Semi-synthesis and anti-lung cancer activity evaluation of aryl dihydrothiazol acyl podophyllotoxin ester derivatives

Article abstract of DOI:10.1039/c5ra01871d

Lung cancer is the leading cause of cancer death worldwide, making it one of the biggest concerns for chemoprevention. In this study, we obtained seventeen potent anticancer agents through semi-synthesis based on a natural product, podophyllotoxin. Despite prior studies of podophyllotoxin derivatives being focussed on DNA-topoisomerase II, we now turn our attention to their effect on tubulin. The MTT assay screened out the most potent anticancer agent, S12 (IC₅₀ = 0.18 μM against A549 cell line), and it showed lower cytotoxicity against normal cells. Next, the flow cytometry analysis result demonstrated that it can cause a remarkable cell cycle arrest at G2/M phase but the effect on apoptosis is not very significant. In addition, docking simulation results showed that S12 can nicely bound to the colchicine binding site of tubulin. Furthermore, we confirmed that S12 can really inhibit tubulin polymerization through confocal microscopy and protein expression determination assay.

Full text of DOI:10.1039/c5ra01871d

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Semi-synthesis and anti-lung cancer activity
evaluation of aryl dihydrothiazol acyl
Cite this: RSC Adv., 2015, 5, 27775
podophyllotoxin ester derivatives†
Hong-Yan Lin,ab Li-Fei Bai,c Fang Wang,ab Xun Wu,ab Lu-Jing Han,ab
ab
ab
Shahla Karim Baloch,ab Yong-Hua Yang
and Xiao-Ming Wang
*
*
Lung cancer is the leading cause of cancer death worldwide, making it one of the biggest concerns for
chemoprevention. In this study, we obtained seventeen potent anticancer agents through semi-synthesis
based on a natural product, podophyllotoxin. Despite prior studies of podophyllotoxin derivatives being
focussed on DNA-topoisomerase II, we now turn our attention to their effect on tubulin. The MTT assay
screened out the most potent anticancer agent, S12 (IC50 ¼ 0.18 mM against A549 cell line), and it
showed lower cytotoxicity against normal cells. Next, the flow cytometry analysis result demonstrated
that it can cause a remarkable cell cycle arrest at G2/M phase but the effect on apoptosis is not very
significant. In addition, docking simulation results showed that S12 can nicely bound to the colchicine
binding site of tubulin. Furthermore, we confirmed that S12 can really inhibit tubulin polymerization
through confocal microscopy and protein expression determination assay.
Received 30th January 2015
Accepted 12th March 2015
DOI: 10.1039/c5ra01871d
www.rsc.org/advances
increasing serious air pollution.8 In consideration of the seri-
ousness of lung cancer, we should not only protect the envi-
Introduction
ronment but endeavour to prepare a new drug that is more
effective and less toxic for lung cancer patients.
Lung cancer is one of the malignant tumors which have the
fastest growing morbidity and mortality and pose the biggest
threat to people’s health and life.1,2 Histopathologically, it is
classied as a small-cell carcinoma and the non-small cell
carcinoma types of adenocarcinoma, squamous-cell carcinoma,
and large-cell undifferentiated carcinoma.3 Lung cancer is
currently the leading cause of cancer deaths in both men and
women worldwide, with over a million deaths annually and a
dismal 16% ve-year survival rate.4,5
Tobacco smoking is the most predominant risk factor for the
disease, and accounts for about 87% of lung cancer cases.6
Carcinogens present in tobacco smoke might bond covalently to
DNA at certain specic sites, forming bulky adducts, creating a
microenvironment that facilitates oncogenic mutations and
lung cancer initiation and promotion.7 Additionally, a fraction
of lung cancers arise in patients who have never smoked. Apart
from a hereditary factor, the main reason is long-term exposure
to a polluted environment, including second-hand smoke and
Natural products have long been an important source of
treatments for cancer.9 At present, there are hundreds of plants
that have been found to possess signicant antitumor proper-
ties.10 Podophyllotoxin (1) (Fig. 1), a naturally occurring cyclo-
lignan which is the main component of podophyllum resin,
shows strong cytotoxic activity against various cancer cell lines
but failed to be used in human clinical trials because of unac-
ceptable toxic side-effects.11–14 In order to overcome the limita-
tions, numerous researchers focus on structural modication at
the 4 position of cycloparaffin (C ring) to generate derivatives
with superior pharmacological proles.15–17 Etoposide (2) and
teniposide (3), two semi-synthetic epipodophyllotoxin deriva-
tives, are successful in clinical use as antitumor agents by
inhibiting the enzyme DNA-topoisomerase II.18,19 However,
reports on podophyllotoxin derivatives, which exhibit anti-
tumor activities by inhibiting cancer cellular microtubule
assembly, are rare. In this study, we aim to discover some more
potent and selective drugs targeting tubulin based on the
podophyllotoxin scaffold.
aState Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of
Plant Molecular Biology, Nanjing University, Nanjing, 210023, China. E-mail:
Yangyh@nju.edu.cn; Wangxm07@nju.edu.cn; Fax: +86-25-89681381; Tel: +86-25-
89681381
In order to reduce the toxic side effect of podophyllotoxin
itself and improve drug targeting, we plan to introduce an aryl
dihydrothiazol moiety, which can improve the water solubility
and pharmacokinetic parameters of the drug into the podo-
phyllotoxin skeleton.20 Some researchers have indicated that
aryl dihydrothiazole compounds can also disrupt tubulin poly-
merization, therefore inhibiting the production of functional
bCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry
University, Nanjing, 210037, China
cSchool of Life Sciences and Chemistry, Jiangsu Second Normal University, Nanjing
210013, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra01871d
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Fig. 1 Chemical structures of podophyllotoxin (1), etoposide (2) and teniposide (3).
microtubules and cell mitosis.21 For this purpose, we synthe- in Table 2, the anti-proliferative activities of S3 (IC50 ¼ 0.56 mM),
sized a series of novel aryl dihydrothiazol acyl podophyllotoxin S7 (IC50 ¼ 1.94 mM), S8 (IC50 ¼ 0.93 mM), S12 (IC50 ¼ 0.18 mM),
ester derivatives and evaluated their anticancer activities. We S14 (IC50 ¼ 0.79 mM) and S17 (IC50 ¼ 0.95 mM) are better than
hope our study will pave the way for exploring new anticancer podophyllotoxin (IC50 ¼ 6.57 mM) itself and the positive control,
mechanisms of podophyllotoxin derivatives.
Combretastatin A-4 (CA-4) (IC50 ¼ 2.78 mM) against A549 cells.
For Calu-1 cells, the anti-proliferative effects of S2 (IC50 ¼ 2.34
mM), S7 (IC50 ¼ 3.46 mM), S12 (IC50 ¼ 3.03 mM), S14 (IC50 ¼ 3.45
mM) and S15 (IC50 ¼ 3.37 mM) are superior to podophyllotoxin
(IC50 ¼ 9.47 mM) and CA-4 (IC50 ¼ 3.65 mM). In the case of 973
cells, S4 (IC50 ¼ 3.12 mM), S7 (IC50 ¼ 1.94 mM), S8 (IC50 ¼ 2.95
Results and discussion
The synthetic routes for the novel aryl dihydrothiazol acyl
podophyllotoxin ester derivatives S1–S17 are outlined in
Scheme 1. These compounds were obtained by two steps,22,23
which are elucidated in the experimental section, and their
structures are shown in Table 1. All of them are rst reported
and characterized by 1H NMR, elemental analysis, melting test
and mass spectra, which were in full accordance with their
depicted structures. The structure of S12 was also characterized
by 13C NMR spectrum.
All the synthesized aryl dihydrothiazol acyl podophyllotoxin
ester derivatives S1–S17 were evaluated for their anti-
proliferative activities against three lung cancer cell lines,
A549, Calu-1, 973 and two normal cell lines, Vero (African green
monkey kidney cells) and L02 (human normal liver cells) by
MTT assay and the results are shown in Table 2. Compared with
podophyllotoxin itself, the introduction of aryl dihydrothiazol
moieties generally attenuated the cytotoxicity of podophyllo-
toxin against the normal cells. However, the lethality of podo-
phyllotoxin derivatives to lung cancer cells was not reduced
apparently. Conversely, most dihydrothiazol moieties even
improved the anti-proliferative activities of the agents. As listed
mM), S11 (IC50 ¼ 2.49 mM), S12 (IC50 ¼ 1.63 mM) and S15 (IC50
¼
3.54 mM) displayed good anti-proliferative properties. Through
comparison, we found that S12 is the best agent, which should
be selected for further study.
Aer that, we investigated the effect of S12 on cell apoptosis.
We treated A549 cells with varying concentrations (0, 0.18, 0.37,
0.75 mM) of S12 for 24 h and analysed cells for changes in
apoptotic markers by ow cytometer in vitro. As is shown in
Fig. 2, aer treatment with increasing concentrations of S12, the
percentage of apoptotic cell increased slightly. In view of the
fact that the increase is not very signicant, we speculated that
the apoptosis inducing effect of S12 is moderate.
We next assessed the cell cycle distribution of A549 cells by
ow cytometry. Treatment of A549 cells with S12 at various
concentrations (0, 0.09, 0.18, 0.37 mM) for 8 h and treated cells
with 0.18 mM S12 for different times (0, 4, 8, 12 h) could both
arrest cells at G2/M phase. From Fig. 3(A), we can see that
treatment of A549 cells with varying doses of S12 for 8 hours
resulted in increased accumulation of the cells in G2/M phase.
Scheme 1 The synthetic routes for S1–S17.
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Table 1 Chemical structures of S1–S17
Table 2 The cytotoxicity of compounds S1–S17 against a panel of
human lung cancer cell lines and two normal cell linesb
Cytotoxicity (IC50a, mM)
Compound
A549
Calu-1
973
L02
Vero
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
7.36
14.62
0.56
9.57
16.41
13.88
1.94
0.93
10.9
3.01
6.49
0.18
8.18
0.79
13.62
11.7
0.95
6.57
2.78
12.5
2.34
11.7
9.71
14.29
16.26
3.46
13.8
28.9
8.25
12.3
3.03
24.6
3.45
3.37
6.02
18.16
9.47
3.65
31.4
9.19
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.16
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.04
7.23
3.12
28.5
21.2
1.94
2.95
6.23
16.9
2.49
1.63
36.5
13.7
3.54
31.4
16.68
5.58
4.76
Compound
R1
R2
R3
R4
R5
S1
S2
S3
S4
S5
S6
S7
S8
H
H
H
H
H
H
H
H
F
H
CH3
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
CH3
H
OCH3
CF3
OCF3
CH(CH3)2
H
H
F
Cl
H
Br
I
Cl
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
S17
Podophyllotoxin
CA-4
4.23
2.56
a
b
Data are the mean of three independent experiments. CA-4:
Combretastatin A-4.
S9
S10
S11
S12
S13
S14
S15
S16
S17
F
Cl
H
Cl
H
H
Cl
H
group, S12 treated cells induced substantial destabilization of
microtubules and formed polymorphonuclear cells. The
cellular morphology of S12 treated A549 is the same as or even
more remarkable than the positive control group colchicine
treated cells.
To further investigate the effect of S12 on microtubule
organization, we did an in vitro microtubule assembly assay. As
shown in Fig. 6, S12 caused a decrease in the microtubule
assembly (the curve shis to the le when compared with the
control group), which was the same as colchicine and in
contrast to paclitaxel. To sum up, our results demonstrate that
S12 inhibits tubulin polymerization, blocks mitosis, and causes
cell death.
When the drug concentration rose to 0.37 mM, 74.06% of cells
were arrested at G2/M phase. In a time-dependent experiment,
maximum accumulation (84.56%) of cells in the G2/M phase
was observed aer treatment of cells with 0.18 mM S12 for 12
hours (Fig. 3(B)). Remarkably, S12 can cause a signicant cell
cycle arrest at G2/M phase at a low dose within a short time.
For better understanding of the potency of S12 and to guide
further SAR studies, we examined the interaction of S12 with
tubulin (PDB code: 1SA0). All docking runs applied the
Lamarckian genetic algorithm of Auto-Dock 4.0. The interaction
of S12 with tubulin amino acid residues is depicted in Fig. 4(A).
All the amino acid residues of tubulin which had interactions
with S12 were exhibited. In the binding model, S12 is nicely
bound to the colchicine binding site of tubulin via one p bond
Experimental
General information and materials
All chemicals (reagent grade) used were purchased from Nanj-
ing Chemical Reagent Co. Ltd (China). Melting points (uncor-
rected) were measured on a XT4 MP Apparatus (Taike Corp.,
Beijing, China). All 1H NMR and 13C NMR spectra were recorded
on a Bruker DPX 300 or DRX 500 spectrometer in CDCl3.
ꢀ
with LYS 254 (distance ¼ 4.56 A) and two hydrogen bonds with
ꢀ
ꢀ
1
CYS 241 (distance ¼ 2.47 A and 1.92 A). In Fig. 4(B), 3D models
of the interaction between S12 and tubulin are depicted. The
molecular docking results argue that S12 may be a potential
tubulin polymerization inhibitor.
Chemical shis (d) for H NMR spectra were reported in ppm
(d). ESI mass spectra were obtained on a Mariner Bio-
spectrometry Workstation (ESI-TOF) mass spectrometer.
Elemental analyses were performed on a CHN-O-Rapid instru-
ment and were within 0.4% of the theoretical values. TLC was
In order to observe the phenotypic effect of S12 on the
cellular cytoskeletal network of tubulin, A549 cells were
immunostained and analysed under a confocal microscope. As
illustrated in Fig. 5, substantial de-polymerization of microtu-
bules is demonstrated in this assay. Compared with the control
ꢀ
carried out on the glass-backed silica gel sheets (silica gel 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
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Fig. 2 AnnexinV/PI dual-immuno-fluorescence staining after treatment with different concentrations of S12 for 24 h revealed a significantly
increased number of apoptotic and necrotic cells (measured with Annexin V+/PI+ cells). Cells treated with 0, 0.18, 0.37 and 0.75 mM S12 for 24
hours were collected and processed for analysis. The percentage of early 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 repre-
sentative of three independent experiments. Data are mean ꢂ S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.
antibody (#2146) was purchased from Cell Signaling Technology apoptosis assay kit (#BA11100) was purchased from BIO-BOX
(Beverly, MA). Anti-tubulin (#AT819), Cy3-labeled goat anti- (Nanjing, China).
mouse IgG (H + L) (#A0521) were purchased from Cytoskel-
eton, Inc. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) were purchased from Beyotime Institute of Biotech-
nology (Haimen, China). RNase A (#EN0531) was purchased
from Thermo Scientic, Fermentas (USA). AnnexinV-FITC cell
General procedure for preparation of compounds a1–a17
A mixture of aryl nitrile (30 mmol), L-cysteine (60 mmol), and
NaHCO3 (120 mmol) in EtOH (1.5 mL mmolꢀ1) was stirred at
100 ꢁC, until TLC analysis indicated disappearance of the
Fig. 3 Effect of S12 on the cell cycle distribution of A549 cells. (A) Cells treated with 0, 0.09, 0.18 and 0.37 mM S12 for 8 hours were collected and
processed for analysis. (B) Cells treated with 0.18 mM S12 for different times were collected and analysed.
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nitrile. The solvent was evaporated under reduced pressure and
H2O (5 mL mmolꢀ1) was added to the residual white solid. The
cold solution was then acidied with HCl to pH 2. The precip-
itated solid was collected by ltration and washed with a small
amount of H2O.
General procedure for preparation of compounds S1–S17
Aryl dihydrothiazol acids derivatives a1–a17, podophyllotoxin,
4-dimethyaminopyridine (DMAP) and N,N0-dicyclohexyl-
carbodiimide (DCC) were dissolved in dichloromethane and
stirred for 12 h. A proper amount of silica gel was added and the
solvent was condensed by vacuum concentration. Then, the
target compounds were collected by column chromatography
(V(acetone) : V(dichloromethane) ¼ 50 : 1). Chemical structures of the
target compounds (S1–S17) are shown in Table 1.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-
phenyl-4,5-dihydrothiazole-4-carboxylate (S1). White powder,
yield 75%; mp: 84.9–85.7 ꢁC; 1H NMR (300 MHz, CDCl3) d 7.89–
7.80 (m, 2H, Ar-H), 7.54–7.46 (m, 1H, Ar-H), 7.42 (dd, J ¼ 8.0, 6.6
Hz, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.55 (s, 1H, Ar-H), 6.39 (s, 2H,
Ar-H), 6.00 (t, J ¼ 3.2 Hz, 3H, O–CH2–O, CH–O–C]O), 5.40 (t,
J ¼ 8.7 Hz, 1H, O]C–CH–N), 4.61 (s, 1H, CH-Ar), 4.42 (dd, J ¼
9.1, 6.5 Hz, 1H, CH–CH2–O), 4.22 (t, J ¼ 9.9 Hz, 1H, CH–CH2–O),
3.81 (s, 3H, 40-OCH3), 3.74 (dd, J ¼ 7.7, 2.3 Hz, 2H, S–CH2), 3.71
(s, 6H, 30,50-OCH3), 2.99–2.91 (m, 2H, O]C–CH, O–CH2–CH).
ESI-TOF, calcd for C32H29NO9S ([M + Na]+) 626.1563, found
626.1162. Anal. Calcd for C32H29NO9S: C, 63.67; H, 4.84; N, 2.32;
Fig. 4 Molecular docking analysis of S12, showing proposed binding
modes at the colchicine binding pocket b-tubulin (PDB code: 1SA0).
Hydrogen bonds are displayed as lime dashed lines and p bond is
displayed as an orange line. (A) Interaction of S12 with the amino acid
residues of colchicine binding site (carbon atom, gray; oxygen atom,
red; nitrogen atom, purple; sulphur atom, yellow, chlorine atom,
green). (B) Binding position of S12 in the protein surface of tubulin
(carbon atom, gray; oxygen atom, red; nitrogen atom, purple; sulphur
atom, yellow, chlorine atom, green; hydrogen atom, white).
Fig. 5 Effect of S12 (0.5 mM) on interphase microtubules of A549 cells. Microtubules tagged with rhodamine (red) and nuclei tagged with DAPI
(blue) were observed under a confocal microscope.
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Fig. 6 S12 affected microtubule assembly in vitro. After 24 hours treatment with S12 (0.5 mM), paclitaxel (1 mM) and colchicine (1 mM), polymerized
tubulin on the cell membrane was immunoblotted with b-tubulin antibody and detected by flow cytometry.
O, 23.85; S, 5.31. Found: C, 62.33; H, 4.89; N, 2.56; O, 23.99; S, OCH3), 3.73 (d, J ¼ 10.3, 5.1 Hz, 2H, S–CH2), 2.93 (d, J ¼ 14.8 Hz,
5.16.
2H, O]C–CH, O–CH2–CH), 2.40 (d, J ¼ 5.6 Hz, 3H, –CH3). ESI-
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6, TOF, calcd for C33H31NO9S ([M + Na]+) 640.1720, found
8,8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-
640.1562. Anal. Calcd for C33H31NO9S: C, 64.17; H, 5.06; N, 2.27;
(m-tolyl)-4,5-dihydrothiazole-4-carboxylate (S2). White powder, O, 23.31; S, 5.19. Found: C, 63.35; H, 5.29; N, 2.34; O, 23.46; S,
yield 85%; mp: 119.5–120.8 ꢁC; 1H NMR (300 MHz, CDCl3) d 5.15.
7.75–7.57 (m, 2H, Ar-H), 7.31 (t, J ¼ 5.5 Hz, 2H, Ar-H), 6.88 (d, J ¼
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
26.2 Hz, 1H, Ar-H), 6.54 (s, 1H, Ar-H), 6.39 (d, J ¼ 3.8 Hz, 2H, Ar- 8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(p-
H), 5.97 (d, J ¼ 11.7 Hz, 3H, O–CH2–O, CH–O–C]O), 5.40 (td, tolyl)-4,5-dihydrothiazole-4-carboxylate (S3). White powder,
1
ꢁ
J ¼ 8.8, 3.4 Hz, 1H, O]C–CH–N), 4.61 (s, 1H, CH-Ar), 4.42 (dt, yield 79%; mp: 76.8–77.8 C; H NMR (300 MHz, CDCl3) d 7.75
J ¼ 14.4, 7.3 Hz, 1H, CH–CH2–O), 4.24 (dd, J ¼ 19.2, 9.1 Hz, 1H, (dd, J ¼ 13.1, 8.1 Hz, 2H, Ar-H), 7.22 (t, J ¼ 7.2 Hz, 2H, Ar-H),
CH–CH2–O), 3.81 (s, 3H, 40-OCH3), 3.72 (d, J ¼ 5.9 Hz, 6H, 30,50- 6.87 (d, J ¼ 22.7 Hz, 1H, Ar-H), 6.54 (d, J ¼ 2.0 Hz, 1H, Ar-H),
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6.38 (d, J ¼ 1.9 Hz, 2H, Ar-H), 5.97 (d, J ¼ 11.5 Hz, 3H, O–CH2–O,
CH–O–C]O), 5.39 (t, J ¼ 8.0 Hz, 1H, O]C–CH–N), 4.61 (s, 1H,
CH-Ar), 4.42 (d, J ¼ 5.1 Hz, 1H, CH–CH2–O), 4.29–4.16 (m, 1H,
CH–CH2–O), 3.81 (s, 3H, 40-OCH3), 3.87–3.61 (m, 2H, S–CH2),
3.71 (d, J ¼ 4.0 Hz, 6H, 30,50-OCH3), 2.95 (s, 2H, O]C–CH,
O–CH2–CH), 2.40 (d, J ¼ 4.0 Hz, 3H, –CH3). ESI-TOF, calcd for
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
(triuoromethoxy)phenyl)-4,5-dihydrothiazole-4-carboxylate (S7).
White powder, yield 87%; mp: 78.9–80.3 ꢁC; 1H NMR (300 MHz,
CDCl3) d 7.98–7.86 (m, 2H, Ar-H), 7.32–7.22 (m, 2H, Ar-H), 6.85
(d, J ¼ 23.3 Hz, 1H, Ar-H), 6.55 (d, J ¼ 2.2 Hz, 1H, Ar-H), 6.39 (s,
2H, Ar-H), 5.98 (t, J ¼ 6.3 Hz, 3H, O–CH2–O, CH–O–C]O), 5.39
(tt, J ¼ 25.9, 12.8 Hz, 1H, O]C–CH–N), 4.62 (s, 1H, CH-Ar), 4.42
(dd, J ¼ 9.2, 6.2 Hz, 1H, CH–CH2–O), 4.24 (dd, J ¼ 19.7, 9.4 Hz,
1H, CH–CH2–O), 3.81 (s, 3H, 40-OCH3), 3.80–3.75 (m, 2H,
S–CH2), 3.74 (d, J ¼ 3.6 Hz, 6H, 30,50-OCH3), 2.95 (d, J ¼ 1.9 Hz,
2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd for C33H28F3NO10S
C
33H31NO9S ([M + Na]+) 640.1720, found 640.1562. Anal. Calcd
for C33H31NO9S: C, 64.17; H, 5.06; N, 2.27; O, 23.31; S, 5.19.
Found: C, 63.35; H, 5.29; N, 2.34; O, 23.46; S, 5.15.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(3-
methoxyphenyl)-4,5-dihydrothiazole-4-carboxylate (S4). White
powder, yield 82%; mp: 105.6–106.9 ꢁC; 1H NMR (300 MHz,
CDCl3) d 7.51–7.29 (m, 3H, Ar-H), 7.05 (s, 1H, Ar-H), 6.87 (d, J ¼
19.0 Hz, 1H, Ar-H), 6.55 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.98 (s,
3H, O–CH2–O, CH–O–C]O), 5.39 (d, J ¼ 8.2 Hz, 1H, O]C–CH–
N), 4.61 (s, 1H, CH-Ar), 4.42 (s, 1H, CH–CH2–O), 4.24 (d, J ¼ 6.1
Hz, 1H, CH–CH2–O), 3.91–3.76 (m, 5H, S–CH2, 40-OCH3), 3.71 (s,
6H, 30,50-OCH3), 2.95 (s, 2H, O]C–CH, O–CH2–CH). ESI-TOF,
calcd for C33H31NO10S ([M + Na]+) 656.1669, found 656.1325.
Anal. Calcd for C33H31NO10S: C, 62.55; H, 4.93; N, 2.21; O, 25.25;
S, 5.06. Found: C, 62.30; H, 5.01; N, 2.32; O, 25.33; S, 5.00.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
methoxyphenyl)-4,5-dihydrothiazole-4-carboxylate (S5). White
powder, yield 72%; mp: 96.7–98.2 ꢁC; 1H NMR (500 MHz, CDCl3)
d 7.92 (dd, J ¼ 23.1, 8.7 Hz, 2H, Ar-H), 7.28 (t, J ¼ 8.8 Hz, 2H, Ar-
H), 6.86 (d, J ¼ 39.4 Hz, 1H, Ar-H), 6.56 (d, J ¼ 3.6 Hz, 1H, Ar-H),
6.41 (s, 2H, Ar-H), 5.99 (t, J ¼ 8.4 Hz, 3H, O–CH2–O, CH–O–C]O),
5.41 (t, J ¼ 8.7 Hz, 1H, O]C–CH–N), 4.63 (s, 1H, CH-Ar), 4.43 (dd,
J ¼ 7.6, 5.1 Hz, 1H, CH–CH2–O), 4.25 (dt, J ¼ 15.4, 9.9 Hz, 1H,
CH–CH2–O), 3.83 (s, 3H, 40-OCH3), 3.78 (dd, J ¼ 13.8, 5.3 Hz, 2H,
S–CH2), 3.75 (s, 3H, –OCH3), 3.75–3.69 (s, 6H, 30,50-OCH3), 2.96
(s, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd for C33H31NO10S
([M
33H28F3NO10S: C, 57.64; H, 4.10; F, 8.29; N, 2.04; O, 23.27; S,
+
Na]+) 710.1386, found 710.1082. Anal. Calcd for
C
4.66. Found: C, 57.45; H, 4.43; F, 8.43; N, 2.47; O, 21.97; S, 4.74.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
isopropylphenyl)-4,5-dihydrothiazole-4-carboxylate (S8). Light
yellow powder, yield 74%; mp: 104.3–105.7 ꢁC; 1H NMR (300
MHz, CDCl3) d 7.77 (dd, J ¼ 13.3, 8.3 Hz, 2H, Ar-H), 7.27 (dd, J ¼
8.0, 6.5 Hz, 2H, Ar-H), 6.82 (s, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 6.38
(s, 2H, Ar-H), 5.97 (d, J ¼ 4.8 Hz, 3H, O–CH2–O, CH–O–C]O),
5.38 (t, J ¼ 8.6 Hz, 1H, O]C–CH–N), 4.59 (s, 1H, CH-Ar), 4.41
(dd, J ¼ 9.1, 6.3 Hz, 1H, CH–CH2–O), 4.22 (dd, J ¼ 19.9, 9.8 Hz,
1H, CH–CH2–O), 3.80 (s, 3H, 40-OCH3), 3.71 (s, 6H, 30,50-OCH3),
3.77–3.62 (m, 2H, S–CH2), 3.02–2.87 (m, 3H, O]C–CH, O–CH2–
CH, –CH–(CH3)2), 1.26 (s, 3H, –CH–(CH3)2), 1.24 (s, 3H, –CH–
(CH3)2). ESI-TOF, calcd for C35H35NO9S ([M + Na]+) 668.2033,
found 668.1937. Anal. Calcd for C35H35NO9S: C, 65.10; H, 5.46;
N, 2.17; O, 22.30; S, 4.97. Found: C, 64, 25; H, 5.58; N, 2.33; O,
22.57; S, 4.86.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(2-
uorophenyl)-4,5-dihydrothiazole-4-carboxylate (S9). White
powder, yield 75%; mp: 93.9–95.7 ꢁC; 1H NMR (300 MHz, CDCl3)
d 7.93 (dtd, J ¼ 9.2, 7.5, 1.7 Hz, 1H, Ar-H), 7.47 (dt, J ¼ 13.3, 4.6
Hz, 1H, Ar-H), 7.25–7.08 (m, 2H, Ar-H), 6.86 (d, J ¼ 24.8 Hz, 1H,
Ar-H), 6.55 (d, J ¼ 3.2 Hz, 1H, Ar-H), 6.39 (s, 2H, Ar-H), 6.05–5.93
(m, 3H, O–CH2–O, CH–O–C]O), 5.38 (td, J ¼ 9.0, 3.9 Hz, 1H,
O]C–CH–N), 4.62 (d, J ¼ 2.6 Hz, 1H, CH-Ar), 4.43 (dd, J ¼ 9.6,
5.4 Hz, 1H, CH–CH2–O), 4.24 (dd, J ¼ 19.9, 9.6 Hz, 1H, CH–i2–O),
3.81 (d, J ¼ 3.0 Hz, 3H, 40-OCH3), 3.73 (s, 6H, 30,50-OCH3), 3.77–
3.65 (m, 2H, S–CH2), 3.00–2.88 (m, 2H, O]C–CH, O–CH2–CH).
ESI-TOF, calcd for C32H28FNO9S ([M + Na]+) 643.1469, found
643.1232. Anal. Calcd for C32H28FNO9S: C, 61.83; H, 4.54; F,
3.06; N, 2.25; O, 23.16; S, 5.16. Found: C, 61.01; H, 4.64; F, 3.02;
N, 2.43; O, 23.27; S, 5.04.
([M
33H31NO10S: C, 62.55; H, 4.93; N, 2.21; O, 25.25; S, 5.06. Found:
C, 62.30; H, 5.01; N, 2.32; O, 25.33; S, 5.00. ESI-TOF, calcd for
33H28F3NO10S ([M + Na]+) 694.1437, found 694.1089. Anal.
+
Na]+) 656.1669, found 656.1325. Anal. Calcd for
C
C
Calcd for C33H28F3NO10S: C, 59.01; H, 4.20; F, 8.49; N, 2.09; O,
21.44; S, 4.77. Found: C, 58.72; H, 4.32; F, 8.47; N, 2.17; O, 21.59;
S, 4.75.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
(triuoromethyl)phenyl)-4,5-dihydrothiazole-4-carboxylate (S6).
White powder, yield 78%; mp: 185.9–187.5 ꢁC; 1H NMR (300
MHz, CDCl3) d 7.96 (d, J ¼ 8.1 Hz, 2H, Ar-H), 7.69 (d, J ¼ 8.2 Hz,
2H, Ar-H), 6.80 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.39 (s, 2H, Ar-
H), 6.00 (d, J ¼ 6.1 Hz, 3H, O–CH2–O, CH–O–C]O), 5.42 (t, J ¼
8.9 Hz, 1H, O]C–CH–N), 4.62 (s, 1H, CH-Ar), 4.42 (dd, J ¼ 9.2,
6.4 Hz, 1H, CH–CH2–O), 4.28–4.16 (m, 1H, CH–CH2–O), 3.81 (s,
3H, 40-OCH3), 3.80–3.75 (m, 2H, S–CH2), 3.73 (s, 6H, 30,50-OCH3),
3.01–2.90 (m, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd for
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(2-
uoro-6-methoxyphenyl)-4,5-dihydrothiazole-4-carboxylate (S10).
White powder, yield 65%; mp: 67.8–68.7 ꢁC; 1H NMR (500 MHz,
CDCl3) d 7.36 (dd, J ¼ 15.0, 8.3 Hz, 1H, Ar-H), 6.85 (s, 1H, Ar-H),
6.79–6.70 (m, 2H, Ar-H), 6.56 (s, 1H, Ar-H), 6.41 (s, 2H, Ar-H),
6.09–5.96 (m, 3H, O–CH2–O, CH–O–C]O), 5.52–5.43 (m, 1H,
O]C–CH–N), 4.63 (s, 1H, CH-Ar), 4.45 (d, J ¼ 6.2 Hz, 1H,
CH–CH2–O), 4.22 (d, J ¼ 9.1 Hz, 1H, CH–CH2–O), 3.82 (s, 3H,
C
33H28F3NO9S ([M + Na]+) 694.1437, found 694.1089. Anal.
Calcd for C33H28F3NO9S: C, 59.01; H, 4.20; F, 8.49; N, 2.09; O,
21.44; S, 4.77. Found: C, 58.72; H, 4.32; F, 8.47; N, 2.17; O, 21.59;
S, 4.75.
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–OCH3), 3.78 (s, 3H, 40-OCH3), 3.71 (s, 6H, 30,50-OCH3), 3.49 (m,
2H, S–CH2), 2.97 (s, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd
for C33H30FNO10S ([M + Na]+) 674.1574, found 674.1346. Anal.
Calcd for C33H30FNO10S: C, 60.82; H, 4.64; F, 2.92; N, 2.15; O,
24.55; S, 4.92. Found: C, 60.08; H, 4.92; F, 3.00; N, 2.33; O, 24.77;
S, 4.84.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
bromophenyl)-4,5-dihydrothiazole-4-carboxylate (S14). White
powder, yield 81%; mp: 108.4–109.6 ꢁC; 1H NMR (300 MHz,
CDCl3) d 7.72 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.56 (d, J ¼ 8.4 Hz, 2H, Ar-
H), 6.81 (s, 1H, Ar-H), 6.55 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 6.04–
5.93 (m, 3H, O–CH2–O, CH–O–C]O), 5.38 (t, J ¼ 8.6 Hz, 1H,
O]C–CH–N), 4.61 (s, 1H, CH-Ar), 4.48–4.32 (m, 1H, CH–CH2–
O), 4.28–4.17 (m, 1H, CH–i2–O), 3.81 (s, 3H, 40-OCH3), 3.77 (dd,
J ¼ 8.9, 4.4 Hz, 2H, S–CH2), 3.72 (s, 6H, 30,50-OCH3), 3.05–2.87
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(2-
chloro-4-uorophenyl)-4,5-dihydrothiazole-4-carboxylate (S11).
White powder, yield 70%; mp: 123.1–124.8 ꢁC; 1H NMR (300
MHz, CDCl3) d 7.76–7.59 (m, 1H, Ar-H), 7.18 (d, J ¼ 6.5 Hz, 1H,
Ar-H), 7.02 (d, J ¼ 7.5 Hz, 1H, Ar-H), 6.85 (d, J ¼ 20.1 Hz, 1H, Ar-
H), 6.55 (s, 1H, Ar-H), 6.39 (s, 2H, Ar-H), 5.99 (m, 3H, O–CH2–O,
CH–O–C]O), 5.40 (t, J ¼ 8.6 Hz, 1H, O]C–CH–N), 4.61 (s, 1H,
CH-Ar), 4.40 (s, 1H, CH–CH2–O), 4.22 (s, 1H, CH–CH2–O), 3.77
(d, J ¼ 15.2 Hz, 5H, 40-OCH3, S–CH2), 3.71 (s, 6H, 30,50-OCH3),
(m, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd for C32H28
BrNO9S ([M + Na]+) 704.0668, found 704.0274. Anal. Calcd for
32H28BrNO9S: C, 56.31; H, 4.13; Br, 11.71; N, 2.05; O, 21.10 S,
-
C
4.70. Found: C, 55.82; H, 4.43; Br, 11.68; N, 2.15; O, 21.19 S, 4.64.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
iodophenyl)-4,5-dihydrothiazole-4-carboxylate (S15). White
powder, yield 76%; mp: 154.6–155.9 ꢁC; 1H NMR (300 MHz,
CDCl3) d 7.81–7.70 (m, 2H, Ar-H), 7.64–7.47 (m, 2H, Ar-H), 6.83
(d, J ¼ 23.0 Hz, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H),
5.97 (t, J ¼ 7.8 Hz, 3H, O–CH2–O, CH–O–C]O), 5.37 (td, J ¼ 8.8,
3.9 Hz, 1H, O]C–CH–N), 4.60 (s, 1H, CH-Ar), 4.45–4.33 (m, 1H,
CH–CH2–O), 4.22 (dd, J ¼ 19.5, 9.7 Hz, 1H, CH–CH2–O), 3.80 (s,
3H, 40-OCH3), 3.72 (d, J ¼ 4.3 Hz, 6H, 30,50-OCH3), 3.48 (t, J ¼
10.3 Hz, 2H, S–CH2), 2.91 (d, J ¼ 11.6 Hz, 2H, O]C–CH, O–CH2–
CH). ESI-TOF, calcd for C32H28INO9S ([M + Na]+) 752.0529,
found 752.0341. Anal. Calcd for C32H28INO9S: C, 56.68; H, 3.87;
I, 17.40; N, 1.92; O, 19.71 S, 4.40. Found: C, 56.12; H, 3.97; I,
17.42; N, 2.02; O, 19.97 S, 4.41.
2.95 (s, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd for C32H27
ClFNO9S ([M + Na]+) 678.1079, found 678.1002. Anal. Calcd for
32H27ClFNO9S: C, 58.58; H, 4.15; Cl, 5.40; F, 2.90; N, 2.13; O,
-
C
21.95; S, 4.89. Found: C, 58.03; H, 4.22; Cl, 5.49; F, 3.05; N, 2.36;
O, 22.41; S, 4.72.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-(4-
chlorophenyl)-4,5-dihydrothiazole-4-carboxylate (S12). White
powder, yield 77%; mp: 92.4–93.7 ꢁC; 1H NMR (300 MHz, CDCl3)
d 7.78 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.40 (d, J ¼ 8.5 Hz, 2H, Ar-H),
6.81 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.39 (s, 2H, Ar-H), 6.00
(d, J ¼ 2.4 Hz, 3H, O–CH2–O, CH–O–C]O), 5.38 (t, J ¼ 8.7 Hz,
1H, O]C–CH–N), 4.61 (s, 1H, CH-Ar), 4.48–4.36 (m, 1H, CH–
CH2–O), 4.29–4.14 (m, 1H, CH–CH2–O), 3.82 (s, 3H, 40-OCH3),
3.78–3.74 (m, 2H, S–CH2), 3.73 (s, 6H, 30,50-OCH3), 3.01–2.86 (m,
2H, O]C–CH, O–CH2–CH). 13C NMR (CDCl3, 300 MHz): d
173.34 (13 C), 171.16 (14 C), 170.29 (17 C), 152.56 (30,50-C),
148.23 (7 C), 147.6 (6 C), 138.05 (40-C), 137.07 (400-C), 134.52 (10-
C), 132.45 (100-C), 130.78 (9 C), 129.74 (200, 600-C), 128.79 (300, 500-
C), 127.56 (10 C), 109.71 (8 C), 107.96 (20, 60-C), 106.79 (5 C),
101.58 (13 C), 78.24 (15 C), 75.02 (4 C), 71.13 (11 C), 60.66 (40-
OCH3), 56.01 (30,50-OCH3), 45.43 (1 C), 43.63 (2 C), 38.43 (3 C),
33.84 (16 C). ESI-TOF, calcd for C32H28ClNO9S ([M + Na]+)
660.1173, found 660.1084. Anal. Calcd for C32H28ClNO9S: C,
60.23; H, 4.42; Cl, 5.56; N, 2.20; O, 22.57; S, 5.03. Found: C,
59.79; H, 4.63; Cl, 5.54; N, 2.29; O, 22.74; S, 5.00.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-
(2,4-dichlorophenyl)-4,5-dihydrothiazole-4-carboxylate
(S16).
White powder, yield 75%; mp: 121.2–122.6 ꢁC; 1H NMR (300
MHz, CDCl3) d 7.61 (dt, J ¼ 14.4, 7.1 Hz, 1H, Ar-H), 7.46 (dd, J ¼
4.7, 1.8 Hz, 1H, Ar-H), 7.33–7.27 (m, 1H, Ar-H), 6.89–6.76 (m, 1H,
Ar-H), 6.54 (d, J ¼ 3.2 Hz, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.99 (d,
J ¼ 4.7 Hz, 3H, O–CH2–O, CH–O–C]O), 5.40 (t, J ¼ 8.7 Hz, 1H,
O]C–CH–N), 4.61 (s, 1H, CH-Ar), 4.42 (dd, J ¼ 9.0, 6.2 Hz, 1H,
CH–CH2–O), 4.23 (dd, J ¼ 16.5, 9.5 Hz, 1H, CH–CH2–O), 3.86–
3.73 (m, 5H, 40-OCH3, S–CH2), 3.71 (s, 6H, 30,50-OCH3), 2.92 (d,
J ¼ 14.7 Hz, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd for
C
32H27Cl2NO9S ([M + Na]+) 694.0784, found 694.0039. Anal.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-
Calcd for C32H27Cl2NO9S: C, 57.15; H, 4.05; Cl, 10.54; N, 2.08; O,
21.41 S, 4.77. Found: C, 58.92; H, 4.23; Cl, 10.64; N, 2.19; O,
21.75; S, 4.52.
(2,5-dichlorophenyl)-4,5-dihydrothiazole-4-carboxylate
(S13).
Light yellow powder, yield 68%; mp: 89.6–90.7 ꢁC; 1H NMR (300
MHz, CDCl3) d 7.63 (dd, J ¼ 5.1, 2.1 Hz, 1H, Ar-H), 7.40–7.30 (m,
2H, Ar-H), 6.84 (d, J ¼ 12.5 Hz, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.39
(s, 2H, Ar-H), 6.00 (d, J ¼ 2.8 Hz, 3H, O–CH2–O, CH–O–C]O),
5.49–5.36 (m, 1H, O]C–CH–N), 4.62 (s, 1H, CH-Ar), 4.49–4.37
(m, 1H, CH–CH2–O), 4.31–4.16 (m, 1H, CH–CH2–O), 3.88–3.74
(m, 2H, S–CH2), 3.80 (s, 3H, 40-OCH3), 3.71 (s, 6H, 30,50-OCH3),
2.97 (d, J ¼ 9.7 Hz, 2H, O]C–CH, O–CH2–CH). ESI-TOF, calcd
for C32H27Cl2NO9S ([M + Na]+) 694.0784, found 694.0039. Anal.
Calcd for C32H27Cl2NO9S: C, 57.15; H, 4.05; Cl, 10.54; N, 2.08; O,
21.41 S, 4.77. Found: C, 58.92; H, 4.23; Cl, 10.64; N, 2.19; O,
21.75; S, 4.52.
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-2-
(3,4-dimethoxyphenyl)-4,5-dihydrothiazole-4-carboxylate (S17).
1
Yellow powder, yield 87%; mp: 203.3–205.1 ꢁC; H NMR (300
MHz, CDCl3) d 7.57–7.46 (m, 1H, Ar-H), 7.43–7.34 (m, 1H, Ar-H),
6.89 (ddd, J ¼ 17.4, 8.5, 4.2 Hz, 2H, Ar-H), 6.55 (s, 1H, Ar-H), 6.39
(d, J ¼ 7.5 Hz, 2H, Ar-H), 6.01–5.91 (m, 3H, O–CH2–O, CH–O–
C]O), 5.44–5.34 (m, 1H, O]C–CH–N), 4.61 (s, 1H, CH-Ar),
4.48–4.38 (m, 1H, CH–CH2–O), 4.26 (t, J ¼ 9.6 Hz, 1H, CH–
CH2–O), 3.94 (dd, J ¼ 6.0, 2.5 Hz, 6H, –OCH3), 3.81 (d, J ¼ 2.2 Hz,
3H, 40-OCH3), 3.72 (d, J ¼ 12.0 Hz, 6H, 30,50-OCH3), 3.48 (ddd,
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J ¼ 10.4, 9.2, 3.9 Hz, 2H, S–CH2), 2.95 (m, 2H, O]C–CH, In the time-dependent assays, exponentially growing cells were
O–CH2–CH). ESI-TOF, calcd for C34H33NO11S ([M + Na]+) incubated with 0.18 mM S12 at 37 ꢁC for 4, 8 and 12 h. Aer that,
ꢁ
686.1774, found 684.1243. Anal. Calcd for C34H33NO11S: C, cells were centrifuged and xed in 70% ethanol at 4 C for at
61.53; H, 5.01; N, 2.11; O, 26.52 S, 4.83. Found: C, 60.92; H, 5.23; least 12 hours and subsequently resuspended in PBS containing
N, 2.29; O, 26.75; S, 4.72.
0.1 mg mLꢀ1 RNase A and 5 mg mLꢀ1 propidium iodide (PI).
Cellular DNA content, for cell cycle distribution analysis, was
measured by ow cytometry using a 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 Flowjo 7.6.1 soware aer cell debris
exclusion.
Cell lines and culture conditions
A549, Calu-1, 973, Vero and L02 cells were purchased from
Nanjing Keygen Technology (Nanjing, China). Cells were
maintained in Dulbecco’s modied Eagle’s medium (DMEM,
Hyclone) (High Glucose) with L-glutamine supplemented with
10% fetal bovine serum (FBS, BI), 100 U mLꢀ1 penicillin and 100
Docking simulation
mg mLꢀ1 streptomycin (Hyclone), and incubated at 37 C in a
ꢁ
The three-dimensional X-ray structure of tubulin (PDB code:
1SA0) was chosen as the template for the modelling study of S12
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
using Ligand Fit protocol within Auto-Dock 4.0. For ligand
preparation, the 3D structure of S12 was generated and mini-
mized using Auto-Dock 4.0. For protein preparation, hydrogen
atoms were added, and the water and impurities were removed.
The whole tubulin was dened as a receptor and the site sphere
was selected based on the ligand binding location of taxol, then
the taxol molecule was removed and S12 was placed during the
molecular docking procedure. The types of interactions of the
docked protein with ligand were analysed aer the end of
molecular docking.
humidied atmosphere containing 5% CO2.
Anti-proliferation assay
Target tumor cell lines were grown to log phase in DMEM
medium supplemented with 10% fetal bovine serum. Aer
diluting to 2 ꢃ 104 cells per mL with the complete medium, 100
mL of the obtained cell suspension was added to each well of
96-well culture plates and then allowed to adhere for 12 hours at
ꢁ
37 C, 5% CO2 atmosphere. Tested samples at pre-set concen-
trations (0.1, 1, 10, 100 mM) were added to 96 wells with podo-
phyllotoxin and CA-4 as positive references.
Aer 24 hours exposure period, 20 mL PBS containing 2.5
mg mLꢀ1 MTT was added to each well. Plates were then incu-
bated for a further 4 hours, and then were centrifuged (1500 rpm
at 4 ꢁC for 10 minutes) to remove the supernatant. 150 mL DMSO
was added to each well for coloration. The plates were shaken
vigorously to ensure complete solubilization for 10 minutes 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 experiments three replicate wells were used for
each drug concentration. Each assay was carried out at least
three times. The results are summarized in Table 2.
Confocal microscopy assay
A549 cells were grown on round cover slips to 70% conuence
and incubated with 0.5 mM S12 and 2 mM colchicine for 12
hours, respectively. Aer incubating, cells were washed with
PBS three times and xed with 4% paraformaldehyde for 20
minutes, permeabilized with 1% TritonX-100 for another 10
minutes. Then, the cells were blocked with 3% BSA for 1 hour.
Subsequently, the cells were washed once with PBS, and incu-
bated with anti-tubulin antibody (1 : 500, Cytoskeleton, Inc.) in
3% BSA overnight at 4 ꢁC. Aer being washed with 0.5%
TritonX-100 (incubate for 5 minutes), each coverslip was added
to 200 mL Cy3-labeled goat anti-mouse IgG (H + L) (1 : 1500,
Cytoskeleton, Inc.) in 3% BSA and incubated for 1 hour at room
temperature followed by DAPI (5 ng mLꢀ1). Cells were then
observed under an Olympus confocal microscope and data was
analysed using FV-10-ASW 1.7 viewer.
Cell apoptosis assay
For Annexin V/PI assays, A549 cells were stained with Annexin V-
FITC and PI and then monitored for apoptosis by ow cytom-
etry. Briey, 5 ꢃ 103 cells were seeded in 6-well plates for 24
hours and then were treated with S12 (0, 0.18, 0.37, 0.75 mM) for
24 hours. Then cells were collected and washed twice with PBS
and stained with 5 mL Annexin V-FITC and 2.5 mL PI (5 mg mLꢀ1
)
in 1ꢃ binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5
mM CaCl2) for 15 minutes at room temperature in the dark.
Apoptotic cells were quantied using a BD Accuri C6 Flow
Cytometer (BD, USA). Statistical analysis was done using Flowjo
7.6.1 soware. Both early apoptotic (AnnexinV-positive, PI-
negative) and late apoptotic (double positive of Annexin V and
PI) cells were detected.
Flow cytometry analysis of the expression of extracellular
polymerized tubulin
A549 cells were seeded in 6-well plates and treated with 0.5 mM
S12, 1 mM colchicine and 1 mM paclitaxel for 24 hours. Then,
cells were harvested, washed once with PBS and blocked with
1% BSA for 30 min on ice, then were centrifuged (2800 rpm at
4 ꢁC for 5 minutes) to remove supernatant. Aer that, cells were
Cell cycle analysis
A549 cells were plated in 6-well plates (5.0 ꢃ 103 cells per well) incubated with b-tubulin antibody for 1 h on ice and were
ꢁ
and incubated at 37 ꢁC for 24 hours. Exponentially growing cells centrifuged (2800 rpm at 4 C for 5 minutes) to remove super-
were then incubated with S12 at 0.09, 0.18 and 0.37 mM for 8 h. natant. Next, cells were incubated with secondary antibody
This journal is © The Royal Society of Chemistry 2015
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(IgG) on ice for 1 h and were also centrifuged (2800 rpm at 4 ꢁC
for 5 minutes) to remove supernatant. Finally, cells were then
resuspended in PBS and the expression level of polymerized
tubulin was analysed by ow cytometry (BD, USA).
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Conclusion
In this paper, a series of novel aryl dihydrothiazol acyl podo-
phyllotoxin ester derivatives S1–S17 were obtained through a
two-step semi-synthetic route. Their anticancer biological
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value of 0.18 mM, which is better than podophyllotoxin (IC50
¼
6.57 mM) and CA-4 (IC50 ¼ 2.78 mM), and showed lower cyto-
toxicity against normal cells. Cell cycle distribution assay and
apoptosis assay demonstrated that S12 can cause a remarkable
cell cycle arrest at G2/M phase but the effect on apoptosis is not
very signicant. Furthermore, the docking simulation result
enabled us to determine that tubulin is a target for S12.
Confocal microscopy assay and extracellular polymerized
tubulin expression analysis further conrmed that S12 really
displayed its’ anticancer effect by inhibiting tubulin polymeri-
zation. To sum up, these ndings prompt us to consider it as a
potent anti-cancer agent.
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Abbreviations
CA-4
NMR
TLC
TMS
DMSO
PBS
Combretastatin A-4
Nuclear magnetic resonance spectrum
Thin layer chromatography
Tetramethylsilane
Dimethyl sulfoxide
Phosphate-buffered saline
40,6-Diamidino-2-phenylindole
Bovine serum albumin
DAPI
BSA
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Acknowledgements
The authors are grateful to the Program for Changjiang Scholars
and Innovative Research Team in University (IRT_14R27).
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27784 | RSC Adv., 2015, 5, 27775–27784
This journal is © The Royal Society of Chemistry 2015