Synthesis and Anticancer Activity of Glucosylated Podophyllotoxin Derivatives Linked via 4β-Triazole Rings

A series of 4β-triazole-linked glucose podophyllotoxin conjugates have been designed and synthesized by employing a click chemistry approach. All the compounds were evaluated for their anticancer activity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assays. Most of these triazole derivatives have good anticancer activity. Among them, compound 35 showed the highest potency against all five cancer cell lines tested, with IC50 values ranging from 0.59 to 2.90 μM, which is significantly more active than the drug etoposide currently in clinical use. Structure-activity relationship analysis reveals that the acyl substitution on the glucose residue, the length of oligoethylene glycol linker, and the 4'-demethylation of podophyllotoxin scaffold can significantly affect the potency of the anticancer activity. Most notably, derivatives with a perbutyrylated glucose residue show much higher activity than their counterparts with either a free glucose or a peracetylated glucose residue.


Introduction
Podophyllotoxin (1, Figure 1), which is a lignan mainly isolated from Podophyllum peltatum and Podophyllum hexandrum [1,2], shows strong cytotoxic activity against various cancer cell lines by inhibiting tubulin polymerization and preventing microtubule formation. Due to its complicated side effects such as nausea, vomiting, and damage of normal tissues, attempts to use podophyllotoxin in the treatment of human neoplasia have been mostly unsuccessful [3]. The unique cyclolignan scaffold of 1 has however drawn a lot of attention for the discovery and development of new anticancer agents. Extensive structural modifications, particularly at the C-4 and C-4' position of podophyllotoxin have led to the development of many semisynthetic derivatives of podophyllotoxin [4][5][6]. Among them, five semisynthetic derivatives, etoposide (2), teniposide (3), etopophos (4), GL-331 (5) and TOP-53 (6) ( Figure 1) are currently used in the chemotherapy for a variety of cancers, including small-cell lung cancer, non-Hodgkin's lymphoma, leukemia, Kaposi's sarcoma, neurobslastoma and soft tissue sarcoma. These derivatives display binding activity to DNA topoisomerase II during the late S and early G2 cell cycle stages and are potent inhibitors of the enzyme [7][8][9][10][11][12][13][14]. Their anticancer activity proceeds through a mechanism of action entirely different from that of their parent compound podophyllotoxin (1). Etoposide (2), teniposide (3), and etopophos (4) are three semisynthetic glucosidic cyclic acetals of 1, and in particular, etoposide (2) is considered to be one of the most successful pharmaceuticals derived from plants. Both GL-331 (5) and TOP-53 (6) are more active than etoposide (2) and are currently under clinical investigation [12]. Recently, novel podophyllotoxin hybrids obtained by covalently linking another biologically active molecule to podophyllotoxin have been reported. For example, thiocolchicine-podophyllotoxin conjugates were reported to have improved solubility and anticancer activity [15]. In addition, a series of conjugates of podophyllotoxin with 5-fluorouracil (5-FU) were reported to have better cytotoxic activity than VP-16 [16]. Structure-activity relationship (SAR) studies [17] have demonstrated that C-4 is the molecular area tolerable to significant structural diversification.
Chemotherapeutic agents such as the podophyllotoxin derivatives 2-4 are often associated with undesirable side effects and the development of multi-drug resistance by cancer cells. Thus, structural modification of podophyllotoxin for developing new antitumor drugs with increased selectivity and reduced toxicity is highly desirable. In recent years, the altered glucose metabolism in cancer cells has been explored for targeted cancer therapy [18]. Glucose is the main source of metabolic energy of animal cells, generating ATP through glycolysis and oxidative phosphorylation. Cancer cells are well known to display an enhanced uptake and consumption of glucose, which is metabolized primarily through the fermentative pathway instead of tricarboxylic acid cycle and oxidative phosphorylation in the mitochondria of normal cells [19]. The transport of glucose across the plasma membrane into the cytosol is mediated by a family of glucose transporters (GLUTs) [20,21]. Due to their enhanced glucose consumption, cancer cells generally express higher levels of GLUTs than normal cells [22]. For example, glucose transporter class 1 (GLUT1) has been found to be overexpressed in a variety of both solid and hematological malignancies such as large B-cell lymphoma, colorectal carcinomas, hepatocellular carcinoma, head and neck cancer, gastrointestinal stromal tumor (GIST), prostate carcinoma, thyroid carcinoma, renal cell cancer, lung cancer, pancreatic cancer, sarcomas and laryngeal carcinomas [19]. Thus, in this study we planned to covalently link a glucose residue to podopyllotoxin so the resulting cytotoxic agents may be preferably taken up by cancer cells through the mediation of GLUTs.
Recently, the click reaction has been widely used to covalently link two molecular fragments in creating a wide variety of drug-like molecules [23,24]. Typically, a terminal alkyne and an azide undergo a copper-catalyzed [3+2]-cycloaddition to generate a substituted 4β-triazole ring [25,26]. Podophyllotoxin derivatives containing the featured 4β-triazole ring have also been reported as potential DNA topoisomerase-II inhibitors [27], including a few compounds bearing a sugar residue [28]. Through click reactions we have now synthesized a series of glucose-podophyllotoxin conjugates in order to systematically study the effect of: (a) the length of the linker; (b) the substituent on the glucose; (c) the configuration of the anomeric carbon of glucose residue; and (d) the substituent on the 4-position of the E-ring of the podophyllotoxin scaffold on the anticancer activity of such conjugates. Herein we report the synthesis, the preliminary anticancer activity and the structure activity relationship of these conjugates.
The glycosylated terminal alkynes 12-23 were allowed to react with azide 7 or 8 in the presence of copper (II) acetate and sodium ascorbate to yield a series of 4β-triazole-linked glucose-podophyllotoxin conjugates (Scheme 2, Table 1).

Scheme 2.
Click-chemistry strategy for the synthesis of 4β-triazole-linked glucose podophyllotoxin conjugates. δ 5.9-6.3 ppm, usually with a coupling constant J 3,4 < 5.0 Hz, indicating a cis-relationship between H-3 and H-4. The formation of the triazole ring was confirmed by the resonance of its C 5" -H signal (δ 7.8-8.2 ppm) in the aromatic region in the 1 H-NMR spectra, which was further supported by two characteristic carbon signals at around 145 ppm and 126 ppm in the 13 C-NMR spectra. The coupling constant of the anomeric proton of the glucose residue (J 1"',2"' ) is typically <4.0 Hz for the α-linkage and >7.6 Hz for the β-linkage.
In addition, the anomeric carbon of an α-glucoside always has a lower chemical shift than the corresponding β-glucoside in the 13 C-NMR spectra. Poor water-solubility is a common problem in developing podophyllotoxin derivatives for therapeutic use. For the glucose-podophyllotoxin conjugates described in this study, compounds with a peracetylated or perbutyrylated glucose residue are slightly soluble in water. In general, the conjugates with a free glucose residue are soluble in water and methanol, while those with a peracetylated or a perbutyrylated glucose residue are more soluble in chloroform. For example, at room temperature compound 29 with a perbutyrylated glucose residue has a solubility of 1.7 mg/mL in water while compound 30 with a free glucose residue has a solubility of 13.3 mg/mL in water. Overall, the inclusion of the triazole-ring and the multiple triethylene glycol units improved the aqueous solubility of these compounds, and in the case when a free glucose residue is present, the compound becomes fairly soluble in water.

Evaluation of Biological Activity
All the 4β-triazole-linked glucose-podophyllotoxin conjugates 24-44 were tested for their anticancer activity against five human cancer cell lines, including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer). Etoposide and cisplatin were taken as control drugs and the anticancer activity data are presented in Table 2. Our first observation is that compounds having a free glucose residue (compounds 24, 27, 30, 33, 36, 39, and 42-44) mostly show weak activity (all having IC 50 > 40 μM, except 30), while several derivatives containing a peracetylated glucose residue (compounds 28, 31 and 37) show improved activity. The length of the linking spacer between the glucose moiety and the 1,2,3-triazole residue does not exhibit a uniform effect on the cytotoxic potency of these compounds. For example, among the set of compounds with a perbutyrylated sugar moiety and a methoxyl group at the C-4'-position (compounds 26, 32 and 38), compound 38 with the longest linking spacer (six ethylene glycol repeating units) is the most active compound against four out of five cancer cell lines tested, followed by 32 which has a shorter linker (three ethylene glycol repeating units), and 26 which has no linking spacer in-between is the least active. However, among the set of compounds with a perbutyrylated sugar moiety and a hydroxyl group at the C- Previously, Reddy and co-workers [28] reported the cytotoxic activity of several 4β-triazole-linked sugar-podophyllotoxin conjugates. The cytotoxic potency of compound 42 against A-549 cells reported in their paper was quite similar to our data, however, our findings that peracetylation and perbutyrylation of the glucose residue lead to increased activity are different from their observation [28]. Among all the synthesized compounds, several compounds (i.e., 32, 35 and 38) display significantly higher activity than etoposide (2), and in general derivatives containing a perbutyrylated D-glucose moiety are more active than other derivatives. n-Butyrate, a naturally occurring short-chain fatty acid, is a well known histone deacetylase (HDAC) inhibitor [37]. In recent years, HDAC inhibition has attracted much attention for the development of anticancer drugs because HDAC inhibitors are able to disrupt cell cycle progression or selectively induce apoptosis via depression of certain genes [38][39][40][41]. Thus, the butyrate species liberated from the hydrolysis of these butyrylated derivatives may have contributed to the apparently higher potency of these compounds. Further studies are needed in order to confirm the potential role of the butyryl substituents on the sugar residue.

General
Melting points were uncorrected. MS data were obtained in the ESI mode on API Qstar Pulsar instrument. HRMS data were obtained in the ESI mode on LCMS-IT-TOF (Shimadzu, Kyoto, Japan). NMR spectra were acquired on Bruker AV-400 or DRX-500 or Bruker AVANCE Ш-600 (Bruker BioSpin GmbH, Rheinstetten, Germany) instruments, using tetramethylsilane (TMS) as an internal standard. Column chromatography (CC) was performed on flash silica gel (200-300 mesh; Qingdao Makall Group Co., Ltd., Qingdao, China). All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates.

General Procedure for Fisher Glycosylation Catalyzed with H 2 SO 4 -on-Silica Gel (Preparation of 12-17)
cooling to room temperature, the reaction mixture was transferred to a short silica gel column and eluted using CHCl 3 /CH 3 OH = 9:1. The preparation of 12, 13 and 15 using the same method has been reported in the literature [32,33]. (14). 1

General Procedure for Acetylation and Butyrylation of the Glucoside (Preparation of 18-23)
To a solution of a propargyl glycosides 12-17 (1 mmol) in pyridine (4.0 mL) at 0 °C, acetic anhydride (or butyryl anhydride) (4.0 mL) was added. The reaction mixture was stirred overnight until the starting material disappeared as indicated by TLC. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The organic layer was washed with 10% aqueous hydrochloric acid (20 mL) and brine (20 mL). The organic layer was dried over magnesium sulfate and evaporated to give a residue, which was chromatographed on silica gel with petroleum ether/acetone = 4:1→2:1 to give the peracetylated or perbutyrylated product. The preparation of 18 using the same method has been reported in the literature [34]. (19). Yield: 96%.  (22). Yield: 92%.

Conclusions
In conclusion, we have used an effective and facile Fisher glycosylation strategy to prepare glucose-bearing terminal-alkynes with a catalyst of H 2 SO 4 -silica. Then, all glycosides were subjected to peracetylation or perbutyrylation, and the resulting glycosylated terminal alkynes underwent click-reactions with azide derivatives of podophyllotoxin to yield a series of 4β-triazole-linked glucose-podophyllotoxin conjugates in high yields. All conjugated derivatives were screened for anticancer activity against a panel of five human cancer cell lines including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer). All these derivatives display different level of anticancer activity which can be affected by the nature of substituents on the glucose residue, the length of the linking spacer between the sugar and the triazole ring, and the substituent on the 4'-position of the E-ring of podophyllotoxin scaffold. Derivatives with a perbutyrylated glucose residue generally display higher anticancer activity than other derivatives. The two most active compounds 29 and 35, both having a perbutyrylated glucose residue and a 4'-OH on the E ring, are significantly more active than etopodide or cisplatin. Further investigation of these compounds in in vivo tumor models is necessary in order to evaluate their therapeutic potential for cancer treatment.