Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives

Abstract

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGlo^TM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.

Keywords: apoptosis; cell cycle; melanoma; shikonin derivatives; synthesis.

Figure 1

Acylation of shikonin (1).

Figure 2

Synthesis of the bicyclic acetic acid p1.

Figure 3

Synthesis of 3-cyclopropylpropanoic acid (p2).

Figure 4

Synthesis of precursor acids p3 and p4.

Figure 5

Results of the XTT assay. For clarity reasons, only the results of the treatment with 5.0 µM for 72 h are shown. The complete results can be found in the Supplementary Material (Figures S1 and S2). 2 was tested as a reference at 5.0 µM. The strongest cytotoxicity was found for 5 (n = 6, mean ± sem). Vinblastine was used as positive control. At a concentration of 0.01 nM, it reduced the cell viability compared to control cells to: WM9 cells: 23.8 ± 1.5%, WM164 cells: 59.4 ± 4.4 %, and MUG-Mel2 cells: 65.0 ± 6.9 % (n = 6, mean ± sem, 72 h of treatment).

Figure 6

Results of the ApoToxGlo™ Triplex Assay. WM9 and WM164 cells were treated with 1 µM, 5 µM, 10 µM, and 20 µM of 5 for 4 h, 24 h, or 48 h (n = 6, mean ± sem). (A) Viability of the cells measured as relative fluorescence of control cells. (B) Cytotoxicity of 5 towards the cells measured as relative fluorescence of control cells. (C) Activity of caspases 3 and 7 indicative for apoptosis induction. Staurosporine (25 µM) served as positive control (apoptosis increase in WM9 cells after 24 h: 1193.9 % and after 48 h: 297.4%; in WM164 cells after 24 h: 989.1% and after 48 h: 362.6%).

Figure 7

Results of the LDH assay. WM9 and WM164 cells were treated with 1 µM, 5 µM, 10 µM, and 20 µM of 5 for 24 h, 48 h, or 72 h (n = 9, mean ± sem). Results are displayed as percentage of cell lysis. Control = vehicle treated cells (0.5% EtOH). Only at 20 µM, slight increases in LDH release were found.

Figure 8

Effects of 5 on the cell cycle. (A) WM9 and (B) WM164 cells were treated with 10 µM and 20 µM of 5 for 24 h or 48 h (n = 6, mean). Results are displayed as percentage of cell cycle distribution. Control = vehicle treated cells (0.5% EtOH). 5 influence the distribution of the cell cycle only at high concentrations.