Benzopyrane, pharmaceutical composition and method of treatment


(57) Abstract:

The invention relates to certain CIS - and TRANS-benzopyrane having substituted benzamide in position C-4, and to their use for the treatment and/or prevention (prophylaxis) of certain CNS disorders. 3 c.p. f-crystals.

This invention relates to new compounds, to processes for their preparation and to their use as therapeutic agents.

In the published application for the European Patent N 0126311 disclosed substituted benzopyrano compounds with the ability to lower blood pressure and including 6-acetyl-TRANS-4-4-(4-perbenzoate)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3-ol.

In the documents EP-A-0376524, EP-A-0205292, EP-A-0250077, EP-A-0093535, EP-A-0150202, EP-A-0076075 and WO/89/05808 (Beecham group PLC) also described some benzopyrane derivatives, which have antihypertensive activity.

Some benzopyrane derivatives, which are believed to possess antihypertensive activity are described in EP-A-0350805 (Biersdorf), EP-A-0277611, EP-A-0277612, EP-A-0337179 and EP-A-0355565 (Hoechst Aktiengesellschaft); EP-A-0466131 (Nissan chemical industries Ltd), EP-A-0339562 (Ashitomi pharmaceuticals), EP-A-415065 (E. Merck), EP-A-450415 (Squib intermediates, useful to obtain the compounds described in the aforementioned patent applications.

In EP-A-0139992 (Beecham group PLC) describes some benzopyrane derivatives with CIS-isomerism in positions 3 and 4, which have antihypertensive activity. In PCT/GB92/01045 (SmithKline Beecham PLC) describes some corbusierinspired, pyrenopeziza and tetrahydronaphthalene, in which the substituents in the 3 and 4 positions are in the TRANS configuration to each other. For these compounds also showed the presence of anxiolytic and anticonvulsant activity.

In PCT/GB93/02512 and PCT/GB93/02513 described further group of compounds with anticonvulsant activity. These patent applications were not published at the priority date of this application.

Unexpectedly, it was found that some of these compounds have anticonvulsant activity and are therefore useful in the treatment of epilepsy and, as is also considered to be useful for the treatment and prevention of anxiety, manic syndrome, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the conditions associated with rejecting a number of harmful substances, disease, Parkinson asevich disorders and/or anxiety and/or aggressive conditions.

Accordingly, the present invention provides the following connections:

CIS-6-acetyl-4S-(2,3-dichloraniline)-3,4-dihydro-2H-1 - benzopyran-3S-ol,

TRANS-6-acetyl-4S-(2,3-dichloro-4-perbenzoate)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4S-(3,5-dichloraniline)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4S-(3,5-differentiating)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4-(2 - thiophenecarboxylate)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol,

TRANS-6-acetyl-4S-(3-thiophenecarboxylate)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4S-(2,5-dichloro-3 - thiophenecarboxylate)-3,4-dimethyl-2H-1-benzopyran-3R-ol,

CIS-6-acetyl-4S-(2,5-dichloro-3-thiophenecarboxylate)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3S-ol,

TRANS-6-acetyl-4S-(2,3,5-trichlorosilane)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4S-(2,3,4-triptoreline)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4S-benzoylamine-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol,


TRANS-6-acetyl-4S-(5-fluoro-2-methylbenzylamino)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-benzoylamino)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3-ol,

CIS-6-acetyl-4S-(2,3-dichloro-4 - perbenzoate)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3S-ol,

CIS-6-acetyl-4S-(3-chloro-4-perbenzoate)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol,

TRANS-6-acetyl-4S-(2-fluoro-5 - pyridylcarbonyl-amino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran - 3R-ol,

TRANS-6-acetyl-4-(2-phenoxyethylamine)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3-ol,

TRANS-7-acetyl-4-(4 - perbenzoate)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol,

TRANS-6-acetyl-4S-(2-chloro-5-fluoro-2-thiophenecarboxylate)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3R-ol,

CIS-6-acetyl-4S-(2 - chloro-5-fluoro-2-thiophencarboxylic-amino)-3,4-dihydro-2,2-dimethyl-2H - 1-benzopyran-3S-ol, CIS-6-acetyl-4S-(2,3,4-triptoreline)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3S-ol

or their pharmaceutically acceptable salts.

Such compounds will be further called compounds of formula (I).

It should be understood that the compounds of formula (I) may have chiral carbon atoms at positions 3 and 4 and can therefore exist as enantiomers. The present invention covers each enantiomer and mixtures thereof, including racemates.

It should also be noted that for different use cases preferred private or individual anytitle to use 3R, 4S and 3S, 4S enantiomers, and after subarachnoid haemorrhage or neural shock preferred 3S, 4S or 3R, 4R enantiomers.

It should also be noted that the compound of formula (I) or its pharmaceutically acceptable salt also include a solvate of such compounds, for example, the hydrate.

The present invention relates also to the compound of formula (I) or its pharmaceutically acceptable salts within the above definitions, which are mostly 3S, 4S or 3R, 4S enantiomeric form, depending on whether the connection is in the CIS - or TRANS-configuration.

For example, the term is mostly 3S, 4S enantiomeric form" means that the enantiomer is 3S, 4S is more than 50% in comparison with the enantiomer 3R, 4R.

More preferably, the specified enantiomer is 3S, 4S was present in quantities greater than 60%, even more preferably the presence of the enantiomer is 3S, 4S more than 70%, even more preferably the presence of the enantiomer is 3S, 4S more than 80%, much more preferably the content 3S, 4S enantiomer more than 90%. It is most preferable that was attended by more than 95% 3S, 4S enantiomer relative 3R, 4R enantiomer.

The purpose of mammals for admission may be made orally, parenterally, sublingually, under the tongue or transdermal.

The amount effective to treat the above disorders, depends on the usual factors such as the nature and severity of the disease to be treated, and the weight of the mammal. However, a single dose usually contains from 1 to 1000 mg, suitably from 1 to 500 mg, for example, in the range from 2 to 400 mg, in particular, 2, 5, 10, 20, 30, 40, 50, 100, 200, 300 and 400 mg of active compound. A single dose usually assigned to receive one or more times a day, for example, 1, 2, 3, 4, 5 or 6 times a day, more often from 1 to 4 times per day, the total daily dose for an adult weighing 70 kg is usually in the range from 1 to 1000 mg, for example from 1 to 500 mg, or about 0.01 to 15 mg/kg/day, and more often from 0.1 to 6 mg/kg/day, for example, 1-6 mg/kg/day.

Very preferably, the compound of formula (I) was administered as a single dose composition, such as a single oral dose, including the sublingual form, rectal, local, or tonic, or parenteral (especially intravenous) composition.

Such compositions are prepared by mixing and appropriately adapt e liquid preparations, powders, granules, pellet, reconstituirea powders, injectable and infusium solutions, or suspensions, or suppositories. Preferred oral compositions, in particular compositions having a certain kind, because they are more convenient for General use.

Tablets and capsules for oral administration are usually presented in the form of single dose and contain conventional excipients such as binders, fillers, diluents, substances used for the manufacture of tablets, lubricants, disintegrators, coloring agents, flavoring agents and wetting agents. Tablets may also have a coating applied well-known methods.

Suitable for use fillers include cellulose, mannitol, lactose and other similar agents. To suitable disintegrators include starch, polyvinylpyrrolidone and derivatives of starch such as sodium starch-glycollate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents or moisturizers include sodium lauryl sulfate.

These solid oral compositions may be prepared by conventional methods of blending, Naya distribution of the active agent throughout the composition in the case when it involves large quantities of fillers. Such operations are well known in practice.

Oral liquid compositions can be, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be a dry product, ready for reconstitution before use with water or a suitable carrier. Such liquid compositions may contain conventional additives such as suspendresume agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, gel, aluminum stearate or hydrogenated edible fats, emulsifying agents, for example lecithin, monooleate sorbitan or gum acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring substances or coloring agents.

Oral formulations of compositions also include traditional prolonged forms, such as tablets or granules having enterocele floor.

Parenteral suspensions, essentially, is prepared in the same manner, except that the connection suspendered in the media instead of dissolution, and before suspendirovanie in a sterile medium is sterilized by exposure to the action of ethylene oxide. Favorable is the inclusion in the composition of a surfactant or humidifier to facilitate uniform distribution of the compounds of the present invention.

As is customary in practice, the compositions are accompanied usually written or printed instructions for use with appropriate medical treatment.

The present invention further precollege syndrome, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the conditions associated with the rejection of the abuse of harmful substances, such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and/or prevention of anticonvulsant drugs; such as epilepsy, Parkinson's disease, psychosis, migraine, cerebral ischaemia, Alzheimer's disease, schizophrenia and/or aggressive state, which includes the compound of formula (I) or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

The present invention also provides a method of treatment and/or prevention of anxiety, manic syndrome, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the conditions associated with the rejection of the abuse of harmful substances, such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and/or prevention of anticonvulsant drugs, such as epilepsy, Parkinson's disease, psychosis, migraine, cerebral ischaemia, Alzheimer's disease, schizophrenia and/or aggressive state, which includes the introduction of suffering from these seski acceptable salt.

According to an additional aspect, the present invention relates to the use of compounds of formula (I) or its pharmaceutically acceptable salt for the manufacture of medicaments for the treatment and/or prevention of anxiety, manic syndrome, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the conditions associated with the abandonment of the use of harmful substances, such as cocaine, nicotine, alcohol and benzodiazepines, disorders that can be treated and/or prevention of anticonvulsant drugs, such as epilepsy, Parkinson's disease, psychosis, migraine, cerebral ischaemia, Alzheimer's disease, schizophrenia and/or aggressive state.

According to another aspect of the present invention relates to pharmaceutical compositions containing a compound of the formula (I) or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

According to further aspect the present invention relates to the use of compounds of formula (I) or its pharmaceutically acceptable salt as a therapeutic agent, in particular for the treatment and/or prevention of anxiety, manic syndrome, depression, R. the use of harmful substances, such as cocaine, nicotine, alcohol and benzodiazepines, disorders which can be subjected to treatment or prevention of anticonvulsant drugs, such as epilepsy, Parkinson's disease, psychosis, migraine, cerebral ischaemia, Alzheimer's disease, schizophrenia and/or aggressive state.

Such compositions can be obtained by the method described previously.

Usually CIS-compounds of formula (I) can be obtained from the corresponding TRANS compounds, the procedures for obtaining them are described in EP-0126311, EP-0376542, EP-205292, EP-0250077, EP-0093535, EP-0150202, EP-0076075, WO/89/05808, EP-0350805, EP-0277611, EP-0277612, EP-0337179, EP-0339562, EP-0355565, EP-A-415065 (E. Merck), EP-A-450415 (Squibb) EP-0466131, EP-A-0482934, EP-A-0296975, JO-2004-791 and WO/89/07103.

CIS-compounds of formula (I) can be obtained using the procedures described or similar to those described in EP-A-0139992.

CIS-compounds of formula (I) can be obtained using the procedures described Borrelli et al. (G. Burrelli et al., Tet. Letters, 31, 3649-3652, 1990) or by using the procedures described by Custom and Vilhauer (U. Quast and E. Villhauer, Eur. J. Pharmacol, Molecular Pharmacology Section 245, 165-171, 1993).

It should be clear that the racemates of the compounds of formula (I) can be separated or purified enantiomeric compounds of formula (I) can be obtained with the use of the="ptx2">

It should also be noted that preferably, the compounds of formula (I) can be obtained in the desired enantiomeric form through education pure chiral epoxide with the use of catalysts and conditions described mainly in WO91/14694 or WO93/17026 with subsequent conversion of epoxides to the desired compound of formula (I) using the procedures described here.

TRANS-compounds of formula (I) can be obtained using the procedures described in PCT/GB92/01045, which is included in the request for information, or TRANS-compounds of formula (I) can be obtained using methods similar to those described in one of the above-mentioned patents.

Using methods similar to those described in the above patent publications and literary sources, were obtained the following compounds.

The following descriptions, examples and results of pharmacological tests illustrate the present invention.

Description 1

2,3-Dichloro-4-fluoro-benzoic acid

2,3-Dichlorobenzoyl (5 g), trichloride aluminum (6,91 g) and acetylchloride (3,85 ml) is heated at a temperature of 130oC for 20 hours in an argon atmosphere. The black residue is poured into a mixture of concentrated HCl-ATA sodium and salt solution and dried over anhydrous magnesium sulfate. Filtration and evaporation to give a black residue which is treated with n-hexane. A solution of n-hexane was filtered and the solvent is evaporated to obtain 2,3-dichloro-4-fortetienne (1.04 g).

Specified raw material (0,69 g), 5% sodium hypochlorite solution (23 ml) and dioxane (20 ml) heated at boiling temperature under reflux for 16 hours. The solution is cooled, evaporated and water is added (20 ml) with conc. HCl to pH 1, and the solid is cooled, filtered off and recrystallized from a mixture of acetone-water to obtain 2,3-dichloro-4-fermenting acid (0.36 g).

Description 2

CIS-8-acetyl-2-(3-chloro-4-forfinal)-3a, 9b-dihydro-4,4-di-methyl-4H-benzo[b]pyrano[4,3-d]oxazol

A solution of TRANS-6-acetyl-4S(H-chloro-4-perbenzoate)-3,4 - dihydro-2,2-dimethyl-2H-benzo[1b] Piran-3R-ol (example 62 in WO 9413656) (3,37 g, 8.6 mmole) in dry dichloromethane (90 ml) and DAST (1,40 ml; 10,59 mmole) was kept at room temperature for 2 days. Evaporation under vacuum, followed by chromatography of the residue on Kieselgel 60 in a mixture of 25% ethylate/n-hexane gives the target oxazoline as a colorless gum (2.50 g; 78%).

1H NMR (CDCl3) : of 1.35 (3H, s), of 1.59 (3H, s), 2,60 (3H, s), 4,80 (1H, d ), to 5.35 (1H, d), of 6.90 (1H, d), to 7.15 (1H, T.),-3S-ol

TRANS-6-acetyl-4S-(2,3-dichloraniline)-3, 4-dihydro-2H-1-benzopyran-3R-ol (0.84 g) was dissolved in dichloromethane (50 ml). Add diethylamino-sulfuretted (DAST) (0,325 ml) and stirred solution over night. The mixture is evaporated to dryness and chromatographic on Kieselgel 60. Gradient elution using CH2Cl2-EtOAc gives a pale yellow gum (0.71 g), which stand in dioxane (20 ml) and water (6 ml) containing 5 standards. H2SO4(3 ml) for 2 days. The mixture is alkalinized with solid sodium bicarbonate, stirred for 2 hours and evaporated to dryness. The mixture is distributed between ethyl acetate and water and the organic layer washed with brine. The organic layer is dried over anhydrous sodium sulfate, filtered and evaporated to obtain a pale yellow gum (0,639 g). Combine the products of the previous reaction (0.3 g) and chromatographic on Kieselgel 60. Elution with a mixture of dichloromethane to 1% methanol gives a white solid (0.47 in), and recrystallization from ethyl acetate-hexane and then from acetone-hexane gives the target connection with so pl. 104-107oC.

[]D= -7,50o(MeOH, c=1,0)

Example 2

TRANS-6-acetyl-4S-(2,3-dichloro-4-perbenzoate-3,4-dihydro-2,2-dimethyl-2R-1-benzop is the W 1) with D-(-)-mandelate TRANS-6-acetyl-4S-amino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran - 3R-ol according to the method, used in example 1. The target compound are obtained by recrystallization from acetone-hexane in the form of crystals with so pl. 205-206oC;

[]2D0= +17,6o(MeOH, C=0,44)

Example 3

TRANS-6-acetyl-4S-(3,5-dichloraniline)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol

So pl. 220oC; []2D0= +22,2o(MeOH, C=1,18)

Example 4

TRANS-6-acetyl-4S-(3,5-differentiating)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol

So pl. 203oC; []2D0= +28,9o(MeOH, C=1,0)

Example 5

TRANS-6-acetyl-4-(2-thiophenecarboxylate)-3,4-dihydro-2,2-dimethyl - 2H-1-benzopyran-3-ol

So pl. 175,5-177oC;

Example 6

TRANS-6-acetyl-4S-(3-thiophenecarboxylate)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol

So pl. 211-213oC;

Example 7

TRANS-6-acetyl-4S-(2,5-dichloro-3-thiophenecarboxylate)-3,4 - dimethyl-2H-1-benzopyran-3R-ol

So pl. 194oC; []2D0= -1,0o(MeOH, C=1,03)

Example 8

TRANS-6-acetyl-4S-(2,5-dichloro-3-thiophencarboxylic-amino)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3R-ol

Yellowish-brown foam.

NMR (CDCl3) : of 1.40 (3H), and 1.54 (3H, to 2.06 (1H, d), a-3.84 (1H, DD)), the ceiling of 5.60 (1H, DD. ), 6,92 (1H, d), 7,22 (1H, sm.), 7,30 (1H), a 7.85 (1H, DD)), of 7.96 (1H).


Example 10

TRANS-6-acetyl-4S-(2,3,4-triptoreline)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3R-ol

So pl. 167oC; []2D0= +3.0Vo(MeOH, c=0,945)

Example 11

TRANS-6-acetyl-4S-benzoylamine-3,4-dihydro-2,2-dimethyl - 2H-1-benzopyran-3R-ol

So pl. 188-191oC;

Example 12

TRANS-6-acetyl-4S-(3-iodobenzylamine)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3R-ol

So pl. 171-173oC; []2D0= +15,5o(MeOH, C=1,00)

Example 13

TRANS-6-acetyl-4S-(5-fluoro-2-methylbenzylamino)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3R-ol

So pl. 194-196oC;

Example 14

TRANS-6-acetyl-4S-(5-chloro-2-methoxybenzylamine)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3R-ol

So pl. 191-194oC;

Example 15

TRANS-6-acetyl-4-(2,3-dimethylbutylamino)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3-ol

So pl. 188-189oC;

Example 16

CIS-6-acetyl-4S-(2,3-dichloro-4-perbenzoate)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3S-ol

So pl. 130oC;

Example 17

CIS-6-acetyl-4S-(3-chloro-4-perbenzoate)-3,4-dihydro - 2,2-dimethyl-2H-1-benzopyran-3S-ol

The solution oxazoline (D2) (2.5 g; 668 mmole) in 1,4-dioxane (45 ml), water (15 ml) and 5 standards. sulfuric acid (6 ml) was kept at room temperature is 2 hours. The mixture is concentrated under vacuum and then distributed between ethyl acetate and water. The organic layer is washed with water, brine and dried (Na2SO4).

Evaporation under vacuum gives a pale yellow gum, which chromatographic on Kieselgel 60 in a mixture of 25% ethyl acetate/n-hexane. Combining the appropriate fractions followed by recrystallization from a mixture of acetone/n-hexane gives the target compound as white crystals (1.20 g; 46%).

So pl. 151-153oC;

Found: C-61,04; H-4,91; N-3,86. For C20H19ClFNO4calculation: C-61,31; H-4,89 and N IS 3.57%

v max (KBr): 3360, 3310, 2950, 1680, 1640, 1260 and 840 cm-1< / BR>
1H NMR (CDCl3) : of 1.40 (3H, s), and 1.56 (3H, s), 2,12 (1H, d, ex D2O) of 2.50 (3H, s), a-3.84 (1H, DD)), 5,62 (1H, DD)), 6,91 (1H, d), to 6.95 (1H, sm. ), of 7.23 (1H, T.), of 7.75 (1H, m), 7,83 (1H, DD)), a 7.92 (1H, d), of 7.96 (1H, DD.).

m/z: 391 (M+1%), 373 (4), 358 (88), 203 (50), 157 (100).

Example 18

CIS-6-acetyl-4-(2,3,4-triptoreline)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3-ol

NMR (CDCl3) : of 1.41 (3H, s), of 1.55 (3H, s), of 2.50 (3H, s), 3,85 (1H, DD)), 5,67 (1H, DD)), 6,92 (1H, d), 7.03 is-7,20 (1H, m), 7,78-of 7.90 (2H, m), 95 (1H, s).

Example 19

TRANS-7-acetyl-4-(4-perbenzoate)-3,4-dihydro-2,2-dimethyl - 2H-1-benzopyran-3-ol

So pl. 189-193oC;

Example 20

TRANS-6-acetyl ->/BR>TRANS-6-acetyl-4-(2-phenoxyethylamine)-3,4-dihydro-2,2 - dimethyl-2H-1-benzopyran-3-ol

So pl. 208oC;

Compounds of the following examples were obtained using procedures similar to the above.

Example 22

TRANS-6-acetyl-4-(2-chloro-5-fluoro-2-thiophenecarboxylate)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol

Example 23

CIS-6-acetyl-4-(2-chloro-5-fluoro-2-thiophencarboxylic-amino)-3,4 - dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol


Test for social interaction in rats

The compounds of formula (I) or their pharmaceutically acceptable salts can be tested with the aim of further use as a pharmaceutical in accordance with the following procedure.

Potential anxiolytic properties evaluated using a procedure that determines the level of social interaction in rats, which is based on the method originally described by File, 1980, J. Neurosci. Methods 2, 219-238). In this model, anxiolytic means selectively enhance social interaction, independently of any effect on locomotor activity.


Male rats sprag-Share (Sprague-Dawley) (Charles river, UK, 250-300 g) p is ocinum way in groups of 8-16 animals which is administered orally in the amount of 1 ml/kg of various doses of the compounds (1) (1-300 mg/kg) or the media. On the 60th minute after a dose rats are placed in a pair with a male, chosen in accordance with weight and treatment group (this was the first contact of animals) in the box used for the assessment of social interaction under the bright light in unusual circumstances. The box is made of white plexiglass size HH cm with transparent front wall is also made of plexiglass. The floor is divided into 24 equal squares and brightly lit (Suite 115). The time spent in active social communication (seconds), (obnyukhivanie, courtship, walking one behind the other, standing up, carabini up or down, wrestling, biting) is a remote monitoring ("blind method") according to the number of crossed rats squares (as an index of locomotor activity).

Mean and standard error for time spent in social intercourse, and the number of crossed squares is then calculated for each group of treatment, and changes induced by drug, expressed as a percent increase or decrease relative to control values. Statistical comparisons between groups the wow comparisons with Dunnett subsequent assessment of significance by analysis of options. Drug suspended in 1% methylcellulose.

Map test

The test to determine the threshold of development of the maximum electroshock seizure (IEP) in rodents is particularly sensitive to detect anticonvulsant activity ( see Losher W. and Schmidt D. (1988), Epilepsy Res., 2, 145-181). In this model, anti-convulsants the threshold increases induced electric attacks, while proconvulsant reduce the seizure threshold.


Mice (male, Charles river, UK. CD-1 strain, 25-30 g) are distributed randomly in groups of 10-20 animals, and injected them intraperitoneally in an amount of 10 ml/kg of various doses of the compounds (0.3 to 300 mg/kg) or media. Then after 30 or 60 minutes after a dose of mice subjected to the action of a single shock (0.1 sec, 50 Hz sinus wave) supplied through corneal electrodes. The average current value (and the value of standard error) required to induce an epileptic fit at 50% (CC50) mice in the treatment group is determined using the method of "up-down" ("up and down") of Dickson and Muda (Dixon and Mood) (1948) (see Dixon W. J. and Mood A. M. (1948), J. Amer. Stat. Assn., 43, 109-126). Statistical sravnenie and Wilcoxon (Litchfield and Wilcoxon (1949) (see Litchfield, J. T. and F. Wilcoxon (1949), J. Pharmacol. Exp. Ther., 96, 99-113).

For control animals, CC50is usually 14-18 mA. When this first animals in the control group subjected to the action of the current strength 16 mA. If this does not occur tonic seizure, the current value increases next investigated mouse. If there is a tonic spasm, the action of the current is lower, and so on, until, while all animals in the group will not be tested. Calculate the percentage decrease or increase the value CC50for each group compared to control.

Conduct research using generator shock DC Hugo Sachs Electronics (Hugo Sachs Electronic Constant Current Shock Generator with a remote level control shock from 0 to 300 mA, and typically use a step value of 2 mA. The drug is suspended in 1% methylcellulose.


The compound of example 17 increases the threshold of shock on 105% of the oral dose of 10 mg/kg


The compounds of formula (I) or their pharmaceutically acceptable salts can be investigated with the aim of further therapeutic use, using the procedure below.


The test for fear of the AI, in which there are entries (open passages) and relatively anxiogenic (covered walkways) areas. Selective increase the ability to survey open passages after administration of the medicinal product allows to make a conclusion about the presence aksiomaticheskoi steps.


X-maze, raised on 70 cm above the floor, consists of two closed aisles size: 45 cm (length) x 15 cm (width) x 10 cm (height) and two outdoor walkways size: HH cm, arranged in such a way that both the passage of each type are located against each other. Passages of both types has been divided into two equal sections. Rats are placed in the center of the X-maze and watching them for 10 minutes, measuring and recording the following parameters:

1) the number of entries and time spent on the site (a) with an open passage, and (b) with a closed passage, (b) at the end of the open passages and (g) at the end of the closed passages;

2) the number of cross-sections.

Fear of open areas greater than the fear of enclosed areas, and rats typically show a distinct preference for the closed passages. Anxiolytic drug increase the number of entries and time spent on the outer half opened the matter of the level of fear, as well as the total number of cross-sections, calculated for each animal. Drugs administered intraperitoneally or orally to the animals, divided into groups comprising from 6 to 12 rats for 30 to 60 minutes before the test. Statistical comparison between groups receiving the carrier and drug are using U-test Mann-Whitney (Mann-Whitney, U-test) (two-sided test) (see S. L. Handley and S. Mithani, Arch. Pharmacol., 1984, 327 1-5).

Delayed cerebral vasospasm in mongrel dogs

The compounds of formula (I) or their pharmaceutical salts can be investigated for their pharmaceutical use, using the procedures below.

In these studies used a 25 male mongrel dogs weighing from 9 to 12 kg. Animals are placed in cages and care for them in accordance with the guide for the care and use of laboratory animals [DHEW (DHHS), publication N (N1H) 85-23, revised in 1985]. All procedures on animals were approved by the Committee on animal care and use, Smithklein Pests Pharmaceutical (Institutional Animal Care Committee of SmithKline Beecham Phamaceutical). Each animal is subjected to anesthesia pentobarbital (35 mg/kg, in/in) and the Ute (tubocurarine; 0.1 mg/kg, I/V) and subjected to artificial ventilation of indoor air. At the end of the free exhalation conduct ongoing assessment of the level of CO2(et CO2and periodically analyze arterial blood gas composition to confirm the stability and adequacy of ventilation during each experiment. In the left external jugular vein and right femoral artery and vein was placed a polyethylene cannula to enter a drug, controlling blood pressure and taking blood samples. Through the left femoral artery using a French bow catheter Lehmann N5 (5 frenchLehman dakron catheter) (Bard, Tewksbury MA) conduct transperency catheterization of the left vertebral artery. Before the beginning of the experiment support as needed anesthesia introduction of pentobarbital (5 mg/kg, in/in).

The effect of the compounds of the present invention on acute cerebral vasospasm was evaluated in 15 dogs. For all animals receive after vnutriploschadnoj injection of a radiopaque material [omnipaque 300 (Omnipaque 300)] control digital angiogram of the anterior spinal artery and basilar artery. Each dog using puncture occipital membrane selected 4 ml of cerebrospinal LM is abdominal injection of blood and identify and quantify acute vasospasm in the primary and the anterior spinal artery. Infusion media (10% polyethylene glycol 200) for 30 minutes has no effect on acute vasospasm. Action 30-minute infusion of the studied compounds in reversible acute vasospasm was shown on both primary and anterior spinal arteries.

The effect of the compounds of the invention was also demonstrated on the model of the dogs that have the long introduction is delayed cerebral vasospasm (two models of cerebral vasospasm with hemorrhage). In this model, the receive control vertebral angiogram and enter vnutripolostno in day 1 (as described above) autologous blood. On day 3 repeat intracavitary injection of blood, and on the 7th day all animals evaluated quantitatively on the angiogram severe delayed vasospasm. Infusion media (10% polyethylene glycol) within 60 minutes had no effect on delayed vasospasm observed on the main and anterior spinal arteries (n=5). The effect of infusion of the studied compounds on the reversibility significantly delayed cerebral vasospasm indicates the potency of the compound.

The compounds of formula (I) or their pharmaceutically acceptable salts can be investigated with the aim of pharmacy is Sona

The model rats with lesions caused by 6-hydroxydopamine

The specified test (Ungerstedt U, 1971, Acta Physiol. Scand 367, 49-68, and/or Ungerstedt U, 1971, Acta Physiol. Scand 367, 69-93) can be used to determine whether compounds of the formula (I) or their pharmaceutically acceptable salts activity against Parkinson's disease.

2) Antipsychotic activity

The model rats with hyperdirichlet activity induced by amphetamine

The above test (Kokkindis L. and Anisman, M, 1980, Psyhological Bulletin, 88, 551-579) can be used to determine whether compounds of the formula (I) or their pharmaceutically acceptable salts antipsychotic activity.

3) Activity against migraine

Cortical spreading depression and migraine

The above test (Wahl et al., 1987, Brain Researh, 411, 72-80) can be used to determine whether antimigrainous the activity of compounds of the formula (I) or their pharmaceutically acceptable salts.

Preparation of animals

Experiments conducted on the female or male cats (2.5 to 3.0 kg), which were kept without food during the night, but were provided free access to water. Anaesthesia was induced 4-5% halothane gas and supported further through intravenous alpha-XCOM range. The right femoral artery and vein kateteriziruyut for blood pressure measurement, sampling of arterial blood and injection of drugs. Determine heart rate on the basis of the blood pressure signal and recorded. Hold the left parietal craniotomy bone and directoey and cover the brain layer heated to a temperature of 37oC mineral oil. To determine the changes in the diameter of blood vessels by lifetime videomicroscopy and record the results on videotape.

Induction of cortical spreading depression(CSD)

CSD was induced by introduction of small amount (30 mg, crystalline) KCl in the region nadsolevoe gyrus, remote from the recording electrodes, the observed vessels and other blood vessels. Because the brain is covered with a layer of warm mineral oil, KCl dissolves slowly in the brain within 5 minutes after application. After that the remaining KCl is removed from the surface of the brain, using moistened in saline swab.

Changing the extracellular potential and the diameters of the arteries and veins of the record for 120 minutes.

Treatment drug

The compound of example 17 in the form of a suspension (10 m the and observations from control animals (n=4) tracked reproducible phenomena CSD, induced by KCl. In contrast, in animals treated with the product of example 17 (n=3), in response to the introduction of KCl was observed only primary phenomenon CSD. The overall mean (minimum-maximum) number of phenomena CSD in the control and test groups was 5.5 (4-9) and 2 (1-2), respectively. The total duration of CSD similar phenomena were 60,36 minutes (n= 4) in control and was significantly reduced (p<0,05) to 13,70,3 minutes from animals treated with compound of example 17 (1=3).

Cerebral ischemia

a) Test Mongolian gerbils

The in vivo experiments were performed on adult Mongolian gerbils (Tumblebrook Farm (MA) weighing 60-80 grams. Temporary forebrain ischemia caused by legirovaniem bilateral carotid artery under anesthesia with 2.5% isoflurane in 100% O2and animals were placed on a heated blanket to maintain body temperature 37oC degrees. Expose the common carotid artery and placed the clamps on both arteries for the period of time specified in the manual to create aneurysm. PBN, dissolved in saline solution, injected intraperitoneally in the form of a bolus 30 minutes before occlusion (pre-treatment), or immediately after, and again on the 6th hour of reperfusion, followed by the introduction of the same ghormley on day 7 after induction of ischemia and spend perfusion buffered formalin. The brain is removed, kept in formalin for 3 days, immersed in formalin, cut into coronal sections with a thickness of 7 μm (1.5 to 1.9 mm after brahmi15and paint tinina. Count for each animal the number of intact neurons 750-μm length of the CA1 layer on both sides of the hippocampus in 3 sections.

b) MCAO method

Three strains of Mature male rats (SHR) are obtained from commercial suppliers [Taconic the farms, Germantown, NY, Charles river, Danvers, MA; and the Charles river, respectively) (Taconic Farmz, Germantown, NY; Charles River, Danvers, MA; and Charles River)] at the age of 18 weeks (weighing 250-300 g) and placed before use in these studies for 2-4 weeks in a cage. To confirm that the studied strains of animals really are hypertensive and normotensive type, group of animals of each strain anaesthetize with 2% izoflurana (Uniquest Madison W1 (Anaquest, Nadison, W1)] and prepared in aseptic conditions for long-term control of blood pressure. The femoral artery kateteriziruyut with polyethylene tube [RE 60; clay Adams. Parsippany, NJ (PE-60; Clay Adams. Parsippany, NJ)] , which reaches the descending aorta. Tube passes subcutaneously from the artery and output between the scapula under the rear frequent thread and process 5% lidocaine ointment [Astra pharmaceuticals Of Westboro, M. A.(Astra Pharmaceuticals, Westborough, M. A.)]. Animals come to life after surgery/anesthesia within 5 minutes. After 4-5 hours after the operation record mean arterial block pressure for 5 minutes/rat when connecting derived out of the tube, each rat with the pressure sensor of Statham (Statham pressure transducer) [P2.3Db; Statham medical instruments. Los Angeles (Statham Medical Instruments. Los Angeles, CA)] with access to the polygraph [Model R 711: Beckman instruments, Inc. Fullerton. CA (Model R 711: Beckman Instruments, Inc., Fullerton. CA)].

The procedure of focal cerebral stroke

MSAA or simulation surgery performed on rats, SHR, SD under anesthesia by nutrientlimited (65 kg/kg, in/in, with the addition if necessary). All animals shall have free access to water and food before and after the surgery. Body temperature is maintained at 37oC using heated pads during all surgical procedures. The surgery is conducted by the method similar to the previously described (2-4). Right, dorsal head relative to the surface is shaved away and handled povidon-iodine, after which the rat was placed in a stereotactic device [David Kopf instruments, Tujunga, CA (David Kopf Instruments, CA)] , so that the operated (right) one hundred and the output passage. The temporal muscle is dissected from the skull and pull without damaging the auditory connection or mandibular nerve. Under microscopic control, irrigation with saline perform the craniotomy size of 2-3 mm Rostral to scolopacidae suture of the skull. Cut above the artery of the Dura using a modified needle tip 30 size. In the case of permanent right MCAO at the same time spend occlusion of the artery with the use of electrocautery [Electrosurgical generator, power 2, CO (Force 2 Electrosurgica Generator, Valley Lab Inc., Boulder, CO)] then the specified artery cut dorsally much farther towards at the side of the olfactory pathways at the level of the bottom of the cerebral veins of the cerebrum. A small piece of the specified artery after immersion in sterile saline solution Gelfoam [cleared, Kalamazoo, M1 (Gelfoam decision Upjohn, Kalamazoo, M1)] then placed above the craniotomy, and the temporal muscle and the skin is closed with two layers. Animals allow you to recover from anesthesia under the heating lamp, and then return them to the cells. 2 hours after holding MCAO animals kill and prepare the brain for reactive histological examination.

Measurement of ishemic the m overdose sodium-pentobarbital. Within 2-3 minutes, remove the brain and make 6 coronal sections of the forebrain (2 mm thick) at the level of the olfactory bulbs towards the cortical-cerebellar connection using the slicer for the brain of rats [(59); zivic-Miller laboratories, Inc., Allison Park (Zivic-Miller Laboratories Inc., Allison Park, PA)]. These sections of the forebrain then immediately immersed in a 1% solution of chloride of triphenyltetrazolium (TTX) in phosphate buffer at a temperature of 37oWith 20-30 minutes (6,78). Then the dyed fabric is fixed filtering in 10% formalin, buffered with phosphate. Both sides of each TTX slice photographed in color with the use of Polaroid. The pictures examined for the presence of ischemic damage in quantitative terms using image analysis [Amersham RAS 3000, Loats associates, Inc. (Amersham Ras 3000; Loats Associates, Inc.)]. Morphological changes caused by surgery, evaluate in General the forebrain (only 11 flat surfaces) for each animal. 11 planar images are obtained from each side 6 slice thickness of 2 mm, each of which corresponds to approximately 1 mm slice surface with fluctuations of +5 mm to -5 mm from bregma (97) and include full forebrain. Such a flat is agenia planar identify damaged by necrosis zone and swelling. For each slice define 2 parameters that characterize ischemic damage associated with MCAO in accordance with the previously given definition (2, 4,98,122).

"Swelling of the hemisphere" is expressed as the percentage of increase in the size of the ipsilateral (i.e., on the operated side) hemisphere over the contralateral (normal) hemisphere and is calculated as follows:

< / BR>
"Infarction" is expressed as the percentage of tissue damaged by necrosis, relative to the contralateral (normal) hemisphere and is calculated as follows:

< / BR>
The swelling and the size of infarction zone expressed relative to the contralateral hemisphere (i.e., it ipsilaterally located ischemic damage, normalized with respect to the normal contralateral hemisphere). These parameters are determined for each slice to assess the profile of the damage around the front brain (i.e., "profile forebrain") and "full" changes forebrain, summarizing the data for all slices obtained using the above formulas.

The presence of brain edema associated with swelling hemisphere after conducting MCAO determine when comparing wet/dry weight in accordance with the previously Hirurgii or MCAO. The brain is quickly removed, isolated forebrain in the zone approaches cortical connections and cut into 2 hemispheres, then each hemisphere of the forebrain measured within 2 minutes after decapitate using the device for determining the chemical balance (Mettler Types H5 chemical balance) [Mettler instruments Corp. Hightstown, NJ (Mettler Instruments Corp., Hightstown, NJ)] . Dry weight is measured using the same scale after drying hemispheres in a drying Cabinet at a temperature of 80oC for 48-72 hours. The water content in each hemisphere is calculated as the difference between wet and dry weight as a percentage fraction of wet weight:

1. Benzopyrane selected from the group consisting of:







TRANS-6-aceti is ylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3S-ol,















or their pharmaceutically acceptable salts.

2. Pharmaceutical composition having anticonvulsant activity, including the active agent and a carrier, wherein the active means it has a connection on p. 1 or its pharmaceutically acceptable salt in an effective amount.

3. The method of treatment demonological compulsive disorder and/or panic, and/or aggressive States by introducing ingredient, wherein the active means introduce a connection on p. 1 or its pharmaceutically acceptable salt.


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