Abstract Background: Non-small cell lung (NSCLC) constitutes approximately 80% of all diagnosed cases of lung cancer. Up to 30% of NSCLC acquire mutations that result in constitutive NRF2 pathway activity, which correlates with worse patient prognosis. NRF2 regulates antioxidant genes that confer cytoprotection and chemoresistance. Inhibiting NRF2 activity increases tumor susceptibility to chemotherapy in preclinical lung cancer models; however, there are no FDA-approved NRF2 inhibitors available. Because current inhibitors have low potency and specificity, we developed novel compounds that target the NRF2 pathway. Previously, a high throughput screen identified MSU-38225, which was efficacious in sensitizing chemoresistant lung cancer to chemotherapy in a mouse xenograft model. We sought to improve the poor pharmacokinetics of this compound and developed and characterized a series of novel derivatives with increased solubility. Hypothesis: Derivatives of MSU-38225 inhibit the NRF2 pathway and decrease cancer cell viability. Methods: The efficacy of our NRF2 pathway inhibitors was assessed in A549 and H460 human lung cancer cells with constitutive NRF2 pathway activity using western blotting, RT-PCR, and MTT or Cell Titer Glow viability assays. Cell death and cell cycle analysis were evaluated using flow cytometry. Data were evaluated with one or two-way ANOVA and the Dunnett post-hoc test. Results: The NRF2 pathway inhibitors decreased NRF2 protein level in A549 and H460 cells by approximately 60%, as measured by western blotting. As a result, NRF2-mediated transcription of cytoprotective genes NQO1, GCLC, GCLM, and AKR1C2 were decreased by 30-60% (p<0.05) by qPCR. Further, these compounds decreased lung cancer cell viability, as measured in an MTT assay, by 50% in A549 cells and by 90% in H460 (p<0.05). We then evaluated a specific inhibitor, MSU-71, to determine the cause of decreased viability and found it did not induce annexin-V positive apoptosis. However, subsequent analysis of the cell cycle with propidium iodide univariate analysis showed MSU-71 arrests lung cancer cells. 50% of H460 and 75% of A549 cells were arrested in G2/M (p<0.05). Finally, we developed several “click” chemistry tool compounds that have equitable NRF2 pathway inhibition, which will aid in target identification of the new inhibitors. Conclusion: Taken together, the new NRF2 pathway inhibitors are efficacious in blocking NRF2 activation. Optimization of these inhibitors could lead to a new therapy to treat lung cancer. Citation Format: Christopher John Occhiuto, Lizbeth Lockwood, Zhilin Hou, Aaron Odom, Karen Liby. Novel inhibitors of the NRF2 pathway also arrest lung cancer cells in G2/M [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 529.