Facile total synthesis of lysicamine and the anticancer activities of the RuII, RhIII, MnII and ZnII complexes of lysicamine

Abstract

Lysicamine is a natural oxoaporphine alkaloid, which isolated from traditional Chinese medicine (TCM) herbs and has been shown to possess cytotoxicity to hepatocarcinoma cell lines. Reports on its antitumor activity are scarce because lysicamine occurs in plants at a low content. In this work, we demonstrate a facile concise total synthesis of lysicamine from simple raw materials under mild reaction conditions, and the preparation of the Ru(II), Rh(III), Mn(II) and Zn(II) complexes 1-4 of lysicamine (LY). All the compounds were fully characterized by elemental analysis, IR, ESI-MS, ¹H and ¹³C NMR, as well as single-crystal X-ray diffraction analysis. Compared with the free ligand LY, complexes 2 and 3 exhibited superior in vitro cytotoxicity against HepG2 and NCI-H460. Mechanistic studies indicated that 2 and 3 blocked the cell cycle in the S phase by decreasing of cyclins A2/B1/D1/E1, CDK 2/6, and PCNA levels and increasing levels of p21, p27, p53 and CDC25A proteins. In addition, 2 and 3 induced cell apoptosis via both the caspase-dependent mitochondrial pathway and the death receptor pathway. in vivo study showed that 2 inhibited HepG2 tumor growth at 1/3 maximum tolerated dose (MTD) and had a better safety profile than cisplatin.

Keywords: antitumor activity; apoptosis; lysicamine; metal complexes.

Figure 1. Synthetic routes of lysicamine (LY)

Reagents and conditions are as follows: a: (i) SOCl₂, CHCl₃ (75 °C reflux, 2 h); (ii) CHCl₃, NaHCO₃ (ice-bath, 2 h); b: (i) POCl₃, toluene (80 °C reflux, 3 h); (ii) (CH₃COO)₃BHNa, CHCl₃ (room temperature, 1 h); c: Methyl chloroformate (ClCOOCH₃), NaOH, CHCl₃ (room temperature, 1 h); d: tricyclohexyl phosphine [P(cy)₃], Pd(OAc)₂, K₂CO₃, DMA (120 °C, N₂,5 h); e: LiAlH₄, THF (reflux, 6 h); f: Mn(Ac)₃, glacial acetic acid (80 °C, 12 h).

Figure 2. Crystal structures of complexes 1–4

Figure 3. The effects of 2 and 3 treatment in HepG2 cells on cell cycle

(A) Cell cycle analysis of 2, 3, LY and metal salt in HepG2 cells. (B, D) Effects of 2 treatment in HepG2 cells on cell cycle regulatory proteins at 3.5, 7.0 and 14.0 μM for 24 h, respectively. (C, E) The relative protein expression of each band = (density of each band/density of β-Actin band). Mean ± SD was from three independent measurements.

Figure 4. Apoptosis of HepG2 cells induced by 2 and 3

(A) The apoptotic nuclear morphological analysis by Hoechst-33258 staining(magnification 100×) and (B) the apoptosis of HepG2 cells analysis by flow cytometry after 24 h treatment with 2 and 3 at variously concentrations for 24 h. (C, E) Western blot analysis of apoptosis associated proteins after treatment of HepG2 cells with 2 (3.5, 7.0 and 14.0 μM) and 3 (7.0, 14.0 and 28.0 μM) for 24 h, respectively. (D, F) Densitometry analysis from part C and E. The relative expression of each band = (density of each band/density of β-actin band). Mean and SD values were from three independent measurements.

Figure 5. The effect of 2 or 3 on the levels of ROS, intracellular Ca²⁺, loss of ΔΨm and the activated caspase-3/8/9 expression after HepG2 cells were treated with 2 and 3 for 24 h, respectively

(A) The images of fluorescence microscope (magnification 100×). (B) The change of ROS, Ca²⁺ and ΔΨm examined by flow cytometry assay. (C) Flow cytometry analysis of activated caspase-3/8/9 expression in HepG2 cells after incubated with 2 and 3 at IC₅₀ values for 24 h.

Figure 6. Relative expression profiles of 89 genes in HepG2 cells after being treated with 2 (7.0 μM) for 24 h

(A) Cell cycle related genes and (B) apoptosis-related genes.

Figure 7

(A) Circular dichroism spectra of ct-DNA bound to LY, 2 and 3 with [DNA]/[each compound] ratios range were 10:0. 5, 10:1.0, 10:1.5 and 10:2.0 (the concentration of ct-DNA bases alone of 1×10⁻⁴ M, dashed line). (B) Agarose gel electrophoresis mobility shift assay of pBR322 plasmid DNA (0.5 μg/μL) when interacted with LY, 2 and 3 with increasing concentrations from 10 to 100 μM.

Figure 8. In vivo anticancer activity of 2 in HepG2 xenograft model

(A) Tumour volume vs days of treatment with 2 (7.6, 3.8 mg/kg/2 days), cisplatin (2.0 mg/kg/2days), or vehicle. Tumour growth is tracked by the mean tumour volume (mm³) ± SD (n=6) and calculated as tumour growth rate [TGI%] values. (B) Body weight change. Relative body weight by considering the body weight at the start of the treatment as 100%, the percent weight loss or gain was calculated on subsequent days of treatment.