Unveiling a novel pyrazolopyrimidine scaffold as a dual COX-2/5-LOX inhibitor with immunomodulatory potential: Design, synthesis, target prediction, anti-inflammatory activity, and ADME-T with docking simulation.

Dual-target COX-2/5-LOX inhibitors are regarded as a rational strategy for the design of potent anti-inflammatory agents with favorable safety profiles. In this study, novel pyrazolo[1,5-a]pyrimidine derivatives were synthesized, developed, and screened for their ability to inhibit the cyclooxygenase-2 enzyme in vitro, with comparisons made to the established inhibitors Celecoxib and Meloxicam. Spectroscopic analyses confirmed the structure of the designed derivatives. The target prediction using AI was performed to identify potential targets that could be engaged through Swiss target prediction database. The SAR study was established by incorporating various substituents and nuclei into the pyrazolopyrimidine pharmacophore. The synthesized pyrazolopyrimidines exhibited IC values ranging from 53.32 ± 4.43 to 254.90 ± 6.45 nM, in comparison to Celecoxib (IC = 6.73 ± 5.69 nM) and Meloxicam (IC = 52.35 ± 6.66 nM). Notably, compound 5a was identified as the most active derivative, demonstrating an IC of 53.32 ± 4.43 nM. The three most prominent pyrazolopyrimidine derivatives, 3a, 5a, and 6a, were subsequently evaluated for their ability to inhibit the COX-1 and 5-LOX enzymes. Compounds 3a, 5a, and 6a demonstrated inhibitory activity against COX-1, with IC values of 476.45 ± 16.56, 757.51 ± 2.61, and 169.13 ± 5.77 nM, respectively. These derivatives 3a, 5a, and 6a showed significant selectivity index values of 7.91, 14.20, and 2.80, respectively, toward COX-2 rather than COX-1 in comparison to Meloxicam (SI = 0.75) and Celecoxib (SI = 2.35). Moreover, compound 5a exhibited 86 % inhibition compared to Zileuton's 88 %, while compounds 3a and 6a displayed inhibition rates of 84 % and 80 %, respectively, at a concentration of 100 μM. The most potent compound 5a, demonstrated the highest 5-LOX inhibitory activity, with IC of 2.292 ± 0.14 μM. The most promising pyrazolopyrimidine derivative 5a demonstrated a down-regulation of TNF-α and IL-6 gene expression by approximately 0.3826-fold and 0.2732-fold, respectively, when compared to Celecoxib, which induced reductions of 0.2320-fold and 0.2730-fold in these cytokines to promote apoptosis in RAW264.7 cells. Finally, in-silico ADME-T and docking simulations were conducted to predict the oral bioavailability, toxicity, and binding interactions with binding affinity.