Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents through microtubule stabilization

Article abstract of DOI:10.1016/j.bcp.2015.04.021

The high incidence of cancer and the side effects of traditional anticancer drugs motivate the search for new and more effective anticancer drugs. In this study, we synthesized 17 kinds of aryl dihydrothiazol acyl shikonin ester derivatives and evaluated their anticancer activity through MTT assay. Among them, C13 showed better antiproliferation activity with IC₅₀ = 3.14 ± 0.21 μM against HeLa cells than shikonin (IC₅₀ = 5.75 ± 0.47 μM). We then performed PI staining assay, cell cycle distribution, and cell apoptosis analysis for C13 and found that it can cause cell arrest in G2/M phase, which leads to cell apoptosis. This derivative can also reduce the adhesive ability of HeLa cells. Docking simulation and confocal microscopy assay results further indicated that C13 could bind well to the tubulin at paclitaxel binding site, leading to tubulin polymerization and mitotic disruption.

Full text of DOI:10.1016/j.bcp.2015.04.021

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BCP-12238; No. of Pages 14
Biochemical Pharmacology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as
anticancer agents through microtubule stabilization
a,b
a,b
c
a,b
a,b
Hong-Yan Lin , Zi-Kang Li , Li-Fei Bai , Shahla Karim Baloch , Fang Wang
,
a,b
a,b
a,b
a,b
a,b,
Han-Yue Qiu , Xue Wang , Jin-Liang Qi , Raong-Wu Yang , Xiao-Ming Wang *,
a,b,
Yong-Hua Yang
*
a
State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of Plant Molecular Biology, School of Life Sciences, Nanjing University,
Nanjing 210023, China
b
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
c
Jiangsu Key Laboratory of Biofunction Molecule, Jiangsu Second Normal University, Nanjing 210013, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The high incidence of cancer and the side effects of traditional anticancer drugs motivate the search for
new and more effective anticancer drugs. In this study, we synthesized 17 kinds of aryl dihydrothiazol
acyl shikonin ester derivatives and evaluated their anticancer activity through MTT assay. Among them,
C13 showed better antiproliferation activity with IC50 = 3.14 ꢀ 0.21 mM against HeLa cells than shikonin
Received 6 February 2015
Accepted 28 April 2015
Available online xxx
(
IC50 = 5.75 ꢀ 0.47 mM). We then performed PI staining assay, cell cycle distribution, and cell apoptosis
Chemical compounds studied in this article:
analysis for C13 and found that it can cause cell arrest in G2/M phase, which leads to cell apoptosis. This
derivative can also reduce the adhesive ability of HeLa cells. Docking simulation and confocal microscopy
assay results further indicated that C13 could bind well to the tubulin at paclitaxel binding site, leading to
tubulin polymerization and mitotic disruption.
Shikonin (PubChem CID: 479503)
L
-Cysteine (PubChem CID: 5862)
Colchicine (PubChem CID: 6167)
Paclitaxel (PubChem CID: 36314)
Benzonitrile (PubChem CID: 7505)
ß 2015 Elsevier Inc. All rights reserved.
4
-(Dimethylamino)benzonitrile (PubChem
CID: 70967)
4
7
4
4
1
2
7
-Methoxybenzonitrile (PubChem CID:
0129)
-Methylbenzonitrile (PubChem CID: 7724)
-Chlorobenzonitrile (PubChem CID:
2163)
,3-Dichlorobenzonitrile (PubChem CID:
36567)
Keywords:
Tubulin polymerization
Mitotic arrest
Shikonin ester derivatives
instability are the two established hallmarks of cancer [2]. Micro-
1
. Introduction
tubules (MTs) are cytoskeletal filaments comprising
tubulin proteins and an individual MT is a long and hollow cylinder
3]. However, the collective structure of MTs may assume various
a- and b-
Mitosis is a complicated process that ensures the faithful
[
inheritance of genetic material by cellular progeny while
preventing aneuploidy [1]. Mitosis disruption and chromosomal
configurations. For example, MT aster is a star-shaped structure
where MTs radiate from a centrally located centrosome [4]. The
mitotic spindle that forms during cell division is composed of two
asters located at opposite poles of the cell. MTs emanate from
asters toward the spindle midzone and exert forces to separate
chromosomes between two daughter cells. The formation of MTs is
a dynamic process that involves the polymerization and de-
Abbreviations: MTT, (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide); NMR, nuclear magnetic resonance; TLC, thin layer chromatography;
TMS, tetramethylsilane; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered
0
saline; DAPI, 4 ,6-diamidino-2-phenylindole; BSA, bovine serum albumin; SAR,
structure–activity relationship.
polymerization of a- and b-tubulin heterodimers [5]. Disruption of
*
Corresponding authors. Tel.: +86 25 89681381; fax: +86 25 89681381.
the dynamic equilibrium blocks the cell division machinery at
mitosis and leads to cells cycle arrest in metaphase, resulting in cell
E-mail addresses: Wangxm07@nju.edu.cn (X.-M. Wang), Yangyh@nju.edu.cn
(
Y.-H. Yang).
http://dx.doi.org/10.1016/j.bcp.2015.04.021
006-2952/ß 2015 Elsevier Inc. All rights reserved.
0
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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death [5,6]. Given the vital role of MTs in cell growth and function,
it has become an important target for the design and development
of new anticancer agents.
2.2. Bioactivity
2.2.1. C13 selectively inhibited cancer-cell proliferation
Tubulin-binding agents, also named ‘‘spindle poisons’’ can bind
to tubulin and alter MT polymerization, perturb MT dynamics,
disrupt mitotic spindle function, and block cell cycle progression
All synthesized derivatives C1–C17 were evaluated for their
antiproliferation activities against five cancer cell lines [human
hepatoma cell line (HepG2), human lung adenocarcinoma
epithelial cell line (A549), carcinoma of cervix cell line (HeLa),
human breast cancer cell line (MCF-7) and squamous cell
carcinoma (SSC-4)], and two non-cancer cell lines [African green
monkey kidney cell (VERO) and human normal liver cell (L02)] by
MTT assay. Results shown in Table 3 indicated that all compounds
had lower toxicity against the non-cancer cells VERO and L02 than
shikonin itself. However, the introduction of dihydrothiazol
moiety also generally attenuated the antiproliferation activity of
shikonin against cancer cells as well. Fortunately, we obtained
some good compounds with strong toxicity toward cancer cells
while avoiding VERO and L02 cells.
[
7]. Historically, taxanes and vinca alkaloids represent tubulin-
binding agents [7–10]. The former promotes MT polymerization,
whereas the latter inhibits MT polymerization. These agents
induce sustained mitotic arrest in metaphase/anaphase transition
and subsequent apoptotic cell death [11,12].
Shikonin and its derivatives are active naphthoquinone
compounds isolated from the root of the Chinese herbal medicine
Lithospermum erythrorhizon [13]. Shikonin is attracting consider-
able attention in the field of natural product chemistry because it
has extensive pharmacological activities [14–16], especially good
anticancer effects [17–20]. However, the side effects and toxicity of
shikonin hinder its development as a new clinical anticancer agent
Table 3 shows that most of the target compounds can also
effectively inhibit the proliferation of the five tumor cells, but the
effects werenotbetter than those of the shikonin. The main reason is
dihydrothiazol moiety reduced shikonin cytotoxicity. Interestingly,
the introduction of some moieties reduced only the toxicity of
shikonin toward the non-cancer cells except for cancer cells, such as
C2 (IC50 = 15.6 ꢀ 1.00 mM), C5 (IC50 = 9.91 ꢀ 0.64 mM), and C7
(IC50 = 10.4 ꢀ 0.87 mM) against HepG2 cells (IC50 = 9.36 ꢀ 0.57 mM
for shikonin); C1 (IC50 = 7.95 ꢀ 0.32 mM), C12 (IC50 = 9.66 ꢀ 0.37 mM),
[
21]. In our previous study, we found that changing the side chain
hydroxyl group into an ester group may reduce the toxicity of
shikonin toward non-cancer cells; thus, shikonin ester derivatives
may be good anticancer agents [19,20,22–25]. Meanwhile, some
researchers have reported aryl thiazole compounds that can
disrupt tubulin polymerization, thereby inhibiting the production
of functional MTs and cell mitosis [26]. Thiazole also improves the
water solubility and pharmacokinetic parameters of the drugs
itself [26,27]. Based on the aforementioned results, we proposed in
this work that theintroduction of thiazole moiety into shikonin
may improve its water solubility and tumor targeting. Based on
these studies, we synthesized a series of aryl dihydrothiazol acyl
shikonin ester derivatives and evaluated them as tubulin-binding
agents. The underlying mechanism was also studied.
and C16 (IC50 = 7.93 ꢀ 0.47 mM) against HeLa cells (IC50
=
5.75 ꢀ 0.47 mM for shikonin); C13 (IC50 = 6.52 ꢀ 0.68 mM), C7
(IC50 = 7.24 ꢀ 0.77 mM), and C2 (IC50 = 6.41 ꢀ 0.38 mM) against
MCF-7 cells (IC50 = 4.99 ꢀ 0.36 mM for shikonin); and C1
(IC50 = 16.2 ꢀ 1.14 mM), and C12 (IC50 = 11.1 ꢀ 1.00 mM) against
SCC-4 cells (IC50 = 10.1 ꢀ 0.49 mM for shikonin). We also obtained
C13 (IC50 = 3.14 ꢀ 0.21 mM) and C8 (IC50 = 5.69 ꢀ 0.42 mM), which
showed better antiproliferation activity against HeLa cells than
shikonin (IC50 = 5.75 ꢀ 0.47 mM).
2
. Results
2.1. Chemistry
To further determine the effect of the compounds on HeLa cell
viability, we performed PI staining assay for C13. HeLa cells
5
The routes to synthesizing the novel aryl dihydrothiazol acyl
shikonin ester derivatives C1–C17 are outlined in Fig. 1. These
(1 ꢁ 10 per well) were seeded in 12-well plates and treated with
C13 of various concentrations (0, 1, 3, and 5 mM) at 37 8C and 5%
compounds were obtained in three steps, as elucidated in Section
CO for 0, 12, 24, and 36 h. Cells were then collected and analyzed
2
3
. All synthesized compounds were reported and characterized for
using PI staining and flow cytometry analysis. From the results
1
the first time by H NMR, elemental analysis, melting test, and
mass spectroscopy, and results were in accordance with their
depicted structures.
shown in Fig. 2, we observed that C13 induced dose- and time-
dependent death of cancer cells. Therefore, C13 could efficiently
inhibit cancer cell growth.
Fig. 1. Synthesis routes of C1–C17. The detailed process was described in Section 3.
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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Fig. 2. Effects of C13 in killing HeLa cells was analyzed using PI staining and flow cytometry analysis. Each image is representative of three experiments. Data shown are the
mean ꢀ S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.
5
2
.2.2. C13 reduced the adhesive ability of HeLa cells
Cell adhesion is an important indicator of cancer cells. Herein,
HeLa cells (2 ꢁ 10 per well) were seeded in 24-well plates and
treated with C13 and shikonin of different concentrations for 24 h.
After harvesting the cells, their adhesive ability to fibronectin and
laminin was detected. The data shown in Fig. 3 indicated that C13
we examined the adhesive ability of HeLa cells as influenced by
C13 and shikonin, which was used as positive control in this assay.
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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Fig. 3. Influence of C13 and shikonin on HeLa cell adhesive to fibronectin and laminin (A) Influence of C13 on HeLa cell adhesive to fibronectin; (B) Influence of C13 on HeLa cell
adhesive to laminin; (C) Influence of shikonin on HeLa cell adhesive to fibronectin; (D) Influence of shikonin on HeLa cell adhesive to laminin. Data shown are the
mean ꢀ S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.
could significantly reduce the adhesive ability of HeLa cells to
fibronectin and laminin but the effects of shikonin were not
obvious.
phase increases with increased drug concentration, and 30.16% of
cells were arrested in G2/M phase upon exposure to 5 M C13 for
24 h. Meanwhile, data shown in Fig. 5(B) demonstrated that the
maximum accumulation of cells in G2/M phase was observed after
m
2.2.3. C13 caused cell apoptosis in HeLa cells in a dose- and time-
treatment with 5 mM C13 for 48 h.
dependent manner
Subsequently, we investigated the effect of C13 on cell
apoptosis using shikonin as positive control. We treated HeLa
cells with various concentrations of C13 and shikonin for 24 h and
analyzed cells for changes in apoptotic markers with a flow
cytometer in vitro. As shown in Fig. 4(A), the percentage of
apoptotic cell significantly increased after treatment with high
2.2.5. Molecular docking of C13 and tubulin
To better understand the potency of C13 and further guide
structure–activity relationship (SAR) studies, we examined the
interaction of C13 and shikonin with tubulin (PDB code: 1JFF). All
docking runs were applied using the Lamarckian genetic
algorithm of Auto-Dock 4.0. The interaction of C13 with tubulin
amino acid residues is depicted in Fig. 6(A). All amino acid
residues of tubulin that interacted with C13 were exhibited. In
doses of C13. Meanwhile, we treated HeLa cells with 4
, 12, 24 and 36 h and found that cells were induced to apoptosis in
a time-dependent manner (Fig. 4(B)). Likewise, we treated HeLa
cells with different doses of shikonin and found that the 5 M- and
M-treated groups displayed apoptosis (Fig. 4(C)). We also
treated HeLa cells with 4 M shikonin for 12, 24, and 36 h and
mM C13 for
0
the binding mode, C13 bound well to the paclitaxel site of tubulin
˚
m
through three
p
bonds with ARG284 (distance = 4.81 A) and HIS
˚
1
0
m
229 (distance = 4.18 and 4.04 A). The CDOCK interaction energy
value for C13 and tubulin was ꢂ40.17 kcal/mol. Fig. 6(B) depicts
the 3D models of the interaction between C13 and tubulin. We
further investigated the binding mode of shikonin and tubulin at
the paclitaxel site. Fig. 6(C) and (D) showed that shikonin bound
m
found that the cells did not undergo significant apoptosis until the
exposure time was extended to 36 h (Fig. 4(D)). By comparison, we
drew the conclusion that C13 caused cell apoptosis more
effectively than shikonin.
to the paclitaxel site of tubulin through one
p
bond with HIS 229
˚
(
distance = 4.90 A) and two hydrogen bonds with PRO 274
˚
˚
2
.2.4. C13 induced cell cycle arrest in HeLa cells in a dose- and time-
(distance = 1.98 A), as well as THR276 (distance = 2.21 A). The
CDOCK interaction energy value for shikonin and tubulin was
dependent manner
We next assessed the cell cycle distribution of HeLa cells by flow
cytometry. HeLa cells treated with C13 of different concentrations
ꢂ27.36 kcal/mol. We found that C13 could bind to
b-tubulin at
the paclitaxel site better than shikonin by comparing binding
modes and interaction energy. Molecular docking results
indicated that C13 could be a potential tubulin polymerization
stabilizer.
(0, 1, 3, and 5 mM) for 24 h and cells treated with 5 mM C13 for
different times (0, 12, 24, and 48 h) could both arrest cells in G2/M
phase. As shown in Fig. 5(A), the accumulation of cells in G2/M
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
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Fig. 4. Annexin V/PI dual-immuno-fluorescence staining after treatment with different concentrations of C13 and shikonin for different times revealed significantly increased
number of apoptotic and necrotic cells (measured with Annexin V+/PI+ cells). (A) Cells treated with 0, 1, 3, 5, and 10 M C13 for 24 h were collected and processed for analysis.
B) Cells treated with 4 M C13 for different times (0, 12, 24, and 36 h) was collected and analyzed. (C) Cells treated with 0, 1.25, 2.5, 5, and 10 M shikonin for 24 h were
collected and processed for analysis. (D) Cells treated with 4 M shikonin for different times (0, 12, 24, and 36 h) was collected and analyzed. The percentage of early
m
(
m
m
m
apoptotic cells in the lower right quadrant (Annexin V-FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant (Annexin V-FITC
positive/PI positive cells). Images are representative of three independent experiments. Data are mean ꢀ S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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Fig. 5. Effect of C13 on the cell cycle distribution of HeLa cells. (A) Cells treated with 0, 1, 3, and 5
mM C13 for 24 h were collected and processed for analysis (G1 phase, green; S
phase, yellow and G2/M phase, blue). (B) Cells treated with 3 M C13 for different times (0, 12, 24, and 48 h) were collected and analyzed (G1 phase, green; S phase, yellow
m
and G2/M phase, blue). Images are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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Fig. 6. Molecular docking analysis of C13, showing proposed binding modes at the paclitaxel binding pocket
b
-tubulin (PDB code: 1JFF). (A) Interaction of C13 with the amino
acid residues of paclitaxel binding site (carbon atom, gray; oxygen atom, red; hydrogen atom, white; sulfur atom, yellow, chlorine atom, green and nitrogen atom; light blue).
B) Binding position of C13 in the protein surface of tubulin (carbon atom, gray; oxygen atom, red; hydrogen atom, white; sulfur atom, yellow, chlorine atom, green and
(
nitrogen atom; light blue). (C) Interaction of shikonin with the amino acid residues of paclitaxel binding site (carbon atom, blue; oxygen atom, red; hydrogen atom, white). (D)
Binding position of shikonin in the protein surface of tubulin (carbon atom, blue; oxygen atom, red; hydrogen atom, white). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
2
.2.6. C13 enhanced tubulin polymerization and disrupted cell
division
To observe the phenotypic effect of C13 on the cellular
3. Experimental
3.1. Materials and measurements
cytoskeletal network of tubulin, HeLa cells were immunostained
and analyzed under a confocal microscope. Fig. 7 clearly shows
the substantial stabilization of MTs with bundle-like appearance
All chemicals (reagent grade) used were purchased from
Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Shikonin,
in paclitaxel (1
control in this assay. By contrast, the cell membrane MT of
colchicine (1 M)-treated cells (negative control) showed de-
m
M)-treated cells that were used as positive
paclitaxel, colchicine and L-cysteine were purchased from Sigma–
Aldrich (St. Louis, MO). Aromatic aldehydes were purchased from
1
m
Shanghai Shaoyuan Co. Ltd. (Shanghai, China). All the H NMR
polymerization and solubility. With reference to these two
groups and the control group, the stabilization of tubulin
network in C13-treated cells was not apparent up to the level
of the paclitaxel-treated group, but stabilization was more
obvious than in the control and colchicine-treated groups.
Specifically, substantial stabilization of MTs appeared and
formed polymorphonuclear cells when HeLa cells were treated
spectra were recorded on a Bruker DPX 300 or DRX 500 spectrom-
1
eter in CDCl
in ppm (
3
. Chemical shifts (
). Melting points (uncorrected) were measured on a XT4
d
) for H NMR spectra were reported
d
MP Apparatus (Taike Corp., Beijing, China). ESI mass spectra were
obtained on a Mariner Biospectrometry Workstation (ESI-TOF)
mass spectrometer. Elemental analyses were performed on a CHN-
O-Rapid instrument and were within 0.4% of the theoretical values.
TLC was carried out on the glass-backed silica gel sheets (silica gel
with 3
mM C13. With increased dose to 5 mM, we can observe
˚
that a cell (enlarged image on the right in the third line) was
arrested in metaphase, and the spindle fiber was unable to form.
Meanwhile, the control group also had a cell (enlarged image on
the right in the first line) that was going through metaphase.
After the comparison, we found that the spindle fiber was unable
60 A GF254) and visualized in UV light (254 nm).
Goat anti-mouse IgG (H + L) was purchased from Invitrogen
Trading (Shanghai) Co., Ltd (Shanghai, China). b-Tubulin antibody
(#2146) was purchased from Cell Signaling Technology (Beverly,
MA). Anti-tubulin (#AT819), Cy3-labeled goat anti-mouse IgG
(H + L) (#A0521) were purchased from Cytoskeleton, Inc. 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were pur-
chased from Beyotime Institute of Biotechnology (Haimen, China).
to form in the 5
mM C13-treated group, whereas the spindle fiber
was distinct and strong in the control group. These results
suggested that C13 can enhance MT polymerization.
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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Fig. 7. Effects of C13 (0, 3, and 5
mM), paclitaxel (1 mM), and colchicine (1 mM) on interphase MTs of HeLa cells. Microtubules tagged with rhodamine (red) and nuclei tagged
with DAPI (blue) were observed under a confocal microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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9
RNase A (#EN0531) was purchased from Thermo Scientific,
Fermentas (USA). AnnexcinV-FITC cell apoptosis assay kit
Methold B: A mixture of aryl nitrile (30 mmol),
(60 mmol), and NaHCO
L
-cysteine
3
(120 mmol) in EtOH (1.5 mL/mmol) was
(#BA11100) was purchased from BIO-BOX (Nanjing, China).
stirred at 100 8C, until TLC analysis indicated disappearance of the
Fibronectin (#F1056) and laminin (#L2020) were purchased from
Sigma–Aldrich (St. Louis, MO).
nitrile. The solvent was evaporated under reduced pressure and
H
2
O (5 mL/mmol) was added to the residual white solid. The cold
solution was then acidified with HCl to pH 2. The precipitated solid
was collected by filtration and washed with a small amount of H O.
3.2. General procedure for preparation of compounds a1–a17
2
A mixture of aromatic aldehydes (50 mmol) (1–17), hydrox-
3.4. General procedure for the preparation of compounds C1–C17
ylamine hydrochloride (62.5 mmol), sodium acetate (125 mmol)
were dissolved in the mixture of formic acid and water (60:40)
and stirred at 80 8C until TLC analysis indicated the disappear-
ance of aromatic aldehydes. Then, cooling the reaction
system to room temperature and put it into water to obtain
the target compounds. Some desired products which were
dissolved in the mixture of water and formic acid can be
obtained by salting out. Then, solid target compounds were
obtained by filtration and recrystallized by alcohol, and then
dried under vacuum. While, some target compounds are oily.
These oily compounds were obtained by extraction with ethyl
acetate and the solvent was removed at the vacuum to afford
aryl nitriles (a1–a17).
Aryl dihydrothiazol acids b1–b17, shikonin, 4-dimethyamino-
pyridine (DMAP) and N,N -dicyclohexylcarbodiimide (DCC) were
0
dissolved in dichloromethane and cooled and stirred for 12 h.
Adding proper amount of silica gel and condensing solvent by
vacuum concentration. Then, collecting target compounds by
column chromatography. Chemical structures of the target
compounds (C1–C17) were shown in Table 1 and the eluent
composition and proportion was shown in Table 2.
3.4.1. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-phenyl-4,5-dihydrothiazole-4-
carboxylate (C1)
1
Red powder, yield 75%; mp: 97.5–99.7 8C; H NMR (500 MHz,
3.3. General procedure for preparation of compounds b1–b17
3
CDCl ) d: 12.56 (s, 1H, –OH), 12.40 (s, 1H, –OH), 7.90 (d, J = 7.3 Hz,
2H, Ar–H), 7.46 (dt, J = 14.5, 7.1 Hz, 3H, Ar–H), 7.21 (s, 1H, Ar–H),
Methold A: Aryl nitrile (30 mmol),
NaHCO (45 mmol) were subsequently added to a degassed
mixture of MeOH (2.6 mL/mmol), H O (1.7 mL/mmol), and a
catalytic amount of 1 M NaOH (5 mol%). The reaction mixture,
which remained suspended throughout, was stirred at room
temperature until TLC analysis indicated disappearance of the
nitrile. MeOH was then removed from the mixture by evaporation
under reduced pressure (40–50 8C/300–50 mbar).
L
-cysteine (45 mmol),
7.16 (s, 2H, Ar–H), 6.14 (dd, J = 7.5, 4.4 Hz, 1H, –O–CH), 5.40 (t,
J = 8.5 Hz, 1H, N–CH), 5.17 (m, 1H, –C 55 CH), 3.79–3.71 (m, 1H, S–
3
2
CH
(dd, J = 14.6, 7.4 Hz, 1H, C–CH
(s, 3H, C 55 C–CH ), 1.38–1.21 (m, 3H). ESI-TOF, calcd for C26
([M+Na] ) 500.1246, found 500.1162. Anal. Calcd for C26
2
), 3.64 (t, J = 10.2 Hz, 1H, S–CH
2
), 2.69 (s, 1H, C–CH
2
–C 55 C), 2.56
2
–C 55 C), 1.69 (s, 3H, C 55 C–CH
3
), 1.60
3
6
H23NO S
+
H
23NO S:
6
C, 65.39; H, 4.85; N, 2.93; O, 20.10; S, 6.71. Found: C, 64.83; H, 4.89;
N, 2.96; O, 20.14; S, 6.66.
Table 1
Chemical structures of C1–C17.
Compound
R
1
R
2
R
3
R
4
R
5
C1
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
C2
OCH
H
3
3
H
H
C3
OCH
OCH
H
3
3
H
C4
OCH
H
C5
CH
H
H
H
H
H
H
H
Cl
H
H
H
H
3
H
C6
CH
OCF
3
H
C7
3
H
C8
CH(CH
3
)
2
H
C9
H
H
F
OCH
H
3
C10
C11
C12
C13
C14
C15
C16
C17
F
Cl
H
Cl
Cl
Cl
H
H
H
Cl
H
Cl
H
Br
I
H
H
H
H
H
H
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

G Model
BCP-12238; No. of Pages 14
1
0
H.-Y. Lin et al. / Biochemical Pharmacology xxx (2015) xxx–xxx
Table 2
3
.4.5. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
Eluent composition and proportion.
yl)-4-methylpent-3-en-1-yl 2-(m-tolyl)-4,5-dihydrothiazole-4-
carboxylate (C5)
Compound
Eluent
1
Red powder, yield 80%; mp: 109.9–111.2 8C; H NMR (500 MHz,
C1
C2
Ethyl acetate:petroleum ether (V:V = 1:7)
Ethyl acetate:petroleum ether:methyl alcohol
CDCl
3
) d: 12.56 (s, 1H, –OH), 12.40 (s, 1H, –OH), 7.77 (s, 1H, Ar–H),
(
V:V:V = 1:12:0.25)
Ethyl acetate:petroleum ether:methyl alcohol
V:V:V = 1:15:0.025)
7.69 (s, 1H, Ar–H), 7.33 (d, J = 4.0 Hz, 2H, Ar–H), 7.22 (s, 1H, Ar–H),
C3
7
1
.16 (s, 2H, Ar–H), 6.15 (s, 1H, –O–CH), 5.42 (m, 1H, N–CH), 5.18 (s,
H, –C 55 CH), 3.70 (dt, J = 20.5, 10.9 Hz, 2H, S–CH ), 2.59 (dd,
–C 55 C), 2.42 (s, 3H, –CH ), 1.69 (s, 3H,
), 1.60 (s, 3H, C 55 C–CH ). ESI-TOF, calcd for C27
[M+Na] ) 514.1403, found 514.1252. Anal. Calcd for C27
(
2
C4
C5
Ethyl acetate:petroleum ether (V:V = 1:3)
J = 40.5, 33.1 Hz, 2H, C–CH
C 55 C–CH
(
2
3
Ethyl acetate:petroleum ether:methyl alcohol
(
V:V:V = 1:15:0.025)
Ethyl acetate:petroleum ether:methyl alcohol
V:V:V = 1:15:3.2)
Ethyl acetate:petroleum ether:methyl alcohol
V:V:V = 1:12:0.25)
Ethyl acetate:petroleum ether:methyl alcohol
V:V:V = 1:15:1.6)
Ethyl acetate:petroleum ether:methyl alcohol
V:V:V = 1:7:3.2)
3
+
3
H
25NO
6
S
C6
C7
C8
C9
H25NO
6
S:
(
C, 65.97; H, 5.13; N, 2.85; O, 19.53; S, 6.52. Found: C, 65.55; H, 5.36;
N, 2.89; O, 20.06; S, 6.15.
(
(
3.4.6. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(p-tolyl)-4,5-dihydrothiazole-4-
(
carboxylate (C6)
C10
C11
C12
Methyl alcohol:dichloromethane (V:V = 1:50)
Ethyl acetate:petroleum ether (V:V = 1:7)
Ethyl acetate:petroleum ether:methyl alcohol
1
Red oil, yield 58%; H NMR (300 MHz, CDCl
3
)
d
: 12.58 (s, 1H, –
OH), 12.40 (s, 1H, –OH), 8.01 (d, J = 8.1 Hz, 2H, Ar–H), 7.71 (d,
J = 8.0 Hz, 2H, Ar–H), 7.23–7.15 (m, 3H, Ar–H), 6.15 (dd, J = 7.3,
(
V:V:V = 1:15:0.4)
C13
C14
C15
Ethyl acetate:petroleum ether (V:V = 1:5)
Ethyl acetate:petroleum ether (V:V = 1:7)
Ethyl acetate:petroleum ether:methyl alcohol
4
.4 Hz, 1H, –O–CH), 5.51–5.39 (m, 1H, N–CH), 5.24–5.07 (m, 1H, –
C 55 CH), 3.89–3.62 (m, 2H, S–CH ), 2.78–2.46 (m, 2H, C–CH –C 55 C),
), 1.61 (s, 3H, C 55 C–CH ), 1.26 (s, 3H, –CH ).
([M+Na] ) 514.1403, found
S: C, 65.97; H, 5.13; N, 2.85;
2
2
(
V:V:V = 1:15:0.03)
1.70 (s, 3H, C 55 C–CH
ESI-TOF, calcd for
3
3
+
3
C16
C17
Ethyl acetate:petroleum ether (V:V = 1:7)
Ethyl acetate:petroleum ether (V:V = 1:7)
C
27
H
6
25NO S
5
14.1252. Anal. Calcd for C27
H
25NO
6
O, 19.53; S, 6.52. Found: C, 65.55; H, 5.36; N, 2.89; O, 20.06; S, 6.15.
3.4.2. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(3-methoxyphenyl)-4,5-
3.4.7. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(4-(trifluoromethoxy)phenyl)-4,5-
dihydrothiazole-4-carboxylate (C7)
Red powder, yield 72%; mp: 96.5–97.7 8C; H NMR (500 MHz,
3
CDCl ) d: 12.56 (s, 1H, –OH), 12.39 (s, 1H, –OH), 7.94 (d, J = 8.5 Hz,
2H, Ar–H), 7.28 (d, J = 8.5 Hz, 2H, Ar–H), 7.20 (s, 1H, Ar–H), 7.16 (s,
2H, Ar–H), 6.14 (dd, J = 7.3, 4.3 Hz, 1H, –O–CH), 5.40 (t, J = 8.6 Hz,
1H, N–CH), 5.17 (t, J = 6.9 Hz, 1H, –C 55 CH), 3.87–3.59 (m, 2H, S–
dihydrothiazole-4-carboxylate (C2)
1
Red oil, yield 68%; H NMR (300 MHz, CDCl
3
)
d
: 12.57 (s, 1H, –
1
OH), 12.40 (s, 1H, –OH), 7.54–7.41 (m, 2H, Ar–H), 7.38–7.29 (m, 1H,
Ar–H), 7.22–7.14 (m, 3H, Ar–H), 7.06 (m, 1H, Ar–H), 6.14 (dd,
J = 7.4, 4.4 Hz, 1H, –O–CH), 5.45–5.36 (m, 1H, N–CH), 5.16 (dd,
J = 16.3, 9.1 Hz, 1H, –C 55 CH), 3.89 (s, 3H, –OCH
S–CH ), 2.78–2.47 (m, 2H, C–CH –C 55 C), 1.69 (s, 3H, C 55 C–CH
.60 (s, 3H, C 55 C–CH ). ESI-TOF, calcd for C27
30.1352, found 530.1103. Anal. Calcd for C27
3
), 3.80–3.58 (m, 2H,
2
2
3
+
),
1
5
4
2
3
H
25NO
7
S ([M+Na] )
S: C, 63.89; H,
CH
C 55 C–CH
2
), 2.61 (ddd, J = 22.5, 14.9, 8.5 Hz, 2H, C–CH
), 1.61 (s, 3H, C 55 C–CH ). ESI-TOF, calcd for C27
([M+Na] ) 584.1069, found 584.0836. Anal. Calcd for
2
–C 55 C), 1.69 (s, 3H,
H25NO
7
3
+
3
H
22
F
3
NO S
7
.96; N, 2.76; O, 22.07; S, 6.32. Found: C, 63.55; H, 5.06; N, 2.81; O,
2.01; S, 6.25.
C
27
H
22
F
3
7
NO S: C, 57.75; H, 3.95; F, 10.15; N, 2.49; O, 19.94; S,
5.71. Found: C, 57.03; H, 4.06; F, 10.07; N, 2.94; O, 20.02; S, 5.23.
3.4.3. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(4-methoxyphenyl)-4,5-
3.4.8. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(4-isopropylphenyl)-4,5-
dihydrothiazole-4-carboxylate (C3)
1
Red oil, yield 62%; H NMR (500 MHz, CDCl
3
)
d
: 12.56 (s, 1H, –
dihydrothiazole-4-carboxylate (C8)
1
OH), 12.40 (s, 1H, –OH), 7.85 (d, J = 8.2 Hz, 2H, Ar–H), 7.23–7.13 (m,
Red oil, yield 65%; H NMR (300 MHz, CDCl
3
)
d
: 12.58 (s, 1H, –
3
5
H, Ar–H), 6.93 (d, J = 8.2 Hz, 2H, Ar–H), 6.13 (m, 1H, –O–CH), 5.42–
.28 (m, 1H, N–CH), 5.15 (m, 1H, –C 55 CH), 3.86 (s, 3H, –OCH ), 3.66
), 2.58 (dd, J = 40.7, 33.5 Hz,
), 1.60 (s, 3H, C 55 C–CH ).
OH), 12.42 (s, 1H, –OH), 7.89–7.80 (m, 2H, Ar–H), 7.30 (d, J = 8.2 Hz,
2H, Ar–H), 7.22–7.11 (m, 3H, Ar–H), 6.14 (dd, J = 7.4, 4.3 Hz, 1H, –
O–CH), 5.49–5.34 (m, 1H, N–CH), 5.16 (dd, J = 16.1, 8.5 Hz, 1H, –
3
(ddd, J = 30.7, 19.5, 10.6 Hz, 2H, S–CH
2
2
H, C–CH
2
–C 55 C), 1.69 (s, 3H, C 55 C–CH
3
3
C 55 CH), 3.69 (dt, J = 20.5, 11.2 Hz, 2H, S–CH
6.7 Hz, 1H, –CH(CH ), 2.77–2.48 (m, 2H, C–CH
C 55 C–CH ), 1.60 (s, 3H, C 55 C–CH ), 1.27 (d, J = 7.0 Hz, 6H–
CH(CH ). ESI-TOF, calcd for C29
found 542.1542. Anal. Calcd for C29
2
), 2.97 (dd, J = 13.6,
+
ESI-TOF, calcd for
30.1103. Anal. Calcd for C27
O, 22.07; S, 6.32. Found: C, 63.55; H, 5.06; N, 2.81; O, 22.01; S, 6.25.
C
27
H
25NO
7
S
([M+Na] ) 530.1352, found
3
)
2
2
–C 55 C), 1.69 (s, 3H,
5
H25NO
7
S: C, 63.89; H, 4.96; N, 2.76;
3
3
+
3
)
2
H29NO
6
S ([M+Na] ) 542.1716,
6
S: C, 67.03; H, 5.63; N,
H
29NO
3
.4.4. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
2.70; O, 18.47; S, 6.17. Found: C, 66.54; H, 5.82; N, 2.83; O, 18.54; S,
6.01.
yl)-4-methylpent-3-en-1-yl 2-(3,4-dimethoxyphenyl)-4,5-
dihydrothiazole-4-carboxylate (C4)
1
Red oil, yield 66%; H NMR (500 MHz, CDCl
3
)
d
: 12.56 (s, 1H, –
3.4.9. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(2-fluoro-6-methoxyphenyl)-4,5-
OH), 12.40 (s, 1H, –OH), 7.21 (s, 2H, Ar–H); 7.17–7.15 (m, 1H, Ar–H,
Ar–H); 7.11 (d, J = 1.5 Hz, 1H, Ar–H); 7.08 (s, 1H, Ar–H); 6.91 (d,
J = 9.0 Hz, 1H, Ar–H); 6.14 (m, 1H, –O–CH); 5.43–5.35 (m, 1H, N–
dihydrothiazole-4-carboxylate (C9)
1
Red oil, yield 83%; H NMR (300 MHz, CDCl
3
)
d
: 12.58 (s, 1H, –
CH), 5.16 (dd, J = 16.3, 9.1 Hz, 1H, –C 55 CH), 3.89 (s, 3H, –OCH
s, 3H, –OCH ), 3.80–3.58 (m, 2H, S–CH ), 2.78–2.47 (m, 2H, C–
CH –C 55 C), 1.69 (s, 3H, C 55 C–CH ), 1.60 (s, 3H, C 55 C–CH ). ESI-TOF,
calcd for C28 S ([M+Na] ) 560.1457, found 560.1303. Anal.
Calcd for C28 S: C, 62.56; H, 5.06; N, 2.61; O, 23.81; S,
.96. Found: C, 62.05; H, 5.24; N, 2.73; O, 23.88; S, 6.06.
3
), 3.86
OH), 12.44 (s, 1H, –OH), 7.36 (td, J = 8.4, 4.3 Hz, 1H, Ar–H), 7.18 (s,
2H, Ar–H), 7.15 (s, 1H, Ar–H), 6.77 (t, J = 9.1 Hz, 2H, Ar–H), 6.23–
6.08 (m, 1H, –O–CH), 5.53–5.36 (m, 1H, N–CH), 5.16 (d, J = 6.1 Hz,
(
3
2
2
3
3
+
H
H
27NO
27NO
8
1H, –C 55 CH), 3.88 (s, 3H, –OCH
(m, 2H, C–CH –C 55 C), 1.67 (s, 3H, C 55 C–CH
CH ). ESI-TOF, calcd for C27 24FNO S ([M+Na] ) 548.1258, found
3
2
), 3.72 (m, 2H, S–CH ), 2.74–2.45
8
2
3
), 1.58 (s, 3H, C 55 C–
+
5
3
H
7
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

G Model
BCP-12238; No. of Pages 14
H.-Y. Lin et al. / Biochemical Pharmacology xxx (2015) xxx–xxx
11
5
2
2
48.1053. Anal. Calcd for C27
.67; O, 21.31; S, 6.10. Found: C, 61.11; H, 4.73; F, 3.64; N, 2.73; O,
1.36; S, 5.49.
H
24FNO
7
S: C, 61.71; H, 4.60; F, 3.61; N,
J = 9.1,4.5 Hz, 2H, S–CH
1.26 (s, 3H, C 55 C–CH
2
), 1.63(dd, J = 28.3, 7.5 Hz, 2H, C–CH
2
–C 55 C),
3
3
), 1.22 (s, 3H, C 55 C–CH ). ESI-TOF, calcd for
+
C
C
26
H
21Cl
21Cl
2
NO
NO
6
S ([M+Na] ) 568.0467, found 568.0218. Anal. Calcd for
S: C, 57.15; H, 3.87; Cl, 12.98; N, 2.56; O, 17.57; S,
26
H
2
6
3
.4.10. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(2-fluorophenyl)-4,5-dihydrothiazole-
-carboxylate (C10)
Red oil, yield 61%; H NMR (500 MHz, CDCl
OH), 12.41 (s, 1H, –OH), 8.02 (t, J = 7.5 Hz, 1H, Ar–H), 7.52–7.42 (m,
H, Ar–H), 7.20 (s, 1H, Ar–H), 7.19 (s, 1H, Ar–H), 7.17 (d, J = 5.5 Hz,
H, Ar–H), 6.14 (dd, J = 7.3, 3.6 Hz, 1H, –O–CH), 5.34 (dd, J = 17.9,
.0 Hz, 1H, N–CH), 5.18 (dd, J = 27.6, 20.3 Hz, 1H, –C 55 CH), 3.78–
5.87. Found: C, 56.52; H, 4.15; Cl, 13.05; N, 2.74; O, 17.79; S, 5.56.
4
3.4.15. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(2,5-dichlorophenyl)-4,5-
1
3
) d: 12.57 (s, 1H, –
dihydrothiazole-4-carboxylate (C15)
1
2
2
9
3
Red oil, yield 69%; H NMR (300 MHz, CDCl
3
)
d
: 12.59 (s, 1H, –
OH), 12.41 (s, 1H, –OH), 7.72–7.66 (m, 2H, Ar–H), 7.37 (s, 1H, Ar–
H), 7.17 (t, J = 3.3 Hz, 3H, Ar–H), 6.20–6.10 (m, 1H, –O–CH), 5.44–
5.35 (m, 1H, N–CH), 5.18 (dd, J = 14.0, 6.7 Hz, 1H, –C 55 CH), 3.82–
.60 (m, 2H, S–CH
), 1.60 (s, 3H, C 55 C–CH
2
), 2.75–2.49 (m, 2H, C–CH
). ESI-TOF, calcd for C26
[M+Na] ) 518.1152, found 518.1003. Anal. Calcd for C26
2
–C 55 C), 1.69 (s, 3H,
22FNO
22FNO
C 55 C–CH
(
3
+
3
H
H
6
S
S:
3.65 (m, 2H, S–CH
2
), 2.77–2.48 (m, 2H, C–CH
). ESI-TOF, calcd for
S ([M+Na] ) 568.0467, found 568.0218. Anal. Calcd
NO S: C, 57.15; H, 3.87; Cl, 12.98; N, 2.56; O, 17.57; S,
.87. Found: C, 56.52; H, 4.15; Cl, 13.05; N, 2.74; O, 17.79; S, 5.56.
2
–C 55 C), 1.69 (s, 3H,
6
C 55 C–CH ), 1.60 (s, 3H, C 55 C–CH
3
3
+
C, 63.02; H, 4.48; F, 3.83; N, 2.83; O, 19.37; S, 6.47. Found: C, 62.21;
H, 4.63; F, 3.76; N, 2.98; O, 19.42; S, 6.05.
C
26
H21Cl
2 6
NO
for C26 21Cl
H
2
6
5
3.4.11. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(2-chloro-4-fluorophenyl)-4,5-
3.4.16. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(4-bromophenyl)-4,5-dihydrothiazole-
4-carboxylate (C16)
Red powder, yield 87%; mp: 106.4–107.9 8C; H NMR (300 MHz,
3
CDCl ) d: 12.57 (s, 1H, –OH), 12.40 (s, 1H, –OH), 7.78 (d, J = 8.4 Hz,
dihydrothiazole-4-carboxylate (C11)
1
Red oil, yield 63%; H NMR (500 MHz, CDCl
3
)
d
: 12.60 (s, 1H, –
1
OH), 12.43 (s, 1H, –OH), 7.79 (d, J = 8.6 Hz, 1H, Ar–H), 7.26–7.17 (m,
H, Ar–H), 7.11 (d, J = 22.6 Hz, 2H, Ar–H), 6.18 (s, 1H, –O–CH), 5.40
dd, J = 18.6, 9.5 Hz, 1H, N–CH), 5.26–5.11 (m, 1H, –C 55 CH), 3.76
ddd, J = 39.8, 20.1, 10.9 Hz, 2H, S–CH ), 2.79–2.53 (m, 2H, C–CH
), 1.63 (s, 3H, C 55 C–CH ). ESI-TOF, calcd
S ([M+Na] ) 552.0762, found 552.0105. Anal.
21ClFNO S: C, 58.92; H, 3.99; Cl, 6.69; F, 3.58; N, 2.64;
3
(
(
2H, Ar–H), 7.58 (d, J = 8.4 Hz, 2H, Ar–H), 7.22–7.13 (m, 3H, Ar–H),
6.14 (dd, J = 7.5, 4.5 Hz, 1H, –O–CH), 5.39 (t, J = 8.4 Hz, 1H, N–CH),
2
2
–
C 55 C), 1.72 (s, 3H, C 55 C–CH
for C26 21ClFNO
Calcd for C26
3
3
5.16 (dd, J = 16.3, 8.9 Hz, 1H, –C 55 CH), 3.79–3.64 (m, 2H, S–CH
2.78–2.45 (m, 2H, C–CH –C 55 C), 1.69 (s, 3H, C 55 C–CH ), 1.60 (s, 3H,
C 55 C–CH ). ESI-TOF, calcd for C26
found 578.0203. Anal. Calcd for C26
Br, 14.36; N, 2.52; O, 17.25; S, 5.76. Found: C, 55.43; H, 4.28; Br,
4.48; N, 2.76; O, 17.39; S, 5.45.
2
),
+
H
6
2
3
+
H
6
3
H
22BrNO
6
S ([M+Na] ) 578.0351,
S: C, 56.12; H, 3.99;
O, 18.11; S, 6.05. Found: C, 58.24; H, 4.15; Cl, 6.65; F, 3.59; N, 2.72;
O, 18.24; S, 6.00.
H
22BrNO
6
1
3
.4.12. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(4-chlorophenyl)-4,5-dihydrothiazole-
-carboxylate (C12)
Red powder, yield 65%; mp: 36.5–37.7 8C; H NMR (500 MHz,
CDCl :12.59(s, 1H,–OH), 12.42(s, 1H,–OH),7.86(d, J = 8.0 Hz,2H,
Ar–H), 7.44 (d, J = 8.0 Hz, 2H, Ar–H), 7.21 (d, J = 18.8 Hz, 3H, Ar–H),
3.4.17. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(4-iodophenyl)-4,5-dihydrothiazole-4-
carboxylate (C17)
Red powder, yield 79%; mp: 98.2–99.5 8C; H NMR (300 MHz,
3
CDCl ) d: 12.58 (s, 1H, –OH), 12.41 (s, 1H, –OH), 7.79 (d, J = 8.5 Hz,
4
1
1
3
)
d
6
1
.17 (d, J = 4.3 Hz, 1H, –O–CH), 5.42 (t, J = 8.3 Hz, 1H, N–CH), 5.20 (m,
H, –C 55 CH), 3.74 (dt, J = 59.9, 10.3 Hz, 2H, S–CH ), 2.80–2.52 (m, 2H,
–C 55 C), 1.72(s, 3H, C 55 C–CH ), 1.63(s, 3H, C 55 C–CH ). ESI-TOF,
S ([M+Na] ) 534.0856, found 534.0148. Anal.
S:C, 60.99;H, 4.33; Cl, 6.92; N, 2.74; O, 18.75;S,
.26. Found: C, 60.01; H, 4.52; Cl, 6.65; N, 2.94; O, 18.94; S, 6.07.
2H, Ar–H), 7.62 (d, J = 8.5 Hz, 2H, Ar–H), 7.19 (dd, J = 6.3, 2.4 Hz, 3H,
Ar–H), 6.14 (dd, J = 7.5, 4.4 Hz, 1H, –O–CH), 5.40 (t, J = 8.5 Hz, 1H,
N–CH), 5.16 (dd, J = 16.3, 8.9 Hz, 1H, –C 55 CH), 3.82–3.60 (m, 2H, S–
2
C–CH
calcd for C26
Calcd forC26
2
3
3
+
H
22FNO
6
CH
(s, 3H, C 55 C–CH
626.0213, found 626.0048. Anal. Calcd for
1.75; H, 3.67; I, 21.03; N, 2.32; O, 15.91; S, 5.31. Found: C,
2
), 2.78–2.48 (m, 2H, C–CH
2
–C 55 C), 1.70 (s, 3H, C 55 C–CH
22INO S ([M+Na] )
22INO S: C,
3
), 1.61
+
H22FNO
6
3
). ESI-TOF, calcd for C26
H
6
6
C
26
H
6
5
3.4.13. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
51.13; H, 4.00; I, 21.06; N, 2.46; O, 15.98; S, 5.17.
yl)-4-methylpent-3-en-1-yl 2-(2,3-dichlorophenyl)-4,5-
dihydrothiazole-4-carboxylate (C13)
3.5. Cell culture
1
Red oil, yield 64%; H NMR (500 MHz, CDCl
3
)
d: 12.60 (s, 1H, –
OH), 12.43 (s, 1H, –OH), 7.59 (dt, J = 15.7, 7.8 Hz, 2H, Ar–H), 7.35–
.30 (m, 1H, Ar–H), 7.25–7.17 (m, 3H, Ar–H), 6.23–6.15 (m, 1H, –O–
CH), 5.44 (dd, J = 19.0, 10.1 Hz, 1H, N–CH), 5.19 (dd, J = 13.9, 6.9 Hz,
H, –C 55 CH), 3.90–3.72 (m, 2H, S–CH ), 2.78–2.53 (m, 2H, C–CH
C 55 C), 1.72 (s, 3H, C 55 C–CH ), 1.63 (s, 3H, C 55 C–CH ). ESI-TOF, calcd
S ([M+Na] ) 568.0467, found 568.0218. Anal. Calcd
S: C, 57.15; H, 3.87; Cl, 12.98; N, 2.56; O, 17.57; S,
.87. Found: C, 56.52; H, 4.15; Cl, 13.05; N, 2.74; O, 17.79; S, 5.56.
Human hepatoma cell line (HepG2), human lung adenocarci-
noma epithelial cell line (A549), carcinoma of cervix cell line
(HeLa), human breast cancer cell line (MCF-7), squamous cell
carcinoma (SSC-4), African green monkey kidney cell (VERO) and
human normal liver cell (L02) were purchased from Nanjing
Keygen Technology (Nanjing, China). Cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) (High
7
1
2
2
–
3
+
3
for C26
H
H
21Cl
2
NO
NO
6
for C26
21Cl
2
6
5
Glucose) with
serum (FBS, BI), 100 U/mL penicillin and 100
(Hyclone), and incubated 37 8C in a humidified atmosphere
containing 5% CO
L-glutamine supplemented with 10% fetal bovine
m
g/mL streptomycin
3.4.14. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-4-methylpent-3-en-1-yl 2-(2,4-dichlorophenyl)-4,5-
2
.
dihydrothiazole-4-carboxylate (C14)
1
Red powder, yield 71%; mp: 131.6–132.7 8C; H NMR (300 MHz,
3.6. Anti-proliferation assay
CDCl
H, Ar–H), 7.45 (dd, J = 15.6, 5.4 Hz, 1H, Ar–H), 7.36–7.28 (m, 1H,
Ar–H), 7.18 (s, 3H, Ar–H), 6.15 (dd, J = 11.5, 6.9 Hz, 1H, –O–CH), 5.39
t, J = 8.7 Hz, 1H, N–CH), 5.17 (t, J = 7.6 Hz, 1H, –C 55 CH), 3.71 (qt,
3
) d: 12.59 (s, 1H, –OH), 12.42 (s, 1H, –OH), 7.68 (t, J = 10.8 Hz,
1
Target tumor cell lines were grown to log phase in DMEM
medium supplemented with 10% fetal bovine serum. After diluting
4
(
to 2 ꢁ 10 cells/mL with the complete medium, 100
mL of the
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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BCP-12238; No. of Pages 14
1
2
H.-Y. Lin et al. / Biochemical Pharmacology xxx (2015) xxx–xxx
Table 3
Inhibition of cell proliferation against HepG2, A549, HeLa, MCF-7, SCC-4, VERO and L02 cells by C1–C17 and shikonin.
Compt
IC50
(mM)
HepG2
A549
HeLa
MCF-7
SCC-4
VERO
L02
C1
31.7 ꢀ 1.61
15.6 ꢀ 1.00
29.3 ꢀ 1.01
38.7 ꢀ 1.72
9.91 ꢀ 0.64
19.26 ꢀ 1.26
10.4 ꢀ 0.87
32.8 ꢀ 1.65
42.2 ꢀ 2.86
43.4 ꢀ 1.98
37.6 ꢀ 1.56
47.4 ꢀ 2.76
35.7 ꢀ 1.23
39.7 ꢀ 2.06
28.9 ꢀ 1.86
52.1 ꢀ 2.87
54.6 ꢀ 3.04
9.36 ꢀ 0.57
10.1 ꢀ 0.48
30.3 ꢀ 2.24
32.7 ꢀ 1.64
27.1 ꢀ 1.08
30.0 ꢀ 2.00
17.4 ꢀ 1.54
28.8 ꢀ 1.42
43.2 ꢀ 3.42
53.8 ꢀ 3.65
27.8 ꢀ 1.23
12.3 ꢀ 0.57
34.7 ꢀ 1.69
14.7 ꢀ 0.77
25.5 ꢀ 1.22
38.6 ꢀ 1.68
17.9 ꢀ 1.05
23.6 ꢀ 1.11
3.13 ꢀ 0.25
7.95 ꢀ 0.32
54.6 ꢀ 3.32
29.1 ꢀ 1.17
24.2 ꢀ 0.99
40.1 ꢀ 2.04
27.6 ꢀ 1.04
24.7 ꢀ 1.01
5.69 ꢀ 0.42
53.0 ꢀ 3.25
43.7 ꢀ 1.23
34.9 ꢀ 1.48
9.66 ꢀ 0.37
3.14 ꢀ 0.21
36.5 ꢀ 2.29
38.1 ꢀ 2.54
7.93 ꢀ 0.47
18.8 ꢀ 1.48
5.75 ꢀ 0.47
21.2 ꢀ 1.06
6.41 ꢀ 0.38
34.1 ꢀ 2.07
24.8 ꢀ 1.25
18.4 ꢀ 1.42
12.4 ꢀ 1.43
7.24 ꢀ 0.77
33.7 ꢀ 2.03
51.4 ꢀ 3.08
28.1 ꢀ 1.78
27.9 ꢀ 1.22
16.3 ꢀ 1.27
6.52 ꢀ 0.68
31.2 ꢀ 1.65
25.5 ꢀ 1.75
41.22 ꢀ 2.99
26.8 ꢀ 2.37
4.99 ꢀ 0.36
16.2 ꢀ 1.14
45.3 ꢀ 2.98
34.5 ꢀ 1.68
29.9 ꢀ 1.27
41.2 ꢀ 2.69
35.9 ꢀ 2.27
38.2 ꢀ 1.95
37.3 ꢀ 2.24
21.6 ꢀ 1.09
30.1 ꢀ 1.69
45.0 ꢀ 3.53
11.1 ꢀ 1.00
36.4 ꢀ 1.77
22.7 ꢀ 1.56
31.7 ꢀ 1.22
35.1 ꢀ 2.23
46.2 ꢀ 3.01
10.1 ꢀ 0.49
86.2 ꢀ 5.67
124 ꢀ 6.04
121 ꢀ 5.78
157 ꢀ 7.89
108 ꢀ 5.41
99.6 ꢀ 4.49
101 ꢀ 5.52
103 ꢀ 5.55
89.2 ꢀ 4.01
115 ꢀ 6.47
99.1 ꢀ 4.89
96.1 ꢀ 5.29
92.2 ꢀ 4.48
187 ꢀ 6.88
97.9 ꢀ 3.77
253 ꢀ 7.89
104 ꢀ 6.35
6.76 ꢀ 0.85
105 ꢀ 3.91
167 ꢀ 4.89
106 ꢀ 4.97
99.1 ꢀ 3.36
115 ꢀ 3.36
106 ꢀ 2.98
98.7 ꢀ 2.27
136 ꢀ 4.65
142 ꢀ 4.68
93.4 ꢀ 3.60
102 ꢀ 3.92
118 ꢀ 4.55
107 ꢀ 4.46
147 ꢀ 3.35
108 ꢀ 4.57
189 ꢀ 3.78
113 ꢀ 4.39
83.8 ꢀ 2.26
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
Shikonin
3
obtained cell suspension was added to each well of 96-well culture
plates and then allowed to adhere for 12 h at 37 8C, 5% CO
atmosphere. Tested samples at pre-set concentrations (0.1, 1, 10,
and 100 M) were added to 96 wells with shikonin as positive
reference.
After 24 h exposure period, 20
Briefly, 5 ꢁ 10 cells were seeded in 6-well plates for 24 h and then
were treated with C13 (0, 1, 3, 5, and 10 M) or shikonin (0, 1.25,
2.5, 5, and 10 M) for 24 h and treated cells with 4 M C13 or
shikonin for 0, 12, 24, and 36 h. Then cells were collected and
washed twice with PBS and stained with 5 L of Annexin V-FITC
and 2.5 L of PI (5
g/mL) in 1ꢁ binding buffer (10 mM HEPES, pH
7.4, 140 mM NaOH, 2.5 mM CaCl ) for 30 min at room temperature
2
m
m
m
m
m
m
L of PBS containing 2.5 mg/mL
m
m
of MTT was added to each well. Plates were then incubated for
2
further 4 h, and then were centrifuged (1500 rpm at 4 8C for
in the dark. Apoptotic cells were quantified using BD Accuri C6
Flow Cytometer (BD, USA). Statistical analysis was done using
Flowjo 7.6.1 software.
Both early apoptotic (Annexin V-positive, PI-negative) and late
apoptotic (double positive of Annexin V and PI) cells were detected.
1
0 min) to remove supernatant. 150 mL of DMSO was added to
each well for coloration. The plates were shaken vigorously to
ensure complete solubilization for 10 min at room temperature.
The absorbance was measured and recorded on an ELISA reader
(ELx800, BioTek, USA) at a test wavelength of 570 nm. In all the
experiments three replicate wells were used for each drug
concentration. Each assay was carried out at least three times.
The results are shown in Table 3.
3.10. Cell cycle analysis
3
HeLa cells were seeded in 6-well plates (5.0 ꢁ 10 cells/well)
and incubated at 37 8C for 24 h. Exponentially growing cells were
3
.7. PI staining assay
then incubated with the C13 at 1, 3, and 5
dependent assays, exponentially growing cells were incubated
with 5 M C13 at 37 8C for 12, 24, and 48 h. Cells treated with the
compounds solvent (DMSO) were included. DMSO was used at the
highest concentration used in the experiments. After then, cells
were centrifuged and fixed in 70% ethanol at 4 8C for at least 12 h
and subsequently resuspended in PBS containing 0.1 mg/mL RNase
mM. And in the time-
5
Cells were seeded into 12-well plate (1 ꢁ 10 per well) and
incubated at 37 8C for 12 h. Then the cells were treated with
different concentrations (0, 1, 3, and 5 M) of C13 and continue to
incubate at 37 8C, 5% CO for 0, 12, 24, and 36 h. Then, cells were
collected and analyzed using PI staining and flow cytometry
analysis (BD, USA).
m
m
2
A and 5
mg/mL propidium iodide (PI). Cellular DNA content, for cell
cycle distribution analysis, was measured by flow cytometry using
BD Accuri C6 Flow Cytometer (BD, USA) plotting 10,000 events per
sample. The percentage of cells in the G1, S and G2/M phases of the
cell cycle were determined using the Flowjo 7.6.1 software after
cell debris exclusion.
3
.8. Cell adhesion assay
Cell adhesion assay was performed using method published by
Qian et al. [33] with some modifications. Briefly, 96-well flat-
bottom plates were coated with 50 L fibronectin and laminin
10 g/mL) in PBS overnight at 4 8C and then blocked with 0.2%
m
(
m
3.11. Docking simulation
BSA for 2 h at room temperature followed by three times washing.
Then, HeLa cells, which had been presented by C13 or shikonin for
The three-dimensional X-ray structure of tubulin (PDB code:
1JFF) was chosen as the template for the modeling study of C13 and
shikonin bound to tubulin. The crystal structure was obtained from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). The molecular docking procedure was performed by
using Ligand Fit protocol within Auto-Dock 4.0. For ligand
preparation, the 3D structure of C13 was generated and minimized
using Auto-Dock 4.0. For protein preparation, the hydrogen atoms
were added, and the water and impurities were removed. The
whole tubulin was defined as a receptor and the site sphere was
selected based on the ligand binding location of taxol, then the
taxol molecule was removed and C13 or shikonin was placed
3
2
4 h, were added to each well (5 ꢁ 10 per well) in triplicate, and
2
incubated at 37 8C, 5% CO for 40 min. Next, plates were washed
twice with PBS to remove unbound cells. Cells remained adhering
to the plated were determined by MTT assay. The binding cells
were calculated by dividing the optical density of initial input cells.
Each assay was carried out at least three times.
3.9. Cell apoptosis assay
For Annexin V/PI assays, HeLa cells were stained with Annexin
V-FITC and PI and then monitored for apoptosis by flow cytometry.
Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents
through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

G Model
BCP-12238; No. of Pages 14
H.-Y. Lin et al. / Biochemical Pharmacology xxx (2015) xxx–xxx
13
during the molecular docking procedure. Types of interactions of
the docked protein with ligand were analyzed after the end of
molecular docking.
meant that the binding of C13 with tubulin was more stable than
that of shikonin. In view of the binding mode and the interaction
energy, we can conclude that C13 can bind well to the paclitaxel
site of
b-tubulin, and this effect was more potent than that of
3
.12. Confocal microscopy assay
shikonin. Therefore, C13 was the best anticancer agent in this
study; it enhanced MT polymerization by binding to the paclitaxel
site of tubulin, and the effect was better than that of shikonin.
HeLa cells were grown on round cover slips to 70% confluence
and incubated with 3
mM or 5 mM C13, 1 mM paclitaxel and 1 mM
colchicine for 24 h, respectively. After incubating, cells were
washed with PBS three times and fixed with 4% paraformaldehyde
for 20 min, permeabilized with 1% Triton X-100 for another
Acknowledgements
The authors are grateful to the National Natural Science
Foundation of China (NSFC) (Nos. 31470384, 31170275,
1
0 min. Then, the cells were blocked with 3% BSA for 1 h.
Subsequently, the cells were washed once with PBS, and incubated
with anti-tubulin antibody (1:500, Cytoskeleton, Inc.) in 3% BSA
overnight at 4 8C. After being washed with 0.5% Triton X-100
3
1171161), the Program for Changjiang Scholars and Innovative
Research Team in University (IRT_14R27), the Project of New
Century Excellent Talents in University (NCET-11-0234) and the
Natural Science Foundation of the Jiangsu (BK2011414).
(
incubate for 5 min), each coverslip was added 200
labeled goat anti-mouse IgG (H + L) (1:1500, Cytoskeleton, Inc.) in
% BSA and incubated for 1 h at room temperature followed by
mL of Cy3-
3
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