Novel isatin derivatives of podophyllotoxin: synthesis and cytotoxic evaluation against human leukaemia cancer cells as potent anti-MDR agents

Abstract

Multidrug resistance (MDR) is a major cause of chemotherapy failure in cancer therapy. In this study, a series of isatin derivatives of podophyllotoxin were synthesized and evaluated for their cytotoxic activity against human leukemia K562 cells and adriamycin-resistant K562/ADR cells using CCK-8 assay in vitro. All derivatives exhibited higher potency of antiproliferative effects against K562 and K562/ADR cell lines than the control drugs etoposide and adriamycin at nanomolar range, and markedly reduced the resistant factors. Among them, the cytotoxicities exhibited by compounds 8c and 8i were found to comparable or more effective than podophyllotoxin. In particular, 8c exhibited significant cytotoxicity against resistance K562/ADR cells with IC₅₀ value of 0.067 ± 0.010 μM. Furthermore, cell cycle analysis revealed that 8c could remarkably induce K562/ADR cell cycle arrest in the G2/M phase. Meanwhile, the effect of 8c on apoptosis inducing was also observed notably by flow cytometry and Hoechst 33342 staining. Moreover, western blotting analysis suggested that 8c had the potential to overcome the resistance of K562/ADR cells by down-regulating the expression levels of multi-drug resistance-related proteins, such as Pgp, MRP-1 and GST-π.

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Introduction

Multidrug resistance (MDR), a cross-resistance of tumor cells to seemingly unrelated chemotherapeutic drugs, continues to be a major cause of failure in cancer therapy and is one of the greatest challenges in the development of anticancer drugs.¹ It was reported that MDR could cause chemotherapy fails in over 90% of patients with metastatic cancer.² Although the mechanisms of MDR are multifaceted, overexpressions of P-glycoprotein (Pgp) and MDR related protein-1 (MRP-1), belonging to the ATP-binding cassette family, have been shown to be involved in MDR, which extrude anticancer drugs from the cytoplasm and thus reduce cellular toxicity.³ Another strategy by which resistant tumor cells could circumvent the toxicity action of chemotherapeutic agents is the increased detoxification by overexpressing drug metabolizing enzymes, such as glutathione S-transferases (GSTs). Particularly, GST-π, a cytosolic GST isozyme, is overexpressed in diverse MDR cancer cell lines.⁴ At present, one strategy for overcoming MDR is combined use of anticancer drugs with chemosensitizers, for instance, inhibitors of Pgp could be used to enhance the therapeutic effects of various antineoplastic agents. Down-regulating Pgp, MRP-1 or circumventing other MDR mechanisms is other approach to reversing MDR. In the past few years, with the aim to overcome MDR, intense efforts have focused on the development of novel antitumor agents with cytotoxicity against resistance tumor cells.⁵

Podophyllotoxin⁶ (1, Fig. 1), a well-known arylnaphthalene lignan lactone, isolated from natural plants including Podophyllum peltatum, has been used as a traditional Chinese medicine for hundreds of years, and is recently found to be a promising therapeutic agent for various cancer cells⁷ as well as for viral infections.⁸ Particularly, it was demonstrated that podophyllotoxin was a tubulin polymerization inhibitor.⁹ However, because of the unacceptable toxicity, poor water solubility and side effects of podophyllotoxin, its use has been hampered in cancer therapy.¹⁰ In spite of podophyllotoxin displaying several shortcomings, it still was a magnetic natural product for chemical modifications to develop new antineoplastic agents.¹¹ Up to now, many podophyllotoxin derivatives have been synthesized, including etoposide (2) and teniposide (3) (Fig. 1), used in the treatment of several cancers.¹² Meanwhile, several other derivatives (e.g. GL-331, NK-611 and TOP-53) had entered phase II clinical trials for the treatment of diverse neoplasm.¹³ Significantly, it was found that many derivatives of podophyllotoxin possessed the distinctive ability to inhibit topoisomerase II¹⁴ and other mechanisms,¹⁵ such as cell cycle arrest, apoptosis, antiangiogenesis and DNA cleavage. So far, significant progress has been obtained in the area of chemical modifications to develop new anticancer agents based on podophyllotoxin, however, the semisynthetic derivatives approved clinically are still frequently accompanied by drug resistance,¹⁶ and this has drawed great attention from many researchers in the development of novel podophyllotoxin derivatives with cytotoxicity against MDR cancer cell lines.¹⁷

Fig. 1 Structures of podophyllotoxin, etoposide and teniposide.

Prior studies indicated that podophyllotoxin derivatives exhibited significant anticancer activity against several MDR tumor cell lines,¹⁸ for example, vincristine-resistant KBvin and adriamycin-resistant MCF-7/ADR cells. In 2014, Chen and co-works reported that furan derivatives of podophyllotoxin could inhibit tumor cell proliferation by down-regulating the expression of Pgp in K562/ADR cells.¹⁹ Recently, the same group demonstrated that CIP-36, an indole derivative of podophyllotoxin, had the potential to overcome the multidrug resistance of K562/ADR cells by mediating topo IIα activity.²⁰ More recently, our group has also found that the podophyllotoxin–artesunate conjugate could efficiently down-regulate the levels of P-glycoprotein in Pgp overexpressing K562/ADR cells.²¹

On the other hand, isatin²² (4, Fig. 2), a well-known heterocyclic scaffold, found in plants of genus Isatis and in Couropita guianancis, shows various biological activities,²³ such as antitumor, anti-amyloid-β and anti-inflammatory. Recent literatures showed that isatin-based molecules exhibited a diverse array of pharmacological effects,²⁴ for instance, antitumor, antitubercular and anti-HIV. Furthermore, isatin is a core skeleton of many natural substances²⁵ and clinic drugs,²⁶ such as indirubin (5) and sunitinib (6) (Fig. 2). In the past few decades, great efforts have been made to promote the development of novel isatin-based antitumor agents.²⁷ For example, Gottesman and co-workers demonstrated that isatin-thiosemicarbazones showed significant toxicity in Pgp-expressing cancer cells.²⁸

Fig. 2 Structures of isatin, indirubin and sunitinib.

It was found from the literatures that molecular hybridization strategy²⁹ represented one of the most promising approaches for the development of new podophyllotoxin-³⁰ and isatin³¹-based antitumor agents. Herein, we reported the synthesis and antitumor activity of a series of podophyllotoxin derivatives bearing substituted isatins at C4 position against sensitive and resistance human leukaemia cancer cells. Meanwhile, we investigated the potential anti-MDR mechanisms of new podophyllotoxin derivatives on multidrug resistance K562/ADR cells.

Results and discussion

The routes to synthesizing the isatin derivatives of podophyllotoxin 8a–8j were outlined in Scheme 1. Initially, podophyllotoxin was reacted with chloroacetic acid to provide the intermediate 7.³² Then, compound 7 was reacted with different isatins under basic condition to generate the target products 8a–8j. Their structures were fully characterized by IR, ¹H NMR, ¹³C NMR and high-resolution mass spectra.

Scheme 1 Synthesis of the isatin derivatives of podophyllotoxin. Reagents and conditions: (a) chloroacetic acid, DCC, benzene, 61 °C; (b) appropriate isatin, K₂CO₃, DMF, 80 °C.

The synthesized compounds were tested for in vitro anticancer activity against two human leukemic cell lines (K562 and adriamycin-resistance K562/ADR) by CCK-8 assay. Podophyllotoxin, etoposide and adriamycin (ADR) were taken as positive drugs and the results are presented in Table 1. From IC₅₀ values, we found that all the compounds, except 8c and 8i, lost their cytotoxic effects against K562 and K562/ADR cells, and the anti-proliferative activities were lower than those of podophyllotoxin. However, all of the derivatives exhibited much better anticancer activities than etoposide and ADR against the two cancer cell lines at nanomolar range. The isatin derivatives possessed promising inhibitory effects with IC₅₀ values of 0.019 ± 0.007–0.084 ± 0.030 μM and 0.067 ± 0.010–0.245 ± 0.025 μM against K562 and K562/ADR cells, respectively. Among them, compounds, 8c and 8i, showed significant anticancer activity against sensitive K562 cells with IC₅₀ values of 0.019 ± 0.007 and 0.025 ± 0.009 μM, respectively. The IC₅₀ values of 8c and 8i against K562 were a little weaker than that of podophyllotoxin (IC₅₀ = 0.006 ± 0.005 μM), whereas the two derivatives, 8c and 8i, showed 21.73 and 16.52-fold higher anticancer activity than etoposide (IC₅₀ = 0.413 ± 0.067 μM), as well as 11.57 and 8.80-fold higher cytotoxicity than ADR (IC₅₀ = 0.220 ± 0.044 μM), respectively. Meanwhile, in K562/ADR cells, compound 8i showed comparable IC₅₀ value of 0.086 ± 0.007 μM with podophyllotoxin (IC₅₀ = 0.085 ± 0.056 μM), and 8c showed better anticancer activity (IC₅₀ = 0.067 ± 0.010 μM) than podophyllotoxin. Moreover, compounds 8c and 8i showed much higher inhibitory effects against K562/ADR cells than etoposide (IC₅₀ = 2.025 ± 0.476 μM) and ADR (IC₅₀ = 18.779 ± 3.069 μM). Namely, 8c exhibited 280.28 and 30.22-fold higher cytotoxic activity against K562/ADR cells than adriamycin and etoposide, and 8i showed 218.36 and 23.54-fold higher cytotoxic activity than adriamycin and etoposide, respectively. The data indicated that ADR exhibited very weak cytotoxicity against MDR K562/ADR cells and its resistance factor (RF) was 85.359. The RFs of 8c and 8i were 3.526 and 3.44, respectively, which were dramatically lower than those of podophyllotoxin (14.166), etoposide (4.903) and ADR (85.359).

Table 1 Effects of new isatin derivatives of podophyllotoxin on proliferation of K562 and K562/ADR cancer cell lines

Compound	IC50^a^,^b (μM)		RF^c
	K562	K562/ADR
a Data represent as mean ± SD of three independent experiments.b CCK-8 methods, drug exposure was for 72 h.c RF: resistance factor was calculated from the ratio of the growth inhibition constant (IC50) of K562/ADR cells to that of K562 cells.
8a	0.069 ± 0.038	0.241 ± 0.042	3.492
8b	0.031 ± 0.013	0.152 ± 0.081	4.903
8c	0.019 ± 0.007	0.067 ± 0.010	3.526
8d	0.067 ± 0.042	0.216 ± 0.128	3.223
8e	0.046 ± 0.009	0.151 ± 0.018	3.282
8f	0.039 ± 0.021	0.146 ± 0.051	3.743
8g	0.042 ± 0.009	0.152 ± 0.011	3.619
8h	0.028 ± 0.010	0.108 ± 0.016	3.857
8i	0.025 ± 0.009	0.086 ± 0.007	3.440
8j	0.084 ± 0.030	0.245 ± 0.025	2.916
1	0.006 ± 0.005	0.085 ± 0.056	14.166
2	0.413 ± 0.067	2.025 ± 0.476	4.903
ADR	0.220 ± 0.044	18.779 ± 3.069	85.359

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The preliminary structure–activity relationship trends were also performed from the results in Table 1. It was found that the positions 4 and 6 on the isatin scaffold appeared to accommodate various substituents, while positions 5 and 7 were not suitable for substitution. In position 4, bromo-substitution showed higher activity than chloro-substitution (8c vs. 8b). However, in position 6, chloro-substitution exhibited higher inhibition activity than bromo-substitution (8i vs. 8g). Finally, substitute groups in the positions 5 and 7 decreased anticancer activity, such as 8d and 8j. It was found that there were some differences among the derivatives according to the results, however, their IC₅₀ values were kept in the same range which indicated the isatin derivatives showed quite similar anticancer activities. The further study on the structure–activity relationship will be carried out in due course.

Recently, Chen et al. indicated that podophyllotoxin derivatives had the potential to inhibit K562/ADR cells proliferation by cell cycle arrest and apoptosis.^19,20,33 To determine the effect of compound 8c on the cell cycle, in this study, K562/ADR cells were incubated with 8c in comparison to podophyllotoxin for 48 h. The data obtained obviously demonstrated that 8c and podophyllotoxin resulted in an accumulation of cells in the G2/M phase as compared to the control cells (Fig. 3). Compound 8c showed 33.88 ± 5.89% and 34.92 ± 2.22% of cell accumulation in G2/M phase at concentrations of 0.05 μM and 0.1 μM, respectively, whereas podophyllotoxin induced 34.35 ± 5.61% of cell accumulation in G2/M phase at concentrations of 0.05 μM, compared with 4.56 ± 0.43% of cell accumulation in G2/M phase in untreated cultures. Nevertheless, when the concentration of 8c increased from 0.05 μM to 0.1 μM, remarkable increase of the percentage of G2/M cells was not observed. As shown in Fig. 3, the DNA cell cycle analysis revealed that 8c could induce MDR K562/ADR cells arrest in G2/M phase.

Fig. 3 Effects of compounds (1 and 8c) on the K562/ADR cells cycle. Cells were treated with vehicle, 0.05 μM 1, 0.05 μM 8c and 0.1 μM 8c for 48 h. (A) Effects of 1 and 8c on the cell cycle distribution of K562/ADR cells. (B) Quantitative analysis of cell cycle phase. **P < 0.01 compared with the control.

Subsequently, the ability of 8c to trigger apoptosis in K562/ADR cells was investigated. Cells were treated with vehicle, 0.05 μM podophyllotoxin, 0.05 μM 8c and 0.1 μM 8c for 48 h, respectively. The cells were harvested and stained with annexin V-APC and 7-AAD, and the percentages of apoptotic K562/ADR cells were determined by flow cytometry analysis. As seen in Fig. 4, the percentage of apoptotic cells was 3.46 ± 0.49% in control group. However, 29.12 ± 1.47%, 25.54 ± 1.60% and 34.68 ± 1.00% cells were detected to be undergoing apoptosis following treatment with 0.05 μM podophyllotoxin, 0.05 μM 8c and 0.1 μM 8c, respectively. After treating K562/ADR cells with different concentrations (0.05 μM and 0.1 μM) of 8c, K562/ADR cells displayed notable sensitivity to 8c in a dose-dependent manner. From the data, it was revealed that compound 8c could induce apoptosis in K562/ADR cells in a dose-dependent manner.

Fig. 4 Induction of apoptosis on K562/ADR cells by compounds (1 and 8c). The K562/ADR cells were treated with vehicle, 0.05 μM 1, 0.05 μM 8c and 0.1 μM 8c for 48 h. (A) Apoptosis was determined by Annexin V-APC/7-AAD staining. (B) Quantitative analysis of apoptotic cells. **P < 0.01 compared with the control.

To confirm the effect of 8c on induction of apoptosis, the morphology of K562/ADR cells was stained with Hoechst 33342. It was clear from Fig. 5 that control cells were normal and the nuclei were stained weak homogeneous blue. Nevertheless, the cells treated with 0.05 μM 8c and 0.1 μM 8c exhibited the characteristics of apoptosis with cell shrinkage, nuclear condensation and fragmentation, and relative fluorescence. A similar effect was observed when cells were incubated with 0.05 μM podophyllotoxin. The results distinctly proved that compound 8c was effective in inducing K562/ADR cells apoptosis.

Fig. 5 Hoechst 33342 staining in K562/ADR cells. (A) Control K562/ADR cells; (B) K562/ADR cells treated with 0.05 μM 1; (C) K562/ADR cells treated with 0.05 μM 8c; (D) K562/ADR cells treated with 0.1 μM 8c. Magnification 200 × .

Previous studies showed that K562/ADR cells could express high levels of MDR-related proteins, such as Pgp,³⁴ MRP-1 (ref. 35) and GST-π.³⁶ It was also revealed that podophyllotoxin is not a substrate for Pgp,³⁷ and Li et al. have disclosed that podophyllotoxin could exhibite significant activity against several P-glycoprotein mediated MDR tumor cell lines.³⁸ Also, Chen et al. showed that 4-substituted derivatives of podophyllotoxin may overcome MDR by reducing the expression of Pgp in K562/ADR cells.^19,33 Thus, in this work, western blotting analysis was used to determine the expressions of Pgp, MRP-1 and GST-π in sensitive K562 and MDR K562/ADR cells. The results showed K562 cells displayed low levels of Pgp, MRP-1 and GST-π, while K562/ADR cells expressed high levels of Pgp, MRP-1 and GST-π, as shown in Fig. 6. These results were consistent with aforementioned literatures. To further explore the mechanism of anti-multidrug resistance activity, the effects of 8c and podophyllotoxin on the expression of Pgp, MRP-1 and GST-π were also measured by western blotting. After the cells were treated with 0.1 μM 8c and 0.1 μM podophyllotoxin, the expressions of Pgp, MRP-1 and GST-π were significantly decreased, compared to the untreated K562/ADR cells. The results indicated that 8c could suppress the expression of Pgp in K562/ADR cells, which was coincident with literatures.^19,33,38 Moreover, we found that podophyllotoxin and its derivative (8c) had the potential to overcome the resistance of K562/ADR cells by down-regulating the expression levels of other multi-drug resistance-related proteins, such as MRP-1 and GST-π, which were not reported in the literatures before.

Fig. 6 Effect of compounds (1 and 8c) on the expression levels of Pgp, MRP-1 and GST-π in K562/ADR cells by western blotting using β-actin as a control. (A) Control K562 cells; (B) control K562/ADR cells; (C) K562/ADR cells treated with 0.1 μM 1; (D) K562/ADR cells treated with 0.1 μM 8c.

Experimental

General

Melting points were determined on an SGWX-4 binocular microscope melting-point apparatus. ¹H NMR and ¹³C NMR spectra were recorded using an Agilent-NMR-vnmrs 400 instrument at 400 MHz or 100 MHz, respectively, using TMS as internal standard. High-resolution mass spectra (HRMS) were taken on Agilent Accurate-Mass-Q-TOF-MS 6520 (Agilent Technologies, USA). IR spectra were recorded on Varian 1000 FT-IR spectrometer on KBr pellets and absorptions are reported in cm⁻¹. Compound 7 was synthesized as previously described.³²

Procedure for the synthesis of compounds 8a–8j

To a solution of podophyllotoxin (0.2 mmol, 1 eq.), potassium carbonate (1 eq.) in DMF (2 mL), an appropriate isatin (0.95 eq.) was added and the mixture was stirred at 80 °C under nitrogen atmosphere. After completion of reaction as evidenced by thin layer chromatography, the mixture was quenched with H₂O (20 mL) and extracted with CH₂Cl₂ (10 mL × 3). The organic phase was washed with saturated NH₄Cl (10 mL) and brine (10 mL), then the organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude was purified by silica gel column chromatography (EtOAc/petroleum ether = 1 : 4) to obtain the pure product.

4α-[2-(1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8a). Orange solid, yield 73%; mp: 115–117 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.63–7.69 (m, 2H, Ar–H), 7.21 (t, J = 7.0 Hz, 1H, Ar–H), 6.84 (d, J = 6.6 Hz, 1H, Ar–H), 6.59 (s, 1H, Ar–H), 6.52 (s, 1H, Ar–H), 6.33 (s, 2H, Ar–H), 5.99 (d, J = 9.6 Hz, 2H, O–CH₂–O), 5.94 (d, J = 8.1 Hz, 1H, CH–O–C=O), 4.70 (d, J = 17.2 Hz, 1H, O=C–CH₂–N), 4.59 (s, 1H, CH–Ar), 4.53 (d, J = 17.2 Hz, 1H, O=C–CH₂–N), 4.33–4.36 (m, 1H, CH–CH₂–O), 4.16 (t, J = 8.8 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.72 (s, 6H, 3′,5′-OCH₃), 2.84–2.99 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 181.80, 173.23, 167.48, 158.06, 152.67, 149.88, 148.46, 147.73, 146.34, 138.61, 134.47, 132.50, 126.88, 125.99, 124.62, 117.70, 109.84, 109.63, 107.91, 106.57, 101.76, 75.85, 71.03, 63.99, 60.78, 56.16, 45.43, 43.59, 41.49, 38.46, 35.14, 29.70; IR (KBr, cm⁻¹) 3470, 1741, 1615, 1505, 1485, 1420, 1376, 1340, 1240, 1190, 1126, 1037, 999, 930, 860, 757; HRMS-ESI (m/z): calcd for C₃₂H₃₁N₂O₁₁ [M + NH₄]⁺ 619.1922, found 619.1926.

4α-[2-(4-Chloro-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8b). Orange solid, yield 70%; mp: 116–118 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.55 (t, J = 8.0 Hz, 1H, Ar–H), 7.15 (d, J = 8.2 Hz, 1H, Ar–H), 6.75 (d, J = 7.9 Hz, 1H, Ar–H), 6.55 (s, 1H, Ar–H), 6.52 (s, 1H, Ar–H), 6.32 (s, 2H, Ar–H), 5.99 (dd, J = 6.1, 1.1 Hz, 2H, O–CH₂–O), 5.93 (d, J = 8.6 Hz, 1H, CH–O–C=O), 4.72 (d, J = 17.8 Hz, 1H, O=C–CH₂–N), 4.59 (d, J = 4.0 Hz, 1H, CH–Ar), 4.52 (d, J = 17.2 Hz, 1H, O=C–CH₂–N), 4.32–4.36 (m, 1H, CH–CH₂–O), 4.16 (t, J = 9.6 Hz, 1H, CH–CH₂–O), 3.80 (s, 3H, 4′-OCH₃), 3.72 (s, 6H, 3′,5′-OCH₃), 2.86–2.95 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 178.85, 173.23, 167.23, 157.19, 152.66, 150.94, 148.49, 147.72, 138.69, 137.10, 134.60, 134.45, 132.53, 126.78, 126.21, 114.88, 109.86, 107.88, 106.49, 101.81, 75.97, 70.99, 60.77, 56.13, 45.40, 43.57, 41.54, 38.43, 29.70; IR (KBr, cm⁻¹) 3455, 2926, 1743, 1604, 1506, 1485, 1456, 1420, 1366, 1329, 1240, 1191, 1126, 1036, 999, 930, 871, 781; HRMS-ESI (m/z): calcd for C₃₂H₂₇ClNO₁₁ [M + H]⁺ 636.1267, found 636.1271.

4α-[2-(4-Bromo-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8c). Orange solid, yield 63%; mp: 145–146 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.46 (t, J = 8.0 Hz, 1H, Ar–H), 7.35 (d, J = 8.2 Hz, 1H, Ar–H), 6.79 (d, J = 7.8 Hz, 1H, Ar–H), 6.54 (s, 1H, Ar–H), 6.53 (s, 1H, Ar–H), 6.33 (s, 2H, Ar–H), 5.99 (d, J = 5.7 Hz, 2H, O–CH₂–O), 5.93 (d, J = 8.4 Hz, 1H, CH–O–C=O), 4.71 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.59 (d, J = 3.8 Hz, 1H, CH–Ar), 4.51 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.32–4.36 (m, 1H, CH–CH₂–O), 4.16 (t, J = 9.7 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.73 (s, 6H, 3′,5′-OCH₃), 2.86–2.95 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 179.27, 173.09, 167.16, 157.10, 152.72, 151.39, 148.51, 147.75, 138.41, 137.34, 134.37, 132.59, 129.36, 126.81, 122.43, 116.63, 109.87, 108.32, 108.07, 106.44, 101.77, 76.01, 70.92, 60.73, 56.18, 45.44, 43.61, 41.46, 38.44, 29.67; IR (KBr, cm⁻¹) 3463, 2927, 2839, 1744, 1601, 1506, 1485, 1454, 1419, 1367, 1329, 1240, 1191, 1126, 1035, 999, 929, 866, 781; HRMS-ESI (m/z): calcd for C₃₂H₂₇BrNO₁₁ [M + H]⁺ 680.0762, found 680.0753.

4α-[2-(5-Methyl-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8d). Orange solid, yield 46%; mp: 155–157 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.48 (s, 1H, Ar–H), 7.45 (d, J = 8.1 Hz, 1H, Ar–H), 6.73 (d, J = 8.0 Hz, 1H, Ar–H), 6.55 (s, 1H, Ar–H), 6.52 (s, 1H, Ar–H), 6.33 (s, 2H, Ar–H), 5.98 (dd, J = 9.1, 1.1 Hz, 2H, O–CH₂–O), 5.92 (d, J = 8.6 Hz, 1H, CH–O–C=O), 4.68 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.58 (d, J = 4.0 Hz, 1H, CH–Ar), 4.49 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.32–4.35 (m, 1H, CH–CH₂–O), 4.11–4.18 (m, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.72 (s, 6H, 3′,5′-OCH₃), 2.83–2.93 (m, 2H, CH–CH₂–O, O=C–CH), 2.37 (s, 3H, Ar-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 182.13, 173.15, 167.53, 158.22, 152.71, 148.45, 147.75, 138.90, 137.30, 134.59, 134.41, 132.53, 126.94, 126.26, 117.78, 109.82, 109.36, 108.04, 106.55, 101.70, 75.78, 70.97, 60.73, 56.16, 45.44, 43.63, 41.49, 38.46, 29.67, 20.70; IR (KBr, cm⁻¹) 3456, 2935, 2838, 1778, 1736, 1621, 1598, 1492, 1460, 1420, 1376, 1337, 1240, 1199, 1122, 1033, 1000, 871, 798; HRMS-ESI (m/z): calcd for C₃₃H₃₃N₂O₁₁ [M + NH₄]⁺ 633.2079, found 633.2086.

4α-[2-(5-Chloro-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8e). Orange solid, yield 67%; mp: 158–159 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.65 (s, 1H, Ar–H), 7.62 (d, J = 8.4 Hz, 1H, Ar–H), 6.82 (d, J = 8.1 Hz, 1H, Ar–H), 6.58 (s, 1H, Ar–H), 6.53 (s, 1H, Ar–H), 6.34 (s, 2H, Ar–H), 6.00 (d, J = 8.6 Hz, 2H, O–CH₂–O), 5.94 (d, J = 8.3 Hz, 1H, CH–O–C=O), 4.69 (d, J = 17.6 Hz, 1H, O=C–CH₂–N), 4.59 (d, J = 3.3 Hz, 1H, CH–Ar), 4.51 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.33–4.36 (m, 1H, CH–CH₂–O), 4.15 (t, J = 9.4 Hz, 1H, CH–CH₂–O), 3.82 (s, 3H, 4′-OCH₃), 3.73 (s, 6H, 3′,5′-OCH₃), 2.85–2.94 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 180.87, 173.07, 167.19, 157.52, 152.73, 148.52, 148.14, 147.76, 137.90, 137.41, 134.37, 132.61, 130.51, 126.81, 125.84, 118.56, 110.96, 109.90, 108.15, 106.47, 101.78, 76.03, 70.90, 60.74, 56.21, 45.45, 43.61, 41.52, 38.44, 29.67; IR (KBr, cm⁻¹) 3447, 2936, 2838, 1741, 1607, 1507, 1484, 1446, 1419, 1377, 1336, 1240, 1202, 1120, 1031, 991, 868; HRMS-ESI (m/z): calcd for C₃₂H₂₇ClNO₁₁ [M + H]⁺ 636.1267, found 636.1265.

4α-[2-(5-Bromo-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8f). Orange solid, yield 71%; mp: 154–156 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 1H, Ar–H), 6.78 (d, J = 8.0 Hz, 1H, Ar–H), 6.59 (s, 1H, Ar–H), 6.52 (s, 1H, Ar–H), 6.33 (s, 2H, Ar–H), 5.98 (d, J = 8.1 Hz, 2H, O–CH₂–O), 5.93 (d, J = 8.0 Hz, 1H, CH–O–C=O), 4.69 (d, J = 17.8 Hz, 1H, O=C–CH₂–N), 4.57 (s, 1H, CH–Ar), 4.51 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.32–4.36 (m, 1H, CH–CH₂–O), 4.14 (t, J = 9.2 Hz, 1H, CH–CH₂–O), 3.80 (s, 3H, 4′-OCH₃), 3.71 (s, 6H, 3′,5′-OCH₃), 2.84–2.89 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 180.80, 173.15, 167.22, 157.37, 152.68, 148.64, 148.48, 147.72, 140.75, 137.32, 134.44, 132.58, 128.60, 126.86, 118.84, 117.40, 111.48, 109.86, 108.12, 106.52, 101.77, 75.98, 70.93, 60.72, 56.17, 45.40, 43.59, 41.47, 38.42, 29.66; IR (KBr, cm⁻¹) 3454, 2968, 2937, 2837, 1740, 1604, 1506, 1483, 1442, 1420, 1373, 1334, 1240, 1202, 1120, 1031, 1002, 954, 928, 869, 767; HRMS-ESI (m/z): calcd for C₃₂H₂₆BrNNaO₁₁ [M + Na]⁺ 702.0581, found 702.0589.

4α-[2-(6-Bromo-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8g). Orange solid, yield 48%; mp: 126–128 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.38 (d, J = 7.5 Hz, 1H, Ar–H), 6.83 (dd, J = 8.5, 3.3 Hz, 1H, Ar–H), 6.57 (s, 1H, Ar–H), 6.53 (s, 1H, Ar–H), 6.34 (s, 2H, Ar–H), 5.99 (d, J = 8.4 Hz, 2H, O–CH₂–O), 5.94 (d, J = 8.4 Hz, 1H, CH–O–C=O), 4.70 (d, J = 17.8 Hz, 1H, O=C–CH₂–N), 4.59 (d, J = 3.8 Hz, 1H, CH–Ar), 4.51 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.33–4.36 (m, 1H, CH–CH₂–O), 4.13–4.18 (m, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.72 (s, 6H, 3′,5′-OCH₃), 2.86–2.94 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.12, 167.31, 160.86, 158.40, 157.85, 152.71, 148.50, 147.74, 145.98, 137.37, 134.40, 132.60, 126.85, 125.07, 124.83, 113.10, 112.86, 110.95, 110.88, 109.88, 108.13, 106.48, 101.76, 75.97, 70.93, 60.73, 56.19, 45.44, 43.60, 41.53, 38.44, 29.67; IR (KBr, cm⁻¹) 3447, 2934, 1743, 1624, 1588, 1487, 1465, 1419, 1368, 1331, 1240, 1190, 1126, 1037, 999, 930, 868, 786; HRMS-ESI (m/z): calcd for C₃₂H₂₆BrNNaO₁₁ [M + Na]⁺ 702.0581, found 702.0586.

4α-[2-(5-Fluoro-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8h). Orange solid, yield 50%; mp: 245–246 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.52 (d, J = 8.0 Hz, 1H, Ar–H), 7.36 (d, J = 7.9 Hz, 1H, Ar–H), 7.03 (s, 1H, Ar–H), 6.64 (s, 1H, Ar–H), 6.53 (s, 1H, Ar–H), 6.34 (s, 2H, Ar–H), 5.99 (d, J = 6.9 Hz, 2H, O–CH₂–O), 5.96 (s, 1H, CH–O–C=O), 4.66 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.59 (d, J = 3.1 Hz, 1H, CH–Ar), 4.52 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.35–4.39 (m, 1H, CH–CH₂–O), 4.14–4.19 (m, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.73 (s, 6H, 3′,5′-OCH₃), 2.87–2.95 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 180.65, 173.15, 167.18, 157.86, 152.70, 150.64, 148.51, 147.78, 137.34, 134.43, 133.79, 132.61, 127.77, 126.87, 126.79, 116.41, 113.49, 109.89, 108.11, 106.56, 101.75, 76.05, 70.93, 60.73, 56.19, 45.45, 43.62, 41.57, 38.42, 29.67; IR (KBr, cm⁻¹) 3442, 2936, 1773, 1736, 1606, 1507, 1487, 1458, 1420, 1376, 1331, 1241, 1125, 1032, 999, 885, 768; HRMS-ESI (m/z): calcd for C₃₂H₂₇FNO₁₁ [M + H]⁺ 620.1563, found 620.1563.

4α-[2-(6-Chloro-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8i). Orange solid, yield 39%; mp: 256–257 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 8.0 Hz, 1H, Ar–H), 7.19 (d, J = 8.0 Hz, 1H, Ar–H), 6.86 (s, 1H, Ar–H), 6.63 (s, 1H, Ar–H), 6.54 (s, 1H, Ar–H), 6.35 (s, 2H, Ar–H), 5.99 (d, J = 7.4 Hz, 2H, O–CH₂–O), 5.96 (s, 1H, CH–O–C=O), 4.66 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.60 (d, J = 3.2 Hz, 1H, CH–Ar), 4.52 (d, J = 17.7 Hz, 1H, O=C–CH₂–N), 4.35–4.39 (m, 1H, CH–CH₂–O), 4.09–4.19 (m, 1H, CH–CH₂–O), 3.82 (s, 3H, 4′-OCH₃), 3.74 (s, 6H, 3′,5′-OCH₃), 2.83–2.95 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 180.34, 173.11, 167.16, 157.97, 152.73, 150.86, 148.52, 147.79, 145.08, 137.38, 134.40, 132.62, 126.92, 126.85, 124.78, 116.05, 110.60, 109.90, 108.13, 106.52, 101.75, 76.08, 70.90, 60.74, 56.20, 45.47, 43.63, 41.56, 38.41, 29.67; IR (KBr, cm⁻¹) 3443, 2935, 2850, 1777, 1738, 1609, 1506, 1487, 1457, 1421, 1379, 1331, 1241, 1125, 1032, 999, 892, 796; HRMS-ESI (m/z): calcd for C₃₂H₂₇ClNO₁₁ [M + H]⁺ 636.1267, found 636.1271.

4α-[2-(7-Fluoro-1H-indole-2,3-dione-1-yl)-acetate]-4-desoxy-podophyllotoxin (8i). Orange solid, yield 63%; mp: 125–127 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.52 (d, J = 7.4 Hz, 1H, Ar–H), 7.39 (dd, J = 11.1, 8.6 Hz, 1H, Ar–H), 7.16–7.19 (m, 1H, Ar–H), 6.73 (s, 1H, Ar–H), 6.55 (s, 1H, Ar–H), 6.37 (s, 2H, Ar–H), 5.99–6.01 (m, 3H, O–CH₂–O, CH–O–C=O), 4.87 (d, J = 18.0 Hz, 1H, O=C–CH₂–N), 4.69 (d, J = 18.0 Hz, 1H, O=C–CH₂–N), 4.60 (s, 1H, CH–Ar), 4.36–4.39 (m, 1H, CH–CH₂–O), 4.16 (t, J = 9.5 Hz, 1H, CH–CH₂–O), 3.82 (s, 3H, 4′-OCH₃), 3.74 (s, 6H, 3′,5′-OCH₃), 2.88–2.96 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 180.99, 173.21, 167.95, 157.44, 152.71, 149.32, 148.45, 147.76, 146.89, 137.37, 134.48, 132.54, 127.08, 126.30, 126.11, 125.28, 125.22, 121.79, 120.11, 109.84, 108.18, 106.69, 101.71, 75.67, 70.92, 60.74, 56.20, 45.45, 43.67, 43.22, 38.49, 29.67; IR (KBr, cm⁻¹) 3448, 2933, 2840, 1749, 1646, 1634, 1589, 1558, 1540, 1506, 1488, 1464, 1419, 1373, 1338, 1241, 1190, 1126, 1035, 999, 849, 768; HRMS-ESI (m/z): calcd for C₃₂H₃₀FN₂O₁₁ [M + NH₄]⁺ 637.1828, found 637.1828.

Cytotoxic activity

Cells were seeded (3000 cells per well) in a 96-well plate and grown for 24 h at 37 °C in 5% CO₂/95% air. Subsequently, the cells were exposed to vehicle, with or without various concentrations of test compounds for 72 h before addition of 10 μL CCK-8 to the culture medium in each well. After 3 h of incubation at 37 °C, the absorbance at 450 nm of each well was determined with a fluorimeter. The IC₅₀ (concentration that inhibits 50% of cell growth) values were determined by linear and polynomial regression.

Cell cycle analysis

K562/ADR cells were cultured overnight and incubated with compound 8c (0.05 or 0.1 μM), podophyllotoxin (0.05 μM) or vehicle for 48 h. After washing once with PBS, the cells were harvested, fixed with 70% ethanol for 2 h, incubated with 100 μL RNase-A at 37 °C for 30 min, and then stained with 400 μL PI at 4 °C for 30 min. The cell cycles were determined by a flow cytometer (Becton-Dickinson FACS Calibur).

Apoptosis analysis

K562/ADR cells were cultured overnight and treated with vehicle or various concentrations of 8c (0.05 or 0.1 μM), podophyllotoxin (0.05 μM) for 48 h. Cells were harvested with 0.25% trypsin, washed twice with ice-cold PBS, resuspended with 500 μL binding buffer and, then stained with Annexin V-APC and 7-AAD for 15 min at room temperature in the dark. Stained cells were determined by a FACS Calibur flow cytometer (Becton Dickinson).

Hoechst 33342 staining

K562/ADR cells were treated with vehicle or various concentrations of 8c (0.05 or 0.1 μM), podophyllotoxin (0.05 μM) for 48 h, fixed, washed thrice with PBS, stained with Hoechst 33342, followed by examining under a fluorescent microscope.

Western blot analysis

K562 cells were treated with vehicle, and K562/ADR cells were treated with vehicle, podophyllotoxin (0.1 μM) and compound 8c (0.1 μM). After 48 h of treatment, cells were harvested and lysed. After protein quantification using the BCA assay, equal amounts of the cell lysates were subjected to electrophoresis in SDS-polyacrylamide gels and transferred to nitrocellulose (NC) membranes. After the membranes were blocked in 5% fat-free milk, the target proteins were probed with the desired primary antibody: anti-Pgp, anti-MRP-1, anti-GST-π and anti-β-actin. Subsequently, the membranes were incubated with appropriate secondary antibody at room temperature for 2 h. The relative levels of target proteins to the control were performed using a G:BOX Chemi XR5 scanner (Syngene, Cambridge, United Kingdom).

Conclusions

In summary, a series of podophyllotoxin derivatives containing isatin were synthesized and tested for their anticancer potential against K562 and K562/ADR cancer cell lines. All the compounds exhibited remarkable anticancer activity at nanomolar concentration. Among them, compound 8c showed significant anticancer activity in K562 and K562/ADR cells. Furthermore, 8c could block cell cycle progression at G2/M phase and induce apoptosis in K562/ADR cells. In addition, 8c could reverse the MDR of K562/ADR cells via mechanisms of down-regulating the expressions of Pgp, MRP-1 and GST-π. The potential of compound 8c in reversing MDR is worth further study.

Acknowledgements

This work was financially supported by the Department of Science and Technology of Guizhou Province (No. [2014]7565, [2014]7557, [2014]4002) and the Education Department of Guizhou Province (No. QJHRCTDZ-2012-03).

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