IE892932L - Novel organic nitrates and processes for their preparation - Google Patents

Abstract

Organic nitrates with a specific structure, consisting of nitrato fatty acids (nitratoalkanoic acids) and sulphur-containing amino acids or peptides, of the general formula <IMAGE> in which the symbols have the meanings specified in the claims. Because of the general structural principle - presence of sulphhydryl groups - they prevent nitrate tolerance or attenuate a tolerance which has already occurred. The compounds are used in medicaments for treating circulatory disorders, high blood pressure, heart failure and for dilating peripheral vessels.

Description

6591 8 - i - Description This invention is concerned with new organic nitrates and with a method for their preparation.

Organic nitrates (nitric acid esters) have been proven useful in the therapy of cardiac diseases.

They act both by alleviating the before and after effects of a load on the heart, as well as through improvement of the oxygen supply to the heart by dilation of the coronary vessels.

In any case, it has been found in -.recent years that the organic nitrates which have been used so far in therapy, such as glycerol trinitrate (GTN), is osorbid-5-mononitrate or isosor-bid dinitrate, because of nitrate tolerance, exhibit a clear drop in efficacy in a relatively short time when continuous high dosages are administered to the patient. Numerous experiments indicate that the presence of sulfhydryl (~SH) groups prevents the development of nitrate tolerance and that an existing tolerance can be reduced.

The development of tolerance is presently understood to be as follows: According to the present state of knowledge, the pharmacological action of organic nitrate compounds depends on the presence of cysteine. The organic nitrate forms a common precursor with cysteine and, when it decomposes, -NO radicals among others, are liberated and activate soluble guanylate cyclase, the target enzyme, of the smooth muscle cells. Subsequent reactions triggered by the formation of cGMP lead to relaxation or dilation of the vessels.

The reactive and short-lived, and so far, only hypothectical intermediate product would have to be a thioester of nitric acid or a thionitrate. Through intramolecular rearrangement and other subsequent reactions which have not yet been estab- lished, the final formation of a nitroso thiol is postulated, from which nitrogen monoxide or nitrite ions are liberated. On the other hand, the enzyme-dependent degradation with the aid 5 of GSH reductase would not be of significance for the pharmacological action, because it leads exclusively to the formation of nitrite ions. As already stated, the nonenzymatic . degradation needs . cysteine and can thus be exhausted in a dose-dependent manner (exhaustion of the SH group pool), so 10 that over a long term sufficient NO, which is the actual activator of guanyl cyclase, can no longer be formed so that the clinical effectiveness will be reduced.

The compounds provided by the invention for the therapy of 15 " cardiac diseases have a specific structure which includes a group from a nitrato fatty acid (nitratoalkanoic acid) and a group from a sulfur-containing amino acid or peptide.

Therefore, an object of the present invention is to provide 20 new organic nitrates which, based on the general structural principle that they include a sulfhydryl group, are characterized by the fact that they prevent or resist development of nitrate tolerance and/or reverse existing nitrate tolerance when used for the therapy of cardiac diseases.

This object is achieved by the fact that compounds are provided having the general formula: 1 3 4 -,A R O R R O I II I I II °2N_0_C—, CH2 )rC_N—(CH2 ,_C_(CH2 )5-C—R wherein the symbols have the following meaning: R-represents hydroxy, c^- C^-alkoxy, amino; A R represents hydrogen, alkyl with 1 to 6 carbon atoms; R2"represents hydrogen or methyl; 3 R represents hydrogen; lL ' R represents hydrogen, phenyl or methoxyphenyl; \ K5" represents mercaptomethyl, mercaptoethyl, methylthioethyl, particularly S-acetate, S-propionate, S-butyrate, S-caproate, S-caprylate, S-isobutyrate, S-pivalate, S-benzoate, groups in which R and R5" are bonded together in the form of thiolactone, groups in which R and R^ are bonded together in the form 20 of an ester or am amide, groups in which K3 and R1" are bonded together in the form of an alkylene bridge with 2 to 4 carbon atoms, an 25 alkylene bridge with 2 to 3 carbon atoms and a sulfur atom, an alkylene bridge with 3 to 4 carbon atoms, which contains a double bond or an alkylene bridge with 2 to 3 carbon atoms which can.be substituted with a member of the group consisting of hydroxy;' m, n and o are whole numbers from 0 to 10, and pharmaceutical^ acceptable salts thereof.

According to a further feature of the invention, the 5 nitrato alkanoic acid components can have a chain length of CL- , they may be straight-chain or branched chain, and they may be racemic or optical isomers.

Preferably, the amino acids cysteine, methionine or homocysteine are used in making the organic nitrates so that groups from such amino acids are present therein.

Advantageously, the amino acids are of the stereochemical L-form.

Cysteine and/or methionine are desirably present in the form of their methyl, ethyl or propyl esters.

The SH group of cysteine can be esterified with a lower alkanoic acid having 2 to 8 carbon atoms.

According to an especially advantageous further development the invention provides compounds having the following chemical names: 2-nitratoacetyl) -cysteine ethyl ester 2-nitratoacetyl)-S-acetyl-cysteine ethyl ester 2-nitratoacetyl )-S-propionyl-cysteine ethyl ester 2-nitratoacetyl )-S-pivaloyl-cysteine ethyl ester 2-nitratoacetyl)-methionine methyl ester 2-nitratopropionyl)-cysteine 2-nitratopropionyl)-cysteine ethyl ester 2-nitratopropionyl)-methionine ethyl ester 2-nitratobutyryl)-cysteine ' 2-nitratobutyryl)-cysteine ethyl ester 2-nitratobutyryl)-S-acetyl-cysteine ethyl ester 2-nitratobutyryl)-S-butyryl-cysteine ethyl ester 2-nitratobutyryl )-metionine ethyl ester 2-nitratoisobutyryl)-cysteine 2-nitratoisobutyryl)-cysteine ethyl ester 2-nitratoisobutyryl )-S-benzyol-cysteine ethyl ester 2-nitratoisobutyryl)-S-acetyl-cysteine ethyl ester 2-nitratoisobutyryl )-S-pivaloyl-cysteine ethyl ester 2-nitratoisobutyryl)-methionine ethyl ester 3-nitratobutyryl)-cysteine 3-nitratobutyryl)-cysteine ethyl ester 3-nitratobutyryl) -S-acetyl-cysteine-ethyl ester 3-nitratobutyryl)-S-propionyl-cysteine ethyl ester 3-nitratobutyryl)-methionine ethyl ester 3-nitratobutyryl)-homocysteine thiolactone 3-nitratopivaloyl 3-nitratopivaloy1 3-nitratopivaloyl 3 -nitratopivaloyl 3-nitratopiva1oy1 3-nitratopivaloyl 3 -nitratopivaloyl 3-nitratopivaloyl 3-nitratopivaloyl 3-nitratopivaloyl 3-nitratopivaloyl 3-nitratopivaloyl -cysteine -cysteine ethyl ester -cysteine ethyl ester-S-carbonate -S-acetyl-cysteine ethyl ester -S-propionyl-cysteine ethyl ester -S-butyryl-cysteine ethyl ester -S-isobutyryl-cysteine ethyl ester -S-pivaloyl-cysteine ethyl ester -S-benzoyl-cysteine ethyl ester -methionine ethyl ester -methionine -homocysteine thiolactone N- 2-nitratohexanoyl -cysteine ethyl ester N- 2-nitratohexanoy 1 -S-propionyl-cysteine ethyl ester N- 3 -nitra tohexanoyl -cysteine ethyl ester N- 3 -ni tratohexanoyl -methionine methyl ester N- 12-nitratolauroyl -cysteine N- 12 -nitratolauroyl -cysteine ethyl ester N- 12-nitratolauroyl -S-acetyl-cys.teine N- 12-nitratolauroyl -S-pivaloyl-cysteine According "to another aspect of the invention, drugs or medicines containing one or more of the compounds aye pro-15 vided.

These organic nitrates, desirably in the form of a pharmaceutical composition, can be used for the treatment of cardiac disease as manifested by circulatory diseases, for example, as 20 coronary dilators, as agents for the treatment of high blood pressure, cardiac insuffiency and for dilating the peripheral vessels, including the vessels of the brain and kidneys.

Finally, the compounds can be prepared by condensing the cor-25 responding nitrato fatty acids or their reactive derivatives with the amino group of an amino acid or a peptide. If desired, the compounds obtained can be subjected to side-chain alkylation or side-chain acylation in a subsequent reaction step.

Reactive derivatives of the nitrato fatty acids which can be used as reactants according to the invention are, for example, acid halides, acid anhydrides, activated amides and activated esters. Preferabyl, acid chlorides acid azides, symmetrical 35 acid anhydrides, activated esters and mixed anhydrides with organic or inorganic acids can be used.

The condensation reaction of a nitrato fatty acid with an amino group of an amino acid can also be carried out in an inert solvent and in the. presence of a condensing agent which promotes the formation of an acid amide bond, a carbodiimide such as N,N'-dicyclohexyl carbodiimide or a similar carbodiimide, an imine compound such as diphenylketene-N-cyclo-hexylimine or. pentamethylene-ketene-N-cyclohexylimine, or. a phosphate or phospite such as triethyl phosphite, ethyl poly-10 phosphate or isopropyl polyphosphate, over a period of 1-48 hours at temperatures from -10 °C to the refluxing temperature of the solvent used.

The following examples illustrate without limiting the inven-15 tion.

Example 1 Preparation of N-(3-nitratobutvrvl^ -cysteine ethyl ester First Step Saponification of 3-hvdroxvbutvric acid ethvl ester 3-Hydroxybutyric acid ethyl ester (Aldrich), 13.2 g (0.1 25 mole), was reacted with 4.0 g (0.1 mole) of NaOH dissolved in 100 ml of water. The reaction was complete when the solution became homogeneous.

. The reaction mixture was acidified with 10 ml of concentrated 30 HC1 and then extracted twice using 100 ml of ethyl acetate each time. Then the solution was evaporated on a rotary evaporator; a thin flowing oil remained.

The yield was 8.81 g (therory: 10.4 g) of 3-hydroxybutyric 35 acid.

Second Step Nitration of the 3-hydroxybutyric acid 3-Hydroxybutyric acid, 8.81 g (0.08 mole), and 50 mg of urea were dissolved in 50 ml of acetic acid at 5°C. First 6.27 ml (0.15 mole) of HN03 was added dropwise and then 14.17 ml (0.15 mole) of AC2O was added under cooling. The reaction mixture was stirred overnight.

The mixture was worked up by adding. 200 ml of ice water to the solution obtained and then extracted with ethyl acetate. The organic phase was then extracting it with NaHC03. The NaHC03 phase was acidified with concentrated HCl and extracted with ethyl acetate. Finally, the solution was evaporated on a rotary evaporator, whereupon a thin flowing oil remained.

The yield was 9.4 g (therory 11.9 g) of 3-nitratobutyric acid.

Third Step Preparation of N-( 3-nitratobutvrvl^ -cysteine ethvl ester 3-Nitratobutyric acid, 16.6 g (0.11 mole), was dissolved in 100 ml of dichloromethane. While passing N2 through the mixture, 17.9 g (0.12) of cysteine ethyl ester was added slowly at 15°C. Then 24.7 g (0.12 mole) of dicyclohexyl carbodiimide (DCC) dissolved in 80 ml of dichloromethane was added dropwise slowly at 15°C while N2 was passed through. The dicyclohexy-lurea formed was filtered off at the end of the reaction under suction and the solution was washed with 150 ml of 0.1 N HCl. Then the solution was evaporated on a rotary evaporator.

A purified sample of the substance was prepared by column chromatography and recrystallization from ethanol/n-hexane.

Yield: 6.88 g (therory: 30.83 g) M.p. 77.8°C.

Example 2 Preparation of N-f 3-nitratobutvrvH -methionine ethvl ester 3-Nitratobutyric acid, 6.35 g (0.043 mole), 7.47 g (0.043 mole) of methionine ethyl ester and an amount of dimethyl- aminopyridine (DMAP),.that the tip of a spatula holds, were .dissolved in 100 ml of dichloromethane with stirring and 10 cooling to 10°C. Then 10.31 g (0.05 mole) of DdC was dissolved in 80 ml of CH2CI2 ^and added dropwise slowly under simultaneous introduction of nitrogen. After the reaction was completed, the solution was filtered off under suction, washed with NaHCO^ and finally with HCl.

The solution was evaporated in a rotary evaporator, whereupon an oil remained.

Purification of a sample was carried out by column chromato-20 graphy or by crystallization in the cold.

The yield was 1.95 g (theory 12.05 g) of N-(3-nitratobutyryl)-methionine ethyl ester as a colorless oil.

Example 3 Preparation of N-f 3-nitroatopivalovH -cysteine ethvl ester First Step Preparation of nitratopivalic acid methvl ester Hydroxypivalic acid methyl ester, 25.0 g (0.19 mole) and 0.12 g of urea were dissolved at room temperature in 250 ml of CH2CI2 and cooled to 5°C under stirring. Then 23.8 g (0.38 35 mole) of HN03 (100 %) was added dropwise with stirring, so that the temperature did not exceed 10°C. The mixture was cooled to 5°C and 38.6 g (0.38 mole) of acetic anhydride was added dropwise with stirring in such a way that the temperature did not exceed 10°C, the mixture was then stirred into an ice bath with cooling for a 15 minute period and then slowly warmed to room temperature and further stirred overnight at room temperature. The batch was introduced slowly into 500 ml of ice water under stirring. The CH2CI2 phase was separated and was washed once with each of 100 ml of distilled H20,.100 ml of. saturated aqueous NaHC03 solution and again with 100 ml V of distilled H2O. Then the CH2CI2 extract was evaporated to dryness on a Rotavapor at a maximum bath temperature of 40°C in the vacuum of a water jet pumpv The light-yellow oily residue distilled at the vacuum of an oil pump at a bath temperature of 60°C as a clear thin flowing oil.

Yield: 31.5 g, corresponding to 94.0% of theory.

Second Step Preparation of nitratopivalic acid NaOH, 14.0 g (0.350 mole), was dissolved in E^O and cooled to about 10°C. Then a solution of 31.0 g (0.175 mole) of nitratopivalic acid methyl ester in 250 ml of methanol was added with stirring whereupon the reaction mixture developed a yellow color and the temperature increased to about 25°C.

After 90 minutes of stirring, the batch was neutralized with 29.5 ml (0.35 mole) of 37% HCl and the methanol was completely distilled off in a Rotavapor. The aqueous phase was extracted twice using 200 ml of methylene chloride each time. The combined methylene chloride extracts were washed once with 50 ml of distilled H2O and the methylene chloride phase was evaporated to dryness on the Rotavapor. The colorless, oily residue was dissolved in 100 ml of ethyl acetate and again evaporated to dryness on the Rotavapor, whereupon a white solid residue remained from which the solvent residues were removed at the vacuum of an oil pump (0.4 torr) at a bath temperature of about 40°C over a period of 15- minutes in a Rotavapor. The solid, white "residue of 25.44 g (89.1% of theoi^) was dissolved in 100 ml of boiling n-hexane and 2 ml of diisopropyl ether was added. After cooling to room temperature and 5 addition of nucleating crystals, the product crystallized out. The product was allowed to "stand at 0°C for 72 hours, the crystals were filtered off under suction and, after washing twice using 10 ml of n-hexane each time, it was dried in a vacuum drying oven to a constant weight at room temperature at 10 about 2 torr. » M.p. 54.2°C Yield: 23.66 g, corresponding to 82.9% of the theory.

Third Step Preparation of .N-(3-nitratopivaloyl^ -cysteine ethvl ester L-cysteine ethyl ester base, 10.7 g (71.7 mmoles), was dissolved in 200 ml of methylene chloride at room temperature under stirring in an N2 .atmosphere. Then 11.4 g (70.0. mmoles) of crystalline nitratopivalic acid were added "and dissolved under stirring at room temperature. Then a solution of 14.8 g (71.7 25 mmoles) of N,N-dicyclohexylurea (DCC) in 50 ml of methylene chloride was added to this mixture with stirring in a nitrogen atmosphere at room temperature. The addition was carried out dropwise over a period of about 15 minutes whereupon the temperature •increased to 35°C. After further stirring, white di-30 cyclohexylurea precipitated. The batch was cooled to room temperature and stirred overnight under a nitrogen atmosphere. The dicyclohexylurea was then filtered off through a sintered glass filter and was washed once with 50 ml of CH2C12. The combined methylene chloride solutions were washed once with 35 100 ml of 1 N HCl and twice using 100 ml of distilled H20 each time (under an N2 atmosphere) and then evaporated in a Rotava- por at a bath temperature of about 40°C in the vacuum of a water jet pump at an initial pressure of 550 mbar to approximately 20 mbar." A yellow-brown oil was obtained.

Yield: 21.2 g, corresponding to 102.9% of theory.

The substance was purified by recrystallization from 10 ethanol/hexane in the cold. > Yield: 13.42 g of N-(3-nitratopivaloyl)-cysteine ethyl ester as a light-pink oil, corresponding to 65.1 % of theory.

Fourth Step Preparation of N-(3-nitratopivaloyl S-acetvl-cvsteine ethvl ester To 10.3 g (35.0 mmoles) of N-(3-nitratopivaloyl)-cysteine ethyl ester dissolved in 70 ml of dichloromethane was added a solution of 4.3 g (42.0 mmoles) of acetic anhydride in 10 ml of dichloromethane in the cold dropwise with stirring. Then a solution of 5.0 g (49.0 mmoles) of triethylamine in 20 ml of 25 dichloromethane was added dropwise in the cold with stirring. After the end the reaction, the batch was washed with 1 N HCl, 10% aqueous sodium bicarbonate solution and water. The dichloromethane extract was evaporated to dryness on the Rotavapor. 11.6 grams of a light-yellow, oily product thus was 30 obtained from which 7.8 g of crystalline product (66.3% of theory) was obtained by recrystallization from ethanol/water in the cold and with the addition of nucleating crystals.

M.p. < 5°C.

Fourth Step/Variation 1 Preparation of N-f3-nitratopivaloyl^-S-butrvrvl-cysteine ethvl ester If one uses 6.7 g (42.0 mmoles) of butyric acid anhydride instead of acetic anhydride as descrive in Step 4, and if the reaction and the work-up is conducted in the same.way, 13.0 g of a light-yellow, oily product is obtained froih which 9 .7' g of crystalline product (corresponding to 76.2% of theory) was obtained by recrystallization in trie cold, as described in Step 4.

M.p.: < 5°C.

Fourth Step/Variation 2 Preparation of N-(3-nitratopivalovl)-S-pivalovl-cvsteine ethvl ester If one-uses 7.8 g (42.0 mmoles) of pivalic acid anhydride instead of the acetic anhydride descrived in Step 4, and if one conducts the reaction and the work-up in the same way, 14.1 g of a light-yellow, oily product is obtained from which one obtains 10.5 g of crystalline product (corresponding to 79.5% of theory) by recrystallization as described in Step 4.

M.p. 45°C.

Fourth Step/Variation 3 Preparation of N'-( 3-nitratopivalovl^-cvsteine ethvl ester-S-carbonate If one uses 4.3 g (42.0 mmoles) of ethyl chloroformate instead of the acetic anhydride described in Step 4, and if one conducts the reaction and the work-up in the same way, 11.5 g of a light-yellow, oily product is obtained from which one ob tains 9.5 g of crystalline product (corresponding to 74.1% of theory) by recrystallization as described in Step 4.

M.p. 36°C.

Example 4 Preparation of N-f 3-nitratopivaloyl 10 methionine ethyl ester L-methionine ethyl ester base, 12.4 g (70.0 mmoles), was dissolved in 250 ml of methylene chloride at room temperature in 15 an N2 atmosphere. Then 11.4 g (70.0 mmoles) of crystalline ni-tratopivalic acid was added and dissolved with stirring at room temperature. To the mixture was added dropwise a solution of 14.8 g (71.7 mmoles). of N,N-dicyclohexylurea (DCC) in 50 ml of methylene chloride in about 15 minutes at room temperature 20 under stirring and in a nitrogen atmosphere, whereupon the temperature increased to 35°C. After further stirring, white dicyclohexylurea precipitated. The batch was cooled to room temperature and was stirred overnight in a nitrogen atmosphere. The DCC urea was then filtered off through a sin-25 tered glass filter and was washed once with 50 ml of CH2C12. The combined methylene chloride solutions were washed once with 100 ml of 1 N HCl and twice with 100 ml of distilled H20 each time (under am N2 atmosphere) and then evaporated in the Rotavapor at a bath temperature of about 40°C and in the 30 vacuum of a water jet pump at an initial pressure of 550 mbar, which went to about 20 mbar. A light-yellow oil was obtained.

Yield: 24.9 g of crude N-(3-nitratopivaloy])-L-methionine methyl ester, corresponding to 110.3% of theory.

The crude product was purified by sample chromatography on a column.

Yield: 17.6 g of N-( 3-nitratopivaloyl)-methionine ethyl ester as a colorless oil, corresponding to 78.0% of theory.

Exemple 5 N- (12-nitratolaurovl ^ -S-acetvl-cvsteine First Step Preparation of 12-nitratolauric acid 12-Hydroxylauric acid, 54.1 g (0.250 mole), and 0.3 g of urea were dissolved in 1.3 liter of CHCI3 with slight warming and then cooled to 20°C with stirring. Then, with stirring, 23.6 g (0.375 mole) of HNO3 (100%) was added slowly, dropwise, where-20 upon the temperature rose to 27°C. The mixture was cooled to 20°C and 38.3 g. (0.375 mole) of acetic acid anhydride was added dropwise under stirring and cooling, whereby- a temperature limit of 25°C was observed. The mixture was stirred overnight at room temperature. Finally, it was washed five 25 times using 0.5 liter of distilled H2O each time. The CHCI3 phase, which-had been dried over Na2S0^ and clarified with pulverized activated carbon, was evaporated to dryness in a Rotavapor at a bath temperature of 50 °C at the vacuum of a water jet pump. The oily'residue of 60.8 g was dissolved in 500 30 ml of boiling n-hexane and, after cooling to room temperature, it was allowed to stand overnight in a refrigerator at 0°C. The crystalline product was precipitated and was washed twice using 50 ml of n-hexane each time. Finally, a product was evaporated to constant weight in a vacuum drying, oven at room temperature and at about 2 torr.

M.p. 29°C.

Yield: 39.4 g, corresponding to 60.3% of theory.

% Second Step Preparation of 12-nitratolau:ric acid chloride Nitratolauric acid, 2.61 g (10 mmoles), was dissolved in 50 ml of methylene chloride and 4.44 g (35 .mmoles) of oxyalyl chlo-15 ride in 50 ml of methylene chloride was added dropwise at room temperature with stirring. The mixture was stirred overnight. Finally, the product was evaporated to dryness in a rotary evaporator.

Yield: 3 g, corresponding to 93.2% of theory.

Third Step Preparation of N-/12-nitratolaurovl^ -cysteine Under a nitrogen atmosphere, 6.06 g (50 mmoles) of L-cysteine was introduced into 300 ml of DMF with stirring. Then 5.60 g (20 mmoles) of 12-nitratolauric acid chloride in 50 ml of dichloromethane was added dropwise. Since a clear solution was 30 not obtained, the mixture was heated to 60°C. Finally, 100 ml of distilled H2O was added and the mixture was stirred overnight at room temperature. Finally, the mixture was diluted with 300 ml of H2O and was extracted four times using 200 ml of ethyl acetate each time. The organic phase was dried 35 over Na2S0^ and then evaporated. The residue was taken up in 100 ml of ether and was allowed to stand in a refrigerator overnight at 0°C. White crystals were obtained.

M.p. 74-75°C.

Yield: 4.1 g of N-(12-nitratolauroyl)-cysteine.

Fourth Step Preparation of N-f 12-nitratolaurovl ^-S-acetvl-cvsteine Under a nitrogen atmosphere, 1.82 g (5 mmoles) of N-(12-nitra-tolauroyl)-cysteine was placed in 20 ml of ethyl acetate. The mixture was cooled to 0°C and 2.5 ml of acetic anhydride was added dropwise. Then at -5°C/ 1.52 g (15 mmoles) of 10 triethylamine dissolved in 5 ml of ethyl acetate was added dropwise. The reaction solution was washed with water and evaporated to dryness.

M.p.i oil at room temperature.

Yield: 2 g, corresponding to 98.4% of theory.

Example 6 Preparation of N-f 12-nitratolaurovl^ -cysteine ethvl ester Cysteine ethyl ester base, 4 g (26.8 mmoles), was dissolved in 50 ml of methylene chloride and 2.8 g (10 mmoles). of 12-nitra-25 tolauric acid chloride dissolved in 50 ml of methylene chloride was added dropwise with stirring and the mixture then was stirred overnight. The precipitated cysteine ethyl ester HCl salt was filtered off under suction and the solvent was removed in a Rotavapor. The oily residue (6 g) was dissolved in 30 100 ml of ether and was allowed to stand in a refrigerator overnight at 0°C. The precipitated product was filtered off.

M.p. 59-60°C.

Yield: 1.6 g, corresponding to 40.0% of theory.

Example 7 Preparation of N-f 2-nitratopropiorivH -cysteine ethvl ester First Step Preparation of nitratolactic acid ethvl ester Lactic acid ethyl ester, 33 g (0.28 mole), was dissolved.in 300 ml of dichloromethane. After addition of 100 mg of urea, 10 22.5 ml (0.56 mole) of 100% nitric acid was added dropwise at a temperature of 5-10°C. The solution.was cooled to 0°C. Then 52.8 ml (0.56 mole) of acetic anhydride was added dropwise in such a way that the temperature did not rise above 5°C. The solution was allowed to stand overnight at room temperature 15 and then was washed with 250 ml of water. The organic phase was separated and dried over sodium sulfate. After filtration, the dichloromethane was distilled off. The oily residue obtained was processed by distillation.

Yield: 30.34 g, corresponding to 66.4% of theory.

B.p.: 34°C (0.25 torr).

Second Step Preparation of nitratolactic acid Nitratolactic acid ethyl'ester, 30 g (0.18 mole), was dis-30 solved in 80 ml of dioxane. Then 30 ml of water and 2 g (0.02 mole) of sulfuric acid were added to the solution and the mixture was refluxed for 19 hours. The solution was evaporated to a volume of about 50 ml and then diluted with 300 ml of water. The pH value was adjusted to 7-8 by the addition of sodium bi-35 carbonate. The unreacted ester was removed by extraction with dichloromethane.

The aqueous phase was adjusted to pH 1 with concentrated hydrochloric acid and extracted three times using 150 ml of ethyl acetate each time. The extracts were combined and dried 5 over sodium sulfate. After filtration the ethyl acetate was removed completely in a rotary evaporator.

Yield: 14.6 g of colorless oil, corresponding to 59.2%. of theory.

Third Ste'p Preparation of N-f2-nitratopropionvl^-cysteine ethvl ester In a nitrogen atmosphere, 17 g (0.13 mole) of nitratolactic acid and 18.9 g (0.13 mole) of cysteine ethyl ester were dissolved in 200 ml of dichloromethane at 10-15°C. At 15-20°C a solution of 28.6 g (0.14 mole) of N,N-dicyclohexyl carbodiimide 20 and 75 ml of dichloromethane was added dropwise. After 1 hour, the precipitated N,N-dicyclohexylurea was filtered off and was washed with 75 ml of dichloromethane. The filtrate .was extracted twice using 50 ml of 0.1 N hydrochloric acid each time. The organic phase was evaporated completely on a 25 rotary evaporator. The crude crystalline product (22.4 g) was recrystallized from 100 ml of ethanol/n-hexane (1:1).

Yield: 7.6 g, corresponding to 22.6% of theory.

M.p.: 92.8 °C.

PHARMACOLOGICAL ACTIVITY TESTING The pharmacological action of the compounds provided by the 35 invention is illustrated further by the following experiments.

Pharmacological experiment: method 1 Administration of new organic nitrates to detect the nitrate 5 action on the circulatory parameters of a dog that is awake.

The purpose of the test is to determine how the new organic nitrates act on various circulatory parameters of a dog that is awake after intravenous or oral administration. All experi-10 ments were carried out on trained beagle dogs; the circulatory parameters were measured with eui arterial catheter tip manometer and a ballon, catheter introduced, into the V. jugularis. In order to describe the action on the arterial system, the systolic (SAP systolic arterial pressure), mean (MAP mean ar-15 terial pressure) and diastolic (DAP diastolic arterial pressure) blood pressure (BP) and heart rate (HR) were measured. Prom these, the peripheral resistance (TPR total periphere resistance) and the extensibility of the arterial air chamber (COMPL) were calculated. The low pressure system was measured through the central venous 2Q pressure (CVP) and the pulmonary arterial pressure (PAP). The control or reference product was isosorbid-5-mononitrate (ISM-5).

DESCRIPTION OF THE DRAWINGS The drawing figures illustrate graphically the activity spectrum of specific organic nitrates. The abbreviations used in the drawings have the following meanings: CO means cardiac output 30 SV means stroke volume BP means diastolic blood pressure HR means heart rate CVP means central venous pressure PAP means pulmonary arterial pressure 35 COMPL means extensibility of the arterial air chamber TPR means peripheral resistance In "Mean +. sera.N = 5" for example, mean is mean value, sem. is standard error of the mean, and N is' the number of errors of the mean value.

Icon 1 : Figures 1 to 8 show the action of orally and intravenously administered ISM-5. After administration by each route", the ISM-5 caused a slight decrease in systolic blood pressure, the mean pressure was hardly effected, the extensibility of the air chamber increased significantly and the low pressure system pressure decreased. » Icon 2 : Figures 1 to 8 show the corresponding effects of N-(3-nitratopivaloyl)-methionine ethyl ester (Nitrato-Piv-Meth-Et) for the corresponding circulatory areas. Here, again, the comparison between intravenous and oral admjLnistration indicates good bioavailability.

The novel substance N-(3-nitratopivaloyl)-cysteine ethyl ester was also tested and it shows good bioavailability and a course of action typical of a nitrate.

The results for both of the tested novel, compounds show they have an action which is comparable to that of ISM-5 and have good bioavailability.

Pharmacological experiment method 2 Determining the lack of tolerance to the action of new organic nitrates on increasing coronary flow in isolated perfused heart.

The purpose of the present investigation was to study the action and the development of tolerance to new organic nitrate compounds on isolated perfused rat heart. For this purpose, a rat heart was isolated and prepared as a "working heart".

In. this experimental arrangement, the heart performs defined circulatory work from which a defined oxygen consumption and coronary flow result. The action of nitrate-like compounds can be measured in this model by the druginduced coronary flow increase .

The resistance of the coronary vessels on an isolated working rat heart was chosen as the parameter for the detection of the action of the nitrate. A rat heart with a weight of about one gram is perfused through the-ledt auricle with a plasma-like solution which contains nutrients and is saturated with oxygen. The left ventricle pumps the solution into the aorta against a defined pressure. Corresponding to the physiological conditions, a part of this solution flows through the coronary vessels for supplying the heart itself. At a definded work of the heart, this fraction, from wich the coronary resistance can be calculated, is constant. The addition of a nitrate or other coronary dilating drug causes a drop in this coronary restistance. Therefore, if a constant concentration of an organic nitrate is introduced to the heart, then, after an initial decrease of the resistance, a partial ioss of activity occurs within 20 minutes. On this model, the substances also show a coronary dilating effect, but this is not followed by a loss in activity. The maximum drop of the coronary resistance is still completely present after 60 minutes. The tested compounds were compared in an equimolar dosage with 10"^ M nitroglycerin trinitrate. Continuous infusion of nitroglycerin causes a rapid coronary flow increase by 7.6 + 1.88 ml/min^g^^ (x + SD). Within 20 minutes, the flow decreases by 55.9%. Upon further perfusion, the action of nitroglycerin is unchanged. In this experimental model, the new nitrates also show a coronary flow increase, but this is followed by only a very slight drop in the activity. This result indicates that the described new compounds do not show the tolerance behavior as do conventional nitrates.

Claims (1)

1.Claims: 10 15 1. Compounds of the general formula (I) R1 OR3 R4 °2n—0—9—(<»2) in—C—N—( ch2 ) n—9—(ch2 > Q—C—R (I) R2 R5 wherein 20 R represents hydroxy, Cj - C3 -alkoxy, amino; R^represents hydrogen, alkyl with 1 to 6 carbon atoms; B? represents hydrogen or methyl; 25 R3represents hydrogen; R^represents hydrogen, phenyl or methoxyphenyl; 30 R? represents mercaptomethyl, mercaptoethyl, methyl-thioethyl its derivatives of S-acyl, particularly S-acetate, S-propionate, S-butyrate, S-caproate, S-caprylate, S-isobutyrate, S-pivalate, S-benzoate, 35 groups in which R and R5are bonded together in the form of a thiolactone, - 26 - iracterized in :ing of: ester lyl ester '1 ester :h R and K4 are - bonded together in the form of 1 aiaide, "3 i :h R and R*are bonded together in the form of ridge with 2 to 4 carbon atoms, an alkylene .to 3 carbon atoms and a sulfur atom, an e with 3 to 4 carbon atoms, which contains a an alkylene bridge with 2 to 3 carbon atoms substituted with a member . of the group hydroxy; jr are whole numbers from 0 ly acceptable salts thereof. to 10, and . ester rl ester according to claim 1 characterized in that .kanoic acid components have a chain length i are selected from the group consisting of n, branched chain, racemic or optical 3thyl ester :hyl ester ethyl ester :er ccording to claims 1 and 2 characterized in o acid is a member of the group " consisting ithionine or homocysteine. L ester thyl ester tie ethyl ester ccording to claims 1 to 3 characterized in acids are in the stereochemical L-form. zcording to claims 1 to 4 characterized in acids selected from the group consisting of >r methionine are present as an ester the group consisting of methyl, ethyl and er :cording to claims 1 to 4 characterized in is esterified on the SH - group with an aving a chain length of C2" Cq. - 29 - 13. A compound of the general formula given and defined in claim 1 or a pharmaceutically acceptable salt thereof, whenever prepared by a process claimed in a preceding claim. 14. A pharmaceutical composition according to claim 9, substantially as hereinbefore described. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.