Podophyllotoxin (1, 0.7 mmol) was added to a solution of triethylamine (14 mmol) in dichloromethane (10 ml), and then chloroacetyl chloride (7 mmol) in dichloromethane (4 ml) was added dropwise to the solution. The reaction solution was stirred at room temperature for 2 h, quenched by ammonium chloride solution, and extracted with dichloromethane. The organic layer was washed with water, dried over Na₂SO₄, filtered, and concentrated by evaporation in vacuo to give the crude intermediate, which was further purified via column chromatography (DCM/MeOH = 100:1) to give the key intermediate 11.

11: brown solid, yield 89%, ¹H NMR (400 MHz, DMSO-d₆) δ 7.02 (s, 1H, Ar–H), 6.56 (s, 1H, Ar–H), 6.28 (s, 2H, Ar–H), 5.99 (d, J = 6.4 Hz, 2H, O–CH₂-O), 5.91 (d, J = 9.2 Hz, 1H, CH–Ar), 4.54 (d, J = 14.8 Hz, 1H, CH₂-Cl), 4.53 (d, J = 4.4 Hz, 1H, CH–Ar), 4.45 (d, J = 15.2 Hz, 1H, CH₂-Cl), 4.31 (t, J = 7.2 Hz, 1H, CH–CH₂–O), 4.16 (t, J = 8.8 Hz, 1H, CH–CH₂–O), 3.60 (s, 6H, 3′,5′-OCH₃), 3.57 (s, 3H, 4′-OCH₃), 3.37 (dd, J = 4.8 Hz, 1H, CH–CH₂-O), 3.76–3.66 (m, 1H, O-C–CH); ¹³C NMR (100 MHz, DMSO-d₆) δ 174.24, 168.39, 152.45, 147.87, 147.33, 136.73, 135.94, 132.78, 128.51, 109.66, 108.30, 107.76, 101.85, 74.77, 71.07, 60.32, 56.10, 44.46, 43.17, 41.95, 38.61.

To a solution of intermediate (11, 0.2 mmol) and coumarin (7 or 8, 0.2 mmol) in acetonitrile (3 ml), catalytic amount potassium iodide and cesium carbonate (0.2 mmol) were added. After reflux for 4 h, the mixture was filtered and washed with dichloromethane. Then, the solution was removed by evaporation in vacuo, and the crude product was purified by flash column chromatography (DCM/MeOH = 200:1) to yield the target compound.

12a: white solid, yield 48%, ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (d, J = 8.8 Hz, 1H, Ar–H), 6.98 (d, J = 2.4 Hz, 1H, Ar–H), 6.96 (dd, J = 8.8, 2.4 Hz, 1H, Ar–H), 6.87 (s, 1H, Ar–H), 6.56 (s, 2H, Ar–H), 6.52 (s, 1H, Ar–H), 6.25 (d, J = 1.2 Hz, 1H, Ar–H), 5.99 (d, J = 7.6 Hz, 2H, O–CH₂-O), 5.80 (d, J = 5.6 Hz, 1H, CH–Ar), 4.94 (dd, J = 29.6, 16.4 Hz, 2H, O-C–CH₂-O), 4.46 (dd, J = 9.6, 6.8 Hz, 1H, CH–Ar), 4.34 (dd, J = 9.6, 2.4 Hz, 1H, CH–CH₂–O), 4.30 (d, J = 4.8 Hz, 1H, CH–CH₂–O), 3.74 (s, 6H, 3′,5′-OCH₃), 3.64 (s, 3H, 4′-OCH₃), 3.63–3.59 (m, 1H, CH–CH₂-O), 3.10–3.04 (m, 1H, O-C–CH), 2.41 (s, 3H, Ar–CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.70, 168.59, 160.92, 160.54, 155.00, 153.82, 153.22, 147.94, 146.74, 138.77, 136.57, 132.83, 127.05, 114.27, 112.69, 112.03, 109.26, 108.14, 106.20, 102.08, 73.48, 70.66, 65.29, 60.42, 56.38, 55.35, 44.06, 43.80, 18.58; HRMS-ESI (m/z): calcd for C₃₄H₃₀O₁₂Na [M+Na]⁺ 653.1635, found 653.1638. High-performance liquid chromatography (HPLC), 99.49%.

12b: white solid, yield 50%, ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 9.6 Hz, 1H, Ar–H), 7.65 (d, J = 8.8 Hz, 1H, Ar–H), 6.99 (d, J = 2.4 Hz, 1H, Ar–H), 6.95 (dd, J = 8.8, 2.4 Hz, 1H, Ar–H), 6.88 (s, 1H, Ar–H), 6.56 (s, 2H, Ar–H), 6.52 (s, 1H, Ar–H), 6.34 (d, J = 9.6 Hz, 1H, Ar–H), 5.99 (d, J = 6.8 Hz, 2H, O–CH₂-O), 5.80 (d, J = 6.0 Hz, 1H, CH–Ar), 4.93 (dd, J = 26.8, 16.4 Hz, 2H, O-C–CH₂-O), 4.45 (dd, J = 9.6, 6.8 Hz, 1H, CH–Ar), 4.34 (dd, J = 9.6, 2.0 Hz, 1H, CH–CH₂–O), 4.29 (d, J = 4.8 Hz, 1H, CH–CH₂–O), 3.74 (s, 6H, 3′,5′-OCH₃), 3.64 (s, 3H, 4′-OCH₃), 6.63–6.59 (m, 1H, CH–CH₂-O), 3.10–3.04 (m, 1H, O-C–CH); ¹³C NMR (100 MHz, DMSO-d₆) δ 177.70, 168.57, 161.02, 160.68, 155.64, 153.22, 147.93, 146.74, 144.68, 138.77, 136.57, 132.83, 130.05, 126.88, 113.48, 113.00, 106.20, 102.06, 73.47, 70.65, 65.30, 60.42, 56.38, 44.04, 43.79; HRMS-ESI (m/z): calcd for C₃₃H₂₈O₁₂Na [M+Na]⁺ 639.1478, found 639.1474. HPLC, 99.16%.

The in vitro antiproliferative activities of target compounds were investigated against human oral squamous carcinoma HSC-2, SCC-9, and A253 cells and human renal tubular epithelial HK-2 cells by Cell Counting Kit-8 (CCK-8) assay. The HSC-2, SCC-9, and A253 cell lines were purchased from Cobioer Biosciences (Nanjing, China), and HK-2 cell line was from KeyGen Biotech (Nanjing, China). The cells were placed into 96-well plates. After 24 h, cells were treated with 0.1% DMSO or different doses of test compounds for 72 h at 37°C in a CO₂ incubator. Subsequently, 100 μl of CCK-8 test solution was added and incubated for 3 h at 37°C. The absorbance value of each well was measured at 450 nm.

HSC-2 cells were incubated in six-well plates and then treated with 0.1% DMSO or different concentrations of test compounds for 48 h. Subsequently, cells were fixed with 70% ethanol at 4°C overnight. After that, cells were washed with phosphate-buffered saline (PBS) and stained with 100 μl of RNase A at 37°C for 30 min and then 400 μl of propidium iodide (PI) at 4°C for 30 min. The cell cycle was measured by flow cytometry.

HSC-2 cells were seeded into six-well plates, grown overnight at 37°C in a 5% CO₂ incubator, and then treated with 0.1% DMSO or different concentrations of test compounds for 48 h. Cells were washed twice with ice-cold PBS and then suspended with 500 μl of binding buffer. Subsequently, 5 μl of Annexin V-APC and 5 μl of 7-AAD were added, and the cells were incubated at room temperature for 15 min in the dark. The samples were analyzed by flow cytometry.

HSC-2 cells were seeded on six-well plates and grown overnight at 37°C in a 5% CO₂ incubator. Then, cells were incubated with 0.1% DMSO or different concentrations of compounds at 37°C for 48 h. Subsequently, cells were trypsinized, washed with PBS, and resuspended in incubation buffer containing JC-1 at 37°C for 20 min in a 5% CO₂ incubator. The dyeing cells were rinsed, suspended in incubation PBS, and then analyzed d by flow cytometry.

HSC-2 cells were seeded on six-well plates and grown overnight at 37°C in a 5% CO₂ incubator. Then, cells were incubated with 0.1% DMSO or different concentrations of compounds at 37°C for 48 h. Subsequently, cells were trypsinized, collected, washed with PBS, and stained with 100 μl of AO/EB dye at room temperature for 15 min in the dark. The dyeing cells were rinsed, suspended in incubation PBS, and then observed under a fluorescence microscope.

HSC-2 cells were seeded on 24-well plates, grown overnight at 37°C in a 5% CO₂ incubator, and then incubated with 0.1% DMSO or different concentrations of compounds at 37°C for 48 h. Cells were washed with PBS, fixed with 4% paraformaldehyde, and then further permeabilized with 0.5% Triton X-100 for 10 min. Subsequently, cells were blocked for 20 min by 50–100 μl of goat serum albumin at room temperature and then treated with α-tubulin primary antibody at 4°C for 2 h. Then, cells were treated with the Alexa Fluor 488-labeled goat anti-rabbit fluorescence secondary antibody at 37°C for 1 h in the dark, and the cell nuclei were labeled by DAPI at room temperature for 5 min. Finally, cells were visualized under a confocal microscope.

HSC-2 cells were seeded on six-well plates and grown to 80% at 37°C in a 5% CO₂ incubator. Then, cells were scratched using the tip of the pipette, then washed with PBS to remove the non-adherent cells, and incubated with 0.1% DMSO or different concentrations of compounds at 37°C for 48 h. The images of cell migration were photographed with an inverted fluorescent microscope at 0 and 48 h.

After being cultured, HSC-2 cells were treated with 0.1% DMSO or different concentrations of compounds at 37°C for 48 h. Then, cells were collected, washed twice with PBS, and lysed with cold lysis buffer, followed by centrifugation. Then, the total proteins were collected, and protein concentration was quantified using Bradford assay. Equal amounts of proteins were electrophoresed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) membrane. Membranes were blocked with 5% non-fat milk for 2 h, then washed, and incubated with primary antibodies overnight at 4°C. After being washed, the membranes were treated with secondary antibodies at room temperature for 2 h and then visualized using an ECL Western blotting kit.

The antiproliferative activities of target compounds (12a–b), lead compound podophyllotoxin (1), 4-methylumbelliferone (7), and 7-hydroxycoumarin (8) against human oral squamous carcinoma HSC-2, SCC-9, and A253 cells and human renal tubular epithelial HK-2 cells were screened using CCK-8 assay in vitro. 5-FU, etoposide, and cisplatin were used as positive compounds. The IC₅₀ values were calculated and shown in Table 1. Firstly, two hybrids exhibited significant cytotoxicity against all three human oral cancer cell lines with IC₅₀ values varying from 0.226 ± 0.021 to 0.349 ± 0.063 μM, which were more potent than positive compounds 5-FU, etoposide, and cisplatin. Moreover, compound 12b containing the 7-hydroxycoumarin group possessed more potent cytotoxicity than 12a against HSC-2, SCC-9, and A253 cells with IC₅₀ values of 0.226 ± 0.021, 0.231 ± 0.05, and 0.25 ± 0.019 μM, respectively. As lead compounds, 4-methylumbelliferone (7) and 7-hydroxycoumarin (8) were observed to have no antiproliferative effect on all three oral cancer cell lines. And as expected, lead podophyllotoxin displayed excellent antiproliferative activity against not only all human oral cancer cell lines but also human normal cell line. Although the IC₅₀ values of podophyllotoxin against all human oral cancer cell lines were stronger than 12b, 12b showed less inhibition effect (IC_{50 =} 0.626 ± 0.043 μM) on the viability of HK-2 cells than podophyllotoxin (IC_{50 =} 0.04 ± 0.006 μM), which indicated that 12b had good selectivity between tumor and normal cells, and it exhibited much less toxicity on human normal cells. Moreover, hybrid 12b in the present study showed more anticancer potential than the conjugates of 4′-demethylepipodophyllotoxin and coumarin (Hao et al., 2019).

To detect whether cytotoxicity potency of compound 12b resulted from cell cycle progression, the effects of 12b on the cell cycle of HSC-2 cells were analyzed by flow cytometry after labeling with PI. HSC-2 cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. As seen in Figure 3, compared with the control group, treatment of HSC-2 cells with 0.1 μM of 12b showed a significant S accumulation, while higher concentrations (0.25 and 0.5 μM) of 12b caused cell cycle arrest at both S and G2 phases in a dose-dependent manner. In short, the cell cycle arrest of 12b could be responsible for the cytotoxicity in cancer cells.

Further efforts were conducted to conform whether the cytotoxicity of compound 12b in the oral cancer cells was related to the induction of apoptosis. The effects of 12b on the induction of apoptosis in HSC-2 cells were analyzed by flow cytometry after labeling with Annexin V-FITC/7-AAD. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. As shown in Figure 4, compared with the control group, 12b obviously induced apoptosis in HSC-2 cells in a dose-dependent manner.

In order to further confirm the effects of 12b on the mitochondrial membrane potential (MMP), which is an important marker of apoptosis (Tsujimoto and Shimizu, 2007), fluorescent probe JC-1 was used to detect the MMP. HSC-2 cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h, and then cells were dyed with JC-1 and further analyzed by flow cytometry. As shown in Figure 5, compared with the control group, MMP was significantly decreased in HSC-2 cells treated with 12b in a dose-dependent manner.

As compound 12b induced HSC-2 cells death via the apoptotic pathway, we further evaluated the morphological change using AO/EB staining assay. HSC-2 cells were treated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h, then stained with AO/EB, and tested by fluorescence microscopy. As depicted in Figure 6, few cells underwent apoptosis in the control group; however, after treatment with 12b, the cells were stained to light-orange fluorescence and showed chromatin condensation and shrinkage, indicating that 12b could induce apoptosis in HSC-2 cells.

To study whether the hybrid 12b was able to disrupt the organizations of microtubule network in HSC-2 cells, we investigated the effect of 12b on the microtubule network by immunofluorescence assay analysis. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. The microtubules were dyed with anti-tubulin antibody, and cell nuclei were stained with DAPI. As shown in Figure 7, the microtubule network in the control cells exhibited normal organization with slim and fibrous microtubules (green) around the cell nucleus (blue). However, in the 12b-treated cells, intracellular microtubules became notably stabilized and disordered in a dose-dependent manner, indicating the disruption of microtubule network organizations. Taken together, these data indicated that 12b could induce the depolymerization of the microtubule network, which may lead to cell cycle arrest and apoptosis.

Then, we further investigated the effect of 12b on the migration of HSC-2 cells using wound healing assay. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. The images were photographed at 0 and 48 h. And the migration rates of test compounds were quantified and calculated relative to untreated cells. In wound healing assay (Figure 9), compared with the control group, 12b inhibited HSC-2 cell migration in a dose-dependent manner. These results indicated that 12b had the ability to inhibit oral cancer cell migration.

Next, we explored the molecular mechanism behind 12b-induced cell cycle arrest in HSC-2 cells. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. Western blot was used to detect the relative levels of cycle-related proteins CDK2 and cyclin A2, which were essential for driving S and G2 cell-cycle phases (Hochegger et al., 2008), and β-actin was used as internal standard. Compared with the control group, the CDK2 and cyclin A2 in 12b-treated HSC-2 cells were suppressed in a concentration-dependent manner (Figure 10). Interestingly, a low concentration (0.1 μM) of 12b slightly inhibited the expression of CDK2 and cyclin A2, while higher concentrations (0.25 and 0.5 μM) of 12b significantly downregulated the levels of CDK2 and cyclin A2 in HSC-2 cells, which maybe cause the above results that a low concentration (0.1 μM) of 12b showed a significant S accumulation and higher concentrations (0.25 and 0.5 μM) of 12b caused cell cycle arrest at both S and G2 phases.

In order to characterize the molecular mechanisms of 12b-induced apoptosis, we explored the expression levels of cleaved caspase3 and cleaved PARP, two critical apoptosis-related proteins. HSC-2 cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. Western blot was used to detect the relative levels of cleaved caspase3 and cleaved PARP. As shown in Figure 11, we observed that 12b upregulated the relative levels of cleaved caspase3 and cleaved PARP in a concentration-dependent manner.

As mentioned above, 12b inhibited HSC-2 cell migration in wound healing assay. Thus, we evaluated the effects of 12b on the migration-related proteins in HSC-2 cells. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. Western blot was used to detect the relative levels of E-cadherin and MMP-2, which were associated with tumor proliferation and metastasis (Grelewski and Bar, 2013; Hu et al., 2016). As shown in Figure 12, it was observed that 12b obviously enhanced the expression of E-cadherin and reduced the expression of MMP-2 compared with the control group. These data showed that the antimigratory effects of 12b on HSC-2 cells were mediated by regulating the expression of E-cadherin and MMP-2.

Considering autophagy is another crucial programmed cell death (Levy and Thorburn, 2020), we next evaluated whether autophagy was involved in 12b-reduced HSC-2 cell death. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. Western blot was used to detect the relative levels of Beclin1 and LC3. It was found that 12b significantly induced autophagy by increasing the aggregation of Beclin1 and LC3 (Figure 13). Moreover, we observed that 12b upregulated the level of cleavage of LC3 (LC3-II). These results indicated that 12b induced autophagy in HSC-2 cells.

Many signaling pathways, such as AMPK and AKT/mTOR, play important roles in the regulation of autophagy and maintenance of cellular homeostasis and survival. AMPK acted as a positive regulator of autophagy (Mihaylova and Shaw, 2011), whereas AKT/mTOR pathway was reported to contribute to inhibiting autophagy (Xu et al., 2020). According to the above results, we next detected the effects of 12b on the autophagy-related pathways in HSC-2 cells. Cells were incubated with vehicle and 12b (0.1, 0.25, and 0.5 μM) for 48 h. Western blot was used to detect the relative levels of p-AMPK, p-AKT, and p-mTOR. As shown in Figure 14, we found that p-AMPK expression was upregulated in HSC-2 cells following incubation with 12b in a dose-dependent manner. Further investigation of the AKT/mTOR signaling pathway indicated that 12b downregulated phosphorylation of AKT, leading to downstream inhibition of mTOR phosphorylation in HSC-2 cells.