NO152650B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE PROLINE DERIVATIVES. - Google Patents

Description

This invention relates to an analog method for . preparation of new thioether-mercaptoacylprolines with the formula

where the group S-R^ is attached to the 3- or 4-position of the ring;

is C 1 -C 4 alkyl or phenyl optionally substituted with a C 1 -C 4 alkyl, C 1 -C 4 alkoxy or halogen;

R 2 is hydrogen or C 1 -C 4 alkyl;

R 4 is hydrogen, R 4 -CO- or

R 5 is C 1 -C 4 alkyl, phenyl or phenyl-C 1 -C 4 alkyl; and

n is 0 or 1;

and basic salts thereof.

The new thioether mercaptoacylprolines of formula I are

therapeutically active, and in particular they are active as antihyper-

tensive agents.

Among "C-1-C4 alkyl", methyl and ethyl are particularly preferred.

The term "halogen" includes the four common halogens,

i.e. chlorine, bromine, fluorine and iodine, as chlorine, bromine and fluorine

is drawn.

The term "phenyl-C₁-C₁ alkyl" as used in defining

tion of the symbol R,., includes such groups as

where m is an integer from 1 to 4.

Preferred phenyl-C₁-C₁ alkyl groups are phenylmethyl and phenylethyl,

especially phenylmethyl.

The lower alkanoyl groups represented by R^-CO- are

the acyl radicals of the lower (C2-C5) fatty acids, e.g. acetyl,

propionyl, butyryl and isobutyryl, and acetyl are particularly

drawn. Similarly, when R5 in the group R5~CO- is phenyl or

phenyl-C 1 -C 4 alkyl, benzoyl is particularly preferred.

The asterisk in formula I denotes an asymmetric center present in the proline ring. Of course, a further asymmetric center can be present in the mercapto side chain depending on the substituent R 2 • The compounds of formula I thus exist in stereoisomeric forms or as racemic mixtures thereof. In the synthesis described below, the racemate or one of the enantiomers can be used as starting materials. When the racemic starting material is used in the synthesis, the stereoisomers obtained in the final product are separated by usual chromatographic methods or by fractional crystallization. The group S-R^ can also lead to cis-trans isomerism.

The asymmetric center in the proline ring is in the L configuration, and if there is an asymmetric center in the mercaptoacyl side chain, it is in the D configuration.

Preferred compounds of formula I are those where R 1 is

alkyl of 1 to 3 carbon atoms or

where R, is

D

hydrogen, methyl, methoxy, chlorine or fluorine;

R 2 is hydrogen or methyl; and

R 4 is hydrogen.

Most preferred are the compounds of formula I where R 1 is methyl or ethyl, especially methyl; n is 1;

R 2 is hydrogen or methyl, especially methyl; R 4 is hydrogen; and the group -SR-^ is in the 4-position in the proline ring, especially where the group -SR^ is in cis-configuration.

The thioethers with formula I are prepared according to the invention by combining the thioether-proline derivative with the formula: with an acid with the formula

or a chemical equivalent thereof, wherein R'4 is hydrogen or

R.--CO-, to form a product of the formula

This reaction can be carried out in the presence of a coupling agent such as dicyclohexylcarbodiimide, or the acid can be activated by forming its mixed anhydride, symmetrical anhydride, acid halide, active ester or by using e.g. Woodward reagent K, or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. An overview of acylation methods is given in Methoden der Organischen Chemie (Houben-Weyl), vol. XV, Part II, pp. 1 et seq. (1974). Preferably, the acid halide, especially the acid chloride, of formula III is reacted with the acid of formula II.

The product of formula IV is preferably isolated and purified by crystallization, e.g. by forming the dicyclohexylamine salt and then converting the salt to the free acid form by treatment with an aqueous solution of an acid, such as potassium hydrogen sulfate.

The product of formula IV, which contains the acyl group R^-CO-, can be converted into the compounds of formula I where R^ is hydrogen, by ordinary hydrolysis or by ammonolysis.

The products of formula I where R^ is

is obtained by oxidizing a product of formula I where R^ is hydrogen directly with iodine. The proline starting materials of formula II can be prepared in various ways. E.g. can a hydroxy- or mercapto-proline with the formula

acylated with an acylating agent such as acetic anhydride,

4

acetyl chloride, propionic anhydride, butyric anhydride, benzyl chloroformate or the like, to protect the nitrogen. The group is then introduced by reacting the N-protected form of the compound of formula V with a halide, R 2 -hal, where hal means a halogen, preferably iodine, in the presence of e.g. silver oxide, sodium hydride, or sodium hydroxide, to form an intermediate of the formula

Alkaline hydrolysis of the intermediate of formula VI with a base such as barium hydroxide, sodium hydroxide or potassium hydroxide first gives the free acid (COOH), and hydrolysis with a mineral acid such as sulfuric acid then gives the starting material of formula II.

Another method for preparing the proline starting materials of formula II is treatment of the N-protected tosyloxy-proline ester, preferably the methyl ester of the formula

with the sodium salt of the formula to form the intermediate of the formula I formula VII represents the symbol Ts

and the N-protecting group is benzyloxycarbonyl, which pre-

is drawn, or other commonly used acyl protecting groups. If the tosylate group in this reaction is in the cis configuration, the SF^ group will be in the trans configuration, and if the tosyloxy group is in the trans configuration, the SR^ group will be in the cis configuration. The intermediate of formula IX is then treated to remove the alkyl ester group and then reacted with hydrogen bromide to form the HBr salt of the proline starting material of formula II, which can then be coupled with the acid, preferably the acid chloride, of formula III.

The proline starting materials with formula II where the SR^ group is bound in the 3-position in the proline, can also be obtained by a 1,2-dehydroproline ester, preferably the t-butyl ester, with the formula

treated with an acylating agent such as benzyl chloroformate, acetyl chloride, etc., to form the 4,5-dehydro compound which is then reacted with the mercaptan R^-SH to give

where the substituent -S-R-^ is in the trans configuration. The N-protecting group and the alkyl group are then removed to

provide the desired starting material.

The proline starting materials of formula II where R-^ is phenyl or substituted phenyl can also be obtained by reacting the benzyl ester of the N-protected proline of formula V with the alcohol R-^-OH in the presence of triphenylphosphine and diethyl azodicarboxylate by the method of Bittner et al., Chemistry and Industry, March 15, 1975, page 281. Removal of the N-protecting group and the benzyl ester group gives the starting material

with formula II.

Reference must also be made to the following publications

which provides further illustrative information with respect to. the preparation of starting materials and intermediates: US Patents 4,046,889, 4,105,776 and 4,154,935; Neuberger,

J. Chem/Soc. 1945, pp. 429-432; Patchett et al., J. Amer. Chem. Soc. 7_ 9, pp. 185-192 (1975); Bauer et al., Can. J. Biochem. and Phys., 37, pp. 583-587 (1959); Sheehan et al.,

J. Amer. Chem. Soc. 85, pp. 3863-3865 (1963); Magerlein, J. Med. Chem. 10, pp. 1161-1163 (1967). The methods illustrated there can be used as general methods for synthesis and stereoconversion of the compounds that can be used in the method according to the invention.

Additional experimental details are shown in the examples which represent preferred embodiments and also serve as models for the preparation of other compounds from the same group.

The new compounds form basic salts with a variety of inorganic or organic bases. The salt-forming ion derived from such bases may be metal ions, e.g. aluminum, alkali metal ions such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion, a number of which are known for this purpose, e.g. aralkylamines such as dibenzylamine, N,N-dibenzylethylenediamine, lower alkylamines such as methylamine,. t-butylamine, procaine, lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines such as cyclohexylamine or dicyclohexylamine, 1-adamantanamine, benzathine, or salts derived from amino acids such as arginine or lysine. The physiologically acceptable salts such as the sodium or potassium salts can be used medicinally as described below, and are preferred. These and other salts which are not necessarily physiologically acceptable are useful for the isolation and purification of a product which is acceptable for the purposes described below, as illustrated by the dicyclohexylamine salt and the cyclohexylamine salt in the examples. The salts are produced by reacting the acid form of the compound with an equivalent of the base that gives the desired basic ion, in a medium in which the salt is precipitated, or in an aqueous medium, after which the product is lyophilized. The free acid form can be obtained from the salt by normal neutralization, e.g. with potassium bisulphate, hydrochloric acid, etc.

The compounds produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II

and are therefore useful in reducing or alleviating hypertension. The compounds produced according to the invention intervene in the course of the reaction renin -»■ angiotensinogen -> angiotensin I angiotensin II by inhibiting the angiotensin-converting enzyme and reducing or eliminating the formation of the pressor compound angiotensin II. By administering a hypotensively effective amount of a preparation containing one or a combination of the novel compounds of formula I or physiologically acceptable salts thereof, hypertension in mammals suffering from it can be reduced or alleviated.

A single dose or preferably 2-4 divided daily doses, given in an amount of approx. 0.1 to approx. 100 mg per kg per day, preferably approx. 1 to approx. 50 mg per kg per day, is suitable for reducing blood pressure as shown by the animal model experiments described by S.L. Engel, T.R. Schaeffer, M.H. Waugh and B. Rubin, Proe. Soc. Exp. Biol. With. 143, 483 (1973). The active compound is preferably administered orally, but parenteral administration such as subcutaneous, intramuscular, intravenous or intraperitoneal may also be used.

The new compounds of formula I can be used to achieve a reduction in blood pressure by being incorporated into such preparations as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About. 10 to approx. 500 mg of a compound or a mixture of compounds of formula I or physiologically acceptable salts thereof are mixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. in a unit dosage form according to accepted pharmaceutical practice. The amount of active substance in these preparations is such that a suitable dosage is obtained in the indicated area.

The potency of angiotensin-converting enzyme (ACE) inhibitors is expressed as the I^^ value, which is the amount of inhibitor

in yg/ml or ymol required to inhibit by 50% the activity of angiotensin-converting enzyme as measured by spectrophotometric measurement.

EC^q, sometimes referred to as IC^q (the concentration of the test compound that produces 50% inhibition of the contractile action of angiotensin I) is determined graphically or biometrically. A minimum of concentrations of the test compound is typically used to construct the concentration-inhibition response curve.

In both compounds the 2-methyl group is in the D configuration.

The following examples shall serve to further illustrate the invention and represent preferred embodiments. They also serve as models for the preparation of other compounds from the same group which can be prepared by replacing the indicated reaction components with suitably substituted analogues. All temperatures are in °C.

Example 1

(a) 1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl] cis- 4-methylthio- L-proline

A solution of 4.67 g (0.029 mol) of cis-4-methylthio-L-proline in 50 ml of water is stirred, cooled to 5°, and 3 g of sodium carbonate are added. This solution is treated with 5.2 g (0.029 mol) of D-3-(acetylthio)-2-methylpropionyl chloride in 5 ml of ether during 10 minutes with occasional addition of 3 g of sodium carbonate to keep the pH at about 8.0. This mixture is stirred in an ice bath for 1.5 hours, 25 ml of water is added,

and then a solution of 6 ml of concentrated hydrochloric acid in 25 ml of water (CO,, evolution). The resulting strongly acidic solution is extracted with 50 ml of ethyl acetate (four times).

The organic phases are combined, dried (MgSO 4 ), filtered, and the solvent evaporated to give the title product.

(b) 1-( D- 3- mercapto- 2- methyl- l- oxo- propyl) cis- 4- methylthio-L- proline

To 1-[D-3-(acetylthio)-2-methyl-1-oxo-propyl]-cis-4-methylthio-L-proline (3.2 g) is added a cold solution of 8 ml of concentrated ammonia in 20 ml water. The base dissolves within approx. 10 minutes, and the resulting solution (under argon) is allowed to stand at room temperature for 2 hours. The solution is cooled, extracted with 20 ml of ethyl acetate (2X), covered with 20 ml of ethyl acetate and acidified with 15 ml of 1:1 hydrochloric acid. This mixture is saturated with sodium chloride, the layers are separated, and the aqueous phase is extracted with 20 ml of ethyl acetate (3X). The organic phases are combined, dried (MgSO 4 ), filtered, and the solvent evaporated to give the title product, m.p. as 1-adamantanamine salt 198-201°C (decomp.); [a]p<0> -20°

(c=1% in methanol).

Example 2

1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- cis- 4- phenylthio- L- proline (a) N- carbobenzyloxy- cis- 4- phenylthio- L- proline- methyl ester

Sodium metal (0.85 g, 0.037 mol) is dissolved in 40 ml of absolute ethanol. To this solution is added, with stirring, 3.7 ml (0.036 mol) of thiophenol followed by 7.5 g (0.017 mol) of N-carbobenzyloxy-trans-4-tosyloxy-L-proline methyl ester [J. Am. Chem. Soc, 7-9, 191 (1957)]. The latter gradually dissolves, but soon afterwards a solid product is secreted. After stirring for 4 hours and standing overnight, most of the ethanol is removed on a rotary evaporator. The mainly solid residue is stirred with 120 ml of dichloromethane and 60 ml of water. The layers are separated (some methanol is added to break up emulsions), and the aqueous phase is extracted with additional dichloromethane (2 x 60 ml). The combined organic phases are washed with 100 ml saturated sodium chloride solution, dried (MgSO4),

and the solvent is evaporated to give 6.5 g (100%) of N-carbobenzyloxy-cis-4-phenylthio-L-proline methyl ester as a pale yellow viscous oil.

(b) N-carbobenzyloxy-cis-4-phenylthio-L-proline

The methyl ester product from part (a) (6.5 g, 0.017 mol) is dissolved in 55 ml of methanol, treated portionwise at -1 to 4° with 13 ml (0.02 6 mol) of 2N sodium hydroxide, stirred at 0° for 1 hour and kept at room temperature for approx. 16 hours. After approx. half of the solvent is removed on a rotary evaporator, the cooled solution is diluted with 100 ml of water, washed with 60 ml of ether (the washings are discarded), covered with a layer of 70 ml of ethyl acetate, stirred, cooled and acidified with 4.8 ml of 1:1 hydrochloric acid. After separation, the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml), and the combined organic layers are dried (MgSO 4 ) and evaporated to give 5.9 g of a pale yellow viscous oil. The latter is dissolved in 30 ml of ethanol, treated with 1.9 g of cyclohexylamine in 3 ml of ethanol and diluted to 330 ml with ether. During inoculation, the crystalline cyclohexylamine salt is secreted. After cooling for approx. 16 hours, the latter weighs 5.3 g, m.p. 148-151° (p. 135°). This material is mixed with 1.5 g of identical product from a previous experiment, stirred with 200 ml of boiling acetonitrile and cooled to give 6.3 g of colorless cyclohexylamine salt; sm.p. 152-155°

(p. 137°) [ a]* 6 -24° (c, 1% in ethanol).

This cyclohexylamine salt is suspended in 25 ml of ethyl acetate, stirred and treated with 25 ml of N hydrochloric acid. When two clear layers are obtained, they are separated and the aqueous phase is extracted with additional ethyl acetate (3 x 25 mL). The dried organic layers are dried (MgSO 4 ) and the solvent is evaporated to give 5.0 g (65%) of N-carbobenzyloxy-cis-4-phenylthio-L-proline as an almost colorless, very viscous syrup.

(c) (cis)-4-phenylthio-L-proline hydrobromide

N-Carbobenzyloxy-cis-4-phenylthio-L-proline (4.9 g, 0.014 mol) is treated with 25 ml of hydrogen bromide in acetic acid (30-32%), loosely covered and magnetically stirred. After 1 hour, the orange-yellow solution is diluted to 250 ml with ether to precipitate the product as a heavy oil which gradually crystallizes on seeding, rubbing and cooling. After stirring in an ice bath for 1 hour, the material is filtered under nitrogen, washed with ether, suspended in fresh ether, cooled for approx. 16 hours and filtered again to give 3.2 g (77%) colorless solid (cis)-4-phenylthio-L-proline hydrobromide, m.p. 106-109° (p. 99°), [a]^ -3° (c, 1% in methanol).

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

Reaction of 3.0 g (0.0094 mol) (cis)-4-phenylio-L-proline hydrobromide and 2.0 g (0.011 mol) D-3-acetylthio-2-methyl-propionyl chloride in 25 ml of water as described in Example 1, part (a) (using about 15 ml of 20% sodium carbonate solution to maintain the pH at 8.0 to 8.4), gives 3.8 g of a pale yellow viscous oil . The dicyclohexylamine salt (prepared in 30 ml of ethyl acetate using 1.8 g of dicyclohexylamine) weighs 2.9 g (isolated in two portions), m.p. 184-188° (p. 180°). After rubbing with 15 ml of acetonitrile, 2.4 g of colourless, solid dicyclohexylamine salt are obtained, m.p. 184-186° (p. 180°), [α]p<6> -75° (c, 1% in ethanol).

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

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

1-[D-3-(acetylthio)-2-methyl-l-oxopropyl]-cis-4-phenylthio-L-proline (2.0 g, 0.0042 mol) is treated with 3.5 ml of concentrated ammonia for 8 .5 ml of water by the procedure of Example 1, part (b) (the solid ammonium salt of the product is separated from the reaction mixture) to give 1.35 g (100%) of viscous, syrupy 1-(D-3-mercapto-2 -methyl-1-oxopropyl)-cis-4-phenylthio-L-proline, [α]<ø6> -43° (c, 1% in ethanol). Sm.p. 126-128°C,

[a]^ -43.5° (c=1% in methanol), as the L-arginine salt (1:1).

Example 3 (Racemic, must be separated to give the L-proline product)

( trans)- 1-( 3- mercaptol- l- oxopropyl)- 3- methylthio- D, L- proline (a) 1, 2- dehydroproline- t- butyl ester

21.7 g (23.9 ml, 0.20 mol) freshly prepared t-butyl hypochloride [Org. Syn., Coll. Vol. V, 184, (1973)]. During the addition, the temperature is maintained at -5 to 0°. After

that the addition is complete, the solution is stirred at this temperature for a further 5 minutes.

Add quickly (approx.

3-5 min.) in a solution of 7.8 g (0.20 mol) potassium in freshly distilled dry (CaH2) t-butanol. After the addition, the temperature of the reaction mixture is approx. 18°. The reaction vessel is removed from the cooling bath, and stirring is carried out for 30 minutes. The reaction mixture is filtered through "Celite" (date earth),

and the filtrate is concentrated in vacuo. The residue is taken up in ether

and washed with several portions of water. The ether solution is dried and concentrated in vacuo to 31.6 g of yellow liquid. A trace of hydroquinone is added and the crude product is distilled to give 22.4 g of 1,2-dehydroproline-t-butyl ester (66%), m.p. 60-62°/-

0.1 mm.

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

A solution of 16.9 g (0.1 mol) of 1,2-dehydroproline-t-butyl ester in 70 ml of dichloromethane is cooled to -10° under argon.

A solution of freshly distilled benzyl chloroformate (14.2 ml,

0.1 mol, b.p. 62-64° (0.4 mm)) in 70 ml of dichloromethane is added dropwise over a period of 30 minutes. After stirring while cooling for a further 30 minutes, a solution of 15.22 g (0.1 mol) of 1,5-diazabicyclo[5.4.0]undec-5-ene in 70 ml of dichloromethane is added over a period of 20 minutes. The cooling bath is then removed, and the mixture is stirred at room temperature for 1 hour. After washing twice with cold, diluted hydrochloric acid and a

once with saturated sodium carbonate solution, the solution is dried in vacuo to give 18.2 g (60%) of 1-benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid t-butyl ester as a pale oil.

(c) ( trans )- 1- benzyloxycarbonyl- 3- methylthio- D, L- proline- t -butyl ester

A solution of 18.2 g (0.06 mol) of 1-benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid t-butyl ester in 180 ml of dry methanol is treated with 3.24 g (0.06 mol ) sodium methoxide and cooled in an ice bath. Methanethiol is slowly bubbled into the solution for 30 minutes. The mixture is stirred overnight under argon at room temperature. Dilute aqueous acetic acid is added until the solution is slightly acidic. Argon is bubbled through the solution for 1 hour before the solution is brought to near dryness in vacuum. Ethyl acetate is added and the solution is washed twice with saturated sodium carbonate solution, dried and desolvated in vacuo to give 17 g of yellow oil. This oil is chromatographed using 300 g of silica gel and petroleum ether: ether (4:1) to give 11.1 g of (trans)-1-benzyloxycarbonyl-3-methylthio-D,L-proline as a colorless oil.

(d) (trans)-3-methylthio-D,L-proline

8.4 g (0.024 mol) (trans)-1-benzyloxycarbonyl-3-methyl-

thio-D,L-proline is treated with 45 ml of 4N hydrobromic acid in acetic acid. After stirring for 1 hour at room temperature, the solution is dried in a vacuum. A small amount of water is added and washing is carried out twice with ether. The aqueous solution is applied to a column containing 300 ml of an ion exchange resin, and water is passed through until the eluate is no longer strongly acidic. The product is then eluted with pH 6.5 (aqueous pyridine acetate) buffer. Fractions positive for ninhydrin are combined and lyophilized to yield 3.4 g (88%)

of a white, downy substance. A small sample of this material is crystallized from methanol to give (trans)-3-methylthio-D,L-proline, m.p. 196-200° (spltn.) (p. 192°).

Analysis:

Calculated for CgtL^O^NS: C 44.70, H 6.88, N 8.69, S 19.89 Found: C 44.53, H 7.10, N 8.61, S 19.95.

(e) (trans)- 1-[ 3-( acetylthio)- 1- oxopropyl]- 3- methylthio- D, L-proline

3.05 g (0.019 mol) (trans)-3-methylthio-D,L-proline is dissolved

in 19 ml IN sodium carbonate and dilute with 10 ml water. The solution is cooled in an ice bath, and a solution of 3-acetylthiopropionyl chloride is quickly added while stirring. in 20 ml of ether. The pH value is maintained at 8 by adding IN sodium carbonate. After 30 minutes, the pH value remains constant,

and 4 5 ml sodium carbonate solution is added. The layers are separated, and the aqueous layer is washed once with ether. The aqueous layer is then acidified with 10% potassium bisulfate solution, and the product is extracted into ethyl acetate, dried and desolvated in vacuo to give 5.2 g of oil. The material is chromatographed on 150 g of silica gel using ethyl acetate for elution. 3.85 g of partially crude (trans)-1-[3-(acetyl-thio)-1-oxopropyl]-3-methylthio-D,L-proline are obtained.

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

Analysis:

Calculated for C^H^O^S' (CgH^) 2NH: C 58.44, H 8.53, N 5.83,

S 13.57

Found: C, 58.77, H 8.57, N 5.68,

S 13.74.

(-f) (tran~ s1^ 1^( 3- mercapto^ l- oxopropyl) - 3- methylthio- D, L-proline

2.05 g (0.007^mol) (trans)-1-[3-(acetylthio)-1-oxopropyl]-3-methylthio-D,L-proline is cooled in an ice bath under argon and treated with a cold, argon-saturated mixture of 7 ml of water and 7 ml of concentrated ammonia. After stirring for 30 minutes at 0°, the solution is acidified with hydrochloric acid. The product is extracted into ethyl acetate, dried and freed from solvent in vacuo to give 1.85 g of material which becomes partially crystalline on standing. Trituration with ether gives 1.0 g (57%) of white, crystalline product. Recrystallization from ethyl acetate (5 ml) gives 0.85 g of (trans)-1-(3-mercapto-1-oxopropyl)-3-methylthio-D,L-proline, m.p. 89-93°.

Analysis:

Calculated for C9H15<0>3NS2: C 43.35, H 6.06, N 5.62, S 25.72 Found: C 43.35, H 6.27, N 5.54, S 25.91. Example 4 (cis)-4-[(4-fluorophenyl)thio]-1-(D-3-mercapto-2-methyl-1-oxopropyl)-L-proline, L-arginine (1:1) salt (a ) ( trans)- 1-[ D- 3~( benzoylthio)- 2- methyl- l- oxopropyl]- 4-hydroxy- L- proline, methyl ester (D)-3-(benzoylthio)-2-methylpropanoic acid (56, 05 g, 0.25 mol) is suspended in 62.5 ml of toluene. The temperature drops to 15°, and then 0.386 ml of dimethylformamide is added. Then 32.7 g (0.25 mol + 10% excess) of thionyl chloride are added all at once while stirring. The temperature of the reaction mixture drops to 12°, and the container used to measure out the thionyl chloride is filled with 21.2 ml of toluene which is then added to the reaction mixture. The temperature is gradually raised to 35° and held there for 1 hour. The reaction mixture is stirred at room temperature overnight. The solvent and excess thionyl chloride are removed, and the residue is treated twice with 100 ml of toluene, after which the toluene is removed to give 63.4 g of (D)-3-(benzoylthio)-2-methylpropanoic acid chloride. L-4-hydroxyproline (32.7 g, 0.25 mol) is dissolved in 250 ml of water at pH 5.8. About. 60 ml of 10% sodium carbonate is added to bring the pH to 9.3. The solution is heated to 30°, and a toluene solution (75 ml) containing 63 g of (D)-3-(benzoylthio)-2-methylpropanoic acid chloride is added simultaneously with 10 % aqueous sodium carbonate over 1 hour at 30° while maintaining a pH of 9.0. The solution is stirred at pH 9.0 for 1.5 hours, and the toluene layer is separated. The aqueous layer is made strongly acidic with concentrated HCl, and the crystalline solid is filtered, washed with water and air dried to give 71.4 g of (trans)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-hydroxy-L- proline, m.p. 195-196°. Recrystallization from alcohol gives m.p. 196-197°; [a]^5 -139° (c=l,. methanol). Analysis: Calculated for C16H19N05S: N 4.15, C 56.95, H 5.67, S 9.50 Found: N 3.96, C 56.56, H 5.77, S 9.50.

(b) N-(p-fluorophenylthio)succinimide

By following the procedure of Y. Abe et al., Bull. Chem. Soc. Japan, Vol. 46, p. 1898 (1973), a suspension of 12.1 g (90.8 mmol) of N-chlorosuccinimide in 500 ml of benzene is treated under nitrogen with a solution of 9.7 g (75.7 mmol) of p-fluorothiophenol dissolved in 90 ml of benzene. A moderate increase in temperature is observed, and the reaction mixture turns yellow-orange. After 75 minutes, the solution is cooled to 15°C and treated with 9.19

g (90.8 mmol) of triethylamine in 90 ml of benzene added over a period of 25 minutes. The cooling bath is removed and the reaction mixture is stirred for 45 minutes, then filtered and the organic solution is rinsed with three 150 ml portions of water, brine and dried (MgSO 4 ). Removal of the solvents in vacuo gives 15.8 g of crude product. Recrystallization from chloroform/hexane gives 11.6 g of N-(p-fluorophenylthio)succinimide, m.p. 130-138°.

(c) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-[( 4- fluorophenyl) thio]- L- proline, methyl ester

A solution of 11.0 g (48.8 mmol) of N-(p-fluorophenylthio)-succinimide in 200 ml of benzene under an atmosphere of nitrogen is treated with 9.87 g (48.8 mmol) of tri-n-butylphosphine in 25 ml benzene. The resulting dark solution is stirred for 10 minutes at room temperature, and then 15.6 g

(44.4 mmol) (trans)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-hydroxy-L-proline, methyl ester. After stirring overnight at room temperature, the reaction mixture is diluted with 200 ml of ether and rinsed with four 75 ml portions of water, saline,

and dried (MgSO^). Removal of the solvents in vacuo

gives 32.6 g of raw product which is absorbed in approx. 70 g of silica gel and chromatographed on 450 g of Baker silica gel packed and eluted with two liters of 1:1 Et20:hexanes, then eluted with two liters of 2:1, Et20:hexanes and finally with 100% ether. The fractions containing the best product are combined to give 16.7 g of (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-[(4-fluorophenyl)thio] -L-proline, methyl ester as an oil;

2 5

[α]D -80.2 (c=1, chloroform).

(d) ( cis )- 4-[( 4- fluorophenyl) thio]- 1-( D- 3- mercapto- 2- methyl- l-oxopropyl)- L- proline, L- arginine ( 1:1) salt

A solution of 15.7 g (34.0 mmol) of (cis)-1-[D-3-(benzoyl-thio)-2-methyl-1-oxopropyl]-4-[(4-fluorophenyl)thio]- L-proline, methyl ester in a mixture of 50 ml of methanol and 150 ml of tetrahydrofuran is cooled in an ice bath under an atmosphere of nitrogen. The cold solution is treated with 71.4 mL of 1N aqueous sodium hydroxide added over a period of 10 minutes. The cold bath is removed and the reaction mixture is stirred at room temperature for 5.5 hours, then diluted with 300 ml of water and washed with three 200 ml portions of ether. The aqueous layer is cooled under nitrogen in an ice bath and acidified to pH 1-2 with concentrated aqueous HCl, and then extracted with three 200 mL portions of ether. The combined organic extracts are rinsed with 200 ml of water and saline and dried (MgSO 4 ). Removal of solvents in vacuo gives 11.7 g of crude product. This material is dissolved in ether and treated with a solution of 5.66 g (37.4 mmol) of 1-adamantanamine in methanol. Dilution with ether gives 15.2 g of the adamantanamine salt which is recrystallized by dissolution in (1:1) chloroform:methanol and dilution with ether. This procedure leads to 13.3 g of the adamantanamine salt; sm.p. 238-240° (d). Two further recrystallizations from chloroform-methanol-ether give 10.6 g of (cis)-4-[(4-fluorophenyl)thio]-1-(D-3-mercapto-2-methyl-1-oxopropyl)-L-proline , adamantamine salt, m.p. 242-243° (d); [α]^<5> -51.8° (c=0.5, methanol).

A sample of this adamantamine salt is extracted with ethyl acetate from 1N aqueous HCl to give (cis)-4-[(4-fluorophenyl)thio]-25 o 1-(D-3-mercapto-2-methyl-1-oxopropyl)- L-proline; [a]D -42.5 (c=1, absolute ethanol).

A rapidly stirred mixture of 20 mL of 1N HCl, 40 mL of water, and 60 mL of ethyl acetate is treated with 8.0 g (16.2 mmol) of (cis)-4-[(4-fluorophenyl)thio]-1-(D -3-mercapto-2-methyl-1-oxopropyl)-L-proline, adamantanamine salt which is added portionwise. The method described above is carried out under nitrogen with ice bath cooling. The aqueous layer is extracted with two further 60 ml portions of ethyl acetate, and the combined organic extracts are washed with dilute aqueous HCl, water and saline. After drying (MgSO 4 ), removal of the solvents in vacuo gives 5.78 g of the liberated acid as a crude oil. The oil is dissolved in a mixture of 40 ml of ethyl acetate and 10 ml of ether. This solution is shaken in a rinsing funnel with 2.68 g (15.4 mmol) of L-arginine dissolved in 50 ml of water. The aqueous layer is rinsed again with ether, placed in a rotary evaporator for 15 minutes at low vacuum and finally lyophilized overnight to give 7.97 g of (cis)-4-[(4-fluorophenyl)thio]-1-(D -3-mercapto-2-methyl-1-oxopropyl)-L-proline, L-arginine salt (1:1);

25

[a]D -42.7 (c=1, water).

Analysis:

Calculated for C15HlgFN03S2•C6H14N4O2: C 47.08, H 6.40, N 13.08,

S 11.97, F 3.55, SH 6.17. Found: C 47.21, H 6.74, N 13.28,

S 11.71, F 3.38, SH 5.98.

Example 5

( cis )- 4-[( 4- chlorophenyl) thio]- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)-L- proline, L- arginine salt ( 1:1)

(a) N-(p-Chlorophenylthio)succinimide

Following the procedure of Y. Abe et al., supra, 4.3 g (33.2 mmol) of N-chlorosuccinimide and 1800 ml of benzene are treated with 40.0 g (27.7 mmol) of p-chlorothiophenol dissolved in 360 ml benzene. After one hour, the resulting orange solution is cooled

to 15°, and 33.6 g (33.2 mmol) of triethylamine in 360 ml of benzene are added over a period of 15 minutes. The cooling bath is removed, and the reaction mixture is stirred at room temperature for a further 45 minutes. The reaction mixture is then filtered, and the organic solution is washed with three 500 ml portions of water,

500 ml salt solution and dried (MgSO 4 ). Removal of the solvents in vacuo gives 55.1 g of crude product. Trituration with 900 ml of petroleum ether gives 42.0 g of N-(p-chlorophenylthio)succinimide, m.p. 135-152°.

(b) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-[( 4-chlorophenyl) thio]- L- proline, methyl ester

12.6 g (62.3 mmol) of tri-n-butylphosphine dissolved in 25 ml

benzene is added to a solution of 15.1 g (62.3 mmol) of N-(p-chlorophenylthio)succinimide in 250 ml of benzene at room temperature under a nitrogen atmosphere. After 10 minutes, 20 g (56.9 mmol) of (trans)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-hydroxy-L-proline, methyl ester are added, and the reaction mixture is kept at room temperature under nitrogen for 2 days. At the end of this time, the reaction mixture is diluted with 300 ml of ether and washed with four 100 ml portions of water, brine and dried (MgSO 4 ). Removal of the solvents in vacuo gives 46 g of crude material which is absorbed in approx. 75 g of silica gel and chromatographed on a 450 g Baker silica gel column.

The column is packed and eluted with ether, and the fractions containing the best product are collected to give 27.2 g of product.

A small amount (0.48 g) of the above is flash chromatographed on 25 g EM9385 silica gel packed and eluted first with methylene chloride followed by 18:1 methylene chloride:ethyl acetate to give 0.28 g of (cis)-1-[D-3- (benzoylthio)-2-methyl-1-oxopropyl]-4-[(4-chlorophenyl)-2 5

thio]-L-proline, methyl ester; [<*]D -86.6 (c=1, chloroform).

(c) ( cis )- 4-[( 4- chloro-phenyl) thio]- 1-( D- 3- mercapto- 2- methyl- l-oxopropyl)- L- proline, L- arginine salt ( 1:1)

A solution of 24.3 g (50.8 mmol) of (cis)-1-[D-3-(benzoyl-thio)-2-methyl-1-oxopropyl]-4-[(4-chlorophenyl)thio]- L-proline, methyl ester in a mixture of 80 ml of ethanol and 220 ml of tetrahydrofuran is cooled in an ice bath under nitrogen and treated with 106 ml of IN aqueous sodium hydroxide which is added over a period of 10 minutes. The cooling bath is removed, and the reaction mixture is stirred at room temperature for 5.5 hours, then diluted with 500 ml of water and washed with three 300 ml portions of ether. The aqueous* solution is cooled in an ice bath under nitrogen and acidified to pH 1-2 with concentrated HCl and extracted three times with 500 ml portions of ether. The combined ether extracts are washed with 400 ml of water and brine, then dried (MgSO 4 ) and concentrated in vacuo to 18.2 g of crude product. This material is dissolved in ether and treated with a solution of 8.5 g (55.9 mmol) of 1-adamantanamine in methanol. The mixture is diluted to approx. 1 liter of ether, cooled for 1.5 hours and filtered to give 20.1 g of adamantanamine salt, m.p. 239-242°. This material is dissolved in 1:1 chloroform:methanol and diluted with ether to give 15.3 g of (cis)-4-[(4-chlorophenyl)thio]-(D-3-mercapto-2-methyl-1-oxopropyl )-L-proline, 1-adamantanamine salt; sm.p. 240-241 o (d); [a]Q 2 5 -55.4<C>

(c=0.5, methanol).

A small sample of this adamantanamine salt (0.5 g,

0.98 mmol) is partitioned between aqueous HCl and ethyl acetate to give the corresponding free acid; ta^5 -38.1° (c=1, absolute ethanol).

To a rapidly stirred mixture of 18.8 mL of 1N aqueous HCl,

8 g (15.6 mmol) of the adamantanamine salt are added in portions to 30 ml of water and 50 ml of ethyl acetate. The solution is kept in an ice bath and covered with a layer of nitrogen. After all the solid has dissolved, the aqueous layer is extracted with two further 50 ml portions of ethyl acetate. The organic extracts are mixed and washed with 30 ml of 0.5N aqueous HCl, 30 ml of water and finally saline solution. The solution is dried (MgSO 4 ) and concentrated in vacuo to give 5.6 g of liberated acid. The acid is dissolved in a mixture of 50 ml of ethyl acetate and 15 ml of ether and shaken with a solution of 2.57 g (14.8 mmol) of L-arginine dissolved in 65 ml of water. The aqueous layer is washed with 50 ml of ether and lyophilized overnight to give 8.0 g of (cis)-4-[(4-chlorophenyl)thio]-1-(D-3-mercapto-2-methyl-1-oxopropyl )-L-proline,

25

L-arginine salt (1:1); [a]D -44.3 (c=1, water).

Analysis:

Calculated for C15HlgClN03S2•cgH14N4°2:

C 45.09, H 6.27, N 12.52, S 11.46, Cl 6.34, SH 5.91 Found: C 45.34, H 6.34, N 12.57, S 11.48 , Cl 6.12, SH 5.44.

Example 6

( cis )- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[( 4- methylphenyl)-thio]- L- proline, L- arginine- salt ( 1:1)

(a) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-[( 4- methylphenyl) thio]- L- proline, methyl ester

A solution of 12.6 g (62.5 mmol) of tri-n-butylphosphine

in 20 ml of benzene is added over a five-minute period to a solution of 20 g (56.8 mmol) of (trans)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-hydroxy- L-proline, methyl ester and 15.4 g (62.5 mmol) of p-tolyl disulfide in 120 ml of benzene at room temperature under an atmosphere of nitrogen. After stirring overnight, the reaction mixture is diluted with 300 ml Et 2 O, and the organic solution is washed with three 100 ml portions of water, brine and dried (MgSO 4 ). Concentration in vacuum gives 49 g of crude product mixture. This material is absorbed on 75 g Baker silica gel and added to a 450 g Baker silica gel column packed in 1:1,

hexanes: ether. The column is eluted with 3 liters of 1:1 followed by 2 liters of 2:1, ether:hexanes and finally with 100% ether. Collection of the fractions containing the best product gives 14.6 g of (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-[(4-methylphenyl)thio]-L- proline, methyl ester. A small sample is purified by flash chromatography on EM9385 silica gel using methylene chloride: ethyl acetate; [α]^ -70.8° (c=1, chloroform).

(b) ( cis )- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[( 4- methylphenyl) thio]- L- proline, L- arginine salt ( 1:1)

An ice-cold solution of 13.0 g (28.4 mmol) of (cis)-l-[D-3-(benzoylthio)-2-methyl-l-oxopropyl]-4-[(4-methylphenyl)thio]-L -proline, methyl ester in a mixture of 40 ml of methanol and 130 ml of tetrahydrofuran is continuously flushed with nitrogen while 59.6 ml of IN aqueous sodium hydroxide is added over a period of 10 minutes. At the end of this time, the cooling bath is removed, and the reaction mixture is stirred under nitrogen at room temperature for 5.5 hours. The reaction mixture is then diluted with 250 ml of water and washed twice with 200 ml portions of ether. The aqueous layer is then cooled in an ice bath under nitrogen and acidified to pH 1-2 with concentrated HCl and extracted with three 200 mL portions of ether. The combined organic extract is washed with 200 ml of water and brine, dried (MgSO 4 ) and concentrated in vacuo to 10.6 g of colorless oil which is almost homogeneous at tic. The product is dissolved in ether and treated with 4.72 g (31.2 mmol) of 1-adamantanamine dissolved in 20 ml of methanol. Clearing occurs quickly, and the solution is diluted with ether to approx. 700 ml total volume, and the salt is collected. New trituration with hot methanol-ether gives 12.4 g of adamantanamine salt, m.p. 237-240° (d). This salt is recrystallized by dissolving in a minimum amount of 1:1 chloroform:methanol and diluting with ether. After 10 minutes at room temperature, the solid is collected and dried to give 8.2 g of adamantanamine salt,

sm.p. 240-241° (d); [α]^<5> -58.0° (c=0.5, methanol).

A small sample of this adamantanamine salt is partitioned between aqueous HCl and ethyl acetate to give (cis)-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[(4-methylphenyl)thio]- L-25 etc

proline as an oil; [a]D -45.1 (c=1, absolute ethanol).

To a mixture of 17.1 ml of aqueous 1N HCl, 30 ml of water and 50 ml of ethyl acetate, 7.0 g (14.3 mmol) of the above-mentioned adamantanamine salt are added in several portions. During the addition, the solution is cooled in an ice bath and kept under a blanket of argon. When all the salt has dissolved, the layers are separated, and the ethyl acetate layer is mixed with two further 50 ml extractions of the aqueous solution. The combined ethyl acetate extract is washed with dilute HCl, water and brine, dried (MgSO 4 ) and concentrated in vacuo to give the liberated acid. The acid is dissolved in a mixture of 40 ml of ethyl acetate and 10 ml

ether and shake with a solution of 2.37 g (13.6 mmol) of L-arginine in 50 ml of water. The aqueous layer is washed with 50 mL of ether and then lyophilized to give (cis)-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[(4-methylphenyl)thio]-L-proline , L-arginine salt (1:1); [alp<5> -45.3° (c=1, water).

Analysis:

Calculated for ci6H2iN03S2'C6H14<N>4°<2:>

C 49.69, H 7.01, N 13.17, S 12.06, SH 6.22 Found: C 49.55, H 7.04, N 13.28, S 11.73, SH 5.97 .

Example 7

( cis )- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[( 4- methoxy-phenyl) thio]- L- proline, L- arginine- salt ( 1:1)

(a) N-(p-Methoxyphenylthio)succinimide

A mixture of 17.1 g (128 mmol) of N-chlorosuccinimide and

700 ml of benzene is treated with 15 g (107 mmol) of p-methoxythiophenol dissolved in 125 ml of benzene. After 75 minutes, the resulting orange solution is cooled to 15°, and 13.0 g

(128 mmol) of triethylamine in 125 ml of benzene is added over a period of 25 minutes. The cooling bath is removed, and the reaction mixture is stirred at room temperature for a further 45 minutes. The reaction mixture is then filtered, and the organic solution is washed with three 200 ml portions of water, 200 ml of brine and dried (MgSO 4 ). Removal of the solvents in vacuo gives 27.0 g of crude product. Recrystallization from chloroform-hexane gives 21.2 g of N-(p-methoxyphenylthio)succinimide; sm.p. 100-106°.

(b) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-[( 4- methoxy) phenylthio]- L- proline, methyl ester

12.0 g (59.1 mmol) of tri-n-butylphosphine dissolved in 25 ml of benzene. After 10 minutes, 18.9 g (53.7 mmol) of (trans)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-hydroxy-L-proline, methyl ester, and the reaction mixture are added allowed to stand at room temperature under nitrogen for 25 hours. At the end of this time, the reaction mixture is diluted with 300 ml of ether

and washed with three 100 ml portions of water, saline and dried (MgSO4). Removal of the solvents in vacuo gives 40 g of crude material which is absorbed in approx. 80 g of silica gel and chromatographed on a 450 Baker silica gel column. The column is packed and eluted with a 4:1 mixture of hexane:ether, followed by 2:1 hexane:ether and finally 1:1 hexane:ether. The fractions containing the best product are pooled to give 20.6 g of product. A small sample (0.5 g) of the product is further purified by flash chromatography on 50 g EM9385 silica gel packed and eluted with ether. The fractions containing the best product are collected and give 480 mg of (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxo-propyl]-4-[(4-methoxyphenyl)thio]-L- proline, methyl ester; [Q]D

31 66.9° (c=1, chloroform).

(c) ( cis )- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[( 4-methoxyphenyl) thio]- L- proline, L- arginine- salt ( 1:1)

A solution of 20.1 g (42.4 mmol) of (cis)-1-[D-3-(benzoyl-thio)-2-methyl-1-oxopropyl]-4-[(4-methoxyphenyl)thio]- L-proline, methyl ester in a mixture of 70 ml methanol and 200 ml tetrahydrofuran is cooled in an ice bath under nitrogen and treated with 89 ml 1N aqueous sodium hydroxide over a period of 10 minutes. The cooling bath is removed, and the reaction mixture is stirred at ambient temperature for 5.5 hours, then diluted with 500 ml of water and washed with three 300 ml portions of ether. The aqueous solution is cooled in an ice bath under nitrogen and acidified to pH 1 with concentrated HCl and saturated with three 500 ml portions of ether. The combined ether extracts are washed with 400 ml of water and brine, dried (MgSO 4 ) and concentrated in vacuo to give 14.8 g of crude product. This material is dissolved in ether and treated with a solution of 6.34 g (42

mmol) 1-adamantanamine in methanol. The mixture is diluted to approx. 1 liter of ether, allowed to stand at room temperature for 10 minutes and filtered to give 17.8 g of adamantanamine salt, m.p. 235-236° (decomp.). This material is dissolved in 1:1 chloroform:methanol with gentle heating and diluted with ether.

This recrystallization is repeated two more times to

yield 12.2 g of (cis)-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[(4-methoxyphenyl)thio]-L-proline, 1-adamantanamine salt; sm.p. 237-239° (d); [a]<*5> -52.8°; (c=0.5, methanol).

A small sample of this 1-adamantanamine salt (500 mg, 0.99 mmol) is partitioned between aqueous HCl and ethyl acetate to

give the corresponding free acid, CcJq"' -36.8° (c=1, absolute ethanol).

11.5 g (22.7 mmol) of the above-mentioned 1-adamantanamine salt are added in portions to a rapidly stirred mixture of 2 7.2 ml IN aqueous HCl, 4 5 ml water and 7 5 ml ethyl acetate. The solution is kept cold in an ice bath and covered with a layer of nitrogen. After all the solid has dissolved, the aqueous layer is extracted with two further 75 ml portions of ethyl acetate. The organic extracts are collected and washed with 50 ml of 0.5N aqueous HCl, 50 ml of water and finally saline solution. The solution is dried (MgSO 4 ) and concentrated in vacuo to give 8.2 g of liberated acid. The acid is dissolved in a mixture of 75 ml of ethyl acetate and 20 ml of ether and shaken with a solution of 3.76 g (21.6 mmol) of L-arginine dissolved in 100 ml of water. The aqueous layer is washed with 75 ml of ether and lyophilized overnight to give 11.2 g of (cis)-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[(4-methoxyphenyl)thio ]-L-proline, L-arginine salt (1:1); [a]" -46.0° (c=l, water).

Analysis:

Calculated for cigH21N04S2*C6H14<N>4°2<:>

C 47.93, H 6.84, N 12.70, S 11.63, SH 6.00. Found: C 47.99, H 6.61, N 12.66, S 11.62, SH 6.00.

Example 8

(a) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-( phenylthio)- L- proline

9.9 g (0.031 mol) of cis-4-phenylthio-L-proline is suspended in 100 ml of water (pH 5.6), and the pH is adjusted to 10.2 by adding approx. 20 ml of 10% sodium bicarbonate to give a clear solution. The pH is then adjusted to 9.5 by adding approx. 4.5 ml of concentrated HCl. The solution is maintained at 30° while 8.1 g (0.033 mol) of (D)-3-(benzoylthio)-2-methylpropanoic acid chloride in 30 ml of toluene is added simultaneously with 100 ml of 10% sodium bicarbonate to maintain the pH at 9.3. After approx. 1/4 of the acid chloride has been added, a slimy precipitate begins to form which remains during the reaction. After stirring the reaction mixture at pH 9.3 for 2.5 hours, it is made strongly acidic by the addition of 20% HCl in the presence of ethyl acetate. The

aqueous layers are extracted twice with 350 ml portions of ethyl acetate, and the combined organic layers are washed with 300 ml of saturated saline and dried (MgSO 4 ). The solvent is removed to give 11.8 g of foamy, solid, crude product.

Add approx. 6 g of dicyclohexylamine in 25 ml of ether. A white, crystalline precipitate forms immediately. After standing overnight in a cold room, the solid is filtered off and washed with ether to

give (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-(phenylthio)-L-proline, dicyclohexylamine salt (1:1).

Analysis:

Calculated for C22H23N04S2-C^H^N: C 66.85, H 7.59, N 4.59. Found: C 66.33, H 7.30, N 4.48.

The slightly moist dicyclohexylamine salt is stirred for 2.5

hours in a mixture of 300 ml ethyl acetate and 200 ml 10% potassium bisulphate. Two clear layers are formed. The aqueous layer is extracted with two 200 ml portions of ethyl acetate, and the combined organic layers are dried (MgSO 4 ). The solvent is removed to give 10.1 g of foamy solid (cis)-1-[D-3-(benzoyl-thio)-2-methyl-1-oxopropyl]-4-(phenylthio)-L-proline; sm.p. 42-44°; [α]" -36.5° (c=1, methanol).

Analysis:

Calculated for C22H23N04S2: C 60.87, H 5.46, N 3.23.

Found: C 60.75, H 5.35, N 3.17.

(b) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-( phenylthio)- L- proline, L- arginine- salt ( 1:1)

2.3 g (0.0054 mol) of (cis)-1-[D-3-(benzoylthio)-2-methyl-l-oxopropyl]-4-(phenylthio)-L-proline dissolved in 100 ml of methanol are treated with 0.941 g (0.0054 mol) of L-arginine. A clear solution is formed after stirring for approx. 10 minutes. The reaction mixture is stirred at room temperature for approx. 5 hours, the solvent is removed, and the resulting solid is triturated with ether and filtered to give 2.5 g of (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]- 4-(phenylthio)-L-proline, L-arginine salt (TM^soft at 105°, m.p. 110-112° (d) ; [a]^<5> -278° (c=l, methanol).

Analysis:

Calculated for C22H23N04S2•<C>6<H>14<N>4<0>2<:>

C 54.88, H 6.25, N 11.43, S 10.47.

Found: C 54.44, H 6.21, N 11.60, S 10.40.

Example 9

( cis)- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-( phenylthio)- L- proline- potassium salt ( 1:1)

(a) N-benzoyl-cis-4-phenylthio-L-proline

N-benzoyl-trans-4-tosyloxy-L-proline, methyl ester [prepared by the method of Portoghese et al., Tetrahedron, Vol. 27, pp. 961-967] (750 g, 1.86 mol) is added to 2, 5 liters of methanol, and the mixture is heated and stirred at 35°. 200 ml (1.95 mol) of thiophenol are added, followed by a solution of 78 g of sodium hydroxide (1.95 mol) in 500 ml of methanol. The mixture is heated to reflux temperature until thin layer chromatography shows that the reaction is complete. The mixture is allowed to cool to room temperature, and a solution of 80 g of sodium hydroxide in 500 ml of water is added with continued stirring for 1 hour. The solvent is removed in vacuo, and the oily concentrate is diluted with 2.5 liters of water and extracted with 0.7 liters of isobutyl alcohol. The pH in the aqueous phase is adjusted to below 2 with 0.2 liters of concentrated HCl. The precipitated oil is extracted with 2 liters of isobutyl acetate in three portions. The organic layer is filtered and vacuum-concentrated to 650 g of N-benzoyl-cis-4-phenylthio-L-proline as a thick syrup.

(b) cis-4-phenylthio-L-proline

The above N-benzoyl-cis-4-phenylthio-L-proline is heated

on a steam bath, and 0.5 liters of glacial acetic acid are added. The mixture

is rotated, and 0.5 liters of water followed by 100 ml of concentrated sulfuric acid are added. The cloudy mixture is heated at reflux temperature at an oil bath temperature of approx. 12 5° and kept at reflux temperature until the reaction is complete. The liquid reaction mixture is cooled to room temperature, and 0.75 liters of isobutyl acetate and 3 kg of crushed ice are added with vigorous stirring. 50 g of concentrated aqueous sodium hydroxide solution is added, and the product is collected and washed with methanol. The product is further purified by dissolution in aqueous sodium hydroxide, treatment with activated carbon, filtration and careful crystallization with HCl. The crystals are collected, washed with water and dried to give cis-4-phenylthio-L-proline as a white crystalline powder, m.p. 240° (d) ; [<*]D -118°

(c=1, N sodium hydroxide).

Analysis:

Calculated for C^H^NC^S: C 59.17, H 5.87, N 6.27, S 14.36. Found: C 58.58, H 5.63, N 6.28, S 14.07.

(c) ( cis )- 1-[ D- 3-( benzoylthio)- 2- methyl- l- oxopropyl]- 4-( phenylthio)- L- proline, potassium salt ( 1:1)

cis-4-phenylthio-L-proline is acylated in water at pH 8.3 with

an equimolar amount of (D)-3-(benzoylthio)-2-methylpropanoyl chloride. The pH is kept at 8-8.5 by adding 5N sodium hydroxide solution. The reaction mixture is acidified with hydrochloric acid, and the product is extracted into isobutyl acetate. Concentration of the brine-washed extract gives crude (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-(phenylthio)-L-proline as a tough resin.

This resinous material is dissolved in isopropanol, and a solution of potassium 2-ethylhexanoate in isopropanol is added slowly at an elevated temperature. The isopropyl adduct of the desired potassium salt crystallizes.

The adduct is collected, washed with isopropanol and vacuum-dried. When the adduct is exposed to moist air, isopropanol is replaced by water, and (cis)-1-[D-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-(phenylthio)- L-proline, potassium salt (1:1).

Analysis:

Calculated for C^H^NO^K-1 1/2- H20:

C 53.41, H 5.09, N 2.83, S 12.96, K 7.88.

Found: C 53.55, H 4.89, N 2.90, S 12.88, K 8.03.

Example 10

(a) 1,1'-[Dithiodi-(1-D-3-mercapto-2-methyl-1-oxopropyl)]-bis[(cis)-4-(phenylthio)-L-proline]

A mixture of 2.9 g (0.089 mol) (cis)-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-(phenylthio)-L-proline and 9 ml of 1N sodium hydroxide in 70 ml water is stirred and treated dropwise with a solution of 1.2 g (0.047 mol) of iodine in 10 ml of 95% ethanol while the pH of the solution is maintained at 5.5 to 6.5 with IN sodium hydroxide. After approx. After 15 minutes, the slight excess of iodine color is removed with dilute sodium thiosulphate, and the reaction mixture is acidified with 6N HCl and extracted with ethyl acetate (3 x 70 ml). The organic phases are combined, dried (MgSO 4 ), filtered, and the solvent evaporated to give 2.4 g of a yellow foamy solid, m.p. 75-85° (p. 47°).' This material is dissolved in 40 ml of ethyl acetate and treated with a solution of 1.4 g of dicyclohexylamine in 5 ml of ethyl acetate. The product is rapidly excreted as a gelatinous material. After standing overnight at room temperature, the solid is filtered to give 4.2 g of light yellow solid, m.p. 205-207°.

This material is crystallized from 40 mL of ethanol to give

2.8 g colorless solid 1,11-[dithiodi-(1-D-3-mercapto-2-methyl-1-oxopropyl)]bis[(cis)-4-(phenylthio)-L-proline], dicyclohexylamine salt ; sm.p. 207-209°.

Analysis: Calculated for c30H36<N>2<O>6<S>4'<2>^<C>12H23N): C 64.12, H 8.17, N 5.54, S 12.68.

Found: C 63.87, H 8.30, N 5.45, S 12.46.

The above dicyclohexylamine salt is crushed in a mortar, suspended in 30 ml of ethyl acetate, treated with 30 ml of 10% potassium bisulfate and shaken to give two layers. The organic phase is separated, and the aqueous phase is extracted with ethyl acetate (3 x 30 ml). The organic phases are combined, washed twice with 10 mL water, dried (MgSO 4 ), filtered, and the solvent evaporated to give a foamy, colorless solid. The latter is treated with 25 ml of ether (solid becomes an oil), and the solvent is removed on a rotary evaporator to give 1.8 g of colorless, granular 1,1'-[dithiodi-(1-D-3-mercapto-2 -methyl-1-oxopropyl)]bis[(cis)-4-(phenylthio)-L-proline]; sm.p. 75-90°

(p. 65°); [a]<25> -42° (c=1, ethanol).

Analysis:

Calculated for C3oH36N2°6S4 ' 1// 2 H20:

C 54.77, H 5.67, N 4.26, S 19.50.

Found: C 54.79, H 5.95, N 4.01, S 19.52.

(b) 1, 1'-[Dithiodi-(1-D-3-mercapto-2-methyl-1-oxopropyl)]-bis[(cis)-4-(phenylthio)-L-proline], potassium salt

A stirred solution of 7.0 g (0.0106 mol) 1,1'-[dithiodi-(1-D-3-mercapto-2-methyl-1-oxopropyl)]bis[(cis)-4-(phenylthio)- L-proline] in 200 ml of ethanol is treated with 21.3 ml of IN potassium hydroxide (2 equivalents) in ethanol. The resulting solution is seeded, allowed to crystallize at room temperature for several hours and placed in a cold room overnight. The solid is filtered, washed with ethanol and ether to give 5.2 g of colorless solid, m.p. 275° (decomp.). The material is then air-dried overnight to give 5.3 g of 1,11-[dithiodi-(1-D-3-mercapto-2-methyl-1-oxopropyl)]bis[(cis)-4-(phenylthio) -L-proline], dipotassium salt; sm.p. 275° (decomp.); [a]<2>^ -227°

(c=l, water).

Analysis:

Calculated for <C>3Q<H>34<N>2<0>6<S>4•<2>K-3 H20:

C 46.24, H 5.17, N 3.59, S 16.46, K 10.04 Found: C 45.98, H 5.11, N 3.36, S 16.10, K 10.03 .

Claims (1)

Analogous method for the preparation of therapeutically active compounds of the formula and basic salts thereof where the group S-R-^ is in the 3- or 4-position in the proline ring; R 1 is C 1 - C 4 alkyl or phenyl optionally substituted with a C 1 - C 4 alkyl, C 1 - C 4 alkoxy or halogen; R 2 is hydrogen or C 1 -C 4 alkyl in D configuration n is 0 or 1; R 4 is hydrogen, R 4 -CO- or to form a symmetrical product, R5 is C1~C4 alkyl, phenyl or phenyl-C^-C4 alkyl, and the proline ring is in L configuration, characterized in that a proline compound with the formula is coupled with an acid with the formula or a functional derivative thereof, where R 4' is hydrogen or R 4 -CO-, to form a product with formula I where R4 is hydrogen or R5CO, and optionally the product where R is R5CO is hydrolysed to form the product where R4 is hydrogen, and optionally a product where R4 is hydrogen is oxidized with iodine to form the symmetrical bis product, and a racemic mixture is separated into its stereoisomers, and optionally a prepared compound is converted to a basic salt thereof.