Exploration of anti-inflammatory activity of pyrazolo[3,4-d]pyrimidine/1,2,4-oxadiazole hybrids as COX-2, 5-LOX and NO release inhibitors: Design, synthesis, in silico and in vivo studies.

New pyrazolo[3,4-d]pyrimidine derivatives 7a-h and 8a-h were synthesized and evaluated for their in vitro inhibitory potential against COX-1, COX-2, 5-LOX along with the NO release inhibitory activity to assess their anti-inflammatory potential. Most compounds confered inhibitory activity at a micromolar level and exhibited prominent selectivity towards COX-2 especially in the 8a-h series. The most useful compound 8e as a COX-2/5-LOX dual inhibitor, exhibited IC50 results of; 1.837 µM for COX-2, 2.662 µM for 5-LOX with an acceptable NO release inhibition rate of 66.02 %. Compounds 7e, 7f, 8e and 8f proved their efficiency as 5-LOX/NO release dual inhibitors; with IC50 values of 2.833, 1.952, 2.662 and 1.573 µM, respectively for 5-LOX biotarget, and with superior NO inhibitory ratio of 73.85, 65.57, 66.02 and 72.28 %, respectively. The in vivo anti-inflammatory assay explored that 7e is the most effective with minimal gastric ulceration prevalence. Molecular docking in the active site of both COX-2 and 5-LOX showed that, the most active 8e and 7e are correctly oriented inside the COX-2 binding pocket with unique binding mode independently on the reference celecoxib. Also, they demonstrated superior binding affinities to the 5-LOX enzyme over both the Zileuton as a reference drug and the normal ligand 30Z. The stability of the complex formed between the most promising candidates 7e or 8e with the COX-2 and 5-LOX active sites, was considered using a typical atomistic 100 ns dynamic simulation study. Investigation of the SAR revealed the importance of both the sulfonamide group in the 8a-h series and the substituents of the 3-phenyl ring tethered on the 1,2,4-oxadiazole core.