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Article

Novel Substituted Azoloazines with Anticoagulant Activity

by
Alexander A. Spasov
1,*,
Olga V. Fedorova
2,
Nikolay A. Rasputin
2,*,
Irina G. Ovchinnikova
2,
Rashida I. Ishmetova
2,
Nina K. Ignatenko
2,
Evgeny B. Gorbunov
2,
Gusein A. o. Sadykhov
2,3,
Aida F. Kucheryavenko
1,
Kseniia A. Gaidukova
1,
Victor S. Sirotenko
1,
Gennady L. Rusinov
2,4,
Egor V. Verbitskiy
2,3 and
Valery N. Charushin
2,3
1
Department of Pharmacology & Bioinformatics, Scientific Center for Innovative Drugs, Volgograd State Medical University, Volgograd 400131, Russia
2
I. Ya. Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences, Ekaterinburg 620108, Russia
3
Department of Organic and Biomolecular Chemistry, Ural Federal University Named after the First President of Russia B. N. Yeltsin, Ekaterinburg 620002, Russia
4
Department of Technology & Organic Synthesis, Ural Federal University Named after the First President of Russia B. N. Yeltsin, Ekaterinburg 620002, Russia
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(21), 15581; https://doi.org/10.3390/ijms242115581
Submission received: 2 October 2023 / Revised: 21 October 2023 / Accepted: 23 October 2023 / Published: 25 October 2023
(This article belongs to the Section Molecular Pharmacology)
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Abstract

:
Hypercytokinemia, or cytokine storm, often complicates the treatment of viral and bacterial infections, including COVID-19, leading to the risk of thrombosis. However, the use of currently available direct anticoagulants for the treatment of COVID-19 patients is limited due to safety reasons. Therefore, the development of new anticoagulants remains an urgent task for organic and medicinal chemistry. At the same time, new drugs that combine anticoagulant properties with antiviral or antidiabetic activity could be helpfull in the treatment of COVID-19 patients, especially those suffering from such concomitant diseases as arterial hypertension or diabetes. We have synthesized a number of novel substituted azoloazines, some of which have previously been identified as compounds with pronounced antiviral, antibacterial, antidiabetic, antiaggregant, and anticoagulant activity. Two compounds from the family of 1,2,4-triazolo[1,5-a]pyrimidines have demonstrated anticoagulant activity at a level exceeding or at least comparable with that of dabigatran etexilate as the reference compound. 7,5-Di(2-thienyl)-4,5-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine has shown the highest ability to prolong the thrombin time, surpassing this reference drug by 2.2 times. This compound has also exhibited anticoagulant activity associated with the inhibition of thrombin (factor IIa). Moreover, the anticoagulant effect of this substance becomes enhanced under the conditions of a systemic inflammatory reaction.

1. Introduction

Anticoagulant drugs are widely used for the treatment and prevention of thrombosis in case of patients with sepsis of various etiologies. However, modern direct oral anticoagulants are not always compatible with new antiviral agents, as has recently been established in the treatment of COVID-19 patients [1]. Therefore, the development of novel anticoagulants remains an urgent task for organic and medicinal chemistry. Drugs that combine anticoagulant properties with antiviral or antibacterial activity [2] are of particular interest. In this regard, substituted azoloazines, also exhibiting antidiabetic activity as a decisive factor in their use for treatment of COVID-19 and other viral and bacterial infections [3], comprise a promising family of biologically active compounds. Intra- or extracellular accumulation of glycation end products is considered to be an important factor for the pathogenesis of such diseases as atherosclerosis, rheumatoid arthritis, inflammation, atherosclerosis, and heart failure [4]. The latter is directly related to the hematopoietic system, in particular, with an increased formation of platelets.
Azoloazines are azaheterocyclic compounds containing fused five- and six-membered rings. These compounds can be regarded as structural analogues of purine bases. Indeed, these compounds can act as antimetabolites or enzyme inhibitors, and are known to have a wide spectrum of biological activity [5,6] (Figure 1). In particular, they exhibit antiviral activity, and some compounds of this series are characterized by activity against the main 3CLpro-2 protease of the coronavirus, which is involved in the maturation and replication of the SARS-CoV-2 virus [7]. In addition, azoloazines have demonstrated a pronounced protective effect against septic shock and exhibit antibacterial, antidiabetic, and anticoagulant activities [8,9,10]. It is worth mentioning that substituted 1,2,4-triazolo[1,5-a]pyrimidin-7-ones have a higher anticoagulant activity than that of the well-known drug dabigatran etexilate, which also contains azole and azine moieties [2].
We have found polyvalent antidiabetic activity in one of these derivatives [11], and have shown that 7-(4-methoxyphenyl)-5-phenyl-4,5-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine appears to be a glucokinase activator and dipeptidyl peptidase type 4 inhibitor. Also, we have identified amino derivatives of 5,7-diaryl substituted [1,2,4]triazolo[1,5-a]pyrimidines, which are micromolar inhibitors of IL-6 secretion and NO synthesis, capable of preventing LPS-induced acute lung injury [12]. Previously, a series of substituted azolo[1,2,4,5]tetrazines have been synthesized, including compounds which have exhibited pronounced antibacterial and antiglycating activities [13]. Considering all these data, it appears to be worth testing analogues of the mentioned azoloazines as potential anticoagulants.
In this communication, we wish to describe new derivatives of the azoloazine family with varied numbers and positions of nitrogen atoms in the azine moiety (two, three, or four atoms), and in the azole fragment (one, two, or three atoms) as potential anticoagulants. These compounds involve 6-(2-Alkyl)-6H-indolo[2,3-b]quinoxalines, substituted azolo[1,5-a]pyrimidines, and 1,2,4-triazolo[5,1-c]triazines, as well as azolo[1,2,4,5]tetrazine derivatives.

2. Results and Discussion

2.1. Chemistry

Substituted indolo[2,3-b]quinoxalines are known to be DNA-intercalating antiviral agents [14] and can also exhibit antidiabetic activity [15]. It is interesting to study the anticoagulant activity of substituted indolo[2,3-b]quinoxalines. Therefore, a new series of indolo[2,3-b]quinoxaline derivatives 1a–j have been obtained from 2-(2-bromophenyl)quinoxalines through a microwave-assisted Buchwald−Hartwig cross-coupling reaction, followed by intramolecular oxidative cyclodehydrogenation (Scheme 1) [16].
Substituted [1,2,4]triazolo[1,5-a]pyrimidines, the structural isomers of natural purines, belong to another important class of nitrogen-containing heterocycles which has received great attention during recent decades due to its wide application in pharmaceuticals [17]. They have been proven to exhibit antiviral [18], antibacterial [19], tuberculostatic [20], and antitumor [21] activities. In addition, there are references in the literature indicating that compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine core appear to be anti-inflammatory agents [22].
To find out the “structure–activity” relations for substituted azolo[1,5-a]pyrimidines, we synthesized compounds bearing aryl, hetaryl or the nitro substituents in the pyrimidine ring [6]. Hydrogenated analogues of azolo[1,5-a]pyrimidines, as well as their salts with guanidine, which are an important fragment of the known anticoagulants, have been obtained.
The following approaches have been used for the synthesis of new representatives of 5,7-di(hetero)aryl-substituted [1,2,4]triazolo[1,5-a]pyrimidines. Compounds 5a–d have been synthesized using the so-called SNH methodology (nucleophilic substitution of hydrogen) [23,24,25]. Bromination of [1,2,4]triazolo[1,5-a]pyrimidine has produced bromo compound 2; its further reaction with a Grignard reagent led to the formation of intermediate σH-adducts 3a,b, which on treatment with triethylamine (TEA) were converted into aromatic compounds 4a,b (Scheme 2). Subsequent functionalization at C-5 position of compounds 4a,b with a second Grignard reagent afforded the target 5,7-di(hetero)aryl-substituted-4,5-dihydro-[1,2,4]triazolo[1,5-a]pyrimidines 5a–d [26].
The three-step synthesis of amino derivatives of di(hetero)aryl-substituted azolo[1,5-a]pyrimidines 7a,b from commercially available reagents is shown in Scheme 2. The interaction of aromatic aldehydes with aromatic ketones results in the formation of the corresponding nitrochalcones 6a,b [27,28]. These nitrochalcones react with aminoazoles to produce nitro derivatives of di(hetero)aryl-substituted azolo[1,5-a]pyrimidines 4c,d. Amino derivatives of di(hetero)aryl-substituted azolo[1,5-a]pyrimidines 7a,b have been synthesized by reduction of the nitro group in the presence of hydrazine hydrate and Raney nickel.
Dihydro derivatives of [1,2,4]triazolo[1,5-a]pyrimidines 10a,b have been obtained from compounds 8a,b derived from a three-component Biginelli reaction [29], while compound 8c could not be subjected to further modification. Amino derivatives 8a,b have been condensed with Boc-protected L-glycine, with TBTU as a coupling agent (Scheme 2).
Guanidinium salts of 6-nitro1,2,4-triazolo[1,5-a]pyrimidines 12a,b and 6-nitro-1,2,4-triazolo[5,1-c]-triazine 14 have been synthesized through the reaction of the corresponding 6-nitro-1,2,4-triazoloazines 11a,b or 13 with guanidine in methanol (Scheme 3).
In addition to azoloazines containing two and three nitrogen atoms in the azine fragment, a number of substituted azolo[1,2,4,5]tetrazines have also been synthesized. It should be emphasized that compounds bearing the 1,2,4,5-tetrazine ring can exhibit antiaggregant activity [30].
Imidazo[1,2-b][1,2,4,5]tetrazines and [1,2,4]triazolo[4,3-b][1,2,4,5]tetrazines containing guanidine residue have been synthesized. The latter appear to be an important structural fragment providing anticoagulant activity to FXIa inhibitors [31]. The interaction of imidazo[1,2,4,5]tetrazine with free guanidine takes place in a mixture of acetonitrile and methanol without heating, thus affording 3-guanidino-6-alkylthioimidazo[1,2-b][1,2,4,5]tetrazines 15a,b (Scheme 4). 3-Guanidino-6-alkylthio-imidazo[1,2-b][1,2,4,5]tetrazines 16a–c have been obtained in a similar way (Scheme 5) [17].

2.2. Biology

2.2.1. Anticoagulant Activity of the Target Compounds In Vitro

The effect of these compounds on coagulogram parameters was determined using in vitro experiments. We observed that dabigatran etexilate at a concentration of 100 μM increased the activated partial thromboplastin time (APTT) by 1.6 times relative to the control (Table 1). Dabigatran etexilate in the studied concentration increased the thrombin time (TT) by 6.0 times, which corresponds to the mechanism of its anticoagulant action—the disruption of the final stage of coagulation without changing the prothrombin time.
In the similar way, our study of targeted novel azoloazine derivatives was carried out with an emphasis on their effect on the coagulogram parameters of rabbit blood. The results are summarized in Table 1. It was shown that compound 5d increased the APTT by 1.3 times relative to the control and had the greatest ability to prolong the thrombin time, exceeding the comparison drug dabigatran etexilate by 13.0 times. Compound 7a was comparable to dabigatran etexilate in terms of activity. Compounds 1f, 5a, and 10a prolonged the thrombin time by 3.5 times relative to the control. Other substances also significantly prolonged the thrombin time relative to control, but to a lesser extent than the comparison drug. None of the studied compounds affected the prothrombin time.
In this regard, compound 5d, which demonstrated the greatest antithrombin activity in the in vitro experiment, was investigated further for its effect on the coagulogram parameters of rabbit blood treated with LPS to mimic the conditions of hypercytokinemia (Table 2). The coagulation parameters of blood treated with LPS changed, with the exception of PT. The APTT was lengthened by 1.2 times and TT was decreased by 1.3 times compared to the intact blood sample. At the same time, the reference drug dabigatran etexilate reliably prolonged the APTT and TT by 2.3 and 14.0 times, respectively. Compound 5d had a significant effect only on the thrombin time, reliably exceeding the control values of LPS-treated blood by 17.0 times.
Due to the high antithrombin effect of the compound and reference drug, they were studied in a concentration range from 10 to 0.1 μM to calculate EC50 both in intact and LPS-treated PPP.
Thus, the comparison drug dabigatran etexilate at a concentration of 10 μM significantly prolonged this index up to 46.8 s, which corresponds to a reliable increase of 302.0% relative to the control. When the concentration was further reduced 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. The EC50 antithrombin activity of the comparison drug was 1.4 μM.
Compound 5d at a concentration of 10 μM significantly prolonged the thrombin time up to 467.1% more than the control values. Further reductions to 5 and 1 μM resulted in differences relative to the control of 194.1 and 46.5%, respectively. At a concentration of 0.1 μM, compound 5d had no effect on this parameter. The EC50 of antithrombin activity was equal to 1.25 μM.
Effect of compound 5d and the comparison drug on coagulogram parameters were studied under conditions of hypercytokinemia in vitro. The thrombin time index in control samples of blood treated with lipopolysaccharide amounted to 9.0 s, which is significantly lower than these data in the intact blood group (Table 2). Dabigatran etexilate at a concentration of 10 μM significantly prolonged this index up to 292.0% relative to the control (Table 3). When the concentration was further reduced to 5 and 1 μM, the thrombin time was also significantly prolonged by 208.6 and 51.7%, respectively. At a concentration of 0.1 μM, dabigitran etexilate had no effect on this parameter. The EC50 of antithrombin activity of the comparison drug under conditions of hypercytokinemia was 0.76 μM (Table 3).
Compound 5d at a concentration of 10 μM significantly prolonged the thrombin time index up to 354.1% relative to the control. When reducing the concentration of the substance to 5 and 1 μM, the thrombin time statistically significantly increased by 234.1 and 56.3%, respectively, relative to control values. At a concentration of 0.1 μM, compound 5d did not affect this parameter. The EC50 of antithrombin activity under conditions of hypercytokinemia was equal to 0.78 μM (Table 3).
Thus, under conditions of hypercytokinemia, compound 5d and dabigatran etexilate were comparable in terms of EC50 of antithrombin activity.

2.2.2. Ecarin Clotting Time

The ecarin time of PPP control samples was 39.95 ± 0.96. The study of PPP with the addition of the comparison drug dabigatran etexilate at a concentration of 100 μM resulted in a 2.3-fold lengthening of the time of stable clot formation compared to control values. In another step of the experiment, the concentration of dabigatran etexilate was reduced to 10 and 1 μM, and as a result, there was a linear shortening of the studied index to 54.1 and 41.6 s, respectively.
Compound 5d with an initial concentration of 100 μM showed a prolongation of the clotting time to 81.3 secs. At concentrations of 10 and 1 μM, there was a change in the clotting time to 50.5 and 40.2 secs, respectively.
Based on the data obtained, the EC50 was calculated. The EC50 of the comparison drug was 10.5 μM, while the EC50 of the compound under study was 17.9 μM. As a result of this study, the direct antithrombin effect of compound 5d was shown to be comparable with the comparison drug dabigatran etexilate in terms of EC50 (Figure 2).
On the basis of the obtained data, it is possible to state that both of the tested compounds exhibit a pronounced direct antithrombin action.

2.2.3. An Animal Study of Anticoagulant Activity

Dabigatran etexilate showed activity in blood for both group of intact animals, having hypercytokinemia and not. Thus, compound 5d was administrated intragastricaly to these both groups of intact animals in a series of experiments in amount equal to dabigatran etexilate. Parameters of the coagulograms obtained in experiments using animal blood are presented in Table 4.
In experiments on intact control rats, the APTT was 38.3 s (Table 4). At the same time, dabigatran etexilate at a dose of 12 mg/kg significantly prolonged this indicator by 3.6 times within 2 h after intragastric administration. Compound 5d at a dose equimolar to dabigatran etexilate of 5.5 mg/kg significantly increased this indicator compared to the control by 1.5 times within 2 h after administration.
The thrombin time of the blood of the intact control group of rats was 57.7 min. The reference drug dabigatran etexilate and compound 5d increased this indicator by 10.5 and 5.6 times, respectively.
The prothrombin time did not change under the influence of the test substance and dabigatran etexilate (Table 4).
Coagulogram parameters in control blood samples changed significantly under conditions of a systemic inflammatory reaction. The APTT decreased by 2.1 times compared to intact control animals, and the thrombin time also decreased by 1.3 times, indicating the activation of coagulation hemostasis in rats.
Dabigatran etexilate significantly prolonged the APTT in the blood of LPS-injected rats, increasing it by 2.2 times in relation to the control group. At the same time, the thrombin time statistically significantly increased by 12.8 times. Compound 5d also significantly prolonged the APTT by 1.6 times. At the same time, the thrombin time in the rat blood increased 14.5 times.
However, neither the test compound nor dabigatran etexilate had an effect on the prothrombin time. Thus, under conditions of a systemic inflammatory reaction, compound 5d showed a more pronounced antithrombin effect than in its absence, which indicated the possible effect of this substance on the inflammatory components of blood (Table 4).

3. Materials and Methods

3.1. Chemistry

Commercial reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), Acros Organics (Waltham, MA, USA), or Alfa Aesar (Haverhill, MA, USA) and used without any further purification. All workup and purification procedures were carried out using analytical-grade solvents. 1H NMR spectra were recorded in DMSO-d6 solution on “Bruker DRX-400” devices using TMS and DMSO-d6 as an internal standard. The following abbreviations were used for NMR signals: s—singlet, d—doublet, t—triplet, q—quartet, dd—double doublet, m—multiplet, br—broaded. Electrospray ionization mass spectra were recorded for positive ions on a qTOF maXis Impact HD ultra-high resolution mass spectrometer (Bruker Daltonics, Billerica, MA, USA), with a standard ionization source in the mass range 50–2500 Da, by injection analysis for sample solutions in acetonitrile using a syringe pump inlet (model No. 601553 kdScientific Inc., Holliston, MA, USA); solution infusion rate of 240 µL/h) using a modified “Direct_Infusion 100–1000” preset method. Calibration of the mass scale was external, according to the signals of a lithium acetate solution using HPC or improved quadratic methods. All data were collected and processed using the Compass for oTof series 1.7 software package (oTOF Control 3.4; Bruker Compass DataAnalysis 4.2). IR spectra were recorded on a Spectrum One FT-IT spectrometer (Perkin Elmer, Waltham, MA, USA) in a range of 4000–400 cm−1 using a diffuse reflectance attachment. Elemental analysis was performed on a PerkinElmer PE 2400 elemental analyzer. Melting points were determined on a Stuart SMP3 and were uncorrected. The monitoring of reaction progress was performed using TLC on Silufol UV254 plates. Column chromatography was performed on Chromagel (silica gel, 400 mesh).
Compounds 1a–j, 5a–d, 8a,b, 9a,b, 8c, and 15a,b were synthesized in accordance with literature data: 1a–j [16], 5a–d [26], 8a,b, 9a,b [12], 8c [29], and 15a,b [30]. All synthesized compounds were >96% pure by elemental analysis. 1H and 13C NMR spectra of compounds 7a,c, 10a,c, 12a,c, 14, and 16a–c are given in the Supplementary Materials section (see in Figures S1–S10).

3.1.1. General Procedure for the Preparation of Nitro Derivatives of Azolo[1,5-a]pyrimidines 4c,d (Scheme 2)

K2CO3 (1.0 mmol) was added to chalcone solution 1 (0.7 mmol), obtained by method [5] or [32] for 1a or 1b, and to aminoazole (1.0 mmol) in 10 mL of dimethylformamide. The mixture was stirred at 100 ℃ for 32 h. Water was added to the mixture upon completion of the process, and the formed precipitate was filtered. The product, eluting with chloroform, was purified chromatographically on a preparative column (SiO2) and crystallized from CH3CN:
5,7-Bis(4-nitrophenyl)imidazo[1,2-a]pyrimidine 4c: light yellow solid; mp = 334–336 °C. Yield: 200 mg (83%). 1H NMR (400 MHz, DMSO-d6) δ, 8.61 (m (AA’BB’), 2 H, phenyl), 8.81 (m (AA’BB’), 2 H, phenyl), 8.41 (m (AA’BB’), 2 H, phenyl), 8.25 (m (AA’BB’), 2 H, phenyl), 8.05 (s, 1 H, CH, imidazopyrimidine), 8.00 (d, 1 H, J = 1.4 Hz, imidazopyrimidine), 7.94 (d, 1 H, J = 1.4 Hz, imidazopyrimidine). 13C NMR (101 MHz, DMSO-d6) δ 152.93, 148.73, 148.67, 148.45, 144.27, 142.58, 137.73, 136.79, 130.24, 128.41, 124.23, 123.97, 110.35, 106.74. HRMS(ESI): calculated for C18H12N5O4 [M + H]+: 362.0884, found: 362.0885; IR νmax (cm−1): 3145, 3105, 3073, 1601, 1587, 1515, 1483, 1340, 1278, 1255, 1140, 1102, 8492, 830, 754, 732, 696. Anal. calcd. for C18H11N5O4: C, 59.84; H, 3.07; N, 19.38. Found: C, 59.87; H, 3.05; N, 19.40.
5-(4-Nitrophenyl)-7-(thiophen-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine 4d: light yellow solid; mp = 246–247 °C. Yield: 210 mg (84%). 1H NMR (400 MHz, DMSO-d6) δ, 8.89 (s, 1 H, CH, triazolopyrimidine), 8.85 (dd, 1 H, J = 1.0, 4.0 Hz, thienyl), 8.69 (m (AA’BB’), 2 H, phenyl), 8.66 (s, 1 H, CH, triazolopyrimidine), 8.45 (m (AA’BB’), 2 H, phenyl), 8.26 (dd, 1 H, J = 1.0, 4.9 Hz, thienyl), 7.49 (dd, 1 H, J = 4.0, 4.9 Hz, thienyl). 13C NMR (101 MHz, DMSO-d6) δ 157.63, 156.38, 155.39, 148.84, 142.10, 141.79, 136.22, 133.77, 129.83, 129.07, 128.21, 123.93, 103.49. HRMS(ESI): calculated for C15H10N5O2S [M + H]+: 324.0550, found: 324.0551; IR νmax (cm−1): 3110, 3067, 2937, 1543, 1519, 1488,1417, 1344, 1318, 1288, 1261, 1247, 1188, 1108, 1089, 865, 854, 838, 825, 757. Anal. calcd. for C15H9N5O2S: C, 55.72; H, 2.81; N, 21.66. Found: C, 55.70; H, 2.84; N, 21.63.

3.1.2. General Procedure for the Preparation of Di(het)aryl-Substituted Azolo[1,5-a]pyrimidines 7a,b (Scheme 2)

The reduction of nitro derivatives to anilines was carried out according to the standard procedure. Compounds 4c,d (0.2 g) were dissolved in a heated mixture of THF and ethanol (1:1); Raney nickel was added, followed by dropwise addition of hydrazine hydrate (1–2 mL). The reduction was carried out until the complete disappearance of the starting compound, testing by TLC (eluent: chloroform/ethanol, 50:1). The following products were crystallized from ethanol:
4,4′-(Imidazo[1,2-a]pyrimidine-5,7-diyl)dianiline 7a: light yellow solid; mp = 169–171 °C. Yield: 145 mg (87%). 1H NMR (400 MHz, DMSO-d6) δ, 7.99 (m (AA’BB’), 2 H, phenyl), 7.79 (d, 1 H, J = 1.4 Hz, imidazopyrimidine), 7.60 (d, 1 H, J = 1.4 Hz, imidazopyrimidine), 7.57 (m (AA’BB’), 2 H, phenyl), 7.34 (s, 1 H, CH, imidazopyrimidine), 6.75 (m (AA’BB’), 2 H, phenyl), 6.66 (m (AA’BB’), 2 H, phenyl), 5.82 (s, 2 H, NH2) 5.68 (s, 2 H, NH2) (Figure S1-1). 13C NMR (101 MHz, DMSO-d6) δ 156.22, 151.36, 151.21, 149.79, 146.33, 134.06, 129.32, 128.44, 124.05, 118.50, 113.56, 113.50, 108.78, 102.65 (Figure S1-2). HRMS(ESI): calculated for C18H16N5 [M + H]+: 302.1400, found: 302.1401; IR νmax (cm−1): 3444, 3334, 3204, 3029, 1594, 1530, 1498, 1471, 1385, 1293, 1273, 1241, 1178, 1139, 825, 708, 684. Anal. calcd. for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found: C, 71.71; H, 5.06; N, 23.20.
4-(7-(Thiophen-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)aniline 7b: light yellow solid; mp = 211–214 °C. Yield: 150 mg (83%). 1H NMR (400 MHz, DMSO-d6) δ, 8.72 (dd, 1 H, J = 1.0, 4.0 Hz, thienyl), 8.65 (s, 1 H, CH, triazolopyrimidine), 8.27 (s, 1 H, CH, triazolopyrimidine), 8.16 (m (AA’BB’), 2 H, phenyl), 8.15 (dd, 1 H, J = 1.0, 4.9 Hz, thienyl), 8.43 (dd, 1 H, J = 4.0, 4.9 Hz, thienyl), 6.71 (m (AA’BB’), 2 H, phenyl), 5.92 (s, 2 H, NH2) (Figure S2-1). 13C NMR (101 MHz, DMSO-d6) δ, 160.51, 155.70, 155.25, 152.23, 140.36, 134.59, 132.61, 130.28, 129.33, 127.95, 122.82, 113.45, 101.46 (Figure S2-2). HRMS(ESI): calculated for C15H12N5S [M + H]+: 294.0808, found: 294.0806; IR νmax (cm−1): 3559, 3421, 3326, 3215, 3099, 1545, 1504, 1383, 1317, 1302, 1245, 1197, 1178, 825, 773, 727. Anal. calcd. for C15H11N5S: C, 61.42; H, 3.78; N, 23.87. Found: C, 61.45; H, 3.76; N, 23.88.

3.1.3. General Procedure for the Preparation of 7-Aryl-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylates 10a,b (Scheme 2)

A mixture of 20 mL of anhydrous CH2Cl2 and 0.6 mmol of Boc-glycine was heated to 45 °C; then, 10.1 mmol of TBTU and 0.9 mmol of DIPEA were added and stirred for 25–30 min. After that, 1 mmol of compound 8a or 8b was added, and the mixture was continuously stirred for 48 h at 45 °C. Further, the reaction mixture was kept without heating or stirring for another 48 h. The reaction mixture was filtered through thin layer of silicaand washed with CH2Cl2/ethyl acetate (1:1). To remove the protective group, intermediate compound 9a or 9b was dissolved in 1 mL of CF3COOH and left in air until the acid completely evaporated. The precipitate was washed with NaHCO3 solution and water, then dried in air. If necessary, product 10 was crystallized from THF.
Ethyl 7-(3-(2-aminoacetamido)phenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimi-dine-6-carboxylate 10a: yellow powder; mp = 270–272 °C. Yield: 320 mg (63%). 1H NMR (400 MHz, DMSO-d6) δ, 10.84 (br.s, 1H, NH), 10.46 (br.s, 1H, NH), 7.88 (br.s, 2H, NH2), 7.65 (s, 1H, CH), 7.50–7.48 (m, 2H, Ar), 7.28 (t, J = 7.8 Hz, 1H, Ar), 7.00 (d, J = 7.7 Hz, 1H, Ar), 6.24 (s, 1H, CH), 4.00–3.93 (m, 2H, CH2, OEt), 3.71 (s, 2H, CH2), 2.42 (s, 3H, Me), 1.06 (t, J = 7.1 Hz, 3H, CH3, OEt) (Figure S3-1). 13C NMR (101 MHz, DMSO-d6) δ, 165.4, 165.2, 157.7, 150.1, 146.4, 141.3, 140.1, 126.9, 122.4, 119.3, 116.5, 97.1, 59.5, 59.1, 41.2, 18.4, 14.0 (Figure S3-2). FT-IR (neat) νmax (cm−1): 2947, 1644, 1596, 1509, 1289, 1100. Anal. calcd. for C17H20N6O3: C 57.29; H 5.66; N 23.58. Found: C 57.27; H 5.16; N 23.59.
Ethyl 7-(4-(2-aminoacetamido)phenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimi-dine-6-carboxy-late 10b: yellow powder; mp = 270–272 °C. Yield: 320 mg (63%). 1H NMR (400 MHz, DMSO-d6) δ, 10.80 (br.s, 1H, NH), 10.46 (br.s, 1H, NH), 7.94 (br.s, 2H, NH2), 7.65 (s, 1H, CH), 7.51 (d (AA’BB’), J = 8.4 Hz, 2H, Ar), 7.21 (d, (AA’BB’), J = 8.4 Hz, 2H, Ar), 6.23 (s, 1H, CH), 4.00–3.92 (m, 2H, CH2, OEt), 3.73 (s, 2H, CH2, 2.41 (s, 3H, Me), 1.04 (t, J = 7.1 Hz, 3H, CH3, OEt) (Figure S4-1). 13C NMR (101 MHz, DMSO-d6) δ, 165.2, 165.1, 157.8, 150.1, 146.7, 137.8, 137.6, 127.7, 119.1, 97.1, 41.2, 59.3, 59.0, 18.4, 13.9 (Figure S4-2). FT-IR (neat) νmax (cm−1): 2947, 1644, 1596, 1509, 1289, 1100. Anal. calcd. for C17H20N6O3: C 57.29; H 5.66; N 23.58. Found: C 57.12; H 5.69; N 23.60.

3.1.4. The General Synthesis of 6-Nitro-7-methoxy-4,7-dihydro-azolyl-4 guanidates 12a,b, 14 (Scheme 3)

A 1 M solution of free guanidine in methanol (1 mL) was added to a solution of 1 mmol of the starting 6-nitroazolopyrimidine in 5 mL of methanol. The solution was stirred at 60 °C for 10 min; the precipitated product was separated by filtration, washed with cold methanol, and dried in a vacuum desiccator.
2-Trifluoromethyl-6-nitro-7-methoxy-4,7-dihydro[1,5-a]pyrimidyl-4 guanidate 12a: yellow powder; mp = 167–168 °C. Yield: 240 mg (74%). 1H NMR (400 MHz, DMSO-d6) δ, 8.51 (s, 1H, H5), 6.97 (br.s, 6H), 6.70 (s, 1H, H7), 3.26 s, 3H, CH3) (Figure S5-1). 13C NMR (126 MHz, DMSO-d6) δ, 157.95, 157.15, 151.57, 151.25 (q), 119.72 (q), 116.89, 83.24, 55.31 (Figure S5-2). IR νmax (cm−1): 1000 (C-F); 1670, 1686 (C=N), 3141, 3359 (NH). Anal. calcd. for C8H11N8O3F3C: C 29.63; H 3.40; N 34.22. Found: 29.50; H 3.46; N 34.44.
2-Cyclopentylthio-6-nitro-7-methoxy-4,7-dihydro[1,5-a]pyrimidyl-4 guanidate 12b: yellow powder; mp = 164–166 °C. Yield: 275 mg (77%). 1H NMR (500 MHz, DMSO-d6) δ, 8.43 (s, 1H, H5), 6.59 (br.s, 6H), 6.51 (s, 1H, H7), 3.83–3.78 (m, 1H), 3.21 (c, 3H, CH3), 2.13–2.06 (m, 2H), 1,70–1.55 (2m, 6H) (Figure S6-1). 13C NMR (126 MHz, DMSO-d6) δ, 158.66, 157.86, 156.54, 150.95, 116.36, 82.52, 54.90, 43.83, 33.51, 33.23, 24.31, 24.28 (Figure S6-2). IR νmax (cm−1): 1682 (C=N), 3107, 3325 (NH). Anal. calcd. for C12H20N8O3S: C 40.45; H 5.62; N 31.46. Found: C 40.59; H 5.57; N 31.30.
6-Nitro-7-hydroxy-4,7-dihydro-1,2,4-triazolo[5,1-c]triazinyl-4 guanidate 14: burgundy powder; mp > 300 °C. Yield: 165 mg (68%). 1H NMR (500 MHz, DMSO-d6) δ, 7.80 (s, 1H, H2), 7.07 (br.s, 6H), 6.91 (s, 1H, H7) (Figure S7-1). 13C NMR (126 MHz, DMSO-d6), δ, 157.91, 154.47, 150.99, 140.66, 72.06 (Figure S7-2). IR νmax (cm−1): 1680 (C=N), 3107, 3318 (NH). Anal. calcd. for C5H9N9O3: C 24.50; H 3.79; N 51.71. Found: C 24.69; H 3.70; N 51.85.

3.1.5. The General Synthetic Method of 6-Guanidino-3-alkylthio-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazines 16a–c (Scheme 5)

1.0 mL (C = 1.0 mmol·mL−1) of freshly prepared solution of free guanidine in methanol was added dropwise to a mixture of 1 mmol of the corresponding [1,2,4]triazolo[1,2,4,5]tetrazine in 10 mL of acetonitrile while stirring on a magnetic stirrer. The precipitate of 6-guanidino-3-alkylthio-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine that formed after 15 min was filtered, washed on the filter with acetonitrile, and crystallized from water or acetonitrile.
6-Guanidino-3-octylthio-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine 16a: yellow powder; mp > 260 °C. Yield: 266 mg (82%). 1H NMR (400 MHz, DMSO-d6) δ, 0.83 (t, 3H, CH3, J = 7.0 Hz); 1.23–1.26 (m, 8 H, 4 CH2); 1.36–1.39 (m, 2 H, CH2); 1.63–1.69 (m, 2 H, CH2); 3.22–3.25 (t, 2 H, SCH2, J = 7.0 Hz); 7.20 (br.s, 4H guanidino) (Figure S8-1). 13C NMR (101 MHz, DMSO-d6) δ, 13.9, 22.0, 27.8, 28.3, 28.5, 29.4, 31.1, 31.4, 142.3, 149.9, 158.4, 159.8 (Figure S8-2). IR νmax (cm−1): 1639 (C=NH, guanidino), 3414,3447 (N-H, guanidino). Anal. calcd. for C12H21N9S: C, 44.56; H, 6.54; N, 38.98. Found: C, 44.26; H, 6.70; N, 39.23.
6-Guanidino-3-cyclopentylthio-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine 16b: orange powder; mp = 252–253 °C. Yield: 142 mg (51%). 1H NMR (400 MHz, DMSO-d6) δ, 1.58–1.67, 1.70–1.77, 2.05–2.12 (3 m, 8 H, 4 CH2); 3.96–4.01 (m, 1 H, CH, cyclopentyl); 7.24 (br.s, 4H guanidino) (Figure S9-1). 13C NMR (101 MHz, DMSO-d6) δ, 24.1, 33.4, 45.4, 142.1, 149.8, 158.5, 159.8. IR νmax (cm−1): 1644 (C=NH, guanidino), 3381,3416 (N-H, guanidino) (Figure S9-2). Anal. calcd. for C9H13N9S: C, 38.70; H, 4.69; N, 45.13. Found: C, 38.31; H, 4.70; N, 45.23.
6-Guanidino-3-(3-fluorophenylmethylthio[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine 16c: yellow powder; mp = 235–237 °C. Yield: 280 mg (87%). 1H NMR (400 MHz, DMSO-d6) δ, 4.49 (c, 2 H, SCH2); 7.05–7.33 (m, 8H, guanidino, Ph) (Figure S10-1). 13C NMR (101 MHz, DMSO-d6) δ, 34.7, 114.3 (d, J =20.9), 125.6 (d, J = 21.8 Hz), 124.9 (d, J =2.6 Hz), 130.30 (d, J= 8.4 Hz), 139.9 (d, J = 7.7 Hz), 141.3, 149.9, 158.4, 159.7, 161.8 (d, J = 243.8 Hz) (Figure S10-2). IR-spectra, IR νmax (cm−1): 1645 (C=NH, guanidino), 3382, 3424 (N-H, guanidino). Anal. calcd. for C11H10FN9S: C, 41.37; H, 3.16; N, 39.48. Found: C, 41.62; H, 3.30; N, 39.38.

3.2. Biology

3.2.1. Animals

All animal procedures were carried out under the generally accepted ethical standards for animal testing adopted by the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (1986) and taking into account the International Recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental Research (1997). This study was approved by the Local Ethics Committee of Volgograd State Medical University (registration no. IRB 00005839 IORG 0004900, OHRP), certificate no. 2021/056, 15 June 2021. All sections of this study adhered to the ARRIVE Guidelines for reporting animal research [33]. The experiments were carried out on 10 male Chinchilla rabbits weighing 3.0–3.5 kg and 60 outbred albino male rats weighing 250–270 g. Animals were kept under standard vivarium conditions (22–24 °C, 40–50% humidity, ambient light) during the study. The control and experimental groups included 10 samples each.

3.2.2. In Vitro Anticoagulant Assay

The study was performed on platelet-poor plasma (PPP) stabilized with a 3.8% sodium citrate solution in a ratio of 9:1 according to the method described in [34]. Dabigatran etexilate (Boehringer Ingelheim Pharma GmbH and Co., Ingelheim am Rhein, Germany) was used as the reference drug. Test compounds and the reference drug were evaluated at a concentration of 100 μM. Effect on rabbit blood coagulograms in vitro was determined chronometrically with a SOLAR hemocoagulometer (Minsk, Belarus) using commercial kits (Technology-Standard, Barnaul, Russia) as per the manufacturer’s instructions. The following parameters were determined: activated partial thromboplastin time, thrombin time, and prothrombin time.
Hypercytokinemia conditions were modeled by incubation of whole blood with S. typhimurium LPS (Sigma Aldrich, St. Louis, MO, USA) at a final concentration of 20 ng/mL and subsequent preparation of PPP. Compounds that showed high dose-dependent prolongation of thrombin time with LPS treatment and without were assessed for IC50 values using the regression analysis method in the Microsoft Excel 2007 program (Microsoft Corporation, Albuquerque, NM, USA).

3.2.3. Ecarin Clotting Time

The ecarin clotting time was designed for the determination of antithrombin anticoagulant activity in platelet-poor plasma [35]. Venous blood was collected into a 15 mL Falcon type plastic tube with 3.8% sodium citrate solution (0.5 mL of sodium citrate per 5 mL of blood), centrifuged on a CM-6M centrifuge (Elmi, Riga, Latvia) at room temperature from 18 to 25 °C for 15 min at 3000 rpm (1200× g). The obtained plasma was withdrawn using an automatic pipette and transferred to a separate clean, dry plastic tube. After that, the obtained plasma was recentrifuged under the same conditions and the upper third of the plasma was taken for analysis.
The ecarin stock solution (STA-ECT, Stago, Düsseldorf, Germany) was prepared according to the manufacturer’s instructions and diluted to 4 units/mL with HEPES-buffered saline (0.2 M) containing 0.025 M calcium chloride.
The most active compound was selected as the study sample and the anticoagulant drug dabigatran etexilate was chosen as the comparison drug. The tested samples were investigated in the concentration range from 100 to 1 μM for IC50 determination.
Prepared reagents and platelet-poor plasma were heated to 37 °C. The assay was performed on a Solar CGL2120 coagulometer (Belarus), adding 50 µL of the reagent ecarin to 100 µL of PPP and recording the clotting time. As for substances exhibiting antithrombin action, the test was performed by adding 10 µL of the compound to 100 µL of PPP followed by an incubation for 5 min, after which 50 µL of the reagent ecarin was added and the clotting time was recorded.

3.2.4. Anticoagulant Assay in Animals

The most active compound was studied on male rats in vivo, in a single intragastric administration in a volume of no more than 2 mL. Distilled water was used as a solvent. In all experiments, control animals were injected with an equivalent volume of a vehicle. The reference drug dabigatran etexilate was administered to rats 2 h before the study at a 12 mg/kg dose (equivalent to the human dose, taking into account the interspecies conversion factor). Compound 5d was administered to rats 2 h before the study in a 5.5 mg/kg dose, equimolar to the dose of dabigatran etexilate.
Blood was taken from the inferior vena cava of rats anesthetized with 50 mg/kg pentobarbital intraperitoneally. To stabilize the blood, a 3.8% aqueous solution of sodium citrate (pH 6.0) was used in a ratio of 9:1. Coagulogram parameters of platelet-poor plasma were measured with a SOLAR coagulometer according to the methods described above.
Hypercytokinemia conditions were modeled by an intravenous injection of S. typhimurium LPS (Sigma Aldrich, St. Louis, MO, USA) at a final dose of 2 mg/kg [36] into the tail vein of the rats 2 h before the administration of the substances. The evaluation of anticoagulant activity was conducted according to the methods described above.

3.2.5. Statistical Analysis

Biological data were analyzed with one-way ANOVA using Bonferroni’s multiple comparison correction in the Microsoft Excel 2007 spreadsheet editor, STATISTICA 5.0 (StatSoft, Inc., Tulsa, OK, USA) for Windows, and Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Data were presented as M + m, where m is a SEM. Changes were statistically significant if p < 0.05.
The calculation of EC50 (effective concentration that prolongs the time of clot formation by 50%) was performed using linear regression analysis.

4. Conclusions

Venous thromboembolism (VTE) is a serious clinical problem, associated with significant morbidity and mortality, including in viral and bacterial infections [37]. The process of thrombosis prevention is a delicate balance between the inhibition of hypercoagulability, stasis, and damage of the vascular wall. The basis for protecting the body from the effects of external and internal environments is inflammation and blood clotting, known as immunocoagulation, which is a part of innate immunity and can serve as the first line of defense against infection [38]. However, uncontrolled inflammation is associated with a pronounced reaction of the immune system, which leads to a hypercoagulable state, which is manifested by the development of macro- and microvascular thrombosis of the venous and arterial beds [39]. It is known that coagulation can be activated by extrinsic and intrinsic pathways, leading to the formation of fibrin. Preclinical and clinical studies have confirmed the pathological role of tissue factor, the initiator of the extrinsic pathway, in the development of endotoxemia [40]. It has been experimentally shown that exogenous lipopolysaccharide (LPS) can induce the expression and release of tissue factor on the cell surface and lead to septic death in mice [41].
In addition, hypercytokinemia, observed in sepsis, not only causes the activation of coagulation factors, but also suppresses anticoagulant pathways, such as the antithrombin system, activated protein C, and tissue factor inhibitor, thus leading to disseminated intravascular coagulation and fibrin deposition in the blood vessels and tissues. So, the interaction between increasing immune dysfunction and abnormal blood clotting is a major event favoring the complications of sepsis and multiple organ failure in humans and should be strategically targeted for therapeutic purposes [42]. Therefore, the use of anticoagulants for viral and bacterial infections helps prevent blood clots.
Direct thrombin inhibitors are important anticoagulant drugs, acting through the inhibition of the common pathway via factor IIa. These drugs can selectively bind to the active site of thrombin, inhibit thrombin activity, and exhibit a strong action and high specificity, and are important for the clinical treatment of thrombus diseases. Direct thrombin inhibitors are more efficient than other anticoagulants (such as heparin and warfarin), due to their higher capacity for inhibiting both free and bound thrombin, having a relatively safe pharmacological profile, and lacking the need for cofactors [43]. Therefore, the search for new direct thrombin inhibitors is currently in a great demand. A series of 27 novel azoloazine derivatives were evaluated in vitro for their anticoagulant properties by estimating the coagulogram parameters of rabbit blood. As a result, 26 compounds proved to prolong the thrombin time relative to the control, but to a lesser extent than the reference drug dabigatran etexilate. None of the studied compounds affected the prothrombin time.
It was established that the most active compound, 5d, had the highest ability to prolong the thrombin time, exceeding the reference drug dabigatran etexilate by 13.0 times, but it did not affect the prothrombin time, thus indicating its ability to inhibit thrombin.
At present, the gold standard for the study of direct oral anticoagulants is the ecarin clotting time, wherein ecarin maximizes thrombin activity and clotting time is evaluated to assess direct thrombin-inhibiting anticoagulation capability. Ecarin is a metalloprotease enzyme derived from the venom of the Indian saw-scaled viper. Ecarin cleaves prothrombin’s Arg320 peptide bond at the alanine–arginine–aspartic acid peptide motif, yielding the thrombin intermediate meizothrombin. Meizothrombin has up to 97% thrombin activity (compared with 10% without ecarin), and it converts fibrinogen to fibrin much more efficiently. Because direct thrombin inhibitors can inhibit not only thrombin, but also meizothrombin, ecarin-based assays are quite appropriate to estimate anticoagulation capability under optimal conditions, compared with conventional coagulation analyses [44]. Therefore, as the next step of our study, the effect of compound 5d on ecarin time was estimated. The results of the ecarin time study show that the direct antithrombin effect of the compound under investigation is comparable with the reference drug dabigatran etexilate in terms of EC50. This study demonstrates that the novel compound 5d (dimethyl-4,5-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine) exhibits direct antithrombin anticoagulant activity. Thus, among the class of compounds studied, substituted triazolopyrimidines and their hydrogenated analogues should be recognized as the most promising for further research on anticoagulants.
The antithrombin activity of compound 5d was confirmed using LPS-treated rabbit blood to mimic the conditions of cytokine release syndrome. Under conditions of hypercytokinemia, both compound 5d and dabigatran etexilate showed higher activity than those obtained for the blood of intact animals, and the EC50 values of their antithrombin activity proved to be comparable.
Compound 5d was also evaluated in animals via a single intragastric administration of 5d to rats in doses equimolar to those of dabigatran etexilate with and without hypercytokinemia conditions. Compound 5d and dabigatran etexilate proved to increase the thrombin time for normal animals by 5.6 and 10.5 times, respectively. In animals under conditions of a systemic inflammation reaction, the antithrombin activity of dabigatran etexilate did not change. The antithrombin effect of compound 5d was twice as pronounced than in the absence of a systemic inflammation reaction.
Thus, the enhancement of the anticoagulant effect of compound 5d under conditions of a systemic inflammatory response indicates the presence of anti-inflammatory activity. Consequently, this compound can make a significant contribution by influencing the pathogenetic links of immunocoagulation and thereby reducing the risk of thrombosis, including in conditions of viral and bacterial infections. It is difficult to predict the structural modifications that could lead to improved activity of compound 5d, but there should probably be a substituent on the thiophene rings.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242115581/s1.

Author Contributions

Conceptualization and methodology, A.A.S., G.L.R., O.V.F., E.V.V. and V.N.C.; funding acquisition, A.A.S. and V.N.C.; synthesis of organic compounds, N.A.R., I.G.O., R.I.I., N.K.I., E.B.G. and G.A.o.S.; studying of the anticoagulant activity, A.F.K., K.A.G. and V.S.S.; data curation, A.F.K., O.V.F. and N.A.R.; writing—review and editing, A.A.S., O.V.F., A.F.K., I.G.O., E.V.V. and N.A.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Higher Education of the Russian Federation (agreement on the provision of grants from the federal budget in the form of subsidies under paragraph 4 of article 78.1 of the Budget Code of the Russian Federation, Moscow, 1 October 2020, no. 075-15-2020-777).

Institutional Review Board Statement

This study was approved by the Local Ethics Committee of Volgograd State Medical University (registration no. IRB 00005839 IORG 0004900, OHRP), certificate no. 2021/056, 15 June 2021.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

Analytical studies were carried out using equipment from the Center for Joint Use “Spectroscopy and Analysis of Organic Compounds” at the Postovsky Institute of Organic Synthesis of the Ural Branch of the Russian Academy of Sciences.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Testa, S.; Prandoni, P.; Paoletti, O.; Morandini, R.; Tala, M.; Dellanoce, C.; Giorgi-Pierfranceschi, M.; Betti, M.; Danzi, G.B.; Pan, A.; et al. Direct Oral Anticoagulant Plasma Levels’ Striking Increase in Severe COVID-19 Respiratory Syndrome Patients Treated with Antiviral Agents: The Cremona Experience. J. Thromb. Haemost. 2020, 18, 1320–1323. [Google Scholar] [CrossRef] [PubMed]
  2. Manjunath, S.H.; Thimmulappa, R.K. Antiviral, immunomodulatory, and anticoagulant effects of quercetin and its derivatives: Potential role in prevention and management of COVID-19. J. Pharm. Anal. 2022, 12, 29–34. [Google Scholar] [CrossRef] [PubMed]
  3. Singh, A.K.; Gupta, R.; Ghosh, A.; Misra, A. Diabetes in COVID-19: Prevalence, pathophysiology, prognosis and practical considerations. Diabetes Metab. Syndr. Clin. Res. Rev. 2020, 14, 303–310. [Google Scholar] [CrossRef] [PubMed]
  4. Busch, M.; Franke, S.; Rüster, C.; Wolf, G. Advanced glycation end-products and the kidney. Eur. J. Clin. Investig. 2010, 40, 742–755. [Google Scholar] [CrossRef]
  5. Pinheiro, S.; Pinheiro, E.M.C.; Muri, E.M.F.; Pessôa, J.C.; Cadorini, M.A.; Greco, S.J. Biological Activities of [1,2,4]triazolo[1,5-a]pyrimidines and Analogs. Med. Chem. Res. 2020, 29, 1751–1776. [Google Scholar] [CrossRef]
  6. Gurevich, K.G.; Urakov, A.L.; Rozit, G.A.; Klen, E.É.; Samorodov, A.V.; Khaliullin, F.A. Synthesis and Antiplatelet and Anticoagulant Activity of Thietane-Containing 2-(5-Bromo-2,4-Dihydro-3-Oxo-1,2,4-Triazolyl-4)Acetate Salts. Pharm. Chem. J. 2021, 55, 417–422. [Google Scholar] [CrossRef]
  7. Muhammad, Z.A.; Farghaly, T.A.; Althagafi, I.; Al-Hussain, S.A.; Zaki, M.E.A.; Harras, M.F. Synthesis of antimicrobial azoloazines and molecular docking for inhibiting COVID-19. J. Heterocycl. Chem. 2021, 58, 1286–1301. [Google Scholar] [CrossRef]
  8. Rusinov, V.L.; Sapozhnikova, I.M.; Spasov, A.A.; Chupakhin, O.N. Fused azoloazines with antidiabetic activity. Russ. Chem. Bull. 2022, 71, 2561–2594. [Google Scholar] [CrossRef]
  9. Ilin, I.; Lipets, E.; Sulimov, A.; Kutov, D.; Shikhaliev, K.; Potapov, A.; Krysin, M.; Zubkov, F.; Sapronova, L.; Ataullakhanov, F.; et al. New factor Xa inhibitors based on 1,2,3,4-tetrahydroquinoline developed by molecular modelling. J. Mol. Graph. Model. 2019, 89, 215–224. [Google Scholar] [CrossRef]
  10. Savateev, K.V.; Ulomsky, E.N.; Fedotov, V.V.; Rusinov, V.L.; Sivak, K.V.; Lyubishin, M.M.; Kuzmich, N.N.; Aleksandrov, A.G. 6-Nitrotriazolo[1,5-a]pyrimidines as Promising Structures for Pharmacotherapy of Septic Conditions. Russ. J. Bioorg. Chem. 2017, 43, 421–428. [Google Scholar] [CrossRef]
  11. Esteban-Parra, G.M.; Sebastián, E.S.; Cepeda, J.; Sánchez-González, C.; Rivas-García, L.; Llopis, J.; Aranda, P.; Sánchez-Moreno, M.; Quirós, M.; Rodríguez-Diéguez, A. Anti-diabetic and anti-parasitic properties of a family of luminescent zinc coordination compounds based on the 7-amino-5-methyl-1,2,4-triazolo[1,5-a]pyrimidine ligand. J. Inorg. Biochem. 2020, 212, 111235. [Google Scholar] [CrossRef] [PubMed]
  12. Wang, B.S.; Huang, X.; Chen, L.Z.; Liu, M.M.; Shi, J.B. Design and synthesis of novel pyrazolo[4,3-d]pyrimidines as potential therapeutic agents for acute lung injury. J. Enzym. Inhib. Med. Chem. 2019, 34, 1121–1130. [Google Scholar] [CrossRef] [PubMed]
  13. Ishmetova, R.I.; Ignatenko, N.K.; Korotina, A.V.; Ganebnykh, I.N.; Slepukhin, P.A.; Babkova, V.A.; Gerasimova, N.A.; Evstigneeva, N.P.; Zilberberg, N.V.; Kungurov, N.V.; et al. Synthesis and biological activity of 3-guanidino-6-R-imidazo[1,2-b]- and 6-guanidino-3-R-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazines. Russ. Chem. Bull. 2018, 67, 2079–2087. [Google Scholar] [CrossRef]
  14. Klimenko, K.; Lyakhov, S.; Shibinskaya, M.; Karpenko, A.; Marcou, G.; Horvath, D.; Zenkova, M.; Goncharova, E.; Amirkhanov, R.; Krysko, A.; et al. Virtual screening, synthesis and biological evaluation of DNA intercalating antiviral agents. Bioorg. Med. Chem. Lett. 2017, 27, 3915–3919. [Google Scholar] [CrossRef]
  15. Kunjiappan, S.; Theivendren, S.; Pavadai, P.; Govindaraj, S.; Sankaranarayanan, M.; Somasundaram, B.; Arunachalam, S.; Pandian, S.R.K.; Damodar, N.A. Design and in silico modeling of Indoloquinoxaline incorporated keratin nanoparticles for modulation of glucose metabolism in 3T3-L1 adipocytes. Biotechnol. Prog. 2020, 36, e2904. [Google Scholar] [CrossRef]
  16. Sadykhov, G.A.; Belyaev, D.V.; Vakhrusheva, D.V.; Eremeeva, N.I.; Khramtsova, E.E.; Pervova, M.G.; Rusinov, G.L.; Verbitskiy, E.V.; Chupakhin, O.N.; Charushin, V.N. New Approach to Biologically Active Indolo[2,3-b]quinoxaline Derivatives through Intramolecular Oxidative Cyclodehydrogenation. ChemistrySelect 2022, 7, e202200497. [Google Scholar] [CrossRef]
  17. Oukoloff, K.; Lucero, B.; Francisco, K.R.; Brunden, K.R.; Ballatore, C. 1,2,4-Triazolo[1,5-a]pyrimidines in drug design. Eur. J. Med. Chem. 2019, 165, 332–346. [Google Scholar] [CrossRef]
  18. Yu, W.; Goddard, C.; Clearfield, E.; Mills, C.; Xiao, T.; Guo, H.; Morrey, J.D.; Motter, N.E.; Zhao, K.; Block, T.M.; et al. Design, synthesis, and biological evaluation of triazolo-pyrimidine derivatives as novel inhibitors of hepatitis B virus surface antigen (HBsAg) secretion. J. Med. Chem. 2011, 54, 5660–5670. [Google Scholar] [CrossRef]
  19. Girasolo, M.A.; Canfora, P.; Sabatino, D.; Schillaci, E.; Foresti, S.; Rubino, G.; Ruisi, G.S. Synthesis, characterization, crystal structures and in vitro antistaphylococcal activity of organotin(IV) derivatives with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine. J. Inorg. Biochem. 2012, 106, 156–163. [Google Scholar] [CrossRef]
  20. Bhatt, J.D.; Chudasama, C.J.; Patel, K.D. Pyrazole clubbed triazolo[1,5-a]pyrimidine hybrids as an anti-tubercular agents: Synthesis, in vitro screening and molecular docking study. Bioorg. Med. Chem. 2015, 23, 7711–7716. [Google Scholar] [CrossRef]
  21. Gamal-Eldeen, A.M.; Hamdy, N.A.; Abdel-Aziz, H.A.; El-Hussieny, E.A.; Fakhr, I.M.I. Induction of intrinsic apoptosis pathway in colon cancer HCT-116 cells by novel 2-substituted-5,6,7,8-tetrahydronaphthalene derivatives. Eur. J. Med. Chem. 2014, 77, 323–333. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, T.Y.; Li, C.S.; Li, P.; Bai, X.Q.; Guo, S.Y.; Jin, Y.; Piao, S.J. Synthesis and evaluation of ursolic acid-based 1,2,4-triazolo[1,5-a]pyrimidines derivatives as anti-inflammatory agents. Mol. Divers. 2022, 26, 27–38. [Google Scholar] [CrossRef] [PubMed]
  23. Mąkosza, M.; Wojciechowski, K. Nucleophilic Substitution of Hydrogen in Heterocyclic Chemistry. Chem. Rev. 2004, 104, 2631–2666. [Google Scholar] [CrossRef]
  24. Mąkosza, M. Nucleophilic substitution of hydrogen in electron-deficient arenes, a general process of great practical value. Chem. Soc. Rev. 2010, 39, 2855–2868. [Google Scholar] [CrossRef] [PubMed]
  25. Verbitskiy, E.V.; Rusinov, G.L.; Chupakhin, O.N.; Charushin, V.N. Recent Advances in Direct C–H Functionalization of Pyrimidines. Synthesis 2018, 50, 193–210. [Google Scholar] [CrossRef]
  26. Rasputin, N.A.; Demina, N.S.; Irgashev, R.A.; Rusinov, G.L.; Chupakhin, O.N.; Charushin, V.N. Direct (het)arylation of [1,2,4]triazolo[1,5-a]pyrimidines: Both eliminative and oxidative pathways. Tetrahedron 2017, 73, 5500–5508. [Google Scholar] [CrossRef]
  27. Bhagat, S.; Sharma, R.; Sawant, D.M.; Sharma, L.; Chakraborti, A.K. LiOH·H2O as a Novel Dual Activation Catalyst for Highly Efficient and Easy Synthesis of 1,3-Diaryl-2-Propenones by Claisen–Schmidt Condensation under Mild Conditions. J. Mol. Catal. A Chem. 2006, 244, 20–24. [Google Scholar] [CrossRef]
  28. Wei, W.; Qunrong, W.; Liqin, D.; Aiqing, Z.; Duoyuan, W. Synthesis of Dinitrochalcones by Using Ultrasonic Irradiation in the Presence of Potassium Carbonate. Ultrason. Sonochem. 2005, 12, 411–414. [Google Scholar] [CrossRef]
  29. Fedorova, O.V.; Zhidovinova, M.S.; Rusinov, G.L.; Ovchinnikova, I.G. Aminoazoles in the three component synthesis of 7substituted 6ethoxycarbonyl5methyl4,7dihydroazolo[1,5a]pyrimidines. Russ. Chem. Bull. Int. Ed. 2003, 52, 1768–1769. [Google Scholar] [CrossRef]
  30. Ishmetova, R.I.; Babkov, D.A.; Kucheryavenko, A.F.; Babkova, V.A.; Sirotenko, V.S.; Ignatenko, N.K.; Tolschina, S.G.; Vassiliev, P.M.; Rusinov, G.L.; Spasov, A.A. In silico consensus activity prediction, rational synthesis, and evaluation of antiglycation and antiplatelet activities of 3,6-disubstituted 1,2,4,5-tetrazines. Russ. Chem. Bull. 2020, 69, 768–773. [Google Scholar] [CrossRef]
  31. Quan, M.L.; Pinto, D.J.P.; Smallheer, J.M.; Ewing, W.R.; Rossi, K.A.; Luettgen, J.M.; Seiffert, D.A.; Wexler, R.R. Factor XIa Inhibitors as New Anticoagulants. J. Med. Chem. 2018, 61, 7425–7447. [Google Scholar] [CrossRef]
  32. Abd El-Sattar, N.E.A.; Badawy, E.H.K.; Abdel-Mottaleb, M.S.A. Synthesis of Some Pyrimidine, Pyrazole, and Pyridine Derivatives and Their Reactivity Descriptors. J. Chem. 2018, 2018, 8795061. [Google Scholar] [CrossRef]
  33. Du Sert, N.P.; Ahluwalia, A.; Alam, S.; Avey, M.T.; Baker, M.; Browne, W.J.; Clark, A.; Cuthill, I.C.; Dirnagl, U.; Emerson, M.; et al. Reporting Animal Research: Explanation and Elaboration for the ARRIVE Guidelines 2.0. PLoS Biol. 2020, 18, e3000411. [Google Scholar] [CrossRef]
  34. Spasov, A.A.; Kucheryavenko, A.F.; Gaidukova, K.A.; Kosolapov, V.A.; Zhukovskaya, O.N. Antiplatelet Activity of New Derivatives of Benzimidazole Containing Sterically Hindered Phenolic Group in Their Structure. Res. Result. Pharmacol. 2020, 6, 1–9. [Google Scholar] [CrossRef]
  35. Nowak, G. The ecarin clotting time, a universal method to quantify direct thrombin inhibitors. Pathophysiol. Haemost. Thromb. 2003, 33, 173–183. [Google Scholar] [CrossRef] [PubMed]
  36. Fu, H.Q.; Yang, T.; Xiao, W.; Fan, L.; Wu, Y.; Terrando, N.; Wang, T.L. Prolonged Neuroinflammation after Lipopolysaccharide Exposure in Aged Rats. PLoS ONE 2014, 9, e106331. [Google Scholar] [CrossRef] [PubMed]
  37. Franchini, M.; Mannucci, P.M. Direct oral anticoagulants and venous thromboembolism. Eur. Respir. Rev. 2016, 141, 295–302. [Google Scholar] [CrossRef]
  38. Jackson, S.P.; Darbousset, R.; Schoenwaelder, S.M. Thromboinflammation: Challenges of therapeutically targeting coagulation and other host defense mechanisms. Blood 2019, 133, 906–918. [Google Scholar] [CrossRef] [PubMed]
  39. Wong, J.P.; Viswanathan, S.; Wang, M.; Sun, L.Q.; Clark, G.C.; D’Elia, R.V. Current and future developments in the treatment of virus-induced hypercytokinemia. Future Med. Chem. 2017, 9, 169–178. [Google Scholar] [CrossRef]
  40. Pawlinski, R.; Pedersen, B.; Schabbauer, G.; Tencati, M.; Holscher, T.; Boisvert, W.; Andrade-Gordon, P.; Frank, R.D.; Mackman, N. Role of Tissue Factor and Protease-Activated Receptors in a Mouse Model of Endotoxemia. Blood 2004, 103, 1342–1347. [Google Scholar] [CrossRef]
  41. Zhang, H.; Zeng, L.; Xie, M.; Liu, J.; Zhou, B.; Wu, R.; Cao, L.; Kroemer, G.; Wang, H.; Billiar, T.R.; et al. TMEM173 Drives Lethal coagulation in Sepsis. Cell Host Microbe 2020, 27, 556–570. [Google Scholar] [CrossRef] [PubMed]
  42. Behrens, E.M.; Koretzky, G.A. Review: Cytokine Storm Syndrome: Looking Toward the Precision Medicine Era. Arthritis Rheumatol. 2017, 69, 1135–1143. [Google Scholar] [CrossRef] [PubMed]
  43. Sun, Z.-G.; Liu, Y.; Zhang, J.-M.; Cui, S.-C.; Zhang, Z.-G.; Zhu, H.-L. The Research Progress of Direct Thrombin Inhibitors. Mini-Rev. Med. Chem. 2020, 20, 1574–1585. [Google Scholar] [CrossRef] [PubMed]
  44. Alouidor, B.; Sweeney, R.E.; Tat, T.; Wong, R.K.; Yoon, J.-Y. Microfluidic Point-of-Care Ecarin-Based Clotting and Chromogenic Assays for Monitoring Direct Thrombin Inhibitors. J. Extracorpor. Technol. 2019, 51, 29–37. [Google Scholar] [CrossRef]
Ijms 24 15581 g001
Figure 1. Examples of biologically active azoloazines.
Figure 1. Examples of biologically active azoloazines.
Ijms 24 15581 g001
Ijms 24 15581 sch001
Scheme 1. Synthesis of indolo[2,3-b]pyrazine derivatives 1a–j.
Scheme 1. Synthesis of indolo[2,3-b]pyrazine derivatives 1a–j.
Ijms 24 15581 sch001
Ijms 24 15581 sch002
Scheme 2. Synthesis of azolo[1,5-a]pyrimidines.
Scheme 2. Synthesis of azolo[1,5-a]pyrimidines.
Ijms 24 15581 sch002
Ijms 24 15581 sch003
Scheme 3. Synthesis of guanidinium salts of 6-nitroazolo[1,5-a]pyrimidines 12a,b and 6-nitro-1,2,4-triazolo[5,1-c]triazine 14.
Scheme 3. Synthesis of guanidinium salts of 6-nitroazolo[1,5-a]pyrimidines 12a,b and 6-nitro-1,2,4-triazolo[5,1-c]triazine 14.
Ijms 24 15581 sch003
Ijms 24 15581 sch004
Scheme 4. Synthesis of 6-alkylthio-3-guanidinoimidazo[1,2-b][1,2,4,5]tetrazines 15a,b.
Scheme 4. Synthesis of 6-alkylthio-3-guanidinoimidazo[1,2-b][1,2,4,5]tetrazines 15a,b.
Ijms 24 15581 sch004
Ijms 24 15581 sch005
Scheme 5. Synthesis of 6-guanidino-3-alkylthio-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazines 16a–c.
Scheme 5. Synthesis of 6-guanidino-3-alkylthio-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazines 16a–c.
Ijms 24 15581 sch005
Ijms 24 15581 g002
Figure 2. Study of the effect of compound 5d and dabigatran etexilate on ecarin clotting time in vitro. n—number of animals.
Figure 2. Study of the effect of compound 5d and dabigatran etexilate on ecarin clotting time in vitro. n—number of animals.
Ijms 24 15581 g002
Table 1. Effect of novel azoloazine derivatives on the coagulogram parameters of rabbit blood in vitro at a concentration of 100 μM (M ± m, n = 6).
Table 1. Effect of novel azoloazine derivatives on the coagulogram parameters of rabbit blood in vitro at a concentration of 100 μM (M ± m, n = 6).
No.CompoundStructureCoagulogram Parameters
APTT, sTT, sPT, s
1Control 46.23 ± 0.2211.65 ± 0.0614.60 ± 0.10
2Dabigatran etexilate 75.26 ± 2.61 *70.23 ± 3.15 *14.20 ± 0.38 *
31aIjms 24 15581 i00122.1 ± 5.922.7 ± 2.0 *14.5 ± 0.0
41bIjms 24 15581 i00226.8 ± 2.423.9 ± 0.9 *13.3 ± 0.2
51cIjms 24 15581 i00327.1 ± 2.026.2 ± 1.0 *13.8 ± 0.4
61dIjms 24 15581 i00418.2 ± 0.526.7 ± 1.3 *13.4 ± 0.3
71eIjms 24 15581 i00529.6 ± 0.238.0 ± 0.5 *13.4 ± 0.1
81fIjms 24 15581 i00624.8 ± 4.239.3 ± 1.8 *13.1 ± 0.3
91gIjms 24 15581 i00730.6 ± 1.634.1 ± 0.9 *11.7 ± 0.4
101hIjms 24 15581 i00825.7 ± 3.135.6 ± 1.1 *13.9 ± 0.1
111iIjms 24 15581 i00932.1 ± 4.821.9 ± 1.2 *13.8 ± 0.3
121jIjms 24 15581 i01032.5 ± 0.122.0 ± 2.7 *14.2 ± 0.4
135aIjms 24 15581 i01126.3 ± 2.943.8 ± 4.3 *13.4 ± 0.1
145bIjms 24 15581 i01225.9 ± 2.221.0 ± 0.0 *15.8 ± 0.3
155cIjms 24 15581 i01327.6 ± 1.941.4 ± 3.1 *13.9 ± 0.2
165dIjms 24 15581 i01461.78 ± 4.58 *154.43 ± 11.14 *14.08 ± 0.4
177aIjms 24 15581 i01556.23 ± 5.0566.58 ± 4.64 *11.62 ± 1.00
187bIjms 24 15581 i01635.0 ± 0.232.3 ± 0.9 *13.6 ± 0.5
198cIjms 24 15581 i01734.6 ± 0.734.7 ± 0.7 *14.2 ± 0.1
2010aIjms 24 15581 i01830.9 ± 0.648.6 ± 22.4 *13.2 ± 1.0
2110bIjms 24 15581 i01930.6 ± 2.627.1 ± 1.4 *12.7 ± 0.2
2212aIjms 24 15581 i02025.1 ± 0.824.3 ± 3.5 *14.2 ± 0.1
2312bIjms 24 15581 i02126.2 ± 1.525.6 ± 3.9 *14.8 ± 0.4
2414Ijms 24 15581 i02224.5 ± 0.823.8 ± 3.5 *14.5 ± 0.5
2515aIjms 24 15581 i02334.0 ± 0.632.8 ± 0.8 *14.5 ± 0.3
2615bIjms 24 15581 i02434.1 ± 0.533.7 ± 0.6 *14.0 ± 0.2
2716aIjms 24 15581 i02529.1 ± 1.434.3 ± 4.7 *12.2 ± 0.2
2816bIjms 24 15581 i02624.7 ± 1.222.7 ± 1.7 *13.9 ± 0.5
2916Ijms 24 15581 i02726.3 ± 1.125.9 ± 1.6 *14.1 ± 0.3
Notes: APTT—activated partial thromboplastin time; TT—thrombin time; PT—prothrombin time. *—(p ≤ 0.05) changes are statistically significant vs. control, 1-way ANOVA; n—number of animals. The bold font is used to highlight the highest scores.
Table 2. Effect of compound 5d and dabigatran etexilate on the coagulogram parameters of rabbit blood treated with LPS in vitro in a concentration of 100 μM (M ± m, n = 6).
Table 2. Effect of compound 5d and dabigatran etexilate on the coagulogram parameters of rabbit blood treated with LPS in vitro in a concentration of 100 μM (M ± m, n = 6).
No.CompoundCoagulogram Parameters
APTT, sTT, sPT, s
1Control46.23 ± 0.2211.65 ± 0.0614.60 ± 0.10
2LPS control56.28 ± 1.60 *9.01 ± 0.37 *15.10 ± 0.07
3Dabigatran etexilate135.13 ± 15.17 *#125.45 ± 1.54 *#15.73 ± 0.20
45d61.78 ± 4.58 *154.43 ± 7.14 *#14.08 ± 0.4
*—(p < 0.05) changes are statistically significant vs. control, 1-way ANOVA; #—(p < 0.05) changes are statistically significant vs. LPS-control, 1-way ANOVA; n—number of animals.
Table 3. IC50 of compound 5d and dabigatran etexilate of thrombin time prolongation with and without LPS in vitro (M ± m, n = 6).
Table 3. IC50 of compound 5d and dabigatran etexilate of thrombin time prolongation with and without LPS in vitro (M ± m, n = 6).
CompoundΔ% of Thrombin Time Prolongation Relative to ControlEC50, μM
10 μM5 μM1 μM0.1 μM
Dabigatran etexilate302.0 ± 34.4 *176.1 ± 15.3 *42.3 ± 5.6 *1.4 ± 0.4 *1.4
5d467.1 ± 14.4 *#194.1 ± 10.0 *#46.5 ± 6.7 *4.3 ± 1.0 *1.3
CompoundΔ % of Thrombin Time Prolongation Relative To Control + LPSIC50, μM
10 μM5 μM1 μM0.1 μM
Dabigatran etexilate292.0 ± 0.6 #292.0 ± 0.6 #51.7 ± 0.516.3 ± 0.2 #0.76
5d354.1 ± 6.5 #$234.1 ± 4.2 #$56.3 ± 1.1 *8.4 ± 1.60.78
Notes: *—(p < 0.05) changes are statistically significant vs. control, 1-way ANOVA; #—(p < 0.05) changes are statistically significant vs. LPS control, 1-way ANOVA; $—(p < 0.05) changes are statistically significant vs. dabigatran etexilate, 1-way ANOVA; n—number of animals.
Table 4. Effect of a single intragastric administration of compound 5d and dabigatran etexilate in equimolar doses on the coagulograms of rats with hypercytokinemia and without. (M ± m, n = 6).
Table 4. Effect of a single intragastric administration of compound 5d and dabigatran etexilate in equimolar doses on the coagulograms of rats with hypercytokinemia and without. (M ± m, n = 6).
CompoundCoagulogram Parameters of Animals without Hypercytokinemia
APTT, sTT, sPT, s
Control group38.3 ± 1.457.7 ± 1.828.1 ± 1.4
Dabigatran etexilate137.5 ± 2.2 *603.9 ± 14.2 *31.2 ± 1.3
5d59.2 ± 8.3 *325.3 ± 5.1 *24.5 ± 1.1
CompoundCoagulogram Parameters of Animals with Hypercytokinemia
APTT, sTT, sPT, s
LPS group18.4 ± 0.844.1 ± 1.721.2 ± 1.3
Dabigatran etexilate41.0 ± 2.0 #566.1 ± 11.5 #28.1 ± 1.0
5d28.7 ± 1.0 #640.3 ± 7.4 #$26.4 ± 1.4
Notes: *—(p < 0.05) changes are statistically significant vs. control, 1-way ANOVA; #—(p < 0.05) changes are statistically significant vs. LPS control, 1-way ANOVA; $—(p < 0.05) changes are statistically significant vs. dabigatran etexilate, 1-way ANOVA; n—number of animals.
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Spasov, A.A.; Fedorova, O.V.; Rasputin, N.A.; Ovchinnikova, I.G.; Ishmetova, R.I.; Ignatenko, N.K.; Gorbunov, E.B.; Sadykhov, G.A.o.; Kucheryavenko, A.F.; Gaidukova, K.A.; et al. Novel Substituted Azoloazines with Anticoagulant Activity. Int. J. Mol. Sci. 2023, 24, 15581. https://doi.org/10.3390/ijms242115581

AMA Style

Spasov AA, Fedorova OV, Rasputin NA, Ovchinnikova IG, Ishmetova RI, Ignatenko NK, Gorbunov EB, Sadykhov GAo, Kucheryavenko AF, Gaidukova KA, et al. Novel Substituted Azoloazines with Anticoagulant Activity. International Journal of Molecular Sciences. 2023; 24(21):15581. https://doi.org/10.3390/ijms242115581

Chicago/Turabian Style

Spasov, Alexander A., Olga V. Fedorova, Nikolay A. Rasputin, Irina G. Ovchinnikova, Rashida I. Ishmetova, Nina K. Ignatenko, Evgeny B. Gorbunov, Gusein A. o. Sadykhov, Aida F. Kucheryavenko, Kseniia A. Gaidukova, and et al. 2023. "Novel Substituted Azoloazines with Anticoagulant Activity" International Journal of Molecular Sciences 24, no. 21: 15581. https://doi.org/10.3390/ijms242115581

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