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 IC₅₀ 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 IC₅₀ values of 1.11 ± 0.04 and 0.70 ± 0.02 µM, respectively, compared to celecoxib (IC₅₀ = 0.87 ± 0.03 µM). Furthermore, these promising derivatives exhibited COX-1 inhibitory activity, with IC₅₀ 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 (IC₅₀ = 167.65 ± 6.65 pg/mL) and TNF-α (IC₅₀ = 816.59 ± 28.01 pg/mL) compared to celecoxib's IC₅₀ (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.