Synthesis and Biological Activities of 11- and 12-Substituted Benzophenanthridinone Derivatives as DNA Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1 Inhibitors

Abstract

Herein, a series of 11- or 12-substituted benzophenanthridinone derivatives was designed and synthesized for the discovery of dual topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Enzyme-based assays indicated that two compounds 12 and 38 showed high TOP1 inhibitory potency (+++), and four compounds 35, 37, 39 and 43 showed good TDP1 inhibition with IC₅₀ values ranging from 10 to 18 μM. 38 could induce cellular TOP1cc formation, resulting in the highest cytotoxicity against HCT-116 cells (0.25 μM). The most potent TDP1 inhibitor 43 (10 μM) could induce cellular TDP1cc formation and enhance topotecan-induced DNA damage and showed strong synergistic cytotoxicity with topotecan in both MCF-7 and MCF-7/TDP1 cells.

Keywords: DNA repair; anticancer agents; inhibitors; topoisomerase; tyrosyl-DNA phosphodiesterase.

Figure 1.

The structures of the reported dual TOP1 and TDP1 inhibitors and the representative oxynitidine derivatives.

Figure 2.

Representative gels of TOP1-mediated DNA cleavage assay. Lane 1, DNA alone; lane 2, DNA and TOP1; lane 3, DNA and TOP1 with CPT (1 μM); lane 4, DNA and TOP1 with LMP744 (1 μM); lanes 5–20, DNA and TOP1 with the tested compounds at 0.1, 1.0, 10, and 100 μM concentration, respectively. The arrows at left indicate the cleavage site positions.

Figure 3.

Representative gels of TDP1 assay. Lane 1, DNA alone; lane 2, DNA and recombinant TDP1; lanes 3–26, DNA, recombinant TDP1 and the compounds at the concentrations increasing from 0.051 to 111 μM. The imidazopyridine XZ664 was tested as the positive control at the concentrations increasing from 0.15 to 333 μM. Cy5–14Y and Cy5–14P are the substrate and product of TDP1, respectively.

Figure 4.

Cellular TOP1ccs induced by 38 (A) and TDP1cc induced by 43 (B) in MCF-7 cells, detected through ICE assays. (A) Lane 1, untreated control; lanes 2 and 3, cells treated with TPT at 25 and 50 μM, respectively; lanes 4–6, cells treated with 38 at 25, 50, and 100 μM, respectively. (B) Lane 1, untreated control; lanes 2–4, cells treated with TPT (10 μM) and 43 at 10 and 100 μM, respectively; lanes 5 and 6, cells co-treated with TPT (10 μM) and 43 (10 and 100 μM). (C) Hypothetical binding mode of 38 in the ternary TOP1-DNA-drug cleavage complex (PDB 1K4T). 38 is shown as yellow carbon atoms ball and stick representation. Key residues are highlighted in green ball and stick representation. All distances are measured from heavy atom to heavy atom.

Figure 5.

The synergistic effects and the combination index (CI) of 35 or 43 with TPT in the MCF-7 (A, B) and MCF-7/TDP1 (C, D) cells. The cells were co-incubated with drugs for 96 h.

Figure 6.

(A) Histone γH2AX foci (red) induced by 43 and TPT alone or combined in MCF-7 cells. The cells were incubated with drugs for 5 h at the indicated concentrations. DNA was stained with DAPI (blue). (B) Quantification of the number of γH2AX foci on the figure (A). P < 0.001 (***) and P < 0.0001 (****). Every experiment was repeated at least three times independently. (C) Flow cytometry histograms of apoptosis in MCF-7 cells induced by 43 at 1, 2, and 4 μM, respectively.

Scheme 1.

Synthesis of 10-23. Reagents and conditions: (a) NH₂Me, MeOH, rt. (b) i) N₂, Ni(cod)₂, P(o-Tol)₃, MeCN; ii) CsOH, K₃[Fe(CN)₆], MeOH, H₂O. (c) (COCl)₂, DMSO, TEA, DCM. (d) i) NaClO₂, NaH₂PO₄·2H₂O, 2-methyl-2-butene, acetone; ii) Ac₂O, AcOK. (e) KOH, H₂O, MeOH. (f) R(CH₂)_nCl, KI, K₂CO₃, DMF.

Scheme 2.

Synthesis of 33-45. Reagents and conditions: (a) i) ICl, AcOH, rt; ii) MeOH, H₂SO₄. (b) N₂, TMSA, PdCl₂(PPh₃)₂, CuI, NEt₃, THF. (c) K₂CO₃, MeOH. (d) i) AgNO₃, I₂, MeOH; ii) KMnO₄, H₂O/MeCN. (e) i) SOCl₂, reflux; ii) NH₃/H₂O, THF. (f) PdCl₂(PPh₃)₂, NEt₃, MeCN. (g) i) Me₃OBF₄, DCM; ii) CF₃COOAg, DCM. (h) NaBr, TsOH·H₂O, MeOH. (i) MeI, K₂CO₃, DMF. (j) i) K₂CO₃, H₂O, MeOH; ii) Ac₂O, AcOK; iii) K₂CO₃, H₂O, MeOH. (k) R(CH₂)_nCl, KI, K₂CO₃, DMF.