prrolidine-tert-butyl

-ester

6.2 g of dicyclohexylcarbodiimide and 4.86 g of 3- (acetyl-20-thio) -butyric acid are added with stirring to a solution of 5.1 g of L-proline tert-butyl ester in 60 ml of dichloromethane cooled in an ice bath. . The ice bath was removed after 15 minutes and the mixture was stirred at room temperature for 16 hours. The precipitate is filtered off, the filtrate is evaporated to dryness and the residue is chromatographed on a silica gel column to give 5.2 g of 1- [3- (acetylthio) butyryl] -L-proline tert-butyl ester.

B) 1- [3- (Acetylthio) butyryl] -L-proline 30

5.2 g of 1- [3- (acetylthio) butyryl] -L-proline tert-butyl ester prepared above are dissolved in a mixture of 60 ml of trifluoroacetic acid and 30 ml of anisole and the solution is left for one hour at room temperature. we. The solvents were evaporated in vacuo and the remaining 1- [3- (acetylthio) -butyryl] -L-proline was repeatedly precipitated several times from ether-hexane to give 4 g. The dicyclohexylammonium salt was prepared according to the procedure of Example 18; 175-176 °. 40

Example 21

- (3-Mercapto-butyryl) -L-proline 45

0.86 g of 1- [3- (acetylthio) butyryl] -L-proline prepared in Example 20 is dissolved in 20 ml of 5.5N methanolic ammonia solution and the reaction mixture is kept at room temperature for 2 hours. The solvent was removed in vacuo and the residue was chromatographed on a column packed with Dowex 50 ion exchange resin with water. Fractions containing the desired 1- (3-mercaptobutyryl) -L-proline were combined and lyophilized to give 0.6 g. The dicyclohexylammonium salt was prepared according to the procedure of Example 18; mp 183-184 °.

Example 22 1- [3- (Ethoxy-thiocarbonylthio) -propionyl] -L-proline in ml aqueous 2N sodium hydroxide solution and 8.5 g of (3-bromopropionyl) chloride were added with stirring.

To a solution of 5.75 g of L-proinine in 50 ml of 1 N sodium hydroxide was cooled in a 65 µl ice bath. After 5 minutes, the ice bath was removed and stirring continued at room temperature. After 3 hours, potassium O-ethyl dithiocarbonate (9.6 g) was added and the mixture was stirred overnight at room temperature. The solution was acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer was evaporated to dryness and the residue was chromatographed on a silica gel column with benzene-acetic acid (7: 1) to give 1- [3- (ethoxythiocarbonylthio) -propionyl] -L-proline, m.p. 95 °.

Example 23

3 - [(Methylamino) thiocarbonylthio] propionic acid A solution of 5.3 g of 3-mercaptopropionic acid in a mixture of 5.3 ml of a solution of 250 ml of pyridine and 100 ml of 0.5 N sodium hydroxide was added. The solution was kept at 40 for two hours and then evaporated to dryness in vacuo. The residue was dissolved in water (100 ml), acidified with concentrated hydrochloric acid and extracted with ether. The organic phase is evaporated to dryness to give 3 - [(methylamino) thiocarbonylthio] propionic acid, m.p. 86-87 °.

Example 24 1- [3 - {(Methylamino) thiocarbonylthio} propionyl] -L-proline tert-butyl ester

To a solution of 1.71 g of L-proline tert-butyl ester and 1.35 g of hydroxybenzotriazole in 10 ml of dichloromethane cooled in an ice bath are added 2.06 g of dicyclohexylcarbodiimide and 1.79 g of - [(methylamino) thiocarbonyl] thio-propionic acid. The bath was removed after 15 minutes and stirring was continued overnight. The precipitate was filtered off and the filtrate was diluted with ethyl acetate and washed neutral. The organic phase is evaporated to dryness to give 1- [3 - {(methylamino) thiocarbonylthio} propionyl] -L-proline tert-butyl ester, m.p. 129-130 °.

Example 25 1- [3 - ((Methylamino) thiocarbonylthio} propionyl] -L-proline

0.98 g of 1- [3 - {(methylamino) thiocarbonylthio} propionyl] -L-proline tert-butyl ester is dissolved in 3.6 ml of anisole and

7.5 ml of a mixture of trifluoroacetic acid. After standing at room temperature for one hour, the mixture was evaporated to dryness in vacuo and the residue was extracted three times with ether / hexane. The resulting material was chromatographed on a silica gel column with benzene-acetic acid (75:25) to give 1- [3 - {(methylamino) -thiocarbonylthio} -propionyl] -L-proline. Rf = 0.4 [silica gel: benzene-acetic acid (75:25)]. The dicyclohexylammonium salt has a melting point of 127-129 °.

22-25. Any of the compounds of Examples 1 to 4 is hydrolyzed to give 1- (3-mercaptopropionyl) -L-proline, m.p. 68-70 ° C.

-8181965

Example 26 1- [3- (Methylthio) propionyl] -L-proline methyl [3- (methylthio) propionate] in 150 ml of 10% sodium hydroxide solution at 100 ° for 30 minutes hydrolyzing. The cooled solution was extracted with ether and then acidified. The crude acid thus obtained is distilled and converted to the acid chloride with thionyl chloride.

A solution of 11.5 g of L-proline in 100 ml of 1N sodium hydroxide was cooled in an ice bath and 6.9 g of 3- (methylthio) propionyl chloride was added dropwise with vigorous stirring over 10 minutes. After 5 hours, the reaction mixture was acidified and extracted with ether to give 1- [3- (methylthio) propionyl] -L-proline. The dicyclohexylammonium salt is prepared by adding dicyclohexylamine to a solution of the free acid in ethyl acetate; m.p. 169-171 °.

Example 27

- [(methylthio) acetyl] -L-proline

In the procedure of Example 26, using methyl [(methylthio) acetate] instead of methyl [3- (methylthio) propionate] gives 1 - [(methylthio) acetyl] -L-proline, mp 123-124 °.

Example 28 1 - [(Benzylthio) acetyl] -L-proline

In the procedure of Example 26, 3- (methylthio) propionyl chloride is replaced by (benzylthio) acetyl chloride to give 1 - [(benzylthio) acetyl] -L-proline, m.p. 86-88 °.

Example 29

1,1 '- [Dithiobis (3-propionyl)] - bis-L-proline

Dissolve 0.95 g of 1- (3-mercaptopropionyl) -L-proline in 20 ml of water and adjust the pH to 1 π with sodium hydroxide solution.

Set to 6.5. Ethanolic iodine solution was added dropwise while the pH was maintained at 6.5 by careful addition of 1N sodium hydroxide solution. When a permanent yellow color is obtained, the iodine addition is stopped and the color is removed with a little sodium thiosulfate. The reaction mixture was acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with water, dried and evaporated to dryness to give 1,1 '- [dithiobis (3-propionyl)] - J-bis-L-proline. The dicyclohexylammonium salt is prepared by adding dicyclohexylamine to a solution of the free acid in acetonitrile; mp 179-180 °.

Example 30 1,1 '- [Dithiobis (2-D-methyl-3-propionyl)] - bis-L-proline

In the procedure of Example 110, 1- (3-mercapto-propionyl) -L-proline is replaced by 1- (3-mercapto-2-D-methylpropionyl) -L-proline, 1,1 '- [dithiothiol (2-D-methylpropionyl)] bis-L-proline is obtained; mp 236-237 °.

Example 31

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

In the procedure of Example 13A, methacrylic acid was used

Substitution with 2-phenylacrylic acid gives 3- (acetylthio) -2-phenylpropionic acid.

Β) 1- [3- (Acetylthio) -2-phenylpropionyl] -L-proline tert-butyl ester

In the procedure of Example 13B, 3- (acetylthio) -2-methylpropionic acid is substituted with 3- (acetylthio) -2-phenylpropionic acid 1- [3- (acetylthio) -2-phenyl -propionyl] -L-proline tert-butyl ester is obtained. Converted to the free acid [α] D = + 2.3 ° (c = 1, ethanol).

Example 32 1- (3-Mercapto-2-phenylpropionyl) -L-proline

In the procedure of Example 14, 1 - [(3-acetylthio) -2-methylpropionyl] -L-proline tert-butyl ester is 1 - [(3-acetylthio) -2-phenyl- propionyl] -L-proline tert-butyl ester and then the product is ammonolized according to Example 15 to give 1 - [(3-acetylthio) -2-phenylpropionyl] -L-proline or 1- (3) -mercapto-2-phenylpropionyl) -L-proline is obtained. [α] D ⁼ + 2.5 ° (c = 1 in ethanol) and [α] η = + 2.5 ° (c = in methanol).

Melting point 49-52 ° C.

Example 33

3 - [(4-Methoxyphenyl) methylthio] -2-methylpropionic acid

15.4 g (0.1 mol) of p-methoxy-α-toluene thiol are added to a solution of 8.6 g (0.1 mol) of methacrylic acid in 50 ml of 2N sodium hydroxide solution. The mixture was heated in a hot water bath for 3 hours, then refluxed for 2 hours and cooled. After extraction with ether, the aqueous phase is acidified with concentrated hydrochloric acid and extracted with dichloromethane. The acidic extracts were washed with brine, dried over anhydrous magnesium sulfate and evaporated in vacuo. The resulting semi-solid was dissolved in dichloromethane (50 mL), diluted with hexane (50 mL) and cooled. 5.5 g of 3 - [(4-methoxyphenyl) methylthio] -2-methylpropionic acid are obtained as white crystals, m.p. 74-82 ° C.

Example 34 1- [3- (4-Methoxyphenyl) methylthio] -2-methylpropionyl-L-proline tert-butyl ester

3.6 g (0.015 mol) of 3 - [(4-methoxyphenyl) methylthio] -2-methylpropionic acid, 2.6 g (0.015 mol) of L-proline tert-butyl).

-9181965

ester and dicyclohexylcarbodiimide (3.1 g, 0.015 mol) were dissolved in dichloromethane (50 ml) and stirred at 0 ° for 30 minutes. The cooling bath was then removed and the mixture was stirred overnight (sixteen hours). The resulting suspension was filtered and the filtrate was washed with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and brine, then dried over anhydrous magnesium sulfate and concentrated in vacuo. The resulting light oil was loaded onto a silica gel column (250 mL) and chromatographed eluting with 20% ethyl acetate / hexane. The main fraction _(Rf = 0.70, silica gel, ethyl acetate), concentrated to give 5.5 g (93%) of l- [3- (4-methoxyphenyl) methylthio] -2-methyl-propionyl L-proline tert-butyl ester was obtained as a light oil. _Rf = 0.70 (silica gel, ethyl acetate); R _f = 0.60 (silica gel, ether).

Example 35 1- (3-Mercapto-2-methyl-propionyl) -L-proline

1.2 g (0.003 mol) of the ester obtained in Example 34, 5 ml of anisole and 0.5 ml of trifluoromethanesulfonic acid are dissolved in 20 ml of trifluoroacetic acid under nitrogen and the resulting red solution is allowed to stand for one hour at room temperature. The solution was concentrated in vacuo to give a red residue, which was dissolved in ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate and evaporated. The residue was triturated again with hexane and the remaining hexane was evaporated; 0.4 g of an oily residue is obtained. 180 mg of the material was separated on 2 mm thick silica gel plates by preparative thin layer chromatography, eluting with benzene-acetic acid 75; Using 25 mixtures. The main nitroprussiate positive band _(Rf = 0.40) was worked up, 135 mg of l- (3-mercapto-2-methyl-propionyl) -L-proline as an oil. _Rf = 0.40 (benzene-acetic acid 75: 25) and R _f = 0.62 (chloroform: methanol: acetic acid 50: 40; 10).

Example 36

- (3-Mercapto-2-D-methyl-1-propionyl) -L-proline

Under argon, 10.0 g of 1- [3- (acetylthio) -2-D-methylpropionyl] -L-proline is suspended in 150 ml of water at 10 °. To the mixture was added 5N sodium hydroxide solution and the pH of the solution was maintained at 13 ° for 1.5 hours. The sodium hydroxide solution is then discontinued and the solution is acidified to pH 2.0 with concentrated sulfuric acid. The aqueous solution was then extracted with dichloromethane (3 x 150 mL) and the combined dichloromethane fractions were evaporated to an oil density. The concentrate was dissolved in ethyl acetate, filtered, and the filtrate was diluted with 30 mL of hexane. After an additional amount of hexane is added ^l / ₂ hours and then cooled to 10 ° for 1 hour.

The crystals were filtered off and washed with hexane (2 x 25 mL) and dried to constant weight to give 6.26 g of white crystalline 1- (3-mercapto-2-D-methylethylpropionyl) -L-proline, m.p. 100-102 °.

Example 37

A) 1- [3- (tosyloxy) -2-methylpropionyl] -L-proline

In the procedure of Example 14B, 3- (acetylthio) -2-methylpropionyl chloride is replaced with 3- (tosyloxy) -2-methylpropionyl chloride in 1- [3- (tosyloxy) -2-methylpropionyl] -L-proline is obtained.

B) 1- [3- (Acetylthio) -2-methylpropionyl] -L-proline

3.5 g of 1- [3- (tosyloxy) -2-methylpropionyl] -L-proline are added to a solution of 1.14 g of thioacetic acid and 3.5 ml of triethylamine in 20 ml of ethyl acetate. . The solution was heated at 50 ° C for three hours, cooled, diluted with 100 mL of ethyl acetate and washed with dilute hydrochloric acid. The organic layer was dried and evaporated to dryness in vacuo. The residue was dissolved in acetonitrile and dicyclohexylamine was added. The crystalline precipitate is recrystallized from isopropanol to give 1- [3- (acetylthio) -2-D-methylpropionyl] -L-proline-dicyclohexylammonium salt, m.p. 187-188 ° C; [α] D = -67 ° (c = 1.4, ethanol). The salt was converted to the free acid, m.p. 83-85 °. (The crystallization is followed by inoculation of the solution with a high melting point to give an isomorphic form having a melting point of 104-105 °.)

Example 38 1- (3-Mercaptopropionyl) -L-proline tert-butyl ester

A solution of 1.71 g (10 mmol) of L-proline tert-butyl ester and 1.35 g (10 mmol) of 1-hydroxybenzotriazole hydrate in 20 ml of dimethylformamide with stirring at 0-5 ° C, Dicyclohexylcarbodiimide (06 g, 10 mmol) was added. After stirring for 10 minutes, a solution of 3-mercaptopropionic acid (1.06 g, 10 mmol) in dimethylformamide (2 mL) was added. The mixture was then stirred at 0-5 ° C for one hour and then at room temperature overnight.

The precipitated dicyclohexylurea was filtered off and the filtrate was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed thoroughly with a saturated aqueous sodium bicarbonate solution, dried and evaporated in vacuo to give 2.5 g of an oil.

The oil was dissolved in 1: 1 ethyl acetate-hexane and applied to a column containing 100 g of silica gel. Elution with ethyl acetate-hexane (1: 1) gave 1.40 g (54%) of 1- (3-mercaptopropionyl) -L-proline tert-butyl ester as an oil which crystallized on standing. Recrystallization from ether-hexane gave 0.9 g of a colorless crystalline solid, m.p. 55-60 °, identical to the compound of Example 9.

Example 39 1- (3-Mercaptopropionyl) -L-proline mg (0.27 mmol) 1- [3 - ((ethylamino) carbonylthio} propionyl] -L-proline 1 ml concentrated ammonium A solution of hydroxide solution in water (1 ml) was allowed to stand at room temperature under argon for 18 hours, diluted with a little water and extracted with ether, acidified with cold concentrated hydrochloric acid and extracted with ethyl acetate. and evaporated in vacuo to give the same compound as in Example 10. R _f = 0.4 [silica gel, benzene-acetic acid (7: 3).

Example 40

Methacryloyl-L-proline

L-proline (23.0 g, 0.2 mol) was dissolved in water (100 mL) and stirred in an ice bath. A solution of 19.6 ml (0.2 mol) of methacryloyl chloride in 25 ml of methyl isobutyl ketone is added dropwise over 3 hours. Meanwhile, the pH of the reaction mixture was maintained at 7.0 by the simultaneous addition of 2N sodium hydroxide. The addition of the aliquot was continued for another four hours after the addition of the acid chloride was completed. The reaction mixture was adjusted to pH 5 with concentrated hydrochloric acid and extracted with ethyl acetate. The aqueous layer was then acidified to pH 2.5 and extracted thoroughly with ethyl acetate. The acidic extracts were washed with brine and dried over anhydrous magnesium sulfate. The ethyl acetate solution was combined with 40 ml of dicyclohexylamine and cooled overnight. The resulting white precipitate was filtered and dried. 29 g (39%) of white crystals are obtained, m.p. 202-210 °. The solid

Recrystallization from 1.5 liters of a 3: 1 mixture of acetonitrile / isopropanol gives 19.7 g of methacryloyl-L-proline-dicyclohexylammonium salt as fine white needles, m.p. 202-210 °.

The salt was dissolved in a mixture of water and ethyl acetate and the mixture was acidified with concentrated hydrochloric acid. The resulting suspension was filtered and the fine white precipitate was washed thoroughly with ethyl acetate. The filtrate was saturated with sodium chloride and thoroughly extracted with ethyl acetate. The extracts were washed with brine, dried over anhydrous magnesium sulfate, and evaporated to give a light oil which solidified. Crystallization from ethyl acetate-hexane gave 7.5 g (83%) of methacryloyl L-proline as a white crystalline solid, m.p. 89-93 ° C. An analytical sample was obtained by recrystallization, m.p. 95-98 °.

Example 41 1- [3- (Acetylthio) -2-D-methylpropionyl] -L-proline

Methacryloyl L-proline (183 mg, 0.001 mol) was dissolved in 0.5 ml of thioacetic acid and allowed to stand at room temperature for sixteen hours. The solution was concentrated in vacuo to give a yellow residue. Preparative thin layer chromatography [silica gel; dichloromethane: methanol: acetic acid = 90: 5: 5], the main fraction was isolated as a light oil (240 mg). TLC [dichloromethane: methanol: acetic acid = 90: 5: 5] to give 1- [3- (acetylthio) -2-DL-methylpropionyl] -L corresponding to the product of Example 14B. proves to be proline; R _f O, 35; benzene: acetic acid (75: 25) R _f = O, 38.

The oil was dissolved in acetonitrile (3 mL), dicyclohexylamine added until alkaline and cooled. 106 mg of white crystals are obtained, m.p. 175-181 °. Crystallized from isopropanol 1- [3

- (acetylthio) -2-D-methylethylpropionyl] -L-proline dicyclohexylammonium salt, m.p. 187-88 ° C, identical with the product of Example 14A.

Example 42

- (3-Mercapto-2-methyl-propionyl) -L-proline n

Zinc powder (10.0 g) was added to a suspension of 5.0 l- [dithiobis (2-methylpropioyl)] - bis-L-proline in 100 ml of 1.0 N sulfuric acid and the reaction mixture was stirred at 18 ° for 4 hours. nitrogen atmosphere. The solution was filtered, the zinc washed with water (20 mL) and the combined filtrates extracted with dichloromethane (3 x 75 mL). The dichloromethane washes were back-extracted with 25 mL of water and the organic solution was concentrated to an oil. The oil was dissolved in ethyl acetate (20 mL) and filtered. To the filtrate X was added 15 ml of hexane and the mixture was stirred for 15 minutes. An additional 30 mL of hexane was added and the solution was cooled to 5 ° for 1 h. The mixture was filtered and the product was washed with hexane (2 x 10 mL) and dried. 4.17 g of white crystalline 1- (3-mercapto-2-methylpropionyl) -L-proline are obtained. R _f = 0.60 [benzene: acetic acid (75:25)].

Example 43

A) 3- (Benzylthio) -2-methylpropionic acid

In the procedure of Example 33, using α-toluene thiol instead of p-methoxy-α-toluene thiol, 3- (benzylthio) -2-methylpropionic acid is obtained.

B) 1- [3- (Benzylthio) -2-methylpropionyl] -L-proline tert-butyl ester

In the procedure of Example 34, using 3- [benzylthio) -2-methylpropionic acid instead of 3- [4-methoxyphenyl) methylthio] -2-methylpropionic acid, 1- [3- (benzylthio) - 2-methylpropionyl] -L-proline tert-butyl ester is obtained.

C) 1- [3- (Benzylthio) -2-methylpropionyl] -L-proline

7.8 g of 1- [3- (benzylthio) -2-methylpropionyl] -L-proline tert-butyl ester are dissolved in a mixture of 55 ml of anisole and 110 ml of trifluoroacetic acid. After one hour at room temperature, the solvent was removed in vacuo, the residue dissolved in ether, washed several times with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness under reduced pressure to give 1- [3- (benzylthio) 2-methylpropionyl]. -L-proline is obtained. R _f = 0.5 [silica gel; benzene-acetic acid (3: 1)]; R _f = 0.5 [silica gel; methylethyl ketone-acetic acid-pyridine-water = 14: 1: 2: 1].

Example 44

1- (3-Mercapto-2-methyl-propionyl) -L-proline

0.1 g of 1- [3- (benzylthio) -2-methylpropionyl] -L-proline is suspended in 10 ml of boiling liquid ammonia, and small pieces of sodium are added with stirring until it becomes blue. The color

-11181965 is removed with some crystals of ammonium sulfate and allowed to distil in a constant stream of nitrogen. The residue was dissolved in a mixture of dilute hydrochloric acid and ethyl acetate. The organic phase is dried and evaporated to dryness under vacuum to give 1- (3-mercapto-2-methylpropionyl) -L-proline. R _f = 0.35 [silica gel; benzene-acetic acid (3: 1)]; R _f = 0.5 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)], which is identical to the compound of Example 15.

Example 45

3- (triphenylmethyl) thio-2-methylpropionic acid

A solution of 3-mercapto-2-methylpropionic acid (1.2 g) and trityl chloride (2.9 g) in dichloromethane (50 ml) was kept at room temperature for 2 hours. The mixture was heated on a hot water bath for 20 minutes and then evaporated to dryness in vacuo. The residue was dissolved in saturated aqueous sodium bicarbonate solution and washed with ethyl acetate. The aqueous phase was acidified to pH 3 and extracted with ethyl acetate. The organic phase was dried and evaporated to dryness to give 3- (triphenylmethylthio) -2-methylpropionic acid. R _f = 0.8 [silica gel; benzene-acetic acid (3: 1)].

Example 46

A) 1- [3- (Trifenylmethylthio) -2-methylpropionyl] -L-proline tert-butyl ester

In the procedure of Example 34, 3 - [(4-methoxyphenyl) methylthio] -2-methylpropionic acid is substituted with 3- [triphenylmethylthio) -2-methylpropionic acid 1- [3- ( yields triphenylmethylthio) -2-methylpropionyl] -L-proline tert-butyl ester.

B) 1- [3- (Triphenylmethylthio) -2-methylpropioryl] -L-proline

1.8 g of 3- (triphenylmethylthio) -2-methylpropionic acid and

0.8 g of Ν, Ν'-carbonyldiimidazole is dissolved in 10 ml of tetrahydrofuran with stirring at room temperature. After 20 minutes, the solution was added to a solution of 0.6 g of L-proline and 1 g of N-methylmorpholine in 20 ml of dimethylacetamide. The resulting mixture was stirred at room temperature overnight, evaporated to dryness and the residue was dissolved in a mixture of ethyl acetate and 10% aqueous potassium bisulfate. The organic phase was separated and, after drying under vacuum, concentrated to dryness in vacuo to give 1- [3- (triphenylmethylthio) -2-methylpropionyl] -L-proline. R _f = 0.4 [silica gel; benzene-acetic acid (3: 1)], R _f = l, 0 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)]. 55

Example 47 1- (3-Mercapto-2-methylpropionyl) -L-proline 60 g of 1- [3- (triphenylmethylthio) -2-methylpropionyl] -L-proline tert-butyl The ester is dissolved in a mixture of 55 ml of anisole and 110 ml of trifluoroacetic acid. After standing for one hour at room temperature, the solvents were evaporated in vacuo and the residue chromatographed on a silica gel column moistened with benzene-acetic acid (75:25) using the same solvent. The 0.4 _Rf -U [silica gel; in the same solvent] fractions corresponding to component 1 are combined and evaporated to dryness to give 1- (3-mercapto-2-methylpropionyl) -L-proline. R _f = 0.62 [silica gel; chloroform / methanol-acetic acid-water (50:40:10)], which is identical to the compound of Example 15.

Example 48

A) 3- (Tetrahydro-2-pyranol-thio) -2-methyl-propionic acid

To a solution of 2.4 g of 3-mercapto-2-methylpropionic acid and 1.9 g of freshly distilled 2,3-dihydro-4H-pyran in 60 ml of benzene are added 2.8 g of boron trifluoride etherate. After 2 hours, potassium carbonate (4 g) was added and the mixture was stirred and filtered. The filtrate was evaporated to dryness to give 3- (tetrahydro-2-pyranylthio) -2-methylpropionic acid.

Β) 1- [3- (Tetrahydro-2-pyranyl-thio) -2-methyl-propionyl] -L-proline

In the procedure of Example 47, 3- (triphenylmethylthio) -2-methylpropionic acid was substituted with 3- (tetrahydro-2-pyranylthio) -2-methylpropionic acid 1- [3- (tetrahydro-2-) pyranylthio) -2-methylpropionyl] -L-proline is obtained. _Rf = 0.8 [szili30 gel; benzene-acetic acid (3: 1)]; R _f O, 75 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)].

Example 49 1- (3-Mercapto-2-methylpropionyl) -L-proline g 1- [3- (tetrahydro-2-pyranylthio) -2-methylpropionyl] -L-proline in 25 mL of methanol and A solution of 25 ml of concentrated hydrochloric acid 40 was allowed to stand at room temperature for 30 minutes. The solvents were evaporated in vacuo to give 1- (3-mercapto-2-methylpropionyl) -L-proline. R _f = 0.35 [silica gel; benzene-acetic acid (3: 1)]; R _f = 0.5 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)] 45, which is identical to the compound of Example 15.

Example 50

A) 3- (Acetamido-methyl-thio) -2-methyl-propionic acid

2.4 g of 3-mercapto-2-methylpropionic acid and 1.8 g of N-hydroxymethylacetamide are dissolved in trifluoroacetic acid and the solution is kept at room temperature for one hour. The trifluoroacetic acid was evaporated in vacuo and the residue dried in vacuo over potassium hydroxide to give 3- (acetamidomethylthio) -2-methylpropionic acid.

B) 1- [3- (Acetamido-methyl-thio) -2-methyl-propionyl] -L-proline

In the procedure of Example 48, 3- (tetrahydro-2-pyranylthio) -2-methylpropionic acid was substituted with 3- (acetamidomethylthio) -2-methylpropionic acid to give 1- [3- (acetamidomethyl) acid. 12181965 (thio) -2-methylpropionyl] -L-proline is obtained. R _f = 0.2 [silica gel; benzene-acetic acid (3: 1], _Rf -0.3 [silica gel; methyl-Ethyl Ketone acid pyridin-water (14: 1: 2: 1)].

Example 51 1- (3-Mercapto-2-methylpropionyl) -L-proline

1.4 g of 1- [3- (acetamidomethylthio) -2-methylpropionyl] -L-proline and 1.93 g of mercury (II) acetate are dissolved in a mixture of 25 ml of acetic acid and 25 ml of water. Stir in a hot water bath for one hour, then add hydrogen sulphide until no more mercury (II) sulfide precipitates. The mixture was filtered, the precipitate washed with ethanol and the filtrate evaporated to dryness in vacuo to give 1- (3-mercapio-2-methylpropionyl) -L-proline. R _f = 0.35 [silica gel; benzene-acetic acid (3: 1)], _Rf = 0.5 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)], which is identical to the compound of Example 15.

Example 52

1- (3-Mercapto-2-methyl-propionyl) -L-proline t-butyl ester

1.2 g (10 mmol) of 3-mercapto-2-methylpropionic acid and

To a cold (5 °) solution of L-proline tert-butyl ester (1.7 g, 10 mmol) in dichloromethane (25 ml) was added portionwise a solution of dicyclohexylcarbodiimide (2.26 g) in dichloromethane (5 ml). After standing for two hours at room temperature, 5 drops of acetic acid are added, the mixture is filtered and the filtrate is evaporated to give an oily residue. This residue was dissolved in 20 ml of petroleum ether / ethyl acetate (3: 1) and applied to a 150 ml silica gel column previously moistened with petroleum ether. The fraction eluted with petroleum ether-ethyl acetate (1: 1) contains the product, 1- (3-mercapto-2-methylpropionyl) -L-proline tert-butyl ester. This 0.6 g fraction was dried in vacuo over phosphorus pentoxide for 12 hours. R _f = 0.6 [silica gel; benzene-acetic acid (3: 1)] _Rf = 0.8 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)].

Example 53 1- (3-Mercapto-2-methylpropionyl) -L-proline

In the procedure of Example 10J, 1- (3-mercaptopropionyl) -L-proline tert-butyl ester with 1- (3-mercapto-2-methylpropionyl) -L-proline tert-butyl ester substituted to give 1- (3-mercapto-2-methylpropionyl) -L-prooline. R _f = 0.35 [silica gel; benzene-acetic acid (3: 1)] _Rf = 0.3 [silica gel; methyl ethyl ketone-acetic acid-pyridine-water (14: 1: 2: 1)], which is identical to the compound of Example 15.

The racemic form of the final product of any of the above examples is prepared using the DL form of the starting amino acid instead of the L form.

Similarly, the D-form of the final product of any of the above examples is prepared using the D-form of the starting amino acid instead of the L-form.

Example 54 1- (3-Mercapto-2-D-methyl-propionyl) -4-cis-methyl-L-proline

A) 1- (3-Acetylthio-2-D-methylpropionyl) -4-cis-methyl-L-proline

To a solution of 2.87 g (17.4 mmol) of 4-cis-methyl-L-proline in 10.8 ml of 10 3.2 N sodium carbonate in ice-cooled solution is added 2.88 g (16 mmol) of 3-acetylthio 2-methylpropionyl chloride in 3 ml ether. The mixture was stirred vigorously for 35 minutes while maintaining the pH at 8.5 in 12 mL of 4N sodium carbonate solution. After stirring for one hour at room temperature, the mixture was extracted with ether and the aqueous solution was acidified with 4.5 ml of concentrated hydrochloric acid. The acidified solution is extracted extensively with ethyl acetate, the ethyl acetate is removed in vacuo and the residue is chromatographed on 175 g silica gel (Merek) under a pressure of 10: 1 benzene / acetic acid. Further purification of the product is accomplished by formation of the dicyclohexylamine salt in ethyl acetate. From the dicyclohexylamine salt, the desired title compound is obtained in pure form as an oil. Yield: 1.8 g (42%). [α] D = -171.5 ° (c = 1.9, in chloroform). _Rf: 0.54 (silica gel, benzene: acetic acid 7: 3 eluent). An analytical sample of the dicyclohexylamine salt was obtained by recrystallization from acetonitrile. [α] D = -69.3 ° (c = 1.76, in chloroform); 183-184 °.

B) 1- (3-Mercapto-2-D-methylpropionyl) -4-cis-methyl-L-proline

To a solution of the compound obtained in (A) (1.67 g, 6.1 mmol) in methanol (10 ml) was added sodium hydroxide solution (15.25 ml) and the reaction mixture was allowed to stand at room temperature under argon for 15 minutes. The solution was centrifuged to remove the precipitated solid and the methanol was removed in vacuo from the supernatant.

The resulting aqueous solution was acidified with 5 mL of 3N hydrochloric acid and extracted intimately with ethyl acetate. The ethyl acetate was removed in vacuo and the residue was chromatographed on silica gel (70 g, Merek) under a pressure of 7: 3 4; and eluted with a mixture of benzene and acetic acid.

The resulting material was dissolved in chloroform, filtered and the chloroform removed in vacuo to give the desired product as a viscous oil. Yield: 950 mg (67%). [.alpha.] D @ 20 = -139.7 DEG (c = 1.26 in chloroform). 5 (i R _r value: 0.54 (silica gel., 7: 3 mixture of benzene and acetic acid) dicyclohexylamine salt of a compound formed by the ethyl acetate solution of [a] D = -62.6 ° (c = 07th (in chloroform), m.p. (175 °) 177-179.

Example 55 1- (3-Mercapto-2-D-methylpropionyl) -4-cis-heptyl-L-proline

A) 1- [3- (Acetylthio) -2-D-methylpropionyl) -4-cis-heptyl-L-proline

A solution of 3.0 g (0.013 mol) of cis-4-heptyl-L-proline in 1 ml of sodium carbonate in 50 ml of water was obtained.

The suspension of -13181965 was cooled with stirring to 10 ° and a solution of 2.6 g (0.014 mol) of D-3-acetylthio-2-methylpropionyl chloride in 5 ml of ether was added portionwise over 10 minutes. During this time, additional 2.5 g of sodium carbonate was added in portions to maintain the pH at about 8. A clear solution is obtained. The ice bath was removed, the mixture was stirred at room temperature for 90 min, cooled, ethyl acetate (50 mL) was added and acidified with 10 mL of 6N hydrochloric acid. The layers were separated and the aqueous layer was extracted with ethyl acetate (25 mL). The organic layers were combined, dried over anhydrous magnesium sulfate, filtered and the solvent evaporated to give 5.5 g of an almost colorless viscous residue. This was dissolved in ethyl acetate (25 mL) and reacted with a solution of dicyclohexylamine (2.7 g) in ethyl acetate (5 mL). The dicyclohexylamine salt crystallizes rapidly upon inoculation of the resulting solution. After standing overnight, 6.0 g (79%) of a colorless solid are obtained. Melting point 157-159 °, color gradient from 151 °. Recrystallization from 80 ml of acetonitrile gave 5.4 g (71%) of a colorless material. Mp 161-163 °. [α] D = -64.5 ° (c = 1, in chloroform).

5.35 g of the dicyclohexylamine salt obtained are converted into the free acid by suspension in 60 ml of ethyl acetate and treated in portions with 60 ml of 10% potassium hydrogen sulfate solution. The layers were separated and the aqueous layer was extracted twice with 30 mL of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered and the solvent was evaporated to give 3.6 g (74%) of a nearly colorless syrup, [α] 105 DEG (c = 1, in ethanol). _Rf: 0.26 (silica gel, 85% toluene and 15% acetic acid).

B) 1- (3-Mercapto-2-D-methylpropionyl) -4-cis-heptyl-L-prooline

3.3 g of the product obtained in A are treated with a cold solution of 10 ml of concentrated ammonia in 25 ml of water under argon. After stirring for a few minutes, a solution was formed which was allowed to stand at room temperature for 2 hours, cooled and extracted twice with 20 ml of ethyl acetate. The aqueous phase was stirred, ethyl acetate (20 ml) was added, the mixture was acidified with 6N hydrochloric acid and the layers were separated. The aqueous phase was extracted twice with ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered and the solvent was evaporated to give 2.87 g (98%) of a nearly colorless syrup. [α] D = -74 ° (c = 1, in ethanol). _Rf: 0.22 (silica gel, 85% toluene and 15% acetic acid); _Rf: 0.72 (silica gel, 95: 5: 5 mixture of dichloro- 50 methane, methanol and acetic acid).

The 1-adamantanamine salt of this product melts at 177-179 ° C, crystallized from ethyl acetate.

Example 56 1- [2- (Mercaptomethyl) -9-phenyl-nonanoyl] -L-proline

A) 1- [2- (Acetylthiomethyl) -9-phenyl nonanoyl] -L-proline

5.0 g of 3- (acetylthio) - (2-phenyl-n-heptyl) propionic acid and

2.38 g of hydroxybenzotriazole are stirred in 23 ml of tetrahydrofuran in an ice bath. 3.2 g of dicyclohexylcarbodiimide were added followed by 2.65 g of L-proline tert-butyl ester in 100 ml of tetrahydrofuran. The reaction mixture is stirred overnight at room temperature, the urea by-product is filtered off, the filtrate is concentrated to dryness in vacuo, the residue is taken up in ethyl acetate and washed with 10% potassium bisulfate and saturated sodium bicarbonate , the ethyl acetate was removed and the residue was purified by silica gel column chromatography (9: 1 chloroform: ethyl acetate). The tert-butyl ester of the title compound is obtained which is treated with 11.5 ml of anisole and 25 ml of 10 trifluoroacetic acid to give the free acid. Yield 4.2 g.

B) 1- [2- (Mercaptomethyl) -9-phenyl-nonanoyl] -L-proline

To the product obtained in (A) under cold argon is added 15 ml of cold concentrated ammonium hydroxide. After 30 minutes, the mixture was acidified with concentrated hydrochloric acid and extracted with ethyl acetate to give 4 g of a clear oil. This title compound is converted to its adamantylamine salt in ether / hexane, m.p. 104-110 ° C.

Example 57

- [2- (mercaptomethyl) -nonanoil] -L-proline

A) 1- [2- (Acetylthio-methyl) -nonanoyl] -L-proline

2.46 g of 3-acetylthio-2-n-heptylpropionic acid and 1.53 g of 3o-hydroxybenzotriazole are stirred in 15 ml of tetrahydrofuran in an ice bath. To the mixture was added dicyclohexylcarbodiimide (2.06 g) followed by L-proline tert-butyl ester (1.71 g) in tetrahydrofuran (5 ml). After 15 minutes, the ice bath was removed and the mixture was stirred overnight at room temperature. The urea by-product was filtered off and the filtrate was evaporated to dryness. The residue was taken up in ethyl acetate, washed with 10% potassium bisulfate solution and saturated sodium bicarbonate solution and the ethyl acetate was evaporated. of the title compound as tert-butyl ester. This ether was added to 11.3 ml of anisole and 20 ml of trifluoroacetic acid, allowed to stand for one hour, and evaporated to dryness in vacuo to give an oil which was purified on a silica gel column eluting with 7: 1 benzene: ethyl acetate. Yield: 1.8 g.

B) 1- [2- (Mercapto-methyl) -nonanoyl] -L-proline

1.7 g of the product obtained in A are treated with 8 ml of cold concentrated ammonium hydroxide solution in 8 ml of cold methanol under argon. After 30 minutes, the reaction mixture was acidified with concentrated hydrochloric acid, extracted with ethyl acetate and evaporated to give 1.5 g of the title compound as an oil. The adamantylamine salt has a melting point of 159-162 °.

Example 58 1- (3-Mercapto-decanol) -L-proline

A) 1- [3- (Acetylthio) decanoyl] -L-proline tert-butyl ester

6.75 g (0.05 mole) of hydroxybenzotriazole are dissolved in 500 m. The reaction mixture was cooled in ethyl acetate (65 ° C) at 50 ° C and then to room temperature

-14181965

(±) -3-Acetylthio-decanoic acid (12.3 g, 0.05 mol) was added followed by dicyclohexylcarbodiimide (10.3 g, 0.05 mol). After a few minutes crystalline material begins to precipitate. After stirring at room temperature for 2.5 hours, L-proline tert-butyl ester (8.6 g, 0.05 mol) was added and the mixture was stirred overnight at room temperature. The resulting 9.6 g of dicyclohexylurea is filtered off. The ethyl acetate filtrate was washed twice with 200 ml of 5% sodium bicarbonate solution, once with 200 ml of saturated sodium chloride solution, twice with 200 ml of 5% hydrochloric acid, and once with 200 ml of saturated sodium chloride solution. wash and dry over anhydrous magnesium sulfate. Removal of the solvent gave 17.8 g (89%) of product.

Elemental composition: Calculated for C ₂₁ H ₃₇ NO ₄ S: C, 63.12; H, 9.33; N, 3.51; Found: C, 64.01; H, 9.66; N, 3.94.

B) 1- [3- (Acetylthio) decanoyl] -L-proline

The product obtained in (A) (17.3 g, 0.043 mol) was stirred with trifluoroacetic acid (135 ml) and anisole (6 ml) under nitrogen at room temperature for 4 hours. Excess trifluoroacetic acid was removed at 40 ° and 3 torr

25.6 g of a viscous residue (14.8 g of theory) are obtained. This residue is dissolved in 150 ml of ether and a solution of 10 g of dicyclohexylamine in 50 ml of ether is added. Immediately a white crystalline precipitate formed, which was accompanied by warming. After cooling, the solid was filtered off to give 15 g of trifluoroacetic acid dicyclohexylamine salt (m.p. 225-230 °). The filtrate was partitioned between 10% potassium bisulfate solution and water, and the organic layer was washed with saturated sodium chloride solution and dried over anhydrous magnesium sulfate. Removal of the solvent gave 15 g of the title compound as a dark viscous residue.

Elemental composition of C ₁₇ H ₂₉ NO ₄ S Calculated O: C 59.44%, H 8.51%, N 4.08%, S 9.33%; Found: C, 59.64; H, 8.60; N, 3.81; S, 9.56.

C) 1- (3-Mercapto-decanoyl) -L-proline g (0.043 mol) of the product obtained in A) was stirred in 160 ml of water with 65 ml of concentrated ammonium hydroxide solution under nitrogen at room temperature for 3 hours. Some insoluble material was removed by extraction with ethyl acetate. The light yellow aqueous layer was layered with ethyl acetate and strongly acidified with 20% hydrochloric acid. The layers were separated and the aqueous layer was extracted with ethyl acetate (200 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solvent was removed

8.7 g (67%) of a residue is obtained which has a strong SH content. This material was dissolved in benzene (200 mL) and freeze dried to give 8.4 g (62%) of the title compound as a viscous liquid.

Elemental composition according to the formula C ₁₅ H ₂₇ NO ₃ S.0.5 mol H ₂ O:

Calculated: C 58.02%, H 9.09%, S 10.33%,

N 4.51%, SH 100%;

Found: C, 57.77; H, 9.37; S, 10.24;

N, 4.47%, SH, 98.9%.

Example 59

1000 tablets each containing 100 mg of 1- (2-mercaptopropionyl) -L-proline are prepared from the following chair:

1- (2-mark apto-propion I) -L-prol 100 g, cornstarch 50 g, gelatine 7.5 g, Avicel (microcrystalline cellulose) 25 g, magnesium stearate 2.5 g. 1- (2-mercapto-prolionyl) -L-proline and the corn

starch is mixed with an aqueous solution of gelatin. The mixture is dried and the magnesium stearate is then added to the granulate. This is then compressed into 15000 tablets each containing 100 mg of active ingredient.

Example 60

In Example 59, 1- (2-mercapto-propionyl) -L-proline is replaced by 100 g of 1- (3-mercapto-2-D-methylpropionyl) -L-proline per 1000 units of 100 mg of 1- (3- 25 tablets containing mercapto-2-D-methylpropionyl) -L-proline were prepared.

Example 61

1000 tablets each containing 200 mg of 1- (2-mercaptoacetyl) -L-proinine are prepared from the following ingredients:

1- (2-mercaptoacetyl) -L-proline 200g, lactose 100 g, Avicel 150g cornstarch 50g magnesium stearate 5g 1- (2-mercaptoacetyl) -L-proline, a lactose and that

Avicel is mixed and then corn40 is added. Magnesium stearate is added. The dry mixture is compressed in a tablet press to 1000 tablets of 505 mg each containing 200 mg of active ingredient. The tablets are coated with Methocel E 15 (methylcellulose) solution containing N 6 6 45 as a dye.

Example 62

Two pieces No. 1 a gelatin capsule is filled with a mixture of the following ingredients to obtain capsules containing 250-250 mg of 1- (2-mercaptopropionyl) -L-proline:

1- (2-mercaptopropionyl) -L-proline 250mg, magnesium stearate 7mg, lactose (USP grade) 193mg.

Example 63

The solution for injection is prepared from:

l- (2-mercaptopropionyl) -L-proline 500 mg p-hydroxybenzoic acid methyl ester 5 g, p-hydroxybenzoic acid propyl ester 1 g, NaCI 25 g, 65 water for injections 5 liters.

-15181965

The active ingredient, preservatives and sodium chloride are dissolved in 3 liters of water for injections and then made up to 5 liters. The solution is filtered through a sterile filter and aseptically filled into pre-sterilized vials, which are then sealed with pre-sterilized rubber caps. Each vial contains 5 ml of solution containing 100 mg of active substance per ml of solution for injection.

IC

Example 64

In Example 59, 1- (2-mercaptopropionyl) -L-proline was replaced by 100 µl of l'-[dithiobis (2-D-methyl-3-propionyl)] - bis-L-proline, 1000 each. 100 mg of 15.1.1 '- [dithiobis (2-D-methyl-3-propionyl)] - bis-L-proline tablet is prepared.

Pharmacological studies

2C

One of the factors that increase the blood pressure in the human body is the renin-angiotensin-aldosterone system. The renin enzyme is produced in and released from the muscle surface cells of the muscular tissues adjacent to the cortex in the kidney cortex. The mechanism regulating renin secretion is likely to be influenced primarily by the salt and water content of the blood and the pressure of the blood flowing through the kidneys. When these factors are attenuated, renin is released directly into the blood. Here, it encounters a _2- globulin, the angiotensinogen (active 3C protein), as a substrate. Renin cleaves the angiotensinogen into two equally inactive components, a tetrapeptide and an angiotensin I (A-1) decapeptide. When circulating in the capillary vessels (primarily the lungs), A-I contacts the angiotensin-converting 3: enzyme (ACE) in the squamous cells of the capillaries. ACE cleaves A-I into a dipeptide and into the octapeptide, most commonly known as the blood pressure-raising substance in the human body, angiotensin II (A-II). The blood pressure-raising effects of A-II occur locally in the muscles as well as centrally (stimulated by A-II stimulation), and the brain transmits stimuli that increase blood pressure throughout the body. It is also physiologically stimulated by the excretion of aldosterone, a mineralalkorticoid, in the adrenal cortex, which increases the absorption of sodium ions from the kidney, increasing blood volume and increasing blood pressure by this second mechanism.

5C

In addition to converting A-I to A-ΙΙ, ACE also has a role to play. ACE converts bradykinin, a vasodilator, into inactive components. For this function, ACE is also accounted for as kininase II. 55

In the person suffering from hypertension, the renin-angiotensin-aldosterone system is disturbed and this results in significant hypertension. ACE inhibitors can produce the following effects: 6C

1. It inhibits the formation of A-II and thus (a) directly inhibits vasoconstriction and (b) inhibits aldosicron secretion (i.e., excretion of water and excess salt) from the breast tissue. 65

2. The inhibition of ACE inhibits the breakdown of bradykinin, thus maintaining its vasodilator effect (lowering blood pressure).

None of the current antihypertensive drugs inhibit ACE.

The proline derivatives of formula I of the present invention are potent ACE inhibitors. They inhibit the conversion of angiotensin I decapeptide to angiotensin ΙΙ, thereby reducing or eliminating the angiotensin II-induced hypertension. The compounds of the present invention intervene in the angiotensinogen (renin) angiotensin I to angiotensin II conversion process by deactivating ACE, thereby reducing or preventing the formation of angiotensin II, which is responsible for the increase in blood pressure. The activity of the compounds of formula I is completely different from that of conventional antihypertensive drugs. The compounds of the present invention allow direct intervention in the causes of hypertension, rather than the symptomatic treatment of prior art agents.

Thus, the therapeutic utility of the compounds of formula I is based on the inhibition of the action of the angiotensin converting enzyme. The inhibitory effect was determined in vitro by the effect on the angiotensin converting enzyme extracted from the rabbit lung and the effect of the angiotensin I contraction of the guinea-pig gut. These are assay methods known in the art.

Inhibition of angiotensin converting enzyme isolated from rabbit lung

The efficacy of the angiotensin converting enzyme of a cellular extract from rabbit lung was measured by spectrophotometric determination of hippuryl-L-histidyl-L-leucine (HHL) released by the enzyme by Cushman and Cheung, Biochem. Pharmacol. 20, 1637 (1971)].

C ₆ H ₄ —Gly — His — Leu -> - C ₆ H ₄ —Gly — f — His — Leu.

In this spectrophotometric assay, the following medium was used to measure inhibition of the angiotensin converting enzyme at 37 ° C in 0.25 mL volumes:

100 mmol phosphate buffer, pH 8.3

300 mmol of sodium chloride

0.001-1000 μg / ml inhibitor (with or without inhibitor)

5-10 m units of enzyme.

The medium of the above composition was incubated for 30 minutes with or without various inhibitor concentrations and the enzyme reaction was stopped with 0.25 mL of n hydrochloric acid. A control assay was also performed by inactivating the enzyme immediately (at time 0) with hydrochloric acid before adding the enzyme.

The enzyme-released hippuric acid was extracted with 1.5 ml of ethyl acetate, after stirring vigorously for 20 seconds and separating the ethyl acetate layer by centrifugation, a 1.0 ml aliquot of the solvent was removed and the hippuric acid was evaporated to dryness at 100 ° C. dissolved in water and quantitated at 228 nm by its absorbance.

The potency of the angiotensin converting enzyme inhibitor is expressed as T ₅₀ , i.e., the amount of inhibitor in µg / ml or [J. mol which is 50% of the effect of the angiotensin converting enzyme.

-16181965, determined by spectrophotometry. This value was obtained by plotting the% inhibition obtained with 2-3 fold concentrations of inhibitor in the range of 0.001 µg / ml to 1000 µg / ml. Percent inhibition was calculated using the following formula:

Inhibition,% = (A-A) - (B-B °) (A-A °)

Absorption of a hippuric acid in a 30 minute experiment without inhibitor;

A background absorption at time 0;

B is the absorbance of hippuric acid in the 30 minute experiment at the appropriate concentration of inhibitor tested;

Background B in the presence of an absorption inhibitor but at time 0.

In vitro study of angiotensin-1 converting enzyme (ACE) inhibitory activity in guinea pig colon

This method was used to determine whether a compound is considered to be an inhibitor of the angiotensin I converting enzyme due to its effect on angiotensin I contractions in the guinea pig's gut. ACE inhibitors typically inhibit angiotensin I contraction.

300-400 g of both sexes, stunned guinea pigs (Hartley strain) were killed by a blow to the head. Hips removed by cs Vane method (1)

We cut it into strips of 2-3 cm. Each intestinal strip was placed in a bath filled with 10 ml of Krebs solution (2) at 37 ° C with 95% oxygen and 5% carbon dioxide bubbling. Muscle activity was measured isotonically with a load of 1 g using a cardiac smooth muscle transducer (Harvard 356) coupled to a dinograph (Beckmann R type).

After the tissues were equilibrated for 90 minutes, 2 minute signals were measured with 10 min intervals with the following aganist:

angiotensin I (A-I) 0.025 mcg / ml final bath concentration.

The contractions induced by this concentration of A-T correspond to a maximum of 50-75%.

The test compound was generally dissolved in water or sodium chloride solution and diluted 10-fold with water. The agonist was pretreated with the test compound for 2 minutes and the percentage change in contractions was recorded. The active compounds, i.e., which may be considered as ACE inhibitors, typically inhibited angiotensin I-induced contraction.

The EC ₅₀ value, also referred to as IC ₅₀ (the concentration of test compound that inhibits A-15 contractile activity by 50%), was estimated graphically and bionetrically; the test compound was tested at a few concentrations to construct a concentration-inhibitory curve.

The assay methods were based on the following publications:

(1) Vane, J.R.: Brit. J. Pharmacol. 23, 360, 1964;

(2) HANDSCHUMACHER, R.C. Vane, J.R.: Brit. J. Pharmacol. 29, 105, 1967.

(3) Rubin, B., O'Keefe, E., Kotler, D.G., DeMaio D.

A. and Cushman, D.W., Fed. Proc. 34, 770, 1975;

C4) O'Keefe, Ε. H., D. G. Kotler, Waugh, Μ. H., Rubin, B.: Fed. Proc. 31, 511, 1972;

(5) Rubin, B., Laffan, R. J., Kotler, D. G., & O'Keefe, Ε. H., DeMaio, D. A., Goldberg, Μ. E. J. Phar30 macol. Exper. Therap. 204, 271, 1978.

The formula for the percentage inhibition is:

A B ------. 100 A

The contraction (mm) I-effect agent néikü ^1B contraction (mm) I-effect agent.

The following table summarizes the data determined with some representative compounds of formula I of the invention (R _{2 is} hydrogen 40 atoms).

R Ri r ₄ rj η Ϊ50 (Μβ / πίΐ) ECso (μβ / τπΐ) OH H H 0 0.19  0,036 OH -CH ₃ (DL) - H 0 0.23 0,064 OH -CH ₃ (L) - H 0 0,035 0.0029 OH H H H 1 0.05 0,022 OH H H OH 1 0.21 0,056 OH -CH ₃ (D) H H 1 0,005 0,008 OH -CH ₃ (L) H H 1 0.52 1.7 —Nh ₂ -CH ₃ (D) H H 1 0.18 0.26 OH —Bz (DL) H H 1 0,018 0.113 OH —CH ₃ (DL) H OH 1 0.011 0,007 OH -CH ₃ (D) H OH 1 0,002 0.0017 OH H H H 2 2.1 2.7 OH H —CH ₃ (DL) H 1 0.23 0.5 OH —Ph (DL) H H 1 0.56 0.4 OH H H H 1 0.18 0.56 —OC (CH ₃ ) ₃ -CH ₃ (D) H H 1 2.9 - -OH Ad CH ₃ (D) H H 1 0,009 0.0015 OH C ₂ H ₅ (L) H H 1 0.06 0.046 OH -C ₂ H ₅ (D) H H 1 0,004 0,006

-1718/965

R Ri f ₄ ^R 3 η Iso (Μβ / ηΊ1) EC50 (gg / ml) OH —NC ₃ H ₇ (DL) H H 1 0.02 0.46 OH -CH ₃ (D) Η —Ch ₃ 1 0,003 0,007 OH —CH ₃ (D) H nC ₇ H ₁₅ I 0,014 0.0006 OH —CH ₃ (DL) H H 1 0,017 0.042 OH -ch ₃ (D) H Η 1 0,006 0,005 —OnC ₃ H ₇ -CH ₃ (D) H Η 1 0.16 0.16 —OiC ₃ H ₇ -ch ₃ (D) H Η 1 0.60 0,058 —Och ₂ —c ₃ h ₅ —Ch ₃ (D) H Η 1 0.24 0.25 —OnC ₇ H ₍₅ -ch ₃ (D) H Η 1 3.2 - —OnC ₄ H ₉ -ch ₃ (D) Η Η 1 0,365 0.30 - and ₃ -ch ₃ (D) H Η 1 0.22 0.13 OH —NC ₇ H _(J (DL) H Η 1 0,054 0,068 -OH Ad —NC ₇ H ₁₅ (DL) H Η 1 0,050 0.66 OH —Ph-nC ₇ HP ₄ (DL) Η Η 1 0.33 0.13 OH H -nC ₇ H ₁₅ (Dl.) Η 1 1.4 0.69

Ad = adamantylamine salt Bz = benzyl group Ph = phenyl group

Patent claims

Claims (17)

Patent claims 1. A process for the preparation of the substituted proline derivatives of the formula I and their isomers and their salts with bases R is hydroxy or C 1-4 alkoxy, R 1 and R ₄ independently of one another are hydrogen, C 1-7 -alkyl or phenyl (C 1-7) -alkyl, but R ₁ may also be phenyl, R ₂ is hydrogen, C 1-4 alkyl, phenyl (C 1-4) alkyl or triphenyl (C 1-2) alkyl; R _{5 is} -C- P II, or (a) general formula group and the above groups _R5 is C1-4 alkyl or phenyl, and (a) a group of formula R, R, R ₃ and R ₄ are as defined herein, and simultaneously with the same R , R 1, R ₃ and R ₄ I R ₃ is hydrogen, hydroxy or C 1-7 alkyl and n is 0.1 or 2, characterized in that a) proline or isomer of a general formula TI - RCS R ₃ is as defined above - one of the formula III mercapto-alkanoic acid or its isomer - R, R ₄ and n as defined above, _R2 represents the same _two above as R or a protecting sulfur group - or reacting a reactive derivative thereof, preferably an acid chloride, and then removing any protecting group present, or b) a proline or isomer thereof of formula II, wherein R and R _{3 are} as defined above, or a carboxylic acid derivative of formula IIa or a reactive derivative of R ₁ , R ₄ and na, X is halogen, preferably chlorine or bromine or toluenesulfonyloxy; , preferably acid chloride, or when n is only 1, IIIb formula dehidrohalogénezett derivative or isomer - R and R ₄ are as defined above - or a reactive derivative thereof, preferably an acid chloride thereof, and one R ₂ S ^_ sulfide of the formula - R ₂ is ) and remove the protecting group, if present, or c) for the preparation of compounds of formula I wherein n is 1 or 2 and R _{2 is} hydrogen, R, R 1, R ₄ and R _{3 are} proline or isomers of formula II as defined herein - R and R _{3 are} as defined above - an IV of thiolactone of formula - as defined above, n is R, and R ₄ is 1 or 2 - is reacted, if desired, the preparation of a compound of formula I, wherein R ₂ (a) wherein, R, R, R _3, R ₄ and n are oxidized by iodine to a compound of any one of processes a) to c) wherein R _{2 is} hydrogen; optionally separating a mixture of isomers into its isomers; optionally converting a compound of formula I wherein R 1 -C 4 alkoxy is free acid, optionally converting a free acid to a base thereof. (Priority: December 22, 1976) 2. A process for the preparation of the substituted proline derivatives of the formula I, their isomers and their salts with bases R is hydroxy or C 1-4 alkoxy, R _{4 and} R ₄ independently of one another are hydrogen, C 1-7 -alkyl or phenyl (C 1-7) -alkyl, R ₂ is hydrogen or a group of formula -CO-R _s, and R ₅ in this group from 1 to 4 carbon atoms or phenyl, R ₃ is hydrogen, hydroxy or C 1-7 alkyl -18181965 n is Ο, 1 or 2 - characterized in that a) for the preparation of compounds of the formula I in which R _{4 is} hydrogen, R, R _p R ₂ , R ₃ and a proline or isomer thereof of the general formula II - R _{3 is} a C 1-4 alkoxy group as defined above; represents - a III general aciltioalkánsavval or isomer of formula - R, and n as defined above, R ₄ is hydrogen, R ₂ R _s CO is a group of formula wherein _R5 is as defined above - in the presence of an activating agent, then if desired, R ₅ -CO- group of formula removed, and the resulting ester is converted to free acid; obsession b) a proline or isomer thereof of formula II, R or R ₃ as defined above, or a reactive derivative thereof with a halocarboxylic acid or isomer of formula IIa, R _p R ₄ and na, X halogen, preferably chlorine or bromine; preferably or acid chloride thereof, when n is only 1, and R ₄ is hydrogen, IIIb formula dehidrohalogénezett derivative or isomer - R, as defined above, R ₄ a hydrogen atom - reacting a R ₅ -CO-S ^_ reacting a sulfide of formula, the desired optionally removing the R ₅ -CO- group or converting the resulting ester to the free acid, optionally separating the isomeric mixture into its isomers; optionally converting a free acid into its base salt. (Priority: February 13, 1976) 3. Process for the preparation of the substituted proline derivatives of the formula I and their isomers and their salts with the bases R is hydroxy or C 1-4 alkoxy, R; and R ₄ independently of one another are hydrogen, C 1-7 alkyl or phenyl (C 1-7) alkyl, but R 4 may also be phenyl, R ₂ is hydrogen, (C 1 -C 4) -alkyl, phenyl (C 1 -C 4) -alkyl or triphenyl (C 1 -C 2) -alkyl, R _{5 is} -CO- or a group of the formula: R ₅ is C 1 -C 4 alkyl or phenyl and in the formula (a) R, R _p R ₃ and R ₄ are as defined herein and at the same time as R, R _p R ₃ and R ₄ I, R ₃ is hydrogen, hydroxy or C 1-7 alkyl and n is 0.1 or 2, characterized in that a) for the preparation of compounds of the formula I in which R _{4 is} hydrogen, R, R _p R ₂ , R ₃ and a proline or isomer thereof of the general formula II - R _{3 is} a C 1-4 alkoxy group as defined above; represents - a III general aciltioalkánsavval or isomer of formula - R, and n as defined above, _R4 represents a hydrogen atom, R ₂ R ₅ -CO group of formula wherein _R5 is as defined above - in the presence of an activating agent, if desired, the R and _{5 is} removed or the resulting ester is converted to the free acid, or b) a proline or isomer thereof of formula II, R or R ₃ as defined above, or a reactive derivative thereof with a halocarboxylic acid or isomer of formula IIa, R ₁ , R ₄ and na, X halogen, preferably chlorine or bromine; , preferably with an acid chloride, or when n is only 1 and R _{4 is a} hydrogen atom, a dehydrohalogenated derivative of formula IIIb or an isomer thereof - R ₍ R ₄ hydrogen atom as defined above) and then a sulfide of formula R ₅ -CO-S in the case of R 1 -CO-, the ester obtained is converted to the free acid, or c) for the preparation of compounds of formula I wherein n is 1 or 2 and R _{2 is} hydrogen, R, R _p R ₃ and R _{4 are} as defined in the foregoing, a proline or isomer thereof of the general formula - and R _{3 is} as defined above - a general thiolactone of formula IV, - the value of _R, and R ₄ is as hereinbefore defined, n is 1 or 2 - is reacted, if desired, a resulting ester into the free acid, if desired in the preparation of a compound of formula I, wherein R ₂ (a) of formula a compound of formula (I) wherein R _{2 is} hydrogen; optionally separating a mixture of isomers into its isomers; optionally converting a free acid into its base salt. (Priority: June 21, 1976) A process for the preparation of 1- (3-mercaptopropionyl) -L-proline according to claim 1, wherein the L-proline is reacted with 3-bromopropionyl chloride followed by thiobenzoic acid and the resulting - (3-Benzoylthiopropionyl) -L-proline is hydrolyzed. (Priority: December 22, 1976) Process for the preparation of 1- (2-mercaptoacetyl) -L-proline according to claim 2, characterized in that L-proline is reacted with chloroacetyl chloride followed by thiobenzoic acid and the resulting 1- (2- benzoylthioacetyl) -L-proline is hydrolyzed. (Priority: February 13, 1976) 6. A process for the preparation of 1- (3-mercaptopropionyl) -L-proline according to claim 3, wherein the L-proline (C 1-4) alkyl ester is reacted with propiothiolactone and the resulting ester is hydrolyze to the free acid. (Priority: June 21, 1976) 7. A process according to claim 2 for the preparation of 1- (3-mercapto-2-D-methylpropionyl) -L-proline, wherein the L-proline tert-butyl ester is 3- (acetyl). -thio) -2-methylpropionic acid in the presence of an activating agent, and after conversion to the free acid, 1- [3- (acetylthio) -2-D-methylpropionyl] -L-proline is hydrolyzed. (Priority: February 13, 1976) 8. The process according to claim 2, for the preparation of 1- (3-mercaptobutyryl) -L-proline, which comprises activating 3- (acetylthio) -butyric acid with L-proline tert-butyl ester. in the presence of an agent, and after conversion to the free acid, 1- [3- (acetylthio) butyryl] -L-proline is hydrolyzed. (Priority: February 13, 1976) 9. The process of claim 1 for the preparation of 1- (3-mercapto-2-methylpropionyl) -L-proline, characterized in that 3 - [(4-methoxyphenyl) methylthio] -2-methylpropionic acid is reacted with L-proline tert-butyl ester and the sulfur protecting group is removed simultaneously with conversion to the free acid. (Priority: December 22, 1976) -19181965 10. A process according to claim 1 for the preparation of 1- [3- (acetylthio) -2-methylpropionyl] -L-proline, characterized in that 3- (tosyloxy) -2-methyl -propionyl chloride is reacted with L-proline followed by thioacetic acid. (Priority: December 22, 1976) 11. A process according to claim 1 for the preparation of 1- (3-mercapto-2-D-methylpropionyl) -L-proline, characterized in that L-proline is coupled with methacryloyl chloride followed by thioacetic acid. and hydrolyzing the resulting 1- [3- (acetylthio) -2-D-methylpropionyl] -L-proline. (Priority: December 22, 1976) Mode of implementation b) 12. A process according to claim 1 of formula I is substituted with L-prolinszármaz.ék thereof, wherein R is hydroxy, R is hydrogen or C1-4 alkyl szcnatomos, R _2, R ₃ and R ₄ are hydrogen, n 1 is characterized in that L-proline is reacted with a chloride of an acid of formula III wherein X is a chlorine or bromine atom, R 1 is hydrogen or C 1-4 alkyl, R _{4 is} hydrogen, n is 1 and R _{2 is} S- and hydrolyzed with a sulfide of the formula R ₂ as defined in claim 1. (Priority December 22, 1976) 13. A process according to claim 1 for the preparation of 1- (3-mercapto-2-methylpropionyl) -L-proline, characterized in that the sulfur atom contains a lower alkanoyl group, a benzyl group, a triphenylmethyl group, a tetrahydro-2 pyranyl, acetamidomethyl-protected 3-mercapto-2-methyl-propionic acid is reacted with L-proline or C1-C4 alkyl ester and optionally deprotected to convert the ester to the free acid. (Priority: December 22, 1976) 14. A process for the preparation of a medicament containing a substituted proline derivative of the formula I or an isomer thereof or a physiologically acceptable salt thereof with a base, wherein R, R ₁ , R ₂ , R ₃ , R ₄ and n are as defined in claim 1. The active ingredient produced by any of the methods of claim 1 is processed into a pharmaceutical composition in admixture with a carrier, diluent, excipient, and / or other excipients conventionally used in the manufacture of a medicament. (Priority: December 22, 1976) 0 15. The process of claim 13 wherein the active ingredient is 1- (3-mercapto-2-D-methylpropionyl) -L-proline. (Priority: December 22, 1976) J5 16. A process for the preparation of a medicament containing a substituted proline derivative or a isomer thereof or a physiologically acceptable salt thereof with an active ingredient as defined in the claims R, R ₁ , R ₂ , R ₃ , R ₄ and na 2 as an active ingredient. to process the active ingredient produced by any of the methods of claim 2 in admixture with a carrier, diluent, excipient and / or other excipient in the pharmaceutical formulation to form a pharmaceutical composition. (Priority: February 13, 1976) 17. A process for the preparation of a medicament containing a substituted proline derivative of the Formula I or an isomer thereof or a physiologically acceptable salt thereof with a base as defined in the claims R, R ₁ , R ₂ , R ₃ , R ₄ and na 3, characterized in that the active ingredient produced by any of the methods of claim 3 being processed into a pharmaceutical composition in admixture with a carrier, diluent, excipient and / or other excipient in the pharmaceutical formulation. (Priority: June 21, 1976) 4 pieces of drawing Responsible for publishing: Director of Economic and Legal Publishing 86.5589.66-4 Alföldi Nyomda, Debrecen Chief Executive Officer: István Bcnkő CEO