In the present study, the reagents and solvents were purchased from Sigma Aldrich; Merck Millipore (Darmstadt, Germany). TLC (0.25 mm silica gel 60 F254; Merck Millipore) plates were used to monitor the reaction progress. Compound purification was performed by column chromatography using silica gel 60–120 mesh. Bruker DPX 410 and DPX 540 NMR instruments were used to record ¹H NMR and ¹³C NMR spectra, TMS as the internal standard and CDCl₃ as the solvent. The chemical shifts are expressed in d ppm and coupling constants in Hertz.

In a typical procedure, the apigenin-7-methyl ether (1 mmol), propargyl bromide (1.1 mmol) and K₂CO₃ (1.5 mmol) were added to a round-bottom flask containing methanol (10 ml). The reaction mixture was then vigorously stirred on a magnetic stirrer at 80°C until the initial material had completely disappeared, which was monitored by TLC. Following completion, the reaction mixture was partitioned with EtOAc and water three times. The collected organic layers were concentrated in a vacuum and purified by column chromatography using silica gel (60–120 mesh) and EtOAc: hexane as eluting solvents to produce compound 2 at 98% yield.

Compound 3 (1 eq) and respective organic azides (1.1 eq) were added to a round bottom flask containing 15 ml of 1:1 water: ethanol mixture, to which 10 mol% each of sodium ascorbate and CuSO₄.5H₂O was added. The reaction mixture was stirred on a magnetic stirrer at room temperature until its completion. The crude reaction mixture was then partitioned using aqueous ethylacetate. The collected ethylacetate layer was dried over anhydrous magnesium sulphate and subjected to column chromatography using EtOAc: hexane as eluting solvents to produce the pure desired products (3a-g) in quantitative yields.

The details of product 3a were as follows: White crystalline solid, yield: 93%; 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J=6.3 Hz, 1H), 7.49 (d, J=7.5 Hz, 2H), 7.37 (s, 1H), 7.23–7.17 (m, 3H), 7.01 (d, J=7.4 Hz, 2H), 6.33 (s, 1H), 6.19 (d, J=1.5 Hz, 1H), 6.13 (d, J=1.4 Hz, 1H), 5.16 (s, 2H), 4.76 (s, 2H), 3.81 (s, 3H). 13C NMR (125 MHz,) δ 182.28, 166.02, 163.74, 161.60, 159.47, 140.09, 137.73, 136.17, 132.36, 129.19, 127.52, 127.24, 126.97, 125.85, 125.31, 115.31, 105.68, 104.77, 98.01, 93.64, 63.36, 57.74, 56.03.

The details of product 3b were as follows: White solid, yield 89%; 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J=7.5 Hz, 2H), 7.42–7.31 (m, 3H), 7.02 (d, J=7.6 Hz, 2H), 6.82 (d, J=7.5 Hz, 2H), 6.30 (s, 1H), 6.21 (d, J=1.4 Hz, 1H), 6.13 (d, J=1.4 Hz, 1H), 5.23 (s, 1H), 3.81 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 182.28, 166.02, 163.74, 161.60, 161.27, 159.47, 136.00, 133.10, 128.4, 128.06, 127.24, 125.3, 117.03, 115.3, 105.68, 104.77, 98.01, 93.64, 57.74, 56.03.

The details of product 3c were as follows: White solid, yield: 89%; 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J=7.5 Hz, 2H), 7.41–7.31 (m, 3H), 7.02 (d, J=7.5 Hz, 2H), 6.81 (d, J=7.5 Hz, 2H), 6.27 (s, 1H), 6.19 (d, J=1.4 Hz, 1H), 6.09 (d, J=1.4 Hz, 1H), 5.27 (s, 1H), 3.81 (s, 3H), 3.80 (s, 3H).

The details of product 3d were as follows: White solid, yield: 93%; 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J=7.5 Hz, 2H), 7.48 (m, 1H), 7.39 (s, 1H), 7.15 (m, 1H), 7.10 (m, 1H), 7.07–6.97 (m, 3H), 6.52 (s, 2H), 6.26 (d, J=1.4 Hz, 1H), 6.11 (d, J=1.6 Hz, 1H), 5.18 (s, 2H), 3.81 (s, 3H).

The details of product 3e were as follows: Yellowish solid, yield: 93%; 1H NMR (400 MHz, CDCl3) δ 7.98 (m, 1H), 7.92 (d, J=1.5 Hz, 1H), 7.51 (d, J=7.5 Hz, 2H), 7.42–7.32 (m, 3H), 7.03 (d, J=7.5 Hz, 2H), 6.32 (s, 1H), 6.21 (d, J=1.4 Hz, 1H), 6.14 (d, J=1.4 Hz, 1H), 5.22 (s, 2H), 3.79 (s, 3H).

The details of product 3f were as follows: Yellow amorphous powder, yield: 93%; 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J=7.5 Hz, 2H), 7.11 (s, 1H), 7.03 (d, J=7.5 Hz, 2H), 6.30 (s, 1H), 6.25 (d, J=1.4 Hz, 1H), 6.21 (d, J=1.4 Hz, 1H), 5.22 (s, 2H), 3.82 (s, 3H), 2.93 (m, 2H), 1.26–1.37 (m, 4H), 0.91 (t, 3H, J=7.2 Hz).

The details of product 3g were as follows: Yellow amorphous powder, yield: 93%; ¹H NMR (400 MHz, CDCl₃) δ 7.50 (d, J=7.4 Hz, 2H), 7.09 (s, 1H), 7.03 (d, J=7.4 Hz, 2H), 6.31 (s, 1H), 6.27 (d, J=1.4 Hz, 1H), 6.22 (d, J=1.4 Hz, 1H), 5.22 (s, 2H), 3.82 (s, 3H), 2.95 (m, 2H), 1.29–1.38 (m, 6H), 0.92 (t, 3H, J=7.2 Hz).

The antiproliferation effect of the novel triazole analogs of apigenin-7-methyl ether against three human ovarian cancer cell lines, OVCAR-3, Caov-3, and SKOV3, (Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) was investigated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were cultured at the density of 1×10⁶ cells/well in 96-well plates for a time period of 12 h at 37°C. The cells were then subsequently treated with 0–200 µM doses of the apigenin-7-methyl ether derivatives for 24 h at 37°C. Following this, 20 µl of MTT solution was added to each well. Prior to the addition of 500 µl of DMSO, the medium was completely removed. For solubilizing the MTT formazan crystals, dimethylsulfoxide (500 µl) was added. The absorbance at 570 nm was measured using an ELISA plate reader. As the derivative 3d was found to be most active, only this molecule was used for further experimentation.

To investigate the effect of the 3d derivative on the colony formation potential of SKOV3 cells, the cells were collected at the exponential growth phase and then counted using a hemocytometer. The platting of the cells was performed at 200 cells/well, and the plates were then incubated at 37°C for 48 h to permit the cells to adhere. This was followed by the addition of various concentrations (0, 5, 10, and 20 µM) of 3d. Following treatment with 3d, the cells plates were incubated for 6 days at 37°C. Following 6 days of incubation, the cells were washed with PBS and fixed with methanol. Subsequently, the cells were treated with crystal violet for 30 min at room temperature and then counted under a light microscope (magnification, ×200).

The SKOV3 cells were seeded at the density of 1×10⁶ cells/well in 6-well plates and then treated with 0, 10, 20 and 40 µM 3d for the time period of 24 h at 37°C. This was immediately followed by 4′,6-diamidino-2-phenylindole (DAPI) staining at 25°C for 5 min. The cell samples were then examined and images were captured with a fluorescence microscope (magnification, ×200). To estimate the apoptotic cell populations, the SKOV3 cells were seeded at a density of 1×10⁶ cells/well in 6-well plates and treated with varied concentrations (0, 5, 10, and 20 µM) of 3d for 24 h at 37°C. The cells were then collected and washed with PBS. The cells were then incubated with Annexin V/FITC and PI for 15 min and the apoptotic cell populations were estimated by flow cytometry (BD Biosciences, San Jose, CA, USA). The estimated percentage of cells in each phase of the cell cycle was quantified using WinMDI software v2.0 (Informer Technologies, Inc., Los Angeles, CA, USA).

The SKOV3 cells were seeded at a density of 2×10⁵ cells/well in a 6-well plate and incubated at 37°C for 24 h and treated with 0, 5, 10, and 20 µM of 3d for 24 h at 37°C in 5% CO₂ and 95% air. Subsequently, the cells from all samples were collected, washed twice PBS and resuspended in 500 µl of DCFH-DA (10 µM) for ROS estimation and DiOC₆ (1 µmol/l) for MMP at 37°C in a dark room for 30 min. The samples were then examined immediately using a flow cytometer and BD FACSuite software v1.0 (BD Biosciences, San Jose, CA, USA).

Protein expression was determined by western blot analysis. Briefly, the cells were lysed in a lysis buffer (20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% Nonidet P 40, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 mM protease inhibitor cocktail and 10% phosphatase inhibitor cocktail). The proteins present in the cell extracts were quantified using a BCA assay and proteins (50 µg/lane) from each sample were resolved by SDS-PAGE on a 10% gel. This was followed by transference onto a nitrocellulose membrane. The membrane was then treated with non-fat milk (5%) in PBS, and then incubated with a suitable primary antibody: B-cell lymphoma 2-associated X protein (Bax; cat. no. sc-6236) and Bcl-2 (cat no. sc-509) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) overnight at 4°C (dilution 1:1,000), followed by incubation with horseradish peroxidase-conjugated (cat. no. 9003-99-0) and anti-rabbit secondary antibody (cat. no. sc-2372) (dilution 1:1,000) for 1 h at room temperature. The western blots were then observed in an ECL western blot analysis system (GE Healthcare Life Sciences, Chalfont, UK).

The experiments were repeated three times and results are presented as the mean ± standard deviation. The significance was determined, compared with the untreated control, using one way analysis of variance and Tukey's test with GraphPad prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.01 was considered to indicate a statistically significant difference.

In the present study, apeginin-7-methylether (compound 1) was isolated from the ethanolic extract of leaves of Aquilaria sinensis. The isolated natural product (compound 1) was subjected to propargylation using propargyl bromide in presence of base K₂CO₃ to give compound 2. Compound 2 was then reacted with different substituted organic azides under click chemistry conditions (Fig. 1) to produce desired 1,2,3-triazole products in quantitative yields. In the ¹H NMR, products were easily identified by a characteristic singlet for H-5 in the 1,2,3-triazole moiety, which appeared as singlet downfield (~7.5 ppm) with other aromatic protons. All the prepared triazolyl analogs were characterized by ¹H NMR, ¹³CNMR and MS spectroscopic analysis.

To investigate the antiproliferative role of synthesized compounds on three human ovarian cancer cell lines (OVCAR-3, Caov-3, and SKOV3), the cells were treated with different concentrations of the synthesized compounds and the IC₅₀ was determined for all compounds (Table I). Compound 3d exhibited a potent antiproliferative effect against SKOV3 cells in a dose-dependent manner with an IC₅₀ of 10 µM (Fig. 2). In the formazan crystal assay, it was revealed that administering 3d to cells reduced the number of formazan crystals in a concentration-dependent manner (Fig. 3). As 3d exhibited highest activity against the SKOV3 cells, this cell line was used for further experimentation.

Following treatment with the different concentrations of compound 3d, apoptosis was detected by DAPI staining. The results indicated that compound 3d caused apoptosis in a concentration-dependent manner, as evident from the increased density of white-colored nuclei (Fig. 4). The apoptotic cell populations were further estimated by annexin V/PI staining and it was observed that the apoptotic cell populations increased from 0.05% in the control to 39.62% at 20 µM concentrations of 3d (Fig. 5). In addition, this was associated with the increase in the expression of Bax and a decrease in the expression of Bcl-2 (Fig. 6).

The potential of 3d to induce apoptosis, as observed through DAPI staining, indicated that 3d may trigger the production of intracellular ROS. Therefore, the present study estimated the ROS level at different concentrations of 3d for 48 h. The results showed that the intracellular ROS levels of the treated cells increased up to 255%, compared with the untreated cells (Fig. 7). This result suggested that compound 3d is an effective molecule for stimulating the generation of ROS in SKOV3 cells.

The generation of ROS causes mitochondrial mutilation and disrupts the outer mitochondrial potential, ultimately leading to the discharge of death-promoting proteins (16). Therefore, the present study investigated whether compound 3d decreased the MMP in the SKOV3 cells administrated with various doses (0–20 µM). The compound 3d-administrated SKOV3 cells showed a considerable decrease in MMP in a dose-dependent manner. The MMP decreased up to 63% at 20 µM of compound 3d, compared with that in the untreated control (Fig. 8).