HK1000093B - Dextrorotatory enantiomer of alpha-(4, 5, 6, 7-tetrahydrothienod3, 2-capyrid-5-yl)(2-chlorophenyl)methyl acetate, process for its preparation and pharmaceutical c. - Google Patents

Description

The present invention relates to a salt of the dextrogyre enantiomer of alpha-tetrahydro-4,5-6,7 thieno (3,2-c) pyridyl-5 (chloro-2 phenyl) methyl acetate, its preparation process and the pharmaceutical compositions containing it.

This enantiomer has the following formula (I): - What? In the case of a compound containing C* asymmetrical carbon, this formula represents both the dextrogyre molecule and its levogyre enantiomer. The racemic mixture corresponding to this formula was described in the French patent application published under No 2 530 247.

It is known that the rotational power of a compound depends on the solvent in which it is measured and its concentration in that solvent.

Unsurprisingly, only the dextrogyre enantiomer Id exhibits platelet anti-aggregant activity, the levogyre enantiomer Il being inactive.

The compound (Id) is an oil, while its hydrochloride is a white powder.Oily products are generally difficult to purify, and crystalline products which can usually be purified by recrystallization are preferred for the preparation of pharmaceutical compositions.

In the present case, however, it has been found that some salts of the compound (Id) generally precipitate in amorphous form and/or are hygroscopic, making their handling at the industrial stage difficult. Therefore, salts of conventional carboxylic and sulphonic acids used in pharmacies were prepared, such as acetic, benzoic, fumaric, maleic, citric, tartaric, gentan, methanesulphonic, ethanesulphonic, benzene-sulphonic and laurylsulphonic acids, as well as the salt of benzoyl acid (F = 70°C) and that of para-toluenesulphonic acid (F = 51°C), which proved to be difficult to purify.

Among the salts of organic and mineral acids of the dextrogyre isomer of the compound with formula Id, salts have been found which crystallize easily, are not hygroscopic and are sufficiently water soluble to be particularly advantageous for use as active ingredients in medicinal products.

The present invention therefore relates more particularly to hydrogen sulfate of the dextrogyl enantiomer of alpha-tetrahydro-4,5,6,7 thieno-3,2-c) pyridyl-5) (chloro-2 phenyl) methyl acetate.

This salt is conventionally prepared by the action of the corresponding acid on the base in solution in a solvent into which it precipitates spontaneously or after addition of a salt non-solvent.

The dextrogyre isomer of alpha-tetrahydro-4,5,6,7 thieno (3,2-c) pyridyl-5) methyl-acetate can be prepared by salivation of the racemic compound with an optically active acid in a solvent, successive recrystallizations of the salt until a constant rotational power product is obtained, then release of the dextrogyre isomer from its salt by a base; where appropriate the dextrogyre isomer is salivated with a pharmaceutically acceptable acid.

The optically active acid may be advantageously camphosulfonic-10 levogyre acid.

Only one solvent can be used for salification and recrystallization: in this case, acetone is perfectly suited.

Camphosulfonic acid-10 chiral levogyr deformul (IIl) is reacted in an inert solvent with the racemic mixture of formula (I) according to the following reaction scheme: - What?

Salification can be carried out in solvents such as alcohols, ketones, dimethylformamide. The salt precipitates spontaneously or is isolated by re-loading or evaporation of the solvent. A mixture of the two diastereoisomers of formula (IIIa) is formed. By successive recrystallizations in a solvent such as acetone, the precipitate is enriched with salt of the dextrogyre isomer of the compound (I). $\frac{\text{20}}{\text{D }}$ The precipitate is measured at 20°C in methanol at a concentration of 1,5 to 2 g/100 ml. As soon as [α] $\frac{\text{20}}{\text{D }}$ The base of the formula (Id) is released from the salt (IIIa) by the action of a base such as sodium or potassium hydrocarbonate in an aqueous medium at temperatures between 5°C and 20°C.

Evaporation of the filtrate from the 1st recrystallization IV after filtration of the precipitated salt crystals (IIIa) gives a salt-enriched mixture of the enantiomer (Il). The alkalinization of this mixture of diastereoisomeric salts, with a weak base such as sodium or potassium hydrocarbonate, in aqueous solution at temperatures between 5°C and 20°C leads to a mixture of the two enantiomers (Id) + (Il) enriched in levodygyr enantiomer (Il).

This mixture (Id) + (Il) enantiomerized (Il) is reacted with camphosulfonic acid-10 dextrogyre, which we shall designate as (IId), in a solvent, as follows: - What? The resulting crystalline diastereoisomeric (IIIb) salt mixture is recrystallized in acetone until the rotational power [α] $\frac{\text{20}}{\text{D }}$ The rotational power [α] is determined as before. $\frac{\text{20}}{\text{D }}$ Diasteroisomeric salt (IIIb) after each recrystallization.

The release of the stereoisomer (Il) from its salt is carried out in the classic manner, as is the case with the compound (Id).

Camphosulfonic acid-10 levogyre (IIl) can be obtained from commercial ammonium camphosulfonate-10 of formula (V) according to the reaction scheme: - What?

An aqueous solution of ammonium salt (V) is chromatographed on an Amberlite IRN-77 resin.

The whole process is outlined below:

Each of the pure enantiomers (Id) and (Il) can be salivated by the conventional methods: for example, hydrochlorides are prepared by adding a solution of hydrochloric gas in diethyl ether to a solution of (Id) or (Il) in diethyl ether.

Determination of the enantiomeric purity of the dextrogyre (Id) and levogyre (Il) enantiomers

Two methods were used: Proton MRI spectroscopy with addition of rare earths high pressure liquid chiral chromatography using a chiral stationary phase.

(a) Proton MRI spectroscopy with addition of chiral rare earth

Enantiomeric purity (optical purity) was determined by RMN1H 60 MHz spectroscopy, in the presence of rare earth chiral complex, using the method described by G.M. WHITESIDES et al. (J. Am. Chem. Soc. 1974, 96, 1038).

On the racemic product (I), the hydrogen fixed to the asymmetry center, in α of the ester function, appears as a singlet (chemical displacement δ = 4.87 ppm, in CDCl3 as solvent. The addition of the rare earth complex Eu(tfc) 3 [tris (trifluoromethyl hydroxymethylene-3) -d-camphorate of europium (III) ] in the probe, containing the solution of the racemic (I) in CDCl3, leads to the splitting of the initial singlet into two well-separated singlets, corresponding to the proton of each of the enantiomers (Id) and (Il). For a complex/compound molar ratio (I) = 0.4, the separation between the two singlets is 6 Hz.

With each of the two prepared enantiomers (Id) and (Il), the same procedure as for the racemic (I) was used.

The accuracy of the method was determined by comparing spectra of RMN1H (60 MHz), obtained with or without addition of rare earth complex, for each of the two enantiomers (Id) and (Il), pure or mixed with increasing amounts of the other enantiomer.

(b) high pressure liquid chromatography using a steady-state chiral

The study was conducted with a HP-1084 liquid chromatograph using a UV detector at 215 nm. The stationary chiral phase was DEAE (10 micron) silica grafted with alpha-1 acid glycoprotein (0.4 x 100 mm) (ENANTIOPAC R-LKB). The mobile phase was a buffered aqueous mixture of phosphate (NaH2PO4/Na2HPO4)-8mM, containing 0.1 M NaCl, adjusted at pH = 7.4 and containing 15% isopropanol (V/V). The flow rate was set at 0.3 ml/minute and the column temperature was maintained at around 18-20°C.

Under these conditions, the dextrogyre enantiomer (Id) has a retention time of 45 minutes and the levogyre enantiomer (Il) has a retention time of 35 minutes.

The precision of the determination of the optical purity of the two enantiomers was estimated by chromatographing each of the two enantiomers (Id) and (Il) prepared either alone or in combination with increasing amounts of the other enantiomer. The test chemical is used to determine the concentration of the enantiomer in the enantiomer (Id) and the enantiomer (Il) in the enantiomer (Id).

Under these conditions, it can be concluded that the optical purity of the two enantiomers (Id) and (Il) obtained from the examples is at least 96% for the dextrogyre enantiomer (Id) and at least 98% for the levogyre enantiomer (Il).

The following non-limiting examples are given as illustrations of the present invention.

EXAMPLE 1 - Salts of alpha-tetrahydro-4,5,6,7 thieno-3,2-c pyridyl-5) (chloro-2 phenyl) methyl dextrogyre acetate. (a) Camphosulfonic acid-10

The ammonium levo-glycerate camphosulfonate-10 is dissolved in a minimum of water and deposited on the pre-prepared resin column. The electrolyte is removed with water. The electrolyte fractions containing the acid are lyophilized. White crystals, F = 198°C; [α] $\frac{\text{20}}{\text{D }}$ -20,53 (C: 2,075 g/100 ml, water) and the other

(b) 1-camphosulfonic acid-10 salt of alpha-tetrahydro-4,5,6,7 thieno-3,2-c) pyridyl-5 (chloro-2 phenyl) methyl acetate (SR 25990 B).

32 g (0.0994 mole) of alpha-tetrahydro-4,5,6,7 thieno (3,2-c) pyridyl-5) (chloro-2 phenyl) methyl acetate of racemic acid is dissolved in 150 ml of acetone. 9.95 g (0.0397 mole) of camphosulfonic acid-10 monohydrate levogyr is added. The homogeneous medium is left at room temperature. After 48 hours, some crystals appear. The reaction medium is concentrated at 50 ml and left at room temperature for 24 hours. The resulting crystals are filtered, washed with acetone and dried (Rdt. The test chemical is a white crystal, F = 165°C, [α] $\frac{\text{20}}{\text{D }}$ The total volume of the product shall be calculated as follows:

The crystals obtained are dissolved in the minimum boiling acetone (50 ml) and the crystals obtained after cooling are filtered, washed with acetone and dried (efficiency: 88%). The test chemical is a white crystal, F = 165°C, [α] $\frac{\text{20}}{\text{D }}$ The total volume of the product shall be calculated as follows:

(c) Alpha-tetrahydro-4,5,6,7 thieno-3,2-c) pyridyl-5) (chloro-2 phenyl) methyl dextrogyre acetate.

The aqueous solution is alkalized with a saturated aqueous solution of sodium hydrogen carbonate. The aqueous alkaline phase is extracted with dichloromethane. The organic extracts are dried on anhydrous sodium sulfate. The evaporation of the solvent leaves a colorless oil (quantitative yield). $\frac{\text{20}}{\text{D }}$ The total volume of the product shall be calculated as follows:

(d) Hydrogen sulphate of alpha-tetrahydro-4,5,6,7 thieno-3,2-c) pyridyl-5) (chloro-2 phenyl) methyl dextrogyre acetate (SR 25990 C) and its salts

800 ml of a saturated aqueous solution of sodium bicarbonate is added to a 200 g suspension of SR 25990 B in 800 ml of dichloromethane. After stirring, the organic phase is settled, dried on sodium sulphate and the solvent removed at reduced pressure. The residue is dissolved in 500 ml of acetone cooled in ice and 20,7 ml of concentrated sulphuric acid (93,64 % -d=1,83) is added by drip. The precipitate is washed by filtration and washed with 1000 ml of acetone and then dried in a vacuum oven at 50°C.

This results in 139 grams of analytically pure white crystals with a melting point of 184°C. The following table shows the total number of samples of the product:

EXAMPLE 2 - Salts of alpha-tetrahydro-4,5,6,7 thieno-3,2-c pyridyl-5) (chloro-2 phenyl) methyl acetate of levogyr. (a) Salt of d-camphosulfonic acid-10 (SR 25989 B)

Evaporate the solvent from the acetone solution in example 1-b after separation of SR 25990 B.

The ether phase is decanted, the aqueous phase is cooled to 5°C and alkalized with a saturated aqueous solution of sodium bicarbonate, the aqueous phase is extracted with diethyl ether, the ether extracts are joined and dried on anhydrous sodium sulfate, and the ether phase is removed.

Evaporation leaves behind an oil which is purified by filtration on a bed of silica (elevent: diethyl ether).

A colourless oil is obtained, consisting of a mixture of approximately 65% levogyre enantiomer and 35% dextrogyre enantiomer, proportions determined by RMN1H spectroscopy (60 MHz) with the addition of a chiral rare earth complex.

The resulting mixture is dissolved in 70 ml of acetone in the form of 16.66 g (0.0517 moles) and 7.77 g (0.0310 moles) of camphosulfonic acid-10 dextrogyre monohydrate is added. The homogeneous medium is left overnight at room temperature. The resulting crystals are filtered, washed with acetone and dried (Rdt.

The resulting crystals are dissolved in the minimum acetone at reflux (60 ml). The precipitate obtained after cooling at room temperature is filtered, washed with acetone and dried. $\frac{\text{20}}{\text{D }}$ - = 24.85 (C = 1.79 g/100 ml, methanol) and the other

(b) Alpha-tetrahydro-4,5,6,7 thieno-3,2-c) pyridyl-5) (chloro-2 phenyl) methyl levohyre acetate.

The aqueous solution obtained is alkalized at 5°C with an aqueous solution saturated with sodium hydrocarbonate. The aqueous alkaline phase is extracted with dichloromethane. The organic solution is dried and the solvent is evaporated. A colorless oil is isolated (quantitative yield).

Oil [α] $\frac{\text{20}}{\text{D }}$ - = 50,74 (C = 1,58 g/100 ml, methanol) and the other

(c) Chloride of alpha-tetrahydro-4,5, 6,7 thieno-3,2-c) pyridyl-5) (chloro-2 phenyl) methyl levogyre acetate (SR 25989 A) and its salts

Prepared in the manner described in Example 1d. Efficiency: 94%.

The test chemical is a white crystal, F = 117°C, [α] $\frac{\text{20}}{\text{D }}$ - = 62.56 (C = 1.80 g/100 ml of methanol) and the other

(d) Hydrogen sulphate of alpha-tetrahydro-4,5,6,7 thieno-3,2-c) pyridyl-5) (chloro-2 phenyl) methyl levohyre acetate (SR 25989 C) and its salts

70 g (0,126 moles) of camphosulfonate SR 25989 B obtained in accordance with (a) above is basicised with an aqueous solution saturated with sodium bicarbonate in the presence of dichloromethane.

The residue in 300 ml of acetone is added 7.2 ml (0.126 mole) of concentrated sulphuric acid, after stirring, filtered and washed with acetone to obtain 47.8 g of white crystals. The temperature of the water is not less than 180 °C. $\frac{\text{20}}{\text{D }}$ The following table shows the results of the analysis: Analysis (C,H,N) is consistent.

The pharmaceutical industry

The anti-aggregating activity and toxicity of the new compounds were compared with those of the racemic mixture described in French patent No 82.12599 (publication No 2.530 247).

The results of this study, which highlights another advantage of the invention, are described below, namely that the salts of the dextrogyre isomer have a better therapeutic indication than the salt of the racemic mixture, as the levogyre isomer has virtually no antiplatelet activity and is significantly more toxic than its dextrogyre counterpart.

The antiplatelet and antithrombotic activities of the compounds have been studied in rats by conventional methods.

Activity on platelet aggregation induced by ADP or collagen was determined ex vivo.

Products in ethanol solution (200 mg/ml) diluted in water containing gum arabic (5% w/v) were administered orally to batches of 5 female rats of CD-COBS strain weighing 250-300 g at a rate of 10 ml suspension per kilogram two hours before blood sampling.

Samples of sodium citrate in 3.8% aqueous solution (1 vol/9 volumes of blood) were taken from animals anesthetized with diethyl ether by puncture into the abdominal aorta.

Aggregation is induced by the addition of 2 l of agglutinating solution to 400 l of platelet rich plasma. l The agglutinating solutions used were: an aqueous solution of ADP marketed by Boehringer Mannheim in a concentration of 500 M (final concentration 2.5 M), and a collagen solution marketed by Sigma (type 1) in 0.25 g/100 ml in 3% acetic acid (v/v) (final concentration 12.5 g/ml).

The aggregation of platelets was followed, according to the method described by G.V.R. Born in Nature 194 p. 927 (1967), with a Coultronics R aggregator at 37°C, with a shaking rate of 900 rpm.

For ADP aggregation, the aggregator provides a curve representing platelet aggregation as measured by a change in optical density. The height of this curve is defined as aggregation height. The percentage of aggregation is the ratio of the measured aggregation height to the height corresponding to 100% of aggregation x 100. The percentage of inhibition is determined by the ratio: - What? The results obtained on ADP aggregation for the racemic mixture hydrochloride (PCR 4099), the hydrogen sulphates of the isomers dextrogyre (SR 25990 C) and levogyre (SR 25989 C), on the one hand, and PCR 4099 and the hydrochlorides of the isomers dextrogyre (SR 25990 A) and levogyre (SR 25989 A), on the other hand, are shown in Table I and demonstrate that the levogyre isomer is inactive and that the dextrogyre isomer is at least as active as the racemic isomer. TABLEAU I

PRODUIT	DOSE mg/Kg P.O	QUANTITE de base administrée	% D'AGREGATION	% INHIBITION	P**
Témoins			42,4 +/-1,5
PCR 4099 (racémique)	4,48	3,84	29,8 +/-2,4	30	0,01
	8,97	7,69	17,2 +/-2,2	59	0,001
	17,9	15,38	11,1 +/-2,3	74	0,001
SR 25989C	20	15,38	41,0 +/-1,5	3	n.s
	40	30,76	37,1 +/-1,7	13	n.s
SR 25990C	1,25	0,96	39,4 +/-1,3	7	n.s
	2,5	1,92	28,4 +/-2,3	33	0,01
	5	3,84	14,0 +/-1,6	67	0,001
	10	7,69	8,5 +/-1,6	80	0,001

TABLEAU I

* moyenne des résultats +/- écart standard moyen (ESM)

** test de Student

For collagen aggregation, the inhibition percentage is the difference between the slopes of the curves representing the change in optical density with respect to time for the control and the test product divided by the slope for the control, multiplied by 100. The results in Table II further demonstrate that only the dextrogyre isomer is active, whereas salts have comparable activities. TABLEAU II

PRODUIT	DOSE mg/Kg P.O	QUANTITE de base administrée	PENTE	% INHIBITION	P**
Témoins			4,8 +/-0,3
PCR 4099 (racémique)	4,48	3,84	3,6 +/-0,2	25	0,05
	8,97	7,69	2,7 +/-0,3	44	0,01
	17,9	15,38	1,5 +/-0,3	69	0,001
SR 25989C	20	15,38	4,3 +/-0,2	10	n.s
	40	30,76	4,0 +/-0,2	17	n.s
SR 25990C	1,25	0,96	4,5 +/-0,3	6	n.s
	2,5	1,92	4,1 +/-0,2	15	n.s
	5	3,84	2,3 +/-0,1	52	0,001
	10	7,69	1,7 +/-0,3	65	0,001

TABLEAU II

** test de Student

n.s non significatif.

The antithrombotic activity of the compounds has also been studied in the test for thrombosis of the veins on the spleen described by Kumada T. et al. in Thromb. Res. 18 p. 189 (1980).

Female rats of the same type as before, at a rate of 10 animals per batch, were anesthetized with diethyl ether and their vena cava isolated after abdominal incision.

A metal rod consisting of a 21 mm long toothpaste drill, marketed by Dyna (France) size No 30, was inserted into the light of this vein just below the renal bifurcation and down to the iliac veins without damaging the wall. The rod is implanted on a length of 19 to 20 mm and extends 1 mm beyond the outside and the closed abdomen.

The thrombuses form quickly, and five hours later, under pentobarbital anesthesia, the abdomen is reopened and ligatures are placed up and down the spleen, which is removed after longitudinal incision of the vein and the isolated thrombus is weighed.

The results in Table III show that the levogyre isomer is inactive in this test, unlike the dextrogyre isomer and the racemic. TABLEAU III

PRODUIT	DOSE mg/Kg P.O administrée	QUANTITE de base	POIDS des thrombi *	VARIATION %	P**
Témoins			3,9 +/-0,3
PCR 4099 (racémique)	4,48	3,84	2,17 +/-0,24	44	0,001
	8,97	7,69	1,39 +/-0,15	64	0,001
	17,9	15,38	1,00 +/-0,19	74	0,001
SR 25989C	40	30,76	4,17 +/-0,42	-7	n.s
SR 25990C	1,25	0,96	3,11 +/-0,32	20	n.s
	2,5	1,92	2,29 +/-0,22	41	0,01
	5	3,84	1,71 +/-0,24	56	0,01
	10	7,69	1,26 +/-0,19	67	0,01
	20	15,38	1,20 +/-0,13	69	0,01

TABLEAU III

* = poids des thrombi en mg +/- écart standard moyen

P = test U de Kruskal-Wallis

For the toxicological study, the compounds were administered orally as suspension in the same volume of water with 10% (w/v) gum arabic to batches of 10 female Sprague Dawley rats weighing 120-135 grams on an empty stomach.

The deaths were determined 14 days after administration of the test product. The lethal doses determined at that time, expressed in terms of the salt administered, are given in Table IV. These results show that the toxicity of the racemic mixture is close to that of the levogyre isomer, while the dextrogyre isomer is significantly less toxic, and that the toxicity depends on the nature of the acid used for salivation. - What? TABLEAU IV

PRODUITS	D 10	D 50 (   )	D 90	DOSE LETHALE ABSOLUE
PCR 4099 (racémique)	1318	1615 (1448-1747)	1979	2000
SR 25989 A	1259	1702 (1443-1797)	2299	2000
SR 25990 A	3055	4316 (3569-5705)	6137	5000
SR 25990 C	2257	2591 (2372-2805)	2974	4000
(   ) = intervalle de confiance

Claims (4)

1. Process for preparing the hydrogen sulphate of the dextrorotatory isomer of alpha-(4,5,6,7-tetrahydrothieno[3,2-c]pyrid-5-yl)(2-chlorophenyl)methyl acetate, characterised in that racemic alpha-(4,5,6,7-tetrahydrothieno[3,2-c]pyrid-5-yl)(2-chlorophenyl)methyl acetate is converted into a salt in acetone with laevorotatory 10-camphosulphonic acid, successive recrystallisations of the salt are carried out in acetone until a product having a constant rotatory power is obtained, then the dextrorotatory isomer is liberated from its salt by a base and converted into a salt with sulphuric acid.
2. Process for preparing a pharmaceutical composition, characterised in that a compound obtained according to claim 1 is put into a pharmaceutically acceptable form.