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

Article abstract of DOI:10.1016/j.ejmech.2020.112105

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.

Full text of DOI:10.1016/j.ejmech.2020.112105

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
EJMECH 112105
Reference:
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: cpu_lykong@126.com (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: cpu_lykong@126.com (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)
1
Violet powder. Yield: 35.7%. m. p: 88.7-90.3℃. H 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). 13C 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)
1
Violet powder. Yield: 36.6%. m. p: 113.4-115.2℃. H 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)
1
Violet powder. Yield: 32.1%. m. p: 102.9-105.4℃. H 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). 13C 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)

1
Violet powder. Yield: 33.2%. m. p: 107.6-108.9℃. H 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). 13C 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)
1
Violet powder. Yield: 30.2%. m. p: 102.9-105.2℃. H 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). 13C 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)
1
Violet powder. Yield: 29.1%. m. p: 100.1-101.9℃. H 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,
13
3H, CH3), 1.62 (s, 3H, CH3). 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)
1
Violet powder. Yield: 28.9%. m. p: 105.4-107.0℃. H 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)
1
Violet powder. Yield: 33.6%. m. p: 106.7-108.8℃. H 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)
1
Violet powder. Yield: 23.7%. m. p: 101.2-103.4℃. H 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)
1
Violet powder. Yield: 26.6%. m. p: 111.2-113.1℃. H 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,
13
3H, CH3), 1.65 (s, 3H, CH3). 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)
1
Violet powder. Yield: 27.9%. m. p: 108.3-110.2℃. H 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). 13C 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)
1
Violet powder. Yield: 31.6%. m. p: 116.6-118.0℃. H 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)
1
Violet powder. Yield: 36.4%. m. p: 103.0-104.9℃. H 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). 13C 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)
1
Violet powder. Yield: 20.1%. m. p: 116.1-118.3℃. H 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). 13C 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),
13
2.65 (dt, J = 14.9, 7.4 Hz, 1H, CH2), 1.69 (s, 3H, CH3), 1.62 (s, 3H, CH3). C 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 (B18056) and the "Double First-Class"
University Project (CPU2018GF03).

References
[1] G.M. Alushin, G.C. Lander, E.H. Kellogg, R. Zhang, D. Baker, E. Nogales,
Highresolution microtubule structures reveal the structural transitions in α/β tubulin
upon GTP hydrolysis, Cell 157 (2014) 1117-1129.
[2] K. Harindranath, R.V. Hofele, B. Jacek, K. Satish, T. Katharina, U. Henning, M.
Eckhard, Z. Markus, Tau stabilizes microtubules by binding at the interface between
tubulin heterodimers, P. Natl. Acad. Sci. USA 112 (2015) 7501-7506.
[3] A. Anna, M.O. Steinmetz, Control of microtubule organization and dynamics: two
ends in the limelight, Nat. Rev. Mol. Cell Bio. 16 (2015) 711-726.
[4] A. Muroyama, T. Lechler, Microtubule organization, dynamics and functions in
differentiated cells, Development 144 (2017) 3012-3021.
[5] D. Charles, J. Mary Ann, Microtubule-binding agents: a dynamic field of cancer
therapeutics, Nat. Rev. Drug Discov. 9 (2010) 790-803.
[6] G. Wang, F. Peng, D. Cao, Z. Yang, X. Han, J. Liu, W. Wu, L. He, L. Ma, J. Chen,
Y. Sang, M. Xiang, A. Peng, Y. Wei, L. Chen, Design, synthesis and biological
evaluation of millepachine derivatives as a new class of tubulin polymerization
inhibitors, Bioorg. Med. Chem. 21 (2013) 6844-6854.
[7] M.A. Jordan, K. Wendell, S. Gardiner, W.B. Derry, H. Copp, L. Wilson, Mitotic
block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in
abnormal mitotic exit and apoptotic cell death, Cancer Res. 56 (1996) 816-825.
[8] I. Ojima, K. Kumar, D. Awasthi, J.G. Vineberg, Drug discovery targeting cell
division proteins, microtubules and FtsZ, Bioorg. Med. Chem. 22 (2014) 5060-5077.
[9] J. Xi, X. Zhu, Y. Feng, N. Huang, G. Luo, Y. Mao, X. Han, W. Tian, G. Wang, X.
Han, Development of a novel class of tubulin inhibitors with promising anticancer
activities, Mol. Cancer Res. 11 (2013) 856-864.
[10] R.B.G. Ravelli, G. Beno T, P.A. Curmi, J. Isabelle, L. Sylvie, S. André, K.
Marcel, Insight into tubulin regulation from a complex with colchicine and a
stathmin-like domain, Nature 428 (2004) 198-202.
[11] B. Bhabatarak, P. Dulal, G. Suvroma, B. Mithu, Anti-mitotic activity of
colchicine and the structural basis for its interaction with tubulin, Med. Res. Rev. 28
(2010) 155-183.
[12] L. Ling, S. Jiang, X. Li, L. Yao, S. Jing, J. Chen, Recent advances in
trimethoxyphenyl (TMP) based tubulin inhibitors targeting the colchicine binding site,
Eur. J. Med. Chem. 151 (2018) 482-494.

[13] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An overview of tubulin inhibitors
that interact with the colchicine binding site, Pharm. Res. 29 (2012) 2943-2971.
[14] H.Y. Lin, H.W. Han, L.F. Bai, H.Y. Qiu, D.Z. Yin, J.L. Qi, X.M. Wang, H.W. Gu,
Y.H. Yang, Design, synthesis and biological evaluation of shikonin thioglycoside
derivatives: new antitubulin agents, RSC Adv. 4 (2014) 49796-49805.
[15] J. Guo, X.F. Chen, J. Liu, H.Y. Lin, H.W. Han, H.C. Liu, S.C. Huang, B.K.
Shahla, A. Kulek, J.L. Qi, X.M. Wang, L.J. Liang, Y.H. Yang, Novel shikonin
derivatives targeting tubulin as anti-cancer agents, Chem. Biol. Drug Des. 84 (2014)
603-615.
[16] F. Yang, Y. Chen, W.H. Duan, C. Zhang, H. Zhu, J. Ding, SH-7, A new
synthesized shikonin derivative, exerting its potent antitumor activities as a
topoisomerase inhibitor, Int. J. Cancer 119 (2006) 1184-1193.
[17] L.S. Yoshida, T. Kawada, K. Irie, Y. Yuda, T. Himi, F. Ikemoto, H.
Takanoohmuro, Shikonin directly inhibits nitric oxide synthases: possible targets that
affect thoracic aorta relaxation response and nitric oxide release from RAW 264.7
Macrophages, J. Pharmacol. Sci. 112 (2010) 343-351.
[18] W. Liang, A. Cai, G. Chen, H. Xi, X. Wu, J. Cui, K. Zhang, X. Zhao, J. Yu, B.
Wei, L. Chen, Shikonin induces mitochondria-mediated apoptosis and enhances
chemotherapeutic sensitivity of gastric cancer through reactive oxygen species, Sci.
Rep. 6 (2016) 38267-38278.
[19] X. Mao, C.R. Yu, W.H. Li, W.X. Li, Induction of apoptosis by shikonin through a
ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia
(CML) cells, Cell Res. 18 (2008) 879-888.
[20] K.B. Shahla, L.J. Ling, H.Y. Qiu, L. Ma, H.Y. Lin, S.C. Huang, J.L. Qi, X.M.
Wang, G.H. Lu, Y.H. Yang, Synthesis and biological evaluation of novel shikonin
ester derivatives as potential anti-cancer agents, RSC Adv. 4 (2014) 35588-35596.
[21] J. Chen, J. Xie, Z. Jiang, B. Wang, Y. Wang, X. Hu, Shikonin and its analogs
inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2, Oncogene 30
(2011) 4297-4306.
[22] H.Y. Lin, W. Chen, J.Shi, W.Y. Kong, J.L. Qi, X.M. Wang, Y.H. Yang, Design,
synthesis and biological evaluation of cinnamic acyl shikonin derivatives, Chem. Biol.
Drug Des. 81 (2013) 275-283.
[23] X.M. Wang, H.Y. Lin, W.Y. Kong, J. Guo, H.S. Huang, J.L. Qi, Synthesis and
biological evaluation of heterocyclic carboxylic acyl shikonin derivatives, Chem. Biol.
Drug Des. 83 (2014) 334-343.

[24] J. Cao, J. Zhang, W. Peng, A phase I study of safety and pharmacokinetics of
fruquintinib, a novel selective inhibitor of vascular endothelial growth factor receptor
-1, -2, -3 tyrosine kinases in Chinese patients with advanced solid tumors, Cancer
Chemoth. Pharm. l78 (2016) 259-269.
[25] D.D. Wang, G.G. Chen, L.L. Ren, Preparation and characterization of the
sulfobutylether-β-cyclodextrin inclusion complex of amiodarone hydrozhloride with
enhanced oral bioavaliability in fasted state, AAPS Pharmscitech 18 (2017)
1526-1535.
[26] B.L. Flynn, G.S. Gill, D.W. Grobelny, J.H. Chaplin, D. Paul, A.F. Leske,
Discovery
of
7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)
tubulin polymerization inhibitor with potent
benzo[b]furan (BNC105),
a
antiproliferative and tumor vascular disrupting properties, J. Med. Chem. 17 (2011)
6014-6027.
[27] B.L. Flynn, E. Hamel, M.K. Jung, One-pot synthesis of benzo[b]furan and indole
inhibitors of tubulin polymerization, J. Med. Chem. 45 (2002) 2670-2673.
[28] R. Romagnoli, P.G. Baraldi, T. Sarkar, M.D. Carrion, O.C. Lopez, C.L. Cara,
Synthesis and biological evaluation of 2- (3’,4’,5’-trimethoxybenzoyl)
-3-N,N-dimethylamino benzo[b]furan derivatives as inhibitors of tubulin
polymerization, Bioorg. Med. Chem. 16 (2008) 8149-8426.
[29] R. Romagnoli, P.G. Baraldi, M.D. Carrion, C.L. Cara, O.C. Lopez, M. Tolomeo,
Design, synthesis and structure-activity relationship of 2- (3’,4’,5’-trimethoxybenzoyl)
-benzo[b]furan derivatives as a novel class of inhibitors of tubulin polymerization,
Bioorg. Med. Chem. 17 (2009) 6862-6871.
[30] M.J. Pérez-Pérez, E.M. Priego, O. Bueno, M.S. Martins, M.D. Canela, S. Liekens,
Blocking blood flow to solid tumors by destabilizing tubulin: an approach to targeting
tumor growth, J. Med. Chem. 59 (2016) 8685-8711.
[31] J.H. Byun, H.Y. Kim, Y.S. Kim, I.M. Jung, D.J. Kim, W.K. Lee, K.H. Yoo,
Aminostyrylbenzofuran derivatives as potent inhibitors for Aβ fibril formation,
Bioorg. Med. Chem. Lett. 18 (2008) 5591-5593.
[32] L.D. Luca, G. Giacomelli, G. Nieddu, Synthesis of substituted benzofurans via
microwave-enhanced catch and release strategy, J. Comb. Chem. 10 (2008) 517-520.
[33] P.K. Working, M.S. Newman, J. Johnson, J.B. Cornacoff, Safety of poly(ethylene
glycol) and poly(ethylene glycol) derivatives, ACS Sym. Ser. 680 (1997) 45-57.

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
H
6-F
6a
6b
6c
6d
6e
6f
6j
4-CH3
3-OCH3
5-Br
5-Cl
6k
6l
3-Br-5-Cl
5-CH3
6m
6n
6o
6p
6q
2-Cl
4-Br
2-Br
3-OCH2CH3
3,5-C(CH3)3
5-OCH3
5-F
6g
6h
6i
3,5-Cl
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.
IC50 ± SD (µM)a
CC50 ± SD (µM)b
Compounds
HepG2
HT29
HCT116
MDA-MB-231 A549
293T
LO2
46.75±6.96 7.16±0.81
75.33±3.23 1.22±0.09
73.20±4.03 0.18±0.04
71.83±5.19 7.62±0.85
10.27±1.60 6.00±0.51
24.91±2.29
19.05±1.83
0.57±0.79
18.53±1.43
47.20±2.24
27.33±1.34
42.43±5.35
30.77±2.11
48.81±5.66
28.19±2.53
23.27±2.65
21.55±3.26
22.24±3.83
16.99±0.66
9.11±2.21
--
169.08±17.58 155.31±9.20
6a
2.28±0.32
0.58±0.11
1.91±0.34
0.81±0.13
--
--
6b
184.86±9.88
154.76±9.98
6c
10.68±0.18 8.22±0.38
158.90±19.48 151.52±8.23
-- --
6d
--
52.38±5.27 1.03±0.17
-- 10.37±0.44
44.60±4.88 5.89±0.77
-- 0.82±0.08
6.06±0.16
3.86±0.47
2.21±0.40
3.59±0.33
2.63±0.13
6e
162.38±17.77 197.07±11.64
170.07±18.85 --
6f
20.36±1.82 10.42±0.61
6g
9.16±0.44
1.21±0.07
9.30±0.76
4.09±0.75
1.51±0.10
10.74±0.60
158.61±9.77
--
143.36±9.88
--
6h
6i
70.68±6.96 4.04±0.29
82.60±9.68 9.14±0.65
74.81±8.50 7.55±0.44
80.92±9.13 16.35±1.06
77.71±5.81 1.12±0.11
81.32±8.76 0.73±0.09
194.98±10.79 107.54±9.93
121.92±9.86 165.62±11.98
148.46±18.78 178.54±8.50
6j
11.61±0.91 4.88±0.12
11.99±1.21 15.25±0.22
23.01±0.97 18.33±0.03
6k
6l
149.38±7.86
--
104.74±9.85
--
6m
3.57±0.50
1.27±0.34
--
2.01±0.06
1.33±0.44
24.68±0.56
6n
--
--
6o
--
--
--
--
6p
90.78±9.73 10.94±0.90
11.28±0.94 6.21±0.03
19.13±1.89
10.25±1.37
1.17±0.09
0.23±0.006
108.40±9.14
7.00±0.89
8.43±0.71
1.44±0.011
137.4±8.81
11.77±0.14
9.18±0.62
0.36±0.003
6q
Shikonin c
Colchicine c
CA-4 c
2.92±0.09
1.47±0.07
2.80±0.26
1.81±0.06
2.47±0.19
2.13±0.13
2.77±0.29
2.94±0.12
0.27±0.007 0.31±0.02
0.11±0.006 0.09±0.008
a
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
0.53±0.09
5.40±0.05
0.11±0.03
0.25±0.01
0.66±0.06
2.51±0.55
23.41±0.16
0.52±0.08
0.49±0.85
107.11±1.81
4.73
0.87±0.09
20.29±0.47
0.44±0.11
0.61±0.02
0.21±0.03
9.34±0.84
120.54±1.65
6.59±0.13
6.15±0.38
16.31±0.51
10.73
5.94
6c
4.33
shikonin
colchicine
CA-4
4.72
14.97
10.08
77.66
1.96
162.28
doxorubicine
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
IC50 ± SD (µM)a
(% inhibition ± SD)b
Compounds
2µM
4µM
0.98±0.014
4.13±0.26
2.37±0.21
2.11±0.32
1.12±0.13
82.36±0.88
92.42±0.79
6c
--
--
6f
--
--
6i
--
--
colchicine
CA-4
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
0.81±0.13
A549
73.20±4.03
2.92±0.09
>80
0.18±0.04
2.80±0.26
47.38±3.46
4.16±0.47
0.58±0.11
2.47±0.19
61.42±4.62
4.83±0.34
0.57±0.79
10.25±1.37
>80
6c
2.77±0.29
Shikonin
58.14±3.97
5.63±0.59
4c
Shikonin+4cb
3.11±0.22
7.18±0.51
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.