DK156054B - ANALOGY PROCEDURE FOR PREPARING N- (3-ACYLTHIO-2-METHYLPROPANOYL) PROLINE COMPOUNDS OR SALTS THEREOF - Google Patents

Description

DK 156054 B

The present invention relates to an analogous process for the preparation of novel N- (3-acyl-thio-2-methylpropanoyl) -proline compounds of the formula CH

R-A-S-N-CHoCHCO

V-1 'I

COOH

wherein R is an acyl group linked to an α-amino group in A and is selected from a cyclopropanecarbonyl, cyclohexanecarbonyl and adamantanecarbonyl group, and A is a glycine, sarcosine, D-alanine, D-leucine, D -phenyl-alanine, D-tryptophan, D-phenylglycine, D-methionine or D-glutamine residue, whose α-carbonyl group forms a thiol ester bond with the sulfur atom, or pharmaceutically acceptable salts thereof with bases.

Similar compounds such as those represented by formula I, e.g. those wherein R is a benzoyl, acetyl or t-butyloxycarbonyl group and A is an L-α amino acid are described in British Patent Application No. 2,050,359A. European Patent Application (EP) 9.898A1 discloses compounds wherein R is a benzoyl, acetyl, t-butyloxycarbonyl or cyclopentanecarbonyl group or the like and A is an L-α amino acid. In European Patent Application No. 35,383A1, compounds are disclosed wherein R is a benzoyl group and A is a D-phenylalanyl group. However, there is no prior art literature that specifically describes the compounds of formula I. They are therefore non-described, novel compounds and useful as pharmaceuticals as will be described below.

The group CH 2 in structural formula I has an asymmetric -ch 2 -chrotic carbon atom such that the group can be in D-form, L-form or DL-form, i.e. a mixture of D and L forms. Any of these forms fall within the scope of the present invention and the D and DL precursors are preferred for their biological action.

DK 156054B

2

The proline residue -N of formula I may be in D-

VJ

/

COOH

form, L-form or DL-form, and any of these forms 5 fall within the scope of the present invention, the L and DL forms being preferred for their biological activity.

Examples of pharmaceutically acceptable salts of the proline derivative of formula I include salts 10 with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with basic amino acids such as arginine and lysine. Calcium salts and lysine salts are preferred.

The process according to the invention is characterized in that:

(a) reacting a compound of formula II

CH3 R-A -S-CH2CHC00H 11

Wherein R and A have the above meanings, or a reactive derivative thereof, with a compound of formula III

HN III

N '- 1 COOR' wherein R 'is a hydrogen atom or a carboxyl protecting group, or a reactive derivative thereof in an inert organic solvent at between -50 and -20 ° C and removing any protecting groups from the reaction product, or

b) reacting a compound of formula IV

R-A-OH IV

wherein R and A have the above meanings, or a reactive derivative thereof having a compound of formula V

DK 156054B

3 CH3 \ n HS-CH2CHC0-N v

COOR

Wherein R 1 is a hydrogen atom or a carboxyl-protected group, or a reactive derivative thereof in an inert organic solvent at between -50 and 20 ° C, and removes any protecting groups from the reaction product, whereupon converting an obtained compound of formula I into a pharmaceutically acceptable salt thereof with a base.

When R 'of formula III or formula V is a carboxyl protecting group, it may be a t-butyl group or a lower alkyl substituted silyl group and may be removed by treatment with hydrogen fluoride, trifluoroacetic acid, hydrogen chloride or water.

Various convenient embodiments of the method according to the invention can be used as set forth in claims 2-5.

The reactive derivative of the compound of formula II is such that the carboxyl group included in the reaction is activated. The carboxyl group can be activated to any of the forms commonly used in peptide syntheses such as activated amides, acid halides, activated esters and mixed acid anhydrides. Of these forms, activated esters are preferred with N-hydroxysuccinimide, mixed acid anhydrides with carbonic acid monoesters, and activated amides with carbonyl diimidazole. Carbodiimides, such as dicyclohexylcarbodiimide, can be used as a condensing agent to form an amide bond between the carboxyl group and the imino group.

The reactive derivative of the compound of formula III is such that the imino group of said compound is activated. The imino group may be activated at any time.

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4 are sensitive to the methods conventionally used in the field of peptide synthesis, e.g. the method of introducing a silyl group, such as a trimethylsilyl group, the "phosphazo method" using a phosphorus compound such as phosphorus trichloride [Ann. Chem., 572, 96 (1951)], the phosphoric acid ester method using a phosphoric acid ester such as the tetraethyl ester of pyro phosphoric acid, or the "N-carboxy anhydride method" (NCA method).

The reaction of process a) can be carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphoric triamide, chloroform, dichloromethane or acetonitrile. The reaction is carried out as mentioned at -50 to 20 ° C, preferably -15 at -30 to 10 ° C. The reaction time varies with the reaction temperature, the compounds being reacted and the solvent, and it is generally between 0.5 and 48 hours, preferably between 1 and 6 hours.

After the amide formation reaction, all protecting groups in the reaction product are removed by any of the appropriate methods.

The final product can be isolated from the reaction mixture and purified by any conventional method, e.g. by various types of chromatography using silica gel, dextran crosslinked polymer and porous polymers such as styrene divinylbenzene or acrylic acid esters. A suitable developing solvent may be selected from chloroform, ethyl acetate, methanol, ethanol, tetrahydrofuran, benzene, water and acetonitrile. Alternatively, the final product may be obtained by first isolating the reaction product in the form of an organic salt, such as a dicyclohexylamine salt, which is then treated with an acid such as hydrochloric acid or potassium hydrogen sulfate.

The compound of formula I thus obtained has a carboxyl group at the proline site so that, if desired, it is capable of forming salts with various basic compounds.

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5 spoons. Salts with pharmaceutically acceptable basic substances are particularly important. Such salts can be prepared by a conventional process, i.e. by treating said carboxyl group with an equimolar amount of any of the bases listed above (e.g., alkali metals, alkaline earth metals and basic amino acids).

The starting material of process a) according to the present invention of formula II can be readily prepared by the following process:

A compound of formula VI

R-OH VI

wherein R is as defined above, or a reactive derivative thereof in the carboxyl group is reacted with an amino acid of formula VII

h2n-a "-coor" VII

wherein A "is an amino acid residue equivalent to A minus the group NH or C = O, and R" is a hydrogen atom or a carboxyl protecting group, or a reactive derivative thereof, followed by removal of R "if it is in a protecting group, to form a compound of formula VIII

R-NH-A "-COOH VIII

wherein R and A "have the above meanings, wherein the compound of formula VIII or a reactive derivative thereof is reacted with a compound of formula IX

CH

'IX

HS-CH2 CHCOOR "" wherein R "" is a hydrogen atom or a carboxyl protected group, followed by removal of R "" if it is a protecting group.

Examples of the reactive derivative in the carboxyl group of the compound of formula VI include those commonly used in the field of peptide synthesis, such as activated amides, acid halides, activated esters and mixed acid anhydrides.

If R "in the amino acid of formula VII is a protecting group, then as advantageous examples,

DK 156054 B

6, there is mentioned a t-butyl group (which can be removed by hydrogen fluoride, trifluoroacetic acid or hydrogen chloride), a benzyl group (which can be removed by catalytic reduction with palladium or the like), and lower alkyl groups such as methyl and ethyl ( which can be removed by hydrolysis under alkaline conditions).

The reactive derivative of the amino acid of formula VII is such that the amino group is activated and such a derivative is prepared by any of the activation methods commonly used in the peptide synthesis domain, such as introduction of a silyl group (e.g., a trimethylsilyl group). ), the "phosphazo method", the phosphoric acid ester method, and the N-carboxyanhydride method).

A carbodiimide such as dicyclohexylcarbodiimide can be used as a condensing agent to form an amide bond between the compound of formula VI and the amino acid of formula VII.

The reaction can be carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphoric triamide, chloroform, dichloromethane or acetonitrile. The reaction is usually carried out under cooling or at room temperature (-50 25 to 20 ° C), preferably from -30 to 10 ° C. The reaction time varies with the reaction temperature, the compounds to be reacted, and the solvent, and it is generally between 0.5 and 48 hours, preferably between 1 and 6 hours.

If R "of formula VII is a protecting group, it can be removed from the reaction product by any of the conventional methods described above.

The compound of formula VIII can be isolated from the reaction mixture and purified by any conventional method, e.g. by recrystallization of ethyl acetate, n-hexane, acetone or water, or by various types of chromatography as described in connection with the isolation and purification of the compound of formula I, or by decomposition of an organic

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7 salt of the reaction product with an acid.

The compound of formula VIII can be reacted with the compound of formula IX by reacting the carboxyl group of the former with the mereapto group of the latter compound in the presence of a condensing agent (ie, carbodiimides such as dicyclohexylcarbodiimide). A reactive derivative of the compound of formula VIII can be reacted with a compound of formula IX.

Examples of the reactive derivative of compound 10 of formula VIII include activated amides, acid halides, activated esters and mixed acid anhydrides. Activated amides with carbonyl diimidazole are preferred.

The carboxyl group of the compound of formula IX may be protected or unprotected. Preferred protecting groups are t-butyl and other groups that can be removed under acidic conditions.

The thiol ester formation reaction is carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphorotriamide, chloroform, dichloromethane and acetonitrile. The reaction is usually carried out under cooling or at room temperature (-50 to 20 ° C), preferably at -30 to 10 ° C when the compound of formula VIII is used in the form of an activated amide or acid halide, and at between -10 and 25 ° C. ° C when said compound is used in the form of an acid anhydride. The reaction time varies with the reaction temperature, the compounds to be reacted, and the solvent, and it is generally between 0.5 and 48 hours, preferably between 1 and 6 hours. After the thiol ester 3Q formation reaction, R "1 is removed from the reaction product if it is a protecting group. If the protecting group is a t-butyl group, it can be removed by a conventional process, i.e. by reaction with hydrogen fluoride, trifluoroacetic acid or hydrogen chloride.

The final compound can be isolated from the reaction mixture and purified by any conventional process such as recrystallization of an organic solvent such as ethyl acetate or n-hexane

DK 156054 B

8 types for chromatography as described in connection with the isolation and purification of the compound of formula I, or by decomposition of an organic salt of the reaction product with an acid.

The reactive derivative of the compound of formula IV is such that the carboxyl group involved in the reaction is activated. The carboxyl group can be activated to a suitable form, such as activated amides, acid halides, activated esters or mixed acid anhydrides. Activated amides with carbonyl diimidazole are preferred. Carbodiimides, such as dicyclohexylcarbodiimide, can be used as a condensing agent to form an amide bond from the carboxyl group and the mercapto group.

The reaction of process b) according to the invention between the compound of formula IV or its reactive derivative and the compound of formula V is carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphorotriamide, chloroform, dichloromethane or acetonitrile. The reaction is carried out as mentioned at -50 to 20 ° C, and preferably at -30 to 10 ° C when the compound of formula IV is used in the form of an activated amide or acid halide, and at between -10 and 10 ° C. C when said compound is used in the form of an acid anhydride. The reaction time varies with the reaction temperature, the compounds to be reacted and the solvent, and it is generally between 0.5 and 48 hours, preferably between 1 and 6 hours. After the thiol ester formation reaction, all protecting groups are removed from the reaction product by any of the procedures specified for the respective protecting groups.

The final compound can be isolated from the reaction mixture and purified by any of the conventional methods described above, such as various types of chromatography using silica gel, dextran crosslinked polymer and porous polymers such as styrene-divinobenzene or acrylic acid esters. An appropriate developmental solvent may be selected from chloroform,

DK 156054 B

9 ethyl acetate, methanol, ethanol, tetrahydrofuran, benzene, water and acetonitrile. Alternatively, the final compound can be obtained by first isolating the reaction product in the form of an organic salt, such as a dicyclohexylamine salt, which is then treated with an acid such as hydrochloric acid or potassium hydrogen sulfate.

The present end compounds, namely the proline derivative of formula I and pharmaceutically acceptable salts thereof, prevent the formation of angiotensin II from angiotensin I by inhibiting the action of angiotensin converting enzyme. Therefore, they are useful for the treatment of hypertension derived from angiotensin II and as a means of treating heart failure.

The effect of some of the aforementioned final compounds in inhibiting the angiotensin converting enzyme was measured.

(1) Method.

An angiotensin converting enzyme was extracted from a rabbit lung. A 0.111 M boric acid Na 2 CO 2 buffer solution 20 (pH 8.3, 0.6 ml), 0.2 ml 0.111 M boric acid Na 2 CO 2 buffer solution (pH 8.3) containing 25 mM benzoylglycylhistidylleucine (substrate) and 0.1 ml of 0.111 M boric acid Na9 CO-.-Ft-ft buffer buffer (pH 8.3) containing 10 to 10 M of test compounds (13 of the final compounds listed in the table below) are placed in tube glass and preincubate at 37 ° C for 5 to 10 minutes. Then, 0.1 ml of a solution of the enzyme (acetone powder) was added to each tube and each mixture was incubated at 37 ° C for 30 minutes. The benzoylglycine produced by the enzyme was extracted with ethyl acetate in the presence of hydrochloric acid and the amount thereof was determined by UV absorption at 228 nm. The enzyme activity in the presence of the test compounds relative to the activity in the absence of the test compounds (100) was determined. The concentration of each test compound at which the relative activity of the enzyme was 50% was designated as the activity of each test compound that inhibits the activity of the enzyme and was given as an IQ value.

10

DK 156054B

(2) Results.

Test compound I ^ g - 5 Compound No. 1 1.6 x 10 _ "" 2__ 4.8 x 10 ~ 7_ "_" 3__1.3 x 10 ~ 7_ _ "_» 4__4.0 x 10 ~ 7_ _ "" 5__1 , 8 x 10 ~ 7_ 10 "_" 6__2.4 x 10 ~ 7_ _ "_" 7__1.0 x 10 ~ 7_ _ "_" 8__1.5 x 10 ~ 7_ _I_ "9__1.7 x 10 ~ 7_" _ "10 4.8 x 10 ~ 8_ 15" _ "11 1.7 x 10 ~ 7_" _ "12 5.4 x 10 ~ 7_ 13 4.4 x 10 ~ 7

Compound # 1: N- [3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline # 2: N- [3- (N-cyclopropanecarbonyl-D-alanine) nylthio) -2-D-methylpropanoyl] -L-proline # 3: N- [3- (N-cyclohexanecarbonylglycylthio) -2-D-methylpropanoyl] -L-proline # 4: N- [3- (N-cyclohexanecarbonyl-N-methylglycylthio) -2-D-methylpropanoyl] -L-proline # 5: N- [3- (N-cyclopropanecarbonylglycylthio) -2-D-methylpropanoyl] -L-pro - Lin in # 6: N- [3- (N-adamantanecarbonyl) glycylthio) * 2-D-methylpropanoyl] -L-proline # 7: N- [3- (N-cyclohexanecarbonyl-D-phenylalanylthio) ) -2-D-methylpropanoyl] -L-proline 11

DK 156054B

Compound No. 8: N- [3- (N-cyclohexanecarbonyl-D-leucylthio) -2-D-methylpropanoyl] -L-proline # 9: N- [3- (N-cyclohexanecarbonyl-D-trypropylene) 5 tophylthio) -2-D-methylpropanoyl] - L-proline # 10: N- [3- (N-cyclohexanecarbonyl-D-phenylglycylthio) -2-D-methylpropanoyl] -L-proline # 11: N- [3- (N-cyclohexanecarbonyl-D-methionylthio) -2-D-methylpropanoyl] -L-proline # 12: N- [3- (N-cyclohexanecarbonyl-D-glutaminylthio) -2- D-methylpropanoyl] -15 L-proline # 13: N- [3- (N-adamantanecarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline.

The activity of the present end compounds is longer than that of known compounds having a similar activity, e.g. 3-mercapto-2-D-methylpropanoyl-L-proline (conventionally designated Captopril) so that the desired blood pressure regulation can be achieved with fewer daily administrations. Captopril and other known compounds cause a sudden drop in blood pressure during the first period after administration, and this may develop into orthostatic hypotensive asthenia [Lancet, vol.

1, No. 8, 115, p. 557 (March 10, 1979)], but the end-products in question have only a mild hypotensive effect 30 in the first period after administration, and there is little possibility of it developing into orthostatic hypotensive asthenia. Medications such as Captopril having a free mercapto group give various side effects that can be attributed to this mercapto group. To name a few, disorders of taste, production of albumin in the urine, agranulocytosis and skin diseases accompanied by fever have been reported in Lancet, vol. 1, 12

DK 156054B

No. 8160, p. 150 (January 19, 1980), Lancet, vol. 2, no.

8186, p. 129 (July 19, 1980), and the South African Medical Journal, vol. 58, 172 (1980). On the other hand, the thiol ester linkage of the subject compounds is susceptible to hydrolysis in vivo and therefore there is little likelihood of forming a mercapto group.

Therefore, there is little possibility that the final compounds in question cause the aforementioned side effects attributable to the mercapto group.

In addition to the above, it should be noted that the known compounds most closely related to the formula I are those known from EP-9898A1, and in particular the compounds known from Examples 35-43 of ΕΡ-9898ΑΊ. The 15 structural differences between the compounds thus known and those herein will be shown in Table 1 below.

Table 1 CH3 r-a-s-ch9-ch-co-n ^

COOH

-1-- 25 '| RAW_

Comparative / h \ -CO- glycine up- P ^ s-CO- sarcosine finding α-D-amino- (> -CO-; \ _y acid 30 I (more specifically I specified in ____ claim 1)

Ex.35 E> C0- L-Phe

Ex.36 __ "_ j___glycine__ Ex.37 _ _;" L-Trp_ EP-r Ex. 38 "_i_L-Tyr_ 35 9898A1 Ex.39 __" __ L-Ile_

Ex.40 "L-Leu

Ex. 41 "L-His

Ex. 42__L-Val_ In Ex.43 "L-Ala,

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13

The chemical and biological properties of a representative compound prepared according to the present invention and the closely related compound known from EP-9898A1 are listed in Table 5 below.

Table 2

CH.J

I 3 - r-a-s-ch9-ch-co-n ^ 10 - "

COOH

Porbin (T) (2) In Vitro R A I Hydrolysis Aeise K A ± 50 Remaining Percent Amount _____10 30 60 (min)

In accordance with the invention r- <_7 "<H> -CO- D-Ala 2, .6x10 '100 98 97

Ex.3 or \ _ / '20 ^ 5, 5_____ ΕΡ-9898ΑΊ v _7

Ex.43 [HV-CO- L_Ma 2.4x10 '71 56 53

From the test results set forth in Table 2, it can be seen that the compounds of the invention compared to the compounds known from EP-9898A1 have approximately the same level of activity in inhibiting angiotensin converting enzyme, but are much more stable against hydrolysis by the AS binding. In the general formula I. As above, it is believed that the presence of the free mercapto group -SH formed by the hydrolysis causes undesirable side effects such as orthostatic hypotensive asthenia, taste disturbance, production of albumin in the urine, granulocytosis and skin disorders. Thus, the compounds of the invention exhibit much lower side effects than the compounds known from EP-9898A1.

14

DK 156054B

The test φ for the determination of Ι, -q in Table 2 was performed as described above on page 9 under the heading (1) Method. The test for determining the degree of hydrolysis in Table 2 was performed as described below.

5

Test method φ

Colorimetry / In vitro enzymatic hydrolysis

Incubation mixture: 1 ml 5% rat liver homogenate (0.1 M potassium phosphate buffer, pH 7.4) plus 1 ml -2 test compound 10 Mi the same phosphate buffer solution.

Test Method: 100 µl incubation mixture was taken from the medium and then 6 drops of 1.0 N HCl and 5 ml of ethyl acetate were added. After mechanical mixing for 10 15 min. and spin for 10 min. the ethyl acetate layer was removed and evaporated. The residue was dissolved in 1 ml of water and then 6.5 μΐ of 0.4 M phosphate buffer (pH 6.85) and 5/5 of 5 x 10 B M BIPM * ^ were added and the mixture was allowed to stand for 40 minutes. at 0 ° C.

The assessment was made by 310 nm excitation and 365 nm emission (limit> 0.1 µg SQ) X) BIPM = N- [p- (2-benzimidazoyl) phenyl] -maleimide o X sr

'O ^ H3 ~ 0 SR

Η O H

BIPM fluorescence

The proline derivatives of formula I and their pharmaceutically acceptable salts may be formulated into preparations 30 for oral administration such as tablets, capsules, granules, powders, syrups and elixirs, or for sterile solutions or suspensions for parenteral administration. For this purpose, pharmaceutical compositions can be prepared from one or more of the present final compounds (as the active ingredient) and pharmaceutically acceptable adjuvants such as excipients, carriers, binders, stabilizers and flavoring agents. The daily

DK 156054 B

15 doses of the subject compounds of the present invention range from 0.5 mg to 2 g, preferably from ca. 1 to 500 mg, for oral administration, and from 0.1 to 600 mg, preferably from ca. 0.3 to 300 mg, for parenteral administration.

The process of the invention is further illustrated by the following Examples, Examples 1 and 2 of which describe the preparation of starting compounds, while Examples 3-19 describe the preparation of final compounds.

10

Example 1

Preparation of N-substituted amino acids.

(a) 4.5 g of D-alanine was dissolved in 230 ml of aqueous 1 N Na 2 CO 3 solution with stirring. To the solution was added dropwise 100 ml of tetrahydrofuran containing 9.0 g of cyclohexanecarbonyl chloride at 5 to 10 ° C, at which temperature the mixture was stirred for 30 minutes and then stirred at room temperature for 1.5 hours. Then, 2N HCl was added to the reaction mixture to adjust its pH to between 1 and 2. The reaction mixture was extracted with ethyl acetate and the organic layer was washed with saturated NaCl solution and dried over magnesium sulfate. The filtrate was evaporated in vacuo to give a crude compound. Recrystallization of ethyl acetate / n-hexane yielded 4.65 g of N-cyclohexanecarbonyl-D-alanine. [α] D = + 26.6 °.

(b) 3.0 g of an ester of adamantanecarboxylic acid and N-hydroxysuccinimide were dissolved in 30 ml of tetrahydrofuran. To the solution was added 5 ml of water containing 0.89 g of D-alanine and 1.1 g of triethylamine and the mixture was stirred at 5 ° C overnight. After removal of tetrahydrofuran, water was added to the residue and further 2N HCl was added to the mixture to adjust its pH to between 1 and 2. The mixture was then treated as under (a) above to give 0.38 g of N-adamantanecarbonyl -D-alanine. [α] D = + 11.6 ° (C = 1.0 MeOH).

The compounds listed below were prepared by one of the processes described under (a) and (b).

DK 156054B

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Example 1 (a) 3- (N-Cyclohexanecarbonyl-D-alanylthio) -2-D-methyl-propanoic acid.

5.97 g of N-cyclohexanecarbonyl-D-alanine was dissolved in 5 100 ml of dry tetrahydrofuran. To the solution was added 5.84 g of carbonyl diimidazole at between -20 and -15 ° C and the mixture was stirred at this temperature for 1 hour. Then 3.60 g of 3-mercapto-2-D-methylpropanoic acid was added and the mixture was stirred at between -15 and -10 ° C for 1 hour and then at room temperature for another 1 hour. The mixture was evaporated in vacuo to remove the solvent.

To the residue was added 40 ml of water and to the mixture was added 2N HCl to adjust its pH to between 1 and 2. The mixture was extracted with ethyl acetate and the organic layer was washed twice with saturated NaCl solution and dried over magnesium sulfate. The filtrate was evaporated in vacuo to give a crude compound. Recrystallization of ethyl acetate / n-hexane afforded 7.50 g (83%) of the title compound as colorless 20 prismatic crystals.

Mp. 149-152 ° C

[α] D = + 46.4 ° (C = 1.07, MeOH) NMR (CD 3 OD, 6): 1.20 (3H, d), 1.35 (3H, d), 1.20 - 2, 0 (11H, m), 2.40 - 2.80 (1H, m), 3.05 (2H, m), 4.50 (1H, m) (b) 3- (N-Cyclohexanecarbonyl-D phenylglycylthio) -2-D-methylpropanoic acid.

Process (a) above was repeated using 2.61 g of N-cyclohexanecarbonyl-D-phenylglycine, 1.95 g of carbonyl diimidazole and 1.20 g of 3-mercapto-2-D-methylpropanoic acid. 2.07 g (57%) of the title compound was obtained as a yellow oil. [α] D = -48.7 ° (C = 1.19, MeOH) NMR (CDCl 3, δ): 1.24 (3H, d), 1.20 - 2.10 (11H, 2.40 - 2.80 (1H, m), 3.10 (2H, m), 5.75 (1H, d), 6.40 - 6.80 (1H, m), 7.30 (5H, S), 8 , 90 (1H, S).

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19 (c) 3- (N-Cyclohexanecarbonyl-D-leucylthio) -2-D-methyl-propanoic acid.

The procedure under (a) was repeated using 2.41 g of N-cyclohexanecarbonyl-D-leucine, 1.95 g of carbonyl diimidazole and 1.20 g of 3-mercapto-2-D-methylpropanoic acid. 2.40 g (70%) of the title compound was obtained as an oil.

[α] D = + 12.2 ° (C = 1.03, MeOH) NMR (CDCl3, δ): 0.95 (6H, d), 1.28 (3H, d), 1.20 - 2.10 (11H, m), 2.40 - 2.80 (1H, m), 3.10 (2H, m), 4.70 (1H, m), 6.00 - 6.30 (1H, m), 9.20 (1H, S).

(d) 3- (N-Cyclohexanecarbonyl-D-glytaminylthio) -2-D-methyl-propanoic acid.

The procedure under (a) above was repeated using 2.56 g of N-cyclohexanecarbonyl-D-glutamine, 1.95 g of carbonyl diimidazole and 1.20 g of 3-mercapto-2-D-methylpropanoic acid. A sample of 0.70 g (20%) of the title compound was obtained.

M.p. 146-149 ° C

[α] D = + 10.8 ° (C = 1.05, MeOH) NMR (CD 3 OD, δ): 1.20 (3H, d), 1.20 - 2.10 (11H, ir), 2, (4H, m), 2.40 - 2.80 (1H, m), 3.05 (2H, m), 4.50 (1H, m).

(E) 3- (N-Cyclopropanecarbonyl-glycylthio) -2-D-methylpropanoic acid.

The procedure under (a) above was repeated using 0.78 g of N-cyclopropanecarbonylglycine, 1.06 g of carbonyl diimidazole and 0.66 g of 3-mercapto-2-D-methylpropanoic acid. 0.70 g (52%) of the title compound was obtained as an oil.

[α] D = -33.8 ° (C = 1.01, MeOH) NMR (CDCl 3, δ): 0.70 - 1.20 (4H, m), 1.25 (3H, d), 1, -30 - 1.80 (1H, m), 2.40 - 2.80 (1H, m), 3.14 (2H, m), 4.20 (2H, d), 7.30 (1H, S).

20

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(f) 3- (N-Adamantanecarbonyl-glycylthio) -2-D-methylpropanoic acid.

The procedure under (a) above was repeated using 2.38 g of N-adamantanecarbonylglycine, 1.95 g of carbonyl diimidazole and 1.20 g of 3-mercapto-2-D-methylpropanoic acid. A sample of 1.08 g (32%) of the title compound was obtained.

Mp. 132 ° (decomposition).

[? x] D = -8.3 ° (01.01, MeOH) NMR (CD 3 OD, δ): 1.25 (3H, d), 1.65 - 2.30 (15H, m), 2 , 40 - 2.80 (1H, m), 3.14 (2H, m), 4.06 (2H, d).

(g) 3- (N-Cyclohexanecarbonyl-D-phenylalanylthio) -2-D-methylpropanoic acid.

The procedure under (a) above was repeated using 1.37 g of N-cyclohexanecarbonyl-D-phenylalanine, 1.0 g of carbonyl diimidazole and 0.60 g of 3-mercap-to-2-D-methylpropanoic acid. 1.5 g (79%) of the title compound was obtained as an oil.

[α] D = + 13.9 ° (01.02, MeOH) NMR (CDCl 3, δ): 1.25 (3H, d), 1.20 - 2.0 (10H, m), 2.1-3 , 0 (2H, m), 3.0 - 3.4 (4H, m), 4.95 (1H, t), 6.19 (1H, d), 7.24 (5H, S), 10, 12 (1H, S).

(h) 3- (N-Cyclohexanecarbonyl-D-methionylthio) -2-D-methyl-propanoic acid.

The process under (a) was repeated using 0.325 g of N-cyclohexanecarbonyl-D-methionine, 0.25 g of carbonyl diimidazole and 0.15 g of 3-mercapto-2-D-methylpropanoic acid. 0.29 g (64%) of the title compound was obtained as an oil.

[Α] = -7.9 ° (01.0, MeOH) NMR (CDCl 3, δ): 1.26 (3H, d), 1.2 - 2.0 (10H, m), 2.09 ( 3H, S), 2.0 - 3.2 (8H, m), 4.5 - 5.0 (1H, m), 6.62 (1H, d), 10.07 (1H, S).

(I) 3- (N-Cyclohexanecarbonyl-D-tryptophylthio) -2-D-methyl-propanoic acid.

The procedure under (a) above was repeated using 0.157 g of N-cyclohexanecarbonyl-D-tryptophan, 0.1 g of carbonyl diimidazole and 0.06 g of 3-mercapto-2

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21 D-methylpropanoic acid. Q, 17 g (82%) of a gum-like sample of the title compound was obtained.

[α] D = -7.1 ° (O = 1.0, MeOH) NMR (CDCl 3, δ): 1.26 (3H, d), 1.2 - 2.4 (11H, m), , 5 - 2.9 (1H, m), 3.0 - 3.3 (2H, m), 3.35 (2H, d), 4.87 (1H, t), 6.4 - 6.9 (2H, broad S), 6.9 - 7.8 (5H, m), 10.5 (1H, S).

Example 3 (a) N- [3- (N-Cyclohexanecarbonyl-D-alanylthio) -2-D-methyl-propanoyl] -L-proline.

1.51 g of 3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoic acid was dissolved in 40 ml of dry tetrahydrofuran.

To the solution was added 0.61 g of triethylamine and 0.65 g of ethyl chloroformate at -5 ° C with stirring. Five minutes later, a solution of 0.58 g of L-proline and 0.61 g of triethylamine in 5 ml of water was added and the mixture was stirred at 0 ° C for 1 hour and then at room temperature for 30 minutes. The mixture was evaporated in vacuo to remove the solvent. After adding water to the residue, 2N HCl was added to the mixture to adjust its pH to between 1 and 2. The reaction mixture was extracted with ethyl acetate and the organic layer was washed twice with saturated NaCl solution and dried over magnesium sulfate. . The filtrate was concentrated in vacuo to remove ethyl acetate and the residue subjected to chromatography on silica gel using a mixture of chloroform and methanol (100: 1 to 100: 2) as eluent. Fractions containing the final compound were collected and evaporated in vacuo to give 0.3 g of a rubbery sample of the title compound.

(b) The same compound could be prepared by the following procedure.

6.03 g of 3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoic acid and 2.3 g of N-hydroxysuccinimide were dissolved in 50 ml of dry tetrahydrofuran. To the solution was added 4.3 g of dicyclohexylcarbodiimide at between 0 and 5 ° C, and 22

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the mixture was stirred overnight at this temperature.

After the reaction, the precipitate was filtered off and the insoluble material was washed with a small amount of tetrahydrofuran. The filtrate and wash were combined and concentrated in vacuo. The residue was added ethyl acetate and the mixture was filtered. The ethyl acetate solution was washed with 0.5 N HCl, water, aqueous Na 2 CO 3, and saturated NaCl solution in the above order. The solution was dried over magnesium sulfate and evaporated in vacuo and the residue 10 solidified after the addition of a mixture of ethyl acetate and hexane (1:10). the yield of the solid product was 6.90 g (87%).

Mp. 113 - 116 ° C

[α] D = 14.2 ° (C = 1.05, MeOH) NMR (CDCl3, δ): 1.38 (6H, d), 1.20 - 2.20 (11H, m), 2, 82 (4H, S), 2.8 - 3.30 (3H, m), 4.75 (1H, m), 6.30 (1H, broad d).

3.98 g of the activated ester was dissolved in 40 ml of tetrahydrofuran. To the solution was added a solution of 1.15 g of L-proline and 1.26 ml of N-ethylmorpholine in 5 ml of water and the mixture was stirred overnight. The mixture was evaporated in vacuo to remove the solvents and water was added to the residue. To the mixture was added 2N HCl to adjust its pH to between 1 and 2. The reaction mixture was extracted with ethyl acetate and the organic layer was washed with saturated NaCl solution and dried over magnesium sulfate. The filtrate was evaporated in vacuo. The residue was subjected to column chromatography on silica gel using a mixture of chloroform 30 and methanol (100: 1 to 100: 2) as eluent. The product obtained by evaporation of the eluate in vacuo (1.45 g) was found by NMR analysis to be identical to the title compound.

(c) The title compound could also be prepared by the following procedure.

4.52 g of 3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoic acid were dissolved in 5 ml of thionyl chloride. To the solution was added a drop of dimethylformamide, and bis-23

DK 156054B

the mixture was stirred at room temperature overnight. The mixture was evaporated in vacuo to remove the solvents and to the residue was added 2 ml of dry toluene and the mixture was evaporated again in vacuo to remove the solvent. To the residue was added 10 ml of dry tetrahydrofuran with stirring. The tetrahydrofuran solution containing the acid chloride was added dropwise at a temperature of between 5 and 10 ° C with stirring to 25 ml of water, dissolving 2.3 g of L-proline and 2.5 g of 10 Na 2 CO 3. The mixture was stirred at this temperature for 1 hour. at room temperature for an additional 2 hours. The reaction mixture was subjected to extraction with chloroform. To the aqueous layer was added 2N HCl to adjust its pH to between 1 and 2. After extraction with chloroform, the chloroform layer was washed twice with saturated NaCl solution and dried over magnesium sulfate. After removal of the chloroform, the residue was subjected to column chromatography on silica gel using a mixture of chloroform and methanol (100: 1 to 100: 2) as eluent. After evaporation in vacuo, 1.6 g of a gummy substance was obtained. The substance was found by NMR analysis to be identical to the title compound.

(d) The title compound could be prepared by another process as described below.

3.01 g of 3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoic acid and 1.71 g of t-butyl ester of L-proline were dissolved in 50 ml of dry dichloromethane and 2.15 g of dicyclohexylcarbodiimide was added. the solution while stirring and cooling with ice. The mixture was stirred at the same temperature for 1/2 hour and allowed to stand overnight at 5 ° C. The reaction mixture was filtered and the insoluble material washed with dichloromethane. The filtrate and wash liquid were combined and washed with 1N HCl, water, 1N NaHCO 3 and saturated NaCl solution in the above order, dried over MgSO 4 and evaporated in vacuo to give 4.3 g of a rubbery sample of t-butyl ester 24

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of N- [3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methyl-propanoyl] -L-proline. A 4.0 g sample of the ester was dissolved in 30 ml of anisole and 10 ml of trifluoroacetic acid was added to the solution. The mixture was stirred at room temperature for 5 hours and evaporated in vacuo to remove excess trifluoroacetic acid. The residue was subjected to column chromatography on silica gel (column: 2 x 35 cm) using a mixture of methanol and chloroform (1: 100 to 3: 100) as eluent. Fractions containing the final compound were collected and evaporated in vacuo to give 3.4 g of a gummy substance. This substance was found to be identical to the title compound by NMR and thin layer chromatography.

The reactants used, reaction solvents 15, and methods for preparing the title compound of Example 3 are listed in the table below, along with data on its physical properties. Other compounds were prepared using one of the methods described in Examples 3 (a) to (d), and details of the particular process are given in the table below, along with data on the physical properties of the final compounds. In the table, the numbers after the numbers (1) and (2) in the column of "Rf" indicate the Rf-25 values obtained by thin-layer chromatography using CHCl3 / MeOH / AcOH (2: 1: 0.003) and n-BuOH, respectively. / AcOH / 1 O (4: 1: 1) as eluent.

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30

Example 4

Various salts were formed from the samples of N- [3- (N-cyclohexane-carbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline prepared in Examples 3 and 5.

(A) Ca salt.

A 3.98 g sample of the compound of Example 3 or 5 was dissolved in 40 ml of methanol. To the solution was added 0 * 84 g of calcium acetate hydrate and the mixture was heated under reflux for 1 hour. The insoluble matter was filtered off and the filtrate was evaporated in vacuo. To the residue was added chloroform and the mixture was filtered and evaporated in vacuo. Diethyl ether was added to the residue and the mixture was filtered and air dried to give 3.40 g of a potassium salt of the compound of Example 3 or 5. [α] D = -47.2 ° (C = 1.0, MeOH).

(b) Mg salt.

The process under (a) was repeated using 3.09 g of the compound of Example 3 or 5 and 0.772 g of magnesium acetate tetrahydrate.

2.42 g of a magnesium salt of the compound of Example 3 or 5. was obtained. [Α] D = -46.6 (C = 1.0, MeOH)

(c) Lysine salt.

A 1.19 g sample of the compound of Example 3 or 5 was dissolved in 22 ml of methanol. To the solution was added 0.416 g of lysine and the mixture was stirred at room temperature for 1 hour and evaporated in vacuo.

To the residue was added chloroform and the mixture was filtered and evaporated in vacuo. To the residue was added dimethyl ether and the product filtered off with suction to give 1.54 g of a lysine salt of the compound of Examples 3 or 5. [α] β = -23.1 ° (C = 1.0,

MeOH).

(d) Na salt.

A 2.27 g sample of the compound of Example 3 or 5 was dissolved in 25 ml of methanol. To the solution was added 0.514 g of sodium acetate and the mixture was stirred 31

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was stirred at room temperature for 30 minutes and evaporated in vacuo. To the residue was added 200 ml of a mixture of methanol and chloroform (3: 100 v / v) and the resulting mixture was filtered and evaporated in vacuo. The residue was dissolved in methanol and the solution filtered and evaporated in vacuo. To the residue was added ethyl acetate and the product filtered with suction to give a Nasal of the compound of Example 3 or 5 (1.85 g).

[α] Q = -26.7 ° (C = 1.0, MeOH).

(E) Dicyclohexylamine salt.

A 13.1 g sample of the compound of Example 3 or 5 was dissolved in 120 ml of acetonitrile. To the solution was added 6 ml of dicyclohexylamine with stirring. The mixture was further stirred for 30 minutes and then allowed to stand overnight. The precipitate was filtered off with suction and air dried. The obtained crude crystals were suspended in 300 ml of acetonitrile and the suspension was heated under reflux for 30 minutes. The suspension was cooled and the crystals were filtered off with suction and air-dried to give a dicyclohexylamine salt of the compound of Example 3 or 5 (12.2 g). [α] D = -24.5 ° (C = 1.0,

MeOH).

Example 5 N- [3- (N-Cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline.

5.98 g of N-cyclohexanecarbonyl-D-alanine was dissolved in 80 ml of dry tetrahydrofuran. To the solution was added 5.84 g of carbonyl diimidazole at -18 ° C with stirring while the mixture was cooled with ice. The mixture was stirred at this temperature for 1 hour and then 6.29 g of N- (3-mercapto-2-D-methylpropanoyl) -L-proline was added to the mixture, then stirred at -18 ° C for 30 minutes. minutes and thereafter at room temperature for 1 hour. After completion of the reaction, the mixture was evaporated in vacuo to remove the solvent. To the residue was added 50 ml of water and 2N HCl was added to the mixture to adjust its pH to between 1 and 2. The mixture was extracted with ethyl acetate,

., DK 156054 B

32 and the ethyl acetate layer was washed with saturated NaCl solution, dried over MgSO4 and evaporated in vacuo. To the residue was added 120 ml of acetonitrile and then 6 ml of dicyclohexylamine (DCHA). The mixture was stirred at room temperature 5 for 1 hour. After standing overnight, the bunch was filtered off and air dried to give a crude DCHA salt (14.35 g). The crude salt was suspended in 300 ml of acetonitrile and the suspension was heated under reflux for 30 minutes. After cooling, the precipitate was collected by suction and air dried to give a white DCHA salt (12.20 g). The DCHA salt (12.20 g) was suspended in 90 ml of ethyl acetate and to the suspension was added 60 ml of 0.5 N aqueous KHSO 2 solution and the mixture was shaken. The organic layer was washed with distilled water, dried over 15 MgSO 4 and evaporated in vacuo to give a gummy substance (8.64 g). The substance was found to be identical to the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl3, δ) Rf-value L

1.20 (3H, broad d, CHg), 1.38 (3H, d, OL), 1.55-2.0 (10H, m, (¾) 2.0-2.5 (4H, m, OU) , 0.66 2.5-3.3 (4H, m, qf & OL,).

3.4-3.9 (2H, m, 0¾), XV 0.71 4.0 - 5.0 (2H, m, CH), 6.0-6.5 (1H, m, Mi), 7, 31 (1H, S, CO 2 H),

Example 6 N- [3- (N-Cyclohexanecarbonyl-glycylthio) -2-D-methylpropanoyl] -L-proline.

1.02 g of N-cyclohexanecarbonylglycine was dissolved in 10 ml of dry tetrahydrofuran. To the solution was added 20 ml of dry tetrahydrofuran containing 1.07 g of carbonyl diimidazole at -20 ° C with stirring, while the mixture was cooled with ice. The mixture was stirred at this temperature for 1 hour and then 6 ml of dry tetrahydrofuran was added.

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containing 1.09 g of N- (3-mercapto-2-D-methylpropanoyl) -L-proline, then stirred at -20 ° C for 30 minutes and then at room temperature for 1 hour. After completion of the reaction, the mixture was evaporated in vacuo to remove the solvent. The residue was subjected to column chromatography on silica gel (column: 2 x 35 cm) using a mixture of methanol and chloroform (1: 100 to 3: 100) as eluent. Fractions containing the final compound were pooled and evaporated in vacuo to give a gummy substance (1.16 g). The substance was found to be identical to the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl3, δ) Rf value.

1.21 (3H, broad d, QU, 1.35-2.5 (14H, m, qg), 2.6-3.3 (4H, m, (¾ & Og) / 2) 0.60 3, 4-3.9 (2H, m, OLJ, 4.0-4.7 (3H, m, QT & qU / 2) 0.68 6.35- 7.1 (1H, m, NH), 9.01 (1H, S , C0? H_20

Example 7 N- [3- (N-Cyclopropanecarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline, 635 mg of N-cyclopropanecarbonyl-D-alanine and 0.70 ml of triethylamine dissolved in 14 ml dry tetrahydrofuran. To the solution was added 0.48 ml of ethyl chloroformate at -15 ° C with stirring while the mixture was cooled with ice. The mixture was stirred at this temperature for 15 minutes and then 1.09 g of N- (3-mercapto-2-D-methyl-propanoyl) -L-proline and 10 ml of dry tetrahydrofuran containing 0.70 ml of triethylamine were added. stirred at ~ 15 ° C for 15 minutes and then at 5 ° C overnight.

After completion of the reaction, the mixture was evaporated in vacuo at 30 to 35 ° C to remove the solvent. To the residue was added 10 ml of water and 2N HCl was added to the mixture to adjust its pH to between 1 and 2.

The mixture was extracted with chloroform and chloroform-34

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the layer was washed with saturated NaCl solution, dried over MgSO4 and evaporated in vacuo. The residue was subjected to column chromatography on silica gel (column: 2 x 35 cm) using a mixture of methanol and chloroform 5 (1: 100 to 3: 100) as eluent. Fractions containing the final compound were pooled and evaporated in vacuo to give a gummy substance (395 mg).

This substance, by NMR analysis and thin layer chromatography, was found to be identical to the title compound.

NMR (CDCl 3, δ) R f value 0.6-1.0 (4H, m, (¾), 1.20 (3H, broad d, CH),

1.42 (3H, d, OL), J

1.4-1.9 (1H, mT CH), φθ, 53 1.9-2.4 (4H, m, (¾), 2.5-3.3 (3H, m, qf & OL) J00 , 60 3.4 - 3.8 (2H, m, OL), Δ4.1-5.0 (2H, m, CHJ / 6.4 - 7.0 (1H, m, NH), 7.45 (1H) , S, 002H)

Example 8 N- [3- (N-Cyclohexanecarbonyl-N-methylglycylthio) -2-D-methylpropanoyl] -L-proline.

The procedure of Example 6 was repeated except that N-cyclohexanecarbonylglycine was replaced by N-cyclohexanecarbonyl-N-methylglycine (1.09g), carbonyl diimidazole (1.09g) and N- (3-mercapto-2-D methyl-propanoyl) -L-proline (1.09 g). A rubbery substance (0.76 g) was obtained and it was found to be identical to the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl 3, δ) R f value 1.21 (3H, broad d%) δ 1.3-2.4 (14H, m,% 7, 2.4-3.3 (4H, m, 0.66 3.22 (3H, S, CEL), 3.3-3.8 (2H, m, cL), <2) 0.62 4.0-4.7 (3H, m, ΟΓ & OL) 9.75 (1H, S, CO

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35

Example 9 N- [3- (N-Cyclopropanecarbonylglycylthio) -2-D-methylpropanoyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-cyclopropanecarbonylglycine (0.96g), carbanyldiimidazole (1.09g) and N- (3-mercapto-2-D-methylpropane). noyl) -L-proline (1.09 g). A gummy substance (0.4 g) was obtained and, by NMR analysis and thin layer chromatography, was found to be identical to the title compound.

NMR (CDCl3, δ) Rf activity.

0.6-1.0 (4H, m, OL), 1.20 (3H, broad d, CH,), 1.4-1.9 (1H, m, CH), 1.9-2.4 (4H) , m, OL), δ 0.47 2.6-3.3 (3H, m, CH & QU, 3.3-3.9 (2H, m, OL), δ 0.53 3.9-4.7 (3H, m, Cf (%), 7.2 - 8.0 (1H, m, NH),

8.48 (1H, S, G02hF

20

Example 10 N- [3- (N-Adamantanecarbonylglycylthio) -2-D-methylpropanoyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-adamantanecarbonylglycine (1.42 g), carbonyl diimidazole (1.09 g) and N- (3-mercapto-2-D-methylpropanoyl) -L-proline (1.09 g). A gummy substance (1.7 30 g) was obtained and it was found to be identical to the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl 3

1.21 (3Η, broad d, OL.), 1.6-2.4 (19H, m, CH5CH-) 2.2-3.2 (3H, m, CH &CT) 0.58 3.4-3.8 (2H, m, (¾), 3.9 - 4.7 (3H, m, qT & CH -) δ 0.72 6.3-7.0 (1H, m, I®, 8.19 (1H, S, CO 2 H)

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36

Example 11 N- [3- (N-Cyclohexanecarbonyl-D-phenylalanylthio) -2-D-methyl-propanoyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-cyclohexanecarbonyl-D-phenylalanine (1.54g), carbonyl diimidazole (1.09g) and N- (3-mercapto-2-D methyl-propanoyl) -L-proline (1.09 g). A gummy substance (0.97 g) was obtained and it was shown by NMR analysis and thin layer chromatography to be identical to the title compound.

NMR (CDCl 3, δ) R f value 1.20 (3H, broad d, CHJ, 1.2-2.5 (14H, m, O € f, 2.5 2.5-3.3 (6H, m, CH & CEL Δ 0.64 3.3-3.6 (2H, m, (fL), δ 4.0-5, 3 (2H, m, c | 7, δ 0.82 5.7-6.4 (1H, m, 1®}, 7.29 (5H, S, aromatic), 7.5-7.9 (1H, broad S, CD

Example 12 N- [3- (N-C1-Clohexanecarbonyl-D-leucylthio) -2-D-methylpropanoyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-cyclohexanecarbonyl-D-leucine (1.20g), carbonyl diimidazole (0.97g) and N- (3-mercapto-2-D methylpropano-yl) -L-proline (0.98 g). A gummy substance (1.17 g) was obtained and, by NMR analysis and thin layer chromatography, was found to be identical to the title compound.

37

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NMR (CDCl3, δ) Rf value 0.93 (6H, df OL), 1.21 (3H, width, CHJ, 1.30-2.5 (17H, m, CH & CH , 3 (4H, m, CH & CH.S, 3.3-3.9 (2H, m, (¾),) 0.73 4.0-5.0 (2H, m, CH), 95-6.5 (1H, m, NH), 7.30 (1H, S, 002g) Example 13 N- [3- (N-Cyclohexanecarbonyl-D-tryptophylthio) -2-D-methyl-propanoyl] - L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-cyclohexanecarbonyl-D-tryptophan (1.75 g), carbonyl diimidazole (1.09 g) and N- (3-mercapto 2-D-methylpropanoyl) -L-proline (1.09 g). A gummy substance (0.8 g) was obtained and, by NMR analysis and thin layer chromatography, was found to be identical to the title compound.

NMR (CDCl3, δ) Rf value

1.15 (3H, broad d, _q | X)

1.3-2.4 (14H, m, C₂,, 2.5-3.9 (8H, m, CH₂S CEL), 4.1-4.7 (1H, m, CH), 58 4.7-5.2 (1H, m, CH), 6.0-6.4 (2H, m, NH), δ 0.82 6.95-7.9 (5H, m, aromatic) , 8.80 (1H, broad S, COH) Example 14 N- [3- (N-Cyclohexanecarbonyl-D-phenylglycylthio) -2-D-methylpropanoyl] -L-proline.

The procedure of Example 6 was repeated except that N-cyclohexanecarbonylglycine was replaced by N -cyclohexanecarbonyl-D-phenylglycine (1.30g), carbonyl diimidazole (0.97g) and N ~ (3-mercapto-2 -D-methyl-propanoyl) -L-proline (0.98 g). A rubber sheet was obtained.

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38 (0.35 g) and by NMR analysis and thin layer chromatography were found to be identical to the title compound.

NMR (CDCl 3, δ) R f value δ 1.18 (3H, broad d, (¾), 1.3-2.5 (14H, m, CoJ, 2.5-3.8 (6H, m, 0.58 4.2-4.7 (1H, m, CH), 5.62 (1H, d, CH), 0.83 6.53 (1H, d, MI), 7.37 (5H, S, aromatic), 8.90 (1H, S, CO

Example 15 N- [3- (N-Cyclohexanecarbonyl-D-methionylthio) -2-D-methyl-propanoyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-cyclohexanecarbonyl-D-methionine (1.30 g), carbodiimidazole (1.09 g). and N- (3-mercapto-2-D-methylpropanoyl) -L-proline (1.09 g). A gummy substance (0.59 g) was obtained and, by NMR analysis and thin layer chromatography, was found to be identical to the title compound.

NMR (CDCl 3, δ) R f

1.23 (3H, broad d, OL), 1.5-2.4 (16H, m, OL ·), 2.10 (3H, S, CH.), * 2.59 (2H, t , CEL ·), 277-3.3 (4Η, φθ, 60 m, CH CH & cfu), 3.3-3.9 (2H, m, QU, 470-5.1 (2H, m, © 0.77 CH), 5.49 (1H, d, NH), 7.81 (1H, S, CO 2 g)

Example 16 N- [3- (N-Cyclohexanecarbonyl-D-glutaminylthio) -2-D-methyl-propanoyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine substituted 39

DK 156054 B

for example, with N-cyclohexanecarbonyl-D-glutamine (0.5 g), carbonyl diimidazole (0.41 g) and N- (3-mercapto-2-D-methylpropanoyl) -L-proline (0.41 g) ).

A gummy substance (0.3 g) was obtained and it was found to be identical to the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl3) Rf value.

1.20 (3H, broad d, CEL), 1.3-2.5 (16H, m, CH?), Φθ, 42 2.5-3.2 (6H, m, OJ_ & UL), 3.3-3 , 9 (2H, m, OL) © 0.48 4.1-4.9 (2H, m, Oif, 5.6- 7.6 (4H, m, NH & CO 2 H) Example 17 N- [3- ( N-adamantanecarbonyl-D-alanylthio) -2-D-methylpropa-noyl] -L-proline.

The procedure of Example 6 was repeated with the difference that N-cyclohexanecarbonylglycine was replaced with N-adamantanecarbonyl-D-alanine (1.2 g), carbonyl diimidazole (0.97 g) and N- (3-mercapto 2-D-methylpropanoyl) -L-proline (0.98 g). A gummy substance (1.02 g) was obtained and it was found to be identical to the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl3, δ) R f value 1.21 (3H, broad d, CEL), 1.4, (3H, d, OL), 176-2.4 (19H, m, æ,), 1.4 -3.2 (3HJO0.63 m, CH & OLJ, 3.4-3.9 (2H, wide, cS), 4.1-5.0 (2Η, φθ, 67 m, CH), 675- 6.8 (1H, broad S, NH), 8.10 (1H, S, CO 2 H)

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40

Example 18

Dicyclohexylamine salt of N- [3- (N-cyclohexanecarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline.

(1) 3- (N-Cyclohexanecarbonyl-D-alanylthio) -2-D-methyl-propanoic acid (241 g) was suspended in dry dichloromethane (1.3 liters). To the suspension was added dropwise triethylamine (117 ml) at -15 ° C with stirring, and then pivaloyl chloride (104 ml) was added dropwise at: -5 ° C with stirring, and the mixture was further stirred for 10 30 minutes.

(2) L-Proline (101 g) was suspended in dry dichloromethane (1.2 L). To the suspension was added dropwise trimethylsilyl chloride (112 ml) at -15 ° C with stirring, and then triethylamine (122 ml) was added dropwise at below -5 ° C with stirring, and the mixture was further stirred at -15 ° C for 30 minutes. . To the obtained mixture was added the solution prepared under (1) and the mixture was stirred under cooling for 15 minutes and then at room temperature for 1 hour. After completion of the reaction, cold distilled water (1 liter) was added to the reaction mixture under cooling, and then concentrated hydrochloric acid (90 ml) was added dropwise. The mixture was stirred for 10 minutes and the organic layer was separated, washed twice with saturated NaCl solution and dried over magnesium sulfate. To the filtrate was added dicyclohexylamine (326 ml) and acetonitrile (800 ml) and the mixture was stirred for 30 minutes. Then, 4 800 ml portions of acetonitrile were added every 10 minutes and the resulting mixture was allowed to stand overnight at 5 ° C.

The precipitate was filtered off with suction and air dried to obtain a crude sample of the title compound. The crude sample was dissolved in dichloromethane (3 liters) in hot state and filtered. The filtrate was stirred at room temperature for 30 minutes, and another 4 750 ml portions of acetonitrile were added every 10 minutes and the mixture obtained was allowed to stand overnight at 5 ° C. The precipitate was filtered off and air dried to obtain a colorless sample of the title

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41 compound (404 g). Mp. 19Q-191 ° C, [α] = -24.5 ° (01.0, MeOH).

Example 19

Calcium salt of N- [3- (N-cyclohexanecarbonyl-D-alanylthio) -5-2-D-methylpropanoyl] -L-proline.

A 404 g sample of the dicyclohexylamine salt prepared in Example 18 was suspended in ethyl acetate (2 liters) and to the suspension was added 0.5 N KHSO 2 solution (2.5 liters) and the mixture was stirred for 10 minutes. The organic layer was separated, washed with distilled water and dried over magnesium sulfate. The filtrate was evaporated in vacuo and the residue was dissolved in acetone (1.2 L).

To the acetone solution was added distilled water (1.2 liters) and calcium carbonate (36.6 g) and the mixture was stirred vigorously at 40 ° C for 1 hour. After cooling, the mixture was filtered and the filtrate was evaporated in vacuo. The syrup obtained was suspended in acetone (1 liter) and the solution was stirred for 5 hours. The precipitate was filtered with suction, air dried and then vacuum dried to give the title compound as a white powder (212 g). [α] β = -47.2 ° (0 = 1.0, MeOH).

Claims (5)

An analogous process for the preparation of N- (3-acylthio-2-methylpropanoyl) -proline compounds of Formula I 5 f3 RAS-CH2CHCO-N in COOH wherein R is one of an α among a cyclopropanecarbonyl, cyclolohexanecarbonyl and adamantanecarbonyl group, and A is a glycine, sarcosine, D-alanine, D-leucine,. D-phenyl-alanine, D-tryptophan, D-phenylglycine, D-methionine or D-glutamine residue, whose α-carbonyl group forms a thiol ester bond with the sulfur atom, or pharmaceutically acceptable salts thereof with bases, characterized in that a) reacting a compound of formula II 20 ch3 RA-s-ch2chcooh II wherein R and A have the above meanings, or a reactive derivative thereof having a compound of formula III HN3 - III COOR 'wherein R1 is a hydrogen atom carboxyl protecting group, or a reactive derivative thereof in an inert organic solvent at between -50 and 20 ° C and removing any protecting groups from the reaction product, or b) reacting a compound of formula IV R-A-OH IV wherein R and A have the above meanings, or a reactive derivative thereof having a compound of formula V CH 3 i3 r ~ \ HS-CH 2 CHCO-N V 5 COOR 'wherein R' is a hydrogen atom or a carboxyl protected group, or a reactive derivative thereof in an inert organic solvent at between -50 and 20 ° C and removes any protecting groups from the reaction product and then, if desired, converting an obtained compound of formula I into a pharmaceutically acceptable salt thereof with a base. Process according to claim 1, characterized in that the reactive derivative of the compound of formula II or IV is an activated amide, acid halide, activated ester or mixed acid anhydride. Process according to claim 1, characterized in that the compound of formula II or IV is reacted with, respectively, the compound of formula III or a reactive derivative thereof or the compound of formula V in the presence of a carbodiimide. The process of claim 1a), characterized in that the reactive derivative of the compound of formula III has a silyl group introduced into the imino group of said compound. Process according to claim 1, characterized in that the inert organic solvent is tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphorotriamide, chloroform, dichloromethane or acetonitrile.