SAR Studies around the SULT1A1-Activated Alkylator YC-1 as Cytotoxic Agents against Biliary Tract Cancer Cells

Abstract

A quantitative high-throughput screen using biliary tract cancer cell lines had identified the small-molecule YC-1 as being selectively cytotoxic against the IDH1-mutant cell lines with high expression of SULT1A1. We discuss the structure-activity relationship study of YC-1 analogs and identify the key structural motifs that are essential for this cytotoxicity. We highlight the narrow SAR around the furfuryl alcohol that has been reported as a critical motif that is activated to a reactive electrophile by the sulfotransferase enzyme SULT1A1. Drug-like properties of key analogs are evaluated, including a close look at YC1 hepatic metabolism. We also show the SAR of a smaller subset of 2-choloro-4-amino benzyl alcohols from the NCI compound collection with a similar benzyl alcohol motif. We also demonstrate the ability of key analogs to act as substrates of SULT1A1 in a colorimetric biochemical assay.

Figure 1.

A) Representative IC₅₀ curves of initial HTS hit YC-1 against RBE and SNU-1079, cell lines with mIDH1 and high SULT1A1 expression, and CCLP1 and HUCCT1 cell lines with wt IDH1 and low SULT1A1 expression (N=3). B) Key regions of the YC-1 scaffold for SAR exploration.

Figure 2.

Proposed mechanism of SULT1A1-mediated bioactivation of YC-1.

Figure 3.

A) Plasma conc. vs. time plots for YC-1, analogs 15 and 58 in male CD1 mice after a administration of a single dose of 5, 10, 30 mpk via IV, PO and IP routes. B) Separation of metabolites by HPLC after incubation of amine 15 (10 μM) in male CD1 mouse liver microsomes at 0, 30, 60 min with NADPH (2 mM) that shows the two major metabolites. MS/MS fragmentations pattern of parent 15 and the metabolites I and II that identifies them as acid 14a and YC-1 (1).

Figure 4.

Stability and half-lives of YC1 (1) in human and mouse liver microsomes (hLM and mLM) in the presence of cofactors NADH, UDPGA and both; and in mouse and human hepatocytes.

Figure 5.

A) Absorbance at 405 nm from p-nitrophenolate was measured while various concentrations of YC-1 were added to SULT1A1 in the biochemical colorimetric assay. B) Absorbance at 405 nm from p-nitrophenolate was measured as 100 μM of each small molecule was added to SULT1A1 in the biochemical colorimetric assay.

Figure 6.. Evaluating YC-1 and analogs as SULT1A1 substrates.

Quantified absorbance from the colorimetric SULT1A1 activity assay at two hours to identify substrates of SULT1A1 sulfonation at 100 μM concentration. *p ≤ 0.05 vs DMSO; **p ≤ 0.01 vs DMSO; ***p ≤ 0.001 vs DMSO; ****p ≤ 0.0001 vs DMSO. Table includes reference IC₅₀s from RBE assay.

Figure 7.

Schematic for the colorimetric SULT1A1 activity assay.

Scheme 1.

General procedures to access YC-1 analogs.

Scheme 2–5.

Procedures to access oxazole (7-9) and tetrahydrofuran (12) replacements of the furan in YC-1.