BackgroundThe quinoline‐3‐carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on‐target mechanism‐of‐action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF‐1α transcriptional activation and repressing MEF‐2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration‐resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time‐to‐progression, but not overall survival.MethodsTasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT‐PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate.ResultsHere, we document that, on this regimen (1 mg/day), TasQ blood levels are 10‐fold lower than the optimal concentration (≥2 μM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 μM to produce unwanted off‐target side effects. Therefore, we screened a library of TasQ analogsto maximize on‐target versus off‐target activity. Using this approach, we identified ESATA‐20, which has ~10‐fold lower AHR agonism and 5‐fold greater potency against prostate cancer patient‐derived xenografts.ConclusionThis increased therapeuticindex nominates ESATA‐20 as a lead candidate forclinical development as an orally active third generation quinoline‐3‐carboxamide analog thatretains its on‐target ability to disrupt HDAC4/HIF‐1α/MEF‐2‐dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.