Potential anti-MDR agents based on the podophyllotoxin scaffold: synthesis and antiproliferative activity evaluation against chronic myeloid leukemia cells by activating MAPK signaling pathways

Abstract

One of the main causes for chemotherapeutic failure is the development of multidrug resistance (MDR), which is also one of the major obstacles to successful cancer chemotherapy. To overcome multidrug resistance, in this study, we synthesized several acrylic esters of podophyllotoxin and evaluated their antiproliferative activities in sensitive and resistant human chronic myeloid leukemia cells, K562 and K562/ADR, by CCK-8 method in vitro. All of the synthesized compounds demonstrated potent anticancer effects with IC₅₀ values in the nM range. In particular, compound 9k exhibited the most potent activity towards drug-resistant K562/ADR cells with an IC₅₀ value of 0.055 ± 0.014 μM, which was more effective than podophyllotoxin, doxorubicin and etoposide. Furthermore, flow cytometry results revealed that 9k could obviously induce cell cycle arrest in the G2/M phase in K562/ADR cells. Meanwhile, it was found that 9k could induce apoptosis in K562/ADR cells by up-regulating the expression of Bax, caspase-3 and p53, and inhibiting the expression of Bcl-2 and survivin. Western blot analysis revealed that 9k significantly inhibited Pgp protein expression in K562/ADR cells. Additionally, further studies demonstrated that 9k could dose-dependently stimulate the MAPK signaling pathways in K562/ADR cells by up-regulating the expression levels of p-ERK1/2, p-JNK and p-p38. The results indicate that 9k has the potential to be a novel MDR reversal agent in human chronic myeloid leukemia therapy.

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Introduction

The development of multidrug resistance (MDR), a phenomenon of cross-resistance to a variety of structurally diverse and mechanistically unrelated chemotherapeutic agents, is one of the prime obstacles to successful cancer chemotherapy.¹ The mechanisms underlying MDR in cancer cells are complex, including drug efflux, cell cycle effects, apoptosis evasion and drug metabolism.² Classic MDR is the consequence of overexpression of P-glycoprotein (Pgp) belonging to the ATP-binding cassette (ABC) family, which is encoded by the mdr1 gene.³ The function of Pgp is to extrude antineoplastic drugs (e.g. taxol, doxorubicin, etoposide or cisplatin) and other types of xenobiotics from the cytoplasm by the ATP-dependent efflux, thus reducing lower intracellular drug accumulation.⁴ Currently, the increased expression of P-gp has been closely related with the development of MDR in many human cancers such as leukemias and multiple myelomas.⁵

One strategy for overcoming MDR is the combined use of anticancer drugs with chemosensitizers,⁶ such as inhibitors of Pgp. Several agents,⁷ including verapamil, cyclosporine A, zosuquidar and tariquidar, have been demonstrated to reverse P-gp mediated MDR. However, because of the side effects and unacceptable toxicity of these compounds, their clinical use has been hampered in MDR cancer therapy.⁸ Down-regulating Pgp is another approach to overcoming MDR, and this approach has drawed great attention in the development of novel anti-MDR agents in the past few years.⁹ For example, Ren and coworkers reported that chabamide, a dimeric alkaloid isolated from Piper chaba Hunter, could induce apoptosis and cell cycle arrest and inhibit the expression of Pgp in K562/ADR cells.¹⁰ Meanwhile, dasatinib was studied by Liu et al. to reverse the multidrug resistance of MCF-7 cells to doxorubicin by downregulating Pgp expression.¹¹

Natural products are an important source of various bioactive lead compounds for the development of antineioplastic drugs.¹² Podophyllotoxin (1, Fig. 1),¹³ a naturally occurring cyclolignan found in the plants of Podophyllum peltatum and Podohyllum hexandrum, exhibits strong cytotoxic activity. Podophyllotoxin is well known to display anticancer properties by inhibiting microtubule assembly.¹⁴ However, podophyllotoxin failed to be used in cancer therapy because of undesirable toxic side-effects.¹⁵ In order to overcome the limitations, numerous attempts have been devoted to reduce its toxicity while retaining antitumor activity, which has resulted in the development of clinical anticancer agents, such as etoposide (2), teniposide (3) and etopophos (4) (Fig. 1), the podophyllotoxin analogues used extensively in the treatment of a variety of malignancies such as lymphoma, testicular cancer, small cell lung cancer and Hodgkin's disease.¹⁶ Meanwhile, some other derivatives, such as TOP-53, GL-331 and NK-611, are in late stage clinical development. Interestingly, the cytotoxic mechanism of these semisynthetic derivatives was distinct from that of podophyllotoxin, which was explained by the inhibition of DNA-topoisomerase II.¹⁷ Unfortunately, the new anticancer drugs were not devoid of drug-resistance, which prompted a great deal of interest in the area of chemical modifications for the development of new anti-MDR candidates based on podophyllotoxin scaffold to kill resistant tumor cells.^18–20 For example, L1EPO (5, Fig. 1), an indole derivative of podophyllotoxin, was reported to induce apoptosis and reverse the expression of Pgp in K562/ADR cells.²¹ Lately, it was reported that the 5-nitrofuran derivative of podophyllotoxin (6, Fig. 1) showed potent antitumor activity against K562/ADR cells by down-regulating Pgp expression.²² More recently, our group have reported that the conjugate of artesunate and podophyllotoxin inhibited the proliferation of K562/ADR cells though inducement of apoptosis, disruption of the microtubule network and down-regulation of Pgp.²³ At the same time, besides the Pgp protein, we also found that the 4-bromisatin derivative of podophyllotoxin (7, Fig. 1) had the potential to overcome the resistance of K562/ADR cells by down-regulating the expression levels of other multidrug resistance-related proteins such as MRP-1 and GST-π.²⁴

Fig. 1 Chemical structures of podophyllotoxin derivatives and cinnamic acid.

On the other hand, cinnamic acid (8, Fig. 1), a well-known derivative of acrylic acid, found in lots of natural products,^25,26 shows various biological activities such as anticancer,²⁷ antibacterial²⁸ and antiviral.²⁹ During the last decade, results from several studies revealed that introduction the cinnamic acid scaffold to natural product or other heterocyclic pharmacophore represented one attractive approach to developing anticancer candidates.^30,31 For example, acrylic ester of podophyllotoxins displayed potent antiproliferative effects against several sensitive cancer cell lines such as P388, SGC-7901 and A549.^32,33 Recently, 4β-cinnamido linked podophyllotoxins have been reported by Kamal et al. and some of these compounds displayed potent antiproliferative activity against A549 cells.³⁴

To our best knowledge, no papers have been focused on the anticancer activities of acrylic esters of podophyllotoxin against drug-resistant tumor cells. In continuation of our work to find more anticancer agents with anti-MDR property,^23,24 in this study, we described the synthesis and cytotoxicity of the acrylic ester analogues of podophyllotoxin against sensitive and resistance human leukaemia cells, and the inducement of apoptosis with cell cycle arrest properties in K562/ADR cells was also evaluated. To understand the mechanism involved, MAPK signaling pathways in K562/ADR cells were further investigated.

Results and discussion

The strategies for the synthesis of the target compounds 9a–9k were outlined in Scheme 1. Podophyllotoxin (1) coupled with aromatic and heterocyclic acrylic acid catalyzed by EDCI and DMAP generated corresponding products 9a–9j in good yields. Then, the reaction of podophyllotoxin with acryloyl chloride under basic condition (triethylamine in CH₂Cl₂) afforded 9k. The acrylic ester derivatives of podophyllotoxin were fully characterized by IR, ¹H NMR, ¹³C NMR and high-resolution mass spectra (HR-MS).

Scheme 1 Reagents and conditions: (a) appropriate cinnamic acid or furylacrylic acid (0.95 eq.), EDCI (2 eq.), DMAP (1.2 eq.), DMF, 30 °C, for 9a–9j; (b) acryloyl chloride (2 eq.), Et₃N (3 eq.), CH₂Cl₂, r.t., for 9k.

The preliminary antiproliferative activities of the synthesized compounds were investigated by CCK-8 assay against human leukemic K562 cells and adriamycin-resistance human leukemic K562/ADR cells in vitro using podophyllotoxin, adriamycin and etoposide as positive controls, and the results are shown in Table 1. All of the tested compounds exhibited potent anticancer effects against the two cell lines with nanomolar IC₅₀ values. Interestingly, most of the targeted compounds lost their antiproliferative activities and the anticancer activities were lower than those of podophyllotoxin against K562 and K562/ADR. However, most of the acrylic esters derivatives showed better activities than two anticancer drugs commonly used in clinic, adriamycin and etoposide. The synthesized compounds exhibited potent antiproliferative effects on K562 and K562/ADR cells with IC₅₀ values ranging from 0.011 ± 0.005 to 0.343 ± 0.071 μM and from 0.055 ± 0.014 to 0.509 ± 0.086 μM, respectively. Notably, the compound 9k showed comparable (IC₅₀ = 0.011 ± 0.005 μM in K562) or stronger (IC₅₀ = 0.055 ± 0.014 μM in K562/ADR) anticancer activity than podophyllotoxin (IC₅₀ = 0.006 ± 0.005 μM in K562 and 0.085 ± 0.056 μM in K562/ADR). The IC₅₀ values of 9k against K562 are 20 and 37.545-fold higher than those of adriamycin (IC₅₀ = 0.220 ± 0.044 μM) and etoposide (IC₅₀ = 0.413 ± 0.067 μM), respectively. Meanwhile, 9k showed 1.545, 341.436 and 36.818-fold higher cytotoxic activity against K562/ADR cells than podophyllotoxin (IC₅₀ = 0.085 ± 0.056 μM), adriamycin (IC₅₀ = 18.779 ± 3.069 μM) and etoposide (IC₅₀ = 2.025 ± 0.476 μM), respectively. Meanwhile, the data showed that adriamycin exhibited much lower antiproliferative effect against adriamycin-resistance K562/ADR cells with resistance factor of 85.359. Especially, the resistance factor of 9k was 5, which was obviously lower than that of podophyllotoxin (14.166) and adriamycin (85.359), comparable to etoposide (4.903). Those results revealed that 9k may overcome the multidrug resistance of the K562/ADR cells.

Table 1 Cytotoxicity against K562 and K562/ADR cells in vitro

Compound	IC50^a^,^b (μM)		RF^c
	K562	K562/ADR
a CCK-8 methods, drug exposure was for 72 h.b Data represent as mean ± SD of three independent experiments.c RF: resistance factor was calculated from the ratio of the growth inhibition constant (IC50) of K562/ADR cells to that of K562 cells.d Literature values.^23,24
9a	0.078 ± 0.006	0.189 ± 0.041	2.423
9b	0.285 ± 0.098	0.481 ± 0.237	1.687
9c	0.343 ± 0.071	0.509 ± 0.086	1.483
9d	0.186 ± 0.031	0.255 ± 0.073	1.370
9e	0.258 ± 0.061	0.362 ± 0.070	1.403
9f	0.341 ± 0.021	0.397 ± 0.078	1.164
9g	0.097 ± 0.018	0.139 ± 0.042	1.432
9h	0.162 ± 0.060	0.299 ± 0.045	1.845
9i	0.081 ± 0.035	0.160 ± 0.057	1.975
9j	0.057 ± 0.021	0.141 ± 0.040	2.473
9k	0.011 ± 0.005	0.055 ± 0.014	5
1^d	0.006 ± 0.005	0.085 ± 0.056	14.166
2^d	0.413 ± 0.067	2.025 ± 0.476	4.903
Adriamycin^d	0.220 ± 0.044	18.779 ± 3.069	85.359

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From the data, we observed some interesting trends. Among the targeted molecules, 9k, without any substituent in the acrylic skeleton, showed the best anticancer effects against K562 and K562/ADR cells. However, furyl- and phenyl groups dramatically decreased the inhibitory activities with a concomitant decrease the resistance factor. Meanwhile, the derivatives with furyl group exhibited higher inhibition activity than the compound with phenyl group. Among the compounds with phenyl group, substitute group in the benzene ring slightly decreased anti-K562 proliferation activity, except compound 9i with electron-withdrawing nitro group at 4-position of the benzene ring. On the other hand, halogen substituent slightly decreased anti-K562/ADR proliferation activity, such as 9e, 9f and 9i. Meanwhile, introduction of one electron-donating group (e.g. 4-OCH₃, 4-CH₃ or 3-OCH₃) slightly decreased anti-K562/ADR proliferation activity, however, introduction of two electron-donating groups (e.g. 3,4-(OCH₃)₂) dramatically increased anti-K562/ADR proliferation activity. The deep study on the structure–activity relationship will be carried out in future.

To evaluate the effect of compound 9k on the cell cycle, we investigated cell cycle accumulation by FACS analysis in adriamycin-resistant K562/ADR cancer cells treated with 9k for 48 h using podophyllotoxin as a positive control. As shown in Fig. 2, in K562/ADR cells treated with 0.05 μM 9k, 0.1 μM 9k and 0.05 μM podophyllotoxin, 31.66 ± 3.68%, 33.73 ± 3.23% and 33.28 ± 3.36% of the cells were in G2/M phase, respectively, compared with 6.63 ± 2.48% in untreated cultures. These results indicated that 9k could induce K562/ADR cells arrest in G2/M phase. However, remarkable increase in the accumulation of cells in the G2/M phase was not observed when the concentration of 9k increased from 0.05 μM to 0.1 μM.

Fig. 2 Effects of compounds (1 and 9k) on the K562/ADR cells cycle. (A) Effects of 1 and 9k on the cell cycle distribution of K562/ADR cells. (B) Quantitative analysis of cell cycle phase. *P < 0.05, **P < 0.01 compared with the control.

To validate whether compound 9k could cause the growth inhibition of K562/ADR cells by apoptosis, the percentages of apoptotic k562/ADR cells were determined by flow cytometry. K562/ADR cells were treated with vehicle, 0.05 μM 9k, 0.1 μM 9k and 0.05 μM podophyllotoxin for 48 h, respectively. As shown in Fig. 3, the percentage of apoptotic cell was 3.19 ± 0.35% in the vehicle control group. Meanwhile, 28.21 ± 0.47%, 36.31 ± 0.71% and 29.03 ± 1.24% cell apoptosis on cancer cells were observed following treatment with 0.05 μM 9k, 0.1 μM 9k and 0.05 μM podophyllotoxin, respectively. These data suggested that 9k could induce apoptosis in K562/ADR cells.

Fig. 3 Effects of compounds (1 and 9k) on the apoptosis of K562/ADR cells. (A) Apoptosis was determined by Annexin V-APC/7-AAD staining by flow cytometry. (B) Quantitative analysis of apoptotic cells. **P < 0.01 compared with the control.

K562/ADR cells were examined using Hoechst 33342 staining to confirm the effect of compound 9k on induction of apoptosis. As seen in Fig. 4, control cells displayed excellent growth characteristic and the nuclei were stained weak homogeneous blue after 48 h incubation. However, K562/ADR cells treated with 0.05 μM 9k, 0.1 μM 9k and 0.05 μM podophyllotoxin evoked typical characteristics of apoptosis, such as cell shrinkage, nuclear fragmentation, condensation and relative fluorescence. These results revealed that 9k could inhibit the cell proliferation of K562/ADR by arresting the cell cycle and induce G2/M arrest accompanied by apoptosis.

Fig. 4 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 9k; (D) K562/ADR cells treated with 0.1 μM 9k. Magnification 200×.

The process of apoptosis is regulated by several biomolecules such as caspases, Bcl-2, Bax, p53 and several other proteins.³⁵ To further explore the apoptotic mechanism of 9k in K562/ADR cells, the effects of 9k on the expression of Bcl-2, Bax, caspase-3, p53 and survivin were performed by western blotting using podophyllotoxin as a positive control. As shown in Fig. 5, in comparison with those of the vehicle treated control, the expression of Bax, caspase-3 and tumor-suppressor protein p53 was significantly increased, while Bcl-2 and anti-apoptosis protein survivin expression were decreased by 9k and the effects of 9k were significantly stronger than those of podophyllotoxin in K562/ADR cells. These findings suggested that 9k induced apoptosis by means of multiple pathways.

Fig. 5 Effect of compounds (1 and 9k) on the expression levels of Bcl-2, Bax, caspase-3, p53 and survivin in K562/ADR cells by western blotting using β-actin as a control. (A) Control K562/ADR cells; (B) K562/ADR cells treated with 0.05 μM 1; (C) K562/ADR cells treated with 0.05 μM 9k; (D) K562/ADR cells treated with 0.1 μM 9k.

Previous study showed that K562/ADR cells expressed high levels of Pgp.²⁴ To further explore the mechanism of anti-MDR properties, the effect of 9k on the expression of Pgp in K562/ADR cells was measured by western blotting. Podophyllotoxin was used as positive control. After the cells were treated with 0.05 μM 9k and 0.1 μM 9k for 48 h, the expression of Pgp was significantly decreased (Fig. 6), compared to the untreated K562/ADR cells, and 9k showed more stronger effect than podophyllotoxin. These results indicated that 9k could suppress the expression of Pgp in K562/ADR cells.

Fig. 6 Effect of compounds (1 and 9k) on the expression levels of Pgp in K562/ADR cells by western blotting using β-actin as a control. (A) Control K562/ADR cells; (B) K562/ADR cells treated with 0.05 μM 1; (C) K562/ADR cells treated with 0.05 μM 9k; (D) K562/ADR cells treated with 0.1 μM 9k.

Mitogen-activated protein kinases (MAPKs) are serine–threonine kinases, including extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), which are associated with various cellular activities such as survival and death.³⁶ To gain further insights into the molecular mechanisms involved in K562/ADR cells, the role of the MAPK pathways were investigated. The K562/ADR cells were treated with vehicle, various concentrations of 9k or podophyllotoxin for 48 h, respectively. The expression and activation of ERK1/2, JNK and p38 were determined by Western blotting. As shown in Fig. 7, treatment with 9k significantly increased the levels of phosphorylated ERK1/2, JNK and p38 in K562/ADR cells. Meanwhile, no differences in the total levels of ERK1/2, JNK and p38 were observed. Similarly, treatment with podophyllotoxin also increased the levels of p-ERK1/2, p-JNK and p-p38, however, the effects of podophyllotoxin were significantly less than those of 9k. These findings indicated that 9k had the potential to overcome the resistance of K562/ADR cells probably by activation of the MAPK pathways, which was coincident with some literatures.^37,38 Ding and co-worker showed that salvicine, a diterpenoid quinone compound, could promote the phosphorylation of c-Jun-N-terminal kinase and c-Jun protein to overcome the MDR in K562/ADR cells.³⁷ Recently, Han et al. reported that downregulation the expression of Pgp by cepharanthine hydrochloride, a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, was related to the activation of c-Jun/JNK in K562/ADR cells.³⁸ However, some other studies obtained different conclusions. For example, Chen et al. had reported that inhibition of p38 diminished doxorubicin-induced drug resistance associated with the down-regulation of Pgp in K562/ADR cells.³⁹ In addition, Pang and co-workers showed that Pgp expression was down-regulated by the intracellular acidification through inhibition of p38 and the activation of JNK in K562/ADR cells, which suggesting that cross-talk within MAPKs was important for the regulation of Pgp.⁴⁰ Here, our paper for the first time indicated that podophyllotoxin and its derivative 9k could stimulate the MAPK signaling pathways in resistance K562/ADR cells by up-regulating the expression levels of p-ERK1/2, p-JNK and p-p38, which need to be investigated with further functional studies.

Fig. 7 Effects of compounds (1 and 9k) on MAPK pathway-related proteins in K562/ADR cells by western blotting using β-actin as a control. (A) Control K562/ADR cells; (B) K562/ADR cells treated with 0.05 μM 1; (C) K562/ADR cells treated with 0.05 μM 9k; (D) K562/ADR cells treated with 0.1 μM 9k.

Experimental

General

Melting point was measured on SGWX-4 microscope melting-point apparatus. High-resolution mass spectra (HR-MS) was taken on Agilent Accurate-Mass-Q-TOF-MS 6520 (Agilent Technologies, USA). ¹H NMR and ¹³C NMR spectra were performed using an Agilent-NMR-vnmrs 400 MHz instrument using TMS as internal standard. IR spectra were recorded on Varian 1000 FT-IR spectrometer on KBr pellets and absorptions are reported in cm⁻¹.

General procedure for the synthesis of compounds 9a–9j

To a solution of podophyllotoxin (0.48 mmol, 1 eq.), an appropriate cinnamic or heterocyclic acrylic acid (0.95 eq.), 4-(dimethylamino)pyridine (DMAP) (1.2 eq.) in DMF (3 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2 eq.) was added under the condition of ice water bath and the mixture was stirred at 30 °C, until TLC (silica gel, EtOAc/petroleum ether = 1 : 1) indicated the end of reaction. The reaction solution was poured into water (20 mL) and solid was collected by filtration, washed with water and dried by vacuum drying. The crude product was purified by column-chromatography (EtOAc/petroleum ether = 1 : 4) to obtain the pure product.

General procedure for the synthesis of compound 9k

To a solution of podophyllotoxin (0.48 mmol, 1 eq.) and triethylamine (3 eq.) in CH₂Cl₂ (5 mL), acryloyl chloride (2 eq.) was dropped in slowly while stirring in an ice-salt bath and the reaction mixture was stirred at room temperature, until TLC indicated the end of reaction. The mixture was quenched with saturated NH₄Cl (15 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 MgSO₄ and evaporated under reduced pressure. The crude was purified by silica gel column chromatography (EtOAc/petroleum ether = 1 : 2) to obtain the pure product.

4α-[(E)-3-Phenyl-2-acrylate]-4-desoxy-podophyllotoxin (9a). White power, yield 64%; mp: 108–110 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J = 16.0 Hz, 1H, CH=CH), 7.55 (s, 2H, Ar-H), 7.43 (s, 3H, Ar-H), 6.85 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.50 (d, J = 16.0 Hz, 1H, CH=CH), 6.43 (s, 2H, Ar-H), 6.03 (d, J = 8.4 Hz, 1H, CH–O–C=O), 5.99 (d, J = 6.4 Hz, 2H, O–CH₂–O), 4.63 (s, 1H, CH-Ar), 4.42–4.46 (m, 1H, CH–CH₂–O), 4.28 (t, J = 9.2 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.79 (s, 6H, 3′,5′-OCH₃), 2.89–2.99 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.72, 167.26, 152.62, 148.13, 147.62, 146.46, 137.13, 134.89, 133.90, 132.32, 130.86, 129.03, 128.50, 128.24, 116.87, 109.74, 108.12, 107.09, 101.60, 73.63, 71.44, 60.76, 56.18, 45.62, 43.77, 38.81; IR (KBr, cm⁻¹) 3433, 2926, 2837, 1779, 1709, 1635, 1588, 1505, 1484, 1379, 1333, 1239, 1162, 1126, 1037, 996, 928, 861, 768; HRMS-ESI (m/z): calcd for C₃₁H₃₂NO₉ [M + NH₄]⁺ 562.2072, found 562.2076.

4α-[(E)-3-(4-Chlorophenyl)-2-acrylate]-4-desoxy-podophyll-otoxin (9b). White power, yield 76%; mp: 112–114 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 15.9 Hz, 1H, CH=CH), 7.48 (d, J = 6.8 Hz, 2H, Ar-H), 7.39 (d, J = 7.7 Hz, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.47 (d, J = 16.0 Hz, 1H, CH=CH), 6.42 (s, 2H, Ar-H), 5.94–6.07 (m, 3H, CH–O–C=O, O–CH₂–O), 4.63 (s, 1H, CH-Ar), 4.41–4.42 (m, 1H, CH–CH₂–O), 4.27 (t, J = 9.0 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.92–3.00 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.68, 167.03, 152.62, 148.15, 147.61, 144.99, 136.82, 134.87, 132.33, 129.39, 129.34, 128.37, 117.44, 109.77, 108.13, 107.03, 101.61, 73.78, 71.39, 60.77, 56.19, 45.60, 43.74, 38.77; IR (KBr, cm⁻¹) 3425, 2935, 2904, 2837, 1780, 1711, 1635, 1589, 1505, 1484, 1419, 1332, 1239, 1167, 1126, 1037, 997, 928, 862, 823, 767; HRMS-ESI (m/z): calcd for C₃₁H₂₇Cl NaO₉ [M + Na]⁺ 601.1236, found 601.1239.

4α-[(E)-3-(4-Fluorophenyl)-2-acrylate]-4-desoxy-podophyllo-toxin (9c). White power, yield 75%; mp: 109–111 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 16.0 Hz, 1H, CH=CH), 7.56 (d, J = 5.2 Hz, 1H, Ar-H), 7.54 (d, J = 5.2 Hz, 1H, Ar-H), 7.12 (d, J = 16.0 Hz, 1H, CH=CH), 7.11 (s, 1H, Ar-H), 6.84 (s, 1H, Ar-H), 6.57 (s, 1H, Ar-H), 6.43 (t, J = 8.0 Hz, 3H, Ar-H), 6.03 (d, J = 8.0 Hz, 1H, CH–O–C=O), 5.99 (d, J = 6.9 Hz, 2H, O–CH₂–O), 4.64 (d, J = 3.7 Hz, 1H, CH-Ar), 4.41–4.45 (m, 1H, CH–CH₂–O), 4.27 (t, J = 9.5 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.90–3.00 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.70, 167.15, 165.44, 162.93, 152.62, 148.14, 147.61, 145.13, 137.15, 134.88, 132.33, 130.23, 130.15, 128.44, 116.62, 116.36, 116.14, 109.76, 108.14, 107.04, 101.61, 73.70, 71.41, 60.77, 56.19, 45.60, 43.76, 38.79; IR (KBr, cm⁻¹) 3442, 2934, 2837, 1779, 1710, 16 363, 1600, 1509, 1484, 1461, 1418, 1332, 1238, 1159, 1127, 1038, 997, 930, 862, 833; HRMS-ESI (m/z): calcd for C₃₁H₃₁FNO₉ [M + NH₄]⁺ 580.1977, found 580.1973.

4α-[(E)-3-(4-Methoxyphenyl)-2-acrylate]-4-desoxy-podophy-llotoxin (9d). White power, yield 79%; mp: 97–99 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 15.9 Hz, 1H, CH=CH), 7.50 (d, J = 8.4 Hz, 2H, Ar-H), 6.93 (d, J = 8.4 Hz, 2H, Ar-H), 6.85 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.43 (s, 2H, Ar-H), 6.36 (d, J = 15.9 Hz, 1H, CH=CH), 5.98–6.03 (m, 3H, CH–O–C=O, O–CH₂–O), 4.62 (s, 1H, CH-Ar), 4.41–4.43 (m, 1H, CH–CH₂–O), 4.27 (t, J = 9.4 Hz, 1H, CH–CH₂–O), 3.85 (s, 3H, 4-OCH₃ in cinnamic acid), 3.81 (s, 3H, 4′-OCH₃), 3.79 (s, 6H, 3′,5′-OCH₃), 2.89–2.99 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.79, 167.60, 161.81, 152.60, 148.07, 147.59, 146.11, 137.10, 134.94, 132.28, 129.99, 128.68, 126.63, 114.45, 114.19, 109.71, 108.11, 107.13, 101.57, 73.39, 71.49, 60.76, 56.17, 55.42, 45.61, 43.78, 38.84; IR (KBr, cm⁻¹) 3432, 2934, 2838, 1778, 1706, 1632, 1602, 1512, 1484, 1461, 1421, 1332, 1289, 1240, 1157, 1126, 1036, 997, 929, 830; HRMS-ESI (m/z): calcd for C₃₂H₃₄NO₁₀ [M + NH₄]⁺ 592.2177, found 592.2177.

4α-[(E)-3-(4-Methylphenyl)-2-acrylate]-4-desoxy-podophyll-otoxin (9e). White power, yield 79%; mp: 106–108 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 16.0 Hz, 1H, CH=CH), 7.44 (d, J = 8.0 Hz, 2H, Ar-H), 7.22 (d, J = 7.9 Hz, 2H, Ar-H), 6.85 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.43–6.47 (m, 3H, CH=CH, Ar-H), 5.98–6.03 (m, 3H, CH–O–C=O, O–CH₂–O), 4.63 (d, J = 3.8 Hz, 1H, CH-Ar), 4.44 (dd, J = 9.1, 6.6 Hz, 1H, CH–CH₂–O), 4.27 (t, J = 9.6 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.89–3.00 (m, 2H, CH–CH₂–O, O=C–CH), 2.39 (s, 3H, Ar-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 173.77, 167.46, 152.60, 148.09, 147.60, 146.47, 141.43, 134.91, 132.29, 131.17, 129.76, 128.58, 128.25, 115.70, 109.72, 108.08, 107.12, 101.58, 73.50, 71.48, 60.77, 56.17, 45.62, 43.77, 38.82, 21.54; IR (KBr, cm⁻¹) 3429, 2935, 2837, 1779, 1707, 1632, 1588, 1507, 1483, 1460, 1419, 1332, 1239, 1159, 1127, 1037, 997, 929, 862, 814, 766; HRMS-ESI (m/z): calcd for C₃₂H₃₄NO₉ [M + NH₄]⁺ 576.2228, found 576.2226.

4α-[(E)-3-(3-Chlorophenyl)-2-acrylate]-4-desoxy-podophyll-otoxin (9f). White power, yield 77%; mp: 105–106 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J = 16.0 Hz, 1H, CH=CH), 7.54 (s, 1H, Ar-H), 7.33–7.43 (m, 3H, Ar-H), 6.83 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.51 (d, J = 16.0 Hz, 1H, CH=CH), 6.42 (s, 2H, CH=CH), 5.99–6.03 (m, 3H, CH–O–C=O, O–CH₂–O), 4.64 (d, J = 3.9 Hz, 1H, CH-Ar), 4.43 (dd, J = 9.0, 6.8 Hz, 1H, CH–CH₂–O), 4.27 (t, J = 9.5 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.90–3.00 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.66, 166.86, 152.63, 148.17, 147.63, 144.81, 135.68, 135.07, 134.85, 132.34, 130.70, 130.28, 128.30, 127.90, 126.47, 118.36, 109.78, 108.11, 107.02, 101.62, 73.88, 71.39, 60.77, 56.19, 45.60, 43.74, 38.77; IR (KBr, cm⁻¹) 3426, 2936, 2837, 1779, 1711, 1638, 1588, 1504, 1484, 1419, 1331, 1170, 1038, 997, 929, 862, 787; HRMS-ESI (m/z): calcd for C₃₁H₂₇ClNaO₉ [M + Na]⁺ 601.1236, found 601.1237.

4α-[(E)-3-(3,4-Dimethoxyphenyl)-2-acrylate]-4-desoxy-podo-phyllotoxin (9g). White power, yield 75%; mp: 111–112 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J = 15.9 Hz, 1H, CH=CH), 7.12 (d, J = 8.3, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.89 (d, J = 8.3 Hz, 1H, Ar-H), 6.86 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.34 (s, 2H, Ar-H), 6.36 (d, J = 15.9 Hz, 1H, CH=CH), 6.02 (d, J = 8.8 Hz, 1H, CH–O–C=O), 5.98 (d, J = 5.2 Hz, 2H, O–CH₂–O), 4.63 (d, J = 4.1 Hz, 1H, CH-Ar), 4.43 (dd, J = 9.2, 6.7 Hz, 1H, CH–CH₂–O), 4.28 (t, J = 9.6 Hz, 1H, CH–CH₂–O), 3.93 (s, 6H, 3,4-OCH₃ in cinnamic acid), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.89–3.00 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.77, 167.51, 152.60, 151.58, 149.27, 148.08, 147.58, 146.38, 137.17, 134.94, 132.29, 128.65, 126.86, 123.11, 114.39, 111.02, 109.75, 109.53, 108.22, 107.08, 101.58, 73.46, 71.47, 60.77, 56.22, 56.00, 55.92, 45.60, 43.77, 38.83; IR (KBr, cm⁻¹) 3448, 2936, 2906, 2837, 1778, 1706, 1630, 1596, 1512, 1484, 1420, 1332, 1239, 1127, 1037, 998, 929, 805, 765; HRMS-ESI (m/z): calcd for C₃₃H₃₂NaO₁₁ [M + Na]⁺ 627.1837, found 627.1843.

4α-[(E)-3-(3-Methoxyphenyl)-2-acrylate]-4-desoxy-podophy-llotoxin (9h). White power, yield 44%; mp: 106–107 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 16.0 Hz, 1H, CH=CH), 7.33 (t, J = 7.9 Hz, 1H, Ar-H), 7.14 (d, J = 7.6 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.97 (d, J = 8.2, 1H, Ar-H), 6.85 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.48 (d, J = 16.0 Hz, 1H, CH=CH), 6.43 (s, 2H, Ar-H), 6.03 (d, J = 8.7 Hz, 1H, CH–O–C=O), 5.99 (d, J = 6.0 Hz, 2H, O–CH₂–O), 4.64 (d, J = 3.8 Hz, 1H, CH-Ar), 4.44 (dd, J = 8.9, 6.8 Hz, 1H, CH–CH₂–O), 4.28 (t, J = 9.6 Hz, 1H, CH–CH₂–O), 3.85 (s, 3H, 3-OCH₃ in cinnamic acid), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.92–2.97 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.74, 167.23, 159.93, 152.61, 148.13, 147.61, 146.40, 137.10, 135.22, 134.89, 132.32, 130.04, 128.46, 120.94, 117.14, 116.59, 113.15, 109.75, 108.09, 107.08, 101.60, 73.66, 71.45, 60.77, 56.18, 55.35, 45.61, 43.76, 38.80, 29.70; IR (KBr, cm⁻¹) 3424, 2932, 2836, 1785, 1702, 1633, 1588, 1506, 1483, 1462, 1421, 1333, 1239, 1166, 1126, 1038, 999, 932, 847, 786; HRMS-ESI (m/z): calcd for C₃₂H₃₄NO₁₀ [M + NH₄]⁺ 592.2177, found 592.2176.

4α-[(E)-3-(4-Nitrophenyl)-2-acrylate]-4-desoxy-podophyllot-oxin (9i). Light yellow power, yield 68%; mp: 118–120 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 8.7 Hz, 2H, Ar-H), 7.79 (d, J = 16.0 Hz, 1H, CH=CH), 7.71 (d, J = 8.7 Hz, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.63 (d, J = 16.0 Hz, 1H, CH=CH), 6.57 (s, 1H, Ar-H), 6.42 (s, 2H, Ar-H), 6.05 (d, J = 8.7 Hz, 1H, CH–O–C=O), 6.00 (d, J = 7.2 Hz, 2H, O–CH₂–O), 4.64 (d, J = 3.9 Hz, 1H, CH-Ar), 4.43 (dd, J = 8.9, 6.8 Hz, 1H, CH–CH₂–O), 4.28 (t, J = 9.5 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.91–3.01 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.56, 166.37, 152.64, 148.77, 148.24, 147.66, 143.43, 139.89, 137.21, 134.79, 132.41, 128.85, 128.03, 124.29, 121.18, 109.86, 108.18, 106.93, 101.67, 74.26, 71.28, 60.78, 56.22, 45.58, 43.71, 38.72; IR (KBr, cm⁻¹) 3448, 2937, 2904, 2837, 1779, 1712, 1638, 1589, 1507, 1522, 1483, 1419, 1343, 1239, 1170, 1126, 1037, 997, 929, 847, 767; HRMS-ESI (m/z): calcd for C₃₁H₃₁N₂O₁₁ [M + NH₄]⁺ 607.1922, found 607.1923.

4α-[(E)-3-(2-Furyl)-2-acrylate]-4-desoxy-podophyllotoxin (9j). White power, yield 77%; mp: 106–107 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.51–7.52 (m, 1H, furyl-H), 7.49 (d, J = 15.7 Hz, 1H, CH=CH), 6.83 (s, 1H, Ar-H), 6.68 (d, J = 3.4 Hz, 1H, furyl-H), 6.55 (s, 1H, Ar-H), 6.51 (dd, J = 3.4, 1.8 Hz, 1H, furyl-H), 6.41 (s, 2H, Ar-H), 6.37 (d, J = 15.7 Hz, 1H, CH=CH), 5.97–6.01 (m, 3H, CH–O–C=O, O–CH₂–O), 4.62 (d, J = 4.1 Hz, 1H, CH-Ar), 4.42 (dd, J = 9.2, 6.7 Hz, 1H, CH–CH₂–O), 4.26 (t, J = 9.6 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.78 (s, 6H, 3′,5′-OCH₃), 2.88–2.99 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.75, 167.35, 152.60, 150.52, 148.09, 147.58, 145.32, 137.05, 134.88, 132.40, 132.28, 128.53, 115.89, 114.35, 112.55, 109.69, 108.03, 107.11, 101.58, 73.53, 71.47, 60.76, 56.14, 45.61, 43.76, 38.81; IR (KBr, cm⁻¹) 3426, 2937, 2904, 2837, 1779, 1707, 1635, 1588, 1505, 1484, 1420, 1330, 1240, 1208, 1158, 1126, 1037, 997, 929, 860, 764; HRMS-ESI (m/z): calcd for C₂₉H₂₆NaO₁₀ [M + Na]⁺ 557.1418, found 557.1423.

4α-Podophyllotoxin-4-acrylate (9k). Light yellow power, yield 82%; mp: 128–130 °C; ¹H NMR (400 MHz, CDCl₃) δ 6.79 (s, 1H, Ar-H), 6.55 (s, 1H, Ar-H), 6.49 (d, J = 17.3 Hz, 1H, CH=CH₂), 6.40 (s, 2H, Ar-H), 6.20 (dd, J = 17.3, 10.4 Hz, 1H, CH=CH₂), 5.55–5.99 (m, 4H, Ar-H, CH=CH₂), 4.62 (d, J = 4.1 Hz, 1H, CH-Ar), 4.40 (dd, J = 9.2, 6.8 Hz, 1H, CH–CH₂–O), 4.24 (t, J = 9.7 Hz, 1H, CH–CH₂–O), 3.81 (s, 3H, 4′-OCH₃), 3.76 (s, 6H, 3′,5′-OCH₃), 2.86–2.98 (m, 2H, CH–CH₂–O, O=C–CH); ¹³C NMR (100 MHz, CDCl₃) δ 173.64, 166.41, 152.60, 148.15, 147.60, 137.05, 134.78, 132.36, 128.20, 127.65, 109.71, 107.99, 107.06, 101.60, 73.72, 71.39, 60.76, 56.10, 45.59, 43.72, 38.72; IR (KBr, cm⁻¹) 3428, 2936, 2903, 2838, 1780, 1721, 1587, 1505, 1486, 1466, 1415, 1330, 1261, 1239, 1184, 1126, 1040, 999, 933, 873, 809, 769; HRMS-ESI (m/z): calcd for C₂₅H₂₈NO₉ [M + NH₄]⁺ 486.1759, found 486.1764.

Cytotoxic activity

Cells at 3 × 10³ cells per well were cultured in 10% FBS DMEM in 96-well flat-bottom microplates. After culturing for 24 h at 37 °C and with 5% CO₂, cells were incubated with vehicle and scalar concentrations of the test compounds for 72 h. Then the CCK-8 (10 μL) was added to each well. After 3 h of incubation, the absorbance at 450 nm was determined with a plate reader. IC₅₀ (concentration that inhibits 50% of cell growth) was calculated from a log plot of percent of control versus concentration.

Cell cycle analysis

K562/ADR cells were seeded in six-well plates and then treated with different concentrations of test compounds for 48 h. After incubation, trypsinized adherent cells were collected, washed with PBS, and fixed overnight in 70% ethanol at 4 °C. Then cells were centrifuged to remove the fixative, washed with PBS, incubated with 100 μL RNase-A for 30 min at 37 °C and stained with 400 μL PI for 30 min at 4 °C in the dark. The cell cycle distribution was detected by a flow cytometer (Becton–Dickinson FACS Calibur).

Apoptosis analysis

K562/ADR cells were incubated in six-well plates overnight. Cells were then treated with vehicle and different concentrations of test compounds for 48 h. After incubation, cells were collected and washed twice with PBS. Resuspended cells were incubated with 500 μL binding buffer containing 5 μL of Annexin V-APC and 5 μL 7-AAD in the dark for 15 min at room temperature. The percentage of apoptotic cells was determined by flow cytometry (Becton–Dickinson FACS Calibur) analysis.

Hoechst 33342 staining

The K562/ADR cells were treated with vehicle and different concentrations of test compounds for 48 h. Subsequently, cells were fixed, washed thrice with PBS and stained with Hoechst 33342 staining solution for 10 min, followed by examining under a fluorescent microscope (OLYMPUS, IX51).

Western blot analysis

After the treatment of K562/ADR cells with vehicle or different concentrations of tested compounds for 48 h, the proteins were extracted with cell lysis buffer on ice for 30 min, and then lysis buffer was collected and centrifuged at 13 000g, 4 °C, for 10 min. After the protein concentration measured using the bicinchoninic acid (BCA) protein assay. Equal amount of proteins were separated by electrophoresis in a 12% SDS-polyacrylamide and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skimmed milk for 2 h. Then, membranes were incubated with the primary antibodies Bcl-2, Bax, caspase-3, p53, survivin, Pgp, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p-38 and β-actin overnight at 4 °C. After three washings with TBST, the membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. The membranes were washed with TBST and antibody-reactive proteins were visualised using the enhanced chemiluminescence reaction (ECL) detection system.

Conclusions

In summary, a series of acrylic esters of podophyllotoxin were synthesized and biologically evaluated. The most active compound 9k exhibited strong antiproliferative effects against both drug-sensitive K562 and drug-resistant K562/ADR cells in vitro. In addition, treatment with 9k induced K562/ADR cell cycle arrest at G2/M phase and apoptosis by up-regulating the expression of Bax, caspase-3 and p53, and inhibiting the expression of Bcl-2 and survivin. Furthermore, treatment with 9k stimulated the MAPK signaling pathways and reduced the expression of Pgp protein in K562/ADR cells. Together, the results indicated that 9k had the potential to be a potential anti-MDR agent in chronic myeloid leukemia therapy.

Acknowledgements

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

Notes and references

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