Warhead exploration. Though the medicinal chemistry campaign had utilized cytotoxicity against cell lines with mIDH1 to drive structure-activity relationship studies, we had discovered that this activity was strongly correlated to the expression of SULT1A1. Our SAR studies suggested that the furfuryl alcohol was the key element for activity. To that end, we decided to examine if compounds with similar motifs could elicit cytotoxicity via SULT1A1-mediated sulfonation as proposed in Figure . We approached this analysis by investigating the annotated cytotoxicity profiles of >22,000 compounds in the NCI60 8 and used the CellMiner NCI-60 tool to identify molecules whose cytotoxicity positively correlated with SULT1A1 expression across the sixty cell lines. We have reported the findings of our analysis, by which we found within the top 150 compounds, candidates whose activities have been linked to SULT1A1; these included analogs of oncrasin-1 (N-benzyl indole carbinol (N-BIC), RITA (reactivating p53 and inducing tumor apoptosis, compound 104 in Table ) and amino flavones. Another group of compounds that was very well represented in this set was a series of 88 amino halogenated benzyl alcohols (AHBA). We obtained representative compounds from the NCI compound collection library and tested them for cytotoxicity against the same cell lines we used to drive the SAR studies around YC1; Tables and shows a snapshot of the SAR in RBE cells where most compounds belong to a 2-choloro 4-amino benzyl alcohol series. With a constant 4-N-ethyl group, we found a variety of substitutions were tolerated in the phenyl ring attached to the Nbenzyl substituent (Table ). Analogs 65-75 showcase a systematic evaluation of a single F, Cl, Me and OMe substitution and analogs 76-83 show the effects of double substitutions. Several compounds in this set (highlighted in green) had IC50s <200 nM and >75% activity at the top 46 M concentration tested. Replacing this phenyl ring with a pyridine ring led to >2-fold loss on the potencies compared to parent 64. Then we looked at some compounds where we maintained a 4-fluorobenzyl nitrogen substitution and scanned alkyl groups at the 4-amine (Table ). Smaller substitutions linked with a methylene were tolerated (analogs 87-89), but immediate branching (cyclobutyl 90) and a larger benzyl group (91) were less active. Cyclization of the benzyl amine to tetrahydroisoquinolines 92-94 was possible and produced analogs 92 and 93 with <200 nM IC50. The activity of the known di-thiophenyl alcohol RITA 95 is also shown in Table . IC50s are reported as an average of N=3 from a 11-pt dose response CellTiter-Glo™ assay in RBE cells and are not determined (ND) when response in cell viability is <50% of DMSO control at the maximum compound concentration of 46 M tested in the assay SULT1A1 activity. To directly characterize the ability of compounds to serve as SULT1A1 substrates, we established a biochemical colorimetric assay that utilized E. coli-derived human SULT1A1 protein. The assay utilizes the canonical cofactor 3'-phosphoadenosine-5'phosphosulfate (PAPS) as the sulfate donor which is converted to the byproduct 3'phosphoadenosine-5'-phosphate (PAP). PAP can be quantitated by its conversion back to PAPS by SULT1A1 using p-nitrophenyl sulfate. This biochemical reaction produces p-nitrophenolate that can be quantified by measuring absorbance at 405 nm.