Dual-functional abeo-taxane derivatives destabilizing microtubule equilibrium and inhibiting NF-κB activation

Abstract

Taxchinin A, with a 11(15→1)-abeo-taxane skeleton, is a major, but inactive taxoid contained in leaves of Taxus chinensis . In our design of dual-functional antitumor abeo-taxane derivatives, two fragments from antitumor agents with different molecular targets (the N-acyl-3'-phenylisoserine side chain from the antimitotic agent paclitaxel and an α,β-unsaturated carbonyl system from NF-κB inhibitors) were incorporated into the scaffold of taxchinin A. The resulting compounds displayed broad inhibitory effects against proliferation of tumor cell lines, with notable selectivity toward colon cancer, melanoma, and renal cancer, when evaluated in the NCI-60 human tumor cell line screening panel. On the basis of the NCI-60 assay data, structure-activity relationship (SAR) correlations were elucidated. Mechanistic studies indicated that this new compound type can both destabilize microtubules and inhibit NF-κB activation, thereby inducing tumor cell apoptosis. This first report of the dual-functional taxoid-core compounds thus provides new opportunities for future drug development based on natural axoid scaffolds.

Figure 1

Structures of paclitaxel, docetaxel, taxchinin A and parthenolide

Figure 2

Design strategy for new dual-functional abeo-taxoid derivatives

Figure 3

SAR of taxoid-based derivatives

Figure 4

Compounds 11 and 12 induce apoptosis of HCT-116 cells. Cells were treated with 11 or 12 for 24 h. Treatment with 11 and 12 increased the early apoptotic (Annexin V +/PI -, lower right quadrant) and late apoptotic (Annexin V +/PI +, upper right quadrant) cells.

Figure 5

Effects of compounds on microtubule assembly. (A) Effects of compounds on tubulin polymerization in a cell-free system. Tubulin polymerization was detected in a fluorescence based cell-free system in the presence of DMSO (control), 500 μM CaCl₂, 3 μM paclitaxel, 15 μM 11, 15 μM 12, or 30 μM 17. Tubulin polymerization was measured by fluorescence intensity at 420 nm emission and 340 nm excitation. Data are representative of three independent experiments performed in triplicate. (B) Effects of compounds on microtubule dynamics in PC-3 cells. PC-3 cells were cultured and treated with DMSO (control), 2 μM compounds 11, or 5 μM compound 12 for 24 h. Cells were stained with monoclonal antibody to α-tubulin and DAPI for DNA. The insert (upper panel) shows a typical image of metaphase cell with spindles. Cells with abnormal spindles and microtubules were detected among the cells treated with compound 11 or 12. Although both compounds gave similar defects on tubulin dynamics, significant effects were not seen in cells treated with 2 μM 12. Additional images are available in Supporting Information Figure S2. Bar, 25 μm.

Figure 6

Compound 11 and 12 induce cell cycle arrest at G2/M phase. HCT-116 cells were treated with 1 or 2 μM compounds 11 or 12 for 12 h. Cells were fixed, stained with propidium iodide (PI), and were assessed by flow cytometry. The percentages of cells in different DNA ploidy of 2N (G0/G1), between 2N and 4N (S) and 4N (G2/M) are indicated on the right of each cell cycle profile. Data shown are from one of three independent experiments.

Figure 7

Inhibitory effects of compounds 11 and 12 on TNF-α–induced NF-κB activation. HEK 293T cells transiently transfected with pNFκB-luc and pRL-TK vectors, were pretreated with DMSO, 0.31, 0.62, 1.25 μM compound 11 or 0.62, 1.25, 2.50 μM compound 12 for 1 h before TNF-α stimulation (25 ng/mL) for 18 h. The NF-κB reporter was assayed by measuring the luciferase activity as described under Materials and Methods. Data shown are from one of three independent experiments. *p<0.05, **p<0.01

Scheme 1

Reagents and conditions. (a) TBDMSCl, DMF, −40°C; (b) PCC, CH₂Cl₂ (c) TBAF, THF, rt; (d) HF/CH₃CN (95/5); (e) (i) NaHMDS, 7 or 8, THF, −78°C; (ii) 0.5 N HCl, THF, 0°C to rt.