NO148673B - ANALOGY PROCEDURE FOR THE PREPARATION OF PHARMACOLOGICALLY EFFECTIVE TIOFENDER DERIVATIVES - Google Patents

Description

The present invention relates to an analogous method for the production of >pharmacologically active compounds with formula

where is lower alkyl or phenyl; R 1 is hydrogen, hydroxy, lower alkoxy or amino; R-, and R^ lower alkyl or hydrogen, and salts thereof.

The compounds of formula I and their salts have value as anti-obesity agents and for lowering blood lipids. They should also have value in the treatment of arteriosclerosis and related cardiovascular diseases, which are related to elevated blood lipid levels.

The term lower alkyl used here, alone or in combination, such as lower alkoxy, denotes straight-chain or branched saturated aliphatic alkyl groups with 1 to 8 C atoms such as methyl, ethyl, propyl and isopropyl.

Preferred compounds of formula I are those mentioned

in claims 2 and 3.

The compound of formula I and its salts are obtained according to the invention by treating a compound of formula II

where R<1>2 is lower alkoxy and R^ has the above-mentioned meaning, with an acid, and if desired in the thus obtained compound of formula Ia

where R' and R^ have the previously stated meanings,

transfers the lower carbolic group into a carboxy, formyl or carbamoyl group and/or if desired lower alkyler-er the amino group, and if desired transfers a compound of formula I in a salt.

The compound of formula Ia can be obtained by treating the oxime of formula II with an acid, preferably a hydrogen halide, especially hydrogen chloride, in an inert organic solvent, e.g. an ether, especially a di-lower alkyl ether such as diethyl ether; a cyclic ether such as tetrahydrofuran or dioxane; a lower alkanol or water. Temperature and pressure during the reaction are not critical. The reaction can conveniently be carried out at temperatures between approx. 0 and 70°C, preferably at room temperature and at atmospheric pressure.

The compound of formula Ia can be converted into the corresponding aldehyde, corresponding acid, amide or other esters of formula I or into their salts by conventional methods. The lower carboxyl carboxyl group can be converted by basic hydrolysis in a conventional inert organic solvent, preferably a lower alkanol, especially methanol or ethanol; an aqueous ether, preferably di-lower alkyl ether, especially diethyl ether} or an aqueous cyclic ether, preferably tetrahydrofuran or dioxane, in a carboxy group. The preferred bases include alkali metal hydroxides such as sodium, potassium and lithium hydroxide and alkaline earth metal hydroxides such as barium, calcium and magnesium hydroxide, especially the alkali metal hydroxides. In this hydrolysis, temperature and pressure are not critical. The reaction can advantageously be carried out at between approx. 0 and 100°C, preferably during reflux of the reaction mixture, in particular at 70°C and under atmospheric pressure. When treating an ester of formula Ia with a reducing agent, e.g. lithium aluminum hydride gives the corresponding primary alcohol which can then be oxidized to the corresponding aldehyde with formula I, e.g. with Mn02- When an ester of formula Ia is treated with ammonia, the corresponding amide of formula I is obtained, in which R2 is amino. If R 3 and/or R 4 means lower alkyl, the residues can be converted in a manner known per se

way for the transfer of aromatic primary amines in N-substituted derivatives. Thus, a primary amine of the formula Ia can be reacted with a lower alkylating agent, e.g. a lower alkyl halide.

The compounds of formula II can be obtained by reacting a compound of formula III

with a compound of formula IV to form a compound of formula V

wherein R-^ and R'2 have the above meanings,

R is lower alkyl and Rg is halogen, mesyloxy or tosyloxy.

This reaction can be carried out in the presence of a lower alkanol and an alkali metal alkoxide, preferably methanol and sodium methoxide. Although temperature and pressure are not critical, the reaction is generally carried out at atmospheric pressure and at temperatures between 15 and 60°C, preferably at 25°C.

v

The compound of formula V is then treated with an alkali metal alkoxide, preferably sodium methoxide, in the presence of an aromatic hydrocarbon, preferably benzene, to form a compound of formula VI

wherein R 1 and R 2 have the above meanings.

Although temperature and pressure are not critical, this reaction is generally carried out at atmospheric pressure and temperatures of 15

to 60°C, preferably at 25°C.

The compound of formula VI is then transferred into an oxime of formula II after the conversion of ketones to oximes in a manner known per se. Preferably, the ketone VI is treated with a hydroxylamine hydrohalide, preferably hydroxylamine hydrochloride, in a nitrogenous base. Any conventional nitrogen-containing base, preferably an amine, can be used. Suitable amines are primary amines, such as lower alkylamines, especially methylamine, ethylamine or aniline; secondary amines such as di-lower alkylamines, especially dimethylamine or diethylamine; or pyrrole; and tertiary amines, such as tri-lower alkylamines, especially trimethylamine and triethylamine; or pyridine or picoline. Temperature and pressure are not critical. The reaction is preferably carried out at temperatures between room temperature and reflux, preferably at approx. 22°C and atmospheric pressure in an inert organic solvent, such as an aliphatic or aromatic hydrocarbon, e.g. N-hexane or benzene. Preferably, the reaction is carried out in an excess of nitrogenous base which serves as solvent.

As previously mentioned, the thiophene derivatives of formula I and their pharmaceutically usable salts are hypolipemic active agents, i.e. they lower the blood lipid level in mammals. This property was demonstrated in normal, female Charles River rats weighing 150 to 180 g. The animals were first fed a corn oil-glucose mixture for a few days, then given the test compounds orally or parenterally in dimethyl sulfoxide.

Comparison of the blood triglyceride, fatty acid and cholesterol levels of the rats that had received the test compounds showed a significant reduction of the corresponding values in untreated animals. Similar results were obtained with rat hepatocytes.

Fatty acid and cholesterol synthesis in isolated hepatocytes

Female Charles River rats were fasted for 48 hours, after which they were fed 7 to 14 days from 8 to 11 am on a diet containing 1% corn oil and 70% glucose. The isolated rat hepatocytes were obtained by in situ perfusion of the liver. The hepatocytes were incubated for 60 minutes at 37°C. Each sample contained a total volume of 2.1 ml, consisting of 1 ml of isolated rat hepatocytes

(10 to 20 mg dry cells), 1 ml Krebs-Henseleit bicarbonate buffer pH 7.4, 16.5 mM glucose, 1 µM L-alanine (1 /tCi), 1 mCi <3>HjO and 2 mM inhibitor in water or dimethyl sulfoxide at pH 7.4.

All experiments were repeated 2 times and performed with 3 tests

ver every time. The medium containing the cells was mixed with 0.4 ml of 62.5% citric acid and incubated for 45 minutes. The evolved CO 2 was collected in 0.3 ml of a mixture of ethanolamine/2-methoxy-ethanol (1:2). At the end of the experiment, in this solu-

14

ning the CO2 content determined using a scintillator counter. The cell medium was saponified, acidified (for lipogenesis rate determination only) and extracted with hexane. At this step, the lipids were either counted (to determine the rate of lipogenesis) or precipitated with digitonin, washed and counted (to determine the rate of cholesterol esterogenesis). The conversion of 3 14

H 2 O and [ C]alanine in fatty acids or sterols were determined in a liquid scintillation counter. The results are given in Table I as nmol H 2 O and alanine which were converted into fatty acids or cholesterol and nmol alanine which was oxidized to CO 2 , per mg dry cells per 60 minutes.

Fatty acid and cholesterol synthesis in vivo

Rats were fasted for 48 hours and then fed for 5 to 15 days a diet containing 1% corn oil and 70% glucose. On the day of the experiment, the test compound was given 30 to 60 minutes before the 3-hour feeding period by oral intubation or 60 minutes after the 3-hour feeding period by intraperitoneal injection. 30 minutes later, the animals received an injection of 1 mCi <3>H 2 O, 12.3 mg alanine (5 uCi) and 30.6 mg α-keto-glutaric acid in 0.25 ml saline into the tail vein. 30 minutes later, the animals were decapitated, the liver was quickly excised, saponified and acidified (only for determination of lipogenesis rate) and extracted with hexane. The lipids were either counted (for the determination of lipogenesis rate) or precipitated with digitonin, washed and counted (for the determination of cholesterogenesis). The conversion of H 2 O and alanine into fatty acids or sterols was carried out in a liquid scintillation counter. The results are reproduced in tables II to V.

As appears from the subsequent trial report, the present compounds are better than their positional isomers, e.g. the compounds described in British patent 1,278,084 as hypolipemic agents. It has thus been shown that the compound of formula I, A and B,

A 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester hydrochloride

and

B 4-amino-5-methyl-3-thiophenecarboxylic acid methyl ester hydrochloride, already causes a considerable inhibition of fatty acid synthesis at a dose of 10 pM, while their positional isomers C and D which fall under formula IV of the British patent, and are

C 3-amino-5-ethyl-2-thiophenecarboxylic acid methyl ester hydrochloride and

D 3-amino-5-methyl-2-thiophenecarboxylic acid methyl ester hydrochloride,

are practically inactive at this dosage.

COMPARATIVE TRIAL REPORT

Methodology

The methodology for carrying out the comparison experiment

are described in Sullivan, A.C., Triscari, J. and Hamilton, J.G., Lipids, vol. 12, pp. 1-9 (1977)

According to this method, the effect of the above compounds was recorded in comparison with a control on fatty acid synthesis in isolated rat liver cells. The reduction in fatty acid synthesis was determined by using tritiated water ( 3H_0) and

14

carbon 14 ([C]alanine). A compound that shows statistically significant reduction of fatty acid synthesis compared to control at low concentrations is useful as a triglyceride-lowering agent. Triglyceride is a specific blood lipid.

0

Compounds A, B, C and D were tested at a concentration of 10 µM. Compound A was also previously investigated in concentrations of 0.5 - 10 uM. The results are shown in Table I.

In Table I, a positive value indicates a decrease in fatty acid synthesis compared to the control and a negative value indicates an increase (negative decrease) of the variable compared to the control. A statistically significant reduction from the control is indicated by an asterisk.

Results:

The results of the above experiments were as follows:

<a>^ Data are expressed as % reduction from control. Each value is an average of at least triplicate measurements. A positive value indicates a decrease compared to the control and a negative value indicates an increase from the control, b) Each bottle contained 8-10 mg of dry cells, and the concentration of each test compound in 25 (al dimethylsulfoxide.

f cf- p< 0 , 01; statistically significant reduction from control. fff p<0.001; statistically significant reduction from control.

The compounds of formula I and their pharmaceutically acceptable salts can be administered parenterally and orally. For parenteral administration, solutions and suspensions of the compounds in dimethylsulfoxide, water or gum arabic can be used. Particularly suitable are sterile aqueous solutions of corresponding water-soluble salts.

The dosage required to lower the blood lipid level depends on the type and degree of the symptoms. With oral administration, larger quantities of the active substance are needed than with parenteral administration. In general, dosages of approx. 0.1 to 1.2 mg of active ingredient per body weight relevant to achieve a significant reduction in the blood lipid level.

The following examples illustrate the invention. All temperatures are given in degrees Celsius.

EXAMPLE 1

Into a solution of 100 g of 4-carbomethoxy-3-keto-2-propyltetrahydro-thiophene oxime in 1 liter of anhydrous ether, gaseous hydrogen chloride was introduced at 0° for 1 hour. The reaction vessel was closed with a drying tube and stirred overnight at room temperature. The solvent was evaporated until the product crystallized. The white solid was filtered off and washed with ether. 60 g of 3-amino-4-carbomethoxy-2-n-propylthiophene hydrochloride with a melting point of 178-180°C were obtained. Recrystallization from methanol/ether gave a product with melting point 180-181°C.

The starting material can be prepared as follows:

a) To a solution of 116.55 g of 3-mercaptopropionic acid methyl ester in 220 ml of dry methanol was added 52.46 g of sodium methoxide at -20°C. After 20 minutes, a solution of 203 g of 2-bromovaleric acid ethyl ester in 150 g of dry methanol was added dropwise. The reaction mixture was warmed with stirring overnight to room temperature. The methanol was evaporated and the residue partitioned between ether and water. The organic phase was washed with 10% bicarbonate solution with water, dried over magnesium sulfate and evaporated. 130 g of 4-thia-5-carbomethoxyoctanoic acid methyl ester was obtained as a colorless oil. b) To a suspension of 54.0 g of sodium methoxide in 500 ml of anhydrous benzene, 130 g of 4-thia-5-carbomethoxyoctanoic acid methyl ester was added dropwise at 25°C. The mixture was stirred overnight and poured into ice water. The aqueous phase was extracted with benzene/ether (1:1) and then acidified by addition of 6 N HCl to pH 1. The product which partially separated from the aqueous solution was taken up in methylene chloride. The aqueous phase was then extracted with methylene chloride. The combined organic phases were dried and evaporated to give 94 g of pure 4-carbomethoxy-3-keto-2-propyl-tetrahydrothiophene as a colorless oil. c) A solution of 94.0 g of 4-carbomethoxy-3-keto-2-propyl-tetrahydrothiophene in 250 ml of dry pyridine was added at 25°C to 40.0 g

hydroxylamine hydrochloride. The reaction mixture was stirred

overnight at room temperature, the solvent evaporated and the residue partitioned between 1 N hydrochloric acid and methylene chloride. The organic phases were dried over sodium sulfate and evaporated to give 100 g of pure 4-carbomethoxy-3-keto-2-propyl-tetrahydrothiophene-oxime as a colorless oil.

EXAMPLE 2

A solution of 41.4 g of 4-carbomethoxy-3-keto-2-methyltetrahydro-thiophene oxime in 600 ml of anhydrous ether was saturated with hydrogen chloride at 0° and then stirred overnight at 25°C. The separated solid was collected, washed with ether and dried. After crystallization and working up of the mother liquors, a total of 37.4 g of 3-amino-4-carbomethoxy-2-methylthiophene hydrochloride was obtained with a melting point of 191-192°C.

In a similar manner, 49.12 g of 4-carbomethoxy-2-isopropyl-3-keto-tetrahydrothiophene oxime was transferred into 18.49 g of 3-amino-4-carbomethoxy-2-isopropylthiophene hydrochloride with a melting point of 185°C (splitting ning) .

The starting material can be prepared as follows:

a) A solution of 66.29 g of 3-mercaptopropionic acid methyl ester in 50 ml of anhydrous methanol was added at 0° to 120 ml of a 25% sodium methoxide solution in methanol. The solution was added dropwise to a solution of 100 g of 2-bromopropionic acid ethyl ester in 100 ml of anhydrous methanol. The reaction mixture was allowed to warm to 25°C overnight. The solvent was evaporated and the residue partitioned between ether and 10% sodium bicarbonate solution. The aqueous phase was then extracted with ether. The combined organic extracts were dried over magnesium sulfate and evaporated to give 121.40 g of 2-methyl-3-thia-adipic acid 1-ethyl-6-methyl ester as a pale yellow oil.

In a similar manner, 61.4 g of 3-mercaptopropionic acid methyl ester was reacted with 106.8 g of 2-bromovaleric acid ethyl ester to 120.91 g of 2-isopropyl-3-thia-adipic acid-1-ethyl-6-methyl ester. b) A solution of 121.4 g of 2-methyl-3-thia-adipic acid-1-ethyl-6-methyl ester in 90 ml of dry benzene was added dropwise to a suspension of 30 g of anhydrous sodium methoxide in 200 ml of dry benzene The reaction mixture was heated overnight to room temperature, and then partitioned between water and ether. The aqueous phase was post-extracted with benzene. The aqueous phase was then acidified by addition of 6 N HCl to pH 1 and extracted three times with methylene chloride. The methylene chloride extracts were dried over sodium sulfate and evaporated to give 79.17 g of pure 4-carbomethoxy-3-keto-2-methyltetrahydrothiophene as a colorless oil.

In a similar manner, 120.91 g of 2-isopropyl-3-thia-adipic acid 1-ethyl-6-methyl ester was converted to 91 g of 4-carbomethoxy-2-isopropyl-3-keto-tetrahydrothiophene. c) To a solution of 37.2'6 g of 4-carbomethoxy-3-keto-2-methyl-tetrahydrothiophene in 100 ml of anhydrous pyridine was added 18.0 g of hydroxylamine hydrochloride. The mixture was stirred for 24 hours at 25°C, then evaporated and partitioned between 1 N hydrochloric acid and methylene chloride. The aqueous phase was extracted twice with methylene chloride, the combined organic extracts dried and evaporated to give 40.1 g of pure 4-carbomethoxy-3-keto-2-methyltetrahydrothiophene oxime as a colorless oil.

Similarly, 52.8 g of 4-carbomethoxy-2-isopropyl-3-keto-tetrahydrothiophene was converted to 49.0 g of 4-carbomethoxy-2-iso-propyl-3-keto-tetrahydrothiophene oxime.

EXAMPLE 3

A solution of 2.07 g of 3-amino-4-carbomethoxy-2-methyl-thiophene hydrochloride in 35 ml of methanol was treated with 23 ml of 1 N caustic soda. The mixture was heated for half an hour to reflux, cooled and poured into sodium chloride solution. The pH of the solution was adjusted to 5 and the solution was extracted seven times with methylene chloride/methanol (4:1). The organic extracts were dried and evaporated to give 1.23 g of pure 3-amino-4-carboxy-2-methylthiophene with a melting point of 162-164°C. An analytical preparation was recrystallized from ethyl acetate/pentane, melting point 163-164°C.

In a similar manner, 5 g of 3-amino-4-carbomethoxy-2-isopropylthiophene hydrochloride was converted to 3.3 g of 3-amino-4-carboxy-2-isopropylthiophene with melting point 117-118°C and 1.41 g 3-amino-4-carbomethoxy-2-propylthiophene hydrochloride to 0.625 g of 3-amino-4-carboxy-2-propylthiophene with melting point 144-145°c<->

EXAMPLE 4

Into a solution of 80.0 g of 4-carbomethoxy-3-keto-2-phenyl-tetrahydrothiophene-oxime in 600 ml of anhydrous ether, gaseous hydrogen chloride was introduced at 0° over the course of 1 hour. The suspension was added to 300 ml of methanol and stirred overnight at 25°C. The reaction product was filtered off and washed with ether. 70.0 g of 4-amino-5-phenylthiophene-3-carboxylic acid methyl ester hydrochloride was obtained as a pale yellow solid with a melting point of 181-182°C. This compound can be recrystallized from methanol.

The starting material can be prepared as follows:

a) A solution of 104.95 g of 3-mercaptopropionic acid methyl ester in 200 ml of methanol was added to 207.5 g of a 25% sodium methoxide solution in methanol at 0°. The homogeneous solution obtained was added dropwise and under argon to a solution of 200 g of α-bromo-phenylacetic acid methyl ester in 200 ml of methanol. The reaction mixture was stirred overnight at 25°C, the solvent evaporated and the residue partitioned between water and methylene chloride. 234 g of 2-phenyl-3-thia-adipic acid dimethyl ester were obtained as a colorless oil. b) A solution of 234 g of 2-phenyl-3-thia-adipic acid dimethyl ester in 300 ml of dry benzene was added dropwise at 25°C to 54.05 g of sodium methoxide. The reaction mixture was stirred overnight and poured into water. The solid was filtered off and the filtrate extracted twice with diethyl ether. The solid was added to the aqueous phase, which was then adjusted to pH 1 by addition of 6 N HCl. The mixture was extracted three times with methylene chloride, the organic extracts were dried over sodium sulfate and evaporated to give 145.24 g of 4-carbomethoxy-3-keto-2-phenyl-tetrahydrothiophene as a yellow oil. c) To a solution of 82.24 g of 4-carbomethoxy-3-keto-2-phenyl-tetrahydrothiophene in 120 ml of anhydrous pyridine was added 28.85 g of hydroxylamine hydrochloride. The solution was stirred for 2 days at 25°C, the solvent evaporated under reduced pressure and the residue distributed between 1 N hydrochloric acid and methylene chloride. The aqueous phase was post-extracted with methylene chloride. The organic extracts were dried over sodium sulfate and evaporated to give 90.0 g of 4-carbomethoxy-3-keto-2-phenyltetrahydrothiophene oxime as a colorless oil.

EXAMPLE 5

A solution of 10.0 g of 4-amino-5-phenylthiophene-3-carboxylic acid methyl ester hydrochloride in 80 ml of methanol was added to 82 ml of 1 N caustic soda. The mixture was heated to reflux for 30 minutes, cooled to room temperature and adjusted to pH 5. The precipitated product was filtered off and dried to give 8.2 g of pure 4-amino-5-phenylthiophene carboxylic acid, mp 201-202°C (ethyl acetate/ pen-tan) .

EXAMPLE 6

Preparation of 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester hydrochloride.

A solution of 125 g of methyl-3-mercapto-propionate in 75 ml of dry methanol was added dropwise to 249 ml of 25% sodium methoxide-containing methanol at 0°. At 0°, 200 g of ethyl 2-bromobutyrate in 75 ml of dry methanol was added to the mixture. The mixture is stirred overnight at 25°, then concentrated and distributed between water and methylene chloride. The organic phase is dried and concentrated, whereby 229 g of diester are obtained as a colorless oil.

A suspension of 63.5 g of sodium methoxide in 300 ml of dry benzene is added dropwise at 25° to 229 g of the obtained diester in 200 ml of dry benzene. The mixture is stirred overnight at room temperature, then poured into 800 ml of water and the benzene layer is extracted with 200 ml of water. The aqueous phases are combined, carefully acidified with 6 N

HCl and extracted three times with methylene chloride/methanol. The organic extracts are dried and evaporated, whereby 149.7 is obtained

g pure ketone as colorless oil.

A solution of 276.1 g of this ketone in 500 ml of anhydrous pyridine is added portionwise to 121.6 g of hydroxylamine hydrochloride. The mixture is allowed to stand for 20 hours at 25°, then concentrated and distributed between methylene chloride and 3N HCl. The aqueous phase is washed twice with methylene chloride/methanol 5/1. The organic phases are dried and evaporated, whereby 253 g of pure oxime is obtained as a yellowish oil.

Into a solution of 253 g of the oxime obtained in 2 liters of anhydrous ether, gaseous hydrogen chloride is introduced at 25° and over the course of 1 hour. The mixture is inoculated with 0.5 g of genuine product and stirred overnight at 25°. The crude product is filtered, washed with anhydrous ether and recrystallized from methanol-ether, thereby obtaining 173 g of pure aminothiophene hydrochloride with a melting point of 161°.

EXAMPLE 7

A sample of 10.0 g of 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester hydrochloride in 100 ml of methanol was treated with 105 ml of 1N sodium hydroxide and heated under reflux for 1 hour. The resulting mixture was cooled and partitioned between water (pH 4.5) and methylene chloride/methanol (4:1). The aqueous phase was further extracted with methylene chloride/methanol (4:1) five additional times. The organic extracts were combined, dried over sodium sulfate and evaporated to give 6.4 g of 4-amino-5-ethyl-3-thiophenecarboxylic acid. The product was recrystallized from ethyl acetate/pentane to give a pure sample, m.p. 132-133°C.

Claims (3)

1. Analogy method for the production of pharmacologically active compounds with formula wherein R 1 is lower alkyl or phenyl; R 2 is hydrogen, hydroxy, lower alkoxy or amino; R^ and R^ lower alkyl or hydrogen, and salts thereof, characterized in that one treats a compound with formula where R'2 is lower alkoxy and R^ has the above-mentioned meaning, with an acid and in the thus obtained compound of formula Ia where R'2 and R^ have the above-mentioned meaning, if desired, transfers the lower caralkyloxy group into a carboxy-, formyl- or carbamoyl group and/or if desired lower alkylates the amino group, and if desired transfers a compound of formula I into a salt. 2. Process according to claim 1 for the production of 4-amino-5-ethyl-3-thiophenecarboxylic acid, or a salt or methyl ester thereof, characterized in that correspondingly substituted starting materials are used. 3. Process according to claim 1 for the production of 3-amino-4-carbomethoxy-2-n-propylthiophene hydrochloride, characterized in that correspondingly substituted starting materials are used.