RU2798587C1 - MEDICINAL PRODUCT WITH ANTICOAGULANT (INHIBITOR OF FACTOR IIA), ANTITHROMBOTIC, ANTI-INFLAMMATORY ACTIVITIES AND CONTAINING 5,7-DI(THIOPHEN-2-IL)-4,5-DIHYDRO-[1,2,4]TRIAZOLO[1,5-a ]PYRIMIDINE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a medicinal products with anticoagulant, antithrombotic, anti-inflammatory activities. A medicinal products that has anticoagulant factor IIa inhibitor, antithrombotic and anti-inflammatory activities and contains 5,7-di(thiophen-2-yl)-4,5-dihydro-[1,2,4]triazolo[1,5-a] pyrimidine.

EFFECT: above invention allows to expand the arsenal of medicinal products to prevent thrombosis.

1 cl, 12 dwg, 14 tbl, 3 ex

Description

The invention relates to the field of biologically active compounds and concerns a drug containing 5,7-di(thiophen-2-yl)-4,5-dihydro-[1,2,4]triazolo[1,5- a ]pyrimidine, showing anticoagulant (factor IIa inhibitor), antithrombotic and anti-inflammatory activity, intended for the treatment and prevention of thrombosis and thrombosis-associated diseases, and can be used in medical institutions, research laboratories.

According to the WHO, thrombosis-associated diseases occupy one of the leading positions in the structure of morbidity and mortality in the world's population. Venous thrombosis and its most dangerous complication - pulmonary embolism - is one of the most common pathological conditions that many patients are susceptible to [Schulman S., Ageno W., Konstantinides S.V. Venous thromboembolism: Past, present and future.//Thromb Haemost..- 2017.-117(7).-P.219-1229]. The most significant risk factors include surgery and trauma, malignant neoplasms, prolonged immobilization and paralysis of the limbs, pregnancy, the postpartum period, oral contraceptives, hormone replacement therapy, the presence of varicose veins of the lower extremities, atrial fibrillation, chronic heart failure, obesity , viral and bacterial infections, the installation of intravenous catheters and old age [Friedman A.M. Introduction: Obstetric venous thromboemboembolism//Semin Perinatol.-2019.-43(4).-P.187-188; Spehlmann M.E., Frey N., Müller O. J. Herz J. Prevention and treatment of venous thromboembolism in cancer patients. - 2020.- 45(7). - P.652-658; Tritschler T., Aujesky D. Venous thromboembolism in the elderly: A narrative review//Thromb Res. -2017.-155.-P.140-147]. Also, increased intravascular coagulation occurs with sepsis, which is one of the severe complications of various microbial and viral infections, including COVID-19 (SARS-CoV-2). [Mitchell W.B. ThromboinflammationinCOVID-19 acute lung injury //Paediatric Respiratory Reviews.-2020).-35.- P. 20-24; Skeik N., Smith J.E., Patel L., Mirza A.K, Manunga J.M., Beddow D. Risk and Management of Venous Thromboembolism in Patients with COVID-19//Ann. Vasc. Surg.-2021/-73/-P.78-85]. Normally, this so-called immunocoagulation is part of innate immunity and can serve as the first line of defense against infection. It is known that coagulation can be activated by extrinsic and intrinsic pathways, resulting in the formation of fibrin. Preclinical and clinical studies confirmed the pathological role of the initiator of the external pathway - tissue factor in the occurrence of endotoxemia [Wu, C. et al. (2019) Inflammasome activation triggers blood clotting and host death through pyroptosis. Immunity 50, 1401-1411; Pawlinski, R. et al. (2004) Role of tissue factor and proteaseactivated receptors in a mouse model of endotoxemia. Blood 103, 1342-1347].

Among the means to prevent thrombosis, anticoagulants occupy a leading place. Until recently, vitamin K antagonists were the only oral anticoagulants available, and warfarin remains the most commonly prescribed oral anticoagulant worldwide. [Witt D.M., Clark N.P., Kaatz S., Schnurr T., Ansell J.E. Guidance for the practical management of warfarin therapy in the treatment of venous thromboembolism//J. Thromb Thrombolysis. Jan 2016 41(1):187-205]. However, its serious disadvantage is the slow development of the effect, the long-term effect of coagulation recovery and the small breadth of therapeutic action, which require constant monitoring of the degree of coagulation [Krylov A.I. Shulutko A.M., Prasolov N.V., Petrovskaia A.A., Khmyrova S.E. Coagulological aspects of treatment of complications of prevention therapy with warfarin// Angiol Sosud Khir. - 2016.-22(3).-P.33-41].

Currently, indirect and direct inhibitors of factor IIa and factor Xa are used as new oral anticoagulants (NOACs). Dabigatran is an oral direct thrombin/factor IIa inhibitor [Antonijevic N.M., Zivkovic I.D., Jovanovic L.M., Matic D.M., Kocica M.J. et al Dabigatran - Metabolism, Pharmacologic Properties and Drug Interactions// Curr Drug Metab. - 2017.-18(7). -P.622-635]. Three other drugs, rivaroxaban, apixaban, and edoxaban, are oral selective factor Xa inhibitors [Reiffel J.A. Novel oral anticoagulants// Am J Med.-2014.-127(4). -P.16-7; Chan N.C., Weitz J.I. Rivaroxaban for prevention and treatment of venous thromboembolis//Future Cardiol. - 2019.-15(2).-R.63-77]. Their anticoagulant effect is more predictable and stable and does not require routine laboratory monitoring. As a class, new oral anticoagulants show comparable efficacy and a significantly lower risk of bleeding compared to warfarin [Hulle T., Kooiman J., Exter P.L., Dekkers O.M., F.A. Klok et al., Effectiveness and safety of novel oral anticoagulants as compared with vitamin K antagonists in the treatment of acute symptomatic venous thromboembolism: a systematic review and meta-analysis // J Thromb Harmost .-2014.-12:.-P. 320-8]. However, this group of drugs has its drawbacks, as it enters into undesirable pharmacodynamic interactions with foods, herbal supplements and drugs [Frigerio B., Spadarella G., Ravani A., Sansaro D., Amato M. et al., Old and new oral anticoagulants: Food, herbal medicines and drug interactions// Blood Reviews.-2017.-32(4). -P.193-203]. In addition, most of the drugs currently used to prevent thrombosis are produced abroad.

As an invention, a drug is proposed that contains a biologically active component that has anticoagulant (factor IIa inhibitor), antithrombotic, anti-inflammatory activities, and is 5,7-di(thiophen-2-yl)-4,5-dihydro-[1, 2,4]triazolo[1,5- a ]pyrimidine of formula (1). In addition to compound (1), the medicinal product may contain pharmaceutically acceptable components: excipients, diluents and/or other excipients.

To assess the anticoagulant, antithrombotic, anti-inflammatory activities in vitro and in vivo without and in conditions of sepsis of 5,7-dimethyl-4,5-dihydro-[1,2,4]triazolo[1,5- a ]pyrimidine (1) and A reference drug, dabigatran ethixilate (Boehringer Ingelheim Pharma GmbH & Co., Germany), a direct anticoagulant for oral administration and a thrombin inhibitor (factor IIa) was used to prepare a drug based on it.

Compound (1) was obtained by reacting 7-(thiophen-2-yl)-[1,2,4]triazolo[1,5- a ]pyrimidine (2) with thiophen-2-ylmagnesium bromide (3).

Compound (1) is a pale beige powder, soluble in ethanol, dimethyl sulfoxide, sparingly soluble in ethanol and ethyl acetate, insoluble in hexane.

Elemental analysis and ¹ H NMR data of Compound (1) fully correspond to the attributed structure (see Example 1).

The drug can be used either orally or by parenteral injection in solution. It can be used alone, for example in the form of microcapsules, or with suitable auxiliaries and/or excipients. Suitable solid or liquid dosage forms include, for example, granules, powders, coated tablets, microcapsules, suppositories, syrups, elixirs, suspensions, emulsions, drops or injectables, as well as targeted delivery preparations of the active substance, in the production of which excipients such as fillers, disintegrators, binders, coating agents, disintegrants, lubricants, flavorings or sweeteners. Suitable excipients are, for example, titanium dioxide, lactose, mannitol and other sugars, talc, milk albumin, gelatin, flour, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents such as sterile water, and monohydric and polyhydric alcohols. such as glycerin.

Example 1 . Synthesis

In a flask equipped with a magnetic stirrer, a thermometer, a reflux condenser, an inert, dry atmosphere is created and 36.5 mg of magnesium (1.5 mmol), freshly distilled tetrahydrofuran (THF) and 145.0 μl of 2-bromothiophene (1.5 mmol) are placed . After magnesium is dissolved, the reaction mixture is cooled to 0°C and 226.0 mg of 7-(thiophen-2-yl)-[1,2,4]triazolo[1,5- a ]pyrimidine (1 mmol) is added, heated to 50 °C for 2 hours. Then, while maintaining an inert atmosphere, the reaction mixture is neutralized with a solution of 107.0 mg (2 mmol) ammonium chloride in 2 ml of water and the THF is removed on a rotary evaporator. The resulting precipitate of compound 1 is filtered and recrystallized from ethanol. Yield 223.6 mg (78.1%) as a crystalline light beige precipitate with a melting point of 206°C, insoluble in water, soluble in hot ethanol and dimethyl sulfoxide (DMSO).

5,7-di(thiophen-2-yl)-4,5-dihydro-[1,2,4]triazolo[1,5- a ]pyrimidine - Compound (1) has the following physicochemical characteristics: ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 8.44 (s, 1H, N H ), 7.75 (d, J = 3.7 Hz, 1H, C=C H -CH), 7.67 (d , J = 5.1 Hz, 1H, CH=C H -S), 7.63 (s, 1H, N=C H -N), 7.47 (d, J = 5.0 Hz, 1H, CH =C H -S), 7.13 (t, J = 4.4 Hz, 1H, C H =CH-S), 7.06 (d, J = 3.5 Hz, 1H, C=C H - CH), 7.01 (t, J =4.4 Hz, 1H, CH =CH-S), 5.83 (d, J =5.3 Hz, 1H, NH-CH- CH ), 5 .73 (d, J = 5.5 Hz, 1H, NH-C H -CH). ¹³ C NMR (126 MHz, DMSO- d ₆ ) δ 152.8; 149.5; 147.5; 132.3; 129.0; 128.3; 128.2; 127.3; 127.1; 125.8; 124.3; 105.0; 49.5. Calculated for C ₁₃ H ₁₀ N ₄ S ₂ (286.37): C: 54.52; H: 3.52; N: 19.56 Found: C: 54.44; H: 3.48; N: 19.60.

The physicochemical characteristics of Compound (1) fully correspond to the attributed structure.

Example 2 . Study of cytotoxicity

Studying spent on HepG2 cells (human hepatocellular carcinoma) (CLS Cell Lines Service), which were cultured in a culture flask in a complete growth medium F-12 in CO₂- an incubator at a temperature of 37 ° C in an atmosphere of 5% CO₂. To wash the monolayer, Hanks' solution was used; to remove the monolayer, a 0.05% trypsin-EDTA solution was used (5 min). The cell suspension was seeded into a 24-well plate followed by incubation for 24 hours for cell adhesion to the bottom of the plate. After the incubation time and change of the growth medium, the Compound (1) and the reference drug were introduced into the wells (n=3-5) at final concentrations of 0.01-100 μM (multiplicity ×10). Control wells were filled with only an equivalent volume of complete growth medium. The compounds were incubated for 48 h, after which the compounds were removed together with the growth medium, and the MTT reagent was added (incubation 2 h). The formed formazan crystals were solubilized using DMSO in a plate thermoshaker (5 min, 24°C, 1200 RPM).

Using a tablet reader, according to the optical density, cell viability was determined, which correlated with the activity of mitochondrial dehydrogenases. Absorbance measurements were made at 555 nm.

The cytotoxicity of Compound (1) and the reference drug dabigatran etexilate was evaluated at concentrations of 10 nM, 0.1 μM, 1 μM, 10 μM and 100 μM.

In the course of the study, absorbance values at 555 nm (reference λ = 650 nm) were obtained, reflecting the quantitative assessment of the converted MTT reagent into formazan by mitochondrial dehydrogenases. According to the results of the study, relative to the control (only cells with complete growth medium F-12), the percentage of metabolic activity of the cells was calculated, reflecting the number of viable cells (survival) in % relative to the control values (Fig. 1).

As a result of the study, it was found that for Compound (1) in the range of studied concentrations of 0.01-100 μM, there is no statistically significant effect on the survival of HepG2 cells after 48 hours of incubation. The data obtained allow us to conclude that this substance has no cytotoxic properties in the concentration range of 0.01-100 μM, the level of CC ₅₀ >>100 μM.

For the reference drug dabigatran etexilate, a dose-dependent effect on the survival of HepG2 cells was found in the concentration range of 1-100 μM. At concentrations of 0.01 and 0.1 μM, no statistically significant cytotoxic effect was detected for dabigatran etexilate.

The data obtained for the suspension of the drug dabigatran etexilate made it possible to calculate the concentration that causes a 50% cytotoxic effect on intact cells (CC ₅₀ ), which was 5.4±1.2 μM (R ² =0.92).

The data obtained correspond to the absence of changes in the morphological pattern of cells after 48 hours of incubation at the maximum concentration of 100 μM studied with Compound (1) samples, but not with dabigatran drug granulate (Fig. 2). The integrity of the cellular structure during incubation with the latter is not traced.

Cytotoxicity data for Compound (1) and dabigatran etexilate made it possible to calculate their conditional therapeutic index. For the reference drug, it was 3.8, and for Compound (1) - >>80.0 (Table 1).

Table 1 - Conditional Therapeutic Index (UTI) score of Compound (1) and dabigatran etexilate. N
p/n Samples tested EC ₅₀ , μM Level of SS ₅₀ , μM UTI 1. Dabigatran etexilate 1.4 5.4 3.8 2. Connection (1) 1.25 >>100 >>80.0

Thus, according to the value of the conditional therapeutic index, Compound (1) exceeds the reference drug dabigatran etexilate by more than 21 times.

Example 3 .

1. Study of the effect of Compound (1) on the coagulogram parameter - thrombin time in vitro.

The study was performed on rabbits of the Chinchilla breed. The analysis was carried out chronometrically, on a SOLAR hemocoagulometer (Belarus) using reagent kits manufactured by Technology Standard, Russia. The material for the study was platelet-poor animal plasma, which was obtained by centrifugation of blood taken from the ear vein of a rabbit at 3000 rpm for 15 minutes. To stabilize the blood, a 3.8% aqueous solution of sodium citrate (pH 6.0) was used in a ratio of 9:1. To determine the thrombin time, 100 μl of the investigated, platelet-poor plasma and a magnetic anchor were introduced into the cuvette. After incubation of the sample (120 seconds at 37°C), 100 µl of thrombin working solution standardized for activity was added to it. To study the effect on thrombin activity, Compound (1) in a volume of 10 μl was introduced into a coagulometer cuvette containing 100 μl of thrombin working solution and incubated for 5 minutes at room temperature. Compound I and dabigatran etexilate were studied in the concentration range of 10-0.1 μM with the calculation of EC ₅₀ and conditional therapeutic index (UTI). Distilled water was used as a solvent for Compound (1) and a reference drug, and saline was used as a solvent for the reagents.

The thrombin time in the control was 11.65 seconds. The comparison drug dabigatran etexilate at a concentration of 10 μm significantly lengthened this indicator to about 46.8 seconds, which corresponds to a significant increase in its relative to the control by 302.0% (table 1). With a further decrease in the concentration to 5 and 1 μM, the thrombin time was also statistically significantly prolonged by 176.1 and 42.3%, respectively. At a concentration of 0.1 μM dabigitran etexilate had no effect on this parameter. EC ₅₀ antithrombin activity of the comparison drug was 1.4 μm (table 2).

Compound (1) at a concentration of 10 μM significantly lengthened the thrombin time to 66 seconds, which was 467.1% more than the control. With a decrease in the concentration of the substance to 5 and 1 μM, the thrombin time also decreased to 34.3 and 16.6 seconds, but at the same time, the relative control values increased statistically significantly by 194.1 and 46.5%, respectively. At a concentration of 0.1 μm Compound (1) did not affect this parameter. EC ₅₀ antithrombin activity was equal to 1.25 μm (table 2).

Table 2 - EC ₅₀ Compound (1) and reference drug dabigatran etexilate on the effect on rabbit blood thrombin time in vitro (M±m) (n=6). No. p / p Sample being tested Δ% prolongation of thrombin time
regarding control EC ₅₀ , μM 10 µM 5 µM 1 µM 0.1 µM 1. Dabigatran
etexilate 302.0±0.3 ^* 176.1± 2.1 ^* 42.3±0.6 ^* 1.4±0.2 1.4 2. Connection (1) 467.1 ±1.4 ^*# 194.1 ± 3.2 ^*# 46.5±1.5 ^* 4.3±1.1 1.25 Note:
^* -data are significant relative to control, ANOVA test (p<0.05);
^# - (p≤0.05) changes are statistically significant in relation to the reference drug dabigatran etexilate, ANOVA criterion;
n is the number of tested animals.

2. Study of the effect of Compound (1) on coagulogram parameters in vivo.

Studies were performed on 25 outbred male rats. The reference drug dabigatran etexilate was studied at a dose of 12 mg/kg (dose equivalent to the human dose, taking into account the interspecies conversion factor). Compound (1) was tested at a dose equimolar to the reference drug, which was 5.5 mg/kg. To determine the time of onset of the greatest antithrombin activity in Compound (1), it was administered once intragastrically for 1; 2 and 4 hours before the start of the experiment. Dabigatran etexilate was administered 2 hours (time of peak blood concentration) prior to the study. Distilled water was used as a solvent. Blood was taken from the abdominal aorta of rats, which were previously anesthetized (chloral hydrate 400 mg/kg intraperitoneally). To stabilize the blood, a 3.8% aqueous solution of sodium citrate (pH 6.0) was used in a ratio of 9:1. All studies were performed on platelet-poor rat plasma, which was obtained as described above. Coagulogram parameters were measured on a SOLAR coagulometer according to the methods described above.

As a result of the studies in the control, the APTT was 38.3 seconds (Table 3). At the same time, dabigatran etexilate at a dose of 12 mg/kg significantly lengthened this indicator by 3.6 times. Compound (1) at a dose of 5.5 mg/kg after 1 and 2 hours after administration significantly increased this indicator in relation to the control by 1.3 and 1.5 times, respectively, and after 4 hours no effect on this parameter was detected. (Table 3).

Thrombin time in the blood of control rats was 57.7 minutes. The reference drug dabigatran etexilate increased this indicator by 10.5 times. Under the influence of Compound I, 1 hour after its administration, the thrombin time was extended by 4.4 times, 2 hours after administration by 5.6 times, and after 4 hours this indicator corresponded to the control values. The prothrombin time index did not change either under the action of Compound (1) or under the influence of dabigatran etexilate (Table 3).

Table 3 - Effect of Compound (1) and dabigatran etexilate in equimolar doses on coagulogram parameters in rats after a single intragastric injection (M±m) (n=5) No. p / p Tested
sample Dose, mg/kg Time
fence
blood, h Coagulogram parameters APTT, s Thrombin time, s prothrombin time, s 1. Control 38.3±1.7 57.7±3.8 28.1±1.4 2. Dabigatran etexilate 12.0 ¹ 2 137.53±2.79 ^* 603.9±18.2 ^* 31.20 ± 1.17 3. Connection (1) 5.5* 1 48.8±1.4 ^* 255.5±10.8 ^* 23.3±1.8 2 59.2±1.6* 325.3±7.1 ^* 24.5±1.2 4 40.4±1.9 76.2 ± 7.4 22.5±0.4 Note:
¹ - dose obtained by recalculation using the interspecies coefficient;
² - dose equimolar to the dose of dabigatran etexilate 12.0 mg/kg;
^* - data are significant relative to control, ANOVA criterion (p<0.05).

3. Study of the effect of Compound (1) on the ability to bind factor IIa on the chromogenic substrate S-2238 in vivo.

Studies were performed on 35 outbred male rats. Compound (1) was administered once intragastrically in doses of 2.5; 5.5 and 11 mg/kg. The reference drug dabigatran etexilate was studied at doses of 3.0; 6.0 and 12.0 mg/kg. Blood was taken from the abdominal aorta of rats that were previously anesthetized (chloral hydrate 400 mg/kg ip) 2 hours after the administration of the test samples. The resulting blood was collected in a blood collection tube and centrifuged at room temperature for 15 minutes at 3000 rpm (1200g). Plasma was collected and re-centrifuged under the same conditions. Only the upper third of the plasma was used for analysis. The analysis was performed according to the instructions for the "Renaparin - plasma test" kit. The degree of binding of the studied substances with factor IIa was assessed.

The ability to bind factor IIa (thrombin) on the chromogenic substrate of Compound (1) and the reference drug dabigatran etexilate is shown in Table 4.

Compound (1) at a dose of 2.5 mg/kg did not bind factor IIa, since the level of luminescence on the chromogenic substrate corresponded to the level of luminescence of the plasma-calibrator S1 - 0.36. With an increase in the dose of the test compound to 5.5 mg/kg, the luminescence level decreased to 0.31, while thrombin binding was 44.7%. At a dose of 11.0 mg/kg, the luminescence level was 0.25, which corresponded to the interaction of Compound (1) with factor IIa by 67%. The ED ₅₀ of this compound was 9.9 mg/kg (Table 4).

When studying the effect of dabigatran etexilate on thrombin binding at a dose of 3.0 mg/kg, the luminescence level was 0.46, indicating no effect. When the dose of the reference drug was increased to 6.0 mg/kg, the luminescence level corresponded to the luminescence of the S2 calibrator plasma, which corresponded to 25% thrombin binding. At a dose of 12 mg/kg, binding to thrombin occurred by 64.5%. The ED ₅₀ of dabigatran etexilate was reported to be 8.1 mg/kg (Table 4). Thus, Compound (1) has the ability to bind factor IIa on the chromogenic substrate S2238, which confirms its antithrombin activity.

Table 4 - ED ₅₀ Compound (1) and comparator dabigatran etexilate by ability to bind factor IIa on chromogenic substrate S-2238 in vivo test (n=5) No.
p/n Sample being tested Dose mg/kg Glow level % binding to factor IIa ED ₅₀ , mg/kg 1. buffer solution 0.06±0.0 2. S1 0.36±0.01 0 3. S2 0.33±0.01 25 4. S3 0.30±0.01 50 5. S4 0.28±0.01 75 6. S5 0.23±0.01 100 7. Platelet-poor plasma
+
Connection (1) 2.5 0.36±0.01 0 9.9 5.5 0.31±0.01 44.7 11.0 0.25±0.03 67 8. Platelet-poor plasma
+
dabigatran etexilate 3.0 0.46±0.03 0 8.1 6.0 0.33±0.02 25 12.0 0.28±0.01 64.5 Note: S1 - S5 plasma calibrator; n is the number of animals.

4. Study of the effect of Compound (1) on the survival of mice under conditions of thrombin-induced pulmonary thrombosis.

The study was performed on 30 outbred male mice. The introduction of solutions of Compound (1) and the reference drug was carried out once intragastrically 2 hours before modeling thrombosis at doses of 13.0 and 27.0 mg/kg, respectively. The control group of animals was injected with a solvent (distilled water). Modeling of acute thrombosis in mice was carried out according to the method [Zyablitsky, R.M. To the pathogenesis of acute intravascular coagulation / R.M. Zyablitsky, D.P. Pavlovsky, O.Yu. Tokarev // Pathological physiology and experimental therapy. -1983. - No. 2 S. 33-35]. Mice were fixed in a specialized house with access to the tail, and thrombin at a dose of 40 units, dissolved in saline, was injected into the tail vein with an insulin syringe. Animal behavior was assessed (mobility, convulsions, etc.). Lungs were taken from animals for histological evaluation. The group of surviving animals was observed for 7 days for remote death.

The morphological study was carried out as follows: the extracted material (lung tissue samples) was placed in a 10% solution of neutral buffered formalin (pH 7.4). Less than 2 minutes passed from the moment of organ removal to the immersion of the tissue sample in the fixative. The residence time in the fixative was 1-2 days at 4°C. After fixation, the material was cut out. Subsequently, the material was carried out according to the generally accepted histological method through a battery of alcohols of increasing concentration, chloroform, and embedded in paraffin. Then, sections 4 μm thick were made on a rotary microtome and mounted on glass slides.

Subsequently, they were stained with hematoxylin and eosin according to generally accepted histological methods (deparaffinization in xylene, deparaffinization in alcohols of decreasing concentration to water, staining with Harris's hematoxylin, differentiation with hydrochloric acid alcohol, staining with eosin, dilution of alcohols of increasing concentration to absolute ethanol, clarification in acid, conclusion in mounting Wednesday) [Sarkisov D.S., Perov Yu.L. Microscopic technique: a guide for physicians and laboratory assistants. - M.: Medicine 1996. - 542 p.], and also used the Carstairs trichrome staining technique to detect fibrin [Eitzman DT, Bodary PF, Shen Y, Khairallah CG, Wild SR, Abe A, Shaffer-Hartman J, Shayman JA Fabry disease in mice is associated with age-dependent susceptibility to vascular thrombosis. / J Am Soc Nephrol. // Feb 2003; 14(2):298-302.]. To analyze the morphological parameters, microphotography was performed with an Axiocam 105 color digital camera (CarlZeiss, Germany) based on an Axiocamplus microscope (CarlZeiss, Germany) using x10 objectives; x40. The resulting photographs were processed using the ZENpro 2012 program (ZEISS, Germany).

Data from studies of the antithrombotic activity of Compound (1) and dabigatran etexilate are presented in Table 5.

As a result of the experiments in the group of control animals, out of 10, 7 died. Thus, the survival rate of mice was 30%. At the same time, characteristic signs of a violation of the respiratory function of the lungs were noted: an increase in the frequency and shallow nature of breathing, tetanic convulsions, paresis of the hind limbs, the animals took a characteristic posture in which the hind legs were straightened and laid back. Within 10-15 minutes after intravenous administration of the thrombotic agent thrombin at a dose of 40 units. animals died.

Dabigatran etexilate with a single intragastric administration prevented the death of 60% of animals compared with the control group. Compound (1) was comparable in antithrombotic activity to dabigatran etexilate. Animals that died on the background of the administration of the study drugs, when visually observed after the intake of thrombotic agents in their body, were more active than the dying animals of the control group. They observed a decrease in the severity of motor disorders, while the external manifestations of generalized thrombosis developed gradually (within 15-30 minutes) in contrast to the control group (10-15 minutes). The group of surviving animals was observed for 7 days for remote death. Thus, in terms of antithrombotic activity in the model of thrombin-induced thrombosis, Compound (1) showed the same effect as the reference drug dabigatran etexilate.

Table 5 - The effect of Compound (1) and dabigatran etexilate with a single intragastric administration on the survival of white outbred mice in the model of thrombin-induced thrombosis (40 units) (M±m) (n=10) N
p/n Samples tested Dose,
mg/kg ¹ Number
tested
animals Death toll
animals Survivors
animals, % 1. Control
(physical solution) 10 7 30.0 2. Dabigatran etexilate 27.0 10 4 60.0* 3. Connection (1) 13.0 ² 10 4 60.0* * - (p≤0.01) data are statistically significant in relation to the control group of animals, chi-square with Yates correction;
¹ - dose obtained by recalculation using the interspecies coefficient;
² - dose equimolar to the dose of dabigatran etexilate 12.0 mg/kg.
Note: n is the number of animals.

4.1. Morphological examination of the lungs

Microscopic examination of the lung tissue 24 hours after the administration of the thrombotic agent showed that the experimental mice developed focal hemorrhages. In a significant part of the vessels of the microvasculature, erythrocyte sludges were found, dilation of the capillaries of the interalveolar septa was noted, as well as hemorrhagic exudate in the walls and lumens of the alveoli. Small and medium-sized alveoli predominated, dystelectasis was present (Fig. 3).

When examining paraffin sections of lung tissue stained by Carstair, red thrombi were detected, consisting mainly of adherent erythrocytes and fibrin strands, both in the lumens of large and small vessels (Fig. 4A). In addition, the presence of fibrin filaments was also noted in the lumen of the alveoli as part of the exudate (Fig. 4B).

Microscopic examination of the lung tissue of mice treated with dabigatran etexilate revealed focal hemorrhages, as well as erythrostasis in the capillaries of the interalveolar septa. At the same time, the thickness of most of the interalveolar septa corresponded to the thickness of normal alveoli. In addition, slugged erythrocytes adhered to the vascular wall were recorded (Fig. 7).

Thus, according to the results of a morphological study, reference drugs dabigatran etexilate and Compound (1) on the model of thrombin-induced thrombosis contributed to the correction of model disturbances in the rheological properties of blood, inhibiting the formation of intravascular fibrin thrombi.

5. Study of the antithrombotic activity of Compound (1) in a model of inferior vena cava thrombosis.

Studies were performed on 45 male rats according to the method [Peter K., Henke et al. Fibrotic injury after inperimental deep vein thrombosisis determined by the mechanism of thrombogenesis//Thromb.Haemost.- 2007.-98.-P.1045-1055]. In rats anesthetized with chloral hydrate (400 mg/kg), the inferior vena cava was isolated and ligated 1 cm above the bifurcation site. The abdominal aorta should be unaffected. The abdominal cavity was sutured, and after 24 hours the rats were re-anesthetized and laparotomy was performed, followed by the removal of blood clots from the inferior vena cava. The test samples were administered once intragastrically at the above doses 2 hours before the ligation of the inferior vena cava. The control group of animals was injected with the solvent in an equivalent volume. The group of sham-operated animals was also injected with a solvent, laparotomy and suturing of the abdominal cavity were performed in order to exclude the effect of surgical manipulation on the formation of a thrombus. Antithrombotic activity was assessed by weighing the removed blood clots.

A study of the antithrombotic activity of Compound (1) in a venous thrombosis model showed that it was reliably able to prevent venous thrombosis of the inferior vena cava.

A group of sham-operated animals was needed in order to show that surgical manipulations with animals do not cause the formation of a thrombus in the inferior vena cava. This was confirmed by experiments (Table 6). In the group of control animals, after ligation of the inferior vena cava, the formation of thrombi was observed in a day, the average weight of which was 82.3 mg. In the experimental group of animals, which were administered dabigatran etexilate at a dose of 12.0 mg/kg two hours before ligation of the inferior vena cava, the average thrombus weight was significantly lower compared to the values obtained in the control by 98.7%. The average weight of blood clots taken from the veins of animals that received Compound (1) was also statistically significantly less than the control values by 82.2% (Table 6).

To determine the dose-dependent activity of the reference drug dabigatran etexilate and Compound (1), in order to calculate the ED ₅₀ , they were additionally studied at doses of 3.0; 6.0 mg/kg and 1.25; 2.5; 11.0 mg/kg, respectively. The masses of vein thrombi in the group of animals treated with the reference drug at doses of 3.0 and 6.0 mg/kg were significantly less than the control values by 86.6% and 30.5%, respectively (Table 6). The ED ₅₀ of antithrombotic activity of dabigatran etexilate was 3.8 mg/kg.

In the group of animals treated with Compound (1) at a dose of 11 mg/kg, no thrombi were detected. After a halving of the equimolar dose of dabigatran etexilate to 2.5 mg/kg, venous thrombus weights were significantly reduced by 74.2%. With a further decrease in the dose of the test substance by a further two times to 1.25 mg/kg, the average weight of blood clots removed from the veins of animals decreased statistically significantly relative to the group of control animals by 39.3%. The ED ₅₀ of the antithrombotic activity of Compound (1) was 1.5 mg/kg.

Table 6 - Antithrombotic activity of triazolopyrimidine derivative and dabigatran etexilate in the model of inferior vena cava thrombosis with a single intragastric injection (M±m) (n=5) No.
p/n Tested
sample Dose, mg/kg Average weight
blood clots, mg ED ₅₀ , mg/kg 1 Sham operated rats - 0 - 2 Control group (solvent) - 82.3 ± 2.4 3 Connection (1) 1.25 50.0±2.6* 1.5 4 2.5 21.2 ± 7.4* 5 5.5 ² 14.7 ± 3.1* 6 11.0 0.0* 7 Dabigatran etexilate 3.0 57.2 ± 1.8* 3.8 8 6.0 11.0 ± 3.3* 9 12.0 ¹ 1.1 ± 0.9* * - data are significant relative to control, one-way ANOVA test with Bonferroni correction (p<0.0001);
¹ - dose equivalent to doses used for humans in clinical practice, taking into account the interspecies conversion factor;
² - dose equimolar to the dose of dabigatran etexilate.
Note: n is the number of animals in the group.

Therefore, in terms of the ability to reduce the weight of a thrombus obtained by ligation of the inferior vena cava and, in particular, in terms of ED ₅₀ value, Compound (1), which exhibits anticoagulant activity in vitro and in vivo , exceeds the well-known direct-acting anticoagulant dabigatran etexilate by 2.5 times.

6. Study of the antithrombotic activity of Compound (1) in a model of rat femoral vein thrombosis induced by application of ferric chloride.

The studies were performed on 35 male rats. The model of venous thrombosis of the femoral vein in rats caused by surface application of a 50% iron (III) chloride solution was reproduced according to the method [Röttger C., Madlener K., Heil M., Gerriets T., Walberer M., Wessels T. et al., Is heparin treatment the optimal management for cerebral venous thrombosis? Effect of abciximab, recombinant tissue plasminogen activator, and enoxaparin in experimentally induced superior sagittal sinus thrombosis// Stroke. - 2005.-36(4). - P.841], 2 hours after a single intragastric administration of the test compounds at the above doses. The group of control animals was injected with the solvent in an equivalent volume. Rats were anesthetized with chloral hydrate (400 mg/kg ip) and the femoral vein was isolated, on which a cotton pad moistened with 50% iron (III) chloride solution was applied. For the study, an ultrasound dopplerograph "Minimax-Doppler-K" (St. Petersburg) was used. Blood flow was recorded until complete occlusion of the vessel. The antithrombotic activity of the tested samples was judged by the time of thrombus formation (the time of stopping blood flow). (Δ%) prolongation of the time of thrombus formation was calculated by the formula:

Δ% prolongation = (B/A) × 100%-100;

where A is the time of thrombus formation (control sample);

B is the time of formation of a thrombus during the introduction of prototypes.

When the femoral vein of rats is treated with a solution of iron (III) chloride, a thrombus is formed, an indicator of which is the time of complete occlusion of the vessel (stopping blood flow).

The mean time of occlusion of the femoral vein in the control group of animals that were injected with the solvent was 9.3 minutes. The antithrombotic effect of the test samples administered 2 hours before the start of thrombosis modeling in equimolar doses is presented in Table 7.

At the same time, dabigatran etexilate at a dose of 3 mg/kg increased the time of onset of occlusion of the femoral vein to 13.3 minutes, which is 42.5% more than the values obtained in the control group of animals. With an increase in the dose of the reference drug to 6 and 12 mg/kg, the occlusion time was 18.3 and 29.7 minutes, respectively. Thus, at a dose of 6 mg/kg of dabigatran etexilate, the time of thrombus formation was prolonged by 96.2% relative to the control group of animals, and when the dose was increased to 12 mg/kg, by 219%. The ED ₅₀ of antithrombotic activity of dabigatran etexilate was 3.5 mg/kg.

Compound (1) at a dose of 1.25 mg/kg extended the time of thrombus formation to 12.8 minutes, which is 37.1% more than the control values. At a dose of 2.5 mg/kg, test Compound (1) increased the time to complete occlusion of the femoral vein to 18.8 minutes, and at a dose of 5.5 mg/kg, to 24.1 minutes. At the same time, the time of thrombus formation Compound (1) at doses of 2.5 and 5.5 mg/kg was prolonged by 101.6 and 158.1%, respectively. The ED ₅₀ of the antithrombotic activity of the test compound was 1.4 mg/kg.

Thus, in the model of femoral vein thrombosis induced by 50% ferric chloride, Compound (1) in terms of ED ₅₀ antithrombotic activity exceeded the reference drug by 2.5 times.

Table 7 - Antithrombotic activity of Compound (1) and dabigatran etexilate after a single intragastric administration in a model of rat femoral vein thrombosis induced by 50% iron (III) chloride solution (M±m) (n=5). No. p / p Tested
sample Dose, mg/kg Occlusion time
femoral vein, min Δ% prolongation
occlusion time
femoral vein ED ₅₀ , mg/kg 1 Control 9.3±0.2 2 Connection (1) 1.25 12.8±0.8 37.1 ± 8.1 1.4 2.5 18.8 ± 0.3* 101.6 ± 2.7* 5.5 ² 24.1 ± 1.0* 158.1 ± 10.8* 3 Dabigatran etexilate 3.0 13.3±0.3 42.5 ± 2.7 3.5 6.0 18.3 ± 1.3* 96.2 ± 13.4* 12.0 ¹ 29.7 ± 1.5* 219.0 ± 15.9* * - data are significant relative to control, one-way ANOVA criterion (p <0.05);
¹ - dose equivalent to doses used for humans in clinical practice, taking into account the interspecies conversion factor;
² - dose equimolar to the dose of dabigatran etexilate.
Note: n is the number of animals in the group.

7. Study of the effect of Compound (1) on coagulogram parameters under conditions of hypercytokinemia in vitro and in vivo .

The study was performed on 6 rabbits of the Chinchilla breed. The analysis was carried out chronometrically, on a SOLAR hemocoagulometer (Belarus) using reagent kits manufactured by Technology Standard, Russia. Hypercytokinemia was reproduced by incubation of lipopolysaccharide (LPS, E. coli O111:B4; Sigma, USA) with whole blood until experimental samples of platelet-poor plasma were obtained at a final concentration of 10 μM, and then measurements were made according to the methods described above. Compound I and dabigatran etexilate were studied in the concentration range of 10-0.1 μM with the calculation of EC ₅₀ and conditional therapeutic index (UTI).

Experiencesin vivoperformed on 20 male rats. Hypercytokinemia was created by intravenous administration of LPS at a dose of 2 mg/kg [QunFu H., Yang T., Xiao W., Fan L., Wu Y., Terrando N., Long Wang T. Prolonged Neuroinflammation after Lipopolysaccharide Exposure in Aged Rats. PLOS One. 2014; 9(8):106331] into the tail vein of a rat 2 hours after the administration of Compound (1) and the reference drug at doses of 5.5 and 12.0 mg/kg, respectively. 4 hours after the administration of LPS, blood was taken from the abdominal aorta and the anticoagulant activity was assessed according to the methods described above.

The thrombin time in control samples of blood treated with LPS was 9.3 seconds, which is significantly lower than these data in the group of intact blood (see section 2.1). The comparison drug dabigatran etexilate at a concentration of 10 μM significantly lengthened this indicator to 27.2 seconds, which corresponds to a significant increase in its relative to the control by 292.0% (Table 8). With a further decrease in the concentration to 5 and 1 μM, the thrombin time was also statistically significantly prolonged by 208.6 and 51.7%, respectively. At a concentration of 0.1 μM dabigitran etexilate had no effect on this parameter. EC ₅₀ antithrombin activity of the reference drug under conditions of hypercytokinemia was 0.76 μM (Table 8).

Compound (1) at a concentration of 10 μM significantly extended the thrombin time to 33 seconds, which was 354.1% more than the control. With a decrease in the concentration of the substance to 5 and 1 μM, the thrombin time increased statistically significantly relative to the control values by 194.1 and 46.5%, respectively. At a concentration of 0.1 μm Compound (1) did not affect this parameter. EC ₅₀ antithrombin activity under conditions of hypercytokinemia was equal to 0.78 μM (Table 8).

Thus, under conditions of hypercytokinemia, the EC ₅₀ antithrombin activity of Compound (1) and dabigatran etexilate were comparable. It was also shown that under conditions of a systemic inflammatory reaction, this activity of Compound (1) and dabigatran etexilate exceeded that on intact blood by 1.6 and 1.8 times, respectively. Conditional therapeutic index Compound (1) is 18 times superior to dabigatran etexilate (Table 9).

Table 8 - EC ₅₀ Compound (1) and reference drug dabigatran etexilate on the effect on blood thrombin time in rabbits under conditions of hypercytokinemia (M±m) (n=6). No. p / p Sample being tested Δ% prolongation of thrombin time
regarding control EC ₅₀ , μM 10 µM 5 µM 1 µM 0.1 µM 1. Dabigatran
etexilate 292.0±0.6 ^* 208.6±0.7 51.7±0.5 ^* 16.3±0.2 ^* 0.76 2. Connection (1) 354.1 ± 6.5 ^*# 234.1 ± 4.2 ^*# 56.3±1.1 ^* 8.4 ± 1.6 0.78 Note:
^* - data are significant relative to control, ANOVA criterion (p<0.05);
^# - (p≤0.05) changes are statistically significant in relation to the reference drug dabigatran etexilate, ANOVA test;
n is the number of tested animals.

Table 9 - Conditional Therapeutic Index (UTI) score of NAR-0278b compound and dabigatran etexilate under conditions of hypercytokinemia N
p/n Samples tested EC ₅₀ , μM Level of SS ₅₀ , μM UTI 1. Dabigatran etexilate 0.76 5.4 7.1 2. Connection (1) 0.78 >>100 >>128.2

Data on anticoagulant activity in vivo under conditions of hypercytokinemia Compound (1) are presented in table 10.

Coagulogram parameters significantly changed under conditions of systemic inflammatory response. APTT decreased by 2.1 times compared to the control of intact animals, and the thrombin time also decreased by 1.3 times, which indicates the activation of coagulation hemostasis in rats.

The reference drug significantly reduced the APTT relative to the control blood of rats injected with LPS by 2.2 times. At the same time, thrombin time was statistically significantly extended by 12.8 times. Compound (1) also significantly prolonged the APTT by 1.6 times. At the same time, the thrombin time in the blood of rats increased by 14.5 times. Compound (1) and dabigatran etexilate had no effect on prothrombin time. Thus, under conditions of a systemic inflammatory reaction, Compound (1) exhibits a more pronounced antithrombin effect than in its absence, which indicates the effect of this substance on inflammatory components of the blood.

Table 10 - Effect of Compound (1) and reference drug dabigatran etexilate at equimolar doses on coagulogram parameters in rats after 2 hours of single intragastric administration under conditions of hypercytokinemia (M±m) (n=5) No. p / p Sample being tested Dose, mg/kg Coagulogram parameters APTT, s Thrombin time, s prothrombin time, s 1. Control (physical solution) 38.3±1.7 57.7 ± 3.8 28.1 ± 1.4 2. Control (administration of LPS) 18.4±0.8 ^# 44.1±1.7 ^# 21.2 ± 1.3 3. Dabigatran etexilate 12.0 ¹ 41.0 ± 2.0* 566.1 ± 45.5* 31.1 ± 1.0 4. Connection (1) 5.5 ² 28.7 ± 1.0* 640.3 ± 7.4* 26.4 ± 1.4 Note:
¹ - dose obtained by recalculation using the interspecies coefficient;
² - dose equimolar to the dose of dabigatran etexilate 12.0 mg/kg;
^* - data are significant relative to the control with LPS, ANOVA criterion (p<0.05);
^# - data, reliable relative to control (phys. solution).

8. Study of the effect of Compound (1) on the formation of extracellular traps of neutrophils.

Determination of extracellular neutrophil traps induced by LPS-stimulated platelets was performed according to the method [Clark SR et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood // Nature medicine. - 2007. - T. 13. - No. 4. - S. 463-469]. Blood stabilized with 3.8% sodium citrate (1:10) was used. Blood was centrifuged at 1000 g for 10 min, platelet-rich plasma was separated and brought to a concentration of 1.0×10 ⁶ cells/ml in autologous plasma. platelets and leukocytes by gradient centrifugation on Ficoll (GE Healthcare) with a density of 1.077 g/ml and 1.091 g/ml (1:1). Platelets with test compounds or DMSO and 10 μg/ml E. coli O26:B6 LPS were incubated for 30 min. A 1% aqueous DMSO solution was used as a solvent. Activated platelets were combined with autologous leukocytes in a slide vial. Fixed according to May-Grunwald and stained according to Romanovsky-Giemsa. The number of neutrophil traps per 100 neutrophils was counted at ×400 magnification.

Incubation of resting neutrophils with autologous platelets does not lead to the formation of extracellular traps. At the same time, during incubation with LPS-stimulated platelets, a time-dependent increase in the proportion of neutrophils that released traps was observed (Table 11). Pre-treatment of cells with Compound (1) revealed an almost complete suppression of the formation of neutrophil traps after 1 hour of incubation with LPS-stimulated platelets. After 2 hours of incubation, the proportion of neutrophils with traps did not significantly differ from samples with LPS. The reference drug dabigatran etexilate under the same conditions inhibited the formation of extracellular traps at both time points.

Table 11 - Effect of Compound (1) and dabigatran etexilate at a concentration of 100 μM on the proportion of neutrophils that ejected extracellular traps in response to LPS-stimulated platelets, % (m ± SD) No.
p/n Samples tested Time 60 min. 120 min. 1. Solvent 0.00±0.00 0.00±0.00 2. LPS + solvent 14.00±5.66 28.00±2.83 3. LPS + Compound (1) 2.50±2.12 26.50±2.12 4. LPS + dabigatran etexilate 0.00±0.00 1.50±2.12* * p < 0.05 relative to LPS + vehicle, 2-way ANOVA.

9. Study of the effect of Compound (1) on the formation of tissue factor by LPS-stimulated monocytes

Blood stabilized with 3.8% sodium citrate (1:10) was centrifuged at 1000 g for 10 min. and plasma was collected. Solutions of test compounds in 1% DMSO or DMSO alone at an equal concentration and E. coli O26:B6 LPS were added to test tubes with 5 ml of whole blood to a concentration of 1 μg/ml. Samples were incubated for 4 hours at 37°C in an ES-20/60 orbital shaker (Biosan, Latvia). Monocytes were isolated by layering on Phicoll (GE Healthcare) at a density of 1.077 g/ml [Drake TA et al. Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line // The Journal of cell biology. - 1989. - T. 109. - No. 1. - S. 389-395]. The homogenate was added in 100 µl to a 96-well transparent plate, 100 µl of 25 mM calcium chloride solution and 100 µl of platelet-poor blood plasma were added. The activity of tissue factor on monocytes was assessed by the kinetics of fibrin polymerization at a wavelength of 365 nm using an Infinite M200 PRO microplate reader (Tecan, Austria) [Miserez R., Jungi TW LPS-induced, but not interferon-gamma-induced procoagulant activity of suspended human macrophages is followed by a refractory state of low procoagulant expression // Thrombosis research. - 1992. - T. 65. - No. 6. - S. 733-744].

Peripheral blood monocytic cells under the influence of LPS expressed active tissue factor (TF, also known as coagulation factor VIIa), which is reflected in the acceleration of the formation of fibrin filaments (a two-fold decrease in T _1/2 ). Incubation of monocytes in the presence of dabigatran etexilate and Compound (1) led to a significant slowdown in TF-mediated fibrin polymerization, which is characterized by an increase in T _1/2 by 5.5 and 10.1 times relative to the sample with LPS, respectively (Table 12).

Table 12 - Effect of Compound (1) and dabigatran etexilate at a concentration of 100 μM on the procoagulant activity of LPS-stimulated monocytes No.
p/n Samples tested Fibrin polymerization T _1/2 , sec. 95% D.I. 1. Solvent 116.9* 114.7-119.0 2. LPS + solvent 61.09 58.62-63.58 3. LPS + Compound (1) 619.2* 608.7-631.1 4. LPS + dabigatran etexilate 335.8* 333.2-338.4 * p <0.05 relative to LPS + solvent, F-test;
T _1/2 - the time of half-maximal polymerization of fibrin;
95% D.I. - 95% confidence interval.

10. Study of the effect of Compound (1) on the synthesis of nitric oxide by thrombin-stimulated macrophages

Peritoneal macrophages (PM) were isolated from the peritoneal exudate of outbred mice after injection of 1 ml of 3% peptone solution. The cell concentration was adjusted to 1.0×10 ⁶ cells/ml in complete DMEM (Gibco) supplemented with 2 mM L-glutamine (Gibco), 10% heat-inactivated fetal bovine serum (BioClot, Germany), supplemented with 100 U/ ml penicillin and 100 mg/ml streptomycin (Gibco). Seeded 200 μl/well in 96-well transparent plates (SPL Life Sciences Co., Ltd., Korea). Left for 2 h at 37°C in a humidified atmosphere with 5% CO ₂ , after which the wells were washed to remove non-adherent cells. After 24 hours of incubation, 20 μl of the supernatant were taken and 20 μl of solutions of the test compounds in 1% DMSO or DMSO alone in equal concentration were added. The determination of nitric oxide in supernatants was determined using the standard Griess method [Weissman BA, Gross SS Measurement of NO and NO synthase // Current Protocols in Neuroscience. - 1998. - V. 5. - No. 1. - S. 7.13. 1-7.13. 22].

Thrombin stimulation of macrophages leads to their pro-inflammatory activation, one of the consequences of which is the synthesis and accumulation of nitric oxide in the culture medium. Preincubation with Compound (1) leads to concentration dependent inhibition of nitric oxide synthesis in response to thrombin stimulation. Dabigatran etexilate does not have this effect. Compound (1) and the reference drug did not show cytotoxic properties in the experiment in the form of a decrease in metabolic activity or a violation of the integrity of the cytoplasmic membrane (Table 13).

Table 13 - Effect of Compound (1) and reference drug dabigatran etexilate on nitric oxide prosynthesis and viability of thrombin-stimulated macrophages No.
p/n Samples tested Nitric oxide synthesis, IC ₅₀ (95% DI), μM Cell viability according to the MTT test, CC ₅₀ , μM Cell viability according to LDH test, CC ₅₀ , μM 1. Connection (1) 20.86 (7.21-26.8) >100 >100 2. Dabigatran etexilate >100 >100 >100

Determination of cytotoxicity on peritoneal macrophages

To determine lactate dehydrogenase activity, 10 µl of supernatants taken 24 hours after incubation of PM with test compounds and LPS were mixed with 250 µl of 0.194 nM/l NADH solution dissolved in 54 mM phosphate buffer, pH 7.5, and 25 µl of 6.48 was added. mM sodium pyruvate solution. Optical density was determined at a wavelength of 340 nm for 20 min. using an Infinite M200 PRO microplate reader (Tecan, Austria) [Cummings BS, Schnellmann RG Measurement of cell death in mammalian cells // Current protocols in pharmacology. - 2004. - T. 25. - No. 1. - S. 12.8. 1-12.8. 22]. After 24 incubation of cells with test compounds, 20 μl of MTT solution was added to each well, incubated for 4 hours at 37°C in a humidified atmosphere containing 5% CO ₂ . The culture medium was removed, the cells were lysed, and formazan crystals were dissolved in 150 µl of DMSO. The plates were shaken at room temperature for 10 min. and measured the optical density at a wavelength of 565 nm in a microplate reader Infinite M200 PRO (Tecan, Austria) [Ehrich M., Sharova L. In vitro methods for detecting cytotoxicity // Current protocols in toxicology. - 2000. - Vol. 3. - No. 1. - S. 2.6. 1-2.6. 27].

11. Study of the effect of Compound (1) on mouse survival in a mouse model of LPS-induced pulmonary thrombosis.

The study was performed on 30 outbred male mice. The introduction of solutions of Compound (1) and the reference drug was carried out once intragastrically 2 hours before modeling thrombosis at doses of 13.0 and 27.0 mg/kg, respectively. The control group of animals was injected with a solvent (distilled water). Modeling of acute thrombosis in mice was carried out by administering LPS at a dose of 2 mg/kg, which was dissolved in saline. The control group of animals was injected with a solvent (distilled water). Lungs were taken from dead animals for histological evaluation. The group of surviving animals was observed for 7 days for remote death.

Morphological examination of lung tissue was carried out according to the method described above.

As a result of the performed studies in the group of control animals, the death of 100% of mice was observed (Table 14). At the same time, before death, the animals showed an increase in breathing, convulsions appeared, the animals assumed a characteristic posture, in which the hind legs were straightened and laid back. During the day after intravenous administration of LPS at a dose of 2 mg/kg, the animals died.

The evaluation of the effect of Compound (1) and dabigatran etexilate, which were administered 2 hours before the creation of hypercytokinemia, was performed one day after the administration of LPS. Dabigatran etexilate with a single intragastric administration significantly prevented the death of 6 out of 10 animals compared with the control group, in which 10 mice died. In the group of animals treated with Compound (1), 2 out of 10 mice died (Table 14).

Animals that died on the background of the introduction of the studied compounds, when visually observed after the intake of thrombotic agents in their body, were more active than the dying animals of the control group. They showed a decrease in the severity of motor disorders compared with the control group. The group of surviving animals was observed for 7 days for remote death.

Table 14 - Effect of Compound (1) and reference drug dabigatran etexilate on animal survival in a mouse model of LPS-induced pulmonary thrombosis (LPS - 2 mg/kg) (n=10) N
p/n Samples tested Dose,
mg/kg ¹ Death toll
animals Survivors
animals, % 1. Control
(physical solution) 10 0 2. Dabigatran etexilate 27.0 ¹ 4 60* 3. Connection (1) 13.0 ² 2 80* * - (p≤0.01) data are statistically significant in relation to the control group of animals, chi-square with Yates correction;
¹ - dose obtained by recalculation using the interspecies coefficient;
² - dose equimolar to the dose of dabigatran etexilate 12.0 mg/kg.
Note: n is the number of animals.

Morphological examination of the lungs

Microscopic examination of the lung tissue of mice 24 hours after intravenous administration of bacterial lipopolysaccharide at a dose of 2 mg/kg showed that circulatory disorders prevailed in the organ, realized by focal hemorrhages, venous plethora, hemorrhagic and hemorrhagic-serous exudate in the walls and lumens of the alveoli. There were signs of dystelectasis. In a significant part of the vessels of the microvasculature, erythrocyte sludges were found (Fig. 8).

When studying paraffin sections of the lung tissue of mice stained by Carstair, 24 hours after intravenous administration of bacterial lipopolysaccharide at a dose of 2 mg/kg, venous plethora and sluggish erythrocytes were recorded, forming primary red thrombi obturating the lumen of large and small vessels (Fig. 9A) . In the parietal part of the vessels, adherent formed fibrin clots were found, in the composition of which an insignificant collagen component was determined. In addition, small deposits of fibrin were deposited in the walls of the alveoli (Fig. 9B).

Microscopic examination of the lung tissue of mice treated with Compound (1) at a dose of 13 mg/kg against the background of LPS-induced generalized acute injury, in most experimental animals, the pathological process was less pronounced and was characterized mainly by diapedesis of erythrocytes into the interalveolar septa, in the cavity of the alveoli, focal small hemorrhages and the phenomena of a distelectasis. Hemorrhagic exudate was present in the alveolar cavities (Fig. 10). At the same time, the general histoarchitectonics of the lung tissue was preserved.

Staining of paraffin sections of the lung tissue of experimental mice with Carstair trichrome staining showed that in most cases, thrombi were represented by sluggish erythrocytes obturating the lumen of large vessels (Fig. 11A). At the same time, in some cases, a fibrin component in the form of thin filaments was detected in the lumen of small vessels (Fig. 11B).

Microscopic examination of the lung tissue of mice treated with dabigatran etexilate revealed focal and confluent hemorrhages in half of the experimental animals, as well as plethora of capillaries of the interalveolar septa and venous plethora. Hemorrhagic exudate was present in the alveolar cavities. The lumens of small and large vessels were almost completely covered with glued erythrocytes (Fig. 12A).

In the second half of the animals, the pathological process was less pronounced and was characterized mainly by diapedesis of erythrocytes into the interalveolar septa, in the cavity of the alveoli, focal small hemorrhages and dystelectasis. Slugged erythrocytes obturated the lumen of small vessels (Fig. 12B).

According to the results of a morphological study, a single administration of dabigatran etexilate and Compound (1) in acute LPS-induced lung injury weakly contributed to the restoration of impaired blood circulation in the lung tissue, but protected the alveoli from acute damage. At the same time, Compound (1) had the greatest potential.

Thus, a medicinal product containing Compound (10) - 5,7-di(thiophen-2-yl)-4,5-dihydro-[1,2,4]triazolo[1,5- a ] as an active ingredient pyrimidine, has anticoagulant (factor IIa inhibitor), antithrombotic and anti-inflammatory activity, exceeding the activity of known drugs, and can be used for the treatment and prevention of thrombosis and thrombosis-associated diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates the effect of Compound (1) and the reference drug dabigatran etexilate on the metabolic activity (viability) of HepG2 cells during a 48 hour incubation in the MTT assay.

Fig. 2 shows micrographs of HepG2 cells after 48 hours of incubation with samples of Compound (1) and dabigatran etexilate at a concentration of 100 μM (phase contrast microscopy, ×400): A - Control, growth medium F-12; B - Compound (1), 100 µM; B - Dabigatran etexilate (granulate), 100 µM.

Fig. 3 illustrates sections of histological samples of the lungs of mice of the control group after administration of a thrombotic agent. Hematoxylin-eosin staining. Total magnification ×50 (A); ×200 (B).

Fig. 4 illustrates sections of histological specimens from the lungs of mice in the control group after administration of a thrombotic agent. Trichrome stain by Carstair. Total magnification ×100 (A); ×400 (B).

Fig. 5 illustrates sections of histological lung samples from mice treated with Compound (1) in a thrombin-induced pulmonary thrombosis model. Hematoxylin-eosin staining. Total magnification ×100 (A); ×400 (B).

Fig. 6 illustrates sections of histological samples of the lungs of mice, with a single intragastric administration of Compound (1) in a model of thrombin-induced pulmonary thrombosis. Trichrome stain by Carstair. Total magnification ×100 (A); ×200 (B).

Fig. 7 illustrates sections of histological lung specimens from mice following a single intragastric administration of dabigatran etexilate in a thrombin-induced pulmonary thrombosis model. Hematoxylin-eosin staining. Total magnification ×50 (A); ×200 (B).

Fig. 8 illustrates sections of histological samples of the lungs of a control group of mice 24 hours after administration of LPS at a dose of 2 mg/kg. Hematoxylin-eosin staining. Total magnification ×100 (A); ×400 (B).

Fig. 9 illustrates sections of histological samples of the lungs of a control group of mice 24 hours after administration of LPS at a dose of 2 mg/kg. Trichrome stain by Carstair. Total magnification ×100 (A); ×400 (B).

Fig. 10 illustrates sections of histological samples of the lungs of mice after a single intragastric administration of Compound (1) against the backdrop of lipopolysaccharide intoxication. Hematoxylin-eosin staining. Total magnification ×100 (A); ×400 (B).

Fig. 11 illustrates sections of histological samples of the lungs of mice after a single intragastric administration of Compound (1) against the backdrop of lipopolysaccharide intoxication. Trichrome stain by Carstair. Total magnification ×100 (A); ×400 (B).

Fig. 12 illustrates sections of histological specimens of the lungs of mice after a single intragastric administration of dabigatran etexilate against the background of intoxication with lipopolysaccharide. Hematoxylin-eosin staining. Total magnification ×50 (A); ×200 (B).

Claims (2)

A drug that has anticoagulant factor IIa inhibitor, antithrombotic and anti-inflammatory activities and contains 5,7-di(thiophen-2-yl)-4,5-dihydro-[1,2,4]triazolo[1,5- a ] pyrimidine of formula I

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