Design and synthesis of new disubstituted benzoxazolone derivatives that act as iNOS inhibitors with potent anti-inflammatory activity against LPS-induced acute lung injury (ALI)

[Display omitted] •New benzoxazolone derivatives with high iNOS inhibitory activity were synthesized.•2d was a promising iNOS inhibitor (IC50 = 3.342 µM) with anti-inflammatory activity.•Disubstitution at 4, N-position of benzoxazolone increased iNOS inhibitory activity.•iNOS inhibitors might be an effective therapeutic strategy for acute lung injury. Acute lung injury (ALI) is a pulmonary disease that acts as a severe acute inflammatory response with no specific drugs. iNOS, a catalyst of the excessive production of NO, has been demonstrated to participate in the inflammatory process, and targeting iNOS may be a promising therapeutic pathway to alleviate ALI. In our research, eighteen new disubstituted benzoxazolone derivatives were synthesized, characterized, and evaluated for activity against NO production in an LPS-induced RAW264.7 cell. The results showed that these compounds could obviously inhibit the over-generation of NO and disubstitution at the 4, N-position of the benzoxazolone ring, presenting better potency than substitution only at the 4-position. Among the analogues generated, compounds 2c, 2d, and 3d showed NO inhibitory activity with IC50 values of 16.43, 14.72, and 13.44 µM and iNOS inhibitory activity with IC50 values of 4.605, 3.342, and 9.733 µM, respectively. Meanwhile, compounds 2c, 2d, and 3d could also inhibit the release of IL-6, IL-1β in vitro and suppress xylene-induced ear edema in vivo to realize anti-inflammatory activity. Furthermore, compound 2d could significantly protect the LPS-induced ALI, presenting as decreased inflammatory cytokines and obvious pathological changes. Immunohistochemistry and molecular modeling demonstrated that compound 2d significantly inhibited the expression of iNOS in vivo and interacted with iNOS through two hydrogen bindings with the MET368 and ILE195 residues of the iNOS protein. These results demonstrated that compound 2d could be a promising lead structure for iNOS inhibitors, with anti-inflammatory activity to treat LPS-induced acute lung injury.