NO149241B - ANALOGY PROCEDURE FOR THE PREPARATION OF HYPOLIPIDEMIC ACTIVE 4 (2-TIENYLMETHYLAMINO) BENZOIC ACID COMPOUNDS - Google Patents

Description

The present invention relates to the production of hypolipidemically active 4-(2-thienylmethylamino)benzoic acid derivatives with the formula:

and pharmaceutically acceptable salts thereof, where R is hydrogen or (C-^^alkyl, and X, Y and Z independently are hydrogen, chlorine or bromine, - provided that at least one of X, Y and Z is chlorine or bromine. 4- (2-thienylmethylamino)benzoic acids and the corresponding ethyl ester are described in Izo. Akad. Nauk SSR, Ser. Khim. 1967 (9), 2049 - 55 (CA. 68; 104869K). No use for the compounds is described Dutch Patent Application No. 7602332 describes 4[aryl(alkyl or alkenyl)-amino]-benzoic acid compounds with hypolipidemic activity.

Pharmaceutically acceptable salts of said p-aminobenzoic acid compounds of formula I are formed when R is hydrogen. Pharmaceutically acceptable salts are understood to mean acid addition salts of those bases which will form a salt with a carboxylic acid and which will not produce harmful physiological effects when the preparation or compound is administered to an animal in doses which are consistent with good pharmacological activity. Suitable bases thus include alkali metal and alkaline earth metal hydroxides, carbonates and bicarbonates such as sodium hydroxides, potassium hydroxides, calcium hydroxide, potassium carbonate, sodium carbonate or magnesium carbonate, ammonia, primary, secondary and tertiary amines. You can also prepare aluminum salts by treating the corresponding sodium salt with a suitable aluminum complex, e.g. aluminum chloride hexahydrate.

Compounds prepared according to the present invention have been shown to have hypolipidemic activity in animals, and especially in mammals. By hypolipid€;mic activity is understood here the effect of lowering the lipid content of the blood, especially the cholesterol and triglyceride content in the serum. The compounds are therefore suitable for the treatment of serum hyperlipidaemia in mammals, and especially for the treatment of hypercholesterolaemia and hypertriglyceridemia, i.e. such conditions where one has an abnormally high content of lipids, cholesterol or triglycerides, respectively. The compounds can be administered orally or parenterally by subcutaneous, intravenous or intraperitoneal injection or by implantation, however, oral administration is preferred.

The effective hypolipidaemic amount of compounds of formula I to be administered to the patient, i.e. the amount which effectively and significantly lowers the level of lipids in the serum, will depend on factors such as the patient's condition, the particular compound to be used, the lipid level one wishes to obtain whether or not the patient is hyperlipidemic, the mode of administration and the period of administration. Generally, an effective daily dose will range from 1 to 200 mg/kg body weight, and it is preferred to use a daily dose ranging from 10 to 100 mg/kg body weight.

For oral administration, preparations of the p-amino-benzoic acid compounds can be prepared in a commonly known manner. One can e.g. use granulation and compression, and if necessary, to mix and dissolve or suspend the components, depending on how the final product is desired. One can thus use numerous pharmaceutical forms of the compounds. E.g. the pure compound can be used as such or mixed with a solid carrier or diluent. Generally, it is preferred to use inorganic pharmaceutical diluents and especially solid inorganic diluents. One reason for this is that a large number of inorganic materials are known which are pharmaceutically safe and acceptable, apart from the fact that they significantly facilitate the processing of the preparations. The preparations can be in the form of tablets, powders, capsules, suspensions or solutions, and the preparations can be prepared by commonly known standard pharmaceutical techniques. Tablet preparations may be coated or uncoated, and they may be effervescent or non-effervescent. Common diluents for preparing tablets can be used. One can e.g. use inert diluents such as magnesium carbonate or lactose, disintegrants such as corn starch or alginic acid, and lubricants such as magnesium stearate.

If a liquid diluent is used, the preparation may be in the form of a soft gelatin capsule, a syrup, a liquid solution or suspension.

The hydrocarbon solubility of most compounds according to the present invention is sufficiently high that pharmaceutically acceptable oils and diluents can be used. One can e.g. use vegetable or animal oils

such as sunflower seed oil, corn oil or regular cod liver oil. Glycerin can also be used. In connection with the latter solvent, 2-30% by weight of water can be added. When water alone is used as diluent, or when the solubility of the compound in the oil is low, the preparations can be supplied in the form of a suspension.

Emulsion preparations can be prepared by using emulsifiers such as sorbitan trioleate, polyoxyethylene sorbitan monooleate, lecithin, gum acacia or tragacanth. Aqueous-based suspensions can be prepared using wetting agents such as polyethyleneoxy condensation products of alkyl phenols, of fatty alcohols or fatty acids with a suspending agent, e.g. a hydrophilic colloid such as polyvinylpyrrolidone. The emulsions and suspensions may contain commonly known additives such as sweeteners, coloring agents or preservatives. The compounds can also be incorporated into foodstuffs, e.g. butter, margarine, edible oils, casein or carbohydrates. Such specially prepared foodstuffs can be supplied as a whole or as part of a diet or as a supplement to a diet. Such preparations preferably contain 0.02-2.0% of the active ingredient when the food is supplied as the total diet. The preparations may contain higher concentrations of the active ingredient when added as a supplement.

For parenteral use, the compounds can be prepared with sterile ingredients, after which they can be processed and packaged aseptically. They can be administered intravenously or intramuscularly. Useful solvents for such preparations are polyaliphatic alcohols and mixtures thereof. Particularly satisfactory are pharmaceutically acceptable glycols such as propylene glycol and mixtures with this compound. Glycerin is particularly useful. 25-30 volume-% water can be added to the liquid if this is desired. An 80% aqueous propylene glycol solution is a particularly suitable solvent system. It is desirable to adjust the pH to approx. 7.4 and otherwise the solution is isotonically compatible with the isotonicity of the body. The basicity can be regulated by adding a base as needed, and a particularly suitable base is monoethanolamine. It may often be desirable to add a local anesthetic of the type used for such purposes.

The percentage of compound used in the pharmaceutical carrier or diluent may vary. It is necessary that the preparation contains such a large amount that a suitable dose is obtained, and it is preferred to use pharmaceutical preparations containing at least 10% by weight of the compound. The activity increases with the concentration of the agent in the carrier, but it is preferred to use preparations containing a significant amount of the carrier, i.e. at least 1% and preferably at least 5%, as this facilitates the delivery of the compound.

The above-defined compounds with formula I" are produced according to the present invention by reacting a 4-aminobenzoic acid compound with the formula: where R has the above meaning, with a 2-thiophene carboxyaldehyde with the formula:

where X, Y and Z have the meaning indicated above, followed by reduction of the Schiff base thus obtained, and, if desired, conversion of a compound obtained into pharmaceutically acceptable salts thereof.

A suitable method is thus to mix said Schiff's base with an excess of ethanol and water. For-

diluted aqueous sodium hydroxide may optionally be added to the mixture. Sodium borohydride is added at room temperature and stirred until a complete solution is obtained. The mixture is then heated, poured over ice and acidified. The product can be filtered off as a precipitate and further purified in a known manner.

Several of the halogenated aldehydes could not be obtained and were prepared by methods that are generally known from the literature. Thus, e.g. 4-Chloro-2-thiophenecarboxaldehyde prepared by chlorinating 2-thiophenecarboxaldehyde using a large excess of the aluminum chloride catalyst.

See J. Org. Chem., 21, 381 (1956) and J. Heter. Chem., 393

(1976). Using the same procedure, 4,5-dichloro-2-thiophenecarboxaldehyde can be prepared from 5-chloro-2-thiophenecarboxaldehyde. See CA. 57; 16526i.

The following examples illustrate the invention.

Example 1

4-(3,5-dichloro-2-thienylmethylamino)benzoic acid A 50% oil dispersion (5.8 g, 0.12 mol) of sodium hydride was washed with hexane under nitrogen. To the dispersion was then added 200 ml of dry dimethylformamide and 29.5 g (0.113 mol) of solid ethyl N-trifluoroacetyl-4-aminobenzoate. The reaction is weakly exothermic. The mixture was cooled to 20°C and stirred for 15 minutes. 27.7 g of 3,5-dichloro-2-bromomethylthiophene were then added. The reaction mass was then kept at 85°C for 18 hours. The mixture was then poured into 1 liter of cold water and extracted with methylene chloride. The product was dried with anhydrous sodium sulfate and the dry solution was concentrated to a brown oil. The oil was refluxed for 2 hours with 50 ml of ethanol and 200 ml of 5-N-sodium hydroxide. The cooled brown solution was diluted with water and acidified with acetic acid. The crude 4-(3,5-dichloro-2-thienylmethylamino)benzoic acid was obtained as a brown gum, and a recrystallization from propylene glycol methyl ether gave 17 g of brown crystals, mp 169-170.5°C.

Example 2

4-(5-chloro-2-thienylmethylamino)benzoic acid

A mixture containing 46.6 g (0.34 mol) of p-aminobenzoic acid, 50 g (0.34 mol) of 5-chloro-2-thiophenecarboxaldehyde and 400 ml of toluene was heated under reflux for approx. 3.5 hours. The water that formed was collected in a Dean-Stark trap. Reak's ion mass was cooled and Schif f.' s base collected as a faint brown solid. A mixture of the intermediate Schiff base and 1.2 liters of glacial acetic acid was added to 21.2 g

(0.35 mole) of dimethylaminoborane. The reaction mass was kept at 40°C for half an hour and poured into 2 liters of ice water. The white precipitate of 4-(5-chloro-2-thienylmethylamino)benzoic acid

was washed with water, dried and recrystallized from toluene. Yield 60 g (70%), melting point 177-178°C.

Example 3

4-(4,5-dichloro-2-thienylmethylamino)benzoic acid

Step 1: Synthesis of 4,5-dichloro-2-thiophenecarboxaldehyde.

A mechanically stirred solution of 31.8 g (0.217 mol) of 5-chloro-2-thiophenecarboxaldehyde in 150 ml of methylene chloride was added in small portions to 65 g (0.487 mol) of anhydrous aluminum chloride. The mixture reacts exothermicly and turns purplish red. To it was then added 20.6 g (0.29 mol) of chlorine in 250 ml of carbon tetrachloride, and the addition was carried out dropwise. The reaction mass was boiled under reflux for approx. 15 hours, after which a further 15 g (0.21 mol) of chlorine in 200 ml of carbon tetrachloride were added. The mixture was then refluxed for a further 7 hours. The reaction was then stopped by pouring the mixture into 1 liter of ice water. The aqueous layer was extracted with chloroform. The chloroform solution was washed with water and sodium bicarbonate. After drying with anhydrous sodium sulfate and concentration under vacuum, a tan oil was obtained. The impure 4,5-dichloro-2-thiophenecarboxaldehyde crystallized on standing and was later recrystallized from hexane.

Step 2: The product 4-(4,5-dichloro-2-thienylmethylamino)-benzoic acid, melting point 225-227°C was prepared from the above-mentioned intermediate, namely 4,5-dichloro-2-thiophenecarboxaldehyde in the same manner as described in example 2 above. Yield 88%.

In addition to the compounds described in the above examples, other compounds were prepared using essentially the same technique. These compounds are indicated in Examples 4, 5 and 6 as follows:

Example 4 4-(5-chloro-2-thienylmethylamino)benzoic acid ethyl ester, melting point 108-109°C was prepared from ethyl p-aminobenzoate and 5-chloro-2-thiophenecarboxaldehyde. Yield 87%. Example 5 4-(5-bromo-2-thienylmethylamino)benzoic acid, melting point 188-190°C. Yield 61%.

Example 6

4-(4-chloro-2-thienylmethylamino)benzoic acid, melting point 165-168°C. Yield 96%.

The hypolipidemic effect of compounds of formula I was demonstrated in rats. The compound was dissolved in acetone, absorbed on silica gel and mixed with normal food so that a concentration of 0.125% of the compound was obtained in the food. The treated food was fed to male rats weighing 150-160 g over 14 days. After this period, the rats were euthanized and blood samples were taken. The liver was removed, weighed and frozen for further analysis. The relative levels of serum cholesterol in the blood samples were determined by Henly's method, A.A. Henly, Analyst, 82, 286 (1957). Liver cholesterol was measured by the Sperry-Webb method. Journal of Biological Chemistry 187, 97 (1950). The relative levels of triglycerides in blood and liver were determined by the Von Handel and Zilversmit method, J. Lab. Clin. With. 50, 152 (1957)

and Clin. Chem. 7, 249 (1961). By taking the average level of a number of control rats as standard, one could determine the results in the treated groups.

The results are shown in Table I.

Data in Table I clearly indicate that compounds according to the present invention are very effective as hypolipidemic agents for lowering serum cholesterol and serum triglycerides while at the same time only minimal changes are obtained with regard to liver weight and total body weight. The compounds 4-(4,5-dichloro-2-thienylmethylamino)benzoic acid (Example 3) and 4-(5-chloro-2-thienylmethylamino)benzoic acid ethyl ester (Example 4)

have been shown to be particularly useful in lowering cholesterol levels in mammals. Furthermore, it has been shown that the compounds 4-(5-chloro-2-thienylmethylamino)benzoic acid ethyl ester (Example 4) and 4-(4-chloro-2-thienylmethylamino)benzoic acid (Example 6) have been shown to be particularly effective for lowering serum triglycerides.

Comparison experiment

The test compounds were dissolved in acetone, taken up on a silica gel and mixed with normal ground flour to obtain concentrations of 0.125% of the relevant compounds in the animal feed. The treated feed was administered to male Sprague-Dawley rats weighing 150-160 g over a 14 day period. A group of six rats was used for the assessment of each of the three compounds tested. Additional groups of control rats that received untreated treatment were used as control animals. After the 14-day feeding period, the rats were weighed and euthanized. Blood samples were collected and the liver removed.

The relative levels of serum cholesterol and diglycerides in the blood samples were determined either using the manual methods described by Henly (Analyst 82, 286 (1957)) and by Van Handel and Zilversmidt (J. Lab. Clin. Med. 50, 152 (1957 ) and Clin. Chem. 7, 249 (1961)) or by using an automated method using a Technicon Autoanalyzer. By taking the average levels in the control rats as a standard, the average results obtained in the treated groups were thereby ascertained. The results are shown in the table below expressed as relative change in values for the treated animals compared to the control group.

It appears from the above data that the compounds produced according to the present invention consistently have about 30% greater hypocholesterolemic activity than the known compound. Even more unexpected is the increase in hypotriglycerolaemic activity which is more than doubled for the compounds of the present invention compared to the previously known compound.

Claims (1)

Analogous method for the preparation of hypolipidemically active 4-(2-thienylmethylamino)benzoic acid derivatives with the formula: and pharmaceutically acceptable salts thereof, wherein R is hydrogen or (C^_-j) alkyl, and X, Y and Z are independently hydrogen, chlorine or bromine, provided that at least one of X, Y and Z is chlorine or bromine, characterized in that a 4-amino-benzoic acid compound with the formula: where R has the above meaning, is reacted with a 2-thiophene carboxyaldehyde with the formula: where X, Y and Z have the meaning indicated above, followed by reduction of the Schiff base thus obtained, and, if desired, conversion of a compound obtained into pharmaceutically acceptable salts thereof.