RU2641107C1 - SODIUM SALT OF 3-NITRO-4-OXO-1,4-DIHYDROPYRAZOLO[5,1-c]-1,2,4-TRIAZINE-8-CARBOXYLIC ACID, DIHYDRATE - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo[5,1-c]-1,2,4-triazine-8-carboxylic acid, dihydrate,.EFFECT: new compound showing antiglating properties is produced.2 tbl, 3 ex

Description

1. The technical field to which the invention relates.

The invention relates to the field of biologically active compounds - the sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5.1-s] -1,2,4-triazine-8-carboxylic acid, a dihydrate having antiglycant activity, intended for the treatment and prevention of the effects of diabetes.

The invention can be used in medical institutions and research laboratories.

2. The level of technology

1. The accumulation of end products of protein glycation proceeding in the framework of the Maillard reaction (Aldini G., Vistoli G., Stefek M., Chondrogianni N., Grune T., Sereikaite J. Molecular strategies to prevent, inhibit, and degrade advanced glycoxidation and advanced lipoxidation end products. Free Radical Res. - 2013. - N 47. - P. 93.), is one of the main causes of the development of cardiovascular diseases such as atherosclerosis, ophthalmopathy, renal failure, damage to the nervous system, impaired peripheral blood circulation etc. (Emelyanov V.V., Maksimova N.E., Mochulskaya H.N. Non-enzymatic glycosylation of proteins: chemistry, pathophysiology, prospects for correction. Issues of biological, medical and pharmaceutical chemistry. - 2010. - N 1. - P. 50).

The reaction of non-enzymatic protein glycation is especially intense in the body of patients with diabetes mellitus due to the hyperglycemia associated with this disease. Numerous complications of diabetes are directly related to this process (Balabolkin MI The role of glycation of proteins, oxidative stress in the pathogenesis of vascular complications in diabetes. Diabetes mellitus. - 2002. - N 4. - C. 8).

. Arterial stiffness and cardiovascular therapy. Biomed. Res. Int. - 2014. - P. 1-11).The main mechanisms through which CNG effects are realized are glycation of intracellular proteins, cross-linking, and interaction with CNG receptors (G. Pugliese, Do advanced glycation end products contribute to the development of long-term diabetic complications? Nutr. Metab. Cardiovasc. Dis . - 2008. - No. 18 (7). - P. 457-460). The formation of stable inter- and intramolecular cross-links is pathogenetically more significant for structural, long-lived proteins that are updated within a few months, years, for example, proteins of the basement membrane (collagen, elastin, myelin), and lens proteins of the eye (crystallins). Impaired structural proteins cause subsequent functional changes in the vascular wall (decreased elasticity, altered response to the vasodilating effect of nitric oxide) (M. Janic, M. Lunder, and M.

. Arterial stiffness and cardiovascular therapy. Biomed. Res. Int. - 2014 .-- P. 1-11).

Glycation of structural proteins of the basement membrane of the renal glomeruli (type IV collagen, laminin, heparan sulfate, etc.) is a key factor in the development of nephropathy - one of the main causes of disability and mortality in patients with diabetes mellitus. In conditions of chronic hyperglycemia, an increased concentration of CNG leads to the accumulation of intercellular matrix proteins and a change in their composition, a thickening of the glomerular basement membranes, preceding such irreversible changes as glomerulosclerosis and tubulointerstitial fibrosis. These processes characterize the final stages of nephropathy development (S.-Y. Goh, M.E. Cooper. The Role of Advanced Glycation End Products in Progression and Complications of Diabetes. J. Clin. Endocrinol. Metab. - 2008. - No. 93 ( 4). - P. 1143-1152).

The receptor-dependent effects of CNG are mediated by their interaction with specific receptors, which leads to the activation of secondary transmitters, such as protein kinase C and nuclear factor NF-κB, which travels to the nucleus and leads to increased transcription of proteins such as intercellular adhesion molecules-1, E-selectin , endothelium-1, vascular endothelial growth factor, pro-inflammatory cytokines (R. Ramasamy, SF Yan, AM Schmidt. Receptor for AGE (RAGE): signaling mechanisms in the pathogenesis of diabetes and its complications. Ann NY Acad Sci. - 2011 Dec; 1243: 88-102.). All these mechanisms underlie the pathogenesis of such consequences of diabetes mellitus (NA Ansari, Z. Rasheed. Non-enzymatic glycation of proteins: from diabetes to cancer. Biomed Khim. - 2010. - No. 56 (2). - P. 168-1781 ), such as diabetic atherosclerosis, nephro-, neuro-, retino-, cardio-, angiopathies, which are the cause of a high risk of disability and mortality among patients with diabetes mellitus.

To date, pharmacological correction of the reaction of formation of the final products of glycation of proteins in the patient's body is an important task in medicine. Clinical drugs have not been developed that are capable of neutralizing CNG or inhibiting their formation, however, the scientific interest in non-enzymatic protein glycation (GBS) reactions is growing every year, and compounds that have proved to be promising inhibitors of CNG formation have already been obtained.

The first and most studied substance that inhibits the glycation of proteins is aminoguanidine 2 (AG), selected as a prototype. It prevents the formation of fluorescent CNG and glucose-derived cross-linked collagen molecules. The mechanism of the anti-glycation action of aminoguanidine is based on its interaction with intermediate products of protein glycation, which are carbonyl compounds with high reactivity. However, due to the toxicity and low efficacy of the compound, clinical trials were stopped (Freedman BI, Wuerth J.-P., Cartwright K., Bain RP, Dippe S., Hershon K., Mooradian AD, Spinowitz BS Design and baseline characteristics for the aminoguanidine clinical trial in overt type 2 diabetic nephropathy (ACTION II). Control. Clin. Trials - 1999.- N 20 (5) .- P. 493; Bolton WK, Cattran DC, Williams ME, Adler SG, Appel GB, Cartwright K. , Foiles PG, Freedman BI, Raskin P., Ratner RE, Spinowitz BS, Whittier FC, Wuerth J.-P. Randomized trial of an inhibitor of formation of advanced glycation end products in diabetic nephropathy. Am. J. Nephrol. - 2004 . - N 24. - P. 32).

In the series of azolo-1,2,4-triazines, compounds having antiviral action are known. Antiviral drug Triazavirin - 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one, trihydrate 3 (Chupakhin O.N. , Rusinov V.L., Charushin V.N., Ulomsky E.N., Petrov A.Yu., Kiselev O.I., RF Patent 2294936 dated 03/10/2007), is registered in the register of medicines of the Russian Federation as a drug for treatment influenza infection (state registration number LP-002604).

3. The invention

The essence of the invention is the sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5.1-s] -1,2,4-triazine-8-carboxylic acid, a dihydrate having antiglyculation activity, of the formula (1).

4. Information confirming the possibility of carrying out the invention

4.1. The inventive compound is the sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5.1-s] -1,2,4-triazine-8-carboxylic acid, dihydrate 1, obtained according to the following scheme: diazotization 5- aminopyrazole-4-carboxylic acid 4 to form diazopyrazole 5, azo coupling with ethyl nitroacetate, followed by cyclization of the intermediate hydrazone in the presence of sodium carbonate.

Example 1. The synthesis of the sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5.1-s] -1,2,4-triazine-8-carboxylic acid, dihydrate:

1.50 g (0.012 mol) of 5-aminopyrazole-4-carboxylic acid 4 is dissolved in 2.4 ml of hydrochloric acid and 1.2 ml of water. The solution was cooled to -10 ° C and diazotized with a solution of 0.84 g of NaNO ₂ in 1.2 ml of water. The resulting diazonium salt solution is poured into a mixture of 14 ml of a 2M solution of Na ₂ CO ₃ and 1.3 ml (0.012 mol) of ethyl nitroacetate, the pH should be 8-10. The reaction mass is kept for 1 hour at a temperature of from -10 to 0 ° C and 1 hour at room temperature. The precipitate is filtered off, dried and recrystallized first from 50% acetic acid, then from 50% ethanol. Yield 1.02 g (31%).

The inventive compound is the sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5.1-s] -1,2,4-triazine-8-carboxylic acid, dihydrate 1, has the following physicochemical characteristics: T _pl 248-250 ° C. ¹ H NMR (400 MHz, DMSO-d ₆ ): 8.19 (1H, s, CH). ¹³ C NMR (100 MHz, DMSO-d ₆ ): 103.97 (C = C), 144.20 (C-NO ₂ ), 144.31 (C = 0), 146.94 (C-H), 152.12 (C-N), 164.14 (COOH). Found,%: C - 25.62; H - 2.23; N, 24.70; C ₆ H ₂ N ₅ NaO ₅ * 2H ₂ O; Calculated,%: C - 25.45; H - 2.12; N - 24.74.

The physicochemical characteristics of compound 1 are fully consistent with the attributed structure.

4.2. Determination of the anti-glycation activity of compound 1 in vitro

Example 2. The glycation reaction was reproduced according to the method (Jedsadayanmata A. In Vitro Antiglycation Activity of Arbutin. Naresuan University Journal - 2005. - No. 13 (2). - P. 35-41). The reaction mixture contained solutions of bovine serum albumin (BSA) (1 mg / ml) and glucose (500 mM) in phosphate buffer (pH 7.4). Solutions of the studied substances in various concentrations were added to the experimental samples. Phosphate buffer was added to the control samples in the same volume. Sodium azide at a final concentration of 0.02% was added to the buffer solution to avoid bacterial growth. All experimental samples were incubated for 24 hours at 60 ° C. At the end of the incubation period, specific fluorescence of glycated bovine serum albumin (BSA) was determined on an F-7000 spectrofluorimeter (Hitachi, Japan) at an excitation wavelength of 370 nm and an emission of 440 nm. Aminoguanidine was used as the reference substance. The inhibitory effect of the compound on BSA glycation was calculated using the formula:

Δ% = 100- (V / A) × 100%,

where A is the fluorescence intensity of the control samples;

In - the fluorescence intensity of samples containing the studied substance.

Statistical processing of the results was carried out using the nonparametric Mann-Whitney test, the spreadsheet editor Microsoft Excel 2007 and the program GraphPad Prism 5.0.

As a result, it was found that the compound (1) showed a high antiglycan activity exceeding the activity of the reference substance (Table 1). In this regard, at the next stage, the concentration dependence of the antiglyating effect of compound (1) was studied. The results obtained allowed us to calculate the concentration of substances that cause a decrease in the fluorescence of glycated BSA by 50% (IC ₅₀ ). The IC ₅₀ values for compound (1) were 104.11 μM, and for aminoguanidine - 765.00 μM.

Example 3. Determination of LD ₅₀ in the study of acute toxicity in laboratory animals. The acute toxicity of compound 1 and the aminoguanidine comparison substance were studied according to the Guidelines for preclinical studies of drugs (Mironov A.N. Guidelines for preclinical studies of drugs. Part One, Grif and K, Moscow, 2012, 944 p.). Acute daily toxicity was determined on white nonlinear female mice weighing 21-24 g with intraperitoneal administration. The value of the toxicological indicator - LD _{50 was} calculated according to the method of Miller and Teitner (Belenky ML Elements of a quantitative assessment of the pharmacological effect. L .: Medgiz, 1963, 146 S.). The calculation of the effective concentration of IC ₅₀ and LD ₅₀ was performed by regression analysis. As an indicator of the breadth of therapeutic action used conditional therapeutic index (UTI), which was calculated as the ratio of LD ₅₀ to IC ₅₀ .

When studying the acute toxicity of compound (1), it was found that the LD ₅₀ for this substance was 1200.00 mg / kg (Table 2). Based on the results obtained, the UTI index was calculated, the value of which compound (1) exceeds aminoguanidine by 6.12 times.

Thus, the claimed compound — the sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5.1-s] -1,2,4-triazine-8-carboxylic acid, the dihydrate, is superior in antiglycation activity and conditional therapeutic index comparison substance (prototype).

Claims (2)

Sodium salt of 3-nitro-4-oxo-1,4-dihydropyrazolo [5,1-s] -1,2,4-triazine-8-carboxylic acid, dihydrate,