Discovery of a Potential Multi-Target Anti-Tumor Agent via Structural Modification on Flavonoid

Abstract A series of flavonoid derivatives bearing trimethoxyphenyl and benzimidazole were designed, synthesized and evaluated as potential anti-tumor agents. Compound 5g showed remarkable inhibitory activity against SGC-7901, HGC-27 and MCF-7 (IC50 values of 20.5 ± 5.93, 3.3 ± 2.53 and 16.6 ± 0.75 μM, respectively), and had great selectivity to cancer cells. The results of Hoechst 33258 staining and Annexin V-FITC/PI dual staining confirmed that compound 5 g induced apoptosis of the HGC-27 cells in a concentration-dependent manner. After treatment with compound 5 g, the expression of HIF-1α, VEGF and PKM2 were down-regulated, accompanied by weakened the levels of lactate. Further research revealed that compound 5 g could obviously inhibit tubulin assembly. In summary, compound 5 g possessed a promising potential for further development into anti-tumor drug candidates. Graphical Abstract


Biological activity
Anti-proliferation evaluation Table 1 summarized the anti-proliferative effects of 5,6,7-trimethoxy flavonoid benzimidazole derivatives 5a-5r against five human cancer cell lines and two human normal cell lines, using 5-Fluorouracil (5-Fu) as the reference compound. On the whole, compounds 5a-5r had weak antiproliferative activity against SGC-7901, MGC-803, HCT-116 and MCF-7 cell lines. Compounds 5a-5r had good anti-proliferative activity against the HGC-27 cell line. Compounds 5c and 5e-5m exhibited better anti-proliferative activity against the HGC-27 cell line than 5-Fu. For the HGC-27 cell line, compound 5 g-5l with a medium length linker (n ¼ 3) had a good anti-proliferative activity and more potent than 5-Fu, indicating that suitable linker length between flavonoid and benzimidazole was favorable for anti-proliferative activity. When the linker length was three carbons, compared to strong electron-withdrawing group (2-CF 3 and 2-Cl), R2-position of benzimidazole was electron-donating group (2-CH 3 ), weak electron-withdrawing group (2-Ph) or unsubstituted (2-H) group which was more conducive to enhance the activity of compounds. When the linker length was four carbons, R2-position of hydrogen or phenyl seemed to be the more suitable. However, for two linker length, the law of electron-induced effects was not obvious. Among them, compound 5 h was the most potent one having good cytotoxic properties against five cancer cell lines, but also have strong cytotoxicity on two human normal cell lines (against GES-1 and L-02, with IC 50 values of 6.0 ± 2.31 and 1.9 ± 1.25 lM, respectively). Furthermore, compound 5 g showed remarkable inhibitory activity against the three cell lines (against SGC-7901, HGC-27 and MCF-7, with IC 50 values of 20.5 ± 5.93, 3.3 ± 2.53 and 16.6 ± 0.75 lM, respectively), and had great selectivity to cancer cells. Thus, we selected compound 5 g for further analysis to learn the mechanism of growth inhibition on HGC-27 cells.

Colony formation assay
To further research the long-term cytotoxic effects of compound 5 g, colony formation assay was performed to investigate whether 5 g would affect the infinite replication potential of HGC-27 cells. As shown in Figure 2, the colonies formed by HGC-27 cells were decreased by treatment with compound 5 g.

Cell migration
The wound-healing assay is one of the earliest developed methods to study cell migration in vitro. The results of wound-healing assay showed that compound 5 g concentration-dependently inhibited the ability of cells to close a wound, demonstrating the inhibition of cell migration in vitro ( Figure 3).

Cell apoptosis analysis
Apoptotic cells possess morphological characteristics, such as cell shrinkage, nuclear fragmentation and apoptotic body formation. To investigate the effect of compound 5 g on the induction of apoptosis, HGC-27 cells treated with compound 5 g for 48 h at different concentrations (2, 4 and 8 lM) were stained with Hoechst 33258 and observed by fluorescence microscopy. As observed in Figure 4, with the concentration of compound 5 g increased, HGC-27 cells displayed stronger blue fluorescence and exhibited typical apoptotic characteristic morphologies such as cell shrinkage and nuclear fragmentation, indicating that compound 5 g induced apoptosis in a concentration-dependent manner on HGC-27 cells.
In order to further research the effect of compound 5 g on cell apoptosis, we analyzed HGC-27 cells treated with different concentrations of compound 5 g (2, 4 and 8 lM) by flow cytometry after Annexin V-FITC/PI double staining. As depicted in Figure 5, with the increasing concentration, the early and late apoptotic population increased. The greatest consequent of compound 5 g was at 8 lM with 49.5% early and 34.4% late apoptotic cells while those are 1.59% and 3.99% separately in the control condition. These results confirmed that compound 5 g induced apoptosis of the HGC-27 cells in a concentration-dependent manner.
Screening of potential targets HIF-1a has been reported to play a significant role in the progression of tumor. HIF-1a regulates a series of genes relating angiogenesis and glycolytic energy metabolism. Several studies have confirmed that flavonoids down-regulate the levels of HIF-1a, and therefore the expression of its downstream genes. 20,39 To further explore the biological activity mechanism induced by compound 5 g, the levels of related proteins (HIF-1a, VEGF, HK-2, PFKM and PKM2) were analyzed. In the presence of oxygen, the expression of HIF-1a is repressed. CoCl 2 (100 lM) and serum starvation (0.1% FBS) treatment stimulated the HIF-1a expression. The treatment of compound 5 g significantly down-regulated HIF-1a protein levels under hypoxic conditions ( Figure  6A & C). What's more, compound 5 g decreased the expression of VEGF. HIF-1a and VEGF, as the most important angiogenic factors, played an indispensable role in tumor angiogenesis. It could be speculated that compound 5 g could affect angiogenesis by down-regulating HIF-1a and VEGF expression. Hexokinase (HK), pyruvate kinase (PK) and phosphofructokinase (PFK) are the key rate-limiting enzyme in glycolysis, and also are the target gene of HIF-1a regulation. Furthermore, compound 5 g down-regulated the expression of PKM2, but did not affect the HK-2 and PFKM protein levels ( Figure 6B & D). The results suggested that compound 5 g could inhibit tumor glycolysis. The above results confirmed our hypothesis that compound 5 g might possess the anti-angiogenic and glycolytic inhibitory activity.

Lactate measurement
Cancer cells tend to convert most glucose to lactate via glycolysis regardless of whether oxygen is present. Malignant rapidly-growing tumor cells typically have glycolytic rates that are up to 200 times higher than those of their normal tissues of origin. 40 To further explore the effect of compound 5 g on glycolysis, the levels of lactate after treatment of compound 5 g (2, 4 and 8 lM) for 48 h were detected. As depicted in Figure 7, the levels of lactate treated with compound 5 g were decreased in a concentration-dependent manner. These findings proved that compound 5 g could inhibit glycolysis in the HGC-27 cell line.

Immunofluorescent assay
Microtubules play an important role in various cellular processes, including spindle formation, cellular shape maintenance, and intracellular transportation. MTAs disrupt microtubule dynamics, eventually resulting in cell apoptosis. 33 One of the design targets of 5,6,7-trimethoxy flavonoid benzimidazole derivatives is microtubules. To examine the effect of compound 5 g on the microtubule structure in HGC-27 cells, an immunofluorescent assay was conducted via confocal immunofluorescent microscopy. The results were displayed in Figure 8. The control cells displayed a normal arrangement and organization with clear and fibrous microtubules stretching throughout the cell. In contrast, when treated with compound 5 g at 4 or 8 lM, the microtubule network became disordered and disorganized. Taken together, the results clearly confirmed that compound 5 g could obviously inhibit tubulin assembly on HGC-27 cells.

Docking studies about tubulin
Microtubules are composed of aand b-tubulin heterodimers. Colchicine as one of the representative tubulin inhibitors binds at the interface of A-subunit and b-subunit of tubulin thus lead to microtubule depolymerization. 32 Many methods are used to compare the biological activities of the studied molecules, and the molecular docking is one of the most widely used methods to compare the biological activity of molecules against protein molecules. In this molecular docking simulation, colchicine was selected as the control drug. The results were shown in Figure 9. Interestingly, the binding site of compound 5 g to tubulin is not consistent with that of colchicine.The binding site of compound 5 g existed between the a1-tubulin and b1-tubulin subunit ( Figure 9A). In addition, the oxygen atom on the B-ring ether bond of compound 5 g formed a hydrogen bond with the amine group of ASN-101 ( Figure 9B) (PDB code: 1SA0 for colchicine). Compound 5 g were simultaneously combined with the key amino acid asparagine in their own binding domain to form hydrogen-bonding interactions. The results of these docking studies have certain reference significance for further structural optimization of the compound 5 g.

Chemicals
All material and reagents (reagent grade) were commercially available and were used without further purification unless otherwise stated. Purification of the compounds by column chromatography was carried out with silica gel (100-200 mesh size). Thin-layer chromatography (TLC) was performed using the silica gel 60 F254 plates and visualized under UV light at 254 nm or 365 nm which was used to monitor reactions. Melting points were determined with a Thermo Scientific electrothermal digital melting point apparatus without correction. ESI mass spectra were obtained on a Waters GCT mass spectrometer. 1 H NMR and 13 C NMR spectra were measured on a Bruker AV-400 model spectrometer. Chemical shifts were reported in ppm (d), referenced to tetramethylsilane (TMS).  General procedure for the synthesis of 2-(4-hydroxyphenyl)-5,6,7-trimethoxy-4H-chromen-4one (3) 3,4,5-Trimethoxyphenol (0.024 mol) and 2-chloroacetonitrile (0.048 mol) were dissolved in a mixed solvent of anhydrous diethyl ether and chloroform (60 mL) with a catalytic amount of ZnCl 2 at 0 C, and freshly prepared dry hydrogen chloride gas was bubbled slowly and continuously to the reaction mixture for 4 h. After standing the reaction flask at 4 C for 48 h, a yellow  precipitate was prepared after filtrating, washing and drying. The yellow precipitate was dissolved in hot water and heated at 90 C for 1 h. The crude product after hydrolysis was filtered off, washed with water, and dried in vacuo to obtain a light yellow precipitate 2-chloro-1-(6-hydroxy-2,3,4-trimethoxyphenyl)ethan-1-one (2). 4-Hydroxybenzaldehyde (0.02 mol) and 1 (0.024 mol) were added in one portion to a stirred solution of 6 mL ethanol, the drop 10% NaOH (aq) into the solution. The reaction mixture was stirred at room temperature (RT) for 24 h, and acidification with 10% HCl (aq) gave a crude product, which was filtered off and purified by recrystallized from 95% ethanol to give the 2-(4-hydroxyphenyl)-5,6,7-trimethoxy-4H-chromen-4-one (3) as golden powder.
General procedure for the synthesis of the intermediate 4a-4c To a solution of 2-(4-hydroxyphenyl)-5,6,7-trimethoxy-4H-chromen-4-one (3) (0.04 mol) and (CH 2 ) 2 Br 2 (0.16 mol) in acetone (150 mL), a catalytic amount of K 2 CO 3 was added to this solution at 60 C for 9 h. After the completion of the reaction monitored by TLC, the reaction mixture was concentrated under vacuum. The residue was purified using column chromatography on silica gel [methanol/dichloromethane ¼ 1:200 (v/v)] to give the intermediate 4a. This procedure was also applied to the preparation of compound 4 b and 4c, except that (CH 2 ) 3 Br 2 and (CH 2 ) 4 Br 2 were used as the reactant, respectively. General procedure for the synthesis of the target compounds 5a-5r The intermediate 4a (0.3 mmol), benzimidazole (0.6 mmol) were dissolved in acetone (30 mL), stirred under a catalytic amount of K 2 CO 3 at 60 C for 48 h and monitored by TLC. After the completion of the reaction, the reaction mixture was concentrated under vacuum. The residue was purified using column chromatography on silica gel [methanol/dichloromethane ¼ 1:250 (v/ v)] to give the target compound 5a.
Compounds 5b-5r were prepared in the analogy.      was measured at 490 nm using Wellscan MK-2 microplate reader. The concentration causing 50% inhibition of cell growth (IC 50 ) was calculated using GraphPad Prism 6.0 software. All experiments were independently performed at least three times.

2-[4-(2-
Colony formation assay 1500 HGC-27 cells/well were seeded in 6-well plates for colony formation assay. Cells were cultured for 24 h and then the media were replaced with media added compound 5 g at the indicated concentration (0.5, 1 and 2 lM). The media were changed every three days. After 9 days of the treatment with compound 5 g, the colonies were fixed with 4% paraformaldehyde for 15 min and stained with using 0.1% crystal violet staining for 15 min. After washing three times with PBS, the colonies were then photographed.
Wound-healing assay HGC-27 cells were cultured in a 6-well plate for 24 h. The cell surface was scratched using a 10 lL pipette tip. After treatment with different concentrations of 5 g (2, 4 and 8 lM), the cell wound was photographed on an inverted-phase microscope at 0 h and 48 h.
Cell morphological assessment HGC-27 cells (3 Â 10 4 cells/well) were plated in 12-well plates and incubated for 24 h. Then cells were treated with compound 5 g at different concentrations (2, 4 and 8 lM) for 48 h. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min. Then 1 mL of Hoechst 33258 were added to each well, and the plates were incubated for 10 min in the dark. The cells were observed by a fluorescence microscope (Olympus, Japan) with a peak excitation wavelength of 350 nm.
Annexin V-FITC/PI dual staining HGC-27 cells (2 Â 10 5 cells/well) in 6-well plates were incubated with different concentrations of compound 5 g (2, 4 and 8 lM) for 48 h before the cells were harvested. The harvested cells were washed twice with cold PBS and centrifuged at 800 rpm for 5 min. The harvested cells were resuspended in 100 mL binding buffer, followed by the addition of 5 lL of Annexin V-FITC and 5 lL of PI. After incubation for 15 min at 25 C in the dark, the cells were added an additional 400 ml of binding buffer. The cell apoptosis assay was performed by the FACSCalibur flow cytometry (BD Biosciences, USA) and analyzed using FlowJo software.

Western blotting assay
The expression of HIF-1a was investigated under CoCl 2 treatment (100 lM) and serum starvation Immunofluorescence assay HGC-27 cells incubated with different concentrations of compound 5 g (2, 4 and 8 lM) for 48 h, the cells were fixed with 5% paraformaldehyde for 15 min at RT and blocked with 1% bovine serine albumin (BSA) in PBS for 1 h at RT. Then the cells were incubated with a-tubulin Rabbit Polyclonal Antibody (1: 500; Beyotime, CN) at 4 C overnight, following by incubation with the secondary antibody Cy3 conjugated Goat Anti-mouse IgG (1: 250; Servicebio, CN) at RT for 1 h in dark place. The cell nuclear was stained with DAPI (1: 1000) and cell images were visualized using a confocal laser microscope (Nikon C2, Japan).

Molecular docking
The Docking suit module in SYBYL-X 2.0 software was selected to simulate the binding of small molecules to tubulin. The 3 D tubulin structure containing the colchicine complex was obtained from the protein data bank (PDB) (http://www.rcsb.org/, PDB code 1SA0). The molecular structure of the target compound 5 g and colchicine were made by ChemDraw Ultra 7.0. For the docking simulation, the target and ligands were processed using a custom import setting wizard of the docking program. The docking results were processed with PyMOL (http://www.pymol. org/) to obtain intuitive figures.

Statistical analysis
All data were displayed as mean ± SD from three independently repeated determinations. Statistical analyses were calculated using Origin 8.0 and Prism 6.0 GraphPad software. Student ttest was employed for determining comparisons between two groups. One-way ANOVA was used for multiple group comparisons. The differences were considered significant at Ã P values <0.05 were considered statistically significant.

Conclusion
To summarize, a novel series of 5,6,7-trimethoxy flavonoid benzimidazole derivatives were designed and synthesized based on a hybridization strategy to screen potential multi-target antitumor drugs. In general, most of the target compounds showed a significant cytotoxic effect against HGC-27 cell line. Among them, compound 5 g showed remarkable inhibitory activity against HGC-27 cells with IC 50 values of 3.3 ± 2.53 lM, and had great selectivity for normal cells and cancer cells. Furthermore, the active compound 5 g induce apoptosis in HGC-27 cell line by a concentration-dependent manner. Further biochemical assays demonstrated that compound 5 g had anti-angiogenic and glycolytic inhibitory activity by down-regulating the expression of HIF-1a, VEGF and PKM2, and decreasing the levels of lactate by a concentration-dependent manner. Meanwhile, immunofluorescent assay proved that compound 5 g could obviously inhibit tubulin assembly. The molecular docking results provided more convincing evidence that compound 5 g acted on tubulin. From the obtained results, we concluded that compound 5 g can destroy existing tumor blood vessels while still maintaining anti-angiogenesis and glycolysis inhibitory activities. Taken together, as a promising candidate for cancer therapy, compound 5 g needs further research the specific mechanism of action.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by Hunan Provincial Key Laboratory of tumor microenvironment responsive drug research (Approval number: 2019-56) and Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study.