Design, synthesis, and biological evaluation of stable colchicine binding site tubulin inhibitors as potential anticancer agents

Abstract

To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure-activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring.

Figure 1

Structures of representative tubulin inhibitors bound to the colchicine-binding site.

Figure 2

Proposed approaches to solve metabolic liabilities of lead compounds.

Scheme 1. Synthesis of the Fused D Ring Antitubulin Compounds

Reagents and conditions: (a) TsOH, EtOH, reflux; (b) (3,4,5-trimethoxyphenyl)boronic acid, K₂CO₃, Pd(PPh₃)₄.

Figure 3

Irreversible tubulin binding agents and hypothesis of interactions between Cys-241 and para-position at the C ring.

Scheme 2. Synthesis of Analogues Focused on Modifications at para-Position of the C Ring

Reagents and conditions: (a) MeOH/pH = 6.4 phosphate buffer, RT; (b) EDCI, HOBt, NMM, CH₃OCH₃NH·HCl; (c) CBrCl₃, DBU; (d) 5-bromo-1,2,3-trimethoxybenzene/BuLi, THF, −78 °C; (e) AlCl₃, CH₂Cl₂; (f) ClCH₂COCl, CH₂Cl₂, NEt₃ (12) or (CF₃CO)₂O, CH₂Cl₂, DMAP (13); (g) PhCH₂Br, K₂CO₃, DMF (14); MOMCl, Hunig’s base, CH₂Cl₂ (15); BrCH₂CH₂NHBoc, DMF, Cs₂CO₃ (16) or 2-(2-bromoethyl)isoindoline-1,3-dione, K₂CO₃, DMF 120 °C (17); (h) 4 M HCl in dioxane.

Scheme 3. Synthesis of Analogues Based on PAT Template

Reagents and conditions: (a) BrCN, Et₂O/hexane; (b) CH₃SO₃H, EtOH, reflux, 24 h; (c) (1) 6 M HCl, (2) NaOH 25% conc.; (d) PhSO₂Cl, Et₃N; (e) −78 °C, t-BuLi, 3,4,5-trimethoxybenzoyl chloride; (f) Boc₂O, NaOH; (g) (chloromethanetriyl)tribenzene, Et₃N, CH₂Cl₂; (h) HCl; (i) H₂, Pd–C, 5%, EtOH, 40 psi; (j) PhCOSCN, Me₂CO; (k) MeOH, 1 N NaOH; (l) EtOH, 65 °C; (m) NaOH, MeOH; (n) HBTU, NMM, HNCH₃OCH₃·HCl, CH₂Cl₂; (o) 3,4,5-trimethoxyphenylbromide, n-BuLi, THF.

Figure 4

Potential binding poses for 4 (gold tube model; glide docking score −8.58) and 7 (dark green tube model; glide docking score −8.10) in tubulin α,β-dimer (PDB code 1SA0). The native ligand, DAMA–colchicine (glide docking score of −9.26), is shown in blue thin wire model.

Figure 5

Potential binding poses for 33 (gray tube model; glide docking score of −8.70) and the native ligand DAMA–colchicine (blue thin wire model; glide docking score of −9.26) in tubulin α,β-dimer (PDB Code: 1SA0).

Figure 6

Proposed metabolites and metabolic pathway of 15.

Figure 7

Proposed metabolites and metabolic pathway of 18.

Figure 8

Proposed metabolites and metabolic pathway of 4.

Figure 9

Proposed metabolites and metabolic pathway of 7.

Figure 10

Kinetics of compounds 15, 18, 4, and 7 and their metabolites in human liver microsomes. (A) Compound 15 and its metabolites; (B) compound 18 and its metabolites; (C) compound 4 and its metabolites; and (D) compound 7 and its metabolites; 50 μM of the test compound was incubated with 1 mg/mL microsomal proteins. Samples at various time points were analyzed by Q-TOF LC–MS.

Figure 11

Compounds 4 and 7 inhibit tubulin polymerization in vitro.