N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide as a new scaffold that provides rapid access to antimicrotubule agents: synthesis and evaluation of antiproliferative activity against select cancer cell lines

Abstract

A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamides was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and afforded inhibitors of cancer cell growth. For example, compound 13e had an IC(50) of 46 nM against MCF-7 human breast tumor cells. Structure-activity relationship (SAR) studies demonstrated that (i) meta-phenoxy substitution of the N-1-benzyl group is important for antiproliferative activity and (ii) a variety of heterocyclic substitutions for the aryl group of the arylamide are tolerated. In silico COMPARE analysis of antiproliferative activity against the NCI-60 human tumor cell line panel revealed a correlation to clinically useful antimicrotubule agents such as paclitaxel and vincristine. This in silico correlation was supported by (i) in vitro inhibition of tubulin polymerization, (ii) G(2)/M-phase arrest in HeLa cells as assessed by flow cytometry, and (iii) perturbation of normal microtubule activity in HeLa cells as observed by confocal microscopy. The results demonstrate that N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide is a readily accessible small molecule scaffold for compounds that inhibit tubulin polymerization and tumor cell growth.

Figure 1

Time and concentration dependence of the antiproliferative activity of 13e against MCF-7 tumor cells. Time is in terms of time elapsed after addition of the compound. Cell counts are shown relative to the cell count observed in the vehicle control 96 h after addition of the 0.5% DMSO solution.

Figure 2

Inhibition of tubulin assembly by 4e, 13a, and 13e in vitro. All compounds were tested at a concentration of 10 µM. Effects of compounds on tubulin polymerization were assessed by monitoring the increase in light scattering, measured as optical density (O.D.), at 340 nm. Standards of 10 µM nocodazole (a tubulin assembly inhibitor) and 10 µM paclitaxel (a tubulin assembly promoter) were used for direct comparison.

Figure 3

Effects of 4e, 13a, 13d, 13e, and 14 on the cell cycle distribution of HeLa cells as measured by propidium iodide staining and flow cytometry. HeLa cells were treated with 5 µM compound for 18 h in triplicate.

Figure 4

Confocal microscopy images of HeLa cells after 18 h incubation in the presence of 5 µM compound. Nocodazole is a known tubulin polymerization inhibitor. Nuclear DNA was stained with propidium iodide (red channel) and tubulin was stained with FITC-conjugated anti-α-tubulin antibody (green channel). Compounds 4e and 13e disrupted normal microtubule structures, caused fragmentation of mitotic spindles, and induced M-phase arrest.

Scheme 1

Synthesis of 2-phenyloxazole-4-carboxylic acid.

Scheme 2

Synthesis of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-phenyl-oxazole-4-carboxamide scaffold.

Scheme 3

Synthesis of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold.

Chart 1

Structures of anti-tuberculosis compounds 2 and 3 and antimicrotubule compound 4e, all derived from a fragment of mycobactin S (1). MIC values indicate in vitro anti-tuberculosis activity against M. tuberculosis H37Rv in GAST medium, and IC₅₀ values indicate in vitro antiproliferative activity against human breast cancer cell line MCF-7.