Microtubule Destabilizing Sulfonamides as an Alternative to Taxane-Based Chemotherapy

Abstract

Pan-Gyn cancers entail 1 in 5 cancer cases worldwide, breast cancer being the most commonly diagnosed and responsible for most cancer deaths in women. The high incidence and mortality of these malignancies, together with the handicaps of taxanes-first-line treatments-turn the development of alternative therapeutics into an urgency. Taxanes exhibit low water solubility that require formulations that involve side effects. These drugs are often associated with dose-limiting toxicities and with the appearance of multi-drug resistance (MDR). Here, we propose targeting tubulin with compounds directed to the colchicine site, as their smaller size offer pharmacokinetic advantages and make them less prone to MDR efflux. We have prepared 52 new Microtubule Destabilizing Sulfonamides (MDS) that mostly avoid MDR-mediated resistance and with improved aqueous solubility. The most potent compounds, N-methyl-N-(3,4,5-trimethoxyphenyl-4-methylaminobenzenesulfonamide 38, N-methyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-aminobenzenesulfonamide 42, and N-benzyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-aminobenzenesulfonamide 45 show nanomolar antiproliferative potencies against ovarian, breast, and cervix carcinoma cells, similar or even better than paclitaxel. Compounds behave as tubulin-binding agents, causing an evident disruption of the microtubule network, in vitro Tubulin Polymerization Inhibition (TPI), and mitotic catastrophe followed by apoptosis. Our results suggest that these novel MDS may be promising alternatives to taxane-based chemotherapy in chemoresistant Pan-Gyn cancers.

Keywords: antitumor; breast cancer; combretastatin A-4; gynecologic cancer; sulfonamide; taxane; tubulin.

Figure 1

Representative ligands binding at the colchicine site used as a starting point for the rational design of new Microtubule Destabilizing Sulfonamides (MDS). General structure and structure variations of new MDS.

Figure 2

General synthetic approach. Reagents, conditions, and yields: (a) Pyridine, CH₂Cl₂, rt, 4–8 h, 90–96% (b) R_N = CH₃, CH₃I, KOH, CH₃CN, rt, 24 h, 63–98%; R_N = Ac, acetic anhydride, pyridine, CH₂Cl₂, reflux, 8–12 h, 61–83%; R_N ≠ CH₃ and Ac, R_N-halogen, K₂CO₃, dry DMF, rt, 24–48 h, 40–99% (c) tert-Butyl nitrite, CH₃CN, 45 °C, 24 h, 60% (d) H₂, Pd/C, EtOAc, rt, 48–72 h, 82–98% (e) Formic acid, CH₂Cl₂, rt, 24–48 h, 62–82% (f) Trichloroacetic acid, NaBH₄, dry THF, 0°C, 24 h, 97% (g) Paraformaldehyde, NaBH₃CN, AcOH, MeOH, rt, 72–96 h, 95% (h) Ethyl 2-bromoacetate, NaI, acetone/THF 1:1, reflux, 48 h, 19% (i) NBS, CH₂Cl₂, rt, 6 h, 43%.

Figure 3

Cell cycle analysis. (a) MCF7 cell cycle distribution. Cells were arrested in G₂/M phase. (b) SKOV3 cell cycle distribution. Cells arrested in G₂/M phase experienced time-dependent cell death (SubG₀/G₁ region). (c) HeLa cell cycle distribution. Cells were arrested in the G₂/M phase followed by cell death (SubG₀/G₁ region). Cells were incubated with the lead compounds at 600 nM (38), 50 nM (42), or 400 nM (45) for 24, 48, and 72 h, stained with propidium iodide (PI) and their DNA content was analyzed by flow cytometry. Untreated samples were analyzed in parallel. The different cell cycle populations were quantified, expressed in percentages, and represented in bar charts. Data shown are representative of three independent experiments.

Figure 4

Confocal microscopy experiments in MCF7, SKOV3, and HeLa cells. Cells were incubated in the absence (Control) or the presence of 38 (600 nM) and 45 (400 nM) for 72 h. (a) Visualization of the microtubule network. α-Tubulin was stained in green, and nuclei were stained with DAPI (blue fluorescence). (b) Multilobulated nuclei (blue) resembling bunches of grapes in MCF7 cells. Photomicrographs are representative of three independent experiments. Scale bar: 25 µm.

Figure 5

Docking poses for compounds 38, 42, and 45 at the colchicine site of tubulin. Combretastatin A-4 (CA-4) is shown in diffuse green as a reference.