Biological evaluation and molecular docking studies of nitro benzamide derivatives with respect to in vitro anti-inflammatory activity

- Six (1-6) nitro substituted benzamide derivatives were synthesized.
- Their anti-inflammatory activities were screened by nitric oxide inhibition assay in LPS induced RAW264.7 macrophages.
- Two lead compounds were identified and investigated for NO inhibitory IC50 values and dose-response relations.
- Molecular docking studies on iNOS enzyme supported the biological data.
- The effects of lead compounds on iNOS, COX-2, IL-1β and TNF-α expression were investigated.

A series of nitro substituted benzamide derivatives were synthesized and evaluated for their potential anti-inflammatory activities in vitro. Firstly, all compounds (1-6) were screened for their inhibitory capacity on LPS induced nitric oxide (NO) production in RAW264.7 macrophages. Compounds 5 and 6 demonstrated significantly high inhibition capacities in a dose-dependent manner with IC50 values of 3.7 and 5.3 μM, respectively. These two compounds were also accompanied by no cytotoxicity at the studied concentrations (max 50 μM) in macrophages. Molecular docking analysis on iNOS revealed that compounds 5 and 6 bind to the enzyme more efficiently compared to other compounds due to having optimum number of nitro groups, orientations and polarizabilities. In addition, 5 and 6 demonstrated distinct regulatory mechanisms for the expression of the iNOS enzyme at the mRNA and protein levels. Specifically, both suppressed expressions of COX-2, IL-1β and TNF-α significantly, at 10 and 20 μM. However, only compound 6 significantly and considerably decreased LPS-induced secretion of IL-1β and TNF-α. These results suggest that compound 6 may be a multi-potent promising lead compound for further optimization in structure and as well as for in vivo validation studies.

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