INTRODUCTION:

The Epidermal Growth Factor Receptor (EGFR) is a membrane-bound tyrosine kinase that plays a pivotal role in regulating cellular growth, survival, proliferation and differentiation¹. EGFR belongs to the ErbB family of receptors and upon activation, triggers several downstream signaling pathways, including RAS-RAF-MEK-ERK and PI3K-AKT, which are frequently dysregulated in various malignancies².

Aberrant EGFR signaling, caused by gene amplification, receptor overexpression or activating mutations has been strongly associated with the pathogenesis and poor prognosis of multiple cancers notably non-small cell lung cancer (NSCLC), breast cancer and glioblastoma^3,4. Consequently, EGFR has emerged as a validated molecular target in oncology and its inhibition has led to the development of both small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies⁵.

Despite the success of approved EGFR inhibitors such as gefitinib, erlotinib and osimertinib, several limitations persist including acquired resistance, limited isoform specificity and adverse effects⁶. These challenges underscore the need for new classes of EGFR inhibitors with enhanced potency, selectivity and resistance-overcoming potential⁷. Pyrazole derivatives represent a versatile scaffold in medicinal chemistry and have shown promising pharmacological properties, including kinase inhibition, anticancer, anti-inflammatory and antimicrobial activities⁸. The structural flexibility and heteroaromatic nature of pyrazole enable effective interactions with kinase ATP-binding sites making them suitable candidates for EGFR inhibition⁹.

This study aims to evaluate a series of synthetically derived pyrazole compounds for their inhibitory potential against multiple EGFR kinase variants through molecular docking analysis¹⁰. Five EGFR kinase domain structures PDB IDs: 4HJO¹¹, 5HG7¹², 5XDX¹³, 6S9B¹⁴ and 6Z4B¹⁵ were selected to represent diverse conformational states and resistance-related mutations. Using AutoDock Vina, we conducted a comparative docking study to assess binding energies, hydrogen bond interactions, and binding site preferences across these EGFR structures.

By comparing the molecular interactions of pyrazole derivatives with multiple EGFR isoforms, this research seeks to identify structure-activity relationships (SAR) that could inform the future design of more selective and potent EGFR inhibitors. The insights gained from this in silico study may contribute to the development of next-generation anticancer agents targeting EGFR-driven tumors, particularly those unresponsive to current therapies.

MATERIALS AND METHODS:

Protein and ligand preparation

The crystal structures of mutant EGFR kinase proteins with PDB IDs 4HJO, 5HG7, 5XDX, 6S9B, and 6Z4B were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org)¹⁶. All docking studies were conducted using the monomeric forms of these crystal structures. The AMP-PNP or co-crystallized ligands were removed to prepare a clean and accessible docking site¹⁷. Protein structures were prepared using AutoDock Tools v1.5.7 (Scripps Research Institute, La Jolla, USA). This included deletion of water molecules and addition of polar hydrogens to facilitate hydrogen bond formation. Kollman and Gasteiger charges were applied to accurately represent molecular electrostatics. Kollman charges were derived using quantum mechanical methods, whereas Gasteiger charges were computed using electronegativity equilibration algorithms¹⁸.

A total of 24 pyrazole derivatives were designed using ChemSketch. The structures were converted from SMILES notation to 3D conformers, followed by geometry optimization using Avogadro with the Universal Force Field (UFF). The optimized structures were saved in PDB format, then converted to PDBQT format using AutoDock Tools for compatibility with AutoDock Vina¹⁹. In addition, reference ligands ATP and Gefitinib were obtained from PubChem in SDF format and similarly prepared for docking. All prepared ligands and receptors were visually validated using PyMOL to ensure proper structural integrity before docking²⁰.

Table no 1: The designed pyrazole analogues

Sl No	Ligand	R₁	R₂
1	Compound F1	H	H
2	Compound F2	p-Cl	H
3	Compound F3	2,4-NO₂	H
4	Compound F4	o-NO₂	H
5	Compound F5	H	o-OH
6	Compound F6	p-Cl	o-OH
7	Compound F7	2,4-NO₂	o-OH
8	Compound F8	o-NO₂	o-OH
9	Compound F9	H	p-OH
10	Compound F10	p-Cl	p-OH
11	Compound F11	2,4-NO₂	p-OH
12	Compound F12	o-NO₂	p-OH
13	Compound F13	H	m-OH
14	Compound F14	p-Cl	m-OH
15	Compound F15	2,4-NO₂	m-OH
16	Compound F16	o-NO₂	m-OH
17	Compound F17	H	o-OCH₃
18	Compound F18	p-Cl	o-OCH₃
19	Compound F19	2,4-NO₂	o-OCH₃
20	Compound F20	o-NO₂	o-OCH₃
21	Compound F21	H	p-OCH₃
22	Compound F22	p-Cl	p-OCH₃
23	Compound F23	2,4-NO₂	p-OCH₃
24	Compound F24	o-NO₂	p-OCH₃

Molecular docking:

Molecular docking simulations were executed using AutoDock Vina version 1.2.3 (The Scripps Research Institute, La Jolla, San Diego, USA). Discovery Studio Biovia 2021 (Dassault Systemes, San Diego, California, USA) was utilized for visualizing and refining both protein and ligand structures. All 24 pyrazole derivatives, along with reference ligands ATP and Gefitinib, were docked into the predicted active sites of the selected EGFR variants. Docking results for 4HJO, 5HG7, 5XDX, 6S9B, and 6Z4B were superimposed with ATP and Gefitinib to validate binding site alignment and ligand positioning.

The docking grid was defined using AutoDock Tools, ensuring the active site residues were encompassed within the grid box. The grid dimensions and centers were as follows: for 4HJO at 2.75 Å, grid size was 77 × 114 × 84 with center at 56.8621 × 119.2636 × 73.683; 5HG7 at 1.85 Å, grid size 35 × 70 × 112 centered at 47.465 × 65.755 × 54.450; 5XDX at 1.99 Å, 183 × 205 × 177, center 105.11 × 64.890 × 54.853; 6S9B at 3.25 Å, 144 × 144 × 144, center 111.534 × 74.8681 × 74.301; and 6Z4B at 2.50 Å, 76 × 79 × 89, center 65.527 × 51.640 × 61.739.

Docking was performed using AutoDock Vina, which calculated binding affinities (kcal/mol) and ranked the resulting poses based on interaction strength. The best poses were selected, and protein–ligand complexes were further analyzed using Discovery Studio Visualizer to identify key hydrogen bonding and hydrophobic interactions.

RESULT AND DISCUSSION:

Molecular docking of 24 designed pyrazole derivatives (F1-F24) against five EGFR kinase domains (PDB IDs: 4HJO, 5HG7, 5XDX, 6S9B and 6Z4B) was performed using AutoDock Vina to evaluate their binding affinities and interaction profiles compared with reference ligands ATP and Gefitinib. Across all EGFR variants, most pyrazole derivatives displayed significant binding energies ranging from -7.1 to -10.9kcal/mol, comparable to or exceeding that of ATP and approaching the affinities shown by Gefitinib.

Compounds F3, F4, F6, F11, F15, F18 and F22 exhibited consistently strong binding, forming multiple hydrogen bonds with key catalytic residues such as Met793, Cys797, Asp855, Arg841 and Asn842, which are critical for EGFR inhibition. Notably, compound F4 showed the highest affinity towards 4HJO with -10.9 kcal/mol, closely followed by F8 and F16, indicating strong stabilization within the ATP-binding pocket. Similarly, F11 and F18 formed up to 5 hydrogen bonds in 6Z4B and 6S9B structures, suggesting favorable interactions even with resistant EGFR conformations.

Electron-withdrawing substituents such as nitro (-NO₂) and chloro (-Cl) at ortho or para positions (e.g., F3, F7, F11, F18, F23) generally enhanced binding due to increased hydrogen bonding and π-π stacking interactions, whereas some derivatives with only unsubstituted phenyl rings displayed lower affinities. Furthermore, compounds bearing dual substituents (e.g., -Cl/-OH, -NO₂/-OCH₃) exhibited improved binding energies and stability across multiple EGFR variants, highlighting a preliminary structure–activity relationship favouring electron-withdrawing and hydrogen bond-donating groups.

In summary, the docking analysis revealed several pyrazole derivatives with promising EGFR inhibitory potential. Compounds F4, F11, F15 and F18 emerged as lead candidates based on their high binding affinities and multiple key interactions with active-site residues. These findings suggest that further optimization and synthesis of these derivatives could yield potent EGFR-targeted anticancer agents.

The variation in binding energies across EGFR variants reflects the influence of conformational diversity and active site flexibility. These findings support the potential of pyrazole derivatives as EGFR inhibitors, particularly for 4HJO and 6Z4B targets. The docking outcomes provide structural insights for optimizing lead compounds and lay the groundwork for further in-vitro and in-vivo validation

Figure no 1: Graphical representation of average binding affinities and hydrogen bond counts of pyrazole derivatives against EGFR receptor variants (PDB IDs: 4HJO, 5HG7, 5XDX, 6S9B and 6Z4B)

Table no 2: Binding energies and Hydrogen bond interaction of pyrazole derivatives with five EGFR Kinase variants

Sl. No	Code	4HJO			5HG7			5XDX			6S9B			6Z4B
Sl. No	Code	B E	H B	Amino acids	B E	H B	Amino acids	B E	H B	Amino acids	B E	H B	Amino acids	B E	H B	Amino acids
1	F1	-10.1	1H	Met769	-10.0	-	-	-7.9	1H	Glu762	-9.0	2H	Asp855 Cys797	-9.4	-	-
2	F2	-10.1	1H	Met769	-10.0	-	-	-8.0	-	-	-9.0	2H	Cys797 Asp855	-9.3	2H	Ser720 Thr854
3	F3	-10.5	2H	Met769 Thr766	-9.6	1H	Cys797	-8.4	1H	Cys797	-8.7	3H	Cys797 Asp855 Arg841	-9.3	5H	Asn842 Ala722 Arg841 Met790 Asp855
4	F4	10.9	2H	Met769 Thr766	-9.6	1H	Cys797	-8.4	-	-	-8.8	2H	Glu762 Met793	-8.9	4H	Met790 Asp855 Ala722 Asn842
5	F5	-9.9	1H	Met769	-9.3	1H	Ser720	-7.9	1H	Gly719	-8.8	3H	Gln791 Asn842 Asp855	-9.5	1H	Met793
6	F6	-10.0	1H	Met769	-9.5	-	-	-8.0	1H	Gly724	-8.9	3H	Gln791 Asn842 Asp855	-8.7	1H	Cys797
7	F7	-10.6	2H	Met769 Thr766	-9.0	4H	Cys797 Arg841 Met7934 Asn842	-8.7	2H	Met793 Asp855	-8.9	3H	Glu762 Asp855 Met793	-9.3	5H	Arg841 Ala722 Asn842 Asp855 Met790
8	F8	-10.7	2H	Met769 Thr766	-8.9	2H	Asp800 Ser720	-8.4	1H	Gly724	-8.9	3H	Glu762 Asp855 Met793	-9.5	2H	Met793 Leu788
9	F9	-9.9	1H	Met769	-9.9	-	-	-8.0	-	-	-9.0	3H	Cys797 Asp855 Met793	-9.0	-	-
10	F10	-10.2	2H	Met769 Asp831	-10.0	1H	Asn842	-8.0	-	-	-9.1	2H	Cys797 Asp855	-8.8	2H	Asp855 Met790
11	F11	-10.4	2H	Met769 Thr766	-9.6	1H	Cys797	-8.4	-	-	-8.4	1H	Met793	-9.3	4H	Met790 Asp855 Arg841 Ala722
12	F12	-10.7	2H	Met769 Thr766	-9.4	1H	Cys797	-8.1	-	-	-8.4	2H	Gln791 Met793	-9.0	4H	Met793 Asn842 Ala722 Asp855
13	F13	-9.9	1H	Met769	-9.8			-7.9	-	-	-9.0	3H	Asp855 Cys797 Met793	-9.4	-	-
14	F14	-10.3	2H	Met769 Asp831	-10.0			-8.1	2H	Arg841 Asn842	-9.1	2H	Asp855 Cys797	-9.3	3H	Ser720 Thr854 Lys745
15	F15	-10.3	2H	Met769 Thr766	-9.4	1H	Cys797	-8.3	1H	Cys797	-8.7	3H	Asp855 Arg841 Cys797	-9.1	5H	Asp855 Met790 Ala722 Asn842 Arg841
16	F16	-10.8	2H	Met769 Thr766	-9.0	2H	Cys797 Asn842	-8.0	1H	Cys797	-8.5	4H	Asp855 Asn842 Met793 Arg841	-9.2	4H	Lys745 Ser720 Cys797 Thr854
17	F17	-9.7	1H	Met769	-9.9	-	-	-7.6	-	-	-8.8	2H	Asp855 Cys797	-9.4	1H	Met793
18	F18	-10.0	1H	Met769	-10.0	-	-	-7.9	-	-	-8.9	2H	Asp855 Cys797	-8.7	-	-
19	F19	-10.5	2H	Met769 Thr766	-9.7	1H	Cys797	-8.2	1H	Cys797	-8.4	3H	Cys797 Arg841 Asp855	-9.1	5H	Met790 Asp855 Ala722 Arg841 Asn842
20	F20	-10.6	2H	Met769 Thr766	-9.6	1H	Cys797	-8.1	-	-	-8.4	-	-	-9.2	1H	Lys745
21	F21	-9.9	1H	Met769	-9.9	-	-	-7.9	1H	Glu762	-8.4	-	-	-9.2	1H	Cys797
22	F22	-10.0	2H	Met769 Asp831	-10.1	-	-	-8.0	-	-	-8.7	2H	Asp855 Met793	-9.1	-	-
23	F23	-10.5	2H	Met769 Thr766	-9.6	1H	Cys797	-8.5	-	-	-8.5	4H	Asp855 Asn842 Arg841 Met793	-9.1	5H	Met790 Asp855 Arg841 Asn842 Ala722
24	F24	-10.6	2H	Met769 Thr766	-9.6	1H	Cys797	-8.4	-	-	-8.2	1H	Met793	-8.6	3H	Asp855 Ala722 Asn842
25	ATP	-7.7	5H	Gly700 Phe699 Arg817 Asn818 Asp831	-7.2	2H	Asn842 Met793	-6.4	3H	Ala698 Asn700 Pro699	-6.4	2H	Phe723 Glu746	-7.1	4H	Ala722 Asn842 Ser720 Met793
26	Gefitinib	-9.2	1H	Lys721	-8.1	3H	Asp800 Leu718 Arg841	-7.1	1H	Glu762	-8.0	2H	Asn842 Asp855	-8.2	1H	Leu718

CONCLUSION:

This study presents a comparative molecular docking analysis of pyrazole derivatives against five EGFR kinase variants (4HJO, 5HG7, 5XDX, 6S9B, and 6Z4B), highlighting significant differences in binding affinities and interaction profiles. Among these, 4HJO exhibited the strongest binding affinity (-10.9 kcal/mol), while 6Z4B formed the highest number of hydrogen bonds (4-5), suggesting strong polar interactions. The results demonstrate that receptor conformation significantly influences ligand binding. Pyrazole derivatives show promise as potential EGFR inhibitors, particularly for resistant or mutant forms. These findings support further lead optimization and biological evaluation to validate their anticancer potential against EGFR-driven malignancies.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors express their sincere gratitude to the Department of Pharmaceutical Chemistry, Bharathi College of Pharmacy, Bharathinagara for providing the necessary infrastructure and computational facilities to carry out this research.

REFERENCES:

1. Anita S. Godase, Nayana V. Pimpodkar and Yogita Indalkar R. An Overview on A Pyrazole: Promising Moiety. Asian J Pharm Tech. 2015; 5(4): 201-213.

2. Balaji PN, Prathusha K, Chandu TJ, Sai Sreevani M, Johnsi Rani P and Harini P. Synthesis, Characterization and Antimicrobial Activity of Some Synthesized Isoxazole and Pyrazole Derivatives. Asian J Research Chem. 2011; 4(2): 301-303.

3. Shantaram G. Khanage, Popat Mohite B and Appala Raju S. Synthesis, Anticancer and Antibacterial Activity of Some Novel 1, 2, 4-Triazole Derivatives Containing Pyrazole and Tetrazole Rings. Asian J Research Chem. 2011; 4(4): 567-573.

4. Panneer Selvam T, Saravanan G, Prakash CR and Dinesh Kumar P. Microwave-Assisted Synthesis, Characterization and Biological Activity of Novel Pyrazole Derivatives. Asian J Pharm Res. 2011; 1(4): 126-129.

5. Alhamaky SM, Khalil NA, Bass AK, Osama N and Hassan MS. Design, Synthesis, Docking Studies and Investigation of Dual EGFR/VEGFR‐2 Inhibitory Potentials of New Pyrazole and Pyrazolopyridine Derivatives. Drug Development Research. 2025; 86(1): 70056-60.

6. Raghu MS, Kumar CP, Prashanth MK, Kumar KY, Prathibha BS, Kanthimathi G, Alissa SA, Alghulikah HA and Osman SM. Novel 1, 3, 5-triazine-based pyrazole derivatives as potential antitumor agents and EFGR kinase inhibitors: Synthesis, cytotoxicity, DNA binding, molecular docking and DFT studies. New Journal of Chemistry. 2021; 45(31): 13909-24.

7. Ashok Gadre G, Nawathye VV, Dalavi DB, Upadhyaya DJ, Thorat BR and Yamgar RS. Synthesis and Characterization of Hydrazones, Pyrazoles and Pyrazolones from 8-Amino-6-Chloro Coumarine. Asian J Research Chem. 2011; 4(10): 1621-1624.

8. Sherbiny FF, Bayoumi AH, El-Morsy AM, Sobhy M, and Hagras M. Design, Synthesis, biological Evaluation and molecular docking studies of novel Pyrazolo [3, 4-d] Pyrimidine derivative scaffolds as potent EGFR inhibitors and cell apoptosis inducers. Bioorganic Chemistry. 2021; 116(1): 105325-29.

9. Khalilullah H, Agarwal DK, Ahsan MJ, Jadav SS, Mohammed HA, Khan MA, Mohammed SA and Khan R. Synthesis and anti-cancer activity of new pyrazolinyl-indole derivatives: pharmacophoric interactions and docking studies for identifying new EGFR inhibitors. International Journal of Molecular Sciences. 2022; 23(12): 6548-52.

10. Ahmed MF, Santali EY, El-Deen EM, Naguib IA and El-Haggar R. Development of pyridazine derivatives as potential EGFR inhibitors and apoptosis inducers: Design, synthesis, anticancer evaluation and molecular modeling studies. Bioorganic Chemistry. 2021; 106(1): 104473-77.

11. Abdullahi SH, Uzairu A, Shallangwa GA, Uba S and Umar AB. 2D and 3D-QSAR Modeling of 1H Pyrazole Derivatives as EGFR Inhibitors: Molecular Docking and Pharmacokinetic Profiling. Chemistry Africa. 2023; 6(3): 1381-98.

12. Silvia P, Ongko J and Antonius Y. Epidermal growth factor receptor mutant T790M-L858R-V948R inhibitor from Calophyllum inophyllum L. leaf as potential non-small cell lung cancer drugs. Journal of Pharmacy and Pharmacognosy Research. 2024; 12(5): 881-91.

13. Al-Muntaser SM, Al-Karmalawy AA, El-Naggar AM, Ali AK, Abd El-Sattar NE and Abbass EM. Novel 4-thiophenyl-pyrazole, pyridine, and pyrimidine derivatives as potential antitumor candidates targeting both EGFR and VEGFR-2; design, synthesis, biological evaluations, and in silico studies. RSC advances. 2023; 13(18): 12184-203.

14. Govindarao K, Srinivasan N, Suresh R, Raheja RK, Annadurai S, Bhandare RR and Shaik AB. Quinoline conjugated 2-azetidinone derivatives as prospective anti-breast cancer agents: In-vitro antiproliferative and anti-EGFR activities, molecular docking and in-silico drug likeliness studies. Journal of Saudi Chemical Society. 2022; 26(3): 101471-74.

15. Sepay N, Mondal R, Al-Muhanna MK and Saha D. Identification of natural flavonoids as novel EGFR inhibitors using DFT, molecular docking, and molecular dynamics. New Journal of Chemistry. 2022; 46(20): 9735-44.

16. Vishwajit Patil S and Prithviraj Patil A. Molecular Docking: A useful approach of Drug Discovery on the Basis of their Structure. Asian Journal of Pharmaceutical Research. 2023; 13(3): 191-5.

17. Soham Pawar, Chaitrali Kulkarni, Puja Gadade, Supriya Pujari, Surajkumar Kakade, Rohane SH and Redasani VK. Molecular Docking using different Tools. Asian Journal of Pharmaceutical Research. 2023; 13(4): 292-6.

18. Payal Nagrale R, Payal Gadiwan S, Nitin Gorad Y, Omkar Lahamge D, Omkar Kanse P, Omkar Veer M and Rinaj Attar. Overview of Tools used in Molecular Docking. Asian Journal of Pharmaceutical Research. 2024; 14(3): 326-30.

19. Bhaskar Dawane S, Baseer M Shaikh, Namdev Khandare T, Gajanan Mandawad G, Santosh Chobe S and Shankaraiah Konda G. Synthesis of Some Novel Substituted Pyrazole Based Chalcones and Their In-Vitro Antimicrobial Activity. Asian J Research Chem. 2010; 3(1): 90-93.

20. Vijay Kumar Tirlapur and Kashinath Noubade. Synthesis, Characterization and Biological Activities of New Pyrimidines and Pyrazoles Derivative. Asian J Research Chem. 2010; 3(3): 659-662.

Received on 29.05.2025 Revised on 17.09.2025

Accepted on 22.11.2025 Published on 05.06.2026

Available online from June 06, 2026

Research J. Pharmacy and Technology. 2026;19(6):2621-2628.

DOI: 10.52711/0974-360X.2026.00375

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.