Design, synthesis, in vitro, and in silico evaluation of multi-functionalized pyrimidines as potential multitarget-directed anti-inflammatory agents.

Multi-targeted inhibitors are considered a logical approach for developing effective anti-inflammatory drugs. In this study, a library of pyrimidine derivatives (5a-c, 6, 7a-n) was designed, synthesized, and examined for their in vitro potential to inhibit the activities of COX-1 and COX-2, with comparisons made to the well-known COX inhibitors celecoxib and diclofenac sodium. The most potent and selective COX-2 inhibitor 7j exhibited a higher potency and enhanced selectivity to COX-2 (IC50 = 0.678 μM, SI = 29) compared to celecoxib (IC50 = 0.799 μM, SI = 24.61) and diclofenac sodium (IC50 = 4.812 μM, SI = 2.55). Additionally, compounds 7d and 6 exhibited COX-2 inhibitory effects that exceeded that of diclofenac sodium and were comparable to celecoxib, with IC50 values of 0.724 and 0.824 μM, respectively. Those promising COX-2 inhibitors were further evaluated in vitro for their inhibitory activity against 5-LOX, IL-6, and ROS. Compound 6 demonstrated enhanced 5-LOX inhibitory activity (IC50 = 0.235 μM) compared to the reference zileuton (IC50 = 0.334 μM). Interestingly, compound 6 inhibited IL-6 production in LPS-activated RAW 264.7 macrophages by 96.11%, outperforming celecoxib (86.60%) and diclofenac sodium (67.83%), and reduced ROS production with an IC50 of 28.26 μg/ml, which was lower than the IC50 values for celecoxib (114.16 μg/ml) and diclofenac sodium (87.75 μg/ml). A molecular docking study explored the binding interactions between newly synthesized inhibitors and the active sites of COX-2 and 5-LOX. Additionally, various online tools were utilized to assess the pharmacokinetics, drug likeness, and toxicity of the most active compounds.