Triazole derivatives with anticoagulant activity

Abstract

The present invention discloses a triazole derivative with anticoagulant activity, the general structural formula of which is as follows. The test results show that the compound has higher activity and lower toxicity than acetylsalicylic acid, and is expected to become a new anticoagulant drug. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the fields of chemistry and medicine, in particular to the fields of medicinal chemistry and pharmacology, and aims to enable the synthesis of new drugs for the prevention of thrombosis and thromboembolic complications. Background Art

[0002] Among the previous generation of anticoagulants, the following have been approved for clinical use: L-cysteine, N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl)-L-lysyl-L-α-aspartyl-L-tryptophan-L-prolyl-, cyclic (1-6)-disulfide (eptifibatide, GlaxoSmithKline Operations UK Ltd, UK), N-(butylsulfonyl)-4-[4-(piperidinyl)butoxy]-L-phenylalanine hydrochloride monohydrate (tirofiban, Agrastat Correvio, UK), and the F(ab')2 fragment of mouse monoclonal protein IIb-IIIa of the antiplatelet fibrinogen receptor glycoprotein (GP) ( Framon CJSC, Russia). (LE Holmes, R. Gupta, S. Rajendran, J. Luu, JK French, CP Juergens. A randomized trial assessing the impact of three different glycoprotein IIb / IIIa antagonists on glycoprotein IIb / IIIa plateletreceptor inhibition and clinical endpoints in patients with acute coronary syndromes. Cardiovascular Therapy.2016,no.21.doi:10.1111 / 1755-5922.12203.)

[0003] Although clinical trials have shown that these compounds may be most effective in interventional cardiology, their application is limited due to their high cost and the risk of inducing autoimmune reactions (C. Patrono, F. Andreotti, H. Arnesen. Antiplatelet agents for the treatment and prevention of atherothrombosis. European Heart Journal. 2011. Vol. 32. Pp. 2922-2932.).

[0004] The closest analog and reference drug to the present invention is acetylsalicylic acid (2-acetylhydroxybenzoic acid or aspirin), which has been successfully used in practical medicine and has been shown to be effective in preventing and treating thrombosis and thromboembolic complications (2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association / American Stroke Association. Stroke. 2018; 49: e138.).

[0005] Disadvantages of this original drug are the high frequency of aspirin resistance and adverse clinical phenomena in the form of ulcerogenic and hemorrhagic effects. Summary of the invention

[0006] The object of the present invention is to provide a triazole derivative having anticoagulant activity so as to expand the scope of biologically active substances having anticoagulant activity.

[0007] The technical solution of the present invention is as follows:

[0008] A triazole derivative with anticoagulant activity, the general formula of which is as follows:

[0009]

[0010] The invention also provides an application of the triazole derivative in the preparation of medicines, and the medicines have anticoagulant activity.

[0011] Compared with the prior art, the beneficial effects of the present invention are: providing a triazole derivative with anticoagulant activity, expanding the scope of biologically active substances with anticoagulant activity, and hopefully reducing the defects of existing anticoagulant drugs. DETAILED DESCRIPTION

[0012] Synthesis and toxicity tests of the synthesized compounds.

[0013] The synthesis of the compounds claimed herein is as follows.

[0014] Example 1 Synthesis of 1-[(4-n-octylphenyl)methyl]-1H-benzotriazole (Ia).

[0015] 0.17 g (0.88 mmol) of n-octyl bromide was added to 0.20 g (0.88 mmol) of 4-[(1H-benzotriol-1-yl)methyl]phenol in 6 ml of methyl ethyl ketone, stirred vigorously in argon at 20°C, and then 0.58 g (1.77 mmol) of cesium carbonate was added. The reaction mixture was refluxed and boiled in an inert medium for 6 hours, and then filtered from the inorganic precipitate. The solvent was removed under vacuum. The resulting white residue was washed with hot n-hexane and air-dried.

[0016] Yield: 0.21 g (70%); melting temperature (Tm): 65°C-67°C; retardation factor (Rf): 0.69 (ethanol: carbon tetrachloride, 1:3).

[0017] Elemental analysis: %: C 74.86; H 8.10.C 21 H 27 N3O. Estimated value, %: C 74.74%; H 8.07%.

[0018] Infrared (IR) spectrum, ν, cm -1 :1254(COC,val.),1453,1518,1620(arom.,val.)2873(C8H 17 ,val.).

[0019] High-resolution nuclear magnetic resonance ( 1 HNMR) spectrum (CDCl3), δ, ppm: 0.87–0.90(3H,t,CH3),1.30–1.75(12H,m,6CH2),3.95–3.97(2H,t,CH2),5.82(2H,s,CH2),6 .84–6.85(arom.2H,d,CH=), 7.28–7.30(arom.2H,d,CH=), 7.40–7.51(arom.2H,m,CH=), 8.06–8.10(arom.2H,m,CH=).

[0020] The material was a white solid soluble in DMSO, DMF, chloroform and acetone.

[0021] Example 2. Synthesis of 1-[(4-n-hexadecyloxyphenyl)methyl]-1H-1,2,4-triazole (Ib).

[0022] We added 0.44 g (1.42 mmol) of n-hexadecyl bromide to a solution of 0.25 g (1.42 mmol) of 4-[(1H-1,2,4-triazol-1-yl)methyl]phenol, stirred vigorously under argon at 20°C, and then added 0.93 g (2.86 mmol) of cesium carbonate. The reaction mixture was refluxed in an inert medium for 5 hours and then filtered from the inorganic precipitate. The solvent was removed under vacuum. The resulting white residue was recrystallized from n-hexane and air-dried.

[0023] Yield: 0.40 g (71%); Tm: 68°C-70°C (n-hexane); Rf: 0.44 (ethanol: carbon tetrachloride, 1:7).

[0024] Elemental analysis: Found, %: C 75.28%; H 10.50%.C 25 H 41 N3O. Estimated, %: C75.14%; H10.34%

[0025] Infrared spectrum (KBr), ν, cm -1 :1253(COC,val.),1450,1463,1515,1615(arom.,val.),2848,2917(C 16 H 33 ,val.).

[0026] High-resolution nuclear magnetic resonance spectrum (CDCl3), δppm: 0.89–0.91 (3H, t, CH3), 1.31–1.74 (28H, m, 14CH2), 3.97–3.99 (2H, t, CH2), 5.78 (2H, s, CH2), 6.75–6.76 (arom. 2H, d, CH=), 7.14–7.15 (arom. 2H, d, CH=), 7.95 (arom. 1H, s, CH=), 8.56 (arom. 1H, s, CH=).

[0027] The substance is a white crystalline material which is soluble in DMSO, DMF, chloroform and carbon tetrachloride.

[0028] Example 3 Anticoagulant activity of compounds Ia, b.

[0029] In vitro experiments were performed on blood samples collected from healthy male donors aged 18-24 years (n=22). To investigate the effects of these compounds on hemostasis, BD Blood samples were collected from the cubital vein using a vacuum blood collection system (Becton Dickinson and Company, USA). A 3.8% sodium citrate solution in a ratio of 9:1 was used as a venous blood stabilizer. All experiments were performed on platelet-rich and platelet-poor plasma. Platelet-rich plasma samples were obtained by centrifuging citrated blood at 1000 rpm for 10 minutes, while platelet-free plasma samples were obtained by centrifuging citrated blood at 3000 rpm for 20 minutes. An OPN-3.02 centrifuge (TNK DASTAN OJSC, Kyrgyzstan) was used in this study. The effect of the compound on platelet aggregation was studied using the Born method on an AT-02 aggregometer (NPFMedtech, Russia). The anticoagulant activity of compound I and aspirin was determined at a final concentration of 10 -3 mol / l. Adenosine diphosphate (ADP) at a concentration of 20 μg / ml and collagen at a concentration of 5 mg / ml (Russian standard) were used as aggregation inducers. During ADP-induced platelet aggregation, the maximum aggregation amplitude in the presence of the studied compounds, the aggregation rate, the time to reach the maximum aggregation amplitude and dissociation were evaluated. In collagen-induced platelet aggregation, the latent period was evaluated, during which phospholipase C is activated (leading to the formation of secondary messengers, the secretion of platelet granules and the synthesis of thromboxane A2).

[0030] Acute toxicity studies were conducted on 200 male white mice (2 months of age) with an average body weight of 20–21 g, injected intraperitoneally with the synthesized compounds and aspirin. Prior to the experiments, the animals were isolated in a separate cage in the in vivo facility of the Department of Biochemistry of BSMU for 14 days. The in vitro temperature was maintained at 18°C–22°C, and its lighting was a combination of natural light and artificial. Sterilization was performed weekly using a fixed wall-mounted sterilization irradiator for 20 min. Drinking water was guaranteed 24 hours a day and they were fed a standard diet of natural products (vegetables, cereals) and laboratory animals (mice, rats, rats, hamsters) that met the criteria of the declaration of conformity (No. ROSS RU. PR 98, D 00497, issued on February 7, 2016) containing wheat, barley, soybean meal, fish meal, yeast and bran, as well as vitamin and mineral complexes, vegetable fats and antioxidants.

[0031] The study was conducted in accordance with good laboratory practices and Article 11 of Federal Law No. 61-FZ of April 12, 2010 "On the circulation of medicinal products" and "Guidelines for experimental (preclinical) studies of new drugs" (M., 2012).

[0032] In toxicity studies in mice, the studies were conducted at doses of 100–500 mg / kg, with stepwise increases of 50 mg / kg. The amount of injected substance was calculated based on the body weight of the mice, taking into account the maximum permissible amount of fluid that can be injected. A group of control (untreated) animals was included in the experiments to comparatively evaluate the state and behavior of the individuals in the control and experimental groups. This group of animals was excluded from the experiments at the end of the first day of observation. The experimental groups were observed for a total of 14 days.

[0033] The results were statistically analyzed using the Statistical Software Package t10.0 (StatSoft Inc, USA). The Shapiro–Wilk test was used to test whether the data conformed to the normal distribution. The Kruskal-Wallis test was used for analysis of variance. The critical significance level (p value) of the statistical test was set at 0.05. The probe analysis was performed using BioStat5.9 software (AnalystSoft Inc.), and the lethal dose of 50% (LD50) value was calculated.

[0034] When the compounds under study were added to the cuvette of the aggregometer (Table 1), the platelet release response (lag phase) of Ia and Ib was prolonged by 15.7% (p<0.05) and 10.3% (p<0.05), respectively; the platelet aggregation amplitude in group Ia was reduced by 29.4% (p<0.05), and the platelet aggregation amplitude in group Ib was reduced by 23.5% (p<0.05); compared with the control group, the platelet aggregation rates in group Ia and Ib were reduced by 13.6% (p<0.05) and 7.4% (p<0.05), respectively, and the time required to reach maximum aggregation was increased by 25.7% (p<0.05) and 18.3% (p<0.05), respectively. At the same time, it was found that aspirin had no effect on platelet release, indicating that its effect on platelet aggregation level was more than 2.2 times worse than compound Ia (p<0.05), the platelet aggregation rate index was 28.6% worse than compound Ia (p<0.05), and the time required to reach maximum aggregation was more than 2.4 times worse than compound Ia (p<0.05). In addition, the effect of aspirin on platelet aggregation level was more than 1.7 times lower than compound Ib (p<0.05), the platelet aggregation rate index was 59.4% lower than compound Ib (p<0.05), and the time required to reach maximum aggregation was more than 1.7 times lower than compound Ib (p<0.05).

[0035] Table 1 Anticoagulant activity indexes of compounds Ia, b and acetylsalicylic acid, n=10

[0036]

[0037] Note: Latency of collagen-induced platelet aggregation; other parameters are ADP-induced platelet aggregation. *р≤0.05, **р≤0.001 – compared with control group; #р≤0.05, ##р≤0.001 – compared with acetylsalicylic acid.

[0038] Example 4. Acute toxicity study of compounds Ia and b in mice.

[0039] According to the Berezovskaya system of chemical acute toxicity and the criteria for toxicity of chemical compounds by intravenous injection, the compounds are classified as “low toxicity” (Table 2).

[0040] Table 2 Acute toxicity indexes of the compounds and acetylsalicylic acid, M±SD

[0041] Indicator Acetylsalicylic acid Ia Ib LD50,mg / kg 302,9±32,6 560±27,9 610±39,7

[0042] Therefore, the compound is less toxic and more effectively inhibits platelet activation and aggregation than aspirin.

Claims

1. A triazole derivative having anticoagulant activity, characterized in that: The general structure is as follows:

2. The triazole derivative having anticoagulant activity according to claim 1, characterized in that: It is compound Ia.

3. The triazole derivative having anticoagulant activity according to claim 1, characterized in that: It is compound Ib.

4. Use of a triazole derivative as claimed in any one of claims 1 to 3 in the preparation of a drug, characterized in that: The drug has anticoagulant activity.

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