The inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide with polycyclic derivatives of glucopyranose, the method of production thereof, and pharmaceutical composition

 

(57) Abstract:

The invention relates to new inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of General formula I, where1=R2=CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, polycyclic derivatives of glucopyranose General formula II, where if n= 1, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the formula III, R4=H, R5--D-glucuronidase, R6=R7=H and R8= C(O)OH, or, if n= 7, R3=N, R4and R7- simple connection, R5and R6= H or (CH2CH(CH3)O)mH, where m=1 to 14, and R8=CH2OH or CH2O(CH2CH(CH3)O)mH, where m=1-14, generating nitric oxide and activating the soluble form of guanylate cyclase (RGC), antispasmodic, vasodilator and hypotensive means quick action and platelet aggregation inhibitors, method for their production and pharmaceutical compositions based on them. The products of the present invention obtained by the method lies in the interaction of the source derived 1,2,5-oxadiazol-2-oxide of the above General formula I, where R1and R2have these EIT is 3-R8have the specified values in aqueous-organic medium (for example, in an aqueous solution of dioxane, lower alkanol, in particular ethanol, or a mixture of organic solvents at elevated temperatures (30-60o(C) with subsequent isolation of the target product by evaporation under reduced pressure, washing monopolarly inert organic solvent (diethyl ether or a mixture of hexane and drying under reduced pressure. New inclusione complexes of the present invention are effective NO donors, as well as generate his redactora (nitroxyl) form nitrosothiol and activate RHC and have a strong antispasmodic, vasodilator and fast hypotensive action. Described products are expressed antiplatelet properties. 3 S. and 1 C.p. f-crystals, 4 Il., 6 table.

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The invention relates to new inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide (furoxone) of General formula I:

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where R1= R2= CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, polycyclic derivatives of glucopyranose General formula II:

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The P>--D-glucuronidase, R6=R7=H and R8=C(O)OH, or, if n is 7, R3=H, R4and R7- simple connection, R5and R6= H or (CH2-CH(CH3)O)mN, where m=1-14. and R8= CH2OH or CH2O(CH2-CH(CH3)O)mN, where m=1-14, generating nitric oxide and activating the soluble form of guanylate cyclase (RGC), antispasmodic, vasodilator and hypotensive means quick action and platelet aggregation inhibitors, method for their production and pharmaceutical compositions based on them.

This invention can be used in biochemistry, physiology, pharmacology, and medicine.

Nitric oxide (NO) synthesized by endothelial cells and other cell types and tissues, plays a key role in maintaining the normal tonus of smooth muscles of blood vessels, affecting systemic blood pressure. In addition, NO has an antispasmodic effect on the muscles of the gastrointestinal tract, biliary tract, ureter, uterus, bronchi and other organs (Mashkovsky, M. D. "Drugs", so 1, "Torching", Kharkov, 1998 , page 385). The molecular mechanism of action of endogenous nitric oxide, synthesized NO-synthase from the amino acid L-organovo vessel and smooth muscle cells of the vessel, where the binding heme group RHC. Guanilatziklazu /EC 4.6.1.2; guanosin-5'-triphosphate-pyrophosphatase (cyclessa)/ is the enzyme that catalyzes the biosynthesis of guanosine-3', 5'-cyclophosphate (cGMP) as a secondary messenger in the role of a universal regulator of intracellular metabolism (F. Murad "Regulation of cytosolic guanylyl cyclase by nitric oxide: The NO-cGMP signal transduction system" Adv. Pharmacol. 1994, v 26, p. 19-33). HZ exists in two forms - membrane and soluble. It is now established that the result of the interaction of NO with iron atom of heme, part of the enzyme, and education complex nitrosyl-heme occur conformational changes of the active centre of the enzyme, which lead to activation of the RHC and increased synthesis of cGMP. The increase in intracellular concentration of this second messenger in the smooth muscle cells of the vessel causes the activation of cGMP-dependent protein kinases, which induces the dephosphorylation of myosin molecules, and hence the relaxation of smooth muscles of coronary arteries and other blood vessels (vessels of the brain, abdominal, peripheral vascular), reduced vascular tone and resistance, that is, leads to expansion of the lumen of the blood vessel. Thus, NO SN is

Violations related to the normal course of the above reactions underlie pathophysiological processes for development of various diseases of the cardiovascular system (hypertension, myocardial infarction, heart failure) and other bodies. In such cases there are multiple violations of the synthesis of endogenous NO, its reception RHC, as well as the regulation of the level of cyclic nucleotides and calcium ions.

To the present time has proved the formation of nitric oxide in the biotransformation of trinitroglycerine (glyceryltrinitrate, GHP) and nitrates, which are used for the treatment of cardiovascular diseases as antiischemic and antianginal drugs (Mashkovsky, M. D. "Drugs", so 1, "Torching", Kharkov, 1998, pp. 385-392). However, their major disadvantage is the appearance of tolerance and other side effects with prolonged use. In this regard, is the search for new compounds able to form NO in vivo by non-enzymatic (e.g., spontaneously or diazabicylo), which is regarded as relevant and promising approach to create new, more effective anticipate arousih medicines, used in the treatment of cardiovascular diseases, occupy a special place antihypertensive drugs fast action, the main representatives of which are the GHP and sodium nitroprusside (NNR). It is shown that the half-life (1/2) GHP in plasma is 2-4 minutes recently appeared in the literature data on the physiological activity of the new donor ultrafast NO action (1/2= 1.8 s)-N-diatrizoate l-Proline (PROLI/NO) (I. E. Saavedra, Southan, G. J. , et al. "Localizing antithrombotic and vasodilatory activity with a novel, ultrafast nitric oxide donor" J. Med. Chem. 1996, v. 39, p. 4361-4365). This compound, in contrast to the NNR and other nitric oxide donors, local intravenously in low doses caused selective vasodilation of the smooth muscle of the pulmonary artery (Andrie, C., Hirani, W. M., et al. "Selective pulmonary vasodilation by intravenous infusion of an ultrashort half-life nucleophile/nitric oxide adduct" Anesthesiology 1998, v. 88, 1, R. 190-195), significantly reduced the proliferation of smooth muscle cells (Chen, C. , Hanson, S. R., et al. "Boundary layer infusion of nitric oxide reduces early smooth musle cell prolifertion in the endarterectomized canine artery" J. Surg. Res. 1997, v. 67, 1, p. 26-32), and provided a highly effective effect in protecting against vasospasm and its relief in conditions of cerebral ischemia (Pluta, R. M., et al. "Reversal and prevention of cerebral vasospasm by intracarotid infusion of nitric oxide donors in a primate model of subarachnonile erection by intracavernosal and transurethral administration of novel nitric oxide donors in the cat J. Urol. 1999, v. 161, 6, R. 2013-2019), not calling in all cases, systemic hypotension.

Known 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazin-1,5,6-trioxide (DOPE) of the above General formula I, where R1and R2together with the neighboring carbon atoms form annelirovannymi 3,6-dimethylpyridin-1,2-dioxideis cycle, diazabicylo generating nitric oxide and its active form - nitroxyl and nitrosative, activating RHC and have vasorelaxant, inflammatory and antiplatelet effects (A. Ya.Kots, M. A. Grafov et. al. "Vasorelaxant and antiplatelet activity of 4,7-dimethyl-l,2,5-oxadiazolo[3,4-d] pyridazine 1,5,6-trioxide: role of soluble guanylate cyclase, nitric oxide and thiols" Brit. J. Pharm, 2000, v.129, p. 1163-1177).

Known also another representative class of furoxone - 3,4-dicyano-1,2,5-oxadiazol-2-oxide (3,4-dezineforce, DCF) of the above General formula I, where R1=R2=CN, generating NO in the presence of thiols, activating RHC and have vasorelaxant, inflammatory and antiplatelet effects (Ferioli R., Folco, G. C., S. Ferretti, A. M. Gasco, C. Medana, Fruttero, R. , M. Civelli, Gasco, A. "A new class of furoxan derivatives as NO-donors: mechanism of action and biological activity." Brit. J. Pharmacol. 1995, v. 144, 3, p. 816-820).

A significant drawback DAPTO and DCF are not solubility in water and relatively low solubility DAPTO in the waters of the funds or biologically-active compounds (BASS) with improved physico-chemical properties based on the use of substances, is able to form inclusion complexes (or inclusione complexes) type "host - guest". In accordance with the designation "master" is a molecule with an internal cavity, and a "guest" molecule of another connection, which is included in this cavity and delays due to a combination of intermolecular interactions. In the case of formation of the complex physical and chemical properties "guest" change substantially, which in some cases allows to modify (improve) the pharmacological properties include the BASS.

Currently as complexing agents are widely used cyclic heptamer-D-glucose-cyclodextrin (-CD) the above formula II, where n= 7, R3= N, R4and R7- simple connection, R5=R6=N and R8=CH2HE (Saenger, W., "Cyclodextrin-Einschlussverbindungen in Forschung und Industrie" Angew. Chem. 1980, v.92, p.343-361) and its derivatives, in particular 2-hydroxypropylammonium-cyclodextrin (-GPCD) of the above General formula II, where n=7, R3= N, R4and R7- simple connection, R5and R6=(CH2-CH(CH3)O)mN, where m= 1 to 14, and R8=CH2O(CH2-CH(CH3)O)mN, where m=1-14 (Pitha J., Szabo L,, Fales, H. M. "Reaction of cyclodextrins with propylene oxide or with glycidol: ana is the train 3-[2'-O-(-D-glucuronidase)--D-glucuronosyltransferase]-11-oxo-18, 20-Olean-12-EN-29-OIC acid (glycyrrhizin acid, ha) the above formula II, where n= l, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the above formula III, R4=N, R5--D-glucuronidase, R6= R7= N and R8=C(O)IT (Tolstikov, A., Baltina L. A., and others, "glycyrrhizin acid" Bioorg. chemistry, 1997, T. 23, 9, S. 691-709).

There are many inclusion complexes of different BASS and medicines with the above-mentioned complexing compounds (e.g., Papel, K. E., Degtyarev, S. I., Sugrobov, N. P., "Cyclodextrin" in the middle. "The results of science and technology. Microbiology", VINITI, M., 1988, T. 21, including 2; Degtyarev S. I., Steinhart M. C., and other "prospects for the use of compounds for inclusion based on cyclodextrins in pharmacy" in Proc. "Scientific fundamentals of rozrobka lorski Preparata", ed. "Base", Kharkiv, 1998, S. 225-228; Tolstikov, A., Murinov Y. I., and other "Complexes-glycyrrhizic acid to prostaglandins - a new class uterotonic active compounds" Chem.-Pharm. W. 1991, T. 25, 3, S. 42-44). The resulting products have a lower toxicity, increased solubility, stability and bioavailability compared with the original BASS and pharmaceuticals, as well as, in some cases, are superior Sitel)amino] -4,8-di(piperidine-1-yl)pyrimido[5,4-d] pyrimidine) of formula IV:

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with-CD manifesting antiplatelet and vasodilatory effects (Fregnan G. B., Berte F. "Enhancement of specific biological activity of dipyridamole by complexation with-cyclodextrin" Pharmacology 1990, v. 40, p. 96-102). This complex has a higher solubility (4.5 times) in phosphate buffer (pH 5.5) and improved bioavailability when administered orally in the form of capsules compared with the original dipyridamole. In addition, it was shown that the complex-CD-dipyridamole causes more severe and prolonged coronary vasodilatation in experiments in vivo compared with the effect of the source of the drug.

This complex, both the original ligand, does not have the ability to generate NO, its impact on the RHC has not been studied, and pharmacological properties are manifested in relatively high doses (1-8 mg/kg in terms of active ligand).

Known complexes include nonsteroidal anti-inflammatory drugs (ortofena, aspirin, indomethacin) with GK (copyright certificates of the USSR 1566699, 1566700, C 07 J 63/00, And 61 To 31/705, 1988; 1616925, C 07 J 63/00, And 61 To 31/705, 1989). These products have been obtained by the reaction of the starting components, taken in stoichiometric ratio, in an aqueous-organic medium (aqueous ethanol) at elevated temperature (50oC).

Known for the stabilization of aqueous solutions of the donor NO - N-acetyl-S-nitrosodimethylamine formula V:

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various cyclodextrins and their derivatives, in particular-CD and-GPCD (Bauer, J. A., Fung, H. L., "Chemical stabilisation of a vasoactive S-nitrosothiol with cyclodextrins without loss of pharmacologic activity" Pharm. Res. 1991, v. 8, 10, p. 1329-1334]. It is assumed that this effect is due to the formation of inclusion complexes. N-Acetyl-S-nitrosodimethylamine in the presence of these complexing agents have high stability and their pharmacological (vasorelaxant) properties was bioequivalence source nitrosothiol. The selection of the products obtained and the study of their composition was not conducted.

The common disadvantage of the compounds containing in the molecule nitrosothiol group is their severe toxicity, in particular carcinogenic and mutagenic properties.

It is known that various mono - and bicyclic N-oxides capable of forming inclusione complexes with-CD (Uno, C., Kaida, N. et al. "Spectroscopic study of considered are hydrophobic interaction of heterocyclic amine N-oxides with cyclodextrins" Chem. Pharm. Bull. 1988, v. 36, 10, p. 3753-3759), In the group of such compounds include 4-nitroquinoline-1-oxide of formula VI:

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providing an activating effect on RHC (Craven, P. A., De Rubertis, F. R. , "Inhibition by retinol>The above set inclusion has not been allocated, and its biochemical properties and physiological activity has not been studied.

Known inclusionary complex [(morpholine-4-yl)nitrosoamine]acetonitrile (SIN-1A), formula VII:

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with different cyclodextrins (Vicmon, M., Szente, L.,. et al. "SIN-1A /cyclodextrin complexes. Novel, stable and biologically active NO releasing agents" Portland Press Proc. 1996, v. 10, part 5, p. 188 - prototype). The structure of the obtained products were not investigated. The complexes obtained in this way showed increased stability, spontaneously generated NO and their pharmacological properties have been bio-equivalent of the original SIN-1A, which caused vasodilation in isolated rings of thoracic aorta of rabbit, shortened by the action of norepinephrine (Muramatsu, I., Fujii, K., et al. "Vasorelaxing actions of molsidomine and its metabolites, in comparison with nitroglycerin Can. J. Physiol. Pharmacol. 1983, v. 61, p. 1071-1078), and had an antihypertensive effect for 2.5 hours (European patent 324408, C 07 D 211/98, op. 1991). In addition, SIN-1A effectively inhibited platelet aggregation in vitro (Darius, H., Ahland, C., et al. "The effect of molsidomine and its metabolite SIN-1A on coronary vessel tone, platelet aggregation and eicosanoid formation in vitro inhibition of 12-HPETE biosynthesis" J. Cardiovasc. Pharmacol. 1984, v. 6, p. 115-121).

Toxicity resulting complexes are not Islena (SIN-1), in aerobic conditions is accompanied by the generation of superoxide anion-radical (Feelisch, M., Ostrovski, J., Noack, E., "On the mechanism of NO release from sydnonimines" J. Cardiovasc. Pharmacol. 1989, v. l4, Suppl. 11, p. S13-S22), which, interacting with eye-catching NO, can lead to the formation of highly toxic peroxynitrite.

Known inclusionary complex antianginal organic nitrate-1,4,3,6-dianhydro-D-sorbitol-2,5-dinitrate (isosorbidedinitrate) of the formula VIII:

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with-CD (Japan Patent 63135402 (08 In 37/16) op. 1988). The structure of the obtained product and its pharmacological properties are not investigated. The method of obtaining this complex is that to aqueous solution-CD was added isosorbidedinitrate and the reaction mixture was shaken at room temperature or with mild heating for 48 hours, after which the target product was separated by centrifugation.

A significant disadvantage of this method is the duration of the process.

The closest to this is the well-known inclusion complex stoichiometry (1:1) known organic nitrate fast - acting trinitroglycerine (GHP) of the formula IX:

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with-CD (Stadler-Szoke, A., Szejtii, J., "The inclusion complex of nitroglycerol--cyclodextrin" Acta Pharm. Hung. 1979, v. 49, p. is heated to a temperature of 60oWith 20% solution of TS in ethanol at a molar ratio of the reactants is equal to ~1:1,04, followed by keeping at this temperature, cooled to 4oWith filtering precipitated precipitate, washing with diethyl ether and drying.

A similar product was obtained by other authors in conditions similar to the above-described (Tomono, K., Goton, H., et al. "Interaction of nitroglicerin with 6-O--maltosylcyclomaltoheptaose" Carbohydrate Res. 1989, v. 192, p. 351-356). It has been shown that TS in the composition of this complex has increased stability (continued 80% of the ligand within 6 days) compared to the original TS, in which the same conditions were completely dissolved in 1.5 days. According to known data tablet for sublingual application, obtained on the basis of this product, its pharmacological parameters (pulse pressure shot dogs, systolic blood pressure and content of TS in plasma in healthy people) were bio-equivalent to conventional tablets with the same content GHP (0.3 mg) (Idzu, G., Terada, T., et al. "Stability and bioequivalence of sublingual tablets of nitroglycerin--cyclodextrin complex" Byoin Yakugaku 1989, v. l5, 1, p. 36-42, Chem. Abstr. 1989, v. lll, a). Thus, the inclusion complex as well as the ligand, showed highly Naradaji the HZ.

This complex is the same as the original ligand (HTN), is likely to cause tolerance in the long-term use and shows mild antiplatelet properties.

The purpose of the invention is the creation of new inclusion complexes with improved biochemical and pharmacological properties, the development of the method of their derivation and creation of pharmaceutical compositions based on them.

This goal is achieved by the described new inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of the above General formula I, where R1and R2have the specified values, polycyclic derivatives of glucopyranose the above General formula II, where R3-R8have the specified values, generating NO activating RHC and is antispasmodic, vasodilator, hypotensive drugs fast action and platelet aggregation inhibitors, method for their production and pharmaceutical compositions based on them.

Preferably, the present invention relates to the following new inclusionin complexes:

- 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazin-1,5,6-trioxide with-CD(1:3) (1),

- 4,7-dimethyl-1,2,5-oxadiazole[3R>
- 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazin-1,5,6-trioxide with CC (4),

- 3,4-dicyano-1,2,5-oxadiazol-2-oxide-with-CD(2:3) (5),

- 3,4-dicyano-1,2,5-oxadiazol-2-oxide-GPCD (6).

The products of the present invention obtained by the method lies in the interaction of the source derived 1,2,5-oxadiazol-2-oxide of the above General formula I, where R1and R2have the specified values with the corresponding polycyclic derivatives of glucopyranose the above General formula II, where R3-R8have the specified values, in an aqueous-organic medium (e.g., in an aqueous solution of dioxane, lower alkanol, in particular ethanol, or a mixture of organic solvents at elevated temperatures (30-60oC) followed by distillation of the solvents under reduced pressure, re-evaporation of the residue with ethanol, rinsing the target product monopolarly organic solvent (e.g. , diethyl ether or a mixture of hexane and thorough drying under reduced pressure in the presence of phosphoric anhydride. The significance of differences of this method is determined by the totality of its distinguishing features (including the structure of the initial reagents - derivatives of 1,2,5-oxides asanna the purpose of the invention (see comparative example 7).

According to this invention new inclusione complexes can be used to treat diseases of the cardiovascular system, including the treatment of hypertensive crises, acute myocardial infarction, angina, ischemic heart disease, pulmonary hypertension, acute failure of the left ventricle, toxicogenic spasm of blood vessels, for the prevention and relief of angina attacks and spasms of the coronary arteries during the cardiac catheters, angiography and angioplasty, as well as other diseases of the cardiovascular system, in which a positive therapeutic effect is achieved by reducing vascular resistance, normalization contractility and/or the inhibition of platelet activity, except for those diseases, in which combination blood pressure reduction and/or inhibition of platelet aggregation (for example, arterial hypotension, cardiogenic shock, acute myocardial infarction with low filling pressure of the left ventricle, the General decrease of left ventricular function with low filling pressure, hypertrophic obstructive cardiomyopathy, vascular collapse, toxic Preferably the application of the new compounds of the above General formula I for the prevention and relief of angina, disorders caused by spasm of smooth muscles, and treatment of acute myocardial infarction.

The present invention relates also to pharmaceutical compositions that generates nitric oxide, which activates the soluble form of guanylate cyclase and has antispasmodic, vasodilator, antihypertensive and antiplatelet effect, containing the active ingredient and additives in the desired ratio, as an active ingredient are inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of the above General formula I, where R1and R2have the specified values, polycyclic derivatives of glucopyranose the above General formula II, where R3-R8have the specified values obtained in the described manner.

As additives are the components that are conventional for the preparation of dosage forms antispasmodic, vasodilator and antihypertensive drugs. Depending on the type of drug and the purpose of its use, dosage forms can be made in the form of solutions for injection or infusion, capsules, granules, tablets or pills for gradual dosing (if necessary from a polymeric material, for example from acetosolv for aerosols.

Solid form for oral administration may contain as additives excipient (e.g. lactose, saccharose, sorbitol, starch, mannitol), surfactants (for example, magnesium stearate or calcium, polyvinylpyrrolidone, polyethylene glycol, polypropyleneglycol) and pharmacologically acceptable inorganic salts (e.g. sodium chloride).

Liquid compositions can be obtained by dissolving or dispersing the active ingredient in aqueous or non-aqueous solvent (e.g. ethanol, vegetable oil) in the presence of stabilizing additives (for example, components of the buffer mixtures, surfactants, food carbohydrates, preservatives, food dyes. Liquid compositions can be used for various routes of administration, in particular in the form of drops or aerosols, as well as in ampoule form for intravenous or intramuscular injection.

The above pharmaceutical compositions based on compounds of the above General formula I can be obtained by combination of the inclusion complexes of the present invention with other known active ingredients of pharmaceuticals, in particular, used for the treatment of diseases receptors (prazosin), agonists Central 2-adrenergic and I1imidazoline receptors (clonidine, moxonidine), blockers (1)-adrenergic receptors (propranolol, atenolol, metoprolol), calcium channel blockers (nifedipine, diltiazem), angiotensin converting enzyme inhibitors (lisinopril, fosinopril), koronarorasshiryayuschee drugs (carbokromen), diuretics (dihlotiazid), cardiac glycosides (digitoxin), antihypertensive agents (bezafibrat, gemfibrozil).

The description of the present invention contains 23 example 6 tables and 4 of the drawing, illustrating experimental material.

Examples 1-6 describe the synthesis of new inclusion complexes using the described method, as well as physico-chemical characteristics of the products.

Comparative example 7 confirms the significance of the differences of the described method.

Example 8 contains the results of a study of the products obtained by differential scanning calorimetry, confirming the formation of new inclusion complexes.

Examples 9-11 and table.1 shows the chemical basis of molecular mechanism of pharmacological action of new inclusion Sands

Example 12 and table.2 confirms the formation of NO new inclusionary complexes in the test with oxyhemoglobin.

Example 13 and table. 3 contain the results of the study the reaction rate of new inclusion complexes with low molecular weight thiols, contributing to the formation of NO and its active forms.

Example 14 illustrates the biochemical effect of new inclusion complexes-mediated generation of nitric oxide, activation RHC from the lungs of rats.

Example 15 and table.4 illustrate the pharmacological properties of new inclusion complexes - antispasmodic and vasodilator activity in vitro.

Example 16 and table.5 demonstrate that the new inclusion complexes antihypertensive activity in vivo.

Example 17 shows the ability of the new inclusion complexes to inhibit platelet aggregation in vitro.

Example 18 illustrates the study of acute toxicity of new inclusion complexes.

Example 19 contains a description of some physico-chemical properties of new inclusion complexes (solubility, stability).

Examples 20-23 include a description of the structure of various pharmaceutical conazole[3,4-d]pyridazin-1,5,6-trioxide with-CD(1:3) (1).

It is heated to a temperature of 50oTo a solution of 59 mg (0.3 mmol) DAPTO in a mixture containing 70 ml of 60% aqueous solution of ethanol and 42 ml of dioxane, is added dropwise and with vigorous stirring was added at a temperature of 50oTo a solution of 324 mg (0.25 mmol) -CD in 30 ml of 40% aqueous solution of ethanol. The resulting solution was kept for 2 hours at a temperature of 50oWith, was evaporated to dryness under reduced pressure, re-evaporated with ethanol (35 ml), the residue was washed with diethyl ether (10 ml) and carefully dried under reduced pressure in the presence of phosphoric anhydride. Received 318 mg of the target product (yield to 98.6%). So pl. 149-151oC (decomp.).

UV-spectrum (maxnm): 256 (lg 4.09 to), 359 (lg 3,35). IR spectrum (KBr, cm-1): 3550-3200 (HE), 2800-3000 (CH), 1710, 1625, 1550, 1520 (C=N-O), 1415, 1360 (N-N-O), 1320, 1160, 1080, 1030 (C-O), 950. 860 (C=N-O),760.

Found, %: C 41,06; N 6,70; N 1,50. C132H216N4O10915 NM2ABOUT

Calculated, %: 40,93; N 6,40; N, 1,45.

The resulting complex contains the original components (DAPTO and CD) in a molar ratio of 1:3, respectively.

Example 2. The inclusion complex of 4,7-dimethyl-1,2,5-oxadiazole[3,4-d]pyridazin-1,5,6-trioxide with-CD(1:2) (2).

In the conditions of example 1 were mixed, heated up to temora ethanol and kept at a temperature of 60oC for 2 h the resulting solution was evaporated under reduced pressure, re-evaporated with ethanol, the residue was washed with a mixture (4 ml) of hexane-diethyl ether (1:2) and dried under reduced pressure in the presence of phosphoric anhydride. Received 314 mg of the target product (yield of 97.7%). So pl. 147-149oC (decomp.).

UV-spectrum (maxnm): 256 (lg 6,07), 359 (lg 4,98). IR spectrum (KBr, cm-1): 3380 (OH), 1520 (C=N-O), 1360 (N=N-O), 860 (C=N-O).

Found, %: C 41,86; N 6,40; N 2,20. C90H146O746H2O

Calculated, %:C41,96; N 6,18; N 2,17.

The resulting complex contains the original components (DAPTO and CD) in a molar ratio of 1:2, respectively.

Example 3. The inclusion complex of 4,7-dimethyl-1,2,5-oxadiazole[3,4-d]pyridazin-1,5,6-trioxide-GPCD (3).

In the conditions of example 1 from 60 mg (0.3 mmol) DAPTO and 355 mg (0.25 mmol) is GPCD (average mol. weight 1420) after evaporation under reduced pressure, re-evaporation with ethanol, washing with diethyl ether (10 ml) and drying received 357 mg of the target product. So pl. 164-167oC (decomp.).

UV-spectrum (maxnm): 257 (lg of 7.36), 360 (lg 6,61).

The resulting complex contains the source of the active ingredient (DOPE) in an amount equal to 9.4 wt.% (the moat solutions of complexes DAPTO, obtained in examples 1-3, DMSO-D6shows that these products do not contain appreciable quantities (not more than 0.4 wt.%) used for the synthesis of organic solvents (dioxane, ethanol, hexane or diethyl ether).

Example 4. The inclusion complex of 4,7-dimethyl-1,2,5-oxadiazole[3,4-d]pyridazin-1,5,6-trioxide with CC (4).

In the conditions of example 1 from 60 mg (0.3 mmol) DAPTO and 246 mg (0.3 mmol) of GC after evaporation under reduced pressure, re-evaporation with ethanol, washing with 10 ml of a mixture of hexane-diethyl ether (1:1) and drying received 258 mg of the target product. So pl. 142-145oC (decomp.). IR spectrum (KBr, cm-1): 3550-3200 (HE), 2800-3000 (CH), 1732 (CO), 1680 (CO COOH), 1524 (furostanolic cycle), 1456, 1444 (furostanolic cycle), 1376 (N=N-O).

The resulting complex contains the source of the active ingredient (DOPE) in an amount equal to 18.7 wt.% (conditional mol. the weight of the product in terms of 1 mol of bound peroxidase DAPTO ~ 1060).

Example 5. The inclusion complex of 3,4-dicyano-1,2,5-oxadiazol-2-oxide-PD(2:3) (5).

In the conditions of example 1 were mixed at a temperature of 30oTo a solution of 16 mg (0.12 mmol) DCF in 10 ml of ethanol and 130 mg (0.1 mmol) -CD in 30 ml of 40% aqueous solution of ethanol and kept at a temperature of 30oC.

UV-spectrum (maxnm): 272 (lg 5,49)

IR spectrum (KBr, cm-1): 3550-3200 (HE), 2800-3000 (CH), 22,08 (CN), 1628, 1454 (furostanolic cycle).

Found, %: C 39,28; N. Of 6.52; N, 2.74. C134H210N8O10924N2ABOUT

Calculated, %: C 39,16; N 6,33; N 2,73.

Example 6. The inclusion complex of 3,4-dicyano-1,2,5-oxadiazol-2-oxide-GPCD (6).

In the conditions of example 1 of 41 mg (0.3 mmol) DCF and 355 mg (0.25 mmol) is GPCD (average mol. weight 1420) after evaporation under reduced pressure, re-evaporation with ethanol, washing with 10 ml of a mixture of hexane-diethyl ether (1: 1) and drying received 344 mg of the target product. So pl. 118-120oC. UV range (maxnm): 273 (lg 3.62). IR spectrum (KBr, , cm-1): 3550-3200 (HE), 2800-3000 (CH), 2208 (CN), 1636, 1454 (furostanolic cycle).

The resulting complex contains the source of the active ingredient (DCF) in an amount equal to 9.2 wt.% (average mol. the weight of the product in terms of 1 mol of bound peroxidase DCF ~1470).

Analysis1H-NMR spectra of solutions of complexes DCF obtained in examples 5 and 6, in DMSO-D6shows that these products are not Soana or diethyl ether).

Example 7 (comparative). The study of the interaction benzotrifluoride with-CD.

In the conditions of example 1 from a solution of 75 mg (0.3 mmol) benzotrifluoride in 50 ml of dioxane and 324 mg (0.25 mmol) -CD after evaporation under reduced pressure, re-evaporation with ethanol, washing with 10 ml of dioxane and dried product was received, an analysis using UV spectroscopy showed almost complete absence of maximum absorption in the region 252-258 nm, corresponding to the free ligand.

Example 8. Learning new inclusion complexes by differential scanning calorimetry.

To confirm the formation of inclusion complexes furosemidelasix compounds of General formula I with-CD-CDHP and GC used the method of differential scanning calorimetry (DSC), which is based on the difference in thermal effects that occur when a programmed heating of samples of the mixture components and the system as a whole. In Fig. 2-4 presents the DSC thermograms of individual substances (DAPTO, DCF, -CD, -CDHP), mechanical mix-CD and DOPE, and received new inclusion complexes.

As shown in Fig. 2, the DSC thermogram DAPTO has a clear peak in the region is the development of Azin-N, N'-dioxide group DAPTO, and in the temperature range of 160-180oWith a peak is observed, which reflects the melting of the substance, followed by its decomposition and the release of large amounts of energy. Similar endothermic effect is in the region of 200oWith the DSC thermogram investigated earlier known TNG with-CD.

For CD on the DSC thermogram there are no clear peaks of thermal effects in the temperature range of 60-65oAnd 160-180oC, and the deviation of the temperature curve from the baseline and the presence of a minimum at a temperature of 113oC indicate the process associated with the degradation-CD in this temperature region.

In the case of a mechanical mixture DOPE with-CD, taken in a molar ratio of 1:3, there is total thermal effect, characterized by the presence of a peak in the above (or close) temperatures, which correspond to temperatures above the effects of individual source components.

The DSC thermogram obtained complex 1 /DOPT with-CD (1:3)/ is significantly different from the DSC curves for the individual components and their mechanical mixtures and has a minimum at a temperature of 82oC and maximum at a temperature 147-151

Example 9. The formation of nitric oxide from the new inclusion complexes.

To determine NO used a known method based on the reaction of nitric oxide with oxygen in the aquatic environment with the formation of nitrite, the amount of which was measured by the intensity of staining of the sample with the reaction product of the result with a spectrophotometer. As evidence that this method gives the possibility to measure the nitric oxide released from the compound by chemical reaction and not nitrite, the reaction is carried out in the presence of oxyhemoglobin, which quantitatively reacts with nitric oxide (but not nitrite), forming nitrate, which does not react to the result.

Sample final volume of 1 ml contained 50 mm potassium phosphate buffer (pH 7.4) or 20 mm potassium-citrate buffer (pH 5.0), 0.5 mm cysteine or glutathione, the target compound in a concentration of 0.1 mm and 0.2% dimethyl sulfoxide (DMSO), while incubation with oxyhemoglobin his concentra as a positive control, 0.1 mm sodium nitrite, containing 0.2% DMSO. Samples were incubated for 60 min at 37oWith and added consistently 100 ál of 3 M sodium acetate, 400 µl 0,92% solution of sulfanilic acid in 30% acetic acid and 400 μl of 0.05% N-naphthylethylenediamine. Samples were incubated 10 min and measured the optical density at a wavelength of 554 nm on the spectrophotometer.

In the above conditions at pH 7.4 was not observed the formation of appreciable quantities of nitrite (<0.01 mol of water/mol of complex inclusions) in the absence of thiols. In the presence of thiol complexes 1-6 were generated 0,057-0,411 mol of nitrite per mole of the complex in the presence of cysteine and 0.017-rate £ 0.162 mol of nitrite per mole of the complex in the presence of glutathione (see tab. 1). Further incubation did not lead to additional formation of nitrite. During incubation of the complexes of the present invention in the above-mentioned conditions in the presence of oxyhemoglobin and cysteine or glutathione was observed the formation of methemoglobin (see example 11).

According to the prototype of the NO-generating properties of the known analogue of (complex GHP) in the conditions described, were not studied. It is known that TS in the presence of 1-100 mm 1-cysteine capable of generating nitric oxide (Feelisch, V., Noack, E., Schroder, H. "Explanation of the base currency by the difference between t S-nitrosothiols (S-nitrosoglutathione) new inclusion complexes.

To determine the S-nitrosothiols used a method based on reaction with mercury chloride (II), during which there is a formation of nitrite. To account for the formation of nitrite during the reaction of the compounds of the present invention with glutathione the reaction was carried out as described in example 8, and then nitrite was determined by the method described in example 8, in the absence and in the presence of mercury chloride (II) in a concentration of 0.1%. The difference between the obtained values corresponds to the number of S-nitrosoglutathione.

In the above conditions, the products of the present invention are able to form S-nitrosothiol. Similar properties known complex include GHP has not been studied.

Example 11. The formation of reduced forms of nitrogen oxide new inclusionary complexes.

To determine the reduced forms of nitrogen oxide (NO-/HN) used a known method, based on the fact that formed during the reaction nitroxyl reacts with thiols to form hydroxylamine (or competitive formation of N2O), which, after oxidation of ions of I3-gives nitrite, as defined by azocoupling reaction (see example 8). Because the study secnum stage, as a positive control was used hydroxylamine hydrochloride and sodium nitrite in a concentration of 0.1 mm, and incubation was performed as described in example 1 at pH 7.4. After incubation, the samples were added to 100 ál of 3 M sodium acetate, 400 µl 0,92% solution of sulfanilic acid in 30% acetic acid, 100 μl of 1.25% I22% KI, 30 ál of 0.5 M 2-mercaptoethanol and 400 µl of 0.05% N-naphthylethylenediamine. Samples were incubated 15 min and measured the optical density at a wavelength of 554 nm on the spectrophotometer. In parallel with this experiment was carried out measuring the formation of nitrite, as described in example 8. The content of hydroxylamine was calculated by the formula: X=(A-YN)/H, where X corresponds to the concentration of hydroxylamine, μm; And the optical density of the sample containing the analyzed system; Y is the concentration of the resulting sodium nitrite determined as described in example 1 μm; N is the optical density of the positive control containing sodium nitrite, μm-1; and N is the optical density of the positive control containing hydroxylamine hydrochloride, μm-1.

In the above conditions was observed the formation of a hydroxylamine of the products of the present invention in the presence of cysteine or glutathione. The possibility is you, presented in table.1, show that the products of the present invention are by NO donors, as well as its active form nitrosothiols and NO-/HNO, reflecting the molecular basis of the pharmacological action of the considered complexes.

Example 12. The formation of methemoglobin from oxyhemoglobin under the new inclusion complexes of General formula I.

The definition of generation of NO in the presence of oxyhemoglobin, accompanied by the formation of methemoglobin, studied in a known manner in a spectrophotometric cuvette final volume of 1 ml at 25oC. Samples contained 25 mm potassium phosphate buffer (pH 7.4), 3 μm oxyhemoglobin, 0.5 mm cysteine or glutathione and analyzed complexes at a concentration of 0.1 mm. Had determined the rate of increase of absorbance at 401 nm and linear initial plot was calculated the rate of formation of nitric oxide (molar ratio of absorption of methemoglobin was taken equal to 39.9 mmol-1cm-1). In the absence of thiols noticeable increase in absorbance at 401 nm was not observed. The apparent reaction rate constant of the first order expressed in min-1. From literature data it is known that the process of turning oxigeno is esperimento checked the absence of these forms of hemoglobin. For this purpose, samples were incubated in potassium phosphate buffer pH 7.4 in the presence of 25 μm oxyhemoglobin, 0.2 mm cysteine or glutathione and 20 μm of compounds. Measured optical density at 560, 577, 630 and 700 nm and was calculated concentration of oxyhemoglobin, hemichrome, methemoglobin and hollobone. These experiments showed that incubation of the studied complexes with oxyhemoglobin in the absence of thiols or in the presence of cysteine or glutathione is not observed the formation of hollobone or hemichrome, and occurs exclusively the conversion of oxyhemoglobin to methemoglobin.

In the above conditions, the products of the present invention caused the formation of methemoglobin from oxyhemoglobin, and this effect was enhanced in the presence of cysteine and glutathione. Research results (rate constants of the reaction NO+GM O2-->NO3-+mathm) are presented in table.2. The investigated complexes reacted under similar conditions with methemoglobin.

Example 13. Study of the reaction of new inclusion complexes DOPE with low molecular weight thiols.

The apparent reaction rate constant of the second order was determined in a known manner by measuring the rate of decrease of concentration to the Ufer (pH 7.4), study products at a concentration of 20 μm and 20, 50 and 100 μm solutions of cysteine or glutathione. A control sample contained the corresponding amount of DMSO (0.2 percent). Incubation was carried out at a temperature of 20oC. Decrease the concentration of the complexes was determined from the change in optical density at a wavelength of 360 nm, taking into account the absorption of the reaction products at this wavelength. Check the optical density was started immediately after addition of a solution of the thiol in the reaction mixture. The decrease of optical density was calculated by linear section of the plot of optical density against time. The apparent reaction rate constant of the second order was calculated by the following formula:

k=v/(ctoct)

where k is the apparent reaction rate constant of second order complexes with thiols, (Msec. )-1; v is the reaction velocity, M/sec.; ctothe concentration of the complex, M; ctthe concentration of thiol, M

In an additional experiment, it was found that the apparent reaction rate constant of second order complexes with thiols does not depend on the concentration of thiols.

Example 14. Activation of the soluble form of guanylate cyclase new inclusionary complexes.

The activity of the WG is mg tissue homogenized in 5 volumes of 50 mm Tris-Hcl buffer (pH 7,6), containing 10 mm MgCl2using homogenizer "glass-glass". The homogenate was centrifuged for 30 min at 16000 g, the supernatant was collected and determined the enzyme activity by the number of [32R]cGMP formed from [-32P]GTP, in a known manner. The supernatant (300 μl) was diluted with 450 μl of Tris-HCl buffer (pH 7.6) and an aliquot of 10 µl of ice was added to the incubation mixture (final volume of samples 100 ál) containing (final concentrations) 50 mm Tris-HCl buffer (pH of 7.6), 1 mm 3-isobutyl-1-methylxanthines, 5 mm MgCl2, 0.4 mg/ml creatine phosphokinase, 5 mm creatine phosphate, 2 mm cGMP, 0.2 mm GTP, 10000-20000 pulse/min/pmol [-32P] GTP (Isotope, INP, Obninsk), and 10 mm DTT or other thiols, as indicated in the text later. When defining a trigger actions in the incubation medium was made of the studied compound at a concentration of 10 μm in the form of a solution in aqueous DMSO, and in a control sample was added DMSO to a concentration of 0.02%. The control sample showed no effect of DMSO at the indicated concentrations on the basal activity of the RHC. Samples were incubated at 37oC for 15 minutes the Reaction was stopped by boiling the samples for 2 min After cooling to room temperature the sample was added 0.5 ml of 30 mm Na2CO3whether at 15 000 g for 5 minutes The supernatant was applied to a column with acidified known manner alumina, which was washed with water. The elution of [32P] cgma was performed with 0.2 M ammonium formate in vials for scintillation counting, and the radioactivity was measured using liquid scintillation counter in a known manner Cherenkov.

Determination of protein was carried out according to the method of Lowry using bovine serum albumin as the standard.

As a consequence of the data table. 3, the products of the present invention at a concentration of 29-64 μm 8.2-23.3 fold increased activity RHC in the preparation of rat lung. Biochemical properties of the known analogue in this test has not been studied. From literature data it is known that GHP effectively activates RHC only after biotransformation or in the presence of high concentrations of cysteine.

Example 15. Vasodilator and antispasmodic activity of new inclusion complexes.

Vasodilator and antispasmodic activity of products of the present invention was studied on the model of isometric contraction of smooth muscle of the isolated segment of the aorta in rats under the action of vasoconstrictor (spasmogen)--adrenoagonists familari is in Wistar rats with average weight of 280 g (210-330 g) in a known manner. Rats were decapotable, cut the thoracic aorta and purified from adipose tissue. Then cut the rings with a width of 2.5 mm, which were hung on two parallel hooks stainless steel. One of the hooks fixed to the wall of the chamber, and the other was connected to an isometric transducer DY1 connected with eight-channel data logger (Beckman, USA). The camera contained 30 ml of Krebs solution (130 mm NaCl, of 4.7 mm KCl, 2.5 mm l2, 1,18 mm KN2PO4at 14.9 mm NaHCO3, 1.2 mm MgSO4, 11 mm glucose) at 37oWith that was under constant aeration with 95% O2/5% CO2to maintain pH 7.4. In the Krebs solution was added indomethacin concentrations up to 1 μm. If necessary, the endothelium was removed mechanically. Rings before you experience balanced for one hour under a load of 2.5 g Before the beginning of the experiment ring presacral 10 nm norepinephrine, and after 20 min caused a reduction of the drug by the addition of phenylephrine in a concentration of 0.5 μm. After the stabilization muscles of the vessel was registered, it relaxes in response to cumulative doses of the studied complexes in the concentration range from 0.003 nm to 50,000 nm. After that investigated the complex was washed 6-7 shifts incubation medium for 1 hour connection to the low solubility of compounds in water the effect of solvent (DMSO) on the isometric contraction of the vessel was determined in separate experiments. It was found that DMSO at a concentration of 0.1-0.2% with almost no effect on vascular tone, allowing you to test vasorelaxant active substances in concentrations up to 100 μm. All experiments were repeated at least 3 times. The concentration value of an investigational new complexes corresponding to 50% relaxation of the vessel (IC50) was calculated using the program SigmaPlot version 2.0 standard equation for a sigmoidal dependency

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where R corresponds to the relaxation of the vessel when the concentration of the complex, Rmaxcorresponds to the maximum relaxation under the influence of the same substance, and n represents the virtual coefficient of cooperativity, warioware in the range from 1.1 to 1.8. The reliability of the selection of the parameters (n and IC50) was controlled by the method of least squares, and it was at least 95-97%. Statistical analysis was performed by student's t test (t). The results are presented in table. 5.

The products of the present invention caused the reduction of isometric contraction of the vessel with intact endothelium in the presence of phenylephrine. According to the prototype study known complex include IGT in vitro close to the above, nuna activity new inclusion complexes.

Hypotensive activity of the products of the present invention was determined in vivo in awake rats of Wistar rats with average weight of 280 g in a known manner. For measurement of mean arterial pressure and heart rate and for the introduction of the drug in the bloodstream for days before the experiment, animals were implanted polyethylene catheters (grades D-10, D-50) into the femoral artery and femoral vein. The free ends of the catheter taken out and fixed on the head. The operation was conducted under geksenalovy anesthesia (150 mg/kg). The next day the rat took in the experience. During the experiment were recorded blood pressure sensor company Stadham (USA) with the following registration data using a computer program Bioshell, faculty of fundamental medicine, Moscow state University. University, Russia). Compounds were injected bolus intravenous dose of 2.5 mg/kg in a volume of 0.2 ml in 5% DMSO. During the whole experiment the animals were awake and could move freely in the cage. Within hours the animals were freely adapted to the conditions of the experiment, and then began the registration of hemodynamic parameters, which was conducted continuously throughout the experience.

A bolus of products this is Uchenie ~ 5 sec, which lasted for 6-20 seconds (e.g., 6-11 sec in the case of sets. 1, 7-15 sec - set. 2,6-9 sec compl. 4), and then saw a rapid restoration of the original indicators. A similar indicator for IGT, used as the comparison drug, had a value 22-47 sec. Heart rate (HR) short-term slightly increased only in the peak of fall in blood pressure, and then decreased to the initial level. In control experiments it was found that in the same conditions of the original complexing substances-CD-GPCD and SC doses of 20-140 mg/kg had no significant effect on blood pressure and heart rate.

The results, presented in table. 6, show the effect of bolus intravenous injection of new inclusion complexes in doses 20-140 mg/kg) on mean arterial pressure (BP) in awake rats.

The data table.6 shows the high efficiency of vasodilating and hypotensive action new inclusion complexes when administered intravenously.

According to the prototype effect inclusional complex with NTT-CD on blood pressure in vivo corresponded to similar effect preformed IGT, this comparison is carried out. Research results indicate that NO-generating compounds of General formula 1 (e.g., DOGTO) as part of new inclusion complexes show a stronger hypotensive properties compared to similar effect IGT and effect of the same ligand in the free form. So, according to the data presented in table.6, in the experiment, lowering the average AD under the action of complex 1 containing DOPE at a dose of 40 µg/kg, and complex 2 containing DOPE at a dose of 30 μg/kg, approximately corresponded to changes in the values of this parameter, caused by injection of NTG and DAPTO in doses of 120 mg/kg and 100 mcg/kg, respectively. Thus, the results obtained in vivo, for the first time show that through the formation of inclusion complexes can be significantly improved pharmacological activity (in particular, to enhance the hypotensive properties) NO-generating compounds.

Example 17. Inhibition of aggregation of human platelets under the new inclusion complexes.

The influence of the products of the present invention on the aggregation of human platelets was studied known turbidimetric method of born. This venous blood taken in anie 10 min, using as anticoagulant sodium citrate. The supernatant, i.e. platelet-rich plasma was collected and centrifuged at 650 g for 30 min, receiving platelet-poor plasma. The concentration of platelets was brought in platelet-rich plasma to 2,5108cells/ml with dilution platelet-poor plasma was added and the resulting suspension in a cuvette with a volume of 0.5 ml. Aggregation was induced by the addition of ATP to a concentration of 2-5 microns. The concentration of ADP was selected in each experiment so that the aggregation was reversible and maximum accounted for 2 min after addition of ADP, not exceeding 50%. The scattering suspension of platelets was measured using aggregometry developed in the laboratory of Bioorganic chemistry of Biological faculty of Moscow state University. M. C. Lomonosov (Russia). The studied compounds were added to ADP.

The products of the present invention at a concentration of 1-100 μm inhibited platelet aggregation induced 12.5 μm ADP. For example, the values of the concentrations at which it is achieved premaxillae inhibition (IC50for complexes 3, 4 and 5 were 11.2, 6.3 and 0.9 µm, respectively. According to the prototype antiplatelet properties known analogue of (complex GHP similar DAPTO and DCF were 6.3 and 2.9 μm, respectively.

Example 18. Acute toxicity of new inclusion complexes.

Acute toxicity of the products of the present invention was determined in a known manner by LD50using outbred mice of both sexes with average weight of 21 g at room temperature; standard diet and water was given ad libitum during the whole experiment; the mobility of the animals is not restricted. Solutions of the compounds in DMSO were injected using a sterile syringe intraperitoneally. After injection the animals were observed for 48 hours; after this time the mice were additionally observed within 72 hours (none of the animals died within any additional period of time). The results obtained indicate that the value LD50for complexes 1-4 is ~ 300-500 mg/kg, for complexes 5 and 6 ~150-300 mg/kg

It is known that the acute toxicity of inclusion complexes mainly depends on the toxicity of the bound ligand ("guest"), because commonly used complexing agents having a high value LD50(e.g., LD50for-TP and-CDHP is >10,000 mg/kg). Values LD50for DAPTO and DCF defined in the above way, are ~100 and 50 mg/kg sootvetstvenno RF 2086534 (07 With 203/04) 1994, table. 2), which implies a higher toxicity of inclusion complexes obtained on its basis, compared with the products of the present invention.

Example 19. The solubility and stability of new inclusion complexes.

The solubility of the products examined in the experiment was determined as follows. Prepared the initial solution of the substance in DMSO at a concentration of 50 mm. Then 50 mm solutions in DMSO were diluted with deionized water to concentrations 25, 50, 100, 250, 500, 1000, 2000 µm. To the resulting solution was added DMSO to a final concentration of 4%. Solutions containing substance at a concentration of 100, 250, 500, 1000, 2000 μm, were centrifuged at 14000 g for 15 min Supernatant were carefully selected and diluted to determine the optical density. The optical density of the solutions was determined on spectrophotometer Ultrospec at the corresponding wavelength (360 nm in the case of complexes DAPTO, 272 nm in the case of complexes DCF).

The measurement results show that the obtained products are soluble at all concentrations used in the experiment (25-2000 μm), while the inclusion complexes 1-4 have greater solubility than the ligand (DAPTO). Complete solubility DAPTO in OPA complexes in DMSO were stable for 1 month at room temperature when stored in a place protected from light and frozen at -20oWith up to 6 months. 0.25 mm aqueous solution of complex 1 containing 0.5% DMSO, stable at room temperature when stored protected from light place at a neutral or slightly acidic pH values. Aqueous solutions of the products of the present invention is unstable at alkaline and alkaline pH values (pH>9), in bright light and in the presence of thiols. Dry these substances are highly stable at room temperature when stored in a dark place.

Example 20. The solution inclusional complex for injection.

Components - mg

Active component (complex) - 4

Ethyl alcohol - 500

Water for injection To 1 ml

Example 21. The solution inclusional complex for infusion (concentrate).

Components - mg

Active component (complex) - 25

Ethyl alcohol - 350

Propylene glycol - 300

Water for injection To 1 ml

Example 22. Gelatin capsules of 0.001 and 0.0005 g containing inclusionary complex in refined sunflower oil.

Components wt.%/capsule

Active component (complex) - 5

Refined sunflower oil - 95

Example 23. Powder inclusional complex deposited on an inert carrier, for the Khabibullina (lactose) - 98

The above examples 9-12, 13 and 14 show that the new inclusione complexes of the present invention in the presence of thiols are effective NO donors, as well as generate his redactora (nitroxyl) form nitrosothiol and activate the RHC. From examples 15 and 16, the conclusion must be that these products have a strong antispasmodic, vasodilator and rapid hypotensive action, and their pharmacological effect in vivo (in terms of dose associated ligand) surpasses the similar action of trinitroglycerine - ligand, part of the closest known analogue - its complex with-CD. In addition, the described products, unlike IGT, are expressed antiplatelet properties (example 17).

Thus, the new inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of the above General formula I with polycyclic derivatives of glucopyranose the above General formula II extend the range of the NO donors, activators RHC, highly effective antispasmodic, vasodilator, hypotensive drugs fast action and platelet aggregation inhibitors, and pharmaceutical compositions based on these products assume prob the azole-2-oxide with polycyclic derivatives of glucopyranose, characterized in that the derived 1,2,5-oxadiazol-2-oxide of General formula I:

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where R1= R2= CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, enter into interaction with polycyclic derivatives of glucopyranose General formula II

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where if n= 1, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the formula III

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R4=H;

R5--D-glucuronidase;

R6=R7=H;

R8=C(O)OH,

or, if n= 7, R3= N, R4and R7- simple connection, R5and R6= H or (CH2CH(CH3)O)mH, where m=1 to 14, and R8=CH2OH or CH2O(CH2CH(CH3)O)mH, where m=1 to 14, in an aqueous-organic medium at elevated temperature, followed by separation of the target product, washing monopolarly inert organic solvent or their mixture and drying under reduced pressure.

2. The inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of the above General formula I, where R1=R2=CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis the cycle of polycyclic proizvodi the acid of the above formula III, R4= H, R5--D-glucuronidase, R6=R7=H and R8= C(O)OH, or, if n=7, R3=H, R4and R7- simple connection, R5and R6= H or (CH2CH(CH3)O)mH, where m= 1 to 14, and R8=CH2OH or CH2O(CH2CH(CH3)O)mH, where m=1-14 obtained under item 1, which generates nitric oxide and activating the soluble form of guanylate cyclase, antispasmodic, vasodilator and antihypertensive drugs fast action and platelet aggregation inhibitors.

3. The inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide under item 1 above General formula I, where R1=R2=CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, polycyclic derivatives of glucopyranose the above General formula II, where, if n= 1, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the above formula III, R4=H, R5--D-glucuronidase, R6= R7= H and R8=C(O)HE n or if n=7, R3=H, R4and R7- simple connection, R5and R6=H or (CH2-CH(CH3)O)mH, where m=1 to 14, and R8=CH2HE or CH2O(CH2-CH(CH3)OmH, where m= 1-14, receiving spasms of smooth muscles.

4. Pharmaceutical composition, which generates nitric oxide, which activates the soluble form of guanylate cyclase and has antispasmodic, vasodilator and hypotensive action, containing the active ingredient and additives, characterized in that as the active component are inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of the above General formula I, where R1=R2=CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, polycyclic derivatives of glucopyranose the above General formula II, where, if n=1, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the above formula III, R4=H, R5- -D-glucuronidase, R6=R7=H and R8= C(O)OH, or, if n= 7, R3=H, R4and R7- simple connection, R5and R6=H or (CH2CH(CH3)O)mH, where m=1 to 14, and R8=CH2OH or CH2O(CH2CH(CH3)O)mH, where m=1-14 obtained under item 1.

 

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The invention relates to a steroid compound of General formula I

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whereis = O, -HE, or SIG or ООСR, where R represents an alkyl group having from 1 to 6 carbon atoms; R6represents H or -(CH2)mN, where m = 1 or 2; R7represents H, C1-4-alkyl, C2-4alkenyl or2-4-quinil; R11represents H, C1-4-alkyl, C2-4alkenyl,2-4-quinil; E represents, including the carbon atoms 16 and 17 of the D ring, a 4-7-membered hydrocarbon ring, where the specified ring is in the-position relative to the D-ring, substituted by a group REand optionally contains one endocyclic double bond; RErepresents H, C1-5-alkyl, C2-5alkenyl,2-5-quinil,1-5-alkyliden, -(CH2)n-N3or -(CH2)n-SP, where n = 1 or 2, and where the alkyl group may be substituted by-OR, -OOCR where R is alkyl with 1-6 carbon atoms; R17is-HE-or SIG or ООСR, where R is alkyl with 1-6 carbon atoms, where the aforementioned steroid compound may be, but neeba is either ring may be aromatic

The invention relates to a method of processing betulin and its derivatives, for example Betulinol acid, betulin diacetate, and can be used in cosmetic, pharmaceutical and chemical, in particular in the manufacture of varnishes and paints industries

The invention relates to new biologically active chemical compound, specifically to 3,28-di-O-nicotinate betulin (1), formula

< / BR>
showing hepatoprotective and anti-HIV activity

The invention relates to the synthesis of allobetulin (19, 28-epoxy-oleana-3-Ola) isomerization of Betulinol(loop-20(29)-EN-3, 28-diol) in the presence of catalysts

The invention relates to 14,17-C2-bridged steroids of formula I, where R3- O, R6- Hor-(C1-C4)-alkyl, where R6and R7together form an additional bond; R7-or-(C1-C4)-alkyl, where R6and R6both H, or R9and R10each H or together form a bond, R11and R12each H or together form a bond, R13- CH3or2H5; R15- H or C1-C3-alkyl; R16and R16independently H, (C1-C3)-alkyl or C1-C4alkenyl or together form a (C1-C3-alkyliden; R15and R16together form a cyclewhere n = 1, and h - O and R16- N- H, (C1-C3)-alkyl,- H, (C1-C3)-alkyl,- H, (C1-C3)-alkyl or HE; except 14,17-ethano-19-norpregna-4-ene-3,20-dione

The invention relates to substituted derivatives of 19-norpregnane, methods of producing these compounds and pharmaceutical compositions containing them

Steroid compound // 2160279
The invention relates to extrenely steroids that are associated with neuroepithelial cells in the vomeronasal organ of the human body

The invention relates to compounds of formula (I):

< / BR>
where

-A= B-C= D - represents-CH=CH-CH=CH-group, in which 1 or 2 CH may be replaced by nitrogen;

Ar denotes phenyl or naphthyl, unsubstituted or one-, two - or three-fold substituted with H, Gal, Q, alkenyl with the number of C-atoms up to 6, Ph, OPh, NO2, NR4R5, NHCOR4, CF3, OCF3CN, OR4, COOR4, (CH2)nCOOR4, (CH2)nNR4R5, -N=C=O or NHCONR4R5phenyl or naphthyl;

R1, R2, R3each independently from each other, are absent or represent H, Gal, Q, CF3, NO2, NR4R5, CN, COOR4or CHCOR4;

R4, R5each independently of one another denote H or Q, or together also denote-CH2-(CH2)N-CH2-;

Q denotes alkyl with 1-6 C-atoms;

Ph denotes phenyl;

X denotes O or S;

Gal denotes F, Cl, Br or I;

"n" represents 1, 2 or 3;

and their salts, except 4-methyl-N-(2,1,3-benzothiadiazole - 5-yl)benzosulfimide, 4-nitro-N-(2,1,3-benzothiadiazole-5-yl)- benzosulfimide and 4-amino-N-(2,1,3-benzothiadiazole-5-yl)- benzolsulfonat
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