Semisynthesis and in Vitro Anti-cancer Effect of New Magnolol Derivatives on the Cell Proliferation, Apoptosis, Migration, and Invasion of Human Hepatocellular Carcinoma SMMC-7721 Cells

Abstract

Four new magnolol derivatives were synthesized and evaluated for their in vitro anti-cancer properties. Among these, compound 3 showed the most potent cytotoxic activity against the SMMC-7721, SUN-449, and HepG2 human hepatocellular carcinoma cell lines, with IC₅₀ values of 3.39, 4.11, and 6.88 µM, respectively. Compound 3 also induced apoptosis of SMMC-7721 cells by down-regulating Bcl-2 and Akt protein levels, up-regulating of Bax protein level, and cleaving caspase-9 and -3. In addition, transwell assays showed that compound 3 significantly suppressed the migration and invasion of SMMC-7721 cells, which was confirmed based on the down-regulation of hypoxia inducible factor-1α (HIF-1α), matrix metalloproteinase-2 and -9 (MMP-2, and MMP-9) protein levels.

Introduction

Hepatocellular carcinoma (HCC) is a major type of liver cancer that causes death in humans.^1,2) Owing to the lack of clear symptoms in the early stage of HCC, most patients are in the middle and advanced stages at the time of diagnosis. Although treatments for HCC have become more diverse, chemotherapy remains one of the main therapeutic strategies.³⁾ However, conventional chemotherapeutic drugs are associated with severe side effects and cancer drug resistance.^4,5)

Compared with synthetic anti-cancer drugs, natural products have several advantages, such as chemical diversity, low side effects, and circumvention of cancer drug resistance.^6–8) Magnolol, a natural lignan extracted from Magnolia officinalis (“Hou Po” in Chinese), exhibits various anti-cancer activities, including induction of apoptosis, inhibition of cell proliferation, and suppression of invasion, metastasis, and angiogenesis of cancer cells.^9–12) In a previous study, we reported that mono-substitution of a benzyl group with para-F atoms at the phenolic hydroxyl group of magnolol (compound 1) considerably increased the in vitro anticancer activity against MDA-MB-231 cells.¹³⁾ Nevertheless, compound 1 also has some disadvantages, such as pharmacological activity and poor water solubility, which need to be improved.

The Schiff base is the condensation product of primary amines with carbonyl compounds (RCH = NR′), and the C=N linkage plays an important role in determining its biological activity.¹⁴⁾ Many previous studies have reported that Schiff bases and their complexes show promising and significant antitumor properties.^15–18) Therefore, we hypothesized that the Schiff base moiety could improve the biological activity of the parent compound. In the present study, we modified the Schiff base moiety of compound 1 to obtain a series of new magnolol derivatives and investigated the in vitro anticancer effect of these derivatives on the cell proliferation, apoptosis, migration, and invasion of human hepatocellular carcinoma SMMC-7721 cells.

Results and Discussion

Chemistry

The starting material, 5,5′-diallyl-2-((4-fluorobenzyl)oxy)-1,1′-biphenyl-2′-ol (1), was reacted with paraformaldehyde to obtain compound 2 (Chart 1). Compound 2 was further modified through the Schiff base reaction to obtain compounds 3–5. The chemical structures of compounds 2–5 were characterized using ¹H-NMR, ¹³C-NMR, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

Chart 1. Synthetic Routes for Magnolol Derivatives

i) MgCl₂, POM, THF, TEA, 80 °C, 5 h. ii) Organic base (aminoguanidine carbonate, thiosemicarbazide, and semicarbazide hydrochloride), EtOH, acetic acid, 50 °C, 3 h.

In Vitro Cytotoxic Activity Against HCC Cells

As shown in Table 1, the magnolol derivatives 1–5 showed cytotoxic activity against three human HCC cell lines, namely SMMC-7721, SUN-449, and HepG2, with IC₅₀ values of 3.39 to 27.91 µM. The compound 2, produced by the aldehyde reaction, exhibited moderate cytotoxic activity against the SMMC-7721, SUN-449, and HepG2 cells cell lines, with IC₅₀ values of 7.86, 12.0, and 12.04 µM, respectively. Among these derivatives, compound 3 showed the most cytotoxic activity against the SMMC-7721, SUN-449, and HepG2 cells, with IC₅₀ values of 3.39, 4.11, and 6.88 µM, which was approximately 10.8, 9.3, and 4.2 times stronger than of parent compound 1, respectively. These results indicate that the attachment of the aminoguanidine moiety to the small molecules greatly enhanced their anti-cancer activity, which is consistent with the results of our previous study.¹⁹⁾

Table 1. Cytotoxic Activity of Magnolol Derivatives (1–5) Against for Three Human HCC Cells (IC₅₀, µM, 72 h)

	SMMC-7721	SUN-449	HepG2
Compound 1	36.61 ± 3.46	38.19 ± 2.43	29.16 ± 3.40
Compound 2	7.86 ± 0.95	12.0 ± 1.07	12.04 ± 1.67
Compound 3	3.39 ± 0.66	4.11 ± 0.26	6.88 ± 0.19
Compound 4	6.34 ± 0.28	8.52 ± 0.60	8.40 ± 0.16
Compound 5	16.62 ± 0.66	27.91 ± 1.26	22.31 ± 0.71
Paclitaxel	0.95 ± 0.23	2.49 ± 0.64	0.63 ± 0.11

Anti-proliferative Activity of Compound 3 on SMMC-7721 Cells in Vitro

To further explore the anti-proliferative activity of compound 3 on SMMC-7721 cells, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the cell survival rate after treatment with compound 3. As shown in Fig. 1, compound 3 showed significant anti-proliferative activity on SMMC-7721 cells in a concentration- and time-dependent manner, with IC₅₀ values of 9.13, 6.96, and 3.57 µM at 24, 48, and 72 h, respectively.

Fig. 1. Anti-proliferative Activity of Compound 3 on SMMC-7721 Cells

Compound 3 Induces Apoptosis of SMMC-7721 Cells

To determine whether compound 3 could induce apoptosis in SMMC-7721 cells, we used flow cytometry and Western blotting analysis. After 24 h, the apoptotic rate was 13.47%, 15.48%, and 29.42% for SMMC-7721 cells treated with 2.5, 5, and 10 µM of compound 3, respectively (Fig. 2A). As shown in Figs. 2B and 2C, the protein level of Bcl-2 and Akt decreased and that of the pro-apoptotic protein Bax gradually increased with increasing concentration of compound 3.

Fig. 2. Compound 3 Induced Apoptosis of SMMC-7721 Cells

(A) The apoptosis rate of SMMC-7721 cells treated with different concentrations (0, 2.5, 5 and 10 µM) of compound 3 was determined using flow cytometry analysis with annexin V/PI dual staining. (B) Western blotting analysis was used to detect the expression of Bax, Bcl-2, and Akt in SMMC-7721 cells treated with increasing concentrations (0, 2.5, 5 and 10 µM) of compound 3. (C) Quantitative analysis of Bax, Bcl-2, and Akt protein levels. (D) Western blotting analysis was used to detect the expression of caspase-9 and caspase-3 in SMMC-7721 cells treated with compound 3. (E) Quantitative analysis of cleaved caspase-9 and cleaved caspase-3 protein levels. * p < 0.05, ** p < 0.01 compared with the control (n = 3).

Numerous studies have demonstrated that anticancer agents induce intrinsic apoptosis via the activation of the caspase cascade, such as caspase-3, -8, and -9.^20,21) Consistent with the findings of previous studies, we found that compound 3 significantly increased caspase-9 and -3 activities in a concentration- dependent manner (Figs. 2D, 2E).

Compound 3 Suppresses the Migration and Invasion of SMMC-7721 Cells

Tumor invasion and metastasis are the main causes of treatment failure and death in patients with cancer, including HCC.^22,23) Therefore, transwell assays were performed to investigate the effects of compound 3 on the migration and invasion abilities of SMMC-7721. As shown in Figs. 3A and 3B, the number of cancer cells that crossed the transwell chamber was significantly inhibited in groups treated with 2.5 and 5.0 µM of compound 3 compared with that in the control (p < 0.05). These results suggested that compound 3 suppressed the migration and invasion of SMMC-7721 cells in a concentration-dependent manner.

Fig. 3. Compound 3 Suppressed Migration and Invasion of SMMC-7721 Cells

(A) The migration and invasion of SMMC-7721 cells treated with different concentrations (0, 1.25, 2.5 and 5 µM) of compound 3 was determined through by transwell assays. (B) The results of the quantification analysis are presented as the mean ± standard deviation. (C) Western blotting analysis was used to detect the expression of HIF-1α, MMP-2, and MMP-9 in SMMC-7721 cells treated with different concentrations (0, 2.5, 5, and 10 µM) of compound 3. (D) Quantitative analysis of HIF-1α, MMP-2, and MMP-9 protein levels. * p < 0.05, ** p < 0.01 compared with the control (n = 3).

Hypoxia inducible factor-1α (HIF-1α) plays a key role in tumor growth and processes that lead to epithelial transformation, angiogenesis, and promotes a series of adaptive changes in tumor cells.^24,25) The HIF-1α downstream proteins, matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), also facilitate cancer cell migration and invasion.²⁶⁾ Western blotting data showed that treatment with compound 3 significantly down-regulated the expression of HIF-1α, MMP-2, and MMP-9 protein levels (Figs. 3C, 3D).

Experimental

General Experimental Procedures

Human hepatocellular carcinoma SMMC-7721, SUN-449, and HepG-2 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RPMI-1640 was purchased from GIBCO (California, U.S.A.). Fetal bovine serum (FBS) was purchased from Sijiqing (Hangzhou, China). MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Trypsin, skimmed milk, radio immunoprecipitation assay (RIPA) lysate, phynomethanesulfonyl fluoride (PMSF), bicinchoninic acid (BCA) protein quantitative kit, Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit were purchased from Beyotime (Hangzhou, China). Enhanced chemiluminescence (ECL) solution and polyvinylidene fluoride (PVDF) membrane were purchased from Millipore (MA, U.S.A.). Paclitaxel was purchased from Yuanye BioTechnology Co., Ltd. (Shanghai, China). Transwell inserts were purchased from Corning Life Sciences (Corning, NY, U.S.A.). Matrigel was obtained from BD Biosciences (Bedford, NJ, U.S.A.). The following antibodies were used: anti-Bcl-2, anti-Bax, anti-Akt, anti-MMP-9, anti-MMP-2, and anti-β-actin antibodies were purchased from Proteintech Group (Chicago, IL, U.S.A.); anti-HIF-1α was purchased from Abcam (Cambridge, U.K.); anti-Caspase-9 and anti-Caspase-3 antibodies were purchased from Abclonal (Boston, U.S.A.).

All reactions were monitored by TLC on silica gel F₂₅₄ (Qindao Haiyang Chemical Co., Qindao, China). All NMR spectra were recorded on a Bruker Avance II 300 MHz instrument (Bruker, Billerica, MA, U.S.A.) using DMSO-d₆ as the solvent. Chemical shifts (δ) were reported in parts per million (ppm) and the coupling constants (J) were given in Hertz. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) spectra were recorded on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Bruker, Bremerhaven, Germany) with electrospray ionization.

Semisynthesis and Preparation of Compound 2

Compound 1 (800 mg, 2.14 mM), MgCl₂ (509 mg, 5.35 mM), and paraformaldehyde (POM, 771 mg, 8.56 mM) were dissolved in absolute tetrahydrofuran (THF, 8 mL), and the absolute triethylamine (TEA, 0.74 mL) was added at 80 °C for 5 h under nitrogen protection. The reaction was quenched with water and the product extracted thrice with ethyl acetate (EtOAc). Then the combined extract was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified using silica gel (300–400 mesh) column chromatography with a petroleum ether: EtOAc (50 : 1 to 20 : 1) eluent to afford compound 2.

5,5′-Diallyl-2-((4-fluorobenzyl)oxy)-2′-hydroxy-[1,1′-biphenyl]-3′-carbaldehyde (2). Yield: 27.36%, white oil; IR (KBr) V_max 3079.3, 1645.8, 1509.6, 1448.9, 1266.7, 1221.6, 1146.1, 910.6, 812.1 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆) δ: 10.86 (1H, s, -OH), 10.05 (1H, s, H-10′), 7.56 (1H, m, H-6′), 7.34 (3H, m, H-4, H-6, and H-4′), 7.12 (5H, m, H-3, H-12, H-13, H-15, and H-16), 5.96 (2H, m, H-8 and H-8′), 5.11 (4H, m, H-9 and H-9′), 5.04 (2H, s, H-10), 3.38 (4H, d, J = 12.6 Hz, H-7 and H-7′); ¹³C-NMR (75 MHz, DMSO-d₆) δ: 196.8 (C-10′), 156.5 (C-14), 153.9 (C-2′), 138.8 (C-2), 137.9 (C-8 and C-8′), 137.3 (C-6′), 133.5 (C-5), 131.8 (C-11), 131.7 (C-6), 131.2 (C-5′), 130.6 (C-4′), 129.3 (³J_CF = 8.1 Hz, C-12 and C-16), 128.9 (C-4), 127.3 (C-1′), 125.6 (C-1), 116.2 (C-3′), 115.7 (C-9 and C-9′), 115.2 (²J_CF = 21.0 Hz, C-13 and C-15), 113.1 (C-3), 68.9 (C-10), 38.6 (C-7′), 38.2 (C-7); HR-ESI-MS: Calculated for C₂₆H₂₃FO₃Na⁺ [M + Na]⁺ m/z 425.1523. Found 425.1523; Purity: 95.8% (by HPLC).

Semisynthesis and Preparation of Compounds 3–5

Compound 2 (80 mg, 0.2 mM) and the organic base (aminoguanidine carbonate (0.6 mM), thiosemicarbazide (0.6 mM), and semicarbazide hydrochloride (0.6 mM), respectively) were dissolved in absolute ethanol (EtOH, 4 mL). Acetic acid (AcOH, 0.2 mL) was added and the mixture was stirred for 3 h in an oil bath at 50 °C. The reaction was quenched with water and the product extracted with EtOAc three times. The combined extract was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified using silica gel (300–400 mesh) column chromatography with a petroleum ether: EtOAc (10 : 1 to 1 : 1) eluent to afford compounds 3–5.

(E)-2-((5,5′-Diallyl-2-((4-fluorobenzyl)oxy)-2′hydroxy-[1,1′-biphenyl]-3-yl)methylene)hydrazine-1-carboximid-amide carboximidamide (3). Yield: 82.5%, white powder; IR (KBr) V_max 3336.9, 3238.5, 1631.0, 1509.6, 1403.7, 1221.6, 910.6, 789.1 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆) δ: 8.22 (1H, s, H-10′), 7.36 (2H, m, H-4′ and H-6′), 7.08 (6H, m, H-4, H-6, H-12, H-13, H-15, and H-16), 6.91 (1H, d, J = 2.1 Hz, H-3), 5.93 (6H, m, H-8, H-8′, –NH–, = NH, –NH₂), 5.05 (4H, m, H-9 and H-9′), 5.02 (2H, s, H-10), 3.31 (4H, t, J = 8.0 Hz, H-7 and H-7′); ¹³C-NMR (75 MHz, DMSO-d₆) δ: 173.2 (–C = NH), 158.7 (C-14), 154.0 (C-2′), 153.0 (C-10′), 147.9 (C-2), 138.0 (C-8 and C-8′), 133.7 (⁴J_CF = 2.8 Hz, C-11), 131.6 (C-6′), 131.3 (C-5), 131.1 (C-6), 129.4 (³J_CF = 8.0 Hz, C-12 and C-16), 129.3 (C-5′), 128.6 (C-4′), 128.2 (C-4), 127.6 (C-1), 125.7 (C-1′), 119.6 (C-3′), 115.6 (C-9 and C-9′), 115.1 (²J_CF = 21.2 Hz, C-13 and C-15), 113.0 (C-3), 68.8 (C-10), 22.2 (C-7 and C-7′); HR-ESI-MS: Calcd for C₂₇H₂₇FN₄O₂Na⁺ [M + Na]⁺ m/z 481.2010. Found 481.2006. Purity: 97.6% (by HPLC).

(E)-2-((5,5′-Diallyl-2-((4-fluorobenzyl)oxy)-2′-hydroxy-[1,1′-biphenyl]-3-yl)methylene) hydrazine-1-carbothioamide (4). Yield: 87.0%, white powder; IR (KBr) V_max 3329.6, 3159.7, 1600.7, 1502.2, 1214.2, 994.3, 903.2, 812.1 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆) δ: 11.41 (–OH, s, H-2′), 9.09 (1H, s, –NH–), 8.15 (1H, s, H-10′), 7.99 (1H, s, H-6), 7.38 (2H, s, H-4′ and H-6′), 7.36 (2H, m, –NH₂), 7.08 (6H, m, H-3, H-4, H-12, H-13, H-15, and H-16), 5.96 (2H, m, H-8 and H-8′), 5.06 (4H, m, H-9 and H-9′), 5.02 (2H, s, H-10), 3.32 (4H, d, J = 7.0 Hz, H-7 and H-7′); ¹³C-NMR (75 MHz, DMSO-d₆) δ: 154.0 (–C=S), 152.0 (¹J_CF = 230.2 Hz, C-14), 143.9 (C-2′), 138.0 (C-2 and C-10′), 137.9 (C-8 and C-8′), 131.7 (C-6), 133.5 (⁴J_CF = 2.9 Hz, C-11), 131.3 (C-5′), 133.1 (C-5 and C-6′), 129.4 (³J_CF = 8.1 Hz, C-12 and C-16), 128.6 (C-4′), 128.3 (C-4), 126.9 (C-1 and C-1′), 115.6 (C-9, C-3′, and C-9′), 115.1 (²J_CF = 21.2 Hz, C-13 and C-15), 113.0 (C-3), 68.9 (C-10), 38.5 (C-7 and (C-7′); HR-ESI-MS: Calcd for C₂₇H₂₆FN₃O₂SNa⁺ [M + Na]⁺ m/z 498.1622. Found 498.1621. Purity: 96.8% (by HPLC).

(E)-2-((5,5′-Diallyl-2-((4-fluorobenzyl)oxy)-2′-hydroxy-[1,1′-biphenyl]-3-yl)methylene) hydrazine-1-carboxamide (5). Yield: 80.9%, white powder; IR (KBr) V_max 3458.4, 3336.9, 1676.2, 1434.1, 1221.6, 910.6, 819.5, 758.8 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆) δ: 10.27 (–OH, s, H-2′), 10.06 (1H, s, H-10′), 8.12 (1H, s, H-6), 7.36 (2H, m, H-4′ and H-6′), 7.10 (6H, m, H-3, H-4, H-12, H-13, H-15, and H-16), 6.40 (2H, s, –NH₂), 5.95 (2H, m, H-8 and H-8′), 5.04 (5H, m, H-9, H-9′, and –NH–), 5.02 (2H, s, H-10), 3.32 (4H, t, J = 7.9 Hz, H-7 and H-7′); ¹³C-NMR (75 MHz, DMSO-d₆) δ: 163.1 (¹J_CF = 242.0 Hz, C-14), 155.9 (C-2′), 154.0 (–C=O), 152.0 (C-10′), 142.1 (C-2), 138.0 (C-8′), 137.9 (C-8), 133.5 (C-5), 132.2 (⁴J_CF = 3.1 Hz, C-11), 131.6 (C-6′), 131.3 (C-6), 129.9 (C-5′), 129.4 (³J_CF = 8.2 Hz, C-12 and C-16), 128.4 (C-4′), 128.2 (C-4), 127.1 (C-1′), 126.5 (C-1), 119.1 (C-3′), 115.6 (C-9′, C-9′), 115.1 (²J_CF = 21.3 Hz, C-13 and C-15), 113.0 (C-3), 68.9 (C-10), 38.5 (C-7 and C-7′); HR-ESI-MS: Calcd for C₂₇H₂₆FN₃O₃Na⁺ [M + Na]⁺ m/z 482.1850. Found 482.1851. Purity: 96.3% (by HPLC).

MTT Assay

SMMC-7721, HepG2, and SUN-449 cells were seeded in 96-well plates at a density of 6 × 10³ cells per well and treated with various concentrations of the compounds 1–5 for 72 h. Paclitaxel was used as a positive control. SMMC-7721 cells were seeded in 96-well plates at a density of 6 × 10³ cells per well. After the cells adhered to the wall, they were incubated with different concentrations of compound 3 (0, 1.25, 2.5, 5, and 10 µM) for 24, 48, and 72 h. After incubation, added 10 µL of 5 mg/mL MTT solution was added, and the cells were incubated for 4 h. Subsequently, the liquid was discarded, and 100 µL DMSO was added. The plates were then incubated at 37 °C for 30 min. Cell viability was determined by measuring the absorbance at 490 nm using a microplate reader (Synergy HT, BioTek, Vermont, U.S.A.).

Flow Cytometry Analysis of Apoptosis

SMMC-7721 cells were seeded in 6-well plates at a density of 1.5 × 10⁵ cells per well. After overnight incubation, cells were incubated with different concentrations of compound 3 (0, 2.5, 5 and 10 µM) for 24 h. After incubation, the annexin V-FITC binding solution, annexin V-FITC, and PI staining solution were added to the collected cells and incubated at room temperature in the dark for 20 min. Apoptotic cells were quantified using a flow cytometer and Cell Quest software (Becton-Dickinson, U.S.A.).

Western Blotting

SMMC-7721 cells were seeded in 6-well plates at a density of 3 × 10⁵ cells per well. When the cells density was approximately 80%, the cells were incubated with different concentrations of compound 3 (0, 2.5, 5 and 10 µM) for 24 h. After incubation, cells were collected, lysed, and adjusted to a uniform concentration. Proteins were fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF. The membranes were sealed with 5% skim milk washed twice with tris buffered saline/Tween-20 buffer, and incubated with the corresponding primary antibody at 4 °C for overnight. Membranes were washed again and incubated with the corresponding secondary antibodies at room temperature for 2 h. Images were acquired using a gel imaging system (Bio-Rad, CA, U.S.A.). Anti-β-actin was used as an internal control.

Cell Migration Assay

SMMC-7721 cells were diluted in serum-free medium, seeded in the upper chambers of 24-well plate with 8.0 µm pole cartridge inserts at a density of 3 × 10⁵ cells per well, and incubated with different concentrations of compound 3 (0, 1.25, 2.5, and 5 µM) for 24 h. Culture medium containing fetal bovine serum was added to the lower compartment. After incubation, the liquid in the upper chamber was removed, and residual cells were wiped off. Then, the migrated cells were fixed with 4% paraformaldehyde for 20 min, stained with 0.5% crystal violet for 10 min, washed thrice with phosphate buffered saline (PBS), and dried at room temperature. Thereafter, the cells were photographed in three randomly selected visual fields under a light microscope at 200× magnification, and the number of penetrating cells were counted.

Cell Invasion Assay

The cell invasion assay was performed using a 4-well plate with 8.0 µm pore membrane inserts that were coated with 50 µL of Matrigel (1 : 4 diluted the with serum free medium), and incubated at 37 °C for 1 h. SMMC-7721 cells (3 × 10⁵ cells per well) were added to the upper chambers of 24-well plates and treated with various concentrations (0, 1.25, 2.5, and 5 µM) of compound 3 for 36 h. The remainder of the procedure was the same as that described for the cell migration assay.

Statistical Analysis

Statistical analysis was performed with two samples using SPSS 16.0 software (Armonk, NY, U.S.A.). * p < 0.05 and ** p < 0.01 were considered statistically significant differences.

Conclusion

In conclusion, we demonstrated the semsynthesis of new magnolol Schiff base derivatives that exhibit potent anticancer activities in vitro. The preliminary mechanism action of compound 3 was related to the up-regulation of Bax protein expression, down-regulation of Bcl-2, Akt, HIF-1α, MMP-2, and MMP-9 protein expression, and cleavage of caspas-9 and caspase-3. Thus, compound 3 may be a potential antitumor agent for the discovery and development of anti-hepatocellular carcinoma drugs.

Acknowledgments

This work was financially supported by the Anhui Provincial Natural Science Foundation (2008085MH284); 512 Talent Cultivation Plan of Bengbu Medical College (By51202202); the Natural Science Research Project of Anhui Educational Committee (KJ2019A0326); Anhui University Graduated Research Project (YJS20210533).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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