NO851546L - PROCEDURE FOR THE PREPARATION OF AMINO ACETIC ACID DERIVATIVES - Google Patents

Description

The present invention relates to an analogous process for the production of N-(3-phenylmethylthio-1-oxopropyl)-aminoacetic acid derivatives with therapeutic effect, and with the general formula I

in

in which

R1 stands for one or two halogen atoms, one or two methyl groups, a trifluoromethyl-methoxy-cyano-nitro-carbamoyl or benzoyl group, a benzene nucleus condensed, or when 1*2 is a 3-indolylmethyl group or 2-methylthioethyl group, can also stand for a hydrogen atom, and ;R 2 stands for a hydrogen atom or a straight-chain or branched alkyl group, a carbamoylmethyl group or ;2-carbamoylethyl group, a 2-methylthioethyl group, an optionally hydroxylated benzyl group, or a 3-indolylmethyl group, and their pharmaceutically acceptable salts, and the invention is characterized by a halogenated derivative of formula II; in which R^ has the above-mentioned meaning, is reacted with a mercaptopropionic acid, after which the obtained acid of formula III is reacted with thionyl chloride to obtain the acid chloride of formula IV; which is then substituted with an amino acid of formula;H_NCHCOOH,;2 R ;<R>2;wherein R2 has the above-mentioned meaning, and if desired, salts of the compound of formula (I) are mixed and/or an obtained compound of formula (I) is converted to another of the indicated compounds of formula (I) by changing the substituent R^. These features of the invention appear in the patent claim. The compounds of formula (I) can exist in the form of free acids or salts which they can form with pharmacologically tolerable bases. When R2 is not a hydrogen atom, the molecule of formula (I) comprises an asymmetric carbon atom, and the compounds of formula (I) can then exist in the form of the two enantiomers. The production of these as well as their mixtures form parts of the invention. ;The course of the reaction can be illustrated using the following diagram. ; The method consists of three reaction steps of a known type, first a halogen derivative (II) bearing the selected substituent is subjected to the action of mercaptopropionic acid in an aqueous basic environment, and the acid obtained (III) is then converted to acid chloride (IV), and this is finally subjected the impact an amino acid of formula F^NCHCOOH ;<*>2

under conditions analogous to the conditions in the first step.

The compound of formula (I) which is obtained can then optionally be converted into another compound (I) by modifying the substituent R 1 and, for example, a cyano group can be hydrolysed to a carbamoyl group.

The following examples illustrate the preparation of the compounds of formula (I), the structures having been confirmed by means of analyzes and IR and NMR spectra.

Example 1.

N-^3-(2-chlorophenyl)methylthio-1-oxopropylj-aminoacetic acid.

In a flask, a mixture of 21.2 g (0.2 mol) 3-mercaptopropionic acid, 160 ml water, 16 g (0.4 mol) sodium hydroxide, and 32.2 g (0.2 mol ) 1-chloro-2-chloromethyl-benzene. After 15 minutes, approximately 150 g of crushed ice are added, then 20 ml of 12 N hydrochloric acid, and the formed precipitate is suctioned off on a filter, washed with water and dried.

The mixture is taken up in a mixture of 100 ml of benzene and 21.9 ml (0.3 mol) of thionyl chloride, and heated under reflux for two hours.

The solvent is evaporated under reduced pressure and, with vigorous stirring, the residue at 0°C is poured onto a solution prepared from 100 ml water, 30 g (0.4 mol) glycine and 16 g (0.4 mol) sodium hydroxide. After stirring for 15 minutes at a temperature of approximately 10°C, the mixture is diluted with 1 part by volume of water and 40 ml of 12 N hydrochloric acid is added. The resulting precipitate is filtered, washed, recrystallized from an ethanol/water mixture and dried. Melting point: 101 - 102°C.

Example 2.

N-[[3-(4-carbamoylphenyl)-methylthio-1-oxopropyl]-aminoacetic acid. a) Under analogous conditions as in example 1, N-3-(4-cyanophenyl)methylthio-1-oxopropyl-aminoacetic acid with a melting point of 116 - 118°C is first prepared. b) Then dissolve 12 g (0.043 mol) in 100 ml of formic acid and at between 0 and 10°C chlorine hydrogen gas is bubbled through

30 minutes. The mixture is then allowed to stand for a day under normal conditions

temperature to the exclusion of moisture. The mixture is evaporated under reduced pressure, the residue is taken up in water, filtered off with suction, washed, recrystallized twice from methanol and once from a mixture of dimethylformamide and water, and dried. Melting point: 206 - 208°C.

Example 3.

0( - [3-(2-Chlorophenylmethylthio)-1-oxopropylaminecTJo(-1-methyl-propyl)acetic acid.

In a flask, stir vigorously at 70°C a mixture of 21.2 g

(0.2 mol) 3-mercaptopropionic acid, 160 ml water, 16 g (0.4 mol) sodium hydroxide, and 32.2 g (0.2 mol) 1-chloro-2-chloromethyl-benzene. After 15 minutes, approximately 150 g of crushed ice are added, then 20 ml of 12 N hydrochloric acid, and the resulting precipitate is suctioned off on a filter, washed with water and dried. The mixture is taken up in a mixture of 100 ml of benzene and 21.9 ml (0.3 mol) of thionyl chloride and heated under reflux for two hours.

The solvent is evaporated under reduced pressure and, with vigorous stirring, 14.9 g of the residue is poured out, at a rate such that the temperature does not exceed 10°C over a solution cooled in an ice-water bath and prepared from 50 ml of water, 26.2 g (0.2 mol) L-isoleucine, and 8 g (0.2 mol) sodium hydroxide. The mixture is stirred for 10 minutes without exceeding 10°C and diluted with 500 ml of water and 20 ml of concentrated hydrochloric acid is added. The formed crystals are allowed to settle, suction on a filter, washed with water and suction again on a filter. After recrystallization from an ethanol/water mixture and drying, they melt at 108 - 109°C.'

The following table illustrates the structure and physical properties of compounds produced according to the invention. The compounds that can be prepared by the invention were subjected to pharmacological tests which show their interest as mucolytic, anti-inflammatory, anti-ulcer, anti-secretory and analgesic agents.

For investigation of the mucolytic effect with experiments carried out according to the experimental model with bronchitis against both acetic anhydride in rats according to the method proposed by L. Reid ("An experimental study of hypersecretion of mucus in the bronchial tree", Brit. J. Exp. Pathol. , 1963, 44, 437), and developed by A. Quevauviller and colleagues ("Méthodes d'étydes expérimentale des modificateurs des sécrétions bronchiques", Le Poumon et le Coeur, 1970, 26, No. 1, 71 - 80).

The animals used in the experiment are male Sprague Dawley SPF rats of the Charles River strain (France) with medium weight

300 g distributed randomly in groups using a distribution table. They are submitted for three days for a period of five to six hours each day to an inhalation of sulfuric acid anhydride with a concentration of 500 or 600 ppm. From the fourth day until the thirteenth day, the animals are treated with compounds produced according to the invention in amounts of 100 mg per kg and per day with oral administration. The control animals receive only the solvent which is solution of "Tween 80" 1%.

During the eighth, ninth and eleventh day, all the animals are again subjected to an inhalation of 300 ppm sulfuric acid anhydride in the course of one to two hours, while they receive the compounds that can be prepared by the invention or the solvent.

The animals are killed 24 hours after the last day's treatment by cutting the abdominal aorta after pentobarbital anesthesia.

After removal, the tracheobronchial is fixed together in 10% formaldehyde solution buffered to pH 7.

To assess the tracheobronchial retention, mucus is stained with a solution of 7 p/oo Alsian blue, after sectioning the principal bronchi and trachea along their longitudinal axis after previously curing the tissues in alcohol.

The assessment of endobronchial retention is carried out with the main bronchi in the left lung and right basal lobe, as well as with the trachea. The main bronchus and trachea are randomly divided into three parts (the hilar third, the middle third, and the distal third for the bronchi, and the upper third, the middle third, and the lower third for the trachea), and assessment of retention is carried out for each third using a binoculars.

The retention of mucus in the respiratory tract can be read macroscopically with very different aspects, with four principal forms of obstruction, namely depot, nodulasr arnas, flowing or compact blockage. For each of these forms, with the exception of compact blockage, a distinction is made between several types according to the volume size of the obstruction in question.

For each animal, the retention volume is assessed by giving each observed shape a numerical value corresponding to the volume occupied by this obstruction in the bronchial or tracheal canal of the examined animal. The value 15 corresponds to complete obstruction by a compact obstruction occupying 100% of the bronchial or tracheal channel, and the value corresponds to minimum obstruction for a small depot occupying approximately 1/15 of the bronchial or tracheal channel.

The mucolytic effect of a product on the tracheobronchial retention of pathological mucus is defined as a function of the total reduction of the retention, i.e. the volume of secreted mucus (mean volume per animal and mean volume per animal and per group).

For the bronchi, the total volume is judged for the retention of the two principal bronchi, namely on the basis of a maximum value of 90 (6 third bronchi obstructed by 6 compact obstructions: 6 x 15 = 90).

The total retention volume in the tracheal connection was assessed only on the 3 thirds, namely on the basis of a maximum value of 45 (3 third trachea obstructed by 3 compact obstructions: 3 x 15 = 45), and the numerical indication is multiplied by 2, as that the endotracheal retention and the endobronchial retention in each animal should be expressed in a comparable manner.

The value for the total tracheobronchial retention obtained by adding the value of the endtracheal retention and the value of the endobronchial retention, similarly indicates the two retention levels for pathological mucus.

A statistical analysis of the results is carried out by the non-parametric test of Mann & Whitney.

Results.

Compared to the control animals, the animals treated with the compounds that can be prepared according to the invention show a reduction of up to 50% in the total volume of tracheobronchial retention.

For investigation of the anti-inflammatory effect with conducted trials with the edema test with carrageenan in rats according to the method of Winter et al ("Carrageenin induced edema in hind paw of the rat as an assay for anti-inflammatory drugs". Proe. Soc. Exp. Biol. Med. 1962, 111, 544-547). The animals used are male rats of the strain Sprague Dawley SPF Charles River (France) with an average weight of 150 g distributed in random groups using a distribution table.

The compounds are administered orally in doses of between 20 and 200 mg/kg one hour before injection under the plantar aponeurosis of one of the hind paws of 0.1 ml of carrageenan, in a 1% suspension in sterile physiological serum. The control animals receive only placebo which is a 1% solution of "Tween 80". The increase in paw volume is measured three hours after injection of the carrageenan using a Ugo Basile plethysmometer.

Results.

Compared to the control animals, the animals treated with the compounds that can be prepared according to the invention show a reduction of 40% of the edema volume in doses of 40 to 200 mg/kg.

In order to examine the antiulcerative effect, experiments were carried out with ulcerative wounds introduced into rats using phenylbutazone or ethanol. The animals used for both of these models were female rats of the strain Wistar (I ffa Credo) fasted after 18 hours, distributed randomly using a distribution table.

The wounds are produced by oral administration of phenylbutazone, in solution mole by mole in sodium hydroxide in doses of 200 mg/kg, or 50% ethanol in doses of 5 ml/kg.

The compounds under investigation are administered orally 30 minutes before the ingestion of the ulcerogenic agent. The control animals receive only placebo which is a solution of 1% "Tween 80".

The animals are euthanized by inhalation of chloroform two hours after ingestion of phenylbutazone or one hour after ingestion of ethanol. The stomach contents are removed, the degree of ulceration is judged on a scale from 0 to 3, and the ulceration index (the degree of ulceration x percentage of animals with ulcers) is calculated.

Results.

Compared to the control animals, the animals treated with the compounds that can be prepared by the invention have a 50% reduction in the ulceration index from 4 mg/kg compared to phenylbutazone, and 15 mg/kg compared to ethanol.

In order to investigate the antisecretory effect, experiments were carried out in rats with a ligated pylorus in accordance with the technique of Shay et al. ("A simple method for the uniform production of gastric ulceration in the rat". Gastro-enterology, 1945, 5, 43-61) described by Pascaud et Laubie ("Etude pharmacologique de thiocarboxamides anti-sécrétoires gastriqu et ulcéro-protectrices". Arzneim. Forsch. 1971,

10, 1547-1553).

The animals used are female rats of the strain Wistar (I ffa Credo) with a weight between 200 and 250 g, and fasted after 48 hours, and distributed into random groups using a distribution table. The ligature of the pylorus is carried out under mild anesthesia with ether after washing the stomach with 4 ml of lukewarm physiological serum.

The compounds under investigation are administered intraperitoneally immediately before ligature of the pylorus. The control animals receive only placebo which is a solution of 1% "Tween 80". Four hours later, the animals are killed by inhalation of chloroform. The stomach contents are removed and centrifuged and the volume of the gastric secretion is measured, the free acidity and total acidity are determined by titration.

Results.

Compared to the control animals, the animals treated with the compounds that can be prepared by the invention have a 50-80% reduction in gastric acid secretion from doses of 10 mg/kg.

In order to investigate the analgesic effect, experiments were carried out with the "Writhing test" using acetic acid in mice according to Koster et al. ("Acetic acid for analgesic screening". Fed. Proe. 1959, 18, 412). The animals used are male CD1 mice of the Charles River strain (France), with an average weight of 20 g, and fasted after 18 hours. Intraperitoneal injection of 10 ml/kg of 0.6% acetic acid solution in a 0.2% solution of "Teeen 80" and 0.25% carboxymethyl cellulose produces within a few minutes in mice a syndrome of twitching and torsion (twisting) which can is considered an expression of a diffuse abdominal pain. The withdrawals are counted in the first 15 minutes following this injection.

The compounds under investigation are administered orally 30 minutes before the injection of acetic acid. The control animals receive only placebo which is a 1% solution of "Tween 80".

Results.

Compared to the control animals, the animals treated with the compounds that can be produced by the invention show a 50% reduction in the number of withdrawals produced by the acetic acid from doses of 20 - 200 mg/kg.

The above-mentioned results show that the compounds that can be prepared by the invention can be used as active drug and pharmaceutical preparation substances for the treatment of various disorders in the broncho-respiratory apparatus that are followed by a hypersecretion of mucus, such as bronchitis, tracheo-bronchitis, bronchoinflammation, asthma, etc., and generally disorders associated with disturbances in the secretion or transport of mucus, treatment of inflammation and pain of various origins, as well as treatment of ulcers and gastric hypersecretion.

The compounds can be supplied in suitable forms for enteral or parenteral administration, for example in the form of tablets, gels, dragées, syrups, drinkable or injectable solutions or suspensions, in connection with usual additives.

The daily dose can go up to 0.5 to 100 mg/kg of active substance, i.e. approximately 25 to 7000 mg.

Claims (1)

Analogous process for the preparation of therapeutically active N-(3-phenylmethylthio-1-oxopropyl)-aminoacetic acid derivatives of the general formula I in which represents one or two halogen atoms, one or two methyl groups, a trifluoromethyl group, methoxy group, cyano group, nitro group, carbamoyl group or benzoyl group, a benzene nucleus condensed, or also, when R2 is a 3-indolylmethyl group or a 2-methylthioethyl group, R^ represents also a hydrogen atom, and R 2 stands for a hydrogen atom or a straight-chain or branched C 1 -4 alkyl group, a carbamoylmethyl group or a 2-carbamoylmethyl group, a 2-methylthioethyl group, an optionally hydroxylated benzyl group, or a 3-indolylmethyl group, and their pharmaceutically acceptable salts, characterized by a halogen derivative of formula II in which it has the above meaning, is traded with more- captopropionic acid, and the resulting acid of form III is reacted with thionyl chloride to obtain the acid chloride with formula IV which is finally reacted with amino acid of formula 2 • H_NCHCOOH, 2 wherein R£ has the above-mentioned meaning, and if desired, pharmaceutically acceptable salts are prepared, and/or an obtained compound of formula (I) is converted into another compound of formula (I) by modification of the substituent R^ .