CA1235120A - Proline derivatives and process for producing the same - Google Patents

Abstract

Abstract of the Disclosure Proline derivatives of the formula (I):

Description

Lo o PRALINE DERIVATIVES AND PROCESS FOR
PRODUCING THE SAME

The present invention relates to praline derivatives of the formula (I):
SHEA
R-A-S-CH2CHCO-N (I) COO
(wherein is an azalea group bonded to the Amman group of amino acid and is selected from the group consisting of cycle-propanecarbonyl, cyclohexanecarbonyl and adamantanecarbonyl groups; A is Gleason, sarcosine or Damon acid residue and has an ~-carbonyl group that forms a they'll ester bond with a sulfur atom) and pharmaceutically acceptable salts thereof.
Compounds similar to these compounds represented by formula (I), for example, those wherein R is a bouncily, acutely or t-butyloxycarbonyl group and A is Lyman acid are described in U.S. Patent Application 2,050,3sgA~ European Patent Application (REP) Allah describes compounds wherein R is a bouncily, acutely, t-butyloxycarbonyl, cyclopentane-carbonyl group or the like, and A is Lyman acid. EN
Allah describes compounds wherein R is a bouncily group and A is a D-phenylalanyl group. But there is no prior art refer-once that specifically describes the compounds represented by formula (I). Therefore, they are undocumented novel compounds and are useful as pharmaceuticals as will be described hereunder.
The amino acid residue represented by A in formula (I) ,.,

- 2 _ ~351~

is Gleason, sarcosine or Damon acid residue. Illustrative Damon acids include neutral, acidic or basic amino acids, or aliphatic, aromatic, heterocyclic or alicyclic amino acids such as D-alanine, D-leucine, D-asparagine, D-methionine, D-glutamin~, D-phenylalanine, D-tryptophan, D-ornithine, D-phenylglycine, D-threonine, D-glutamic acid, D-arginine, D-cysteine, D-aspartic acid D-histidine, D-isoleucine, D-praline, D-lysine, D-serine, D-tyrosine, and D-valine. Fun-tonal groups in these amino acts such as hydroxyl, Marquette, amino and carboxyl groups may be substituted by lower alkyd, bouncily and lower alkanoyl groups. Preferred examples of A
include Gleason, sarcosine, D-alanine, D-leucine, D-methionine, D-glutamine, D-phenylalanine, D-tryptophan and D-phenylglycine.
Thy group SHEA in structural formula (I)- has an asymmetric carbon atom, so that the group may be in a D-form, L-form or Deform i.e. a mixture of D- and L-forms. Either form is included in the scope of the present invention, and D- and Deforms are preferred for their biological activity.

The praline residue -No in formula (I) may be in a OH
D-form, L-form or a Deform and either form is included in the scope of the present invention, L- and Deform being preferred for their biological activity.
Illustrative pharmaceutically acceptable salts of the praline derivative of formula (I) include salts with alkali metals such as sodium and potassium, salts with alkaline

- 3 - ~235120 earth metals such as calcium and magnesium, and salts with basic amino acids such as arginine and Lawson. Calcium salts and Lawson salts are preferred.
The present invention also relates to a process for producing the praline derivatives of formula (I) or pharmacy-tidally acceptable salts thereof. The compounds of formula (It are produced by the following process:
A compound of formula (II):

SHEA
¦ (II) R-A'-S-CH2CHCOOH
(wherein A' is Gleason r sarcosine or a protected or unprotected Damon acid residue; R has the same meaning as defined above) or a reactive derivative thereof is reacted with a compound of formula (III~:

HO
(III) COORS

(wherein R' is a hydrogen atom or a carboxyl-protecting group) or a reactive derivative thereof, any protecting group is removed from the reaction product, and the product is option-ally converted to a pharmaceutically acceptable salt.
If A in formula (I) has a free functional group that should not enter into the reaction such as a Marquette, hydroxyl, amino or carboxyl group, the compound of formula (II) means I a derivative wherein such group may be protected.
Any functional group in A' that should not enter into the reaction may be protected by those groups which are convent tonally used in the art of peptize synthesis and which can be _ 4 _ ~35~20 removed under relatively mild conditions. For example, a Marquette group can be protected by an aralkyl group such as tritely, bouncily or p-methoxybenzyl; a hydroxyl group can be protected by a bouncily group; an amino group can be protected 5 by a t-butyloxycarbonyl group; and a carboxyl group can be protected by a t-butyl group. These protecting groups can be removed by treating with hydrogen fluoride, trifluoroacetic acid or hydrogen chloride. Alternatively, a carboxyl group is protected by a loweralkyl-substituted sill group, such as trimethylsilyl group, which is then removed by treatment with water. When R' in formula (III) is a carboxyl-protecting group, it may be a t-butyl group or a loweralkyl-substituted sill group and can be removed by treatment with hydrogen fluoride, trifluoroacetic acid, hydrogen chloride or water.
The reactive derivative of the compound of formula IT
is such that the carboxyl group that enters into the reaction is activated. The carboxyl group can be activated into any of the forms commonly employed in the art of peptize synthesis such as activated asides, acid halides, activated esters and mixed acid androids. Among these forms, activated esters with N-hydroxysuccinimide, mixed acid androids with carbonic acid monstrous, and activated asides with carbonyldiimidazole are preferred. Carbodiimides such as dicyclohexylcarbodiimide may be used as a condensing agent to form an aside bond between the carboxyl group and amino group.
The reactive derivative of the compound of formula (III) is such that the amino group of said compound is activated.
The amino group can be activated by any of the methods that . ,.

_ 5 _ ~35~

are conventionally used in the art OX peptize synthesis, for example, the method of introducing a sill group such as trimethylsilyl group, the "phosphazo method" using a phosphorus compound such as phosphorus trichloride [Ann. Chum., 572 96 (1951)], the phosphorous acid ester method using a phosphorous acid ester such as tetraethyl ester of pyrophosphorous acid, or the "N-carboxyanhydride method" INCA method).
The reaction can be performed in an inert organic solvent such as tetrahydrofuran, Dixon, dimethylformamide, hexamethyl-phosphotriamide, chloroform, dichloromethane or acetonitrile. The reaction is generally performed under cooling or at room temperature (-50 to Z0C), preferably from -30 to 10C. The reaction period 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. -I
After the aside formation reaction, any protecting group in the reaction product is removed by any of the methods specified above for the respective protecting groups.
The end compound can be isolated from the reaction mixture and purified by any conventional method, for example, by various types of chromatography using silica gel r dextran cross linked polymer and porous polymers such as styrenes divinylbenzene or acrylic acid esters. A suitable developing solvent can be selected from among chloroform, ethyl acetate, methanol, ethanol, tetrahydrofuran, Bunsen, water and asset-nitrite. Alternatively, the end compound may be obtained by first isolating the reaction product in the form of an organic , ....~

- 6 - ~35120 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 on the praline site, so it is capable of forming salts with various basic materials if desired. Salts with forum-ceutically acceptable basic materials are particularly import lent. Such salts can be prepared by a conventional method, i.e. 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 compound of formula (II) used as the starting material in the process of the present invention can be pro-pared easily by the following method:
A compound of formula (VI):
R-OH (VI) (wherein R is the same as defined above) or a reactive derive-live 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 COO; R" is a hydrogen atom or a carboxyl-protecting group) or a reactive derivative thereof, followed by removing R" if it is a protecting group, to form a compound of formula (VIII):
R-NH-A"-COOH (VIII) (wherein R and A" are the same as defined above), subsequently the compound of (VIII) or a reactive derivative thereof is , - 7 - ~3~1~0 reacted with a compound of formula (IX):

SHEA
¦ (IX) HS-C~2CHCOOR"' (wherein R"' is a hydrogen atom or a carboxyl-protecting group), followed by removing R"' if it is a protecting group.
Examples of the reactive derivative in the carboxyl group of the compound of formula (VI) include those which are commonly employed in the art of peptize synthesis, such as activated asides, acid halides, activated esters, and mixed acid androids.
If R" in the amino acid of formula (VII) is a protecting group, advantageous examples include a t-butyl group (which can be removed by hydrogen fluoride, trifluroacetic acid or hydrogen chloride), a bouncily group (which can be removed by catalytic reduction with palladium or the like), and lower alkyd 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 derivative is produced by any of the activating methods that are conventionally used in the art of peptize synthesis, such as introducing a sill group (e.g. trimethylsilyl group), the "phosphazo method", the phosphorous acid ester method, and N-carboxyanhydride method.
A carbodiimide such as dicyclohexylcarbodiimide may be used as a condensing agent to form an aside bond between the compound of formula (VI) and the amino acid of formula (VII).

The reaction can be performed in an inert organic solvent such as tetrahydrofuran, Dixon, dimethylformamide, hexamethyl-phosphotriamide, chloroform, dichloromethane or acetonitrile.
The reaction is generally performed under cooling or at room 5 temperature (-50 to 20C), preferably from -30 to Luke. The reaction period 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" in 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, for example, by recrystallization from ethyl acetate, Nixon, acetone or water, or by various types of chromatography as are described in connection with isolation and purification of the compound of formula (I), or by decomposing an organic salt of the reaction product with an acid.
the compound of formula (VIII) may be reacted with the compound of formula (IX) by reacting the carboxyl group in the former compound with the Marquette group in the latter compound in the presence of a condensing agent (i.e. carbide-immediacy such as dicyclohexylcarbodiimide). A reactive derive-live of the compound ox formula (VIII) may be reacted with the compound of formula tip). Examples of the reactive derivative of the compound of formula (VIII) include activated asides, acid halides, activated esters and mixed acid ashy-drives. Activated asides with carbonyldiimidazole are .

9- ~23~2~
preferred.
The carboxyl group in the compound of formula (IX) may or may not be protected. Preferred protecting groups are t-butyl and other groups which can be removed under acidic conditions.
The they'll ester forming reaction is performed in an inert organic solvent such as tetrahydrofuran, Dixon, dip -methylformamide, hexamethylphosphotriamide, chloroform, dip chloromethane and acetonitrile. The reaction is generally performed under cooling or at room temperature (-50 to 20C), preferably at between -30 and 10C when the compound of formula (VIII) is used in the form of an activated aside or acid halide, and at between -10 and 10C when said compound is used in the form of an acid android. The reaction period varies with the reaction temperature, the compounds to be reacted and the solvent, and it is generally between OHS and 48 hours, preferably between 1 and 6 hours. After the they'll ester forming 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 end compound 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 Nixon, various types of chromatography as are described in connection with isolation and purification of the compound of formula I), or by decomposing an organic ,,,,:

3512~

salt of the reaction product with an acid.
nether process for producing the end compound of the present invention comprises reacting a compound of formula (IV):
ROY (IV) (wherein R and A' are the same as define above) or a reactive derivative thereof with a compound of formula (V):

SHEA

HS-CH2CHCO-N~ TV) COORS

(wherein R' is the same as defined above), removing any protecting group from the reaction product, and optionally converting the product to a pharmaceutically acceptable salt.
If A in formula (I) has a free functional group that should not enter into the reaction such as a Marquette, hydroxyl, amino or earboxyl group, the compound of formula (IV) means a derivative wherein such group may be protected.
Any functional group in A' that should not enter into the reaction is protected by those groups which are eonven-tonally used in the art of peptize synthesis and which can be removed under relatively mild conditions For example, a Marquette group can be protected by an aralkyl group such as tritely, bouncily or p-methoxybenzyl group; a hydroxyl group can be protected by a bouncily group; an amino group can be protected by a t-butyloxycarbonyl group; and a carboxyl group can be protected by a t-butyl group. These groups can be removed by treatment with hydrogen fluoride or hydrogen chloride.

L;23S120 Alternatively, a carboxyl group is protected by a lower alkyd=
substituted sill group, such as 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 sill group and can 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 that enters into the reaction is activated. The carboxyl group can be activated into a suitable form such as activated asides, acid halides, activated esters or mixed acid androids. Activated asides with carbon nyldiimidazole are preferred. Carbodiimides such as duskily-By carbodiimide may be used as a condensing agent to form an aside bond from the carboxyl group and Marquette group.
The reaction between the compound of formula (IV) or its reactive derivative and the compound of formula (V) is performed in an inert organic solvent such as tetrahydrofuran, Dixon, dimethylformamide, hexamethylphosphotriamide, sheller-form, dichloromethane or acetonitrile. The reaction is generally performed under cooling or at room temperature t-S0 to 20C), and preferably at between -30 and 10C when the compound of formula (IV) is used in the form of an activated aside or acid halide, and at between -10 and 10C when said compound is used in the form of an acid android. The reaction period 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.

- 12 - ~3~1~0 After the they'll ester forming reaction, any protecting group is removed from the reaction product by any of the methods specified for the respective protecting groups.
The end 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 cross linked polymer and porous polymers like styrene-divinylbenzene or acrylic acid esters. A suitable developing solvent can be selected from among chloroform, ethyl acetate, methanol, ethanol, tetrahydrofuran, Bunsen, water and acetonitrile. Alternatively, the end compound may be obtained by first isolating the reaction product in the form of an organic salt such as dicyclohexylamine salt which is then treated with an acid such as hydrochloric acid or potassium hydrogen sulfate.
The end compounds of the present invention, namely the praline derivative of formula (I) and pharmaceutically accept-able salts thereof, prevent the production of angiotensin II
from angiotensin I by inhibiting the activity of angiotensin converting enzyme. Therefore, they are useful for treating hypertension due to angiotensin II and as an agent to treat cardiac insufficiency.
The activity of some of the end compounds of the present invention to inhibit the angiotensin converting enzyme was measured.
(1) Method An angiotensin converting enzyme was extracted from a rabbit lung. A O.lllM boric acid-Na2CO3 buffer solution - 1 3 - ~:3~20 (pi 8.3, 0.6 ml), 0.2 ml of O.lllM boric acidic buffer solution pi 8.3) containing 25 my benzoylglycylhistidyl Lawson (substrate) and 0.1 ml of O.lllM boric acid-Na2CO3 buffer solution (pi 8.3) containing 10 8 to 10 3 M of test S compounds (13 end compounds of the present invention indicated in the table below) were put in test tubes and preincubated at 37C for 5 to 10 minutes. Then, 0.1 ml of a solution of the enzyme (acetone powder) was added to each test tube and each mixture was incubated at 37C for 30 minutes. The bouncily-Gleason produced by the aid of the enzyme was extracted with ethyl acetate in the presence of hydrochloric acid and its amount was determined by W absorption at 228 no. The enzyme activity in the presence of the test compounds relative to that 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 referred to as the activity of each test compound to inhibit the activity of the enzyme and was indicated as an Is value.
(2) Results 14 51~

Test compound I50 Compound No. 1 1.6 x 10 7 " No. 2 4.8 x 10 7 " No. 3 1.3 x 10~7 " No. 4 4.0 x 10~7 " No. 5 1.8 x 10~
" No. 6 2.4 x 10 7 " No. 7 1.0 x 10~7 " No. 8 1.5 x 10~7 " No. 9 1.7 x 10 7 " No. 10 4.8 x 10~8 " No. 11 1.7 x 10~7 " No. 12 5.4 x 10-7 " No. 13 4.4 x 10~7 Compound No. 1 : N-[3-(N-cyclohexanecarbonyl-D-alanylthio)-2~D-methylpropanoyl]-L-proline No. 2 ; N~[3~(N~cyclopropanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline No. 3 ; N-[3-(N~cyclohexanecarbonylglycylthio)-2-D-methylpropanoyl]~L~proline No. 4 : N~[3~(N~cyclohexanecarbonyl~N-methylglycyl-thio)-2~D-methylpropanoyl]-L-proline No. 5 : N-[3-(N-cyclopropanecarbonylglycylthio) -2-D~methylpropanoyl]~L-proline to No. 6 : N~[3~(N~adamantanecarbonylglycylthio) -2- D-methylpropanoyl]~L~proline - 15 - 1~35~20 Compound No. 7 : N-[3-(N-cyclohexanecarbonyl-D-phenylalanyl-thio)-2-D-methylpropanoyl]-L-proline No. 8 : N-[3-(N-cyclohexanecarbonyl-D-leucylthio)-2-D-methylpropanoyl~-L-proline No. 9 : N-[3-(N-cyclohexanecarbonyl-D-tryptophylthio~-2-D-methylpropanoyl]-L-proline No. 10 : N-[3-(N-cyclohexanecarbonyl-D-phenylglycyl- -thio)-2-D-methylpropanoyl]-L-proline No. 11 : N-[3-(N-cyclohexanecarbonyl-D-methionylthio)-2-D-methylpropanoyl]-~-proline No. 12 : N-[3-(N-cyclohexanecarbonyl-D-glutaminylthio~-2-D-methylpropanoyl]-L-proline c~c/a~c~7c~ne awaken/
A No. 13 : NUN ~-D-alanylthio)-2-D-methylpropanoyl]-L-proline The activity of the end compounds of the present invent lion lasts longer than known compounds having a similar activity r say, 3-mercapto-2-D-methylpropanoyl-L-proline (conventionally named Captopril), so the desired control of blood pressure can be attained by fewer daily administrations. Captopril and other known compounds cause a sudden drop in blood pressure in the initial period of administration and this may develop into orthostatic hypotensive asthenia looniest, vol. 1, No. 8, 115, p. 557 (March 10, 1979)], but the end compounds of the present invention have only a mild hypotensive effect in the initial period of administration and have little chance to develop into orthostatic hypotensive asthenia. Drugs such as Captopril that have a free Marquette group cause various side effects that are attributable to that Marquette group.
To name a few, disorder in the sense of taste, production of albumin in urine, agranulocytosis and dermal diseases accom-panted by fever have been reported in Lance, vol. 1, No. 8160, p. 150 (January 19, 1980), swooper, vol. 2, No. 8186, p. 129 (July 19, 1980), and South African Medical Journal, vol. 58, 172 (1980). On the other hand, the they'll ester bond in the end compounds of the present invention is insusceptible to hydrolysis in viva and hence is little likely to produce a Marquette group. Therefore, the end compounds of the present invention have little chance to cause the above named side effects attributable to the Marquette group.
The praline derivative of formula (I) and its pharmacy-tidally acceptable salts may be formulated in compositions for oral administration such as tablets, capsules, granules, powders, syrups and elixirs, or in sterile solutions or suspensions for parenteral administration. For this purpose, pharmaceutical compositions can be prepared from one or more-of the end of compounds of the present invention (as the active ingredient) and pharmaceutically acceptable adjutants such as excipients, carriers, binders, stabilizers and flavors. The daily dose of the end compounds of the present invention for adults is from 0.5 my to 2 g, preferably from about 1 to 500 my, for oral administration, and from 0.1 to 600 my, preferably 25 from about 0.3 to 300 my, for parenteral administration.
The process for preparing the end compounds of the present invention is now described by reference to the following examples to which the invention is by no means limited.

- 17 - 123S12~

Example l: Preparation of N-substituted amino acids (a) D-alanine (4.5 g) was dissolved in 230 ml of aqueous lN-Na2CO3 under stirring. To the solution, 100 ml of twitter-hydrofuran containing 9.0 g of cyclohexanecarbonylchloride was added drops at 5 - 10C, at which temperature the mixture was stirred for 30 minutes, then stirred at room temperature for 1.5 hours. Thereafter, 2N-HCl was added to the reaction mixture to adjust its pi to between l and 2. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated Nail solution and dried over magnesium sulfate. The filtrate was evaporated under vacuum to obtain a crude compound. Recrystallization from ethyl acetate/n-hexane gave 4.65 g of N-cyclohexanecarbonyl-D-ala nine. Do = +26.6 (b) An ester of adamantanecarboxylic acid and N-hydroxy-succinimide ~3.0 g) was dissolved in 30 ml of tetrahydrofuran.
To the solution, water (5 ml) containing 0.89 g of D-alanine and 1.1 g of triethylamine was added and the mixture was stirred at 5C overnight. After removal of tetrahydrofuran, water was added to the residue, and 2N-HCl was further added to the mixture to adjust its pi to between l and 2. The mixture was subsequently treated as in (a) above to give 0.38 g of N-adamantanecarbonyl-D-alanine. Do = +11.6 (C=1.0 Mesh) The compounds listed below were prepared by either of the methods described in (a) and (b).

..-- 18 - ~L23~;120 _ o Us O O JO In ._ O
a ox $ $ $ o O $ Al $ I
-I o Us o o ox o o o . o o Us u coy o us a) I x I+ I I I + O I
o 11 o 11 o 11 owe o 11 o 11 o 11 o I; _ Q h Pi z, ED no 2~1 a z I

h h h h h h h h I O ' O O O O O O O O
,1 h h h h h So h h o Jo Jo AL: Jo US Jo I Jo Jo Jo O O h lo h (a h Id h do (US h lo h h us O h C) ho h C) h Q) h a) h h Q) I 0 h I 3~3~ I I I I 3 I

I to I o Al R O O O O O O R O
u) 1:: h R Q R R R h R
Al a h h h h h ) h u O
X
x x X a) X ax x a) x o a X Q) h O O O I a a) 'JO O 'I a) h h I Al rJ.C rip S I arc Al or O h O h O h O h O h O h O S-l . O h I O I 0 I 0r~l O Al O Al O Al O Al O
U Al O I U I I I 0 I r.) I
I_ _ __ _ _ __. _ pa o ) S; Q Q R Q R Q

US Al .4 h h - h h h h h h . 11~
V Jo U
O Ox I
us o a OX X axe ax X OX l OX
I O So O
Z (I a) o to Oriole O O I 0-~1 0 0 I 1~-1 0 o O U Id Urn I h I Al I
U
I Jo I II I I I I I I I I I I I I
Z znz;~ I ZCI zap Zoo z~3 Z
.

- lug - 123~2 Us Us Al -I O X
O o Al h I
Us O I O I I:
_ "-I ` 3 --I I
O p;, ,_ h ; I O ` I I I o I o o I or _ 1:: Us h h I O O
Al C h h Jo O
V
So `.
Sol h a h O
us Q h h I
O
X O O
h I -I I
O I 0 h U
I
_ _ .

wreck O 'd Q I
C) h no a Us O
R I Pi Al X O
Us h Z O'er o O " 'I
I I 1 ,1 Z

- 20 - ~35~0 Example 2 (a)3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-methHal--propanolc acid N-Cyclohexanecarbonyl-D-alanine (5.97 g) was dissolved in dry tetrahydrofuran (100 ml). To the solution, carbonyl-diimidazole ~5.84 g) was added at between -20 and -15C, and the mixture was stirred at that temperature for one hour.
Thereafter, 3-mercapto-2-D-methylpropanoic acid (3.60 g) was added, and the mixture was stirred at between -15 and -10C
for one hour, then at room temperature for another owe hour.
The mixture was evaporated under vacuum to remove the solvent.
To the residue, water (40 ml) was added, and 2N-HC1 was added to the mixture to adjust its pi to between 1 and 2. The mix-lure was extracted with ethyl acetate, and the organic layer was washed with saturated Nail solution twice, and dried over magnesium sulfate. The filtrate was evaporated under vacuum to obtain a crude compound. Recrystallization from ethyl acetate/n-hexane gave a colorless prismatic crystal of the titled compound (7.50 g, 83%).
mop. 149 - 152C
Do = +46.4 (C=1.07, Mesh) NOR (CD30D, I): 1.20 (OH, d), 1.35 (OH, d), 1.20 - 2.0 (llH, m), 2.40 - 2.80 (lo, m), 3.05 (OH, m), 4.50 (lo, m) (b) 3-(N-Cyclohexanecarbonyl-D-phenylglycylthio)-2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclo-hexanecarbonyl-D-phenylglycine (2.61 g), car~onyldiimidazole (1.95 go and 3-mercapto-2-D-methylpropanoic acid (1.20 g).

, .

- 21 - ~235120 A yellow oil of the titled compound was produced t2.07 g, 57~).
Do = -48.7 Clue Mesh) NOR (CDC13, I): 1.24 (OH, d), 1.20 - 2.10 (llH, m), 2.40 - 2.80 (lo, m), 3.10 (OH, m), 5.75 (lo, d), Jo 6.40 - 6.80 (lo, m), I (OH, S), 8.90 (lo, S) (c) 3-(N-Cyclohexanecarbonyl-D-leucylthio)-2-D-methyl---propanoic acid The procedure of pa) was reseated using N-cyclohexane-carbonyl-D-leucine (2.41 g), carbonyldiimidazole (1.95 g) and 3-mercapto-2-D-methylpropanoic acid (1.20 g). An oil of the titled compound was produced (2.40 g, 70%).
Do = +12.2 (C=1.03, Mesh) NOR (CDC13, I): 0.95 (OH, d), 1.28 (OH, d), 1.20 - 2.10 (llH, m), 2.40 - 2.80 (lo, m), 3.10 (OH, m), 4.70 (lo, m), 6.00 - 6.30 (lo, m), 9.20 (lo, S) (d) 3-(N-Cyclohexanecarbonyl-D-glutaminylthio)-2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclo-hexanecarbonyl-D-glutamine (2.56 g), carbonyldiimidazole (1.95 g) and 3-mercapto-2-D-methylpropanoic acid (1.20 g).
A 0.70 g sample of the titled compound was produced (20%).
mop. 146 - 149C
Do = +10.8 ~C=1.05, Mesh) NOR (CD30D, I): 1.20 (OH, d), 1.20 - 2.10 (llH, m), 2.30 (OH, m), 2.40 - 2.80 (lo, my, 3.05 (OH, m),

4.50 (lo, m) (e) 3-(N-Cyclopropanecarbonyl-glycylthio)-2-D-methyl-propanoic acid it The procedure of (a) above was repeated using N-cyclo-propanecarbonylglycine (0.78 g), carbonyldiimidazole (1.06 g) and 3-mercapto-2-D-methylpropanoic acid (0.66 g). An oil of the titled compound was produced (0.70 g, 52~).
[rid = -33.8 (C=1.01, Mesh) NOR (CDC13, I): 0.70 - 1.20 (OH, m), 1.25 (OH, d), 1.30 - 1.80 (lo, m), 2.40 - 2.80 (lo, m), 3.14 I m), 4.20 (OH, d), 7.30 (lo, S) (f) 3-(N-Adamantanecarbonyl-glycylthio)-2-D-methylproppanic acid The procedure of (a) above was repeated using N-adamantanecarbonylglycine (2.38 g), carbonyldiimidazole (1.95 g) and 3-mercapto-2-D-methylpropanoic acid (1.20 g). A 1.08 g sample of the titled compound was produced (32~).
mop. 132C (doe.) Do = -8.3 (C=1.01, MOE) NOR (CD30D, I): 1.25 (OH, d), 1.65 - 2.30 (15H, m), 2.40 - 2.80 (lo, m), 3.14 (OH, m), 4.06 (OH, d) (g) 3-(N-Cyclohexanecarbonyl-D-phenylalanylthio)-2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclo-hexanecarbonyl-D-phenylalanine ~1.37 g), carbonyldiimidazole (1.0 g) and 3-mercapto-2-D-methylpropanoic acid (0.60 g).
An oil of the titled compound was produced (1.5 g, 79%).
[ED = +13-9 (C=1.02, Mesh) NOR (CDC13, I): 1.25 (OH, d), 1.20 - 2.0 (lo, m), 2.1 - 3.0 (OH, m), 3.0 - 3.4 (OH, m), 4.95 (lo, t), 6.19 (lo, do, 7.24 (OH, S), 10.12 (lo, S), - 23 - ~3512~

(h) 3-(N-Cyclohexanecarbonyl-D-methionylthio)-2-D-methylpropanoic acid The procedure of (a) was repeater using N-cyclohexane-carbonyl-D-methionine (0.325 g), car~onyldiimidazole (0.25 g) and 3-mercapto-2-D-methylpropanoic acid (0.15 g). An oil of the titled compound was produced (0.29 g, 64~).
Do = ~7-9 (C=1.0, Mesh) NOR (CDC13, I): 1.26 (OH, do, 1.2 - 2.0 (lo, m), 2.09 (OH, S), 2.0 - 3.2 (OH, m), 4.5 - 5.0 (lo, my, 6.62 (lo, d), 10.07 (lo, S) (i) 3-(N-Cyclohexanecarbonyl-D-tryptophylthio)-2-D-methylpropanoic acid The procedure of (a) above was repeated using N-cyclo-hexanecarbonyl-D-tryptophan (0.157 g), carbonyldiimidazole (0.1 g) and 3 mercapto-2-D-methylpropanoic acid (0.06 g).
A gummy sample of the titled compound was produced (0.17 g, 82%).
Do = -7.1 (C=1.0, Mesh) NOR (CDC13, I): 1.26 (OH, d), 1.2 - 2.4 (llH, m), 2.5 - 2.9 (lo, m), 3.0 - 3.3 (OH, m), 3.35 (OH, d), 4.87 (lo, t), 6.4 - 6.9 (OH, broad S), 6.9 - 7.8 (OH, m), 10.5 (lo, S) Example 3 (a) N-[3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline 3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-methyl---propanoic acid (1.51 g) was dissolved in dry tetrahydrofuran (40 ml). To the solution, triethylamine (0.61 g) and ethyl chloroform ate (0.65 g) were added at -5C under stirring.
Five minutes later, a solution having L-proline (0.58 g) and I.

- 24 _ ~23 So O

triethylamine (0.61 g) dissolved in water (5 ml) was added, and the mixture was stirred at 0C for one hour, then at room temperature for 30 minutes. The mixture was evaporated under vacuum to remove the solvent. After adding water to the residue, 2N-HCl was added to the mixture to adjust its pi to between 1 and 2. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated -Nail solution twice, and dried over magnesium sulfate. The filtrate was concentrated under vacuum to remove ethyl acetate, and the residue was subjected to chromatography on silica gel using a mixture of chloroform and methanol (100;1 to 100:2) as an eluant. Fractions containing the end compound were collected and evaporated under vacuum to give a gummy sample of the titled compound (0.3 go.
(b) The same compound could be produced by the following method.
3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-meth~l---propanoic acid (6.03 g) and N-hydroxysuccinimide (2.3 g) were dissolved in dry tetrahydrofuran (50 ml). To the solution, dicyclohexylcarbodiimide (4.3 g) was added at between 0 and 5C, and the mixture was stirred overnight at that temperature.
After the reaction, the precipitate was filtered off and the insoluble matter was washed with a small amount of twitter-hydrofuran. The filtrate and the washings were combined and CC~ f7C e r7z~ra~
cvapor~tc~ under vacuum. To the residue, ethyl acetate was added, and the mixture was filtered. The ethyl acetate soul-lion was washed with 0.5N Hal, water, aqueous Nikko and saturated Nail solution in the order mentioned. The solution ~2~5~20 was dried over magnesium sulfate, evaporated in vacuum, and the residue solidified upon addition of a mixture of ethyl acetate and hexane ~1:10). The yield of the solid product was 6.90 g (87%).
mop. 113 - 116C
Do = +14.2 (C=1.05, Mesh) NOR (CDC13, I): 1.38 (OH, d), 1.20 - 2.20 (llH, m), 2.82 (OH, S), 2.8 - 3.30 (OH, m), 4.75 (lo, m), 6.30 (lo, broad d) The activated ester (3.98 g) was dissolved in twitter-hydrofuran (40 ml). To the solution, water (5 ml) having L-proline (1.15 g) dissolved therein and N-ethylmorpholine (1.26 ml) were added, and the mixture was stirred overnight.
The mixture was evaporated under vacuum to remove the solvents, and water was added to the residue. To the mixture, 2N-HCl was added to adjust its pi to between 1 and 2. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated Nail solution and dried over magnesium sulfate. The filtrate was evaporated under vacuum.
The residue was subjected to column chromatography on silica gel using a mixture of chloroform and methanol (100:1 to 100:2) as an eluant. The product obtained by evaporating the elude (1.45 g) under vacuum was found to be identical to the titled compound by NOR analysis.
(c) The titled compound could also be produced by the following method.
3-tN-cyclohexanecarbonyl-D-alanylthio)-2-D-meth propanoic acid (4.52 g) was dissolved in thionylchloride , "by .

- 26 - ~35~20 (S ml). To the solution, a drop of dimethylformamide was added and the mixture was stirred at room temperature over-night. The mixture was evaporated under vacuum to remove the solvents, and to the residue, dry Tulane (2 ml) was added, and the mixture was again evaporated under vacuum to remove the solvent. To the residue, dry tetrahydrofuran 110 ml) was added under stirring. The tetrahydrofuran solution containing the acid chloride was added drops at a temperature between

5 and 10C under stirring to water (25 ml) having praline (2.3 g) and Nikko (2.5 g) dissolved therein. The mixture was stirred at that temperature for one hour, and at room temperature for two more hours. The reaction mixture was subjected to extraction with chloroform. To the aqueous layer, ON Hal was added to adjust its pi to between l and 2.
After extraction with chloroform, the chloroform layer was washed with saturated Nail solution twice, and dried over magnesium sulfate. After removal of chloroform, the residue was subjected to column chromatography on silica gel using a mixture of chloroform and methanol (100:1 to 100:2) as an eluant. Upon evaporation under vacuum, a gummy substance (1.6 g) was obtained. That substance was found to be identical with the titled compound by NOR analysis.
(d) The titled compound could be produced by yet another method that is described below.
3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-methyl---propanoic acid (3.01 g) and t-butyl ester of L-proline (1.71 g) were dissolved in dry dichloromethane (50 ml), and duskily-hexylcarbodiimide (2.15 g) was added to the solution under ~;~3~120 stirring and cooling with ice. The mixture was stirred at Ct~q A the same temperature for half Lowry and left overnight at 5C. The reaction mixture was filtered and the insoluble matter was washed with dichloromethane. The filtrate and the washings were combined and washed with lo Hal, water, lo Nikko and saturated Nail solution in the order mentioned, dried over McCoy and evaporated under vacuum to provide a gummy sample of t-butyl ester of N-[3-(N-cyclohexanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline (4.3 g). A 4.0 g sample of the ester was dissolved in anisole (30 ml), and trifluoroacetic acid (10 ml) was added to the solution. The mixture was stirred at room temperature for one hour and evaporated under vacuum 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 sheller-form (1:100 to 3:100) as an eluant. Fractions containing the end compound were collected and evaporated under vacuum to obtain a gummy substance (3.4 g). That substance was found to be identical with the titled compound by NOR and thin-layer chromatography.
The reactants, reaction solvents and the methods used for producing the titled compound in Example 3 are listed in the following table together with data on its physical prop-reties. Other compounds were prepared by using one of the methods described in Example 3, (a) to (d), and particulars of the specific method are listed in the following table together with data on the physical properties of the end compounds. In the table, the figures following numbers (1) ~23~;~20 and (2) in the column of "Of value" indicate the Of values obtained in thin-layer chromatography using CHC13/MeOH/AcOH
(2:1:0.003) and n-BuOH/AcOH/H20 (4:1:1) as eluants, respectively.

~:353L~) _ r-t O I N
O O O O O O
(I (I I (I (I
I.
I I I I O

En 1 .. 3 h I è
_ Jo O

I. Lo I O Us . 1 I (I Lo (~) CO I or Lo) I
__~ , . . . , _ Jo . , , _ _, , , . _ .
Jo I) r ) Lo I ¦ to (~) I (I D I =
11~ or o o I ') Jo o I o o Lo O I Lo I

o Us_ O O_ I Ox 'I

by $ so v pa ted a ;

_ 30 - 5120 _ .
Jo r I_ I Jo 1- . ED n CUD Us CO
o o o o o o o N l-- O V --I

Eye ._ OX `^~
X X ZOO I
,,~ 1 o co o us ,1 ox or O us co Jo ` I Jo I I 00 ` CO
.
us ox o I ox I us I

k I MU $

;
_ _ I i - 31 _ ~235j~
_ _ _ . ox ox ox I
I 1 '`'1~1 I ` c l 1 Mel g 8 8 -o 0 - - . . . go - - . . - . . - . - . . . .. -I o I I I n o I I I I I I
a In o or o 1` ED or I a elm o I I Lowe In I I I I o I I I I I ox I_ or Jo . co o us lo b b ..

_ id â "
~'"~ ' ',~
-- 32 - ~L235~1LZO
__ .
o Lnl~
(I ) (I ) (~) ` - I _ I

_ _ 3 ON¦ 1 1 I I

, ,. m _ Q m m m m u, 2 Q m m c m G CO O O I _ o D
O
r ED Us o I or Lo) .
a O O O O O O
on I` l I}

2 #

_' .
:, .

33 lX3~Z

Example 4 Various salts were formed from the samples of NUN

Acyclohexanecarbonyl-D-alanylthio)-2-D-methyl~rcp~Noah -L-proline prepared in Examples 3 and 5.
(a) Cay salt A 3.98 g sample of the compound prepared in Example 3 or 5 was dissolved in methanol (40 ml). To the solution, calcium acetate hydrate (0.84 g) was added, and the mixture was heated under reflex for one hour. The insoluble matter was filtered off and the filtrate was evaporated under vacuum.
To the residue, chloroform was added, and the mixture was filtered and evaporated under vacuum. To the residue, deathly ether was added, and the mixture was filtered and dried with air to give a calcium salt of the compound of Example 3 or 15 5 (3-40 g)- [and = -47.2 (C=1.0, Mesh) (b) My 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. A magnesium salt of the compound of 20 Example 3 or 5 was produced (2.42 g). [and = -46.6 (C=1.0, Mesh) (c) Lawson salt A 1.19 g ox the compound prepared in Example 3 or S
was dissolved in methanol (22 ml). To the solution, Lawson (0.416 g) was added, and the mixture was stirred at room temper-azure for one hour, and evaporated under vacuum. To the residue, chloroform was added, and the mixture was filtered and evapo-rated under vacuum. To the residue, dim ethyl ether was added, and the product was filtered by suction to give a Lawson salt ~235120 of the compound of Example 3 or 5 (1.54 g)- Do = -23.1 (C=1.0, Mesh) (d) No salt A 2 . 27 g sample of the compound prepared in Example 3 or 5 was dissolved in methanol (25 ml). To the solution, 0.514 g of sodium acetate was added, and the mixture was stirred at room temperature for 30 minutes, and evaporated under vacuum. To the residue, 200 ml of a mixture of methanol and chloroform (3:100 v/v) was added, and the resulting mixture was filtered and evaporated under vacuum. The residue was dissolved in methanol, and the solution was filtered and evapo-rated under vacuum. To the residue, ethyl acetate was added, and the product was filtered by suction to give a No salt of the compound of Example 3 or 5 (1.85 g). [and = -26.7 (C=1.0, Mesh) (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, dip cyclohexylamine (6 ml) was added under stirring. The mixture 20 was further stirred for 30 minutes, and then left overnight.
The precipitate was filtered by suction and dried in the air.
The resulting crude crystal was suspended in 300 ml of acetonitrile, and the suspension was heated under reflex for 30 minutes. The suspension was cooled and the crystal was filtered by suction and dried in the air to give a duskily-hexylamine salt of the compound of Example 3 or 5 tl2.2 g).
Do = -24.5 (C=1.0, Mesh) _ 35 _ 123~20 Example 5: N-[3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline N-Cyclohexanecarbonyl-D-alanine (5.98 g) was dissolved in dry tetrahydrofuran (80 ml). To the solution, carbonyl-diimidazole (5.84 g) was added at -18C under stirring while the mixture was cooled with ice. The mixture was stirred at that temperature for one hour, and thereafter, N-(3-mercapto-2-D-methylpropanoyl)-L-proline (6.23 g) was added to the mixture, followed by stirring at -18~C for 30 minutes, then at room temperature for one hour. After completion of the reaction, the mixture was evaporated under vacuum to remove the solvent. To the residue, water (50 ml) was added, and 2N-HCl was added to the mixture to adjust its pi between 1 and 2. The mixture was extracted with ethyl acetate, and the ethyl acetate layer was washed with saturated Nail soul-lion, dried over McCoy and evaporated under vacuum. To Thursday, acetonitrile (120 ml) was added, then 6 ml of dip cyclohexylamine (DCHA) was added. The mixture was stirred at room temperature for one hour. After standing overnight, the precipitate was filtered and dried in the air to give a crude DCHA salt (14.35 g). The crude salt was suspended in asset-nitrite (300 ml) and the suspension was heated under reflex for 30 minutes. After cooling, the precipitate was collected by suction and dried in the air to give a white DCHA salt (12.20 g). The DCHA salt (12.20 g) was suspended in ethyl 25 acetate (90 ml) and to the suspension, 0.5N aqueous KHSO4 (60 ml) was added, and the mixture was shaken. The organic layer was washed with distilled water, dried on McCoy and - 36 ~Z3~1Z~;) evaporated under vacuum to give a gummy substance (8.64 g).
That substance was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 6: N- [3- (~-Cyclohexanecarbonyl-glycylthio) -2-D- -methylpropanoyl]-L-proline N-Cyclohexanecarbonyl-glycine (1.02 go was dissolved in dry tetrahydrofuran (10 ml). To the solution, 20 ml of dry tetrahydrofuran containing 1.07-g of carbonyldiimidazole was added at -20C under stirring while the mixture was cooled with ice. The mixture was stirred at that temperature for one hour, and thereafter, 6 ml of dry tetrahydrofuran contain-in 1.09 g of N-(3-mercapto-2-D-methylpropanoyl)-L-proline was added, followed by stirring at -20C for 30 minutes, then at room temperature for one hour. After completion ox the reaction, the mixture was evaporated under vacuum to remove the solvent. The residue was subjected to column cremate-graph on silica gel (column: 2 x 35 cm) using a mixture of methanol and chloroform (1:100 to 3:100~ as an eluant. Free-lions containing the end compound were combined and evaporated under vacuum to give a gummy substance (1.16 g). The substance was found to be identical with the titled compound by NOR
analysis and thin-layer chromatography.

Example 7: N-~3-(N-Cyclopropanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline N-Cyclopropanecarbonyl-D-alanine (635 my) and in-ethyl amine (0.70 ml) were dissolved in dry tetrahydrofuran 25 (14 ml). To the solution, ethyl chloroform ate (0.48 ml) was added at -15C under stirring while the mixture was cooled with ice. The mixture was stirred at that temperature for , .

37 - ~Z3S120 15 minutes, and thereafter, N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g) and dry tetrahydrofuran (10 ml) containing triethylamine (0.70 ml) were added, followed by stirring at -15C for 15 minutes, then at 5C overnight. After completion of the reaction, the mixture was evaporated under vacuum at 30 - 35C to remove the solvent. To the residue, water (10 ml) was added, and 2N-HCl was added to the mixture to adjust its pi to between 1 and 2. The mixture was extracted with sheller-form, and the chloroform layer was washed with saturated Nail solution, dried over McCoy and evaporated under vacuum. The residue was subjected to column chromatography on silica gel (column: 2 x 35 cm) using a mixture of methanol and sheller-form (1:100 to 3:100) as an eluant. Fractions containing the end compound were combined and evaporated under vacuum to give a gummy substance (395 my). That substance was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 8: N-[3-(N-Cyclohexanecarbonyl-N-methylglycylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-cyclohexane-carbonyl-N-methylglycine (1.09 g), carbonyldiimidazole (1.09 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g).
A gummy substance (0.76 g) was produced and it was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 9: N-~3-(N-Cyclopropanecarbonylglycylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that - 38 lo 3S~2~

N-cyclohexanecarbonyl-glycine was replaced by N-cyclopropane-carbonylglycine (0.96 g), carbonyldiimidazole (1.09 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g). A
gummy substance (0.4 g) was produced and it was found to-be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 10: N-[3-(N-Adamantanecarbonylglycylthio)-2-D- -methylpropanoyl]-L-pro-line The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-adamatane-10 carbonylglycine (1.42 g), carbonyldiimidazole ~1.09 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g). A
gummy substance (1.7 g) was produced and it was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 11: N-[3-(N-Cyclohexanecarbonyl-D-phenylalanylthio)-2-D-methylpropanoyl]-~-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-cyclohexane-carbonyl-D-phenylalanine (1.54 g), carbonyldiimidazole (1.09 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g).
A gummy substance (0.97 g) was produced, and it was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 12: N-[3-(N-Cyclohexanecarbonyl-D-leucylthio)-2-D-methylpropanoyll-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-cyclohexane-carbonyl-D-leucine (1.20 g), carbonyldiimidazole (0.97 g) ,, " ) - 39 - 12 3S~0 and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (0.98 g).
A gummy substance (1.17 g) was produced and was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 13: N-[3-(N-Cyclohexanecarbonyl-D-tryptophylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-cyclohexane-carbonyl-D-tryptophan (1.75 g), carbonyldiimidazole (1.09 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g).
A gummy substance (0.8 g) was produced and it was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 14: N-[3-(N-Cyclohexanecarbonyl-D-phenylglycylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-cyclohexane-carbonyl-D-phenylglycine (1.30 g), carbonyldiimidazole (0.97 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline ~0.98 g).
A gummy substance (0.35 g) was produced and was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 15: N-[3-(N-Cyclohexanecarbonyl-D-methionylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-cyclohexane-carbonyl-D-methionine (1.30 g), carbonyldiimidazole (1.09 g) 25 and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (1.09 g).
A gummy substance (0.59 g) was produced and it was found to ~'~3~20 be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 16: N-[3-(N-Cyclohexanecarbonyl-D-glutaminylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced my N-cyclohexane-carbonyl-D-glutamine (0.5 g), carbonyldiimidazole 10.41 g) and N-(3-mercapto-2-D-methylpropanoyl)-L-proline (0.41 g).
A gummy substance (0.3 g) was produced and was found to be identical with the titled compound by NOR analysis and thin-layer chromatography.

Example 17: N-[3-(N-Adamantanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline The procedure of Example 6 was repeated except that N-cyclohexanecarbonyl-glycine was replaced by N-adamantane-carbonyl-D-alanine ~1.2 g), carbonyldiimidazole Tao g) and 15 N-(3-mercapto-2-D-methylpropanoyl)-L-prollne (0.98 g). A
gummy substance (1.02 g) was produced and was fount to be identical with the titled compound by NOR analysis and thin---layer chromatography.

Example 18: Dicyclohexylamine salt of N-[3-(N-cyclohexane-carbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-praline if) 3-(N-Cyclohexanecarbonyl-D-alanylthio)-2-D-methyl---propanoic acid (241 g) was suspended in dry dichloromethane (1.3 liters). To the suspension, triethylamine (1~7 ml) was added drops at -15C under stirring, then pivaloyl chloride ~104 ml) was added drops at below -5C under stirring, and the mixture was further stirred for 30 minutes.

(2) L-Proline (101 g) was suspended in dry dich~oromethane . I. .

- 41 - ~3~12~

(1.2 liters). To the suspension, trimethylsilyl chloride (112 ml) was added drops at -15C under stirring, then triethylamine (122 ml) was added drops at below -5C under stirring, and the mixture was further stirred at -15C for 30 minutes. To the resulting mixture, the solution prepared in (1) was added, and the mixture was stirred under cooling for 15 minutes, then at room temperature for one 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 drops.
The mixture was stirred for 10 minutes and the organic layer was separated, washed with saturated Nail solution twice, cur/ en A and triad over magnesium sulfate. To the filtrate, duskily-hexcylamine (326 ml) and acetonitrile (800 ml) were added, and the mixture was stirred for 30 minutes. Thereafter, four 800 ml portions of acetonitrile were added at an interval of 10 minutes, and the resulting mixture was left to stand overnight at 5C. The precipitate was filtered by suction and dried in the air to give a crude sample of the titled compound. The crude sample was dissolved in dichloromethane (3 liters) while hot, and filtered. The filtrate was stirred at room temperature for 30 minutes, and another four 750 ml portions of acetonitrile were added at an interval of 10 minutes, and the resulting mixture was left to stand over-night at 5C. The precipitate was filtered and dried in their to give a colorless sample of the titled compound (404 g).
mop. 190-191C, Do = -24.5 (C=1.0, Mesh) - 42 - ~3S120 Example 19: Calcium salt of N- [3- (N cyclohexanecarbonyl-D-alanylthio)-2-D-methylpropanoyl]-L-proline A 404 g sample of the dicyclohexylamine salt prepared in Example 18 was suspended in ethyl acetate (2 livers), and to the suspension, 0.5N XHSO4 (2.5 liters) was added, and the mixture was stirred for 10 minutes. The organic layer was separated, washed with distilled water and dries on magnesium sulfate. The filtrate was evaporated under vacuum, and the residue was dissolved in acetone (1.2 liters). To the acetone solution, distilled water (1.2 liters) and calcium carbonate (36.6 g) were added, and the mixture was stirred vigorously at 40C for an hour. After cooling, the mixture was filtered and the filtrate was evaporated under vacuum.
The resulting syrup was suspended in acetone (1 liter) and the solution was stirred for 5 hours. The precipitate was filtered by suction, dried in the air, then vacuum-dried to give a white powder of the titled compound ~212 g).
Do = -47.2 (C=1.0, Mesh) . _

Claims (21)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of a proline derivative having the general formula:

(I) wherein R is an acyl group bonded to the .alpha.-amino group of amino acid and is selected from the group consisting of cyclopropanecarbonyl, cyclohexanecarbonyl and adaman-tanecarbonyl groups, A is glycine, sarcosine or .alpha.-D-amino acid residue and has an .alpha.-carbonyl group that forms a thiol ester bond with a sulfur atom, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

a) reacting a compound of formula:

(II) wherein A' is glycine, sarcosine or a protected or unprotected .alpha.-D-amino acid residue and R has the afore-said meaning, or a reactive derivative thereof, with a compound of formula:

(III) wherein R' is a hydrogen atom or a carboxyl-protecting group, or a reactive derivative thereof, in an inert organic solvent at a temperature between -50 and 20°C, and removing any protecting group from the reaction product, to obtain the desired derivative of the formula (I); or b) reacting a compound of formula:

R-A'-OH (IV) wherein A' is glycine, sarcosine or a protected or unprotected .alpha.-D-amino acid residue and R has the aforesaid meaning, or a reactive derivative thereof, with a compound of formula:

(V) wherein R' is a hydrogen atom or a carboxyl-protecting group, or a reactive derivative thereof, in an inert organic solvent at a temperature between -50 and 20°C, and removing any protecting group from the reaction product, to obtain the desired derivative of the formula (I); and c) where a pharmaceutically acceptable salt is desired, converting the proline derivative of formula (I) thus obtained into the desired pharmaceutically acceptable salt.

2. A process according to claim 1, wherein a compound of the formula (II) in which A' and R have the aforesaid meanings, or a reactive derivative thereof, is reacted with a compound of the formula (III) in which R' has the aforesaid meaning, or a reactive derivative thereof, in an inert organic solvent at a temperature between -50 and 20°, and any protecting group is removed from the reaction product to obtain a proline derivative of the formula (I) which, if desired, is converted into a pharmaceutically acceptable salt.

3. A process according to claim 2, wherein A' is an amino acid residue selected from the group consisting of glycine, sarcosine, D-alanine, D-leucine, D-methionine, D-glutamine, D-phenylalanine, D-tryptophan and D-phenylglycine.

4. A process according to claim 2, wherein the 2-methylpropanoyl portion of the compound of formula (II) has a D-configuration.

5. A process according to claim 4, wherein R is a cyclohexanecarbonyl group and A' is D-alanine.

6. A process according to claim 2, wherein the reactive derivative of the compound of formula (II) is an activated amide, acid halide, activated ester or a mixed acid anhydride.

7. A process according to claim 2, wherein the compound of formula (II) is reacted with the compound of formula (III) or a reactive derivative thereof in the presence of a carbodiimide.

8. A process according to claim 2, wherein the reactive derivative of the compound of formula (III) has a silyl group introduced in the imino group of said compound.

9. A process according to claim 2, wherein the inert organic solvent is selected from the group consisting of tetrahydrofuran, dioxane, dimethyl-formamide, hexamethylphosphotriamide, chloroform, dichloromethane and acetonitrile.

10. A process according to claim 1, wherein a compound of the formula (IV) in which A' and R have the aforesaid meanings, or a reactive derivative thereof, is reacted with a compound of the formula (V) in which R' has the aforesaid meaning, or a reactive derivative thereof, in an inert organic solvent at a temperature between -50 and 20°C, and any protecting group is removed from the reaction product to obtain a proline derivative of the formula (I) which, if desired, is converted into a pharmaceutically acceptable salt.

11. A process according to claim 10, wherein A' is an amino acid residue selected from the group consisting of glycine, sarcosine, D-alamine, D-leucine, D-methionine, D-glutamine, D-phenylalanine, D-tryptophan and D-phenylglycine.

12. A process according to claim 10, wherein the 2-methylpropanoyl portion of the compound of formula (V) has a D-configuration.

13. A process according to claim 12, wherein R is a cyclohexanecarbonyl group and A' is D-alanine.

14. A process according to claim 10, wherein the reactive derivative of the compound of formula (IV) is an activated amide, acid halide, activated ester or a mixed acid anhydride.

15. A process according to claim 10, wherein the compound of formula (IV) is reacted with the compound of formula (V) in the presence of a carbodiimide.

16. A process according to claims 7 or 15, wherein the carbodiimide is dicyclohexylcarbodiimide.

17. A process according to claim 10, wherein the inert organic solvent is selected from the group consisting of tetrahydrofuran, dioxane, dimethylforma-mide, hexamethylphosphotriamide, chloroform, dichloro-methane and acetonitrile.

18. A proline derivative of the formula (I):

(I) wherein R is an acyl group bonded to the ?-amino group of amino acid and is selected from the group consisting of cyclopropanecarbonyl, cyclohexanecarbonyl and adamantanecarbonyl groups, A is glycine sarcosine or d-D-amino acid residue and has an d-carbonyl group that forms a thiol ester bond with a sulfur atom, or a pharmaceutically acceptable salt thereof.

19. A proline derivative or a pharmaceutically acceptable salt thereof as defined in claim 18, wherein A is an amino acid residue selected from the group consisting of glycine, sarcosine, D-alanine, D-leucine, D-methionine, D-glutamine, D-phenylalanine, D-trypto-phan and D-phenylglycine.

20. A proline derivative or a pharmaceutically acceptable salt thereof as defined in claim 18, wherein the 2-methylpropanoyl portion of the compound of formula (I) has a D-configuration.

21. A proline derivative or a pharmaceutically acceptable salt thereof as defined in claim 18, wherein R is a cyclohexanecarbonyl group and A is D-alanine, the 2-methylpropanoyl portion of the compound having a D-configuration.