Design, green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents^*

Abstract

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.

• 1,2,4-Triazines
• antithrombotic agents
• antiplatelet agents
• aspirin
• green synthesis
• molecular docking

Introduction

Ischemic stroke is the third-leading cause of morbidity and mortality in western countries1. Platelets play a vital role in both thrombosis and hemostasis by maintaining the integrity of the circulatory system and respond rapidly at vascular injury. The activation of platelets leads to a series of responses that impart a critical role in thrombosis2. This activation generally occurs when they are brought in contact with agents, such as collagen, thrombin and adenosine diphosphate (ADP). Currently, acetylsalicylic acid (ASA, aspirin) and thienopyridines, such as clopidrogel and ticlopidine, are widely used as antiplatelet aggregating agents (Figure 1). Nearly half of the global population is resistant to antiplatelet drugs resulting in increased risk of recurrent myocardial infarction and stroke or cardiac related deaths3^,4. Though dual antiplatelet therapy using a combination of aspirin and a thienopyridine drug has been implemented during the primary stage, atherothrombotic disease remains as one of the major causes of morbidity and mortality5. The most widely used antiplatelet drug aspirin inhibits cyclooxygenase (COX) irreversibly6. Aspirin prevents not only the synthesis of thromboxane A₂ (TXA₂) in platelets but also prostaglandin I₂ (PGI₂) in vascular endothelium7. Consequently, it induces stomach ulcers, limiting its clinical use. Development of newer COX inhibitors devoid of side effects of the currently available antiplatelet agents is a major goal in thromboembolic research.

Figure 1. Currently used oral antiplatelet drugs.

Syntheses of small molecules possessing vicinal diaryl scaffold as a privileged substructure have received wide attention in the field of medicinal chemistry8^,9. Molecules incorporating this scaffold have demonstrated biological activity of varying nature, especially as COX inhibitors10 and antiplatelet agents11–13.

Some of the diarylthiazoles were reported to be potent antiplatelet agents with vasodilatory action14. This study helped to conclude that guanidine moiety present in these structures plays a major role in vasodilatation. l-Arginine (S-2-amino-5-guanidinopentanoic acid) is a well-known essential amino acid which acts as a vasodilator through nitric oxide-dependant pathway (NO donor)15. Moreover, agamatine (4-aminobutylguanidine)16 and its derivatives like GYKI-14766 (d-methyl-phenylalanyl-prolyl-arginal)17 were accessed as experimental medicines in anticoagulant and antithrombotic therapy. Timegadine, a trisubstituted guanidine derivative18, and some other acyclic guanidines19 possess potent vasodilatory actions.

Drugs containing morpholinoethyl and morpholinoethylamino groupings have gained noteworthy attention in cardiovascular drug research20–24. Drugs like moricizine20 possess potent antiarrhythmic properties while some antipsychotics like moclobemide21 and aphobazole22 are specially used in patients with cardiovascular diseases. Some of the molecules like sulfonated morpholinoethyl derivatives23 and morpholinoethylamino derivatives of benzothiazoles24 were patented, respectively, for their potent antiplatelet and antiulcer properties.

1,2,4-Triazine scaffold has been associated with diversified pharmacological activities, such as antiplatelet14^,25, anticancer26, thromboxane synthetase inhibition27, anti-inflammatory28 and antimalarial29. Further, the diaryl system attached to 1,2,4-triazine heterocycle has been reported to be a potent anticytokine, mediating through the inhibition of p38 MAPK signaling pathway30. Recent studies on 5,6-diaryl triazines showed neuro-pharmacological applications of this moiety, such as adenosine A_2A antagonism, in Parkinson’s disease31, anti-neuroinflammatory activity in Alzheimer's disease32 and as neuroprotectants33. More recently, potent neuroprotective action of some 1,2,4-triazines by activating Wnt/β-catenin signaling pathway has been demonstrated by our research group34. Some patents have also disclosed the topical anti-inflammatory35 and antithrombotic activity of diaryl triazines35.

Based on the previous findings, it was thought logical to combine the diaryl 1,2,4-triazine scaffold with an amino group at position 3, providing a cyclic guanidine moiety. Such a system could prove beneficial in thromboembolic disorders due to its antiplatelet as well as vasodilatory actions (Figure 2). The well-documented antiplatelet activity associated with the 1,2,4-triazine pharmacophore14^,15^,25 motivated us to synthesize 1,2,4-triazines with concomitantly substituted vicinal diaryl group along with a flexible 3-morpholinoethylamino side chain. As a part of our ongoing research program, aiming at the discovery of novel diaryl heterocycle-based antiplatelet agents, we have recently reported potent antiplatelet effect of compounds containing a vicinal diaryl fragment as a key structural element attached to 1,4-diazepine and 2-aminopyrimidine as the heterocyclic scaffolds36^,37. This paper describes the synthesis of diaryl-1,2,4-triazines with biologically favourable morpholinoethylamino group at 3 position and their in vitro, ex vivo and in vivo pharmacological evaluation as potential antiplatelet/antithrombotic leads.

Figure 2. Designing hypothesis for the new compounds by fusing three pharmacophores in a single molecule for antiplatelet and vasodilatory action without ulcerogenesis.

Materials and methods

General

Melting points were determined in capillaries using Veego programmable melting point apparatus and are uncorrected. IR (in cm⁻¹) spectra in KBr pellets were taken on a Bruker ALPHA-T instrument. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ on a Bruker Avance II spectrometer (400 MHz), using tetramethylsilane (TMS) as an internal standard. Chemical shift is reported in parts per million (δ in ppm) where s, brs, t and m are designated for singlet, broad singlet, triplet and multiplet, respectively. Mass spectra were carried out on Thermoscientific DSQ-II mass spectrometer equipped with an electron spray ionization (ESI) or an electron impact ionization (EI) interface. Flash column chromatography was performed on Combiflash R_F 200 (Teledyne Esco) using flash grade silica gel (230–400 mesh). Elemental analyses were done on a Thermoscientific Flash-2000 CHN analyzer. Single crystals for compounds (3l and 3m) for X-ray crystallography were obtained from mixture of MeOH:CHCl₃ (30:70) and analyzed on Agilent single crystal X-ray model having Xcalibur, Eos, Gemini diffractometer operated at RT. Thin-layer chromatography (TLC) was performed on precoated Silica gel Merck plates. Compounds were visualized by illuminating with UV light (254 nm) or exposure to iodine vapors. All chemicals and solvents were of reagent grade and were purified and dried using standard methods. All the animal experiments were carried out as per the guidelines of Institutional Animal Ethics Committee of Pharmacy Department, The Maharaja Sayajirao University of Baroda, Gujarat, India (IAEC Reg. No. 404/01/a/CPCSEA).

Green synthesis of 5,6-diaryl-N-(2-morpholinoethyl-1,2,4-triazin-3-amines (3a–3q)

General procedure: 5,6-Diaryl-3-methylthio-1,2,4-triazines were synthesized as per the green chemical procedure previously reported by our group38. A mixture of the corresponding 3-methylthiotriazine (1.0 mmol) and 4-(2-aminoethyl)morpholine (4.0 mmol) was stirred at 100–110 °C. The progress of the reaction was monitored by TLC using a mixture of 10% methanol in chloroform as eluent. After completion, the reaction mixture was added to crushed ice. The precipitated product was filtered, washed thoroughly with water and dried. The products showed single spots on TLC. However, for characterization purposes the compounds were further recrystallized in methanol.

N-(2-Morpholinoethyl)-5,6-diphenyl-1,2,4-triazin-3-amine (3a)

Yield 48%, yellow crystals from MeOH, mp: 150–152 °C; FT-IR (KBr, cm⁻¹): 3223, 1580, 1522, 1114; ESI-MS (m/z) [%]: 361.40 (M)⁺ [31.66], 362.40 (M + 1)⁺ [100]; ¹H NMR (CDCl₃): δ 7.20–7.41 (m, 10H, Ar–H), 5.97 (brs, 1H, –NH), 3.58–3.68 (m, 6H, –NH–CH₂ and 2× CH₂–O), 2.61 (t, J = 5.9 Hz, 2H, –NH–CH₂–CH₂), 2.46 (t, J = 4.0 Hz, 4H, 2× –N–CH₂–CH₂–O); ¹³C NMR (CDCl₃): δ 37.65, 53.44, 57.16, 66.99, 128.33, 129.22, 129.56, 130.24, 136.37, 136.45, 149.67, 156.79, 160.29; Anal. Calcd. for C₂₁H₂₃N₅O; C, 69.78%; H, 6.41%; N, 19.38%. Found: C, 69.98%; H, 6.23%; N, 19.19%.

5,6-Bis(4-chlorophenyl)-N-(2-morpholinoethyl)-1,2,4-triazin-3-amine (3b)

Yield 60%, yellow crystals from MeOH, mp: 160–162 °C; FT-IR (KBr, cm⁻¹): 3223, 1595, 1522, 831; ESI-MS (m/z) [%]: 429.40 (M⁺) [100], 431.50 (M + 1)⁺ [87.27], 432.70 (M + 2)⁺ [21.81]; ¹H NMR (CDCl₃): δ 7.21–7.37 (m, 8H, Ar–H), 6.03 (brs, 1H, –NH), 3.60–3.68 (m, 6H, –NH–CH₂ and 2× –CH₂–O), 2.61 (t, J = 5.9 Hz, 2H, –NH–CH₂–CH₂), 2.46 (t, J = 4.1 Hz, 4H, 2× –N–CH₂–CH₂–O); Anal. Calcd. for C₂₁H₂₁Cl₂N₅O; C, 58.61%; H, 4.92%; N, 16.27%. Found: C, 58.87%; H, 4.68%; N, 16.08%.

N-(2-Morpholinoethyl)-5,6-di(p-tolyl)-1,2,4-triazin-3-amine (3c)

Yield 65%, light yellow crystals from MeOH, mp: 159–161 °C; FT-IR (KBr, cm⁻¹): 3221, 1595, 1523, 1115; ESI-MS (m/z) [%]: 390.50 (M + 1)⁺ [100]; ¹H NMR (CDCl₃): δ 7.03–7.32 (m, 8H, Ar–H), 5.91 (brs, 1H, –NH), 3.60–3.67 (m, 6H, –NH–CH₂ and 2× –CH₂–O), 2.59 (t, J = 5.9 Hz, 2H, –NH–CH₂–CH₂), 2.45 (t, J = 4.0 Hz, 4H, 2× –N–CH₂–CH₂–O), 2.27 (s, 6H, 2× Ar–CH₃); ¹³C NMR (CDCl₃): δ 21.30, 21.44, 37.63, 53.43, 57.20, 66.96, 129.01, 129.27, 129.50, 133.67, 138.06, 140.50, 149.62, 156.58, 160.22; Anal. Calcd. for C₂₃H₂₇N₅O; C, 70.92%; H, 6.99%; N, 17.98%. Found: C, 70.72%; H, 6.67%; N, 17.69%.

5-(4-Chlorophenyl)-N-(2-morpholinoethyl)-6-(p-tolyl)-1,2,4-triazin-3-amine (3d)

Yield 53%, light yellow crystals from MeOH, mp: 158–160 °C; FT-IR (KBr, cm⁻¹): 3225, 1595, 1521, 1399, 1118, 830; ESI-MS (m/z) [%]: 409.70 (M)⁺ [100], 411.60 (M + 2)⁺ [53.84]; ¹H NMR (CDCl₃): δ 7.05–7.31 (m, 8H, Ar–H), 5.94 (brs, 1H, –NH), 3.62–3.67 (m, 6H, –NH–CH₂ and 2× –CH₂–O), 2.61 (t, J = 5.9 Hz, 2H, –NH–CH₂–CH₂–N), 2.45 (t, J = 4.1 Hz, 4H, 2× –N–CH₂–CH₂–O), 2.29 (s, 3H, Ar–CH₃); Anal. Calcd. for C₂₂H₂₄ClN₅O; C, 67.55%; H, 6.16%; N, 13.70%. Found: C, 67.28%; H, 6.39%; N, 13.50%.

6-(4-Chlorophenyl)-N-(2-morpholinoethyl)-5-(p-tolyl)-1,2,4-triazin-3-amine (3e)

Yield 22%, yellow crystals from MeOH, mp: 150–152 °C; FT-IR (KBr, cm⁻¹): 3226, 1594, 1522, 1400, 1116, 821; EI-MS (m/z) [%]: 409.60 (M⁺) [100], 411.70 (M + 2)⁺ [54.21]; ¹H NMR (CDCl₃): δ 7.05–7.38 (m, 8H, Ar–H), 5.93 (brs, 1H, –NH), 3.62–3.68 (m, 6H, –NH–CH₂ and 2× –CH₂–O), 2.62 (t, J = 5.9 Hz, 2H, –NH–CH₂–CH₂–N), 2.46 (t, J = 4.0 Hz, 4H, 2× –N–CH₂–CH₂–O), 2.29 (s, 3H, Ar–CH₃); ¹³C NMR (CDCl₃): δ 21.35, 37.65, 53.46, 57.15, 67.01, 128.65, 129.05, 129.26, 130.99, 133.19, 135.05, 136.52, 138.48, 149.51, 155.56, 160.34; Anal. Calcd. for C₂₂H₂₄ClN₅O; C, 67.55%; H, 6.16%; N, 13.70%. Found: C, 67.28%; H, 6.33%; N, 13.45%.

5-(4-Fluorophenyl)-N-(2-morpholinoethyl)-6-phenyl-1,2,4-triazin-3-amine (3f)

Yield 48%, Light yellow crystals from MeOH, mp: 128–130 °C; IR (KBr, cm⁻¹) 3424, 1602, 703; EI-MS (m/z) [%]: 379.14 (M)⁺ [58]; ¹H NMR (CDCl₃): δ (ppm) 6.97–7.58 (m, 9H, Ar–H), 6.05 (brs, 1H, N–H), 3.55–3.92 (m, 6H, 2× CH₂–O and HN–CH₂), 2.68 (t, J = 5.5 Hz, 2H, HN–CH₂–CH₂–N), 2.54 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); ¹³C NMR (CDCl₃): δ 37.64, 53.46, 57.14, 67.00, 115.55, 115.66, 128.50, 129.20, 129.51, 130.38, 131.05, 131.86, 136.31, 156.70, 161.63, 164.10; Anal. Calcd. for C₂₁H₂₂FN₅O: C, 66.47%; H, 5.84%; N, 18.46%; Found: C, 66.65%; H, 5.49%; N, 18.23%.

6-(4-Fluorophenyl)-N-(2-morpholinoethyl)-5-phenyl-1,2,4-triazin-3-amine (3g)

Yield 46%, yellow crystals from MeOH, mp: 121–123 °C; IR (KBr, cm⁻¹) 3434, 1602, 1186, 701; EI-MS (m/z) [%]: 379.28 (M)⁺ [62]; ¹H NMR (CDCl₃): δ (ppm) 6.96–7.56 (m, 9H, Ar–H), 6.09 (brs, 1H, N–H), 3.70–3.74 (m, 6H, 2× CH₂–O and HN–CH₂), 2.67 (t, J = 5.9 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); ¹³C NMR (CDCl₃): δ 37.61, 53.41, 57.10, 66.99, 115.40, 115.61, 128.45, 129.16, 130.33, 130.92, 131.35, 131.73, 136.27, 156.70, 161.63, 164.10; Anal. Calcd. for C₂₁H₂₂FN₅O: C, 66.47%; H, 5.84%; N, 18.46%; Found: C, 66.73%; H, 5.62%; N, 18.28%.

5-(4-Bromophenyl)-6-(4-chlorophenyl)-N-(2-morpholinoethyl)-1,2,4-triazin-3-amine (3h)

Yield 53%, light yellow crystals from MeOH, mp: 152–154 °C; IR (KBr, cm⁻¹) 3441, 1601, 1114, 830; EI-MS (m/z) [%]: 474.08 (M)⁺ [35], 476.11 (M + 2)⁺ [18], 478.00 (M + 4)⁺ [4]; ¹H NMR (CDCl₃): δ (ppm) 7.26–7.48 (m, 8H, Ar–H), 6.10 (brs, 1H, N–H), 3.56–3.74 (m, 6H, 2× CH₂–O and HN–CH₂), 2.67 (t, J = 5.8 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); Anal. Calcd. for C₂₁H₂₁BrClN₅O: C, 53.12%; H, 4.46%; N, 14.75%; Found: C, 53.38%; H, 4.26%; N, 14.43%.

6-(4-Bromophenyl)-5-(4-chlorophenyl)-N-(2-morpholinoethyl)-1,2,4-triazin-3-amine (3i)

Yield 51%, yellow crystals from MeOH, mp: 161–163 °C; IR (KBr, cm⁻¹) 3440, 1602, 1114, 830; EI-MS (m/z) [%]: 473.83 (M)⁺ [72], 478.64 (M + 4)⁺ [57]; ¹H NMR (CDCl₃): δ (ppm) 7.26–7.46 (m, 8H, Ar–H), 6.11 (brs, 1H, N–H), 3.57–3.73 (m, 6H, 2× CH₂–O and HN–CH₂), 2.67 (t, J = 6.0 Hz, 2H, HN–CH₂–CH₂–N), 2.52 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); Anal. Calcd. for C₂₁H₂₁BrClN₅O: C, 53.12%; H, 4.46%; N, 14.75%; Found: C, 53.33%; H, 4.19%; N, 14.50%.

6-(4-Chlorophenyl)-5-(4-fluorophenyl)-N-(2-morpholinoethyl)-1,2,4-triazin-3-amine (3j)

Yield 49%, light yellow crystals from MeOH, mp: 149–151 °C; IR (KBr, cm⁻¹) 3448, 1598, 1115, 839; EI-MS (m/z) [%]: 413.54 (M)⁺ [100], 415.54 (M + 2)⁺ [30]; ¹H NMR (CDCl₃): δ (ppm) 7.00–7.50 (m, 8H, Ar–H), 6.11 (brs, 1H, N–H), 3.70-3.75 (m, 6H, 2× CH₂–O and HN–CH₂), 2.68 (t, J = 5.9 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.3 Hz, 4H, 2× N–CH₂–CH₂O); ¹³C NMR (CDCl₃): δ 37.59, 53.39, 57.05, 66.90, 115.56, 115.68, 128.71, 130.38, 130.89, 131.65, 134.65, 136.67, 148.36, 155.43, 162.77, 165.27; Anal. Calcd. for C₂₁H₂₁ClFN₅O: C, 60.94%; H, 5.11%; N, 16.92%; Found: C, 60.63%; H, 5.33%; N, 16.55%.

5-(4-Chlorophenyl)-6-(4-fluorophenyl)-N-(2-morpholinoethyl)-1,2,4-triazin-3-amine (3k)

Yield 52%, yellow crystals from MeOH, mp: 143–145 °C; IR (KBr, cm⁻¹) 3420, 1603, 1116, 841; EI-MS (m/z) [%]: 413.37 (M)⁺ [100], 415.28 (M + 2)⁺ [30]; ¹H NMR (CDCl₃): δ (ppm) 7.00–7.48 (m, 8H, Ar–H), 6.09 (brs, 1H, N–H), 3.68–3.73 (m, 6H, 2× CH₂–O and HN–CH₂), 2.68 (t, J = 5.2 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); ¹³C NMR (CDCl₃): δ 37.58, 53.40, 57.03, 66.94, 115.58, 115.79, 128.71, 130.38, 130.97, 131.65, 132.16, 134.73, 136.70, 148.31, 162.79, 165.30; Anal. Calcd. for C₂₁H₂₁ClFN₅O: C, 60.94%; H, 5.11%; N, 16.92%; Found: C, 60.68%; H, 5.43%; N, 16.58%.

5-(4-Chlorophenyl)-N-(2-morpholinoethyl)-6-phenyl-1,2,4-triazin-3-amine (3l)

Yield 56%, light yellow crystals from MeOH, mp: 163–165 °C; IR (KBr, cm⁻¹) 3440, 1584, 1119, 705; EI-MS (m/z) [%]: 395.41 (M)⁺ [4], 397.27 (M + 2)⁺ [1]; ¹H NMR (CDCl₃): δ (ppm) 7.26-7.47 (m, 9H, Ar–H), 6.08 (brs, 1H, N–H), 3.55-3.73 (m, 6H, 2× CH₂–O and HN–CH₂), 2.68 (t, J = 5.2 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); ¹³C NMR (CDCl₃): δ 37.60, 53.40, 57.07, 66.96, 128.49, 128.56, 129.44, 130.39, 130.43, 134.41, 134.83, 136.16, 148.53, 156.70, 162.79; Anal. Calcd. for C₂₁H₂₂ClN₅O: C, 63.71%; H, 5.60%; N, 17.69%; Found: C, 63.36%; H, 5.86%; N, 17.38%.

6-(4-Chlorophenyl)-N-(2-morpholinoethyl)-5-phenyl-1,2,4-triazin-3-amine (3m)

Yield 54%, yellow crystals from MeOH, mp: 129–131 °C; IR (KBr, cm⁻¹) 3441, 1596, 1115, 702; EI-MS (m/z) [%]: 395.01 (M)⁺ [90], 397.41 (M + 2)⁺ [40]; ¹H NMR (CDCl₃): δ (ppm) 7.27–7.51 (m, 9H, Ar–H), 6.07 (brs, 1H, N–H), 3.71–3.76 (m, 6H, 2× CH₂–O and HN–CH₂), 2.67 (t, J = 5.8 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O); Anal. Calcd. for C₂₁H₂₂ClN₅O: C, 63.71%; H, 5.60%; N, 17.69%; Found: C, 63.46%; H, 5.86%; N, 17.53%.

N-(2-Morpholinoethyl)-6-(4-nitrophenyl)-5-(p-tolyl)-1,2,4-triazin-3-amine (3n)

Yield 66%, yellow crystals from MeOH, mp: 150–152 °C; IR (KBr, cm⁻¹) 3432, 1585, 1516, 1115; EI-MS (m/z) [%]: 420.26 (M)⁺ [14]; ¹H NMR (CDCl₃): δ (ppm) 7.13–8.19 (m, 8H, Ar–H), 6.22 (brs, 1H, N–H), 3.71–3.76 (m, 6H, 2× CH₂–O and HN–CH₂), 2.69 (t, J = 5.3 Hz, 2H, HN–CH₂–CH₂–N), 2.55 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O), 2.38 (s, 3H, Ar–CH₃); ¹³C NMR (CDCl₃): δ 21.38, 37.58, 53.39, 57.01, 66.88, 123.53, 129.02, 129.68, 130.62, 132.73, 138.86, 141.37, 143.15, 147.43, 153.54, 160.23; Anal. Calcd. for C₂₂H₂₄N₆O₃: C, 62.84%; H, 5.75%; N, 19.99%; Found: C, 62.46%; H, 5.98%; N, 19.68%.

N-(2-Morpholinoethyl)-5-(4-nitrophenyl)-6-(p-tolyl)-1,2,4-triazin-3-amine (3o)

Yield 68%, yellow crystals from MeOH, mp: 141–143 °C; IR (KBr, cm⁻¹) 3440, 1586, 1516, 1115; EI-MS (m/z) [%]: 420.52 (M)⁺ [18]; ¹H NMR (CDCl₃): δ (ppm) 7.13–8.17 (m, 8H, Ar–H), 6.22 (brs, 1H, N–H), 3.73–3.75 (m, 6H, 2× CH₂–O and HN–CH₂), 2.69 (t, J = 5.7 Hz, 2H, HN–CH₂–CH₂–N), 2.54 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O), 2.38 (s, 3H, Ar–CH₃); Anal. Calcd. for C₂₂H₂₄N₆O₃: C, 62.84%; H, 5.75%; N, 19.99%; Found: C, 62.59%; H, 5.98%; N, 19.73%.

5-(3-Chlorophenyl)-N-(2-morpholinoethyl)-6-(p-tolyl)-1,2,4-triazin-3-amine (3p)

Yield 55%, light yellow crystals from MeOH, mp: 142–144 °C; IR (KBr, cm⁻¹) 3450, 1596, 1117, 696; EI-MS (m/z) [%]: 409.56 (M)⁺ [22], 411.25 (M + 2)⁺ [16]; ¹H NMR (CDCl₃): δ (ppm) 7.12–7.55 (m, 8H, Ar–H), 6.06 (brs, 1H, N–H), 3.68–3.74 (m, 6H, 2× CH₂–O and HN–CH₂), 2.67 (t, J = 5.7 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O), 2.36 (s, 3H, Ar–CH₃); Anal. Calcd. for C₂₂H₂₄ClN₅O: C, 64.46%; H, 5.90%; N, 17.09%; Found: C, 64.78%; H, 5.68%; N, 16.82%.

6-(3-Chlorophenyl)-N-(2-morpholinoethyl)-5-(p-tolyl)-1,2,4-triazin-3-amine (3q)

Yield 58%, yellow crystals from MeOH, mp: 138–140 °C; IR (KBr, cm⁻¹) 3447, 1595, 1117, 697; EI-MS (m/z) [%]: 409.02 (M)⁺ [24], 411.37 (M + 2)⁺ [8]; ¹H NMR (CDCl₃): δ (ppm) 7.12–7.55 (m, 8H, Ar–H), 6.09 (brs, 1H, N–H), 3.70–3.74 (m, 6H, 2× CH₂–O and HN–CH₂), 2.67 (t, J = 5.6 Hz, 2H, HN–CH₂–CH₂–N), 2.53 (t, J = 4.0 Hz, 4H, 2× N–CH₂–CH₂O), 2.36 (s, 3H, Ar–CH₃); ¹³C NMR (CDCl₃): δ 21.45, 37.60, 53.41, 57.11, 66.95, 127.40, 128.28, 129.07, 129.17, 129.39, 129.48, 133.03, 134.31, 138.46, 140.94, 148.27, 156.74, 160.34; Anal. Calcd. for C₂₂H₂₄ClN₅O: C, 64.46%; H, 5.90%; N, 17.09%; Found: C, 64.83%; H, 5.68%; N, 16.88%.

Pharmacological (antiplatelet/antithrombotic) activity

In vitro platelet aggregation assay

The newly synthesized compounds were studied for their in vitro platelet aggregation inhibitory activity on whole human blood as previously reported by us37.

Ex vivo antiplatelet study

The suspensions of the test compounds were triturated with minimum quantity of gum acacia (0.9% w/v). Test compounds were orally administered (10 mg/kg) and the first blood sample was withdrawn after 2 h via the retro-orbital puncture. All samples were collected into vaccue tubes containing 1/10 volume of heparin (100 IU/ml) and kept at room temperature until further experimentation, for a period ranging from 5 min to 1 h. Diluted whole blood (490 µl) with isotonic saline (500 µl) was transferred to an aggregometer cuvette placed in the thermostated (37 °C) cuvette holder of the whole-blood aggregometer (Model 592, Chrono-Log Corp., Hawertown, PA). Siliconized stirring bar and electrode were dipped inside and the system was allowed to come to equilibrium. Ten microliters of ADP (10 μM, Chrono-Log Corp.) were added and the change in impedance, which reflected platelet aggregation around the electrodes, is recorded until the maximum extent of aggregation was reached. Each blood sample was tested thrice and the mean was calculated for each group.

Maximal reading (mean ± SEM) of platelet aggregation in whole blood induced by 10 μM ADP was determined without the test sample (Blank, i.e. Maximum Aggregation) and a given concentration of each test sample. The percentage inhibition was calculated considering the blank reading as 100% by the formula as given below: (1)

In vivo antithrombotic study

Ferric chloride induced thrombosis

Rats weighing between 250 and 300 g were anaesthetized with ketamine (100 mg/kg) and a polyethylene catheter (PE-205) was inserted into the trachea via tracheotomy to facilitate breathing. Catheters were also placed in the femoral artery for blood samples and measurement of arterial blood pressure and in the jugular vein for administration of test samples. The right carotid artery was isolated and a small piece of Parafilm “M” was placed under the vessel to isolate it from surrounding tissues throughout the experiment. The test sample was administered by an intravenous injection at a defined time prior to initiation of thrombus formation. Thrombus formation was induced by the application of filter paper (2 × 5 mm), saturated with FeCl₃ solution, to the carotid artery. Concentration of FeCl₃ solution that caused consistent thrombus formation was determined under our experimental conditions and it was found out to be 42.30% w/v. The paper was allowed to remain on the vessel for 10 min before removal. The temperature probe (SS6L, BIOPAC Inc., Goleta, CA) was placed distal to the filter paper piece towards the cephalic end to measure any changes in temperature during the experiment. The experiment was continued for 60 min after the induction of thrombosis. At that time, the thrombus was removed and weighed39. The percentage inhibition of thrombosis was calculated using following formula: (2) where A = thrombus weight of control and A₁ = thrombus weight after treatment with the test sample or standard.

AV shunt model for thrombosis

Rats were anaesthetized and fixed in supine position on a temperature-controlled (37 °C) heating plate to maintain body temperature. The left carotid artery and the right jugular vein were catheterized with short polyethylene catheters. The catheters were filled with isotonic saline solution and clamped. The two ends of the catheters were connected with a 2 cm glass capillary with an internal diameter of 1 mm. At a defined time after administration of the test sample (3d), the clamps which were occluding the AV-shunt were opened. If the blood started flowing, then the temperature would rise from room temperature to body temperature. In contrast, decrease in temperature indicated the formation of an occluding thrombus. The temperature was measured continuously (as mentioned in the procedure for FeCl₃ model) over 30 min after opening of the shunt40.

Bleeding time

The rats were given per oral dose of 10 mg/kg bw of test sample and anesthetized after 2 h. Then they were fixed in supine position on a temperature-controlled (37 °C) heating-table. Catheterization of a carotid artery (for measurement of blood pressure) and a jugular vein was performed. After a defined latency period, the tail of the rat was transected with a razor blade at a distance of 4 mm from the tip of the tail. Immediately after transection, the tail was immersed into a bath filled with isotonic saline solution at 37 °C. The amount of time elapsed from tail transection to cessation of bleeding was measured up to 30 min and was assigned as the bleeding time41.

Gastric ulceration

Female Wistar rats (150–170 g) were fasted for 48 h having access to drinking water ad libitum. Test compounds and standard drug aspirin were orally administered to groups of five rats 5 h before autopsy. The stomachs were macroscopically inspected and the number of ulcers is noted and the severity recorded with the following scores: 0 = no ulcer; 1 = superficial ulcers; 2 = deep ulcers; 3 = perforation14.

Molecular docking studies

The docking studies were performed by using AutoDock442^,43. To validate the docking study, the co-crystallized ligand within the 3D structure of COX-1 and COX-2 (PDB Code: 2OYE and 6COX, respectively) was re-docked into the respective active sites of the enzymes. The docking study of the most active ligand (3d) on active sites of the target enzymes COX-1 and COX-2 was performed to get further insights into the possible interactions between the ligand and the target enzyme. For the ligand–enzyme complex, 10 docking experiments were performed using Lamarckian genetic algorithm to determine the binding energy (docked energy), and by using the obtained binding energy the K_i value was determined. The maximum number of energy evaluations of 25 million was applied for each docking experiment.

Results and discussion

Synthesis

The diaryltriazine derivatives presented in this paper were prepared according to the green synthetic route described in Scheme 1. The starting materials, 1,2-diketones (1a–1j), were prepared as per our previous reported method44. All the reactions were carried out by environment friendly protocols developed in our laboratory without the use of volatile organic compounds (VOCs). Green chemistry proposes optimized synthetic methodologies for high product yields and generation of byproducts and/or waste material that offer little or no harm to environment. The changing paradigm to use alternative reaction media rather than volatile organic solvents emphasizes on the use of ionic liquids and solvent-free solid-phase syntheses. As per our interest in developing green synthetic methodologies for biologically active small molecules34^,38, first step of the synthesis was carried out in ionic liquid and the latter one in solvent-less condition. The ionic liquid 1,3-dibutylimidazolium bromide [Bbim⁺Br⁻]:DMSO (1:10) promoted the one pot reaction of the corresponding diketones (1a–1j) with methyl iodide and thiosemicarbazide offering 3-methylthio-diaryl-1,2,4-triazines (2a–2q) at 70 °C in a very short period of time38. The regioisomers formed were separated using flash chromatography using 5% ethyl acetate in n-hexane as eluent. The separated isomers were unambiguously confirmed by their spectral data and one of them (2f) by single crystal X-ray structure (CCDC 969264)38. In the IR spectra, the 1,2-diketones showed absorption bands for C=O in the range 1650–1670 cm⁻¹. The cyclized products (2a–2q) were confirmed by the appearance of C=N stretching in the IR spectrum at 1590–1610 cm⁻¹ and absence of carbonyl stretching of diketones. Demethylsulfurization and instantaneous amination of these compounds occurred due to sufficient basicity of morpholinoethylamine without the use of any added base/solvent/catalyst. The resulting compounds (2a–2q) when treated with excess of neat 4-(2-aminoethyl)morpholine (4 eq.) at 100–110 °C for 2–4 h afforded 3-morpholinoethylamine derivatives of 1,2,4-triazine (3a–3q) as the desired products.

Scheme 1. Green synthesis of 5,6-diaryl-N-morpholinoethyl-1,2,4-triazin-3-amines (3a–3q). Reagents and conditions: (a) thiosemicarbazide, methyl iodide, ionic liquid [Bbim⁺Br⁻]: DMSO (1:10), 70 °C, 10–110 min; (b) 4-(2-aminoethyl)morpholine (4 eq.), neat, 100–110 °C, 2–4 h.

Compounds (3a–3q) showed characteristic bands at 1610–1590 cm⁻¹ for C=N stretching, asymmetric and symmetric stretching N–H bands at around 3500 and 3350 cm⁻¹, respectively in the IR spectra. In the ¹H NMR spectra, a broad singlet of N–H proton was observed at around δ 6.0. The four morpholinyl protons of the two CH₂ groups directly attached to oxygen and the two CH₂ protons of the side chain attached to NH appeared as multiplet (mixed signal of a broad triplet and a sharp triplet) at δ 3.5–4.0. The CH₂ protons of side chain attached to the tertiary nitrogen of the ring always appeared as a distinct triplet at δ 2.6–2.7. The remaining four morpholinyl CH₂ protons directly attached to the ring nitrogen appeared as a triplet (occasionally broad) at δ 2.4–2.6. The aromatic protons appeared as multiplet at δ 7.0–8.0. In the ¹³C NMR spectra, the δ values of the carbons are in accordance with the structures of the synthesized compounds. The single crystal X-ray structures were also affirmed for two of the compounds 3l and 3m (Figure S1 in Supplementary Information, CCDC 1028877 and 1028686, respectively). The crystal structure data of these positional isomers confirmed their respective structures. All the final compounds (3a–3q) were characterized by spectral and elemental analysis.

Antiplatelet/antithrombotic activity

In vitro platelet aggregation assay

The newly synthesized compounds were studied for their in vitro platelet aggregation inhibitory activity. Our earlier study has shown that this type of compounds does not exhibit any cytotoxicity rather show protective role in Malondialdehyde (MDA), catalase and intracellular reactive oxygen species (ROS) generation assays34. The whole human blood aggregation study was done as per our previously reported procedure37. Each reading was taken in triplicate for the test samples, using control and a standard drug aspirin for comparative reading each time. The percent inhibition of platelet aggregation was determined for each test sample on comparison with the control and the standard aspirin. The inhibitory values were calculated accordingly as shown in Table 1.

Table 1. Structures and in vitro antiplatelet aggregation activity data of the synthesized 3-substituted 5,6-diaryl-1,2,4-triazines (3a–3q)..

Compd. no.	X	Y	Mol. Form.	% Inhibition of platelet aggregation (in vitro, 10 µg/ml)
3a	H	H	C21H23N5O	63.95
3b	4-Cl	4-Cl	C21H21Cl2N5O	34.79
3c	4-CH3	4-CH3	C23H27N5O	71.25
3d	4-CH3	4-Cl	C22H24ClN5O	75.00
3e	4-Cl	4-CH3	C22H24ClN5O	48.02
3f	H	4-F	C21H22FN5O	68.88
3g	4-F	H	C21H22FN5O	66.93
3h	4-Cl	4-Br	C21H21BrClN5O	39.51
3i	4-Br	4-Cl	C21H21BrClN5O	41.93
3j	4-Cl	4-F	C21H21ClFN5O	68.72
3k	4-F	4-Cl	C21H21ClFN5O	70.16
3l	H	4-Cl	C21H22ClN5O	58.79
3m	4-Cl	H	C21H22ClN5O	36.29
3n	4-CH3	4-NO2	C22H24N6O3	49.68
3o	4-NO2	4-CH3	C22H24N6O3	46.77
3p	4-CH3	3-Cl	C22H24ClN5O	58.87
3q	3-Cl	4-CH3	C22H24ClN5O	62.50
Aspirin	–	–	–	34.00

The bold values suggest the noteworthy/comparable activity of respective compounds.

From the in vitro evaluation of the test compounds (3a–3q), 10 compounds were found to have prominent activity. Compounds (3a, 3c, 3d, 3f, 3g, 3j, 3k, 3l, 3p and 3q) produced more than 60% inhibition while compounds (3e, 3h, 3i, 3m, 3n and 3o) were found to be comparable in activity with aspirin. Out of these compounds, 3d was the most potent, being twice as potent as aspirin.

Ex vivo antiplatelet aggregation study

Almost all of the compounds showed good to moderate inhibition of platelet aggregation in the in vitro study. Hence, all the compounds were screened further for their ex vivo platelet aggregation inhibition. The whole blood aggregation method was used for this purpose in which blood was withdrawn from animals, 2 h after pretreatment of the group with test samples by oral dosing, along with the control group and the aspirin-treated group (Table 2).

Table 2. Antithrombotic activity (ex vivo and in vivo) data of the synthesized 3-substituted 5,6-diaryl-1,2,4-triazines (3a–3q) at a dose of 10 mg/kg bw p.o.

Comp. no.	% Inhibition of platelet aggregation (ex vivo)	Thrombus wt. (mg)	% Inhibition of thrombosis	Time to occlusion (min)	Bleeding time (min)
Control	–	6.60	–	15.0	18.0
3a	37.65	4.80	27.27	12.5	17.0
3b	32.53	4.20	36.36	18.0	16.0
3c	19.87	4.00	39.39	21.0	19.5
3d	53.19	4.00	39.39	>30	25.0
3e	44.47	5.00	24.24	12.0	17.5
3f	22.37	4.90	25.75	12.5	24.0
3g	19.11	4.30	34.84	27.0	25.5
3h	23.82	4.90	25.75	20.5	18.0
3i	36.47	4.85	26.51	21.5	20.5
3j	19.94	4.20	36.36	29.0	24.5
3k	17.64	4.25	35.60	29.5	25.0
3l	45.29	4.75	28.03	19.0	20.5
3m	26.17	4.50	31.81	23.5	19.5
3n	36.42	4.25	35.60	21.0	16.5
3o	35.58	4.25	35.60	21.5	16.5
3p	17.64	4.00	39.39	27.0	>30
3q	18.58	4.10	37.87	27.5	>30
Clopidogrel*	84.03	3.00	54.54	>30	>30
Aspirin†	47.80	3.00	54.54	>30	>30

The bold values suggest the noteworthy/comparable activity of respective compounds.

*Clopidogrel used at a dose of 25 mg/kg body weight.

†Aspirin used at a dose of 50 mg/kg body weight.

From the ex vivo study of the tested compounds (3a–3q), six compounds (3a, 3d, 3e, 3i, 3l and 3n) were found with noticeable activity. Out of these five compounds, 3d again was found to be the most potent (53.19% inhibition), which was almost double the activity of aspirin. Compounds (3a, 3e, 3i, 3l and 3n) produced comparable inhibition of platelet aggregation to aspirin. Even though these compounds were found to be less potent in comparison to another standard drug clopidogrel which acted through a different mechanism of action (P2Y₁₂ inhibition), their potential vasodilatory effect could give them an additional advantage.

In vivo antithrombotic study

Ferric chloride induced thrombosis

All the synthesized compounds were evaluated for their antithrombotic activity using FeCl₃-induced thrombosis model, as reported earlier39. From Table 2, it could be seen that almost all the compounds (3a–3q) inhibited the thrombus formation as evidenced from their respective thrombus weight after treatment at a dose of 10 mg/kg bw p.o. Compounds (3b, 3c, 3d, 3j and 3q) decreased the thrombus weight significantly as compared to the standard drug and the control. Two compounds (3c and 3d) showed noteworthy efficacy in this model. The time to occlusion (TTO) was also measured as a parameter indicating obstruction to blood flow after formation of clot/thrombus. Increase in TTO was considered as a preventive measure for prolongation of thrombus formation. Compounds (3d, 3g, 3j, 3k, 3p and 3q) significantly increased TTO as compared to the standard drug and the control (Table 2).

Arterio-venous (AV) shunt thrombosis

Further, to ensure antithrombotic efficacy of the compound (3d), it was evaluated by previously known AV shunt model40. No temperature fall was observed up to 20 min which showed the non-occlusive response indicating the potential antithrombotic action of compound (3d).

Bleeding time

Bleeding time was used as a parameter to evaluate the hemorrhagic properties of antithrombotic drugs. Hence, tail bleeding method41 was used at a dose of 10 mg/kg of body weight for the test samples. Compounds (3d, 3i, 3g, 3j and 3k) showed increased but significantly lower bleeding time than standard drugs clopidogrel and aspirin. The optimum increase in bleeding time by these compounds could be of great advantage since that would overcome the fatal bleeding complications (Table 2).

Gastric ulceration

The gastric ulceration model14 was selected to ascertain the undesired side effects, if any, of the test sample (3d). The results suggested that there was no gastric ulceration in the control (Figure 3a) while a mild ulceration was observed after 10 mg/kg dose (Figure 3b) of the test compound (3d), whereas moderate ulceration was observed at 100 mg/kg dose (Figure 3c). Still, at 100 mg/kg dose the ulceration was much less than the standard drug aspirin which caused severe ulceration at the same dose (100 mg/kg) (Figure 3d).

Figure 3. Effect of compound (3d) and aspirin on gastric mucosa. (a) Control with no gastric lesions; (b) Compound (3d) showing mild gastric lesions (10 mg/kg); (c) Compound (3d) showing moderate gastric lesions (100 mg/kg); (d) Aspirin showing severe gastric lesions (100 mg/kg).

Structure–activity relationships

The effect of various groups on the basic 5,6-diaryl-1,2,4-triazine scaffold was studied with respect to antiplatelet activity of the synthesized compounds (Table 1). Among the compounds in the series, compound (3d) was found to be the most potent inhibitor of in vitro platelet aggregation. This suggests that in the present series, p-methyl substituent on the 6-aryl ring and p-Cl substituent on the 5-aryl ring favor antiplatelet activity. While compound (3d) showed the most prominent activity, its positional isomer (3e) with p-methyl substituent on the 5-aryl ring and p-Cl substituent on the 6-aryl ring showed almost half the activity of 3d. p-Halo substituents on the 5-aryl ring showed better activity in almost all of the compounds except for compound (3b). Halo substitution at these positions increases the lipophilic character of the molecule. This could be one of the reasons for higher in vitro activity of p-halo substituted compounds at the 5-aryl ring. Compounds without halogens on both of the aryl rings showed overall decreased activity (3n and 3o). Compounds with m-Cl substituent on the 5-aryl ring showed lower activity than the p-Cl substituted derivatives (compound 3p versus compound 3d). The compound with p-Br substituent on the 5-aryl ring (3h) showed a drastic reduction in the activity whereas p-F substituent on this position retained the activity (3f and 3j). Surprisingly, a compound with p-Cl substituent on both the aryl rings (3b) has shown diminished activity while p-methyl substituents on at both the aryl rings retained the same level of activity (3c).

In case of ex vivo activity of the synthesized compounds, five of the compounds (3b, 3d, 3e, 3i and 3l) showed better activity than the rest. Compound (3d) again emerged as the most potent compound with 53.19% inhibition of platelet aggregation ex vivo.

In case of bleeding time, compound 3b with p-Cl substituents on both of the aryl rings showed the least bleeding time which was unexpectedly low. Compound (3d) showed the maximum bleeding time of 25 min in the series, in contrast to the reference drugs aspirin and clopidogrel which had still higher bleeding times (>30 min). The increased bleeding time after treatment by the current drugs is considered to be the main drawback, which causes fatal bleeding and loss of preventive mechanism of hemostasis. Hence, compounds with optimum bleeding time, such as 3d, would be beneficial for antiplatelet/antithrombotic therapy.

Thrombus formation is one of the major causes of the thromboembolic diseases. To evaluate the antithrombotic efficacy of the tested compounds, FeCl₃-induced thrombosis model was utilized. FeCl₃ at an optimum concentration of 42.30% w/v causes clot/thrombus formation in vein/artery and the weight of the thrombus formed was measured carefully after isolation. The weight of the thrombus formed was used as one of the parameters to find the preventive role of the synthesized compounds on thrombosis. It was observed that almost all the compounds inhibited thrombus formation to variable extents. Compounds (3c and 3p) were quite active but compound (3d) again was found to be the most potent in this test. Percent inhibition of thrombosis was also calculated using the thrombus weight. The estimated potency of 3d observed in this model was further confirmed by evaluating it using AV shunt model.

Eventually, TTO was measured for all the test compounds. Almost one-third of the compounds significantly inhibited the occlusion for more than 20 min. Compounds (3d, 3g, 3i, 3k, 3m, 3p and 3q) showed better profile in this parameter. The previously hypothesized vasodilatory action of the tested compounds might be responsible for this phenomenon14–18. Compound (3d) showed TTO of more than 30 min which was equal to the standard drugs clopidogrel. Compound (3e) was found to be the least active amongst all the compounds and was comparable to the unsubstituted diaryl compound (3a).

As several COX inhibiting drugs show moderate to severe gastric ulcerations, the most potent compound (3d) was evaluated for its propensity to cause gastric ulceration. It was found that it did not show any undesirable side effect at its normal dose of 10 mg/kg, but at higher dose (100 mg/kg), it showed mild ulceration. It was noteworthy that at the same dose of 100 mg/kg, aspirin, a well-known antithrombotic drug caused severe gastric ulceration (Figure 3).

The newly synthesized compound (3d) increases bleeding time to its optimum level with good in vitro, ex vivo and in vivo profile. These parameters are indicative of fairly good therapeutic profile of compound (3d) and project the compound (3d) worthy of further investigation as a new potential drug candidate for thromboembolic diseases.

Molecular docking studies

Molecular docking studies of the most active compound (3d) with COX-1 and COX-2 enzymes were performed to understand the possible nature of its binding interactions within the active sites (PDB Code: 2OYE45 and 6COX45, respectively).

The most active molecule (3d), when docked into the COX-1 active site, comfortably entered the active site and showed good interactions with binding energy of −10.59 kcal/mole. The p-tolyl group showed hydrophobic interactions with Phe381, Leu384, Tyr385 and Trp387, whereas, the p-chlorophenyl ring depicted hydrophobic interactions with Tyr355, Val349 and Ile523. The in silico binding affinity (K_i) value for the ligand–receptor complex was obtained as 17.16 nM. However, when we studied its interactions with the active site of COX-2 enzyme, the p-tolyl ring oriented itself near the secondary pocket (Leu352, Arg513) of the active site of the receptor. The in silico binding energy for this complex was observed to be −8.23 kcal/mole and the binding affinity value was calculated to be 927.04 nM.

In brief, we compared the binding interactions of the most active molecule (3d) with COX-1 and COX-2 enzymes (Figure 4). The in silico interactions of compound (3d) with COX-1 and COX-2 showed that the compound is having more affinity and selectivity towards COX-1 while moderate activity towards COX-2. These results were in accord with the observations of the previous studies of nonselective COX inhibition as antiplatelet agents7^,46.

Figure 4. Docking simulations of compound (3d) with active sites of (a) COX-1 (PDB: 2OYE) and (b) COX-2 (PDB: 6COX), respectively.

Conclusions

A novel series of 5,6-diaryl-1,2,4-trizines has been reported with several electron withdrawing as well as donating functions attached to the vicinal diaryl rings. In vitro, ex vivo and in vivo evaluations indicated that some of the compounds showed moderate to potent antiplatelet/antithrombotic activities which could be helpful for the development of new antiplatelet drugs. The most promising compound (3d) of the series seems to possess a potentially better therapeutic profile.

Supplementary material available online

Supplementary Figure S1

Declaration of interest

We thank All India Council for Technical Education (AICTE), New Delhi, India for National Doctoral Fellowship to RST [File No. 1–10/RID/NDF/PG/36/2009–2010]. The authors also thank the DST-PURSE Single Crystal X-Ray Diffraction Facility at the Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara for single crystal X-ray crystallographic studies.

The authors declare that they have no conflicts of interest to disclose.

Notes

* This article is dedicated to fond memories of Late Prof. (Mrs.) Rajani Giridhar.