Novel Pyrazole-3-Cyano-2-Pyridinone Hybrids as Multitarget Anti-Inflammatory Agents: Synthesis, Computational Modeling, and Biological Evaluation

Bahgat RM Hussein,1 Ibrahim M Salem,2 Hossameldin A Aziz,3,4 Omar Alshazly,5,6 Heba A Hofny,5,6 Aliaa M Mohassab,7,8 Zuhier A Awan,9 Tarek S Ibrahim,10 Elsayed M Mahmoud,11,12 Stefan Bräse,13 Mamdouh FA Mohamed3– 5 1Department of Chemistry, Faculty of Science, Sohag University, Sohag, 82524, Egypt; 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sphinx University, New Assuit City, Assiut, 71515, Egypt; 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, New Valley University, New Valley, 72511, Egypt; 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, New Valley National University, New Valley, 72511, Egypt; 5Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sohag University, 82524, Sohag, Egypt; 6Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sohag National University, Sohag, 82524, Egypt; 7Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, 61519, Egypt; 8Medicinal Chemistry Department, Faculty of Pharmacy, Minia National University, New Minia, Egypt; 9Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; 10Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 11Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt; 12Course of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, 501-1193, Japan; 13Institute for Biological and Chemical System, Karlsruhe Institute of Technology, Karlsruhe, 76131, GermanyCorrespondence: Mamdouh FA Mohamed, Email [email protected] Stefan Bräse, Email [email protected]: The development of multi-target anti-inflammatory agents represents a promising strategy to improve therapeutic efficacy while minimizing adverse effects associated with single-target drugs. In this study, a rational hybridization approach was employed to design pyrazole/3-cyano-2-pyridinone hybrids aimed at modulating key inflammatory mediators.Methods: A novel series of pyrazole/3-cyano-2-pyridinone hybrids was synthesized and evaluated for anti-inflammatory activity. Nitric oxide (NO) production and iNOS activity were assessed in LPS-stimulated RAW 264.7 macrophages. COX-1/COX-2, LOX (5-LOX and 15-LOX), PGE2, and TNF-α inhibition assays were performed. Cytotoxicity was determined using MTT assays. Molecular docking and 100-ns molecular dynamics (MD) simulations were conducted to investigate binding modes and stability, while in silico ADME profiling was used to predict pharmacokinetic properties.Results: Compounds 5f, 5g, 5k, and 5m significantly inhibited NO production and iNOS activity, with compound 5m showing the strongest effect (IC50 = 203.9 μM). COX inhibition assays revealed selective COX-2 activity, with compound 5k exhibiting the highest potency (IC50 = 0.92 μM) and a selectivity index of 19.6. Compound 5g most effectively suppressed PGE2 production (IC50 = 152.7 pg/mL), while 5m markedly reduced TNF-α levels, comparable to ibuprofen. In LOX assays, compound 5f showed potent inhibition of both 5-LOX (IC50 = 0.34 μM) and 15-LOX (IC50 = 0.21 μM), outperforming zileuton. All tested compounds exhibited low cytotoxicity (IC50 > 85 μM). Docking and MD simulations confirmed stable and favorable binding interactions of 5k, 5f, and 5m with COX-2, 5-LOX, and iNOS, respectively. In silico pharmacokinetic analysis predicted good oral bioavailability and drug-like properties.Conclusion: The synthesized pyrazole/3-cyano-2-pyridinone hybrids demonstrated promising multi-target anti-inflammatory activity with favorable safety and pharmacokinetic profiles, highlighting their potential as lead candidates for further development.Keywords: Pyrazole, 3-Cyano-2-pyridinone, Anti-inflammatory, Healthcare, COX2, iNOS, TNF-α, 5-LOX