Design, Synthesis, and Cytotoxicity of Perbutyrylated Glycosides of 4β-Triazolopodophyllotoxin Derivatives

A series of novel perbutyrylated glycosides of 4β-triazolopodophyllotoxin derivatives were synthesized by utilizing the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Evaluation of cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using the MTT assay shows that some of these glycosylated derivatives have good anticancer activity. Among the synthesized compounds, compound 21a shows the highest activity, with IC50 values ranging from 0.49 to 6.70 μM, which is more potent than the control drugs etoposide and cisplatin. Compound 21a is characterized by a perbutyrylated α-D(+)-galactosyl residue, the absence of an additional linking spacer between the sugar residue and the triazole ring, as well as a 4'-OH group on the E ring of the podophyllotoxin scaffold.


Introduction
Podophyllotoxin (1, Figure 1), a well-known naturally occurring aryltetralin lignan extracted from the roots of Podophyllun peltatum, has been known to inhibit the assembly of tubulin into microtubules through tubulin binding, but the high toxicity of podophyllotoxin has limited its application as a drug in cancer chemotherapy [1][2][3][4]. The potent anticancer activity of 1 has led to extensive structural modifications for the discovery and development of new anticancer agents. Etoposide (2, Figure 1) [5] is a semisynthetic glucosidic cyclic acetal of podophyllotoxin which is in clinical use as an antineoplastic agent against various cancers, including small-cell lung cancer, non-Hodgkin's lymphoma, leukemia, Kaposi's sarcoma, neuroblastoma and soft tissue sarcoma [3,[6][7][8][9][10][11][12]. However, the therapeutic use of 2 is often overcome by the problems of drug resistance, myelo-suppression and poor oral solubility. In order to overcome drug resistance and improve topoisomerase II inhibition, various structure modifications of podophyllotoxin have been made [13,14], novel dimeric podophyllotoxins obtained by condensation of thiocolchicine and/or podophyllotoxin with six different dicarboxylic acids, having a marked ability to inhibit the polymerization of tubulin in vitro and the spacer unit was found to have a significant effect on biological activity [15]. According to structure-activity relationship (SAR) studies, 4 ′ -demethylation, 4-epimerization, trans-lactone D ring with 2α, 3β configuration and free rotation of ring E were essential to maintain the anticancer activity of podophyllotoxin derivatives as topoisomerase-II inhibitors [16,17]. Studies have also demonstrated that substitution at C-4 is tolerable to significant structural diversification. Traditional cancer chemotherapy is often accompanied by systemic toxicity to the patient, therefore the development of new antitumor drugs with increased selectivity and reduced toxicity is highly desirable. Recently, antibody-drug conjugates (ADCs) that use antibodies to deliver a potent cytotoxic compound selectively to tumor cells were approved for cancer therapy: CD30-targeting brentuximab vedotin for use in Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL), and HER2-targeting ado-trastuzumab emtansine (T-DM1) for use in metastatic breast cancer [18]. Carbon nanomaterials are a source of materials that show unique biological applications for their π-electron cloud and structures. Species such as carbon nanotubes (CNTs), fullerenes, graphenes, carbon nanoparticles, nanodiamonds, carbon nanohorns and carbon nanocaps are common in the formulations of these nanomaterials as biosensors, imaging probes, drug and gene delivery systems, and nanomedicine [19]. By combination with other materials, the nanoarchitectures of nanocarbons can be formed into structures of different dimensions and properties for biological applications, especially cell growth, sensing, and control [20].
In recent years, the preparation of glycoconjugates of small molecule anticancer drugs has become an attractive strategy in order to improve drug efficacy. The clinically widely prescribed anticancer drug etoposide (2) is a β-D-glucopyranoside of 4'-demethylepipodophyllotoxin [21][22][23]. The anticancer activity of other types of podophyllotoxin glycosides, e.g., α-glucopyranoside, α/β-galactopyranoside, α/β-mannopyranoside, etc., has not been well studied. In our previous study [24], we reported 4β-triazole-linked glucose podophyllotoxin conjugates as a new class of antitumor compound; it was found that podophyllotoxin derivatives with a perbutyrylated glucose residue showed high activity. Reported here are the chemical synthesis of a series of perbutyrylated glycosides (D-Gal/D-Man/D-Xyl) of 4β-triazolopodophyllotoxin derivatives (3, Figure 1) conjugated with a specific monosaccharide residue and their in vitro 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).
Click chemistry involves a terminal alkyne and an azide that undergo a copper-catalyzed [3+2]-cycloaddition to generate a triazole ring [27,32]. There have been numerous reports documenting the best reaction conditions for this cycloaddition reaction [32,33]. It appears that the type of catalyst (copper species), the additive, the solvent, and the reaction time can all affect the yield of this addition reaction. We did a quick screening for the reaction conditions that would work best for our substrates. Thus alkyne 12a was reacted with 4β-azidopodophyllotoxin 18 [24,34] under different reaction conditions to give the 1,2,3-triazole derivative 20a (Scheme 2). The reaction conditions and the respective yields are listed in Table 1. Note: a nr: no reaction.
As can be seen in Table 1, the reaction occurred with different solvents in the presence of CuSO4·5H2O and sodium L-ascorbate within 2 h (Entries 1-4). It is found that t-BuOH/H2O (1:2) as the solvent provided the highest yield. No transformation occurred in the presence of t-BuOH alone as the solvent (Entry 5). Using the combination of Cu(OAc)2 and sodium L-ascorbate as the source of Cu(I) species [35], the reaction time can affect the yield significantly (Entries 6,7). In the case of CuI-catalyzed reactions [32,33], the solvent was also found to influence the reaction rate (Entries 8-9); however, the reaction yield was not further improved compared to CuSO4·5H2O/sodium L-ascorbate system (Entries 1-4). Subsequently, CuSO4·5H2O/sodium L-ascorbate with t-BuOH/H2O (1:2) as the solvent and the reaction time of 2 h (Entry 2) was chose as the condition for the CuAAC reaction of all substrates reported herein.
The azides 18 and 19 [24,34] were allowed to react with the above terminal alkynes (12a/b-17a/b) in the presence of CuSO4·5H2O, sodium ascorbate in t-butyl alcohol and water (1:2) at room temperature to give glycosylated 4β-triazolopodophyllotoxin derivatives 20a/b-31a/b in excellent yield (Scheme 3).  All the products were characterized by 1 H-NMR, 13 C-NMR, ESI-MS, and HRESI-MS. In the 1 H-NMR spectra, the formation of the podophyllotoxin triazoles was confirmed by the resonance of the C 5" -H signal (δ 7.72-8.33 ppm) of the triazole ring in the aromatic region, which was further supported by two characteristic carbon signals at around 123 ppm and 126 ppm in the 13 C-NMR spectra. The configuration at the C-4 position for target compounds 20a/b-31a/b was confirmed based on the J3,4 coupling constant, which is typically < 5.0 Hz for 4β-substituted compounds due to a cis relationship between H-3 and H-4 [36]. ESI-MS and HRESI-MS of all compounds showed the [M+Na] + or [M+H] + adduct as the molecular ion.
Two representative compounds (21a and 26b) were selected for investigation of the chemical stability in aqueous phase in comparison of podophillotoxin (1). The results indicate that compounds 21a and 26b exhibit better chemical stability under the specific conditions (37 °C, pH = 7.0, Figure 2). Obviously, compound 26b is the most stable one, and having the appropriate length of the linking spacer between the sugar and triazole ring and 4'-OCH3 on the E ring improved the chemical stability of podophillotoxin. These improvements make them much more drug-like than the natural parent podophillotoxin (1), and would be promising for the future further development.

Evaluation of Biological Activity
All the perbutyrylated glycosides of 4β-triazole-podophyllotoxin derivatives 20a/b-31a/b 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 (2) and cisplatin were taken as reference compounds. The screening procedure was based on the standard MTT method [37], and the anticancer activity data are presented in Table 2. Among these compounds 21a shows the most active inhibition against all five cancer cell lines tested, with IC50 values ranging from 0.49 to 6.70 μM. Compound 21a displays higher cytotoxic potency than the control drug etoposide (2) against four of the five cancer cell lines tested. Some other compounds also exhibit promising antitumor potency against one or more cancer cell lines. Against the HL-60 cancer cell line, compounds 20a, 24a and 26b demonstrate cytotoxicity with an IC50 below 10 μM. Most of the other compounds display moderate to weak cytotoxicity against all cancer cells tested.
In our previous study on glucosylated podophyllotoxin derivatives linked via a 4β-triazole ring [24], we have shown that the length of the linker between the glucose moiety and the 1,2,3-triazole residue, the substituents on the glucose residue as well as on the 4 ′ -position of the E ring can significantly affect the anticancer potency of these compounds. Similar structure-activity relationships are also observed for the series of compounds reported here. The present study also shows that different sugar residues conjugated with 4β-triazolopodophyllotoxin also influence the anticancer activity of these compounds. The most active compound (21a) contains a D-galactose residue, and all other compounds containing a D-mannose or D-xylose residue (24a/b-31a/b) display moderate to weak activity. The majority of the compounds with an α-glycosdic linkage are more active than those with a β-linkage (20a vs. 20b, 21a vs. 21b, 24a vs. 24b, 28a vs. 28b).

General
Melting points were uncorrected. MS data were obtained in the ESI mode on API Qstar Pulsar instrument (MDS Sciqaszex, Concord, ON, Canada). HRMS data were obtained in the ESI mode on a LCMS-IT-TOF instrument (Shimadzu, Kyoto, Japan). NMR spectra were acquired on Bruker AV-400 or DRX-500 or Bruker AVANCE Ш-600 instruments (Bruker BioSpin GmbH, Rheinstetten, Germany), 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 the Synthesis of Compounds 12a/b-17a/b
D-sugar (5 mmol) was suspended in propargyl alcohol 4/5 (25 mmol) and stirred at 65 °C. H2SO4-silica (25 mg) was added and stirring was continued until all solids had dissolved (~2.5 h). After cooling to room temperature, the reaction mixture was transferred to a short silica gel column (CHCl3:CH3OH = 15:1→9:1) to afford the desired propargyl glycosides 6-11. Then, to a solution of a propargyl glycosides 6-11 (1 mmol) in pyridine (4 mL) at 0 °C butyryl anhydride (4 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 perbutyrylated product 12a/b-17a/b.

Click Chemistry-General Procedure
To a solution of a terminal-alkyne 12a/b-17a/b (0.1 mmol) and 4β-azidopodophyllotoxin analogues 18 or 19 (0.1 mmol) in t−BuOH-H2O (1:2, 1.0 mL) at room temperature were added copper (II) sulfate pentahydrate (0.01 mmol) and sodium ascorbate (1.0 M in H2O, 3 drops). The reaction mixture was stirred at room temperature for 2 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 × 10 mL), and the combined organic layer was dried over sodium sulfate. The solvent was evaporated and the residue was purified by column chromatography to afford the cycloaddition product 20a/b-31a/b (82%-92%).