Design, Synthesis, and Biological Evaluation of Novel Biotinylated Podophyllotoxin Derivatives as Potential Antitumor Agents

Abstract

Podophyllotoxin has long been used as an active substance for cytotoxic activity. Fourteen novel biotinylated podophyllotoxin derivatives were designed, synthesized, and evaluated for cytotoxic activity for this study. The synthesized compounds were evaluated for cytotoxic activity in the following human cancer cell lines, SW480, MCF-7, A-549, SMMC-7721, and HL-60 by MTT assay. Most of them exhibited potent cytotoxic effects and compound 15 showed the highest cytotoxic activity among the five cancer cell lines tested, having its IC₅₀ values in the range of 0.13 to 0.84 μM. Apoptosis analysis revealed that compound 15 caused obvious induction of cell apoptosis. Compound 15 significantly down-regulated the expression level of the marker proteins (caspase-3 and PARP) in H1299 and H1975 cells, activated the transcription of IRE1α, increased the expression of GRP78 and XBP-1s, and finally induced apoptosis of H1299 cells. In vivo studies showed that 15 at a dose of 20 mg/kg suppressed tumor growth of S180 cell xenografts in icr mice significantly. Further molecular docking studies suggested that compound 15 could bind well with the ATPase domain of Topoisomerase-II. These data suggest that compound 15 is a promising agent for cancer therapy deserving further research.

Keywords: anticancer activity; apoptosis; biotin; podophyllotoxin derivatives; synthesis.

Figure 1

Structure of Podophyllotoxin (1), Etoposide (2), Biotin (3), and 6-biotinylaminocaproic acid (4).

Scheme 1

The synthesis of podophyllotoxin derivatives (5–12). Reagents and reaction condition: (a) MeSO₃H, NaI, CH₃CN/CH₂Cl₂; then, H₂O-Acetone, BaCO₃, rt. 90–92%; (b) NaN₃-TFA, CHCl₃, 67–70%; (c) PPh₃, THF, then H₂O, 72–75%; (d) copper (II) acetate, propargyl alcohol, sodium ascorbate, t-BuOH-H₂O, THF, rt. 85–89%.

Scheme 2

The synthesis of biotinylated podophyllotoxin derivatives (13–26). Reagents and reaction condition: (a) DIC, DMAP, DMF, N₂, rt. 39–65%.

Figure 2

Flow cytometry analysis of lung cancer cell lines H1299 and H1975 after treatment with compound 15. Cells were treated with compound 15 (0.5, 1, and 2 μM) and PPT (1 μM) for 24 h, then stained with Annexin V/7AAD and analyzed by flow cytometry. The ratio of apoptotic cells in each group was expressed as percentage. (A) H1299 cell line; (B) H1975 cell line. The data are presented as the mean ± SD (n = 3). *p < 0.01.

Figure 3

Compound 15 regulates the expression levels of apoptosis-related proteins: H1299 and H1975 cell lines were treated with compound 15 (0.5, 1, and 2 μM) and PPT (1 μM) for 24 h and the expression level of caspase-3, PARP, cleaved-caspase-3, and cleaved-PAPR was detected by western blot (WB). Actin was tested as a loading control.

Figure 4

Compound 15 induces apoptosis through activating the ER stress pathway: (A) H1299 cell line was treated with compound 15 (0.5, 1, and 2 μM) for 24 h, GRP78, CHOP, XBP-1, XBP-1s, ATF4, IRE-1α, and ATF6 were measured by real-time RT-PCR. The data are presented as the mean ± SD (n = 3). *p < 0.01, **p < 0.001, ***p < 0.0001 and ****p < 0.00001. (B) H1299 cell line was treated with compound 15 (0.5, 1, and 2 μM) and PPT (1 μM) for 24 h, and WB was performed to detect the expression levels of protein in the ER stress pathway. Actin was tested as a loading control.

Figure 5

Compound 15 significantly inhibits the growth of S180 tumor xenografts in icr mice: (A) The chemical structure of compound 15. (B) Tumors collected at the end of the treatment (day 7). (C) Mouse's body weights were weighed for those treated with compound 15 (5, 15, 20 mg/kg) compared to TAX control (10 mg/kg) TAX: Taxol. (D) Tumor weights were presented for the groups treated with compound 15 (5, 15, 20 mg/kg).

Figure 6

Proposed binding models of compounds 15 and PPT to the ATPase domain of Topoisomerase-II. Based on the X-ray co-crystal structure of Top-II in complex with Etoposide. (A) Binding mode of compound 15 with Topoisomerase-II. (B) Binding mode of PPT with Topoisomerase-II.