Pharmacological Evaluation of the Anti-Inflammatory Activity and Chemoinformatic Analysis of New Potent 2-Substituted 1-Methyl-5-Nitroindazolinones

New potent analogs of 2-benzyl-1-methyl-5-nitroindazolinone (VA5-13l), a novel anti-inflammatory agent designed and synthesized in a previous study, were investigated in two in vivo anti-inflammatory assays. Those derivatives that demonstrated the higher activity were selected, and in vitro studies were carried out in LPS-stimulated RAW 264.7 macrophages in order to determine their possible mechanism of action. Among the tested compounds in zebrafish (Danio rerio), the products 3, 6, 8, 9 and 10 showed the lowest values of relative leukocyte migration (RLM) at 30 µM (0.14, 0.07, 0.10, 0.13 and 0.07, respectively); lower than the positive control, indomethacin (0.16). All tested compounds significantly inhibited TPA-induced ear edema in mice, showing a significant reduction in myeloperoxidase activity (except 4 and 16). SAR analysis, using the concept of an activity landscape with SARANEA software, provides details on the fine relationship linking structure and activity. It is evident that 5-nitro group of indazole ring plays a relevant role in the anti-inflammatory activity. Furthermore, the change of benzyl group (Bn) by other alkyl moieties reduced modestly the activity. A substituted Bn moiety at N-2 (R) is the best substituent (5-10); nevertheless, the compounds with halogen substituents on the Bn showed the best results. In fact, compounds 7 and 8 (R = 4-FBn and 4-ClBn, respectively) exhibit best activity in both in vivo tests, therefore they appear promising. All compounds, excluding 16, significantly inhibit the production of IL-6 at all doses assayed. Nonetheless, the production of TNF-α was significantly inhibited only by chemical 16 at a concentration of 50 µM. Compound 2 was the best inhibitor of iNOS and COX-2 expression. The inhibition of cytokine release may be one of the mechanisms responsible of this effect for 2-benzyl derivatives while other 2-alkyl derivatives can also inhibit production of NO. Furthermore, this chemical prototype could constitute an important alternative for the search of new anti-inflammatory drugs.