Alkyne-azide cycloaddition analogues of dehydrozingerone as potential anti-prostate cancer inhibitors via the PI3K/Akt/NF-kB pathway

Abstract

Herein, we report the isolation and synthetic modification of dehydrozingerone (DHZ, 1), a secondary metabolite present in the rhizome of Zingiber officinale. We synthesized O-propargylated dehydrozingerone, which was subsequently coupled by alkyne-azide cycloaddition (3-20) using click chemistry. The compounds (1-20) were evaluated for their in vitro cytotoxic activity in a panel of three cancer cell lines. Among all the DHZ derivatives, 3, 6, 7, 8, 9 and 15 displayed potent cytotoxic potential with an IC₅₀ value ranging from 1.8-3.0 μM in MCF-7, PC-3 and HCT-116 cell lines. Furthermore, compound 7 has proven to be the most potent cytotoxic compound in all the three distinct cancer cell lines and also demonstrated significant anti-invasive potential in prostate cancer. The mechanistic study of compound 7 showed that it not only suppressed the AKT/mTOR signalling which regulates nuclear transcription factor-NF-kB but also augmented the expression of anti-invasive markers E-cadherin and TIMP. Compound 7 significantly decreased the expression of pro-invasive markers vimentin, MMP-2 and MMP-9, respectively. This study underscores an efficient synthetic approach employed to evaluate the structure-activity relationship of dehydrozingerone (1) in search of potential new anticancer agents.

Fig. 1. Pharmacophore of (DHZ) 1 for antimutagenic/cytotoxic activity.

Scheme 1. Synthesis of substituted 1-azido derivatives.

Fig. 2. Effects of compound 7 on motility and cell scattering aptitude of PC3 cells. (A and B) The wound healing assay was performed by treating PC-3 cells with increasing concentrations of 7 which were consecutively assessed for the degree of wound healing. Photographs of scratched areas were taken at 20× magnification (C and D). PC-3 cells were treated with indicated concentrations of 7 along with or without VEGF to check its effects on cell scattering. Following 24 h of incubation with 7, individual colonies were observed under an inverted microscope and were photographed. The data shown are representative of three independent experiments. SD ± **P < 0.01.

Fig. 3. Effects of compound 7 on the invasive and colony formation ability of PC-3 cell lines. (A) Boyden chamber assay was performed to check the effect of 7 on the invasive ability of PC-3 cells. Following incubation with compound 7 for the indicated time point, invaded cells were counted and images were captured at 20× magnification. (B) Bar graphs are representative of the number of invasive cells per field. (C and D) To check the effects on colony formation, PC-3 cells were exposed to different concentrations of 7 and incubated for five days. After the incubation, the cells were stained with crystal violet; stained colonies were counted randomly, quantified and images were taken under an inverted microscope at 20× magnification. Data from three independent experiments were subjected to statistical analysis. SD ± **P < 0.01, *p < 0.05.

Fig. 4. Compound 7 inhibits PI3K/Akt/NF-KB signaling. (A and B) PC-3 cells were treated with indicated concentrations of 7. Whole cell lysates were checked for the expressions of indicated proteins by western blotting. β-Actin was used as a loading control and the data shown are representative of three independent experiments.

Fig. 5. Compound 7 abrogates MMP-2 and MMP-9 gelatinase activity and expression. (A) Gelatin zymography was carried out to check the effects of 7 on MMP-2 and MMP-9 activity. BSA was used as a loading control. (B) PC-3 cells were seeded in six-well plates and treated with indicated concentrations of 7. Whole cell lysates were checked for the expressions of indicated proteins by western blotting. β-Actin was used as loading control and the data shown are representative of three independent experiments.

Scheme 2. Synthesis of alkyne–azide cycloaddition derivatives (3–20).