Discovery of potent anticancer agent HJC0416, an orally bioavailable small molecule inhibitor of signal transducer and activator of transcription 3 (STAT3)

Abstract

In a continuing effort to develop orally bioavailable small-molecule STAT3 inhibitors as potential therapeutic agents for human cancer, a series of novel diversified analogues based on our identified lead compound HJC0149 (1) (5-chloro-N-(1,1-dioxo-1H-1λ(6)-benzo[b]thiophen-6-yl)-2-hydroxybenzamide, Eur. J. Med. Chem. 2013, 62, 498-507) have been rationally designed, synthesized, and pharmacologically evaluated. Molecular docking studies and biological characterization supported our earlier findings that the O-alkylamino-tethered side chain on the hydroxyl group is an effective and essential structural determinant for improving biological activities and druglike properties of these molecules. Compounds with such modifications exhibited potent antiproliferative effects against breast and pancreatic cancer cell lines with IC50 values from low micromolar to nanomolar range. Among them, the newly discovered STAT3 inhibitor 12 (HJC0416) displayed an intriguing anticancer profile both in vitro and in vivo (i.p. & p.o.). More importantly, HJC0416 is an orally bioavailable anticancer agent as a promising candidate for further development.

Keywords: Anticancer agents; Breast cancer; Oral bioavailability; Pancreatic cancer; STAT3 inhibitors.

Figure 1

Previous work and drug design strategy for the current work.

Figure 2

Effects of 12 (HJC0416) and stattic on cell growth and cellular morphological changes. Exponentially growing MDA-MB-231 breast cancer cells were incubated with 12 or stattic for 48 h. Cell morphology was evaluated under light microscopy.

Figure 3

Compound 12 (HJC0416) inhibited the STAT3 mediated luciferase reporter activity in MDA-MB-231 cells. STAT3 promoter activity was measured using dual luciferase assay with a STAT3 reporter. Promoter activity obtained from DMSO-treated MDA-MB-231 cells was used as control. Error bars represent standard deviation of triplicate wells. Representative experiment from at least 3 independent experiments is shown. RLU: relative luciferase unit.

Figure 4

Predicted binding modes for HJC0149 (1) and HJC0416 (12). The figures were generated using Pymol.

Figure 5

Western blot analysis of biochemical markers for apoptosis induction and inhibition of STAT3 activity by stattic and compound 12 in the MDA-MB-231 cell line. Cells were treated with stattic or 12 for 12 h, levels of pSTAT3, STAT3, cleaved caspase-3, PARP-1, cleaved PARP-1 and cyclinD1 were probed by specific antibodies. β-actin was used as the loading control.

Figure 6

In vivo efficacy of compound 12 (HJC0416) in inhibiting growth of xenograft tumors (triple-negative breast cancer MDA-MB-231) in mice via A) i.p. or B) oral gavage (p.o.) routes.

Scheme 1^a

^aReagents and conditions: (a) R¹OH, Ph₃P, DIAD, THF, rt, 39–77%; (b) R²H, KI, K₂CO₃, acetone, reflux, 77–98%; (c) (i) Boc-R³OH, Ph₃P, DIAD, THF, rt; (ii) TFA, CH₂Cl₂, 0 °C to rt, 40–52% (two steps).

Scheme 2^a

^aReagents and conditions: (a) 10% NaOH (aq.), MeOH, H₂O, 0 °C to rt, 60%.