NO146985B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE PROLIN-AZETIDINE AND PIPERIDINE DERIVATIVES - Google Patents

Description

The present invention relates to a process for the production of new proline derivatives and related compounds with the general formula (I)

where

R is hydroxy or C₁-C₁ alkoxy,

and R 4 are both hydrogen, C 1 -C 4 alkyl, phenyl or phenyl-

C1-C7 alkyl,

R 2 is hydrogen, C 1 -C 4 alkanoyl or benzoyl,

R^ is hydrogen, hydroxy or C^-C^ alkyl,

m is 1 to 3, and

n is 0 to 2.

The asterisks indicate asymmetric carbon atoms. Each of the carbon atoms bearing a substituent R 1 , R 2 and R 3 is asymmetric when this substituent is different from hydrogen.

The C1~C7 alkyl groups represented by which

preferably of the variables include linear and branched hydrocarbon radicals from methyl to heptyl, e.g. methyl, ethyl, propyl,

isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl and the like.

The C^-Cy alkoxy groups are of the same type bound to oxygen,

e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy and the like.

The C^-C^ alkanoyl groups are those that have acyl radicals of them

lower (C2~ C^) fatty acids, e.g. acetyl, propionyl, butyryl,

isobutyryl and the like.

The products of formula I and the preferred subgroups

can be produced according to different synthesis methods.

In general, the new products are prepared by acylating a compound of formula (III):

with an acid of formula (IV):

or its chemical equivalent.

The end product can thus be prepared not only by direct acylation with an acid of formula IV, but also by means of intermediate products such as e.g.

a) a)-haloalkanoic acids of formula (V):

where X is bromine, chlorine or iodine, or

b) a tosyloxyalkanoic acid, i.e. that X in formula V is tosyloxy

c) a substituted acrylic acid of formula (VI): The product of this acylation is then subjected to displacement or addition with the anion of a thiol or the thioacid of formula (VII):

Acylation can also be carried out using a thiolactone of formula (VIII):

to form a product where F^ is hydrogen.

From a compound of formula (II): where Y is a sulfur protecting group such as e.g. C^-C-y alkanoyl, phenyl-(C1~C7)-alkanoyl, benzoyl, benzyl, triphenylmethyl, or another sulfur protecting group, Y is removed to form a compound where R ? is hydrogen. "Deprotection" can be carried out in the usual way, such as e.g. by treatment with hot trifluoroacetic acid, cold trifluoromethanesulfonic acid, mercury(II) acetate, sodium in liquid ammonia, zinc and hydrochloric acid or the like. For discussion of these methods refer to Methoden der Organischen Chemie (Houben-Weyl), vol. XV, Part I, pp. 736 et seq. (1974).

When the acid of formula IV is used as the acylating agent, the acylation may be carried out in the presence of a coupling agent such as dicyclohexylcarbodiimide or the like, or the acid may be activated by formation of its mixed anhydride, symmetric anhydride, acid chloride, acid ester, or using Woodward's K,N-ethoxy -carbonyl-2-ethoxy-1,2-dihydroquinoline or the like. An overview of acylation methods appears in Methoden der Organischen Chemie (Houben-Weyl), vol. XV, Part II, pp. 1 et seq. (1974).

Compounds of formula III include, for example, proline, hydroxyproline, 4-methylproline, pipecolic acid, 5-hydroxypipecolic acid, azetidine-2-carboxylic acid, their lower alkyl esters and the like. The acylation of such compounds is described in more detail later.

According to a preferred method for preparing compounds of formula I, especially where R_ is alkanoyl or benzoyl, an acid or ester of formula III is coupled with a haloalkanoic acid of formula (V):

where X is halogen, preferably chlorine or bromine. This can be carried out using known methods where the acid IV is activated before the reaction with the acid III, which includes the formation of a mixed anhydride, symmetrical anhydride, acid chloride, active ester, or the use of Woodward's reagent K, EEDQ (N-ethoxycarbonyl-2-ethoxy -1,2-dihydroxyquinoline) or the like.

The product of this reaction is a compound with the formula (X):

The product of formula X is subjected to a displacement reaction with the anion of a thioacid of formula (VII): giving a product of formula (XI):

When R2 is alkanoyl or benzoyl, this product can be converted to the product (XII):

by ammonolysis. When R 2 is a protecting group, the compound of formula XII can be obtained by "deprotection" as described above. When R is an ester group (ie R is lower alkoxy),

can the ester group be removed, e.g. when R is tert-butoxy or tert-amyloxy, by treating the ester of formula XI or XII with trifluoroacetic acid and anisole so that the corresponding free acid is obtained. When other alkoxy groups are present, alkaline hydrolysis will give the corresponding acid.

A variation of this method involves the use of an acrylic acid of the formula (VI): as starting material. This acrylic acid is first converted to the acid halide, and is then allowed to react with a compound of formula III so that a compound of formula (XIII) is obtained:

and this intermediate is subjected to an addition reaction with the thiol or the thioacid VII as described above.

A tosyloxyalkanoic acid of formula (XIV):

can also be used as the agent to acylate the acid of formula III, and then subject the acylation product to transfer reaction, etc., as described above.

Alternatively, the acrylic acid of formula VI can be introduced first

to reaction with the thioacid of formula VII so that a product of formula (XV) is obtained:

which is converted to its acid halide, e.g. with thionyl chloride, is coupled with the compound of formula III and the same sequence as above is followed.

The acid or ester of formula III can also be acylated with a "protected" form of a u-mercaptoalkanoic acid of formula (XVI):

where Rg is the "protecting" group. Such "protecting" groups may have the form described above.

After the acylation, the product can be "deprotected" using one of the known methods referred to above.

Yet another acylating agent can take the form of a thiolactone, e.g. (3-propiothiolactone, α-methyl-(3-propiothiolactone) or the like.

Further details of preferred methods for the preparation of the new compounds can be found in the following and in the following examples.

According to a particularly preferred modification, the acid or ester of formula III is acylated with a halogen-alkanoyl halide of formula (XVII):

where each X is an optional halogen atom, preferably chlorine or bromine, R 1 is hydrogen, lower alkyl or phenyl-lower alkyl and n is 0, 1 or 2. This reaction is carried out in an alkaline medium, e.g. dilute alkali metal hydroxide solution, alkali metal bicarbonate or alkali metal carbonate solution at a reduced temperature, e.g. from approx. 0 to 15°C. The reaction product is subjected to a transfer reaction with the anion of the thiol or thioacid of formula VII above, also in alkaline solution, preferably alkali metal carbonate solution, and is then worked up in the usual way. The product from this reaction, where R 2 in formula I is alkanoyl or benzoyl, is converted to the product where R 2 is hydrogen by ammonolysis, e.g. with alcoholic ammonia or concentrated ammonium hydroxide solution, or alkaline hydrolysis, e.g. with aqueous metal hydroxide. When an acid of formula III is used as starting material, the final product obtained as a free carboxylic acid can be converted to its ester, e.g. by for-es ting with a diazoalkane, such as e.g. diazomethane, 1-alkyl-3-p-tolyl triazene, such as 1-n-butyl-3-p-tolyl triazene or the like. According to another variation, an ester, preferably the t-butyl ester of formula III, is treated in an anhydrous medium such as e.g. dichloromethane, tetrahydrofuran, dioxane or the like, with a thioalkanoic acid of the formula (XVIII)

in the presence of dicyclohexylcarbodiimide, N,N<1->carbonyl-bisimidazole, ethoxyacetylene, diphenylphosphorylazide or similar coupling agents at temperatures in the range from approx. 0 to 10°C. The ester group (R) can then be removed, e.g. by treatment with trifluoroacetic acid and anisole at about room temperature.

When an ester of formula III (e.g. R is lower alkoxy, especially t-butoxy) is acylated with a thiolactone, e.g. (3-propiothiolactone, α-methyl-3-propiothiolactone or the like, the reaction can be carried out in an anhydrous solvent such as tetrahydrofuran, dioxane, methylene chloride or the like at approx.

0°C to approximately room temperature. The ester group can be removed with anisole and trifluoroacetic acid as described above.

Products of formula I have one or more asymmetric carbon atoms. When R1, R3 or R4 is other than hydrogen, the carbon atom to which they are attached is asymmetrical. These carbon atoms are indicated with one asterisk in formula I. The compounds then exist in stereoisomeric forms or in racemic mixtures thereof. The process for producing all of these lies within the scope of the invention. The synthesis described above can utilize the racemate or one of the enantiomers as starting material. When the racemic starting material is used in the synthesis process, the stereoisomers obtained can be separated from each other by conventional chromatographic or fractionation-crystallization methods. In general, the L form with regard to the carbon atom in the amino acid constitutes the preferred isomeric form. Also the D-isomer with regard to the α-carbon atom in the acyl side chain (ie the carbon atom which is connected to R^) is preferred.

The compounds produced according to the invention form

basic salts with various inorganic and organic bases, and the preparation of these is also within the scope of the invention" Such salts include ammonium salts, alkali metal salts

like for example. sodium and potassium salts (which are preferred), alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases, e.g. dicyclohexylamine salt, benzathine, N-methyl-D-glucamine and hydrabamine salts, salts with amino acids

like for example. arginine, lysine and the like. The non-toxic, physiologically acceptable salts are preferred, although other salts are also useful, e.g. by isolating and purifying the product, as illustrated in the examples in the case of the dicyclohexylamine salt.

'The salts are formed in a conventional manner by reacting the free acid form of the product with one or more equivalents of a suitable base which gives the desired cation in a solvent or medium in which the salt is insoluble, or in water and removal of

the water during freeze-drying. By neutralizing the salt with an insoluble acid such as a cation exchange resin in the hydrogen form (eg polystyrene sulfonic acid resin such as "Dowex 50") or with an aqueous acid and extraction with an organic solvent, such as ethyl acetate, dichloromethane or the like, the free acid form can be obtained and, if desired, another salt is formed.

Additional experimental details are found in the examples which are preferred embodiments and also serve as models for the preparation of other members of the group.

The compounds produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful for reducing or alleviating angiotensin-related high blood pressure. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by the angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressure substance that contributes as a causative agent in various forms of high blood pressure in various mammalian species, e.g. rats and dogs. The compounds produced according to the invention intervene in the angiotensin (renin)-»angiotensin I-»angiotensin II sequence by inhibiting the angiotensin-converting enzyme and reducing or eliminating the formation of the pressure substance angiotensin II. By administering a preparation containing one or a combination of compounds of formula I or physiologically acceptable salts thereof, angiotensin-dependent high blood pressure is thus alleviated in mammalian species suffering from it. A single dose, or preferably two to four doses daily at an amount of about 0.1 to 100 mg/kg/day, preferably about 1 to 50 mg/kg/day is appropriate to reduce blood pressure as indicated in the animal model studies which is described by S.L. Engel, T.R. Schaeffer, M.H. Waugh and B. Rubin, Proe. Soc. Exp. Biol. With. 143, 483 (1973). The substance is preferably administered orally, but can also be administered parenterally, e.g. subcutaneously, intramuscularly, intravenously or intra-peritoneally.

The compounds produced according to the invention can be used to achieve a reduction of blood pressure by making preparations from them, e.g. as tablets, capsules or elixirs for oral administration or as sterile solutions or suspensions for parenteral administration. About 10 to 500 mg of a compound or mixture of compounds of formula I or physiologically acceptable salts thereof are mixed together with a physiologically acceptable carrier, binder, preservative, stabilizer, flavoring agent, etc. in the dosage-

unit form to be used in pharmaceutical practice. The amount of active substance in these preparations is such that a suitable dosage is achieved in the specified range.

Illustrative of the aids that can be mixed in

tablets, capsules and the like are the following: a binder such as e.g. tragacanth, gum acacia, corn starch or gelatin, a binder such as dicalcium phosphate, a disintegrating agent such as corn starch, potato starch, alginic acid and the like,

a lubricant such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin, a flavoring such as peppermint, wintergreen oil or cherries. When the dosage unit form is a capsule, in addition to materials of the above type, it may contain a liquid carrier such as "a fatty oil". Various other materials may be present as a coating or to otherwise modify the physical form of the dosage unit. Tablets can, for example, be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, a coloring agent and flavorings such as e.g. cherry or orange flavor.

Sterile preparations for injection can be prepared according to conventional pharmaceutical practice by dissolving or suspending the active substance in a base such as e.g. water for injection,

a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fat base such as ethyl oleate or, the like.

Buffers, preservatives, anti-oxidants and the like can be mixed in if desired.

Pharmaceutical values were determined for a number of representative compounds prepared according to the invention, and the values found are indicated in the following table. I^q is the amount of inhibitor in µg/ml required to inhibit the activity of angiotensin-converting enzyme by 50% as measured by spectrophotometric measurement. EC5q', which °9S^ is denoted as IC^Q, is the concentration of the test compound which produces 50% inhibition of the contractile action of angiotensin 1.

The following examples illustrate the invention and constitute particularly preferred embodiments. All temperatures are given in degrees Celsius.

Example 1

1-(2-benzoylthioacetyl)-L-proline

L-proline (5.75 g) is dissolved in IN sodium hydroxide (50 ml) and the solution is cooled in an ice water bath. Sodium hydroxide 2N (26 ml) and chloroacetyl chloride (5.65 g) are added and the mixture is stirred vigorously at room temperature for three hours. A suspension of thiobenzoic acid (7.5 g) and potassium carbonate (4.8 g) in water (50 ml) is added. , After stirring for 18 hours at room temperature, the reaction mixture is acidified and extracted with ethyl acetate. The ethyl acetate layer is washed with water, dried over magnesium sulfate and concentrated to dryness in vacuo. The residue (14.6 g) is dissolved in ethyl acetate (150 ml) and dicyclohexylamine (11 ml) is added. The crystals are filtered off and recrystallized from ethyl acetate, yield 5.7 g, m.p. 151-152°. To convert the salt into the acid, the crystals are dissolved in a

mixture of 5% aqueous potassium bisulphate (100 ml) and ethyl acetate

(300 ml). The organic phase is washed once with water, dried

over magnesium sulfate and concentrated to dryness in vacuo>

yield 3.45 g,

Example 2

1-(2-mercaptoacetyl)-L-proline

1-(2-benzoylthioacetyl)-L-proline (3.4 g) is dissolved in a mixture of water (10.5 ml) and concentrated ammonia (6.4 ml). After one hour, the reaction mixture is diluted with water and filtered. The filtrate is extracted with ethyl acetate and then acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted twice with ethyl acetate. The ethyl acetate extracts are washed with saturated sodium chloride solution and concentrated to dryness, yield 1.5 g. The product, 1-(2-mercaptoacetyl)-L-proline, is crystallized from ethyl acetate (m.p. 133-135°).

Example 3

1-(2-benzoylthiopropanoyl)-L-proline

L-proline (5.75 g) is dissolved in aqueous IN sodium hydroxide (50 ml), and the solution is cooled in an ice bath with stirring. Sodium hydroxide 2N (25 ml) and 2-bromo-propionyl chloride (8.57 g) are added in the order mentioned and the mixture is removed from the ice bath and stirred at room temperature for one hour. A mixture of thiobenzoic acid (7.5 g) and potassium carbonate (4.8 g) in water (50 ml) is added and the mixture is stirred overnight at room temperature.

After acidification with concentrated hydrochloric acid, the aqueous solution is extracted with ethyl acetate, and the organic phase is washed with water, dried and concentrated to dryness. The residue (14.7 g) is chromatographed on a column with 440 g of silica gel with a mixture of benzene-acetic acid (7:1). The fractions containing the desired material are collected, concentrated to dryness, and the residue precipitated twice with ether-hexane and converted to a dicyclohexylamine salt in ether-hexane, yield 9.4 g, m.p. (142) 148-156°. The dicyclohexylamine salt is reduced to the acid as in example 1, yield 5.7 g.

Example 4

1-(2-mercaptopropanoyl)-L-proline

1-(2-benzoylthiopropanoyl)-L-proline (5.7 g) is dissolved in a mixture of water (12 ml) and concentrated ammonium hydroxide (9 ml) with stirring. After one hour, the mixture is diluted with water (10 ml) and filtered. The filtrate is extracted twice with ethyl acetate, concentrated to one third of the original volume, acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with saturated sodium chloride, dried and concentrated to dryness in vacuo. The residue, 1-(2-mercaptopropanoyl)-L-proline, is crystallized from ethyl acetate-hexane, yield 3 g, m.p. (105) 116-120°.

The 2L-isomer was prepared similarly by acylation of L-proline with 2-(L-acetylthio)propionic acid chloride to form 1-(2-L-thioacetyl-propanoyl)-L-proline,

sm.p. 85-86°C, [a]^<5> -175.5° (c = 1.10, C2H5OH) which was then hydrolyzed to form the 2L isomer of the title compound,

sm.p. (p 138) 143-144°C; [α]^<5> -63.3° (c = 1.50, CH 3 OH).

Example 5

1-(3-benzoylthiopropanoyl)-L-proline

L-proline (5.75 g) is dissolved in normal sodium hydroxide

(50 ml) and the solution is cooled in an ice bath. 3-bromopropionyl chloride (8.5 g) and 2N sodium hydroxide (27 ml) are added and the mixture is stirred for 10 minutes in the ice bath and three hours at room temperature. A suspension of thiobenzoic acid (7.5 g) and potassium carbonate (4.5 g) in water (50 ml) is added and the mixture is stirred for 18 hours at room temperature. After acidification with concentrated hydrochloric acid, the aqueous phase is extracted twice with ethyl acetate. The organic layers are dried over magnesium sulfate and concentrated to dryness in vacuo so that 7.1 g, m.p. 101-102°

(ethyl acetate-hexane) of 1-(3-benzoylthiopropanoyl)-L-proline.

Example 6

1-(3-acetylthiopropanoyl)-L-proline-tert-butyl ester L-proline-tert-butyl ester (5.13 g) is dissolved in dichloromethane (40 ml) and the solution is cooled in an ice water bath. A solution of dicyclohexylcarbodiimide (6.18 g) in dichloromethane (20 mL) is added, followed immediately by 3-acetylthiopropionic acid (4.45 g). After 15 minutes of stirring in the ice water bath and 16 hours at room temperature, the precipitate is filtered off and the filtrate is concentrated to dryness in a vacuum. The residue is dissolved in ethyl acetate and washed neutrally. The organic layer is dried over magnesium sulfate and concentrated to dryness in vacuo so that 9.8 g of 1-(3-acetylthiopropanoyl)-L-proline-tert-butyl ester is obtained.

Example 7

1-(3-acetylthiopropanoyl)-L-proline

1-(3-acetylthiopropanoyl)-L-proline tert-butyl ester (4.7 g) is dissolved in a mixture of anisole (34 ml) and trifluoroacetic acid

(68 ml) and the mixture is kept at room temperature for one hour. The solvents are removed in vacuo and the residue is precipitated from ether-hexane several times. The residue (3.5 g) is dissolved in acetonitrile

(25 ml) and dicyclohexylamine (2.8 ml) are added. The crystalline salt is filtered off and recrystallized from isopropanol. Yield 3.8 g, m.p. 176-177°. The salt is reconverted to l-(3-acetylthiopropanoyl)-L-proline as in example 1, yield 1.25 g, m.p. 89-90° (ethyl acetate-hexane).

Example 8

1-(3-mercaptopropanoyl)-L-proline-tert-butyl ester

To a solution of L-proline tert-butyl ester (3.42 g) in dry tetrahydrofuran (10 ml) cooled in an ice bath is added propiothiolactone (1.76 g). After 5 minutes of storage in the ice bath and three hours at room temperature, the reaction mixture is diluted with ethyl acetate (200 ml) and washed with 5% potassium bisulphate and water. The organic layer is dried over magnesium sulfate and concentrated to dryness in vacuo. The residue, 1-(3-mercaptopropanoyl)-L-proline tert-butyl ester, is crystallized from ether-hexane,

yield 3.7 g, m.p. 57-58°.

Example 9

1-(3-mercaptopropanoyl)-L-proline

Procedure A

1-(3-benzoylthiopropanoyl)-L-proline (4.9 g) is dissolved in a mixture of water (8 ml) and concentrated ammonium hydroxide (5.6 ml) and the solution is kept with stirring under argon for one hour. The reaction mixture is diluted with water, filtered, and the filtrate is extracted with ethyl acetate. The aqueous phase is acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted with ethyl acetate. The organic layers are washed with saturated sodium chloride, dried over magnesium sulfate and concentrated to dryness in vacuo. The residue, 1-(3-mercapto-propanoyl)-L-proline, is crystallized from ethyl acetate-hexane, yield 2.5 g, m.p. 68-70°o Procedure B

1-(3-acetylthiopropanoyl)-L-proline (0.8 g) is dissolved in

5.5 N methanolic ammonia (5 ml) and the solution is kept under argon at room temperature. After 2 hours, the solvent is removed in vacuo, the residue is dissolved in water and applied to an ion exchange column in H+<-> form ("Dowex 50" (analytically pure)) and eluted with water. The fractions which give a thiol-positive reaction are collected and concentrated to dryness, yield 0.6 g. This product is crystallized from ethyl acetate-hexane as in method A and gives 1-(3-mercaptopropanoyl)-L-proline.

Procedure C

1-(3-Mercaptopropanoyl)-L-proline tert-butyl ester (2.3 g) is dissolved in a mixture of anisole (20 ml) and trifluoroacetic acid (45 ml). After one hour's storage at room temperature under argon, the reaction mixture is concentrated to dryness in vacuo and the residue is precipitated from ethyl acetate-hexane several times. The residue (1.9 g) is dissolved in ethyl acetate (30 ml) and dicyclohexylamine (1.85 ml) is added. The crystalline salt is filtered off and recrystallized from isopropanol, yield 2 g, m.p. 187-188°.

The salt is converted to the acid as in example 1, yield 1.3 g. The product is crystallized from ethyl acetate-hexane as in method A.

Salts

Sodium

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

Magnesium

1-(3-mercaptopropanoyl)-L-proline (500 mg), magnesium oxide (49.5 mg) and water (10 ml) are stirred with gentle heating until complete dissolution is achieved. The solvent is then removed by freeze-drying to obtain the magnesium salt.

Calcium

1-(3-mercaptopropanoyl)-L-proline (500 mg) is dissolved in a mixture of calcium hydroxide (91 mg) and water (10 ml), and the solution is freeze-dried to obtain the calcium salt.

Potassium

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

N-methyl-D-glucamine

1-(3-mercaptopropanoyl)-L-proline (500 mg) and N-methyl-D-glucamine (480 mg) are dissolved in water (10 ml) and freeze-dried to obtain the N-methyl-D-glucamine salt.

Example LO

1-(3-mercaptopropanoyl)-L-hydroxyproline

By using L-hydroxyproline instead of L-proline in the method in example 3 and then treating the product by method A from example 9, 1-(3-benzoylthiopropanoyl)-L-hydroxyproline and 1-(3-mercaptopropanoyl)-L-hydroxyproline are obtained, sm.p. 193.5-195°C as dicyclohexylamine salt, [α]^<5> -83.5° (c=2, methanol).

Example 11

1-(3-mercaptopropanoyl)-L-azetidine-2-carboxylic acid

By using L-azetidine-2-carboxylic acid tert-butyl ester instead of L-proline tert-butyl ester in the method in example 6, and treating the product as in example 7 and the thus obtained 1-(3-acetylthiopropanoyl)-L-azetidine -2-carboxylic acid as in method B in example 9, 1-(3-acetylthiopropanoyl)-L-azetidine-2-carboxylic acid tert-butyl ester and 1-(3-mercaptopropanoyl)-L-azetidine-2-carboxylic acid are obtained respectively, the latter which

25 o

a viscous mass, [ot]D -10.1 (c=2, ethanol) .

Example 12

1-(3-mercaptopropanoyl)-L-pipecolic acid

By using L-pipecolic acid tert-butyl ester instead of L-proline tert-butyl ester in the method in example 6 and treating the product according to method C in example 9, 1-(3-mercaptopropanoyl)-L-pipecolic acid tert-butyl ester is obtained and 1-(3-mercaptopropanoyl)-L-pipecolic acid, respectively, the latter as a viscous mass, [α]^ -51.5° (c=1, ethanol).

Example 13

1-(3-mercaptopropanoyl)-D-proline

By using D-proline instead of L-proline in the method from example 5 and then treating the product according to method A from example 9, 1-(3-benzoylthiopropanoyl)-D-proline and 1-(3-mercaptopropanoyl)-D-proline are obtained , sm.p. 68-70°.

Example 14

1-( 3- acetylthio- 2- methylpropanoyl) - L- proline- tert-butyl ester

"L-proline tert-butyl ester (5.1 g) is dissolved in dichloromethane (40 ml) and the solution is stirred and cooled in an ice bath. Dicyclohexylcarbodiimide (6.2 g) dissolved in dichloromethane (15 ml) is added, immediately followed by a solution of 3-acetylthio-2-methylpropanoic acid (4.9 g) in dichloromethane (5 ml). After stirring for 15 minutes in the ice bath and 16 hours at room temperature, the precipitate is filtered off and the filtrate is concentrated to dryness in a vacuum. The residue is dissolved in ethyl acetate and washed neutrally. The organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo. The residue, 1-(3-acetylthio-2-methylpropanoyl)-L-proline-tert-butyl ester, is purified by column chromatography (silica gel-chloroform), yield 7.9 g.

Example 15

1-(3-Acetylthio-2-methylpropanoyl)-L-proline

Procedure A

The 1-(3-acetylthio-2-methylpropanoyl)-L-proline tert-butyl ester from Example 14 (7.8 g) is dissolved in a mixture of anisole (55 ml) and trifluoroacetic acid (110 ml). After one hour's storage at room temperature, the solvent is removed in vacuo and the residue is precipitated several times from ether-hexane. The residue (6.8 g) is dissolved in acetonitrile (40 ml) and dicyclohexylamine (4.5 ml) is added. The crystalline salt is boiled with new acetonitrile

(100 ml), cooled to room temperature and filtered, yield 3.8 g, m.p. (165) 187-188°. This material is recrystallized from isopropanol ta]D -67° (c 1.4, EtOH). The crystalline dicyclohexylamine salt is suspended in a mixture of 5% aqueous potassium bisulphate and ethyl acetate. The organic phase is washed with water and concentrated to dryness. The residue is crystallized from ethyl acetate-hexane so that 1-(3-acetylthio-2-D-methylpropanoyl)-L-proline is obtained, m.p. 83-85° [α]* 5 -162 (c 1.7, EtOH).

Procedure B

3-acetylthio-2-methylpropanoic acid (8.1 g) and thionyl chloride

(7 g) are mixed and the suspension is stirred for 16 hours at room temperature. The reaction mixture is concentrated to dryness and distilled in vacuo (b.p. 80°). To this 3-acetylthio-2-methyl-propanoic acid chloride (5.4 g) and 2N sodium hydroxide (15 ml) are added

to a solution of L-proline (3.45 g) in normal sodium hydroxide

(30 ml) cooled in an ice water bath. After stirring for 3 hours at room temperature, the mixture is extracted with ether, the aqueous phase is acidified and extracted with ethyl acetate. The organic phase is dried over magnesium sulfate and concentrated to dryness so that 1-(3-acetylthio-2-DL-methylpropanoyl)-L-proline is obtained.

Procedure C

Methacryloyl chloride (4.16 g) is added to a solution of L-proline (3.45 g) in a mixture of water (100 ml) and sodium bicarbonate (12 g) cooled in an ice water bath, with vigorous stirring.

When the addition is complete, the mixture is stirred at room temperature for two hours and then extracted with ether. The aqueous phase is acidified with IN hydrochloric acid and extracted with ethyl acetate.

The organic phase is concentrated to dryness in vacuo, the residue is mixed with thiolacetic acid (3.5 g), a few azobisisobutyronitrile crystals are added and the mixture is heated on a steam bath for two hours. The reaction mixture is dissolved in benzene-acetic acid (75:25) and applied to a column of silica gel. Elution with the same solvent gives 1-(3-acetylthio-2-DL-methylpropanoyl)-L-proline.

Example 16

1-(3-benzoylthio-2-D-methylpropanoyl)-L-proline

By using 3-benzoylthio-2-D-methylpropanoic acid instead of 3-acetylthio-2-methylpropanoic acid in the method from example 14, 1-(3-benzoylthio-2-D-methylpropanoyl)-L-proline-tert-butyl ester is obtained.

By using 1-(3-benzoylthio-2-D-methylpropanoyl)-L-proline-tert-butyl ester instead of 1-(3-acetylthio-2-methylpropanoyl)-L-proline-tert-butyl ester in method A from example 15 ,

1-(3-benzoylthio-2-D-methylpropanoyl)-L-proline is obtained,

sm.p. 85-86°C (decomp.) as a crystalline, colorless solid.

Example 17

1-(3-mercapto-2-D-methylpropanoyl)-L-proline

1-(3-Mercapto-2-methylpropanoyl)-L-proline is obtained by treating the product from each of Examples 15 and 16 as follows:

The thioester (0.85 g) is dissolved in 5.5 N methanolic ammonia

and the solution is kept at room temperature for 2 hours. The solvent

is removed in vacuo and the residue is dissolved in water, applied to an ion exchange column in H+<-> form ("Dowex 50", analytically pure) and eluted with water. The fractions that give a positive thiol reaction are collected and freeze-dried. The residue is crystallized from ethyl acetate-hexane, yield 0.3 g. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline has m.p. 103-104°, [<x]D -131 (c 2 , EtOH).

Example 18

1-(3-mercapto-2-benzylpropanoyl)-L-proline

By using 3-acetylthio-2-benzylpropanoic acid instead of 3-acetylthio-2-methylpropanoic acid in the method from example 14, 1-(3-acetylthio-2-benzylpropanoyl)-L-proline-tert-butyl ester is obtained, which is then treated according to method A from example 15 whereby 1-(3-acetylthio-2-benzylpropanoyl)-L-proline is obtained, and this is treated with methanolic ammonia according to the method from example 17 so that 1-(3-mercapto-2-benzylpropanoyl)-L- proline is obtained as an oil, R^ = 0.47 (silica gel, benzene-acetic acid 75:25).

Example 19

1-(3-mercapto-2-methylpropanoyl)-L-hydroxyproline

By using L-hydroxyproline tert-butyl ester in the method from example 14, treating the product according to method A from example 15 and then continuing as in example 17, 1-(3-acetylthio-2-methylpropanoyl)-L-hydroxyproline-tert is obtained -butyl ester, 1-(3-acetylthio-2-methylpropanoyl)-L-hydroxyproline and 1-(3-mercapto-2-methylpropanoyl)-L-hydroxyproline, respectively, the title product as a hygroscopic solid, [&]^ - 114° (c=1, methanol).

Example 20

1-(3-mercapto-2-methylpropanoyl)-L-pipecolic acid

By substituting L-pipecolic acid in the method from example 15, treating the product according to method A from example 16 and then continuing as in example 17, l-(3-acetylthio-2-methylpropanoyl)-L-pipecolic acid tert-butyl ester is obtained, 1 - (3-acetylthio-2-methylpropanoyl)-L-pipecolic acid and 1-(3-mercapto-2-methylpropanoyl)-L-pipecolic acid, respectively. The title product is obtained as the 2-D-methyl isomer, m.p. 85-90°C, [ot]^ -88° (c=l,

ethanol). The 2-L-methyl isomer is recovered from the mother liquor as a

25 o

viscous substance, [alD = 33 (c=1, ethanol).

Example 21

1-(4-benzoylthiobutanoyl)-L-proline

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

E xample 2 2

1-(4-mercaptobutanoyl)-L-proline

1-(4-Benzylthiobutanoyl)-L-proline (1.08 g) is dissolved in a mixture of water (4 ml) and concentrated ammonia (2.7 ml). After stirring for one hour at room temperature, the mixture is diluted with water, filtered, extracted with ethyl acetate and the aqueous phase is concentrated in vacuo. This ammonium salt of 1-(4-mercapto-butanoyl)-L-proline is purified by ion exchange chromatography on a column of diethylaminoethyl "Sephadex" (cross-linked dextran)

with a gradient of ammonium bicarbonate, yield 0.7 g. The ammonium salt is dissolved in water (2 ml) and applied to a column of "Dowex 50" sulphonic acid resin of analytical grade in hydrogen form, and the free acid is eluted with water. The fractions containing the desired material (sulfhydryl reagent and carboxyl reagent positive) are collected and freeze-dried so that 1-(4-mercapto-butanoyl)-L-proline is obtained. The dicyclohexylammonium salt is prepared according to the method from example 21, m.p. 157-158°.

Example 2 3

1-(3-acetylthiobutanoyl)-L-proline-tert-butyl ester

Dicyclohexylcarbodiimide (6.2 g) and 3-acetylthiobutyric acid (4.86 g) are added to a solution of L-proline tert-butyl ester (5.1 g) in dichloromethane (60 ml) stirred in an ice bath. After 15 minutes, the ice bath is removed and the mixture is stirred at room temperature for 16 hours. The precipitate is filtered off, the filtrate is concentrated to dryness and the residue is chromatographed on a column of silica gel with chloroform to obtain 1-(3-acetylthiobutanoyl)-L-proline-tert-butyl ester, yield 5.2 g.

Example 2 4

1-(3-acetylthiobutanoyl)-L-proline

The 1-(3-acetylthiobutanoyl)-L-proline tert-butyl ester from example 23 (5.2 g) is dissolved in a mixture of trifluoroacetic acid (60 ml) and anisole (30 ml) and the solution is kept at room temperature for one hour. The solvents are removed in vacuo, and the remaining 1-(3-acetylthiobutanoyl)-L-proline is precipitated from ether-hexane several times, yield 4 g. The dicyclohexylamine salt is prepared according to the method from example 21, m.p. 175-176°.

Example 2 5

1-(3-mercaptobutanoyl)-L-proline

The 1-(3-acetylthiobutanoyl)-L-proline from Example 24

(0.86 g) is dissolved in 5.5N methanolic ammonia (20 ml) and the reaction mixture is kept at room temperature for two hours. The solvent is removed in vacuo and the residue is chromatographed on an ion exchange column ("Dowex 50") with water. The fractions containing the desired 1-(3-mercaptobutanoyl)-L-proline are collected and lyophilized, yield 0.6 g. The dicyclohexylamine salt is prepared according to the method from example 21, m.p. 183-184°.

Example 2 6

1-(benzylthioacetyl)-L-proline

By reacting benzylthioacetyl chloride with L-proline in a

IN sodium hydroxide solution with cooling, 1-(benzylthio-acetyl)-L-proline is obtained, m.p. 86-88°.

Example 2 7

1-(3-mercapto-2-phenylpropanoyl)-L-proline

By using 3-acetylthio-2-phenylpropanoic acid instead of 3-acetylthio-2-methylpropanoic acid in the method from example 14, 1-(3-acetylthio-2-phenylpropanoyl)-L-proline-tert-butyl ester is obtained.

By using 1-(3-acetylthio-2-phenylpropanoyl)-L-proline-tert-butyl ester instead of 1-(3-acetylthio-2-methylpropanoyl-L-proline-tert-butyl ester in the method from example 15, and subjecting the product ammonolysis as in example 17, 1-(3-acetylthio-2-phenylpropanoyl)-L-proline and 1-(3-mercapto-2-phenyl-propanoyl)-L-proline are obtained. The title product has m.p. 49-52 °C and

[a]j^ +2.5° (c=1, ethanol).

Example 2 8

1-[3-(acetylthio)-DL-propanoyl]-pipecolic acid

Pipecolic acid (6.5 g) is suspended in 200 ml of dimethylacetamide. 3-acetylthiopropanoyl chloride (8.3 g) is added dropwise at 2 3° to the suspension. A clear solution is formed and the temperature rises to 28°. N-methylmorpholine (10.1 g) is added to this clear solution. A precipitate immediately forms and the temperature rises to 34°. The mixture is heated on a steam bath for 1 hour when a clear solution is formed. After cooling, the precipitated solid is filtered off and gives 5.1 g of 1-[3-(acetylthio)-DL-propanoyl]-pipecolic acid, m.p. 190-200°. The solvent is removed and the viscous residue is triturated with isopropyl ether so that 7.8 g of a product melting at 98-101° is obtained. Recrystallization from acetone-hexane gives a constant melting solid,

sm.p. 102-104°; Rf 0.72 (silica gel, benzene, acetic acid (7:2).

Example 29

DL- 1-(3- mercaptopropanoyl) pipecolic acid

12 ml of concentrated ammonium hydroxide are stirred under nitrogen at 10° for approx. 15 minutes, then solid 1-[3-(acetylthio)-DL-propanoyl]-pipecolic acid (6.6 g) is added at 5 to 10°. It is formed

a clear solution after 2-3 minutes. The ice bath is removed and the solution is stirred at room temperature under nitrogen for 45 minutes. The solution is made strongly acidic with 20% HCl (cooling), and the precipitated oil is extracted with 3 x 150 ml of ethyl acetate. The ethyl acetate extracts are dried over magnesium sulfate and the solvent is removed

so that 6.0 g of DL-1-(3-mercaptopropanoyl)pipecolic acid is obtained, Rf 0.77 (silica gel, benzene, acetic acid (7:1)).

Example 30

1-(3-mercaptopropanoyl)-L-pipecolic acid

By using L-pipecolic acid instead of DL-pipecolic acid in the method from example 28 and then subjecting the product to the method in example 29, 1-[3-(acetylthio)propanoyl]-L-pipecolic acid and 1-(3-mercaptopropanoyl)-L-pipecolic acid are obtained ,

20

Rf 0.80 (silica gel, benzene, acetic acid (7:1)), [α]p -51.5 (c 1.0, abs. ethanol).

Example 31

1-[ 3-( acetylthio)- 2- methylpropanoyl- DL- pipecolic acid

6.5 g (0.05 m) of pipecolic acid is suspended in dimethylacetamide (200 ml), 9.0 g (0.05 m) of 3-acetylthio-2-methylpropanoyl chloride is added dropwise. The temperature rises to 29° and a clear solution is formed. Then 10.1 g of N-methylmorpholine are added at once and the temperature rises to 34°. The mixture is heated on a steam bath for 1 hour when a clear solution is formed. This is allowed to stand overnight at room temperature, and the solid which precipitates is filtered so that 6.1 g, m.p. 203-204°. The solvent is removed and the viscous residue is triturated with water and 20% HCl. The yellow oil is extracted with 3 x 150 ml of ethyl acetate. The ethyl acetate extracts are dried over magnesium sulfate and removed so that 14 g of 1-[3-(acetylthio)-2-methylpropanoyl-DL-pipecolic acid is obtained as a viscous oil.

Example 32

1-(3-mercapto-2-methylpropanoyl)-DL-pipecolic acid

Aqueous NH^OH (30 ml water and 20 ml conc. NH^OH) is stirred under nitrogen at 10° for 15 minutes. This is added to 13.0 g (0.05 m) of 1-[3-(acetylthio)-2-methylpropanoyl]-DL-pipecolic acid

and the resulting solution is stirred for 10 minutes under nitrogen and then at room temperature for 50 minutes. It is then treated with water and 20% HCl and the yellow oil is extracted with 3 x 150 ml of ethyl acetate. The ethyl acetate extracts are dried over magnesium sulfate and

is removed so that 11.1 g of 1-(3-mercapto-2-methylpropanoyl)-DL-pipecolic acid is obtained as a viscous oil. Rf 0.62 (silica gel, benzene, acetic acid (7:2)).

Example 33

1-(3-mercapto-2-methylpropanoyl)-L-proline

p-Methoxy-α-toluene-thiol (15.4 g, 0.1 mol) is added to

a solution of methacrylic acid (8.6 g, 0.1 mol) in 50 ml of 2N sodium hydroxide. The mixture is heated on a steam bath for three hours and refluxed for two hours and cooled. The mixture is extracted with ether, the aqueous layer is then acidified with concentrated HCl and extracted with dichloromethane. The acidic extracts are washed with saline, dried (MgSO4) and evaporated in vacuo. The resulting semi-solid is taken up in 50 ml of dichloromethane, diluted with 50 ml of hexane and cooled. 3-[(4-Methoxyphenyl)-methylthio]-2-methylpropanoic acid is collected as a white, crystalline solid, m.p. 74-82° (5.5 g).

3-[(4-Methoxyphenyl)methylthio]-2-methylpropanoic acid (3.6 g, 0.015 mol), L-proline tert-butyl ester (2.6 g, 0.015 mol) and dicyclohexylcarbodiimide (3.1 g, 0.015 mol ) is dissolved in 50 ml of dichloromethane and stirred for 30 minutes at 0°. The cooling bath is removed and the mixture is stirred overnight (16 hours). The resulting suspension is filtered and the filtrate is washed with 5% potassium bisulfate, saturated sodium bicarbonate and brine, then dried (MgSO 4 ) and evaporated in vacuo. The resulting clear oil is applied to a 250 ml silica gel column and chromatographed using 20% ethyl acetate/hexane as eluent. The main fraction (Rf = 0.70, silica gel, ethyl acetate) is evaporated to 5.5 g (9 3%) of 1-[3-(4-methoxyphenyl)methylthio]-2-methylpropanoyl-L-proline-tert-butyl ester as a clear oil. Rf = 0.70 (silica gel, ethyl acetate); Rf = 0.60 (silica gel, ether).

The latter ester (1.2 g, 0.003 mol), anisole (5 ml)

and trifluoromethanesulfonic acid (0.5 ml) were dissolved in 20 ml of trifluoroacetic acid under nitrogen, and the resulting red solution was allowed to stand for one hour at room temperature. The solution is evaporated in vacuo to a red residue which is taken up in ethyl acetate and washed with water, brine, then dried (MgSO4) and evaporated. The residue is triturated repeatedly with hexane and the remaining hexane is evaporated. The oil residue amounts to 0.4 g. One portion (180 mg) of this

material is subjected to preparative thin-layer chromatography on 2 mm silica gel plates using benzene/acetic acid 75:25 as eluent. The major nitroprusside-positive band (Rf = 0.40) is recovered to give 135 mg of 1-(3-mercapto-2-methylpropanoyl)-L-proline as an oil. TLC using benzene/acetic acid 75:25 (Rf = 0.40) and chloroform/methanol/acetic acid 50:40:10 (Rf = 0.62).

Example 34

1-(3-mercapto-2-D-methylpropanoyl)-L-proline

Under an argon blanket, 1-[3-(acetylthio)-2-D-methyl-propanoyl]-L-proline (10.0 g) is slurried in water (150 ml) at 10°. 5N sodium hydroxide is added to this mixture, and the pH of the solution is kept at 13 for 1.5 hours. After this time, when no more sodium hydroxide is absorbed, the solution is acidified to pH 2.0 with concentrated sulfuric acid.

The aqueous solution is then extracted three times with methylene chloride (3 x 150 ml) and the combined methylene chloride fractions are concentrated to an oil. The concentrate is taken up in ethyl acetate, filtered and the filtrate is diluted with hexane (30 ml).

A further amount of hexane is added after 1/2 hour,

and the mixture is then cooled to 10° for one hour.

The crystals are filtered off and washed with hexane (2 x 25 mL) and dried to constant weight to give 1-(3-mercapto-2-D-methyl-propanoyl)-L-proline as white crystals, 6.26 g, sm. p. 100-102°.

Example 35

1-(3-Acetylthio-2-methylpropanoyl)-L-proline

1-(3-Tosyloxy-2-methylpropanoyl)-L-proline (3.5 g) is added to a solution of thiolacetic acid (1.14 g) and triethylamine (3.5 ml) in ethyl acetate (20 ml). The solution is kept at 50° for three hours, cooled, diluted with ethyl acetate (100 ml) and washed with dilute hydrochloric acid. The organic layer is dried and concentrated to dryness in vacuo. The residue is dissolved in acetonitrile and dicyclohexylamine is added. The crystalline precipitate is recrystallized from isopropanol to give 1-(3-acetylthio-2-D-methylpropanoyl)-L-proline-dicyclohexylamine salt, m.p. 187-188°,

25 o

[α]Q -67 (c 1.4, EtOH). This salt is converted into the free acid, m.p. 83-85° (an isomorphic form with m.p. 104-105° is obtained

if high-melting material such as germ is added to the crystallizing solution).

Example 36

1-(3-mercaptopropanoyl)-L-proline-t-butyl ester

To a stirred solution of 1.71 g (10 mmol) proline-t-butyl ester and 1.35 g (10 mmol) 1-hydroxybenzotriazole hydrate in 20 ml N,N'-dimethylformamide at 0-5°, is added 2, 06 g (10 mmol) N,N'-dicyclohexylcarbodiimide. The mixture is stirred for 10 minutes, followed by the addition of 1.06 g (10 mmol) of 3-mercaptopropanoic acid in 2 ml of N,N'-dimethylformamide. The mixture is then stirred at 0-5° for one hour and at room temperature overnight.

The precipitated N,N'-dicyclohexylurea is filtered off and the filtrate is concentrated in vacuo. The residue is taken up in ethyl acetate, washed thoroughly with saturated aqueous sodium bicarbonate, dried and concentrated in vacuo to 2.5 g of oil.

The oil is taken up in 1:1 ethyl acetate-hexane and applied to a silica gel column (100 g). Elution with 1:1 ethyl acetate-hexane gives 1.40 g (54%) of 1-(3-mercaptopropanoyl)-L-proline-t-butyl ester as an oil which crystallizes on standing. Recrystallization from ether-hexane gives 0.9 g of colorless, crystalline solid, m.p. 55-60°, identical to the compound from example 8.

Example 37

1-(3-mercaptopropanoyl)-L-proline

A solution of 75 mg (0.27 mmol) of 1-[3-[[(ethylamino)-carbonyl]thio]propanoyl]-L-proline in 1 ml each of concentrated ammonium hydroxide and water is allowed to stand at room temperature for 18 hours under argon. The solution is diluted with a small amount of water and extracted with ether. The aqueous layer is acidified with cold, concentrated hydrochloric acid and extracted with ethyl acetate. The combined extracts are dried and concentrated in vacuo to give a compound identical to the product from Example 9. TLC (silica gel, benzene:acetic acid 7:3) Rf = 0.4.

Example 38

1-(3-Acetylthio-2-D-methylpropanoyl)-L-proline

L-proline (23.0 g, 0.2 mol) is dissolved in 100 ml of water and stirred in an ice bath. Methacryloyl chloride (19.6 ml, 0.2 mol) in 25 ml of methyl isobutyl ketone is added dropwise over three hours. Sodium hydroxide solution (2N) is added at the same time, whereby the pH of the reaction mixture is kept at 7.0. Addition of base is continued for four hours after the addition of the acid chloride has been completed. The reaction mixture is adjusted to pH 5 with concentrated HCl and extracted with ethyl acetate. The aqueous layer is then acidified to pH 2.5 and extracted thoroughly with ethyl acetate. The acidic extracts are washed with saline and dried (MgSO 4 )„ The ethyl acetate solution is treated with dicyclohexylamine (40 ml) and cooled over the rot. The resulting white precipitate is filtered off and dried to give 29 g (39%) of a white solid, m.p. 202-210°.

The solid is crystallized from 1.5 liters of 3:1 acetonitrile/isopropanol to give 19.7 g of methacryloyl-L-proline-dicyclohexylamine salt as fine white needles, m.p. 202-210°.

The salt is dissolved in water/ethyl acetate and the mixture is acidified with concentrated HCl. The resulting suspension is filtered to remove a white fine precipitate which is washed well with ethyl acetate. The filtrate is saturated with sodium chloride and extracted thoroughly with ethyl acetate. The extracts are washed with salt solution, dried (MgSO^

and evaporated to a clear oil which becomes solid. Crystallization from ethyl acetate/hexane gives 7.5 g (83%) of methacryloyl-L-proline as a white, crystalline solid, mp 89-93°. An analytical sample is obtained by recrystallization, m.p. 95-98°.

Methacryloyl-L-proline (183 mg, 0.001 mol) is dissolved in thiolacetic acid (0.5 ml) and allowed to stand at room temperature for 16 hours. The solution is evaporated in vacuo to a yellow residue. Preparative thin-layer chromatography (silica gel, dichloromethane/methanol/acetic acid 90:5:5) enables the isolation of a clear oil (240 mg) as the main fraction. TLC (dichloromethane/methanol/acetic acid 90:5:5) shows that this material is 1-(3-acetylthio-2-DL-methylpropanoyl)-L-proline corresponding to the product from example 15B. R f = 0.35; (benzene/acetic acid 75:2 5) Rf = 0.38.

The oil is dissolved in 3 ml of acetonitrile, treated with dicyclohexylamine until the solution is basic, and cooled» A white, crystalline solid, m.p. 175-181° (106 mg) is collected. Crystallization from isopropanol gives 1-(3-acetylthio-2-D-methylpropanoyl)-L-proline-dicyclohexylamine salt, m.p. 187-188°,• identical to the corresponding product in example 15A.

Example 39

1-(3-mercapto-2-methylpropanoyl)-L-proline

By using 3,3<1->dithiobis-2-methylpropanoic acid instead of 3-acetylthio-2-methylpropanoic acid in the method from example 15B, 1-[dithiobis-(2-methyl-3-propanoyl)]-bis-L-proline is obtained .

Zinc dust (10.0 g) is added to a slurry of the above product (5.0 g) in 100 ml 1N sulfuric acid, and the mixture is stirred at 18° for four hours under a nitrogen blanket. The solution is then filtered, the zinc is washed with water (20 ml) and the combined filtrates are extracted with methylene chloride (3 x 75 ml). The methylene chloride washes are back-extracted with water (25 ml) and the organic solution is then concentrated to an oil. This oil is taken up in ethyl acetate (20 ml) and filtered. Hexane (15 ml) is added to the filtrate and the mixture is stirred for 15 minutes. After this time, additional hexane (30 ml) is added and the solution is cooled to 5° for 1 hour. The mixture is then filtered and the product is washed with hexane (2 x 10 mL) and dried to give 4.17 g of white crystals of the product, 1-(3-mercapto-2-methylpropanoyl)-L-proline. TLC, Rf = 0.60 (Solvent system: benzene/acetic acid 75:25).

Example 40

1-[3-(benzylthio)-2-methylpropanoyl]-L-proline

By using 3-benzylthio-2-methylpropanoic acid instead of 3-[(4-methoxyphenyl)methylthio]-2-methylpropanoic acid in the method from example 33, 1-[3-(benzylthio)-2-methylpropanoyl]-L-proline tert is obtained -butyl ester.

1-[3-(benzylthio)-2-methylpropanoyl]-L-proline tert-butyl ester (7.8 g) is dissolved in a mixture of anisole (55 ml) and trifluoroacetic acid (110 ml). After 1 hour storage at room temperature, the solvent is removed in vacuo and the residue is dissolved in ether, washed several times with saturated sodium chloride, dried over magnesium sulfate and evaporated to dryness in vacuo to give 1-[3-(benzylthio)-2-methylpropanoyl]-L -proline. Rf = 0.5 (silica gel, benzene/acetic acid 3:1), Rf = 0.5 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1).

Example 41

1-(3-mercapto-2-methylpropanoyl)-L-proline

1-[3-(benzylthio)-2-methylpropanoyl]-L-proline (0.1 g) is suspended in boiling liquid ammonia (10 ml) and small pieces of sodium are added with stirring until a persistent blue color. The color is removed with a few ammonium sulphate crystals and the ammonia is allowed to evaporate under a stream of nitrogen. The residue is dissolved in a mixture of dilute hydrochloric acid and ethyl acetate. The organic layer is dried and concentrated to dryness in vacuo to give 1-(3-mercapto-2-methylpropanoyl)-L-proline. Rf = 0.35 (silica gel, benzene/acetic acid 3:1), Rf = 0.5 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1) identical to the compound from example 17.

Example 42

1-(3-mercapto-2-methylpropanoyl)-L-proline

A solution of 3-mercapto-2-methylpropanoic acid (1.2 g) and trityl chloride (2.9 g) in methylene chloride (50 ml) is kept at room temperature for 2 hours. The mixture is heated on a steam bath for 20 minutes and then evaporated to dryness in vacuo and the residue is dissolved in saturated aqueous sodium bicarbonate and the solution is washed with ethyl acetate. The aqueous phase is acidified to pH 3 and extracted with ethyl acetate. The organic phase is dried and concentrated to dryness to give 3-triphenylmethylthio-2-methylpropanoic acid.

Rf =0.8 (silica gel, benzene/acetic acid 3:1).

By using 3-triphenylmethylthio-2-methylpropanoic acid instead of 3-[(4-methoxyphenyl)methylthio]-2-methylpropanoic acid in the method from example 33, second section, 1-[3-(triphenyl-methylthio)-2-methylpropanoyl]- L-proline tert-butyl ester.

3-Triphenylmethylthio-2-methylpropanoic acid (1.8 g) and N,N'-carbonyldiimidazole (0.8 g) are dissolved in tetrahydrofuran (10 ml) with stirring at room temperature. After 20 minutes, the solution is added to a mixture of L-proline (0.6 g) and N-methylmorpholine (1 g) in dimethylacetamide (20 ml). The resulting mixture is stirred overnight at room temperature, concentrated to dryness and the residue dissolved in a mixture of ethyl acetate and 10% aqueous potassium bisulfate. The organic layer is separated and dried and concentrated to dryness in vacuo to give l-[3-

(triphenylmethylthio)-2-methylpropanoyl]-L-proline.

Rf = 0.4 (silica gel, benzene/acetic acid 3:1), Rf 1.0 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1).

1-[3-(Triphenylmethylthio)-2-methylpropanoyl]-L-proline tert-butyl ester (5 g) is dissolved in a mixture of anisole (55 ml) and trifluoroacetic acid (110 ml). After 1 hour of storage at room temperature, the solvents are removed in vacuo and the residue is applied to a silica gel column that is equilibrated with benzene:acetic acid (75:25) and eluted with the same solvent. The fractions corresponding to the component with Rf 0.40 (TLC silica gel.

with the same system) are collected and concentrated to dryness to give α.[3-mercapto-2-methylpropanoyl)-L-proline. Rf 0.62 (silica gel, fchloroform/methanol:acetic acid:water 50:40:10), identical to the compound from example 17.

/

/ Example 4 3

/

/ 1-(3-mercapto-2-methylpropanoyl)-L-proline

To a solution of 3-mercapto-2-methyl-propanoic acid (2.4 g) and freshly distilled 2,3-dihydro-4H-pyran (1.9 g) in benzene (60 ml) is added boron trifluoride etherate (2.8 g) . After 2 hours, potassium carbonate (4 g) is added, the mixture is stirred and filtered. The filtrate is concentrated to dryness to give 3-(tetrahydropyran-2-ylthio)-2-methylpropanoic acid.

By using 3-(tetrahydropyran-2-ylthio)-2-methylpropanoic acid instead of 3-triphenylmethylthio-2-methylpropanoic acid in the method from example 42 second section, 1-[3-(tetrahydropyran-2-ylthio)-2-methylpropanoyl] is obtained -L-proline. Rf: 0.8 (silica gel, benzene/acetic acid 3:1); Rf: 0.75 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1).

A solution of 1-[3-(tetrahydropyran-2-ylthio)-2-methyl-propanoyl)-L-proline (1 g) in a mixture of methanol (25 ml) and concentrated hydrochloric acid (25 ml) is kept at room temperature in 30 minutes. The solvents are removed in vacuo to give 1-(3-mercapto-2-methylpropanoyl)-L-proline. Rf: 0.35 (silica gel, benzene/acetic acid, 3:1), Rf: 0.5 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water, 14:1:2:1), identical to the compound from example 17.

Example 44

1-(3-mercapto-2-methylpropanoyl)-L-proline

3-mercapto-2-methylpropanoic acid (2.4 g) and N-hydroxymethyl-acetamide (1.8 g) are dissolved in trifluoroacetic acid and the solution is kept at room temperature for 1 hour. The trifluoroacetic acid is removed in vacuo and the residue is dried in vacuo over potassium hydroxide so that 3-acetamidomethylthio-2-methylpropanoic acid is obtained.

By using 3-acetamidomethylthio-2-methylpropanoic acid instead of 3-(tetrahydropyran-2-ylthio)-2-methylpropanoic acid in the method from example 43, 1-[3-(acetamidomethylthio)-2-methylpropanoyl]-L-proline is obtained. Rf 0.2 (silica gel, benzene/acetic acid 3:1), Rf 0.3 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1).

1-[3-(acetamidomethylthio)-2-methylpropanoyl]-L-proline (1.4 g) mercury (II) acetate (1.93 g) is dissolved in a mixture of acetic acid (25 ml) and water (25 ml) . After 1 hour of stirring on a steam bath, hydrogen sulphide is bubbled through until no more precipitation of mercury(II) sulphide is observed. The mixture is filtered, the precipitate is washed with ethanol, and the filtrate is concentrated to dryness in vacuo to give 1-(3-mercapto-2-methylpropanoyl)-L-proline.

Rf: 0.35 (silica gel, benzene/acetic acid 3:1); Rf: 0.5 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1), identical to the compound from example 17.

Example 4 5

1-( 3- mercapto- 2- methylpropanoyl)- L- proline- tert-butyl ester

To the cold (5°) solution of 1.2 g (10 mmol) of 3-mercapto-2-methylpropanoic acid and 1.7 g (10 mmol) of L-proline-tert-butyl ester in 25 ml of dichloromethane is added 2.26 g of dicyclohexylcarbodiimide in 5 ml of dichloromethane in portions. After 2 hours at room temperature, 5 drops of acetic acid are added, the mixture is filtered and the filtrate is evaporated to an oil residue. This residue is taken up in 20 ml of petroleum ether-ethyl acetate (3:1) and applied to a 150 ml silica gel column prepared in petroleum ether. The fractions eluted with petroleum ether-ethyl acetate (1:1) contain the product 1-(3-mercapto-2-methylpropanoyl)-L-proline-tert-butyl ester. This fraction (0.6 g) is dried over P2°5 1 vacuum 1 12 hours. Rf 0.6 (silica gel, benzene/acetic acid 3:1), Rf: 0.8 (silica gel, methyl ethyl ketone/

acetic acid/pyridine/water 14:1:2:1).

Example 46

1-(3-mercapto-2-methylpropanoyl)-L-proline

By using 1-(3-mercapto-2-methylpropanoyl)-L-proline-tert-butyl ester instead of 1-(3-mercapto-propanoyl)-L-proline-tert-butyl ester in the method from example 9C, 1-(3 -mercapto-2-methylpropanoyl)-L-proline. Rf 0.35 (silica gel, benzene/acetic acid 3:1), Rf 0.5 (silica gel, methyl ethyl ketone/acetic acid/pyridine/water 14:1:2:1), identical to the compound from example 17.

Example 47

A) 1-(3-acetylthio-2-L-methylpropanoyl)-L-proline

The mother liquor from Example 15A was evaporated to dryness and the residue was triturated with acetonitrile and the resulting crystalline dicyclohexylamine salt was filtered and dried, m.p. 136-138°C. The crystalline dicyclohexylamine salt was converted to the free acid according to Example 15A and recrystallized from 1:1 ethyl acetate-hexane as a crystalline solid with a melting point of 96-97°C.

B) 1-(3-mercapto-2L-methylpropanoyl)-L-proline

Using the product from part A by the method described in Example 17, the title product is obtained which is recrystallized from ethyl acetate-hexane, m.p. 104-105°C,

[α]<p5> -41.1° (c = 2.32, C₂OH).

Example 48

1-( 3- mercapto- 2- D- methylpropanoyl)- L- hydroxyproline

By using the 1-(3-acetylthio-2-DL-methylpropanoyl)-L-hydroxyproline tert-butyl ester from Example 19 and to

following the procedure according to examples 15 and 17, the title product is obtained as a hygroscopic material, [cOD <-1>14°

(c = 1, methanol).

Example 49

1-( 3- mercapto- 2- methylpropanoyl)- L- piperidine- 2- carboxylic acid

By using the method according to example 14 and

using L-piperidine-2-carboxylic acid t-butyl ester instead of L-proline-t-butyl ester, 1-(3-acetylthio-2-methylpropanoyl)-L-piperidine carboxylic acid is obtained which is separated by the method according to example 15 and further as in Example 17 to form the 2-D-methyl isomer, m.p. 85-90°C; CoOq5 -88° (c = 1, ethanol) and the 2-L-methyl isomer as a viscous substance [ci]^ -33° (c=1, ethanol).

Isomer separation was performed on the racemic 1-(3-acetylthio-2-D,L-methylpropanoyl)-L-piperidine-2-carboxylic acid step by forming the dicyclohexylamine salt in acetonitrile,

whereby the 2-D-methyl isomer was precipitated, m.p. 163-165°C as the dicyclohexylamine salt; and the 2-L-methyl isomer was recovered from the acetonitrile filtrate as a viscous residue.

Both isomers were then converted back to

the acid and hydrolyzed to give the D and L isomers defined above.

By using the previously described methods

the following compounds were prepared:

a. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline-adamantyl-amine salt, m.p. 219-221°C (sinters, 227°C). [a]^<5> -53°

(c = 1; C2H5OH).

b. 1-(3-mercapto-2-L-ethylpropanoyl)-L-proline,

sm.p. 120-122°C, [α]^<0> -66.8° (c = 0.6, C^OH).

c. 1-(3-mercapto-2-D-ethylpropanoyl)-L-proline,

sm.p. 100-102°C, [α]p° -79.6° (c = 0.5, C₂OH).

d. 1-(3-mercapto-2-DL-propylpropanoyl)-L-proline, oil, [α]^° -63.1° (c = 0.44, CH 3 OH). e. 1-(3-mercapto-2-D-methylpropanoyl)-4-methyl-L-proline, oil, [a]" -139.7° (c = 1.26, Ct^OH) , f. 1 -(3-mercapto-2-D-methylpropanoyl)-4-heptyl-L-proline, colorless oil, [a]p<5> -74° (c = 1, C^OH) . g. 1-(3 -mercapto-2-DL-methylpropanoyl)-trans-4-hydroxy-L-proline, highly hygroscopic material, -73.5° (c = 0.98, CH3OH). h. 1-(3-mercapto-2- D-methylpropanoyl)-trans-4-hydroxy-L-proline, semisolid. The dicyclohexylamine salt melts at 183-185°C, [a]<25> -119.2° (c = 2, CH3OH). i. 1- (3-Mercapto-2-DL-methylpropanoyl)-trans-5-hydroxy-2-piperidinecarboxylic acid, semisolid, very hygroscopic substance, [α]<25> -44.0° (c = 1, CH3OH).

j. 1-(3-Mercaptopropanoyl)-5-butyl-2-piperidinecarboxylic acid, oil, R- = 0.4 (toluene/acetic acid 9:1).

k. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline-n-propyl ester, liquid, b.p. 148-150°C/10~<4> Dry, [a]^<5> -117.7°

(c = 11.8, C-H..OH).

1. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline-isopropyl ester, liquid, [α] -131.1° (c = 14.3; C2Hj-OH) . m. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline cyclopropyl methyl ester, liquid, [α]^2 -184.9° (c = 15.85, C^OH).

h. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline-n-heptyl ester, liquid, b.p. 167-169°C/0.05 Torr, [a]^<2> -108.7°

(c = 22.65, C2H5OH).

o. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline-n-butyl ester, liquid, b.p. 143-145°C/10~<4> Dry, -118.1°

(c = 12.8, C2H5OH).

p. 1-(3-mercapto-2-D-methylpropanoyl)-L-proline methyl ester, liquid, b.p. 116-118°C/10<-4> Dry, [α]^<5> -132.5° (c = 1.67, C2H5OH).

q. 1-(3-mercapto-2-DL-n-heptylpropanoyl)-L-proline; oil. The adamantylamine salt melts at 159-162°C.

r. 1-(3-mercapto-2-DL-phenylheptylpropanoyl)-L-proline, oil. The adamantylamine salt melts at 104-110°C

pp. 1-(3-mercapto-3-DL-n-heptylpropanoyl)-L-proline, oil. The adamantylamine salt melts at 131-133°C (with sintering).

t. 1-(3-mercapto-propanoyl)-3-heptyl-2-azetidine carboxylic acid as a hygroscopic solid. The adamantyl amine salt melts at 164-167°C.

Claims (4)

1. Analogous method for the preparation of a therapeutically active compound of the formula where R is hydroxy or C₁-C₁ alkoxy; R 1 and R 4 are hydrogen, C 1 -C 4 alkyl, phenyl or phenyl-(C 1 -C 4 )-alkyl; R 2 is hydrogen, C 1 -C 4 alkanoyl or benzoyl; R 1 is hydrogen, hydroxy or C 1 -C 6 alkyl; m is 1, 2 or 3; and n is 0, 1 or 2, characterized in that A) a compound with the formula is reacted with a compound with the formula where X is chlorine, bromine or iodine, followed by reaction with the anion of a thioacid of the formula followed by reaction with the anion of a thioacid of the formula (d) a thiolactone of the formula to form a product where R~ is hydrogen, or (e) a tosyloxyalkanoic acid of the formula followed by reaction with the anion of a thioacid of the formula or B) the sulfur protecting group Y is removed according to conventional methods from a compound with the formula wherein Y is a conventional sulfur-protecting group such as C 1 -C 4 alkanoyl, phenyl 1-(C 2 -C 4 )-alkanoyl, benzoyl, benzyl, methoxybenzyl, triphenylmethyl, tetrahydropyranyl or acetamido-methyl, to form a compound where R 1 is hydrogen. 2. Process as stated in claim 1 for the production of 1-(3-mercapto-2-D-methylpropanoyl)-L-proline, characterized in that starting materials where R is OH, R^ is methyl, R2, R^ and R4 are hydrogen, m is 2 and n is 1, or R, R 2 and/or R 3 are converted thereto. 3, Process as stated in claim 1 for the production of 1-(3-mercapto-2-D-methylpropanoyl)-L-hydroxyproline, characterized in that starting materials are used where R is OH, R1 is methyl, R2 and R4 are hydrogen, R₂ is —OH, m is 2 and n is 1, or R, R₂ and/or R₂ are converted thereto. 4. Process as stated in claim 1 for the production of 1-(3-mercapto-2-D-methylpropanoyl)-trans-4-hydroxy-L-proline, characterized in that starting materials are used where R is OH, R is methyl, R 2 and R 3 are hydrogen, R-j is —OH, m is 2 and n is 1, or R, R 2 and/or R 3 are converted thereto.