Derivatives of 1,2,5-oxadiazole-[3,4-d]-pyridazin-5,6 - dioxide as activators of soluble form of guanylate cyclase and means for the treatment of diseases of the cardiovascular system and pharmaceutical compositions based on them

 

(57) Abstract:

The invention relates to medicine, in particular it is proposed to use derivatives of the formula I, where R = CH3or6H5and n = 0 or 1, as vasodilator, hypotensive, antispasmodic, anti-means and platelet aggregation inhibitors and pharmaceutical composition thereof. Connections implement the specified activity through activation of the soluble form of guanylate cyclase. 2 S. and 1 C.p. f-crystals, 4 tables, 4 Il.

The invention relates to biochemistry and pharmacology, in particular to the application of the known derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I

(I)

where R = CH3or C6H5and n = 0 or 1, for the specific activation of the soluble form of guanylate cyclase (RGC) and treatment of diseases of the cardiovascular system and pharmaceutical compositions based on them.

Guanilatziklazu/EC 4.6.1.2; guanosin-5'-triphosphate-pyrophosphatase (cyclessa)/is the enzyme that catalyzes the biosynthesis of guanosine-3',5'-cyclophosphate (cGMP) is a universal regulator of intracellular metabolism [1].

HZ exists in two forms - membrane and soluble. In the common of nitrovasodilators (nitroglycerin, nitrosorbid, sodium nitroprusside) and plays a key role in the regulation of these physiological processes such as contraction and relaxation of smooth muscles of blood vessels and platelet aggregation. It is shown that therapeutic effect of the above pharmaceuticals related to the stimulation of the activity of the RHC as a result of interaction of nitric oxide formed during biotransformation, with the iron atom of heme, part of the enzyme, and education complex nitrosyl-heme.

A significant drawback of the known vasodilator on the basis of organic nitrates is the emergence of tolerance in their long-term use. In this regard, the study of the mechanism of action of new compounds capable of generating NO in vivo and/or to cause activation of the enzyme NO-independent way, is a promising approach for the discovery and development of more effective antihypertensive and antiplatelet medicines.

Known pharmaceuticals "Minoxidil" - 6-(1-piperidinyl)pyrrolidine-2,4-diamine 3-oxide of formula II

(II)

which peripheral vasodilatation [2] and increases the level of cGMP in smooth muscle cells of blood vessels [3].

There are various N-oxidum (restored form - NO-/HNO, nitrosothiols) and activators RHC and have a pharmacological effect on the cardiovascular system [4].

Thus, the known 3,4-disubstituted furoxane, in particular 1,2,5-oxadiazole-3,4-dinitrile-2-oxide, 3-phenyl-1,2,5-oxadiazol-4-nitrile-2-oxide and its isomer of General formula III

(III)

where R1= CN and R2= CN or C6H5or R1= C6H5and R2= CN, which is a nitric oxide donors and activators RHC from the lungs of rats (12-37 times), and also has a pronounced vasorelaxant action on the rings of rabbit aorta, reduced under the action of phenylephrine (IC50= from 0.005 to 0.016 μm) and inhibiting platelet aggregation caused by the action of collagen (IC50= 0,24 is 0.84 μm) [5].

However, these compounds have high toxicity (see example 12), and their synthesis is quite complicated.

Known salts of N-nitroso-N-substituted hydroxylamine derivatives of the General formula IV

(IV)

where R = O-, SO3-C6H5and others, which NO donors in vitro and possess antihypertensive activity [6].

According to presented data, the most activity in vivo had salt Angeli/K2(O350for this connection amounted to 3,1610-7M [7].

The disadvantages of this connection are its instability due to rapid hydrolysis at physiological pH values and relatively low antihypertensive activity.

Known salts of deadenylylation(NONO) of General formula V

(V)

where R and R1> is alkyl, substituted alkyl, representing dimeric adducts with NO nitrogen-containing nucleophiles [4]. These compounds are spontaneous NO donors or NO-increases the level of cGMP in the cell and have vasorelaxant action.

Their disadvantages are the relative complexity of the synthesis (autoclaving under pressure in an atmosphere of NO within 1-3 days), comparative instability in aqueous medium at physiological pH values, as well as the possibility of formation of carcinogenic N-nitrosodialkylamines during their decomposition in aerobic conditions [4].

Known substituted 1,2-diacetin-1,2-dioxides of the General formula VI

(VI)

where R1- H or Br, R2- alkyl or phenyl, R3- alkyl and R4Is H or alkyl, generating nitric oxide when heated (80oC), aktiviranega platelets under the action of ATP [10]. The most active compound, 1-bromo-6-methyl-7,8-diazabicyclo[4,2,0]octene-7,8 - oxide in vitro on isolated rings of rat aorta, reduced under the action of norepinephrine, had IC50in 2 times above the IC50for nitroglycerin.

In General, these compounds exhibit high enough inflammatory and antiplatelet activity, and possess low stability when stored at room temperature, which is related to their ability to spontaneously generate nitric oxide.

Known 3,6-disubstituted pyridazine-1,2-dioxides of the General formula VII

(VII)

where R1and R2- CH3or C6H5[II].

However, the biochemical properties and the pharmacological action of these compounds has not been studied.

Patented substituted pyrrolo[2,3-d] pyridazin-5,6 - dioxide in a number of other analogues of General formula VIII

(VIII)

where A, R1- R5X are specified in the patent values, m = 0 or 1 and n = 0 or 1, as a means to treat stomach ulcers caused by Helicobacter pylori [12].

The impact of these compounds on the activity of RHC and applicability for the treatment of diseases of the cardiovascular system have not been studied.

ETBE photoactivated donor of nitric oxide [13].

However, the biochemical properties and pharmacological activity of this compound has not been studied.

Closest to the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the above General formula I are 3,5-disubstituted pyrazole-3-he-1,2-dioxides of the General formula X

(X)

where R1- alkyl (CH3), aryl (C6H5), substituted aryl, etc., R2aryl (C6H5), substituted aryl and others, which are thiol-dependent NO donors, activators RHC and means for the treatment of diseases of the cardiovascular system [14].

The disadvantages of these compounds, in particular 3,5-diphenylphenol-3-one-1,2-dioxide (compound 5), are the low degree of activation RHC (2.5-fold at a concentration of 10 µm, see example 6), inhibition of aggregation of human platelets using relatively high concentrations (IC50= 1.5 μm in the model with collagen as an inducer of aggregation [15]), and low efficiency vasodilatory action in vitro on isolated rings of rat aorta, reduced under the action of phenylephrine (IC50= 1.5 μm).

Known derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine - 5,6-dioxide of the General formula I as the products chemicly of the invention is to find new activators RHC and means for the treatment of diseases of the cardiovascular system, with more severe biochemical and pharmacological properties.

This goal is achieved by the application of known derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the above General formula I as activators RHC and medicines for the treatment of diseases of the cardiovascular system.

Specific compounds according to General formula I are 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazin-1,5,6-trioxide (4,7-dimethylpyrazolo[3,4-d] pyridazin-1,5,6-trioxide; compound 1); 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide (4,7-dimethylpyrazolo[3,4-d]pyridazine-5,6-dioxide; compound 2); 4,7-diphenyl-1,2,5-oxadiazole[3,4-d]pyridazin-1,5,6-trioxide (4,7-diphenylpyrazine[3,4-d]pyridazin-1,5,6-trioxide; compound 3); 4,7-diphenyl-1,2,5-oxadiazole[3,4-d]pyridazine - 5,6-dioxide(4,7-diphenylpyrazine[3,4-d]pyridazine-5,6-dioxide; compound 4).

Preferably, the derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the above General formula I are 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazin-1,5,6 - trioxide (compound 1) and 4,7-dimethyl-1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide (compound 2).

This invention relates to the use of derivatives of 1,2,5- -oxadiazole[3,4-d] peridas the Noah hypertension, congestive heart failure in myocardial infarction, hypertensive crises, refractory heart failure, myocardial infarction and acute myocardial infarction, angina, ischemic heart disease, chronic heart failure, pulmonary hypertension, acute failure of the left ventricle, lung heart, lung edema, peripheral arterial embolism, 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 blood pressure, normalization contractility and/or the inhibition of platelet activity, except for those diseases which risk reduction in blood pressure 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 cardiomyo diathesis, gipoprotrombinemii).

This invention also relates to the use of 4,7-dimethyl-1,2,5-oxadiazole[3,4-d] pyridazin-1,5,6-trioxide for the prevention and relief of angina and for the treatment of acute myocardial infarction.

Compounds 1, 3, and 4 were synthesized in 1925-1927, [16, 17] (compound 2 in 1899 [18]), but their chemical structure was unambiguously established much later [19, 20].

In General, compounds 1, 3, 4 is obtained by oxidation of dioximes corresponding 3,4-decylphosphonic of furoxan or furazan (scheme 1). Depending on the structure of the original dioxide as oxidant use cityregional nitrogen, nitric acid, or a dilute solution of potassium permanganate.

Another method of synthesis of compounds 1 and 3 based on the reaction of 2-substituted 1-galogenarenov with silver nitrite in the medium of ether [21, 22].

Compound 2 was obtained by processing dioxime 3,4 - diacetylpyridine aqueous solution of sodium hydroxide [16, 20] (scheme 1).

Source dioxime 3,4-diacylhydrazines synthesized from hydroxylamine and the corresponding diacylhydrazines, which, in turn, is obtained in high yield by treatment of acetone chetyrehokisi nitrogen or when the mini-compositions, comprising as an active ingredient derived 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the General formula I together with appropriate additives. As additives are the components that are conventional for the preparation of dosage forms for the treatment of diseases of the cardiovascular system.

Depending on the type of drug and the purpose of its use, dosage forms can be made in the form of tablets, capsules, granules, pills, pills for gradual dosing (if necessary from a polymeric material, for example from acetylcellulose, or any other suitable for this purpose material, for example gelatin), as well as powders, solutions, suspensions or emulsions.

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 water or buffer components of the mixtures, surface-active substances, food carbohydrates, preservatives, food dyes. Liquid compositions can be used for various routes of administration, particularly in the form of drops or aerosols, as well as in ampoule form for intravenous or intramuscular injection.

Another possible form of application of the derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the General formula I are transdermal therapeutic composition in the form of ointments and plasters, including metered percutaneous flow of active components in the body.

The above pharmaceutical compositions based on derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the General formula I can be obtained by combination of compounds 1-4 with other known active ingredients of pharmaceutical preparations for the treatment of diseases of the cardiovascular system. In particular, for this purpose you can use blockers1-adrenergic receptors (prazosin), agonists Central2- adrenergic and I1imidazoline receptors (clonidine, moxonidine), blockers -(1-) adrenergic receptors (propranolol, atenolol, metoprolol), calcium channel blockers (nyati (carbokromen), diuretics (dihlotiazid), cardiac glycosides (digitoxin), antihypertensive agents (bezafibrat, gemfibrozil).

The description of the present invention contains 16 examples and Fig. 1-4, illustrating the experimental material.

Examples 1-5 reflect the chemical basis of molecular mechanism of action of derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I, mainly consisting in the generation of nitric oxide and its active forms (restored form and S-nitrosothiols), modification of sulfhydryl groups and the interaction with the heme iron atom of the protein group.

Examples 6 and 7 show the biochemical effects of derivatives of 1,2,5-oxadiazole[3,4-D] pyridazine-5,6 - dioxide of the General formula I-mediated generation of nitric oxide and its active forms, the activation of RHC and increasing the level of cGMP in human platelets. Examples 8-11 and Fig. 1-4 illustrate the pharmacological properties of derivatives of 1,2,5-oxadiazole[3,4 - d]pyridazine-5,6-dioxide of the General formula I - vasodilating and hypotensive activity in vitro and in vivo, and inhibition of platelet aggregation caused under the action of ATP, and the disaggregation of aggregated platelets, i.e. the ability of ignoreme the research acute toxicity derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I.

Example 13 describes some not previously studied physico-chemical properties of derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I (solubility, stability in aqueous solutions and other).

Examples 14-16 describe the composition of various pharmaceutical compositions based on derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6 - dioxide of the General formula I.

Example 1. The formation of nitric oxide from derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide.

For the determination of nitric oxide 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 of 7.4) or 20 mysurface (DMSO), and during incubation with oxyhemoglobin concentration was 0.1 mm. As a negative control was used an aqueous solution of DMSO at a concentration of 0.2%, and as a positive control, 0.1 mm sodium nitrite containing 0.2% DMSO. Samples were incubated 30 min at 37oC was added sequentially with 100 µl 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 a pH of 7.4 was not observed the formation of appreciable quantities of nitrite (< 0.01 mol of water/mol of starting compound (ex.with.)) in the absence of thiols. In the presence of a thiol compound 1 was generated 0.32 mol of nitrite per mole ex.with. in the presence of cysteine and 0.43 mol of nitrite per mole ex. C. in the presence of glutathione; compound 2 was generated 0.02 mol of nitrite per mole ex.with. in the presence of cysteine and 0.01 mol of nitrite per mole ex.with. in the presence of glutathione. In the same conditions, but at pH 5.0 were formed from compound 1 to 0.72 mol of nitrite per mole ex.with. in the presence of cysteine and 0.75 mol of nitrite per mole ex.with. in the presence of glutathione; in the case of compound 2 countername the formation of nitrite. During incubation in the above conditions at pH 7.4 in the presence of oxyhemoglobin connection 1 generated 0.04 and 0.05 mol of nitrite per mole ex.with. in the presence of cysteine and glutathione, respectively, and the optical density of the positive control sample was not changed. This suggests that up to 90% of nitrite determined by this method, is the product of the oxidation of nitric oxide at a pH of 7.4. At pH 5.0 the nitrite reacts with oxyhemoglobin, which complicates the definition of formation of oxide of nitrogen at this pH value. Known analogue (compound 5) in the specified conditions in the presence of thiols generated 0.08 mol of nitrite per mole ex.with.

Example 2. The formation of S-nitrosothiols (S-nitrosoglutathione) derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide.

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 in the reaction of compound 1 with glutathione the reaction was carried out as described in example 1, and then nitrite was determined by the method described in example 1 in the absence and in the presence of mercury chloride (II) in a concentration of 0.1%. The difference between the obtained values with the mol S-nitrosoglutathione per mole of compound 1. The formation of S-nitrosothiols known analogue (compound 5) are not investigated.

Example 3. The formation of reduced forms of nitrogen oxide derivatives of 1,2,5 - oxadiazole[3,4-d]pyridazine-5,6-dioxide.

To determine the reduced forms of nitrogen oxide (NO-/HNO) 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 1). Since compounds 1 and 2 also generate and nitrite, which reacts result, at the final 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 a pH of 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. PA is lamina was calculated by the formula X = (A - YN)/H, where X corresponds to the concentration of hydroxylamine, μm; A is the optical density of the sample containing the analyzed compound, 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 H is the optical density of the positive control containing hydroxylamine hydrochloride, μm-1.

In the above conditions, the formation of 0.18 mol of hydroxylamine per mole of compound 1 in the presence of cysteine and 0.34 mol of hydroxylamine per mole of compound 1 in the presence of glutathione. In these conditions, from compound 2 in the presence or in the absence of thiols no formation of hydroxylamine. The possibility of generating a known analog (compound 5) reduced forms of nitrogen oxide have not been investigated.

Example 4. The formation of methemoglobin from oxyhemoglobin under the action of the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6 - dioxide.

The formation of methemoglobin from oxyhemoglobin studied in a known manner using a spectrophotometer.

Sample final volume of 1 ml contained 50 mm potassium phosphate buffer (pH 7,4), 40 μm oxyhemoglobin, 50 μm compounds for 60 min at 37oC and measured the optical density at wavelengths of 577, 630 and 700 nm, and calculating the concentration of oxyhemoglobin, methemoglobin and hollobone (see tab. 1).

In the above conditions, compounds 1 and 2 was caused by the formation of methemoglobin from oxyhemoglobin, and this effect was enhanced in the presence of cysteine. This demonstrates the ability of these compounds to interact with the heme iron atom of the group of proteins. A similar effect is known analogue (compound 5) was not studied.

Example 5. Modification of sulfhydryl groups of low molecular weight thiols under the action of the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide. The concentration of SH groups was determined in a known manner of Ellman. Sample final volume of 0.2 ml contained 50 mm potassium phosphate buffer (pH 7,4), bovine serum at a concentration of 0.5 mm and connections 1 or 2 at a concentration of 0.05 mm. A control sample contained the corresponding amount of DMSO (0.2 percent). Incubation was carried out for 20 min at 37oC and was added to 1.8 ml of 0.5 mm solution of 5,5'-dithiobis(2-nitrobenzoic) acid in 50 mm potassium phosphate buffer (pH of 7.4). After 5 min was measured by optical density at a wavelength of 412 them expected concentration of SH-groups (see tab. 2)

Vzaimodeystviya soluble form of guanylate cyclase derivatives of 1,2,5 - oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I.

The RHC activity was measured in preparations of cytosole of human platelets and rat lung by known methods.

In the first case, to obtain the drug RHC were isolated platelets from venous blood of healthy donors by a known method using separation of blood corpuscles in the density gradient ficoll. The platelets were washed 3 times with 20 mm Tris-HCl-buffer (pH 7.5) containing 150 mm NaCl and 5 mm EDTA, centrifuger at 1 000 g for 15 min and then suspended in 50 mm Tris-HCl-buffer (pH of 7.6) containing 0.2 mm dithiothreitol, and voiced by ultrasound by the ultrasonic generator MSE 7-78 (UK) for 20 sec at 4oC. Suspension of damaged cells was centrifuged at 100 000 g for 1 h, the supernatant was diluted to a protein concentration of 1 mg/ml, was added dithiothreitol to a concentration of 0.2 mm and was determined by the enzyme activity by the number of cGMP formed from guanosin-5'-triphosphate (GTP), enzyme immunoassay method using reagent kits for the quantitative determination of cGMP (JSC "Biogen", Russia). The incubation mixture for determining the activity of the final volume of 0.15 ml was prepared at 4oC and contained 50 mm Tris-HCl (pH of 7.6), 1 mm GTP, 4 mm MgCl2, 4 mm phosphocreatine, 10 the relocation 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 DMSO and water, if necessary, other compounds, such as 0.3 microns 1H-[1,2,4]oxadiazole[4,3-a]cinoxacin-1-he (HBS; specific inhibitor of heme-dependent activation of RHC) or 1 mm cysteine, 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 followed by cooling in an ice bath. After separation denaturirovannogo protein by centrifugation (10 min at 1 500 g) in the supernatant was determined by the amount of cGMP formed by the above method.

To obtain drug RHC from rat lung tissue homogenized in 5 volumes of 50 mm Tris-HCl buffer (pH of 7.6) containing 10 mm MgCl2using homogenizer "glass-glass". The homogenate was centrifuged for 30 min at 30 000 g, the supernatant was collected and determined the enzyme activity by the number of [32P]cGMP formed from [-32P]GTP in a known manner. The incubation mixture a final volume of 100 μl contained 50 mm Tris-HCl buffer (pH of 7.6), 5 mm MgCl2, 5 mm creatine phosphate, 0.4 mg/mg creatine phosphokinase, 1 mm 3-isobutyl-1-methylxanthines, 2 mm cGMP, 0.2 mm GTP, the food incubation was made of the studied compound at a concentration of 10 μm in the form of a solution in DMSO and water, if necessary, other compounds, such as 3 μm in HBS or 5 mm cysteine, 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 Na2CO3and 0.6 ml of 36 mm Zn(CH3COO)2were mixed , incubated at 4oC for 10 min and centrifuged at 15000 g for 5 min the Supernatant was applied to a column with acidified known manner alumina, which was washed with water. The elution of [32P]cGMP was performed with 0.2 M ammonium formate in vials for scintillation account and 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.

Compounds 1 and 2 activated RHC from human platelets and rat lung. Activation was decreased in the presence of HBS, which indicates that heme-dependent nature of the effect. Activation Pranich derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the General formula I on the RHC in cells with reduced content of free thiols. In the above described conditions known analogue (compound 5) activated RHC of human platelets in 2.5 times (250%).

Example 7. Increased levels of cGMP in human platelets derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I.

Determination of cGMP in platelets were performed in a known manner. Platelet-rich plasma was diluted with depleted platelets plasma to a concentration of 109cells/ml Sample final volume of 4 ml contained 1 ml suspension of platelets (2,5108cells/ml), 3 ml of depleted platelets, plasma, compounds 1 or 2 at a concentration of 10 μm or 0.02% DMSO. Samples were incubated 10 min at 37oC, centrifuged 5 min at 1 500 g, the residue is suspended in 100 mm Tris-HCl-buffer (pH 7,6) containing 8 mm EDTA, voiced by ultrasound for 20 sec at 4oC, boiled for 2 min and centrifuged at 2 000 g for 10 min the Concentration of cGMP was determined in the supernatant ELISA method using reagent kits for the quantitative determination of cGMP (JSC "Biogen", Russia).

In the control sample, containing 0,02% DMSO, the cGMP content was 10.2 pmol/109platelet. In samples that contain compounds 1 and 2, mA 2 increase the levels of cGMP in intact human platelets. A similar effect of compound 5 has not been studied.

Example 8. The vasodilator action of derivatives of 1,2,5 - oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I.

Under vasodilator here and hereinafter understood the reduction of isometric contraction of smooth muscle of the isolated segment oarty rats. Activity of derivatives of 1,2,5-oxadiazole [3,4-d]pyridazine-5,6-dioxide of the General formula I were determined in vitro on the rings of thoracic aorta of male rats of 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 ring thickness of 3.5 mm, which was 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, coupled with dual Gemini recorder (Ugo Basile, Italy). The camera contained 30 ml of Krebs solution (130 mm NaCl, of 4.7 mm KCl, 2.5 mm CaCl2, 1,18 mm KH2PO4at 14.9 mm NaHCO3, 1.2 mm MgSO4, 11 mm glucose) at 37oC, who was under constant aeration with 95% O2/5% CO2to maintain a pH of 7.4. In the Krebs solution was added indomethacin concentrations up to 1 μm. If necessary F. The reduction of the drug caused by the addition of norepinephrine at a concentration of 0.5 µm, was added to 0.1 mm N-G-nitro-L-arginine(L-NNA; inhibitor of endothelium-dependent formation of nitric oxide), and after the stabilization muscles of the vessel was registered, it relaxes in response to cumulative doses of the compounds in the concentration range from 0.003 nm to 50,000 nm. In separate experiments to establish the role of NO or RHC in the mechanisms of action of compounds were added 0.01 mm oxyhemoglobin (trap nitric oxide) or 3 μm 1H-[1,2,4]oxadiazole[4,3-a]cinoxacin-1-he (HBS; specific inhibitor of heme-dependent activation of soluble guanylate cyclase; see Example 6). The presence or absence of endothelium controlled expansion of isolated segments in response to 1 μm acetylcholine, and the intact state of the smooth muscle of the vessel was controlled using 0.5 µm of sodium nitroprusside.

Compounds 1 and 2 was caused by the reduction of isometric contraction of the vessel with intalnim endothelium in the presence of noradrenaline and L-NNA (Fig. 1 and 2). The concentration of the compounds 1, corresponding to a 50% reduction in the initial vascular tone (IC50), was 3,510,5 nm (average standard deviation according to the track Abijah had IC50= to 2.29 μm, and the relaxation of 100% was observed at a concentration of 50 μm. Data on the effect of intact endothelium, oxyhemoglobin and HBS on the IC50and maximum relaxation under the action of compounds 1 and 2 are presented in table 4. The obtained data indicate that compounds 1 and 2 are endothelium-independent relaxant, act directly on the smooth muscle cells of the aorta, and the mechanism of their action is due to the formation of nitric oxide (inhibition by oxyhemoglobin) and activation of the soluble form of guanylate cyclase (inhibition of HBS).

According to the prototype, the connection 5 in vitro close to above, when used as a vasoconstrictor of phenylephrine had a vasodilating effect; the value of the IC50was 1.5 μm. The influence of additional factors on the activity of compounds 5 were not studied.

Example 9. Hypotensive activity of derivatives of 1,2,5-oxadiazole [3,4-d] pyridazine-5,6-dioxide of the General formula I.

Activity of derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6 - dioxide of the General formula I were determined in vivo in male Wistar rats with average weight of 320 g in a known manner. To measure the average arter the and animals implanted polyethylene catheters (grades D-10, RE -50) in 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). Compound 1 was administered 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 carried out continuously during the entire experience. The original values of mean arterial pressure and heart rate was 98,3 mm RT. Art. and 315,6 beats/min, respectively.

A bolus of compound 1 at a dose of 2.5 mg/kg caused a biphasic response of mean arterial pressure quickly decreases to 16.1 mm RT. century, which was followed by a compensatory increase in blood pressure to 121,4 mm RT. Art. 2.5 minutes after weaving, rising to 663,6 beats/min in 10 sec after the injection, and then decreasing up to KZT 205.7 beats/min after 3 min after injection. Thus, the apparent high efficiency and absence of time delay vasodilator action of compound 1 when administered intravenously. The known analogue (compound 5) on blood pressure in vivo has not been studied.

Example 10. Inhibition of aggregation of human platelets under the action of the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I.

The impact of derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine - 5,6-dioxide of the General formula I on the aggregation of human platelets was studied known turbidimetric method of born. This venous blood taken at 8 a.m. in healthy donors was centrifuged at 450 g at room temperature in plastic dishes for 10 min, using as an 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 the cuvette is home experiment so in order aggregation was reversible, and the 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, Biological faculty of Moscow state University. M. C. Lomonosov (Russia). Compounds 1, 2 and their known analogue (compound 5) was added to ADP.

Compounds 1 and 2 inhibited platelet aggregation, as shown in Fig. 3. The corresponding values of the concentrations at which it is achieved premaxillae inhibition (IC50), was of 0.43 and 0.49 μm, respectively. Maximum inhibition of 100% was observed when the concentrations of both compounds, equal to 10 μm. Known analogue (compound 5) in the above-described conditions mattered IC20= 10 μm. According to published data, in the model of platelet aggregation caused by the action of collagen, the value of the IC50for compound 5 was 1.5 μm.

Example 11. Disaggregation of aggregated platelets under the action of the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6 - dioxide of the General formula I.

The impact of derivatives of 1,2,5 - oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I on the disaggregation of aggregated thrombocytemia the maximum reversible aggregation.

Compounds 1 and 2 was caused disaggregation of aggregated platelets, as shown in Fig. 4. The corresponding values of the concentrations at which it is achieved premaxilla disaggregation, amounted to 1.4 and 1.2 μm, and the maximum (full) disaggregation was observed when the concentrations of both compounds, equal to 10 μm. A similar effect is known analogue (compound 5) are not investigated.

Example 12. Determination of acute toxicity derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I.

Acute toxicity 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 h (none of the animals died within any additional period of time).

The data indicate that LD50for connection 1 is 100 mg/kg, to connect 2 - from 200 to 250 mg/kg Acute t is Uchenie LD50intraperitoneal injection to mice of both sexes were 104-110 mg/kg [23]. One of the most active vasodilator in a series of furoxone - 1,2,5-oxadiazole-3,4-dinitrile-2-oxide(dezineforce) [5] in the above-mentioned conditions had LD50< 50 mg/kg Comparison of physiological activity and toxicity of compounds 1 and 2 shows that therapeutic concentrations of these compounds are significantly lower than LD50.

Example 13. The solubility of the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I and the stability of aqueous solutions and solutions prepared with different organic solvents.

The solubility of the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6 - dioxide of the General formula I and the known analog (compound 5) was determined by adding an aliquot of the concentrated 50 mm solution miscible with water, an organic solvent (DMSO, dimethylformamide, dioxane, acetone, methanol) in water to the final concentration at which there was a formation of nerastvorim particles and flakes.

It is established that under these conditions the solubility of compound 1 is 250 µmol/l, compounds 2 to 1000 µmol/l, known analogue (with the e 50 mm solution of compound 1 in DMSO and 0.25 mm solution in water, containing 0.5% DMSO. The decrease in concentration of the compound was evaluated by means of spectroscopy, using values of molar absorption coefficient at 353 nm ( = 5 320 M-1cm-1) in water.

A solution of compound 1 in DMSO was stable for 1 month at room temperature when stored in a place protected from light and frozen at -20oC for up to 6 months. An aqueous solution of compound 1 is quite stable at room temperature when stored protected from light place at a neutral or slightly acidic pH values ( 1/2 > 4.2 months). An aqueous solution of compound 1 is unstable at alkaline and alkaline pH values (pH > 9), in bright light and in the presence of thiols. Dry compound 1 is stable at room temperature when stored in a dark place for at least two years.

Example 14. A solution of compounds 1-4 sublingual injection.

Components, wt.%

Active component - 1

Ethyl alcohol - 99

Example 15. Tablets of compounds 1-4 for ingestion.

Components - mg/1 tablet

The active ingredient is 0.5

Lactose - 50

Microcrystalline cellulose - 50

Magnesium stearate and 0.5

Polyvinylpyrrolidone - 10

Components wt.%/capsule

Active component - 1

Refined sunflower oil - 99

From the above examples 1-5, the conclusion must be that the connections 1 and 2 are significantly more effective nitric oxide donors than the well-known analogue (compound 5). In addition, the connection 1 generates the biologically active form of nitrogen oxide, including the restored form NO and nitrosothiol, as well as compound 2, is a modifier of SH-groups and the iron atom of heme protein group. From examples 6 and 7 it follows that the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I heme-dependent activate RHC significantly more efficient than their well-known analogue (compound 5), and increase the level of cGMP in platelets. From examples 8 and 9, the conclusion must be that the derivatives of 1,2,5-oxadiazole[3,4-d]pyridazine-5,6-dioxide of the General formula I have a pronounced vasodilator and hypotensive action, and the connection 1 in vitro surpasses the known analogue (compound 5) and the known pharmaceuticals - trinitroglycerine (cf. IC50= 3,5 nm for compounds 1, 1500 nm for compounds 5 and 11.3 nm for trinitroglycerine). The vasodilator effect of compound 2 in the above conditions would the 10 and 11 should that the derivatives of 1,2,5-oxadiazole[3,4-d] pyridazine-5,6-dioxide of the General formula I is superior to antiplatelet activity similar to the effect of compound 5 (cf. IC50= 0,43, 0,49 and > 10 μm, respectively), and also have desegregated effect on the aggregated platelets.

Example 12 shows that the acute toxicity of the compounds of General formula I is comparable to or lower than the known toxicity of pharmaceuticals - trinitroglycerine.

Compounds of General formula I is obtained from the available source products of acetone and acetophenone.

Thus, the use of derivatives of 1,2,5-oxadiazole[3,4-d]pyridazin- -5,6-dioxide of the General formula I may expand the range of effective activators RHC and medicines for the treatment of diseases of the cardiovascular system.

1. Use of derivatives of formula I

< / BR>
where R = CH3or C6H5and n = 0 or 1,

as vasodilator, hypotensive, antispasmodic, anti-means and platelet aggregation inhibitors.

2. The use of compounds on p. 1 for the prevention and relief of angina and acute myocardial infarction.

3. Pharmaceutical composition, obladaushi the action, containing the active ingredient and additives, characterized in that as the active component used derivatives of the formula I, where R = CH3or6H5and n = 0 or 1.

 

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