Blocking the Kelch-like epichlorohydrin-related protein 1 (Keap1)-nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway represents as a promising strategy to reduce oxidative stress and related-inflammation, including acute lung injury (ALI). NXPZ-2, a naphthalensulfonamide derivative, was previously reported to effectively inhibit the Keap1-Nrf2 protein-protein interaction (PPI) by our group. In the present work, a series of novel isothiocyanate-containing naphthalensulfonamides with the thioether, sulfoxide and sulfone moieties were designed by a structure-based molecular hybridization strategy using NXPZ-2 and the Nrf2 activator sulforaphane. They possessed good Keap1-Nrf2 PPI inhibitory activity and low cytotoxicity. The molecular docking study was performed to further explain the different activity of the thioether-, sulfoxide- and sulfone-containing naphthalensulfonamides. Among these new derivatives, 2-((N-(4-((N-(2-amino-2-oxoethyl)-4-((3-isothiocyanatopropyl)sulfinyl)phenyl)sulfonamido) naphthalen-1-yl)-4-methoxyphenyl)sulfonamido)acetamide (SCN-16) showed a good K<sub>D2</sub> value of 0.455 μM to disrupt the PPI. In an LPS-induced peritoneal macrophage cell model, this compound could cause a significant increase in the nuclear Nrf2 protein, decrease in the cytosolic Nrf2 protein, and further elevate the downstream protective enzymes HO-1 and NQO-1, which were better than the lead compound NXPZ-2 and sulforaphane. What's more, the production of ROS and NO and the expression of pro-inflammatory cytokine TNF-α were also suppressed. In the LPS-induced ALI model, SCN-16 could significantly reduce LPS-induced inflammations and alleviate lung injuries by triggering Nrf2 nuclear translocation. Collectively, our results suggested that SCN-16 could be a novel lead compound targeting Keap1-Nrf2 protective pathway for clinical treatment of ALI.