RU2240321C2 - Derivatives of 3,4-bis-(furazan-3-yl)furoxane producing nitrogen oxide, activating guanylate cyclase soluble form, inhibiting platelet aggregation and eliciting spasmolytic, vasodilating, hypotensive effect and pharmaceutical composition based on thereof - Google Patents

Abstract

FIELD: organic chemistry, medicine, biochemistry.

SUBSTANCE: invention relates, in particular, new chemical compounds - derivatives of 3,4-bis-(furazan-3-yl)furoxane of the general formula (I):

wherein R = R¹ mean hydroxyl group (OH), NH₂, N₃, lower alkoxyl or group of the general formula: NR²R³ wherein R² = R³ mean hydrogen atom (H); or R and R³ with nitrogen atom form piperidine ring; or R means NH₂ and R¹ means (lower alkanoyl)-amino-group under condition that R and R¹ don't mean methoxy-group eliciting pharmacological activity. Also, invention describes pharmaceutical compositions based on their base. Claimed compounds are able to generate nitrogen oxide in body, to inhibit aggregation of platelets and to activate the soluble form of guanylate cyclase that provides their spasmolytic, vasodilating and hypotensive effects that allows their applying for prophylaxis and arresting attacks of stenocardia, disorders caused by smooth muscle spasms and for treatment of acute myocardium infarction or acute thrombosis.

EFFECT: valuable medicinal properties of compounds.

3 cl, 4 tbl, 12 dwg, 18 ex

Description

The invention relates to chemistry and medicine, in particular, to new chemical compounds - derivatives of 3,4-bis (furazan-3-yl) furoxan of the general formula I:

where R = R ¹ = OH, NH ₂ , N ₃ , a lower alkoxyl or a group of the general formula NR ² R ³ , where R ² = R ³ = H or R ² and R ³ together with the nitrogen atom form a piperidine ring, or R = NH ₂ and R ¹ - (lower alkanoyl) amino group with pharmacological activity, and pharmaceutical compositions based on them. The claimed compounds, in particular, are able to generate nitric oxide in the body, activate the soluble form of guanylate cyclase (rHC), which causes their antispasmodic, vasodilating, hypotensive effect and the ability to inhibit platelet aggregation.

The new compounds can be used to treat diseases of the cardiovascular system, including the treatment of arterial hypertension, congestive heart failure with myocardial infarction, hypertensive crises, refractory heart failure, myocardial infarction and acute myocardial infarction, angina pectoris, coronary heart disease, chronic heart failure, pulmonary hypertension , acute failure of the left ventricle, pulmonary heart, pulmonary edema, peripheral arterial embolism, toxicogenic spasms of the for the prevention and relief of angina attacks and spasms of coronary arteries when using cardiac catheters, angiography and angioplasty, as well as other diseases of the cardiovascular system, in which a positive therapeutic effect is achieved by lowering blood pressure, normalizing myocardial contractility and / or inhibiting activity platelets, with the exception of those diseases in which a decrease in blood pressure and / or inhibition of platelet aggregation is contraindicated.

The use of new compounds for the prevention and relief of angina attacks, disorders caused by smooth muscle spasm, and the treatment of acute myocardial infarction or acute thrombosis is preferred.

It is known that nitric oxide (NO), synthesized by endothelial cells and other types of cells and tissues, plays one of the key roles 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 (M.D. Mashkovsky "Medicines", v.1 , "Torsing", Kharkov, 1997, p. 385). The molecular mechanism of the endogenous nitric oxide synthesized by the enzyme NO synthase from the amino acid L-arginine in the endothelium of blood vessels and in other types of cells, organs and tissues includes its diffusion through plasma membranes into the cavity of the blood vessel and its smooth muscle cells, where NO binding occurs with heme group 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 guanyl 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 nitric oxide NO with the heme iron atom, which is part of the enzyme, and the formation of the nitrosylgem complex, conformational changes in the active center of the enzyme occur, which lead to activation of the RHC and increased synthesis of cGMP. As a result of the interaction of nitric oxide with platelets and leukocytes, their aggregation and adhesion on the walls of blood vessels are reduced. Therefore, NO inhibits the processes of thrombosis and has an anti-inflammatory effect. On the other hand, an increase in the intracellular concentration of cGMP in vascular smooth muscle cells causes activation of cGMP-dependent protein kinases, which induces dephosphorylation of the myosin molecule and, consequently, relaxation of the smooth muscles of coronary and other blood vessels (brain vessels, abdominal cavity, peripheral vessels), and a decrease in vascular tone and resistance, that is, leads to an expansion of the lumen of the blood vessel. Thus, NO reduces blood pressure, increases blood flow and helps restore vascular function (figure 1). Similarly, NO acts on other, non-vascular smooth muscles, including the muscles of the gastrointestinal tract, bile ducts, ureters, uterus, bronchi, and other organs.

Disorders associated with the normal course of the above reactions underlie the pathophysiological processes characteristic of the development of various diseases of the cardiovascular system (hypertension, myocardial infarction, heart failure, acute thrombosis) and other organs. In such diseases, there are multiple violations of the synthesis of endogenous NO, its reception of RHC, as well as the regulation of the level of cyclic nucleotides.

To date, the formation of nitric oxide as a result of enzymatic biotransformation of trinitroglycerin and other nitrates, which are used for the treatment of cardiovascular diseases as anti-ischemic and antianginal drugs (MD Mashkovsky "Medicines", v.1, "Torsing", Kharkov, has been proved) , 1997, pp. 385-392). However, their significant drawback is the emergence of tolerance and other side effects with prolonged use. In this regard, a search is underway for new compounds that can generate NO in a living organism in a non-enzymatic way (for example, spontaneously or thiol-dependent), which is considered as an urgent and promising approach for creating new, more effective antihypertensive and antiplatelet drugs with antianginal and anti-ischemic activity.

Various heterocyclic N-oxides are known, the biochemical and pharmacological properties of some representatives of this class of compounds are studied and described in the literature.

So, the known drug "minoxidil" -6- (1-piperidinyl) pyrrolidin-2,4, -diamine-3-oxide of the formula II:

being a peripheral vasodilator (MD Mashkovsky "Medicines", t.1, Kharkov, "Torsing", 1997, p. 429). This compound does not have the ability to generate NO, exhibits weak antiplatelet properties, and its effect on RHCs has not been studied.

Derivatives of 2- (2,2-dimethyl-1,3-benzoxazin-4-yl) pyridin-1-oxide of the general formula III are known:

where R and R ¹ = F, Cl, Br, etc., with vasorelaxant and hypotensive effects (S. Yamamoto, S. Hashiguchi et al. "Synthesis and biological activity of novel 1,3-benzoxazine derivatives as K ⁺ channel openers" Chem. Pharm. Bull. 1996, v. 44, p. 734-745). These compounds do not have the ability to generate NO, their effect on RHC and antiplatelet properties have not been studied.

Known substituted quinazolin-3-oxides of the general formula IV:

where RR ⁵ = H, lower alkyl, etc., exhibiting cardiotonic and bronchodilating effects (US patent No. 4745118, 1988). These compounds do not have the ability to generate NO, their effect on RHC, as well as hypotensive and antiplatelet properties have not been studied.

Various 3,4-disubstituted furoxanes (1,2,5-oxadiazole-2-oxides) of the general formula V are known:

where R ¹ and R ² - (substituted) lower alkyl, (substituted) phenyl, phenylsulfonyl, alkoxycarbonyl, (substituted) carboxamide group and others, with a pronounced vasorelaxant effect on the rings of the pulmonary artery of a guinea pig, previously reduced by KCl, and showing antihypertensive activity (laid out application of Germany No. 4401150, 1995, European patents No. 0054872, 1984; No. 0054873, 1984; No. 0575782, 1993, No. 0683159, 1995, No. 0687256, 1994) . The NO-generating properties of these compounds, their effect on the activity of the RGC and the ability to inhibit platelet aggregation have not been studied.

Disubstituted trifuroxanes of general formula VI are known:

where R ¹ and R ² are (substituted) methyl, phenyl, etc., which are nitric oxide donors and cause vasodilation on isolated rat aortic rings, previously reduced by norepinephrine (AMGasco, C. Cena et al. "Synthesis and structural characterization of the trimeric furoxan (= furazan-2-oxide) system, a new potent vasodilating moiety "Helv. Chim. Acta 1996, v. 79, p. 1803-1817). The effect of these compounds on the activity of RHCs and their ability to inhibit platelet aggregation (with the exception of one derivative) has not been studied. In addition, the vast majority of disubstituted trifuroxanes had extremely low solubility under experimental conditions, which made their study difficult.

Closest to the claimed compounds of the present invention is 4,4'-diphenyl-3,3 ': 4', 3 '' - trifyrazan-2,2 ', 2''- trioxide of the above formula VI, where R ¹ = R ² = C ₆ H ₅ , which is a NO donor, causes vasodilation on isolated rabbit thoracic aortic rings previously contracted by norepinephrine (IC ₅₀ = 0.12 μM) and inhibits platelet aggregation (IC ₅₀ = 1.8 μM) (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).

The effect of this trifuroxan on the activity of rHC and its hypotensive activity have not been studied. In addition, it has an insufficiently effective vasorelaxant and anti-aggregate action, as well as low solubility in experimental conditions.

The search for new compounds that generate nitric oxide in the body and activate the soluble form of guanylate cyclase (RHC), which have good solubility and lack of tolerance, and the creation of drugs based on them is an urgent task.

The invention is based on the task of expanding the arsenal of chemical compounds acting on a living organism, in particular, generating nitric oxide in the body and activating the soluble form of guanylate cyclase (RHC) and having improved solubility and lack of tolerance, and the creation of drugs based on them.

The problem is solved in that according to the invention, new chemical compounds are created - derivatives of 3,4-bis (furazan-3-yl) furoxan of the general formula I,

where R = R ¹ = OH, NH ₂ , N ₃ , a lower alkoxyl or a group of the general formula NR ² R ³ , where R ² = R ³ = H or R ² and R ³ together with the nitrogen atom form a piperidine ring, or R = NH ₂ and R ¹ - (lower alkanoyl) amino group with pharmacological activity.

The invention is also the fact that the claimed compounds of general formula I are able to generate nitric oxide, activate the soluble form of guanylate cyclase, and inhibit platelet aggregation.

The invention is also a pharmaceutical composition having antispasmodic, vasodilating, hypotensive effect and inhibiting platelet aggregation, contains, as an active component, a compound of general formula I in an amount sufficient to achieve a pharmacological effect, and a pharmaceutically acceptable excipient.

The ability of the claimed compounds to generate nitric oxide in the body, as well as activate RHC, causes their antispasmodic, vasodilating, hypotensive effect and inhibition of platelet aggregation. In addition, the proposed compounds and pharmaceutical compositions based on them have improved solubility and lack of tolerance.

A brief description of the drawings illustrating the invention:

Figure 1 - mechanism of action of nitric oxide.

Figure 2 - cumulative relaxation curves of the rat thoracic aorta, previously reduced by phenylephrine, in the presence of compounds 1, 4 and trinitroglycerin.

Figure 3 - the effect of a bolus intravenous administration of compound 1 in doses of 0.1; 0.5 and 1 μM / kg per blood pressure (BP) in awake rats.

Figure 4 - effect of bolus intravenous administration of compound 1 in doses of 0.1; 0.5 and 1 μM / kg per heart rate (heart rate) in awake rats.

Figure 5 - effect of bolus intravenous administration of compound 2 in doses of 0.1; 0.5 and 1 μM / kg on blood pressure in awake rats.

6 - the effect of a bolus intravenous administration of compound 2 in doses of 0.1; 0.5 and 1 μM / kg on heart rate in waking rats.

Fig.7 - the effect of a bolus intravenous administration of compound 3 in doses of 0.1; 0.5 and 1 μM / kg on blood pressure in awake rats.

Fig - the effect of a bolus intravenous administration of compound 3 in doses of 0.1; 0.5 and 1 μM / kg on heart rate in waking rats.

Fig.9 - the effect of a bolus intravenous administration of compound 5 in doses of 0.1; 0.5 and 1 μM / kg on blood pressure in awake rats.

Figure 10 - the effect of a bolus intravenous administration of compound 5 in doses of 0.1; 0.5 and 1 μM / kg on heart rate in waking rats

11 - the effect of alternating periods of infusion of compound 1 (0.5 μm / min / kg) and recovery on blood pressure in awake rats with arrows and solid lines indicate the beginning of the infusion, and arrows and dashed lines indicate the cessation of infusion.

Fig. 12 shows the effect of alternating infusion periods of compound 1 (0.5 μM / min / kg) and recovery on heart rate in awake rats with arrows and solid lines showing the beginning of the infusion, and arrows and dashed lines showing the termination of the infusion

The invention is illustrated by the following examples.

According to the invention, derivatives of 3,4-bis (furazan-3-yl) furoxane of the above general formula I are proposed having pharmacological activity. Physico-chemical and pharmacological properties of the derivatives of the proposed compounds of General formula I according to the invention are shown in the examples of the following compounds:

-3,4-bis (4-aminofurazan-3-yl) furoxan (compound 1),

-3,4-bis (4- (piperidin-1-yl) furazanil] furoxan (compound 2),

-3,4-bis (4-hydroxyfurazan-3-yl) furoxan (compound 3),

-3,4-bis (4-azidofurazan-3-yl) furoxan (compound 4),

-3- [4- (acetylamino) furazan-3-yl) -4- (4-aminofurazan-3-yl) furoxan (compound 5).

The above compounds according to the invention were synthesized by a method based on the known reaction of directed dimerization of nitriloxides generated on the basis of chloroximes and processing of the corresponding 3-substituted 4- [chloro (hydroxyimino) methyl] furazan with a base (for example, low concentration NaHCO ₃ solution) in an organic solvent (ether, ethyl acetate) (L.I. Khmelnitsky, S. S. Novikov, T. I. Godovikova "Chemistry of furoxans. Structure and synthesis" M., Nauka, 1996, p.165-178). Amide derivatives were obtained by selective mono-N-acylation of compound 1 with the corresponding lower carboxylic acid anhydride (e.g., acetic acid) in the presence of the corresponding alkali metal salt (e.g. sodium acetate) under conditions analogous to the known method of acylation of amino derivatives 1,2,5- oxadiazole (L.I. Khmelnitsky, S. S. Novikov, T. I. Godovikova "Chemistry of Furoxans. Reactions and Applications" M., Nauka, 1996, p. 310).

We give specific examples of the preparation of the above derivatives of the compounds of general formula I.

Example 1. The preparation of compound 1 (3,4-Bis (4-aminofurazan-3-yl) furoxan)

To a suspension of 1.62 g (0.01 mol) of 4-amino-3 - [(hydroxyimino) chloromethyl] furazan in 35 ml of ethyl acetate with vigorous stirring at a temperature of 0-3 ° C, 18 ml of 5% NaHCO solution were added in small portions. ₃ . After that, the reaction mixture was stirred for 2 hours at a temperature not exceeding 5 ° С, the precipitate was filtered off, washed with 10 ml of cold water and dried in air. 1.72 g (69%) of compound 1 was obtained. Mp 167-168 ° C. IR spectrum (in a thin layer on a NaCl substrate), ν, cm ^-1 : 3480, 3440, 3340, 1650.1620, 1470, 1420, 1360, 1220.1150, 1130, 1020, 1000, 970, 930, 880, 840. NMR ¹ H spectrum, δ, ppm: 6.64 (2H, NH ₂ ) and 6.59 ( 2H, NH ₂ ). ¹³ C NMR spectrum, δ, ppm: 156.12; 155.20; 146.66; 136.29; 133.41; 104.33. Mass spectrum, m / z: 252 (M +), 222 (M-NO), 192 (M-NO-NO). Found,%: C 28.41; H 1.75; N, 44.56; MM 258. C ₆ H ₄ N ₈ O ₄ . Calculated,%: C 28.57; H 1.59; N, 44.44; MM 252.

Example 2. Obtaining compound 2 (3,4-Bis [4- (piperidin-1-yl) furazan-3-yl] furoxan)

Compound 2 was synthesized from 2.3 g (0.01 mol) of 3 - [(hydroxyimino) chloromethyl] -4- (piperidin-1-yl) furazan under conditions similar to Example 1. Yield of the final product -2.93 g (76 %). T. pl. 89-90 ° C. IR spectrum, ν, cm ^-1 : 2980, 2960, 1640, 1610, 1570, 1475, 1405, 1305, 1290, 1235, 1190, 1250, 1230, 1020, 980. NMR ¹ H spectrum, δ, ppm: 3.15 (4H, CH ₂ ); 1.5 (4H, CH ₂ ). ¹³ C NMR spectrum, δ, ppm: 159.59; 159.55; 144.84; 137.79; 134.26; 105.06; 50.04; 49.57; 24.43; 24.36; 22.92. Found,%: C 49.89; H 5.49; N, 29.20. MM 386. C ₁₆ H ₂₀ N ₈ O ₄ . Calculated,%: C 45.90; H 5.20; N, 33.50. MM 418.

Example 3. Obtaining compound 3 (3,4-Bis (4-hydroxyfurazan-3-yl) furoxan)

Under the conditions of Example 1, from 1.62 g (0.01 mol) of 4-hydroxy-3 - [(hydroxyimino) chloromethyl] furazan after recrystallization from chloroform, 0.35 g (14%) of compound 3 was obtained. 88-90 ° С. IR spectrum, ν, cm ^-1 : 3632, 3566, 1651, 1622, 1563, 1545, 474, 1429, 1391, 1351, 1220, 1151, 1075, 1000, 976, 930, 899, 891 828. NMR ¹ H spectrum (chloroform -d6), δ, ppm: 4.76 (2H, OH). ¹³ C NMR spectrum, δ, ppm: (see table 1). Found,%: C 28.81; H 0.95; N, 33.42. MM 252. C ₆ H ₂ N ₆ O ₆ . Calculated,%: C 28.35; H 0.79; N, 33.07. MM 254.

Example 4. Obtaining compound 4 (3,4-Bis (4-azidofurazan-3-yl) furoxan)

Under the conditions of Example 1, from 1.88 g (0.01 mol) of 4-azido-3 - [(hydroxyimino) chloromethyl] furazan, 0.72 g (24%) of compound 4 was obtained. 51 ° C (from ethanol). IR spectrum, ν, cm ^-1 : 2144, 1632, 1456, 1328, 1232, 992, 960. ¹ C NMR spectrum, δ, ppm: (see table 1). Found,%: C 23.80; H 0.50; N, 55.37. C ₆ N ₁₂ O ₄ . Calculated,%: C 23.68; H 0.00; N, 55.26.

Example 5. Obtaining compound 5 (4- (4-Aminofurazan-3-yl) -3- [4- (acetylamino) furazan-3-yl] furoxan)

A mixture of 0.6 g (0.0024 mol) of compound 1 and 0.5 g of freshly prepared sodium acetate in 10 ml of acetic anhydride was kept under stirring at room temperature until the starting compound 1 completely disappeared (TLC control; reaction time about 2 hours), after which it was evaporated to air to dryness. The obtained solid residue was washed with water, 5% soda solution and again with water. After crystallization from methanol with charcoal, 0.4 g (57%) of compound 6 was obtained. 134-135 ° С. IR spectrum, ν, cm ^-1 : 3976, 3752, 3744, 3688, 3648 (NH), 3336 (NH ₂ ), 1744 (С = O), 1692, 1664, 1632, 1580, 1552 , 1480, 1450, 1424, 1384, 1276, 1256, 1168, 1148, 1024, 992, 973. NMR ¹ H spectrum, δ, ppm: 10.5 br. (1H, NH); 6.1 ears (2H, NH ₂ ); 2.07 s (3H, CH ₃ ) (solvent-acetone-d6). Found,%: C 32.05; H 1.96; N, 38.78. MM 300. C ₈ H ₆ N ₈ O ₅ . Calculated,%: C 32.65; H 2.04; N, 38.10. MM 294.

The characteristics of the ¹³ C NMR spectra of the obtained derivatives are presented in table 1.

Further, the chemical foundations of the molecular mechanism of action of the proposed compound of general formula I, mainly consisting in the selective generation of nitric oxide, are shown using the obtained derivatives as an example.

Example 6. The study of the formation of nitric oxide from derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I.

To determine nitric oxide, we used a known method 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.

The sample with a final volume of 1 ml contained 50 mM potassium phosphate buffer (pH 7.4), 0.5 mM cysteine or glutathione, the studied compound at a concentration of 0.1 mM and 0.2% dimethyl sulfoxide (DMSO). 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 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 successively added. 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, the formation of a noticeable amount of nitrite (<0.01 mol of nitrite / mol of the starting compound (ref.s)) was not observed in the absence of thiols. In the presence of thiols of compound 1-5, 0.057-0.411 mol of nitrite per mol of the starting compound in the presence of cysteine and 0.017-0.162 mol of nitrite per mol of the starting compound were generated. in the presence of glutathione. The experimental results are presented in table 2.

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

Example 7. The study of the formation of S-nitrosothiols (S-nitrosoglutathione) derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I.

To determine S-nitrosothiols, a known method was used, based on the reaction with mercury (II) chloride, during which nitrite is formed. To take into account 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 6, and then nitrite was determined by the method described in example 6, in the absence and 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 compounds of the general formula I does not occur. The possibility of generation by a known analogue of S-nitrosothiols has not been investigated.

Example 8. The study of the formation of the reduced form of nitric oxide derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I.

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 ions $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ gives nitrite, determined by the azo coupling reaction (see example 7). Since the studied compounds also generate nitrite, which likewise undergoes 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 6 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 were added successively at intervals of 20 s. M 2-mercaptoethanol and 400 μl of 0.05% N-naphthylethylenediamine. Samples were incubated for 15 min and 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 6. 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 the formed sodium nitrite, determined as described in example 6, 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, the compounds of the present invention are selective NO donors and do not form oxidized or reduced forms of NO.

Example 9. The study of the formation of methemoglobin from oxyhemoglobin under the action of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I.

The determination of NO generation in the presence of oxyhemoglobin, accompanied by the quantitative conversion of oxyhemoglobin to methemoglobin during the reaction, was studied in a known manner in a spectrophotometric cuvette with a final volume of 3 ml at 25 ° C. Samples contained: 25 mM potassium phosphate buffer (pH 7.4), 3 μM oxyhemoglobin, 0.5 mM cysteine or glutathione, and test compounds 1-5 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 mM ^-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 hemichrom 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 test compounds. The optical density was measured at 560, 577, 630 and 700 nm, and the concentrations of oxyhemoglobin, hemichrom, 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 cholelobin or hemichrome is observed, and only the conversion of oxyhemoglobin to methemoglobin occurs.

Under the above conditions, test compounds 1-5 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 (reaction rate constants NO + oxyhemoglobin → NO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ + methemoglobin) are presented in table 2. Compounds did not react under similar conditions with methemoglobin.

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

The results of the above examples 6-9, presented in table 2, show that the new derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I are effective donors of nitric oxide.

Example 10. The study of the effect of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I on the activation of the soluble form of guanylate cyclase (rHC)

Activation of rHC under the action of derivatives of 3,4-bis (furazan-3-yl) furoxan of general formula I was studied using compounds 1-5 as an example using a known method using partially purified rHC preparation from bovine lungs. Also, the activity of rHC was measured by a known indirect method for the accumulation of cGMP in preparations of an isolated rabbit aorta.

The activity of rHC was measured in preparations from bull lungs obtained in a known manner. The lungs were ground at 4 ° C, homogenized in five volumes of buffer A (50 mM triethanolamine-Hcl pH 7.6 containing 5 mM DTT and 75 mM NaCl) and centrifuged at 100,000 g for 1 h. The supernatant was applied to a DEAE-Toyoperl column 650M (Toyosoda, Japan), equilibrated with buffer A, and eluted with a linear NaCl gradient (0.075 to 0.5 M) in buffer A. The peak of the enzyme activity eluted at an ionic strength corresponding to a 0.22 M NaCl concentration. Fractions containing the maximum activity, concentrated and gel-filtered on a Sephacryl S-300 column (Pharmacia, Schwe Ia) equilibrated in buffer A. Active fractions were combined and concentrated and column chromatography was conducted with Blue agarose CL-4B (HFZ Tallinn, Estonia) equilibrated in 50 mM triethanolamine-HCl pH 7.6, 10 mM DTT and 10 nM hemin. The protein was eluted with a linear NaCl gradient (0.05 to 1 M). The active fractions were combined, concentrated and chromatographed on a Q-Sepharose column (Fast Flow, Pharmacia, Sweden) equilibrated with buffer A. The enzyme was eluted with a linear NaCl gradient from 0.075 to 1.5 M. The purified rHC was stored in a nitrogen atmosphere in 50 mM triethanolamine- HCl pH 7.6 containing 30% glycerol, 10 mM DTT and 1 M NaCl at -70 ° C.

The 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 (Isotope, INP, Obninsk), as well as 10 mM DTT or other thiols, as indicated in the text additionally. 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, mixed, incubated at 4 ° C for 10 min and centrifuged at 15,000 g for 5 minutes. The supernatant was applied onto alumina columns acidified in a known manner, which were washed with water. Elution of [ ³² P] GMF was carried out with 0.2 M ammonium formate in scintillation counting bottles, and radioactivity was measured using a liquid scintillation counter in a known manner by Cherenkov.

Protein determination was carried out according to the known Lowry method using bovine serum albumin as a standard.

The results of the experiments are presented in table 3.

Analysis of the data presented in table 3 shows that the compounds of the present invention, depending on the concentration, activated 2.09-35.8 times in the RHC from the lungs of a bull.

Thus, derivatives of 3,4-bis (furazan-3-yl) furoxan of the general formula I are effective activators of rHC.

The influence of the known structural analogue on the activity of rHCs has not been studied.

To determine the accumulation of cGMP in preparations of an isolated rabbit aorta, the aortic segment was incubated for 30 min in a 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 ° C in the presence of 1 mM 3-isobutyl-1-methylxanthine, and then compound 1 was added to a final concentration of 10 μM. The incubation was carried out for 3 min, then the tissue was frozen in liquid nitrogen, 20 mg of powder was weighed and triturated with a mortar with liquid nitrogen. 0.5 ml of a 1 M solution of perchloric acid was extracted for 60 min at 4 ° C. The mixture was centrifuged at 10000 g for 5 min, the precipitate was discarded, and the supernatant was neutralized by adding 0.25 ml of a 1 M solution of K ₂ CO ₃ . The precipitate was removed by centrifugation, the supernatant was collected and the amount of cGMP was determined in a known manner using cGMP determination kits manufactured by Bioimmunogen (Moscow, Russia). The results were expressed in pmol cGMP / mg protein. In the absence of compound 1 (0.02% DMSO), the level of cGMP was 2.55 pmol cGMP / mg protein, and in the presence of compound 1-3.9 pmol cGMP / mg protein. Therefore, compound 1 increases the level of cGMP in the preparation of an isolated rabbit aorta. The influence of the known structural analogue on the level of cGMP in smooth muscles has not been studied.

Example 11. Determination of vasodilator and antispasmodic activity of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I

, 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) контролировали по методу наименьших квадратов и она составляла не менее 95%. Статистическую обработку результатов проводили по тесту Стьюдента (t). Результаты представлены в таблице 4 и на фиг.2.The vasodilator and antispasmodic activity of the compounds of the present invention 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. The activity of the proposed derivatives of general formula I was determined by the example of compounds 1-5 in vitro on the rings of the thoracic aorta of male rats of the Wistar strain 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 ° C, which was under 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 compounds 1-5 in the concentration range from 0.003 nM to 50,000 nM. After that, the studied compound was washed with 6-7 changes in the incubation medium for 1 h and the intact state of the smooth muscle of the vessel was controlled using 0.5 μM sodium nitroprusside. Due to the fact that the solvent of the studied compounds in the present experiment was DMSO, we determined the effect of the solvent on the isometric contraction of the vessel in individual experiments. It was found that DMSO in a concentration of up to 0.1-0.2% has virtually no effect on vascular tone, which allows testing the vasorelaxant activity of the studied compounds at a concentration of up to 100 μM. All experiments were repeated at least 3 times. The concentration of the studied compounds corresponding to 50% relaxation of the vessel (IC ₅₀ ) was calculated using the SigmaPlot software version 2.0 (Jandel Scientific Corp., USA) 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 IC50) was controlled by the least squares method and it was at least 95%. Statistical processing of the results was carried out according to Student's test (t). The results are presented in table 4 and figure 2.

Analysis of the data presented in table 4 shows that the compounds of the present invention caused a decrease in the isometric contraction of the vessel with intact endothelium in the presence of phenylephrine (IC ₅₀ ranged from 0.45 to 100.5 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 (trinitroglycerin) used as a reference standard under the above conditions, the value of this parameter was 5.3 nM.

Thus, the antispasmodic activity of the compounds of the present invention is comparable to the activity of known preparations.

Example 12. The study of the hypotensive activity of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I

The antihypertensive activity of the compounds of the present invention of the general formula I was determined in vivo in waking rats of the Wistar strain with an average weight of 280 g in a known manner. To measure the average blood pressure and heart rate and to introduce 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 fixed on 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 cage. Within an hour, the animals were allowed to freely adapt to the experimental conditions, and then registration of hemodynamic parameters was started, which was carried out continuously throughout the experiment. Baseline mean arterial pressure and heart rate were 98.3 mmHg. Art. and 315.6 bpm, respectively. The results obtained are illustrated in drawings 3-12, showing the effect of bolus intravenous administration of compounds of the general formula I at doses of 0.1.0.5 and 1 μmol / kg on blood pressure (BP) and heart rate (heart rate) in awake rats.

So, the bolus administration of compound 1 in doses of 0.1-1 mg / kg caused a rapid decrease in mean arterial pressure for 5 minutes, and then a quick restoration of the initial parameters occurred. The heart rate briefly increased only at the peak of a drop in blood pressure, and then decreased to the initial level. Thus, the high efficiency and lack of time delay of the vasodilating effect of compound 1 with intravenous administration is obvious.

The effect of the known analogue on blood pressure in vivo has not been investigated.

Example 13. Tolerance test with repeated re-introduction of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I in vivo

We studied the effect of alternating infusion periods of the compounds of the present invention (in particular, compound 1 at a dose of 0.5 μmol / min / kg) on blood pressure (BP) and heart rate (HR) in awake rats. The results are illustrated in FIGS. 11 and 12. the indicated figures, the arrows and solid lines indicate the beginning of the infusion, and the arrows and dashed lines indicate the termination of the infusion. The data obtained show a lack of tolerance with repeated re-administration of derivatives of 3,4-bis (furazan-3-yl) furoxan of the general formula I in vivo.

Example 14. The study of the effect of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I on the aggregation of human platelets

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 min 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 was adjusted to 2.5 × 10 ⁸ cells / ml in platelet-rich plasma 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. Lomonosova (Russia). Test compounds were added to ADP.

The compounds of the present invention inhibited platelet aggregation. For example, the concentrations at which half-maximal inhibition is achieved (IC ₅₀ ) for compounds 1, 3, and 4 were 0.85, 0.17, and 0.82 μM, respectively. The known closest analogue has an IC ₅₀ value of 1.8 μM.

Example 15. The study of the solubility of derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I

The solubility of the test compounds in the experimental conditions was checked as follows. An initial solution of the substance in DMSO at a concentration of 50 mM was prepared. Then 20 μl of this solution was introduced into a glass tube containing 5 ml of deionized water, which was stirred at a speed of about 1000 rpm, to a final concentration of 0.2 mm. With this dilution method, in the case of compounds 1-5, a clear, true solution was obtained.

Solutions of 3,4-bis (furazan-3-yl) furoxane derivatives of general formula I in DMSO were stable for 1 month at room temperature when stored in a dark place and frozen at -20 ° C for up to 6 months. 0.25 mM An aqueous solution of compound 1 containing 0.5% DMSO is quite stable at room temperature when stored in a dark place at neutral or slightly acidic pH values. Aqueous solutions of the compounds of the present invention are unstable at weakly alkaline and alkaline pH values (pH> 9), in bright light and in the presence of thiols. When dry, these compounds are highly stable at room temperature when stored in a dark place.

This invention also relates to pharmaceutical compositions generating nitric oxide, activating a soluble form of guanylate cyclase and having antispasmodic, vasodilating, hypotensive and inhibiting platelet aggregation action, containing 3,4-bis (furazan-3-yl) furoxane derivatives of the general formula as an active component I and pharmaceutically acceptable additives in the required ratio. As additives, components are used that are conventional for the preparation of dosage forms of antispasmodic, vasodilator, antihypertensive and antiplatelet agents. Depending on the method of administration of the drug and the purpose of its use, the dosage forms can be in the form of tablets, capsules, granules, dragees, pills for gradual dosing (if necessary, from a polymeric material, for example, acetyl phthaloyl cellulose, or other material suitable for this purpose, for example gelatin ), as well as in the form of powders, solutions, suspensions or emulsions.

A solid oral dosage form may contain, as additives, an inert excipient (e.g. lactose, sucrose, sorbitol, starch, mannitol), surfactants (e.g. magnesium or calcium stearate, polyvinylpyrrolidone, polyethylene glycol, polypropylene glycol) and pharmacologically acceptable inorganic salts ( e.g. sodium chloride).

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

Another possible form of application of the derivatives of 3,4-bis (furazan-3-yl) furoxan of the general formula I are transdermal therapeutic compositions in the form of ointments and plasters, including those providing a dosed transdermal supply of active components into the body.

The above pharmaceutical compositions based on the compounds of the present invention can be obtained by combining the latter with the active components of other known pharmaceuticals, in particular those used to treat diseases of the cardiovascular system. For example, for this purpose, α ₁ -adrenergic receptor blockers (prazosin), central α ₂ -adrenergic and I ₁ imidazoline receptor agonists (clofelin, moxonidine), β - (β ₁ -) adrenergic receptor blockers (propranolol, atenolol, metoprolol can be used) ), calcium channel blockers (nifedipine, diltiazem), angiotensin-converting enzyme inhibitors (lisinopril, fosinopril), coronary expansion drugs (carbocromene), diuretics (dichlorothiazide), cardiac glycosides (digitoxin), antihypertensive agents ( gemfibrozil).

Example 16. The composition of the solution for receiving sublingually containing derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I as an active component:

Components wt.%

Active component 1

Ethyl alcohol 99

Example 17. The composition of the tablets for oral administration, containing derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I as an active component:

Ingredients mg / 1 tablet

Active component 0.5

Lactose 50

Microcrystalline Cellulose 50

Magnesium Stearate 0.5

Polyvinylpyrrolidone 10

Total weight 111

Example 18. Gelatin capsules of 0.001 and 0.0005 g each containing 3,4-bis (furazan-3-yl) furoxan derivatives of the general formula I in refined sunflower oil:

Components% wt./capsule

Active component 1

Refined Sunflower Oil 99

The above examples 6-11 show that the new derivatives of 3,4-bis (furazan-3-yl) furoxan of general formula I are effective nitric oxide donors and activators of RHC. From examples 12-14, it follows that these compounds have a strong antispasmodic, vasodilator and hypotensive effect, and their pharmacological effect in vitro exceeds the similar action of the known structural analogue and is comparable to the activity of the basic pharmaceutical drug - trinitroglycerin, without the occurrence of tolerance in experimental conditions with repeated administration of drugs. From example 15 it follows that the derivatives of 3,4-bis (furazan-3-yl) furoxan of the general formula I are superior in antiplatelet activity to the analogous effect of the closest known compound.

Thus, derivatives of 3,4-bis (furazan-3-yl) furoxan of the above general formula I expand the spectrum of NO donors, activators of RHC, antispasmodic, vasodilator, hypotensive agents and platelet aggregation inhibitors, and pharmaceutical compositions based on these compounds suggest their possibility medical applications.

Claims (3)

1. Derivatives of 3,4-bis (furazan-3-yl) furoxan of General formula I

where R is R ¹ - OH, NH ₂ , N ₃ , a lower alkoxyl or a group of the general formula NR ² R ³ , where R ² - R ³ - H or R ² and R ³ together with the nitrogen atom form a piperidine ring, or R - NH ₂ and R ¹ - (lower alkanoyl) amino group, provided that R and R ¹ do not denote methoxy having pharmacological activity. 2. The compounds according to claim 1, characterized in that they generate nitric oxide, activate the soluble form of guanylate cyclase, inhibit platelet aggregation and have antispasmodic, vasodilating and hypotensive effects. 3. A pharmaceutical composition having an antispasmodic, vasodilating, hypotensive effect and inhibiting platelet aggregation, containing an active substance and a pharmaceutically acceptable excipient, characterized in that as an active substance it contains the compounds of claim 1 in an effective amount.

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