Int J App Pharm, Vol 13, Issue 6, 2021, 157-169Original Article

STRUCTURE-BASED MULTITARGETED MOLECULAR DOCKING ANALYSIS OF PYRAZOLE-CONDENSED HETEROCYCLICS AGAINST LUNG CANCER

JAINEY P. JAMES1*, AISWARYA T. C.1, SNEH PRIYA1, DIVYA JYOTHI1, SHESHAGIRI R. DIXIT2

1Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences (NGSMIPS), Deralakatte, Mangaluru 575018, Karnataka, India, 2Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru 570015, Karnataka, India
Email: jaineyjames@nitte.edu.in

Received: 02 Aug 2021, Revised and Accepted: 03 Sep 2021

ABSTRACT

Objective: The significant drawbacks of chemotherapy are that it destroys healthy cells, resulting in adverse effects. Hence, there is a need to adopt new techniques to develop cancer-specific chemicals that target the molecular pathways in a non-toxic fashion. This study aims to screen pyrazole-condensed heterocyclics for their anticancer activities and analyse their enzyme inhibitory potentials EGFR, ALK, VEGFR and TNKS receptors.

Methods: The structures of the compounds were confirmed by IR, NMR and Mass spectral studies. The in silico techniques applied in this study were molecular docking and pharmacophore modeling to analyse the protein-ligand interactions, as they have a significant role in drug discovery. Drug-likeness properties were assessed by the Lipinski rule of five and ADMET properties. Anticancer activity was performed by in vitro MTT assay on lung cancer cell lines.

Results: The results confirm that all the synthesised pyrazole derivatives interacted well with the selected targets showing docking scores above-5 kcal/mol. Pyrazole 2e interacted well with all the four lung cancer targets with its stable binding mode and was found to be potent as per the in vitro reports, followed by compounds 3d and 2d. Pharmacophore modeling exposed the responsible features responsible for the anticancer action. ADMET properties reported that all the compounds were found to have properties within the standard limit. The activity spectra of the pyrazoles predicted that pyrazolopyridines (2a-2e) are more effective against specific receptors such as EGFR, ALK and Tankyrase.

Conclusion: Thus, this study suggests that the synthesised pyrazole derivatives can be further investigated to validate their enzyme inhibitory potentials by in vivo studies.

Keywords: Lung cancer, Pyrazolopyrimidines, Pyrazolopyridines, Molecular docking, Pharmacophore modeling, Anticancer activities

© 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2021v13i6.42801. Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Lung cancer is one of the leading causes of cancer mortality in men and women, [1, 2] responsible for 1.6 million deaths. Non-small-cell lung cancers (NSCLCs), including large-cell carcinoma, adenocarcinoma, and squamous cell carcinoma, contribute approximately 80-85% of lung cancer.

The major shortcoming of lung cancer chemotherapy is that it causes damages to normal cells, causing surplus adverse effects. Therefore targeted therapies [3] are needed to target only cancer cells, avoiding injuries to the healthy cells. One of the novel methods adopted in lung cancer therapy is developing cancer-specific compounds that can attack the molecular signalling pathways, thus creating non-toxic substances. The significant targets of paramount importance for lung cancers are EGFR (Epidermal growth factor receptor) [4, 5] ALK (Anaplastic lymphoma tyrosine kinase) [6, 7] BRAF (v-raf murine sarcoma viral oncogene homolog B1) [8, 9] VEGFR receptors (Vascular endothelial growth factor) [10, 11], and Wnt signalling pathway [12].

The EGFR receptor is recognised as a significant anticancer target. It belongs to the ErbB (epidermal growth factor) receptor tyrosine kinase family and is expressed at high levels on the surface of some cancer cells. The inhibition of EGFR plays a crucial role in angiogenesis, tumour suppression, and metastasis [13].

In anaplastic non-Hodgkin's lymphoma, the ALK gene was first described as a driver mutation. Dysregulated ALK expression is now an identified driver mutation in nearly twenty different human malignancies. The dysregulated ALK expression is now recognised as the driver mutation, including 4-9% of NSCLC [14].

One of the critical mediators promoting the angiogenesis process is VEGFR, as it has a prominent role in maintaining the vascular supply within the tumour. Its increased levels are a confirmatory factor in diverse human cancers, including NSCLC [15]. The Wnt signalling pathway is another potential target for lung cancer. Effective pharmacological inhibitors of the Wnt pathway have only recently become available. The tankyrase (TNKS), a poly-ADP-ribose polymerase (PARP) enzyme, was the critical mediator of Wnt signalling by the screens for small molecular antagonists of the Wnt pathway. Hence, using the targets mentioned above as partial agonists/antagonists can show promising treatment strategies [16].

The approved therapeutic EGFR inhibitors are gefitinib, erlotinib, afatinib, osimertinib, dacomitinib [17], and ALK inhibitors crizotinib, alectinib, brigatinib, lorlatinib [18], VEGFR inhibitors are axitinib, bevacizumab, sorafenib [19]etc.

Nitrogen-containing heterocycle-pyrazole has a vital role in the development of cancer therapies. The anticancer activity of these compounds is by the inhibition of different types of proteins, receptors and enzymes, which has a crucial role in cell division. Condensed pyrazole rings such as pyrazolopyrimidines, pyrazolopyridines and pyranopyrazole are known for their anticancer properties [20], and the available drugs with these core moieties are depicted in fig. 1.

An extension of previous works on pyrazole scaffolds [21, 22] and in silico studies [23, 24], we have performed an analysis to screen the inhibitory potency of synthesized pyrazole fused derivatives on various targets EGFR, ALK, VEGFR and TNKS by employing molecular docking and pharmacophore modelling techniques.

MATERIALS AND METHODS

Most of the chemicals were purchased from Sigma Aldrich, and further purification was not required. Melting points was determined by the capillary method and were uncorrected. Shimadzu Perkin Ekmer 8201 Pc IR Spectrometer used in recording IR spectra (KBr pellets), and frequencies are expressed in cm-1. Bruker Avance II, 400 NMR spectrometer, recorded NMR spectra and Shimadzu LCMS 8030, Japan Mass spectrometer recorded mass spectra.

Fig. 1: Available drugs with pyrazolopyrimidines and pyrazolopyridines moiety

Preparation of pyrazolopyrimidines (2a-2e) and pyrazolo-pyridines (3a-3e)

A solution of 0.01 mole of malonitrile/diethyl malonate and different pyrazole carbonitrile derivatives 1 (0.01 mole) was prepared in sodium ethoxide and ethanol, which was refluxed for eight hours. The solution was concentrated, and the obtained residue was filtered, washed with ice-cold water [25].

Modeling platform

In silico analysis was carried out on Maestro 11.9 (Schrödinger, 2019-4) [26]. This software package is programmed on DELL Inc.27" workstation machine running on Intel Core i7-7700 CPU@ 3.60 GHz x8, a processor with 8GB RAM and 1000 GB hard disk with Linux–x86_64 as the operating system.

Molecular docking and binding free energy calculation

Based on the literature, the selected targets for lung cancers are EGFR, ALK, VEGFR and tankyrase and their crystal structures EGFR (PDB ID: 4WKQ) [27], ALK (PDB ID: 4Z55) [28], VEGFR (PDB ID: 4AG8) [29], TNKS (PDB ID: 4W5S) [30] were availed from the protein data bank. The downloaded proteins were minimised by Protein Preparation Wizard, using the OPLS-2005 force field of Schrodinger software. The designed fused-pyrazoles were prepared by LigPrep application (Schrödinger, 2019-4) [26] and were used for docking. The minimized protein was employed to generate the grid, and the grid box was developed by applying default parameters. Glide-XP (extra precision) [31] was used for molecular docking computations. The binding free energy MMGBSA (Molecular Mechanics, Generalized Born Model and Solvent Accessibility) dGbind (kcal/mol), between the receptor and ligands, were calculated by the Prime module (Schrödinger, 2019-4) [26]. The docking scores and the 2D and 3D conformations were generated to analyse further the affinities and binding interactions of the selected ten fused-pyrazole molecules.

The docking procedure was confirmed by redocking the co-crystal ligand of the proteins into the binding sites, respectively. The docking poses of the co-crystals in XP method and experimental conditions were compared and found to be similar with RMSD, thus validating the docking results.

Pharmacophore modeling

Pharmacophore modeling was performed by Phase tool (Schrödinger, 2019-4) [26]. In this model, six pyrazoles were considered active (≥ 69 %), and four were inactive based on their percent inhibition on lung cancer cells. Common pharmacophore hypotheses (CPH) were searched, and the best CPH was selected based on the survival score until at least one hypothesis was found and scored successfully. Pharmacophore-matching tolerance was set to 2 A◦.

Drug-likeness, ADMET property and prediction of activity spectral studies

The compounds were screened for drug-likeness properties by checking with the Lipinski Rule of five [32] and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) property prediction by the QikProp tool [26]. The features considered for ADMET studies are the following: QPlogHERG, QPPCaco Caco-2 cell permeability, QPlogKhsa, Percent Human Oral Absorption. Further, to validate them as appropriate drug candidates, an online tool, prediction of activity spectra for substances (PASS), was used, which evaluate the biological activity based on their structural data [33]. This tool gives the values for the probability of activity (Pa) and inactivity (Pi) by comparing more than 300 pharmacological effects and biochemical mechanisms of compounds.

In vitro anticancer study by MTT assay

We procured A-549 (Human small-cell lung carcinoma) cell culture from National Centre for Cell Sciences (NCCS), Pune, India. Ten compounds were incubated with different concentrations (25, 50, 100, 200 µM) to screen the cytotoxic activity of the compounds against human small-cell lung carcinoma (A-549). The cell viability was then determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after 24 h of incubation. Percent inhibition was calculated from the absorbance as % growth inhibition [34].

RESULTS

Chemistry

The fused pyrazole derivatives were synthesized from substituted aminopyrazoles cyclising with malononitrile and diethyl malonate to yield pyrazolopyridines and pyrazolopyrimidines. IR, NMR and mass spectroscopic techniques were used to confirm the structures (table 1).

Table 1: Structure and spectral data of pyrazole derivatives

S. No. Compound code Structure IR (KBr, cm-1) 1H NMR (400 MHz, DMSO, δ/ppm LC-MS (m/z)
2a 1625.78 (C=N), 1583.61 (C=C), 3463.56 and 3296.45 (NH2), 2213.54 (CN) 3.67 (s, 2H, CH2), 7.74 (s, 2H, NH2), 8.19 (s, 1H, CH), 7.51-7.60 (m, 5H, Ar-H) (M+) 250
2b 1666.38 (C=N), 1592.32 (C=C), 3423.76 and 3265.45 (NH2), 2219.34 (CN), 767.97 (C-Cl) 3.12 (s, 2H, CH2), 7.68 (s, 2H, NH2), 8.33 (s, 1H, CH), 7.52-7.61 (m, 4H, Ar-H) (M+) 284
2c 1635.89 (C=N), 1593.82(C=C), 3421.45 and 3288.32 (NH2), 2214.43 (CN), 786.23 (C-Cl) 3.31 (s, 2H, CH2), 7.58 (s, 2H, NH2), 8.35 (s, 1H, CH), 7.53-7.60 (m, 4H, Ar-H) (M+) 284
2d 1646.32 (C=N), 1594.69 (C=C), 3408.08 and 3269.67 (NH2), 2245.76 (CN), 732.43 (C-Cl) 3.52 (s, 2H, CH2), 7.64 (s, 2H, NH2), 8.42 (s, 1H, CH), 7.54-7.63 (m, 4H, Ar-H) (M+) 284
2e 1654.21 (C=N), 1591.81 (C=C), 3414.34 and 3285.67 (NH2), 2249.76 (CN), 1447.93 (C-F) 3.61 (s, 2H, CH2), 7.73 (s, 2H, NH2), 8.39 (s, 1H, CH), 7.57-7.64 (m, 4H, Ar-H) (M+) 268
3a 1653.08 (C=N), 1588.63 (C=C), 3444.54 and 3281.32 (NH2), 2243.76 (CN) 3.34 (s, 2H, CH2), 7.78 (s, 2H, NH2), 8.41 (s, 1H, CH), 7.55-7.59 (m, 5H, Ar-H), 11.52 (s, 1H, OH) (M+) 298
3b 1687.24 (C=N), 1589.11 (C=C), 3451.65 and 3256.78 (NH2), 2234.31 (CN), 778.98 (C-Cl) 3.51 (s, 2H, CH2), 7.62 (s, 2H, NH2), 8.37 (s, 1H, CH), 7.52-7.58 (m, 4H, Ar-H), 11.23 (s, 1H, OH) (M+) 332
3c 1632.58 (C=N), 1589.31 (C=C), 3454.44 and 3268.91 (NH2), 2247.04 (CN), 778.12 (C-Cl) 3.11 (s, 2H, CH2), 7.55 (s, 2H, NH2), 8.39 (s, 1H, CH), 7.37-7.57 (m, 4H, Ar-H), 11.05 (s, 1H, OH) (M+) 332
3d 1651.32 (C=N), 1598.12 (C=C), 3464.07 and 3256.17 (NH2), 2241.75 (CN), 756.76 (C-Cl) 3.52 (s, 2H, CH2), 7.77 (s, 2H, NH2), 8.47 (s, 1H, CH), 7.50-7.56 (m, 4H, Ar-H), 11.41 (s, 1H, OH) (M+) 332
3e 1643.13 (C=N), 1591.32 (C=C), 3401.67 and 3239.31 (NH2), 2208.89 (CN), 1432.76 (C-F) 3.12 (s, 2H, CH2), 7.67 (s, 2H, NH2), 8.31 (s, 1H, CH), 7.48-7.60 (m, 4H, Ar-H), 11.41 (s, 1H, OH) (M+) 316

Molecular docking

The docking and binding free energy scores obtained from their respective receptor targets EGFR, ALK, VEGFR, and TNKS, confirmed the molecular interactions. The co-crystallised structures of gefitinib, ceritinib, axitinib, 3J1, which are active against lung cancer with the corresponding PDB IDs 4WKQ, 4Z55, 4AG8 and 4W5S, were obtained and found to have docking scores-8.80 kcal/mol,-11.36 kcal/mol,-14.41 kcal/mol, and-13.95 kcal/mol respectively; and their binding free energies are-95.15 kcal/mol,-100.94 kcal/mol,-123.86 kcal/mol,-101.34 kcal/mol towards their respective receptors EGFR, ALK, VEGFR and TNKS, (table 1). The RMSD values of the crystallised structures showed RMSD values as 1.231 Å, 1.321 Å, 1.412 Å, 1.114 Å, respectively, which validated the docking results.

All the ten pyrazole derivatives screened for lung cancer targets exhibited docking values above-5 kcal/mol. The top pyrazole derivatives were 2e, 2d and 3d towards EGFR, ALK, VEGFR and TNKS. Their docking scores and binding affinity were given in table 2. In these top evaluations, 2e showed the best docking conformation with docking scores-7.75,-7.23,-8.52 and-8.31 kcal/mol and binding free energy-62.77,-53.42,-77.78, and-63.67 kcal/mol against, followed by pyrazoles 2d (-7.70,-7.13,-8.47,-8.22 kcal/mol) and 3d (-7.51,-7.20,-8.30,-8.01 kcal/mol) EGFR, ALK, VEGFR and TNKS respectively (table 2).

Table 2: Structures, docking score and MMGBSA dG bind of reference compounds

Reference compounds available in PDB Structures Target receptors Docking scores MMGBSA dG Bind
Gefitinib 4WKQ -8.806 -95.15
Ceritinib 4Z55 -11.362 -100.94
Axitinib 4AG8 -14.414 -123.86
3J1 4W5S -13.953 -101.34

To validate the chemical interactions, the analysis of co-crystals conformations are as follows; in enzyme EGFR, the common amino acids that make interactions with standard geftinib and pyrazole derivatives are Gln 791, Thr 790 and Thr 854 (hydrophobic); Met 793 (hydrogen bond); Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Leu 788 (polar). Further, the common amino acids for the standard ceritinib and pyrazole derivatives that make interactions with enzyme ALK are Asn 1254; polar interactions are Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180 and hydrogen bond with the same amino acid Met 1199. Similarly, for the enzyme, VEGFR, the common amino acids for axitinib and pyrazole derivatives that bond by hydrophobic interactions is Asn 923 and polar interactions are Cys 919, Phe 918, Val 916, Leu 1035, Ala 866, Val 899, Phe 1047, Cys 1045, Leu 840, Val 848. In the case of the TNKS enzyme, for the ligand 3J1 and pyrazoles, the amino acids that frequently make hydrophobic interactions are Ser 1221, Hid 1184, Ser 1186; Tyr 1224, Tyr 1213, Phe 1214, Ala 1215, Phe 1188, Pro 1187; polar interactions are Ala 1215, Phe 1214, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224.

The finest docking conformations were also examined to reveal the primary interacting amino acid residues in the active pockets of EGFR, ALK, VEGFR and TNKS (table 3-5 and fig. 2-5).

Table 3: Docking score and MMGBSA dG bind of pyrazole derivatives

Compounds Docking scores (in kcal/mol) MMGBSA dG bind (in kcal/mol)
EGFR-4WKQ ALK–4Z55
2a -6.11 -6.75
2b -6.08 -6.04
2c -5.25 -5.39
2d -7.70 -7.13
2e -7.75 -7.23
3a -6.97 -7.04
3b -6.35 -5.91
3c -7.44 -7.06
3d -7.51 -7.20
3e -7.21 -6.75

Table 4: Molecular interactions of reference compounds with the active site of protein

Reference

compounds

Protein and

PDB IDs

 Nature of interactions Amino acids on active sites with
Gefitinib EGFR-4WKQ Hydrophobic Interaction Gln 791,Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Pro 794, Phe 795, Leu 788
H-Bond Met 793, Cx 797
Halogen Bonding Leu 788, Lys 745
Pi-Pi Stacking --
Pi Cation --
Ceritinib ALK–4Z55 Hydrophobic Interaction Asn 1254
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Ala 1200, Val 1180
H-Bond Met 1199, Glu 1197, Lys 1150
Halogen Bonding --
Pi-Pi Stacking --
Pi Cation --
Axitinib VEGFR–4AG8 Hydrophobic Interaction --
Polar Interactions Phe 921, Cys 919, Phe 918, Leu 1035, Val 916, Ala 866, Val 899, Cys 1045, Leu 840, Val 848, Phe 1047, Val 867, Val 914, Leu 889
H-Bond Asp 1046, Glu 885, Glu 917
Halogen Bonding --
Pi-Pi Stacking Phe 1047
Pi Cation --
3J1 TNKS 1-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Tyr 1203, Ile 1228, Met 1207, Tyr 1224, Tyr 1213, Phe 1214, Ala 1215, Phe 1188, Pro 1187
H-Bond Gly 1185, Glu 1291, Ser 1221
Halogen Bonding --
Pi-Pi Stacking Tyr 1224
Pi Cation --

Table 5: Molecular interactions of the pyrazole derivatives with the active site of protein

Compounds

Protein and

PDB IDs

 Nature of Interactions Amino acids on active sites
2a EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Ile 744, Ile 789, Leu 788, Leu 777, Met 766
H-Bond Met 793
ALK–4Z55 Hydrophobic Interaction Hid 1124
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1126
H-Bond Met 1199
VEGFR–4AG8 Hydrophobic Interaction Asn 923
Polar Interactions Cys 919, Phe 918, Val 916, Leu 1035, Ala 866, Val 899, Phe 1047, Cys 1045, Leu 840, Val 848
H-Bond Cys 919
Pi-Pi Stacking Phe 1047
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186
Polar Interactions Ala 1215, Phe 1214, Tyr 1213, Met 1207, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224
H-Bond Tyr 1224, Tyr 1203
Pi-Pi Stacking Tyr 1224
2b EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Ile 744, Ile 789, Leu 788, Leu 777, Met 766
H-Bond Met 793
Halogen Bonding Asp 855
ALK–4Z55 Hydrophobic Interaction Hid 1124
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1126
H-Bond Lys 1150, Ala 1126, Hid 1124
Halogen Bonding Met 1199
VEGFR–4AG8 Hydrophobic Interaction Asn 923
Polar Interactions Cys 919, Phe 918, Val 916, Leu 1035, Ala 866, Val 899, Phe 1047, Cys 1045, Leu 840, Val 848
H-Bond Cys 919
Halogen Bonding Glu 917
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Ala 1215, Phe 1214, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ala 1202, Phe 1188, Phe 1183
H-Bond Tyr 1213, Hid 1201
Pi-Pi Stacking Hid 1184, Tyr 1224
2c EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Ile 744, Leu 788, Met 766, Phe 856
H-Bond Met 793
Halogen Bonding Leu 788, Lys 745, Ala 743
ALK–4Z55 Hydrophobic Interaction Hid 1124
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180
H-Bond Glu 1197
VEGFR–4AG8 Polar Interactions Cys 919, Phe 918, Val 916, Leu 1035, Ala 866, Val 899, Phe 1047, Cys 1045, Leu 840, Val 848, Val 914, Ile 915, Val 867, Leu 889, Val 898, Ile 1044
Pi Cation Lys 868
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Phe 1214, Tyr 1213, Met 1207, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ile 1212, Ala 1202
H-Bond Hid 1201, Tyr 1213
Halogen Bonding Ser 1221, Gly 1185
Pi-Pi Stacking Tyr 1224, Hid 1184
2d EGFR-4WKQ Hydrophobic Interaction Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Pro 794
H-Bond Csx 797, Met 793
Halogen Bonding Cys 745
ALK–4Z55 Hydrophobic Interaction Hid 1124
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180
H-Bond Glu 1197
VEGFR–4AG8 Hydrophobic Interaction Asn 923, Thr 926
Polar Interactions Cys 919, Phe 918, Val 916, Leu 1035, Ala 866, Val 899, Phe 1047, Cys 1045, Leu 840, Val 848
H-Bond Cys 919
Halogen Bonding Asp 1046
Pi-Pi Stacking Phe 1047
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186
Polar Interactions Ala 1215, Phe 1214, Tyr 1213, Met 1207, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224
H-Bond Tyr 1224, Tyr 1203
Pi-Pi Stacking Tyr 1224
2e EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Pro 794
H-Bond Csx 797, Met 793
ALK–4Z55 Hydrophobic Interaction Hid 1124
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1126
H-Bond Ala 1126, Lys 1150, Hid 1124
VEGFR–4AG8 Hydrophobic Interaction Asn 923, Thr 926
Polar Interactions Cys 919, Phe 918, Val 916, Leu 1035, Ala 866, Val 899, Phe 1047, Cys 1045, Leu 840,Val 848
H-Bond Cys 919
Pi-Pi Stacking Phe 1047
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Ala 1215, Phe 1214,Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ala 1202, Phe 1188, Ile 1212
H-Bond Hid 1201, Tyr 1213
Pi-Pi Stacking Hid 1184, Tyr 1224
3a EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Leu 788, Pro 794, Cys 775
ALK–4Z55 Hydrophobic Interaction Ser 1206
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1200
VEGFR–4AG8 Hydrophobic Interaction Thr 926, Asn 923
Polar Interactions Phe 921, Cys 919, Phe 918, Leu 1035, Val 916, Ala 866, Val 899, Cys 1045, Leu 840, Val 848, Phe 1047
H-Bond Cys 919, Leu 840
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Ala 1215, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Phe 1188, Ala 1202, Ile 1212, Phe 1214
H-Bond Hid 1201, Tyr 1213
Pi-Pi Stacking Hid 1184, Hid 1201
3b EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Ile 789, Met 766, Leu 788, Pro 794, Ile 744, Leu 777
H-Bond Met 793, Glu 791
Halogen Bonding Lys 745
ALK–4Z55 Hydrophobic Interaction --
Polar Interactions Leu 1122, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1200
H-Bond Met 1199
VEGFR–4AG8 Hydrophobic Interaction Asn 923
Polar Interactions Phe 921, Cys 919, Phe 918, Leu 1035, Val 916, Ala 866, Val 899, Cys 1045, Leu 840, Val 848, Phe 1047, Val 867, Val 914, Leu 889
H-Bond Cys 919
Pi-Pi Stacking Phe 1047
Pi Cation Lys 868
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Ala 1215, Phe 1214, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ala 1202, Ile 1212, Phe 1188, Phe 1183
Halogen Bonding Tyr 1224, Tyr 1213
3c EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Leu 788, Pro 794
H-Bond Met 793
ALK–4Z55 Hydrophobic Interaction Asn 1254
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1200, Ala 1126
H-Bond Lys 1150, Met 1199
VEGFR–4AG8 Hydrophobic Interaction Asn 923
Polar Interactions Phe 921, Cys 919, Phe 918, Leu 1035, Val 916, Ala 866, Val 899, Cys 1045, Leu 840, Val 848, Phe 1047, Val 914, Leu 889
H-Bond Cys 919
Pi-Pi Stacking Phe 1047
Pi Cation Lys 868
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Phe 1214, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ile 1212, Phe 1188, Phe 1197, Ile 1192
H-Bond --
Halogen Bonding Gly 1185, Ser 1221
Pi-Pi Stacking Tyr 1224
3d EGFR-4WKQ Hydrophobic Interaction Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Pro 794, Phe 795
H-Bond Met 793
Halogen Bonding Asp 855
Pi-Pi Stacking --
Pi Cation --
ALK–4Z55 Hydrophobic Interaction Asn 1254
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1200
H-Bond Met 1199, Lys 1150
Halogen Bonding --
Pi-Pi Stacking --
Pi Cation --
VEGFR–4AG8 Hydrophobic Interaction Thr 926, Asn 923
Polar Interactions Phe 921, Cys 919, Phe 918, Leu 1035, Val 916, Ala 866, Val 899, Cys 1045, Leu 840, Val 848, Phe 1047
H-Bond Cys 919, Leu 840
Halogen Bonding Asp 1046
Pi-Pi Stacking --
Pi Cation --
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Ala 1215, Phe 1214, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ile 1212, Phe 1188, Phe 1197, Ile 1192, Ala 1191
H-Bond Ser 1186
Pi-Pi Stacking Tyr 1224
3e EGFR-4WKQ Hydrophobic Interaction Gln 791, Thr 790, Thr 854
Polar Interactions Leu 718, Leu 844, Met 793, Leu 792, Val 726, Ala 743, Met 766, Leu 788, Phe 794
H-Bond Met 793
Halogen Bonding --
Pi-Pi Stacking --
Pi Cation --
ALK–4Z55 Hydrophobic Interaction Asn 1254
Polar Interactions Leu 1122, Val 1130, Met 1199, Leu 1198, Leu 1256, Leu 1196, Val 1180, Ala 1148, Val 1180, Ala 1200, Ala 1126
H-Bond Met 1199, Lys 1150
Halogen Bonding --
Pi-Pi Stacking --
Pi Cation --
VEGFR–4AG8 Hydrophobic Interaction Asn 923
Polar Interactions Cys 919, Phe 918, Leu 1035, Val 916, Ala 866, Val 899, Cys 1045, Leu 840, Val 848, Phe 1047
H-Bond Cys 919, Leu 840
Halogen Bonding --
Pi-Pi Stacking Phe 1047
Pi Cation --
TNKS-4W5S Hydrophobic Interaction Ser 1221, Hid 1184, Ser 1186, Hid 1201
Polar Interactions Ala 1215, Phe 1214, Tyr 1213, Tyr 1203, Ile 1228, Pro 1187, Tyr 1224, Ile 1212, Phe 1188, Phe 1197, Ile 1192, Ala 1191
H-Bond Ser 1186
Halogen Bonding --
Pi-Pi Stacking Tyr 1224
Pi Cation --

Fig. 2: Molecular docking (a) 2D (b) 3D interactions of pyrazolopyrimidine 2e with 4WKQ

Fig. 3: Molecular docking (a) 2D (b) 3D interactions of pyrazolopyrimidine 2e with 4Z55

Fig. 4: Molecular docking (a) 2D (b) 3D interactions of pyrazolopyrimidine 2e with 4AG8

Fig. 5: Molecular docking (a) 2D (b) 3D interactions of pyrazolopyrimidine 2e with 4W5S

Pharmacophore hypothesis generation and modeling

The results of all featured pharmacophore hypotheses are in table 6. DHRRR_1 is having the best survival score of 5.1979 in this study, which consists of one hydrophobic group (H), one hydrogen bond donor (D), and three aromatic rings (R). The distances between the sites in the common pharmacophore hypothesis DHRRR_1 are given in fig. 6 (a-b) and table 7.

Table 6: Score hypothesis

Hypothesis ID Survival score Site score Vector score Volume Selectivity
DRRR_1 4.753541 0.967441 0.897639 0.778978 1.331332
DRRR_2 4.742687 0.966377 0.897544 0.777513 1.323101
ADRR_1 4.638511 0.966887 0.897754 0.779634 1.216084
ADRR_2 4.622626 0.970017 0.898106 0.777694 1.198658
ADRR_3 4.508706 0.942376 0.90864 0.690508 1.18903
DHRRR_1 5.197949 0.90054 0.861293 0.812639 2.021416
DHRRR_2 5.164714 0.882849 0.854915 0.809522 2.015368
ADHRR_1 5.000595 0.864909 0.861097 0.813654 1.858875
ADHRR_2 4.968628 0.864643 0.858113 0.807504 1.836308
ADHRR_3 4.915803 0.853138 0.86407 0.814323 1.782212
ADHRR_4 4.904614 0.868992 0.865852 0.813708 1.754002
ADHRR_5 4.898018 0.851548 0.85963 0.807438 1.777342
ADHRR_6 4.893939 0.846438 0.886796 0.781327 1.777319
ADHRR_7 4.881754 0.844744 0.859158 0.814788 1.761005
DHRR_5 4.529072 0.70509 0.955143 0.688846 1.481023
DHRR_1 4.712077 0.988826 0.852928 0.775829 1.492433
DHRR_2 4.684691 0.934964 0.862256 0.786716 1.498695
DHRR_3 4.682611 0.917492 0.865663 0.781164 1.51623
DHRR_4 4.620457 0.867853 0.852025 0.783564 1.514955
DHRRR_3 4.901226 0.663248 0.935309 0.690003 2.010606

Table 7: Distances between different sites of model DHRRR_1

S. No. Site 1 Site 2 Distance
H8 D6 5.12
H8 R11 3.16
H8 R9 5.09
H8 R10 6.53
D6 R11 3.41
D6 R9 4.57
D6 R10 8.34
R11 R9 2.15
R11 R10 5.12
R11 H8 3.16
R11 D6 3.41
R9 R10 3.97
R9 H8 5.09
R9 D6 4.57
R9 R11 2.15
R10 H8 6.53
R10 D6 8.34
R10 R11 5.12
R10 R9 3.97

Fig. 6: a) Pharmacophore hypothesis DHRRR_1 b) Distances in the pharmacophore hypothesis DHRRR_1

Table 8: Physicochemical and ADMET properties of pyrazole derivatives

S. No.

Comp-

ounds

 MW Log P donorHB

Accpt

HB

 PSA QPlogHERG

QPP

Caco

QPlog

Khsa

Percent human oral

absorption
Ceritinib 577.743 4.838 2 9.75 119.604 -7.51 54.854 1.146 73.44
Axitinib 386.47 4.721 2 4.5 74.603 -6.767 861.397 0.728 100
Gefitinib 446.908 4.314 1 7.7 61.213 -7.105 1044.67 0.351 100
3J1 332.361 2.438 2 6.7 89.242 -6.288 352.29 0.097 86.808
2a 250.262 1.41 2 5 89.799 -5.273 236.605 -0.209 77.689
2b 284.707 1.859 2 5 90.018 -5.241 237.627 -0.107 80.355
2c 284.707 1.897 2 5 89.767 -5.223 236.907 -0.108 80.553
2d 284.707 1.895 2 5 89.789 -5.208 236.913 -0.109 80.543
2e 268.253 1.638 2 5 89.79 -5.153 237.079 -0.173 79.045
3a 298.301 3.232 1 3 104.222 -5.703 299.715 0.471 90.197
3b 332.746 3.679 1 3 105.318 -5.612 282.256 0.597 92.346
3c 332.746 3.728 1 3 104.218 -5.626 299.505 0.59 93.095
3d 332.746 3.728 1 3 104.227 -5.624 299.536 0.591 93.099
3e 316.291 3.468 1 3 104.233 -5.583 299.429 0.515 91.575

Table 9: PASS prediction of anticancer properties

Compounds Activity Pa
1. 2a Antineoplastic (melanoma) 0.155
Antineoplastic antimetabolite 0.108
Epidermal growth factor receptor kinase inhibitor 0.142
ALK inhibitor 0.107
Tankyrase inhibitor 0.254
2. 2b ALK inhibitor 0.101
Antineoplastic (melanoma) 0.139
Tankyrase inhibitor 0.174
Epidermal growth factor receptor kinase inhibitor 0.133
3. 2c Epidermal growth factor receptor kinase inhibitor 0.138
ALK inhibitor 0.100
Tankyrase inhibitor 0.182
4. 2d Tankyrase inhibitor 0.192
Epidermal growth factor receptor kinase inhibitor 0.146
ALK inhibitor 0.108
5. 2e Tankyrase inhibitor 0.275
ALK inhibitor 0.115
Epidermal growth factor receptor kinase inhibitor 0.145
6. 3a Antineoplastic (melanoma) 0.148
Antineoplastic antimetabolite 0.113
ALK inhibitor 0.097
Antileukemic 0.205
7. 3b Antineoplastic (multiple myeloma) 0.269
Antineoplastic (melanoma) 0.136
ALK inhibitor 0.094
8. 3c Antileukemic 0.152
ALK inhibitor 0.093
9. 3d Antineoplastic (multiple myeloma) 0.223
ALK inhibitor 0.098
10. 3e Antineoplastic antimetabolite 0.102
ALK inhibitor 0.104
Antileukemic 0.186
Tankyrase inhibitor 0.175

Drug-likeness, ADMET and prediction of activity spectral studies

The synthesized ten pyrazoles have good drug-likeness properties, as shown in table 8. We evaluated the physicochemical properties to fit into the Lipinski rule of five, which is a way to determine if they are orally bioavailable. The compounds have shown no violations for the Lipinski rule of 5. Their ADMET properties were analysed, and reported that all the compounds checked were found to have all the properties within the standard limit (table 8). The activity spectra for anticancer activity of the pyrazoles were predicted to find out the inhibitory effect on the particular enzymes (table 9). The compounds bearing pyrazolopyridines (2a-2e) are more effective against specific receptors such as EGFR, ALK and tankyrase.

In vitro anticancer study by MTT assay

The results of the cytotoxicity studies were presented in table 10. Compound 2e, at the highest concentration, 200 µM, exhibited the most increased activity, which was 92% cytotoxic in nature and compounds 2d and 3d showed moderate cell growth inhibition around 80%. On correlating with their docking scores, these compounds have excellently interacted with the four lung cancer targets. Thus the results interpret that the synthesized derivatives might inhibit any of the four targets discussed and exert their anti-cancer action. On further analysis of the top interacted pyrazole 2e, they have maximum interaction with the VEGFR receptor, which proves their mechanism.

Table 10: Cytotoxicity studies of the pyrazole derivatives

S. No. Compound ID % Cytotoxicity
Concentration (µM)
25 50 100 200
2a 08 18 33 48
2b 15 32 48 71
2c 13 31 44 69
2d 17 34 51 84
2e 40 58 78 92
3a 09 20 30 45
3b 17 34 51 78
3c 08 19 36 53
3d 11 26 51 85
3e 10 22 38 56

DISCUSSION

We found that the pyrazole condensed derivatives interacted with four lung cancer targets EGFR, ALK, VEGFR and TNKS, and their cytotoxicity action was proved against lung cancer. The compound 2e was the most active in both in silico and in vitro studies, followed by 3d and 2d. Top compound 2e interacted with the VEGFR receptor excellently with stable binding mode and affinity. The best pharmacophore hypothesis, DHRRR_1 reveals the importance of the hydrogen bond donors, hydrophobic and aromatic groups essential for the anticancer action. Thus, validating the hydrogen bonds, hydrophobic groups and pi-pi interactions, which were showed by molecular docking. As per the cytotoxicity studies, the anticancer activity of the compounds 2e, 3d and 2d might be due to the introduction of electron-withdrawing fluorine and chlorine atoms in the benzene ring attached to the pyrazole ring.

Lung cancer development is stimulated by specific signaling pathways produced by receptors such as EGFR, ALK, VEGFR and TNKS. Much research has been performed to prove the anticancer efficacy of pyrazolopyrimidines on lung cancer [35], and some reported their inhibitory potentials on specific targets such as EGFR [36], VEGFR [37], tankyrase inhibitors [38] etc. We have screened the anticancer action by in vitro studies using A549 cell lines as a preliminary evaluation. Some reports are interfering in EGFR [39, 40] /VEGFR [41] /ALK [42] /Wnt [43, 44] /pathways inhibits the proliferation of A549 cell lines, and with this proof, we have carried the MTT assay. Cucurbitacin [39] and diazole [40] have been reported in proliferation inhibition in A549 cells by interfering EGFR signaling pathway. A study was performed to evaluate the TNKS small molecule inhibitor XAV939 on the proliferation and migration of lung adenocarcinoma A549 cells and found that XAV939 intervention inhibited A549 cell proliferation [43]. Determination of the appropriate target should be performed by analysing the enzyme antagonistic potential further, authenticating the mechanism of inhibition.

CONCLUSION

The synthesized pyrazole derivatives interacted well with the selected lung cancer targets-EGFR, ALK, VEGFR and TNKS; with their docking scores above-5 kcal/mol equivalent with their standards. The molecular interactions are based on various parameters such as glide score, binding free energy, polar interactions, hydrophobic interactions, and hydrogen bond interactions. Further, the in vitro results exhibit compounds 2e as the best anti-lung cancer agents followed by 3d and 2d, which was in agreement with their docking results. ADMET properties reported that all the compounds were found to have properties within the standard limit. The activity spectra of the pyrazoles predicted that pyrazolopyridines (2a-2e) are more effective against specific receptors such as EGFR, ALK and Tankyrase. Thus, this study suggests that the synthesized pyrazole derivatives can be further investigated to validate their enzyme inhibitory potentials by in vivo studies.

ACKNOWLEDGEMENT

We acknowledge Nitte Deemed to be University, Mangaluru, for the funding to carry out this project (University Research Grant No. NUFR1/2017/06/16). Also, thankful to the authorities of the NGSM Institute of Pharmaceutical Sciences, Mangaluru and NGSMIPS CADD lab for providing requirements for this work. Thanks to CUSAT, Cochin for NMR, Mysore University, Mysuru for Mass and Yenepoya Research Centre for anticancer studies.

AUTHORS CONTRIBUTIONS

All authors have contributed equally.

CONFLICT OF INTERESTS

The authors declare no conflict of interest, financial or otherwise.

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