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Off-target aerobic activation of PR-104A by human aldo-keto reductase 1C3 (AKR1C3) has confounded the development of this dual hypoxia/gene therapy prodrug. Previous attempts to design prodrugs resistant to AKR1C3 activation have resulted in candidates that require further optimization. Herein we report the evaluation of a lipophilic series of PR-104A analogues in which a piperazine moiety has been introduced to improve drug-like properties. Octanol-water partition coefficients (LogD 7.4 ) spanned >2 orders of magnitude. 2D antiproliferative and 3D multicellular clonogenic assays using isogenic HCT116 and H1299 cells confirmed that all examples were resistant to AKR1C3 metabolism while producing an E. coli NfsA nitroreductase-mediated bystander effect. Prodrugs 16 , 17 , and 20 demonstrated efficacy in H1299 xenografts where only a minority of tumor cells express NfsA. These prodrugs and their bromo/mesylate counterparts ( 25 - 27 ) were also evaluated for hypoxia-selective cell killing in vitro . These results in conjunction with stability assays recommended prodrug 26 (CP-506) for Phase I/II clinical trial., Competing Interests: The authors declare the following competing financial interest(s): A.A., A.M.M., C.P.G., A.V.P., and J.B.S. are listed as inventors on the patents DK2888227T3, EP2888227B1, US10202408B2, CA2886574C, US9873710B2, AU2013/306514B2, and US9505791B2 issued and assigned to Health Innovation Ventures. As such, they potentially stand to benefit financially from the commercial development of this intellectual property. J.B.S. and A.V.P. report receipt of consultancy fees from Convert Pharmaceuticals., (© 2023 American Chemical Society.)