Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide

Abstract

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.

Figure 1.

Reported TDP2 inhibitors. Deazaflavin 4 is the most potent and best characterized TDP2 inhibitor type.

Figure 2.

Novel deazaflavin subtypes 11-12 designed to improve lipophilicity by adding a second phenyl ring (B) directly off ring A (for subtype 11) or at N-3 site (for subtype 12).

Figure 3.

Chemoselectivity of the Chan-Lam coupling reaction. Under reaction conditions, the arylation occurred exclusively at the N-3 site to yield subtype 12 with O-arylated products 11 not observed.

Figure 4.

Effect of TDP2 inhibitors 4a, 12a, 12b, 12h, 11a and 11e on potentiating toxic action of ETP.

Figure 5.

Molecular modeling of 11k and 12b. (A) Binding mode of 4c within the crystal structure of catalytic domain of humanized mouse TDP2 (PDB code: 5J42). (B) Potential vectors for designing novel deazaflavin inhibitor types. (C) Predicted binding mode of 11k within the catalytic domain of humanized mouse TDP2. (D) Predicted binding mode of 12b within the catalytic domain of humanized mouse TDP2. Key residues are highlighted in green sticks. H-bond interactions are depicted as black dotted lines. Cation- π and π-π interaction are represented as double headed arrow in black. Water molecule and magnesium ion were represented as red and blue non-bonded sphere. All the residue numberings are based on the human TDP2.

Scheme 1.

Synthesis of deazaflavin scaffold 4 and new subtypes 11-12 ^aReagents and conditions: a) EtOH, 150 °C, 30 min, MW, 60–85%; b) DMF, 110 °C, MW, 30 min, 40–60%; c) K₂CO₃, DMF, r. t., 12 h; d) TFA, DCM, r. t., 6h, 60–75% over two steps; e) K₂CO₃, DMF, 110°C, 12h, 59%; f) 10% Pd/C, EtOAc, rt, o. n. 87%; g) EtOH, NaOH 1N, reflux, 3h, 85% h) Cu(OAc)₂, DMF, air, r. t., 24–48 h, 40–52%.

Scheme 2.

Alternative linear synthesis for subtype 12 ^aReagents and conditions: a) Diethylmalonate, NaOEt, EtOH, reflux, o. n., 86%; b) POCl₃, BnEt₃NCl, 50 °C, 6 h, 70%; c) aniline derivative, EtOH, 150 °C, 30 min, MW, 55–80%; d) DMF, 110 °C, 30 min, MW, 50–69%.