Synthesis and Antiplatelet Potential Evaluation of 1,3,4-Oxadiazoles Derivatives.

A novel series of 2-(3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-5-aryl-1,3,4-oxadiazoles (4a–4h) has been synthesized from corresponding hydrazones (3a–3h) and evaluated their antiplatelet aggregation effect induced by arachidonic acid and collagen. Spectral data and elemental evaluation were used to confirm the structure of the compounds while molecular docking against cyclooxygenase 1 and 2 (COX1 & COX2) and quantitative structure-activity relationship (QSAR) were performed in describing their antiplatelet potential. All synthesized compound exhibited more than 50% platelet aggregation inhibition against both arachidonic acid and collagen. Antiplatelet activities results showed that 4b and 4f compounds have highest % inhibition against arachidonic acid. High Egap and ionization potential values showed that the compound 4d, 4e and 4f were supposed to be more active and good electron donor while 4b, 4c, 4d, 4e, 4g and 4h might be more active due to more electrophilic sites. Interaction with more than one residues in the binding pocket of COX-1 in comparison with aspirin and ligand efficacy (LE) consequences showed that compounds have excellent action potential for COX-1. Computational evaluations are in good agreement with antiplatelet activities of the compounds. All compounds might be promising antiplatelet agents especially 4b, 4f and helpful in the synthesis of new drugs for the treatment of cardiovascular diseases (CVDs).

Keywords: computational; cyclization; density functional theory; molecular docking study and pyrazolopyridines

Cardiovascular disease (CVD) is one of the most common cause of death in the western countries. In the United States of America (USA), more than 25 million people have been diagnosed with CVD which causes a lot of expenditure in health care [[1]], [[2]]. Platelet aggregation plays an important role in thrombosis and atherosclerosis formation, leading to heart attack and strokes [[3]], [[4]]. During these diseases platelets functioning is altered which leads to many pathogenesis involving the lipid deposition in the artery wall and the cellular response interaction which is an inflammatory response characteristic. The atherosclerosis in platelet increased adhesiveness in the platelet endothelial wall and anticoagulant properties lost, so the atherosclerosis plaque forms. The rupturing of this plaque is the most important event for sudden vascular disease preparation [[5]]. As the rupturing of plaque, exposes the platelet endothelial cells matrix protein and cascade of reactions started [[6]]. Arachidonic acid present in the platelet phospholipid membranes liberated and react with oxygen to form the prostaglandin, which is then converted to a potent platelet aggregation agonist, thromboxane A2 [[7]], [[8]], [[9]].

Asprin, clopidogrel and naproxen, anti-aggregating agents are being in used for the treatment of cardiovascular diseases (CVDs) even though they showed complications during treatment. There is a need of some agents which may lower the risks of strokes and heart attack and also have low collateral effects [[10]], [[11]], [[12]].

1,3,4-Oxadiazole ring containing compounds are widely reported in literature and claimed to possess activities such as anti-inflammatory [[13]], [[14]] and anticancer [[15]]. They have also been used as integrase inhibitor of HIV and angiogenesis. The effect of solvatochromic behaviour and solubilization was also studied for NSAIDS at various concentrations in various solvents [[16]], [[17]], [[18]]. NSAIDS are known to have their role in the treatment of pain, fever and irritation [[19]]. Oxadiazole ring has become an important moiety of biologically important compounds by virtue of its involvement in hydrogen bond formation and also due to its role in the human metabolic profile [[20]]. The oxadiazoles are found to have in vivo antithrombotic, antitussive and in vitro antiplatelet activities [[21]]. There is also reported that nitric oxide caused antiplatelet aggregation by generating cyclic guanidine monophosphate via activation of soluble guanylate cyclase [[22]], [[23]]. Pyrazolo[3,4-b] pyridine ring system is considered to be isoelectronic with quinoline and its derivatives such as (1a) and (1b) have been found to exhibit antichagasic and excellent antiplatelet activities [[24]], [[25]], [[26]].

A variety of methods has been used for the preparation of oxadiazoles [[27]], [[28]], [[29]], [[30]], [[31]], [[32]] and biologically active pyrazolo[3,4-b]pyridine ring system [[33]], [[34]]. In the present work, a series of 1, 3, 4-oxadiazoles derivatives (4a–4h) containing a pyrazole[3,4-b]pyridine ring system has been synthesized and screened for their antiplatelet potential augmented with in-silico studies.

Graph

All chemicals were purchased from Sigma-Aldrich and well reputed international companies. The chemicals were used mostly as such or purified by common techniques i.e. distillation and recrystallization if required. The melting point of synthesized compounds were determined on Gallenkamp Melting Point Apparatus. Fourier transform infrared spectroscopy (FTIR) spectra, nuclear magnetic resonance (NMR) data (1H and 13C), high resolution mass spectra (HRMS), and elemental analyses (CHN) were recorded on Agilent Technologies 41630, Bruker DPX 400 (100) and 500 (125) MHz instruments, Fisons VG Platform II Spectrometer and Perkin Elmer 2400 Series II, respectively. To monitor the reaction and confirm the purity of compounds thin layer chromatography (TLC) silica gel 60 F254 plate (Sigma Aldrich) was used in every step of the reaction scheme. N′-benzylidene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazones (3a–3h) were prepared by the already reported method [[24]], [[25]].

The compounds (4a–4h) were prepared by the two methods.

• Equimolar mixture of 3-methyl-1,6-diphenyl-1 H -pyrazolo[3,4- b ]pyridine-4-carbohydrazide (_B_2) and respective benzoic acid in POCl 3 was refluxed for 6 h.
• Equimolar mixture of compound (_B_2) and an aldehyde in acetic acid was refluxed for 1–2 h. On cooling the precipitates of resultant hydrazone were filtered out and refluxed without purification in o -phosphoric acid for 4–5 h. During above methods for the preparation of all compounds (4a–4h), the reaction mixtures were cooled to room temperature and poured over crushed ice with vigorous stirring. The resulting precipitates were filtered and dried. The crude products were recrystallized from ethanol.

(method a) 74% (method b) 60%. m.p. 269–271 °C. IR (v-cm−1): 1655 (C=O); 1593 (C–C); 1542 (C=N); 1504 (C–O); 1069 (C–O). 1HNMR (400 MHz, DMSO-d6): δ 2.82 (s, 3H; CH3), 7.46–7.49 (m, 2H; 2Ar–H), 7.66–7.73 (m, 8H; 8Ar–H), 8.25 (s, 1H; H–5), 8.33 (d, 2H, J = 8.2 Hz; 2Ar–H), 8.39 (d, 2H, J = 8.4 Hz; 2Ar–H). 13CNMR (100 MHz, DMSO-d6): δ13.9 (CH3), 111.3, 114.4, 114.5, 120.7, 120.8, 125.8, 125.9, 127.2, 129.0, 129.1, 130.0, 130.1, 134.6 (C–Cl), 136.1, 137.5, 137.6, 138.8, 142.1, 151.4, 156.1, 164.9, 166.5 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 463 (C27H18N5ClO); M+2: 465. CHN (C27H18N5ClO): Calculated C 69.97, H 3.88, N 15.11%; Found C 69.89, H 3.86, N 15.07%.

(method a) 60% (method b) 59%. m.p. 269–270 °C. IR (v-cm−1): 1655 (C=O); 1593 (C–C); 1544 (C=N); 1526, 1354 (NO2) 1500(C–O); 1072(C–O). 1HNMR (300 MHz, DMSO-d6): δ 2.71 (s, 3H; CH3), 7.33–7.38 (m, 2H; 2Ar–H), 7.52–7.62 (m, 7H; 7Ar–H), 8.17 (s, 1H; H–5), 8.24 (d, 2H, J = 7.8 Hz; 2Ar–H), 8.31 (d, 2H, J = 8.1 Hz; 2Ar–H).13CNMR (75 MHz, DMSO-d6): δ 15.5 (CH3), 111.5, 114.4, 120.7, 123.2, 125.1, 125.8, 126.7, 127.3, 127.7, 129.0, 129.1, 130.1, 130.5, 136.1 (C–Cl), 137.5, 138.8, 142.2 (C–NO2), 151.4, 156.1, 164.2, 166.5 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 508 (C27H17N6O3Cl); M+2: 510. CHN (C27H17N6O3Cl): Calculated C 63.77, H 3.34, N 16.53%; Found C 63.71, H 3.31, N 16.49%.

(method a) 66% (method b) 60%. m.p. 248–250 °C. IR (v-cm−1): 1655 (C=O); 1593 (C–C); 1542 (C=N); 1504 (C–O); 1069 (C–O). 1HNMR (400 MHz, CDCl3): δ 2.78 (s, 3H; CH3), 7.29–7.32 (m, 2H; 2Ar–H), 7.48–7.55 (m, 8H; 8Ar–H), 8.13 (s, 1H; H–5), 8.18 (d, 2H, J = 7.8 Hz; 2Ar–H), 8.34 (d, 2H, J = 7.9 Hz; 2Ar–H). 13CNMR (100 MHz, CDCl3): δ 16.2 (CH3), 112.5, 115.0, 115.1, 121.2, 125.6, 127.4, 128.7, 128.8, 129.7, 129.8, 134.2, 138.2, 139.2, 142.4, 142.5, 151.9 (C–F), 152.0, 156.5, 164.1, 165.5 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 447 (C27H18N5FO) M+. CHN (C27H18N5FO): Calculated C 72.48, H 4.02, N 15.65%; Found C 72.42, H 3.99, N 15.63%.

(method a) 73% (method b) 70%. m.p. 238 °C. IR (v-cm−1):1650 (C=O); 1581 (C–C); 1572 (C=N); 1505 (C–O); 1072 (C–O). 1HNMR (400 MHz, DMSO-d6): δ 2.81 (s, 3H; CH3), 7.47–7.50 (m, 2H; 2Ar–H), 7.66−7.76 (m, 8H; 8Ar–H), 8.29 (s, 1H; H–5), 8.34 (d, 2H, J = 8.0 Hz; 2Ar–H), 8.40 (d, 2H, J = 8.2 Hz; 2Ar–H). 13C NMR(100 MHz, DMSO-d6): δ 13.5 (CH3), 111.5, 114.3, 114.5, 120.6, 120.7, 125.7 (C–Br), 125.8, 127.2, 127.3, 128.9, 129.0, 129.9, 130.0, 134.6, 136.0, 137.6, 138.8, 142.2, 151.4, 156.1, 164.1, 166.4 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 507 (C27H18N5BrO); M+2: 509. CHN (C27H18N5BrO): Calculated C 63.90, H 3.55, N 13.80%; Found C 63.82, H 3.51, N 17.75%.

(method a) 85% (method b) 80%. m.p. 272–274 °C. IR (v-cm−1): 1686 (C=O); 1591 (C–C); 1573 (C=N); 1504(C–O); 1059(C–O).1HNMR (400 MHz, CDCl3): δ 1.50 (s, 3H; 4–CH3–Ar), 2.82 (s, 3H; CH3), 7.29–7.32 (m, 2H; 2Ar–H), 7.47–7.55 (m, 8H; 8Ar–H), 8.13 (s, 1H; H–5), 8.19 (d, 2H, J = 8.0 Hz; 2Ar–H), 8.34 (d, 2H, J = 8.3 Hz; 2Ar–H). 13CNMR (75 MHz, DMSO-d6): δ 14.4 (4–CH3–Ar), 15.5 (CH3), 111.6, 113.0, 114.4, 120.5, 120.7, 120.8, 125.8, 127.3, 127.4, 129.1, 129.2, 130.1, 130.5, 137.7, 137.9, 138.9, 142.3, 151.0, 156.1, 156.2, 164.7, 166.5 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 443 (C28H21N5O) M+; CHN (C28H21N5O): Calculated C 75.84, H 4.74, N 15.80%; Found C 75.88, H 3.73, N 15.82%.

(method a) 72% (method b) 70%. m.p. 284–285 °C. IR (v-cm−1): 1655 (C=O); 1593 (C–C); 1542 (C=N); 1504 (C–O); 1069 (C–O). 1HNMR (300 MHz, DMSO-d6): δ 2.69 (s, 3H; CH3), 7.31–7.36 (m, 2H; 1Ar–H, 1Fu–H), 7.51–7.61 (m, 7H; 5Ar–H, 2Fu–H), 8.14 (s, 1H; H–5), 8.22 (d, 2H, J = 7.6 Hz; 2Ar–H), 8.28 (d, 2H, J = 7.8 Hz; 2Ar–H). 13CNMR (75 MHz, DMSO-d6): δ 15.5 (CH3), 111.5, 114.4, 120.5, 120.6, 125.7, 127.2, 129.0, 129.1, 130.0, 136.0, 137.5, 138.8, 142.2, 151.3, 156.0, 166.4 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 419 (C25H17N5O2) M+. CHN (C25H17N5O2): Calculated C 71.59, H 4.05, N 16.70%; Found C 71.63, H 4.07, N 16.75%.

(method a) 75% (method b) 70%. m.p. 265–267 °C. IR (v-cm−1): 1653 (C=O); 1589 (C–C); 1539 (C=N); 1500 (C–O); 1072 (C–O). 1HNMR (300 MHz, DMSO-d6): δ 2.70 (s, 3H; CH3), 7.32–7.37 (m, 2H; 2Ar–H), 7.52–7.62 (m, 8H; 8Ar–H), 8.16 (s, 1H; H–5), 8.23 (d, 2H, J = 7.8 Hz; 2Ar–H), 8.30 (d, 2H, J = 8.1 Hz; 2Ar–H). 13CNMR (75 MHz, DMSO-d6): δ 15.5 (CH3), 111.5, 114.4, 120.5, 120.6, 120.7, 125.7, 125.8, 127.2, 127.4, 129.0, 129.1, 130.0, 130.1, 136.0, 137.5, 138.8, 142.2, 151.3, 156.0, 166.5 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 430 (C26H18N6O) M+. CHN (C26H18N6O): Calculated C 72.55, H 4.18, N 19.53%; Found C 72.47, H 4.13, N 19.49%.

(method a) 60% (method b) 55%. m.p. 267–269 °C. IR (v-cm−1): 1665 (C=O); 1587 (C–C); 1542 (C=N); 1530, 1340 (NO2), 1504 (C–O); 1069 (C–O). 1HNMR (300 MHz, DMSO-d6): δ 2.71 (s, 3H; CH3), 7.33–7.38 (m, 2H; 2Ar–H), 7.52–7.62 (m, 7H; 7Ar–H), 8.17 (s, 1H; H–5), 8.24 (d, 2H, J = 7.8 Hz; 2Ar–H), 8.31 (d, 2H, J = 8.1 Hz; 2Ar–H). 13CNMR (75 MHz, DMSO-d6): δ 15.5 (CH3), 111.5, 114.4, 120.7, 123.2, 125.1, 125.8, 126.7, 127.3, 127.7, 129.0, 129.1, 130.1, 130.5, 136.1 (C–Cl), 137.5, 138.8, 142.2 (C–NO2), 151.4, 156.1, 164.2, 166.5 (C–2 & C–5 of oxadiazole). MS (EI+): m/z 508 (C27H17N6O3Cl); M+2: 510. CHN (C27H17N6O3Cl): Calculated C 63.77, H 3.34, N 16.53%; Found C 63.71, H 3.31, N 16.49%.

Antiplatelet activity against arachidonic acid and collagen was determined by previously reported method [[35]] with slight modification. Female healthy volunteers (20–25 years) with no clinical history of cardiovascular diseases, no medication was used in last month were included with the signed consent in the present study. The experimental protocol and consent were approved by Institutional Ethical Committee, institute of Chemistry, university of the Punjab, New Campus, Lahore. Blood was drawn from healthy volunteer by careful venipuncture. 5 mL blood containing 0.5 mL tri-sodium citrate (3.8%) was taken in falcon tube, and incubated at 37 °C for 30 min. 460 μL aliquots of this pre-incubated blood was taken in falcon tube and 600 μL fixing solution (0.5% formaldehyde in PBS) was added to take the baseline platelet count at t = 0 min. 20 μL of antagonist i.e. DMSO/Aspirin/compounds (4a–4f) were dispensed in falcon tube containing 460 μL whole blood and stirred for 2 min at 37 °C. Platelets count was recorded after 6 min on platelet counter (sysmex XT-1800i) after mixing and adding 20 μL of agonist i.e. arachidonic acid/collagen at 37 °C. Aspirin was used as a control while % Inhibition was calculated by the following formula. All assays were completed within 1.5 h of venipuncture.

Graph

$$\begin{array}{ccccc}\text{\% Inhibition =}\hfill & \left(\text{Agonist Platelet Count/Baseline Platelet Count}\right)\text{*100}\hfill & & & \\ \text{\% Aggregation =}\hfill & [\left(\text{Baseline Platelet Count–Agonist Platelet Count}\right)/\hfill & & & \\ & \text{Baseline Platelet Count}]\text{*100}\hfill & & & \end{array}$$

(Where Baseline Platelet count is the first blood draw count at t = 0 min).

In order to understand the insight mechanism of binding mode of synthesized compound with receptor residues, molecular docking simulations were performed for compounds (4a–4h & Aspirin). Molecular docking studies were performed by using MOE 2009.10 [[36]], [[37]], [[38]]. The human PGH-2 synthase 3D model of COX-1 (PDB-ID: 1Q4G) & COX-2 (PDB-ID: 3NT1) were retrieved from PDB (source www.rcsb.org//pdb/).

AMBER99 force field function of MOE was used for 3D protonation and energy minimization was done by receptor molecule optimization. The slope was set at 0.5 and receptor was reduced till its root mean square slope reached below point 0.05. After 3D protonation of receptor protein and hide molecule option was used to hydrogen molecule. Again MOE option of surface and map was used for pinpointing the pocket residues and docking site. This 3D protonated and energy minimized receptor molecules were used for molecular docking analysis.

The scaffolds of synthesized compounds (4a–4h) were constructed under chem3D pro version 12.0. Their 3D structure were then saved in.mol file for docking analysis the ligands structure prepared by addition of hydrogen atom and energy minimization at 0.05 gradient was done with the help of MMFF94X force field option in MOE. These ligands were then added into a database and were used for docking against the targeted receptor protein.

Molecular docking analysis was executed after receptor protein and ligands molecule preparation..mbd format was used for saving the docking output database file containing receptor ligands complexes. In order to evaluate the ligands interactions with receptor protein active sites residue, complexes with minimum S values were taken. MOE ligX option was used for analysis of best docked pose having highest negative binding energy value (S value), highest hydrogen bonding and π-π interaction.

To understand the radical scavenging procedure H-atom transfer and one-electron transfer mechanisms are frequently used [[39]], [[40]]. Recently, B3LYP functional has been proved proficient method that reproduces the experimental data [[41]], [[42]], [[43]], [[44]], [[45]], [[46]]. Thus in the present study ground state geometries have been optimized at B3LYP/6-31G** level of theory. Details about methodology can be found in the reference [[47]]. Ionization potential (IP) was calculated from the following equation.

Graph

$$\text{IP}=-{\text{E}}_{\text{HOMO}}$$

The initial ground state geometries were performed by using Gaussian 09 then all the calculations were executed by Spartan '14 v1.1.8' software including QSAR investigations.

All the data are presented as Mean ± Standard error mean (SEM). Whole data was analyzed by graph pad prism 6 software. One way ANOVA was applied followed by Tukey's test. The values were considered significantly different when P < 0.05.

The synthetic routes for the pyrazole derivatives are summarized in (Scheme 1). 2-(3-Methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-5-aryl-1,3,4-oxadiazoles (4a–4h) were synthesized by the cyclization of their respective hydrazones (3a–3h) using o-phosphoric acid as an oxidative cyclization agent. The percentage yield, melting point data was shown in Table 1.

Graph: Scheme 1: Synthesis of the target 2-(3-Methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-5- aryl-1,3,4-oxadiazoles. Reagents and Conditions: (a) POCl3, reflux, 6 h; (b) Acetic acid, reflux, 2–3 h; (c) o-phosphoric acid, reflux, 4 h.

Tab. 1: Physical and Mass Spectral data of 2-(3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-5-aryl-1,3,4-oxadiazoles.

Whole blood was used to determine the antiplatelet potential of synthesized compounds (4a–4f) against arachidonic acid and collagen induced platelet aggregation. We found that compound 4a, 4b, 4c, 4d, 4e and 4f showed good antiplatelet activity induced by arachidonic acid and exhibited more than 50% collagen and inhibited more than 50% platelet aggregation inhibition when whole human blood was used. Furthermore, the compounds 4b and 4f exhibited higher inhibitory effect 72.34 and 77.17%, respectively against collagen induced platelet aggregation. In compassion with aspirin when used arachidonic acid for platelet aggregation, compound 4b, 4d, 4e and 4f showed more that 93% inhibition (Table 2).

Tab. 2: Antiplatelet activity of 2-(3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-5-aryl-1,3,4-oxadiazoles.

1 adata is shown as the Mean ± SEM. bP < 0.001 shown difference between the compounds (4a, 4b, 4d, 4e and 4f) when compared with compound 4c.

Molecular docking study was performed to study the binding affinity of compounds (4a–4h). Results depicted that all the compounds could occupy two hydrophobic pockets of COX with impressive interaction energy value except 4a and 4h (Tables 3 and 4) against Arg83 and Arg120 residues of COX-1. No distinctive interaction energy was found with COX-2 protein when compounds were docked by using MOE 2009.10 (Figures 1 and 2).

Tab. 3: Docking result with the residue of Naproxen Pocket.

Tab. 4: Docking results of synthesized compound with selected residues.

Graph: Fig. 1: Two and three dimensional interaction diagrams of 4c, 4f and Aspirin using Naproxen Pocket. Interaction diagrams were attained by using ligand interaction analysis feature of MOE. Two dimensional structures of 4c (A), 4f (B) and aspirin (C). Three dimensional structures of 4c (D), 4f (E) and aspirin (F).

Graph: Fig. 2: Two and three dimensional interaction diagrams of 4b, 4c, 4d, 4e, 4f, 4g, and Aspirin using selected residues. Interaction diagrams were attained by using ligand interaction analysis feature of MOE while Arg120, Arg83 and Tyr355 residue of COX-1 were used. Where A and B; represents 2&3D interaction diagram of 4b, C and D: 2&3D interaction diagram of 4c, E and F; 2&3D interaction diagram of 4d, G and H; 2&3D interaction diagram of 4e, I and J; 2&3D interaction diagram of 4f, K and L; 2&3D interaction diagram of 4g, and M and N = 2&3D interaction diagram of Aspirin, respectively.

The distribution pattern of the frontier molecular orbitals (FMOs); highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) has been used to determine the stability of the synthesized compounds. The HOMO is delocalized on the phenyl pyrazolopyridines units in case of all 1,3,4-oxadiazole derivatives (4a–4h), while the LUMO is localized on the oxadiazole-pyrazolopyridines moiety except 4b and 4h in which nitro phenyl oxadiazole moieties are participating. The intra-molecular charge transfer (ICT) has been observed from phenyl group to the oxadiazole units in all the studied compounds except 4b and 4h. In 4b and 4h, significant ICT was seen from phenylpyrazolopyridines moiety to the nitrophenyl oxadiazole units, respectively. Moreover, 4d and 4e derivatives exhibited larger Egap energy. The HOMO energies (EHOMO), LUMO energies (ELUMO) and HOMO–LUMO energy gaps (Egap) at the B3LYP/6-31G** level of theory have been tabulated in Table 5. The trend in the EHOMO is 4e > 4f > 4c > 4a > 4d > 4g > 4b > 4h, ELUMO 4d > 4e > 4f > 4c > 4a > 4g > 4b > 4h and Egap 4d > 4e > 4c > 4f > 4a >4g > 4b > 4h was calculated.

Tab. 5: HOMO energies (EHOMO), LUMO energies (ELUMO) and energy gaps (Egap) in eV of oxadiazole derivatives obtained at B3LYP/6-31G** level of theory.

To understand the molecular interactions usually molecular electrostatic potential (MEP) is being used especially this 3-D mapping is good tool to know the relative reactivity sites for nucleophilic and electrophilic attack. The red and blue color parts represent negative and positive electrostatic potential (ESP) regions while green color parts denote the zero potential regions Figure 3. In addition, negative regions can be observed on the phenylpyrazole moiety in 4c, 4d, 4e and 4f as well as on nitro group in 4b and 4g. The negative charge on pyrazole moiety and nitrogen of end-core pyridine can also be seen (Figure 4).

Graph: Fig. 3: The distribution pattern of the HOMOs and LUMOs of the studied compounds (4a–4h) at ground states.

Graph: Fig. 4: The molecular electrostatic potential surfaces of the oxadiazole derivatives (4a–4h). Red, blue and green color regions represented the negative, positive and zero electrostatic potential of derivative (4a–4h).

Structure–activity relationship (SAR), structure–property–activity relationship (SPAR) and quantitative structure–activity relationship (QSAR) was used to understand the medicine behavior, nature of antiplatelet drugs and the correlations between the biological activity of the compounds and their physicochemical properties. Present all compound showed polar surface area ¡120. While lipophilicity of the studied compounds ranges from 5.96 to 8.66 in the following order 4f < 4g < 4b < 4c < 4e < 4a < 4h < 4d Table 6. It is widely accepted that the compounds having very high/low log P values do not have good bio-availability as they cannot cross hydrophilic and lipophilic barricades, respectively.

Tab. 6: Different SAR descriptors of oxadiazole derivatives obtained at B3LYP/6-31G** level of theory.

2 μ D = dipole moment; HBD = hydrogen bond donor; HBA = hydrogen bond acceptor; PSA = polar surface area; S.E. = solvation energy; Pol. = Polarizability.

Whole blood platelet aggregometry (WBI) enables the platelet functioning within their physiologic environment that is the RBCs and WBCs, as they are known to have platelet function modulation [[48]]. Arachidonic acid and collagen are naturally agonist in body and routinely used for the platelet aggregation to study the Storage Pool Disease and also to examine the effect of aspirin on platelet aggregation [[7]], [[8]], [[49]]. Aspirin inhibits the COX protein generation and thus acts as antagonist. All the synthesized compounds (4a–4f) showed more than 50% inhibition against both arachidonic acid and collagen induced platelet aggregation. Platelet aggregation inhibition results exhibited that our newly synthesized compounds efficiently targeted the arachidonic acid pathways compared with collagen pathway because the compound showed higher anti-platelet aggregation potential against arachidonic acid compared with collagen. Arachidonic acid present in the platelet phospholipid membranes, liberated and react with oxygen to form the prostaglandin which is then converted to a potent platelet aggregation agonist, thromboxane A2 [[7]], [[8]], [[9]]. The improved ICT in 4b and 4h is due to the significant push-pull effect in which phenylpyrazolopyridines moiety is acting as donor and nitrophenyl oxadiazole unit as strong acceptor. The negative/positive ESP regions are associated with electrophilic/nucleophilic reactivity, i.e. site would be more favorable for the electrophile/nucleophile attack. Careful analysis of the MEP revealed that oxadiazole moiety would be favorable site for electrophile attack in all the studied derivatives. The PSA of all the studied oxadiazole derivatives is less than 120 A2 showing that these would be orally active drugs and would be better for brain penetration. Linear relationship between the PSA and brain penetration [[50]] that is in contrast to the Palm and coworkers study for oral absorption in which they reported sigmoidal curve [[51]]. When the PSA increases then brain penetration decreases. Previously it has been shown that PSA should not exceed 120 A2 for the orally active drug which are transported by transcellular route [[50]], [[52]] and <100 A2 for brain penetration [[52]]. More electrophilic reactive sites in 4b, 4c, 4d, 4e, 4f and 4g derivatives might lead to more reactivity and biological activity.

All compound showed impressive antiplatelet activity when compared with 4c. Moreover, in comparison of Aspirin, 4b, 4d, 4e and 4f exhibited good antiplatelet activity. The docked compound (4c, 4f and Aspirin) showed impressive binding interaction with the COX-1 using naproxen pocket residues while (4b, 4c, 4d, 4e, 4f, 4g and Aspirin) showed the binding interaction with the COX-1 using selective residues pocket. The results for antiplatelet activity are found to be in good agreement with theoretical results which suggested that the oxadiazoles might have useful implications and potential as antiplatelet drugs for treatment of thromboembolic diseases.

We used ecofriendly method to synthesized oxadiazoles from hydrazones. All compound showed impressive antiplatelet activity when compared with 4c. Moreover, in comparison of Aspirin, 4b, 4d, 4e and 4f exhibited good antiplatelet activity. Comprehensible intra-molecular charge transfer has been observed in (4a–4h) derivatives. The higher HOMO energy of 4d and 4e is revealing that these derivatives would have higher electron donor ability as compared to the other studied compounds. The main focus of the present work was sorting of most potent compound for antiplatelet activity from the series (4a–4h) via Molecular Dockings and computational screening with targeted protein. The docked compound (4c, 4f and Aspirin) showed the binding interaction with the COX-1 using naproxen pocket residues while (4b, 4c, 4d, 4e, 4f, 4g and Aspirin) showed the binding interaction with the COX-1 using selective residues pocket. Present work also showed that 6 compounds out of 8 compounds involved in the hydrogen bond formation with the interacting residues of (Cox-1 & Cox-2) for antiplatelet activity.

In conclusion, a series of 2-(3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-5-aryl-1,3,4-oxadiazoles (4a–4h) derivatives has been synthesized. Computational, docking studies and antiplatelet potential of the synthesized compounds were determined. The result suggest that 1, 3, 4-oxadiazoles derivatives have potential to be used antiplatelet drugs for treatment of thromboembolic diseases. The 4b and 4f compounds were found to be most potent and might be used for the treatment of CVDs but required to do mechanistic evaluation at molecular level.

We are thankful to Higher Education Commission of Pakistan for research funding and Professor Dr. Makshoof Athar, Director, Institute of Chemistry, University of the Punjab, Lahore for his support and providing facilities to carry out this research work. A. Irfan extends his appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through the research groups program under grant number R.G.P.2/15/40.