NO153569B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE BRAND CAPACETYL-PROLIN DERIVATIVES. - Google Patents

Abstract

ANALOGICAL PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE MARKAPTOACETYL-PROLIN DERIVATIVES.

Description

The present invention relates to the preparation of new mercapto-acetyl-proline and pipecolic acid derivatives of formula I and salts thereof:

R and Rg are hydrogen or lower alkyl.

R 1 and R 2 are hydrogen, lower alkyl or halogen-substituted

lower alkyl.

X, and X2 is oxygen or sulfur.

R 1 and R 2 are lower alkyl, or R 1 and R ? form together

an optionally lower alkyl-substituted polymethylene chain to complete a 5- or 6-membered ring.

When R^ and R£ together form a polymethylene chain with 2

or 3 carbon atoms, these cyclic ketals and thioketals can be illustrated as follows:

where t is 2 or 3 and Rg and Rq are both hydrogen or both are lower alkyl, or one is hydrogen and the other is lower alkyl.

Preferably, only one carbon atom in the polymethylene chain is substituted.

Rj. is hydrogen, lower alkanoyl, benzoyl or a sulfide group

with the formula

m is 0, 1 or 2.

The star in the above formula indicates an asymmetry center

in the ring. This center is in L configuration.

Centers of asymmetry may also be present in the mercaptoacyl side chain, depending on the meaning of R 1 , R 2 , and R 3 . Another center of asymmetry may also be present in the ring when X^-R^

and X2-R2 are different. The synthesis described below can use the racemate or one of the enantiomers as starting material. When the racemic starting material is used in the synthesis, the stereoisomers obtained in the final product are separated in the usual way by chromatography or fractional crystallization. If there is an asymmetry center in the mercaptoacyl side chain, this is in the D configuration.

In its broadest scope, the present invention relates to the production of mercaptoacyl derivatives of proline with formula I and salts thereof, which can be used as anti-hypertensive agents.

The term lower alkyl as used in the definition

of the symbols R, R^, R2, R4 0<3 R6 denote*" linear or branched hydrocarbon radicals of up to 7 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, etc. Preferred lower alkyl groups are those of up to 4 carbon atoms, of which methyl and ethyl are most preferred.Similarly, the terms lower alkoxy and lower alkylthio denote such lower alkyl groups bonded to oxygen or sulfur.

The term halogen-substituted lower alkyl denotes such lower alkyl groups as described above where one or more of the hydrogen atoms are replaced by chlorine, bromine or fluorine, such as trifluoromethyl, pentafluoroethyl, 2,2,2,-trichloroethyl chloromethyl, bromomethyl, etc.

In the case of the mercaptoacyl side chain, preferred end products are those compounds where Rj. is hydrogen, m is 0

or 1, R^ is hydrogen, and R^ and Rg are both lower alkyl of 1 to 4 carbon atoms, especially both are methyl, or Rg is hydrogen and R^ is hydrogen, lower alkyl of 1 to 4 carbon atoms, especially methyl, or trifluoromethyl. Also preferred, both as intermediates and final products, are the above side chains where R5 is lower alkanoyl with 1 to 4 carbon atoms, especially acetyl or benzoyl.

Particularly preferred as end products are the compounds

of formula I having a mercaptoacyl side chain where R,, is hydrogen,

m is 1, R^ and Rg are hydrogen, and R^ is methyl.

Preferred compounds with respect to the substituents

on the proline ring, are those wherein R 1 and R 2 are independently selected from lower alkyl of 1 to 4 carbon atoms, especially methyl or ethyl; or X^-R^ and X2_R2 are joined together to form

where Rg and R^q are both hydrogen or both lower alkyl with 1 to 4 carbon atoms, in particular both are hydrogen or both

methyl, or Rg is hydrogen and R^q is lower alkyl of 1 to 4 carbon atoms, especially methyl.

The most preferred compounds with regard to the substituents on the proline ring are those where X and X2 are the same, especially those where Xl-Rl°^ X2-R2 are both methoxy or both ethoxy or X1~R1°^ X2-R2 is only <^et together to form

The compounds of formula I are obtained by substituting proline or pipecolic acid with the formula

(II)

is coupled with an acid or its chemical equivalent such as an acid chloride of the formula where R,.1 is lower alkanoyl or benzoyl, to give the product of the formula

The reaction can be carried out in the presence of a coupling agent such as dicyclohexylcarbodiimide or the like, or the acid can be activated by the formation of its mixed anhydride, symmetrical anhydride, acid halide, active esters or by using Woodward's reagent K, N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline or the like. For an overview of acylation methods, reference is made to Methoden der Organischen Chemie (Houben-Weyl), Vol. XV, part II, page 1 et seq. (1974). Preferably, the acid halide, in particular the acid chloride of formula III, is reacted with the acid of formula II.

If the proline of formula II is reacted in

the ester form, the resulting ester product of formula IV, i.e. R is alkyl, can be converted to the free acid, i.e. R is hydrogen, by conventional methods. For example, when R is ethyl this ester protecting group can be removed by saponification.

The product of formula IV is isolated and purified preferably 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 bisulfate.

The product of formula IV can be converted into the products

with formula I where R,, is hydrogen by ordinary hydrolysis or by ammonolysis.

The products of formula I where R^ is

is obtained by direct oxidation with iodine of a product of formula I where R,, is hydrogen.

The esters of formula I where R is lower alkyl can be obtained from the carboxylic acid compounds, i.e. where R is hydrogen, by usual esterification methods, e.g. by esterification with a diazoalkane such as diazomethane, a 1-alkyl-3-p-tolyl triazene, then

such as 1-n-butyl-3-p-tolyltriazene or the like.

The disubstituted prolines with

formula II where X-^_R^ and X2_R2 are -'•-i-ke' ^an is obtained by reacting an N-protected keto-preparation with the formula

where Cbz means carbobenzyloxy, with an alcohol or thiol of the formula

in the presence of an orthoformate or thioformate of the formula HCCX^-R^)^ and an acid such as concentrated sulfuric acid or p-toluenesulfonic acid. This reaction can be carried out in an inert organic solvent such as benzene, acetic acid, ether, cyclohexane etc., preferably without heating, e.g.

at about reflux temperature. See Buehler et al.,

Survey of Organic Syntheses (Wiley & Sons, 1977) vol. 1,

pages 516-519. The product from this reaction is the N-protected intermediate with the formula

The N-protected intermediate of formula VII can be treated with one molar equivalent of an alcohol or thiol of the formula

according to the conditions described above to give the intermediate By using a molar excess of the alcohol or thiol of formula VIII, one obtains the intermediate

The N-protecting group can be removed by conventional methods, e.g. when X^ and X are both oxygen, by hydrogenolysis in the presence of a palladium-charcoal catalyst, or when one or both of X^ and X_ is sulfur, by treatment with HBr and acetic acid to obtain the disubstituted compounds of formula II.

Similarly, the spiro compounds of formula II (i.e.

and R2 is bonded together in a polymethylene chain) is obtained by reacting the keto compound of formula V with an alcohol or thiol of the formula

where Rg, R^0 and t are as above in the presence of an acid such as p-toluenesulfonic acid, to give the intermediate

Alternatively, the disubstituted compound of formula VII may be treated directly with a molar excess of the alcohol or thiol of formula XI to give the intermediate of formula XII. This method is particularly useful when one or both of Rg and R1Q is different from hydrogen.

As indicated above, the N-protecting group can then be removed to give the spiro compound of formula II.

As an alternative method, the introduction of the X^-R^ and X2-R2 groups can be carried out later in the course of the reaction. According to this modification, the protected keto compound of formula V is treated to remove the protecting group, e.g. with hydrogen bromide, resulting in an intermediate of the formula which is then acylated with the acid, preferably the acid halide, of formula III to give the compound of the formula

can then be introduced at this point, by the methods described above, to give the product of formula IV.

Reference is also made to the following publications for additional illustrative methodology for the preparation of starting materials and intermediates: US patents 4,046,889, 4,105,776,

4,154,935 and 4,116,962; Can. J. Biochem. & Physiol. J37, 584

(1959) ; J.A.C.S. 79, 189 (1957); J. Med. Chem. 21, 445 (1978); Aus. J. Chem. 20, 1493-1509 (1967); Buehler et al., Survey of Organic Syntheses (Wiley & Sons, 1977), vol. 1, pages 516-519, vol. 2, pages 461-470; Chem. Pharm. Bull., Tokyo 26, 2209 and 2217 (1978); Can. J. Chem. 47, 860 (1969); J. Amer. Chem.

Soc, 80, 6350 (1968); Harrison et al., Compendium of

Organic Synthetic Methods, (Wiley-Interscience, New York, 1971), pages 449-456; J. Amer. Chem. Soc, 79^, 192 (1956); Bull. Soc. Chem., 1965(8) pages 2253-2259; J. Org. Chem. 2J5, pp. 521-530

(1960).

The methods illustrated therein can be used as general methods for synthesizing the compounds and separating the isomers that can be used in the present invention. Further illustrative details are provided in the examples which serve as models for the preparation of other compounds in the group.

The compounds produced according to the present invention form basic salts with various inorganic or organic bases. The salt-forming ion from such bases can 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, many 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 of amino acids such 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 isolating or purifying a product which is acceptable for the purposes described below, as illustrated by the dicyclohexylamine 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 where the salt is precipitated or in an aqueous medium, and subsequent lyophilization. The free acid form can be obtained from the salt by usual neutralization techniques, e.g. with potassium bisulphate, hydrochloric acid, etc.

The compounds of formula I are useful as

hypotensive agents. They inhibit the conversion of

the decapeptide angiotensin I to angiotensin II, and are therefore useful in alleviating angiotensin-associated hypertension. The action of the enzyme renin on angiotensinogen, a pseudo-globulin in blood plasma, produces angiotensin I. Angiotensin I is converted by an angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance that is believed to be. the cause of different forms of hypertension in different mammalian species, e.g. rats and dogs. The compounds produced according to the invention intervene in the angiotensinogen ->■ (renin) -> angiotensin I -> (ACE) ->• angiotensin II course by inhibiting the angiotensin-converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin II. When administering a preparation containing one or a combination of compounds of formula I, angiotensin-

dependent hypertension in mammalian species suffering from this, be alleviated. A single dose, or preferably two to four daily

doses, which are given on the basis of 0.1 to 100 mg per kg body weight per day, preferably approx. 1 to 15 mg per kg body weight per day is appropriate to reduce blood pressure. The compound is preferably administered orally, but parenterally, such as.

subcutaneous, intramuscular, intravenous or intraperitoneal administration may also be used.

The compounds produced according to the invention can also

used in combination with a diuretic 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 to a mammal in need of it. Examples of diuretics that can be used in combination with a compound produced according to the invention are thiazide diuretics, e.g. chlorothiazide, hydrochlorothiazide, flumethiazide, hydroglumethiazide, benzdroflumethiazide, methchlothiazide, trichloromethiazide, polythiazide or benzthiazide, as well as ethacrynic acid, ticrynafen, chlorthalidone, furosemide, musolimine, bumetanide, triamterene, amiloride and spironolactone, and salts of such compounds.

The compounds of formula I can, for the reduction of blood pressure, be prepared in preparations such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About. 10 to 500 mg of a compound or a mixture of compounds of formula I is mixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer,

flavoring etc. in the unit dosage form required by acceptable pharmaceutical practice. The amount of active substance in these mixtures or preparations is such that a suitable dose in the specified range is achieved.

Illustrative process details are given in the following examples for the various reactions. These examples are preferred embodiments and also serve as a model for the preparation of other compounds of formula I. The temperatures are given in °C.

The proline ring is in each case in the L configuration and the side chain in the D configuration.

(a) Ij-0 is the concentration of the test compound (ug/ml)

which produces 50% inhibition of angiotensin-converting enzyme isolated from rabbit lung. (b) % inhibition of angiotensin-converting enzyme recovered after 24 hours in rats, as measured by blood pressure after intravenous infusion of Angiotensin I.

Dose of the test compound - 10 mg/kg equivalents of captopril.

Example 1

[ 7( S), 8S]- 7-'( acetylfib)- 2- methyl- 1-oxbpropyl]- 1, 4- dioxo-7- azaspiro [ 4. 4] nonane- 8- carboxylic acid

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

26.5 g (0.20 mol) 4-hydroxy-L-proline and 32.8 ml (0.23 mol)

benzyl chloroformate is reacted in 200 ml of water and 100 ml of acetone in the presence of 20 g (0.02 mol) of potassium bicarbonate and 69.2 g (0.50 mol) of potassium carbonate and worked up with 90 ml of concentrated hydrochloric acid as described in Can. J. Biochem. & Physiol. _37, 584 (1959) to obtain N-carbobenzyloxy-4-hydroxy-L-proline. This product is reacted with cyclohexylamine to form the cyclohexylamine salt in a yield of 69 g, m.p. 193-195°. The salt (34 g) is neutralized with N-hydrochloric acid to obtain 27 g of the free acid as a colorless glass [a]^<6> -70° (c, 1% in chloroform).

b) N-carbobenzyloxy-4-keto-L-proline

21.5 g (0.81 mol) N-carbobenzyloxy-4-hydroxy-L-proline

oxidized in 1.2 liters of acetone with 83 ml of 8N chromic acid in sulfuric acid as described in J.A.C.S. 79 , 189 (1957). To facilitate the subsequent filtration of chromium salt, 30 g of Celite (diatomaceous earth) is added to the acetone solution before introducing the oxidizing agent. Air ducts are used. The reaction mixture is filtered,

and the acetone filtrate is concentrated to approx. 300 ml before dilution with 1 liter of chloroform. The solution is washed with 300 ml saturated sodium chloride (four times), dried (MgSO 4 ), filtered and the solvent evaporated to give N-carbobenzyloxy-4-keto-L-proline (22.8 g) which is crystallized from ether (50 ml) -

hexane (150 mL) to obtain 17.2 g (81%) of the product,

sm.p. 99-101°, [a]^<6> +17° (c, 1% in chloroform).

c) N-carbobenzyloxy-4, 4-ethylenedioxy-L-proline

A stirred mixture of 12.8 g (0.049 mol) of N-carbobenzyloxy-4-keto-L-proline, 53 ml (0.095 mol) of ethylene glycol and 0.35 g of p-toluenesulfonic acid'H 2 O in 1.31 liters of benzene is heated, and the resulting solution is refluxed for 7 hours (water formed is collected in a Dean-Stark apparatus). After standing overnight at room temperature, the lower glycol layer is separated and the benzene solution is washed with 150 ml of saturated sodium chloride, dried (MgSO 4 ) and the solvent is evaporated to give 14.6 g of N-carbobenzyloxy-4,4-ethylenedioxy-L-proline as a syrupy residue. This is dissolved in 60 ml of ethanol, filtered, treated with 5 g of cyclohexylamine and diluted with ether. During inoculation and rubbing, the crystalline cyclohexylamine salt is secreted; weight after cooling overnight, 9.0 g, m.p. 179-180° (p. 173°). The material is recrystallized from acetonitrile, m.p. 182-184° (p. 179°), [ct]^<6> -21° (c, 1% in EtOH).

The cyclohexylamine salt (8.4 g) is suspended in 40 ml of ethyl acetate, stirred, cooled and treated with 40 ml of IN hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 40 mL), the combined organic layers are dried (MgSO 4 ), and the solvent is evaporated, finally at 0.2 mm.

The syrupy residue which begins to crystallize is triturated under ether and the ether evaporated to give 6.4 g (42%) of almost colorless N-carbobenzyloxy-4,4-ethylenedioxy-L-proline, m.p. 101-103° (p. 98°), [a]p<6> -34° (c, 1% in CHCl 3 ).

d) 4, 4-ethylenedioxy-L-proline

A solution of 3.2 g (0.0104 mol) of N-carbobenzyloxy-4,4-ethylenedioxy-L-proline in 100 ml of methanol-water (2:1) is treated with 1 g of 5% palladium charcoal 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 1.7 g (94%) of a colorless solid, 4,4-ethylenedioxy- L-proline, m.p. 245-247° (dec.); [a]^ -32°

(c, 0.5% in 1:1 MeOH-H 2 O).

e) [ 7( S ), 8 S ]- 7-[ 3-( acetylthio )- 2- methyl- l- oxopropyl]- 1, 4-dioxo- 7- azaspiro[ 4. 4] nonan- 8- carboxylic acid

A stirred solution of 3.2 g of 4,4-ethylenedioxy-L-proline (0.0185 mol) in 50 ml of water is cooled to 5° and treated portionwise with solid sodium carbonate to pH 8.5. A solution of 3.7 g (0.020 mol) of D-3-acetylthio-2-methylpropanoyl chloride in 5 ml of ether is then added in portions under

continued stirring and cooling, while the pH value is kept at 8.5

with 25% sodium carbonate solution (approx. 14 ml). After 1 1/4 hours, the solution is treated with 50 ml of ethyl acetate, stirred, cooled, carefully acidified with hydrochloric acid (1:1) to pH 2.0, saturated with sodium chloride and the layers separated. The aqueous phase is extracted with further ethyl acetate (3 x 50 ml), the combined organics

layer is dried (MgSO4) and the solvent is finally evaporated at 0.2 mm. The solid residue is triturated under ether, and evaporation is repeated to obtain 5.9 g (100%) of [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1, 4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid,

sm.p. 108-111°.

The product is converted to the dicyclohexylamine salt with 3.4 g of dicyclohexylamine in 70 ml of ethyl acetate. During inoculation and rubbing, the crystalline salt is precipitated and recrystallized from 95 ml of acetonitrile; yield 6.7 g, m.p. 187-189° (p. 184°), [a]j?,<5> -59°

(c, 1% in EtOH).

The dicyclohexylamine salt is converted to the free acid by

that it is suspended in ethyl acetate and treated with 75 ml of 10% potassium bisulphate and stirred until two layers are obtained. After separation, the aqueous phase is extracted with ethyl acetate

(4 x 75 ml), they . organic layers are combined, dried (MgSO 4 ), and the solvent evaporated to give 4.1 g of colorless [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1, 4-dioxo-7-azaspiro-[4.4]nonane-8-carboxylic acid, m.p. 120-122° (p. 117°) [a]^5 -118°

(c, 1% in EtOH).

Example 2

[7(S)-8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid

Argon is passed through a cold solution of 8.5 ml of concen-

triturated ammonium hydroxide in 20 ml of water. 4.0 g (0.013 mol)

[7 (S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro-[4.4]nonane-8-carboxylic acid from example le to-

is then added, and the mixture is stirred in an ice bath for a few

minutes and at room temperature under argon for 2 hours. Up-

the solution is treated with 30 ml of ethyl acetate, cooled, stirred and acidified with 16 ml of hydrochloric acid (1:1). The layers are separated, the <0 >aqueous phase is extracted with a further 30 ml of ethyl acetate (twice), the ethyl acetate extracts are combined, dried (MgSO 4 )

and the solvent is evaporated to give [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid as a solid residue. The product is triturated under ether and evaporation is repeated. The product is then triturated with 30 ml of hexane, cooled for 1 hour, filtered under argon and dried in vacuo to give 2.7 g of colorless solid [7(S),8S]-7-(3-mercapto-

2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, m.p. 131-133° (p. 125°), [a]D<5> -66°

(c, 1% in EtOH).

Example 3

( S )- 1-[( 3- acetylthio)- 2- methyl- l- oxopropyl]- 4, 4- dimethoxy-L- proline

a) N- carbobenzyloxy- 4, 4- dimethoxy- L- proline methyl ester

A stirred solution of 7.8 g (0.03 mol) of N-carbobenzyloxy-4-keto-L-proline from Example 1 in 60 ml of methanol is treated with 96 ml of trimethyl orthoformate, followed by 0.6 ml of concentrated sulfuric acid and allowed to stand overnight above at room temperature.

The pale yellow solution is stirred, treated with 1.5 g of potassium carbonate, followed by 30 ml of water, and most of the solvent is removed on a rotary evaporator to give a syrupy residue which is shaken with 30 ml of water and 30 ml of chloroform. After separation of the layers, the aqueous phase is extracted with additional chloroform (3 x 30 ml), and the combined organic layers are washed with 45 ml of saturated sodium chloride solution and dried

(MgSO 4 ). Evaporation of the solvent gives 8.4 g (88%) of N-carbobenzyloxy-4,4-dimethoxy-L-proline methyl ester.

b). N-carbobenzyloxy-4, 4-dimethoxy-L-proline

The ester (8.4 g, 0.026 mol) from part a) is dissolved in 80 ml

methanol, treated dropwise at -1 to 4° with 18 ml (0.036 mol) of 2N sodium hydroxide, kept at 0° for 1 hour and at room temperature overnight'. After removing approx. half of the solvent on a rotary evaporator, the solution is diluted with 150 ml of water, washed with 100 ml of ether (the washing liquid is discarded), acidified under cooling with 63 ml of 1:1 hydrochloric acid to pH 2 and extracted with ethyl acetate (4 x 750 ml). The combined extracts are washed with 50 ml saturated sodium chloride solution, dried (MgSO 4 ) and the solvent evaporated to give 8.0 g of a pale yellow viscous oil. The oil is dissolved in 35 ml of ethanol, treated with 3.0 g of cyclohexylamine in 10 ml of ethanol and diluted to 500 ml with ether. During seeding and rubbing, the crystalline N-carbobenzyloxy-4,4-dimethoxy-L-proline-cyclohexylamine salt is secreted; weight after cooling overnight, 7.0 g, m.p. 157-159° (p, 151),

26 o

[α]Q -34 (c, 1% in EtOH). This material is recrystallized from

in Example 4 to give 2.4 g (92% (S)-4,4-diethoxy-1-(3-mercapto-2-methyl-1-oxopropyl)-L-proline as an almost colorless viscous syrup [ α]^ -64° (c, 1% in ethanol).

Analysis:

Calculated for C13H23N05-0.25 H2O: C 50.38, H 7.64, N 4.52, S 10.35 Found: C 50.68, H 7.96, N 4.78, S 10.07.

Example 7

[ 2( S ), 3 S ]- 2-[ 3-( acetylthio )- 2- methyl- l- oxopropyl]- 6, 10- dioxo-2- azaspiro[ 4. 5] decane- 3- carboxylic acid

a) N-carbobenzyloxy-4, 4-trimethylenedioxy-L-proline

Reaction between 8.2 g (0.031 mol) N-carbobenzyloxy-4-keto-L-proline and 45 ml (0.62 mol) 1,3-propanediol in 450 ml benzene

in the presence of 500 mg of p-toluenesulfonic acid gives 12.3 g of crude, viscous ester product. This product is saponified with 70 mL of 1N sodium hydroxide to give 10.6 g of crude N-carbobenzyloxy-4,4-trimethylenedioxy-L-proline as a yellow oil. This is dissolved in 40 ml of ethanol - 400 ml of ether and treated with 3.2 g of cyclohexylamine to give 10.1 g of N-carbobenzyloxy-4,4-trimethylenedioxy-L-proline-cyclohexylamine salt, m.p. 163-165° (p. 160°),

[α]j^ -27° (c, 1% in ethanol). Crystallization of 9.8 g of the salt from 300 ml of acetonitrile gives 9.5 g of colourless, solid cyclohexylamine salt, m.p. 165-167° (s, 162°) la]* 5 -27° (c, 1% in ethanol).

The cyclohexylamine salt (9.0 g) is suspended in 40 ml of ethyl acetate, stirred, cooled and treated with 45 ml of IN hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 40 mL), the combined organic layers are dried (MgSO 4 ), and the solvent is evaporated to give 7.1 g (75%) of a glassy N-carbobenzyloxy-4, 4-trimethylenedioxy-L-proline.

b) 4, 4-trimethylenedioxy-L-proline

A solution of 7.1 g (0.022 mol) of N-carbobenzyloxy-4,4-trimethylenedioxy-L-proline in 200 ml of 2:1 methanol-water is hydrogenated in the presence of 2 g of a 5% palladium-charcoal catalyst to give 3.8 g (93%) almost colorless 4,4-trimethylenedioxy-L-proline,

sm.p. 234-236° (decomposition), after previous gradual darkening and sintering; [a]^<5> -36° (c, 0.5% in 1:1 methanol-water).

Analysis: Calculated for CgH^NC^: C 51.33, H 7.00, N 7.48

Found: C 51.42, H 7.11, N 7.40.

c) [ 2 ( S ), 3 S ]- 2-[ 3-( acetylthio )- 2- methyl- 1- oxopropyl]- 6, 10-dioxb- 2- azaspiro[ 4, 5] decah- 3- carboxylic acid

4,4-trimethylenedioxy-L-proline (3.7 g, 0.02 mol) is acylated

with 4.0 g (0.022 mol) of D-3-acetylthio-2-methylpropionyl chloride in 50 ml of water in the presence of sodium carbonate according to the procedure of Example 1(e) to give 7.3 g of a glassy crude product.

The product is converted to the dicyclohexylamine salt with 3.6 g of dicyclohexylamine in 70 ml of ethyl acetate. By inoculation and rubbing, the crystalline salt precipitates and gives 7.5 g of dicyclohexylamine salt, m.p. 168-170° (p. 166°), [ a]^ 6 -59° (c, 1% in ethanol). Recrystallization from 30 ml of acetonitrile gives 6.5 g colorless,

solid salt, m.p. 169-171°, [ a.]* 5 -63° (c, 1% in ethanol).

The dicyclohexylamine salt is converted to the free acid by

that 6.4 g is suspended in 75 ml of ethyl acetate and treated with 75 ml of 10% potassium bisulphate and stirred, until two layers are obtained. After separation, the aqueous phase is extracted with ethyl acetate

(4 x 75 mL), the organic layers are combined, dried (MgSO 4 ), and the solvent evaporated to give 4.3 g (6 7%) of glassy [2(S),3S]-2-[3-(acetylthio) -2-methyl-1-oxopropyl]-6,10-dioxo-2-azaspiro[4.5]decane-3-carboxylic acid.

Example 8

[ 2( S ), 3 S ]- 2-( 3- mercapto- 2- methyl- l- oxopropyl)- 6, 10- dioxo- 2-azaspiro[ 4. 5] decane- 3- carboxylic acid

[2(S),3S]-2-[3-(acetylthio)-2-methyl-1-oxopropyl]-6,10-dioxo-2-azaspiro[4.5]decane-3-carboxylic acid (4.3 g, 0.013 mol) is hydrolysed with 8.5 ml of concentrated ammonia in 20 ml according to the procedure of Example 2 to give 0.9 g of a colorless solid product; ta]D -64° (c, 0.5% in ethanol). A further 0.8 g of product is obtained by extraction of the aqueous phase with chloroform; [a]^ -66°. The two portions are dissolved in chloroform, evaporated, triturated under ether and evaporated again to give 1.7 g (46%) of [2(S),3S]-2-(3-mercapto-2-methyl-1-oxopropyl) -6,10-dioxo-2-azaspiro[4.5]decane-3-carboxylic acid, m.p. 169-171° (p. 167°), [a]^<6> -71° (c, 1% in methanol).

Analysis:

Calculated for C12HigN05S: C 49.81, H 6.62, N 4.84, S 11.08 Found: C 49.67, H 6.67, N 4.93, S 11.10.

Example 9

[ 7( S ), 8 S ]- 7-[ 3-( acetylthio )- 2- methyl- l- oxopropyl]- 7- aza- l, 4-dithiaspiro[ 4. 4] nonane- 8- carboxylic acid

a) N- carbobenzyloxy- 4, 4- ethylenedithio- L- proline- methyl ester

A stirred solution of 3.9 g (0.014 mol) of N-carbobenzyloxy-4-keto-L-proline methyl ester in 60 ml of methylene chloride is treated with 3 ml (0.036 mol) of ethanedithiol, cooled to 8° and treated under a blanket of argon with 3 ml (0.02 4 mol) boron trifluoride etherate. After removing the cooling bath, the pale yellow solution is stirred

for a further 1 hour and kept overnight at room temperature. The solution is stirred, treated with several pieces of crushed ice, followed by 20 ml of water. After 30 minutes, the layers are separated and the aqueous phase (50 ml) is extracted with further methylene chloride (3 x 30 ml). The combined organic layers are washed with 50 mL saturated sodium chloride solution, dried (MgSO 4 ), and the solvent removed on a rotary evaporator to give 6 g (100%) of a pale yellow oil, N-carbobenzyloxy-4,4-ethylenedithio-L -proline methyl ester.

b) N- carbobenzyloxy- 4, 4- ethylenedithio- L- proline The methyl ester product from part (a) (7.4 g, about 0.018 mol)

dissolved in 65 ml methanol, treated dropwise at -1 to 4° with 14.5 ml (0.029 mol) 2N sodium hydroxide, kept at 0° for 1 hour and at room temperature overnight. After approx. half of the solvent is removed on a rotary evaporator, the solution is diluted with 125 ml of water, washed with ether (the washing liquid is discarded), acidified under cooling with 5 ml of 1:1 hydrochloric acid to pH 2, and extracted with ethyl acetate (4 x 50 ml).

The combined extracts are washed with 50 mL saturated sodium chloride, dried (MgSO 4 ), and the solvent evaporated to give 6 g of a pale yellow viscous oil. This oil is dissolved in 25 ml of ethanol, treated with 1.8 g of cyclohexylamine in 5 ml of ethanol, and diluted to 300 ml with ether. By inoculation and rubbing, the crystalline cyclohexylamine salt is separated to give, after cooling overnight, 5.7 g of N-carbobenzyloxy-4,4-ethylenedithio-L-proline-cyclohexylamine salt, m.p. 205-207° (p. 201°). Recrystallization from 50 ml of ethanol - 400 ml of ether gives 4.9 g

of a colourless, solid salt, m.p. 207-209° (p. 201°),

[a]^ -15° (c, 1% in chloroform).

The cyclohexylamine salt (4.8 g) is suspended in 25 ml of ethyl acetate, stirred and treated with 25 ml of IN hydrochloric acid. When two clear layers are obtained, these are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 25 mL), the combined organic layers are dried (MgSO 4 ) and the solvent is evaporated to give 3.8 g (62%) of N-carbobenzyloxy -4,4-ethylenedithio-L-proline as a pale yellow viscous syrup.

c) 4, 4-ethylenedithio-L-proline hydrobromide

N-carbobenzyloxy-4,4-ethylenedithio-L-proline (3.7 g, 0.011 mol)

treated with 20 ml hydrogen bromide in acetic acid (30-32%),

cork loosely and stir magnetically. Mixing is difficult

due to the viscosity of the starting material, and this is broken up as much as possible with a spatula. Meanwhile, the crystalline product begins to be secreted. Further amounts of hydrogen bromide in acetic acid are added after 15 minutes (10 ml) and after 25 minutes (5 ml) and stirring is continued for a total of 35 minutes. Ether (250 mL) is added to complete the precipitation of the product, and after cooling for 15 minutes, the cream-colored material is filtered under nitrogen, washed with ether, and dried in vacuo to give 2.7 g of 4,4-ethylenedithio-L-proline- hydrobromide, m.p. 240-242° (dec.); sintering and darkening from approx. 200°;

[α]p6 -40° (c, 0.5% in 1:1 chloroform-methanol).

d) [ 7( S ), 8 S ]- 7-[ 3-( acetylthio )- 2- methyl- l- oxopropyl]- 7- aza-1, 4- dithiaspiro[ 4. 4] nonane- 8- carboxylic acid

A stirred solution of 2.6 g (0.0091 mol) of 4,4-ethylenedithio-L-proline hydrobromide in 25 ml of water is cooled to 5° and brought

to pH 8.2 by adding 25% sodium carbonate (weight/volume). Add in portions while continuing to stir and cool

a solution of 1.9 g (0.01 mol) of D-3-acetylthio-2-methyl-propionyl chloride in 2.5 ml of ether while maintaining the pH at 7.5-8.2 by dropwise addition of 25% sodium carbonate. When the pH value is stabilized at 8.2-8.5 (after approx. 15 minutes), stirring and cooling are continued for a total of 1 hour. The solution is then washed with 25 ml of ethyl acetate (the washing liquid is discarded), covered with a

layer with 25 ml of ethyl acetate, cool, stir, acidify carefully with 1:1 hydrochloric acid to pH 2.0, saturate with sodium chloride, and separate the layers. The aqueous phase is extracted with further ethyl acetate (3 x 25 ml), the combined organic layers are dried

(MgSO^) and the solvent is evaporated, finally at 0.2 mm,

to give 2.6 g of a syrupy product which begins to crystallize. This is treated in 30 ml of ethyl acetate with 1.5 g of dicyclohexylamine to give 3.0 g of colorless dicyclohexylamine salt; sm.p. 176-178° (p, 170°); [a]^<6> -55° (c, 1% in ethanol). This material is ground in a mortar under 15 ml of acetonitrile, cooled for 1 hour, filtered, washed with 5 ml of cold acetonitrile and with ether, and dried to give 2.9 g of dicyclohexylamine salt, m.p. 177-179° (p, 172°); [a]^<6> -56° (c, 1% in ethanol).

Analysis :

Calculated for C13HigN04S•C12H23<N:> C 56.56, H 7.98, N 5.28, S 18.12 Found: C 56.21, H 8.18, N 5.05, S 18.00.

The above-mentioned dicyclohexylamine salt is converted to

the free acid by suspending 2.8 g in 30 ml of ethyl acetate, cooling and treating with 30 ml of 10% potassium bisulphate to

give two clear layers. After separation, the aqueous phase is extracted with ethyl acetate (3 x 30 mL), the combined organic layers are dried (MgSO.), and the solvent is evaporated, finally at 0.1-0.2 mm and 45° to give 2.0 g (63%) colorless solid [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-7-aza-1,4-dithiaspiro[4.4]-nonane-8 -carboxylic acid, m.p. 125-126° (p. 122°), [ct]^<6> -101

(c, 1% in ethanol).

Example 10

[ 7( S ), 8 S ]- 7-( 3- mercapto- 2- methyl- l- oxopropyl)- 7- aza- l, 4-dithiaspiro[ 4. 4] nonane- 8- carboxylic acid

Argon is passed through a cold solution of 3.5 ml of concentrated ammonia in 8.5 ml of water for 15 minutes. The mixture is then placed under cooling and under a blanket of argon,

to 1.9 g (0.0054 mol) [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-7-aza-1,4-dithiaspiro[4.4] nonane-8-carboxylic acid and the mixture is vortexed in an ice bath until a solution is obtained. Stirring under argon is continued at room temperature for a further 2 hours, then the solution is extracted with 15 ml of ethyl acetate under an argon atmosphere. The aqueous layer

cooled, stirred, covered with a layer of 15 ml of ethyl acetate and acidified in portions with 6.5 ml of 1:1 hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 15 ml), the combined acetate layers are dried (MgSO4),

and the solvent is evaporated to give a glassy residue which solidifies on rubbing under ether. Evaporation is repeated,

and the colorless product is suspended in 30 ml of hexane,

filtered and dried in vacuo to give 1.4 g (84%) of [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-7-aza-1,4-dithiaspiro [4.4]-nonane-8-carboxylic acid; sm.p. 116-118° (p, 105°); [a]^<6> -44°

(c, 1% in ethanol).

Analysis:

Calculated for C^H^NO-jS: C 42.97, H 5.57, N 4.56, S 31.29, SH 100%. Found: C 42.70, H 5.71, N 4.54, S 31.16, SH 100%.

Example 11

( 8S)- 7-[ 3-( acetylthio)- 2D- trifluoromethyl- 1- oxopropyl]- 1, 4- dioxo-7- azaspiro[ 4. 4] nonan- 8- carboxylic acid

a) D,L-3-(acetylthio)-2-trifluoromethylpropionic acid α-trifluoromethylacrylic acid (10 g, 0.071 mol) [prepared

according to the procedure set forth in J. Chem. Soc, 1954 ,

p. 371] is cooled in a salt-ice-water bath, stirred and treated in portions with 5.7 ml (0.075 mol) 97% thiolacetic acid. After the addition, the yellow liquid is stirred cold for 1 hour, allowed to warm to room temperature and distilled to give 14 g (91%) of D,L-3-(acetylthio)-2-trifluoromethylpropionic acid as a pale yellow

oil, k.p. 149-153°/13 mm. The material becomes solid when stored cold. b) D,L-3-(acetylthio)-2-trifluoromethylpropionyl chloride D,L-3-(acetylthio)-2-trifluoromethyl-propionic acid (7 g,

0.032 mol) is treated with 18 ml (0.25) of redistilled thionyl chloride and the mixture is refluxed for 3 hours. After removal of excess thionyl chloride on a rotary evaporator,

distill the residue to give 6.8 g of D,L-3-(acetylthio)-2-trifluoromethylpropionyl chloride as a pale yellow oil, m.p. 80-82°/16 mm.

c) (8S)-7-[3-(acetylthio)-2D-trifluoromethyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid

4,4-ethylenedioxy-L-proline (2.4 g, 0.014 mol) is reacted

with 3.4 g (0.014 mol) of D,L-3-(acetylthio)-2-trifluoromethyl-propionyl chloride in 40 ml of water in the presence of sodium carbonate according to the procedure of Example 1(e) to give 4.5 g of almost colorless solid product; sm.p. 126-145° (p. 115°),

[d]p<5> -34° (c, 1% in ethanol).

The mixture of diastereoisomers (4.2 g) is suspended in

45 ml of ether, stirred for 2 hours, cooled for 20 minutes and the undissolved solid filtered, washed with cold ether and air-dried to give 2.7 g of the product; sm.p. 166-172° (p. 140°),

25' o

[a]D -62 (c, 1% in ethanol). The material is then ground in a mortar under 25 ml of ether, filtered after 15 minutes, washed with some ether and air-dried again to give 2.1 g of the product;

sm.p. 172-177° (p. 143°). Subsequent crystallization from 11 ml of boiling isopropanol and cooling overnight gives 1.55 g of colorless (8S)-7-[3-(acetylthio)-2D-trifluoromethyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4 ]nonane-8-carboxylic acid;

sm.p. 192-194° (s, 183°), [a]^<5> -32° (c, 1% in ethanol). A sample is recrystallized from isopropanol; sm.p. 193-195°

(p. 184°), [α]p<5> -134° (c, 1% ethanol).

Example 12

(8S)-7-(3-mercapto-2D-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.

The product from Example 16 (1.45 g, 0.0039 mol) is hydrolyzed with 2.5 ml of concentrated ammonia in 6 ml of water over a period of 1 hour as described in Example 2, to give 1.25 g (97%) colorless (8S)-7-(3-mercapto-2D-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, isomer A, as a glassy product, [a] p<5> -61° (c, 1% in ethanol).

TLC: Rf 0.40 (95:5:5 methylene chloride-methanol-acetic acid;

show. SH reagent, PMA and heat).

Analysis: chh;l4F3N05S:

Calculated: C 40.12, H 4.28, N 4.25, S 9.74, F 17, 31

Found: C 40.10, H 4.43, N 4.51, S 9.63, F 17.10.

The above acid is dissolved in ethyl acetate and treated with 1-adamantanamine to give the 1-adamantanamine salt; sm.p. 213-215°

(dec.), [α]p<5> -47° (c, 1% in methanol).

Example 13

1-(3-mercaptol-l-oxopropyl)-4,4-dimethylthio-L-proline

a) 4-keto-L-proline hydrobromide

To 4.0 g (0.015 mol) N-carbobenzyloxy-4-keto-L-proline

add 20 ml of hydrogen bromide in acetic acid (30-32%). The mixture is vortexed frequently for a period of 8 minutes. At the end of this period (effervescence has ceased), the yellow-orange solution is covered with a layer of 25 ml of ether, and the gummy product is rubbed off. The ether layer is discarded and the resulting sticky solid is triturated with fresh ether and finally with 50 mL of acetonitrile to give 4-keto-L-proline hydrobromide as a crystalline solid weighing 2.7 g (85%). sm.p. 153-155° (dec.),

[a]-49° (c, 1% in water).

b) 1-[3-(acetylthio-l-oxopropyl]-4-oxo-L-proline

A stirred solution of 4.1 g (0.0195 mol) of 4-keto-L-proline hydrobromide in 50 ml of water is cooled to 5° and treated portionwise with solid sodium carbonate (foaming is controlled by the addition of a few drops of ether) to pH 8.0 (about 2 g is needed). During continued stirring and cooling, a solution of 3.5 g (0.012 mol) of 3-acetylthiopropanoyl chloride in 5 ml of ethyl acetate is then added in portions using a pipette while the pH value is kept at 7.0-8.0 by dropwise addition of 25% (weight/volume) sodium carbonate solution (approx. 10 ml). After approx. After 10 minutes, the pH value stabilizes at 8.0-8.4. After continued stirring and cooling for a total of 1 hour, the solution is washed with ethyl acetate (2 x 50 ml), covered with a layer of 50 ml ethyl acetate, stirred, cooled, acidified carefully with concentrated hydrochloric acid to pH 2.0, saturated with sodium chloride ,

and the layers are separated. The aqueous phase is extracted with additional ethyl acetate (3 x 50 ml), the combined organic layers are dried (MgSO4)

and the solvent is evaporated, finally at 0.2 mm to give 4.8 g of a yellow-orange glassy residue. This residue is dissolved in 35 ml of ethyl acetate and treated with a solution of 3.5 g of dicyclohexylamine in 5 ml of ethyl acetate. By inoculation and rubbing, the crystalline 1-[3-(acetylthio-l-oxo-propyl)]-4-oxo-L-proline-dicyclohexylamine salt is separated, weight after cooling overnight, 2.7 g (almost colorless), m.p. 191-193°

(dec.) [a]p<6> -24° (c, 1% in CHCl3).

This dicyclohexylamine salt is converted to the free acid using potassium bisulphate as described in Example 1 (e), to give 3.7 g of 1-[3-(acetylthio)-1-oxopropyl]-4 H -oxo-L-proline as a pale yellow glassy solid.

c) 1-[3-(acetylthioyl-l-oxopropyl]-4,4-dimethylthio-l-proline

1-[3-(acetylthio)-1-oxopropyl]-4-oxo-L-proline is reacted

with methylthio according to the procedure from example 9

(a) to give 1-[3-(acetylthio)-1-oxopropyl]-4,4-dimethylthio-L-proline.

d) 1-(3-mercaptol-l-oxopropyl)-4,4-dimethylthio-L-proline

The product from part (b) is hydrolysed with concentrated

ammonia according to the procedure from example 2 to

give 1-(3-mercapto-1-oxopropyl)-4,4-dimethylthio-L-proline.

Example 14

[ 7 ( s ), 8S]- 7-( 3- mercapto- 2- methyl- l- oxopropyl)- 1, 4- dioxo- 7- azaspiro[ 4. 4] nonan- 2- methyl- 8- carboxylic acid

a) N- carbobenzyloxy- 4, 4-( 1- methylethylenedioxy)- L- proline methyl ester

A stirred mixture of 8 g (0.025 mol) N-carbobenzyloxy-4,4-dimethoxy-L-proline methyl ester from Example 3a, 2.4 g (0.032 mol) 1,2-propanediol, 0.4 g p-toluenesulfonic acid -monohydrate and 400 ml of toluene are heated to reflux temperature (110-112°). The backflow treatment is regulated so that

solvent is distilled slowly using a Dean-

Stark tube into a graduated cylinder. When 80 ml of solvent has been collected, an equal volume of fresh solvent is added to the reaction flask through an addition funnel. This method of removing and adding 80 ml of the solvent is repeated four times during a total reflux treatment time of 1.25 hours.

After standing overnight, the mixture is washed with water

(2 x 100 mL), the combined washings back-extracted with 100 mL toluene, the combined organic layers dried (MgSO 4 ), and the solvent removed on a rotary evaporator, finally at 0.2 mm, to give 8.2 g (99 %) N-carbobenzyloxy-4,4-(1-methylethylenedioxy)-L-proline methyl ester as a yellow, viscous oil.

b) N- carbobenzyloxy- 4, 4-( 1- methylethylenedioxy)- L- proline

The crude methyl ester product from part (a) (8.2 g, 0.025 mol)

dissolve in 80 ml methanol, treat dropwise at -1 to 4° with 18 ml (0.036 mol) 2N sodium hydroxide, keep at 0° for 1 hour,

and at room temperature overnight. After removing approx. half of the solvent on a rotary evaporator, the solution is diluted with 150 ml of water, washed with 100 ml of ether (the washing liquid is discarded), acidified while cooling with 6.3 ml

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 8 g of a red-orange viscous oil. This oil is dissolved in 50 ml of acetonitrile, heated, stirred and treated with 3.8 g of 1-adamantanamine. The solid salt is excreted quickly. After cooling overnight, the material is filtered, washed with cold acetonitrile and with ether and dried in vacuo to give 10.3 g of crude adamantamine salt, m.p. 202-204° (dec.),

[2]D 26 -13 o (c, 1% in methanol). After trituration with 50 ml of boiling acetonitrile and cooling, the light brown solid salt weighed 9.4 g, m.p. 202-204° (dec.), [a]^<6> -13° (c, 1% in methanol).

The above adamantanamine salt is suspended in 40 ml of ethyl acetate, stirred and treated with 1N hydrochloric acid. When two clear layers are obtained, these are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml), the combined organic layers are dried (MgSO 4 ) and the solvent is evaporated, finally at 0>2 mm and 40 , to give 5>8 g (72%) of N-carbobenzyloxy-4,4-(1-methylethylenedioxy)-L-proline as a yellow-orange viscous syrup.

c) 4, 4-(1-methylethylenedioxy)-L-proline

A solution of the above N-carbobenzyloxy-4,4-(1-methylethylenedioxy)-L-proline (5.6 g, 0.017 mol) in 150 ml of 2:1 methanol-water is treated with 1.6 g of 5% palladium -coal catalyst and shaken under 3 atmospheres of hydrogen for 5 hours. The catalyst from raf is evaporated under nitrogen, washed with methanol,

and the combined filtrates are evaporated, finally at 0.1-0.2 mm to give a crystalline residue. This is suspended in 200 ml of methanol and evaporation is repeated. The solid residue is triturated under ether (evaporation repeated) to give 3.0 q (94%)

of a light brownish 4 ,4-(1-methyl -

ethylenedioxy)-L-proline , m.p. 219-221° (decomposition), after previous darkening and sintering; tot]^ -22° (c, 1% in 1:1 ethanol-water) .

d) [ 7( S), 8S]- 7-[ 3-( acetylthio)- 2- methyl- l- oxopropyl]- 1, 4- dioxo-7- azaspiro[ 4. 4] nonan- 2- methyl- 8 - carboxylic acid

4,4-(i-Methylethylenedioxy)-L-proline (2.8 g, 0.015 mol) is reacted with 3.0 g (0.017 mol) of D-3-acetylthio-2-methylpropionyl chloride in 40 ml of water according to the procedure of Example 1 (e) to give 5.0 g of a viscous yellow product. This is treated with 2.8 g of dicyclohexylamine in 45 ml of ethyl acetate to give 4.2 g of almost colorless [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxo-propyl] - 1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid dicyclohexylamine salt; sm.p. 170-172° (p. 168°), M^<5> -58°

(c, 1% in ethanol). After crystallization from 12 ml of acetonitrile, the colorless solid salt weighs 3.85 g (51%), m.p. 170-172°

(p. 168°), [a]^<5> -57°, (c, 1% in ethanol).

Analysis: c14H2iN06<S>"<C>12H23<N:>

Calculated: C 60.90, H 8.65, N 5.46', S 6.26

Found: C 60/93, H 8.72, N 5.43, S 6.35.

The dicyclohexylamine salt is converted to the free acid by suspending 3.8 g in ethyl acetate and treating with 45 ml of 10% potassium bisulphate while stirring, until two layers are obtained. After

separation, the aqueous phase is extracted with ethyl acetate

(4 x 40 mL), the organic layers are combined, dried (MgSO 4 ) and the solvent evaporated to give 2.5 g (51%) of almost colorless solid [7(S),8S]-7-[3 -(acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid,

sm.p. 65-68° (p. 48°); [a]" -100° (c, 1% in ethanol).

e) [ 7 ( S ), 8S]- 7-( 3- mercapto- 2- methyl- l- oxopropyl)- 1, 4- dioxo-7- azaspiro[ 4. 4] nonan- 2- methyl- 8- carboxylic acid

The product from part (d) is hydrolysed with concentrated ammonia according to the procedure from example 2 to

give 2.05 g [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonan-2-methyl-8- carboxylic acid as a

25 o

viscous, colorless oil, [a]D -57 (c, 1% in ethanol).

Example 15

( S, S, S, S)- 7, 7'- [ dithiobis ( ; 2- methyl- l- oxo- 3, 1- propanediyl) ] - bis[ 1, 4- dioxa- 7- azaspiro[ 4. 4 ] nohan- 8- carboxylic acid]

]7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid (3.0 g, 0, 0109 moles)

from example 2 is dissolved in 80 ml of water and the pH value is adjusted to 6.5 with IN sodium hydroxide. To this stirred up-

solution, a total of 11 ml of 0.5 M iodine solution in 95% ethanol (6>34 g iodine/50 ml solution) is added dropwise while the pH is kept at 5>5 to 6.5 with IN sodium hydroxide. After 15 minutes, traces of excess iodine are removed with diluted sodium thiosulphate and the solution is concentrated to approx. 50 ml, cool and acidify with 1:1 hydrochloric acid. Methylene chloride (30 ml) is added, and the mixture is saturated with sodium chloride, stirred and the layers are separated. The aqueous phase is extracted with further methylene chloride (3 x 20 ml), the combined organic layers are dried

(MgSO^) and the solvent is evaporated, finally at 0.2 mm.

The friable residue is triturated under ether and evaporation is repeated

to give 2.8 g of a pale yellow solid residue. The material is redissolved in 50 ml of methylene chloride, washed with water (3 x 10 ml), the combined aqueous layers are back-extracted with 20 ml of methylene chloride, and the combined layers are dried (MgSO 4 ). Evaporation and trituration with ether as above gives 2.2 g (73%) of a cream-colored, amorphous solid (S,S,S,S)-7,71-[dithiobis(2-methyl-l-oxo -3,1-propanediyl)]bis[1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid]; sm.p. 61-63° (foaming), (p. 50°), [a]p6 -92°,

(c, 1% in ethanol).

Analysis, S^<N>^<S>^<O:>

Calculated: C 46.63, H 6.05, N 4.94, S 11.31

Found: C 46.52, H 6.29, N 4.63, S 10.96.

Example 16

(S, S, S, S)- 7, 7'-[ di ti obis( 2- methy1- oxo- 3, 1- propandiy1) j bi s-[ 7- aza- l, 4- dithiaspiro[ 4. 4 ] nonane- 8- carboxylic acid]

[7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid from Example 10

reacted with iodine according to the procedure of Example 125 to give (S,S,S,S)-7,7'-[dithiobis(2-methyl-1-oxo-3,1-propanediyl)]-

bis-[7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid].

Example 1^.

(S,S,S,S)-1,1'-[dithiobis(2-methyl-l-oxo-3,1-propanediyl)]-bis[4,4-dimethoxy-L-proline]

The (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline of Example 4 is reacted with iodine according to the procedure of Example 125 to give (S,S, S,S)-1,1'-[dithiobis(2-methyl-1-oxo-3,1-propanediyl)]-bis-[4,4-dimethoxy-L-proline].

Example 18

[ 7( S ), 8( S )]- 7-[ 3-( acetylthio )- 2- methyl- l- oxopropyl]- 1, 4- dioxo-7- azaspiro[ 4. 4] nonan- 8- carboxylic acid- methyl ester

A solution of the product from Example 1 in ether is treated with a small excess of diazomethane. After standing at room temperature for 2 hours, the solvent is evaporated to give [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[ 4.4]nonane-8-carboxylic acid methyl ester.

Example 19 i

[ 7( S ), 8 S ]- 7-( 3- mercapto- 2- methyl- l- oxopropyl)- 1, 4- dioxo- 7- azaspiro[ 4. 4] nonane- 8- carboxylic acid methyl ester

The product from example 12 8 is hydrolysed with concentrated ammonia according to the method according to example 2

to give [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid methyl ester.

Example 2 0

( S)- 1-[ 3-( acetylthio)- 2- methyl- l- oxopropyl]- 4, 4- dimethoxy-L- proline methyl ester

A solution of the product from Example 3 in ether is treated with a small excess of diazomethane. After standing at room temperature, the solvent is evaporated to give (S)-1-[3-(acetylthio)-2-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline methyl ester.

in

Example 21

( S )- 1-( 3- mercapto- 2- methyl- l- oxopropyl)- 4, 4- dimethoxy- L- proline methyl ester

The product from example 130 is hydrolysed with concentrated ammonia according to the method according to example 4

to give (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline methyl ester.

Example 22

[ 7( S ), 8 S ]- 7-( 3- mercapto- 2- methyl- l- oxopropyl)- 1, 4- dioxo- 7-azaspiro[ 4. 4] nonane- 8- carboxylic acid sodium salt

A solution of 1.0 g of the product from example 2 is dissolved in 10 ml of water and treated with an equivalent of sodium bicarbonate. The solution is lyophilized to give [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid sodium salt.

Similarly, using potassium bicarbonate,

the corresponding potassium salt is obtained.

Example 23

( S )- 1-( 3- mercapto- 2- methyl- l- oxopropyl)- 4, 4- dimethoxy- L- proline sodium salt

A solution of 1.0 g of the product from example 4 is dissolved in 10 ml of water and treated with an equivalent of sodium bicarbonate. The solution is lyophilized to give (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline sodium salt.

In a similar way, by using potassium bicarbonate,

one the corresponding potassium salt.

Claims (1)

Analogy method for the preparation of therapeutic active compounds with the formula and physiologically acceptable salts thereof, where R and R 1 are hydrogen or lower alkyl; R 1 and R 2 are hydrogen, lower alkyl or halogen-substituted lower alkyl; X 1 and X 2 are oxygen or sulfur; R 1 and R 2 are lower alkyl, or R 1 and R 2 are linked together to an optionally lower alkyl-substituted polymethylene chain to complete a 5- or 6-membered ring; m is 0, 1 or 2; R 1 is hydrogen, lower alkanoyl, benzoyl or a sulfide group with the formula so that a bis-symmetric disulfide product is formed, and the proline ring is in L-configuration and the side chain in D-configuration, characterized in that a substituted proline with the formula is coupled with an acid, or its chemical equivalent such as an acid chloride, with the formula wherein R,.<1> is lower alkanoyl or benzoyl, and said group is removed to form a product wherein R,, is hydrogen, and this product is oxidized with iodine to form a bls-symmetrical disulfide product, and a racemic mixture is separated into its stereoisomers, and optionally the compound produced is converted into a physiologically acceptable salt.