FI75804B - FOERFARANDE FOER FRAMSTAELLNING AV TERAPEUTISKT AKTIVT PROLINDERIVAT. - Google Patents

Description

1 75804

A process for the preparation of therapeutically active proline derivatives

The present invention relates to a process for the preparation of proline derivatives of formula (I)

f »3 / —I

R-A-S-CH, CHCO-N (I) 10 \ -

Dooh

(where R is a cyclohexylcarbonyl group attached to the Δ-amino group of the amino acid; A is glycine, sarcosine, D-alanine, D-leucine, D-methionine, D-glutamine, D-phenyl-alanine , A D-tryptophan or D-phenylglycine residue and has a Λ-carbonyl group which forms a thiol ester bond with a sulfur atom) and pharmaceutically acceptable salts thereof.

Compounds similar to these compounds of formula (I), for example compounds in which R is a benzoyl, acetyl or t-butyloxycarbonyl group and A is an L-Λ "amino acid, are described in GB patent application. No. 2,050,359 A. European Patent Application (EP) 9,898A1 describes compounds in which R is a benzoyl, acetyl, t-butyloxycarbonyl, cyclopentylcarbonyl or the like group and A is an L-Λ amino acid. 35383A1 describes compounds in which R is a benzoyl group and A is a D-phenylalanyl group, however, there is no prior art reference which specifically describes the compounds represented by formula (I) .Therefore, they are novel compounds not described in the literature and are useful as pharmaceuticals as described below.

The group of structural formula (I): 35 fH 3: -CH 2 CHCO- 2 75804 has an asymmetric carbon atom, so this group may be in the D-form, the L-form or the DL-form, i. As a mixture of D and L forms. Each form is within the scope of the present invention, and the D and DL forms are preferred due to their biological activity.

Proline group in formula (I) 10

COOH

may be in the D-form, the L-form, or the DL-form, and each form is within the scope of the present invention, with the L- and DL-forms being preferred for their biological activity.

Illustrative examples of pharmaceutically acceptable salts of proline derivatives of formula (I) include salts formed with alkali metals such as sodium and potassium, salts formed with alkaline earth metals such as calcium and magnesium, and salts formed with basic amino acids such as arginine and lysine. Calcium salts and lysine salts are preferred.

Proline derivatives of formula (I) as defined above, or pharmaceutically acceptable salts thereof, are prepared according to the invention by a) providing a compound of formula (II) ΪΗ3 R-A'-S-CH2CHCOOH (II) 30 (wherein A ' is a group A or a protected derivative thereof as defined above, and R is as defined above), in the presence of a carbodiimide or a reactive amide, acid halide, activated ester or mixed anhydride thereof to react with a compound of formula (III) 3 75804 HN (III) COOR '5 (where R' is a hydrogen atom or a carboxyl protecting group) or a derivative containing a silyl group thereof in an inert organic solvent at a temperature between -20 ° C and 10 ° C and any protecting groups in the reaction product are removed, or b) reacting a compound of formula (IV) R-A'-OH (IV) 15 (wherein A1 and R are as defined above) in the presence of a carbodiimide or a reactive amide thereof, an acid halide, an activated ester reacting a different or mixed anhydride with a compound of formula (V) 20 CHo j- I 3 / HS-CHoCHCO-N (V) vi COOR '25 (where R' is as defined above) in an organic solvent at a temperature between - From 20 ° C to 10 ° C, and any protecting groups present in the reaction product are removed and the product obtained by method a) or method b) is optionally converted into a pharmaceutically acceptable salt.

If the group A of the formula (I) has a functional group which should not react, such as an amino or carboxyl group, the compound of the formula (II) denotes a derivative in which such a group may be protected.

4 75804

Any functional group in A 'that should not participate in the reaction can be protected with groups commonly used in peptide syntheses that can be removed under relatively mild conditions. The amino group of Example 5 may be protected with a t-butyloxycarbonyl group; the carboxyl group may be protected with a t-butyl group. These protecting groups can be removed by treatment with hydrogen fluoride, trifluoroacetic acid or hydrogen chloride. Alternatively, the carboxyl group is protected with a lower alkyl-substituted silyl group such as trimethylsilyl, which is then removed by treatment with water.

When R 'in the formula (III) 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 hydrofluoric acid, trifluoroacetic acid, hydrogen chloride or water.

The reactive derivative of the compound of formula (II) is such that the carboxyl group involved in the reaction is activated. The carboxyl group can be activated by converting it to any of the forms commonly used in peptide synthesis, such as activated amides, acid halides, activated esters, and mixed anhydrides of acids. Of these forms, activated esters formed with N-hydroxysuccinimide, mixed acid anhydrides with carbonic acid monoesters and activated amides with carbonyldiimidazole are preferred. Carbodiimides such as dicyclohexylcarbodiimide can be used as condensing agents 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 can be activated by any method commonly used in the field of peptide syntheses, for example, a method of attaching a silyl group such as a trimethylsilyl group, a "phosphato method" using a phosphorus compound such as phosphotrichloride / Ann. Chem. 572 96 (1951) 71 by a method using 5,758,804 phosphoric acid esters such as tetraethyl ester of pyrophosphoric acid or by the "N-carboxyanhydride method" (NCA method).

The reaction may be carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphoramide, chloroform, dichloromethane or acetonitrile. The reaction is carried out at a temperature of -20 ° C to 10 ° C. The reaction time varies depending on the temperature, the reactants and the solvent, and is generally between 0.5 hours and 48 hours, preferably from 1 hour to 6 hours.

After the amide formation reaction, any protecting groups present in the reaction product are removed by any of the methods specific to the particular protecting group mentioned above.

The final product can be isolated from the reaction mixture and purified by any conventional method, for example, various chromatographic methods using silica gel, dextran network polymer, or porous polymers such as styrene-divinylbenzene or acrylic acid esters. A suitable elution solvent can be selected from chloroform, ethyl acetate, methanol, ethanol, tetrahydrofuran, benzene, water and acetonitrile. Alternatively, the final product can be obtained by first isolating the reaction product as an organic salt such as a dicyclohexylamine salt, which is then treated with an acid such as hydrochloric acid or potassium bisulfate.

The compound of formula (II) used as a starting material in process variant a) can be easily prepared by the following method: A compound of formula (VI): R-OH (VI) (where R is as defined above) or a carboxyl group thereof for which the reactive derivative is reacted with an amino acid of formula (VII): 6 75804 H2N-A "-COOR" (VII) (where A "is an amino acid residue which is the same as A 'except for the group NH or C = O; R "is a hydrogen atom or a carboxyl protecting group) or a reactive derivative thereof, followed by removal of R" if it is a protecting group to give a compound of formula (VIII): 1Q R-NH-A "- COOH (VIII) (wherein R and A 'are as defined above), then reacting a compound of formula (VIII) or a reactive derivative thereof with a compound of formula (IX): CHO I 3 HS-CH 2 CHCOOR' "(IX) (where R '" is a hydrogen atom or a carboxyl protecting group), followed by the removal of R * 1 if it is protecting group.

Examples of the reactive derivatives for 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 anhydrides of acids.

When R "in an amino acid of formula (VII) has a protecting group, preferred examples thereof include a t-butyl group (which can be removed with 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 time conditions).

The reactive derivative of the amino acid of formula (VII) is such that its amino group is activated and such derivative is prepared by any activation method commonly used in the field of peptide syntheses, such as by attaching a silyl group (e.g. a trimethylsilyl group). ", by 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 a compound of formula (VI) and an amino acid of formula (VII).

The reaction may be carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphotriazide, chloroform, dichloromethane or acetonitrile. The reaction is generally carried out under cooling or at room temperature (-50 ° C to 20 ° C), preferably -30 ° C to 15 ° C. The reaction time varies depending on the reaction temperature, the reactants and the solvent, and is generally from 0.5 hour to 48 hours, preferably from 1 to 6 hours.

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

The compound of formula (VIII) may be isolated from the reaction mixture and purified by any conventional method, for example by recrystallization from ethyl acetate, n-hexane, acetone or water or by various types of chromatographic methods as described for the isolation and purification of the compound of formula (I). or by decomposing the organic salt of the reaction product with an acid.

A compound of formula (VIII) may be reacted with a compound of formula (IX) by reacting the carboxyl group of the former compound with the mercapto group of the latter compound in the presence of a condensing agent (i.e. carbodiimides such as dicyclohexylcarbodiimide). A reactive derivative of a compound of formula (VIII) may be reacted with a compound of formula (IX) 8 75804. Examples of reactive derivatives of a compound of formula (VIII) are activated amides, acid halides, activated esters and mixed anhydrides of acids. Activated amides formed with carbonyl diimidazole are preferred.

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

The reaction to form a thiol ester is performed in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosotriamide, chloroform, dichloromethane or acetonitrile. The reaction is generally carried out under cooling at 15 or room temperature (-50 ° C to 20 ° C), preferably between -30 ° C and 10 ° C when the compound of formula (VIII) is used as its activated amide or acid. as a halide, and between -10 ° C and 10 ° C when said compound is used in the acid anhydride form. The reaction time 20 varies depending on the reaction temperature, the reactants and the solvent, and is generally from 0.5 to 48 hours, preferably from 1 to 6 hours. After the thiol ester formation reaction, R "'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 method, i.e., by reaction with hydrogen fluoride, trifluoroacetic acid or hydrogen chloride.

The final product can be isolated from the reaction mixture and purified by any conventional method such as recrystallization from an organic solvent such as ethyl acetate or n-hexane, various types of chromatographic methods as described for isolating and purifying a compound of formula (I) or decomposing the organic salt of the reaction product.

In process variant b) of the invention, a compound of formula (IV) or a reactive derivative thereof is reacted with a compound of formula (V) as described above.

If the group A of the formula (I) has a free functional group which should not participate in the reaction, such as an amino-5 or a carboxyl group, the compound of the formula (IV) denotes a derivative in which such a group may be protected.

Any functional group in A 'that should not participate in the reaction is protected with groups commonly used in peptide synthesis that can be removed under relatively mild conditions. For example, an amino group may be protected with a t-butyloxycarbonyl group and a carboxyl group may be protected with a t-butyl group. These groups can be removed by treatment with hydrogen fluoride or hydrogen chloride.

Alternatively, the carboxyl group may be protected with a lower alkyl substituted silyl group such as a trimethylsilyl group which is then removed by treatment with water. When R 'in 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.

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 anhydrides of acids. Activated amides formed with carbonyl-diimidazole are preferred. Carbodiimides such as dicyclohexylcarbodiimide can be used as condensing agents to form an amide bond from a carboxyl group and a mercapto group.

The reaction between a compound of formula (IV) or a reactive derivative thereof and a compound of formula (V) is carried out in an inert organic solvent such as tetrahydrofuran, dioxane, dimethylformamide, hexamethylphosphoric triamide, chloroform or dichloroethane. The reaction is carried out at a temperature in the range of -20 ° C to 10 ° C when the compound of formula (IV) is used as an activated amide or acid-halide, and in the range of -10 ° C to 10 ° C when said compound is used acid anhydride. The reaction time varies depending on the reaction temperature, the reactants and the solvent, and is generally 0.5 to 48 hours, preferably 1 to 6 hours.

After the thiol ester formation reaction, any protecting groups in the reaction product are removed by any method specific for the corresponding protecting groups.

The final product can be isolated from the reaction mixture and purified by any of the conventional methods described above, such as various types of chromatographic methods using silica gel, crosslinked dextran polyol, and porous polymers such as styrene / divinylbenzene or acrylic acid. A suitable elution solvent can be selected from chloroform, ethyl acetate, methanol, ethanol, tetrahydrofuran, benzene, water and acetonitrile. Alternatively, the final product can be obtained by first isolating the reaction product as an organic salt such as a dicyclohexylamine salt, which is then treated with an acid such as hydrochloric acid! or potassium bisulfate.

The compound of formula (I) has a carboxyl group attached to the proline group, so that it can form salts with various basic substances, if desired. Salts formed with pharmaceutically acceptable basic substances are of particular importance. Such salts can be prepared by a conventional method, i. by treating said carboxyl group with an equimolar amount of one of the above bases (e.g., alkali metals, alkaline earth metals and basic amino acids).

11 75804

The proline derivatives of formula (I) and their pharmaceutically acceptable salts prepared according to the present invention inhibit the formation of angiotensin II from angiotensin (I) by inhibiting the action of an angiotensin 5 converting enzyme. Therefore, they are useful in the treatment of hypertension due to the action of angiotensin II and in the treatment of heart failure.

The activity of a few final products of the present invention as inhibitors of the angiotensin converting enzyme was measured.

1) Method

The angiotensin converting enzyme was extracted from the rabbit lung. The test tubes were charged with 0.111 molar boric acid-Na 2 CO 3 buffer solution (pH 8.3, 0.6 mL), 0.2 mL of 0.111 molar boric acid Na 2 CO 3 buffer solution (pH 8.3) containing 25 mmol benzoylglycylhistidyl leucine ( substrate), and 0.1 ml of a 0.111 molar boric acid-Na 2 CO 3 buffer solution (pH 8.3) containing 10 ° -10 moles of test compounds (13 of the final products of the present invention shown in in the table below), and preincubated at 37 ° C for 5-10 minutes. 0.1 ml of enzyme solution (acetone powder) was then added to each test tube and each mixture was incubated at 37 ° C for 30 minutes. The benzoylglycine formed by the enzyme was extracted into ethyl acetate in the presence of hydrochloric acid and measured by UV absorption at 228 nm. The ratio of enzyme activity in the presence of test compounds to enzyme activity without test compounds was determined (100). The concentration of each test compound at which the relative activity of the enzyme was 50% was expressed as the activity of the test compound in question to inhibit the activity of the enzyme and was reported as a value of 150.

12 75804 2) Results -

Test compound I50

Compound No. 1 1.6 x 10 "^ ^ Compound No. 2 1.3 x 10" ^

Compound No. 3 4.0 x 10 "

Compound No. 4 1.0 x 10 ”^

Compound No. 5 1.5 x 10 "?

Compound No. 6 1.7 x 10 "" 7 10 Compound No. 7 4.8 x 10 "8

Compound No. 8 1.7 x 10- * 7

Compound No. 9 5.4 x 10 "Compound No.: 1. N- [3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline 2. N- [3 - (N-cyclohexylcarbonylglycylthio) -2-D-methylpropanoyl-7-L-proline 2Q 3. N- [3- (N-cyclohexylcarbonyl-N-methylglycylthio) -2-D-methylpropanoyl] -L-proline 4. N - E 3- (N-cyclohexylcarbonyl-D-phenylalanylthio) -2-D-methylpropanoyl-7-L-proline 5. N- E 3- (N-cyclohexylcarbonyl-D-leucylthio) -2-D-methylpropanoyl-7-L -proline 6. N- E 3- (N-cyclohexylcarbonyl-D-tryptophylthio) -2-D-methylpropanoyl-7-L-proline 7. N-3- (N-cyclohexylcarbonyl-D-phenylglycylthio) -2-D -methylpropanoyl7' * L ”proline 2q 8. N- [3- (N-cyclohexylcarbonyl-D-methionylthio) -2-D-methylpropanoyl] -L-proline 9. N- [3- (N-cyclohexylcarbonyl-D-glutaminylthio) -2-D-methylpropanoyl-7-L-proline The effect of the final products of the present invention lasts longer than that of known compounds having similar potency, for example, the effect of 3-mercap- 13 75804 to-2-D-methylpropanoyl-L-proline (commonly referred to as Captopril) so that the desired effect on blood pressure can be achieved less frequently at daily doses. Captopril and other known compounds cause a sudden drop in blood-5 pressure at the beginning of the administration period and this may develop into orthostatic hypotensive asthenia. 1, No. 8, 115, p. 557 (March 10, 1979) .7, but the final products of the present invention have only a mild hypotensive effect at the beginning of the administration period and the development of orthostatic hypotensive asthenia is very unlikely. Drugs such as captopril with a free mercapto group produce a variety of side effects that can be attributed to the mercapto group. A few to mention: taste disturbances, urinary albumin 15 excretion, agranulocytosis, and skin diseases associated with fever, have been reported in the Lancet, Vol.

1, No. 8160, p. 150 (19 January 1980), Lancet, vol. 2, No. 8186, pp. 129 (July 19, 1980) and South African Medical Journal, vol. 58, 172 (1980). In contrast, the thiol ester bond of the final products of the present invention is not hydrolyzed in vivo and thus it is unlikely that a mercapto group would form. Therefore, it is very unlikely that the final products of the present invention would cause the above-mentioned side effects due to the mercapto group.

In order to compare the activity of the compounds according to the invention with known compounds of similar structure, comparative experiments were carried out using compounds known from FI patent applications 71749, FI patent applications 810683, 810725 and 810663 and GB application publication 2050359 as known compounds. The experiments determined the angiotensin-converting enzyme inhibitory activity, the in vitro hydrolysis and the antihypertensive effect of angiotensin-I in vivo, as well as the duration thereof. The results are shown in the following Table I. The experimental results show that the compound 14 75804 prepared according to the invention does not differ substantially from the previously known angiotensin-modifying enzyme inhibition. In the other two experiments (hydrolysis, antihypertensive activity) the compound of the invention, on the other hand, is superior to the known compounds; it has significantly fewer side effects and the duration of effects is much longer than with known compounds.

is 75804 <0 -> c * h + j o e

c> rH

<U -H

rH> O <N (Vι VO

fQ 00 VO Γ0 fir-4 ^ (0 -H -

in (N

+ J to o oo eg

<D rj tN in rH

C 3 H

H (0 TJ- Γ ~

<0 -Η ιΗ «H

Λ> O VO

C -H <Tv m 'T

d) -H (N 2 - - T

U -P ιΗ II rn II O '

Jl O II <f C rH CH

> (0 VO C -vr * <#> κβ 0 u ~

UM C

+ J: nJ -H

• H e &> m (0 * C> oi— ro ro (n ον o: (0 • H (U VO (Tv in <Tv Sh »H: (0 HO I * | 0« - c>, :( 0 o oo vo Γ 'm · ** σι fi

>, f-H co σ \ m in σι .X

H H fi 0 0) ^ Η -ι-ι.

Oho o r-ι oo m oo C

> i: n3 (—I o t— v — i> -h vo I (1) ffi -m iH 4-> ________ fi

•B

(0

r- · i'- γ · - γ-- θ 'I — I

o I I I I I (1)

m o o o o o I

H rH rH rH rH iH a) Θχ x x x x o

vo cn cn m, X

%. · * · ** 3 cn cm m co co 11 rH —....... ............ 1 ....... "" 1 1 ..... . . - 1 • H fi

- · («X X X X X X

+ J I I I I I I

- ^ (ö o a) a) <β o

0} I — I iH pfi pfi rH H

> C P4 Λ · <Λ

I I I I I I

fi-P Q Q tfi fil P)

a) to I I I I I I

T3-H OOOO O O I-1 fi -o oouu u u rn! 6 έώ ό ό * *

• H _ __. . _ ___ O

-P-- O

, X ^ I

(0 fi Ä K

(0 (Ö O— CJ

fi · Η> I

HOJiddl σι σι σι cn

• O ^ rH n in ro roinin ro m m ro m m K

0-H rH 0 (Tv 00 (N VO (TiOOfMrO (TlOOCNirO (TiOOCNrO CJ

, ¾¾ 3 in gnu r> vo vo ο ημο γό »id γό i X 4j .f-,: t0 3 ooo Hoom r ~ oom r-oom to ^ tfl d) rHf: rH rH v — I rH rHrHiHO rH rH i —IOI — I rH v — IO |

R_ | ., Η 4J rHd) Γ- 00 00 00 t ^ 00 00 (N I— 00 OO (N Γ "00 00 (N

fi Ό · Η · Η, Χ

(13 x: -H to (0 HHH H HHHCQ HHHffl HHMCQ

fn> ·> W, C & η & ηΡη h, b h h U h Em En O Cm Eh Eh U X

16 75804

The proline derivative of formula (I) and its pharmaceutically acceptable salts may be formulated into compositions for oral administration, such as tablets, capsules, granules, powders, Siira-5 pellets and elixirs, or as sterile solutions or suspensions for parenteral administration. For this purpose, pharmaceutical compositions can be prepared from one or more of the final products of the present invention (as active ingredient) and pharmaceutically acceptable excipients such as excipients, carriers, binders, stabilizers and flavors. The daily dose of the compounds of the present invention for adults is 0.5 mg to 2 g, preferably about 1 to 500 mg administered orally and 0.1 to 600 mg, preferably about 0.3 to 300 mg administered parenterally.

The process for preparing the compounds of the present invention will now be described by way of the following examples, to which the invention is by no means limited.

Example 1 Preparation of 20 N-busted amino acids a) D-Alanine (4.5 g) was dissolved in 230 ml of 1N aqueous Na 2 CO 3 with stirring. To the solution was added dropwise 100 ml of tetrahydrofuran containing 9.0 g of cyclohexylcarbonyl chloride at 5-10 ° C, at which temperature the mixture was stirred for 30 minutes, then stirred at room temperature for 1.5 hours. 2N HCl was then added to the reaction mixture to bring the pH to between 1-2. 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 the crude product. Recrystallization from ethyl acetate / n-hexane gave 4.65 g of N-cyclohexylcarbonyl-1-D-alanine. ~ + 26.6 °.

* The compounds listed below were prepared by the method described above.

17 75804 h.

en en

H -H

I 3 3 • H Λ! 3ί 3 πϊ ro e * M λ; 6 Λ E ^ E - -

O O ^ 0 ^ EK

o— O -— o · -. LO .—. .-. -. ·. ·. · * · ΓΟ (N

+ J ook cr se LO K »® OK OK QK Q 5C - · - ·

(D ^ O ^ O ^ O ro O rH O H O O H -—- O t — I

m oo qj eri qj o <i) ro φ * φ -O γο-η en rorH o lo

• H Hg Hg rH g Hg LO S3 D - - Q - OJ (N

rH I + | I I + U K U '- 3 * V k v V -' LO CNI - 'iJl O rf m 4J II O II O II O H O II O II O K--' K - ^] | «. - «. - *. - g (N g (N «. * • H Ό OH OH OH OH OH OH Z I LO Z | o ro

to β Γ ", II r-. Il Il 4—, Il 1 ^ Il Il I CN σι | rH o rH

> 1 3 U .¾ U Λ5 O «U U .¾1 U K * - K * * *

Puin "vJ - '' -ij'-r VJ - Vj - * <| - H HÖH Hroif

I I I I I I I I

3 3333333

H-) 4H m <4-1 4-1 m MH 4H UH

0) 0 0 000000 ε uuuuuuuu • μ Ό Ό Ό Ό Ό Ό Ό Ό 4- *> 1 ^ 4> 1> 1 ^ 4 ^ 4 ^ 4 4- »,, β Λ Λ Λ Λ A Λ Λ 03 3 333333 β Μ Ρ-ι Μ Μ Μ) Η> Η> Η • μ 4- »4J 4-) 4-) 4-» 4J 4-> 4-> rH 0) φ φ φ φ φ φ φ 0-Ρ 4- > 4J 4- »4-» 4-> 4-> 4-> rl 4-) - -Η - · -Η - · Η - -Η - -Η - · Η - -Η - -Η X -μ β · Η e -Η β -Η β -Η β -rl β -Η β -rl β 3 33 33 33 33 33 33 33 33 φ φ 3 φ 3 0) 3 0) 3 (1) 3 (1) 3 0) 3 (1) 3 Κ> μ> μ>> Η> μ>) Η>) η> Μ> μ

I I I I I I

I I -rl I I · Η I -rl I · Η I -rl I · Η

> 1 I> 1 rH> 1 I> 1 rH> 1 rH> 1Η> 1 rH> 1 rH

> 1> 1> 4> 1> 4> 1> 4> 4> 1> 1> 1> 1> 4> 4> 1> 1 4.) 3> 1-H 3> 1 3> 1- <H 3 > i 3> 4 3> i 3> -i 3> i 3 ί β Ό XC Λί fi Ό Λί β X β XC Λ (β Λί β> φ Ο -Η φ Ο -Η φ Ο -Η φ Ο -Η φ 0 Ή φ Ο Ή φ Ο -Η φ Ο -Η -Η -μ .C Λ μ .β Λ Ό Λ Λ μ ΚΙΛΌ ΛΛΌ ΛΛΌ ΛΛΌΛΛΌ 0 <d ojho ο) η · η oho ο μ -η ο μ - η ομ · η ο> η · ηο) η · η tr 0) '—13' — I ι — I 3 μ ι — I 3 ι — I <—I 3 Μ ι — 13) -1 <—I 3) Η I — I 3 μ '—13) -1 3 β Λί Λί Λί Λί Λ! 0 XXX XX 0 Λ! 0ί 0 Λ! 0ί 0 Λί Λί 0 Λί Λί Ο

φ -rl ^ 1-rl -rl> 4 -rl rH> 1-rl -r | > 4-rl rH> 4-rl r-H> 4-rl rH> 4-rl rH> 4-H rH

K 3 en ri rH en rH Λί w ri h 3 h en rH x en ri x μηλ: wha; i) & β r-H 3 3 3 3 3 3 33

S + * Il I

I> 1 O -rl -rl II ft> 1 -rl I β -rl III -rl II β II> 4 II β II -μ Ι Ι β Ι Ι -Η Ι Ι 3 +> Λί ο ο -η Λίθο Λί ο μ Λίοω-ΗΛίοα), ν ο> -η χο · η) (οο 1 Ο Ο) Λ UH β (1) Λ rH φΛ-μ ΦΛΉ β φ Λ S φ Λ tr Ο) Λ 3 ΦΛϋ -μ & Λ μ I 3 Λμΐ Λ μ I Λ MI -Η Λ MIAMI .β μ> 4, β μ μ 4J -μ 3 Ο 3 Ο Η Ο 3 Q 03Q-rl 03Q-H 0 3 Ο 03Q-HO 3Η 033 3 β Λ rH, lfi I 3 rH χ ι HKI fi HMI η H) (I HXI fi Λ Ό 0 Λί -H -H -HX -H -H -H Λί -H -H 3 Λί -H -H> 4 Λί -H -r | -HX -H -μ -μ Λί -H -μ Λ! -μ -μ

β -μ β> 4 rH ι — I rH> 1 rl rl fi> 4 rH rH 3> 4 rH rH rH> 4 rH rH β> 4 rH rH -μ> 4 rH rH> 4d H

30 -H 3> 4> 4> 4 3> 4> 4-μ 3> 1> (MH 3> 4> 4 tT 3> 4> 4-rt 0)> 4> 1 g 3> i> i 3> 4> 4 I β ε I> 1> 4> 4 · μ I> 4> 4 · μ I> 4> 1 OI> 4> 4 · Η I> 4> 4-μ I> 4> 4 3 I> 1 > 4 I> 1> 4 μ Z -μ 3 Ζ 3 β β β Ζ 3 β 3 Ζ 3 β -μ Ζ 3 β Η ζ 3 β β Ζ 3 β -μ Ζ 3 β Ζ 3 β β is 75804

Example 2 a) 3- (N-Cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoic acid N-cyclohexylcarbonyl-D-alanine (5.97 g) was dissolved in dry tetrahydrofuran (100 ml). To the solution was added carbonyl diimidazole (5.84 g) between -20 ° C and -15 ° C, and the mixture was stirred at that temperature for 1 hour. 3-Mercapto-2-D-methylpropanoic acid (3.60 g) was then added and the mixture was stirred at 10-15 ° C for 10 hours, then at room temperature for 1 hour. The mixture was evaporated in vacuo to remove the solvent. Water (40 mL) was added to the residue, and 2N-HCl was added to the mixture to adjust its pH to 1-2. 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 the crude product. Recrystallization from ethyl acetate / n-hexane gave the title compound as colorless prismatic crystals (7.50 g, 83%). Sp. 149-152 ° C.

Δ δ - + 46.4 ° (C = 1.07, MeOH) NMR (CD 3 OD, δ): 1.20 (3H, d), 1.35 (3H, d), 1.20-2.0 ( UH, m), 2.40-2.80 (1H, m), 3.05 (2H, m), 4.50 (1H, m) b) 3- (N-cyclohexylcarbonyl-D-phenylglycylthio) -2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclohexylcarbonyl-D-phenylglycine (2.61 g), carbonyldiimidazole (1.95 g) and 3-mercapto-2-D- methylpropanoic acid (1.20 g). Obtained the title compound as a yellow oil (2.07 g, 57%).

Δ D = -48.7 ° (C = 1.19, MeOH) NMR (CDCl 3, δ): 1.24 (3H, d), 1.20-2.10 (UH, m), 2.40- 2.80 (1H, m), 3.3, (2H, m), 5.75 (1H, d), 6.40-6.80 (1H, m), 7.30 (5H, S), .90 (1H, S).

c) 3- (N-Cyclohexylcarbonyl-D-leucylthio) -2-D-methylpropanoic acid

The procedure of (a) was repeated using N-cyclohexyl-19-carbonyl-D-leucine (2.41 g), carbonyl-diimidazole (1.95 g) and 3-mercapto-2-D-methylpropanoic acid (1 , 20 g). Obtained the title compound as an oil (2.40 g, 70%).

Z + 7d = + 12.2 ° (C = 1.03, MeOH) δ NMR (CDCl 3, δ): 0.95 (6H, d), 1.28 (3H, d), 1.20-2.10 ( UH, m), 2.40-2.80 (1H, m), 3.10 (2H, m), 4.70 (1H, m), 6.00-6.30 (1H, m), 9 , 20 (1 H, S).

d) 3- (N-Cyclohexylcarbonyl-D-glutaminylthio) -2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclohexylcarbonyl-D-glutamine (2.56 g), carbonyl-di- imidazole (1.95 g) and 3-mercapto-2-D-methylpropanoic acid (1.20 g). 0.70 g of the title compound was obtained (20%). Sp. 146-149 ° C.

15 D = + 10.8 ° (C = 1.05, MeOH) NMR (CD 3 OD, δ) δ 1.20 (3H, d), 1.20-2.10 (UH, m), 2, Δ (4H, m), 2.40-2.80 (1H, m), 3.05 (2H, m), 4.50 (1H, m).

e) 3- (N-Cyclohexylcarbonyl-D-phenylalanylthio) -2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclohexylcarbonyl-D-phenylalanine (1.37 g), carbonyldi- imidazole (1.0 g) and 3-mercapto-2-D-methylpropanoic acid (0.60 g). The title compound was obtained as an oil (1.5 g, 79%).

Δ Zd = + 13.9 ° (C = 1.02, 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).

f) 3- (N-Cyclohexylcarbonyl-D-methionylthio) -20-D-methylpropanoic acid

The procedure of (a) was repeated using N-cyclohexylcarbonyl-D-methionine (0.325 g), carbonyldiimidazole (0.25 g) and 3-mercapto-2-D-methylpropanoic acid (0.15 g). . Obtained the title compound as an oil (0.29 g, 64%).

35 D = -7.9 ° (C = 1.0, MeOH) δ 75804 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) .

g) 3- (N-Cyclohexylcarbonyl-D-tryptophylthio) -5 2-D-methylpropanoic acid

The procedure of (a) above was repeated using N-cyclohexylcarbonyl-D-tryptophan (0.157 g), carbonyldiimidazole (0.1 g) and 3-mercapto-2-D-methylpropanoic acid (0.06 g). . A gummy portion of the title compound (0.17 g, 82%) was obtained.

Δ 7d = -7.1 ° (C = 1.0, MeOH) NMR (CDCl 3, δ): 1.26 (3H, d), 1.2-2.4 (11H, m), 2.5 -2.9 (1H, m), 3.03-3.3 (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) β- [β- (N-Cyclohexylcarbonyl-D-alanylthio) -2-β-methylpropanoyl] -L-proline 3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoic acid (1.51 g) was dissolved in dry tetrahydrofuran (40 ml). To the solution were added triethylamine (0.61 g) and ethyl chloroformate (0.65 g) at -5 ° C with stirring. After 5 minutes, a solution of L-proline (0.58 g) and triethylamine (0.61 g) dissolved in water (5 ml) was added, and the mixture was stirred at 0 ° C for 1 hour, then at room temperature for 30 minutes. . The mixture was evaporated in vacuo to remove the solvent. After water was added to the residue, 2N-HCl was added to the mixture to adjust its pH to 1-2. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed twice with saturated NaCl solution and dried over mangesium sulfate. The filtrate was concentrated in vacuo to remove ethyl acetate and the residue was chromatographed on silica gel using a mixture of chloroform and methanol (100: 1-100: 2) as eluent. The fractions containing the final product were collected and evaporated in vacuo to give a gummy batch of the title compound (0.3 g). ).

2i 75804 b) The same compound could be prepared by the following method: 3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoic acid (6.3 g) and N-hydroxysuccinimide (2.3 g) were dissolved in dry tetrahydrofuran. (50 ml). Dicyclohexylcarbodiimide (4.3 g) was added to the solution at 0-5 ° C, and the mixture was stirred at this temperature overnight. After the reaction, the precipitate was filtered off, and the insoluble matter was washed with a small amount of tetrahydrofuran. The Suo-10 dosage and washings were combined and concentrated in vacuo.

Ethyl acetate was added to the residue, 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 in the order shown. The solution was dried over magnesium sulfate, evaporated in vacuo and the residue solidified by the addition of a mixture of ethyl acetate and hexane (1:10). The yield of solid product was 6.90 g (87%). Sp. 113-116 ° C. D * Jo = + 14.2 ° (C = 1.05, Me OH) NMR (CDCl 3, δ): 1.38 (6H, d), 1.20-2.20 (UH, m), 2, 82 (4H, 20S), 2.8-3.30 (3H, m), 4.75 (1H, m), 6.30 (1H, broad d).

The activated ester (3.98 g) was dissolved in tetrahydrofuran (40 ml). To the solution were added water (5 ml) dissolved in L-proline (1.15 g) and N-ethylmorpholine (1.26 ml), and the mixture was stirred overnight. The mixture was evaporated in vacuo to remove solvents and water was added to the residue. 2N HCl was added to the mixture to adjust its pH to 1-2. 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 silica gel column chromatography using a mixture of chloroform and methanol (100: 1 to 100: 2) as an eluent. The product (1.45 g) obtained by evaporating the eluate in vacuo was found to be identical to the title compound by NMR analysis.

C) The title compound could also be prepared by the following method: 3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoic acid (4.52 g) was dissolved in thionyl chloride (5 ml). A drop of dimethylformamide was added to the solution, and the mixture was stirred at room temperature overnight. The mixture was evaporated in vacuo to remove solvents and dry toluene (2 ml) was added to the residue, and the mixture was re-evaporated in vacuo to remove the solvent. Dry tetrahydrofuran (10 ml) was added to the residue with stirring. A solution of tetrahydrofuran containing the acid chloride was added dropwise at 5-10 ° C with stirring in water (25 ml) dissolved in L-proline (2.3 g) and Na 2 CO 3 (2.5 g). The mixture was stirred at this temperature for one hour and at room temperature for another two hours. The reaction mixture was extracted with chloroform. To the aqueous layer was added 2N normal HCl to adjust its pH to 1-2. After the solution was extracted with chloroform, the chloroform layer was washed twice with saturated NaCl solution and dried over magnesium sulfate. After removal of chloroform, the residue was chromatographed on a silica gel column using a mixture of chloroform and methanol (100: 1-100: 2) as eluent. Evaporation of the mixture in vacuo gave a gummy substance (1.6 g). This material was found to be identical to the title compound by NMR analysis.

: d) The title compound could be further prepared by the method described below.

3- (N-Cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoic acid (3.01 g) and L-proline t-butyl ester (1.71 g) were dissolved in dry dichloromethane (50 ml) and dicyclohexylcarboxylate was added to the solution. diimide (2.15 g) with stirring and ice-cooling. The mixture was stirred at the same temperature for half an hour and kept at 5 ° C overnight. The reaction mixture was filtered and the insoluble material was washed with dichloromethane. The filtrate and washings were combined and washed with 1N HCl, water, 1N NaHCO 3 and saturated NaCl solution in that order, dried over MgSO 4 and evaporated in vacuo to give a gummy batch of N- [3- (N- cyclohexylcarbonyl-23-80804 (1-D-alanylthio) -2-D-methylpropyl-7-L-proline t-butyl ester (4.3 g). 4.0 g of this ester was dissolved in anisole (30 g) and trifluoroacetic acid (10 ml) was added to the solution. The mixture was stirred at room temperature for 1 hour and evaporated in vacuo to remove excess trifluoroacetic acid. The residue was chromatographed on a silica gel column (column: 2 x 35 cm) using a mixture of methanol and chloroform (1: 100-3: 100) as eluent. The fractions containing the final product were collected and evaporated in vacuo to give a gum (3.4 g). This material was found to be identical to the title compound by NMR and thin layer chromatography.

The reactants, reaction solvents, and methods used to prepare the title compound of Example 3 are listed in the following table along with its physical properties. The other compounds were prepared using one of the methods (a) to (d) described in Example 3, and the specific features of each method used are listed in the following table, along with the physical properties of the final products. In the table, the numbers following (1) and (2) in the column "Rf value" indicate the Rf values obtained by thin layer chromatography using CHCl 3 / MeOH / AcOH (2: 1: 0.003) and n- BuOH / AcOH / H 2 O (4: 1: 1).

2i 75804 L £ o oo ^ cn g * o vo to to <x> vL o o o o o o

* € D <3> <£ $> <3> O CSD

^ - 'aa

* “TN 'CM fN - Ίν Q

«~ S - ^ B δ" "^ -δ δ - - θρΓ * - r« _ δ ~ ™ »_ 'ΰ« π βθδδδϊ · υ - S S δ ® τ> ° S δΊδ § 1: jj

£ | δΒ.έΒΒέΕ8 3 -f? Ε ε®8 ": $ e'e Β Β s'S I

> ^ * - 88889 "· ι ~ 885 ~“ .5888§ “8 o SS -mm (Τι ο in 3 ίο ^ m σν '" Ί 5 i ^ Lnrnoocn ^ rScjv H cn »« *. »..« - —— · *>, ** «,«. - · „Η Λ ^«. ΛΓϊ4, Τ> <Τ> ιΓ, νΡ, i flri'f i (n π μ ι / no rs n> o oo tn ii II it — ii — itniiiLn> —ioiiiiictii s Nnmoin ^ oon oi na no mnimmoi— nim Ö ^ '* "* * S» * *>' **** »S ***», »*» z H h Hoi oi n ^ io r ri r-ι <nm -a * vo σν HHoin ^ inrr _ O vo Sd so Q vo ος so ~ ^ o ~

& rj "r? ^ o h 3 oo hS

* 5 ΐ JS t & | ‘F ai

+ »Fe S, λ S

2 * «1 | g * g Il 11 l" s p il il is af i§ ύ

Ä ^ tE-tmOOQ) 03Φ QE Q

• h lii -htI. ’1 ägi t & $ h lv 5 +3 1 jj2

Il lii tilli t ^ ^ 1¾ ti iUiiä! Il II Iillä hl 311 II Iin: ^ »s ö 3s s 2 2 lii, || λ || | S |! I * i i! Sliii tlisi, Ifist s llifil Alia * Aiaf *: -: i * s? s iffiAäi ± £ & ss 75804 25 co en o r- oo (N m co 0 VO r »LD 00» -> - - oo 1_ | oooo _ (¾ _ O <0 1 Ö O <0 Θ MH - - Q5 - . Ti - r »_ ro» cn (0 ro »cm · -

ΓΜ »» - »X X 10 S3 - X

- X i «E EX CJ tNU» * * o P CJ cnU - -P

g CJ 2 χ t * Λ Λ Λ m X X ^ Ό »» »» X »» - »- O X X X X td - CJ X X <d e

T> XCO-PCJXX »XTJ CJ CJ CJ 2 - Ό CJ CJ 'EK

VO CN CN T} O - »X '-' 'ON

tj h <^ '--- O Tl £ * · s ^ β H O £ n) -xx g »CJ td E E E E - <o e G o 2 <d u - O ro in en h x o * - P.! ·

t ^ rx »*» rt * P X »» »» X O PK

X »χ ro uo ^ en X ^ XXXXliooo i'fXX'OlJWM

«. MOOl ^ 1 2 3 4 5 I I I — I 00 1 — I I — I (N · - · - H Φ H

ro »i Hinwnooi *» ^ —co »'—''—"' - '- - - ~ »h x m - v» »x x oi x x x x x

CJ ro »cn <N ro ro rr» rH oo en r ~ cn Ί * »- X roincot-HHinH

Q »~ rH - VO ^» r - ^ »cn — 1» »^ - m .... ro ·» »n ro in« i · O <N ro ^ rw ____ __ | __loi lil | tT »4 -> 0 ΟΟΙΙΙΓΜΓΟΓ'-Ο X cm ro (n ror »(n (m—, oo HromHi» o <j \ id Hninnioinrooi a »xxx» xxx »» »» »» »» id »» »» »» » »» 2 i-hcJCJCJcncJCJCJI "ηηιν ^^ όό g te HHN'tiniohoo

O

Q »». ΙΠ »» ro o 0 o -—. »O -»

q .. »X oo» X o »X

r- ^ i — I H O »H o LiOi — 10

.¾ oo il φ r »il ω i il ®

^ i CJ 2 ^ CJ 2 OS

• H ^ W

02 I <U C I

JJ | Q) Il O> -H O

Qj -p 5> · Η * H P 3 P

I g -h ΗΌ Ό »^ O -H C»> 1 · | Η> ι · Η> i-p h-Po-h> i e -cc e

Jj jj 4J rH -Pfl3 föfö · Η rl (o (ö

O <D · Η <Ι) β · Η PtÖ O H M fO

id § «-h e" »-pp ε, ¾ x p Q) -H <P -Η0Φ 0) 3 (ÖT) (U3 PJH Q Ή> En MH 01 OJ E- · Ή-Ι

> i-h i -h LL

> iEJil E -r · $ M -rH O -HI p γ, m L pi -p · O ^, -L -p J o P fl -PHP OH I rH Ή lö X (> i C Sh OHJ · Η> ιΟ> o O XH £ mh · * -P rH -H> 1 m H + j ΗΛΙΛ 4J-HH "PJ | J £ £ 0 0) Λί P 2 Λ5 3P-P <P3> cH O cd t7> Φ Sh <d 3 XO -P> i EC In X Ό

me in X »m O O <d> iP O O

O) -H -H -H -H -H -H OT rH (0 -P O -H H «g

(>; Id QHT) KO ΗΛ E w p gn 5 * 5 -H

1: id o) ε - C r-H _Q ^ a ^ rs a) o »- · id (d u Ό S -P w - - - i i 2

I I I I I 1101 III - »O

3 OOOOIO OO-PQII 0 S ° £

rH X -rl I id P H Λ Λ I Id O H Λ> rH ft I

, X m X CN CU Λ P> ι (Ί ft ii ΛίΡΐΚ-Ρ-HJ

4>. id α> I o I> 1 Id p I o a> i id m -h h i + j ny g »μ j tn4i + i /» μ I>, rv.

0) I H I O ft I I Ή I O (i j * · Ρ JL ^ ^ '' P

a) 2 (JQ-HHN 2CQ-H-HI 2 C Q> 1-P rH c

-P -id I 4J H J - Id I 4JHN - Id I W φ> i-H

0) I Id · Η · Η> 1 rH · Η I Id · Η -r | ^ i-rH-rl I Ifl Ή C ΐ ^ ιΉ • H / n tn rH rH>,> 1 C fi) UIHH> iH e “ί ^ il Λ 2 Tl T3> t> iP> ι · Ρ '-P - *> ι> ι · Ρ> iP> iÖ * QC p

X I I)> 1> 1 dl O H I fl)> i> i <D> i-rl I Φ> ι · Ρ I g P

5 2 X C C ε C rH 2 X C MH g e rH 2 X C rH CM a a 75804

P ι-H ΓΟ VO CS <N CO

p VO Γ * - VO VO ^ 3 * ^ 3 * H · * · · · · <U o o o o o o ^ Θ Θ Θ Θ Θ Θ% <n | * *. k * »^

Sl_ ^ a-61. . a-e1 .s1 suo '. 8 - | * ί8ΐΒ- «§ I ® e s e e rr \ ** M P" * · ** ·· »SÖ ** U S ^ Vj £ C ** * *“ * ·

, Η ΌΛΗΚΚ5ΒΗ10 Λ'ίίωκχω Λ 5 S S S K

Π ^ 'ttcncn ^ iH ^ r 04 m »dSCLilS

R s k m m k a tn tn κ H von · ηο \ ο · Η nvnnooH π in oi <n ouo ςγ> *> - '^ -' (\ | · · · VO * · · w · · w w> .....

g immini (Nm m ^ ^ ^ T ^ gnhoiii in oh ι imti in g *, M i 1 Ä Nm ^ ismoh <nn tn m «h vo ··« ····· · «···« · ·· ···· οΗΗίΝΠΝΜΠ ^ hh oj mmn * HHfNmNMn O oo - * oo ^ ° ° ci O o ~ · S Ί1 »a ^ · 5

x a öl s äI r öI

H ..j Ή in h] in a u r- t: * lilac fi§ ι ii as m 1 g 3 I I ea

I I '4, S-J d ^ I

u, ^, t '0 M I t o ί i Id ii ib I: il s S * tl ι Tipi! account I li «3 H * M H« s SiSiS ö Wlw

· ': | J $ .5. tS S

lii! il, f | $ ssi;: ι ilfäi alli him 27 7 5 8 0 4

Example 4

From the samples prepared in Examples 3 and 5, various salts were formed from N- [3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline.

5 a) Ca salt A 3.98 g portion of the compound prepared in Example 3 or 5 was dissolved in methanol (40 ml). Calcium acetate hydrate (0.84 g) was added to the solution, and the mixture was heated under reflux for 1 hour. The insoluble material was filtered off and the filtrate was evaporated in vacuo. Chloroform was added to the residue, 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 the calcium salt of Example 3 or 5 (3.40 g). 7D = 15 -47 ° (C = 1.0, MeOH).

b) Mg salt

The procedure of (a) was repeated using 3.09 g of the compound prepared in Example 3 or 5 and 0.772 g of magnesium acetate tetrahydrate. The magnesium salt of Example 3 or 5 (2.42 g) was obtained. δ 7D = -46.6 ° (C = 1.0,

MeOH).

c) Lysine salt 1.19 g of the compound prepared in Example 3 or 5 were dissolved in methanol (22 ml). Lysine (0.416 g) was added to the solution and the mixture was stirred at room temperature for 1 hour and evaporated in vacuo. Chloroform was added to the residue, and the mixture was filtered and evaporated in vacuo. Dimethyl ether was added to the residue, and the product was suction filtered to give the lysine salt of Example 3 or 5 (1.54 g). / D = -23.1 ° (C = 1.0, MeOH).

(d) Na salt A 2.27 g portion of the compound prepared in Example 3 or 5 was dissolved in methanol (25 ml). To the solution was added 0.514 g of sodium acetate and the mixture was stirred at room temperature for 30 minutes and evaporated in vacuo.

28 7 5 8 0 4 To the residue was added 200 ml of a mixture of methanol and chloroform (3: 100 by volume) and the resulting mixture was filtered and evaporated in vacuo. The residue was dissolved in methanol and the solution was filtered and evaporated in vacuo. Ethyl acetate was added to the residue, and the product was filtered off with suction to give the Na salt of Example 3 or 5 (1.85 g). = -26.7 ° (C = 1.0, MeOH).

e) Dicyclohexylamine salt A 13.1 g portion of the compound of Example 3 or 5 was dissolved in 120 ml of acetonitrile. Dicyclohexylamine (6 ml) was added to the solution 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 dried in air to give the dicyclohexylamine salt of Example 3 or 5 (12.2 g). (oD = -24.5 ° (C = 1.0, MeOH)).

Example 5 N- [3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline N-cyclohexylcarbonyl-D-alanine (5.98 g) was dissolved in dry tetrahydrofuran (80 ml). To the solution was added carbonyl diimidazole (5.84 g) at -18 ° C with stirring while cooling the mixture on ice. The mixture was stirred at this temperature for 1 hour, and then N- (3-mercapto-2-D-methylpropanoyl) -L-proline (6.29 g) was added to the mixture, followed by stirring at -18 ° C for 30 minutes, then at room temperature for 30 minutes. temperature for one hour. When the reaction was complete, it was evaporated in vacuo to remove the solvent. Water (50 mL) was added to the residue, and 2N HCl was added to the mixture to adjust its pH to 1-2. The mixture was extracted with ethyl acetate and the ethyl acetate layer was washed with saturated NaCl solution, dried over MgSO 4 and evaporated in vacuo. Acetonitrile (120 mL) was added to the residue, followed by 6 mL of dicyclohexylamine (DCHA). The mixture was stirred at room temperature for one hour. After standing overnight, the precipitate was filtered off and dried in air to give 14.35 g of crude DCHA salt. The crude salt was suspended in acetonitrile (300 ml) and the suspension was heated at reflux for 30 minutes. After cooling, the precipitate was collected by suction filtration and air dried to give a white DCHA salt (12.20 g). The DCHA salt (12.20 g) was suspended in ethyl acetate (90 ml), and 0.5N aqueous KHSO 4 solution (60 ml) was added to the suspension, and the mixture was shaken. The organic layer was washed with distilled water, dried over MgSO 4 and evaporated in vacuo to give a gum (8.64 g). This material was found to be identical to the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, 6): 1.20 (3H, broad, d, CH 3), 1.38 (3H, d, CH 3), 1.55-2.0 (10H, m, CH 2), 2.0- 2.5 (4H, m, CH 2), 2.5-3.3 ~ 20 (4H, m, CH & CH 2), 3.4-3.9 (2H, m, CH 2), 4.0- 5.0 (2H, m, CH), 6.0-6.5 "(1Η, m, NH), 7.31 (1H, s, CO 2 H). Rf: (1) 0.66; (2) 0.71.

Example 6 N-3- (N-Cyclohexylcarbonylglycylthio) -2-D-25-methylpropanoyl147-L-proline N-Cyclohexylcarbonylglycine (1.02 g) was dissolved in dry tetrahydrofuran (10 ml). To the solution was added 20 ml of dry tetrahydrofuran containing 1.07 g of carbonyldiimidazole at -20 ° C with stirring while cooling the mixture with ice. The mixture was stirred at this temperature for 1 hour, then 6 ml of dry tetrahydrofuran containing 1.09 g of N- (3-mercapto-2-D-methylpropanoyl) -L-proline was added, followed by stirring at -20 ° C. 30 minutes and then at room temperature for 35 hours. When the reaction was complete, the mixture was evaporated in vacuo to remove the solvent. The residue was chromatographed on a silica gel column (column: 2 x 35 cm) using a mixture of methanol and chloroform (1: 100-3: 100) as eluent. The fractions containing the final product were combined and evaporated in vacuo to give a gum (1.16 g). The substance was found to be identical to the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, δ): 1.21 (3H, broad, d, CH 3), 1.35-2.5 (14H; m, CH 2), 2.6-3.3 (4H, m, CH & CH 2) ), 3.4-1.9 (2H, m, CH 2), 4.0-477 (3H, m, CH & CH 2), 6.35-7.1 (1H, m, NH), 9.01 10 (1H, s, CO 2 H). Rf: (1) 0.60; 72) 0.68.

Example 7 N- [3- (N-Cyclohexylcarbonyl-N-methylglycylthio) -2-D-methylpropanoyl] -L-proline The procedure of Example 6 was repeated except that N-cyclo-15-hexylcarbonylglycine was replaced by N-cyclohexylcarbonyl-N-methylglycine. (1.09 g), carbonyldiimidazole (1.09 g) and N- (3-mercapto-2-D-methylpropanoyl) -L-proline (1.09 g). A gummy substance formed (0.76 g) and was identified with the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl 3, δ): 1.21 (3H, broad d, CH 3), 1.3-2.4 (14H, m, CH 2), 2.4-3.3 (4H, m, CH & CH 2) , 3.22 ~ (3H, s, CH3), 3.3-3.8 (2H, m, CH2), 4.0-4.7 (3H, m, ~ CH & CH2), 9.75 ( 1H, s, CO 2 H). Rf: (1) 0,? 6; (2) 0.62.

Example 8 N- [3- (N-cyclohexylcarbonyl-D-phenylalanylthio) -2-D-methylpropanoyl] -L-proline

The procedure of Example 6 was repeated except that N-cyclohexylcarbonyl-D-phenylalanine (1.54 g), carbonyldiimidazole (1.09 g) and N- (3-mercapto-2-ol) were used instead of N-cyclohexylcarbonylglycine. -D-methylpropanoyl) -L-proline (1.09 g). A gummy substance formed (0.97 g) and was found to be identical to the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, 35 S): 1.20 (3H, broad, d, CH 3), 1.2-2.5 (14H, m, CH 2, 2.5-3.3 (6H, m, CH & CH 2) ), 3.3-3.8 ~ (2H, m, OH2), 4.0-5.3 T2H, m, CH), 3i 75804 5.7-6.4 (1H, m, NH), 7 .29 (5H, s, aromatic), 7.5-7.9 (1H, broad s, CO 2 H). Rf: (1) 0.64; (2) 0.82.

Example 9 Ν-ΖΓ3- (N-cyclohexylcarbonyl-D-leucylthio) -2-D-5-methylthiopropanoyl] -L-proline

The procedure of Example 6 was repeated except that N-cyclohexylcarbonyl-D-leucine (1.20 g), carbonyldiimidazole (0.97 g) and N- (3-mercapto- 2-D-methylpropanoyl) -L-10 proline (0.98 g). A gummy substance formed (1.17 g) and was identified as the title compound by NMR analysis and thin layer chromatography.

NMR (CDCl 3, δ): 0.93 (6H, d, CH 3), 1.21 (3H, broad d, CH 3), 1.30-2.5 (17H, m, CH & CH 2), 2, 5-3.3 (4H, m, CH & CH 2), 3.3-3.9 (2H, m, CH 2), 4.0-5.0 (2H, m, CH), 5.95- 6.5 (1H, m, NH), 7.30 (1H, s, CO 2 H). Rf: (1) 0.61; (2) 0.73.

Example 10 N-Z3- (N-cyclohexylcarbonyl-D-tryptophylthio) -2-D-methylpropanoyl-7-L-proline The procedure of Example 6 was repeated except that N-cyclohexylcarbonyl-D-tryptophan was used instead of N-cyclohexylcarbonylglycine. , 75 g), carbonyl diimidazole (1.09 g) and N- (3-mercapto-2-D-methylpropanoyl) -L-proline (1.09 g). A gummy substance formed (0.8 g) and was identified with the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, δ): 1.15 (3H, broad, CH 3), 1.3-2.4 (14H, m, CH 2), 2.5-3.9 (8H, m, CH & CH 2), 4.1-477 (1H, m, CH), 4.7-5.2_ (1H, M, CH), 6.0-6.4 (2H, m, NH), 6.95-7.9 (5H, m, aromatic), 8.80 (1H, 30 broad s, CO 2 H). Rf: (1) 0.58; (2) 0.82.

Example 11 N-Z3- (N-Cyclohexylcarbonyl-D-phenylglycylthio) -2-D-methylpropanoyl-7-L-proline

The procedure of Example 6 was repeated except that N-cyclohexylcarbonyl-D-phenylglycine (1.30 g), carbonyldi-imidazole (0.97 g) and N- (3-mercapto) were used instead of N-cyclo-35-hexylcarbonylglycine. -2-D-methylpropanoyl-1-L-proline (0.98 g). A gummy substance formed (0.35 g) which was found to be identical to the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, δ): δ 1.18 (3H, broad d, CH 3), 1.3-2.5 (14H, m, CH 2), 2.5-3.8 (6H, m, CH & CH 2) ), 4.2-4.7 (1H, m, CH), 5.62- (1H, d, CH), 6.53 (1H, d, NH), 7.37 5H, s, aromatic), 8.90 (1H), s, CO 2 H). Rf: (1) 0.58, (2) 0.83.

Example 12 N- [3- (N-Cyclohexylcarbonyl-D-methionylthio) -2-D-methylpropanoyl] -L-proline

The procedure of Example 6 was repeated except that N-cyclohexylcarbonyl-D-methionine (1.30 g), carbonyldiimidazole-15 (1.09 g) and N- (mercapto-2) were used instead of N-cyclohexylcarbonylglycine. -D-methylpropanoyl) -L-proline (1.09 g). A gummy substance formed (0.59 g) and was found to be identical to the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, δ): 1.23 (3H, broad d, CH 3), 1.5-2.4 (16H, m, CH 2), 2.10 (3H, s, CH 3), 2.59 ( 2H, t, CH 2), 2.7-3.3 (4H, m, CH 1 CH 2), 3.3-3.9 (2H, m, CH 2), 4.0-5.1_ (2H, m , CH), 6.49 (1H, d, NH), 7.81 (1H, s, CO 2 H). Rf: (1) 0.60; (2) 0.77.

Example 13 N- [3- (N-Cyclohexylcarbonyl-D-glutaminylthio) -2-25-D-methylpropanoyl] -L-proline

The procedure of Example 6 was repeated except that N-cyclohexylcarbonyl-D-glutamine (0.5 g), carbonyldiimidazole (0.41 g) and N- (3-mercapto-glycol) were used instead of N-cyclohexylcarbonylglycine. 2-D-methylpropanoyl) -L-pro-30 (0.41 g). A gummy substance formed (0.3 g) and was identified with the title compound by NMR analysis and thin layer chromatography. NMR (CDCl 3, δ): 1 # 20 (3H, broad d, CH 3), 1.3-2.5 (16H, m, CH 2), 2.5-3.2 (6H, m, CH & CH 2) , 3.3-379 (2H, m, CH 2), 4.1-4.9 (2H, m, CH); 5.6-35 7.6 (4h7 m, NH & CO 2 H). Rf: (I) 0.42; (2) 0.48.

33 75804

Example 14 N- [3- (N-Cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoyl] -L-proline dicyclohexylamine salt 5 1) 3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoic acid ( 241 g) was suspended in dry dichloromethane (1.3 liters). Triethylamine (117 ml) was added dropwise to the suspension at -15 ° C with stirring, then pivaloyl chloride (104 ml) was added dropwise at below -5 ° C with stirring, and the mixture was further stirred for 30 minutes.

2) L-proline (101 g) was suspended in dry dichloromethane (1.2 liters). Trimethylsilyl chloride (112 ml) was added dropwise to the suspension at -15 ° C with stirring, 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 resulting mixture was added the solution prepared in (1), and the mixture was stirred under cooling for 15 minutes, then at room temperature for 20 hours. When the reaction was completed, 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. Dicyclohexylamine (326 ml) and acetonitrile (800 ml) were added to the filtrate, and the mixture was stirred for 30 minutes. Four 800 ml portions of acetonitrile were then added every 10 minutes and the resulting mixture was left overnight at 5 ° C. The precipitate was filtered off with suction and dried in air to give the title compound as a crude product. The crude product was dissolved in hot dichloromethane (3 liters) and filtered. The filtrate was stirred at room temperature for 30 minutes and four more 750 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 and air dried to give a colorless portion of the title compound (404 g). Sp. 190-191 ° C, [α] D = -24.5 ° (C = 1.0, MeOH).

34 75804

Example 15 Calcium salt of N-Z-3- (N-cyclohexylcarbonyl-D-alanylthio) -2-D-methylpropanoyl-L-proline 404 g of the dicyclohexylamine-5 salt prepared in Example 14 were suspended in ethyl acetate (2 liters) and 0.5N KHSO 4 was added to the suspension. (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 liters). To the acetone solution were 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 resulting syrup was suspended in acetone (1 liter) and the solution was stirred for 5 hours. The precipitate was filtered off with suction, air dried, then vacuum dried to give the title compound as a white powder (212 g). Δ D = -47.2 ° (C = 1.0, MeOH).

Claims (3)

A process for the preparation of a therapeutically active proline derivative of formula (I) 5 CH-j, - I-3-R-A-S-CH2CHCO-N (I) V 10 COOH (where R is a cyclohexylcarbonyl group attached to the amino group of the amino acid; A is glycine, sarcosine, D-alanine, D-leucine, D-methionine, D-glutamine, D-phenylalanine -, D-tryptophan or D-phenylglycine residue and having an oi-carbonyl group which forms a thiol ester bond with a sulfur atom) or a pharmaceutically acceptable salt thereof, characterized in that a) a compound of formula (II) is obtained -A'-S-CH 2 CHCOOH (II) (wherein A 'is a group A as defined above or a protected derivative thereof; and R is as defined above), in the presence of a carbodiimide or a reactive amide thereof, an acid halide, an activated ester or mixed anhydride to react with a compound of formula (III) HN (IIIv 30 v_UII) d00R '(wherein R' is a hydrogen atom or a carboxyl protecting group) or a derivative containing a silyl group thereof in an inert organic solvent at a temperature between -20 ° C to 10 ° C and remove the reactant or b) introducing a compound of formula (IV) R-A'-OH (IV) 5 (wherein A 'and R are as defined above) in the presence of a carbodiimide, or its reactive amide, acid halide, activated ester or mixed anhydride to react with a compound of formula (V) 10 CH 0 / -1 / HS-CH 2 CHCO-N (V) 15 & Tr. (wherein R 'is as defined above) in an inert organic solvent at a temperature in the range of -20 ° C to 10 ° C, and any protecting groups in the reaction product are removed and the product obtained by method a) or method b) is optionally made pharmaceutically acceptable. salt. 37 75804 For the preparation of therapeutically active proliferative compounds of formula (I) 5 CH-j, 3 I 3 / RAS-CH9CHCO-N (I) y COOH 10 (color R is not an amino-trans-amino group in cyclo- hexylcarbonyl, glycine, sarcosine, D-alanine, D-leucine, D-methionine, D-glutamine, D-phenylalanine, D-tryptophan or D-phenylglycine (-carbonyl group, 15 waxes and thiol esters with a drug or a pharmaceutical form of the site of action, in the case of a compound, in the form of (a)) 20 CH3 IJ R-A'-S-CH2CHCOOH (II) (color A 'is a group A which is defined as a compound or a compound of the same name) i nervvaro 25 av carbodiimide, eller reactiv amide, cyanohalides, Akti -ester Esters or mixtures of anhydrides with a mixture of formula (III) HN (III) 30 \ _ CÖOR '(color R' is a hydrogen atom or a group of carboxyl groups) or with a derivative of a silyl group, 35 and inert organic heating medium at a temperature of -20 ° C to 10 ° C and a reaction temperature of