RU2208438C1 - Donor of nitrogen oxide activating soluble form of guanylate cyclase, inhibiting platelet aggregation and exhibiting spasmolytic and vasodilating effect - Google Patents

Abstract

FIELD: organic chemistry, biochemistry, medicine. SUBSTANCE: invention proposes an agent donor of nitrogen oxide activating soluble form of enzyme guanylate cyclase. Indicated agent inhibits aggregation of platelets, shows spasmolytic and vasodilating agent and it represents 3,6-bis- (4-aminofurazan-3-yl)-1,4,2,5-dioxadiazine of the formula (I)

Description

в качестве донора оксида азота, активирующего растворимую форму гуанилатциклазы (рГЦ), ингибирующего агрегацию тромбоцитов и обладающего спазмолитическим и сосудорасширяющим действием.The invention relates to biochemistry, physiology and pharmacology, in particular to the use of the known 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine (BAFDOD) of the formula I

as a donor of nitric oxide, activating the soluble form of guanylate cyclase (rHC), inhibiting platelet aggregation and having an antispasmodic and vasodilating effect.

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

 Nitric oxide (NO), synthesized by endothelial cells and other types of cells and tissues, plays a key role in maintaining the normal tone of vascular smooth muscle, which affects the systemic blood pressure. In addition, NO has an antispasmodic effect on the muscles of the gastrointestinal tract, bile ducts, ureters, uterus, bronchi and other organs, and also inhibits the aggregation and adhesion of platelets and neutrophils (MD Mashkovsky "Medicines", v.1 , "Torsing", Kharkov, 1998, p. 385 [1]). The molecular mechanism of the endogenous nitric oxide synthesized from the amino acid L-arginine includes its diffusion through plasma membranes and binding to the heme group of RHC. Guanylate cyclase / CF 4.6.1.2; guanosine-5'-triphosphate pyrophosphatliase (cyclizing) / is an enzyme that catalyzes the biosynthesis of guanosine-3 ', 5'-cyclophosphate (cGMP) - a secondary messenger that acts as a universal regulator of intracellular metabolism (F. Murad "Regulation of cytosolic guanylyl cycl 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 has now been established that as a result of the interaction of NO with the heme iron atom, which is part of the enzyme, and the formation of the nitrosyl-heme complex, conformational changes in the active center of the enzyme occur, which lead to activation of the RHC and increased cGMP synthesis (see drawing).

 To date, the formation of nitric oxide as a result of enzymatic biotransformation of trinitroglycerin and other nitrates, which are used to treat cardiovascular diseases as anti-ischemic and antianginal drugs, has been proven ([1], pp. 385–392). However, their significant drawback is a weak antiplatelet activity, as well as the occurrence of tolerance and other side effects with prolonged use. In addition, these organic nitrates in various dosage forms effectively reduce blood pressure, which significantly complicates their local use (for example, in cases of cerebral vasospasm, pulmonary hypertension, restenosis after angiography, etc.).

 Various heterocyclic compounds are known that have the ability to generate NO, activate RHC and exhibit antiplatelet, antispasmodic and / or vasodilating properties.

где R - фенил, 4-хлорфенил или стирил, которые рассматривались в качестве предшественников (депо-форм) NO-генерирующих соединений (амидоксимов). Данные производные не обладали гипотензивным действием, но ингибировали агрегацию тромбоцитов под действием коллагена в моделях ex vivo и in vivo (K. Rehse, F.Brehme "New NO-donors with antithrombotic and vasodilating activities. Part 26: Amidoximes and their prodrugs" Arch. Pharm. Pharm. Med. Chem. 1998, v. 331, p. 375-379).Thus, 1,2,4-oxadiazol-5-one derivatives of the general formula II are known

where R is phenyl, 4-chlorophenyl or styryl, which were considered as precursors (depot forms) of NO-generating compounds (amidoximes). These derivatives did not have a hypotensive effect, but inhibited platelet aggregation under the action of collagen in ex vivo and in vivo models (K. Rehse, F. Brehme "New NO-donors with antithrombotic and vasodilating activities. Part 26: Amidoximes and their prodrugs" Arch. Pharm. Pharm. Med. Chem. 1998, v. 331, p. 375-379).

 The ability of these compounds to generate NO under in vivo conditions has not been established, their effect on the activity of RHC, as well as antispasmodic and vasodilating properties have not been studied.

где R¹ и R² - метил, фенил, фенилсульфонильная, нитрильная, метокси-, нитро- или аминогруппа, генерирующие NO, активирующие рГЦ, проявляющие антиагрегантные свойства и обладающие выраженным вазорелаксантным действием на изолированных кольцах торакальной аорты кролика, сокращенных под действием норадреналина (R. Ferioli, G.C. Folco et al. "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; заявка РСТ 94/01422, C 07 D 271/08, А 61 К 31/41, 1993 г.).Various 3,4-disubstituted furoxanes (1,2,5-oxadiazole-2-oxides) of the general formula III are known

where R ¹ and R ² are methyl, phenyl, phenylsulfonyl, nitrile, methoxy, nitro, or amino groups generating NO, activating RHCs, exhibiting antiplatelet properties and having a pronounced vasorelaxant effect on isolated rings of the rabbit thoracic aorta, contracted by noradrenaline (R . Ferioli, GC Folco et al. "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; PCT application 94 / 01422, C 07 D 271/08, A 61 K 31/41, 1993).

 The ability of derivatives of furoxanes, as well as organic nitrates, to effectively reduce the systemic blood pressure (by 25-85 mm Hg. Art. When administered intravenously to animals in doses of 0.05-0.1 mg / kg) (for example, European patents 0054872, C 07 D 271/08, A 61 K 31/41, 1984; 0054873, C 07 D 271/08, A 61 K 31/41, 1984) significantly complicates their directed local application.

 Known 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine of the above formula I as a product of chemical synthesis (V.G. Andrianov, V.G. Semenikhina, A.V. Eremeev, "4-Aminofurazan-3-carbohydroximic acid halides. Chemistry heterocycle. Comp. 1992, 5, pp. 687-691 [12]). The biochemical, physiological and pharmacological properties of this compound have not been studied.

Closest to the compound of the present invention is 4,4'-diphenyl-3,3 ', 4', 3 '' - trifurazan-2,2 ', 2''- trioxide of formula III, where R ¹ = R ² = 4- phenylfuroxanyl-3 (2-oxide-4-phenyl-1,2,5-oxadiazol-3-yl), which is an NO donor, inhibiting platelet aggregation by collagen and causing vasodilation on isolated rabbit thoracic aortic rings, contracted by noradrenaline ( AMGasco, A. Di Stilo et al. "Synthesis and structure of a trimer of the furoxan system with high vasodilator and platelet antiaggregatory activity" (Liebigs Ann. Chem. 1993, p. 441-444 - prototype).

 The effect of this compound on the activity of the RHC and its hypotensive activity have not been studied. In addition, it had an insufficiently effective antispasmodic and vasodilating effect under experimental conditions.

 The aim of the invention is the search in a series of derivatives of 1,2,5-oxadiazole for a new donor of nitric oxide and activator RHC, which has an improved antispasmodic and vasodilating effect.

 This goal is achieved by using the well-known chemical compound -3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine of the above formula I as an NO donor, activating RHC, inhibiting platelet aggregation and having antispasmodic and vasodilating effect.

BAFDOD was synthesized in a known manner based on the reaction of stereoselective dimerization of nitrile oxides and consisting in the interaction of acid chloride (4-aminofurazan-3-yl) hydroxamic acid with triethylamine in an inert organic solvent (acetonitrile) at a temperature of 0-5 ^o C [12].

 The description of the present invention contains 9 examples illustrating the experimental material.

 Examples 1-4 show the chemical basis of the molecular mechanism of action of 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine, predominantly consisting in the selective generation of NO.

 Example 5 illustrates the biochemical effect of BAFDOD mediated by the generation of nitric oxide - the activation of partially purified RHC from bovine lungs.

 Examples 6 and 7 illustrate the pharmacological properties of BAFDOD - the ability to inhibit platelet aggregation, as well as antispasmodic and vasodilator activity in vitro.

 Example 8 demonstrates the absence of BAFDOD when administered intravenously at a dose of 0.5 mg / kg of hypotensive activity in vivo.

 Example 9 describes a study of the acute toxicity of BAFDOD.

 Example 1. The formation of nitric oxide from 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

 To determine nitric oxide, a known method was used, based on the reaction of nitric oxide with atmospheric oxygen in an aqueous medium to form nitrite, the amount of which was measured by the intensity of staining of the sample with the product of the azo coupling reaction using a spectrophotometer. As evidence that this method makes it possible to measure precisely nitric oxide released from the compound as a result of a chemical reaction rather than 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 enters into an azo coupling reaction.

A sample with a 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 studied compound at a concentration of 0.1 mM and 0 , 2% dimethyl sulfoxide (DMSO), and when incubated with oxyhemoglobin, its concentration was 0.1 mM. An aqueous solution of DMSO at a concentration of 0.2% was used as a negative control, and 0.1 mM sodium nitrite containing 0.2% DMSO was used as a positive control. Samples were incubated for 60 min at 37 ^° C and 100 μl of 3 M sodium acetate, 400 μl of a 0.92% solution of sulfanilic acid in 30% acetic acid and 400 μl of 0.05% N-naphthylethylenediamine were added sequentially. Samples were incubated for 10 min and absorbance was measured at a wavelength of 554 nm on a spectrophotometer.

 Under the above conditions, at pH 7.4, no appreciable amount of nitrite was formed (<0.01 mol of nitrite / mol of the starting compound (ref. Com.) In the absence of thiols. In the presence of thiols, BAFDOD generated 0.182 mol of nitrite per mole of comp. in the presence of cysteine and 0.154 mol of nitrite per mole of starting compound in the presence of glutathione Further incubation did not lead to additional nitrite formation.

 When BAFDOD was incubated under the above conditions in the presence of oxyhemoglobin and cysteine or glutathione, the formation of methemoglobin was observed (see Example 4).

 According to known data, the NO-generating properties of the closest known analogue under conditions close to those described (in the presence of thiols) were not studied due to its extremely low solubility under experimental conditions.

 Example 2. The formation of S-nitrosothiols (S-nitrosoglutathione) 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

 To determine S-nitrosothiols, a known method was used, based on a reaction with mercury (II) chloride, during which nitrite is formed. To account for the formation of nitrite during the reaction of the compound 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 (II) chloride at a concentration of 0.1%. The difference in the obtained values corresponds to the amount of S-nitrosoglutathione.

 Under the above conditions, the formation of S-nitrosoglutathione from the compound of formula I does not occur. The possibility of generation by the known analogue of S-nitrosothiols has not been investigated.

 Example 3. The formation of a reduced form of nitric oxide from 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

To determine the reduced form of nitric oxide (NO ^- / HNO), a known method was used, based on the fact that the nitroxyl formed during the reaction reacts with thiols to form hydroxylamine (or competitive formation of N ₂ O occurs), which after oxidation with I ₃ ^- ions gives nitrite, determined by the azo coupling reaction (see example 8). Since the studied compounds also generate nitrite, which likewise enters into the azo coupling reaction at the final stage, hydroxylamine hydrochloride and sodium nitrite at a concentration of 0.1 mM were used as a positive control, and the incubation was carried out as described in Example 1 at pH 7.4. After incubation, 100 μl of 3 M sodium acetate, 400 μl of a 0.92% solution of sulfanilic acid in 30% acetic acid, 100 μl of 1.25% I ₂ in 2% KI, 30 μl of 0.5 M 2-mercaptoethanol and 400 μl of 0.05% N-naphthylethylenediamine. Samples were incubated for 15 min and the absorbance was measured at a wavelength of 554 nm on a spectrophotometer. In parallel with this experiment, nitrite formation was measured as described in Example 1. The hydroxylamine content was calculated by the formula: X = (A-Y • N) / H, where X corresponds to the concentration of hydroxylamine, μM; A is the optical density of the sample containing the test compound, Y is the concentration of sodium nitrite formed, 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 .

 Under the above conditions, the formation of hydroxylamine from the compounds of this invention in the presence of cysteine or glutathione was not observed. The possibility of generating a known analog of the reduced form of nitric oxide has not been investigated.

 Thus, BAFDOD is a selective NO donor.

 Example 4. The formation of methemoglobin from oxyhemoglobin under the action of 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

The determination of NO generation in the presence of oxyhemoglobin, accompanied by the formation of methemoglobin, was studied in a known manner in a spectrophotometric cuvette with a final volume of 1 ml at 25 ^° C. The samples contained 25 mM potassium phosphate buffer (pH 7.4), 3 μM oxyhemoglobin, 0.5 mM cysteine or glutathione and BAFDOD at a concentration of 0.1 mM. The rate of increase in optical density at 401 nm was determined, and the rate of formation of nitric oxide was calculated from the linear initial portion (the molar absorption coefficient of methemoglobin was assumed to be 39.9 mmol ^-1 • cm ^-1 ). In the absence of thiols, a noticeable increase in optical density at 401 nm was not observed. The apparent first order reaction rate constant was expressed in min ^-1 . From the literature data it is known that the process of conversion of oxyhemoglobin to methemoglobin can be accompanied by the formation of hemichrome or choleglobin, therefore, the absence of these forms of hemoglobin was checked in separate experiments. For this, the samples were incubated in a potassium phosphate buffer pH 7.4 in the presence of 25 μM oxyhemoglobin, 0.2 mm cysteine or glutathione and 20 μM compounds. The optical density was measured at 560, 577, 630 and 700 nm, and the concentrations of oxyhemoglobin, hemichrome, methemoglobin and cholelobin were calculated. These experiments showed that during incubation of the studied compounds with oxyhemoglobin in the absence of thiols or in the presence of cysteine or glutathione, the formation of choleglobin or hemichrome is observed, and only the conversion of oxyhemoglobin to methemoglobin occurs.

Under the above conditions, BAFDOD caused the formation of methemoglobin from oxyhemoglobin, and this effect was enhanced in the presence of cysteine and glutathione. The reaction rate constant (NO + Gm-O ₂ -> NO ₃ ^- + metGm) in the presence of cysteine was 0.0348 min ^-1 , in the presence of glutathione - 0.0341 min ^-1 . The compound did not react under similar conditions with methemoglobin.

 Example 5. Activation of the soluble form of guanylate cyclase with 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

 The activity of RHC was measured in preparations from bull lungs obtained in a known manner.

To obtain the RHC preparation from bovine lungs, the tissue was homogenized in 5 volumes of 50 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl ₂ using a glass-glass homogenizer. The homogenate was centrifuged for 30 min at 30,000 g, the supernatant was selected and enzyme activity was determined by the amount of [ ³² P] cGMP formed from [α- ³² P] GTP, in a known manner. Protein (about 0.2-0.5 μg protein of partially purified preparation from the lungs of a bull) on ice was added to the incubation mixture (final sample volume 100 μl) containing (final concentration) 50 mM Tris-Hcl pH 7.6, 1 mM 3-isobutyl-1-methylxanthine, 5 mM MgCl ₂ , 0.4 mg / ml creatine phosphokinase, 5 mM creatine phosphate, 2 mM cGMP, 0.2 mM GTP, 10000-20000 cpm / pmol [α- ³² P] GTP (SSC FEI, Obninsk), as well as 10 mM DTT. When determining the activating effect, the studied compound was introduced into the incubation medium at a concentration of 10 μM in the form of a solution in aqueous DMSO, and DMSO was added to control samples to a concentration of 0.02%. A control sample showed no effect of DMSO in the indicated concentration on the basal activity of the RHC. Samples were incubated at 37 ^° C for 15 minutes. The reaction was stopped by boiling samples for 2 minutes. After cooling to room temperature, 0.5 ml of 30 mM Na ₂ CO ₃ and 0.6 ml of 36 mM Zn (CH ₃ COO) ₂ were added to the samples, incubated at 4 ^° C for 10 min and centrifuged at 15,000 g for 5 min The supernatant was applied to chromatographic columns with alumina, equilibrated with 1 M perchloric acid, which were then washed with water. Elution of [ ³² P] cGMP was performed with 0.2 M ammonium formate in scintillation counting bottles, radioactivity was measured using a liquid scintillation counter in a known manner.

 The compound of the present invention depending on the concentration of 11.0 (10 and 50 μm) -16.6 times (100 μm) increased the activity of rhz from rat lungs. The influence of the known structural analogue on the activity of rHCs has not been studied.

 Example 6. Inhibition of aggregation of human platelets caused by 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

The effect of the compounds of the present invention on the aggregation of human platelets was studied by the known Born turbidimetric method. For this, venous blood taken at 8 am from healthy donors was centrifuged at 450 g at room temperature in a plastic dish for 10 minutes using sodium citrate as an anticoagulant. The supernatant, i.e. platelet-rich plasma, was collected and centrifuged at 650 g for 30 minutes to obtain platelet-poor plasma. The platelet concentration in the platelet-rich plasma was adjusted to 2.5 × 10 ⁸ cells / ml by diluting with platelet-poor plasma and the resulting suspension was poured into a 0.5 ml cuvette. Aggregation was induced by the addition of ADP to a concentration of 2-5 μM. The concentration of ADP was selected in each experiment so that the aggregation was reversible and the maximum was 2 min after the addition of ADP, not exceeding 50%. Platelet suspension light scattering was measured using an aggregometer developed at the Laboratory of Bioorganic Chemistry, Faculty of Biology, Moscow State University. M.V. Lomonosov (Russia). The test compound was added to the sample prior to ADP.

The results show that BAFDOD in the described conditions inhibited platelet aggregation. The concentration at which half-maximal inhibition was achieved (IC ₅₀ ) for this compound was 2.2 μM. A well-known analogue, according to the prototype, in close conditions had an IC ₅₀ value of 1.8 μM.

 Example 7. Vasodilator and antispasmodic activity of 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

где R соответствует релаксации сосуда при концентрации соединения с, Rmax соответствует максимальной релаксации под действием этого же соединения, а n представляет собой виртуальный коэффициент кооперативности, варьировавший в пределах от 1,1 до 1,8. Достоверность подбора параметров (n и IC₅₀), которую контролировали по методу наименьших квадратов, составляла не менее 95-97%. Статистическую обработку результатов проводили по тесту Стьюдента (t).The vasodilating and antispasmodic activity of BAFDOD was studied on the model of isometric contraction of smooth muscles of an isolated rat aortic segment under the action of a vasoconstrictor (spasmogen) - phenylephrine. BAFDOD activity was determined in vitro on the rings of the thoracic aorta of male Wistar rats with an average weight of 280 g (210-330 g) in a known manner. Rats were decapitated, the thoracic aorta was excised and purified from adipose tissue. Then 2.5 mm wide rings were cut which were hung on two parallel stainless steel hooks. One of the hooks was fixed on the wall of the chamber, and the other was connected to an isometric sensor DY1 connected to an eight-channel recorder (Beckman, USA). The chamber contained 30 ml of Krebs solution (130 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl ₂ , 1.18 mM KH ₂ PO ₄ , 14.9 mM NaHCO ₃ , 1.2 mM MgSO ₄ , 11 mM glucose ) at 37 ^o C, which was under conditions of constant aeration of 95% O ₂ /5% CO ₂ to maintain a pH of 7.4. Indomethacin was added to Krebs solution to a concentration of 1 μM. If necessary, the endothelium was removed mechanically. The rings were balanced before the experiment for an hour under a load of 2.5 g. Before the start of the experiment, the rings were shortened with 10 nM norepinephrine, and after 20 minutes the drug was reduced by adding phenylephrine at a concentration of 0.3 μM. After stabilization of the state of the vessel muscle, its relaxation was recorded in response to cumulative doses of the compounds in the concentration range from 0.003 nM to 50,000 nM. Then, the studied compound was washed with 6–7 shifts of the incubation medium for 1 hour and the intact state of the smooth muscle of the vessel was monitored using 0.5 μM sodium nitroprusside. Due to the low solubility of the compounds in water, the influence of a solvent (DMSO) on the isometric contraction of the vessel was determined in separate experiments. It was found that DMSO in a concentration of up to 0.1-0.2% has practically no effect on vascular tone, which allows testing the vasorelaxant activity of the compound at a concentration of up to 100 μM. All experiments were repeated at least 3 times. The concentration of the test compound corresponding to 50% relaxation of the vessel (IC ₅₀ ) was calculated using the SigmaPlot program version 2.0 according to the standard equation for sigmoid dependencies of the form

where R corresponds to relaxation of the vessel at a concentration of compound c, Rmax corresponds to maximum relaxation under the action of the same compound, and n represents a virtual coefficient of cooperativity, ranging from 1.1 to 1.8. The reliability of the selection of parameters (n and IC ₅₀ ), which was controlled by the least squares method, was at least 95-97%. Statistical processing of the results was carried out according to Student's test (t).

Under the above conditions, BAFDOD caused a decrease in the isometric contraction of the vessel with intact endothelium in the presence of phenylephrine (IC ₅₀ = 22 nM).

According to the prototype, the known compound in vitro, close to the above, had antispasmodic and vasodilating effects; the IC ₅₀ value being 120 nM. For the basic pharmaceutical preparation (glyceryl trinitrate) used as a reference standard under the above conditions, this parameter had a value of 5.3 nM.

 Example 8. Antihypertensive activity of 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

 The antihypertensive activity of the compounds of the present invention of the general formula I was determined in vivo in awake Wistar rats with an average weight of 280 g in a known manner. To measure the average blood pressure and heart rate and to inject the drug into the bloodstream the day before the experiment, the animals were implanted with polyethylene catheters (PE-10, PE-50 brands) in the femoral artery and femoral vein. The free ends of the catheters were removed and secured to the head. The operation was performed under hexenal anesthesia (150 mg / kg). A day later, the rat was taken into the experiment. During the experiment, blood pressure was recorded with a Stadham sensor (USA) followed by data recording using a computer (Bioshell program, Faculty of Fundamental Medicine, Lomonosov Moscow State University, Russia). Compounds were administered bolus intravenously at a dose of 2.5 mg / kg in a volume of 0.2 ml in 5% DMSO. During the entire experiment, the animals were awake and could freely move around the cell. Within an hour, the animals freely adapted to the experimental conditions, and then began recording hemodynamic parameters, which was carried out continuously throughout the experiment. Baseline mean arterial pressure and heart rate were 98.3 mmHg. Art. and 315.6 beats / min, respectively.

 The bolus administration of BAFDOD at a dose of 0.5 mg / kg did not affect blood pressure for 5 minutes. At the same time, a short-term insignificant increase in the heart rate was observed, followed by a decrease to the initial level. Thus, BAFDOD does not exhibit hypotensive effects when administered to rats at doses up to 0.5 mg / kg. The effect of the known analogue on blood pressure in vivo has not been studied.

 Example 9. Acute toxicity of 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine.

The acute toxicity of the compounds of the present invention was determined in a known manner by LD ₅₀ using outbred mice of both sexes with an average weight of 21 g at room temperature; standard food and water were given ad libitum throughout the experiment; the mobility of animals was not limited. Solutions of the compounds in DMSO were injected intraperitoneally with a sterile syringe. After injection, animals were monitored for 48 hours; after this time, the mice were observed for an additional 72 hours (none of the animals died during the additional time period). The results obtained indicate that the LD ₅₀ value for 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine is ~ 150-300 mg / kg. The acute toxicity of a known analogue has not been studied.

 According to well-known data, a similar indicator for the basic pharmaceutical product - glyceryl trinitrate under close conditions corresponds to 7-9 mg / kg (RF patent 2086534 (C 07 C 203/04) 1994, table 2).

 The above examples 1 to 5 show that 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine is an effective nitric oxide donor and an activator of rHC. From example 7 it follows that this compound has a pronounced antispasmodic and vasodilating effect, superior to the corresponding pharmacological effect in vitro of a known structural analogue. At the same time, unlike the known derivatives of furoxan, BAFDOD did not have a noticeable effect on blood pressure when administered intravenously at a dose of 0.5 mg / kg, which suggests the possibility of its directed local application.

 Thus, 3,6-bis (4-aminofurazan-3-yl) -1,4,2,5-dioxadiazine of the above formula I is a representative of a new class of NO donors, rHC activators, platelet aggregation inhibitors, antispasmodics and vasodilators.

Claims (1)

The use of 3,6-bis (4-aminofurazan-3-yl) 1,4,2,5-dioxadiazine of formula I

as a donor of nitric oxide, activating the soluble form of guanylate cyclase, inhibiting platelet aggregation and having antispasmodic and vasodilating effects.

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