Discovery of New Pyrazole‐Linked Pyridine Derivatives as Multi‐Target Anti‐Inflammatory Agents With Immunomodulatory Potential

ABSTRACT Cyclooxygenase (COX) plays a crucial role in the inflammatory response, making selective COX‐2 inhibition a significant strategy for developing safer anti‐inflammatory medications. Accordingly, developing new pharmacotherapies is a critical objective in anti‐inflammatory drug discovery. In this study, a new pyrazole‐linked pyridine derivatives 2‐7 were synthesized through the reaction of the 4‐(pyridin‐1‐yl)benzohydrazide derivative 1 with substituted‐ketenes, activated olefinic dimers, and α,β ‐unsaturated carbonyl compounds. The synthesized derivatives were characterized and subsequently assessed in vitro for their inhibitory effects on COX‐2. The results of the COX‐2 inhibition assay revealed a broad range of activity, with IC 50 values ranging from 0.70 ± 0.02 to 63.49 ± 2.32 µM. Among the derivatives, 3 and 7b demonstrated the most significant inhibitory effects, with IC 50 values of 1.11 ± 0.04 and 0.70 ± 0.02 µM, respectively, compared to celecoxib (IC 50 = 0.87 ± 0.03 µM). Furthermore, these promising derivatives exhibited COX‐1 inhibitory activity, with IC 50 values of 16.08 ± 0.54 µM (SI cox‐2 = 14.48) and 2.77 ± 0.09 µM (SI cox‐2 = 3.95), respectively, compared to indomethacin (0.199 ± 0.01 µM) and celecoxib (21.62 ± 0.73 µM). Given the extensive COX activity of 7b , further investigations were conducted, as it demonstrated immunomodulatory potential through the suppression of IL‐6 (IC 50 = 167.65 ± 6.65 pg/mL) and TNF‐α (IC 50 = 816.59 ± 28.01 pg/mL) compared to celecoxib's IC 50 (129.50 ± 4.44 pg/mL) and (399.7 ± 13.7 pg/mL), respectively. Additionally, pyrazole‐linked pyridine derivative 7b significantly reduced PGE2 levels to 539.49 ± 17.87 pg/mL and NO production to 4.230 ± 0.159 nM, compared to celecoxib 256.79 ± 8.50 pg/mL for PGE2 and 3.997 ± 0 nM for NO, respectively, suggesting that compound 7b can attenuate both cytokine and eicosanoid signaling, exhibit extensive anti‐inflammatory activity, and may serve as a potential multi‐target therapeutic agent. The molecular docking simulation revealed that compound 7b exhibited a significant binding affinity through various interactions. Finally, the DFT calculation indicated that 7b has a low energy band gap, which suggests lower hardness and higher softness that implies a greater ability to redistribute electron density during binding, favoring the formation of stable polar interactions with protein residues. Overall, these findings demonstrate that compound 7b is a promising multi‐target anti‐inflammatory lead drug with strong COX‐2 inhibition and immunomodulatory properties. Moreover, further studies will concentrate on in vivo anti‐inflammatory evaluation and pharmacokinetic profiling.