DK154553B - METHOD OF ANALOGUE FOR THE PREPARATION OF ETHER OR THIOETHERMERCAPTOACYL PROLINES - Google Patents

Description

in

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The present invention relates to an analogous process for the preparation of novel ether and thio-ether mercaptoacyl prolines of the formula X-If 5 R 2 HC / t> (I 2 | 2 1

R4- S- CH 2- CH - CO- * N-c - COOR

H

wherein the XR group is positioned in the 3 or 4-10 position of the proline ring, X is oxygen or sulfur, R is hydrogen or alkyl, R "*" is C 1-6 alkyl or phenyl, optionally substituted with a halogen atom or a methyl or methoxy group, R is hydrogen or C 1-6 alkyl and R 4 is hydrogen, C 1-6 alkanoyl or benzoyl, or salts thereof.

The ether and thioether mercaptoacyl prolines of the formula I prepared by the process of the invention are useful anti-hypertensive agents.

From German Publication No. 2,703,828, related compounds of the same type of activity are known, namely 20 hypotensive effects. The structure of these known compounds differs from the above Formula I in that they do not exhibit the substituent group XR 2 but instead are either unsubstituted (i.e., have hydrogen) or carry a hydroxy or alkyl group. The most notable of these known compounds 25 is the unsubstituted, commercially sold under the name "Captopril" and having the structure?

HS-CH, -CH-C 0 -C-COOH N_I

1- (3-mercaptopropanoyl) -D-proline ("Captopril") known from DE 2,703,828

This compound has been tested in the following comparative experiments (cf. "Biological test") together with the corresponding 4- (2) prepared according to Example 13 below.

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o phenylthio compound of formula? H3

HS-CH2 “CH-CO-N__COOH

1- (D-3-mercapto-2-methyl-1-oxopropyl) -cis.-4-phenyl-thio-L-proline, the latter compound exhibiting a higher activity level.

alkanoyl groups are acyl groups in fatty acids such as e.g. acetyl, propionyl, butyryl and isobutyryl, of which acetyl is particularly preferred.

The products of formula I occur in stereoisomeric forms or as racemic mixtures thereof. All of these can be prepared by the process of the invention. The synthesis described below may use the racemate or one of the enantiomers as starting material. When the racemic starting material is used for the synthesis, the stereoisomers obtained in the final product can be separated by conventional chromatographic or fractional crystallization methods. The X-R "*" group may also give rise to cis-trans isomerism.

Preferably, the asymmetric center at C2 in the proline ring is in L configuration, and if there is an asymmetric center in the mercaptoacyl side chain, it is in D configuration.

Particularly preferred among the above compounds are those of Formula I wherein X is oxygen, R 1 is methyl or 2 ethyl, especially methyl, R is hydrogen or methyl, especially methyl, 4 1 R is hydrogen and the XR group is in the position in the prolinrin gene, especially when the -XRR group is in cis configuration.

The ethers and thioethers of formula I are obtained by the process of the invention, which is characterized in that a proline compound of formula 35

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r <

HN__COOR

5 wherein R, R1 and X are as defined above are coupled with an acid of formula R2

r, 4-s-ch2-ch-cooh III

Wherein R is as above, R 'is alkanoyl or benzoyl, or a functional derivative thereof, to form 4 a product wherein R is C 1-4 alkanoyl or benzoyl and, if desired, the product is hydrolyzed to form a product, 4 12 15 wherein R is hydrogen and R, R, R and X are as indicated above, whereupon an optionally formed acid is, if desired, transferred into a salt thereof.

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 the mixed anhydride, symmetric anhydride, acid halide, active ester or use of Woodward reagent K, N-ethoxycarbonyl-2-ethoxy. -1,2-dihydroquinoline or the like. Regarding acetylation methods, see the Methods der Organischen Chemie (Houben-25-Weyl), vol. XV, Part II, page 1 et seq. (1974). Preferably, the acid halide, especially the acid chloride, of formula III is reacted with the acid of formula II.

The ester compounds (formula I wherein R is alkyl) can be converted to the free acid (i.e., R is hydrogen) by conventional means.

For example, if R is t-butyl, treatment with trifluoroacetic acid and anisole gives the free acid.

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

4 4

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Products of formula I wherein R is alkanoyl or benzoyl may be converted to products of formula I wherein 4 R is hydrogen by conventional hydrolysis or ammonolysis.

The esters of formula I wherein R is C 1-4 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, 1-alkyl-3-p-tolyltriazine such as 1-n-butyl-3-p-tolyltriazine or the like.

The proline reactants of formula II can be prepared in various ways. Eg. can be a hydroxy or mercapto-proline of the formula

XH

"Ή

HN -> - C-COOH

15 |

H

is acetylated with an acetylating agent such as acetic anhydride, acetyl chloride, propionic anhydride, butyric anhydride, benzyl chloroformate or the like to protect the nitrogen. The R 2 group is then introduced by reacting the N-protected form of the compound of formula 17 with a halide, R 2 hal, where hal is a halogen, preferably iodine, in the presence of silver oxide, sodium hydride, sodium hydroxide or the like, whereby is obtained an intermediate of the formula protected-NC-COOR1 30 '

H

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

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5

Another method of preparing the proline reactants of formula II is to treat the N-protected tosyloxyproline ester, preferably the methyl ester of formula 5

protected-N-C-COOalkyl VI

10 I

H

with a sodium salt of the formula

R1-X-Na VII

15 to give the intermediate of formula

2o Η2ί ^> <Γ VIII

protected-N- (l-COOalkyl

H

In the formula VI, the symbol Ts is -SO 2 + 3 -CH 2, and the N-protecting group is preferred benzyloxycarbonyl or other commonly used acyl protecting groups. If the tosylate group is in cis configuration, the XR 2 group in this reaction will be in the trans configuration, and if the tosyloxy group is in the trans configuration, the XR 2 group will be in the cis 30 configuration. The intermediate of formula VIII is then reacted to remove the alkyl ester group and then reacted with hydrogen bromide to give the HBr salt of the proline reactant of formula II which can then be coupled with the acid, preferably the acid chloride, of formula III.

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the proin starting materials of formula II wherein X is sulfur and the XR 2 group attached to the 3-position of the proline can also be obtained by treating a 1,2-dehydroproline ester, preferably the t-butyl ester, of formula 5

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N ~ == - COOalkyl with an acylating agent such as benzyl chloroformate, acetyl chloride, etc. to give the 4,5-dehydro compound Γ'Ti

protected N-COOalkyl X

15 which is then reacted with the mercaptan R 2 -SH to give rf *

protected N - COOalkyl XI

Wherein the -S-R 2 substituent is in trans-configuration. The N-protecting and alkyl groups are then removed to give the desired starting material.

The proline starting materials of formula II, wherein R "+" is phenyl, substituted phenyl, phenyl-lower alkylene or substituted phenyl-lower alkylene, can also be obtained by treating the benzyl ester of the N-protected proline of formula IV with the alcohol R-OH in presence of triphenylphosphine and diethyl azodicarboxylate according to the method described by Bittner et al. in Chemistry and Industry, March 15, 1975, page 281. Removal of the N-protecting group and the benzyl ester group gives the starting material of formula II.

Reference is also made to the following publications for further information regarding the preparation of starting materials and intermediates: United States Patent Nos. 4,046,889, 4,105,776 and 4,145,935 to Neuberger, J.

Chem. Soc., 1945, pages 429-432, Patchett et al., J. Amer. Chem.

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Soc. 79, pages 185-192 (1957), Baer et al., Can. J. Biochem. and Phys., 37, pp. 583-587 (1959), Sheenan et al., J. Amer.

Chem. Soc. 85, pp. 3863-3865 (1963), and Magerlein, J. Med.

Chem. 10, pp. 1161-1163 (1967). The methods mentioned therein 5 can be used as general methods in the synthesis and stereo-storage of compounds prepared by the process of the invention.

Further experimental details are discussed in the examples which are preferred embodiments and also may serve as models for the preparation of other compounds within the group.

The compounds prepared by the process of the invention form basic salts with various inorganic and organic bases. The salt-forming ion derived from such bases may be metal ions, e.g. aluminum, alkali metal ions such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt, a number 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-adamantinamine, arginine, lysine and the like. The physiologically acceptable salts such as sodium or potassium salts can be used medically as described below and are preferred. These and other salts, which are not necessarily physiologically acceptable, are useful for isolating or purifying a product acceptable for the purposes described below as illustrated by the dicyclohexylamine salt and the cyclohexylamine salt of the Examples. The salts are prepared by reacting the acid form of the compound with 1 equivalent of the base giving the desired basic ion, in a salt precipitating agent, or in aqueous medium and then lyophilizing.

The free acid form can be obtained from the salt by conventional neutralization technique, e.g. with potassium bisulfate, hydrochloric acid etc.

The compounds prepared by the method of the invention inhibit the conversion of the decapeptide by

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giotensin I to angiotensin II and are therefore useful in reducing or curing hypertension. The compounds prepared by the process of the invention interfere with the order of renin - angiotensinogen - angiotensin I -> angiotensin II by inhibiting the angiotensin converting enzyme and reducing or eliminating the formation of the angiotensin II pressor substance. Thus, when a hypotensive effective amount of a composition containing one or a combination of compounds of formula I or 10 is administered a physiologically acceptable salt thereof, hypertension in mammalian species suffering from it is reduced or cured.

A single dose or preferably two to four divided daily doses on the basis of approx. 0.1-ca. 100 mg / kg / day, preferably approx. 1-C. 50 mg / kg / day, blood pressure is lowered as indicated in animal studies described by S.L. Engel, T.R. · Schaeffer, M.H. Waugh and B. Rubin in Proc. Soc. Exp. Biol. With. 143, 483 (1973). The drug is preferably administered orally, but also parenteral routes of administration such as subcutaneous, intramuscular, intravenous or intraperionally can be used.

The compounds prepared by the method of the invention can be used to obtain a reduction in blood pressure by being formulated into preparations thereof, such as tablets, capsules or elixirs for oral administration or sterile solutions or suspensions for parenteral administration. Ca. 10-C. 500 mg of a compound or mixture of compounds of formula I or a physiologically acceptable salt thereof are mixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring, etc. in a unit dosage form according to conventional pharmaceutical practice. The amount of active substance in these agents or compositions is adjusted so as to obtain a suitable dosage within the specified range.

Examples of excipients which can be incorporated into tablets, capsules and the like include the following: a binder such as gum tragacanth, acacia, corn starch or gelatin, excipients such as dicalcium phosphate or

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crystalline cellulose, a disintegrant such as corn starch, potato starch, alginic acid and the like, a lubricant such as magnesium stearate, a sweetener such as saccharose, lactose or saccharin, a flavoring such as peppermint, winter green oil or cherry flavor. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be found such as coatings or other islets which modify the physical form of the dosage unit.

Thus, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor.

15 Sterile injection preparations can be formulated in accordance with current pharmaceutical practice by dissolving or suspending the active ingredient in a carrier such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cotton seed oil, etc.

The following examples serve to further elucidate the process of the invention and to illustrate the activity of the compounds prepared as discussed above.

Example 1 1- (3-Acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline a) N-Acetyl-trans-4-hydroxy-L-proline

A stirred suspension of 26.2 g (0.2 mole) of trans-4-hydroxy-L-proline in 400 ml of acetic acid is treated with 26 ml of acetic anhydride. The solid dissolves gradually after two hours of stirring at room temperature. The solution is poured onto a 2 liter flask and the solvent is removed in a rotary evaporator at a bath temperature of 45 ° C. The syrupy residue (57.5 g) is diluted with 100 ml of ether to give a crystalline solid. After cooling overnight .10

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the solid is filtered off, washed with cold ether and dried in a desiccator. This material weighing 35.7 g is pulverized and suspended in 100 ml of ether, cooled and filtered to give 33.8 g (98%) of N-acetyl-trans-4-hydroxy-5-L-proline, m.p. 128 -131 ° C. Recrystallization of 0.5 g of this material from 5 ml of acetonitrile gives 0.45 g of colorless solid, mp 130-132 ° C: [α] D -92 ° (c, 1% in EtOH).

b) N-Acetyl-trans-4-methoxy-L-proline methyl ester A mixture of 30.0 g (0.17 mol) of N-acetyl-trans-4-hydroxy-L-proline and 130 g of silver oxide is pulverized. in a mortar, and this uniform mixture is added to a 1-liter flask with 300 ml of acetone. The slurry is stirred, treated portionwise with 130 ml of methyl iodide, and the temperature is kept below 40 ° C by cooling with a cold water bath. After stirring for 7 hours, the mixture is left to stand overnight. The solid is filtered off, washed well with actone and the filtrate is evaporated on a rotary evaporator to give 38.3 g of syrupy residue. This is redissolved in 350 ml of acetone and again treated with 130 g of silver-20 oxide and 130 ml of methyl iodide to give 41 g of residue. This is distilled to give 32.2 g of distillate, boiling point 130-140 ° C (0.3 mm). After dissolving in 30 ml of cyclohexane and cooling, the nearly colorless solid N-acetyl-trans-4-methoxy-L-proline methyl ester weighs 31.4 g, mp 71-75 ° C.

Recrystallization from 31 ml of ethyl acetate gives 25.1 g (66%) of colorless solid, mp 76-77 ° C. [α] D -83 ° (c, 1% in EtOH).

c) trans-4-methoxy-L-proline

To a stirred solution of 27.0 g (0.085 mole) of barium hydroxide. To 525 ml of water (about 3.3 N) is added 11.0 g (0.05 mol) of N-acetyl-trans-4-methoxy-L-proline methyl ester.

The resulting solution is stirred at 18-20 ° C for 3 hours, cooled and treated with dilute sulfuric acid (8.8 g of concentrated H 2 SO 4 in 20 ml of water). The acidic suspension is left overnight.

The mixture is filtered through a thick layer of "Celite" to give a "milky" filtrate. This is evaporated on a rotor they evaporate at 50 ° C using a high vacuum pump, giving a milky residue weighing 121 g.

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The material is treated with dilute sulfuric acid (19.0 g concentrated in 75 ml of water) and the resulting mixture is stirred and refluxed for 3 hours. After cooling to 30 ° C, the mixture is treated portionwise with 48 g of barium hydroxide.

5 8H2O ,. and pH is adjusted from 6.0 to 4.0 with dilute sulfuric acid. After standing overnight, the mixture is filtered through a thick layer of "Celite". The milky filtrate is evaporated as above to give 50 g of colorless dry residue. This is dissolved in 200 ml of warm chloroform and filtered through a 10 layer of "Celite" to remove the barium sulfate. The slightly cloudy filtrate is evaporated on a rotary evaporator to give a gelatinous material (17.7 g) suspended in 100 ml of ether and filtered to give 7.5 g (94%) of almost colorless solid, m.p. 185-190 ° C (decomposition). The material is suspended in 30 ml of hot acetonitrile, cooled and filtered to give 4.0 g (50%) of a colorless solid trans-4-methoxy-L-proline, mp 209-211 ° C (dec.); [α] D = -75 ° (c, 1% in EtOH).

d) 1- (3-Acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline A solution of 3.5 g (0.024 mol) of trans-4-methoxy-L-proline in 50 ml of water is stirred. , cool to 5 ° C and add 3 g of sodium carbonate. This mixture is treated with a solution of 4.0 g (0.024 mole) of 3-acetylthiopropionyl chloride in 5 ml of ether over 10 minutes with intermittent addition of 3 g of sodium carbonate to maintain a pH of approx. 8.0.

The mixture is stirred in an ice bath for an additional hour, adding 25 ml of water and then a solution of 5 ml of concentrated hydrochloric acid in 25 ml of water (CC The highly acidic solution is saturated with sodium chloride and extracted with 50 ml of 30 ethyl acetate (four times). The organic phases are combined, dried (MgSO 4), filtered and the solvent evaporated to give 6.0 g (90%) of colorless syrupy 1- (3-acetylthio-1-oxopropyl) -trans-4-methoxy-L proline. This acid is dissolved in 25 ml of ethyl acetate and treated with 4.7 g of dicyclohexylamine to give a solution which quickly becomes a solid mass.

An additional 15 ml of ethyl acetate is added and the mixture is dissolved in a steam bath, cooled and filtered to give 8.7 g of

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dicyclohexylamine salt, mp 170-172 ° C. After crystallization from 60 ml of acetonitrile, the colorless solid weighed 8.3 g (75%), mp 171-173 ° C; [α] D -35 ° (c, 1% in EtOH).

The decyclohexylamine salt is converted to 1- (3-acetylthio-1-oxopropyl) trans-4-methoxy-L-proline by suspending 8.0 g in 60 ml of ethyl acetate cooled in an ice bath and treating it portionwise with 60 ml of 10% potassium bisulfate. The clear layers are separated and the aqueous portion is extracted with 60 ml of ethyl acetate (2 times). The organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated to give 4.6 g (80%) of a colorless syrup.

Example 2 1- (3-Mercapto-1-oxopropyl) -trans-4-methoxy-L-proline

To the 1- (3-acetylthio-1-oxopropyl) -trans-4-methoxy-L-proline prepared in Example 1 (4.6 g, 0.017 mol) is added a cold solution of 9 ml of concentrated ammonia in 22 ml. water. The base dissolves in approx. 30 minutes, and the resulting solution (under argon) is left at room temperature for 2 hours. This solution is cooled, extracted with 25 ml of ethyl acetate (2 times) and the ethyl acetate extract discarded. The solution is extracted again with 25 ml of ethyl acetate and acidified with 17 ml of 1: 1 hydrochloric acid. The mixture is shaken, separated and the aqueous phase is extracted with 25 ml of ethyl acetate (3 times). The organic phases are combined, dried (MgSO 4), filtered and the solvent removed on the rotary evaporator to give 2.3 g (59%) of colorless syrup, 1- (3-mercapto-1-oxopropyl) -trans-4- -methoxy-L-proline, [α] 25 -60 ° (c, 1% in EtOH); R f 0.49 (MeOH 30 on silica gel, developed with nitrosoferricyanide as reagent), Analysis: Calculated for C 1/4 H2 O: C 45.46, H 6.57, N 5.87, S 13.48.

Found: C 45.42, H 6.78, N 5.96, S 13.27.

An additional 1.1 g of product (3.4 g, 87% in total) is obtained by saturating the aqueous phase with sodium chloride and extracting with 25 ml of ethyl acetate (2 times).

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The sodium salt is formed by treating the syrup with aqueous sodium bicarbonate and freeze-drying.

Example 35 (trans) -1 »[D-3- (Acetylthio) -2-methyl-1-oxopropyl] -4-methoxy-L-proline

A solution of 4.3 g (0.029 mol) of trans-4-methoxy-L-proline in 50 ml of water is stirred, cooled to 5 ° C and 3 g of sodium carbonate is added. This solution is treated with 5.2 g (0.029 mol) of D-3- (acetylthio) -2-methylpropionyl chloride in 5 ml of ether over 10 minutes with the intermittent addition of 3 g of sodium carbonate to maintain the pH of ca. 8.0.

This mixture is stirred in an ice bath for 1.5 hours, then 25 ml of water is added and then a solution of 6 ml of concentrated hydrochloric acid in 25 ml of water (CC The resulting strongly acidic solution is extracted with 50 ml of ethyl acetate (four times).

The organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated to give 6.1 g (73%) of (trans) - 1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4-methoxy-L-prolin as a pale yellow syrupy residue. This acid is dissolved in 50 ml of ethyl acetate and treated with a solution of 4.0 g of dicyclohexylamine in 20 ml of ethyl acetate. The product begins to crystallize from the solution over approx. 1 minute. After cooling overnight, the almost colorless solid is filtered and dried, yield 6.7 g, mp 175-177 ° C; [α] D -55 ° (c, 1% EtOH). After crystallization from 60 ml of acetonitrile, the almost colorless solid dicyclohex-ylamine salt weighed 5.6 g (41%), m.p. 179-181 ° C, [α] D -62 ° (c, 1% in EtOH).

The dicyclohexylamine salt is converted to the acid by suspending 5.5 g in 50 ml of ethyl acetate, cooling in an ice bath and treating with 50 ml of 10% potassium bisulfate. The layers are separated and the aqueous portion is extracted with 50 ml of ethyl acetate (2 times).

The organic phases are combined, dried (MgSO 4), filtered and the solvent evaporated to give 3.4 g (41%) of almost colorless (trans) -1- [D-3- (acetylthio) -2-methyl-1 -oxopropyl] -4-methoxy-L-proline as syrup.

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Example 4 (trans) -4-Methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) - -L-proline

To 3.4 g of (trans) -1- [D-e- (acetylthio) -2-methyl-1-5-oxopropyl] -4-methoxy-L-proline is added a cold solution of 8 ml of concentrated ammonia in 20 ml of water. The base dissolves in approx. 10 minutes and the resulting solution (under argon) is left at room temperature for two hours. This solution is cooled, extracted with 20 ml of ethyl acetate (2 times), separated in layers with 20 ml of ethyl acetate and acidified with 15 ml of 1: 1 hydrochloric acid. This mixture is saturated with sodium chloride, the layers are separated and the aqueous phase is extracted with 20 ml of ethyl acetate (3 times). The organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated to give 2.9 g (100%) of almost colorless (trans) -4-methoxy-1- (D-3-mercapto-2 methyl-1-oxopropyl) -L-proline, [α] D -80 ° (c, 1% in EtOH);

Rf 0.53 (MeOH on silica gel developed with nitrosoferricyanide as reagent).

Analysis: Calculated for C ^ qH ^NO ^S. 1/4 O: C 47.69, H 6.83, N 5.56, S 12.73

Found: C, 47.90; H, 6.84; N, 5.85; S, 12.76.

Example 5 (trans) -1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -4-ethoxy-L-proline

Using the procedure described in Example 3, but using an equivalent amount of trans-4-ethoxy-L-protein (J.Med. Chem., 10, 1161 (1967)) instead of (trans) -4- Methoxy-L-proline is obtained (trans) -1- (D-3- (acetylthio) -2-methyl-1-oxopropyl] -4-ethoxy-L-proline. The product is purified as the dicyclohexylamine salt, melt pump 170 -172 ° C (crystallized from isopropyl alcohol); -64 ° (c, 1% in EtOH).

This salt (7.75 g) is converted to the free acid by treatment with potassium bisulfate solution as described in Example 4 to give 4.85 g of almost colorless (trans) -1- [D-3 - (acetylthio) -2- methyl 1-oxopropyl] -4-ethoxy-L-proline as syrup.

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Example 6 (trans) -4-Ethoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L- -proline

To the material of Example 5 (4.85 g) is added a 5 cold solution of 9 ml of concentrated ammonia in 22 ml of water (under argon). The mixture is treated in the same manner as in Example 4 to give 4.2 g (100%) of almost colorless (trans) -4-ethoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L- -proline as syrup, [α] 25 -80 ° (c, 1% in EtOH), 0.64 (MeOH

10 on silica gel, developed with nitrosoferricyanide as reagent). Analysis: Calculated for C- HH ^NO ^S: C 50.55, H 7.33, N 5.36, S 12.27.

Found: C, 50.34; H, 7.34; N, 5.39; S, 12.11.

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Example 7 (cis) -1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -4-methoxy-L-proline a) N-Carbobenzyloxy-cis-4-hydroxy-L-proline N-Carbobenzyloxy-4-keto-L-proline (10 g, 0.038 mol) is dissolved in 300 ml of methanol and reduced with 5.8 g (0.15 mol) of sodium borohydride in 20 ml of water as described in JACS, 79, 189 ( 1957) to give 8.7 g of a foamy product. This material is dissolved in 30 ml of ethanol, treated with 3.5 g of cyclohexylamine in a little ethanol and diluted to 500 ml with ether. By grafting and rubbing, the crystalline cyclohexylamine salt is rapidly excreted and 10.8 g, mp 163-165 ° C are obtained. This cyclohexylamine salt is then treated with 30 ml of 2N HCl and extracted with ethyl acetate (4 x 50 ml) to give 8 g of N-carbobenzyloxy-cis-4-hydroxy-L-proline as a glass-like material.

b) N-Carbobenzyloxy-cis-4-methoxy-L-proline methyl ester 13.9 g of N-carbobenzyloxy-cis-4-hydroxy-L-proline (0.052 mol) are treated with 40 g of silver oxide and 40 ml of methyl iodide (2 times) in acetone (first 100 ml, then 120 ml) as described in Example 1 (b) to give 17.5 g (100%) of N-carbo-benzyloxy-cis-4-methoxy-L-proline methyl ester like a yellow oil.

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c) N-Carbobenzyloxy-cis-4-methoxy-L-proline

The above-mentioned 17.5 g of N-carbobenzyloxy-cis-4-methoxy-L-proline methyl ester (about 0.052 mol) is dissolved in 135 ml of methanol, treated dropwise at -1 ° C to 4 ° C with 32 ml of 5 (0.064 mol) of 2N sodium hydroxide is then 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 300 ml of water, washed with ether (the wash liquid discarded), acidified with cooling with 12.5 ml of 1: 1 hydrochloric acid to pH 2 and extracted with ethyl acetate (4x150). mL). The extracts are combined, dried (MgSC> 4), filtered and the solvent is evaporated to give 15 g of an orange-yellow syrup. This is dissolved in 60 ml of ethanol, treated with 6 g of cyclohexylamine in 10 ml of ethanol and diluted 15 to 900 ml with ether. By grafting and rubbing, the crystalline N-carbobenzyloxy-cis-4-methoxy-L-proline-cyclohexylamine salt is separated: weight after cooling overnight at 10.2 g, mp 148-150 ° C (p. 144 ° C), [ α] -35 ° (c, 1% in ethanol, etc.). After recrystallization from 40 ml of acetonitrile, the nearly colorless solid weighs 8.8 g, m.p. 150-152 ° C (p. 145 ° C), [cc] +6 -34 ° (c, 1% in ethanol ).

The cyclohexylamine salt is treated with hydrochloric acid to give 6.9 g (48%) of N-carbobenzyloxy-cis-4-methoxy-L-procoline as a pale yellow viscous syrup, [α] 32 (c, 1%). ethanol).

d) cis-4-Methoxy-Ir-proline

A mixture of 6.8 g of N-carbobenzyloxy-cis-4-methoxy-L-proline, 210 ml of 2: 1 methanol and water and 2.3 g of 5% palladium on charcoal is placed in a hydrogenator at 3 atm hydrogen. for four hours. The mixture is filtered to remove the catalyst and the filtrate is evaporated to give 3.15 g of a greyish solid, mp 218-220 ° C (dec.). A sample is crystallized from methanol-ether to give colorless cis-4-methxoy-L-proline, m.p. 224-226 ° C (dec.), [Α] 5 ~ 42 ° (c, 1% in methanol) ).

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Analysis: Calculated for CgHH NO NONO: C 49.64, H 7.64, N 9.65.

Pound: C 49.63. H, 7.71; N, 9.54.

e) (cis) -1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -4-methoxy-L-proline 5 g of cis-4-methoxy-L-proline (0.031 and 4.2 g (0.023 mol) of D-3-acetylthio-2-methylpropionyl chloride in 5 ml of ether are reacted in 60 ml of water in the presence of sodium bicarbonate as described in Example 3. 20 ml of 25% sodium carbonate (w / v) to begin with 10. bring the pH of 8.5 and to keep it at 7.5-8.4 during the acetylation. The resulting crude viscous product (6.4 g) is dissolved in 50 ml of ethyl acetate and treated with 3.9 g of dicyclohexylamine in 20 ml of ethyl acetate. The product crystallizes from the solution and is filtered and dried to give 6.6 g of di-cyclohexylamine salt, mp 172-174 ° C (p. 170 ° C).

2 £ Q

[a] p -60 (c, 1% in ethanol). 6.5 g of this material is crystallized from 35 ml of acetonitrile to give 6 g of colorless solid dicyclohexylamine salt, mp 173-175 ° C (p. 170 ° C), [<x] 2d6 -60 ° (c, 1% in ethanol).

Analysis: Calculated for C₂2H] _9 ^O S.S. 22. 23.5 61 c 61/24, H 9.00, N 5.95, S 6.81.

Found: C 61.16, H 8.81, N 5.95, S 6.67.

Using the procedure described in Example 3, the dicyclohexylamine salt is converted to the acid by suspending 5.9 g in 60 ml of ethyl acetate, cooling in an ice bath and treating with 60 ml of 10% potassium bisulfate. The layers are separated and the aqueous phase is extracted with 50 ml of ethyl acetate (4 times).

The organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated to give 3.5 g (60%) of colorless (cis) -1- [D-3- (acetylthio) -2-methyl-1- oxopropyl] -4-methoxy-L-proline, m.p. 90-92 ° C (triturated with ether), [α] -139 ° (c, 1% in ethanol), Rf 0.63 (methanol on silica gel ).

Analysis: Calculated for C ^ HH- ^NO₂S: C 49.81, H 6.62, N 4.84.

Found: C 49.85, H 6.66, N 4.97.

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Example 8 (cis) -4-Methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L- -proline 2.9 g (cis) -1- [D-3- (acetylthio) 2-Methyl-1-oxopropyl] -4-methoxy-L-proline (0.01 mol) is hydrolyzed in 15 ml of water containing 6.5 ml of concentrated ammonia as described in Example 4 to give 2.3 g (93%) highly viscous (cis) - -4-methoxy-1- (D-3-mercap to-2-methyl-1-oxopropyl) -L-proline, which becomes waxy upon standing; [α] D -88 (c, 1% in e-thanol).

Example 8a (cis) -4-Methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline-1-adamantanamine salt A solution of 0.55 g (0.0022 mol) cis-4-methoxy--1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline in 15 ml of ethyl acetate is treated under an argon atmosphere with a hot solution of 0.34 g (0.0022 mole) 1-adamantanamine in 10 ml of ethyl acetate to precipitate the salt. After three hours in the cold, the colorless solid is filtered off under argon (the solvent is persistent), washed with some cold ethyl acetate and dried in vacuo for 20 hours to give 0.7 g (79%) (cis) - 4-Methoxy-1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline-1-adamantanamine salt, mp 215-217 ° C (p. 210 ° C, decomposition 220 ° C), [α] D -60 ° (c, 1% in methanol).

Example 9 (trans) -1- [D-3-Acetylthio) -2-methyl-1-oxopropyl] -4-propoxy-30-L-proline a) N-acetyl-trans-4-propoxy-L-proline propylester

Reaction of 30 g of N-acetyl-trans-4-hydroxy-L-proline from Example 1 (a) with 110 g of silver oxide and 110 ml of propyliodide in 300 ml of acetone as described by the procedure of Example 1 ( b) gives 19.6 g (41%) of pale yellow-orange N-acetyl acid;

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-trans-4-propoxy-L-proline propyl ester, bp 155-165 ° C (0.2 mm).

b) N-Acetyl-trans-4-propoxy-L-proline

A solution of 6 g (0.15 mole) of sodium hydroxide in 5 150 ml of water is added to 19.4 g (0.075 mole) of N-acetyl-trans-4-propoxy-L-proline propyl ester to give a slightly orange solution . After standing overnight at room temperature, the solution is extracted with 60 ml of ethyl acetate (the wash liquid is discarded), then acidified with 1: 1 hydrochloric acid, then saturated with sodium chloride and extracted with 50 ml of chloroform (3 times), dried, (MgSO 4), filtered and the solvent evaporated to give 12.6 g of a brown oil. This is dissolved in 80 ml of ethyl acetate and treated with a solution of 10.7 g of dicyclohexylamine in 15 ml of ethyl acetate. The salt crystallizes at room temperature. After standing overnight during cooling, the dicy clohexylamine salt is filtered and washed with cold ethyl acetate to give 17.3 g of almost colorless solid dicyclohexylamine salt, mp 148-153 ° C. Recrystallization of this material from 125 ml of ethyl acetate gives 14.5 g of colorless dicyclohexylamine salt, mp 157-159 ° C [α] D -30 ° (c, 1% in ethanol).

Analysis: Calculated for C ]oH17NO4 * C12H23H: δ 6/63, H 10.17, N 7.07.

Found: C 66.47, H 10.22, N 7.06.

The 14.4 g of dicyclohexylamine salt is converted to the free acid by pulverizing it, suspending in 100 ml of ethyl acetate and treating the slurry portionwise with 100 ml of 10% potassium bisulfate. The organic phase is separated and the aqueous phase is extracted with 100 ml of ethyl acetate (2 times). The organic phases are combined, dried, (MgSO 4), filtered and the solvent is evaporated to give 6.7 g (41%) of N-acetyl-trans-4-propoxy-L-proline as a slightly brown liquid.

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c) (trans) -1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -4-propoxy-1-proline 6.4 g N-acetyl-trans-4-propoxy-L -proline (0.03 mol) is treated with a solution of 10 g of concentrated sulfuric acid in 5 100 ml of water and the resulting solution is stirred and refluxed for three hours. Then the solution is cooled to 15 ° C, treated portionwise with 12 g of sodium carbonate to bring the pH to 8.0, and then treated with a solution of 5.4 g (0.03 mole) of D-3-acetylthio-2-methylpropionyl chloride in 5 10 ml of ether over 10 minutes, while adding 7 g of sodium carbonate to maintain the pH of approx. 8.0. Then the mixture is stirred in an ice bath for 30 minutes and at room temperature for one hour. The product is then isolated and purified as a dicyclohexylamine salt by the procedure of Example 3 to give 6.3 g of dicyclohexylamine salt, mp 165-167 ° C (from acetonitrile), [α] 5 -56 ° C (c , 1% in ethanol).

Analysis: Calculated for. C 21 F 23: C 62.62, H 9.30, N 5.61, S 6.43.

Found: C, 62.35; H, 9.48; N, 5.88; S, 6.45.

The 6.1 g of dicyclohexylamine salt is converted to the free acid by being suspended in 60 ml of ethyl acetate, cooled in an ice bath and treated with 60 ml of 10% potassium bisulfate. The layers are separated and the aqueous phase is extracted with 60 ml of ethyl acetate (2 times). The organic phases are combined, dried (MgSO 4), filtered and the solvent is evaporated to give 4.0 g (42%) of (trans) -1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4 - propoxy-L-proline as an almost colorless syrup.

Example 10 L- (D-3-Mercapto-2-methyl-1-oxopropyl) -trans-4-propoxy-L-proline

Hydrolysis of 4.0 g of (trans) -1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -4-propoxy-L-proline with 8 ml of concentrated ammonia in 20 ml of water under argon after The procedure given in Example 4 gives 3.42 g (97%) of 1- (D-3-mercapto-2-meth-21

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1-yl-1-oxopropyl) -trans-4-propoxy-L-proline as an almost colorless syrup, [α] D -72 ° (c, 1% in ethanol).

Analysis: Calculated for 1/4 H 2 O: C 51.49, H 9.74, N 5.05, S 11.46.

Found: C, 51.66; H, 7.76; N, 5.94; S, 10.51.

Example H

(cis) -4- (4-Fluorophenoxy) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -10-L-proline a) (cis) -4- (4-Fluorophenoxy) -L proline

To a solution of 8.4 g (0.024 mole) of N-carbobenzoxy-trans-4-hydroxy-L-proline benzyl ester (Baer et al., Can. J. Biochem. & Phys., 37, 583 ( 1959), 4.0 g (0.036 mole) of 4-fluoro-phenol and 9.27 g (0.036 mole) of triphenylphosphine in 75 ml of dry tetrahydrofuran are added dropwise over one hour to 6.2 g (0.036 mole) of diethyl azodicarboxylate. 25 ml of tetrahydrofuran. The solution is left to stir overnight at room temperature. The mixture is evaporated to dryness and 20 ml of ether is added to the residue. A precipitate of triphenylphoshine and diethyl azodicarboxylate is filtered off. Column chromatography (silica gel) separates 8.1 g of a mixture containing approx.

70% N-carbobenzoxy-cis-4- (4-fluorophenoxy) -L-proline-benzyl ester. A solution of 7.5 g of the above-mentioned benzyl ester-containing mixture is hydrogenated at 1 atm (room temperature) with 0.8 g of 10% pd / C. A white precipitate forms during the reaction. After the hydrogen uptake has ceased, the mixture is filtered and the filter cake washed out with three 125 ml portions of hot methanol. The methanol solution is evaporated to dryness to give 3.2 g of cis-4- (4-fluorophenoxy) -L-proline, mp 235-236 ° C.

Analysis: Calculated for C ^H ^FNO ^. 1/3 E + O: C 57.14, H 5.48, N 6.06, F 8.22.

Found: C 56.94, H 5.19, N 5.94, F 7.97.

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b) 1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -cis-4- (4-fluoro-phenoxy) -L-proline

To a suspension of 2.25 g (0.01 mole) of cis-4- (4-fluorophenoxy) -L-proline in 125 ml of dry pyridine at room temperature is added 1.0 g (0.01 mole) of triethylamine. and 2 g (0.011 mole) of D-3- (acetylthio) -2-methylpropionyl chloride. After stirring overnight at room temperature, the mixture is evaporated to dryness. The residue is taken up in water, covered with ether and acidified with 10% hydrochloric acid. The water layer is repeatedly extracted with ether, the ether layers are combined, washed with water, dried (Na 2 SO 4) and evaporated to dryness to give 3.9 g of residue. The residue is chromatographed on 250 ml of silica gel with ether to give a fraction containing the desired product. The column is washed with methanol and the methanol wash liquid is rechromatographed on a short column of silica gel to give further product. This process is repeated again, giving a total of 1.2 g of pure 1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -cis-4- (4-fluorophenoxy) -L-proline.

c) (cis) -4- (4-Fluorophenoxy) -1- (D-3-mercapto-2-methyl-1-oxo-propyl) -L-proline 1.2 g 1- [D-3- ( acetylthio) -2-methyl-1-oxopropyl] - cis-4- (4-fluorophenoxy) -L-proline is dissolved in 25 ml of methanol and treated with 5 ml of concentrated ammonia under argon for 40 minutes. The volatiles are removed and the residue is covered with ethyl acetate. The water layer is acidified with 10% hydrochloric acid.

The layers are separated and the acidic layer is washed with ethyl acetate. The combined organic layers are liberated with water (2 times, saturated sodium chloride solution (2 times) and dried (Na 2 SO 4)). The solvent is evaporated to give 1.1 g of a solid foam residue consisting of cis-4- (4-fluorophenoxy) ) -1- (D-3-mercapto - 2-methyl-1-oxopropy1) -L-proline.

Analysis: Calculated for C- HH ^ gFNO. 1/3 E + O: C 54.04, H 5.70, N 4.20, F 5.70, S 9.60.

Found: C, 54.04; H, 5.71; N, 4.05; F, 5.40; S, 9.61.

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Thin layer chromatography shows the product at 0.30 'as detected by UV light, SH reagent (yellow spot) and vanillin (yellow spot), [α] D -80 ° (c, 1% in chloroform), Example 12 (cis ) -4- (4-Fluorophenylthio) -1- (D-3-mercapto-2-methyl-1-oxopropyl) -L-proline

Using the procedure of Example 11 by using an equivalent amount of 4-fluorophenyl mercaptan instead of the 4-fluorophenol in part (a), (cis) -4- (4-fluorophenylthio) -1- (D-3) is obtained. -mercapto-2-methyl-1-oxo-propyl) -L-proline, m.p. 242-243 ° (dec.) As adamam-tanamine salt after recrystallization from (1: 1) chloroform / methanol mixture diluted with ether. [α] -51.8 (c = 0.5% i

per D

(Methanol). Free acid [α] β -42.5 (c = 1% absolute ethanol).

Example 13 1- (D-3-Mercapto-2-methyl-1-oxopropyl) -cis-4-phenylthio-L-proline a) N-Carbobenzyloxy-cis-4-phenylthio-L-proline methyl ester 0, Dissolve 85 g of sodium metal (© - 037 mol) in 40 ml of absolute ethanol. To this is added 3.7 ml (0.036 mol) of thiophenol, followed by 7.5 g (0.017 mol) of N - carbobenzyloxy-trans-4-tosyloxy-L-proline methyl ester (J. Am. Chem. Soc. 79, 191 (1957)). The latter compound gradually dissolves, but soon after a solid product is separated. After stirring for 4 hours and standing overnight at room temperature, most of the ethanol in the rotary evaporator is removed. The essentially solid residue © m-30 is stirred with 120 ml of Diehlormethane © g of 60 ml of water. The layers are separated (some methanol is added to promote the decomposition of emulsions) and the aqueous phase is extracted with additional dichloromethane (2 x 60 ml). The combined organic phases are washed with 100 ml of saturated sodium chloride solution, dried (MgSO 4) and the solvent evaporated to give

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6.5 g (100%) of N-carbobenzyloxy-cis-4-phenylthio-L-proline - methyl ester as a pale yellow viscous oil.

b) N-Carbobenzyloxy-cis-4-phenylthio-L-proline

The methyl ester product of part (a) (6.5 g, 0.017 mol) is dissolved in 55 ml of methanol, treated portionwise at -1 to 4 ° C with 13 ml (0.026 mol) of 2N sodium hydroxide, stirred at 0 ° C for one hour and kept at room temperature for approx. 16 hours. After of approx. half of the solvent is removed on a rotary evaporator, dilute the cooled solution 10 with 100 ml of water, wash with 60 ml of ether (discard the wash liquid), pour a layer of 70 ml of ethyl acetate, stir, cool and acidify with 4.8 ml 1: 1 hydrochloric acid. After separation, the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml) and the combined organic layers are dried (MgSO 4) and evaporated to give 5.9 g of a light yellow viscous oil.

This is dissolved in 30 ml of ethanol, treated with 1.9 g of cyclohexylamine in 3 ml of ethanol and diluted to 330 ml with ether. After grafting, the crystalline cyclohexylamine salt is separated.

This weighs after cooling for approx. 16 hours 5.3 g, m.p. 148-151 ° C (p. 135 ° C). This material is combined with 1.5 g of identical product from a previous experiment, stirred with 200 ml of boiling acetonitrile and cooled to give 6.3 g of colorless cyclohexylamine salt, mp 152-155 ° C (p. 137 ° C); [.alpha.] @ 6 -24 ° (c, 1% in ethanol).

This cyclohexylamine salt is suspended in 25 ml of ethyl acetate, stirred and treated with 25 ml of N hydrochloric acid.

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

c) (cis) -4-Phenylthio-L-proline hydrochloride 4.9 g of N-carbobenzyloxy-cis-4-phenylthio-L-proline (0.014 mol) are treated with 25 ml of hydrogen bromide in acetic acid.

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re (30-32%), loosely plugged and magnetically stirred. After 1 hour, the orange-yellow solution is diluted to 250 ml with ether to precipitate the product as a heavy oil which gradually crystallizes after grafting, rubbing and cooling. After stirring in an ice bath for one hour, the material is filtered under nitrogen, washed with ether, suspended in fresh ether, cooled for approx. 16 hours and filtered again to give 3.2 g (77%) of colorless solid (cis) -4-phenylthio-L-proline hydrobromide, mp 106-109 ° C (p. 99 ° C), [α] D 20 ° (c, 1% in methanol).

d) 1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -cis-4-phenyl · -thio-L-proline

Reaction of 3.0 g (0.0094 mol) (cis) -4-phenylthio - L-proline hydrobromide and 2.0 g (0.011 mol) of D-3-acetylthio-2-15-methylpropionyl chloride in 25 ml of water as described in Example 1 (d) (using about 15 ml of 20% sodium carbonate solution to maintain the pH of 8.0-8.4) gives 3.8 g of a pale yellow viscous oil. The dicyclohexylamine salt (prepared in 30 ml of ethyl acetate using 1.8 g of dicyclohexylamine) 20o weighs 2.9 g (isolated in two yields), mp 184-188 ° C (p. 180 ° C). After trituration with 15 ml of acetonitil, 2.4 g of colorless solid dicyclohexylamine salt, m.p. 184-186 ° C (p. 180 ° C), [a] p6 -75 ° (c, 1% in ethanol) is obtained.

This cieyclohexylamine salt is treated with 30 ml of 10% potassium bisulfate and extracted into ethyl acetate as described in Example 1 to give 2.0 g (59%) of glass-like 1-ID-3- (acetylthio) -2-methyl-1-oxopropyl ] -cis-4-phenylthio-L-pro-ling.

e) 1- (D-3-Mercapto-2-methyl-1-oxopropyl) -cis-4-phenyl: hiD-30-L-proline 1- [D-3- (Acetylthio) -2-methyl-1- The oxopropyl] -CiLs-4-phenylthio-L-proline (2.0 g, 0.0042 mol) is treated with 3.5 ml of concentrated ammonia in 8.5 ml of water using the procedure described in Example 2 (the solid ammonium salt (35 of the product is separated from the reaction mixture), giving 26

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over 1.35 g (100%) of viscous syrupy 1- (D-3-mercapto-2-methyl-1-oxopropyl) -cis-4-phenylthio-L-proline, [α] 6 -43 ° (c, 1% in ethanol) Example 14 1- (D-3-Mercapto-2-methyl-1-oxopropyl) -cis-4- (4-chlorophenylthio) -L-proline

Using the procedure of Example 13, but using the equivalent of 4-chlorophenylmercaptan instead of the thiophenol in part (a), 1- (D-3-mercapto-2-methyl-1-oxopropyl) is obtained. ) -cis-4- (4-chlorophenylthio) - L-proline, m.p. 241-242 ° C (dec.) As adamantanamine salt after recrystallization from (1: 1) chloroform / methanol-o mixture diluted with ether. [α] η -55.4 (c = 0.5% in methanol). 25 - 15 Free Acid [a] D - 38.1 (c = 1% in absolute ethanol).

Example 15 (trans) -1- (3-Mercapto-1-oxopropyl) -3-methylthio-D, L-proline a) 1,2-Dehydroproline t-butyl ester To a stirred solution of 34.2 g (0.20 mole) of proline t-butyl ester in 600 ml of ether at -5 to 0 ° C is added dropwise over 10 minutes to 21.7 g (23.9 ml, 0.20 mole) of freshly prepared t butyl hypochlorite (Org. Syn., Coll. bd. V, 184 (1973)]. During the addition, the temperature is maintained at -5 to 25 ° C. After completion of the addition, the solution is stirred at this temperature for an additional five minutes.

To the vigorously stirred solution, a solution of 7.8 g (0.20 mol) of potassium in freshly distilled dry (Cal After addition 30, the temperature of the reaction mixture is about 18 ° C. The reaction vessel is removed from the cooling bath and stirred for 30 minutes. The reaction mixture is filtered through "Celite" (diatomaceous earth) and the filtrate is evaporated in vacuo. The residue is taken up in ether and washed with several portions of water. The ether solution is dried and evaporated in vacuo to 31.6 g of yellow liquid.

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Traces of hydroquinone are added and the crude product distilled to give 22.4 g of 1,2-dehydroproline t-butyl ester (66%), boiling point 60-62 ° C / 0.1 mm.

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

A solution of 16.9 g (0.1 mole) of 1,2-dehydroproline-t-butyl ester in 70 ml of dichloromethane is cooled to -10 ° C under argon. A solution of freshly distilled benzyl chloroformate (14.2 ml, 0.1 mol, boiling point 62-64 ° C / 0.4 mm) in 70 ml of dicloro-10-methane is added dropwise over 30 minutes. After stirring in the cold for an additional 30 minutes, a solution of 15.22 g (0.1 mole) of 1,5-d ± azabicyclo [5.4.0] undec-5ene in 70 ml of dichloromethane is added over 20 minutes. . Then, the cooling bath is removed and the mixture is stirred at room temperature for 15 hours. After twice washing with cold dilute hydrochloric acid and once with saturated sodium carbonate solution, the solution is dried in vacuo to give 18.2 g (60%) of 1-benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid. butyl ester as a pale yellow oil.

C) (trans) -1-Benzyloxycarbonyl-3-methylthio-D, L-proline t-butyl ester

A solution of 18.2 g (0.06 mol) of 1-benzyloxycarbonyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid t-butyl ester in 180 ml of dry methanol is treated with 3.24 g (0, (6 mol) sodium methoxide and cool in an ice bath. Methanthiol is slowly bubbled into the solution for 30 minutes. The mixture is stirred overnight under argon at room temperature. Dilute aqueous acetic acid is added until the solution is slightly acidic. Argon is bubbled through the solution for 1 hour before evaporating to near dryness in vacuo. Ethyl acetate is added and the solution is washed twice with saturated sodium carbonate solution, dried and free of solvent in vacuo to give 17 g of yellow oil. This oil is chromatographed using 300 g of silica gel and petroleum ether: ether 35 (4: 1) to give 11.1 g of (trans) -1-benzyloxycarbonyJL-1-methylthio-D, L-proline as a colorless oil.

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d) (trans) -3-Methylthio-D, L-proline 8.4 g (0.024 mol) (trans) -1-benzyloxycarbonyl-3-methylthio-D, L-proline are treated with 45 ml of 4N hydrobromic acid in acetic acid. After stirring for one hour at room temperature, the solution is dried in vacuo. A small amount of water is added and this is washed twice with ether. The aqueous solution is applied to a column of 300 ml ion exchange resin and water is passed through until the eluate is no longer strongly acidic. The product is then eluted with pH 6.5 (aqueous pyridine acetate) 10 buffer. Ninhydrin positive fractions are combined and lyophilized to give 3.4 g (88%) of white lilacs.

A small sample of this material is crystallized from methanol to give (trans) -3- (methylthio-D, L-proline, m.p. 196-200 ° C, decomp. (P. 192 ° C).

Analysis: Calculated for CgHH₂ ^N₂: C 44.70, H 6.88, N 8.69, S 19.89.

Found: C 44.53, H 7.10, N 8.61, S 19.95.

e) (trans) -1- [3- (Acetylthio) -1-oxopropyl] -3-methylthio-D, L-20-proline 3.05 g (0.019 mol) (trans) -3-methylthio-D, L-pro -lin dissolve in 19 ml 1N sodium carbonate and dilute with 10 ml water. The solution is cooled in an ice bath and, with rapid stirring, a solution of 3-acetylthiopropionyl chloride in 20 ml of ether is added. The pH is maintained at 8 by the addition of 1N sodium carbonate. After 30 minutes, the pH is kept constant and 45 ml of sodium carbonate solution is added. The layers are separated and the aqueous layer is washed once with ether.

The aqueous layer is then acidified with 10% potassium bisulfate solution and the product is extracted into ethyl acetate, dried and free of solvent in vacuo to give 5.2 g of oil. This material is chromatographed on 150 g of silica gel using ethyl acetate for elution. 3.85 g of a somewhat crude (trans) -1- (3- (acetylthio) -1-oxopropyl] -3-methyl-thio-D, L-proline is obtained.

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A small sample is dissolved in ether and converted to the di-cyclohexylamine salt which is then recrystallized from ethyl acetate to give (trans) -1- [3- (acetylthio) -1-oxo-propyl] -3-methylthio-D, L proline-dicyclohexylamine salt, mp 153-157 ° C.

Analysis: Calculated for C (CgHH₂ NHNH: C 58.44, H 8.53, N 5.83, S 13.57.

Found: C, 58.77; H, 8.57; N, 5.68; S, 13.74.

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

Analysis: Calculated for CgHH₂ ONO: C 43.35, H 6.06, N 5.62, S 25.72.

Found: G 43.35, H 6.27, N 5.54, S 25.91.

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Example 16 (cis) -4- (1-Dimethylethoxy) -1- (D-3-mercaoto-2-methyl-1-oxopropyl) -L-proline (a) N-Carbobenzyloxy-cis-4- ( 1,1-dimethylethoxy) -L-proline-methyl ester

A solution of 6.0 g (0.021 mol) of N-carbobenzyloxy - cis-4-hydroxy-L-proline methyl ester in 60 ml of methylene chloride is placed in a Parr apparatus, magnetically stirred, cooled in a dry ice / isopropanol bath and treated with approx. 40 ml of liquefied 10 isobutylene followed by 0.75 ml of concentrated sulfuric acid. The apparatus is closed and the reaction mixture is allowed to warm to room temperature with continued stirring. There is no measurable increase in pressure. After stirring at room temperature for 4 days, the apparatus is opened and the solution (80 ml) is diluted with 120 ml of methylene chloride and washed with 5 g of sodium bicarbonate in 100 ml of water.

The aqueous phase is back extracted with 50 ml of methylene chloride, the organic layers are combined and dried (MgSO 4) and the solvent is evaporated to give 7.5 g of N-carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline methyl ester as a pale yellow oil.

[Α] D = 46 ° (c, 1% in chloroform).

TLC (on silica gel): 0.62 (ethyl acetate), 0.43 (7: 1 benzene / acetic acid)

Visible UV, n 2 vapor.

b) N-Carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline A stirred solution of the methyl ester product of section (a) (7.2 g, 0.02 mol) in 15 ml of ether is cooled in ice water and treated portionwise with 42 ml (0.042 mol) of ice-cold N sodium hydroxide. After stirring and cooling for a total of 6 hours, the cooling bath is removed and stirring is continued overnight at room temperature.

30 Wash the clear solution with. 40 ml of ether (wash is discarded), a layer of 40 ml of ethyl acetate is stirred, cooled and acidified with 8 ml of 6N hydrochloric acid. After separation, the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml), the organic layers are combined and dried (MgSO 4) and the solvent is evaporated to give 6.8 g of pale yellow viscous oil. This oil is dissolved in 50 ml of acetonitrile and treated with a warm solution of 3.2 g of 1-adamantanamine in 20 ml of acetonitrile. The solid adamantane

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amine salt is rapidly excreted. After cooling overnight, the colorless salt is filtered under nitrogen, washed with cold ethyl acetate and ether and dried in vacuo to give 8.8 g of N-carbo-benzyloxy-cis-4- (1,1-dimethylethoxy) -L -proline adamantanamine salt, m.p. 228-230 ° C (decomposition), starts at 215 ° C.

[α] p 5 = -28 ° (c, 1% in methanol).

Analysis: Calculated for H 2 O: C 67.96, H 8.56, N 5.87.

Found: C, 67.77; H, 8.54; N, 5.93.

Treatment of the above adamantanamine salt (8.5 g) with acid gives 5.5 g of N-carbobenzyloxy-cis-4- (1,1-dimethylethoxy) -L-proline as an almost colorless viscous syrup.

TLC: 0.20 (85:15 toluene / acetic acid on silica gel, evoked I2 vapor or PMA plus heat).

(C) cis-4- (1,1-Dimethoxyethoxy) -L-proline The N-Carbobenzyloxy product of section (b) (5.5 g, 0.017 mol) is hydrogenated in 165 ml of 2: 1 methanol / water under 3 atm. hydrogen in the presence of 1.7 g of 51 palladium carbon catalyst to give 3.2 g of a sticky partially solid product. This material is suspended in 30 ml of acetonitrile, rubbed and cooled overnight to give 1.35 g of cis-4- (1,1-dimethylethoxy) -L-proline as a colorless solid, m.p. 240-242 ° C (decomposition), prior gradual darkening and sintering.

[Α] D = -36 (c, 0.5% in methanol).

Analysis: Calculated for CgH ^NO3.0.25 H₂O: C 56.37, Έ 9.20, N 7.31.

Found: C 56.26, H 9.27, N 7.26.

(d) 1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -cis-4- (1,1,1-dimethylethoxy) -L-proline cis-4- (1,1 The dimethylethoxy) -L-proline from section (c) (1.3 g, 0.0069 mol) is reacted with 1.4 g (0.0078 mol) of D-5-acetylthio-2-methylpropionyl chloride in 20 ml of water in the presence of sodium bicarbonate as described in Example 3 to give 2.2 g of an almost colorless viscous oil. The resulting crude product is dissolved in 50 ml of ethyl acetate and treated with 1.3 g of dicyclohexylamine in 20 ml of ethyl acetate. The product crystallizes from the solution and is filtered and dried to give 2.65 g of dicyclohexvlamine salt,

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mp 181-183 ° C, starting at 170 ° C.

[α] D = -62 ° (c, 1% in ethanol).

Pouring out with 15 ml of hot acetonitrile followed by cooling gives 2.4 g of colorless solid dicyclohexylamine salt, melting point 183-185 ° C, starts at 175 °.

[α] D = -65 ° (c, 1% in ethanol).

Analysis: Calculated for C 15 H 25 NO 5 S * C 63.24, H 9.44, N 5.46, S 6.25 Found: C 63.22, H 9.61, N 5.41, S 6.02.

Using the procedure of Example 3, the 2.4 g of dicyclohexylamine salt is converted to the acid by suspending in ethyl acetate, cooling in an ice bath and treating with 30 ml of 10% potassium bisulfate. The layers are separated and the aqueous phase is extracted with 25 ml of ethyl acetate (4 times). After removal of the solvent, the material, which becomes partially crystalline upon standing, is rubbed under 10 ml of ether to complete the crystallization, diluted with 20 ml of hexane, rubbed and cooled to give 1.3 g of 1- [D-3- ( acetylthio) -2-methyl-1-oxopropyl] -cis-4- (1,1-dimethyl-ethoxy) -L-proline as a colorless solid, m.p. 96-98 ° C, starting at 93 ° C.

[α] D = -109 ° (c, 1% in ethanol).

Analysis: Calculated for C- IH NO ^N₂S: C 54.36, H 7.60, N 4.23, S 9.68.

Found: C, 53.92; H, 7.73; N, 4.30; S, 9.29.

(e) cis-4- (1,1-Dimethylethoxy) -1- (D-3-mercapto-2-methyl-1-oxo-propyl) -L-proline 1- [D-3- (acetylthio) - 2-Methyl-1-oxopropyl] -cis-4- (1,1- -dimethylethoxy) -L-proline from section (d) (1.3 g, 0.0039 mol) is hydrolyzed with 2.6 ml of concentrated ammonia hydroxide in 6 ml of water to give 1.1 g of a glass-like residue. Upon standing, seed crystals appear in the product. This material is covered with 8 ml of ether, seeded and rubbed. As the glass dissolves, a crystalline solid is gradually separated. After 2 hours at room temperature, 20 ml of hexane is added and the mixture is cooled overnight under argon. The solvent is removed to give 1.0 g of colorless solid cis-4- (1,1-dimethylethoxy) -1- (D-3-mercapto-2-methyl-1-oxo-propyl) -L-proline , mp 106-108 ° C (starts at 103 ° C).

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[α] D = -78 ° (c, 1% ± methanol).

Analysis: Calculated for C 13 H 23 N 4 O · 25 H 2 O: C 53.12, H 8.06, N 4.77, S 10.91.

Found: C 53.30, H 8.28, N 4.76, S 10.70.

5

Example 17 (cis) -1- [D-3- (Benzoylthio) -2-methyl-1-oxopropyl3-4- (phenylthio) -L-proline 9.9 g (0.031 mol) of cis-4-phenylthio-L -proline snowflake in 100 ml of water (pH 5.6), and the pH is adjusted to 10.2 by adding approx. 20 ml of 10% sodium bicarbonate to give a clear solution. The pH is then adjusted to 9.5 by the addition of ca.

4.5 ml of concentrated HCl. The solution is kept at 30 ° C while adding 8.1 g (0.033 mol) of (D) -3- (benzoylthio) -2-methylpropanoic acid chloride in 30 ml of toluene simultaneously with 100 ml of 10% sodium bicarbonate keeping the pH at 9.3. After approx. a quarter of the hydrochloric acid is added, a slimy precipitate begins to form, which stays throughout the reaction. After stirring the reaction mixture at pH 9.3 for 2.5 hours, this is strongly acidified by the addition of 20% HCl in the presence of ethyl acetate. The aqueous layer is extracted twice with 350 ml portions of ethyl acetate and the combined organic layers are washed with 300 ml of saturated brine and dried (MgSO 4). The solvent is removed to give 11.8 g of foamy solid crude product.

To a solution of these 11.8 g (0.027 mol.) Foamy solid in 70 ml of acetonitrile is added approx. 6 g of dicyclohexylamine in 25 ml of ether. A white crystalline precipitate is formed immediately. After standing overnight in cold rooms, the solid is filtered off and washed with ether to give (cis) -1- [D-3-30 - (benzoylthio) -2-methyl-1-oxopropyl] -4- (phenylthio) -L- nroline di-cyclohexylamine salt (1: 1).

Analysis: Calculated for C 22 H 23 NO 4 S 2 C 12 H 23 N: C 66.85, H 7.59, N 4.59.

Found: C, 66.33, Έ. 7.30, N 4.48.

The slightly moist dicyelohexylamine salt is stirred for 2.5 hours in a mixture of 300 ml of ethyl acetate and 200 ml of 10%! s of potassium bisulfate. Two clear layers are formed. The aqueous layer is extracted

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with two 200 ml portions of ethyl acetate and the combined organic layers are dried (MgSO4). The solvent is removed, yielding 10.1 g of foamy solid (cis) -1- [D-3- (benzoyl-thio) -2-methyl-1-oxopropyl] -4- (phenylthio) -L-proline, m.p. 42-44 ° C.

[α] D = -36.5 ° (c = 1, methanol).

Analysis: Calculated for ^22Η23Ν04δ2: C 60.87, H 5.46, N 3.23.

Found: C, 60.75; H, 5.35; N, 3.17.

Example 18 (cis) -1- (D-3-Mercapto-2-methyl-1-oxopropyl) -3-phenoxy-D, L-proline (a) 3-Phenoxy-1,2-dehydroproline-1 butyl ester

A mixture of 9.25 g (50 mmol) of 1,2-dehydroproplin-1-butyl ester in 70 ml of toluene is heated at reflux with 8.9 g (50 mmol) of 15 n-bromosuccinimide while being exposed to a solar lamp ( about 5 cm distance) for one hour. During this time succinimide is produced and the reaction is cooled to room temperature. The succinimide is removed by filtration and the filtrate is evaporated to dryness. The residue is distilled at 80 ° C to yield 20 3.5 g of crude 3-bromo-1,2-dehydroproline-1-butyl ester.

A mixture of this crude 3-bromo-1,2-dehydroproline-1-butyl ester and 3.9 g (13 mmol) of thallium phenoxide in 30 ml of dry dimethylformamide is stirred at room temperature under argon for 24 hours. The solvent is removed in vacuo and the residue is diluted with ether to precipitate thallium bromide which is removed by filtration. The filtrate is evaporated to give an oil which is purified by distillation. After two hours at 0.005 mm and 80 ° C, 1.48 g of 3-phenoxy-1,2-dehydroproline-1-butyl ester is left in the distillation flask.

(B) (cis) -3-Phenoxy-D, L-proline.

A mixture of the 1.48 g of 3-phenoxy-1,2-dehydroproline-1-butyl ester, 5 ml of 1 N sodium hydroxide and 20 ml of 80% dioxane / water is stirred at room temperature for 4 hours. The solvents are removed in vacuo and the residue is dissolved in 25 ml of water and treated with 168 168 mg of sodium borohydride for a period of one hour at room temperature. The mixture is turned to pH 6 by the addition of 2N HCl and the solution is kept at 0 ° C for 16 hours, after which time the precipitate is precipitated.

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stalls (about 25 mg) of (trans) -3-phenoxy-D, L-proline. These are removed by filtration and the filtrate is desalted by passing through a Dowex1® 50 W x 8 column (2.5 x 30 cm) using 1N ammonium hydroxide as the eluant. The UV-active fractions are combined and evaporated, gives 616 mg of solid (cis) -3-phenoxy - D, L-proline.

(c) (cis) -1- [D-3- (Acetylthio) -2-methyl-1-oxopropyl] -3-phenoxy - D, L-proline

The 616 mg (cis) -3-phenoxy-D, L-proline is slurried in 10 ml of water at 5 ° C and the pH is adjusted to 8.0 with solid sodium carbonate. A solution of 550 mg (2.5 mmol) of D-3-acetylthio-2-methyl-propanoyl acid chloride in 1 ml of ether is added and the pH of the reaction mixture is maintained between 7.3 and 8.2 for the next 1.5 hours at addition of sodium carbonate. The mixture is washed with ethyl acetate 15 (2 x 20 ml), acidified to pH 2 by the addition of 10% HCl and extracted with ethyl acetate (3 x 50 ml). The extracts are combined, dried (MgSO 4) and evaporated to yield 810 mg of an oil. This oil is purified by flash chromatography on silica gel (LP-1, 300 ml) using 10-20% acetic acid / toluene mixtures as eluents and the yield is 530 mg (cis) -1- [D-3- (acetylthio ) - 2-methy1-1-oxopropy1] -3-phenoxy-D, L-proline.

(d) (cis) -1- (D-3-Mercapto-2-methyl-1-oxopropyl) -3-phenoxy-D, L-proline

The 530 mg (cis) -1- [D-3- (acetylthio) -2-methyl-1-oxopropyl] -3-phenoxy-D, L-proline is dissolved in 50:50 concentrated ammonia / water (degassed) by bubbling argon through the solution for 15 minutes) under argon and stirring at room temperature for 1.5 hours. The slightly cloudy solution is adjusted to pH 6.5 with concentrated HCl (no heat is present) and extracted with methylene chloride 30 (2 x 25 ml). The pH is then adjusted to 1 and the solution is extracted again with methylene chloride (3x50 ml). The extracts are combined, dried (MgSO4) and evaporated to yield 410 mg of a glassy compound. Chlorine depletion results in crystallization. The solid is recrystallized from ethyl acetate / hexane to yield 261 mg ('c.is)) - 1- (D-3-mercapto-2-methyl-1-oxopropylph-3-phenoxy-D, L-proline , mp 134-135 ° C.

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Analysis: Calculated for C ^ HH ^NO ^S: C 58.23, H 6.19, N 4.53, S 10.36.

Found: C, 58.07; H, 6.18; N, 4.45; S, 10.16.

5 Biological testing

One of the systems that regulate (increase) blood pressure in humans is the system renin / angiotensin / aldosteroon. Renin is an enzyme produced in the myoepithelial cells of the kidney juxtaglomerular apparatus and released therefrom. The mechanism that controls the secretion of renin appears to be primarily regulated by the salt and water content of the blood and by the pressure at which the blood is pumped through the kidney. When these factors are lowered, renin is sent directly into the bloodstream.

There it encounters as a substrate a -globulin, angiotensinogen 15 (an inactive protein). Renin cleaves angiotensinogen into two equally inactive components, a tetrapeptide and a decapeptide called angiotensin I (abbreviated to A-I). As A-I circulates through the capillaries (primarily the capillaries of the lung), it encounters the angiotensin-converting enzyme (abbreviated to ACE) 20 located in the endothelial cells of the capillaries. ACE cleaves A-I into a dipeptide and an octapeptide, angiotensin II (abbreviated to A-II), which is the strongest known blood pressure enhancer in the human body. A-II1's blood pressure boosting action takes place both locally in the musculature and centrally.

25 After stimulation by A-II, the postrema region of the brain sends impulses to the rest of the body that induce blood pressure increase. In addition, A-II produces another effect, namely the physiological stimulation of the secretion from the adrenal cortex of aldosterone, a mineral corticoid. Aldosterone 30 increases the resorption of sodium ions from the renal tubes, thereby increasing the effective blood volume, thus raising the blood pressure further by this secondary mechanism.

In addition to the conversion of A-I to A-II, ACE has yet another effect. Blood contains an octapeptide, bra-dykinin ', which has vasodi later end properties. ACE transformer

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bradykinin for inactive components. For this function, ACE is sometimes referred to as kininase II.

The renin-angiotensin-aldosterone system is disordered in hypertensive individuals and thus contributes significantly to the patient's high blood pressure. Thus, a drug thus involving ACE is capable of producing the following effects: 1. Inhibition of the formation of A-II and thus: a) direct inhibition of vasoconstriction and b) inhibition of the release of aldosterone era adrenal cortex ( i.e., inhibition of excess water and salt resorption), and 2. as a result of the ACE inhibition, an inhibition of bradykinin cleavage is obtained so that its vasodilate-reducing (blood pressure lowering) effect is maintained.

None of the usual antihypertensive agents act as an ACE inhibitor.

The compounds of the above general formula I, on the other hand, are potent ACE inhibitors. They inhibit the conversion 20 of the decapeptide angiotensin I to angiotensin II and thus minimize or eliminate hypertension induced by angiotensin II. The compounds produced by the process of the invention interfere with the conversion process of angiotensinogen (renin) - angiotensin I- ^ angiotensin II by blocking ACE and thus minimizing or eliminating the formation of angiotensin II, which compound is responsible for the increase in blood pressure. . The effective site of action or site of action of the compounds of general formula I is completely different from that known for any of the commonly used antihypertensive drugs. The dressings made by the method of the invention make it possible, unlike the usual antihypertensive agents, to interfere with the causes of hypertension.

35 Thus, the pharmacological utility of the

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the binders of formula I based on their ability to inhibit the action of the angiotensin converting enzyme. This inhibition is determined on an in vitro basis by measuring the effect on the angiotensin converting enzyme extracted from rabbit lung and by determining the inhibitory activity on angiotensin I-induced contractions of guinea pig intestine. These tests are standardized and used as such in this field of medicine and are further described below.

10

Inhibition of angiotensin-converting enzyme isolated from rabbit lung

The activity of the rabbit lung angiotensin converting enzyme in cell-free extract is measured by spectrophotometric determination of hippuric acid released by the action of the enzyme on hippuryl-L-histidyl-L-leucine (HHL) as described by Cushman and Cheung in Biochem. Pharmacol., 20, 1637 (1971), cf. the reaction scheme below.

-Gly-His-Leu - "Gly + His-Leu HHL Hippuric Acid 25 Measurement of the inhibition of the angiotensin converting enzyme using this spectrophotometric biological assay requires incubation of mixtures of the following * components at 37 ° C in a volume of 0.25 ml : 100 mM phosphate buffer (pH 8.3) 300 mM sodium chloride 0.001-1000 µg / ml inhibitor or optionally no inhibitor 5-10 milligrams of enzyme.

Following the incubation of the above mixtures at various inhibitor concentrations or in the absence of inhibitory

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For 30 minutes, the enzyme reaction is stopped by the addition of 0.25 ml of 1 H hydrochloric acid. It is also necessary to run control experiments where the enzyme is inactivated at time 0 upon addition of the acid before the enzyme. 1.5 ml of ethyl acetate are added to each test tube to extract the hippuric acid formed by the enzyme. After 20 seconds vigorous mixing ((in vortex flow) and separation of the ethyl acetate layer by centrifugation, remove from each tube 1.0 ml of the solvent and evaporate to dryness at 100 ° C and dissolve the hippuric acid in 1.0 ml of water and its amount is judged by the basor absorption capacity of the solution at the wavelength of 228 nm.

The force as inhibitors of the angiotensin converting enzyme is expressed as the I50 value, which is the amount of inhibitor in pg / ml or micromolar required to inhibit the activity of the angiotensin converting enzyme by 50% as measured by the spectrophotometric biological assay method. This value is obtained by graphically plotting the percentage inhibition produced by inhibitor concentrations varying from twice to three times the preceding over a range of from 0.001 µg / ml to 1000 µg / ml. Such a percentage inhibition is calculated according to the following terms: percentage inhibition = - ^ A -------- ^ (AA °) where A denotes the absorbency of hippuric acid in a 30 minute biological test without inhibitor, A ° denotes the Jbag basic absorbency in a biological test incubated for 0 minutes, B represents the absorbency of hippuric acid at a 30 minute biological test at the inhibitor concentration tested, and B ° represents the background absorption by biological test containing the inhibitor, but only incubated for 0 minutes.

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Procedure for testing the inhibitory activity of the angiotensin I converting enzyme (ACE) on guinea pig intestine in vitro

This procedure is used to determine whether a compound is an inhibitor of angiotensin I converting enzyme based on its activity set aside as a function of measurement results for angiotensin I-induced contractions of guinea pig colon. ACE inhibitors typically inhibit angiotensin I-induced contractions.

10 Guinea pigs of both species of Hartley that have not fasted and weigh from approx. 300 to approx. 400 g, killed in a blow to the head. The colon is removed and 2-3 cm sections are prepared according to Vane's method, cf. The following literature site 1. Each colon strip is then suspended in a 10 ml 15 tissue bath filled with Krebs's solution, cf. The following list of references is No. 2, and is maintained at 37 ° C and vented at 95% O2 and 5% CO 2. 20 bound with a Beckman type R dynograph.

After the tissues have reached equilibrium position after approx. 90 minutes, a 2 minute control response is achieved at 10 minute intervals using the following agonist.

Angiotensin I (A-I) 0.025 micrograms / ml FBC (final leaf concentration)

The contractions produced at this concentration of A-I represent from 50 to 75% of the maximum.

The test compound is usually dissolved in aqueous or aqueous saline solution and ten-fold dilutions are prepared in H2 O. The agonist 'is pretreated for 2 minutes with the test compound and the percentage change in contraction response calculated. Specifically active compounds, i.e.

35 compounds, which are likely to be ACE inhibitors, include

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typically inhibits angiotensin I-induced contractions.

A-B

Percent inhibition = -x 100 5 where A denotes the contraction imms) caused by A-I before addition of test compound, and B denotes the contraction (mms) caused by A-I after addition of the test compound.

EC50 (the concentration of test compound which produces 50% inhibition of the contractile action of A-I *) is calculated graphically or biometrically. Typically, a few concentrations of the sample compound are used to construct the curve of inhibitory response as a function of concentration.

15

Using the above technique, the following 1 µ and EC 2 q data for "Captopril" and a compound prepared by the method of the invention have been obtained; 20 Activity 'Activity

Mare Vineyards

Rabbit lung ACE ileum Ref_Structure_150 mM_ECgp xnM

Known compare. , 3 Γ I on On "Captopril" HSCH ^ CHCO-N_l-CCØH 20 20 25 S-CfiH, -

Mfr. JL

according to In Example 13 HSCH2CHCO-N_l-COOH 0 30 35

Claims (2)

HN_-COOR wherein R, R 2 and X are as defined above are coupled with an acid of formula R 2 Wherein R is as above and R 'is C 1-4 alkanoyl or benzoyl, or a functional derivative thereof, to give 4 a product wherein R is C 4 alkanoyl or benzoyl, and if desired, the product is hydrolyzed to form a product, 4 12 wherein R is hydrogen and R, R . 35