Abstract Background: Precision oncology is currently restricted to activated oncogenes as drug targets, with tumor suppressor deletions remaining largely in-actionable. We developed a novel method that targets tumor suppressor deletions by exploiting vulnerabilities generated by the co-deletion of neighboring metabolic housekeeping genes with critical but normally redundant functions. Proof-of-principle studies were conducted for the glycolytic enzyme Enolase. Cancers harboring the homozygous deletion of 1p36-localized ENO1 remain metabolically active and viable through redundant action of its paralogue, ENO2. Inhibition of ENO2 in cancer cells harboring ENO1-homozygous deletion discriminately kills ENO1-deleted cells. In accordance, our synthetic efforts have focused on phosphonate chemistry to generate clinically-useful substrate mimics of 2-phosphoglycerate, the natural substrate for Enolase. We have generated a phosphonohydroxamate ENO2-inhibitor termed HEX and a pro-drug of which, POMHEX, kills ENO1-homozygously deleted glioma cells in culture with strong specificity. However, the pharmacokinetics of POMHEX are poor, due to the presence of carboxyesterase in extracellular fluids, including plasma. Results: We have synthesized mechanistically different pro-drugs of HEX, utilizing nitro-aromatics as protecting groups. Nitroaromatic groups are bioactivated by nitroreductases intracellularly, in a manner that is inversely related to oxygen concentration. As such, removal of nitroaromatic pro-drug moieties and release of active drug is favored under low oxygen (hypoxic) conditions. We synthesized a series of nitroaromatic pro-drugs of HEX (the structures will be disclosed at AACR presentation, pending patent filings) and demonstrate that 1) these exhibit selective toxicity to ENO1-deleted glioma cells in culture and 2) this killing is dramatically enhanced by lowering oxygen concentrations as compared to POMHEX. We further find that redox potential of the nitroaromatic moiety correlates with the degree of hypoxia activation with the lower redox potential being associated with great oxygen dependence. Importantly, unlike POMHEX, nitroaromatic HEX pro-drugs are stable in human plasma ex-vivo. We have thus successfully synthesized a pro-drug inhibitor of Enolase that is hypoxia-activated. Conclusion and future directions: We are gearing up synthesis for pre-clinical evaluation of nitroaromatic-HEX in intracranial tumor xenograft models. While the primary purpose of Nitroaromatic HEX is for the precision oncology treatment of ENO1-homozygously deleted tumors, it has not escaped our notice that such drugs have wider applicability within the context of targeting treatment-resistant tumors cells hiding in hypoxic niches. Indeed, we have observed that virtually all glioma cell lines can be killed by nitroaromatic HEX under hypoxia. Note: This abstract was not presented at the meeting. Citation Format: Florian Muller, Victoria C. Yan, Elliot S. Ballato, Kristine L. Yang, Dimitra K. Georgiou, Kenisha Arthur, Pakriti Shrestha, Sunada Khadka, Jeffrey Ackroyd. Synthesis of mixed, hypoxia-activated phosphoramidate esters for the inhibition of Enolase in ENO1-deleted glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 984.