Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines

Abstract

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.

Keywords: Cannabidiol; Capsaicin; Glioblastoma; Ion channel; Resiniferatoxin; Vanilloid.

Figure 1

Structures of selected α,β-unsaturated 1,4-dialdehyde terpenoids.

Figure 2

(A) Demonstration of the feasibility of the modified Paal-Knorr condensation of 1 to form pyrrole 2, (B) Paal-Knorr pyrrole formation implicated in the neurotoxicity of hexane and (C) novel pyrrolylation of primary amines with C12-Wittig derivatives reported herein.

Figure 3

Synthesis of C12-Wittig derivatives 5–13 and formation of pyrrole 14 from 5 and BnNH₂.

Figure 4

(A) The absense of resistant populations in all 5 cultures tested with analogue 5 and contrasting effects on viability of all cells between 5 and standard chemotherapeutic agents paclitaxel and podophyllotoxin in (B) A549 NSCLC and (C) U87 glioblastoma cell cultures. PODO = podophyllotoxin, PAO = phenyl arsine oxide.

Figure 5

Activity of 5 against neurosphere glioma cell cultures with clinically relevant mutations. Transgenic mouse glioma cells of defined molecular subtypes were generated by forced expression of EGFRvIII (classical GBM subtype), PDGFB (proneural GBM subtype) in cdkn2a-deficient and cdkn2a/TRPV1-doubly deficient subventricular neural precursors (NPC). These primary cell cultures were treated for 24 hours either with 10 μM CBD versus a corresponding vehicle-control (containing 0,01% DMSO) or 20 μM of 5 versus vehicle control (0,02% DMSO). Cytotoxicity was measured 24 h after incubation and base-line cytotoxicity levels in the controls were arbitrarily defined as 1. Read-outs from treated cells were normalized to their respective vehicle controls and fold change of relative cytotoxicity was calculated. Each bar represents the mean ± SD from two independent experiments; satistical significance, as determined by unpaired t-tests, is indicated: *** represents p < 0,001; ** p < 0,005; * p=0,0257; ns not significant).

Figure 6

(A) Evaluation of compound 5 in a [³H]-RTX TRPV1 displacement assay. Effects of 1 and 5 at the concentration of 10 μM on the specific binding of [³H]-RTX to the vanilloid site of TRPV1 receptor from rats spinal cord membranes. Results are expressed as mean ± S.E.M from 3 independent experiments, analyzed by one way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test (***p<0.05 and ****p<0.001). (B and C) Evaluation of 5 for TRPV1 activity in MDA-MB-231 breast cancer cells. (B) Effect of 1 (80 μM) on MDA-MB-231 [Ca²⁺]_i. (C) Effect of 5 (20 μM) on MDA-MB-231 [Ca²⁺]_i.

Figure 7

Molecular modeling showing the capsaicin binding region of TRPV1, with the likely binding pose of capsaicin (left). Compound 1 is also well accommodated in this pocket (middle), but the Wittig derivative 5 (right - displayed in orange and overlayed with 1 in green) is required to bind with its apolar ester chain embedded in the polar “southern” region of the pocket (as highlighted by the solvent interpolated charge surface).

Figure 8

In vitro videomicroscopic analysis of the anticancer effects of 1 and its C12-Wittig derivatives 5 and 13. The U373 human glioma cell line was treated with polygodial, 5 and 13 at their mean GI₅₀ concentrations (Table 1) or left untreated. Videomicroscopy enabled taking pictures of the culture field every 4 minutes. The experiment was conducted once in triplicate. While the morphology of cells treated with 1 was fixed over time, 5 and 13 exerted cytostatic effects on U373 cells.

Figure 9

Viability of U373 cells by trypan blue staining. U373 cells were treated for 72 h with 1 or 13 and stained with trypan blue. The experiment was conducted once in triplicate. After having taken pictures, cells were fixed with ice-cold methanol and again stained with trypan blue as internal positive control. While cells treated with 1 were all blue-stained before methanol fixation, the 13-treated cells were still alive after 72 h of treatment.