Synthesis of novel aryl dithian valeryl podophyllotoxin ester derivatives as potential antitubulin agents

Article abstract of DOI:10.1039/c5ra04902d

Microtubules are among the most successful targets for anticancer therapy. In this study, we described the synthesis routes of the lipoyl podophyllotoxin ester derivatives and found that they can selectively inhibit the proliferation of cancer cells without damaging the non-cancer cells. Among them, L4 showed the best antiproliferation activity with IC₅₀ = 2.68 μM against A549 cells. This effect of L4 was similar to that of CA-4 (IC₅₀ = 2.78 μM), a typical microtubule inhibitor, but better than podophyllotoxin (IC₅₀ = 6.57 μM) itself. Furthermore, cell cycle analysis revealed that L4 can remarkably cause cell cycle arrest in the G2/M phase in a time- and dose-dependent manner. But the effect of L4 on apoptosis inducing was not apparent enough. Moreover, confocal microscopy and western blot analysis results indicated that L4 can perturb microtubule polymerization, thus causing tumor growth inhibition.

Full text of DOI:10.1039/c5ra04902d

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Synthesis of novel aryl dithian valeryl
podophyllotoxin ester derivatives as potential
Cite this: RSC Adv., 2015, 5, 47511
antitubulin agents†
Hong-Yan Lin,‡ Zi-Kang Li,‡ Hong-Wei Han, Han-Yue Qiu, Hong-Wei Gu,
*
*
Yong-Hua Yang and Xiao-Ming Wang
Microtubules are among the most successful targets for anticancer therapy. In this study, we described the
synthesis routes of the lipoyl podophyllotoxin ester derivatives and found that they can selectively inhibit the
proliferation of cancer cells without damaging the non-cancer cells. Among them, L4 showed the best
antiproliferation activity with IC50 ¼ 2.68 mM against A549 cells. This effect of L4 was similar to that of
CA-4 (IC50 ¼ 2.78 mM), a typical microtubule inhibitor, but better than podophyllotoxin (IC50 ¼ 6.57 mM)
itself. Furthermore, cell cycle analysis revealed that L4 can remarkably cause cell cycle arrest in the G2/M
phase in a time- and dose-dependent manner. But the effect of L4 on apoptosis inducing was not
apparent enough. Moreover, confocal microscopy and western blot analysis results indicated that L4 can
perturb microtubule polymerization, thus causing tumor growth inhibition.
Received 19th March 2015
Accepted 21st May 2015
DOI: 10.1039/c5ra04902d
www.rsc.org/advances
inspired the researchers to further search for new, effective
anticancer agents with superior pharmacological proles based
on this scaffold.13–17 Herein, we have to note some well-known
Introduction
Tubulins are a universally conserved protein superfamily that
carries out diverse biological roles by assembling laments with
very different architectures.1,2 Microtubules are long, hollow,
cylindrical polymers composed of a- and b-tubulin dimers.3–5
They are highly dynamic structures that are regulated by poly-
merization and de-polymerization of tubulin, and this dynamic
instability property of microtubules is important for carrying out
many cellular functions.6,7 Disruption of microtubule dynamics
or formation of abnormal mitotic microtubules prevents rear-
rangement of the microtubule network, which can induce cell
cycle arrest in G2/M phase and trigger cells death.3,8,9 Addition-
ally, the continuous mitotic division of proliferating cancer cells
showed better sensitivity to the inhibition of mitosis than non-
cancer cells. Based on these, microtubule has become an
attractive molecular target for anticancer therapeutics.
drugs, GL331 (2), NPF (3), TOP53 (4), and NK611 (5), espe-
cially the three most highly prescribed anticancer drugs in the
world, etoposide (6), teniposide (7), and the water-soluble pro-
drug etopophos (8), that base on podophyllotoxin scaffold.13,18–20
According to previous research ndings, the C-4 position of
podophyllum analogs is a variable region and deserves us to
further investigate. The SAR reports also suggested that the 4
position of cycloparaffin can bear bulky groups and thus has
huge scope for functionalization.21
Based on the aforementioned research results, we attempted
to develop new anticancer drugs by introducing a-lipoic acid
derivatives into podophyllotoxin scaffold to improve its phar-
macological activity and drug targeting. It is well known that the
a-lipoic acid is a naturally occurring antioxidant that has been
shown to possess promising anticancer activity because of its
ability to preferentially induce apoptosis and inhibit prolifera-
tion of cancer cells relative to normal cells.22–24 Furthermore, in
Wei's study, a-lipoic acid also plays a role as the carrier of the
anticancer drug, doxorubicin during intracellular transport and
trigger anticancer drug release.25 Based on these, we designed a
series of aryl dithian valeryl podophyllotoxin ester derivatives
and veried they have better application prospects than the
parent drug, podophyllotoxin in cancer chemotherapy.
Podophyllotoxin (1, Fig. 1), a naturally occurring cyclolignan
which is the main component of podophyllum resin, exhibits
pronounced biological activities, especially anticancer effects
through inhibiting microtubule assembly.10–12 However, the
undesirable toxic side-effects of podophyllotoxin hinder its
development as a new clinical anticancer agent, which have
State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of
Plant Molecular Biology, Nanjing University, Nanjing, 210046, China. E-mail:
Yangyh@nju.edu.cn; Wangxm07@nju.edu.cn; Fax: +86-25-89681381; Tel: +86-25-
89681381
Results and discussion
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra04902d
The routes to synthesizing the novel aryl dithian valeryl podo-
‡ These two authors equally contribute to this paper.
phyllotoxin ester derivatives L1–L15 are shown in Scheme 1.
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Fig. 1 Chemical structures of podophyllotoxin derivatives.
These compounds were obtained in three steps which were [HeLa (human cervical cancer cell), A549 (human lung adeno-
elucidated in the experimental section. All synthesized carcinoma cell), Calu-1 (human lung cancer cell)], and two non-
compounds were reported and characterized for the rst time cancer cell lines [L02 (human normal liver cell) and Vero
by melting test, 1H NMR, 13C NMR, mass spectroscopy, and (African green monkey kidney cell)] by MTT assay. The IC50
elemental analysis, and results were in accordance with their values dened as the concentrations that cause a 50% loss of
depicted structures (Table 1).
cell viability and were shown in Table 2. Results shown in
Firstly, all synthesized derivatives L1–L15 were evaluated for Table 2 demonstrated that lipoyl moiety generally reduced the
their antiproliferation activities against three cancer cell lines cytotoxicity of podophyllotoxin toward the non-cancer cells but
the effects on cancer cells were slightly weakened. It may be
because that the continuous mitotic division of proliferating
cancer cells is more sensitive to inhibition of mitosis than non-
cancer cells. And this speculation will be validated in the
following study. It was heartening that several compounds
among them still exhibited the same or even better effect than
podophyllotoxin and CA-4 (combretastatin A-4), a typical
microtubule disruptor. Generally, their antiproliferation activi-
ties against A549 and Calu-1 are better than HeLa cells. To be
specic, L5 (IC50 ¼ 4.57 mM), L7 (IC50 ¼ 7.05 mM) and L9 (IC50
6.22 mM) showed better effects than podophyllotoxin (IC50
8.34 mM) and the effect of L5 even similar to that of CA-4 (IC50
¼
¼
¼
5.86 mM) aer exposure to HeLa cells. We treated Calu-1 cells
with those drugs and found that L3 (IC50 ¼ 9.06 mM), L4 (IC50
¼
6.95 mM), L5 (IC50 ¼ 7.28 mM) and L11 (IC50 ¼ 8.13 mM)
exhibited better activity than podophyllotoxin (IC50 ¼ 9.47 mM)
but their effects are weaker than that of CA-4 (IC50 ¼ 3.65 mM).
For A549 cells, L1 (IC50 ¼ 5.89 mM), L3 (IC50 ¼ 5.91 mM), L4 (IC50
¼ 2.68 mM) and L5 (IC50 ¼ 6.07 mM) are better than podo-
phyllotoxin (IC50 ¼ 6.57 mM). Interestingly, L4 even showed
Scheme 1 The synthetic routes for L1–L15; (a) NaHCO3, NaBH4, H2O,
room temperature, 12 h; (b) aldehyde, boron trifluoride etherate,
nitrogen, dichloromethane, ꢀ25 ꢁC to room temperature, 4 h; (c)
podophyllotoxin, DCC, DMAP, dichloromethane, room temperature,
12 h.
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Table 1 Chemical structures of L1–L15
Compound
R1
R2
R3
R4
R5
L1
L2
L3
L4
L5
L6
L7
L8
H
CH3
H
H
H
OCH3
H
H
Cl
H
Cl
Cl
H
H
H
H
CF3
H
H
OCH3
H
H
H
H
H
CH3
H
CF3
H
OCH3
CH(CH3)2
H
Cl
H
Cl
Br
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
L9
L10
L11
L12
L13
L14
Cl
H
H
H
OH
similar effect as CA-4 (IC50 ¼ 2.78 mM). According to the struc- antiproliferation activity of podophyllotoxin itself. Among them,
ture–activity relationship, we clearly found that tri- triuoromethylphenyl substituted lipoic acids were better than
uoromethylphenyl, tolyl, methoxyphenyl and chlorophenyl other derivatives and m-triuoromethylphenyl substituted lipoic
substituted lipoic acid derivatives can signicantly improve the acid was better than the p-triuoromethylphenyl substituted one.
Taken together, we selected L4, the best candidate for cancer
treatment in this study, for further investigation.
Table
2 The anti-proliferation activities of compound L1–L15,
Subsequently, we investigated the effect of L4 on cell
apoptosis by ow cytometry. A549 cells were seeded in 6-well-
plate for 12 h and then treated with L4 of different concentra-
tions (0, 1, 3, and 9 mM) for 24 h. As shown in Fig. 2, apoptosis
was not obvious when treated cells with L4 at low concentra-
tions. And even at the highest concentration (9 mM, three times
as much as its IC50 value), it only lead to 21.4% cells apoptosis.
Therefore, the effect of L4 on cell apoptosis is not very
signicant.
To determine the effect of L4 on cell cycle distribution, we
treated A549 cells with different doses (0, 0.4, 0.8, and 1.6 mM) of
the drug for 8 h and detect cells by ow cytometry. Results were
shown in Fig. 3(A) and they demonstrated that L4 can cause cell
cycle arrest in G2/M phase remarkably in a dose-dependent
manner. Meanwhile, we also designed a time-dependent assay
to analysis the effect of L4 on cell cycle distribution. As revealed
by Fig. 3(B), the percentage of the cells that arrested in G2/M
phase signicantly increased with extended exposure time.
Specically, 68.19% cells were arrested in G2/M phase when
cells were treated with 0.8 mM L4 for 16 h. Both dose- and time-
dependent cell cycle distribution assay results demonstrated
that L4 can lead to cell cycle arrest in G2/M potently.
podophyllotoxin and CA-4 against three cancer cell lines and two
non-cancer cell linesa
IC50 (mM)
Compound
HeLa
Calu-1
A549
Vero
L02
L1
L2
L3
L4
L5
L6
L7
L8
L9
L10
L11
L12
L13
L14
L15
16.2
17.3
9.61
9.01
4.57
18.9
7.05
15.5
6.22
21.3
8.36
25.7
32.8
27.5
26.8
8.34
5.86
13.6
11.2
9.06
6.95
7.28
19.3
12.4
16.9
10.4
16.9
8.13
26.4
23.3
11.5
18.2
9.47
3.65
5.89
15.0
5.91
2.68
6.07
12.6
10.6
8.47
8.82
28.4
9.38
46.5
20.5
16.8
14.7
6.57
2.78
91.2
>100
93.8
>100
>100
99.5
93.2
>100
91.6
>100
>100
94.5
99.8
>100
>100
1.04
2.56
98.6
92.2
95.8
>100
>100
>100
>100
>100
>100
>100
93.3
90.7
>100
>100
>100
3.16
4.23
Podophyllotoxin
CA-4
a
Pod: podophyllotoxin.
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colchicine. By contrast, taxol signicantly increased the
expression of polymerized tubulin. The results suggested that
L4 can inhibit tubulin polymerization and the effect was similar
to that of colchicine.
Experimental
General information and materials
All chemicals (reagent grade) used were purchased from Nanj-
ing Chemical Reagent Co. Ltd. (China). All the 1H NMR and 13
C
NMR spectra were recorded on a Bruker DPX 300 or DRX 500
spectrometer in CDCl3. Chemical shis (d) for 1H NMR spectra
were reported in ppm (d). ESI mass spectra were obtained on a
Mariner Biospectrometry Workstation (ESI-TOF) mass spec-
trometer. Melting points (uncorrected) were measured on a XT4
MP Apparatus (Taike Corp., Beijing, China). 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 60 A GF254) and visualized in
UV light (254 nm).
Anti-tubulin (#AT819), Cy3-labeled goat anti-mouse IgG (H +
L) (#A0521) were purchased from Cytoskeleton, Inc. b-Tubulin
antibody (#2146) was purchased from Cell Signaling Technology
(Beverly, MA). Goat anti-mouse IgG (H + L) was purchased from
Invitrogen Trading (Shanghai) Co., Ltd (Shanghai, China). 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were
purchased from Beyotime Institute of Biotechnology (Haimen,
China). AnnexcinV-FITC cell apoptosis assay kit (#BA11100) was
purchased from BIO-BOX (Nanjing, China). RNase A (#EN0531)
was purchased from Thermo Scientic, Fermentas (USA).
General procedure for preparation of 6,8-dimercaptooctanoic
acid
Fig. 2 AnnexinV/PI dual-immuno-fluorescence staining after treat-
ment with different concentrations of L4 for 24 h revealed moderate
0.5 mol a-lipoic acid was suspended in water (3000 mL), and 0.5
increased number of apoptotic and necrotic cells (measured with mol NaHCO3 was added. The mixture was stirred to produce a
Annexin V+/PI + cells). 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 representative of three
clear solution. The resulting pale yellow solution was cooled in
an ice bath, and 1 mol solid NaBH4 was added, with stirring, in
small portions. Aer that, the solution was stirred in an ice bath
for another 1 h and then at room temperature for 12 h. The
cloudy solution was cooled in an ice bath, and the pH was
adjusted to 1 by slow addition of concentrated hydrochloric
acid. A vigorous evolution of hydrogen occurred as the excess
NaBH4 decomposed, and an oily liquid was seen to separate.
The mixture was extracted with chloroform for three times. The
combined chloroform extracts were dried over MgSO4, ltered
and the solvent evaporated under reduced pressure at room
temperature. The remaining oil was further dried under
vacuum to remove the residual solvent (Scheme 1).
independent experiments. Data are mean ꢂ S.E.M. of three indepen-
dent experiments. *P < 0.05, **P < 0.01.
To observe the phenotypic effect of L4 on cellular cytoskel-
etal network of tubulin, A549 cells were immunostained and
analyzed under a confocal microscope. As shown in Fig. 4, L4
inhibited tubulin polymerization and formed poly-
morphonuclear cells when compared with the control group.
The effect of L4 (1.5 mM) on cellular cytoskeletal network of
tubulin was similar to that of colchicine (2 mM), but contrary to
taxol (2 mM) treated group.
General procedure for preparation of compounds A1–A15
In order to further investigate the effect of L4 on microtubule
organization, we did an in vitro microtubule assembly assay. As
shown in Fig. 5, A549 cells were treated with L4 (1.5 mM),
colchicine (2 mM) and taxol (2 mM) for 16 h, respectively. Aer
the comparison, we found that L4 reduced the expression of
polymerized tubulin and the effect was similar to that of
6,8-Dimercaptooctanoic acid (0.03 mol) was dissolved in
dichloromethane under a nitrogen atmosphere. To this solu-
tion, the appropriate aldehyde (0.03 mol) was added. The
mixture was stirred at room temperature for 1 h and cooled to
ꢁ
ꢀ25 C. Boron triuoride etherate (0.03 mol) was added, and
the reaction was allowed to warm to room temperature. Aer
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Fig. 3 Effect of L4 on the cell cycle distribution of A549 cells. (A) Cells treated with various concentrations of L4 for 8 h were collected and
processed for analysis. (B) Cells treated with 0.8 mM L4 for 4, 8, and 16 h, respectively was collected and analysed.
evaporation of the solvent, the products were puried by crys- (19-C), 38.67 (3-C), 35.81 (15-C), 34.06 (18-C), 32.70 (20-C), 32.36
tallization with ethyl acetate: petroleum ether (v/v ¼ 1 : 2) as the (16-C), 25.60 (17-C), 24.76 (21-C). ESI-TOF, calcd for C37H40O9S2
solvent (Scheme 1).
([M +
Na]+) 715.2114, found 715.1162. Anal. calcd for
C
37H40O9S2: C, 64.14; H, 5.82; O, 20.78; S, 9.26%. Found: C,
63.28; H, 6.54; O, 21.02; S, 9.05%.
General procedure for preparation of compounds L1–L15
(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-5-(2-(o-tolyl)-
1,3-dithian-4-yl)pentanoate (L2). White powder, yield 77%; mp:
78.0–79.2 ꢁC; 1H NMR (300 MHz, CDCl3) d 7.55 (dd, J ¼ 8.1, 3.3
Hz, 1H, Ar-H), 7.23–7.09 (m, 3H, Ar-H), 6.72 (d, J ¼ 4.9 Hz, 1H,
Ar-H), 6.52 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.95 (d, J ¼ 3.4 Hz,
2H, O–CH2–O), 5.86 (d, J ¼ 8.8 Hz, 1H, CH–O–C]O), 5.30 (s, 1H,
Ar-CH–S2), 4.58 (d, J ¼ 4.0 Hz, 1H, CH-Ar), 4.40–4.30 (m, 1H,
CH–CH2–O), 4.17 (t, J ¼ 9.6 Hz, 1H, CH–CH2–O), 3.80 (s, 3H, 40-
OCH3), 3.74 (s, 6H, 30,50-OCH3), 3.10–2.72 (m, 5H, O]C–CH, O–
CH2–CH, CH2–S, CH–S), 2.42 (s, 3H, –CH3), 2.46–2.37 (m, 2H,
CH2–C]O), 2.18 (dd, J ¼ 14.7, 2.4 Hz, 1H, S–CH2–CH2), 1.76–
1.46 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2–CH2). 13C NMR (126
MHz, CDCl3) d 173.86 (12-C), 173.59 (14-C), 152.64 (30,50-C),
148.12 (7-C), 147.59 (6-C), 137.25 (100-C), 136.80 (40-C), 135.04 (200-
C), 134.83 (10-C), 132.35 (9-C), 130.50 (300-C), 128.38 (600-C),
128.19 (10-C), 127.80 (400-C), 126.64 (500-C), 109.72 (8-C), 108.20
(5-C), 106.95 (20,60-C), 101.59 (13-C), 73.52 (4-C), 71.32 (11-C),
60.72 (40-OCH3), 56.18 (30,50-OCH3), 49.05 (22-C), 46.16 (2-C),
45.54 (1-C), 43.74 (19-C), 38.73 (3-C), 35.89 (15-C), 34.12 (18-C),
32.89 (20-C), 32.59 (16-C), 25.67 (17-C), 24.81 (21-C), 19.08 (200-
CH3). ESI-TOF, calcd for C38H42O9S2 ([M + Na]+) 729.1563, found
729.1212. Anal. calcd for C38H42O9S2: C, 64.57; H, 5.99; O, 20.37;
S, 9.07%. Found: C, 63.83; H, 6.29; O, 20.77; S, 8.96%.
Lipoic acid derivatives A1–A15 (0.015 mol), podophyllotoxin
(0.01 mol), 4-dimethyaminopyridine (DMAP) (0.001 mol) and
N,N0-dicyclohexylcarbodiimide (DCC) (0.02 mol) were dissolved
in dichloromethane (30 mL) and stirred for 12 h at room
temperature. Adding proper amount of silica gel and condensing
solvent by vacuum concentration. Then, collecting target
compounds by column chromatography (v(acetone)
:
(dichloromethane)
¼ 1 : 50). Chemical structures of the target compounds (L1–L15)
were 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-5-(2-phenyl-
1,3-dithian-4-yl)pentanoate (L1). White powder, yield 76%; mp:
76.2–77.7 ꢁC; 1H NMR (300 MHz, CDCl3) d 7.48–7.39 (m, 2H, Ar-
H), 7.35–7.27 (m, 3H, Ar-H), 6.73 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H),
6.37 (s, 2H, Ar-H), 5.95 (s, 2H, O–CH2–O), 5.86 (d, J ¼ 8.8 Hz, 1H,
CH–O–C]O), 5.14 (t, J ¼ 3.4 Hz, 1H, Ar-CH–S2), 4.58 (d, J ¼ 4.1
Hz, 1H, CH-Ar), 4.34 (dd, J ¼ 9.2, 6.8 Hz, 1H, CH–CH2–O), 4.17 (t,
J ¼ 9.7 Hz, 1H, CH–CH2–O), 3.79 (s, 3H, 40-OCH3), 3.74 (s, 6H,
30,50-OCH3), 3.07–2.75 (m, 5H, O]C–CH, O–CH2–CH, CH2–S,
CH–S), 2.46–2.36 (m, 2H, CH2–C]O), 2.17 (d, J ¼ 11.3 Hz, 1H,
S–CH2–CH2), 1.71–1.45 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2–
CH2). 13C NMR (126 MHz, CDCl3) d 173.80 (12-C), 173.58 (14-C),
152.57 (30,50-C), 148.05 (7-C), 147.53 (6-C), 138.72 (100-C), 137.09
(40-C), 134.82 (10-C), 132.29 (9-C), 128.71 (200,600-C), 128.48 (10-C),
128.39 (400-C) 127.66 (300,500-C), 109.65 (8-C), 108.06 (5-C), 106.93
(20,60-C), 101.56 (13-C), 73.45 (4-C), 71.28 (11-C), 60.68 (40-OCH3),
56.10 (30,50-OCH3), 52.35 (22-C), 45.95 (2-C), 45.44 (1-C), 43.67
(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-5-(2-(p-tolyl)-
1,3-dithian-4-yl)pentanoate (L3). White powder, yield 72%; mp:
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Fig. 4 Effect of L4 on the tubulin network of A549 cells. Microtubules tagged with rhodamine (red) and nuclei tagged with DAPI (blue) were
observed under a confocal microscope.
J ¼ 32.4, 16.7 Hz, 5H, O]C–CH, O–CH2–CH, CH2–S, CH–S), 2.36
(d, J ¼ 27.9 Hz, 5H, CH2–C]O, –CH3), 2.16 (d, J ¼ 12.6 Hz, 1H,
S–CH2–CH2), 1.84–1.45 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2–
CH2). 13C NMR (126 MHz, CDCl3) d 173.78 (12-C), 173.56 (14-C),
152.62 (30,50-C), 148.08 (7-C), 147.56 (6-C), 138.20 (400-C), 137.21
(40-C), 135.83 (10-C), 134.87 (100-C), 132.35 (9-C), 129.37 (300,500-C),
128.43 (200,600-C), 127.53 (10-C), 109.67 (8-C), 108.19 (5-C), 106.95
(20,60-C), 101.57 (13-C), 73.49 (4-C), 71.28 (11-C), 60.65 (40-OCH3),
56.13 (30,50-OCH3), 52.10 (22-C), 45.96 (2-C), 45.44 (1-C), 43.72
(19-C), 38.70 (3-C), 35.83 (15-C), 34.09 (18-C), 32.74 (20-C), 32.39
Fig. 5 L4 affected microtubule assembly in vitro. After treating A549
cells with L4 (1.5 mM), colchicine (2 mM), and taxol (2 mM) for 16 h,
cytosolic (S, soluble) and cytoskeletal (P, polymerized tubulin) tubulin
(16-C), 25.62 (17-C), 24.80 (21-C), 21.14 (400-CH3). ESI-TOF, calcd
fractions were separated and immunoblotted with antibody against b-
tubulin.
for C38H42O9S2 ([M + Na]+) 729.1563, found 729.1212. Anal.
calcd for C38H42O9S2: C, 64.57; H, 5.99; O, 20.37; S, 9.07%.
Found: C, 63.79; H, 6.22; O, 20.99; S, 8.94%.
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-5-(2-(3-
(triuoromethyl)phenyl)-1,3-dithian-4-yl)pentanoate (L4). White
powder, yield 70%; mp: 80.3–81.5 ꢁC; 1H NMR (300 MHz, CDCl3)
d 7.72 (s, 1H, Ar-H), 7.64 (d, J ¼ 7.6 Hz, 1H, Ar-H), 7.56 (d, J ¼ 7.8
Hz, 1H, Ar-H), 7.46 (t, J ¼ 7.7 Hz, 1H, Ar-H), 6.73 (s, 1H, Ar-H),
6.54 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.96 (dd, J ¼ 6.5, 5.0 Hz,
ꢁ
72.3–73.4 C; H NMR (300 MHz, CDCl3) d 7.32 (d, J ¼ 6.7 Hz,
2H, Ar-H), 7.13 (d, J ¼ 6.5 Hz, 2H, Ar-H), 6.73 (s, 1H, Ar-H), 6.53
(s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.96 (s, 2H, O–CH2–O), 5.87 (d, J
¼ 8.0 Hz, 1H, CH–O–C]O), 5.12 (s, 1H, Ar-CH–S2), 4.59 (s, 1H,
CH-Ar), 4.35 (s, 1H, CH–CH2–O), 4.18 (t, J ¼ 9.1 Hz, 1H, CH–
CH2–O), 3.80 (s, 3H, 40-OCH3), 3.75 (s, 6H, 30,50-OCH3), 2.88 (dd,
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2H, O–CH2–O), 5.88 (d, J ¼ 8.9 Hz, 1H, CH–O–C]O), 5.19 (s, 1H, 155.41 (20-C), 152.63 (30,50-C), 148.11 (7-C), 147.59 (6-C), 137.16
Ar-CH–S2), 4.60 (d, J ¼ 4.2 Hz, 1H, CH-Ar), 4.35 (dd, J ¼ 9.2, 6.9 (40-C), 134.84 (10-C), 132.32 (9-C), 129.43 (600-C), 129.14 (400-C),
Hz, 1H, CH–CH2–O), 4.19 (t, J ¼ 9.7 Hz, 1H, CH–CH2–O), 3.81 (s, 128.37 (10-C), 126.86 (100-C), 121.05 (500-C), 110.80 (300-C), 109.70
3H, 40-OCH3), 3.75 (s, 6H, 30,50-OCH3), 3.07–2.74 (m, 5H, O]C– (8-C), 108.12 (5-C), 106.96 (20,60-C), 101.59 (13-C), 73.50 (4-C),
CH, O–CH2–CH, CH2–S, CH–S), 2.48–2.31 (m, 2H, CH2–C]O), 71.33 (11-C), 60.73 (40-OCH3), 56.15 (30,50-OCH3, 20-OCH3), 55.74
2.25–2.13 (m, 1H, S–CH2–CH2), 1.98–1.49 (m, 7H, S–CH2–CH2, (22-C), 46.07 (2-C), 45.53 (1-C), 44.37 (19-C), 43.73 (3-C), 38.71
S–CH–CH2–CH2–CH2). 13C NMR (75 MHz, CDCl3) d 173.73 (12- (15-C), 35.90 (18-C), 32.90 (20-C), 32.60 (16-C), 25.67 (17-C),
C), 173.50 (14-C), 152.52 (30,50-C), 148.01 (7-C), 147.48 (6-C), 24.82 (21-C). ESI-TOF, calcd for C38H42O10S2 ([M + Na]+)
139.75 (100-C) 139.66 (40-C), 137.06 (10-C), 134.73 (600-C), 132.22 745.2219, found 745.0016. Anal. calcd for C38H42O10S2: C, 63.14;
(300-C), 131.12 (9-C), 129.18 (500-C), 128.20 (10-C), 125.17 (200-C), H, 5.86; O, 22.13; S, 8.87%. Found: C, 62.53; H, 6.18; O, 22.43; S,
124.61 (300-CF3), 124.57 (400-C), 109.59 (8-C), 108.03 (5-C), 106.84 8.79%.
(20,60-C), 101.48 (13-C), 73.42 (4-C), 71.22 (11-C), 60.59 (40-OCH3),
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
56.03 (30,50-OCH3), 49.69 (22-C), 45.91 (2-C), 45.42 (1-C), 43.61 hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(3,4-
(19-C), 38.63 (3-C), 33.88 (15-C), 32.58 (18-C), 32.41 (20-C), 30.81 dimethoxyphenyl)-1,3-dithian-4-yl)pentanoate (L7). White
(16-C), 26.16 (17-C), 25.51 (21-C). ESI-TOF, calcd for powder, yield 70%; mp: 86.7–87.7 ꢁC; 1H NMR (300 MHz, CDCl3)
C
38H39F3O9S2 ([M + Na]+) 783.1988, found 783.1549. Anal. calcd d 6.99 (d, J ¼ 1.6 Hz, 2H, Ar-H), 6.84–6.77 (m, 1H, Ar-H), 6.74 (s,
for C38H39F3O9S2: C, 59.99; H, 5.17; F, 7.49; O, 18.93; S, 8.43%. 1H, Ar-H), 6.52 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.96 (s, 2H,
Found: C, 59.01; H, 5.92; F, 7.44; O, 20.19; S, 8.22%.
O–CH2–O), 5.86 (d, J ¼ 8.7 Hz, 1H, CH–O–C]O), 5.12 (s, 1H, Ar-
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9- CH–S2), 4.57 (d, J ¼ 3.7 Hz, 1H, CH-Ar), 4.35 (t, J ¼ 7.8 Hz, 1H,
hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(4- CH–CH2–O), 4.18 (t, J ¼ 9.6 Hz, 1H, CH–CH2–O), 3.88 (s, 3H,
(triuoromethyl)phenyl)-1,3-dithian-4-yl)pentanoate
(L5). –OCH3), 3.85 (s, 3H, –OCH3), 3.80 (s, 3H, 40-OCH3), 3.75 (s, 6H,
White powder, yield 79%; mp: 93.8–94.9 ꢁC; 1H NMR (300 MHz, 30,50-OCH3), 3.08–2.73 (m, 5H, O]C–CH, O–CH2–CH, CH2–S,
CDCl3) d 7.63–7.52 (m, 4H, Ar-H), 6.73 (s, 1H, Ar-H), 6.54 (s, 1H, CH–S), 2.43 (dd, J ¼ 11.1, 7.0 Hz, 2H, CH2–C]O), 2.17 (d, J ¼
Ar-H), 6.39 (s, 2H, Ar-H), 5.97 (s, 2H, O–CH2–O), 5.88 (d, J ¼ 9.0 10.8 Hz, 1H, S–CH2–CH2), 1.77–1.47 (m, 7H, S–CH2–CH2, S–CH–
Hz, 1H, CH–O–C]O), 5.19 (d, J ¼ 2.4 Hz, 1H, Ar-CH–S2), 4.60 (d, CH2–CH2–CH2). 13C NMR (126 MHz, CDCl3) d 173.82 (12-C),
J ¼ 4.1 Hz, 1H, CH-Ar), 4.40–4.31 (m, 1H, CH–CH2–O), 4.19 (t, 173.56 (14-C), 152.62 (30,50-C), 149.09 (300,400-C), 148.09 (7-C),
J ¼ 9.7 Hz, 1H, CH–CH2–O), 3.81 (s, 3H, 40-OCH3), 3.76 (s, 6H, 147.56 (6-C), 137.23 (40-C), 134.82 (10-C), 132.34 (9-C), 131.31 (100-
30,50-OCH3), 3.07–2.76 (m, 5H, O]C–CH, O–CH2–CH, CH2–S, C), 128.36 (10-C), 120.03 (600-C), 111.26 (200-C), 110.85 (500-C),
CH–S), 2.42 (dt, J ¼ 12.0, 7.7 Hz, 2H, CH2–C]O), 2.20 (d, J ¼ 109.69 (8-C), 108.19 (5-C), 106.93 (20,60-C), 101.57 (13-C), 73.50
12.9 Hz, 1H, S–CH2–CH2), 1.65 (m, 7H, S–CH2–CH2, S–CH–CH2– (4-C), 71.29 (11-C), 60.69 (40-OCH3), 56.16 (30,50-OCH3), 55.90
CH2–CH2). 13C NMR (126 MHz, CDCl3) d 173.83 (12-C), 173.55 (300,400-OCH3), 52.13 (22-C), 46.06 (2-C), 45.49 (1-C), 43.71 (19-C),
(14-C), 152.65 (30,50-C), 148.14 (7-C), 147.60 (6-C), 142.64 (100-C), 38.71 (3-C), 35.83 (15-C), 34.10 (18-C), 32.69 (20-C), 32.46 (16-C),
137.37 (40-C), 137.18 (10-C), 134.80 (9-C), 132.38 (400-C), 128.32 25.65 (17-C), 24.79 (21-C). ESI-TOF, calcd for C39H44O11S2 ([M +
(200,600-C), 128.23 (10-C), 125.75 (300,500-C), 125.67 (400-CF3), 109.74 Na]+) 775.2325, found 775.0075. Anal. calcd for C39H44O11S2: C,
(8-C), 108.20 (5-C), 106.95 (20,60-C), 101.59 (13-C), 73.55 (4-C), 62.22; H, 5.89; O, 23.38; S, 8.52%. Found: C, 61.64; H, 6.02; O,
71.32 (11-C), 60.73 (40-OCH3), 56.18 (30,50-OCH3), 51.79 (22-C), 23.57; S, 8.53%.
46.01 (2-C), 45.56 (1-C), 43.74 (19-C), 38.75 (3-C), 35.81 (15-C),
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
34.09 (18-C), 32.58 (20-C), 32.33 (16-C), 25.61 (17-C), 24.79 (21- hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(4-
C). ESI-TOF, calcd for C38H39F3O9S2 ([M + Na]+) 783.1988, found isopropylphenyl)-1,3-dithian-4-yl)pentanoate (L8). White
783.1549. Anal. calcd for C38H39F3O9S2: C, 59.99; H, 5.17; F, powder, yield 65%; mp: 82.3–83.6 ꢁC; 1H NMR (300 MHz, CDCl3)
7.49; O, 18.93; S, 8.43%. Found: C, 59.04; H, 5.96; F, 7.44; O, d 7.35 (d, J ¼ 7.8 Hz, 2H, Ar-H), 7.17 (d, J ¼ 7.9 Hz, 2H, Ar-H),
19.12; S, 8.41%.
6.73 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.95
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9- (s, 2H, O–CH2–O), 5.87 (d, J ¼ 8.8 Hz, 1H, CH–O–C]O), 5.13 (s,
hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(2- 1H, Ar-CH–S2), 4.58 (d, J ¼ 3.7 Hz, 1H, CH-Ar), 4.40–4.29 (m, 1H,
methoxyphenyl)-1,3-dithian-4-yl)pentanoate
(L6).
White CH–CH2–O), 4.18 (t, J ¼ 9.6 Hz, 1H, CH–CH2–O), 3.79 (s, 3H, 40-
powder, yield 72%; mp: 84.6–85.7 ꢁC; 1H NMR (300 MHz, CDCl3) OCH3), 3.74 (s, 6H, 30,50-OCH3), 3.05–2.74 (m, 6H, O]C–CH,
d 7.55 (ddd, J ¼ 7.6, 4.0, 1.7 Hz, 1H, Ar-H), 7.25–7.20 (m, 1H, Ar- O–CH2–CH, CH2–S, CH–S, CH–(CH3)2), 2.41 (dd, J ¼ 10.8, 7.0 Hz,
H), 6.94 (td, J ¼ 7.7, 1.4 Hz, 1H, Ar-H), 6.86 (d, J ¼ 8.3 Hz, 1H, Ar- 2H, CH2–C]O), 2.16 (d, J ¼ 13.5 Hz, 1H, S–CH2–CH2), 1.78–1.47
H), 6.73 (s, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), 5.96 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2–CH2), 1.22 (s, 3H, –CH3),
(d, J ¼ 1.3 Hz, 2H, O–CH2–O), 5.87 (d, J ¼ 8.8 Hz, 1H, CH–O–C] 1.20 (s, 3H, –CH3). 13C NMR (126 MHz, CDCl3) d 173.85 (12-C),
O), 5.69 (s, 1H, Ar-CH–S2), 4.59 (d, J ¼ 4.2 Hz, 1H, CH-Ar), 4.35 173.59 (14-C), 152.64 (30,50-C), 149.17 (400-C), 148.11 (7-C), 147.59
(dd, J ¼ 9.2, 6.8 Hz, 1H, CH–CH2–O), 4.18 (t, J ¼ 9.7 Hz, 1H, (6-C), 137.22 (40-C), 136.07 (10-C), 134.84 (9-C), 132.35 (100-C),
CH–CH2–O), 3.85 (s, 3H, –OCH3), 3.80 (s, 3H, 40-OCH3), 3.75 (s, 128.39 (200,600-C), 127.59 (10-C), 126.82 (300,500-C), 109.70 (8-C),
6H, 30,50-OCH3), 3.13–2.75 (m, 5H, O]C–CH, O–CH2–CH, CH2– 108.17 (5-C), 106.97 (20,60-C), 101.59 (13-C), 73.51 (4-C), 71.32
S, CH–S), 2.48–2.34 (m, 2H, CH2–C]O), 2.17 (d, J ¼ 11.9 Hz, 1H, (11-C), 60.71 (40-OCH3), 56.16 (30,50-OCH3), 52.14 (22-C), 46.01
S–CH2–CH2), 1.78–1.47 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2– (2-C), 45.52 (1-C), 43.74 (19-C), 38.72 (3-C), 35.86 (15-C), 34.13
CH2). 13C NMR (126 MHz, CDCl3) d 173.88 (12-C), 173.62 (14-C), (18-C), 33.84 (400-CH–(CH3)), 32.79 (20-C), 32.45 (16-C), 25.66 (17-
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C), 24.81 (21-C), 23.88 (400-CH–(CH3)). ESI-TOF, calcd for (td, J ¼ 7.9, 3.1 Hz, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 6.51 (s, 1H, Ar-
C
40H46O9S2 ([M + Na]+) 757.2583, found 757.1351. Anal. calcd H), 6.37 (s, 2H, Ar-H), 5.99–5.92 (m, 2H, O–CH2–O), 5.86 (d, J ¼
for C40H46O9S2: C, 65.37; H, 6.31; O, 19.59; S, 8.73%. Found: C, 8.8 Hz, 1H, CH–O–C]O), 5.63 (s, 1H, Ar-CH–S2), 4.58 (d, J ¼ 4.0
64.73; H, 6.59; O, 20.09; S, 8.63%.
Hz, 1H, CH-Ar), 4.34 (dd, J ¼ 8.9, 7.0 Hz, 1H, CH–CH2–O), 4.17
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9- (td, J ¼ 9.9, 2.0 Hz, 1H, CH–CH2–O), 3.79 (s, 3H, 40-OCH3), 3.74
hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(2- (s, 6H, 30,50-OCH3), 3.17–2.70 (m, 5H, O]C–CH, O–CH2–CH,
chlorophenyl)-1,3-dithian-4-yl)pentanoate (L9). White powder, CH2–S, CH–S), 2.48–2.36 (m, 2H, CH2–C]O), 2.18 (d, J ¼ 12.3
yield 80%; mp: 78.5–79.7 ꢁC; 1H NMR (300 MHz, CDCl3) d 7.65 (d, Hz, 1H, S–CH2–CH2), 1.79–1.43 (m, 7H, S–CH2–CH2, S–CH–CH2–
J ¼ 6.7 Hz, 1H, Ar-H), 7.35 (d, J ¼ 7.4 Hz, 1H, Ar-H), 7.25–7.15 (m, CH2–CH2). 13C NMR (126 MHz, CDCl3) d 173.84 (12-C), 173.60
2H, Ar-H), 6.73 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 6.38 (s, 2H, Ar-H), (14-C), 152.62 (30,50-C), 148.11 (7-C), 147.58 (6-C), 138.48 (100-C),
5.96 (s, 2H, O–CH2–O), 5.87 (d, J ¼ 8.7 Hz, 1H, CH–O–C]O), 5.62 137.18 (40-C), 134.82 (10-C), 133.23 (300-C), 132.33 (9-C), 130.87
(s, 1H, Ar-CH–S2), 4.59 (d, J ¼ 3.4 Hz, 1H, CH-Ar), 4.42–4.30 (m, (200-C), 130.24 (400-C), 128.33 (10-C), 127.83 (500,600-C), 109.72 (8-
1H, CH–CH2–O), 4.18 (t, J ¼ 9.6 Hz, 1H, CH–CH2–O), 3.80 (s, 3H, C), 108.14 (5-C), 106.95 (20,60-C), 101.60 (13-C), 73.53 (4-C), 71.34
40-OCH3), 3.75 (s, 6H, 30,50-OCH3), 3.16–2.72 (m, 5H, O]C–CH, (11-C), 60.74 (40-OCH3), 56.17 (30,50-OCH3), 49.13 (22-C), 46.18
O–CH2–CH, CH2–S, CH–S), 2.41 (d, J ¼ 5.5 Hz, 2H, CH2–C]O), (2-C), 45.54 (1-C), 43.72 (19-C), 38.72 (3-C), 35.78 (15-C), 34.07
2.19 (d, J ¼ 12.5 Hz, 1H, S–CH2–CH2), 2.00–1.45 (m, 7H, S–CH2– (18-C), 32.66 (20-C), 32.52 (16-C), 25.59 (17-C), 24.77 (21-C). ESI-
CH2, S–CH–CH2–CH2–CH2). 13C NMR (126 MHz, CDCl3) d 173.81 TOF, calcd for C37H38Cl2O9S2 ([M + Na]+) 783.1334, found
(12-C), 173.58 (14-C), 152.63 (30,50-C), 148.11 (7-C), 147.58 (6-C), 783.1095. Anal. calcd for C37H38Cl2O9S2: C, 58.34; H, 5.03; Cl,
137.24 (100-C), 136.22 (40-C), 134.82 (10-C), 132.35 (9-C), 129.62 9.31; O, 18.90; S, 8.42%. Found: C, 57.65; H, 5.75; Cl, 9.42; O,
(200,600-C), 129.46 (500-C), 128.38 (10-C), 127.48 (300,400-C), 109.70 (8- 20.04; S, 8.40%.
C), 108.20 (5-C), 106.94 (20,60-C), 101.58 (13-C), 73.52 (4-C), 71.31
(11-C), 60.70 (40-OCH3), 56.16 (30,50-OCH3), 49.76 (22-C), 48.41 (s), hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(2,4-
46.09 (2-C), 45.53 (1-C), 43.73 (19-C), 38.71 (3-C), 35.81 (15-C), dichlorophenyl)-1,3-dithian-4-yl)pentanoate (L12). White
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
34.08 (18-C), 32.71 (20-C), 32.49 (16-C), 30.92 (s), 25.60 (17-C), powder, yield 66%; mp: 79.1–80.5 ꢁC; 1H NMR (300 MHz, CDCl3)
24.78 (21-C). ESI-TOF, calcd for C37H39ClO9S2 ([M + Na]+) d 7.59 (dd, J ¼ 8.4, 7.0 Hz, 1H, Ar-H), 7.38 (s, 1H, Ar-H), 7.29–7.21
759.1724, found 759.1003. Anal. calcd for C37H39ClO9S2: C, 61.10; (m, 1H, Ar-H), 6.74 (s, 1H, Ar-H), 6.54 (s, 1H, Ar-H), 6.39 (s, 2H,
H, 5.41; Cl, 4.87; O, 19.80; S, 8.82%. Found: C, 59.76; H, 5.88; Cl, Ar-H), 6.01–5.95 (m, 2H, O–CH2–O), 5.88 (d, J ¼ 8.9 Hz, 1H, CH–
4.90; O, 20.32; S, 8.80%.
O–C]O), 5.56 (s, 1H, Ar-CH–S2), 4.60 (d, J ¼ 4.2 Hz, 1H, CH-Ar),
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9- 4.36 (dd, J ¼ 9.3, 6.8 Hz, 1H, CH–CH2–O), 4.19 (t, J ¼ 9.5 Hz, 1H,
hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(4- CH–CH2–O), 3.81 (s, 3H, 40-OCH3), 3.76 (s, 6H, 30,50-OCH3), 3.15–
chlorophenyl)-1,3-dithian-4-yl)pentanoate
(L10).
White 2.80 (m, 5H, O]C–CH, O–CH2–CH, CH2–S, CH–S), 2.49–2.37
powder, yield 77%; mp: 81.5–82.4 ꢁC; 1H NMR (300 MHz, CDCl3) (m, 2H, CH2–C]O), 2.20 (d, J ¼ 11.8 Hz, 1H, S–CH2–CH2), 1.77–
d 7.37 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.28 (d, J ¼ 8.7 Hz, 2H, Ar-H), 1.48 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2–CH2). 13C NMR (126
6.72 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 6.37 (s, 2H, Ar-H), 5.95 MHz, CDCl3) d 173.81 (12-C), 173.56 (14-C), 152.65 (30,50-C),
(s, 2H, O–CH2–O), 5.86 (d, J ¼ 8.8 Hz, 1H, CH–O–C]O), 5.10 (s, 148.12 (7-C), 147.59 (6-C), 134.92 (100-C), 134.80 (40-C), 134.57 (10-
1H, Ar-CH–S2), 4.58 (d, J ¼ 4.0 Hz, 1H, CH-Ar), 4.34 (dd, J ¼ 8.9, C), 133.17 (200-C), 132.37 (9-C), 131.23 (400-C), 130.54 (600-C),
7.0 Hz, 1H, CH–CH2–O), 4.17 (t, J ¼ 9.7 Hz, 1H, CH–CH2–O), 3.79 129.40 (300-C), 128.34 (10-C), 127.84 (500-C), 109.73 (8-C), 108.21
(s, 3H, 40-OCH3), 3.74 (s, 6H, 30,50-OCH3), 3.04–2.78 (m, 5H, O] (5-C), 106.94 (20,60-C), 101.59 (13-C), 73.55 (4-C), 71.32 (11-C),
C–CH, O–CH2–CH, CH2–S, CH–S), 2.39 (m, 2H, CH2–C]O), 2.16 60.72 (40-OCH3), 56.18 (30,50-OCH3), 47.74 (22-C), 46.10 (2-C),
(d, J ¼ 13.3 Hz, 1H, S–CH2–CH2), 1.84–1.44 (m, 7H, S–CH2–CH2, 45.55 (1-C), 43.74 (19-C), 38.73 (3-C), 35.78 (15-C), 34.07 (18-C),
S–CH–CH2–CH2–CH2). 13C NMR (126 MHz, CDCl3) d 173.83 (12- 32.59 (20-C), 32.44 (16-C), 25.59 (17-C), 24.77 (21-C). ESI-TOF,
C), 173.59 (14-C), 152.61 (30,50-C), 148.11 (7-C), 147.57 (6-C), calcd for C37H38Cl2O9S2 ([M + Na]+) 783.1334, found 783.1095.
137.30 (40-C), 137.15 (10-C), 134.83 (9-C), 134.12 (100-C), 132.33 Anal. calcd for C37H38Cl2O9S2: C, 58.34; H, 5.03; Cl, 9.31; O,
(400-C), 129.13 (200,600-C), 128.92 (300,500-C), 128.33 (10-C), 109.71 18.90; S, 8.42%. Found: C, 57.55; H, 5.63; Cl, 9.46; O, 19.56; S,
(8-C), 108.13 (5-C), 106.96 (20,60-C), 101.60 (13-C), 73.52 (4-C), 8.40%.
71.33 (11-C), 60.72 (40-OCH3), 56.15 (30,50-OCH3), 51.51 (22-C),
45.98 (2-C), 45.51 (1-C), 43.71 (19-C), 38.73 (3-C), 35.80 (15-C), hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(3-
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
34.09 (18-C), 32.58 (20-C), 32.34 (16-C), 25.60 (17-C), 24.78 (21- bromophenyl)-1,3-dithian-4-yl)pentanoate
(L13).
White
C). ESI-TOF, calcd for C37H39ClO9S2 ([M + Na]+) 759.1724, found powder, yield 73%; mp: 89.4–90.7 ꢁC; 1H NMR (300 MHz,
759.1003. Anal. calcd for C37H39ClO9S2: C, 61.10; H, 5.41; Cl, CDCl3) d 7.60 (s, 1H, Ar-H), 7.39 (dd, J ¼ 14.3, 7.9 Hz, 2H, Ar-
4.87; O, 19.80; S, 8.82%. Found: C, 60.43; H, 5.82; Cl, 4.95; O, H), 7.19 (t, J ¼ 7.8 Hz, 1H, Ar-H), 6.73 (s, 1H, Ar-H), 6.53 (s, 1H,
20.52; S, 8.81%.
Ar-H), 6.38 (s, 2H, Ar-H), 5.96 (s, 2H, O–CH2–O), 5.87 (d, J ¼ 8.8
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9- Hz, 1H, CH–O–C]O), 5.09 (s, 1H, Ar-CH–S2), 4.59 (d, J ¼ 3.7
hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-(2,3- Hz, 1H, CH-Ar), 4.40–4.29 (m, 1H, CH–CH2–O), 4.18 (t, J ¼ 9.7
dichlorophenyl)-1,3-dithian-4-yl)pentanoate
(L11).
White Hz, 1H, CH–CH2–O), 3.80 (s, 3H, 40-OCH3), 3.75 (s, 6H, 30,50-
powder, yield 75%; mp: 88.0–89.3 ꢁC; 1H NMR (300 MHz, CDCl3) OCH3), 3.08–2.72 (m, 5H, O]C–CH, O–CH2–CH, CH2–S, CH–
d 7.56 (t, J ¼ 7.4 Hz, 1H, Ar-H), 7.38 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.19 S), 2.42 (dd, J ¼ 9.7, 6.2 Hz, 2H, CH2–C]O), 2.17 (d, J ¼ 13.1
47518 | RSC Adv., 2015, 5, 47511–47521
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Hz, 1H, S–CH2–CH2), 1.60 (ddd, J ¼ 26.9, 23.9, 16.1 Hz, 7H, S– TOF, calcd for C37H46O9S2 ([M
+
Na]+) 721.2583, found
CH2–CH2, S–CH–CH2–CH2–CH2). 13C NMR (126 MHz, CDCl3) d 721.1322. Anal. calcd for C37H46O9S2: C, 63.59; H, 6.63; O, 20.60;
173.82 (12-C), 173.60 (14-C), 152.61 (30,50-C), 148.10 (7-C), S, 9.18%. Found: C, 62.55; H, 7.11; O, 21.17; S, 9.11%.
147.57 (6-C), 140.94 (100-C), 137.15 (40-C), 134.85 (10-C), 132.32
(9-C), 131.55 (200-C), 130.84 (400-C), 130.30 (500-C), 128.34 (10-C),
Cell lines and culture conditions
126.45 (600-C), 122.55 (300-C), 109.69 (8-C), 108.12 (5-C), 106.96
HeLa, A549, Calu-1, L02 and Vero cells were purchased from
(20,60-C), 101.60 (13-C), 73.51 (4-C), 71.32 (11-C), 60.70 (40-
Nanjing Keygen Technology (Nanjing, China). Cells were
OCH3), 56.14 (30,50-OCH3), 51.58 (22-C), 45.98 (2-C), 45.49 (1-
maintained in Dulbecco's modied Eagle's medium (DMEM,
C), 43.70 (19-C), 38.72 (3-C), 35.79 (15-C), 34.09 (18-C), 32.59
Hyclone) (high glucose) with L-glutamine supplemented with
(20-C), 32.32 (16-C), 25.61 (17-C), 24.77 (21-C). ESI-TOF, calcd
10% fetal bovine serum (FBS, Transgene), 100 U mLꢀ1 penicillin
for C37H39BrO9S2 ([M + Na]+) 793.1219, found 793.0043. Anal.
and 100 mg mLꢀ1 streptomycin (Hyclone), and incubated at 37
calcd for C37H39BrO9S2: C, 57.58; H, 5.09; Br, 10.35; O, 18.66;
ꢁC in a humidied atmosphere containing 5% CO2.
S, 8.31%. Found: C, 56.63; H, 5.78; Br, 10.42; O, 19.02; S,
8.29%.
Antiproliferation assay
(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-5-(2-(3- Cancer cell lines were grown to log phase in DMEM medium
hydroxy-4-methoxyphenyl)-1,3-dithian-4-yl)pentanoate (L14). supplemented with 10% fetal bovine serum. Aer diluting to 2
White powder, yield 62%; mp: 86.5–87.7 ꢁC; 1H NMR (300 MHz, ꢃ 104 cells per mL with DMEM medium, 100 mL of the obtained
CDCl3) d 7.07 (s, 1H, Ar-H), 7.04–6.91 (m, 1H, Ar-H), 6.86 (d, J ¼ cell suspension was added to each well of 96-well culture plates
8.1 Hz, 1H, Ar-H), 6.73 (s, 1H, Ar-H), 6.54 (s, 1H, Ar-H), 6.38 (s, and then allowed to adhere for 12 h at 37 ꢁC, 5% CO2 atmo-
2H, Ar-H), 5.97 (s, 2H, O–CH2–O), 5.88 (d, J ¼ 8.9 Hz, 1H, CH–O– sphere. Tested samples at various concentrations (0.1, 1, 10,
C]O), 5.14 (s, 1H, –OH), 5.11 (s, 1H, Ar-CH–S2) 4.60 (d, J ¼ 4.2 and 100 mM) were added to the 96-well culture plates with
Hz, 1H, CH-Ar), 4.41–4.32 (m, 1H, CH–CH2–O), 4.19 (t, J ¼ 9.7 podophyllotoxin and CA-4 as positive references.
Hz, 1H, CH–CH2–O), 3.90 (s, 3H, –OCH3), 3.81 (s, 3H, 40-OCH3),
Aer 24 h, 20 mL of PBS containing 2.5 mg mLꢀ1 of MTT was
3.76 (s, 6H, 30,50-OCH3), 3.07–2.74 (m, 5H, O]C–CH, O–CH2– added to each well. Plates were then incubated for further 4 h,
CH, CH2–S, CH–S), 2.48–2.36 (m, 2H, CH2–C]O), 2.18 (s, 1H, S– and then were centrifuged (1500 rpm at 4 ꢁC for 10 min) to
CH2–CH2), 1.81–1.48 (m, 7H, S–CH2–CH2, S–CH–CH2–CH2– remove supernatant. Next, 150 mL of DMSO was added to each
CH2). 13C NMR (126 MHz, CDCl3) d 173.89 (12-C), 173.63 (14-C), well for coloration. The plates were shaken gently to ensure
152.64 (30,50-C), 151.22 (400-C), 148.13 (7-C), 147.59 (6-C), 146.66 complete solubilisation of formazan for 10 min at room
(300-C), 137.56 (40-C), 137.19 (10-C), 134.83 (100-C), 132.33 (9-C), temperature. The absorbance was measured and recorded on
130.72 (600-C), 128.32 (10-C), 120.09 (200-C), 114.48 (500-C), 109.72 an ELISA reader (EPOCH, BioTek, USA) at a test wavelength of
(8-C), 108.15 (5-C), 106.97 (20,60-C), 101.60 (13-C), 73.53 (4-C), 570 nm. In all experiments three replicate wells were used for
71.35 (11-C), 60.74 (40-OCH3), 56.18 (30,50-OCH3), 55.96 (400- each drug concentration. Each assay was carried out at least
OCH3), 52.24 (22-C), 46.07 (2-C), 45.57 (1-C), 43.73 (19-C), 38.74 three times and the results were shown in Table 2.
(3-C), 35.81 (15-C), 34.12 (18-C), 32.50 (16-C), 25.55 (17-C), 24.89
(21-C). ESI-TOF, calcd for C38H42O11S2 ([M + Na]+) 761.2169,
Cell apoptosis assay
found 761.1126. Anal. calcd for C38H42O11S2: C, 61.77; H, 5.73;
Briey, 5 ꢃ 103 cells were seeded in 6-well plates for 24 h and
O, 23.82; S, 8.68%. Found: C, 60.97; H, 6.03; O, 24.16; S, 8.66%.
then were treated with L4 (0, 1, 3, and 9 mM) for 24 h. Then cells
(5R,5aR,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
were collected and washed twice with PBS and stained with 5 mL
hexahydrofuro[30,40:6,7]naphtho[2,3-d][1,3]dioxol-5-yl-5-(2-
of Annexin V-FITC and 2.5 mL of PI (5 mg mLꢀ1) in 1ꢃ binding
cyclohexyl-1,3-dithian-4-yl)pentanoate (L15). White powder, yield
buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2)
66%; mp: 76.4–77.2 ꢁC; 1H NMR (300 MHz, CDCl3) d 6.72 (s, 1H,
for 15 min at room temperature in the dark. Apoptotic cells were
Ar-H), 6.51 (s, 1H, Ar-H), 6.36 (s, 2H, Ar-H), 5.95 (d, J ¼ 3.2 Hz, 2H,
quantied using a BD Accuri C6 Flow Cytometer (BD, USA).
O–CH2–O), 5.86 (d, J ¼ 8.7 Hz, 1H, CH–O–C]O), 4.57 (d, J ¼ 3.1
Statistical analysis was done using Flowjo 7.6.1 soware. Both
Hz, 1H, CH-Ar), 4.34 (t, J ¼ 7.7 Hz, 1H, CH–CH2–O), 4.17 (t, J ¼ 9.6
early apoptotic (Annexin V-positive, PI-negative) and late
Hz, 1H, CH–CH2–O), 4.03 (s, 1H, CH–CH–S2), 3.78 (s, 3H, 40-
apoptotic (double positive of Annexin V and PI) cells were
OCH3), 3.73 (s, 6H, 30,50-OCH3), 2.86 (dd, J ¼ 21.3, 14.4 Hz, 5H,
detected.
O]C–CH, O–CH2–CH, CH2–S, CH–S), 2.40 (s, 2H, CH2–C]O),
2.08 (d, J ¼ 12.9 Hz, 1H, S–CH2–CH2), 1.95–1.42 (m, 18H, S–CH2–
CH2, S–CH–CH2–CH2–CH2, CH–H). 13C NMR (126 MHz, CDCl3) d
Cell cycle analysis
173.89 (12-C), 173.60 (14-C), 152.61 (30,50-C), 148.10 (7-C), 147.57 A549 cells were plated in 6-well plates at 5.0 ꢃ 103 cells per well
(6-C), 137.15 (40-C), 134.81 (10-C), 132.34 (9-C), 128.36 (10-C), and incubated at 37 ꢁC for 12 h. Exponentially growing cells
109.70 (8-C), 108.11 (5-C), 106.95 (20,60-C), 101.59 (13-C), 73.48 (4- were then incubated with the L4 at 0, 0.4, 0.8, and 1.6 mM for 8 h.
C), 71.34 (11-C), 60.71 (40-OCH3), 56.32 (30,50-OCH3), 56.14 (22-C), And in the time-dependent assays, exponentially growing cells
45.52 (2-C), 45.00 (1-C), 43.72 (19-C), 43.16 (100-C), 38.72 (3-C), were incubated with 0.8 mM L4 at 37 ꢁC for 4, 8, and 16 h. Aer
34.13 (15-C), 33.94 (18-C), 31.32 (20-C), 30.55 (200,600-C), 30.42 (16- then, cells were centrifuged and xed in 70% ethanol at ꢀ20 ꢁC
C), 26.24 (400-C), 26.15 (300,500-C), 25.71 (17-C), 24.85 (21-C). ESI- for at least 12 h and subsequently resuspended in PBS
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containing 0.1 mg mLꢀ1 RNase A and 5 mg mLꢀ1 propidium and the effect was similar to that of CA-4, which was the positive
iodide (PI). Cellular DNA content, for cell cycle distribution control in this study. The following ow cytometry analysis
analysis, was measured by ow cytometry using BD Accuri C6 results demonstrated that L4 can potently lead to cell cycle
Flow Cytometer (BD, USA) plotting 10 000 events per sample. arrest at G2/M phase but the effect on apoptosis inducing was
The percentage of cells in the G1, S and G2/M phases of the cell not very signicant. Furthermore, confocal microscopy and
cycle were determined using the Flowjo 7.6.1 soware aer cell western blot analysis results suggested that L4 can potently
debris exclusion.
inhibit tubulin polymerization, thus causing cell cycle arrest
and mitosis disruption.
Confocal microscopy assay
Abbreviations
A549 cells were grown on glass bottom cell culture dish (NEST,
Wuxi, China) to 70% conuence and incubated with 1.5 mM of
L4, 2 mM of colchicine and 2 mM of taxol for 12 h, respectively.
Aer incubating, cells were washed three times with PBS and
xed with 4% paraformaldehyde for 20 min, permeabilized with
1% TritonX-100 for another 10 min. Then, the cells were
blocked with 3% BSA for 1 h at room temperature. Subse-
quently, the cells were washed once with PBS, and incubated
with anti-tubulin antibody (1 : 500, Cytoskeleton, Inc.) in 3%
BSA overnight at 4 ꢁC. Aer being washed with PBS for three
times, each coverslip was added 200 mL of Cy3-labeled goat anti-
mouse IgG (H + L) (1 : 1500, Cytoskeleton, Inc.) in 3% BSA and
incubated for 1 h at room temperature followed by DAPI (5 ng
mLꢀ1). Cells were then observed under an Olympus confocal
microscope.
MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
tetrazolium bromide)
CA-4 Combretastatin A-4
NMR Nuclear magnetic resonance spectrum
TLC
Thin layer chromatography
DMSO Dimethyl sulfoxide
PBS
Phosphate-buffered saline
DAPI 40,6-Diamidino-2-phenylindole
BSA
Bovine serum albumin
EGTA Ethylenebis (oxyethylenenitrilo) tetraacetic acid
PMSF Phenylmethane sulfonyl uoride
Acknowledgements
In vitro microtubule assembly assay
We used an established method to measure soluble (depoly-
merized) and assembled (polymerized) tubulin.26 A549 cells (5
ꢃ 107 per ask) were seeded in 75 T ask and exposed to taxol (2
mM), colchicine (2 mM), and L4 (1.5 mM) for 16 h. Aer treat-
ment, cells were collected and washed twice with PBS then lysed
at 37 ꢁC for 5 min with 50 mL of hypotonic buffer (1 mM MgCl2, 2
mM EGTA, 0.5% NP-40, 2 mM PMSF, 200 units mLꢀ1 aprotinin,
5 mM amino caproic acid, 1 mM benzamidine, and 20 mM Tris–
HCl, pH 6.8). The cell lysates were centrifuged at 13 000 rpm for
10 min at 25 ꢁC. The supernatants containing soluble (cytosolic)
tubulin were separated from the pellets containing polymerized
(cytoskeletal) tubulin. The pellets were resuspended in 100 mL of
hypotonic buffer, sonicated on ice, mixed with 5ꢃ sample
buffer, and heated for 5 min at 100 ꢁC. Equal amounts of the two
fractions were partitioned by SDS-polyacrylamide gel electro-
phoresis. Immunoblots were probed with b-tubulin monoclonal
antibody and secondary HRP-conjugated antibody. The blots
were developed by using an ECL kit and Kodak Bio-MAX MR
lm (Eastman Kodak, Rochester, NY). All results are from three
independent experiments.
The authors are grateful to the Program for Changjiang Scholars
and Innovative Research Team in University (IRT_14R27).
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