Synthesis and biological evaluation of novel shikonin-benzo[b]furan derivatives as tubulin polymerization inhibitors targeting the colchicine binding site

Journal Pre-proof Synthesis and biological evaluation of novel shikonin-benzo[b]furan derivatives as tubulin polymerization inhibitors targeting the colchicine binding site Yu-Ying Shao, Yong Yin, Bao-Ping Lian, Jia-Fu Leng, Yuan-Zheng Xia, Ling-Yi Kong PII:

S0223-5234(20)30072-6

DOI:

https://doi.org/10.1016/j.ejmech.2020.112105

Reference:

EJMECH 112105

To appear in:

European Journal of Medicinal Chemistry

Received Date: 26 November 2019 Revised Date:

17 January 2020

Accepted Date: 27 January 2020

Please cite this article as: Y.-Y. Shao, Y. Yin, B.-P. Lian, J.-F. Leng, Y.-Z. Xia, L.-Y. Kong, Synthesis and biological evaluation of novel shikonin-benzo[b]furan derivatives as tubulin polymerization inhibitors targeting the colchicine binding site, European Journal of Medicinal Chemistry (2020), doi: https:// doi.org/10.1016/j.ejmech.2020.112105. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Synthesis and biological evaluation of novel shikonin-benzo[b]furan derivatives as tubulin polymerization inhibitors targeting the colchicine binding site Yu-Ying Shao†, Yong Yin†, Bao-Ping Lian, Jia-Fu Leng, Yuan-Zheng Xia, Ling-Yi Kong*

Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’ s Republic of China. * Corresponding authors. Tel/Fax: +86-25-83271405; E-mail: [email protected] (Ling-Yi Kong) † Both authors contributed equally to this work.

Synthesis and biological evaluation of novel shikonin-benzo[b]furan derivatives as tubulin polymerization inhibitors targeting the colchicine binding site Yu-Ying Shao†, Yong Yin†, Bao-Ping Lian, Jia-Fu Leng, Yuan-Zheng Xia, Ling-Yi Kong* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’ s Republic of China. * Corresponding authors. Tel/Fax: +86-25-83271405; E-mail: [email protected] (Ling-Yi Kong) † Both authors contributed equally to this work. Abstract A novel series of shikonin-benzo[b]furan derivatives were designed and synthesized as tubulin polymerization inhibitors, and their biological activities were evaluated. Most compounds revealed the comparable anti-proliferation activities against the cancer cell lines to that of shikonin and simultaneously low cytotoxicity to non-cancer cells. Among them, compound 6c displayed powerful anti-cancer activity with the IC50 value of 0.18 µM against HT29 cells, which was significantly better than that of the reference drugs shikonin and CA-4. What’s more, 6c could inhibit tubulin polymerization and compete with [3H] colchicine in binding to tubulin. Further biological studies depicted that 6c can induce cell apoptosis and cell mitochondria depolarize, regulate the expression of apoptosis related proteins in HT29 cells. Besides, 6c actuated the HT29 cell cycle arrest at G2/M phase, and influenced the expression of the cell-cycle related protein. Moreover, 6c displayed potent inhibition on cell migration and tube formation that contributes to the antiangiogenesis. These results prompt us to consider 6c as a potential tubulin polymerization inhibitor and is worthy for further study. Keywords: shikonin-benzo[b]furan derivatives, microtubule, tubulin polymerization inhibitors, colchicine binding site

1. Introduction Microtubules, which are composed of α-and β-tubulin proteins, are the crucial cytoskeletal filaments [1]. It has been implicated that microtubules make an essential role in multiple aspects of cellular process, including cell division, shape maintenance, cell motility and vesicle transport [2-5]. It was found that the formation of microtubules is a dynamic process related to the polymerization and depolymerization of α-and β-tubulin heterodimers [6]. The interruption of the dynamic equilibrium hinders the cell division at mitosis and thus resulting in the cell cycle arrest at metaphase, which leads to the cell death [7]. Therefore, tubulin has become as a promising tumor target [8]. To date, various natural compounds that target the tubulin have been developed and remained a notable component in treating the adult and pediatric malignancies, most of them have been devided into two major groups, one is the microtubule destabilizing agents which binding to vinblastine binding and the colchicine site, the other one is microtubule-stabilizing agents which binding to the tubulin paclitaxel binding site and disturbing the tubulin disassembly [9-12]. However, due to the strong toxicity against non-cancer cells, no candidates binding to the colchicine site has been approved by FDA [13]. Shikonin, a special active naphthoquinone compound which extracted from the roots of Lithospermum erythrorhizon, is found to possess various biological activities such as anti-virus, anti-inflammatory, anti-bacterial, anti-tumor and so on [14,15]. Shikonin can kill cancer cells effectively in multiple aspects like inhibiting topoisomerase [16], inhibiting nitric oxide synthase (NOS) [17], inducing cell apoptosis [18,19], etc. In the last few years, some researchers found that shikonin and its derivatives can inhibit the tubulin polymerization, which contributes to the microtubule collapse, mitosis process disruption and the cell death [20,21]. As shown in Fig. 1, the phenoxyacetic acid and other heterocyclic carboxylic acid pharmacophores were induced to the shikonin and their anti-cancer activities were better than shikonin [22,23]. Benzo[b]furan pharmacophore has been extensively used in many clinical drugs, such

as

fruquintinib

(an

anti-tumor)

and

amiodarone

hydrochloride

(an

cardio-vascular) [24,25]. Moreover, some tubulin polymerization inhibitors also have the benzo[b]furan pharmacophores, like compounds 10-13 (Fig.1) [26-29], which appeared to bind to the colchicine binding domain of tubulin. Therefore, the benzo[b]furan-2-carboxylic acid and shikonin were combined, and in order to achieve the outstanding compounds.

Herein, a series of shikonin-benzo[b]furan derivatives were designed and synthesized, the bioactivities of these compounds were evaluated. The molecular docking was also performed to elucidate the possible model of compound 6c binding to tubulin. 2. Results and discussion 2.1. Chemistry The shikonin-benzo[b]furan derivates 6a-6q were synthesized by four steps as described in Scheme 1. The intermediates 2a-2q were prepared using salicylaldehyde and ethyl bromoacetate as raw materials, KI as the catalyst by the etherification reaction. Then compounds 2a-2q were condensed under the high temperature to obtain the compounds 3a-3q. Subsequently the hydrolysis reaction was produced to get the benzo[b]furan-2-carboxylic acid 4a-4q. After that, the compounds 4a-4q and shikonin were reacted under the catalysts to obtain the desired compounds 6a-6q. 2.2. Antiproliferative activity in vitro and inhibition of colony formation Seventeen compounds 6a-6q were assessed to their anti-proliferative activities against MDA-MB-231, HepG2, HT29, HCT116 and A549 five cancer cell lines using the MTT assay, colchicine and shikonin were treated as the positive drugs. The results were elucidated in Table 1. It was observed that most compounds displayed good anti-proliferation activity against five cancer cells. Particularly for HT29 cells, compound 6c (IC50 = 0.18µM), compound 6f (IC50 = 1.03µM) and compound 6i (IC50 = 0.82µM) revealed much better anti-proliferation effects than that of the positive drugs, shikonin (IC50 = 2.80µM) and colchicine (IC50 = 1.81µM). Notably, compound 6c exhibited the comparable activity with CA-4(IC50 = 0.31µM) against HT29 cells. For HCT116 and MDA-MB-231 cells, the anti-proliferation effects of these three compounds were also better than shikonin but not that obviously. Interestingly, the compounds 6c, 6i and 6o with the 3-position substitution on the benzofuran ring exhibited the better activities than that of shikonin, indicating that the 3-position play an important role in improvement of anti-proliferation activity. The compound 6f with the 2-position substitution also exhibited better activities than that of shikonin and the tendency of the inhibition activity is 3-position > 2-position > 4-position > 6-position > 5-position. Meanwhile, while the substitution was in the 3-position, the anti-proliferation activity of compound was much better than that of

the substitution was in the 3,5-position, such as 6i and 6p, which elucidated that the tendency is monosubstitution > disubstitution. In addition, all the compounds were tested for their cytotoxicity for 293T and LO2 two non-cancer cells, and the results were shown in Table 1, all of them showed low cytotoxicity (the CC50 value >100 µΜ). Based on these results, it could be surmised that the introduction of benzo[b]furan ring promotes the anti-proliferation activity and simultaneously reduce the cytotoxicity comparing to shikonin. What’s more, the 3-position substitution on the benzo[b]furan ring may play a critical role in the anti-proliferation effect. Meanwhile, to evaluate the effects of the designed compounds on the drug resistant cancer cells, 6c was selected to test the anti-proliferation activity against two drug-resistant cancer cells and their parental sensitive cells by the MTT assay and the chemotherapy doxorubicine was treated as the positive drug. As depicted in Table 2, the drug-resistant indexes of 6c (4.73-10.73) were comparable to those of colchicine (4.72-14.97) and CA-4 (1.96-10.08), while the positive drug doxorubicine exhibited 77-167 folds resistance to the corresponding drug-resistant cells, which suggested that 6c exhibit moderate anti-proliferative effect on drug resistant cancer cells. In addition, the colony formation assay in HT29 cells was performed to better evaluate the anti-proliferation activity of compound 6c. The results were displayed in Fig. 2, compound 6c inhibited the colony formation in a dose-dependent manner and the colony formation treated with 6c was inhibited better than that with colchicine and CA-4. 2.3. Inhibition of tubulin polymerization in vitro To

confirm

whether

the

designed

compounds

could

target

the

tubulin-microtubule system, 6c was chose to assess its effect on microtubule dynamics in HT29cells. Meanwhile, colchicine and CA-4(microtubulin-destabilizing agent) and paclitaxel (microtubule-stabilizing agent) were treated as contrast. As depicted in Fig. 3, compound 6c exhibited similar action to that of colchicine, suggesting that 6c was the microtubulin-destabilizing agent, and exhibited the potent inhibition activity of tubulin polymerization (IC50 = 0.98 µM), which was superior to that of colchicine and CA-4. In addition, compound 6c can compete with [3H] colchicine in binding to tubulin. As listed in Table 3, the binding potency to the colchicine binding site was up to 92.42% at 4µM, indicating that compound 6c reveal the tubulin polymerization inhibition and could compete with colchicine binding site.

2.4. Compound 6c induces HT29 cell cycle arrest Considering that most tubulin destabilizing agents could obstruct the cell division at mitosis and lead to the cell cycle arrest at metaphase, the flow cytometry analysis assay was performed to detect the effect of 6c on cell cycle distribution. Similarly, HT29 cells were incubated for 24 h with increased concentration of 6c (0.25, 0.5, 1µM), and as expected, the cell-cycle arrested at G2/M phase (Fig. 4A). By the way, as the positive drugs, shikonin and colchicine could also induce cell cycle arrest in a dose-dependent manner but not that significantly (Fig. 4B and 4C). What’s more, 6c could arrest the cell cycle at G2/M phase in a time-dependent manner (Fig. 4D). In addition, the western blotting assay was employed to assess the effect of 6c on cell cycle related protein expression. As shown in Fig. 4E, 6c obviously increased the expression of P21 and Cyclin B1 and decreased the expression of Cdc2, p-CDC2 and p-Cdc25c, which related well with the results that detected on cell cycle. 2.5. Compound 6c induces HT29 cell apoptosis and depolarized mitochondria Simultaneously, the Annexin V-FITC/PI assay was carried out to confirm the influence of 6c on cell apoptosis in HT29 cells, and the results were displayed in Fig. 5.

To

sum

up,

6c

could

induce

the

cell

apoptosis

not

only

in

a

concentration-dependent but also a time-dependent manner (Fig. 5A and 5D). Besides, shikonin and colchicine were treated as positive drugs, and the influence of them was not that obviously (Fig. 5B and 5C). Furthermore, the effect of 6c on apoptosis related protein expression was also explored. As depicted in Fig. 5E, compound 6c can increase the expression of Bax, cleaved PARP, cleaved caspase 3 and cleaved caspase 9. In contrary, compound 6c decreased the of expression Bcl-2, which accorded with the trend we obtained on cell apoptosis. Meanwhile, the mitochondrial membrane potential of HT29 cells was also analyzed using the fluorescent probe JC-1 to determine whether 6c motivated the disruption of mitochondrial membrane integrity that contributes to cell apoptosis. It was shown in Fig. 6 that untreated cells localized in the upper right region of the plot and while treated with different concentrations of 6c (0.25, 0.5, 1µM) for 24h, the cells showed a progressive loss of red fluorescence and the downward shift. The

results elucidated that 6c induce HT29 cell depolarized mitochondria in the process of apoptosis. 2.6. Compound 6c induces microtubule collapse in HT29 cells Considering that 6c inhibited the tubulin polymerization in vitro, the further investigation was performed to determine whether our compounds inhibit the microtubule dynamics in living cells. So that the immunofluorescent assay was conducted in HT29 cells by different concentrations of 6c (0.25, 0.5, 1µM) for 24h. Likewise, colchicine and paclitaxel were employed as the positive drugs. As shown in Fig. 7, in paclitaxel treated cells, the mircotubules became more stable and even showed as bundle. In a contrast manner, the mircotubule networks of colchicine treated cells were disrupted and even revealed soluble. After comparing the morphologic characteristic of microtubules of the positive drug treated groups and the control group, it could be found that the cells incubated with 6c show the same appearance as that treated with colchicine. What’s more, 6c induced the microtubule collapse in a dose-dependent manner. 2.7. Evaluation of anti-vascular activity in vitro To explore the anti-vascular activity of 6c, the human umbilical vein endothelial cells (HUVECs) tube formation assay was performed. The HUVECs that had been treated with different concentrations of 6c (0.01, 0.02 and 0.04 µM) were seeded on Matrigel. After 6 hours, the capillary-like tubules with multicentric junctions formed at the control group, the 6c treated groups showed the dose-dependent inhibition of HUVEC cord formation (Fig. 8). These results elucidated that 6c can inhibit the HUVEC tube formation effectively. 2.8. Compound 6c inhibits cell migration In order to investigate the anti-migration activity of compound 6c, the A549 cells culture assay was performed. As depicted in Fig. 9, after 24 h, the untreated cells migrated to the area that has been initially scraped while the 6c treated cells incurred migration inhibition in a dose-dependent manner, indicating that compound 6c could inhibit cell migration. 2.9. Molecular modelling studies

In order to elucidate the possible model of compound 6c binding to tubulin, molecular docking was carried out to dock 6c into the colchicine binding site of tubulin (PDB: 1SA0). As represented in Fig. 10, compound 6c could bind well to the colchicine binding pocket of tubulin via two hydrogen bonds, two π-cation interactions and one π-sigma interaction. One hydrogen bond was formed between methoxyl of compound 6c and Cys241, which is a crucial amino acid that anchors most of all CBS inhibitors [30]. What’s more, the same interaction was observed in the binding model of colchicine and tubulin. The other hydrogen bond was formed by hydroxyl and Asn101, which instead of the hydrogen bond formed colchicine and tubulin. Two π-cation interactions were caused by naphthoquinone and Lys254, and the furan ring formed a π-sigma interaction with Leu248. All these results indicated that compound 6c can be considered as a tubulin polymerization inhibitor. 2.10. Metabolic stability study Given the ester linker between the two pharmacophoric moieties, the metabolic stability study was performed to evaluate whether compound 6c remain stable in vivo. The plasma concentration-time profile of 6c, 4c and shikonin is shown in Fig. 11, while the pharmacokinetic parameters of 6c were calculated using non-compartment model analysis, the t1/2 was 3.81 ± 2.14 h, the MRT(0-∞) was 5.12 ± 2.86 h, the AUC(0-∞) was 2156.12 ± 851.88 ng/mL*h and the Vz was 3.348 ± 0.734 L/kg. What’s more, there was no 4c and shikonin in the 6c group, revealing that 6c was relatively stable. Meanwhile, the same amount of an equimolar mixture of shikonin and 4c were evaluate for their anti-proliferative activities comparing to the same amount of 6c, and the results were showed in Table 4, the mixture exhibited the poorer activity than that of compound 6c. It was further indicated that the stable of compound 6c. 3. Conclusion In this study, a novel shikonin-benzo[b]furan derivatives were designed and synthesized as tubulin polymerization inhibitors and their biological activities were evaluated. Out of them, compound 6c exhibited powerful anti-cancer activity with the IC50 value of 0.18 µM against HT29 cells, which was significantly better than that of shikonin. Simultaneously, all the designed compounds showed low cytotoxicity against the normal cells. In addition, 6c could inhibit tubulin polymerization and compete with [3H] colchicine in binding to tubulin. Further biological studies depicted

that 6c can induce the microtubule collapse, cell-cycle arrest at G2/M phase, cell depolarized mitochondria and apoptosis in HT29 cells. Besides, 6c could regulate the protein expression on cell cycle (increase the expression of P21 and Cyclin B1 and decrease the expression of Cdc2, p-CDC2, and p-Cdc25c) and cell apoptosis (increase the expression of Bax, cleaved PARP, cleaved caspase 3, cleaved caspase 9, but decrease Bcl-2 expression). What’s more, 6c displayed potent inhibition on cell migration and tube formation that contributes to the anti-angiogenesis. In addition, The plasma elimination half-life (t1/2), mean retention time (MRT), the area under the curve (AUC0-∞) and distribution volume (Vz) of compound 6c after intravenous administration were 3.81 ± 2.14 h, 5.12 ± 2.86 h, 2156.12 ± 851.88 ng/mL*h and 3.348 ± 0.734 L/kg, respectively. In brief, these results prompt us to consider 6c as a potential tubulin polymerization inhibitor and is worthy for further study. 4. Experimental 4.1. Chemistry 4.1.1. General All the chemical agents and solvents were purchased from commercial suppliers, further purifying and drying were employed by standard methods when necessary. 1H NMR and 13C NMR spectra were measured on a Bruker 500 and 600 spectrometers in the appropriate solvents (TMS as internal standard). Mass spectra were measured on a Agilent 6520B Q-TOF mass spectrometers (ESI-MS). 4.1.2. Synthesis of compounds 4a-4q Benzofuran-2-carboxylic acid derivatives (4a-4q) were synthesized described in the literature [31, 32]. KI (0.5mmol) was slowly added into the solution of DMF (10 mL), and the temperature was kept at 10℃. Subsequently, salicylaldehyde (1mmol) was added and stirred for 30min. After that, ethyl bromoacetate (2mmol) was dropped and reacted for 2 h until the raw material was completely exhausted (monitored by TLC). The precipitation was separated out and purified by recrystallization to get the products 2a-2q. Compound 2a-2q(1mmol) and K2CO3 (3mmol) were suspended in DMF (10 mL), and the reaction mixture was warmed up to 120℃ and refluxed for 3h, and then cooled to room temperature. The precipitation was separated out and purified by recrystallization to get the products 3a-3q.

To a solution of intermediates 3a-3q (1 mmol) in 10 ml DMF, 2.0 mL of 20% sodium hydroxide solution was dropped at 60℃ to carry out the ester hydrolysis reaction for 30min. Afterwards, the mixture was cooled to room temperature, and the PH value of the solution was adjusted to 2 using 10% hydrochloric acid, then the mixture was extracted with ethyl acetate. The organic phase was combined and condensed by vacuum concentration to afford the crude product, which was purified by recrystallization to give the designed compounds (4a-4q). 4.1.3. Synthesis of compounds 6a-6q Shikonin (1mmol), benzofuran-2-carboxylic acid derivatives (4a-4q) (3mmol), 4-dimethyaminopyridine (0.5mmol), and N, N’- dicyclohexylcarbodiimide (0.5mmol) were suspended in the anhydrous DCM (10ml). The reaction mixture was stirred for 8h under 0℃. Afterwards, the targeted compounds 6a-6q were purified by column chromatography. 4.1.3.1. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl benzofuran-2-carboxylate (6a) Violet powder. Yield: 35.7%. m. p: 88.7-90.3℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.42 (s, 1H, OH), 7.72 (d, J = 7.8 Hz, 1H, ArH), 7.62 (d, J = 6.2 Hz, 2H, ArH), 7.49 (t, J = 7.9 Hz, 1H, ArH), 7.34 (t, J = 7.3 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.15 (s, 1H, ArH), 6.32 (dd, J = 6.7, 4.7 Hz, 1H, CH-O), 5.22 (t, J = 7.3 Hz, 1H, CH=C), 2.81 – 2.76 (m, 1H, CH2), 2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 177.38, 175.85, 168.34,

167.81, 158.32, 155.97, 147.43, 144.86, 136.63, 133.25, 133.06, 131.51, 128.03, 126.85, 124.00, 122.96, 117.41, 114.70, 112.50, 111.88, 111.65, 70.54, 32.99, 25.84, 18.05. HRMS (ESI) m/z: 455.1102 [M+Na]+ (calcd for 455.1101, C25H20NaO7). 4.1.3.2. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 6-methylbenzofuran-2-carboxylate (6b) Violet powder. Yield: 32.6%. m. p: 99.1-101.5℃.1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.42 (s, 1H, OH), 7.58 (d, J = 8.6 Hz, 2H, ArH), 7.40 (s, 1H, ArH), 7.19 (s, 2H, ArH), 7.16 (d, J = 8.0 Hz, 1H, ArH), 7.14 (s, 1H, ArH), 6.30 (dd, J = 6.8, 4.9 Hz, 1H, CH-O), 5.21 (t, J =7.4 Hz, 1H, CH=C), 2.80 – 2.75 (m, 1H, CH2), 2.64 (dt, J

= 15.1, 7.5 Hz, 1H, CH2), 2.51 (s, 3H, Ar-CH3), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3).13C NMR (151 MHz, CDCl3): 177.60, 176.09, 168.11, 167.58, 158.41, 156.50, 147.59, 144.32, 138.90, 136.57, 133.16, 132.97, 131.55, 125.74, 124.38, 122.37, 117.46, 114.75, 112.43, 111.88, 111.64, 70.39, 33.00, 25.84, 22.07, 18.05. HRMS (ESI) m/z: 469.1254 [M+Na]+ (calcd for 469.1258, C26H22NaO7). 4.1.3.3. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 6-methoxybenzofuran-2-carboxylate (6c) Violet powder. Yield: 36.6%. m. p: 113.4-115.2℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.42 (s, 1H, OH), 7.56 (d, J = 8.9 Hz, 2H, ArH), 7.19 (s, 2H, ArH), 7.14 (s, 1H, ArH), 7.08 (s, 1H, ArH), 6.97 (dd, J = 8.7, 2.1 Hz, 1H, ArH), 6.30 (dd, J = 6.8, 4.7 Hz, 1H, CH-O), 5.21 (t, J = 7.2 Hz, 1H, CH=C), 3.89 (s, 3H, OCH3), 2.80 – 2.75 (m, 1H, CH2), 2.63 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3).13C NMR (151 MHz, CDCl3): 177.71, 176.20, 168.02, 167.49, 160.84, 158.27, 157.45, 147.70, 144.06, 136.51, 133.12, 132.94, 123.20, 120.11, 117.49, 115.03, 114.42, 111.88, 111.64, 95.67, 70.27, 55.75, 33.00, 29.72, 25.83, 18.05. HRMS (ESI) m/z: 485.1208 [M+Na]+ (calcd for 485.1207, C26H22NaO8). 4.1.3.4. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5-bromobenzofuran-2-carboxylate (6d) Violet powder. Yield: 32.1%. m. p: 102.9-105.4℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.86 (s, 1H, ArH), 7.58 (dd, J = 8.8, 1.8 Hz, 1H, ArH), 7.54 (s, 1H, ArH), 7.50 (d, J = 8.8 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.14 (s, 1H, ArH), 6.31 (dd, J = 7.3, 4.4 Hz, 1H, CH-O), 5.20 (t, J = 7.2 Hz, 1H, CH=C), 2.81 – 2.75 (m, 1H, CH2), 2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 176.90, 175.35, 168.77, 168.25, 157.91,

154.58, 147.08, 145.97, 136.73, 133.42, 133.23, 131.40, 131.04, 128.71, 125.46, 117.30, 117.11, 114.01, 113.73, 111.87, 111.64, 70.81, 32.97, 25.84, 18.05. HRMS (ESI) m/z: 533.0205 [M+Na]+ (calcd for 533.0206, C25H19BrNaO7). 4.1.3.5. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 4-chlorobenzofuran-2-carboxylate (6e)

Violet powder. Yield: 33.2%. m. p: 107.6-108.9℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.68 (s, 1H, ArH), 7.52 (d, J = 8.4 Hz, 1H, ArH), 7.41 (t, J = 8.1 Hz, 1H, ArH), 7.34 (d, J = 7.7 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.15 (s, 1H, ArH), 6.33 (dd, J = 7.1, 4.5 Hz, 1H, CH-O), 5.22 (t, J = 7.2 Hz, 1H, CH=C), 2.81 – 2.76 (m, 1H, CH2), 2.66 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.63 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 176.97, 175.42, 168.70,

168.18, 157.88, 156.00, 147.10, 145.24, 136.74, 133.39, 133.19, 131.41, 128.50, 127.97, 126.63, 123.87, 117.3 2, 112.92, 111.87, 111.64, 111.05, 70.82, 32.99, 25.83, 18.06. HRMS (ESI) m/z: 489.0714 [M+Na]+ (calcd for 489.0712, C25H19ClNaO7). 4.1.3.6. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 4-bromobenzofuran-2-carboxylate (6f) Violet powder. Yield: 30.2%. m. p: 102.9-105.2℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.62 (s, 1H, ArH), 7.56 (d, J = 8.4 Hz, 1H, ArH), 7.50 (d, J = 7.8 Hz, 1H, ArH), 7.35 (t, J = 8.1 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.15 (s, 1H, ArH), 6.33 (dd, J = 7.1, 4.6 Hz, 1H, CH-O), 5.22 (t, J = 7.2 Hz, 1H, CH=C), 2.81 – 2.76 (m, 1H, CH2), 2.66 (dt, J = 15.0, 7.4 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.63 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 176.98, 175.43, 168.70,

168.17, 157.90, 155.54, 147.10, 145.15, 136.75, 133.39, 133.19, 131.41, 128.80, 128.63, 127.01, 117.32, 115.94, 114.44, 111.87, 111.65, 111.57, 70.83, 33.00, 25.84, 18.06. HRMS (ESI) m/z: 533.0206 [M+Na]+ (calcd for 533.0206, C25H19BrNaO7). 4.1.3.7. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5-fluorobenzofuran-2-carboxylate (6g) Violet powder. Yield: 29.1%. m. p: 100.1-101.9℃. 1H NMR (600 MHz, CDCl3): 12.61 (s, 1H, OH), 12.41 (s, 1H, OH), 7.58 (s, 1H, ArH), 7.56 (dd, J = 9.2, 4.0 Hz, 1H, ArH), 7.36 (dd, J = 8.0, 2.5 Hz, 1H, ArH), 7.22 (td, J = 9.0, 2.6 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.14 (s, 1H, ArH), 6.31 (dd, J = 6.8, 4.7 Hz, 1H, CH-O), 5.21 (t, J = 7.3 Hz, 1H, CH=C), 2.80 – 2.76 (m, 1H, CH2), 2.64 (dd, J = 15.0, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 177.01, 175.46, 168.66,

168.14, 162.68(d, J= 241.6 Hz), 157.97, 152.20, 147.16, 146.44, 136.70, 133.37, 133.19, 131.40, 127.53(d, J= 12.1 Hz), 117.32, 116.37(d, J= 27.2 Hz), 114.55,

113.46(d, J= 9.1 Hz), 111.87, 111.64, 107.98(d, J= 25.7 Hz), 70.72, 32.96, 25.83, 18.05. HRMS (ESI) m/z: 473.1003 [M+Na]+ (calcd for 473.1007, C25H19FNaO7). 4.1.3.8. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5,7-dichlorobenzofuran-2-carboxylate (6h) Violet powder. Yield: 28.9%. m. p: 105.4-107.0℃. 1H NMR (600 MHz, CDCl3): 12.61 (s, 1H, OH), 12.40 (s, 1H, OH), 8.10 (s, 1H, ArH), 7.60 (s, 1H, ArH), 7.56 (s, 1H, ArH), 7.49 (s, 1H, ArH), 7.19 (s, 2H, ArH), 7.12 (s, 1H, ArH), 6.30 (dd, J = 7.1, 4.6 Hz, 1H, CH-O), 5.20 (t, J = 7.2 Hz, 1H, CH=C), 2.80 – 2.75 (m, 1H, CH2), 2.65 (dd, J = 14.9, 7.5 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.65 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): 176.68, 175.11, 168.93, 168.41, 157.44, 150.35, 146.91, 136.94, 133.48, 133.28, 131.31, 129.86, 129.75, 128.99, 128.05, 120.88, 118.68, 117.18, 114.26, 111.85, 111.64, 71.03, 32.92, 25.86, 18.06. HRMS (ESI) m/z: 523.0319 [M+Na]+ (calcd for 523.0322, C25H18Cl2NaO7). 4.1.3.9. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 7-(tert-butyl)benzofuran-2-carboxylate (6i) Violet powder. Yield: 32.4%. m. p: 102.1-104.6℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.60 (s, 1H, ArH), 7.56 (d, J = 7.8 Hz, 1H, ArH), 7.37 (d, J = 7.5 Hz, 1H, ArH), 7.26 (t, J = 8.0 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.13 (s, 1H, ArH), 6.30 (dd, J = 6.6, 4.8 Hz, 1H, CH-O), 5.24 (t, J = 7.3 Hz, 1H, CH=C), 2.80 – 2.75 (m, 1H, CH2), 2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.63 (s, 3H, CH3), 1.55 (s, 9H, (CH3)3). 13C NMR (151 MHz, CDCl3): 177.54, 176.00, 168.16, 167.62, 158.32, 154.34, 147.69, 144.13, 136.51, 136.01, 133.18, 132.96, 131.51, 127.41, 124.43, 123.95, 120.75, 117.46, 114.64, 111.86, 111.63, 70.27, 34.49, 32.98, 29.79 (3C), 25.84, 18.05. HRMS (ESI) m/z: 511.1726 [M+Na]+ (calcd for 511.1727, C29H28NaO7). 4.1.3.10. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 4-fluorobenzofuran-2-carboxylate (6j) Violet powder. Yield: 33.6%. m. p: 106.7-108.8℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.68 (s, 1H, ArH), 7.46 – 7.41 (m, 2H, ArH),

7.19 (s, 2H, ArH), 7.14 (s, 1H, ArH), 7.04 – 7.00 (m, 1H, ArH), 6.32 (dd, J = 6.8, 4.7 Hz, 1H, CH-O), 5.21 (t, J = 7.3 Hz, 1H, CH=C), 2.81 – 2.76 (m, 1H, CH2), 2.65 (dd, J = 15.0, 7.5 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.62 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): 176.96, 175.41, 168.71, 168.19, 157.86, 157.38(d, J= 39.3 Hz), 156.57(d, J= 221.9 Hz), 147.12, 144.88, 136.74, 133.39, 133.20, 131.39, 128.66(d, J= 7.55 Hz), 117.31, 116.73(d, J= 22.7 Hz), 111.87, 111.65, 110.75, 109.21(d, J= 18.1 Hz), 108.63(d, J= 4.5 Hz), 70.79, 32.97, 25.83, 18.05. HRMS (ESI) m/z: 473.1165 [M+Na]+ (calcd for 473.1160, C25H19FNaO7). 4.1.3.11. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5-chlorobenzofuran-2-carboxylate (6k) Violet powder. Yield: 23.7%. m. p: 101.2-103.4℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.69 (s, 1H, ArH), 7.55 (t, J = 4.2 Hz, 2H, ArH), 7.44 (dd, J = 8.9, 2.0 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.14 (s, 1H, ArH), 6.31 (dd, J = 6.6, 4.8 Hz, 1H, CH-O), 5.20 (t, J = 7.3 Hz, 1H, CH=C), 2.80 – 2.75 (m, 1H, CH2), 2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): 176.92, 175.37, 168.75, 168.23, 157.93, 154.23, 147.09, 146.15, 136.73, 133.41, 133.22, 131.40, 129.69, 128.40, 128.08, 122.33, 117.30, 113.91, 113.60, 111.87, 111.64, 70.79, 32.97, 25.83, 18.05. HRMS (ESI) m/z: 489.0708 [M+Na]+ (calcd for 489.0712, C25H19ClNaO7). 4.1.3.12. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 7-bromo-5-chlorobenzofuran-2-carboxylate (6l) Violet powder. Yield: 26.6%. m. p: 111.2-113.1℃. 1H NMR (600 MHz, CDCl3): 12.61 (s, 1H, OH), 12.40 (s, 1H, OH), 7.63 (s, 2H, ArH), 7.58 (s, 1H, ArH), 7.18 (s, 2H, ArH), 7.12 (s, 1H, ArH), 6.29 (dd, J = 6.9, 4.6 Hz, 1H, CH-O), 5.20 (t, J = 7.3 Hz, 1H, CH=C), 2.80 – 2.74 (m, 1H, CH2), 2.66 (dt, J = 15.0, 7.5 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.65 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 176.74, 175.18, 168.92,

168.39, 157.46, 151.77, 146.96, 146.81, 136.96, 133.48, 133.27, 131.36, 130.81, 130.14, 128.62, 121.48, 117.22, 114.41, 111.88, 111.66, 105.62, 71.05, 32.95, 25.88, 18.11. HRMS (ESI) m/z: 566.9813 [M+Na]+ (calcd for 566.9817, C25H18BrClNaO7).

4.1.3.13. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5-methylbenzofuran-2-carboxylate (6m) Violet powder. Yield: 27.9%. m. p: 108.3-110.2℃. 1H NMR (600 MHz, CDCl3): 12.61 (s, 1H, OH), 12.41 (s, 1H, OH), 7.54 (s, 1H, ArH), 7.48 (d, J = 8.6 Hz, 2H, ArH), 7.30 – 7.27 (m, 1H, ArH), 7.18 (s, 2H, ArH), 7.14 (s, 1H, ArH), 6.31 (dd, J = 6.6, 4.7 Hz, 1H, CH-O), 5.22 (t, J = 7.3 Hz, 1H, CH=C), 2.80 – 2.75 (m, 1H, CH2), 2.64 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 2.46 (s, 3H, Ar-CH3), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3).

13

C NMR (151 MHz, CDCl3): 177.53, 176.02, 168.19, 167.66, 158.39,

154.52, 147.53, 144.94, 136.58, 133.62, 133.18, 132.99, 131.57, 129.61, 126.97, 122.42, 117.49, 114.48, 111.99, 111.89, 111.66, 70.47, 33.03, 25.84, 21.36, 18.06. HRMS (ESI) m/z: 469.1261 [M+Na]+ (calcd for 469.1258, C26H22NaO7). 4.1.3.14. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 6-bromobenzofuran-2-carboxylate (6n) Violet powder. Yield: 31.6%. m. p: 116.6-118.0℃. 1H NMR (600 MHz, CDCl3): 12.61 (s, 1H, OH), 12.40 (s, 1H, OH), 7.79 (s, 1H, ArH), 7.58 (d, J = 7.7 Hz, 2H, ArH), 7.46 (dd, J = 8.4, 1.4 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.13 (s, 1H, ArH), 6.31 (dd, J = 7.1, 4.5 Hz, 1H, CH-O), 5.20 (t, J = 7.2 Hz, 1H, CH=C), 2.80 – 2.75 (m, 1H, CH2), 2.64 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): 176.96, 175.42, 168.69, 168.16, 157.96, 156.04, 147.13, 145.41, 136.71, 133.38, 133.19, 131.39, 127.73, 125.85, 123.85, 121.66, 117.32, 115.91, 114.38, 111.86, 111.63, 70.75, 32.96, 25.84, 18.05. HRMS (ESI) m/z: 533.0205 [M+Na]+ (calcd for 533.0206, C25H19BrNaO7). 4.1.3.15. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 7-ethoxybenzofuran-2-carboxylate (6o) Violet powder. Yield: 36.4%. m. p: 103.0-104.9℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.42 (s, 1H, OH), 7.59 (s, 1H, ArH), 7.27 (d, J = 7.3 Hz, 1H, ArH), 7.22 (t, J = 7.9 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.13 (s, 1H, ArH), 6.95 (d, J = 7.5 Hz, 1H, ArH), 6.30 (dd, J = 6.6, 4.4 Hz, 1H, CH-O), 5.21 (t, J = 7.4 Hz, 1H, CH=C), 4.29 (dd, J = 7.0 Hz, 2H, OCH2CH3), 2.80 – 2.75 (m, 1H, CH2), 2.65 (dt, J = 14.5, 7.2 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.64 (s, 3H, CH3), 1.53 (t, J = 7.0 Hz, 3H,

OCH2CH3).

13

C NMR (151 MHz, CDCl3): 177.61, 176.10, 168.09, 167.56, 158.14,

147.56, 145.94, 145.32, 144.91, 136.68, 133.15, 132.96, 131.55, 128.57, 124.66, 117.41, 114.98, 114.59, 111.88, 111.64, 110.62, 70.49, 64.81, 32.95, 25.85, 18.06, 14.89. HRMS (ESI) m/z: 499.1360 [M+Na]+ (calcd for 499.1363, C27H24NaO8). 4.1.3.16. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5,7-di-tert-butylbenzofuran-2-carboxylate (6p) Violet powder. Yield: 20.1%. m. p: 116.1-118.3℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.56 (s, 1H, ArH), 7.52 (s, 1H, ArH), 7.44 (s, 1H, ArH), 7.19 (s, 2H, ArH), 7.12 (s, 1H, ArH), 6.29 (dd, J = 6.7, 4.6 Hz, 1H, CH-O), 5.23 (t, J = 7.2 Hz, 1H, CH=C), 2.80 – 2.74 (m, 1H, CH2), 2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.70 (s, 3H, CH3), 1.62 (s, 3H, CH3), 1.55 (s, 9H, (CH3)3), 1.39 (s, 9H, (CH3)3).

13

C NMR (151 MHz, CDCl3): 177.67, 176.15, 168.07, 167.52, 158.41,

152.76, 147.82, 146.90, 136.48, 135.10, 133.14, 132.91, 131.57, 129.76, 127.14, 122.81, 117.51, 116.45, 114.95, 111.90, 111.66, 70.16, 35.00, 34.65, 33.01, 31.77(3C), 29.87(3C), 25.84, 18.06. HRMS (ESI) m/z:567.2355 [M+Na]+ (calcd for 567.2353, C33H36NaO7). 4.1.3.17. (R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 5-methoxybenzofuran-2-carboxylate (6q) Violet powder. Yield: 34.5%. m. p: 106-108℃. 1H NMR (600 MHz, CDCl3): 12.62 (s, 1H, OH), 12.41 (s, 1H, OH), 7.55 (s, 1H, ArH), 7.50 (d, J = 7.0 Hz, 1H, ArH), 7.19 (s, 2H, ArH), 7.14 (s, 1H, ArH), 7.12 – 7.08 (m, 2H, ArH), 6.31 (dd, J = 6.7, 4.8 Hz, 1H, CH-O), 5.21 (t, J = 7.3 Hz, 1H, CH=C), 3.87 (s, 3H, OCH3), 2.80 – 2.75 (m, 1H, CH2), 2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): 177.43, 175.91, 168.29, 167.76, 158.26, 156.67, 147.49, 145.47, 136.58, 133.22, 133.03, 131.54, 129.76, 127.36, 118.06, 117.45, 114.72, 113.14, 111.90, 111.66, 103.76, 70.48, 55.90, 33.02, 25.83, 18.06. HRMS (ESI) m/z: 485.1210 [M+Na]+ (calcd for 485.1207, C26H22NaO8). 4.2. Pharmacology 4.2.1. In vitro antiproliferative assay

Human breast cancer cell line MDA-MB-231, human colon cancer cell line HT29

and

HCT116,

human

liver

carcinoma

cell

line

HepG2,

human

non-small-cell-lung cancer cell line A549, human liver cell L02 and human embryonic kidney cell 293T were occupied to determine the cytotoxicity of the test compounds by MTT assay. All mentioned cancer cell lines and non-cancer cells were purchased from the Cell Bank of the Shanghai Institute of Biochemistry (Shanghai, China). MDA-MB-231, HCT116, HepG2, LO2 and 293T cell lines were cultured in DMEM media, HT29 and A549 cell lines were cultured in RPMI media. The media were supplemented with 10% fetal bovine serum at 37 °C and 5% CO2 in a humidify environment. In a word, the cell lines which were at the logarithmic phase were incubated in 96-microwell plates (0.5×104 each well) for 24h. Then the test compounds at different concentrations dissolved in the culture medium were added to each well. After another 48h, MTT (5 mg/mL in PBS) was added and incubated for 4h. The optical density was detected in SpectraMax Plus384 at the wavelength of 570 nm (reference wavelength 630 nm). Each concentration had three replicates and each plate was detected trice. Then the results were expressed as the IC50 values. 4.2.2. Colony formation assay HT29 cells were trypsinized and seeded in the 6-well plates (1000 cells each well), incubated for 24h and then cells were treated with different concentrations of test compounds for 24h. Afterwards, the cells were incubated for 2 weeks until the colony formed. Then the cells were washed with PBS twice, fixed by 4% paraformaldehyde for 30 min and stained by crystal violet staining solution for 30min. At last, the results were photographed by phones. 4.2.3. In vitro tubulin polymerization inhibitory assay Test compounds in different concentrations were added into a pre-warmed 96-well plate (10µL each well), then a solution of tubulin (Cytoskeleton) in G-PEM buffer was prepared with ice bath freshly and added into plates quickly (90µL each well). Thus, each well contains 2.9 mg/mL tubulin, 9.2% glycerol, 0.9 mM GTP and G-PEM buffer which was composed of 80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM EGTA. Then the plate was immediately transferred to the spectrophotometer (SpectraMax Plus384) and read under 340nm at 37 ℃ every 2 min over 60 min. 4.2.4. Competitive inhibition assay

The radiolabeled [3H] colchicine competition scintillation proximity (SPA) assay was operated to evaluated the competitive binding activity of our compounds. Test compounds in different concentrations were added into the 96-well plate, then a 100 µL buffer which contains 4 µM [3H] colchicine, 1 mM GTP, 1 µM modified tubulin, 80 mM PIPES (pH 6.8), 1 mM MgCl2, 10% glycerol,1 mM EGTA were added and incubated at 37 ℃ for 2 h. Then the radioactive counts were measured by scintillation counter. 4.2.5. Cell cycle assay HT29 cells, in the logarithmic phase, were trypsinized and seeded into 6-well plates (2×105 each well) and incubated for 24 h, and then treated with different concentrations of test compounds for another 24 h. The cells were collected and fixed by 70% ice ethanol at 4℃ for 12-24 h. After that, the cells were resuspended in the staining buffer containing 0.1 mg/ml RNase A and 5 µg/ml of propidium iodide for 30 min. At last, the DNA content of the cells was measured using a FACS Calibur flow cytometer (Bectone Dickinson, San Jose, CA, USA ). 4.2.6. Western blotting HT29 cells initially treated with 6c were suspended in the RIPA lysis buffer containing 1% PMSF and transferred to -80℃ to extract the total proteins, then measured the protein concentrations with the BCA Protein Assay kit. Subsequently, 60µg of total proteins were separated by 8% or 10% SEM Sepolyacrylamide gels and transferred onto the PVDF membranes. After blocking with milk for 2h, membranes were incubated with primary antibodies overnight at 4℃. Afterwards, the membranes were washed and incubated with HRP-conjugated secondary antibodies for 2h at room temperature. At last, the samples were detected using a ChemiDOC™ XRS + system (BioRad Laboratories, Hercules, CA). 4.2.7. Cell apoptosis assay HT29 cells, in the logarithmic phase, were grown in 6-well plates (2×105 each well), and then treated with different concentrations of test compounds for 24h. Afterwards, the cells were resuspended in the Annexin V binding buffer and incubated for 15min at 37℃ in the dark. Then the samples were analyzed using a FACS Calibur flow cytometer (Bectone Dickinson, San Jose, CA, USA ).

4.2.8. Mitochondrial membrane potential analysis HT29 cells, in the logarithmic phase, were grown in 6-well plates (2×105 each well), and then treated with different concentrations of 6c for 24h. Subsequently, the cells were collected and treated with the JC-1 buffer (Keygen Biotech, China), then the samples were analyzed using a FACS Calibur flow cytometer (Bectone Dickinson, San Jose, CA, USA ). 4.2.9. Immunofluorescence staining HT29 cells, in the logarithmic phase, were trypsinized and seeded into the laser confocal petri dish (1×105 each dish), incubated for 24 h and then treated with different concentrations of test compounds for another 24 h. Subsequently, the cells were washed with PBS carefully for three times, fixed by 4% paraformaldehyde for 30 min and then permeabilized with 1% TritonX-100 for another 10 min. After blocking, the cells were incubated with anti-β tubulin antibody (1: 500 dilution) overnight at 4℃. Then the dishes were washed and incubated with Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1: 100 dilution) for 2h at room temperature, followed by DAPI (5mg/ml). At last, the samples were observed under the laser confocal microscope (Olympus FV3000). 4.2.10. Tube formation assay The HUVECs were trypsinized and seeded in the 6-well plates (1×105 each well) and incubated for 24h, then treated with different concentrations of 6c for 24h. The matrigel matrix was thawed at 4℃ overnight, and added into an angiogenesis slide (Ibidi, Germany) 10µL each well, after incubated at 37℃ for 30min, the treated HUVECs suspended in F12K were seeded in the wells at a cell density of 15,000 cells each well. The slide was transferred into the incubator for 6h, subsequently, the tube formation and morphological changes were photographed using a inverted fluorescence microscope (Nikon Ts2R, Japan). 4.2.11. Wound healing assay A549 cells, in the logarithmic phase, were grown in 6-well plates for 24 h, then the scratches were made using 200 µL pipette tip and washed with PBS to remove non-adherent cell debris. Subsequently, the cells were treated with different concentrations of 6c for 24h. The migrations across the wound area were photographed under a phase contrast microscopy.

4.2.12. Molecular docking Docking study was carried out using Discovery Studio 3.5 and the tubulin protein (PDB:1SA0) was downloaded from RCSB Protein Date Bank. The protein and all ligands were prepared by minimization with CHARMM force field. Molecular docking was performed by DS-CDOCKER protocol without constraint, all bound water and ligands were eliminated from the protein and the polar hydrogen was added to the proteins. 4.2.13. Metabolic stability assay The solution of 6c, 4c and shikonin were prepared in PEG400 solution [33]. 18 SD rats, half male and half female, were divided into three groups and injected intravenously with 1.5mg/kg of 6c, 4c and shikonin respectively. Blood samples were collected into heparinized microtubes at certain time points and centrifuged at 8000 rpm for 5 min at 4 °C. The supernatant plasma layer was gathered and analysis by UPLC/MS. Acknowledgments This work was supported by National Natural Science Foundation of China (NO. 81602985), the Fundamental Research Funds for the Central Universities (NO. 2632018ZD19), the Drug Innovation Major Project (2018ZX09711-001-007), the 111 Project from Ministry of Education of China and the State Administration of Foreign

Export Affairs

of

China

University Project (CPU2018GF03).

(B18056)

and

the "Double

First-Class"

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7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)

benzo[b]furan

(BNC105),

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with

potent

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benzo[b]furan

of

2-

derivatives

(3’,4’,5’-trimethoxybenzoyl) as

inhibitors

of

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Caption Scheme 1. Synthesis of the desired compounds 6a-6q. Reagents and conditions: (i) KI/K2CO3, BrCH2COOC2H5, DMF, 10℃ (ii) 120℃, reflux (iii) 20%NaOH aqueous, 60℃ (iv) DCC/DMAP, DCM, 0℃. Table 1. The anti-proliferative activities of 6a-6q, shikonin and colchicine against cancer and non-cancer cell lines. Table 2. The IC50 values of compound 6c, shikonin, colchicine, CA-4 and DOX in different drug-resistant cancer cells. Table 3. Inhibition of tubulin polymerization and colchicine binding to tubulin. Table 4. The anti-proliferative activities of 6c, shikonin, 4c and (shikonin+4c) against cancer and non-cancer cell lines. Fig 1. The structure of tubulin assembly inhibitors and the strategy of molecular design. Fig. 2. Effects of 6c on inhibiting conlony formation. Test compounds (6c at 0.05, 0.1, and 0.2µM), colchicine (0.05µM), CA-4 (0.05µM) and DMSO were added in the dishes and the conlonies were stained with crystal violet. The experiments were performed three times. Fig. 3. Effects of 6c on tubulin polymerization in vitro. Test compounds (6c at 2 or 4µM), colchicine (4µM), CA-4 (4µM), Taxol (4µM) and DMSO were added in the plates and then polymerizations were tested as an increase in fluorescence emission at 340 nm over a 60 min at 37 ℃. The experiments were performed three times. Fig. 4. Compound 6c induced G2/M arrest in HT29 cells. (A) HT29 cells were treated with 0.25, 0.5, and 1 µM of 6c and DMSO for 24 h. Then they were collected and processed by PI staining for cell cycle analysis. (B) Cells treated with 0.25, 0.5, and 1 µM of shikonin and DMSO for 24 h were also collected and analyzed. (C) Cells treated with 0.25, 0.5, and 1 µM of colchicine and DMSO for 24 h were collected and analyzed. (D) HT29 cells were treated with 0.5µM of 6c for 12h,36h and 48h. (E) Western blot analyses of cell cycle-related proteins (P21, Cyclin B1, Cdc2, P-Cdc2, Cdc25c and P-Cdc25c) separated by SDS-PAGE. Relative band intensity was determined by Image Lab software. GAPDH served as a loading control. Data are means ± S.E.M. of three independent experiments. Fig. 5. 6c induced HT29 cell apoptosis. (A) AnnexinV/PI double staining and cytometry analysis of 6c-induced apoptosis in HT29 cells in a dose-dependent manner (0, 0.25, 0.5, and 1 µM). (B) AnnexinV/PI double staining and cytometry analysis of shikonin-induced apoptosis in HT29 cells in a dose-dependent manner (0, 0.25, 0.5,

and 1 µM). (C) AnnexinV/PI double staining and cytometry analysis of colchicine-induced apoptosis in HT29 cells in a dose-dependent manner (0, 0.25, 0.5, and 1 µM). (D) AnnexinV/PI double staining and cytometry analysis of 6c-induced apoptosis in HT29 cells in a time-dependent manner (0.5µM of 6c for 12h, 36h and 48h). (E) Western blot analyses of apoptosis-related proteins (Bax, Bcl-2, Cleaved-PARP, Cleaved Caspase-3 and Cleaved Caspase-9) separated by SDS-PAGE. Relative band intensity was determined by Image Lab software. GAPDH served as a loading control. Fig. 6. 6c induced mitochondrial depolarization of HT29 cells. HT29 cells were incubated with 6c at 0.25, 0.5, and 1 µM and DMSO for 24h and followed by staining with JC-1, then analyzed with the flow cytometry. Fig. 7. The anti-microtubule effects of 6c (0.25, 0.5, 1µM), colchicine (0.5µM), paclitaxel (0.5µM) and DMSO visualized by immunofluorescence in HT29 cells. Nucleus stained by DAPI (blue) and Tubulins tagged with Alexa Fluor 594 (red) were photographed using a confocal fluorescence microscope. Fig. 8. The effects of 6c on inhibiting the formation of HUVEC capillary-like tubular network. HUVEC cells that had been treated with 6c at 0.01, 0.02, 0.04µM and DMSO were incubated in the matrigel matrix for 6h, and then photographed using the inverted fluorescence microscope. Fig. 9. Effects of 6c on inhibiting the cell migrations. A549 cells that had been scratched were incubated with 6c at 0.1, 0.2, 0.4µM and DMSO for 24h. Fig. 10. (A) Molecular docking of 6c with tubulin (PDB ID: 1SA0). (B) Molecular docking of colchicine with tubulin. Fig. 11. The plasma concentration of 6c, 4c and shikonin versus time profile intravenous administration at the dosage of 1.5 mg/kg to rats.

compound

R

compound

R

6a

H

6j

6-F

6b

4-CH3

6k

5-Cl

6c

3-OCH3

6l

3-Br-5-Cl

6d

5-Br

6m

5-CH3

6e

2-Cl

6n

4-Br

6f

2-Br

6o

3-OCH2CH3

6g

5-F

6p

3,5-C(CH3)3

6h

3,5-Cl

6q

5-OCH3

6i

3-C(CH3)3

Scheme 1. Synthesis of the desired compounds 6a-6q. Reagents and conditions: (i) KI/K2CO3, BrCH2COOC2H5, DMF, 10℃ (ii) 120℃, reflux (iii) 20%NaOH aqueous, 60℃ (iv) DCC/DMAP, DCM, 0℃.

Table 1. The anti-proliferative activities of 6a-6q, shikonin and colchicine against cancer and non-cancer cell lines. Compounds

IC50 ± SD (µM)a

CC50 ± SD (µM)b

HepG2

HT29

HCT116

MDA-MB-231

A549

293T

LO2

6a

46.75±6.96

7.16±0.81

10.27±1.60

6.00±0.51

24.91±2.29

169.08±17.58

155.31±9.20

6b

75.33±3.23

1.22±0.09

2.28±0.32

1.91±0.34

19.05±1.83

--

--

6c

73.20±4.03

0.18±0.04

0.58±0.11

0.81±0.13

0.57±0.79

184.86±9.88

154.76±9.98

6d

71.83±5.19

7.62±0.85

10.68±0.18

8.22±0.38

18.53±1.43

158.90±19.48

151.52±8.23

6e

--

6.06±0.16

3.86±0.47

3.59±0.33

47.20±2.24

--

--

6f

52.38±5.27

1.03±0.17

2.21±0.40

2.63±0.13

27.33±1.34

162.38±17.77

197.07±11.64

6g

--

10.37±0.44

20.36±1.82

10.42±0.61

42.43±5.35

170.07±18.85

--

6h

44.60±4.88

5.89±0.77

9.16±0.44

4.09±0.75

30.77±2.11

158.61±9.77

143.36±9.88

6i

--

0.82±0.08

1.21±0.07

1.51±0.10

48.81±5.66

--

--

6j

70.68±6.96

4.04±0.29

9.30±0.76

10.74±0.60

28.19±2.53

194.98±10.79

107.54±9.93

6k

82.60±9.68

9.14±0.65

11.61±0.91

4.88±0.12

23.27±2.65

121.92±9.86

165.62±11.98

6l

74.81±8.50

7.55±0.44

11.99±1.21

15.25±0.22

21.55±3.26

148.46±18.78

178.54±8.50

6m

80.92±9.13

16.35±1.06

23.01±0.97

18.33±0.03

22.24±3.83

149.38±7.86

104.74±9.85

6n

77.71±5.81

1.12±0.11

3.57±0.50

2.01±0.06

16.99±0.66

--

--

6o

81.32±8.76

0.73±0.09

1.27±0.34

1.33±0.44

9.11±2.21

--

--

6p

--

--

--

24.68±0.56

--

--

--

6q

90.78±9.73

10.94±0.90

11.28±0.94

6.21±0.03

19.13±1.89

108.40±9.14

137.4±8.81

Shikonin c

2.92±0.09

2.80±0.26

2.47±0.19

2.77±0.29

10.25±1.37

7.00±0.89

11.77±0.14

1.47±0.07

1.81±0.06

2.13±0.13

2.94±0.12

1.17±0.09

8.43±0.71

9.18±0.62

0.27±0.007

0.31±0.02

0.11±0.006

0.09±0.008

0.23±0.006

1.44±0.011

0.36±0.003

Colchicine CA-4 a

c

c

IC50 values are indicated as the mean ± SD (standard error) of at least three

independent experiments. b

Cytotoxicity in human normal cell.

c

Used as positive control.

Table 2. The IC50 values of compound 6c, shikonin, colchicine, CA-4 and DOX in different drug-resistant cancer cells. IC50 ± SD (µM) Compounds MCF7

MCF7/MDR

DRIa

K562

K562/MDR

DRIa

6c

0.53±0.09

2.51±0.55

4.73

0.87±0.09

9.34±0.84

10.73

shikonin

5.40±0.05

23.41±0.16

4.33

20.29±0.47

120.54±1.65

5.94

colchicine

0.11±0.03

0.52±0.08

4.72

0.44±0.11

6.59±0.13

14.97

CA-4

0.25±0.01

0.49±0.85

1.96

0.61±0.02

6.15±0.38

10.08

doxorubicine

0.66±0.06

107.11±1.81

162.28

0.21±0.03

16.31±0.51

77.66

a

DRI: drug-resistant index = (IC50 of drug resistant cancer cell)/(IC50 of parental

cancer cell). b

ND: not detected.

Table 3. Inhibition of tubulin polymerization and colchicine binding to tubulin. Inhibition of colchicine binding Inhibition of tubulin polymerization

Compounds

IC50 ± SD (µM)a

(% inhibition ± SD)b 2µM

4µM

6c

0.98±0.014

82.36±0.88

92.42±0.79

6f

4.13±0.26

--

--

6i

2.37±0.21

--

--

colchicine

2.11±0.32

--

--

CA-4

1.12±0.13

81.46±0.94

92.96±0.87

a

Data are presented as mean from three independent experiments.

b

Tubulin, 1 µM; [3H]-colchicine, 4 µM; and inhibitors, 2 or 4 µM.

Table 4. The anti-proliferative activities of 6c, shikonin, 4c and (shikonin+4c) against cancer and non-cancer cell lines. IC50 ± SD (µM)a

Compounds

HepG2

HT29

HCT116

MDA-MB-231

A549

6c

73.20±4.03

0.18±0.04

0.58±0.11

0.81±0.13

0.57±0.79

Shikonin

2.92±0.09

2.80±0.26

2.47±0.19

2.77±0.29

10.25±1.37

>80

47.38±3.46

61.42±4.62

58.14±3.97

>80

3.11±0.22

4.16±0.47

4.83±0.34

5.63±0.59

7.18±0.51

4c b

Shikonin+4c a

IC50 values are indicated as the mean ± SD (standard error) of at least three

independent experiments. b

the same amount of an equimolar mixture of shikonin and 4c

Fig. 1. The structure of tubulin assembly inhibitors and the strategy of molecular design.

Fig. 2. Effects of 6c on inhibiting conlony formation. Test compounds (6c at 0.05, 0.1, and 0.2µM), colchicine (0.05µM), CA-4 (0.05µM) and DMSO were added in the dishes and the conlonies were stained with crystal violet. The experiments were performed three times.

Fig. 3. Effects of 6c on tubulin polymerization in vitro. Test compounds (6c at 2 or 4µM), colchicine (4µM), CA-4 (4µM), Taxol (4µM) and DMSO were added in the plates and then polymerizations were tested as an increase in fluorescence emission at 340 nm over a 60 min at 37 ℃. The experiments were performed three times.

Fig. 4. Compound 6c induced G2/M arrest in HT29 cells. (A) HT29 cells were treated with 0.25, 0.5, and 1 µM of 6c and DMSO for 24 h. Then they were collected and processed by PI staining for cell cycle analysis. (B) Cells treated with 0.25, 0.5, and 1 µM of shikonin and DMSO for 24 h were also collected and analyzed. (C) Cells treated with 0.25, 0.5, and 1 µM of colchicine and DMSO for 24 h were collected and analyzed. (D) HT29 cells were treated with 0.5µM of 6c for 12h, 36h and 48h. (E) Western blot analyses of cell cycle-related proteins (P21, Cyclin B1, Cdc2, P-Cdc2, Cdc25c and P-Cdc25c) separated by SDS-PAGE. Relative band intensity was determined by Image Lab software. GAPDH served as a loading control. Data are means ± S.E.M. of three independent experiments.

Fig. 5. 6c induced HT29 cell apoptosis. (A) AnnexinV/PI double staining and cytometry analysis of 6c-induced apoptosis in HT29 cells in a dose-dependent manner (0, 0.25, 0.5, and 1 µM). (B) AnnexinV/PI double staining and cytometry analysis of shikonin-induced apoptosis in HT29 cells in a dose-dependent manner (0, 0.25, 0.5, and 1 µM). (C) AnnexinV/PI double staining and cytometry analysis of colchicine-induced apoptosis in HT29 cells in a dose-dependent manner (0, 0.25, 0.5, and 1 µM). (D) AnnexinV/PI double staining and cytometry analysis of 6c-induced apoptosis in HT29 cells in a time-dependent manner (0.5µM of 6c for 12h, 36h and 48h). (E) Western blot analyses of apoptosis-related proteins (Bax, Bcl-2, Cleaved-PARP, Cleaved Caspase-3 and Cleaved Caspase-9) separated by SDS-PAGE. Relative band intensity was determined by Image Lab software. GAPDH served as a loading control.

Fig. 6. 6c induced mitochondrial depolarization of HT29 cells. HT29 cells were incubated with 6c at 0.25, 0.5, and 1 µM and DMSO for 24h and followed by staining with JC-1, then analyzed with the flow cytometry.

Fig. 7. The anti-microtubule effects of 6c (0.25, 0.5, 1µM), colchicine (0.5µM), paclitaxel (0.5µM) and DMSO visualized by immunofluorescence in HT29 cells. Nucleus stained by DAPI (blue) and Tubulins tagged with Alexa Fluor 594 (red) were photographed using a confocal fluorescence microscope.

Fig. 8. The effects of 6c on inhibiting the formation of HUVEC capillary-like tubular network. HUVEC cells that had been treated with 6c at 0.01, 0.02, 0.04µM and DMSO were incubated in the matrigel matrix for 6h, and then photographed using the inverted fluorescence microscope.

Fig. 9. Effects of 6c on inhibiting the cell migrations. A549 cells that had been scratched were incubated with 6c at 0.1, 0.2, 0.4µM and DMSO for 24h.

Fig. 10. (A) Molecular docking of 6c with tubulin (PDB ID: 1SA0). (B) Molecular docking of colchicine with tubulin.

Fig. 11. The plasma concentration of 6c, 4c and shikonin versus time profile intravenous administration at the dosage of 1.5 mg/kg to rats.

Highlights A novel series of shikonin-benzo[b]furan derivatives were designed and synthesized as tubulin polymerization inhibitors Compound 6c inhibited tubulin polymerization and compete with [3H] colchicine in binding to tubulin Compound 6c exhibited remarkable activity against cancer cell line HT29 Compound 6c displayed the potent inhibition on cell migration and tube formation that contributes to the anti-angiogenesis

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

none