N-alkyl-2-substituted atp and method of production thereof

 

(57) Abstract:

Describes compounds of formula I, where R1and R2independently represent hydrogen or halogen; R3and R4independently represent phenyl or12-6-alkyl, optionally substituted by one or more substituents selected from OR5WITH1-6-alkylthio, NR6R7, COOR8or halogen; R5, R6, R7, R8independently represent a1-6-alkyl; and X is an acid residue, and its pharmaceutically acceptable salts. In addition, the disclosed methods of obtaining these compounds. The compounds of formula I inhibit platelet aggregation. 2 c. and 7 C.p. f-crystals, 1 table.

The present invention relates to new pharmaceutical compounds, methods for their preparation, pharmaceutical compositions containing these compounds, and methods for their use.

Adenosine triphosphate (ATP) has a strong pharmacological effect on various tissues. The activity of ATP and other extracellular Denisovich nucleotides, such as adenosine diphosphate (ADP) and monophosphate (AMP), is mediated by P2-purinoceptors. However, the effect of ATP in some tissues the effect of ectonucleotide, present in these tissues.

In recent works devoted to the study of P2-purinoceptors present in various tissues, were used as biological probes ATP-analogues; which are resistant to dephosphorylation.

In the work Cusack and others (Br. J. Phatmacol., 1987, 90. 791 - 795), carried out using a bladder and tacnia coli of the Guinea pig, describes the activity of such compounds, as monoamide 2-methylthio-5'-Danilovo acid methylenephosphonic acid, monoamide 2-methylthio-5'-Danilovo acid dehloretilifosfamida acid, and monoamide 2-methylthio-5'-Danilovo acid diftormetilirovaniya acid. In the work of Stone and Cusack (Br. J. Phatm., 1989, 97, 631 - 635) describes the use inter alia of monongalia 2-methylthio-5'-Danilovo acid diftormetilirovaniya acid in the study of P2-purinoceptors in the rat hippocampus. Maguire and Satchell in his "Physiological and Regulatory Function of Adenosine and Adenine Nucleotides" (Ed. H. P. Baer & G. I. Drummond, Raven Press, New York, 1979, pp 33 - 43) reveal the inhibition of the taenia coli of the Guinea pig connection monongalia 2-chloro-5'-Danilovo acid methylenephosphonic acid.

In the work Cusack and Hourni (Nucleosides &Nucleosides, 1991, 10 (ingibiruet ATP- -S-induced platelet aggregation.

In International patent application WO 92/17488 (Fisons pls) reveals a number of 2-substituted ATP analogues and their activity as inhibitors of platelet aggregation.

The authors of this application was received by the group of new N-alkyl-2-substituted ATP analogues possessing pharmacological activity.

In accordance with its first variant, the present invention relates to a compound of formula I:

< / BR>
where R1and R2nesasio represent hydrogen or halogen;

R3and R4independently represent phenyl or alkyl, optionally substituted by one or more substituents selected from OR5C1-6-alkylthio, NR6R7, phenyl, COOR3and halogen;

R5, R6, R7and R8independently represent hydrogen or C1-6-alkyl; and

X represents an acid;

and its pharmaceutically acceptable salts.

The compounds of formula I may exist in tautomeric, enantiomeric, and diastereomeric forms, which also violet in the scope of the present invention.

In addition, the present invention relates to a method for producing compounds of formula I and their salts, which SUB> represents a leaving group; and Y represents (i) OH, or (ii) a leaving group L2, is subjected to reaction with the compound of the formula III or its salt

< / BR>
where R1, R2and X is defined above, and then in the case where y is a L2, is subjected to hydrolysis;

b) remove the protected group from the corresponding protected compounds of formula I, which are secured one or more functional groups; and then, if necessary, the obtained compound of formula I or its salt is transformed into a pharmaceutically acceptable salt, or Vice versa.

In stage (a) (i), where Y is OH, leaving groups L1can be amines, for example, dialkylamines, either saturated or unsaturated ciclamini; and preferably, if such leaving groups are morpholinyl, imidazolyl or triazolyl. The above reaction is preferably carried out in a solvent, preferably a polar aprotic solvent, such as pyridine, dimethylformamide, acetonitrile, hexamethylphosphorotriamide, N, N"-dimethylpropylene, or 1-methyl-2-pyrrolidinone. This reaction can be conducted at temperatures from -20oC to 100oC, for example, at 10 - 30oC.

Connect the diversified methods for example, the method described in International patent application WO 92/17499 (Jisons plc. ). For example, the compounds of formula II in which L1is morpholinium can be obtained from the corresponding 5'-monophosphate by treatment with morpholine in the presence of a condensing agent such as dicyclohexylcarbodiimide, preferably in the presence of proton solvent or mixture of solvents such as a mixture of t-butanol and water.

In stage (a) (ii), when Y is a leaving group L2leaving group L1and L2can be a halogen, for example chlorine, L1and L2may be the same or different, but preferably the same. The compounds of formula II in which Y is a L2can be obtained from the corresponding nucleoside by reaction of the nucleoside with fosforiliruyusciye agent, bearing three leaving groups, i.e., POL1L2L3; and in particular, POCl3. The compound obtained of the formula II should not be selected; for example, it may be subjected to reaction in situ with the compound of the formula III, followed by hydrolysis, for example, the basic hydrolysis using Na2CO3.

Nucleosidases, or they can be obtained by known methods from known compounds (see , for example, "Chemistry of Nucleosides and Nucleotides" Vol. 2, Ed. Leroy B. Town send, Plenum Press, 1991).

The compounds of formula III are either known compounds or they can be obtained by the known methods described, for example, in International patent application WO92/17488 (Jisons plc.).

In this reaction it may be necessary to a functional group such as hydroxy or amino group present in the source connections, were secured, and therefore, in stage (b) can be removed by one or more protective groups.

Suitable protective groups and methods for their removal are groups and the methods described, for example, in "Protective Groups in Organic Synthesis", J. Green and P. G. M. Wutts, John Wiley & Sons Inc., 1991. Hydroxy groups can be, for example, protected arylmethylidene groups, such as phenylmethyl, diphenylmethyl, or triphenylmethyl; acyl groups such as acetyl, trichloroacetyl, or triptorelin; or tetrahydropyranyloxy derivatives. Suitable aminosidine groups are arylmethyl groups such as benzyl, (R,S) - phenylethyl, diphenylmethyl, or triphenylmethyl; and acyl groups such as acetyl, trichlo wny hydrolysis or photolysis. Arylmethyl group can be, for example, removed by hydrogenolysis in the presence of a metal catalyst, e.g. palladium carbon. Tetrahydropyranyl groups can be split by hydrolysis in acidic conditions. Acyl groups can be removed by hydrolysis using a base such as sodium hydroxide or potassium carbonate; and a group such as trichloroacetyl, can be removed by restoring, for example, using zinc and acetic acid.

The compounds of formula I or their salts can be isolated from their reaction mixtures using standard equipment.

Descriptions of the above mentioned activities are introduced in the present description as a reference.

Salts of compounds of formula I can be obtained by reaction of the free acid or its salt or the free base or its salt, or a derivative thereof, with one or more equivalents of the appropriate base or acid. This reaction can be carried out in a solvent or medium in which the salt is insoluble; or in a solvent in which the salt is soluble, such as ethanol, tetrahydrofuran, or diethyl ether, which is the battle Metallichesky process or it can be carried on the ion-exchange resin.

Pharmaceutically acceptable salts of the compounds of formula I are alkali metal salts, e.g. sodium and potassium salts; salts of alkaline earth metals, e.g. calcium salts and magnesium salts of elements of Group III; for example, aluminum and ammonium salts. Suitable salts of organic bases are salts formed with hydroxylamine; lower bonds alkylamines, e.g. methylamine or ethylamine; substituted lower alkylamines followed, for example, replacement bonds alkylamines; or monocyclic nitrogen heterocyclic compounds, for example, piperidine or morpholine; and salts formed with amino acids, such as arginine, lysine, etc., or their N-alkylamine derivatives; amino sugars such as N-methyl-D-glucamine or glucosamine. While preferred are non-toxic physiologically acceptable salt, although in some cases, for example, for the isolation or purification of the product can also be used and other salts.

The compounds of formula I can be tautomerism, for example, Imin-enaminones the tautomerism of 6-position of adenine. These connections can teticheskikh isomers and/or diastereoisomers. Diastereoisomer can be separated using standard techniques; for example, chromatography or fractional crystallization. The various optical isomers may be isolated using standard techniques commonly used for the separation of racemic or other mixture of these compounds, such as fractional crystallization or HPLC. Alternatively, the desired optical isomers may be obtained by reaction of the appropriate optically active starting materials under conditions that are not favorable racemization.

Alkyl groups represented by R3- R8can be straight, branched or cyclic, saturated or unsaturated alkyl groups.

The halogen represented by R1and R2are F, Cl, Br, or I. In this case, preferably, R1and R2were the same. Especially preferred are compounds in which R1and R2represent Cl.

Preferred are compounds in which R3and R4represents a C1-6-alkyl, optionally substituted by one or more substituents selected from OR5C1-6-alkylthio, NR6R

Especially preferred are compounds in which R8represents a C1-6-alkyl, optionally substituted C1-6-alkylthio group. Specific examples of alkyl groups represented by R3are propyl, butyl, and especially ethyl. Preferred substituted alkyl groups represented by R3is 2-(methylthio)ethyl.

Especially preferred are compounds in which R4represents a C1-6-alkyl, optionally substituted by one or more, for example, three halogen atoms. Preferred R4groups are propyl and 3,3,3-cryptochromes.

Acid groups represented by X are acid Bronsted-Lowry, i.e., the group, acting as proton donors. These acid groups may be monobasic or polybasic. As specific examples of such acidic groups can serve-P(O)(OH)2, -SO3H,- CO2H.

Predpochtitelnye compounds of formula I are compounds in which a represents-P(O)(OH)2.

The compounds of formula I can be used to treat mlekovita the soup to prevent platelet aggregation.

The effectiveness of compounds of the formula I as inhibitors of platelet aggregation can be evaluated based on their ability to act as antagonists in relation to P2T-receptor, see Example X.

These compounds may be used in any environment where there is aggregation of platelets. Thus, these compounds have antimicrobial activity and can be used for the treatment or prevention of unstable angina, thromboembolic shock and diseases of the peripheral circulation. In addition, these compounds can be used for the treatment or prevention of residual thrombotic complications arising from plastic surgery on vessels, thrombolyse, endarterectomy, transplants heart and blood vessels, kidney, and lung.

Other indications for the compounds of the present invention is the treatment or prophylaxis of disseminated intravascular coagulation, thrombosis grubacic veins, pre-eclampsia/eclampsia, tissue injury after surgery or trauma, vasculitis, arteritis, thrombocythemia, ischemia and migraine.

In certain higher as pharmaceuticals.

In addition, the present invention relates to the use of compounds of the formula I or their farmatsevticheskii acceptable salts for the manufacture of pharmaceutical compositions intended for treating conditions svyazannyh with platelet aggregation.

The dose of the compounds will vary widely depending on various factors such as the structure of the specific compounds of formula I, the method of administration, the physical condition of the patient and severity of the disease. In General, however, the daily dose for humans is from 0.1 mg to 1000 mg, and may be entered as divided doses up to 6 times a day. If this compound is administered by injection, the typical dose for a human is, for example, 0.5 μg/kg/min

Compounds of the present invention are introduced mainly in the form of pharmaceutical compositions.

For example, in accordance with the first variant of the present invention the pharmaceutical composition preferably includes less than 80 wt. %, and more preferably less than 50 wt.%, for example, from 0.1 to 20% of compounds of formula I or its pharmaceutically acceptable salt as defined above, in combination with pharmacoviligance specified pharmaceutical composition by mixing its ingredients.

The following are examples of pharmaceutical preparations which can be used for the purposes of the present invention, and suitable for the purpose of diluents or carriers:

for intravenous injection or infusion - purified water or saline;

for inhalation raw lactose;

for tablets, capsules and pills - microcrystalline cellulose; calcium phosphate; diatomaceous earth; sugars, such as lactose, dextrose or mannitol; talc; stearic acid; starch; sodium bicarbonate and/or gelatin;

for suppositories - natural or hardened oils or waxes.

If this connection is intended for use in aqueous solution, for example, for injection, the composition may include other additives. Such additives can be, for example, hepatoblastoma agents or airing, antioxidants, additives, corrective toychest; pH-modifying agents and buffer additives.

If necessary, the solutions containing the compound of the formula I, can be subjected to evaporation, for example by drying by freezing or drying by spraying with the preparation of solid compositions, which can then be transferred to the rum, it is preferable that it be made in the form, having an average diameter of from 0.01 to 10 μm. These compositions may also contain appropriate preservatives, stabilizing and wetting agents, solubilization, for example, water-soluble cellulose polymer, such as hypromellose, or water-soluble glycol, such as propylene glycol; sweeteners, colorants, and flavoring additives, If necessary, composee of the present invention can be manufactured in the form of medication slow release.

In yet another embodiment, the present invention relates to a method of treatment of diseases associated with platelet aggregation, namely, that the patient is suffering from the specified disease is administered a therapeutically effective amount of the compounds of formula I, defined above.

The advantage of the compounds of the present invention is that they produce far fewer side effects, for example, they have less sposobnostey to induction of hypothermia, as determined using the techniques described in Example V, and, in addition, they have greater duration is e easily removed from the body, detect a broader spectrum of activity or have other predominant pharmacological properties in comparison with known compounds.

Below provoditsya examples illustrating but not limiting the present invention. In this examples, all temperatures are given in degrees Celsius, and the names of the compounds are given in accordance with standard chemical nomenclature.

Example 1

Monoamide N-ethyl-2-(propylthio)-5'-Danilovo acid dichlorodibenzofuran acid, erantzuna salt

a) N-Ethyl-2-(propylthio)adenosine

9-(2,3,5-Tri-O-acetyl -- D-ribofuranosyl)-6-chloro-2-)-(propylthio)purine (1.3 g) and ethylamine (1.6 ml) in dioxane (30 ml) and water (30 ml) was heated in a tightly closed autoclave for 20 hours at a temperature of 110oC. After cooling to room temperature and evaporation obtained residue, which was recrystallized from ethyl acetate. In the purification using chromatography on silica (eluent: methanol/ethyl acetate, 1:15) was obtained target compound, namely N-ethyl-2-(propylthio)adenosine (0,46 g).

MC (FAB): 370 (M+H, 100%), 238 (30%).

in) Monoammonium salt of N-ethyl-2-(propylthio)-5-Danilovo acid

To paramesh the and. After 4.5 hours the reaction mixture was poured into a mixture of ice/water (100 g) containing sodium bicarbonate (1.45 g). After 45 minutes the solution was washed with ether (2 x 100 ml) and loaded on a column with 50WX 8 (H+form). This column was rinsed with water up until the pH of the eluate does not become equal to 6, and then suirable 2 M ammonium hydroxide. After lyophilization received target connection, namely, monoammonium salt of N-ethyl-2-(propylthio)-5'-Danilovo acid (0.32 g).

31P NMR (D2O): 2,03 ().

(C) Monoamide N-ethyl-2-(propylthio)-5'-Danilovo acid dichlorodibenzofuran acid, Terentieva salt

The target product of stage (b) (0,38 g) and tributylamine (0.15 g) were combined in a small volume of pyridine and the resulting solution evaporated to dryness. Then the solution was dried by azeotropic distillation with pyridine (3 x 15 ml) and anhydrous N,N - dimethylformamide (DMF) (2 x 15 ml) and the resulting residue was dissolved in 10 ml of anhydrous DMF. After adding carbonyldiimidazole (0.66 g), the reaction mixture was left for 4 hours at room temperature, and then added methanol (0,209 g). After 30 minutes, was added 2,09 g mono(tributylammonium) salt dichlorodibenzofuran acid in anhydrous DMF (30 ml the Oia received the remainder, which was purified by using chromatography (DEAE-Sephadex, eluent: O M 0.6 M bicarbonate of triethylamine). In the lyophilization received triethylammonium salt, which was converted into the sodium salt by dissolving in methanol (2 ml) and add a solution of sodium iodide (IM suspension in acetone (30 ml)). The precipitate was collected by centrifugation, washed with the above suspension in acetone (4 x 40 ml), and centrifuged Resulting solid was dissolved in water and was liofilizovane, resulting in a received target salt as a colorless powder (0.25 g).

31P-NMR (D2O): 9,00 (d, J = 18.6 Hz), 1,18 (DD, J = 18.6 Hz, J = 30,4 Hz), -9,35 (d, J = 18.6 Hz).

Example 2

Monoamide N-butyl-2-(propylthio)-5'-Danilovo acid dichlorodibenzofuran acid, Terentieva salt

a) N-Butyl-2-(propylthio)adenosine

The target connection, namely, N-butyl-2-(propylthio)-adenosine, was obtained by the method described in Example (1A).

MC (FAB): 398 (M+H+: 100%).

b) Monoamide N-butyl-2-(propylthio)-5'-Danilovo acid dichlorodibenzofuran acid, Terentieva salt

To a solution of the product of stage a) (1.8 g) in triethyl phosphate (50 ml) while cooling with ice, drop by drop added oxychlordane suspension mono(tributylammonium) salt dichlorodibenzofuran acid (5, 76 g) in triethyl phosphate (60 ml) was added tributylamine (2,16 ml). After stirring for one hour at room temperature the resulting solution for 15 minutes was added to the solution described above. Then the resulting mixture was stirred for 4 hours after which the reaction mixture was poured into 5% aqueous sodium bicarbonate solution (113 ml) and was stirred for 18 hours. The resulting solution was washed with ether (4 x 50 ml) and then dried by freezing. In the purification (reverse-phase C18-column with silica; eluent: 4% salt solution, and then water was given to the target salt as a colourless solid (0,69 g).

31P NMR (D2O): to 9.70 (d, J = 18,4 Hz), 3,4 (DD, J = 18,4 Hz, J = 30,5 Hz), 9,19 (d, J = 30,5 Hz).

Example 3

In accordance with the method described in Example 2, were obtained the following compounds:

a) Monoamide N-propyl-2-(propylthio)-5'-Danilovo acid dichlorophenolindophenol acid, Terentieva salt

I) N-Propyl-2-(propylthio)adenosine

MC (FAB): 348 (M+H+, 100%).

II) Monoamide N-propyl-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

31P NMR (D2O): of 8.92 (d, J = 18.5 Hz), with 1.07 (DD, J = 18,7 remotelistsummary acid, demoniaca salt

I) N-(1-Methylethyl)-2-(propylthio)adenosine

MC (FAB): 384 (M+H+, 100%), 252.

II) Monoamide N-(1-methylethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, demoniaca salt

In the purification of the crude product by chromatography (DEAE-Sephadex; eluent 0 M to 0.6 M solution of ammonium bicarbonate) received target Sol.

31P NMR (D2O): 8,71 (d, J = 18.6 Hz), 0,38 (DD, J = 19.1 Hz, J = 28,7 Hz), -9,49 (d, J = 29.0 Hz).

c) Monoamide N-(2-methoxyethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

I) N-(2-Methoxyethyl)-2-(propylthio)adenosine

MC (FAB): 400 (M+H+, 100%), 268.

II) Monoamide N-(2-methoxyethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

31P-NMR (D2O): 9,05 (d, J = 18.7 Hz), the 1.44 (DD, J = 18,8 Hz, J = 29.3 Hz), -9,40 (d, J = 29.5 Hz).

d) Monoamide N-cyclopentyl-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

I) N-Cyclopentyl-2-(propylthio)adenosine

MC (FAB): 410 (M+H+), 278 (100%).

II) Monoamide N-cyclopentyl-2-(propylthio)-5'-Danilovo acid dichlo the SUB>4O12P3S (D2O): 8,98 (d, J = 18.3 Hz), 2,70 (DD, J = 18.3 Hz and J = 30,6 Hz), -9,89 (d, J = 30,6 Hz)

f) Monoamide N-(2,2,2-trifloromethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

I) N-(2,2,2-Trifloromethyl)-2-(propylthio)adenosine

MC (FAB): 424 (M+H+), 292 (100%).

II) Monoamide N-(2,2,2-triptorelin)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

MC (FAB): 822, 820, 818 (M+H+), 115 (100%).

g) Monoamide N-(methoxycarbonylmethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, triammonium salt

I) N-(Methoxycarbonylmethyl)-2-(propylthio)adenosine

Analysis for C16H23N5O6S:

Calculated: C 46,48, H 5,61, N 16,94, S 7,76 (%)

Found: C 46,44, H 5,43, N LS 16.80, S TO 7.67 (%)

II) Monoamide N-(methoxycarbonylmethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid, triammonium salt

The crude product was purified by chromatography

(DEAE-Sephadex; eluent: 0 M to 0.6 M solution of ammonium bicarbonate), and received the target salt.

31P-NMR (D2O): 9,05 (d, J = 18.7 Hz), the 1.44 (DD, J = 18,8 Hz, J = 29.3 Hz), -9,40 (d, J = 29.5 Hz).

h) Monanieba salt

I) N-[2-(Methylthio)ethyl]-2-(propylthio)adenosine

MC (FAB): 416 (M+H+, 100%).

II) Monoamide N-[2-(methylthio)ethyl]-2-(propylthio)-5'- Danilovo acid dehloretilifosfamida acid, triammonium salt

After cleaning the selected crude product by chromatography (DEAE-Sephadex; eluent: O M to 0.6 M solution of ammonium bicarbonate) received target Sol.

31P-NMR (D2O): 8,68 (d, J=18.6 Hz), 0,33 (DD, J=18,9 Hz, J=29.0 Hz); -9,53 (d, J=29.0 Hz).

j) Monoamide N-[2-(N,N-dimethylamino)ethyl]-2-(propylthio)-5 - Danilovo acid dehloretilifosfamida acid, trinacria salt

I) N-[2-(N, N-Dimethylamino)-ethyl] -2-(propylthio)adenosine MC (FAB): 413 (M+H+, 100%).

II) Monoamide N-[2-(N,N-dimethylamino)ethyl]-2-(propylthio)-5'- Danilovo acid dehloretilifosfamida acid, trinacria salt

MC (FAB): 789, 787, 785, (M+H+), 93 (100%).

Example 4

Monoamide 2-(cyclohexylthio)-N-5'-ethyl-5'- Danilovo acid dehloretilifosfamida acid, Terentieva salt

a) 9-(2,3,5-Tris-O-acetyl -- D-ribofuranosyl)-6-chloro-2- (cyclohexylthio)purine

To a solution of 2-amino-9-(2,3,5-tri-O-acetyl -- D-ribofuranosyl-6-chloropurine (10.0 g) in acetonitrile (200 ml) was added to disclaimerthe 60oC for 16 hours. After concentrating the solution, the residue was purified (silica; eluent:ethyl acetate/petroleum ether, 1: 1) and received the target connection, namely 9-(2,3,5-Tri-O-acetyl -- D-ribofuranosyl)-6-chloro-2-N-(cyclohexylthio)purine (3.88 g) as an orange resin. MC (EI): 528, 526 (M+), 43 (100%).

b) 2-(Cyclohexylthio)-N-ethyl-adenosine

Using the product of stage (a) was obtained target compound, namely 2-(cyclohexylthio)-N-ethyl-adenosine, in accordance with the procedure described in Example 1(a). MC (FAB): 510 (M+H+, 100%).

c) Monoamide 2-(cyclohexylthio)-N-ethyl-5'-Danilovo acid dehloretilifosfamida acid, Terentieva salt

The target compound was obtained by the method, optionnum in Example 2(b), except that used the product of stage (b).

31H-NMR (D2O): 9,85 (d, J=18.5 Hz), 3,85 (DD, J=18.5 Hz, J=30,4 Hz), -9,07 (d, J=30,4 Hz).

Example 5

Monoamide N-ethyl-2-[(3,3,3-trifloromethyl)thio]-5'-Danilovo acid dehloretilifosfamida acid, trinacria salt

a) 2-[(3,3,3-Trifloromethyl)thio]adenosine

A suspension consisting of sodium hydride (60%, 1,453 g) and adenosine-2-thinmanager (5.35 g) in DMF (80 ml) was stirred for h is e 5 days the solution was concentrated, and the resulting residue was distributed between ethyl acetate (250 ml) and water (150 ml). The organic phase was dried, and then concentrated. The treatment (SiO2; eluent: dichloromethane/methanol, 9:1) was obtained target compound, namely 2-[(3,3,3-trifloromethyl)thio] adenosine, in the form of a colorless solid (of 5.55 g).

MC (FAB): 396 (M+H+, 100%).

b) N-Acetyl-2-[(3,3,3-trifloromethyl)thio]adenosine-2'-3',5'-triacetate

The product of stage (a) (5,28 g) and anhydrous sodium acetate (0.723 g) in acetic anhydride (42 ml) was stirred for 6.5 hours at a temperature of 80oC. This solution was diluted with water (100 ml) was stirred at room temperature for 18 hours and then was extracted with dichloromethane (I ml). The combined organic extracts were washed with saturated sodium bicarbonate solution (200 ml), then evaporated and the resulting residue was chromatographically on silica (eluent:diethyl ether:methanol, 97:3), resulting in a received target connection (N-acetyl-2-[(3,3,3-trifloromethyl)thio]-adenosine-2',3',5'-triacetate) as a colourless foam (5.35 g).

MC (FAB): 564 (M+H+), 139 (100%).

c) N-Acetyl-N-ethyl-2-[(3,3,3-triftorperasin)thio] adenosine-2', 3',5'-triacetate

Obtained the ml) containing ethyliodide (2.2 ml). After stirring for 2 days, the solution evaporated, and the residue was dissolved in ethyl acetate (300 ml) and washed with water (I ml). The organic phase was concentrated and then the residue was purified (silica; eluent: ethyl acetate:cyclohexane, 1:1) and obtained target compound (N-acetyl-N-ethyl-2-[(3,3,3-trifloromethyl)thio] adenosine-2', 3', 5'-triacetate) as a yellow resin (4,54 g).

MC (FAB): 592 (M+H+), 139 (100%).

d) N-Ethyl-2-[(3,3,3-trifloromethyl(thio]adenosine

The product of stage (c) (4,54 g) in sodium hydroxide solution (0.1 M in methanol, 155 ml) was heated under reflux for 30 minutes. After cooling to room temperature was added to 0.89 ml of glacial acetic acid and the resulting solution was concentrated. In the purification (silica; eluent:dichloromethane/methanol, 95:5) was obtained target compound (N-ethyl-2-[(3,3,3-trifloromethyl)thio]adenosine) as a colourless solid (2,73 g).

MC (FAB): 424 (M+H+, 100%).

e) Monoamide N-ethyl-2-[(3,3,3-trifloromethyl)thio]-5' -Danilovo acid dichloromethanesulfonyl acid, trinacria salt

The target salt was obtained by the method described in Example 2b), using the product of stage (d).


a) Monoamide N-(2,2,2-trifloromethyl)-2-[(3,3,3 - trifloromethyl)thio]-5'-Danilovo acid dichlorotetrafluoroethane acid, triammonium salt

I. N-Acetyl-N-(2,2,2-trifloromethyl)-2-[(3,3,3-trifloromethyl) thio]adenosine-2',3',5'-triacetate

MC (FAB): 646 (M+H+).

II. N-(2,2,2-Trifloromethyl)-2-[(3,3,3-trifloromethyl)thio] adenosine

MC (FAB): 478 (M+H+), 346 (100%).

III. Monoamide N-(2,2,2-trifloromethyl)-2-[(3,3,3 - trifloromethyl)Teal] -5'-Danilovo acid dichloromethanesulfonyl acid, triammonium salt

31P-NMR (D2O: 8,82 (d, J=18.6 Hz) 0,63 (DD, J=18,9 Hz, J=28,9 Hz), -9,43 (d, J=29.0 Hz).

b) Monoamide N-[2-(petitio)ethyl]-2-[(3,3,3-trifloromethyl]-5'-Danilovo acid dichloromethylsilane acid, triammonium salt

I. N-Acetyl-N-[2-(methylthio)ethyl] -2-[(3,3,3-trifloromethyl) thio]adenosine-2', 3', 5'-triacetate

MC (FAB): 638 (M+H+), 139 (100%).

II. N-[2-(Methylthio)ethyl]-2-[(3,3,3-trifloromethyl)thio] adenosine

Analysis for C165H22F3N5O4S2: (%)

Calculated: C 40,90; H 4,72; N 14,92; S 13,70;

Found: C 40,70; H 4,82; N 14,79; S 13,60.

III. Monoamide N-[2-(methylthio)ethyl] -2-[(3,3,3-cryptochromes)thio]-5'-Danilovo acid with dichloromethylene; eluent: O M-0.6 M solution of ammonium bicarbonate), and received the target salt.

31P-NMR (D2O), 8,77 (d, J = 18.7 Hz), 0,38 (DD, J = 18,9 Hz, J = 27.4 Hz), -9,43 (d, J = 28.8 Hz).

Example 7

Monohydric N-(2-methoxyethyl)-2-[(3,3,3-cryptochromes)-thio] -5'- ademiluyi acid dehloretilifosfamida acid, Terentieva salt

a) N-(2-Methoxymethyl)-2-[(3,3,3-cryptochromes)thio]adenosyl

The solution containing the target compound of Example (5b) (4.8 g), 2-bromatology ether (1.2 ml), and potassium carbonate (1.77 g) in dry DMF (190 ml) was stirred at room temperature for 3 days. After adding 2-pomatoleios ether (1.2 ml) and potassium carbonate (1.77 g) and the resulting mixture was stirred for 24 hours at a temperature of 40oC. the Reaction mixture was filtered, then the filtrate was concentrated to obtain an oily substance, which was distributed between ethyl acetate (200 ml) and water (200 ml). The organic phase was dried, and then concentrated. The resulting resin was dissolved in 0.1 M solution of sodium methoxide in methanol (180 ml), after heated under reflux for 45 minutes. This mixture was neutralizable with ispolzovaniem acetic acid and concentrated, and the residue was purified (SiO2, eluent:dichlo izvetnogo solid veshestva (3,41 g).

MC (FAB): 454 (M+H+, 100%.

b) Monoamide N-(2-methoxy ethyl)-2-[(3,3,3-cryptochromes)thio] -5'-Danilovo acid dichlorodibenzofuran acid, Terentieva salt

The target salt was obtained by the method described in Example 2(b) using the connection stage (a).

31P-NMR (D2O): 9,88 (d, J = 19,0 Hz), 3,80 (DD, J = 19,0 Hz, J = 31,0 Hz); -9,12 (d, J = 31,0 Hz).

Example X

Quantitative evaluation of agonistic/antagonistic activity towards P2Tthe receptor using washed human platelets

Venous blood (100 ml) was divided into three equal parts and placed in 3 tubes, each of which contained 3.2% of triacrylate (4 ml) as anticoagulant. Then these tubes were centrifuged for 15 minutes at 240 x g, resulting in a received platelet-rich plasma (PRP), which was added to 300 ng.ml-1prostacyclin (PG12, 3 µl. ml-1Prpc 1/10-breeding in saline solution, obtained from a stock solution of 1 mg ml-1in ethanol), to stabilize the platelets in the washing process. PRP-plasma that does not contain red blood cells, was obtained by centrifugation at 125 x g for 10 minutes, followed tsentrifugirovaniem, not containing calcium solution Tyrode (CFT) [(10 ml composition: 137 mm (8 g/l) NaCl; 11.9 mm (1 g/l NaHCO3; 0.38 mm (0.06 g/l) NaH2PO4; 2,86 mm (1 ml of 20% Rast./l) KCl; 1,05 mm (1 ml of 10% rest. /l MgCl2; 5,55 mm (1 g/l) destroy], which was saturated by gas 95% O2/5% CO2and maintained at 37oC. After adding another 300 ng/ml PG12the suspension was combined and centrifuged at 640 x g for 15 minutes. Then the supernatant was discarded and the platelets resuspendable in 10 ml of CFT and then adding ft. to the final concentration of platelets 2 105/µl. The resulting suspension was stored in a 60-ml syringe with 3oC, with the released air.

Restoration of normal function of platelets after their PG12-inhibition, in order to continue platelet aggregation, carried out not earlier than in 2 hours after the last resuspendable. In all studies, 430 μl-aliquots of a suspension of platelets was added to the silicon cell aggregation, containing CaCl2the solution (10 μl of 45 mm solution, final concentration 1 mm) and stirred at 90 rpm in aggregometry PAP4 (Biodata). Then add the human fibrinogen (Sigma; F 4883) and 8-sulfopropyl (8-SPT, to block any P1- agonistic activity DeeDee) and 3 (10-4M (10 ál 5.6 mg/ml solution in 6% glucose), respectively. Then began the observation of adhesion.

Protocol

a) selecting a submaximal concentrations of ADP

The concentration of ADP, producing directly a submaximal response was chosen by plotting the curve of the concentration/response in the range of 10 to 300 μm. For this purpose, the corresponding solution of ADP was added to the cuvette in a volume of 10 µl in 20 minutes after the start of observation of the aggregation. Response aggregation was evaluated based on the maximum rate of change of transparency, measured by the counter with beveled layer. Submaximal concentrations of ADP, selected at this stage of the Protocol used for the subsequent evaluation of antagonistic activity of the compounds. All measurements for platelets from each donor were carried out twice.

b) Evaluation of agonistic/antagonistic activity

5 minutes after start monitoring the aggregation of the corresponding solution of the test compound or saline solution was added to the cell aggregation in the volume of 30 μl for a final concentration of 0.10 μm or 1000 μm. Aggregation at this stage indicated on the agonistic activity of the adiya's (a).

If the aggregation was not observed, then 15 minutes after the test compounds were added to the pre-selected submaximal concentrations of ADP in a volume of 10 µl. Antagonistic activity was evaluated as % inhibition of the control WDF response, resulting in the obtained approximate values IC50. Compound that completely inhibited ADP-response when the initial concentrations were tested again, but at a lower concentration range. Connection with IC50< 10-8M was also subjected to repeated testing in the absence of 8-SPT to confirm the absence of any agonistic activity towards P1and 2 minutes (instead of 15 minutes) incubation in order to test whether the inhibition from time to time.

Results

Typical results obtained for compounds of formula I are presented in Table 1 as the negative logarithm of antagonistically activity (pIC50).

Example V

Change hypothermic activity using susceptible mice

For the experiment used mice CP/CD 1 (25-45 g). These mice were weighed and the temperature was measured rectally using thermoresistibile using hypodermic needles (27G), connected with plastic kanulau and secured with duct tape in the desired position. Animals were treated with dose used compounds, administered at 10-minute intravenous infusion at a rate of 0.5 ml/kg/min Rectal temperature measurement was performed through 1/3; 1; 1,5; 2; 3; 4; 6 and 24 hours after infusion.

Then build a graph of the dependence of the mean maximum decrease in rectal temperature from the dose of the compound, and determining the dose required for lowering the temperature on the 5o.

1. N-alkyl-2-substituted ATP General formula I:

< / BR>
where R1and R2independently represent hydrogen or halogen;

R3and R4independently represent phenyl,1-C6alkyl, possibly substituted by substituents OR5WITH1-C6alkylthio, NR6R7, R8or halogen, where R5, R6, R7and R8independently represent hydrogen or C1-C6alkyl;

X is an acid residue,

or their pharmaceutically acceptable salts.

2. The connection I on p. 1 or its pharmaceutically acceptable salt, where X Is P(O)(OH)2.

3. Connection under item 1 or 2, or its pharmaceutically acceptable salt, is adidasi points or its pharmaceutically acceptable salt, where R1and R2are Cl.

5. Compound I according to any one of the preceding paragraphs or its pharmaceutically acceptable salt, where R3-C1-C6alkyl, possibly substituted C1-C6alkylthiophene.

6. The connection I on p. 1, which is monoamide N-ethyl-2-(propylthio)-5'-Danilovo acid dichlorodibenzofuran acid, monoamide N-ethyl-2-[(3,3,3-cryptochromes)thio]-5'-Danilovo acid dichlorodibenzofuran acid, monoamide-N-[2-(methylthio)ethyl]-2-[(3,3,3-cryptochromes)thio]-5'-Danilovo acid dichlorodibenzofuran acid, or its pharmaceutically acceptable salt.

7. The connection I on p. 1, which is monoamide N-butyl-2-(propylthio)-5'-Danilovo acid dichlorodibenzofuran acid;

Monoamide N-propyl-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-(1-methylethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-(2-methoxyethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-cyclopentyl-2-(propylthio)-5'-Danilovo acid with sodium dichloro bisphosphonates acid;

Monoamide N-(2,2,2-triptorelin)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-(methoxycarbonylmethyl)-2-(propylthio)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-(2-methylthioethyl)-2-propylthio)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-[2-(N, N-dimethylamino)ethyl] -2-(propitia)-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide 2-(cyclohexylthio)-N-ethyl-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-(2,2,2-triptorelin)-2-[3,3,3-trifloromethyl)thio]-5'-Danilovo acid dehloretilifosfamida acid;

Monoamide N-(2-methoxyethyl)-2-[3,3,3-trifloromethyl)thio] -5'-Danilovo acid dehloretilifosfamida acid; or any of its pharmaceutically acceptable salts.

8. Compound I according to any one of paragraphs.1 - 7 or its pharmaceutically acceptable salt, inhibiting platelet aggregation.

9. Method of producing compounds of the formula I according to any one of paragraphs.1 - 7 or their pharmaceutically acceptable salts, characterized in that

a) compound of General formula II:

< / BR>
where R3and R4UB>2- halogen, is subjected to reaction with a compound of General formula III or its salt

< / BR>
where R1, R2and X have the above values,

and then in the case when Y is L2, is subjected to hydrolysis,

C) removing the protective group from the corresponding protected compounds of formula I, in which is secured one or more functional groups and then, if necessary, translate the resulting compound of formula I in salt.

Priority points and signs -10.02.93 - PP.1 to 9, except for values of R3and R4.

16.12.93 on PP.1 to 9, except for values of R3and R4.

03.02.94 on PP.1 - 9 on all grounds.

 

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< / BR>
where

< / BR>
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< / BR>
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