HU189660B - Process for the separation of enantiomers of 2,3,3a,4,5,6-hexahydro-1h-indolo/3,2,1-de/ 1,5-naphthyridine and for preparing pharmaceutical compositions containing such compounds - Google Patents

Abstract

Enantiomers of the title compound of the formula: <IMAGE> which have hitherto not been characterised specifically, are isolated from their mixtures by preparing addition salts with an enantiomer of a chiral acid. The diastereoisomers obtained are separated by fractional crystallisation and then converted back to the form of the base or of a pharmaceutically acceptable acid addition salt. The enantiomers of the above named compound are useful in therapy as they possess an antianoxic activity.

Description

FIELD OF THE INVENTION The present invention relates to the preparation of enantiomers of 2,3,3a, 4,5,6,1H-hexahydro-4-indolo [3,2,1-de] 1,5-naphthyridine and their pharmaceutically acceptable acid addition salts and pharmaceutical compositions containing these compounds.

French Patent No. 2,494,693 discloses, inter alia, the racemate of 2,3,3a, 4,5,6,1H-hexahydro-4-indolo [3,2,1-de] 1,5-naphthyridine of formula (I).

The structure of this compound has the carbon at the 3a position asymmetrically substituted, resulting in the presence of two optical antipodes.

Each of the enantiomers may be isolated by intermediate preparation of an addition salt of an optically active antipode of a chiral acid, preferably an optically active antipode of 2,3-bis (4-methylbenzyloxy) succinic acid.

The conversion is carried out at a temperature between room temperature and the reflux temperature of the solvent, starting from a mixture of the enantiomers of formula (I), for example the racemate dissolved in a suitable solvent such as an alcohol, preferably ethanol.

The two diastereomeric salts thus obtained are then separated by fractional crystallization.

After isolation, the salts can be separated from the chiral acid and then converted to the addition salt with any physiologically acceptable acid.

The enantiomer of formula (I) may also be prepared by starting from an enriched mixture of one of the enantiomers provided by the mother liquor of a mixture of diastereomeric salts. The chiral acid used to separate such an enriched mixture is then the antipode with which the above mixture was obtained.

The purity of the optical isomers of formula (I) is, for example, by gas-phase (CFV) or liquid-phase (CLHP) chromatography as described by Halpern, B., in Handbook of Derivatives For Chromatography, p. 457476, by Blau, K. and King, G. , Heyden Publisher). _x

The process involves condensing the isomer of formula (I) with the (-) - menthyl chloroformate of formula (II), which is prepared according to the procedure described in the above publication, to give the corresponding menthyl carbamate. The latter, when analyzed by gas or liquid chromatography, gives a single peak if only one of the anantiomers of formula (I) is present and two if the separation is incomplete.

The following examples illustrate the separation of the isomers of the compound of formula (1) and their purity control.

Example 1 (+) - 2,3,3a, 4,5,6,1H-hexahydro-ondolo [3,2,1-de] 1,5-naphthyridine and its methanesulfonate salt

a) Formation of diastereomeric salts with (+) - 2,3-bis (4-methylbenzoyloxy) succinic acid

To a 2 liter Erlenmeyer flask equipped with a magnetic stirrer, 34.41 g of racemic 2,3,3a, 4,5,6,1H-hexahydro-indolo (3,2,1-de) 1,5, oil-consistency, in a 2 liter Erlenmeyer flask naphthyridine base, prepared according to Example 1 of Application No. 8 024 717 and dissolved in 250 ml of anhydrous ethanol, is slowly dissolved in 250 ml of anhydrous ethanol at room temperature under constant stirring. 2,3-Bis (4-methyl-benzoyloxy) succinic acid is added, crystallization begins before the addition is complete, the mixture is slowly warmed to reflux and 200 ml of ethanol are added to dissolve the crystals completely. After that, the insoluble crystals are removed by filtration, the baby is reheated and allowed to cool to room temperature. and dried in vacuo at 50 ° C to give 43.56 g of white crystals.

(b) Reclassification

The crystals obtained above were recrystallized several times from 95% ethanol. At each recrystallization, about 0.25 g of the crystal is removed for sampling and the base is liberated with a mixture of dilute ammonium hydroxide solution and chloroform to determine rotation. After recrystallization four times, the base rotates at about [α] D = 63.29 ° (c = 1, methanol).

The solubility of the salt from which the base was liberated was (a) p = +84.51 '(c = 1, methanol).

(c) methanesulfonate

Into a 250 ml Erlenmeyer flask equipped with a magnetic stirrer was placed a mixture of 5.78 g of salt prepared in (b), 100 ml of 10% ammonium hydroxide solution and 100 ml of ethyl acetate. After stirring for a few minutes, the organic phase is separated off, the aqueous phase is re-treated with 100 ml of ethyl acetate, the combined organic phase is washed with water, dried over anhydrous magnesium sulfate, concentrated by rotary evaporation and finally dried under vacuum. This gives 2.96 g of a base as a mixture of white crystals and oil.

Subsequently, in a 250 ml flask equipped with a magnetic stirrer, the base obtained above is dissolved in 60 ml of ethyl ether and an equivalent amount of 1.34 g of methanesulfonic acid dissolved in 15 ml of anhydrous ethanol is added. Rapid crystallization soon begins. After stirring for another half hour at room temperature, the crystals were filtered off on a frit and dried in vacuo at 50 ° C. After recrystallization from 60 ml of warm, anhydrous ethanol, it was allowed to stand overnight at room temperature, the next day the crystals formed were filtered off and dried in vacuo. This gives 3.03 g of methanesulfonate crystals, m.p. 222-224 ° C.

The rotation, [αρ ^υ η = + 21.58 ° (c = 2, methanol).

Elemental analysis and NMR and IR confirmed the structure of the compound.

(d) Chemical purity check

In a 100 ml flask equipped with a magnetic stirrer and a plug of calcium chloride, 140 mg of (+) - 2,3,3a, 4,5,6,111- in 10 ml of ethyl acetate and 66.7 mg (equivalent) of triethylamine are added. The hexahydroindolo [3,2-1-de]. 1,5-naphthyridine and the contents of the flask were cooled in an ice-water bath. An equivalent amount of (-) - rentyl chloroformate (6.6 10 ⁴ mol / ml) in toluene (1 ml) was added and stirred for 15 minutes at 0 ° C and then for 20 minutes at room temperature. The insoluble material is then removed by filtration, the organic phase is washed with water and then with 5% aqueous sodium bicarbonate solution, then again with water, then dried over anhydrous magnesium sulfate, concentrated in a rotary evaporator and concentrated in vacuo. This gave 251 mg of a yellow oil which was reconstituted with ethyl acetate at a concentration of 0.1 mg / ml. The gas phase chromatogram of this solution shows a single peak.

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Example 2 (-) - 2,3,3a, 4,5,6,1H-Hexahydro-ondolo [3,2,1-de] 1,5-naphthyridine

a) Liberating the base from the mother liquor of the recombinant isomer in the form of 2,3-bis (4-methylbenzoyloxy) succinate

The filtrate from the first recrystallization from Example 1b) was dried in a rotary evaporator, then 200 ml of 10% ammonium hydroxide solution and then 200 ml of ethyl acetate were added. After stirring for a few minutes, the mixture was filtered through a fritted glass, the organic layer was separated, washed with water, dried over anhydrous magnesium sulfate, and concentrated in a rotary evaporator and then in vacuo. This gives about 9.11 g of an orange oil.

b) Formation of diastereomeric salts with (-) - 2,3-bis (4-methylbenzoyloxy) succinic acid

In a 500 mL Erlenmeyer flask, fitted with a magnetic stirrer, cooled and placed in an oil bath, 9.08 g of the compound obtained in Example 2, previously dissolved in 65 mL of anhydrous ethanol, is added after 1 hour at room temperature. A solution of 3-bis (4-methylbenzoyloxy) succinic acid in 65 ml of anhydrous ethanol. Crystal formation it? will begin before the addition is complete. The mixture is then slowly heated to reflux and 140 ml of anhydrous ethanol are added to completely dissolve the crystals. The insoluble crystals are then removed by filtration, the filtrate is heated and then allowed to cool to room temperature. The crystals formed are filtered off on a glass slide and dried in vacuo at 50 ° C. 11.2 g of white crystals are obtained.

(c) Recrystallization

The crystals obtained above were recrystallized several times in 95% ethanol. After each recrystallization an amount of about 0.25 grams is taken from which the base is liberated using ammonium hydroxide solution and chloroform to determine rotation, after five recrystallizations the base has a rotation of about -63.38 ° (c = 1.39, methanol), and the salt has a rotation of about [a] n = -89.21 ° (c = 1, methanol)

Example 3 (-) - 2,3,3a, 4,5,6,1H-phexahydroindolo [3,2,1-de] 1,5-naphthyridine and its methanesulfonate salt

a) Formation of diastereomeric salts of (-) - 2,3-bis (4-methylbenzoyl) oxy) -b-wafers

In Example 1a), 34.41 g of an oily base dissolved in 250 ml of anhydrous ethanol are treated with 32.78 g of anhydrous (-) - 2,3-bis (4-methylbenzoyloxy) succinic acid in 250 ml of anhydrous ethanol. The reaction mixture is heated to reflux and 220 ml of anhydrous ethanol are added, the filtrate is filtered off, and the filtrate is filtered off with suction. The crystals were filtered off and dried in vacuo at 50 ° C to give crystals weighing 35.47 g.

(b) Recrystallization

Following the procedure of Example 1c, after four recrystallization operations, the base had a [α] D 20 = -60.5 ° (c = 1.15 in methanol), a salt of [α] D = -87.9 °, ( c = 1, methanol).

c) Methanesulfonate

Into a 250 ml flask equipped with a magnetic stirrer was added 1.15 g of 20 ml of ethyl ether in a solution of the dissolved (oil) enanthome and rapidly equivalent to 0.52 g of methanesulfonic acid dissolved in 6 ml of ethanol was added. Crystals formed which were further stirred for half an hour before drying at 50 ° C under vacuum. It was then recrystallized from 19 ml of anhydrous ethanol to give 1.12 g of the methanesulfonate salt, m.p.> 219-221 ° C.

lg Rotation: [0 (1-¾ = -22.0 (c = 1, methanol)).

Elemental analysis as well as NMR and IR studies confirm the structure of the compound.

(d) Chemical purity check

The gas phase chromatogram of menthyl carbamate obtained in Example 1d gives a single peak curve.

The compounds of the present invention were subjected to pharmacological tests to ascertain their therapeutic importance.

Low pressure hypoxia

The CDI mouse strain was maintained in an oxygen-deprived atmosphere and was prepared with a partial vacuum of 5.25% oxygen at 190 mmHg.

The survival time of the animals was recorded. This time is increased by the action of agents to enhance oxygenation of tissues, especially brain cells. The test compounds are administered intraperitoneally 10 minutes prior to the experiment in multiple doses! >> k into the body of animals. The percentage increase in survival time was calculated relative to untreated animals. The median dose (DAjqq) to extend the survival time by 100% was plotted. The DA.qq values ranged from 21 to 23 mg / kg and the DA <- _n values ranged from 7 to 10 mg / kg.

General ischemia experiment in mice

The survival time of the animals was measured after injection of 0.1 ml of saturated magnesium chloride solution into the tail vein. Inhibition of cardiac arrhythmia caused by injection causes ischemia of the brain. Survival time is the time elapsed between the administration of magnesium chloride and the last respiratory movement of each mouse, as the last sign of central nervous system function.

The survival time of animals treated intraperitoneally with the ^v * pharmaceutical compositions containing the compounds of the invention 10 minutes prior to injection of inagnesium chloride was compared with the survival time of the animals receiving the vehicle alone.

The animals were treated in groups of ten, and graphically determined as mg / kg body weight of the drug based on a plot of the mean results of each group. effective dose, which prolonged the survival time by 3 seconds (DEj).

An increase in survival time of 3 seconds is also statistically significant and reproducible.

The compounds of the invention have a DE of between 7 and 10 mg / kg.

The pharmacological examination of the compounds of the present invention shows that these compounds have anti-anoxic activity in the treatment of disorders of consciousness, particularly cerebral blood vessels, and behavioral disorders caused by cerebral arteriosclerosis, and in the treatment of metabolic-related depressions and disorders of the brain.

The invention further relates to all pharmaceutical compositions containing the enantiomers of the compounds of formula (I) or salts thereof according to the invention as active ingredient, together with a conventional excipient, which enables the compounds to be used both orally and parenterally.

The formulations may be administered either orally or parenterally.

The daily dosage range is from 10 to 100 mg. Comparative pharmacological studies were performed with the methanesulfonic acid salts of the compounds of Examples 1 and 1 and 3 of British Patent Application 2,037,889. The results of the studies are shown in the table.

Two types of assays were performed as described below

As described in points 1 and 2.

1.) One of the two assays mentioned above was used to test the inhibitory effect of the test compounds on 'H-tryptamine receptor binding to tryptamine receptors in vitro. This test was performed as follows:

Male weighing 150-200 g. Sprague-Dawley rats were used. After decapitation, the brain was excised and the cortex dissected. All further operations were carried out at 0 to 4 ° C unless otherwise stated. The tissue was homogenized in a buffer containing 50 mM Tris-hydroxymethylaminoaminoethane hydrochloride, 5 mM ascorbic acid and 10 mM pargyline. Tissue Hormonisate was washed twice by centrifugation for 10 minutes (48,000xg) and resuspending the solid residue in fresh buffer. The resulting solid was again fevett ÜK such that 50 mg of the wet material was suspended in 1 ml of fresh buffer to give a concentration of _o the initial sample, and then pre-incubated at 37 ° C for 5 minutes. This membrane preparation was incubated with 1 H-tryptamine (New England Nuclear with a specific activity of 39.5 Ci / mmol) diluted to a final volume of 1 ml in the buffer, in the presence or absence of unlabeled drug. After incubation at 0 ° C for 60 minutes, 4 ml of ice-cold buffer was added to the incubated mixture, followed by rapid filtration through a Whatman GF / B porous final filter. The filters were then washed three times with 4 ml portions of ice-cold buffer, dried and placed in counting vials. The membrane-bound radioactivity retained by the filter was determined by liquid scintillation spectroscopy after extracting the filter with toluene-PPO-POROP-based scintillation fluid. The amount of non-specific binding to the tissue and to the filter was determined as the residual binding observed in the presence of 10 mM of unlabeled tryptamine during incubation. Specific binding of '1 H-tryptamine to tryptamine receptors was calculated by subtracting non-specific binding on the filter in the absence of unlabeled compound or from the total amount of radioactivity retained in the presence of the compound being studied. Concentration-dependent inhibition of the specific binding of 1 H-tryptamine by the studied compound was determined by linear regression calculation based on the conventional graph and least squares method. The results are expressed as IC ₅₀ , that is to say concentrations of the active ingredient which inhibit the binding of 1 H-tryptamine by 50%.

2. The second assay that measured binding of anti j -adranorecepírokhoz ^J H-prazosin effect of the compounds in vitro. This test was performed as follows:

Male Sprague-Dewley rats weighing 150-200 g were used. After decapitation, the brain was excised and the cortex dissected. All other procedures were performed at 0 ° C to 4 ° C unless otherwise stated. The tissue was homogenized with polytrone in 50 mM Tris (hydroxymethyl) aminoethane hydrochloride buffer. The tissue homogenate was washed twice by successive centrifugation (30,000 x g) for 10 minutes such that 10 mg of wet weight of each pellet obtained after each centrifugation was resuspended in 1 ml of buffer to obtain an initial concentration of sample. This membrane preparation was incubated with ³ H-prazosin (a 25.4 Ci / mm specific activity formulation of New England Nuclear) in a final volume of 1.05 ml buffer with or without labeled drugs. After incubation for 30 minutes at 25 ° C, the incubated mixture was diluted with 3 mL of ice-cold buffer and rapidly filtered through Whatman GF / B porous glass filters. The filters were washed successively with 2 mL of ice-cold buffer, dried and counted in counting vials. The radioactivity suppressed by the filter was determined by liquid scintillation spectroscopy using a toluene-PPO-POROP-based scintillation fluid. Non-specific binding of < ^{RTI ID} = 0.0> llprazosin </ ^RTI > to tissue and filter was determined as the residual binding observed during incubation in the presence of 10 mM phentolamine.

Specific binding to 1 H-prazosin-adrenergic receptors was calculated by subtracting the non-specific binding from the radioactivity retained by the filter in the absence of unlabeled compound or in the presence of the test compound. The concentration dependence of the effect of the test compound inhibiting the binding of 1 H-prazosin was determined by conventional graphical and least squares linear regression calculations. The results are determined by combining ICjq values, i.e. the concentrations of the drug, which is specific for ^J H-prazosin binding was inhibited 50% of total.

In the table, results are shown for each compound in the form ICjq, inhibitory effect on H-tryptamine (A) signal, the inhibitory effect of ^ for H-prazosin (B) signal, and ^J H-prazosin and ^ Ratios of JCjq for H-trintamine (B | A).

When it ICjq value for the H-pjazosin binding than the value of the inhibitory effect of 9-adrenergic receptors ₅₀ value IC for the ^J H triptaniinra is exerted on triptaminerg receptor inhibitory action as the value of these divided and as effects on triptaminerg neurotransmission szelektivitásaként evaluated. The compound of the first example of the present invention is about 3.5 times more selective than the compound of Example 1 of British Patent 2,087,889 and ta-41.

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The compound of Example 3 of the present invention is nearly 30 times more selective. Spreadsheet 5 3 ^A B / A Compound H-tryptamine H-Prazosin bonding bonding ic ₅₀ 0jM) IC ₅₀ (µM) 10 No. 2,087,889. British Patent Specification Example 1 0,085 9.9 116 cross-chemical lete (racemate) 15 Example 1 0.154 60.0 390 (<) enantiomeric Gyula 20 Example 3 shows .752 10.5 14 rint (-) enantio- mer compound

Claims

Claims (2)

Claims A process for the separation of the enantiomers of 2,3,3a, 4,5,6,1H-hexahydroindolo [3,2,1-de] -1,5-naphthyridine and for the preparation of pharmaceutically acceptable acid addition salts comprising: The racemate of the base of formula (I) or a mixture of the base enantiomers of formula (I), which is the mother liquor of a prior separation, is reacted with one of the enantiomers of 2,3-bis (4-methylbenzoyloxy) succinic acid to fractionate the diastereomeric salt thus obtained. crystallization, liberating the base from the diastereomeric salt and, if desired, converting the resulting base into a pharmaceutically acceptable acid addition salt. Process for the preparation of a pharmaceutical composition, characterized in that the enantiomer of 2,3,3a, 4,5,6,11H4ndolo [3,2,1-de] -naphthyridine of formula (I), or a pharmaceutically acceptable acid addition salt thereof, and its salt mixed with the usual excipients used in pharmaceutical technology to form a pharmaceutical composition.