key: cord-0838122-vhyf99mq
authors: Gurevich, K. G.; Urakov, A. L.; Rozit, G. A.; Klen, E. É.; Samorodov, A. V.; Khaliullin, F. A.
title: Synthesis and Antiplatelet and Anticoagulant Activity of Thietane-Containing 2-(5-Bromo-2,4-Dihydro-3-Oxo-1,2,4-Triazolyl-4)Acetate Salts
date: 2021-08-14
journal: Pharm Chem J
DOI: 10.1007/s11094-021-02437-6
sha: 1facbbe9158181af7636a327dd6a9a23648344d1
doc_id: 838122
cord_uid: vhyf99mq

Sulfone II was synthesized via oxidation of ethyl 2-[5-bromo-2,4-dihydro-3-oxo-2-(thietanyl-3)-1,2,4-triazolyl- 4]acetate (I) by H(2)O(2). Hydrolysis of esters I and II synthesized 2-[5-bromo-2,4-dihydro- 3-oxo-2-(thietanyl-3)-1,2,4-triazolyl-4]- and 2-[5-bromo-2,4-dihydro-3-oxo-2-(1,1-dioxothietanyl-3)-1,2,4- triazolyl-4]acetic acids III and IV, respectively.Water-soluble salts V and VI were prepared by reacting acids III and IV with alkali-metal hydroxides and amines. The structures of the synthesized compounds were confirmed IR and NMR spectroscopic data. The antiplatelet and anticoagulant activity of the synthesized compounds was studied in vitro based on predictions of the PASS computer program. Compounds III and VIb, which showed the absence of predicted toxic risks and were superior to the reference drug in the collagen-induced aggregation test, had the most pronounced antiplatelet activity (comparable to that of acetylsalicylic acid) in the ADP-induced aggregation test. The anticoagulant activity of the compounds was significantly inferior to that of heparin sodium. All synthesized compounds satisfied Lipinski’s rule-of-5.

Thrombohemorrhagic disorders are currently the most common types of human pathologies [1] . Thrombi and associated complications are some of the causes of invalidism and lethality among the total causes of death of the geriatric population [2] . Therapy of clots has become especially critical because of emergent coronavirus infections [3, 4] . Antiplatelet drugs such as ticlodipine, clopidogrel, flurbiprofen, ozagrel, lotrafiban, and acetylsalicylic acid are currently used to treat and prevent thrombi. However, several side effects can be associated with their use [5 -7] . Therefore, the development of new domestic drugs capable of cor-recting the hemostasis system is crucial. Previous research on the synthesis of 1-thietanyl-1,2,4-triazole derivatives demonstrated that they were promising for preparing new biologically active compounds with antiplatelet activity [8, 9] . Therefore, the aim of the present work was to synthesize new thietane-containing 2-(5-bromo-2,4-dihydro-3-oxo-1,2,4-triazolyl-4)acetic acid derivatives and to assess preliminarily their antiplatelet and anticoagulant activity.

The target compounds were synthesized according to Scheme 1.

PMR spectra were recorded in CDCl 3 and DMSO-d 6 on a Bruker AM-300 instrument at operating frequency 300 MHz for 1 H. The internal standards were solvent resonances at 2.50 ppm (DMSO-d 6 ) and 7.26 ppm [CD(H)Cl 3 ]. IR spectra were taken from KBr pellets on an Infralum FT-02 instrument. Melting points were measured on an SMP30 apparatus. The purity of compounds was confirmed by TLC on Sorbfil PTSKh-P-A-UF plates using CHCl 3 -EtOH (9:1, v/v) and hexane-EtOH (5:5). Spots were detected by I 2 vapor in a humid chamber. Elemental analyses were performed on a Hekatech Euro3000 CHNS analyzer. Analyses for C, H, N, and S agreed with the calculated values. Table 1 General method for synthesizing salts Va, b and VIa, b. A solution of KOH or NaOH (3.3 mmol) in i-PrOH (35 mL) and H 2 O (1 mL) was treated with acid III or IV (3 mmol). The mixture was refluxed for 10 min and cooled. The resulting precipitate was filtered off, rinsed with i-PrOH, dried, and purified by crystallization from i-PrOH.

General method for synthesizing salts Vc-e and VIc-e. Acid III or IV (3 mmol) was treated with i-PrOH (15 mL), heated until dissolved, and treated with an amine (6 mmol). The mixture was refluxed for 10 min and cooled. The resulting precipitate was filtered off, rinsed with i-PrOH, dried, and purified by crystallization from i-PrOH.

Toxicity and drug-likeness of the synthesized compounds were predicted by the Osiris DataWarrior program [10] . The biological activity of the synthesized compounds was predicted from the chemical structural formula using the online version of the PASS computer program [11] .

Experiments were conducted according to Good Laboratory Practice Rules of the Eurasian Economic Union on Circulation of Medicines [12] .

Antiplatelet and anticoagulant activity were assessed in vitro using isolated blood samples from 25 healthy male donors aged 18 -24 years. The study was approved by the Ethics Committee of Bashkir State Medical University, Ministry of Health of Russia (No. 2 of Oct. 17, 2012). Informed consent was obtained from all study participants before blood collection. The effect of the compounds on platelet aggregation was studied using the Born method [13] on an AT-02 aggregometer (Medtekh NPF, Russia). Antiplatelet activity of the tested compounds and reference drugs was assessed at a final concentration of 1´10 -3 M. The aggregation inductors were adenosine diphosphate (ADP) at a concentration of 20 mg/mL and collagen at a concentration of 5 mg/mL (Tekhnologiya-Standart, Russia). The reference drugs were pentoxifylline (Pentoxifylline, 20 mg/mL solution for injection, 5-mL ampuls; JSC Dalkhimfarm, Russia) and acetylsalicylic acid (substance-powder; Shandong Xinhua Pharmaceutical Co. Ltd., China).

Anticoagulant activity was determined in clotting tests [14] on a Solar CGL 2110 turbidimetric hemocoagulometer (CSC SOLAR, Belarus). The final concentration of the tested compounds and reference drug was 5´10 -4 g/mL. The activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen concentration were studied according to A. Clauss. The reference drug was heparin sodium (heparin sodium, 5000 IU/mL, solution for injection, 1-mL ampuls; JSC Sintez, Russia).

Statistical analysis used the Statistica 10.0 software (StatSoft Inc., USA). A check for normal distributions of actual data used the Shapiro-Wilk criterion. The distribution of the obtained results was found to differ from normal. Therefore, nonparametric methods were used for further work. The obtained results were given as medians and 25 and 75 percentiles. Dispersion analysis used the Kruskal-Wallis criterion. The critical significance level p for statistical criteria was taken as 0.05 [15] .

Starting ethyl 2-[5-bromo-2,4-dihydro-3-oxo-2-(thietanyl-3)-1,2,4-triazolyl-4]acetate (I) was synthesized by the literature method [16] . Oxidation of I by a 10-fold molar excess of H 2 O 2 in glacial HOAc with heating produced ethyl 2[(5-bromo-2,4-dihydro-3-oxo-2-(1,1-dioxothietanyl-3)-1,2,4triazolyl-4]acetate (II) in 80% yield (Scheme 1). The PMR spectrum of sulfone II showed weak-field shifts of 1.2 and 0.7 ppm for the multiplets of the two S(CH) 2 groups of the thietane dioxide ring and a strong-field shift by 0.4 ppm for the multiplet of the NCH proton relative to the analogous resonances of the unoxidized thietane ring.

Acid III was prepared in 58% yield (Scheme 1) via alkaline hydrolysis of ester I by a two-fold molar excess of aqueous NaOH at room temperature. Hydrolysis of ester II under analogous conditions led to the formation of acid IV in 31% yield (Scheme 1). The yield of IV was increased to 77% by acid hydrolysis of II using HCl solution and heating. The formation of acids III and IV was confirmed by the appearance in their IR spectra of absorption bands for O-H stretching vibrations at 3051 -3105 cm -1 (Table 1) . PMR spectra of acids III and IV lacked resonances for ethoxy protons.

Salts of K (Va and VIa) and Na (Vb and VIb) were synthesized in 58 -77% yields (Scheme 1) by reacting acids III and IV with a slight excess of KOH or NaOH in i-PrOH. Alkylammonium salts Vc-e and VIc-e were synthesized in 83 -95% yields (Scheme 1) by heating acids III and IV with a two-fold molar excess of the amines in i-PrOH. Formation of salts Va-e and VIa-e was confirmed by the presence in their IR spectra (Table 1) Acid III (-14.3%, p £ 0.05) and its K salt Va (-11.5%, p £ 0.05) among thietanyl derivatives III and V showed statistically significant antiplatelet effects that were comparable to that of acetylsalicylic acid in the ADP-induced platelet aggregation test. The effect decreased slightly to -11.4% for acid IV and to -9.4% for K salt VIa if the oxidation state of the S atom was increased to the sulfone. The Na salt VIb (-15.3%, p £ 0.05) showed an antiplatelet effect comparable to that of acetylsalicylic acid. However, the antiplatelet effect of III, Va, and VIb was significantly (by 3.2 -4.2 times) inferior to that of pentoxifylline (Table 3) . All compounds except for VIa exhibited antiplatelet activity from -4.5 to -16.3% (p £ 0.05) in the collagen-induced aggregation test. Pentoxifylline and acetylsalicylic acid did not exhibit biological activity in this test.

Ester II and salts V and VI caused significant hypocoagulation, increasing the APTT by 4.7 -9.3% (p £ 0.05) as compared to the control and did not affect the PT and fibrinogen concentration. The effects of the tested compounds were significantly inferior to that of heparin sodium, which increased the APTT by 54.7%.

The new compounds were analyzed for agreement with Lipinski's rule-of-5 [17, 18] , toxic risks, and the drug-likeness parameter (similarity to a drug) using the Osiris DataWarrior program to discover compounds that could lead to potential drugs (drug candidates) after in vivo testing (Table 4).

A calculation of the toxic risks showed that mutagenic, oncogenic, and irritation properties and a negative effect on reproductive functioning were not predicted for the synthesized compounds.

The calculated physicochemical parameters of II-VI were found to satisfy Lipinski's rule-of-5. The molecular mass of the synthesized compounds was less than 447.2 g/mol. The lipophilicity coefficient fell in the range from -4.05 to -0.24. The number of H acceptors was <9; H donors, 1. The topological polar surface area was 98.51 -135.63 Å 2 , which suggested that the synthesized compounds had good penetrating power through cell membranes. The drug-likeness parameter lay in the range from -14.79 to -2.05, which confirmed the structures of the synthesized compounds were novel.

Thus, the antiplatelet activity in the ADP-induced aggregation test was greatest for III and VIb and comparable to that of acetylsalicylic acid. These compounds typically lacked toxic risks and were superior to the reference drugs in the collagen-induced aggregation test. The calculated drug-likeness parameter led to the conclusion that the search for new compounds with antiplatelet activity among this class of compounds was promising.

Approved by Decision No. 81 of the EAEC Committee of

Handbook for Preclinical Drug Trials

Mathematical Statistics in Experimental and Clinical Pharmacology