Synthesis and biological activity, and molecular modelling studies of potent cytotoxic podophyllotoxin-naphthoquinone compounds

Abstract

A new approach for the synthesis of podophyllotoxin-naphthoquinone compounds using microwave-assisted three-component reactions is reported in this study. Novel podophyllotoxin-naphthoquinone derivatives with modification on ring E were synthesized. All the synthetic compounds were assessed in terms of their cytotoxicity profile against four cancer cell lines (KB, HepG2, A549, and MCF7), and noncancerous Hek-293 cell lines. Notably, treatment of SK-LU-1 cells with compounds 5a and 5b resulted in G2/M phase arrest of the cell cycle, caspase-3/7 activation, and apoptosis. Additionally, molecular docking studies were performed and showed important interaction of two compounds against residues in the colchicine-binding-site of tubulin as well. Taken together, compounds 5a and 5b were identified as potent anticancer agents.

Fig. 1. Structure of podophyllotoxin derivatives, aza-anthraquinones derivatives and podophyllotoxin-naphthoquinone compounds (5a, 5d).

Fig. 2. Design strategy for the potent cytotoxic podophyllotoxin-naphthoquinone compounds.

Scheme 1. Plausible mechanism for the synthesis of podophyllotoxin-naphthoquinone compounds 5.

Fig. 3. Effect of compounds 5a, 5b on cell cycle distribution in SK-LU-1 cells. Cells were treated with tested samples, DMSO 0.05% (negative reference) and positive controls including Ellipticine (1.3 μM), Vincristine (0.4 μM and 4 μM) for 24 h. Cell cycle distribution was evaluated by flow cytometry Novocyte (ACEA Biosciences, San Diego, CA, USA).

Fig. 4. Photomicrograph of apoptotic changes in LU-1 cells treated with compounds 5a, 5b at 0.08 μM, 0.16 μM, and 0.32 μM; negative control (NG); Ellipticine (Ellip) at 1.3 μM; Vincristine (Vin) at 0.4 μM and 4.0 μM. Cells were stained with Hoechst and then observed by fluorescence microscopy. Some cells with fragmented nuclei are enclosed within circles.

Fig. 5. Effect of compounds 5a, 5b on caspase-3/7 activity. Cells were treated with different concentrations of compound 5a, 5b, Ellipticine (1.3 μM), and Vincristine (4 μM), followed by harvesting and mixing with Apo-ONE® Caspase-3/7 reagent. A Tecan GENios Pro microplate reader was used to determine relative fluorescence units (RFU), directly proportional to the activation of caspase-3/7. Caspase activity = RFU value of tested sample – RFU value of the blank.

Fig. 6. Analyses of apoptosis induction in SK-LU-1 cells. Detection of apoptotic cells after Annexin-V/PI staining by flow cytometry. Cells were treated with different concentrations of compound 5a, 5b, control, and references, followed by harvesting and staining with Annexin-V/PI for 24 hours. The diverse cell stages were given as live (Q3), early apoptotic (Q4), late apoptotic (Q2), and necrotic cells (Q1).

Fig. 7. The three-zone structure of colchicine-binding site (CBS) and docking interactions of colchicine, 5a and 5b with tubulin.

Fig. 8. Metal complex and geometries of PAC-1, 5a and 5b with Zn ion bound to the procaspase target (PDB ID: 4FXO).

Fig. 9. Predicted physicochemical properties of 5a (A) and 5b (B) compared with drug optimal ranges. fChar: formal charge; log D, log of octanol partition coefficient at physiological pH 7.4; log P, log of octanol partition coefficient; log S, log of aqueous solubility (mol L⁻¹); MaxRing, number of atoms in the biggest ring; MW, molecular weight; nHA, number of hydrogen bond acceptors; nHD, number of hydrogen bond donors; nHet, number of heteroatoms; nRig, number of rigid bonds; nRing, number of rings; nRot, number of rotatable bonds; TPSA, topological polar surface area (Ų).