Synthesis and Evaluation of New Podophyllotoxin Derivatives with in Vitro Anticancer Activity

A series of novel podophyllotoxin derivatives were designed and synthesized. The cytotoxic activities of these compounds were tested against three tumor cell lines (HeLa, K562, and K562/A02). Most of the derivatives (IC50 = 1–20 μM) were found to have stronger cell growth inhibitory activity than positive control etoposide. Among them, 4β-N-[(E)-(5-((4-(4-nitrophenyl)-piperazin-1-yl)methyl)furan-2-yl)prop-2-en-1-amine]-4-desoxy-podophyllotoxin (9l) demonstrated significant inhibitory activity against HeLa, K562, and K562/A02 cell lines with IC50 values of 7.93, 6.42, 6.89 μM, respectively.


Introduction
Cancer is a major public health problem in the world. In 2008, 7.6 million people died of cancer (around 13% of all deaths), and this number is projected to increase with an estimated 13.1 million in 2030 [1].
Podophyllotoxin (PPT, a), the most abundant naturally occurring cyclolignan isolated mainly from Podophyllum peltatum and P. hexandrum, has important antineoplastic and antiviral properties [2]. However, its antimitotic activity is proved to be of the greatest interest to researchers [3]. Because of its toxic side effects, extensive structural modifications were performed since the 1950s. Podophyllotoxin derivatives possess antitumor activity, such as etoposide (VP-16, b) and teniposide (VM-26, c) ( Figure 1) have been widely used as anticancer drugs for clinical chemotherapy [4]. However, their low water solubility, acquired drug-resistance and severe gastrointestinal disturbances have promoted the search for new derivatives of podophyllotoxin [5]. The structural modifications and mechanism of action of podophyllotoxin have been studied over the years and the C4 position is considered potentially the most modifiable position. Diverse analogs like GL-331 (d), NPF (e), TOP-53 (f), NK-611 (g) (Figure 1), which are presently under clinical trial have been developed [6][7][8].
Investigation of the structure-activity relationships of PPT indicates that the trans-lactone, the 4β-substituted moiety, and the 4′-demethyl moieties are essential for TOP-II inhibitory activity [9,10]. In recent years, our group and others have found that several analogs with N-substitutions at the C4 position show an improved antitumor activity compared with VP-16 [11][12][13][14][15][16][17][18][19][20][21][22][23]. In this study, furfuran amines of 4β-N-substituted podophyllotoxin derivatives were designed and synthesized. The antiproliferative activities of the synthesized compounds against human cervical cancer cell line (HeLa), chronic myeloid leukemia cell line (K562) and red leukemia multi-drug resistance cell line (K562/A02) were evaluated and a preliminary SAR study of these compounds is discussed.

Chemistry
The synthesis of compounds 4a-i is outlined in Scheme 1. Treatment of 1 with NaBH4 in dry methanol yielded compound 2. Compound 3 was prepared by means of a Mannich reaction of 2 with a secondary amine in the presence of glacial acetic acid and formaldehyde. Then, compound 3 was reacted with active manganese dioxide to give the intermediates 4a-i. Reagents  The synthetic route (Scheme 2) to the target compounds 9a-n involved the intermediate 7, which was prepared from 5. In the presence of sodium azide, compound 6 was derived from 5 [24]. Then, compound 7 was derived from 6 through a reduction of azide. Next, 7 was combined with compounds 4a-i, respectively, in the presence of absolute methanol and a catalytic amount of glacial acetic acid to provide the 8. Then, reduction of 8 gave compounds 9a-n, respectively. The structures of the intermediates 4a-i, and 14 target compounds were identified by HRMS, 1 H-NMR, and 13 C-NMR spectral analysis.
In this paper, the C4-configuration of the novel podophyllotoxin derivatives was deduced from the reaction mechanism as well as evidences from NMR data. The nucleophilic substitution occurring at the C4 position was assumed to follow an SN1 mechanism [25]. It was presumed that C4-β-substitution was the main product due to the bulky C1-α-substituted aromatic ring. The configuration of the targeted compounds was identified as C4-β based on their small J3,4 values, because the J3,4 value is larger than 10 Hz in C4-α isomer [26].

Biological Results and Discussion
Cytotoxicities of all target derivatives were evaluated against three human cancer cell lines by the MTT assay. These three cell lines are: HeLa, K562 and K562/A02. The results are summarized in Table 1.
We also deduced the preliminary structure-activity relationships of these compounds. First, the 4′-OH derivatives were more cytotoxic than the corresponding 4′-OMe analogs. This observation is in accord with previously reported activities of closely related structures [11,27]. Second, the introduction of a benzene group (compare 9j and 9f, 9l and 9f, 9m and 9f) such as fluorine substituent at the 4-position of phenyl ring (9j), nitro substituent at the 4-position of phenyl ring (9l), unsubstituted phenyl ring (9m), resulted in a considerably higher increase in cytotoxicity in the MDR cell line, K562/A02, than etoposide. Compound 9l showed outstanding cytotoxicity towards K562/A02. Our previous results indicate that these derivatives were inhibitors of the expression of MDR-1 in K562/A02 cells [11], having crucial research significance. It was suggested that compound 9l may overcome MDR by reducing the expression of MDR-1.

Chemistry
Melting points were determined on an electric X-4 digital visual melting point apparatus. The 1 H-NMR and 13 C-NMR spectra were obtained using a Bruker ARX instrument (300 MHz, 400 MHz and 600 MHz). Chemical shifts are reported in ppm downfield from internal TMS as standard. HRMS were obtained on Agilent 6210 TOP-MS and are reported as m/z. Unless otherwise noted, all common reagents and solvents were obtained from commercial suppliers without further purification.

General Procedure for the Synthesis of Compounds 4a-i
Compounds (4a-i) were synthesized by means of a Mannich reaction. (E)-3-(furan-2-yl) acrylaldehyde (1.0 mmol) and NaBH4 (2.0 mmol) in dry MeOH (15 mL) were added to a 50 mL dried round-bottom flask. The mixture was reacted at room temperature for 2 h. Then the solvent was evaporated to give the intermediate 2. A mixture of 2 (1.0 mmol) and the corresponding secondary amine (1.5 mmol) in glacial acetic acid (20 mL) containing formaldehyde (1.5 mmol) was stirred at 50 °C for 4 h. After completion of the reaction was monitored by thin layer chromatography (TLC), the solvent was removed and the residue was added water (15 mL) before neutralization with saturated aqueous NaOH and extraction with ethyl acetate (3 × 30 mL). The combined organic layer was washed with water followed by brine, dried over Na2SO4, filtered, and concentrated to give compound 3. To a stirred solution of compound 3 (1.0 mmol) in dry CH2Cl2 (20 mL) was added active manganese dioxide (10.0 mmol) at room temperature, and the reaction mixture was stirred for 2 h, After the reaction was completed, the mixture was filtered and concentrated to provide a yellow oil and purified by column chromatography on silica gel using petroleum ether-ethyl acetate to afford the yellow solids 4a-i [11].

General Procedure for the Synthesis of Compounds 9a-n
To a stirred solution of 5 (10 mmol) in dry CH2Cl2 (50 mL), NaN3 (40 mmol) in dry CH2Cl2 (10 mL) was added carefully. CF3COOH (10 mL) was added into the solution dropwise at 0 °C. After stirring for 1 h at room temperature, the mixture was refluxed for 4 h. Saturated aqueous NaHCO3 was added to adjust the pH value to 7. The organic phase was separated and dried with anhydrous Na2SO4 and concentrated. The residue was crystallized from CH2Cl2/acetic ether (1:1) to give a 6. To a solution of 6 (10 mmol) in ethyl acetate (50 mL), 10% Pd/C (1.00 g) and HCOONH4 (40 mmol) were added. The mixture was refluxed for 5 h and filtered. The filtrate was washed with saturated brine three times and concentrated to give white compound 7 [11].

Evaluation of the Biological Activity
The antiproliferative activity of compounds 9a-n was evaluated with human cervical cancer cell line (HeLa), chronic myeloid leukemia cell line (K562) and leukemia multi-drug resistance cell line (K562/A02) by the MTT method in vitro, with etoposide (VP-16) as positive control. The three tumor cell lines were cultured in RPMI-1640 containing 10% FBS, 2 mmol·L −1 glutamine, 100 U·mL −1 penicillin, and 100 µg·mL −1 streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The cells were seeded at a density of 5 × 103 cells/well in 96-well plates and allowed to attach for 24 h. The thiazolyl blue tetrazolium bromide (MTT) assay was performed to quantify cell viability following treatment with the synthetic compounds or reference compound etoposide (VP-16) [28]. After 48 h, 20 μL MTT (5 mg·mL −1 ) solution was added for 4 h at 37 °C. Then, the supernatant was discarded and dimethylsulfoxide (150 μL) was added to dissolve the formazan product. The intensity was measured at a wavelength of 490 nm.