Syntheses and Anticancer Activities of Novel Glucosylated (-)-Epigallocatechin-3-Gallate Derivatives Linked via Triazole Rings

Novel glucosylated (-)-epigallocatechin-3-gallate derivatives 10 – 13 having the EGCG analogues conjugated to the D-glucosyl azide were synthesized by carrying out the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, and were evaluated for their cytotoxicities against a panel of ve human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) using MTT assays. Compounds 10 and 11 showed the highest levels of cytotoxicity against the HL-60 cells with IC 50 values of 4.57 μM and 3.78 μM, respectively, and showed moderate selectivity towards cancer cell lines. Compound 11 was also shown to induce apoptosis in HL-60 cells. Most notably, inclusion of the perbutyrylated glucose residue in an EGCG derivative was concluded to lead to increased anticancer activity.


Introduction
Tea (Camellia sinensis (Linnaeus) O. Kuntze) was rst taxonomically described in 1753 by Carl Linnaeus in SpeciesPlantarum. Two species of tea have been identi ed, namely black tea (Thea bohea) and green tea (Thea viridis) [1]. Four varieties of Camellia sinensis have been identi ed, including Camellia sinensis var. assamica, Camellia sinensis var. sinensis, Camellia sinensis var. dehungensis, and Camellia sinensis var. pubilimba, which are mainly distributed in the understory of forests of broad-leaved evergreen trees at altitudes of between 100-2,200 m [2]. The Chinese have been using tea as a drink since 3000 BC, and the subspecies: var. sinensis (China tea) and var. assamica (Assam tea) are found in China.
Green tea is one of the drinks most widely consumed by people around the world, perhaps second most after water, due to its health, sensory, stimulant, relaxing and cultural properties [3]. Catechins are the primary compounds responsible for the claimed health bene ts of green tea, including its antioxidant and anti-in ammatory properties. The major catechins in green tea including (-)-epicatechin (EC, 1), (-)epicatechin-3-gallate (ECG, 2), (-)-epigallocatechin (EGC, 3) and (-)-epigallocatechin-3-gallate (EGCG, 4) ( Figure 1) have been reported to display numerous biological activities [4][5][6]. EGCG is the most abundant catechin found in green tea and has been reported to display physiological activities stronger than those of the other catechins [7][8][9] and to display many types of biological activities including anti-oxidative, anti-in ammatory, anti-cancer, anti-infection and neuroprotective activities [10][11][12].
However, the use of EGCG is often hindered by problems such as being easily oxidized, readily degraded in aqueous solution and poorly intestinal absorbed in the intestines [13,14]. To obtain more potent analogues and overcome this problem of poor intestinal absorption, many semisynthetic derivatives such as permethyl EGCG [15], peracetyl EGCG [16], EGCG monoester derivatives [17], and EGCG glycosides [13,[18][19][20][21] have been developed. In recent years, the use of glycoconjugates of small-molecule anticancer drugs has become an attractive strategy for improving drug e cacy [22,23]. In our previous study, we reported the syntheses and cytotoxicities of glucosylated EGCG derivatives, we found that, in aqueous solution, EGCG glucosides displayed higher activities against cells of human breast cancer cell lines and higher levels of stability than did EGCG [21].
Due to the ability of terminal alkyne and an azides to undergo copper-catalyzed [3 + 2]-cycloadditions with azides to generate substituted triazole rings [24] and due to butyrate having been shown to be a histone deacetylase (HDAC) inhibitor and to display anticancer effects with promising therapeutic potential [25], we set out in the current work to chemically synthesized glucosylated (-)-epigallocatechin-3gallate derivatives linked via triazole rings and to characterize their in vitro anticancer activities against  ve human cancer cell lines, including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer) and SW480 (colon cancer). In addition, chemical informatics analyses of these compounds were carried out, and the chemical properties of the compounds were correlated with their anticancer activity.
The synthesized compounds 12 and 13 were further analyzed using 2D-NMR spectroscopy (Fig. 2); the heteronuclear multiple bond correlation (HMBC) of compound 12 showed a strong correlation between with C-2 and C-3, those for C 2′ -H and C 6′ -H occurred with C-2 and C 2′ ′ -H, that for C 6′ ′ -H occurred with C-11, and that for C 2 -H occurred with C-11 in both compounds 12 and 13.
In vitro anti-proliferative activity The triazole-linked glucose-(-)-epigallocatechin-3-gallate derivatives 10 -13 were evaluated for their cytotoxicities against ve human cancer cell lines, including HL-60, SMMC-7721, A-549, MCF-7 and SW480. The compounds EGCG and cisplatin were used as positive controls. The screening procedure was based on the standard MTT method [26]. Their activities were expressed as IC 50 values (concentration of drug inhibiting 50% cell growth) and the data are presented in Table 1.
The compounds having a free glucose residue namely compounds 12 and 13 show weaked activity levels (IC 50 > 40 μM) toward cells of the three cancer cell lines SMMC-7721, A-549 and SW480. In contrast, the derivatives containing each a each a perbutyrylated glucose residue, namely compounds 10 and 11 showed higher activity levels, and they showed the highest cytotoxicity levels agaisnt HL-60 cells, with IC 50 values of 4.57 μM and 3.78 μM, respectively; they were also found to more potent than the control drug EGCG, which displayed IC 50 > 40 μM against each of the ve cancer cell lines. Interestingly, all of the EGCG derivatives showed good levels of cytotoxicity against MCF cells with IC 50 values in the range 28.24-39.89 μM. Based on these results taken together, we concluded that perbutyrylation of the glucose residue of the EGCG scaffold lead to increased anticancer activity.

Selectivities of the compounds
To evaluate the degrees of selectivity of the most cytotoxic compounds, namedly 11 and 12, their growth inhibitory effects on cells of a normal human bronchial epithelial cell line (BEAS-2B) were measured ( Table 1). The selectivity index (SI) values of compounds 11, 12 and cisplatin are presented in Table 2. Compounds 11 and 12 showed moderate selectivity toward cancer cell lines with SI values in the range of 1.0-8.4 for all cells tested.

Induction of cell apoptosis
Given that the EGCG derivative 11 exhibited signi cant inhibitory activity of cancerous cell growth in HL-60 cells, we studied further the ability of compound 11 to induce cell death through apoptosis. To carry out this study, the tested HL-60 cells were stained with annexin V, and compound 11 was administered at a concentration of 8 μM. Signi cantly higher amounts of compound 11 were detected in HL-60 cells undergoing apoptosis than in the untreated control ( Fig. 3A-B). We also determined the expression levels of caspase-3 and PARP, which are the hallmarks of apoptosis and play crucial roles in the cellular process. For this purpose, samples of HL-60 cells were treated with compound 11, respectively, concentrations of 2, 4 and 8 μM for 12 h and the expression levels of caspase-3, PARP, cleaved-caspase-3 and cleaved-PARP were monitored using western blot analysis. The treatment of HL-60 cells with compound 11 was found to be associated with increased levels of expression of cleaved-caspase-3 and cleaved-PARP in a dose-dependent manner (Fig. 3C). Compared to the untreated control, compound 11 apparently induced a signi cant increase in the expressed levels cleaved-caspase-3 and cleaved-PARP and a decrease in those of caspase-3 and PARP.

Physicochemical property
As compounds 12 and 13 showed each weak activity in vitro, we calculated the Clogp values of 10 -13 by MarvinSketch version 5.3.8 [29], and the data are shown in Table 3. ClogP values of only 0.50 and -2.08 were calculated for, respectively, compounds 12 and 13. But higher ClogP values of 6.84 and 10.61 were calculated for, respectively, compounds 10 and 11. Based on these results, compounds 12 and 13 were expected to display lower of cell permeability than were compounds 10 and 11.

Solubility
We also determined the water solubilities of the EGCG and EGCG derivatives (10-13), and these results are shown in  . All reagents were commercially available and used without further puri cation unless indicated otherwise. The melting points were measured by using an X-4 melting point apparatus and were uncorrected. Optical rotations data were obtained using a Jasco P-instrument; HRMS data were obtained in ESI mode using LCMS-IT-TOF apparatus (Shimadzu, Kyoto, Japan); 1 H-NMR and 13 C-NMR spectra were recorded using a Bruker DRX-500 instrument (Bruker BioSpin GmbH, Rheinstetten, Germany) with tetramethylsilane (TMS) as an internal standard. Column chromatography (CC) was performed with a 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, which were visualized using ultraviolet light (254 nm) and/or 10% phosphomolybdic acid/EtOH. All cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) were obtained from a Shanghai cell bank in China.
A mixture of EGCG (2.3 g, 5 mmol), DMF (15 mL) and K 2 CO 3 (1.0 g, 7.5 mmol) was made at room temperature under nitrogen and stirred for 0.5 h. Propargyl bromide (0.3 mL, 7.5 mmol) was quickly added to the resulting mixture, which was then stirred at room temperature for 12 h until no starting material was detected according to TLC analysis. The solvent of the resulting mixture was evaporated under vacuum and the residue was puri ed using column chromatography with silica gel (CHCl 3 /CH 3 OH, 9:1→4:1) to afford the EGCG conjugates 8 (1.1 g, 45%) and 9 (0.8 g, 30%). was prepared, and copper(II) acetate (0.01 mmol) and sodium ascorbate (0.01 mmol) were added to this solution. This reaction mixture was stirred at room temperature for 2 h until no starting material was detected according to TLC analysis. The resulting mixture was evaporated under vacuum and the residue was puri ed using column chromatography with silica gel (CHCl 3 /CH 3 OH, 9:1) to afford the target cycloaddition product.    General procedure for the syntheses of α-D-glucopyranosyloxy-1,2,3-triazol-1-yl-(-)-epigallocatechin-3gallates (12 and 13) For each of the target compounds, a solution of 2,3,4,6-tetra-O-butyryl-α-D-glucopyranosyloxy-1,2,3-triazol-1-yl-(-)-epigallocatechin-3-gallate (10or11) (0.05 mmol) in CH 3 OH (1 mL) was prepared, and to this solution was added a KOH solution (0.15 mmol, dissolved in CH 3 OH). This mixture was stirred at 0 ºC for 72 h, and then neutralized with Dowex 50WX4-400 ion-exchange resin to pH = 7. The solvent of the resulting mixture was evaporated in vacuum and resulting the residue was puri ed using column chromatography with silica gel (CHCl 3 /CH 3 OH, 4:1) to afford the product. Cytotoxicity Assay MTT assays were conducted to evaluate the cell viabilities of the triazole-linked glucose-(-)epigallocatechin-3-gallate derivatives. Cells of ve human cancer lines, namely HL-60, SMMC-7721, A-549, MCF-7 and SW480, were seeded in 96-well plates and then exposed to the test compound at various concentrations in triplicate for 48 h. After the incubation, MTT (100 μg) was added to each well, and the incubation was continued for 4 h at 37 °C. After removal of the culture medium, the produced MTT formazan crystals were dissolved in DMSO (150 μL) and the OD of the resulting solution was measured at a wavelength of 492 nm using a microplate reader. The percent inhibition was calculated using the formula inhibition ratio (IR, %) = (1 − OD(sample)/OD(control)) × 100%. The experiments were carried out in triplicate, and the IC 50 (the concentration of drug that inhibits cell growth by 50%) values were determined.

Cell Apoptosis Assay
An annexin V/propidium iodide (PI) detection kit (BD Biosciences, PA, USA) was employed to quantify apoptosis using ow cytometry. For each experiment, HL-60 cells were seeded into each well of a 6-well plate at 5 × 10 5 cells/well and then treated with one of the EGCG derivatives. After the treatment, the collected cells were incubated in 100 μL of binding buffer, and then into the resulting suspension were added 5 μL of FITC annexin V and 10 μL of PI. Each mixture was gently vortexed and then incubated for 15 min at room temperature in the dark before taking the ow cytometry measurements (BD FACSCalibur) within 1 h.

Calculated Partition Coe cient
All structures of EGCG derivatives were built and energy minimized by applying the Tripos force eld. The Gasteiger-Huchel method was used to calculate charges. Energy minimization was performed by applying the Powell method with 2000 iterations. The calculated partition coe cient (ClogP) was obtained from MarvinSketch version 5.3.8. (www.chemaxon.org) [29].

Water Solubility Analysis
All compounds were subjected to water solubility analyses. Each compound was mixed in 200 μL of water in an Eppendorf tube at room temperature. An ultrasonic cleaner was used to maximize the amount of the compound that became dissolved. After 1 h of sonication, each sample was diluted and then ltered through a 0.45 μm MFS membrane for HPLC analysis to determine the concentrations of the tested compound [21]. Analytical HPLC was performed using an Agilent 1260 liquid chromatograph equipped with a ZORBAX SB-C18 (4.6 × 250 mm) column. Here, the detection wavelength was 280 nm, the injection volume was 10 μL. The ow rate was 1 mL/min. Abbreviation: NT = not tested. Table 2 The selectivity index of compounds 10, 11 and cisplatin to cancer cells as compared with BEAS-2B normal cell Line.