NO800077L - PROCEDURE FOR THE PREPARATION OF MERCAPTOACYL HYDROXY PROLINES - Google Patents

Description

This invention relates to the preparation of new carbamate derivatives of mercaptoacylhydroxyprolines with the formula

(IN)

wherein R, R 2 and R 1 are each hydrogen, lower alkyl or trifluoromethyl; Rq and R^ are each hydrogen, lower alkyl, cyclo-lower alkyl, allyl, propargyl, phenyl or substituted phenyl; or Rq and R^ can together with the nitrogen atom form a 5- or 6-membered heterocyclic ring; R 1 is hydrogen or a hydrolyzable organic protecting group of the formula

R<- is lower alkyl, phenyl, substituted phenyl, phenyl-lower alkyl, substituted phenyl-lower alkyl, cycloalkyl, thienyl or furyl;

and n is 0, 1 or 2;

and salts thereof, as well as new intermediate products for their production.

The stars denote centers of asymmetry in The carbon atom in the acyclic side chain is asymmetric when R» and/or R^ is different from hydrogen. Each of the asymmetry centers gives D and L forms which can be separated by common methods as described below. The carbamate group

also entails cis-trans isomerism.

The lower alkyl groups denoted by one of the symbols include linear and branched hydrocarbon radicals with up to 7 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl and the like. The lower alkyl groups with up to 4 carbon atoms and especially those with 1 and 2 carbon atoms are preferred.

The cyclo-lower alkyl groups are the alicyclic groups with up to 7 carbon atoms, ie cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Cyclopentyl and cyclohexyl are preferred.

The substituted phenyl groups comprise monosubstituted phenyl rings where the phenyl substituent is halogen, lower alkyl, lower alkoxy, lower alkylthio or trifluoromethyl. The lower alkoxy and lower alkyl mercapto groups contain lower alkyl groups of the type described above. Examples of such are methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, methylthio, ethylthio, propylthio, isopropylthio and the like. The C -C.°9<" l~(~' 29ruPPs are preferred as indicated above. The halogen substituents are the four common halogens, preferably chlorine and bromine, to form such radicals as o-, m- and p-chlorophenyl, o-, m- and p-bromophenyl etc. Phenyl and substituted phenyl can also be designated as/

where Rg is hydrogen, halogen, lower alkyl, lower alkoxy, lower alkylthio or trifluoromethyl..

The phenyl-lower alkyl groups contain lower alkyl groups of the type described above, attached to the phenyl ring. Phenylmethyl and phenylethyl are the preferred phenyl lower alkyl groups, especially phenylmethyl.

The preferred groups of the formula R^-CO- are those. where R(. is lower alkyl, phenyl or phenyl-lower alkyl.

The lower alkanoyl groups denoted by R,.-CO-, are those which have acyl radicals of the lower (C2~C7) fatty acids, e.g. acetyl, propionyl, butyryl, isobutyryl, etc. The lower alkanoyl groups having up to 4 carbon atoms, and especially acetyl, are preferred. The same preferences apply to the phenyl-lower alkanoyl groups when R^ in the group R^-CO- is phenyl-lower alkyl. Benzoyl is particularly preferred.

The carbamate group on the pyrrolidine ring can be acyclic and contain e.g. the radicals carbamoyl, lower alkylcarbamoyl such as methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl or isopropylcarbamoyl, di(lower alkyl)carbamoyl such as dimethylcarbamoyl or diethylcarbamoyl, propargylcarbamoyl or allylcarbamoyl. It also includes cycloalkylcarbamoyl groups such as cyclopentylcarbamoyl, dicyclopentylcarbamoyl, cyclohexylcarbamoyl, dicyclohexylcarbamoyl and the like. In addition, it includes phenyl and substituted phenylcarbamoyl groups such as phenylcarbamoyl, 4-chlorophenylcarbamoyl, 3-ethylphenylcarbamoyl, 4-methoxyphenylcarbamoyl, 4-(trifluoromethyl)phenylcarbamoyl and the like. Preferably only one of Rq and is a cycloalkyl, phenyl or substituted phenyl radical.

The group

can also form a 5-membered or 6-membered

heterocyclic group such as pyrrolidine, piperidine or morpholine..

Preferably, only one of Rq and R^ is different from hydrogen, except when both Rq and R^ are lower alkyl.

Preferred compounds of formula I are those where R is hydrogen or lower alkyl; R 1 and R 2 are each independently C 1 -C 2 -lower alkyl; R 2 and R 4 are each hydrogen or C 1 -C 4 lower alkyl; R^ is hydrogen, lower alkanoyl or benzoyl, and n is 0 or 1. The carbamate group is in the 3- or 4-position of the pyrrolidine ring, preferably in the 4-position.

Particular preference is given to the compounds of formula I where R is hydrogen; Rq and R^ are each C^-C^ lower alkyl, in particular

C 1 -C 4 lower alkyl; R 1 is methyl; R 1 is hydrogen; R 1 is hydrogen; n is 1, and the carbamate group is in the 4-position.

In the preferred preparation method for the compounds of formula I, a hydroxyproline with . the formula

(II)

First, the nitrogen atom is protected, e.g. with a nitrogen protecting group of the type commonly used in peptide synthesis, such as carbobenzoxy, p-toluenesulfonyl, acetyl or the like to form a protected compound such as where CBz is a carbobenzoxy protecting group.

The protected compound III is then esterified, e.g. by reaction with a diazoalkane, such as diazomethane, to form an ester of the formula

where R is lower alkyl such as methyl or isobutyl, preferably methyl. The carbamate group where Rq is hydrogen and R^ is different from hydrogen can then be introduced by reacting the compound of formula IV with an isocyanate (R^-NCO) in an inert organic solvent such as benzene or the like, to form the next intermediate with the formula

In preparing the cis-isomer, the reaction described above is carried out in the presence of a catalytic amount of a base, such as pyridine or triethylamine.

Alternatively, the compounds of formula V can be prepared by reacting the protected compound IV with phosgene to form an intermediate of formula VI

(which is not necessarily isolated), which is then reacted with the appropriate amine HN XR1 or NH3_, (where both R° and are hydrogen) to form the compound of formula.V.

When RQ and R^ (in formula V) are both different from. hydrogen or together form a heterocyclic group, the protected compound of formula IV is reacted with a carbamoyl halide of the formula

where hal is halogen, preferably chlorine.

Alkaline hydrolysis of the compound of formula V with a base such as sodium hydroxide, barium hydroxide, potassium hydroxide or the like gives the acid of the formula

The compound of formula VIII in acid or ester form can then be deprotected, e.g. by an ordinary hydrogenation in the presence of palladium charcoal to form the compound of the formula

The next step of the synthesis comprises coupling the proline derivative IX with an acid of the formula most conveniently in the form of an acid chloride, to form a product of the formula

The proline derivative XI is preferably isolated and purified by crystallization, e.g. by forming a salt such as 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 XI bearing the acyl group R5~CO,

can optionally be converted to the product of formula I where R^ is hydrogen, by hydrolysis with ammonia, sodium hydroxide. etc.

Esters of formula I where R is lower alkyl can be prepared by usual esterification methods, e.g. by esterification with a diazoalkane such as diazomethane, 1-alkyl-1-3-p-tolyl-triazene such as 1-n-buty1-3-p-tolyltriazene, or the like, preferably after completion of the above-described reaction sequence. However, it is also possible to carry out earlier esterification and omit the alkaline hydrolysis.

The compounds of formula I where R^ forms the symmetrical bis compound are prepared by direct oxidation with iodine of a product of formula I where R^ is hydrogen.

Reference should also be made to the following publications which provide further illustrative information with regard to the preparation of starting materials and intermediates: US patents 4,046,889 and 4,105,776; J. Chem. Soc, 1945, 429-432; J. Amer. Chem. Soc. 79, 185-192 (1957); J. Amer. Soc. 8_5 , 3863-3865 (1963). The methods illustrated there can be used as general methods for synthesis and stereo-conversion of the compounds that can be used in the method according to the invention.

Further 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.

As indicated above, the compounds prepared according to the invention have several centers of asymmetry. These compounds thus exist in stereoisomeric forms or in racemic mixtures thereof. The various stereoisomeric forms and mixtures thereof can all be prepared according to the invention. The synthesis methods described above can be carried out using the racemate or one of the enantiomers as starting material. When a mixture of stereoisomers is obtained as a product, the stereoisomeric forms can optionally be separated by usual chromatographic methods or by fractional crystallization or by conversion to a salt with an optically active base, followed by fractional crystallization or similar known methods. In general, those compounds are preferred where the proline part is in L-configuration, the carbamate group is cis, and the acyl side chain has D-configuration.

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. aralkyl— amines such as dibenzylamine, N,N-dibenzylethylenediamine, lower alkyl amines such as methylamine, triethylamine, t-butylamine, procaine, lower alkyl piperidines such as N-ethylpiperidine, cycloalkyl amines such as cyclohexylamine or dicyclohexylamine, 1- adamantanamine, benzathine, or salts derived from amino acids such as arginine, lysine or the like. 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 acceptable for the purposes described below as well as for the purification and isolation of intermediates, as illustrated in the Examples. The salts are prepared by reacting the acid form of the compound with an equivalent of the base which 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 which is induced by angiotensin-converting enzyme, and are therefore useful in reducing or alleviating hypertension in various mammals, e.g. cats, dogs, mice, rats, etc. with elevated blood pressure. 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 15 mg per kg per day, is appropriate 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 compounds produced according to the invention can also be used in combination with a diuretic agent for the treatment of hypertension. A combination product comprising a compound of the invention and a diuretic can be administered in an effective amount comprising (for a 70 kg mammal) a total daily dose of approx. 30 to 600 mg, preferably approx. 30 to 300 mg, of the new compound and approx. 15 to 300 mg, preferably approx. 15 to 200 mg of a diuretic agent, , to a mammal in need thereof. Examples of diuretics that can be used in combination with a compound produced according to the invention are thiazide diuretics, for example, chlorothiazide, hydrochlorothiazide, flumethiazide, hydroglumethiazide, benzdroflumethiazide, methchlothiazide, trichloromethiazide, polythiazide or benzthiazide, as well as ethacrynic acid, ticrynafen, chlorthalidone, furosemide, bumetanide, triamterene, amiloride and spironolactone, and salts of such compounds.

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 . so that one gets to par.send dosage in the specified area.

Illustrative examples of additives that can be incorporated into tablets, capsules and the like are the following: a binder such as tragacanth gum, acacia gum, corn starch or gelatin; an excipient such as dicalcium phosphate or microcrystalline cellulose; an explosive such as corn starch, potato starch, alginic acid, etc.; a lubricant such as magnesium stearate; a sweetener such as sucrose, lactose or saccharin; a flavoring such as peppermint, wintergreen oil or cherry. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other materials may be present as additions or as materials that otherwise modify the physical form of the dosage unit. E.g. tablets can 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 a flavoring agent such as cherry or orange flavor.

Sterile preparations for injection may be prepared according to ordinary pharmaceutical practice by dissolving or suspending the active ingredient in a vehicle such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc.

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

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[[( methylamino) carbonyl] oxy]- L- proline

a) N-carbobenzyloxy-trans-4-hydroxy-L-proline

26.5 g (0.20 mol) of trans-4-hydroxy-L-proline and 32.8 ml (0.23 mol) of benzyl chloroformate are reacted in 200 ml of water and 100 ml of acetone in the presence of 20 g (0.20 mol ) potassium bicarbonate and 69.2 g (0.50 mol) potassium carbonate and worked up with 90 ml of concentrated hydrochloric acid as described in Can. J. Biochem. & Physiol.<_>37, 584 (1959). The product is reacted with cyclohexylamine to form the cyclohexylamine salt, yield 69 g, m.p. 193-195°. The salt (34 g) is neutralized with 1N hydrochloric acid •to give 27 g of the free acid as a colorless glass [ct]p<6>-70° (c, 1% in chloroform).

b) N-carbobenzyloxy-trans-4-hydroxy-L-proline-methyl ester

12.4 g (0.047 mol) of N-carbobenzyloxy-trans-4-hydroxy-L-proline is esterified with diazomethane in dioxane ether as described in J.A.C.S. 79.' 191 (1957). To avoid freezing. the dioxane begins the addition of diazomethane at 10° and is completed at 0-2°. The yield of N-carbobenzyloxy-trans-4-hydroxy-L-proline methyl ester as an almost colorless, viscous oil is 14.6 g (100%). [a]p<6>-62° (c, 1% in chloroform). c) trans-N-carbobenzyloxy-4-[[(methylamino)carbonyl]oxy]-L-proline methyl ester

To a stirred solution of 6.0 g (0.021 mol) of N-carbobenzyloxy-trans-4-hydroxy-L-proline methyl ester (J.A.C.S. 79 supra) in 120 ml of benzene is added 6 ml (0.10 mol) of methyl isocyanate, and the reaction mixture kept overnight at room temperature. After refluxing for 1 hour, the solvent is removed on a rotary evaporator, finally at 0.2 mm and 50°. The viscous residue is taken up in 150 mL of ether, washed with water (3 x 50 mL), dried (MgSO 4 ), and the ether evaporated to give 6.5 g (90%) of a syrupy product, trans-N-carbobenzyloxy- 4-[ [ (methylamino).carbonyl ] oxy]-L-proline methyl ester .

d) trans-N-carbobenzyloxy-4-[[(methylamino)carbonyl]oxy]-L-proline

The crude ester from part c (7.6 g, 0.023 mol) is dissolved in 60 ml of methanol, treated dropwise at -1 to 4° with 14 ml (0.028 mol) of 2N sodium hydroxide, kept at 0° for 1 h dg at room temperature overnight above. After removing approx. half of the solvent on a rotary evaporator, the solution is diluted with 160 ml of water, washed with ether (the washings are discarded), acidified under cooling with 5.5 ml of 1:1 hydrochloric acid to pH 2 and extracted with ethyl acetate (4 x 75 ml). The combined extracts are washed with 50 ml saturated sodium chloride, dried (MgSO 4 ) and the solvent evaporated to give 7.2 g of a very viscous syrup. The syrup is dissolved in 30 ml of ethanol, treated with 2.3 g of cyclohexylamine in 5 ml of ethanol and diluted to 600 ml with ether. During inoculation and rubbing, the crystalline cyclohexylamine salt is secreted; weight after overnight cooling, 8.5 g; sm.p. 172-174°: ta]^<5>"20° (c, 1% in ethanol)'. After crystallization from 25 ml of isopropanol, the colorless solid trans-N-carbobenzyloxy-4-[[(methylamino)carbonyl] oxy]-L-proline-cyclohexylamine salt 7.8 g, mp 174-176° [α]p<5>-18° (c, 1% in ethanol).

The cyclohexylamine salt is suspended in 60 ml of ethyl acetate, stirred and treated with 40 ml of IN hydrochloric acid. When two clear layers are obtained, they are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 60 ml), the combined organic layers are dried (MgSO 4 ), and the solvent is evaporated. The yield of glass-like, free acid is 5.5 g (81%).

e) 'trans-4-[[(methylamino)carbonyl]oxy]-L-proline

A solution of 2.7 g (0.0084 mol) trans-N-carbobenzyloxy]-4-[[(methylamino)carbonyl]oxy]-L-proline in 100 ml methanol-water (2:1) is treated with 1 g 5% palladium-charcoal and 45 lb. of hydrogen and shaken on a. Parr hydrogenator for 6 hours. The catalyst is filtered off under nitrogen, washed with methanol, and the combined filtrates evaporated, finally at 0.1-0.2 mm, to give 1.5 g (96%) of a residue which gradually crystallizes to give trans-4 -[[(methylamino)carbonyl]oxy]-L-proline as a greyish solid, m.p. 213-215°C (splitting), after a gradual darkening and sintering first occurs. [a]D 2 6 -12 o (c, 0.25% in 1:3 ethanol-methanol).

f) trans- 1-[ D-( 3- acetylthio)- 2- methyl- 1- 1- oxopropyl]- 4-[[( methylamino) carbonyl] oxy]- L- proline

A stirred solution of 2.9 g (0.0154 mol) of trans-4-[[(methylamino)carbonyl]oxy]-L-proline in 45 ml of water is cooled to 5° and treated portionwise with solid sodium carbonate to pH 8 .5 (about 0.4 g needed). With continued stirring and cooling, a solution of 3.1 g (0.017 mol) D-3-acetylthio-2-methylpropanoyl chloride in 4 ml is then added. ether portionwise using a pipette while the pH is maintained at 7.0-8.0 by the dropwise addition of 25% (w/v) sodium carbonate.

After approx. After 10 minutes, the pH value stabilizes at 8.1-8.3 (approx. 14 ml of the sodium carbonate solution has been added). After continued stirring and cooling for a total of 1.25 hours, the solution is washed with ethyl acetate (50 mL), covered with 50 mL of ethyl acetate, stirred, cooled, acidified carefully with 1:1 hydrochloric acid to pH 2.0, saturated with sodium chloride, and the layers separated . The aqueous phase is extracted with additional ethyl acetate

(3 x 50 mL), the combined organic layers are dried (MgSO 4 ), and the solvent is evaporated, finally at 0.2 mm, to give 5.3 g of a glassy residue. The latter is dissolved in 40 ml of ethyl acetate and treated with a solution of 2.8 g of dicyclohexylamine in 15 ml of ethyl acetate. During inoculation and rubbing, the crystalline trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(methylamino)carbonyl]oxy]-L-proline-dicyclohexylamine salt is secreted, weight after cooling overnight 5.8 g (colorless), m.p. 187-189° (p. 183°) [a]^<6>-64° (c, 1% in MeOH). After recrystallization from 15 ml of methanol - 100 ml of ether, the colorless solid weighs 4.5 g, m.p. 190-192°, [α]^5 -67° (c, 1% in MeOH).

The dicyclohexylamine salt is converted to the free acid by suspension in 50 ml of ethyl acetate, cooling, treatment with 50 ml of 10% potassium bisulphate and stirring until two clear layers are obtained. After separation, the aqueous phase is extracted with ethyl acetate (3 x 50 ml), the combined organic layers are dried (MgSO 4 ), and the solvent is evaporated to give 2.8 g (55%) of trans-1-[D-(3- acetylthio)-2-methyl-1-oxopropyl]-4-[[(methylamino)-carbonyl]oxy]-L-proline as a foamy, hygroscopic residue.

Example 2

trans- 1- ( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[[( methylamino)-carbonyl] oxy]- L- proline i

Argon is passed through a cold solution of 6 ml of concentrated ammonium hydroxide in 4 ml of water for 10 minutes. This solution is then added, under cooling and under a blanket of argon, to the product from example 1, and the mixture is stirred in an ice bath until a pale yellow solution is obtained (approx. 3 minutes). Stirring under argon is continued at room temperature for a total of 2 hours, then the solution is extracted with 20 ml of ethyl acetate (this and subsequent operations are carried out as far as possible under an argon atmosphere). The aqueous layer is cooled, stirred, covered with a layer of 20 ml of ethyl acetate and acidified in portions with approx. 13 ml 1:1 hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 20 mL), the combined ethyl acetate layers are dried (MgSO 4 ), and the solvent is evaporated to give trans-1-(D-3-mercapto-2-methyl-1-6xopropyl )-4-[[(methylamino)carbonyl]oxy]-L-proline as a sticky, foamy residue. The latter is triturated under ether and the evaporation repeated, finally at 0.1-0.2 mm, to give 2.2 g (90%) of the product as a colorless, somewhat hygroscopic, amorphous solid, sm. p. 54-57° (p, 45°). [^p6 -53° (c, 1% in EtOH).

The racemic forms of the final products in each of the preceding examples are prepared by using the DL form of the starting amino acid instead of the L form.

Correspondingly, the D-form of the final products in each of the preceding examples is prepared by using the D-form of the starting amino acid instead of the L-form.

Example 3

a) N-carbobenzyloxy-cis-4-hydroxy-L-proline methyl ester

6.5 g (0.024 mol) of N-carbobenzyloxy-cis-4-hydroxy-L-proline [J.A.C.S., 19.'189 (1957)] are dissolved in 65 ml of methanol, stirred and treated with 0.65 ml of concentrated sulfuric acid. After stirring at room temperature for 1/2 hour, the solution is allowed to stand overnight. The main amount of the solvent is removed on a rotary evaporator, and the oily residue (13 g) is taken up in 70 ml of ether and washed with 35 ml of 10% sodium bicarbonate solution. The washing liquid is back-extracted with 35 ml of ether. The combined organic layers are dried (MgSO 4 ), and the ether is evaporated to give 6.5 g (96%) of product as a pale yellow viscous oil. [ct]^<5>-24° (c, 1% in chloroform).

b) cis-N-carbobenzyloxy-4-[[(methylamino)carbonyl]oxy]-L-proline- methyl ester

To a stirred solution of 5.4 g (0.019 mol) of N-carbobenzyloxy-cis-4-hydroxy-L-proline methyl ester in 120 ml of acetonitrile is added 5.4 ml of triethylamine, followed by 5.4 ml of methyl isocyanate. After standing overnight at room temperature and refluxing for 2 hours, the reaction mixture is worked up as in Example 1c to give 5.6 g (86%) of a pale yellow, viscous oil.

c) cis-N-carbobenzyloxy-4-[[(methylamino)carbonyloxy]-L-proline

The crude ester from part b (5.6 g, 0.017 mol) is saponified with 11 ml (0.022 mol) of 2N sodium hydroxide in 45 ml of methanol as in Example 1d to give 5.1 g of a foamy residue. The colorless cyclohexylamine salt, prepared in .25 ml of ethanol and 400 ml of ether using 1.7 g of cyclohexylamine, weighs 4.8 g; sm.p. 171-173°. [ct]^<5>-16° (c, 1% in ethanol), A sample recrystallized from ethanol-ether shows no change in melting point or optical rotation.

The cyclohexylamine salt gives 3.5 g (65%) of the free acid as a colorless, foamy residue.

d) cis-4-[[(methylamino)carbonyl]oxy]-L-proline

3.5 g (0.011 mol) of cis-N-carbobenzyloxy-4-[[(methylamino)-carbonyl]oxy]-L-proline is hydrogenated in 130 ml of 2:1 methanol-water using 1.3 g of 5% palladium -coal as in example le to give 1.9 g (95%) of product as a greyish solid, .m.p. 232-234°C (decomposition), after gradual darkening and sintering. fQJD 2 5 "42 o (c, 0.5% in 1:1 methanol-water).

e) ' cis- 1-[ D-( 3- acetylthio)- 2- methyl- 1- oxdipropyl]- 4-[[( methylamino) carbonyl] oxy]- L- proline

Reaction of 1.85 g (0.0098 mol) cis-4-[[(methylamino)-carbonyl]oxy]-L-proline and 2.0 g (0.011 mol) D-3-acetylthio-2-methylpropanoyl chloride in 30 ml of water in the presence of sodium carbonate which in example 1f gives 3.35 g of a gummy product. The dicyclohexyl amino salt, prepared in 35 ml of ethyl acetate using 1.8 g of dicyclohexylamine, weighs 4.0 g;

sm.p. 177-179°. [alp<5>-54° (c, 1% in methanol). After trituration with 20 ml of acetonitrile and cooling, the colorless solid weighs 3.6 g, m.p. 179-181°. [α]^<5>-54° (c, 1% in methanol). Treatment with 10% potassium bisulphate and extraction into ethyl acetate gives 2.5 g (76%) of the free acid as a colourless, foamy residue.

Example 4

cis- 1- ( D- 3- mercapto- 2- methyl- 1- 1- oxopropyl)- 4-[[( methylamino)-carbonyl] oxy]- L- proline

When treating the material from Example 3 with 5.5 ml of concentrated ammonium hydroxide in 12.5 ml of water according to the procedure described in Example 2, 1.8 g (82%) of the product is obtained as a colorless, hygroscopic, sticky foam . [cx]p<5>-59° (c, 1% in ethanol).

Example 5

cis- 1-[ D-( 3- acetylthio)- 2- methyl- 1- 1- oxopropyl]- 4-[[( ethylamino)-carbonyl] oxy]- L- proline

Using the method according to example 3, but using ethyl isocyanate instead of methyl isocyanate in part b, cis-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(ethylamino )carbonyl]oxy]-L-proline.

Example 6

cis- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[[( ethylamino)-carbonyl] oxy]- L- proline

By treating the material from example 5 with ammonia according to the method described in example 4, cis-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(ethylamino)-carbonyl is obtained ]oxy]-L-proline.

Example 7.

cis- 1-[ D-( 3- acetylthio)- 2- methyl 1- 1- oxopropyl]- 4-[[( propylamino)-carbonyl] oxy]- L- proline

Using the method according to example 3, but using n-propyl isocyanate instead of methyl isocyanate in part b, cis-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[( propylamino)carbonyl]oxy]-L-proline.

Example 8

cis- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[[( propylamino)-carbonyl] oxy]- L- proline

By treating the material from example 7 with ammonia according to the method described in example 4, cis-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(propylamino)-carbonyl] is obtained oxy]-L-proline.

Example 9

cis- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[[( phenylamino) - carbonyl] oxy]- L- proline

Using the method according to example 3, but by using phenyl isocyanate instead of methyl isocyanate in part b, cis-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(phenylamino) carbonyl]oxy]-L-proline.

Example 10

cis- 1-( D- 3- mercapto- 2- methyl 1- 1- oxopropyl)- 4-[[( phenylamino)-carbonyl] oxy]- L- proline

By treating the material from example 9 with ammonia according to the method described in example 4, cis-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(phenylamino)-carbonyl] is obtained oxy]-L-proline.

Example 11

cis- 1-[ D-( 3- acetylthio)- 2- methyl 1- 1- oxopropyl]- 4-[[( 4- chlorophenyl)-carbonyl] oxy]- L- proline

Using the method according to example 3, but by using 4-chlorophenyl isocyanate instead of methyl isocyanate in part b, one obtains cis-1-[D-(3-acetylthio)-2-methyl-1-oxypropyl]-4-[[ (4-Chlorophenyl)carbonyl]oxy]-L-proline.

Example 12

cis- 1-( D- 3- mercapto- 2- methyl- 1- oxopropyl)- 4-[[( 4- chlorophenyl)-carbonyl] oxy]- L- proline

By treating the material from example 11 with ammonia according to the method described in example 4, cis-1-(D-3-mercapto-2-methyl 1-1-oxopropyl)-4- [ [ (4-chloro- rpheny 1) - carbonyl]oxy]-L-proline.

Example 13

cis-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(3-trifluoromethylphenyl)carbonyl]oxy]-L-proline

Using the method according to example 3, but by using 37trifluoromethylphenylisocyanate instead of methylisocyanate in part b, cis-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(3 -trifluoromethylphenyl)carbonyl]oxy]-L-proline.

Example 14

cis- 1-[ D- ( acetylthio)- 2- methyl1- 1- oxopropyl3- 4-.[ [( 2- methoxy-phenyl) carbonyl] oxy]- L- proline

Using the method according to example. 3,. but using 2-methoxyphenyl isocyanate instead of methyl isocyanate in part b gives cis-1-[D-(acetylthio)-2-methyl-1-oxopropyl]-4- [[(2-methoxyphenyl)carbonyl]oxy]-L -proline.

Example 15

trans- 1-[ D-( behzoylthio)- 2- methyl1- 1- oxopropyl]- 4-[[( 2- ethyl-phenyl) carbonyl] oxy]- L- proline

Using the method according to example 1, but by using 2-ethylphenyl isocyanate instead of methyl isocyanate in part c and D-3-benzoylthio-2-methylpropanoyl chloride instead of D-3-acetylthio-2-methylpropanoyl chloride in part f, you get trans-1- [D-(benzoylthio)-2-methyl-1-oxopropyl]-4-[[(2-ethylphenyl)-carbonyl]oxy]-L-proline.

Example 16-trans^- 1-[D-(phenacetylthio)-2-methyl-1-oxopropyl]-4-[[(4-methyl-thiopheny1)carbonyl]oxy]-L-proline

Using the method according to example 1, but by using 4-methylthiophenyl isocyanate instead of methyl isocyanate in part c, and D-phenylacetylthio-2-methylpropanoyl chloride instead of D-(3-acetylthio)-2-methylpropanoyl chloride in part f, you get -trans -1-[D-(phenacetylthio)-2-methyl-1-oxopropyl]-4-[[(4-methylthiophenyl)carbonyl]oxy]-L-proline.

Example 17

trans- 1-[ D-( 3- phenylpropionylthio)- 2- methyl- 1- oxopropyl 1 ]- 4-[[( 3- bromophenyl) carbonyl] oxy]- L- proline

Using the method according to example 1, but by using 3-bromophenyl isocyanate instead of methyl isocyanate in part c, and D-(3-phenylpropionylthio)-2-methylpropanoyl chloride instead of D-(3-acetylthio)-2-methylpropanoyl chloride in part f, one trans-1-[D-(3-phenylpropionylthio)-2-methyl-1-oxopropyl]-4-[[(3-bromophenyl)carbonyl]oxy]-L-proline.

Example 18

trans- 1- [ D- : ( 3- acetylthio)- 2- methyl 1- 1- oxopropyl 1] - 4- [ [ ( cyclo-pentylamino) carbonyl] oxy]- L- proline

Using the method according to example 1, but by using cyclopentyl isocyanate instead of methyl isocyanate in part c, one obtains trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(cyclopentylamino) carbonyl]oxy]-L-proline.

Example 19

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[[( cyclo-hexylamino) carbonyl ] oxy]- L- proline

Using the method according to example 1, but by using cyclohexyl isocyanate instead of methyl isocyanate in part c, you get trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl ] -4- [ [. (cyclohexylamino)carbonyl]oxy]-L-proline.

in Example 20

cis- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[ [ ( allyl-amino) carbonyl] oxy]- L- proline

Using the method according to example 3, but by using allyl isocyanate instead of methyl isocyanate in part b, cis-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(allylamino) carbonyl]oxy]-L-proline.

Example 21

cis- 1- ( D- 3- mercapto- 2- methyl- l- oxopropyl)- 4-[[( allylamino)-carbonyl] oxy]- L- proline

By treating the material from example 20 with ammonia according to the method described in example 4, cis-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(allylamino)-carbonyl] is obtained oxy]-L-proline. . Example 2 2 •

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl] - 4- [[(dimethylamino) carbonyl] oxy]- L- proline

a) trans-N-carbobenzyloxy-4-[[(dimethylamino)-carbonyl]oxy]-L-proline methyl ester

A solution of N-carbobenzyloxy-trans-4-hydroxy-L-proline methyl ester (Example 1, part b) in chloroform is treated. dropwise with an equivalent amount of dimethylcarbamoyl chloride. The mixture is stirred for 2 hours, washed with water, and the organic phase is dried over MgSO 4 . The solution is filtered and the solvent is evaporated to give trans-N-carbobenzyloxy-4-[[(dimethylamino)carbonyl]oxy]-L-proline methyl ester.

b) trans-N-carbobenzyloxy-4-[[(dimethylamino)carbonyl]oxy]-L-proline

Hydrolysis of the methyl ester from part a with sodium hydroxide solution as described in example 1, part d, gives trans-N-carbobenzyloxy-4-[[(dimethylamino)carbonyl]oxy]-L-proline.

c) trans-4-[[(dimethylamino)carbonyl]oxy]-L-proline

Hydrogenation of the material from part b according to the method described in example 1, part e, gives trans-4-[[(dimethylamino)carbonyl]oxy]-L-proline.

d) trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[ [ :( dimethylamino) carbonyl 3 okz , y ] - L- prolih

Treatment of the material from part c with an equivalent amount of D-3-acetylthio-2-methylpropanoyl chloride according to the procedure described in Example 1, part f, gives trans-1-[D-(3-acetylthio)-2-methyl-1 -oxopropyl]-4-[[(dimethylamino)carbonyl]oxy]-L-proline.

Example 2 3

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[[( pyrrolidino)-carbonyl] oxy]- L- proline

Using the method described in example 22, with the use of pyrrolidinocarbamoyl chloride instead of dimethylaminocarbamoyl chloride in part a, trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[( pyrrolidino)carbonyl]oxy]-L-proline.

Example 24

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-,[[( piperidino) carbonyl] oxy]- L- proline

Using the method described in example 22, but using piperidinocarbamoyl chloride instead of dimethylaminocarbamoyl chloride in part a, trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(piperidino )carbonyl]-oxy]-L-proline.

Example 2 5

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[.[ ( morph olino) carbonyl ] oxy ] - L- proline

Using the method described in example 22, with the use of morpholinocarbamoyl chloride instead of dimethylaminocarbamoyl chloride in part a, trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(morpholino )carbonyl]-oxy]-L-proline.

Example 26

trans- 1-( 2- acetyl thio- 1- oxoethyl) - 4- [ [ ( methylamino) carbonyl ].-oxy]- L- proline

Using the method described in example 1, with the use of 2-acetylthioacetyl chloride instead of D-(3-acetylthio)-2-methylpropanoyl chloride in part f, one obtains trans-1-(2-acetylthio-1-oxoethyl)-4- [[(methylamino)carbonyl]oxy]-L-proline.

Example 27

trans- 1-( 4- acetylthio- l- oxobutyl)- 4-[[( methylamino) carbonyl]-oxy]- D- proline

Using the method described in Example 1, but using trans-4-hydroxy-D-proline instead of trans-4-hydroxy-L-proline in part a and 4-acetylthiobutyroyl chloride instead of D-(3-acetylthio)-2- methylpropanoyl chloride in part f, trans-1-(4-acetylthio-1-oxobutyl)-4-[[(methylamino)-carbonyl]oxy]-D-proline is obtained.

Example 2 8

cis- 1-( 4- acetylthio- 4- methyl- l- oxobutyl) - 3- [ [ ( methylamino) - carbonyl] oxy]- L- proline

Using the method described in example 3, with the use of 4-acetylthiovaleroyl chloride instead of D-3-(acetylthio)-3-methylpropionyl chloride in part e, one obtains cis-1-(4-acetylthio-4-methyl-1-oxobutyl )-3-[[(methylamino)carbonyl]-oxy-L-proline.

Example 29

trans- 1-[ L-( 3- acetylthio)- 2- ethyl1- 1- oxopropyl]- 3-[[( methylamino)-carbonyl] oxy]- L- proline

Using the method of Example 1, but using trans-3-hydroxy-L-proline instead of trans-4-hydroxy-L-proline in part a and L-(3-acetylthio)-2-ethyl-propionyl chloride instead of D -3-(acetylthio)-3-methylpropionyl chloride in part-f, one obtains trans-1-[L-(3-acetylthio)-2-ethyl-1<L>oxopropyl]- 3-[[(methylamino )carbonyl]oxy]-L-proline.

Example 30

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[( amino-carbonyl) oxy]- L- proline

a) trans-N-carbobenzyloxy-4-[(aminocarbonyl)oxy]-L-proline-methyl ester

A solution of equivalent amounts of N-carbobenzyloxy-trans-4-hydroxy-L-proline methyl ester from Example' 1, part b,

and dimethylaniline is treated with a solution of phosgene in toluene. After standing overnight, an equivalent amount of ammonia is passed through the solution of trans-N-carbobenzyloxy-4-[(chloro-carbonyl)oxy]-L-proline methyl ester. After standing for 12 hours at room temperature, the solution is washed with water, dried over magnesium sulfate, filtered, and the solvent is evaporated for

to give trans-N-carbobenzyloxy-4-[(aminocarbonyl)oxy]-L-proline methyl ester. b) trans-N-carbobenzyloxy-4-[(aminocarbonyl)oxy]-L-proline Hydrolysis of the methyl ester from part a with sodium hydroxide solution as described in example 1, part d, gives trans-N-carbobenzylbxy-4- [(aminocarbonyl]oxy)-L-proline.

c) trans-4-[(aminocarbonyl)oxy]-L-proline

Hydrogenation of the material from part b according to

the procedure described in Example 1, part e, gives trans-4-[(aminocarbonyl)oxy]-L-proline.

d) trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[( aminocarbonyl) oxy]- L- proline

Treatment of the material from part c with an equivalent amount of D-3-acetylthio-2-methylpropanoyl chloride according to the procedure described in Example 1, part f, gives trans-1-[D-(3-acetylthio ).-2-methyl 1 -1-oxopropyl]-4-[(aminocarbonyl)oxy]-L-proline.

Example 31

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[[(di i so-, ■ propylamino) carbonyloxy]- L- proline

Using the procedure according to example 30, with using diisopropylamine instead of ammonia in part a, trans-1-[D-[3-acetylthio)-2-methyl-1-oxopropyl]-4-[ [(diisopropylamino)carbonyl]oxy]-L-proline.

Example 32

trans- 1-[ D-( 3- acetylthio)- 2- methyl- 1- 1- oxopropyl]- 4-[[( cyclo-propylamino) carbonyl] oxy]- L- proline

Using the method according to example 30, with using cyclopropylamine instead of ammonia in part a, one obtains trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(cyclopropylamino) carbonyl]oxy]-L-proline.

Example 3 3

trans- 1-[ D-( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4-[[(n- butyl-amino) carbonyl] oxy]- L- proline

Using the method according to example 30, with using n-butylamine instead of ammonia in part a, one obtains trans-1-'[D-(3-acetylthio)-2-methyl 1-1-oxopropyl]-4-[ [(n-butylamino)carbonyl]oxy]-L-proline.

Example 34

trans- 1-[ D-( 3- acetylthio)- 2- methyl- 1- 1- oxopropyl]- 4-[[( propargyl-amino(carbonyl] oxy)- L- proline

Using the method according to example 30, but by using propargylamine instead of ammonia in part a, one obtains trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(propargylamino) carbonyl]oxy]-L-proline.

■ Example 35

trans- 1-[ D-( 3- acetylthio)- 2- methyl- 1- 1- oxopropyl]- 4-[[( methylamino)-carbonyl] oxy]- L- proline methyl ester

A solution of the material from Example 1 in ether is treated with a small excess of diazomethane. After stirring at room temperature for 2 hours, the solvent is evaporated to give ^trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(methylamino)-carbonyl]oxy]-L- proline methyl ester.

Example 36

S,S- dimer of trans-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(methylamino)carbonyl]oxy3-L-proline

A solution of the material from example 2 is dissolved in ethanol, stirred and treated with a solution of one equivalent of iodine in ethanol. The pH of the solution is kept at 6-7 by adding 1N sodium hydroxide solution. The solvent is evaporated, and the residue is extracted with ethyl acetate. After drying over MgSO 4 , the solution is filtered and the solvent is removed to give the S,S dimer of trans-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(methylamino)carbonyl]oxy ]-L-proline.

Example 3 7

S,S- dimer of cis-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(methylamino)carbonyl]oxo]-L-proline

Oxidation of the material from example 4 with a solution of iodine according to the procedure used in Example 36 gives the S,S-dimer of cis-1-(D-3-mercapto-2-methyl-1-oxopropyl)-4-[[(methylamino)carbonyl]oxo]-L-proline.

Example 33

Sodium salt of trans-1-[D-(3-acetylthio)-2-methyl1-1-oxopropyl]-4-[[(methylamino)carbonyl]oxy]-L-proline

A solution of 2.9 g of material from Example 1 in 25 ml of water is treated with 0.84 g of sodium bicarbonate. The solution is lyophilized to give the sodium salt of trans-1-[D-(3-acetylthio)-2-methyl-1-oxopropyl]-4-[[(methylamino)carbonyl]oxy]-L-proline.

Claims (10)

1. Procedure for preparing a compound of the formula wherein R, R 2 and R 4 are each hydrogen, lower alkyl or trifluoromethyl; Rq and R^ are each hydrogen, lower alkyl, allyl, propargyl, cyclo-lower alkyl, phenyl or substituted phenyl, or Rq and R^ complete a pyrrolidine, piperidine or morphol ring; R^ is hydrogen, a hydrolyzable, organic protecting group of the formula R^ CO- or R 1 is lower alkyl, phenyl, substituted phenyl, phenyl-lower alkyl, substituted phenyl-lower alkyl, cycloalkyl, thienyl or furyl; where the phenyl substituent in the above-mentioned groups is halogen, lower alkyl, lower alkoxy, lower alkylthio or trifluoromethyl; n is 0, 1 or 2; and pharmaceutically acceptable salts thereof, characterized in that an amino acid with the formula is linked to an acid with the formula to form a product where R^ is CO-, and optionally said product is hydrolyzed to form a product where R^ is hydrogen, and optionally said hydrolyzed product is oxidized to form a product where R^ is and optionally the manufactured product is converted into pharmaceutically acceptable salts thereof. 2. Method as specified in claim 1, character ii-s e r t by using a starting material with the formula where R, Rq and R^ have the meanings specified in claim 1. 3. Method as stated in claim 2, characterized in that starting materials are used where R is hydrogen or lower alkyl, preferably hydrogen; Rq and R₁ are each C₁ -C₁ lower alkyl; R 2 and R 1 are each hydrogen or C 1 -C 3 lower alkyl, preferably hydrogen or methyl; R 1 is or is converted to hydrogen, lower alkanoyl or benzoyl, preferably hydrogen; and n is 0 or 1. 4. Method as stated in claim 2, characterized in that starting materials are used where R, R^, R^ and R4 are each resp. converted into hydrogen; RQ and R^ are each methyl; and n is 1. 5. Method as stated in claim 2, characterized in that starting materials are used where R,. R 1 and R 3 are each hydrogen; Rq and R2 are each methyl; R 1 is acetyl; and n is 1. 6. New compound, characterized in that it has the formula where RQ and R.^ have the same meaning as in claim 1. 7. Compound as set forth in claim 6, characterized in that Rq is lower alkyl, preferably methyl, and Rq is hydrogen. 8. Method for producing an amino acid compound with the formula wherein R is hydrogen or lower alkyl; and Rq and R^ are each hydrogen, lower alkyl, allyl, propargyl, cyclo-lower alkyl, phenyl or phenyl substituted with halogen, lower alkyl, lower alkoxy, lower alkylthio or trifluoromethyl, or R and R^ together complete a pyrrolidine- , piperidine or morpholine ring, characterized in that from the corresponding compound where the amino group is protected, the protecting group is removed. 9. Method as stated in claim 8, characterized in that the protecting group is carbobenzoxy, p-toluenesulfonyl or acetyl. 10. Method as stated in claims 8 and 9, characterized in that Rq is lower alkyl, preferably methyl, and is hydrogen.