DK159115B - A LOGIC PROCEDURE FOR PREPARING A KETAL OR THIOKETAL DERIVATIVE OF A MERCAPTOACYL PROLINE - Google Patents

Description

DK 159115B

in

The present invention relates to an analogous process for the preparation of a ketal or thioketal derivative of a mercaptoacyl proline of the general formula I

5 R1 R2 X1 X2 R3 O f \ (I) * I II I * 1

R1-S- (CH2) m-CH-C-N - C-COOR

10 H

wherein R is hydrogen or C 1 -C 6 alkyl, R 3 is hydrogen, C 1 -C 6 alkyl or halo-C 1 -C 7 alkyl / X 1 and X 2 is oxygen or sulfur, R 1 and R 2 are C 1 R2 together represents a polymethylene chain such that a 5- or 6-membered ring is formed, m represents 0, 1 or 2, and R1 represents hydrogen, C2-C8 alkanoyl, benzoyl or a group 20 R1 R2 I, 15 X1 X2 R3 0 / \

I II I * I

-S- (CH2) m-CH-C-N-C-COOR

25 H

wherein R, R1, R2, R3, X1, X2 and m have the above meanings so as to form a bis-symmetric disulphide compound.

The star of the above formula shows an asymmetric center 30 in the ring. This center has L configuration.

Asymmetric centers may also occur in the mercaptoacyl side chain, depending on the definition of R3. Another asymmetric center may also occur in the ring when X-R1 and X2-R2 are different. The products may therefore be present in stereoisomeric forms or as racemic mixtures thereof. All of these can be prepared by the process of the invention.

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The synthesis described below may use the racemate or one of the enantiomers as starting materials. When racemic starting material is used for the synthesis, the stereoisomers obtained in the final product can be separated by conventional chromatographic methods or fractional crystallization. If an asymmetric center exists in the mercaptoacyl side chain, this is preferably in D configuration.

The compounds prepared by the process of the invention are the mercaptoacyl derivatives of proline 10 of formula I above, which are useful anti-hypertensive agents, having an inhibitory effect on angiotensin-converting enzyme.

Danish Patent Application No. 596/77 discloses closely related compounds having the same effect, especially the compound which is commercially available under the name Captopril. However, the compounds of the invention exhibit a surprisingly increased effect over the compounds thus known.

The term alkyl as used in the definitions 20 of the symbols R, R1, R2 and R3 are straight or branched hydrocarbon groups having up to 7 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and isopentyl. The preferred alkyl groups have up to four carbon atoms, with methyl and ethyl being particularly preferred.

Similarly, the terms alkoxy and alkylthio refer to such alkyl groups attached to an oxygen or sulfur atom.

The term halogen-substituted alkyl refers to such alkyl groups described above, wherein one or more hydrogen atoms are replaced by chlorine, bromine or fluorine atoms such as trifluoromethyl, pentafluoroethyl, 2,2,2-trichloroethyl, chloromethyl and bromomethyl.

As R5, alkanoyl groups having up to four carbon atoms, especially acetyl, and benzoyl are preferred.

Preferred compounds of formula I are the L-proline-containing derivative wherein R is hydrogen.

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With respect to the mercaptoacyl side chain, as final products, such compounds are preferred where R 5 is hydrogen, m is 0 or 1 and R 3 is hydrogen, alkyl of 1-4 carbon atoms, especially methyl, or trifluoromethyl. Also preferred, both as intermediates and end products, are the aforementioned side chains wherein R 5 is alkanoyl of 2-4 carbon atoms, especially acetyl, or benzoyl.

Particularly preferred as end products are compounds of formula I having a mercaptoacyl side chain wherein R 5 is hydrogen, m is 1, R 3 is methyl, and the asymmetric carbon atom to which R 3 is attached is in D-configuration.

Preferred with respect to the substituents on the proline ring are such compounds where X 1 and X 2 are the same, especially those where X 1 -R 1 and X 2 -R 2 are both methoxy or both ethoxy or X 1 R 1 and X 2 -R 2 together form a group H2c - ch2 / h2c - CH2,

0 0 SS

20 CH2 CH2 ^^ CH2 or S ^ S 4 o 25 The process of the invention for preparation

of the compounds of formula I is characterized in that a substituted proline of formula II

R1 R2 γΐ γ2

\ S

(II) h2cX? H2 I λ *

HN-C-C00R

H

wherein R, R1, R2, X1 and X2 have the above meanings,

35 is coupled with an acid of formula III

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4 R 3 R 5-S- (CH 2) m-CH-COOH (III) wherein R 5 represents C 2 -C 8 alkanoyl or benzoyl,

5 or a reactive derivative thereof, such as an acid chloride, to form a compound of formula IV

R1 R2 X1 X2 10 / f i? r J <IV>

R '5-S- (CH 2) m-CH-C-N-C-COOR

in which, if desired, the group R'5 is removed to form a compound of formula I wherein R5 is hydrogen and, if desired, this compound is oxidized with iodine to the bis-symmetric disulfide compound.

This reaction may be carried out in the presence of a coupling agent such as dicyclohexylcarbodiimide or the like, or the acid may be activated by forming its mixed anhydride, symmetric anhydride, acid halide, active ester or using Woodward reagent K, N-ethoxycarbonyl-2-ethoxy. -l, 2-dihydroquinoline. or similar. With regard to acylation methods, see the Method der Organchen 25 Chemie (Houben-Weyl), vol. XV, Part 2, page 1 et seq. (1974). Preferably, the acid halide, especially the acid chloride, of formula III is reacted with an 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 30 alkyl, converted to the free acid, i.e. R is hydrogen, by conventional methods. Thus, if R is ethyl, the ester protecting group can be removed by hydrolysis.

The product of formula IV is preferably isolated and purified by crystallization, e.g. by forming the dicyclohexyl-amine salt and then converting the salt to the free acid form by treatment with an aqueous solution of an acid such as 5

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potassium bisulfate.

The compound of formula IV may be converted to the products of formula I wherein R 1 is hydrogen by conventional hydrolysis or ammonolysis.

The compounds of formula I wherein R 5 is R1 R2 I, I, V *

R3 0 N

I II <* 1

-S- (CH 2) m-CH-C-N-C-COOR

H

is obtained by a compound of formula I wherein R 5 is hydrogen is directly oxidized with iodine.

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

The disubstituted prolines of formula II, wherein χΐ-20 -r! and X 2 -R 2 are the same can be obtained by reacting an N-protected keto compound of formula Λ C CHO 25 I I 2

Cbz-N-C-COOR

wherein Cbz is carbobenzyloxy, with an alcohol or thiol of formula 30 R1-X1-H (VI) in the presence of an orthoformate or thioformate of formula 2 of HC (X1-R1) 3 and an acid such as concentrated sulfuric acid or 35 p-toluenesulfonic acid. This reaction can take place in an inert organic solvent such as benzene, acetic acid, ether,

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6 cyclohexane or the like, preferably under heating, e.g. reflux temperature. See Buehier et al.

Survey of Organic Syntheses (Wiley & Sons, 1977), vol. 1, pages 516-519. The product of this reaction is the N-protected intermediate of the formula R1 R1 f f X \ / χ1 10 h2c ^ h2 (vii)

Cbz-N-xC-COOR

* i

H

The N-protected intermediate of formula VII can be treated with 1 mole equivalent of an alcohol or thiol of formula r2-x2-h (VIII) 20 according to the conditions described above to give the intermediate K1 R2? f '1 f 2

X \ ^ X

25 / C \

I2 <J (IX)

Cb z-N-jC-COOR

"L

H

By using a molar excess of the alcohol or thiol of formula VIII, the intermediate is obtained 2 2 f f k> C.

E0C ^ C.EU (χ) 2 | I 2

Cbz-N-xC-COOR

* 1

H

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The N-protecting group can then be removed by conventional methods, e.g. when X1 and X2 are both oxygen, by hydrogenolysis in the presence of palladium carbon catalyst, or when one or both of the symbols X1 and X2 are sulfur, 5 by treatment with HBr and acetic acid to give the di-substituted compounds of formula II.

Similarly, the germ compounds of formula II (i.e., R 1 and R 2 are joined in a polymethylene chain) can be obtained by reacting the keto compound of formula V with an alcohol 10 or thiol of formula (ch 2) t xlX Xx2 (XI)

I I

Η H

where t means 2 or 3, in the presence of an acid such as p-15-toluenesulfonic acid, giving the intermediate <%> t é x2 \ ^ (XII) (Η2?

20 I

• Cbz-N - C-C00R

H

Alternatively, the disubstituted compound of formula VII can be treated directly with a molar excess of the alcohol 25 or thiol of formula XI to give the intermediate of formula xii.

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

30 As an alternative method, the introduction of groups X-R and X-R can occur later in the sequence. According to this change, the protected keto compound of formula 0 is treated

II

α 35 H, C CH, <XIII> 2 | I 2

H-N - C-COOH

H

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8 which is then acylated with the acid, preferably the acid halide of Formula III, to give a compound of Formula 5 (R3 O 'Η2 ^ Υ2) 9 (XIV)

R 'J-S- (CH 2) -C-C-N -— C-COOH

H

10 112 2

The groups X -R and X -R or the spiro group (CH2> t x1 x2 X / 15 can then be introduced at this time by the methods described above to give a product of formula IV.

Reference is made to the following literature sites for further methods of preparing starting materials and intermediates: United States Patent Nos.

20,046,889, 4,105,776, 4,154,935 and 4,116,962, Can.J.Biochem.

& Physiol. 37, 584 (1959), J.A.C.S. 79, 189 (1957), J.Med.

Chem. 21, 445 (1978), Aus. J. Chem. 20, 1493-1509 (1967),

Buehier et al., Survey of Organic Syntheses (Wiley & Sons, 1977), vol. 1, pages 516-519, vol. 2, pages 461-470, Chem.Pharm.

25 Bull., Tokyo 26, 2209 and 2217 (1978), Can.J.Chem. 47, 860, (1969), J. Amer.Chem.Soc., 80, 6350 (1958), Harrisonm.fi., Compendium or Organic Synthetic Methods (Wiley lnterscience,

New York, 1971), pages 449-456, J.Amer.Chem.Soc., 79, 192 (1956), Bull.Soc.Chem., 1965 (8), pages 2253-2259, J.Org.Chem.

25, pp. 521-530 (1960).

The methods described therein can be used as general methods in the synthesis of the compounds and the separation of the isomers which can be used in the process of the present invention. Further details can be found in the examples which serve as a model for the preparation of other compounds in the group.

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The compounds prepared by the process of the invention form basic salts with a wide variety of inorganic or organic bases. The salt-forming ion emanating 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, several of which are known for this purpose e.g. aralkylamines such as dibenzylamine, Ν, Ν-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 benzathine arginine, lysine or the like. The physiologically acceptable salts can be used medically as described below and preferred.

The salts are prepared by reacting the acid form of the compound with 1 equivalent of the base which gives the desired basic ion in a medium in which the salt precipitates, or in an aqueous medium with subsequent lyophilization. The free acid form can be obtained from the salt by conventional neutralization methods, e.g.

20 with potassium bisulfate or hydrochloric acid.

The compound of formula I, especially when R 5 is hydrogen, is useful as hypotensive agents. They inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in alleviating angiotensin-induced hyper tension. The effect of the enzyme renin on angiotensinogen, a blood plasma pseudoglobulin, produces angiotensin I. Angiotensin I is converted by angiotensin converting enzyme (ACE) to angiotensin II. This is an active pressor substance that has been suspected to be the causative cause of 30 different forms of hypertension in various mammalian species, e.g. rats and dogs. The compounds prepared by the process of the invention interfere with the angiotensinogen ---> (renin) ---> angiotensin I ---> (ACE) ---> angiotensin II sequence by inhibiting the angiotensin II sequence. converting enzyme and reducing or eliminating dan-

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The addition of the pressor substance angiotensin II. Thus, by administration of an agent containing one or a combination of compounds of formula I, better angiotensin-induced hypertension in mammals suffering from it may be obtained. A single 5 dose, or preferably two to four divided daily doses calculated from ca. 0.1-100 mg / kg body weight per day, preferably 1-15 mg / kg / day, is appropriate for lowering blood pressure. The drug is preferably administered orally, but parenteral routes of administration such as subcutaneous, intra-1 ° muscular, intravenous or intraperitoneal can be used.

The compounds prepared by the method of the invention can be combined with a diuretic for the treatment of hypertension. A combined product comprising a compound prepared by the process of the invention and a diuretic may be administered in effective amount, which for a mammal of 70 kg comprises a total daily dose of about 50 mg. 30-600 mg, preferably approx. 30-300 mg, of a compound prepared by the process of the invention, and ca. 15-300 mg, preferably 15-200 mg, of a diuretic for a mammal in need thereof. Examples of diuretics for use in combination with a compound prepared by the process of the invention are thiazide diuretics, e.g. chlorothiazide, hydrochlorothiazide, flumthiazide, hydroglumthiazide, benzdroflumthiazide, methchlothiazide, trichlorothiazide, polythiazide or benzthiazide;

The compounds of formula I may be prepared for use in reducing blood pressure in preparations such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration.

The process according to the invention will now be elucidated by way of example.

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Example 1 [7 (S), 8S] -7- [3- (Acetylthio) -2-methyl-1-oxopropyl] -1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid a) N-Carbobenzyloxy-4-hydroxy-L-proline 26.5 g (0.20 mol) of 4-hydroxy-L-proline and 32.8 ml

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(0.23 mol) of 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. & Phy-10 siol. 37, 584 (1959) to give N-carbobenzyloxy-4-hydroxy-L-proline. This product is reacted with cyclohexylamine to give the cyclohexylamine salt in a yield of 69 g, mp 193-195 ° C. The salt (34 g) is neutralized with N-hydrochloric acid 26 to give 27 g of free acid as a colorless glass. [α] 15 -70 °, (c, 1% in chloroform).

b) N-Carbobenzyloxy-4-keto-L-proline 21.5 g (0.81 mol) of N-carbobenzyloxy-4-hydroxy-L-proline is oxidized in 1.2 liters of acetone with 83 ml of 8N chromic acid in sulfuric acid as described in JACS 79, 189 (1957). To facilitate 2Q the subsequent filtration of chromium salts, 30 g of "Celite" (diatomaceous earth) is added to the acetone solution before introducing the oxidizing agent. An air stirrer is used. The reaction mixture is filtered and the acetone filtrate is evaporated to ca. 300 ml before dilution with 1 liter of chloroform. The solution is washed with 300 ml of saturated sodium chloride (four times), dried (MgSO 4), filtered and the solvent is evaporated to give 22.8 g of N-carbobenzyloxy-4-keto-L-proline crystallized from 50 ether-150 ml hexane to give 17.2 g of product (81%), mp 99-101 ° C, [α] 25 + 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-carbo-benzyloxy-4-keto-L-proline, 53 ml (0.095 mol) of ethylene glycol and 0.35 g of p-toluenesulfonic acid in 1.31 liters benzene is heated, and the resulting solution is refluxed for 7 hours (the resulting water is collected in a Dean-Stark apparatus). After standing

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12 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 5 as a syrupy remnant. This is dissolved in 60 ml of ethanol, filtered, treated with 5 g of cyclohexylamine and diluted with ether. By grafting and rubbing, the crystalline cyclohexylamine salt is separated; weight after cooling overnight 9.0 g, m.p. 179-180 ° C (starting 173 ° C). The material is recrystallized from acetonitrile, m.p. 182-184 ° C (beginning 179 ° C), [α] D -21 ° (c, 1% in EtOH).

8.4 g of the cyclohexylamine salt is suspended in 40 ml of ethyl acetate, stirred, cooled and treated with 1N hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional 3 x 40 ml of ethyl acetate, 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 rubbed under ether and the ether is evaporated to give 6.4 g (42%) of almost colorless N-carbobenzyloxy-4,4-ethylene-20-dioxy-L-proline, m.p. ° C (beginning 98 ° C), [α] D -34 ° (c, 1% in CHCl13).

d) 4,4-Ethylenedioxy-L-proline

A solution of 3.2 g (0.0104 mole) of N-carbobenzyloxy-4,4-ethylenedioxy-L-proline in 100 ml of methanol / water (2: 1) is treated with 1 g of 5% palladium carbon and shaken Parr hydrogenator for 6 hours. The catalyst is filtered off under nitrogen, washed with methanol and the combined filtrates are evaporated, finally at 0.1-0.2 mm to give 1.7 g (94%) of colorless solid 4,4-ethylenedioxy-L-proline. mp 245-247 ° C (dec.), [α] D -32 ° (c, 0.5% in 1: 1 MeOH + O).

e) [7 (S), 8S] -7- [3- (Acetylthio) -2-methyl-1-oxopropyl] -1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid

A stirred solution of 3.2 g of 4,4-ethylenedioxy-L-proline (0.0185 mole) in 50 ml of water is cooled to 5 ° C and treated portionwise with solid sodium carbonate to pH 8.5. Then, with continued stirring and cooling, a solution 13 is added

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3.7 g (0.020 mol) of D-3-acetylthio-2-methylpropanoyl chloride in 5 ml of ether in portions, maintaining the pH of 8.5 with 25% sodium carbonate solution (about 14 ml). After 1 1/4 hour, the solution is treated with 50 ml of ethyl acetate, stirred, cooled, gently acidified with hydrochloric acid (1: 1) to pH 2.0, saturated with sodium chloride and the layers separated. The aqueous phase is extracted with an additional 3 x 50 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is finally evaporated at 0.2 mm. The solid residue is rubbed under 10 ether and the evaporation is repeated to give 5.9 g (100%) of [7 (S), 8S] -7- [3- (acetylthio) -2-methyl-1-oxopropyl] - 1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid, m.p. 108-111 ° C.

The product is converted to the dicyclohexylamine salt with 3.4 g of dicyclohexylamine in 70 ml of ethyl acetate. Upon inoculation and rubbing, the crystalline salt is precipitated and recrystallized from 95 ml of acetonitrile, yield 6.7 g, m.p. 187-189 ° C (beginning at 184 ° C), [.alpha. EtOH).

The dicyclohexylamine salt is converted to the free acid by suspending in ethyl acetate and treated with 75 ml of 10% potassium bisulfate and stirring until two layers are obtained.

After separation, the aqueous phase is extracted with 4 x 75 ml of ethyl acetate, the organic layers are combined, dried (MgSO 4) and the solvent is evaporated to give 4.1 g of colorless [7 (S), - 8S] -7- [3 - (acetylthio) -2-methyl-1-oxopropyl] -1,4-dioxa-7-aza-spiro [4,4] nonane-8-carboxylic acid, mp 120-122 ° C, starts at 117 ° C, [α] 25 D -118 ° (c, 1% in EtOH).

Example 2 [7 (S), 8S] -7- (3-Mercapto-2-methyl-1-oxopropyl) -1,4-dioxa-7-30-azaspiro [4,4] nonane-8-carboxylic acid

Argon is passed through a cold solution of 8.5 ml of concentrated 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 ] nonan-8-carboxylic acid from Example 1 (e) is then added and the mixture is stirred in an ice bath for a few minutes and then at room temperature under argon for 2 hours.

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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 aqueous phase is extracted with an additional 30 ml of ethyl acetate (twice), the ethyl acetate extracts are combined, dried (MgSO 4) and the solvent is evaporated to give E7 (S), - 8S] -7- (3-mercapto-2 methyl-1-oxopropyl) -1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid as a solid residue. The product is rubbed under ether and the evaporation repeated. Then, the product is triturated with 30 ml of hexane, cooled for 1 hour, filtered for 10 ounces of 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-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid, mp 131-133 ° C (beginning 125 ° C), [α] c, 1% in EtOH).

Example 3 (S) -1 - [(3-Acetylthio) -2-methyl-1-oxoprbpyl] -4,4-dimethoxy-L-proline a) N-Carbobenzyloxy-4,4-dimethoxy-L-proline methyl ester

A stirred solution of 7.8 g (0.03 mole) of N-carbo-20-benzyloxy-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 left overnight at room temperature.

The pale yellow solution is stirred, treated with 1.5 g of potassium carbonate followed by 30 ml of water and the majority of the solvent removed on a rotary evaporator to give a syrupy residue which is shaken with 30 ml of water and 30 ml of chloroform. After the layers are separated, the aqueous phase is extracted with additional chloroform (3 x 30 ml) and the 30 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 35.4 g (0.026 mol) of the ester of part (a) is dissolved in 80 ml of methanol, treated dropwise at -1 ° C to 4 ° C with 18 ml (0.036 mol) of 2N sodium hydroxide, maintained at 0 ° C for one hour and

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at room temperature overnight. After approx. half of the solvent is removed on a rotary evaporator, diluted with 150 ml of water, washed with 100 ml of ether (the wash liquid is discarded), acidified with cooling with 63 ml of 5 1: 1 hydrochloric acid to pH 2 and extracted with 4 x 750 ml of ethylene - cetate. The combined extracts are washed with 50 ml of saturated sodium chloride solution, dried (MgSO 4) and the solvent is 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 cyclohexyl-10 amine in 10 ml of ethanol and diluted to 500 ml with ether. Upon grafting and rubbing, the crystalline N-carbobenzyloxy-4,4-dimethoxy-L-proline-cyclohexylamine salt, which after weighing 7.0 g, is separated out, m.p. 157-159 ° C (starts 151 ° C), [ [α] 6 -34 ° (c, 1% in EtOH). This material is recrystallized from 100 ml of acetonitrile to give the salt as a colorless solid, mp 158-160 ° C (s, 154 ° C), [α] 6 -33 ° (c, 1% in EtOH).

The N-Carbobenzyloxy-4,4-dimethoxy-L-proline-cyclohexylamine salt is suspended in 40 ml of ethyl acetate, stirred and treated with 25 ml of 1N hydrochloric acid. When two clear layers are obtained, they are separated, the aqueous phase is extracted with an additional 3 x 40 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated, finally at 0.2 mm and 40 ° C, gives 7.2 g (70%) of N-carbobenzyloxy-4,4-dimethoxy-L-proline as a pale yellow viscous syrup.

c) 4,4-Dimethoxy-L-proline

A solution of 72 g (0.022 mol) of N-carbobenzyloxy - 4,4-dimethoxy-L-proline in 210 ml of methanol / water (2: 1) is treated with 2.3 g of 5% palladium carbon and shaken on a Parr Hydrogenator for 6 hours. The catalyst is filtered off under nitrogen, washed with methanol and the combined filtrates are evaporated, finally at 0.1-0.2 mm to give a partially crystalline residue. This residue is taken up in 200 ml of methanol and the evaporation is repeated. When the solid is rubbed under ether 35 (evaporation is repeated again) 3.6 g (95%) is obtained almost colorless 4,4-dimethoxy-L-proline, mp 192-194 ° C (dec.), [Cc] 6 -47 ° (c, 1% in MeOH).

J

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A sample crystallized from methanol / ether is colorless and melts at 197-198 ° C (dec.) [<-49 °; (c, 1% in MeOH).

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

A stirred solution of 3.3 g (0.019 mol) of 4,4-di-methoxy-L-proline in 50 ml of water is cooled to 5 ° C and brought to pH 8.5 by the addition of 25% sodium carbonate solution (w / v). . Then, with continued stirring and cooling, a solution of 3.8 g (0.021 mole) of D-3-acetylthio-2-methylpropanoyl chloride in 5 ml of ether is added while maintaining the pH of 7.5-8.5 by dropwise addition. addition of 25% sodium carbonate solution. When the pH is stabilized at 8.2-8.4 (after about 15 minutes), stirring and cooling for a total of 15 an hour are continued. The solution is then washed with 50 ml of ethyl acetate (the wash liquid is discarded), covered with a layer of 50 ml of ethyl acetate, cooled, stirred, gently acidified with 1: 1 hydrochloric acid to pH 2.0, saturated with sodium chloride and the layers separated.

The aqueous phase is extracted with an additional 3 x 50 ml of ethyl 20 acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated, finally at 0.2 mm to give 6.7 g of syrupy product. This syrup is treated in 70 ml of ethyl acetate with 3.9 g of dicyclohexylamine to give 6.5 g of colorless (S) -1- [3- (acetylthio) -2-methyl-1-oxopropyl] -4,4-dimeth-25 oxy-L-proline dicyclohexylamine salt in yields of 3.1 g and 3.4 g, m.p. 158-160 ° C (beginning at 145 ° C), [α] 25 -71 ° (c, 1% in EtOH).

After recrystallization from 20 ml of hot ethyl acetate and 60 ml of hexane, the colorless solid salt weighs 6.0 g, m.p. 158-166 ° C (starts 155 ° C), [α] 25 -69 ° (c, 1% in EtOH ).

The dicyclohexylamine salt is converted to the free acid by suspending 5.0 g in 50 ml of ethyl acetate, cooling it and treating with 60 ml of 10% potassium bisulfate solution to give 2 clear layers. After separation, the aqueous phase is extracted with 3 x 50 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated, finally at 0.1-0.2

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mm and 45 ° C to give 4.1 g (69%) of (S) -1- [3- (acetylthio) -2-methyl-1-oxopropyl] -4,4-dimethoxy-L-proline as a viscous 25 o almost glass-like material, [a] -112 (c, 1% in EtOH).

Example 4 (S) -1- (3-Mercapto-2-methyl-1-oxopropyl) -4,4-dimethoxy-L-proline Argon is passed through a cold solution of 8.5 ml of concentrated ammonium hydroxide in 20 ml of water for 0.25 hours. This is added under cooling and argon blanket to 4.1 g (0.013 10 moles) of (S) -1- [3- (acetylthio) -2-methyl-1-oxopropyl] -4,4-dimethoxy-L-proline, and the mixture swirl in an ice bath until a pale yellow solution is obtained (about 15 minutes). Stirring under argon is continued at room temperature for an additional 2 hours, then the solution is extracted with 30 ml of ethyl acetate (these and the following operations are carried out as far as possible under argon atmosphere). The aqueous layer is cooled, stirred, covered with a layer of 30 ml of ethyl acetate, acidified portionwise with 16 ml of 1: 1 hydrochloric acid. The layers are separated, the aqueous phase is extracted with an additional 3 x 30 ml of ethyl acetate, the combined ethyl acetate layers are dried (MgSO 4) and the solvent is evaporated to give 3.5 g (100%) of (S) -1- (3-mercap - (2-methyl-1-oxopropyl) -4,4-dimethoxy-L-proline as a colorless velvic viscous syrup, [α] D -72 (c, 1% in EtOH).

This compound (3.4 g) is triturated with 20 ml of ethyl acetate, rubbed, diluted with 30 ml of hexane and cooled to give a colorless solid weighing 2.6 g, mp 108-110 ° C. ] D -77 ° (c, 1% in EtOH).

Example 5 (S) -1- [3- (Acetylthio) -2-methyl-1-oxopropyl] -4,4-diethoxy-L-proline a) N-Carbobenzyloxy-4,4-diethoxy-L-proline ethyl ester

Following the procedure of Example 3 (a) but using triethyl orthoformate instead of trimethyl orthoformate and ethanol instead of methanol, 10.8 g of N-carbobenzyloxy-4,4-diethoxy-L-proline ethyl ester is obtained as a yellow oil.

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B) N-Carbobenzyloxy-4,4-diethoxy-L-proline

The crude ester of part (a) (10.8 g, 0.03 mol) is hydrolyzed with 70 ml of 1 N sodium hydroxide as set forth in Example 4 (b), to 30 ml of ethanol in 10 ml aliquots to give a solution , which gives 10.5 g of yellow viscous oil. This oil is dissolved in 100 ml of ether and treated with 3.0 g of cyclohexylamine. Upon grafting and rubbing, 8.3 g of the crystalline N-carbobenzyloxy-4,4-diethoxy-L-proline-cyclohexylamine salt is separated, m.p. 123-126 ° C (beginning 114 ° C), [α] 10 -32 ° (c, 1% in ethanol). This material is recrystallized from 20 ml of acetonitrile to give 7.0 g of the salt as a colorless solid, m.p. 125-128 ° C (starts at 115 ° C), [ct] +6 -31 ° (c, 1% in ethanol).

The N-Carbobenzyloxy-4,4-diethoxy-L-proline-cyclohexyl-amine salt is suspended in 40 ml of ethyl acetate, stirred and treated with 20 ml of 1N hydrochloric acid. The layers are separated and the aqueous phase is extracted with an additional 3 x 40 ml of ethyl acetate, the organic layers are combined, dried (MgSO 4) and the solvent is evaporated to give 5.6 g (56%) of N-carbobenzyloxy-4.4-20 -diethoxy-L-proline as a pale yellow oil.

c) 4,4-Diethoxy-L-proline

A solution of the 5.6 g of N-carbobenzyloxy-4,4-di-ethoxy-L-proline (0.017 mol) in 180 ml of 2: 1 ethanol / water is treated with 2 g of a 5% palladium carbon catalyst and shaking 25 below 3 atm. hydrogen for 6 hours. The crude partially solid product is first rubbed under ethanol, then ether, and the evaporation is repeated each time to give 3 g (91%) of almost colorless solid 4,4-diethoxy-L-proline, mp 172-174 ° C (dec. 6 ling) with prior gradual dark staining and sintering, [α] D 30 -40 ° (c, 1% in methanol).

Analysis calculated for C CHH NO NONO · 0.25 E₂O: C 52.03, H 8.49, N 6.74.

Found: C, 52.22; H, 8.59; N, 6.69.

D) (S) -1- [3- (Acetylthio) -2-methyl-1-oxopropyl] -4,4-diethoxy - L-proline 2.9 g 4,4-diethoxy-L-proline (0.014 mol) from part (c)

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19 and 3 g (0.017 mol) of D-3-acetylthio-2-methylpropionyl chloride dissolved in 3.5 ml of ether are reacted in 35 ml of water in the presence of sodium carbonate as indicated in Example 3 (d) to give 5.4 g of pale yellow viscous oil. This oily product is treated in 40 ml of ethyl acetate with 2.6 g of dicyclohexylamine and diluted with 60 ml of hexane to give 4.9 g of (S) - 1 - [(3-acetylthio) -2-methyl in two yields. -1-oxopropyl] -4,4-diethoxy-L-proline dicyclohexylamine salt, mp 135-138 ° C (starts at 132 ° C). After recrystallization from 15 ml of hot ethyl acetate and 45 ml of hexane, the colorless solid salt weighs 4.2 g, m.p. 138-140 ° C (starts at 135 ° C), [α] 25 -63 ° (c, 1 % in ethanol).

Analysis calculated for C 21 H 23 O: C 61.33, H 9.15, N 5.30, S 6.06.

Found: C, 61.48; H, 9.55; N, 5.25; S, 5.91.

The dicyclohexylamine salt is converted to the free acid by suspending 4.2 g in 40 ml of ethyl acetate, cooling and treating with 40 ml of 10% potassium bisulfate solution to give two layers. After separation, the aqueous phase 20 is extracted with 3 x 50 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated to give 3.0 g (61%) of (S) -1- [3- (acetylthio) 2-methyl-1-oxopropyl] -4,4-diethoxy-L-proline as a pale yellow viscous syrup.

Example 6 (S) -4,4-Diethoxy-1- (3-Mercapto-2-methyl-1-oxopropyl) -L-proline Argon is passed through a cold solution of 5.5 ml of concentrated ammonium hydroxide in 13 ml of water. 0.25 hour. This is then added under cooling under argon blanket to 3.0 g of (0.0086 mol) (S) -1- [3- (acetylthio) -2-methyl-1-oxopropyl] -4,4- diethoxy-L- proline, and the mixture is worked up as described in Example 4 to give 2.4 g (92%) of (S) -4,4-diethoxy-1- (3- mercapto-2-methyl-1-oxopropyl) -L- proline as an almost color-26 o loose syrup [a] + -64, (c, 1% in ethanol).

Analysis calculated for C 13 H 23 NO 5 * 0/25 H 2 O: 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.

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20

Example 7 [2 (S), 3S] -2- [3- (Acetylthio) -2-methyl-1-oxopropyl3-6,10-dioxa-2-azaspiro [4,5] decane-3-carboxylic acid a) N-Carbobenzyloxy-4,4-trimethylenedioxy-L-proline Reaction of 8.2 g (0.031 mol) of N-carbobenzyloxy--4-keto-L-proline and 45 ml (0.62 mol) of 1,3-propanediol in 450 ml of benzene in the presence of 500 mg of p-toluenesulfonic acid gives 12.3 g of raw viscous ester product. This product is hydrolyzed with 70 ml of 1 N sodium hydroxide to give 10.6 g of crude N-carbo-10 benzyloxy-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, mp 163-165 ° C Crystallization of 9.8 g of the salt from 300 ml of acetonitrile gives 9.5 g of colorless solid cyclohexylamine salt, mp 165-167 ° C (beginning 162 ° C), [α] 25 D -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 1N hydrochloric acid. The layers are separated, the aqueous phase is extracted with an additional 3 x 40 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated to give 7.1 g (75%) of glass-like N-carbobenzyloxy-4,4-trimethylene di oxy-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 5% palladium carbon catalyst. , giving 3.8 g (93%) of almost colorless 4,4-trimethylenedioxy-L-proline, m.p. 234-236 ° C (dec.) with 25 degrees of gradual darkening of sintering, [α] β -36 (c, 1% in 1: 1 methanol / water).

Analysis calculated for C, 51.33; H, 7.00; N, 7.48; Found: C, 51.42; H, 7.11; N, 7.40.

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C) [2 (S), 3S] -2- [3- (Acetylthio) -2-methyl-1-oxopropyl] -6,10-dioxa-2-azaspiro [4,5] decane-3 Carboxylic acid 3.7 g (0.02 mole) of 4,4-trimethylenedioxy-L-proline is acylated with 4.0 g (0.022 mole) of D-3-acetylthio-2-methylpropionyl chloride in 50 ml of water in the presence of sodium carbonate as set forth in Example 1 (e) to give 7.3 g of glass-like crude product.

The product is converted to the dicyclohexylamine salt with 3.6 g of dicyclohexylamine in 70 ml of ethyl acetate. After grafting 10 and rubbing, the crystalline salt, m.p. 168-170 ° C (starts 166 ° C), [α] D -59 ° (c, 1% in ethanol). Recrystallization from 30 ml of acetonitrile gives 6.5 g of colorless solid salt, m.p. 169-171 °, [α] D -63 °, (c, 1% in ethanol).

The dicyclohexylamine salt is converted to the free acid by suspending 6.4 g in 75 ml of ethyl acetate and treating with 75 ml of 10% potassium bisulfate and stirring 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 (67%) of glass-like [2 (S), 3S] -2 - [3- (acetylthio) -2-methyl-l-oxopropyl] - 6,10-dioxa-2-azaspiro [4.5] decane-3-carboxylic acid.

Example 8 [2 (S), 3S] -2- (3-Mercapto-2-methyl-1-oxopropyl) -6,1-dioxa-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] dican-3-carboxylic acid (4.3 g (0.013 mol) is hydrolyzed with 8.5 ml of concentrated ammonia in 20 ml of the procedure of Example 2 to give 0.9 g of colorless volatile solid, [a] -64 (c, 0.5% in ethanol). ). An additional 0.8 g of product is obtained by extracting the aqueous phase with chloroform, [α] -66. The two yields are dissolved in chloroform, evaporated, rubbed under ether and the evaporation 35 repeated, yielding 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 ° (beginning 167 °), [α] 22

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-71 ° (c, 1% in methanol).

Analysis calculated for 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.

5

Example 9 [7 (S), 8S] -7- [3- (Acetylthio) -2-methyl-1-oxopropyl] -7-aza-1,4-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-carbenzyloxy-4-keto-L-proline methyl ester in 60 ml of methylene chloride treated with 3 ml (0.036 mol) of ethanedithiol, cooled to 8 ° C and treated under argon blanket with 3 ml (0.024 mol) of boron trifluoride etherate. After the cooling bath is removed, the pale yellow solution is stirred for an additional 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 an additional 3 x 30 ml of methylene chloride. The 20 combined organic layers are washed with 50 ml of 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 of part (a) (7.4 g, about 0.018 mol) is dissolved in 65 ml of methanol, treated dropwise at -1 to 4 ° C with 14 , 5 ml (0.029 mol) of 2N sodium hydroxide, is kept at 0 ° C for one hour and at room temperature overnight. After approx. half of the solvent was removed on a rotary evaporator, diluted with 125 ml of water, washed with ether (the washing liquid discarded), acidified with 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 of saturated sodium chloride, dried (MgSO 4) and the solvent is 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. When grafting

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23 and rubbing separates the crystalline cyclohexylamine salt to give, after cooling overnight, 5.7 g of N-carbobenzyloxy - 4,4-ethylenedithio-L-proline-cyclohexylamine salt, mp 205-207 ° C (beginning 201 ° C). Recrystallization from 50 ml of ethanol / 400 ml of ether gives 4.9 g of colorless solid salt, m.p. 207-209 ° C (beginning 201 ° C), [.alpha.

The cyclohexylamine salt (4.8 g) is suspended in 25 ml of ethyl acetate, stirred and treated with 25 ml of 1N hydrochloric acid. When two clear layers are obtained, these are separated, the aqueous phase is extracted with an additional 3 x 25 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated to give 3.8 g (62%) N-carbobenzyloxy-4,4-ethylenedithio - L-proline as a pale yellow viscous syrup.

C) 4,4-Ethylenedithio-L-proline hydrobromide 3.7 g of N-carbobenzyloxy-4,4-ethylenedithio-L-proline (0.011 mol) is treated with 20 ml of hydrogen bromide in acetic acid (30-32%). loosely and magnetically stirred. Mixing is difficult due to the viscosity of the starting material and it is divided as much as possible with a spatula. Meanwhile, the crystalline product begins to secrete. Additional 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. 250 ml of ether 25 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, melting point 240-242 ° C (decomposition), sintering and darkening from approx.

-20 ° (c, 0.5% in 1: 1 chloroform / methanol).

d) [7 (S), 8S] -7- [3- (Acetylthio) -2-methyl-1-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-e-thylenedithio-L-proline hydrobromide in 25 ml of water is cooled to 35 ° C and brought to pH 8.2 by addition of 25% sodium carbohydrate. - night (weight / volume). With continued stirring and cooling, a solution of 1.9 g (0.01 mole) of D-3-acetylthio-2-methylpropionyl chloride in 2.5 ml of ether is added portionwise, keeping the pH of 7.5-8.2 at dropwise addition of 25% sodium carbonate

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24 night. When the pH is stabilized at 8.2-8.5 (after about 15 minutes), stirring and cooling are continued for a total of one hour. The solution is then washed with 25 ml of ethyl acetate (the wash liquid is discarded, covered with a layer of 25 ml of ethyl acetate, cooled, stirred, gently acidified with 1: 1 hydrochloric acid to pH 2.0, saturated with sodium chloride and the layers separated. is extracted with additional ethyl acetate (3 x 25 mL), the combined organic layers are dried (MgSO 4) and the solvent is evaporated, finally at 0.2 mm to give 2.6 g of syrupy product which begins to crystallize. is treated in 30 ml of ethyl acetate with 1.5 g of dicyclohexylamine to give 3.0 g of colorless dicyclohexylamine salt, mp 176-178 ° (s 170 ° C), [α] 6 -55 ° (c, 1% in ethanol). This material is ground in a mortar under 15 ml of acetonitrile, cooled for one hour, filtered, washed with 5 ml of cold acetonitrile and with ether and dried to give 2.9 g of dicyclohexylamine salt, mp 177-179 ° C (p 172 °), [α] 25 -56 (c, 1% in ethanol).

Calcd for C 13 HigNO 4 S; L 2 H 23 N: 20 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 dicyclohexylamine salt is converted to the free acid by suspending 2.8 g in 30 ml of ethyl acetate, cooled and treated with 30 ml of 10% potassium bisulfate to give two clear layers. After separation, the aqueous phase is extracted with 3 x 50 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated, finally at 0.1-0.2 mm and 45 ° C to give 2.0 g ( 63%) colorless solid [7 (S), 8S] -7- [3- (acetylthio) -2-methyl-1-oxopropyl] -7-30 -aza-1,4-dithiaspiro [4,4] nonane -8-carboxylic acid, m.p. 125-126 ° C (s, 122 ° C), [α] 6 -101 ° (c, 1% in ethanol).

Example 10 [7 (S), 8S3-7- (3-Mercapto-2-methyl-1-oxopropyl) -7-aza-1,4-di-thiazpiro [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. This adds

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25 is then added under cooling and argon blanket to 1.9 g (0.054 mol) [7 (S), 8S] -7- [3- (acetylthio) -2-methyl-1-oxopropyl] -7-aza-1, 4-dithiaspiro [4,4] non-8-carboxylic acid and the mixture is swirled in an ice bath until a solution is obtained. Stirring under argon is continued at room temperature for an additional two hours, then the solution is extracted with 15 ml of ethyl acetate under argon atmosphere. The aqueous layer is cooled, stirred, covered with a layer of 15 ml of ethyl acetate and made portionwise acidic with ca. 6.5 ml of 1: 1 hydrochloric acid. The layers are separated, the aqueous phase is extracted with an additional 3 x 15 ml of ethyl acetate, the combined acetate layers are dried (µgSO 4) and the solvent is evaporated to give a glass-like residue which solidifies as rubbed 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%) [7 (S), 8S] - 7- (3-mercapto-2 methyl-1-oxopropyl) -7-aza-1,4-dithiaspiro [4,4] nonane-8-carboxylic acid, m.p. 116-118 ° C (s 105 ° C), [a] p® -44 ° (c, 1% in ethanol).

Analysis calculated for C CH ^ NONO SS: 20 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 31.13, SH 100%.

Example 11

(8S) -7- [3- (Acetylthio) -2-trifluoromethyl-1-oxopropyl] -1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid, isomers A and B

a) D, L-3- (Acetylthio) -2-trifluoromethylpropionic acid 10 g (0.071 mol) of α-trifluoromethylacrylic acid, prepared as set forth in J.Chem.Soc., 1954, page 371, is cooled in a bath of ice, water and salt, stirred and treated portionwise with 5.7 ml (0.075 mol) of 97% thiolacetic acid. After the addition, a yellow liquid is stirred in the cold for one 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, boiling point 149 -153 ° C / 13 mm. The material solidifies when stored in the cold.

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b) D, L-3 ~ (Acetylthio) -2-trifluoromethylpropionyl chloride 7 g (0.032 mol) of D, L-3- (acetylthio) -2-trifluoromethylpropionyl acid are treated with 18 ml (0.25) of redistilled thionyl chloride and the mixture reflux for three hours. After the excess thionyl chloride is removed on a rotary evaporator, the residue is distilled to give 6.8 g of D, L-3- (acetylthio) -2-trifluoromethylpropionyl chloride as a pale yellow oil, boiling point 80-82 ° C / 16 mm.

c) (8S) -7- [3- (Acetylthio) -2-trifluoromethyl-1-oxopropyl] -1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid (isomers A and B ) 2.4 g (0.014 mol) of 4,4-ethylenedioxy-L-proline is reacted with 3.4 g (0.014 mol) of D, L-3- (acetylthio) -2-trifluoromethylpropionyl chloride in 40 ml of water in the presence of sodium carbonate. overnight according to the procedure of Example 1 (e) to give 4.5 15 g of almost colorless solid product, mp 126-145 ° C (s 115 ° C), [cx] 2d5 -34 ° (c, 1% in ethanol) .

The mixture of diastereoisomers (4.2 g) is suspended in 45 ml of ether, stirred for two hours, cooled for 20 minutes and the undissolved solid is filtered, washed with cold ether and dried.

20 is air dried to give 2.7 g of product, mp 166-172 ° C

(s 140 ° C), [α] 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 product, (mp 172-177 ° C (s 143 ° C). from 11 ml of boiling isopropanol and cooling overnight, 1.55 g of colorless (8S) -7- [3- (acetylthio) -2-trifluoromethyl-1-oxopropyl] -1,4-dioxa-7-azaspiro [4 , 4] nonan-8-carboxylic acid, isomer A, m.p. 192-194 ° C, (s 183 ° C), 25 o [a] -32 (c, 1% in ethanol). from isopropanol, mp 193-195 ° C (s 184 ° C), [α] D -134 ° (c, 1% in ethanol).

Isomer B is obtained by combining the above ether and isopropanol filtrates and removing the solvents under reduced pressure to give 2.2 g of a pale yellow solid, mp 108-109 ° C (s 95 ° C), [a] + 30 ° (c, 1% in ethanol). This material is purified by crystallization

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27 from 6 ml of isopropanol to give 1.2 g of almost colorless solid, mp 153-155 ° C (s 130 ° C), [α] + 40 ° (c, 1% in ethanol). After crystallization from 4 ml of ethyl acetate / 6 ml of hexane, 1.1 g of (8S) -7- [3- (acetylthio) -2-trifluoro-5-methyl-1-oxopropyl] -1,4-dioxa-7 is obtained. -azaspiro [4,4] nonan-8-carboxylic acid, isomer B, mp 153-155 ° C (s 141 °), [α] + 41 ° (c, 1% in ethanol).

Example 12 1 q (8S) -7- (3-Mercapto-2-trifluoromethyl-1-oxopropyl) -1,4-dioxa-7-

-azaspiro [4,4] nonane-8-carboxylic acid isomer A

The isomer A product of Example 16 (1.45 g, 0.0039 mol) is hydrolyzed with 2.5 ml of concentrated ammonia in 6 ml of water for one hour as described in Example 2 to give 1.25 g (97%) colorless (8S) -7- (3-mercapto-2-trifluoromethyl-1-oxopropyl) -1,4-dioxo-7-azaspiro [4,4] nonane-8-carboxylic acid, 25 isomer A, as a glass-like product, [α] -61 (c, 1% in ethanol). TLC: Rf 0.40 (95: 5: 5 methylene chloride / methanol / acetic acid, vis. SH reagent, PMA and heat).

Analysis calculated for C 11 H 14 F 3 NO 5 S: 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, mp 213-125 ° (dec., [A] + - 47 ° (c, 1% in methanol).

Example 13 (8S) -7- (3-Mercapto-2-trifluoromethyl-1-oxopropyl) -1,4-dioxide

-7-azaspiro [4,4] nonane-8-carboxylic acid, isomer B

The 3Q Isomer B product of Example 16 (1.05 g, 0.028 mol) is hydrolyzed with 2 ml of concentrated ammonia in 5 ml of water according to the procedure of Example 2 to give 0.9 g (97%) (8S) -7- ( 3-mercapto-2-trifluoromethyl-1-oxopropyl) -1,4-dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid, isomer B as a pale yellow viscous syrup, [a] - 16 ° (c, 1% in ethanol). Must-

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the solid solidifies to a waxy solid, mp 61-64 ° C (s 55 °).

Analysis calculated for C 11 H 14 F 3 NO 5 S.0.25 H 2 O: C 39.58, H 4.38, N 4.20, S 9.61, F 17.01.

Found: C, 39.60; H, 4.28; N, 4.26; S, 9.62; F, 16.89.

Example 14 [8S] -7- (3-Mercapto-2-trifluoromethyl-1-oxopropyl) -7-aza-1,4-dir thiaspiro [4,4] nonane-8-carboxylic acid a) 3 - [[( 4-methoxy) phenylmethyl] thio] -2-trifluormethylpropionyl chloride

A careful mixture of 3.9 g of 1-trifluoromethyl methylene

Crylic acid and 4.3 g of 4-methoxybenzylthiol are stirred at 100-110 ° C

for one hour. The mixture is allowed to cool to room temperature, 15 and the solid is recrystallized from cyclohexane to give 3 - [[(4-methoxy) phenylmethyl] thio] -2-trifluoromethylpropionic acid, mp 72-74 ° C.

Treatment of this acid with thionyl chloride gives 3 - [[(4-methoxy) phenylmethyl] thio] -2-trifluoromethylpropionyl chloride.

b) [8S] -7- [3 - [[(4-Methoxy) phenylmethyl] thio] -2-trifluoromethyl-1-oxopropyl] -7-aza-1,4-dithiaspiro [4,4] nonane The 8-carboxylic acid 3- [[(4-Methoxy) phenylmethyl] thio] -2-trifluoromethylpropionyl chloride from part (a) is reacted with 4,4-ethylenedithio-L-proline according to the procedure of Example 9 (d). ) to give [8S] -7- [3 - [[(4-methoxy) phenylmethyl] thio] -2-trifluoromethyl-1-oxopropyl] -7-aza-1,4-dithiaspiro [4,4] nonan-8-carboxylic acid.

C) [8S] -7- (3-Mercapto-2-trifluoromethyl-1-oxopropyl) -7-aza - 1,4-dithiaspiro [4,4] nonane-8-carboxylic acid

The product of part (b) is mixed with trifluoroacetic acid and anisole under nitrogen. The solvents are removed in vacuo to give, as residue, [8S] -7- (3-mercap-35 * to-2-trifluoromethyl-1-oxopropyl) -7-aza-1,4-dithiaspiro [4,4] nonan 8-carboxylic acid.

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Example 15 1- (3-Mercapto-1-oxopropyl) -4,4-dimethylthio-L-proline a) 4-Keto-L-proline hydrobromide

To 4.0 g (0.015 mol) of N-carbobenzyloxy-4-keto-L-5-proline is added 20 ml of hydrogen bromide in acetic acid (30-32%). The mixture is swirled frequently for a period of 8 minutes.

At the end of this time (the effervescence has ceased), the yellow-orange solution is covered with a layer of ether of 25 ° C and the rubbery product triturated. The ether 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%) , mp 153-155 ° C (dec.), [α] D -49 ° (c, 1% in water).

B) 1- [3- (Aoethylthio-1-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 ° C and treated portionwise with solid sodium carbonate (foaming is controlled by the addition of a few drops of ether). pH 8.0 (about 2 g is needed). Then, with continued stirring and cooling, a solution of 3.5 g (0.012 mol) of 3-acetyl ylthiopropanoyl chloride in 5 ml of ethyl acetate is added by pipette, maintaining the pH of 7.0-8.0 by dropwise addition of 25 ml. % (w / v) sodium carbonate solution (about 10 ml).

25 After approx. For 10 minutes, the pH is stabilized at 8.0-8.4.

After continued stirring and cooling for a total of one hour, the solution is washed with 2 x 50 ml of ethyl acetate, covered with a layer of 50 ml of ethyl acetate, stirred, cooled, gently acidified with concentrated hydrochloric acid to pH 2.0, saturated with sodium chloride, and the layers separate. The aqueous phase is extracted with an additional 3 x 50 ml of ethyl acetate, 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 glass-like 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 grafting and rubbing, crystalline 0 is separated

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1- [3- (acetylthio-1-oxopropyl)] - 4-oxo-L-proline dicyclohexyl amine salt, weight after cooling overnight 2.7 g (almost colorless), m.p. 191-193 ° C (dec.) , [α] D -24 ° (c, 1% in CHCl3).

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

c) 1- [3- (Acetylthio) -1-oxopropyl] -4,4-dimethylthio-L-proline 1- [3- (Acetylthio) -1-oxopropyl] -4-oxo-L-rololine with methylthiol according to the procedure of Example 9 (a) to give 1- [3- (acetylthio) -1-oxopropyl] -4,4-dimethylthio-L-proline.

d) 1- (3-Mercapto-1-oxopropyl) -4,4-dimethylthio-L-proline The product of part (a) is hydrolyzed with concentrated ammonia concentrate according to the procedure of Example 2 to give 1- (3-mercapto- 1-oxopropyl) -4,4-dimethylthio-L-proline.

Example 16 [7 (S), 8Sj-7- (3-Mercapto-2-methyl-1-oxopropyl) -1,4-dioxo-7-azispiro [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 mole) of N-carbobenzyloxy-4,4-dimethoxy-L-proline methyl ester from Example 3 (a), 2.4 g (0.032 mole) of 1,2-propanediol, 0 , 4 g of p-toluenesulfonic acid monohydroate and 400 ml of toluene are heated to reflux (110-112 ° C). The reflux rate is controlled so that the solvent slowly distills by means of a Dean-30 Stark line into a scale cylinder. When 80 ml of solvent has been collected, a corresponding volume of fresh solvent is added to the reaction flask through an addition funnel. This operation of removing and replacing 80 ml of solvent is repeated four times during a total reflux period of 1.25 hours.

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After standing overnight, the mixture is washed with 2 x 100 ml of water, the combined washes extracted with 100 ml of toluene, the combined organic layers are dried (MgSO 4) and the solvent is removed on a rotary evaporator, finally at 0.2 mm. to give 8.2 g (99%) of N-carbobenzyl-oxy-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 of part (a) (8.2 g, 10 0.025 mol) is dissolved in 80 ml of methanol, treated dropwise at -1 to + 4 ° C with 18 ml (0.036 mol) of 2N sodium hydroxide, maintained at 0 ° C one hour and at room temperature overnight. After approx. half of the solvent is removed on a rotary evaporator, diluted with 150 ml of water, washed with 100 ml of ether (the wash liquid discarded), acidified with cooling with 6.3 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 of saturated sodium chloride, dried (MgSO4) and the solvent is evaporated to give 8 g of 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 rapidly excreted. After cooling overnight, the material is filtered, washed with cold acetonitrile and ether and dried in vacuo to give 10.3 g of crude adamantanamine salt, m.p. 202-204 ° C (dec.), [Α] D -13 ° (c , 1% in methanol). After trituration with 50 ml of boiling acetonitrile and cooling, the slightly pale brown salt weighs 9.4 g, m.p. 202-204 ° C, decomp., [Α] D 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 an additional 3 x 40 ml of ethyl acetate, the combined organic layers are dried (MgSO 4) and the solvent is evaporated, finally at 0.2 mm and 40UC, gives 5.8 g (72%) of N-carbobenzyloxy-4,4- (1-methylethylenedioxy) -L-proline as a yellow-orange viscous syrup.

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

A solution of 5.6 g (0.017 mol) of the above N - carbobenzyloxy-4,4- (1-methylethylenedioxo) -L-proline in 150 ml of 2: 1 methanol / water is treated with 1.6 g of 5% palladium carbon. -5 catalyst and shaken for 3 atm. hydrogen for 5 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 a crystalline residue. This is suspended in 200 ml of methanol and the evaporation is repeated. The solid residue is rubbed under ether (evaporation repeated) to give 3.0 g (94%) pale pale brown 4,4- (1-methylethylenedioxy) -L-proline, mp 219-221 ° C (dec.) with prior gradual darkening and sintering, [α] Q -22 (c, 1% in 1: 1 ethanol / water).

D) [7 (S), 8S] -7- [3- (Acetylthio) -2-methyl-1-oxopropyl3-1,4-dioxo-7-azaspiro [4,4] nonan-2-methyl -8-carboxylic acid 2.8 g (Q ', 015 mol) of 4,4- (1-methylethylenedioxy) - The L-proline 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 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-oxopropyl] -1, 4-dioxo-7-azaspiro [4,4] nonane-2-methyl-8-carboxylic acid dicyclohexylamine salt, m.p. 170-172 ° C (s, 168 ° C> [α] 5-58 °, (c After crystallization from 12 ml of acetonitrile, the colorless solid salt weighs 3.85 g (51%), m.p. 170-172 ° C (s, 168 ° C), [.alpha. (c, 1% in ethanol).

Analysis calculated for C- HH ^jNOgS.C ^ I ^N: 30 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 of ethyl acetate and treating with 45 ml of 10% potassium bisulfate and 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),

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And the solvent is evaporated to give 2.5 g (51%) of colorless solid [7 (S), 8 S] -7- [3- (acetylthio) -2-methyl-1-oxopropyl] -1,4 dioxo-7-azaspiro [4,4] nonan-2-methyl-8-carboxylic acid, m.p. 65-68 ° C (s, 48 ° C), [α] 5 -100 ° (c, 1% in ethanol) .

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

The product of part (d) is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to give 2.05 g of [7 (S), 8S] -7- (3-mercapto-2-methyl-1-oxopropyl) -1,4-di 10 oxo-7-azaspiro [4,4] nonan-2-methyl-8-carboxylic acid as a viscous colorless oil, [α] D -57, (c, 1% in ethanol).

Example 17 (S, S, S, S) -7,7 '- [Dithiobis (2-methyl-1-oxo-3,1-propanediyl)] - 15 bis [1,4-dioxa-7-azaspiro [4 , 4] nonan-8-carboxylic acid 3.0 g (0.0109 mol) [7 (S), 8S] -7- (3-Mercapto-2-methyl-1-oxopropyl) -1,4- Dioxa-7-azaspiro [4,4] nonane-8-carboxylic acid from Example 2 is dissolved in 80 ml of water and the pH is adjusted to 6.5 with 1N sodium hydroxide. To this stirred solution, a total of 11 ml of 0.5 molar iodine solution in 95% ethanol (6.34 g iodine / 50 ml solution) is added dropwise, keeping the pH of 5.5-6.5 with 1N sodium hydroxide. After 15 minutes, traces of excess iodine with dilute sodium thiosulfate are removed and the solution is concentrated to ca. 50 ml, cool and acidify with 25 1: 1 hydrochloric acid. 30 ml of methylene chloride are added and the mixture is saturated with sodium chloride, stirred, and the layers are separated.

The aqueous phase is extracted with an additional 3 x .20 ml of methylene chloride, the combined organic layers are dried (MgSO4) and the solvent is evaporated, finally at 0.2 mm. The brittle residue is rubbed under ether and the evaporation repeated, giving 2.8 g of pale yellow solid residue. The material is redissolved in 50 ml of methylene chloride, washed with 3 x 10 ml of water, 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 cream 34

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amorphous solid (S, S, S, S) -7,7 '- [dithiobis (2-methyl-1-oxo-3,1-propanediyl)] bis [1,4-dioxa-7-azaspiro [4 , 4] nonan-8-carboxylic acid], m.p. 61-63 ° C (foaming), (s 50 ° C), [α] -92 °, (c, 1% in ethanol).

Calcd. For Calcd. 222 ^ 32 ^ 2 ^ 10 ^ * 2 ^: 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.

The biological effect of the compounds prepared according to the invention in comparison with captopril, which is known from Danish Patent Application No. 596/77, is shown in the following table.

* 50 ^% Inhibition (k)? H3 ΓΊ

HS-CH.-CH-CO-N L-COOH

2 0.005 3 (Captopril) c «52 r, n | 3 0.0008 10

HS-CH 2 -CH-co-N-1-C00H

CH3 "1 1 o ^ r6 CH3 0.002 16

HS-CH2 ~ CH-CO-N '- CQOH

H? H3 0.002 52 HS-CH2-CH-CO-N-1-COOH 30 (3) 150 is the concentration of the compound under investigation (µg / ml) which provides 50% inhibition of angiotensin converting enzyme isolated from rabbit lung.

(b) Residual percent inhibition of angiotensin-forming enzyme after 24 hours in rats, as measured by blood pressure following intravenous infusion of angiotensin I.

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Dosage of compound - 10 mg / kg equivalents of cap-topril.

5

As can be seen from the results, the compounds prepared by the process of the invention are 3-5 times as effective as captopril, and their effect has a significantly longer duration, leaving a significantly higher percentage inhibition after 24 hours.

Claims (2)

An analogous process for preparing a ketal or thioketal derivative of a mercaptoacyl proline of the general formula 1 R 2 X 1 X 2 X R 3 0 (Λ (I) c i R 5 -S- (CH 2) m-CH-C-N-C-COOR H wherein R is hydrogen or C 1 -C 5 alkyl, R 3 is hydrogen, C 1 -C 7 alkyl or halogen-C 1 -C 6 -alkyl, X1 and X2 represent oxygen or sulfur, R1 and R2 represent C1-C7 alkyl, or R1 and R2 together form a polymethylene chain to form a 5- or 6-membered ring, m means 0, 1 or 2, and R5 means hydrogen, C2-C8 alkanoyl, benzoyl or a group R1 R2 yl v2 R3 O / f 11 f * l -S- (CH2) m-CH-CNC-COOR H wherein R, R1, R2, R3, X1, X2 and m have the meanings given above to form a bis-symmetric disulphide compound, characterized by that a substituted proline of the formula II X2 (II) h2c ce2 HN - C-COOR H wherein R, R1, R2, X1 and X2 have the meanings given above are coupled with an acid of formula III R3 c I Wherein R is as defined above and R 5 is C 2 -C 8 alkanoyl or benzoyl, or a reactive derivative thereof, such as an acid chloride , to form a compound of formula IV, R1, R2, R2, I, * vx2 "fp / ^ (iv) R 5, 5-S- (CH 2) m-CH-CNC-COOR H and then, if desired, the group R 1 is removed to form a compound of formula I wherein R 5 is hydrogen and, if desired, this compound is oxidized with iodine to the bis-25-symmetric disulfide compound. Process according to claim 1, characterized in that R 5 represents C 2 -C 8 alkanoyl or benzoyl and this group is removed by hydrolysis or ammonolysis to give the compound of formula I wherein R 5 represents hydrogen.