FI69834B - PROCEDURE FOR THE FREQUENCY OF THERAPEUTIC ANALYZING ETR- ELLER TIOETERMERKAPTOACYLPROLINER - Google Patents

Description

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(51) Kv.ik.4 / lnt.Cl.4 C 07 D 207/16 FINLAND —FINLAND (21) Patent application - Pitentaniökning 792 * »9δ (22) Application date - Ansöknlngsdag 1 O. 08.79 (23) Start date - Giltighetsdag 10 · 08.79 (41) Become public - Blivit offentlig 12.02.80

Patent, and National Board of Registration Date of approval and publication. -,,,, flc

Patent and registration authorities '' Ansökan utlagd och utl.skriften publicerad 3 *. 1Z.O? (32) (33) (31) Claim claimed — Begärd priority 1 1 .08.78 usa (us) 932883 (71) E. R. Squibb 6 Sons, Inc., Lawrencevi11e-Princeton Road, Princeton,

New Jersey, USA (72) Miguel Angel Ondetti, Princeton, New Jersey,

John Krapcho, Somerset, New Jersey, USA (74) Oy Kolster Ab (5 * 0 Method for the preparation of therapeutically useful ether and thioether mercaptoacyl acylprolines - Förfarande för framstälIning av therapeutiskt användbara eter- Eiler thioetermerkap

The invention relates to a process for the preparation of therapeutically useful ether or thioether mercaptoacyl prolines of the general formula I

XR1 R2 4 I I ^

R-S-CH2-CH-CO-N_C-COOH

wherein the group -XR 4 is attached to the 3- or 4-position of the proline ring, X is an oxygen or sulfur atom, R * is a C 1-4 alkyl or phenyl group which may be substituted by a halogen atom, R 14 is a hydrogen atom or a C 1-4 a C 1-4 alkyl group and R is a hydrogen atom or a C 1-4 alkanoyl or benzoyl group, and to prepare salts thereof.

69834

The ether and thioether mercaptoacyl prolines of formula I prepared according to the invention are useful as antihypertensive drugs.

U.S. Patent No. 4,105,776 discloses structurally related compounds of the invention having the same type of activity. The structure of these known compounds differs from the above-mentioned formula I in that they do not have a substituent XR 1, but are either unsubstituted or have a hydroxy or alkyl group as a substituent. Of the known compounds, the most significant unsubstituted compound is marketed as "Captopril" and has the formula CH, i J!

HS-CH2-CH-CO-N_COOH

1- (3-mercaptopropanoyl) -D-proline ("Captopril") is known from U.S. Patent 4,105,776. This known compound has been used as a reference compound in the therapeutic comparative experiments set forth below and compared to the 4-phenylthio compound of the invention prepared in Example 13 according to the invention.

S-C, HC

| O 3 CH3 i:: hs-ch2-ch-co-n ____ I_cooh 1- (D-3-mercapto-2-methyl-oxopropyl) -cis-4-phenylthio-L-proline.

In this case, it turned out that the latter, i.e. the new compound, had a higher activity. Examples of alkanoyl groups include acetyl, propionyl, butyryl and isobutyryl, with acetyl being most preferred.

The compounds of formula I exist as stereoisomers or as a racemic mixture thereof. All of these can be attenuated by the method of the invention. In the synthesis described below, a racemate or one of the enantiomers can be used as a starting material. If a racemate is used as the starting material, the stereoisomers contained in the final product can be separated by conventional chromatographic or fractional crystallization methods. The XR group also induces cis transisomerism.

The asymmetric center of the proline ring on the C 1-4 atom preferably has the L-configuration. If the mercaptoacyl side chain has an asymmetric center, it has a D-configuration.

Of the novel compounds, those compounds of formula I in which X is oxygen is methyl 2 are very preferred. ...

or ethyl, especially methyl, R is hydrogen or methyl, eno-. 1 ...

methyl, R is hydrogen and the group XR is in the 4-position of the proline ring, especially when the XR1 group has the cis configuration.

The invention is characterized in that the proline compound of formula II

.XR1 c ^ f> <

I 3 II

HN I CCXJH

wherein R 1 and X are as defined above, are attached to an acid of formula III or a reactive derivative thereof R 2, 4-s-ch 2 - !: h-cooh hi 2 4 wherein R is as defined above and R 'is C 1-4 ~ an alkanoyl or benzoyl group, to give a compound in which R is a N-alkanoyl or benzoyl group, and that the compound obtained is, if desired, hydrolyzed to give a compound in which R is a hydrogen atom and R, R and X are as defined above, and that any acid obtained is, if desired, converted into a salt.

This reaction can be carried out in the presence of a coupling reagent such as dicyclohexylcarbodiimide or the like, or the acid can be activated to form a mixed anhydride, a symmetrical anhydride, an acid halide or an active ester, or Woodward's K-reagent, N-ethoxycarbonyl can be used. ethoxy-1,2-dihydroquinoline and the like. Acylation methods are summarized in Methoden der Organischen Chemie (Houben-Weyl), Vol XV, Part II, pp. 1- (1974). It is preferred to react an acid halide of formula III, especially an acid chloride, with an acid of formula II.

The compound of formula I is preferably isolated and purified by crystallization, for example by formation of a dicyclohexylamine salt and subsequent conversion of this salt to the free acid by treatment with an aqueous solution of an acid such as acidic potassium sulphate.

The proline starting materials of formula II can be prepared in various ways. For example, hydroxy or mercaptoproline of the formula

XH

H2C ' "> S

7! (IV)

HN_C-COOH

B

acylating with an acylating reagent such as acetic anhydride, acetyl chloride, propionic anhydride, butyric anhydride, benzyl chloroformate and the like so as to protect the nitrogen. The R 1 group is then attached by reacting a N-protected compound of formula IV with a halide R 1 -hal in which hal is halogen, preferably iodine, in the presence of silver oxide, sodium hydride, sodium hydroxide or the like so that an intermediate of formula XR1 i 1 is obtained, (V)

protected-N-C-COOR

i

B

II

5,69834

When the starting material of formula V is subjected to basic hydrolysis with a base such as barium hydroxide, sodium hydroxide, potassium hydroxide or the like, free acid (COOH) is first obtained, and then hydrolysis with an inorganic acid such as sulfuric acid gives the starting material of formula II.

In another process for the preparation of proline starting materials of formula II, the N-protected tosyloxyproline ester, preferably the methyl ester of the formula OTs Υ ° · ί protected-NC-COO-alkyl

B

is reacted with a sodium salt of formula R1-X-Na VII

to give an intermediate of formula XR1

H C

j 1 (VIII) protected-N-C-COO-alkyl

B

In formula VI, the symbol Ts represents a group -SO 2 - (o 2 -CH 3 and the nitrogen protecting group is preferably a benzyloxycarbonyl group or an ordinary acyl protecting group. If in this reaction the tosylate group has the cis configuration, the group XR 1 will have the trans configuration, and if the tosyloxy group has The intermediate of formula VIII is then deprotected with an alkyl ester group and then reacted with hydrogen bromide to give the hydrobromide salt of the proline reagent of formula II which can be coupled to an acid of formula III or preferably to acid chloride.

Proline starting materials of formula II in which X is sulfur and the group XR1 is attached at the 3-position of the proline ring can also be prepared by preparing a 1,2-dehydroproline ester, preferably a tert-butyl ester of formula! (IX) N 1 -COO-alkyl is reacted with an acylating reagent such as benzyl chloroformate, acetyl chloride, etc. to give a 4,5-dehydro compound of the formula Γ '' il (x) protected-N 1 -C 1 -C 100 alkyl which is then treated with mercaptan R 1 -SH to give a compound of formula XI 'S-r 1 fi (xi) protected-N -----'-COO-alkyl wherein the group -SR 1 - has the trans configuration. The nitrogen protecting group and the alkyl group are then removed to give the desired starting material.

Proline starting materials of formula II wherein R 1 is phenyl, substituted phenyl, phenyl-lower alkylene or substituted phenyl-lower alkylene can also be prepared by treating the N-protected benzyl ester of proline of formula IV with an alcohol R-OH in the presence of triphenylphosphine and diethyl azodicarboxylate following the method described in Bittner et al., Chemistry and Industry, 15 March 1975, 7 69834 p. 281. When the nitrogen protecting group and the benzyl ester group are removed, the starting material of formula II is obtained.

For further information on starting materials and intermediates, reference is also made to the following publications: Ondetti et al., U.S. Patent Nos. 4,046,889, 4,105,776 and 4,154,935, Neuberger, J. Chem. Soc., 1945, p. 429-432, Patchett et al., J. Amer. Chem. Soc. 79, pp. 185-192 (1957), Baer et al., Can.J. Biochem and Phys., 37, pp. 583-587 (1959), Sheenan et al., J. Amer. Chem. Soc. 85, ss. 3863-3865 (1963), Magerlein, J.Med. Chem. 10, ss. 116-1163 (1967). The methods described herein can be used to synthesize and stereoconvert the compounds of the invention.

Further details can be found in the examples, which describe preferred embodiments and are also models for the preparation of other compounds in the group.

The compounds of this invention form basic salts with various inorganic and organic bases. The salt-forming ion derived from such bases may be a metal ion such as aluminum ions, alkali metal ions such as sodium or potassium ions, alkaline earth metal ions such as calcium or magnesium ions or amine salt ions known for this purpose. and are, for example, aralkylamines such as dibenzylamine, Ν, Ν-dibenzylethylenediamine, lower alkylamines such as methylamine, tert-butylamine, procaine, lower alkyl-lipiperidines such as N-ethylpiperidine, cycloalkylamines, cycloalkylamines, cyclohexylamine or cyclohexylamine or salts derived from amino acids such as arginine, lysine and the like. Physiologically acceptable salts such as sodium or potassium salts are preferred because they can be used for medical purposes, as discussed below. These and other salts, which may not be physiologically acceptable, are useful in isolating and purifying products suitable for the purposes described below, such as the dicyclohexylamine salt and cyclohexylamine salt described in the Examples. The salts are prepared by reacting the acid form of the compound with an equivalent amount of the required ion-giving base in a salt-precipitating medium or water and then freeze-drying. The free acid can be obtained from the salt by conventional neutralization methods, for example, sodium bisulfate, hydrochloric acid, etc.

The compounds of this invention inhibit the conversion of decapeptide angiotensin I to angiotensin II and are therefore useful in lowering and alleviating hypertension. The compounds of this invention interfere with the renin -> angiotensinogen -> angiotensin I -> angiotensin II system by inhibiting the angiotensin converting enzyme and thus reducing or eliminating the formation of angiotensin II, which is a pressor. Thus, in a mammal suffering from hypertension, the blood pressure can be lowered or alleviated by administering an effective antihypertensive dose of a mixture containing one or more compounds of formula I or a salt thereof.

Animal experiments have shown (SL Engel, TR Schaeffer, MH Waugh and B. Rubin, Proc. Soc. Exp. Biol. Med. 143,483 (1973)) that a single dose or preferably about 0 divided into 2-4 doses is sufficient to lower blood pressure. 1 to 100 mg per kilogram per day, preferably about 1 to 50 mg per kilogram per day. The agent is preferably administered orally, but may also be administered parenterally, such as subcutaneously, intramuscularly, intravenously or intraperitoneally.

The compounds of this invention may be used for the purpose of lowering blood pressure by formulating them in the form of tablets, capsules or elixirs for oral administration and in the form of sterile solutions or suspensions for parenteral administration. A total of about 10 to 500 mg of one or more compounds of formula I, or a pharmaceutically acceptable salt thereof, is mixed in accordance with standard pharmaceutical practice into a unit dose of a physiologically acceptable excipient, carrier,

II

9 69834 with diluent, binder, preservative, stabilizer, flavor, etc. These mixtures or preparations contain the active ingredient in such an amount that a dose is obtained in the above-mentioned range.

Excipients suitable for tablets, capsules, etc. include e.g. the following: binders gum tragacanth, gum arabic, maize starch or gelatin, diluents dicalcium phosphate or microcrystalline cellulose, maize starch, potato starch, alginic acid and the like as disintegrants, lactic acid or the like, . If the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other substances may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl p-hydroxybenzoic acid as a preservative, a coloring agent and a cherry or orange flavoring agent.

Sterile injectable compositions may be prepared by conventional means for dissolving or suspending the active ingredient in a carrier such as water for injections or naturally occurring vegetable oils such as sesame oil, coconut oil, peanut oil, linseed oil and the like.

Therapeutic Comparative Experiments The inhibitory effect of the compound prepared according to the invention and the known "Captopril" compound according to the invention on augiotensin-converting enzyme isolated from rabbit lung and the inhibitory effect on augiotensin I-converting enzyme (ACE) in guinea pig ileum were determined in vitro.

The results are shown in the following table.

10 69834

Compound Structure Activity Activity

Rabbit lung Guinea pig pulse ACE I_mM ileum ECcrimM

_______________ i> (J_50 CH,

Comparison ; 3 I; "Captopril" HSCH2CHCO-N___ 1-COOH 20 20 S-C, HCl 6 5

Kek.muk. \ example HSCH2CHCO-N _______ 1-COOH 8 3 13

The following examples illustrate the invention and represent preferred embodiments. They are also models for the preparation of other compounds of the group that can be prepared by replacing the given reactants with appropriately substituted analogs. All temperatures are given in degrees Celsius.

Example 1 1- (3-Acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline a) N-Acetyl-trans-4-hydroxy-L-proline To a stirred suspension of 26.2 g (0, 2 moles) of trans-4-hydroxy-L-proline in 400 ml of acetic acid, 26 ml of acetic anhydride are added. The solid gradually dissolves after stirring for 2 hours at room temperature. Transfer the solution to a 2-liter flask and evaporate the solvent on a rotary evaporator in a 45 ° C bath. When the syrupy residue (57.5 g) is diluted with 100 ml of ether, a crystalline solid is obtained. The mixture is kept under cooling overnight, the solid is filtered off, washed with cold ether and dried in a desiccator. This material (35.7 g) was triturated and suspended in 100 ml of ether, cooled and filtered to give 33.8 g (98%) of N-acetyl-trans-4-hydroxy-L-proline, 128- 131 ° C. Recrystallization of 0.5 g of this material from 5 ml of acetonitrile gives 0.45 g of a colorless solid, m.p. 130-132 °, -92 °

II

69834 (c in 1% ethanol).

b) N-acetyl-trans-4-methoxy-L-proline methyl ester

A mixture of 30.0 g (0.17 mol) of N-acetyl-trans-4-hydroxy-L-proline and 130 g of silver oxide is ground in a mortar to a fine mixture and added with acetone (300 ml) to a 1 liter flask. A total of 130 ml of methyl iodide is added in small portions to the stirred suspension and the temperature of the mixture is kept below 40 ° C by cooling in a cold water bath. The mixture is stirred for 7 hours and then allowed to stand overnight. When the solid is filtered off, washed with acetone and the filtrate is evaporated on a rotary evaporator, 38.3 g of a syrupy residue are obtained. When this is redissolved in 350 ml of acetone and treated with a further 130 g of silver oxide and 130 ml of methyl iodide, 41 g of residue are obtained. When the residue is distilled, 32.2 g of distillate are obtained, b.p. 130-140 ° C (0.3 mm). Digestion in 30 ml of cyclohexane and cooling gives 31.4 g of N-acetyl-trans-4-methoxy-L-proline methyl ester as an almost colorless solid, m.p.

71-75 ° C. Recrystallization from 31 ml of ethyl acetate gives 25.1 g (66%) of a colorless solid, m.p.

76-77 ° C, λ max -83 ° (c, 1% in ethanol).

c) trans-4-methoxy-L-proline

To a stirred solution of 27.0 g (0.085 moles) of barium hydroxide octahydrate in 525 ml of water (ca. 3.3 N) is added 11.0 g (0.05 moles) of N-acetyl-trans-4-methoxy-L -proline methyl ester. The resulting solution is stirred at 18-20 ° C for 3 hours, cooled and treated with dilute sulfuric acid (8.85 g of concentrated sulfuric acid in 20 ml of water). The acidic suspension is allowed to stand overnight. When the mixture is filtered through a thick layer of "Celite", a milky filtrate is obtained.

Concentration of the filtrate on a rotary evaporator at 50 ° C using a high vacuum pump gives a milky residue weighing 121 g. To this material is added dilute sulfuric acid (19.0 g of concentrated sulfuric acid in 75 ml of water) and refluxed for 3 hours with stirring. The mixture is cooled to 30 [deg.] C. and then a total of 48 g of barium-12,69834 hydroxide dicohydrate is added in small portions and then the pH is adjusted to between 6.0 and 4.0 with dilute sulfuric acid. The mixture is allowed to stand overnight and then filtered through a thick layer of "Celite". When the milky filtrate is concentrated in the same manner as above, 50 g of a colorless dry residue are obtained. The residue is digested with 200 ml of hot chloroform and the barium sulfate is removed by filtration through a pad of Celite. Concentration of the slightly turbid filtrate on a rotary evaporator gives a gelatinous material (17.7 g) which is suspended in 100 ml of ether and filtered to give 7.5 g (94%) of an almost colorless solid, m.p. 185-190 ° C (decomposes). When this material was suspended in 30 mL of warm acetonitrile, cooled and filtered, trans-4-methoxy-L-proline was obtained as a colorless solid (4.0 g, 50%), m.p. 209-211 ° (decomposes), -75 ° (c, 1% in ethanol).

d) 1- (3-Acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline

A solution of 3.5 g (0.024 mol) of trans-4-methoxy-L-proline in 50 ml of water is cooled to 5 [deg.] C. with stirring and then 3 g of sodium carbonate are added. To this mixture is added over 10 minutes a solution of 4.0 g (0.024 mol) of 3-acetylthiopropionyl chloride in 5 ml of ether, and at the same time 3 g of sodium carbonate are added so that the pH remains at about 8.0. The mixture is stirred in an ice bath for a further 1 hour and then 25 ml of water are added, followed by a solution of 5 ml of concentrated hydrochloric acid in 25 ml of water (carbon dioxide is released). The strongly acidic solution is saturated with sodium chloride and extracted four times with 50 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 6.0 g (90%) of colorless syrupy 1- (3-acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline are obtained. Dissolving this acid in 25 ml of ethyl acetate and adding 4.7 g of dicyclohexylamine gives a solution which solidifies rapidly. After adding 15 ml of ethyl acetate and digesting the mixture with a steam bath, cooling and filtering, 8.7 g of the dicyclohexylamine salt are obtained, m.p. 170-172 °. Crystallization from 13,698,34 of 60 ml of acetonitrile gives 8.3 g (75%) of a colorless solid, m.p. 171-173 ° C, -35 ° (c, 1% ethanol).

The dicyclohexylamine salt is converted into 1- (3-acetyl-thio-1-oxopropyl) -trans-4-methoxy-L-proline by suspending 8.0 g in 60 ml of ice-cooled ethyl acetate and adding in small portions a total of 60 ml of 10% potassium bisulfate. . The clear layers are separated and the aqueous layer is extracted twice with 60 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 4.6 g (80%) of a colorless syrup are obtained.

Example 2 1- (3-Mercapto-1-oxopropyl) -trans-4-methoxy-L-proline

To the 1- (3-acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline (4.6 g, 0.017 mol) obtained in Example 1 is added a cold solution of 9 ml of concentrated ammonia in 22 ml of water. The base dissolves in about 30 minutes and the solution thus obtained is allowed to stand (under an argon atmosphere) for 2 hours at room temperature. This solution is cooled, extracted twice with 25 ml of ethyl acetate and the ethyl acetate extract is discarded. The solution is layered with a further 25 ml of ethyl acetate and acidified with 17 ml of hydrochloric acid (1: 1). The mixture is shaken, separated and the aqueous phase is extracted three times with 25 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is removed on a rotary evaporator to give 2.3 g (59%) of a colorless syrup, 1- (3-mercapto-1-oxopropyl) -trans-4-methoxy-L-proline as a colorless syrup, 25 ° / o <7d -60 (c, 1% in ethanol), 0.49 (methanol on silica gel, visualized with nitroprusside reagent).

Analysis CgH ^ ^ NO ^ S · 1/4 I ^ O

calculated: C 45.46 H 6.57 N 5.87 N 5.87 S 13.48 found: C 45.42 H 6.78 N 5.96 S 13.27.

An additional 1.1 g (3.4 g, 87%) of product is obtained when the aqueous phase is saturated with sodium chloride and extracted twice with 25 ml of ethyl acetate.

14 69834

The sodium salt is obtained by treating the syrup with aqueous sodium bicarbonate solution and lyophilizing.

Example 3 Trans-1- [N> -3- (acetylthio) -2-methyl-1-oxopropyl] -4-methoxy-L-proline

A solution of 4.3 g (0.029 mol) of trans-4-methoxy-L-proline in 50 ml of water is stirred and cooled to 5 [deg.] C. and 3 g of sodium carbonate are added. To this solution is added over 10 minutes 5.2 g (0.029 mol) of D-3- (acetylthio) -2-methylpropionyl chloride in 5 ml of ether and at the same time 3 g of sodium carbonate are added so that the pH remains at about 8.0. This mixture is stirred in an ice bath for about 1.5 hours, 25 ml of water are added, followed by 6 ml of concentrated hydrochloric acid in 25 ml of water (carbon dioxide is released). The strongly acidic solution thus obtained is extracted with 50 ml of ethyl acetate four times. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 6.1 g of trans-1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4-methoxy-L -proline as a pale yellow syrupy residue. This acid is dissolved in 50 ml of ethyl acetate and then a solution of 4.0 g of dicyclohexylamine in 20 ml of ethyl acetate is added. The product begins to crystallize from solution within about a minute. After cooling overnight, the almost colorless solid was collected by filtration and dried to give 6.7 g of product, m.p. 175-177 ° C, -55 ° (c, 1% in ethanol). Crystallization from 50 ml of acetonitrile gives 5.6 g of an almost colorless solid dicyclohexylamine salt (41%), m.p. 179-181 ° C, C 18-62 ° (c, 1% in ethanol).

The dicyclohexylamine salt is converted into the acid by suspending 5.5 g in 50 ml of ethyl acetate, cooling in an ice bath and adding 50 ml of 10% potassium bisulfate. The layers are separated and the aqueous portion is extracted twice with 50 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 3.4 g (41%) of trans-1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4- methoxy-L-proline as an almost colorless syrup.

li 15 69834

Example 4

Trans-4-methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline to 3.4 g of trans-1- [β-3- (acetylthio) -2- methyl 1-oxo-propyl-4-methoxy-L-proline is added 8 ml of concentrated ammonia in 20 ml of water. The base dissolves in about 10 minutes and the solution thus obtained is allowed to stand at room temperature under an argon atmosphere for 2 hours. This solution is cooled, extracted twice with 20 ml of ethyl acetate, layered with 20 ml of ethyl acetate and acidified with 15 ml of hydrochloric acid (1: 1). This mixture is saturated with sodium chloride, the layers are separated and the aqueous phase is extracted three times with 20 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 2.9 g (100%) of almost colorless trans-4-methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) are obtained. ) -L-proline, C ^ J q -80 (c, 1% in ethanol), 0.53 (in methanol on silica gel, visualized with nitro-prusside reagent).

Analysis for C10H17NC> 4S. 1/4 H 2 O calculated: C 47.69 H 6.83 N 5.56 S 12.73 found: C 47.90 H 6.84 N 5.85 S 12.76.

Example 5 Trans-1-Z.D-3- (acetylthio) -2-methyl-1-oxopropyl-1,4-ethoxy-L-proline

Following the procedure of Example 3 but replacing trans-4-methoxy-L-proline with trans-4-ethoxy-L-proline (J. Med. Chem., 10, 1161 (1967)), trans-1-choline is obtained. / D-3- (acetylthio) -2-methyl-l-oxopropyl / -4-ethoxy-L-proline. The product is purified as the dicyclohexylamine salt, m.p. 170-172 ° C (crystallized from isopropyl alcohol), -64 ° (c, 1% in ethanol).

When this salt (7.75 g) is converted to the free acid by treatment with potassium bisulfate solution as described in Example 4, 4.85 g of trans-1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4-ethoxy-L proline as an almost colorless syrup.

16 69834

Example 6 Trans-4-ethoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline

To the material obtained in Example 5 (4.85 g) is added a cold solution of 9 ml of concentrated ammonia in 22 ml of water (under an argon atmosphere). When the mixture is treated as in Example 4, 4.2 g (100%) of trans-4-ethoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline are obtained in an almost colorless manner. o as a syrup, -80 (c, 1% in ethanol), 0.64 (in methanol on silica gel, visualized with nitroprusside reagent).

Analysis for CH 2 Cl 2 NO 2 S

calculated: C 50.55 H 7.33 N 5.36 S 12.27 found: C 50.34 H 7.34 N 5.39 S 12.11 Example 7 cis-1- D-3- (acetylthio ) -2-methyl-1-oxopropyl-4-methoxy-L-proline a) N-carbobenzyloxy-cis-4-hydroxy-L-proline When 10 g (0.038 mol) of N-carbobenzyloxy-4-keto-L-proline is dissolved in 300 ml of methanol and reduced as described in JACS 79.189 (1957) with a solution of 5.8 g (0.15 mol) of sodium borohydride in 20 ml of water to give 8.7 g of a foamy product. This material is dissolved in 30 ml of ethanol, treated with 3.5 g of cyclohexylamine dissolved in a small amount of ethanol and diluted to 500 ml with ether. After seeding and trituration, the crystalline cyclohexylamine salt separates rapidly to give 10.8 g of product, m.p. 163-165 °. This cyclohexylamine salt is then treated with 30 ml of 2N hydrochloric acid and extracted with ethyl acetate (4 x 50 ml) to give 8 g of N-carbobenzyloxy-cis-4-hydroxy-L-proline as a glass.

b) N-carbobenzyloxy-cis-4-methoxy-L-proline methyl ester

When N-carbobenzyloxy-cis-4-hydroxy-L-proline (13.9 g, 0.052 mol) is treated with 40 g of silver oxide and 40 ml of methyl iodide (2 times) in acetone (first 100 ml, n 17 69834 then 120 ml) as described in Example 1 (b), 17.5 g (100%) of N-carbobenzyloxy-cis-4-methoxy-L-proline methyl ester are obtained in the form of a yellow oil.

c) N-carbobenzyloxy-cis-4-methoxy-L-proline

To a solution of 17.5 g (ca. 0.052 mol) of N-carbobenzyloxy-cis-4-methoxy-L-proline methyl ester in 135 ml of methanol is added dropwise between -1 and + 4 ° C 32 ml (0.064 moles) of 2N sodium hydroxide and the mixture thus obtained are kept for 1 hour at 0 ° C and overnight at room temperature. About half of the solvent is removed on a rotary evaporator, the solution is diluted with 300 ml of water, washed with ether (the washings are discarded), the pH is adjusted to 2 with 12.5 ml of hydrochloric acid (1: 1) while cooling and extracted with ethyl acetate (4 x 150 ml). When the extracts are combined, dried (MgSO 4), filtered and the solvent is evaporated, 15 g of an orange-yellow syrup are obtained. The syrup is dissolved in 60 ml of ethanol, a solution of 6 g of cyclohexylamine in 10 ml of ethanol is added and diluted with 900 ml of ether. Upon seeding and trituration, a crystalline cyclohexylamine salt of N-carbobenzyloxy-cis-4-methoxy-L-proline, weighing 10.2 g, separates when cooled overnight, m.p. 148-150 ° (solidification point 144 °), 26 Z ° S7D -35u (c, 1% in ethanol). Recrystallization from 40 ml of acetonitrile gives 8.8 g of an almost colorless solid, m.p. 150-152 ° (solidification point 145 °), Ζ / ΚΖρ6 -34 ° (c, 1% in ethanol).

Treatment of the cyclohexylamine salt with hydrochloric acid gives 6.9 g (48%) of N-carbobenzyloxy-cis-4-methoxy-L-proline as a pale yellow viscous syrup, -32 ° (c, 1% in ethanol).

d) cis-4-methoxy-L-proline

A mixture of 6.8 g of N-carbobenzyloxy-cis-4-methoxy-L-proline, 210 ml of a methanol-water mixture (2: 1) and 2.3 g of 5% Pd-C is placed in a hydrogenator 294 for 4 hours. kPa under hydrogen pressure. When the catalyst is filtered off from the mixture and the filtrate is evaporated, 3.15 g of a brownish solid are obtained, m.p. 218-220 ° (decomposes). When the sample is crystallized from methanol-ether, colorless cis-4-methoxy-L-proline is obtained, 18,69834 m.p. 224-226 ° (decomposes), -42 ° (c, 1% in methanol).

Analysis Calculated for C 9 H 11 NO 2: C 49.64 H 7.64 N 9.65 Found: C 49.63 H 7.71 N 9.54 e) cis-1- [D-3- (acetylthio) -2- methyl 1-oxopropyl-4-methoxy-L-proline

Cis-4-methoxy-L-proline (3 g, 0.021 mol) and a mixture of 4.2 g (0.023 mol) of D-3-acetylthio-2-methylpropionyl chloride in 5 ml of ether are reacted 60 ml of water in the presence of sodium bicarbonate as described in Example 3. About 20 ml of 25% (w / v) sodium bicarbonate solution is required to initially bring the pH to 8.5 and then to maintain it between 7.5 and 8.4 during acylation. The crude viscous product obtained (6.4 g) is dissolved in 50 ml of ethyl acetate and 3.9 g of dicyclohexylamine in 20 ml of ethyl acetate are added to the solution. When the product crystallized from solution is filtered off and dried, 6.6 g of dicyclohexylamine salt are obtained, m.p. 172-174 ° (freezing point 170 °), λ max -60 ° (c, 1% in ethanol). Recrystallization of 6.5 g of this material from 35 ml of acetonitrile gives 6 g of a colorless solid salt of dicyclohexylamine, m.p. 173-175 ° (freezing point 170 °), (-60 ° (c, 1% in ethanol).

Analysis ci2Hi9NO5S · ci2H23N

calculated: C 61.24 H 9.00 N 5.95 S 6.81 found: C 61.16 H 8.81 N 5.95 S 6.67.

Following the procedure of Example 3, the dicyclohexylamine salt is converted to the acid by suspending 5.9 g of the salt in 60 mL of ethyl acetate, cooling in an ice bath, and treating with 60 mL of 10% potassium bisulfate. The layers are separated and the aqueous phase is extracted four times with 50 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 3.5 g (60%) of colorless cis-1-ZP-3- (acetylthio) -2-methyl-1-oxopropyl--4-methoxy-L- proline, m.p. 90-92 ° (triturated with ether), Zp <7q ^ -139 ° (c, 1% in ethanol), 0.63 (in methanol on silica gel).

li 19 69834

Analysis for ci2Hi9NO5S

calculated: C 49.81 H 6.62 N 4.84 found: C 49.85 H 6.66 N 4.97.

Example 8 cis-4-methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline

When cis-1-Zp-3- (acetylthio) -2-methyl-1-oxopropyl-4-methoxy-L-proline (2.9 g, 0.01 mol) is hydrolyzed as described in Example 4 in 15 ml of water, containing 6.5 ml of concentrated ammonia, 2.3 g (93%) of highly viscous cis-4-methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline are obtained, which becomes waxy on standing, -88 ° (c, 1% in ethanol).

Example 8a Adamantanamine salt of cis-4-methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline 1,

When a solution of 0.55 g (0.0022 mol) of cis-4-methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline in 15 ml of ethyl acetate is added under an argon atmosphere, a warm solution of 0.34 g (0.0022 mol) of 1-adamantanamine in 10 ml of ethyl acetate, the salt precipitates. After refluxing for 3 hours, the colorless solid was collected by filtration under argon (solvent removed with difficulty), washed with a small amount of cold ethyl acetate and dried in vacuo for 20 hours to give 0.7 g (79%) of cis-4-methoxy-1- (D 3-mercapto-2-methyl-1-oxopropyl) -L-proline 1-adamantanamine salt, m.p. 215-217 ° (freezing point 210 °, decomposes at 220 ° C), -60 (c, 1% in methanol).

Example 9 Trans-1-ZT> -3- (acetylthio) -2-methyl-1-oxopropyl-4-propoxy-L-proline a) N-acetyl-trans-4-propoxy-L-proline propyl ester

When 30 g of N-acetyl-trans-4-hydroxy-L-proline from Example 1 (a) is reacted with silver oxide (110 g) and propyl iodide (110 ml) in 300 ml of acetone according to Example 1 (b), 19.6 g (41%) of pale yellow N-acetyl-trans-4-propoxy-L-proline propyl ester are obtained, b.p. 155-165 ° (0.2 mm).

b) N-acetyl-trans-4-propoxy-L-proline

When a solution of 6 g (0.15 mol) of sodium hydroxide in 150 ml of water is added to 19.4 g (0.075 mol) of N-acetyl-trans-4-propoxy-L-proline propyl ester, a light orange is obtained. solution. The solution is allowed to stand at room temperature overnight, then extracted with 60 ml of ethyl acetate (the washing solution is discarded), then acidified with hydrochloric acid (1: 1), then saturated with sodium chloride and extracted three times with 50 ml of chloroform. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 12.6 g of a brown oil are obtained, the oil is dissolved in 80 ml of ethyl acetate and a solution of 10.7 g of dicyclohexylamine in 20 ml of ethyl acetate is added. The salt crystallizes at room temperature. After allowing the mixture to stand overnight under cooling and then filtering the dicyclohexylamine salt and washing with cold ethyl acetate, 17.3 g of an almost colorless solid dicyclohexylamine salt are obtained, m.p. 148-153 ° C. Recrystallization of this material from 125 ml of ethyl acetate gives 14.5 g of a colorless dicyclohexylamine salt, m.p. 157-159 °, -30 ° (c, 1% in ethanol).

Analysis for C 12 H 23 NO 2 · C 12 H 23 N

calculated: C 66.63 H 10.17 N 7.07 found: C 66.47 H 10.22 N 7.06.

The dicyclohexylamine salt (14.4 g) is converted into the free acid by first grinding the salt to a powder, then suspending in 100 ml of ethyl acetate and treating the suspension in small portions with a total of 100 ml of 10% potassium bisulphate. The organic phase is separated and the aqueous phase is extracted twice with 100 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 6.7 g (41%) of N-acetyl-trans-4-propoxy-L-proline are obtained in the form of a light brown liquid.

c) trans-1- [E-D- (acetylthio) -2-methyl-1-oxopropyl] -4-propoxy-L-proline to 21,69834 N-acetyl-trans-4-propoxy-L-proline (6.4 g, 0.03 mol) a solution of 10 g of concentrated sulfuric acid in 100 ml of water is added and the solution thus obtained is refluxed for 3 hours with stirring. The solution is then cooled to 15 ° C, 12 g of sodium carbonate are added in small portions to bring the pH to 8.0, then a solution of 5.4 g (0.03 mol) of D-3-acetylthio is added over 10 minutes. -2-methylpropionyl chloride in 5 ml of ether, and at the same time 7 g of sodium carbonate are added so that the pH remains at about 8.0. The mixture is then stirred in an ice bath for 30 minutes and at room temperature for 1 hour. The product is then isolated and purified as the dicyclohexylamine salt by the method of Example 3 to give 6.3 g of product, m.p.

165-167 ° (from acetonitrile), -56 ° (c, 1% ethanol).

Analysis: C 1 H 2 NO 2 S-C 2 H 2 N

calculated: C 62.62 H 9.30 N 5.61 S 6.43 found: C 62.35 H 9.48 N 5.88 S 6.45. The dicyclohexylamine salt (6.1 g) is converted into the free acid by suspending the salt in 60 ml of ethyl acetate, cooling in an ice bath and adding 60 ml of 10% potassium bisulfate. The layers are separated and the aqueous phase is extracted twice with 60 ml of ethyl acetate. When the organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated, 4.0 g (42%) of trans-1- [TD-3- (acetylthio) -2-methyl-1-oxopropyl] -4-propoxy- L-proline as an almost colorless syrup.

Example 10 1- (D-3-Mercapto-2-methyl-1-oxopropyl) -trans-4-propoxy-L-proline

When hydrolyzing 4.0 g of trans-1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4-propoxy-L-proline as in Example 4 with a solution of 8 ml of concentrated ammonia in 20 ml 3.42 g (97%) of 1- (D-3-mercapto-2-methyl-1-oxopropyl) -trans-4-propoxy-L-proline are obtained in the form of an almost colorless syrup, (p c, 1% in ethanol).

69834 22

Analysis for C 12 H 2 N 4 O 4 S 1/4 H 2 O calculated: C 51.49 H 7.74 N 5.05 S 11.46 found: C 51.66 H 7.76 N 5.94 S 10.51.

Example 11 cis-4- (4-fluorophenoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline a) cis-4- (4-fluorophenoxy) -L-proline

To a solution of 8.4 g (0.024 moles) of benzyl ester of N-carbobenzoxy-trans-4-hydroxy-L-proline (Baer et al., Can.J. Biochem. & Phys., 37, 583 (1959)). ). 4.0 g (0.036 mol) of 4-fluorophenol and 9.27 g (0.036 mol) of triphenylphosphine in 75 ml of dry tetrahydrofuran are added dropwise over a period of 6.2 g (0.036 mol) of diethyl azodicarboxylate in 25 ml of tetrahydrofuran. . The solution is stirred at room temperature overnight. The mixture is then evaporated to dryness and 100 ml of ether are added to the residue. The precipitate formed by triphenylphosphine and diethyl azodicarboxylate is filtered off. Separation by column chromatography (silica gel) gives 8.1 g of a mixture containing about 70% of benzyl ester of N-carbobenzoxy-cis-4- (4-fluorophenoxy) -L-proline.

A solution containing 7.5 g of the mixture obtained above is hydrogenated at 98 kPa (room temperature) using 0.8 g of 10% Pd / C as catalyst. During the reaction, a white precipitate forms. When the absorption of hydrogen has ceased, the mixture is filtered and the precipitate cake is extracted three times with 125 ml of hot methanol. Evaporation of the methanol solution to dryness gives 3.2 g of cis-4- (4-fluorophenoxy) -L-proline, m.p. 235-236 °.

Analysis calculated for C 57 H 14 FNO 2 · 1/3 H 2 O: C 57.14 H 5.48 N 6.06 F 8.22 found: C 56.94 H 5.19 N 5.94 F 7.97.

b) 1- [T5-3- (acetylthio) -2-methyl-1-oxopropyl] -cis- 4- (4-fluorophenoxy) -L-proline

To a solution at room temperature of 2.25 g (0.01 mol) of cis-4- (4-fluorophenoxy) -L-proline in 125 ml

II

23 6 9 8 3 4 dry pyridine, 1.0 g (0.01 mol) of triethylamine and 2 g (0.11 mol) of D-3- (acetylthio) -2-methylpropionyl chloride are added. The mixture is stirred overnight at room temperature and then evaporated to dryness. The residue is taken up in water, covered with ether and acidified with 10% hydrochloric acid.

After washing the aqueous layer several times with ether, the ether layers are combined, washed with water, dried (Na 2 SO 4) and evaporated to dryness to give 3.9 g of residue. Chromatography of the residue on 250 ml of silica gel with ether gives a fraction containing the desired product. When the column is washed with methanol and the washings are rechromatographed on a short column of silica gel, more product is obtained. When this procedure is repeated once more, 1.2 g of pure 1-Zd-3- (acetylthio) -2-methyl-1-oxopropyl-7-cis- 4- (4-fluorophenoxy) -L-proline are obtained.

c) cis-4- (4-fluorophenoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline 1.2 g of 1- [D-3- (acetylthio) -2- methyl 1-oxopropyl-cis-4- (4-fluorophenoxy) -L-proline is dissolved in 25 ml of methanol and 5 ml of concentrated ammonia are added under an argon atmosphere over 40 minutes. The volatiles are distilled off and the residue is covered with ethyl acetate. The aqueous layer is acidified with 10% hydrochloric acid. The layers are separated and the acid layer is washed with ethyl acetate. The combined organic layers are distilled with water (2x) and saturated sodium chloride solution (2x) and dried (Na 2 O 4). When the solvent is distilled off, 1.1 g of cis-4- (4-fluorophenoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline are obtained.

Analysis: -H 2 FNO 2. 1/3 ^ 0 calculated: C 54.05 H 5.70 N 4.20 F 5.70 S 9.60 found: C 54.04 H 5.71 N 4.05 F, 5.40 S 9.61 . In the thin layer analysis, the R 2 value of the product became 0.30 when detected by UV light, SH reagent (yellow spot) and vanilla (yellow spot). -80 (c, 1% in chloroform).

24 69834

Example 12 cis-4- (4-fluorophenylthio) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline

Following the procedure of Example 11, but substituting an equivalent amount of 4-fluorophenyl mercaptan in (a) for (a), cis-4- (4-fluorophenylthio) -1- (D-3-mercapto-2-methyl-1-oxopropyl) is obtained. ) -L-proline. Sp. 242-243 ° (decomposes) as the adanamanthamine amine salt after recrystallization from chloroform / methanol (1: 1) diluted with ether.

-51.8 ° (c = 0.5% in methanol. Free acid ZpCl 7 = -42.5 ° (c = 1% abs. In ethanol.

Example 13 1- (D-3-Mercapto-2-methyl-1-oxopropyl) -cis-4-phenylthio-L-proline a) N-Carbobenzyloxy-cis-4-phenylthio-L-proline methyl ester 0.85 g (0.037 mol) of sodium metal are dissolved in 40 ml of absolute ethanol. To the resulting solution, 3.7 ml (0.036 mol) of thiophenol are added with stirring, followed by 7.5 g (0.017 mol) of N-carbobenzyloxy-trans-4-tosyloxy-L-proline methyl ester (J.Am.Chem.Soc. ., 79, 191 (1957)). The latter compound gradually dissolves, but soon after dissolution a solid product separates. The mixture is stirred for 4 hours and then allowed to stand overnight at room temperature and most of the ethanol is removed on a rotary evaporator. Most of the solid residue is mixed with dichloromethane (120 ml) and water (60 ml). The layers are separated (a little methanol is added to break the emulsions) and the aqueous phase is extracted with dichloromethane (2 x 60 ml). After washing the combined organic phase with 100 ml of saturated sodium chloride solution, drying (MgSO 4) and evaporating the solvent, 6.5 g (100%) of N-carbobenzyloxy-cis-4-phenylthio-L-proline methyl ester are obtained in the form of a pale yellow viscous oil.

Il 25 69834 b) N-Carbobenzyloxy-cis-4-phenylthio-L-proline The methyl ester obtained in part (a) (6.5 g, 0.017 mol) is dissolved in 55 ml of methanol, added in small portions at -1- + 4 ° C in 13 ml (0.026 mol) of 2N sodium hydroxide, stirred at 0 [deg.] C. for 1 hour and kept at room temperature for about 16 hours. About half of the solvent is evaporated off on a rotary evaporator, the cooled solution is diluted with 100 ml of water, washed with 60 ml of ether (the washing solution is discarded), partitioned with 70 ml of ethyl acetate, stirred, cooled and acidified with 4.8 ml of 1: 1 hydrochloric acid. The layers are separated, the aqueous phase is extracted with ethyl acetate (3 x 40 ml) and the combined organic layers are dried (MgSO 4) and evaporated to give 5.9 g of a pale yellow viscous oil. The oil is dissolved in 30 ml of ethanol, 1.9 g of cyclohexylamine in 3 ml of ethanol are added and diluted to 330 ml with ether. A crystalline cyclohexylamine salt is separated on seeding. After cooling the mixture for about 16 hours, 5.3 g of product are obtained, m.p. 148-151 ° (pour point 135 °). When this material is mixed with 1.5 g of the product obtained in the previous experiment, stirred in 200 ml of boiling acetonitrile and cooled, 6.3 g of a colorless cyclohexylamine salt are obtained, m.p. 152-155 ° C (freezing point 137 °) "29 ° (c, 1% in ethanol).

This cyclohexylamine salt is suspended in 25 ml of ethyl acetate, stirred and 25 ml of 1N hydrochloric acid are added.

When two clear layers are formed, they are separated and the aqueous phase is extracted with ethyl acetate (3 x 25 ml). Drying of the combined organic layers (MgSO 4) and evaporation of the solvent gives 5.0 g (65%) of N-carbobenzyloxy-cis-4-phenylthio-L-proline as an almost colorless, very viscous syrup.

c) cis-4-phenylthio-L-proline hydrobromide To N-carbobenzyloxy-cis-4-phenylthio-L-proline (4.9 g, 0.014 mol) is added 25 ml of hydrogen bromide acetic acid solution (30-32%), loosely sealed and stirred with a magnet. through.

After diluting the orange-yellow solution after 1 hour with 2 to 50 ml of ether, the product first precipitates as a heavy oil which gradually crystallizes on inoculation, scraping and cooling. After stirring in an ice bath for 1 hour, the precipitate is filtered under 26,698,34 nitrogen, washed with ether, suspended in fresh ether, cooled for about 16 hours and refiltered to give 3.2 g (77%) of cis-4-phenylthio-L-proline hydrobromide as a colorless solid. mp. 106-109 ° (freezing point 99 °), "3 ° (c, 1% in methanol).

d) 1- [3 - (acetylthio) -2-methyl-1-oxopropyl] -cis-4-phenylthio-L-proline

When 3.0 g (0.0094 mol) of cis-4-phenylthio-L-proline hydrobromide and 2.0 g (0.011 mol) of D-3-acetylthio-2-methylpropionyl chloride are reacted together in 25 ml of water as described in Example 1 (d) (using about 15 ml of a 20% solution of sodium carbonate to maintain the pH between 8.0 and 8.4), 3.8 g of a pale yellow viscous oil are obtained. The dicyclohexylamine salt (prepared in 30 ml of ethyl acetate using 1.8 g of dicyclohexylamine) weighs 2.9 g (isolated in two batches), m.p. 184-188 ° (pour point 180 °). Trituration in 15 ml of acetonitrile gives 2.4 g of a colorless solid dicyclohexylamine salt, m.p. 184-186 ° (pour point 180 °), -75 ° (c, 1% in ethanol).

When this dicyclohexylamine salt is treated with 30 ml of 10% potassium bisulfate and extracted into ethyl acetate as described in Example 1, 2.0 g (59%) of glassy 1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -cis-4 -phenylthio-L-proline.

e) 1- (D-3-mercapto-2-methyl-1-oxopropyl) -cis-4-phenylthio-L-proline

When 1-ZD-3- (acetylthio) -2-methyl-1-oxopropyl, 7-cis-4-phenylthio-L-proline (2.0 g, 0.0042 mol) is treated with a solution of 3.5 ml of concentrated ammonia in 8.5 ml of water according to Example 2 (the solid ammonium salt of the product mixes with the reaction mixture) gives 1.35 g (100%) of viscous syrupy 1- (D-3-mercapto-2-methyl-1-oxopropyl) - cis-4-phenylthio-L-proline, -43 ° (c, 1% in ethanol).

Example 14 1- (D-3-Mercapto-2-methyl-1-oxopropyl) -cis-4- (4-chloroethylthio) -L-proline

Following the procedure of Example 13, but thiophenol

II

27 69834 is replaced by an equivalent amount of 4-chlorophenyl mercaptan in (a) to give 1- (D-3-mercapto-2-methyl-oxopropyl) -cis-4- (4-chlorophenylthio) -L-proline.

Sp. 241-242 (decomposes) as an adanamanthamine amine salt after recrystallization from a chloroform / methanol mixture diluted with ether (1 :) - 55.4 ° (c = 0.5% in methanol) free acidic acid = -38.1 ° (c = 1% in absolute ethanol.

Example 15 Trans-1- (3-Mercapto-1-oxopropyl) -3-methylthio-D, L-proline a) 1,2-Dehydroproline tert-butyl ester To a stirred solution of -5 to 0 ° C with 34 , 2 g (0.20 mol) of proline tert-butyl ester in 600 ml of ether are added dropwise over 10 minutes 21.7 g (23.9 ml, 0.20 mol) of freshly prepared tert-butyl hypochlorite (Org. Sym .Coll. Vol. V, 184 (1973)). During the addition, the temperature is maintained between -5-0 ° C. After the addition, the solution is stirred at this temperature for another 5 minutes.

A solution of 7.8 g (0.20 mol) of potassium in freshly distilled dry (CaH2) tert-butanol is added rapidly ('V for 3-5 min) to a vigorously stirred solution. After the addition, the temperature of the reaction mixture is about 18 °. The reaction vessel is removed from the cooling bath and stirred for 30 minutes. The reaction mixture is filtered through "Celite" (diatomaceous earth) and the filtrate is evaporated in vacuo. The residue is taken up in ether and washed several times with water. When the ether solution is dried and concentrated in vacuo, 31.6 g of a yellow liquid are obtained. Add a little hydroquinone and distill the crude product to give 22.4 g of 1,2-dehydropropin tert-butyl ester (66%), b.p. 60-62 ° / 0.1 mm.

b) 1-Benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid tert-butyl ester

A solution of 16.9 g (0.1 mol) of tert-butyl ester of 1,2-dehydropropin in 70 ml of dichloromethane is cooled to -10 ° C under an argon atmosphere. A solution of 14.2 ml (0.1 mol, b.p. 62-64 ° / 0.4 mm) of freshly distilled benzyl chloroformate in 70 ml of dichloromethane is added dropwise over 30 minutes. The mixture is stirred under cooling for a further 28,698,34 minutes and then a solution of 15.22 g (0.1 mol) of 1,5-diazabicyclo [4> 4] is added over a period of 20 minutes. Q7un-dec-5-ene in 70 ml of dichloromethane. The cooling bath is then removed and the mixture is stirred for 1 hour at room temperature. After washing the solution twice with cold dilute hydrochloric acid and once with saturated sodium carbonate solution and evaporating in vacuo, 18.2 g (60%) of 1-benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid tert-butyl ester are obtained in the form of a pale white solid. as an oil.

c) tert-butyl ester of trans-1-benzyloxycarbonyl-3-methylthio-D, L-proline

To a solution of 18.2 g (0.06 mol) of 1-benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid tert-butyl ester in 180 ml of dry methanol is added 3.24 g. g (0.06 moles) of sodium methoxide and cooled in an ice bath. The solution is slowly bubbled with methanethiol for 30 minutes.

The mixture is stirred overnight under argon at room temperature. Add a dilute aqueous solution of acetic acid until the solution is slightly acidic. Argon is bubbled through the solution for one hour and then evaporated to near dryness in vacuo. When ethyl acetate is added to the residue and the solution is washed twice with saturated sodium carbonate solution, dried and the solvent is evaporated off under vacuum, 17 g of a yellow oil are obtained. Chromatography of the oil using 300 g of silica gel and petroleum ether-ether (4: 1) gives 11.1 g of trans-1-benzyloxycarbonyl-3-methylthio-D, L-proline as a colorless oil.

d) trans-3-methylthio-D, L-proline 8.4 g (0.024 mol) of trans-1-benzyloxycarbonyl-3-methylthio-D, L-proline are treated with 45 ml of 4N hydrobromic acid in acetic acid. The mixture is stirred for 1 hour at room temperature and then dried in vacuo. Add a small amount of water and wash twice with ether. The solution is poured onto a column of 300 ml of ion exchange resin and water is passed through until the eluate is no longer acidic. The product is then eluted with pH 6.5 buffer (aqueous pyridine acetate solution). When the ninhydrin-positive fractions are combined and lyc-lysed, 3.4 g (88%) of a white fluff are obtained. When a sample of 29 6 9 8 3 4 taken from this material is crystallized from methanol, trans-3-methylthio-D, L-proline, m.p. 196-200 ° (dec.) (Freezing point 192 °).

Analysis CgH 2 O 2 N 2 NS

calculated: C 44.70 H 6.88 N 8.69 S 19.89 found: C 44.53 H 7.10 N 8.61 S 19.95.

e) Trans-1- [3- (acetylthio) -1-oxopropyl] -3-methylthio-D, L-proline 3.05 g (0.019 mol) of trans-3-methylthio-D, L-proline are dissolved in 19 ml of 1N sodium carbonate. and diluted with 10 ml of water. The solution is cooled in an ice bath and a solution of 3-acetylthiopropionyl chloride in 20 ml of ether is added with rapid stirring. The pH is maintained at 8 by the addition of 1N sodium carbonate solution. After 30 minutes, the pH remains constant after the addition of 45 ml of sodium carbonate solution. The layers are separated and the aqueous layer is washed once with ether. When the aqueous layer is then acidified with 10% sodium bisulfate solution and the product is extracted into ethyl acetate, dried and the solvent is evaporated in vacuo, 5.2 g of oil are obtained. This material is chromatographed on 150 g of silica gel eluting with ethyl acetate. 3.85 g of slightly impure trans-1- [3- (acetylthio) -1-oxopropyl] -3-methylthio-D, L-proline are thus obtained.

A small sample is dissolved in ether and converted to the dicyclohexylamine salt, which is recrystallized from ethyl acetate to give trans-1-Z3- (acetylthio) -1-oxopropyl-3-methylthio-D, L-proline dicyclohexylamine salt, m.p. 153-157 °.

Analysis C ^ H ^ O ^ NS. (CGH ^^ NH

calculated: C 58.44 H 8.53 N 5.89, S 13.57 found: C 58.77 H 8.57 N 5.68 S 13.74.

f) trans-1- (e-mercapto-1-oxopropyl) methylthio-D, L-proline 2.05 g (0.007 moles) trans-1-Z3- (acetylthio) -1-oxopropyl-3-methylthio-D The L-proline is cooled in an ice bath under an argon atmosphere and then a cold argon-saturated mixture containing 7 ml of water and 7 ml of concentrated ammonia is added. The mixture is stirred for 30 minutes at 0 ° C and then acidified with hydrochloric acid. Extraction of the product with ethyl acetate, drying and evaporation of the solvent in vacuo gives 1.85 g of material which partially crystallizes on standing. Trituration with ether gives 1.0 g (57%) of a white crystalline product. Recrystallization from ethyl acetate (5 ml) gives 0.85 g of trans-1- (3-mercapto-1-oxopropyl) -3-methylthio-D, L-proline, m.p. 89-93 °.

Analysis CgH 2 O 2 N 2 NS

calculated: C 43.35 H 6.06 N 5.62 S 25.72 found: C 43.36 H 6.27 N 5.54 S 25.91.

Example 16 (cis) -4- (1,1-dimethylethoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline a) N-carbobenzyloxy-cis-4- (1, 1-dimethylethoxy) -L-proline methyl ester

A solution of 6.0 g (0.021 mol) of N-carbobenzyloxy-cis-4-hydroxy-L-proline methyl ester and 60 ml of methylene chloride was placed in a Parr apparatus, stirred magnetically, cooled in a dry ice / isopropanol bath, and then treated with ca. 40 ml of liquefied isobutylene and then 0.7 5 ml of concentrated sulfuric acid. The apparatus was sealed and the reaction mixture was allowed to warm to room temperature with constant stirring.

There was no noticeable increase in pressure. After stirring at room temperature for 4 days, the device was opened and the solution (80 ml) was diluted with 120 ml of methylene chloride and washed with a solution of 5 g of sodium bicarbonate in 100 ml of water. The aqueous phase is back-extracted with 50 ml of methylene chloride, the organic layers are combined and imaged (MgSO 4), and the solvent is evaporated off to give 7.5 g of N-carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline methyl ester. as a pale yellow oil.

[α] 25 D = 46 ° (c, 1% in chloroform TCL (on silica gel: R

0.62 (ethyl acetate) Rf 0.43 (7: 1 benzene / acetic acid)

Visualized by UV vapor b) N-carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline

A stirred solution of the methyl ester product from a) (7.2 g 0.02 mol) in 15 ml of ether was cooled with ice water 1i 69834 and the solution was treated portionwise with 42 ml (0.042 mol) of ice-cold sodium hydroxide. After stirring and cooling for a total of 6 hours, the cold bath was removed and stirring was continued overnight at room temperature. The clear solution was washed with 40 mL of ether (the washings were discarded), layered with 40 mL of ethyl acetate, stirred, cooled and acidified with 8 mL of 6 N hydrochloric acid. The phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 40 ml), the organic phases were combined and dried (MgSO 4) and the solvent evaporated to give 6.8 g of a pale yellow viscous oil. This oil was dissolved in 50 ml of acetonitrile and the solution was treated with a hot solution containing 3.2 g of 1-adamantanamine and 20 ml of acetonitrile. The solid adamantane amine salt was rapidly separated. The salt was cooled overnight, then this colorless salt was filtered under nitrogen, washed with cold acetonitrile and ether and dried in vacuo to give 8.8 g of N-carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline-adamantanamine salt, m.p. . 228-230 ° C (decomposes), pour point 215 ° C.

- -28 ° (c, 1% in methanol)

Analysis ^ N. 0.25 ^ 0 Calculated: C 67.96 H 8.56 N 5.87 Found: C 67.77 H 8.54 N 5.93.

Treatment of the resulting adamantanamine salt (8.5 g) with acid gave 5.5 g of N-carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline as an almost colorless viscous syrup. TLC: 0.20 (85:15 toluene / acetic acid visualized on silica gel with N-vapor or PMA with heat, c) cis-4- (1,1-dimethylethoxy) -L-proline The N-carbobenzyloxy product from b) (5.5 g 0.017 mol) was hydrogenated in 165 ml of 2: 1 methanol / water mixture 3 atm. in a hydrogen atmosphere in the presence of 1.7 g of a 5% palladium-on-carbon catalyst to give 3.2 g of a sticky, partially solid product. This product was suspended in 30 mL of acetonitrile, triturated, and cooled overnight to give 1.35 g of cis-4- (1,1-dimethylethoxy) -L-proline as a colorless solid 32,69834, m.p. 240-242 ° C (decomposes, before decomposing, gradually staining dark and sintering). mp = -36 ° (c, 0.5% in methanol).

Analysis CgH ^ NOg '0.225H2O

calculated: C 56.37 H 9.20 N 7.31 found: C 56.26 H 9.27 N 7.26.

d) 1- [D-3- (acetylthio) -2-methyloxopropyl] -cis-4- (1,1-dimethylethoxy-L-proline cis-4- (1,1-dimethylethoxy) -L-proline from c ) (1.3 g 0.0069 mol) was reacted with a solution of 1.4 g (0.0078 mol) of D-3-acetylthio-2-methylpropionyl chloride and 20 ml of water in the presence of sodium bicarbonate as in Example 3 is described. This gave 2.2 g of an almost colorless viscous oil. The obtained crude product was dissolved in 50 ml of ethyl acetate, and the solution was treated with a mixture of 1.3 g of dicyclohexylamine in 20 ml of ethyl acetate. The product crystallized from me. The product was filtered off and dried, yielding 2.65 g of the dicyclohexylamine salt, m.p. 181-183 ° C, pour point 170 ° C.

Z067 ^ 5 = -62 ° (c, 1% in ethanol)

Analysis ^ 5H25NO5S · C12H23N

calculated: C 63.24 H 9.44 N 5.46 S 6.25 found: C 63.22 H 9.61 N 5.41 S 6.02.

By proceeding as in Example 3, the 2,4-dicyclohexylamine salt was converted to the acid by suspension in ethyl acetate, cooling in an ice bath and treating with 30 ml of 10% potassium bisulfate. The layers were separated and the aqueous phase was extracted with 25 ml of ethyl acetate (4 times). The solvent was evaporated off and the material which had partially crystallized on standing was triturated with 20 ml of hexane to complete the crystallization and cooled to give 1.3 g of 1-ZD-3- (acetylthio) -2-methyl-1-oxopropyl-7-cis -4- (1,1-dimethyllethoxy) -L-proline as a colorless solid, m.p. 96-98 ° C, pour point 93 ° C.

- -109 ° (c, 1% in ethanol)

II

33 69834

Analysis calculated: C 54.36 H 7.60 N 4.23 S 9.63 Found: C 53.92 H 7.73 N 4.30 S 9.29.

e) cis-4- (1,1-dimethyllethoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline 1- (T) -3- (acetyl) 2-Methyl-1-oxopropyl-7-cis-4- (1,1-dimethylethoxy) -L-proline from d) (1.3 g 0.0039 mol) was hydrolyzed with a mixture of 2.6 ml of concentrated ammonium hydroxide and 6 ml of water to give 1.1 g of a glassy residue. While standing, crystal embryos appeared in the product. This material was covered with 8 ml of ether, seeded and triturated. As the glass dissolved, a crystalline solid gradually separated. After standing for 2 hours at room temperature, 20 ml of hexane was added and the mixture was cooled overnight under argon. The solvent was removed to give 1.0 g of a colorless solid cis-4- (1,1-dimethylethoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline, m.p. 106-108 ° (pour point 10 3 ° C).

= -78 ° (c, 1% in methanol)

Analysis for C 53 H 19 N 2 O 2 S * 0.25H90 Calculated: C 53.12 H 8.06 N 4.77 S 10.91 Found: C 53.30 H 8.28 N 4.76 S 10.70.

Example 17 (cis) -1- (trans) -3- (Benzoylthio) -2-methyl-1-oxopropyl] -4- (phenylthio) -L-proline 9.9 g (0.031 mol) of cis-4-phenylthio L-proline was suspended in 100 ml of water (pH 5.6) and the pH was adjusted to 10.2 by adding about 20 ml of 10% sodium bicarbonate to give a clear solution. The pH was then adjusted to 9.5 by the addition of about 4.5 ml of concentrated hydrochloric acid. The solution was kept at 30 ° C when 8.1 g (0.033 mol) of (D) -3- (benzoylthio) -2-methylpropanoic acid chloride in 30 ml of toluene were added simultaneously with 100 ml of 10% sodium bicarbonate to maintain the pH. 9.3. After about 1/4 of the acid chloride was added, a slimy precipitate began to form which remained throughout the reaction. After stirring at pH 9.3 for 2.5 hours, the reaction mixture was strongly acidified to 34,69834 by the addition of 20% hydrochloric acid in the presence of ethyl acetate. The aqueous phase was extracted twice with 350 ml portions of ethyl acetate and the combined organic phases were washed with 300 ml of saturated sodium chloride solution and dried (MgSO 4). The solvent was removed to give 11.8 g of a crude foamy solid. These 11.8 g of a crude foamy solid were dissolved in 70 ml of acetonitrile and about 6 g of dicyclohexylamine in 25 ml of ether were added to the solution. A free white crystalline precipitate formed. The mixture was allowed to stand overnight in a cold room and then the solid was filtered and washed with ether to give (cis-1-ZD-3- (benzoylthio) -2-methyl-1-oxopropyl) -4- (phenylthio) -L-proline. dicyclohexylamine salt (1: 1).

Analysis calculated for C 66.85 H 7.59 N 4.59: C 66.33 H 7.30 N 4.48.

The slightly moist dicyclohexylamine salt was stirred for 2.5 hours in a mixture of 300 ml of ethyl acetate and 200 ml of 10% potassium bisulfate. 2 bright layers formed. The aqueous layer was extracted twice with 200 ml portions of ethyl acetate. The combined organic phases were dried (MgSO 4). The solvent was removed to give 10.1 g of foamy solid (cis) -1- [D-3- (benzoylthio) -2-methyl-1-oxopropyl] -4- (phenylthio) -L-proline, m.p. 42-44 ° C.

-36.5 ° (c = 1, methanol).

Analysis calculated for C22h23no4s2: C 60.87 H 5.46 N 3.23 Found: C 60.75 H 5.35 N 3.17

Example 18 (cis-1- (D-3-mercapto-2-methyl-1-oxopropyl) -3-phenoxy-D, L-proline a) 3-Phenoxy-1,2-dehydroproline-1-butyl ester Mixture , containing 9.25 g (50 mmol) of 1,2-dehydroproline-1-butyl ester and 70 ml of toluene, was heated together with 8.9 g (50 mmol) of n-bromosuccinimide under reflux with sunlight (about 5 cm: n) for a total of 35 69834 hours. During this time, succinimide was formed; then the reaction mixture was cooled to room temperature. The succinimide was filtered off and the filtrate was evaporated to dryness. The residue was distilled at 80 ° C to give 3.5 g of crude 3-bromo-1,2-dehydro-proline-1-butyl ester.

A mixture of crude 3-bromo-1,2-dehydroproline-1-butyl ester and 3.9 g (13 mmol) of thallium phenoxide in 30 ml of anhydrous dimethylformamide was stirred at room temperature under argon for 24 hours. The solvent was removed in vacuo and the residue was diluted with ether to precipitate thallium bromide which was removed by filtration. The filtrate was evaporated to dryness to give an oil which was purified by distillation. After distilling for 2 hours at 0.005 mm and 80 ° C, 1.48 g of 3-phenoxy-1,2-dehydroproline-1-butyl ester remained on the distillation column.

b) (cis) -3-phenoxy-D, L-proline

A mixture of 1.48 g of 3-phenoxy-1,2-dehydro-proline-1-butyl ester, 5 ml of 1N sodium hydroxide and 20 ml of 80% dioxane (in water) was stirred at room temperature for 4 hours. The solvents were removed in vacuo and the residue was dissolved in 25 ml of water and the solution was treated with 168 mg of sodium borohydride for one hour at room temperature. The mixture was acidified to pH 6 by the addition of 2N hydrochloric acid; the solution was kept at 0 ° C for 16 hours, after which about 25 mg of (trans) -3-phenoxy-D, L-proline crystals precipitated. These were filtered off. The filtrate was desalted on a Dowex® 50 Wx8 column (2.5 x 30 m) using 1N ammonium hydroxide as eluent. The UV active fractions were combined and evaporated to dryness to give 616 mg of solid (cis) -3-phenoxy-D, L-proline c) (cis-1-ZD-3- (acetylthio) -2-methyl-1-oxopropyl) -3-phenoxy-D, L-proline

The obtained 616 mg of (cis) -3-phenoxy-D, L-proline was slurried in 20 ml of water at 5 ° C and the pH was adjusted to 8.0 with solid sodium carbonate. To the slurry was added 550 mg (2.5 mmol) of D-3-acetylthio-2-methylpropanoyl acid chloride in 1 mL of ether. The pH of the reaction mixture was maintained at 7.3-8.2 for the next 1.5 hours by the addition of sodium carbonate. The reaction mixture was washed with ethyl acetate (2 x 20 mL) and acidified to pH 2 by the addition of 10% hydrochloric acid and extracted with ethyl acetate (3 x 50 mL). The extracts were combined, dried (MgSO 4) and evaporated to dryness to give 810 mg of an oil. This oil was purified by flash chromatography on silica gel (LP-1, 300 mL) using 10-20% acetic acid / toluene mixtures as eluents. 530 mg of (cis) -1- [3- (acetylthio) -2-methyl-oxopropyl] -3-phenoxy-D, L-proline d) (cis) -1- (D-3-mercapto-2 -methyl-1-oxopropyl) -3-phenoxy, L-proline 530 mg of (cis) -1- [b-3- (acetylthio) -2-methyl-1-oxopropyl-5-phenoxy] DjL-proline was dissolved in an ammonia / water mixture (50:50) under argon, degassed by passing argon-gas bubbles through the mixture for 15 min. The solution was stirred at room temperature for 1.5 hours. In a slightly identical solution, the pH was adjusted to 6.5 with concentrated hydrochloric acid (some heat was generated). The solution was extracted with methylene chloride (2 x 25 mL). The pH was then adjusted to 1 and the solution was further extracted with methylene chloride (3 x 50 mL). The extracts were combined, dried (MgSO 4) and evaporated to dryness to give 410 mg of the glassy compound. Triturated with chloroform to give crystallization. The solid was recrystallized from a mixture of ethyl acetate and hexane to give 261 mg of (cis) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -3-phenoxy-D, L-proline, m.p. 134-135 ° C.

Analysis calculated: C 58.23 H 6.19 N 4.53 S 10.36 Found: C 58.07 H 6.18 N 4.45 S 10.16.

Example 19 a) 1- [D-3- (Acetyl) -2-methyl-1-oxopropyl, 7-cis-4-methylthio-L-proline

A solution of 4.67 g (0.029 mol) of cis-4-methylthio-L-proline and 50 ml of water was stirred and cooled to 1137,698,345 ° C. 3 g of sodium carbonate were then added to the solution. This solution was treated with 5.2 g (0.029 mol) of D-3- (acetylthio) -2-methylpropionyl chloride in 5 ml of ether for 10 min with the occasional addition of 3 g of sodium carbonate to maintain the pH at about 8.0. The mixture was stirred in an ice bath for 1.5 hours. To the mixture were added 25 ml of water and then a solution of 6 ml of concentrated hydrochloric acid and 25 ml of water (CO 2 gas evolved). The resulting strongly acidic solution was extracted with 50 mL of ethyl acetate (4 times). The organic phases were combined, dried (MgSO 4), filtered and the solvent evaporated to give the title compound.

b) 1- (D-3-Mercapto-2-methyl-1-oxo-propyl) -cis-4-methylthio-L-proline to 3.2 g of 1- [D-3- (acetylthio) -2- methyl 1-oxopropyl-7-cis-4-methylthio-L-proline was added a cold solution of 8 ml of concentrated ammonium hydroxide and 20 ml of water. The base dissolved in about 10 min. The resulting solution was allowed to stand under an argon atmosphere at room temperature for 2 hours. The solution was cooled, extracted with 20 ml of ethyl acetate (2 times), layered with 20 ml of ethyl acetate and acidified with 15 ml of 1: 1 hydrochloric acid. This mixture was saturated with sodium chloride, the layers were separated and the aqueous phase was extracted with 20 ml of ethyl acetate (3 times). The organic phases were combined, dried (MgSO 4), filtered and the solvent evaporated to give the title compound, m.p. As the 1-adamantanamine salt 198-201 ° C (decomposes).

-20 ° (c, 1% in methanol).

Claims (1)

38 69834 A process for the preparation of therapeutically useful ether or thioether mercaptoacyl prolines of the general formula I XR1. f R -S-CH2-CH-CO-N_C-COOH wherein the group -XR1 is attached to the 3- or 4-position of the proline ring, X is an oxygen or sulfur atom, R1 is a C1-4 alkyl group or a phenyl group which may be substituted by a halogen atom , R 12 is a hydrogen atom or a C 1-4 alkyl group and R 3 is a hydrogen atom or a C 1-4 alkanoyl or benzoyl group, and for the preparation of their salts, characterized in that the proline compound of the formula II XR 1 C 3 II HN-COOH wherein R 1 and X is as defined above, or a salt thereof is attached to an acid of formula III or a reactive derivative thereof R1, 4 f R '-s-ch2-ch-cooh III 4 2 io in which R is as defined above and R' is C, an alkanoyl-1-4 yl or benzoyl group to give a compound wherein R is a C 1-4 alkanoyl or benzoyl group, and that the resulting compound is hydrolyzed, if desired, to give a compound wherein R is a hydrogen atom and R , R and X have the same meaning as above, and that the acid obtained is, if desired, converted into a salt.