Synthesis and Anticancer Activity of 4β-Triazole-podophyllotoxin Glycosides

A series of novel 4β-triazole-podophyllotoxin glycosides were synthesized by utilizing the Click reaction. Evaluation of cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assay shows that most of these compounds show weak cytotoxicity. It was observed that compound 16 shows the highest activity with IC50 values ranging from 2.85 to 7.28 μM, which is more potent than the control drugs etoposide and cisplatin against four of five cancer cell lines tested. Compound 16 is characterized with an α-d-galactosyl residue directly linked to the triazole ring and a 4′-OH group on the E ring of the podophyllotoxin scaffold. HPLC investigation of representative compound indicates that incorporation of a sugar moiety seems to improve the chemical stability of the podophyllotoxin scaffold.


Introduction
Podophyllotoxin (1, Fig. 1), a naturally occurring cyclolignan, which is mainly isolated from the roots of Podophyllotoxin peltatum, shows strong cytotoxic activity against various cancer cell lines by inhibiting tubulin through binding with part of its colchicine domain [1,2]. Due to its severe side effects, podophyllotoxin has limited applications as a drug in cancer chemotherapy, but its semisynthetic derivatives etoposide 2 and teniposide 3 ( Fig. 1) are in clinical use for the treatment of a variety of malignancies, including small cell lung cancer, testicular carcinoma, lymphoma, and Kaposi's sarcoma [3,4]. However, their therapeutic uses are often hindered by problems such as acquired drug-resistance and poor water solubility. Numerous studies [5][6][7] have shown that 4b-Nsubstituted derivatives of podophyllotoxin maintain the anticancer activity and function as topoisomerase II inhibitors. Since 1,2,3-triazole ring is a widespread functional group in drug [8], it is intriguing to attach 1,2,3triazoles to podophyllotoxin derivatives.
All the products were characterized by 1 H-NMR, 13 C-NMR, ESI-MS, and HRESI-MS. ESI-MS and HRESI-MS of all compounds showed the [M?Na] ? or [M?H] ? adduct as the molecular ion. In the 1 H-NMR spectra, the formation of the triazole ring was confirmed by the resonance of its C 5 00 -H signal (d 7.81-8.31 ppm) in the aromatic region. The structure was further confirmed by the 13 C-NMR spectra, which showed the two characteristic carbon signals at around 145 ppm (d C-4 00 ) and 126 ppm (d C-5 00 ) corresponding to the triazole residue. The configuration at C-4 position for target compounds 15-26 was confirmed based on the J 3,4 coupling constant, which is typically \ 5.0 Hz for 4b-substituted compounds due to a cis relationship between H-3 and H-4 [23]. In some cases, 4b-substitution The a-linkage in D-mannosides was confirmed by the carbon-proton coupling constant ( 2 J C-H ) of the anomeric carbon by acquiring the non-decoupled 13 C NMR spectra.
For example, the 2 J C-H is 167.9 Hz for the anomeric carbon of the D-mannose residue in compound 23, which confirms that 23 is an a-mannoside since the 2 J C-H of the anomeric carbon for a b-mannoside is typically below 160 Hz [24].

Evaluation of Biological Activity
All cancer), and SW480 (colon cancer). Etoposide (2) and cisplatin were taken as reference compounds. The screening procedure was based on the standard MTT method [25], and the results are reported in the terms of IC 50 values ( Table 1).
As it can be seen in Table 1, most of these compounds show weak cytotoxicity (IC 50 [ 40 lM). However, compound 16 shows strong anticancer activity against all cancer cell lines tested, with IC 50 values ranging from 2.85 to 7.28 lM, which is significantly more potent than the control drug etoposide against four of the five cancer cells. It is interesting to note that the 4 0 -O-methylated analog 15 (IC 50 [ 40 lM) is much less potent than 16, indicating that this 4 0 -O-hydroxy group is perhaps important for the anticancer activity of glycosylated podophyllotoxin derivatives.

Chemical Stability Investigation
Compound 15 was selected for the investigation of chemical stability in aqueous phase with comparison to podophyllotoxin (1) and 4b-azido-4-deoxypodophyllotoxin (13). The results indicated that compound 15 exhibits higher chemical stability under the physiological condition (37°C, pH 7.0, Fig. 3) than both podophyllotoxin (1) and 4b-azido-4-deoxypodophyllotoxin (13). Hydrolysis of the d-lactone is anticipated to be the main degradation pathway under this condition [26]. It appears that the incorporation of the D-galactose moiety slows down the hydrolysis and improves the chemical stability of the podophyllotoxin scaffold.

Conclusions
In conclusion, we have used Fisher glycosylation strategy to prepare glycosylated terminal-alkynes. Then, all the glycosylated terminal-alkynes were reacted with podophyllotoxin-derived azides by the Click reaction to yield a series of 4b-triazole-podophyllotoxin glycosides 15-26 in good yields. All compounds were tested for anticancer activity against five human cancer cell lines. Most of these compounds show weak cytotoxicity while compound 16, having a galactose residue directly linked to the triazole ring and a 4 0 -OH group on the E ring, is more potent than the anticancer drug etoposide against four of the five cancer cell lines tested. In addition, chemical stability investigation indicates that the conjugated sugar residue seems to improve the stability of the podophyllotoxin scaffold under the physiological condition.

Click Chemistry: General Procedure for the Synthesis of Compounds 15-26
To a solution of a terminal-alkyne 7-12 (0.2 mmol) and 4bazido-podophyllotoxin analogues 13 or 14 (0.2 mmol) in t-BuOH-H 2 O (1:2, 1.0 mL) at room temperature were added copper (II) sulfate pentahydrate (0.02 mmol) and sodium ascorbate (1.0 M in H 2 O, 3 drops). The reaction mixture was stirred at room temperature for 4 h until the starting material disappeared as indicated by TLC. Then, the mixture was diluted with water (10 mL) and extracted with ethyl acetate (3 9 10 mL), and the combined organic layer was dried over sodium sulfate. The solvent was evaporated and the residue was purified by CC (CHCl 3 /CH 3 OH, 9:1) to afford the cycloaddition product 15-26 (75-87 %).