The design, synthesis, in vitro biological evaluation and molecular modeling of novel benzenesulfonate derivatives bearing chalcone moieties as potent anti-microtubulin polymerization agents

Article abstract of DOI:10.1039/c4ra12108b

A series of novel 3,4-dimethoxylbenzenesulfonate derivatives containing a chalcone structure were synthesized and evaluated for their anti-proliferative activities against HepG2, HCT-116, MCF-7 and HeLa cell lines, as well as the effects of compound 10b on mitotic arrest and cell cycle of MCF-7 carcinoma cell line. Most importantly, the results of DAPI staining under co-focal microscope justified that compound 10b functioned even at relatively low concentration. The analogues showed a potent bio-activity towards tumor cells with IC₅₀ values at nano-mole class, compared with those of positive control drug Colchicine, whose IC₅₀ were 150.4 nM for MCF-7, 123.9 nM for HepG2, 125.4 nM for HCT-116 and 131.4 nM for HeLa cells. Also, a molecular docking modeling was utilized to reveal the binding mode of derivatives and microtubule. Among all the synthesized compounds, compound 10b stands out as IC₅₀ values against all the selected cell lines were at average 80 nM (in which the values against MCF-7 and HepG2 were similar; about 79.2 nM). In this research, we gave strong evidence upon the optimized stratagem for ligands targeting the colchicine-binding site on microtubules, explaining the attribution that the analogues were designed upon the structure of chalcone and combretastatin A-4.

Full text of DOI:10.1039/c4ra12108b

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Page 1 of 34
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The Design, Synthesis, in vitro Biological Evaluation and Molecular Modeling of
Novel Benzenesulfonate Derivatives Bearing Chalcone Moieties as Potent
Anti-microtubulin Polymerization Agents
Yu-Ning Shen†, Lin Lin†, Han-Yue Qiu†, Wen-Yan Zou, Yong Qian*, Hai-Liang
Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, China.
*Corresponding author. Tel. & fax: +86ꢀ25ꢀ83592672; eꢀmail: zhuhl@nju.edu.cn
((H.ꢀL. Zhu),
† These authors contributed equally to this work.
1

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Abstract
A series of novel 3,4,ꢀdimethoxylbenzenesulfonate derivatives containing a
chalconeꢀstructure were synthesized and evaluated for their antiꢀproliferative activity
against HepG2, HCTꢀ116, MCFꢀ7 and Hela cell lines, as well as the effects of
compound 10b on mitotic arrest and cell cycle of MCFꢀ7 carcinoma cell line. Most
importantly, the results of DAPI staining under coꢀfocal microscope justified that
compound 10b functioned even at relatively low concentration. The analogues
showed a potent bioꢀactivity towards tumor cells with IC50 values at nanoꢀmole class,
compared with those of positive control drug Colchicine, whose IC50 were 150.4 nM
for MCFꢀ7, 123.9 nM for HepG2, 125.4 nM for HCTꢀ116 and 131.4 nM for Hela
cells. Also, a molecular docking modeling was utilized to reveal the binding mode of
derivatives and microtubule. Among all the synthesized compounds, compound 10b
stands out as IC50 values against all the selected cell lines were at average 80 nM (in
which the values against MCFꢀ7 and HepG2 were similar; about 79.2 nM). In this
research, we gave strong evidence upon the optimized stratagem for ligands targeting
the colchicineꢀbinding site on microtubules, explaining the attribution that the
analogues were designed upon the structure of chalcone and Combretastatin Aꢀ4.
Keywords
Microtubluin inhibitors
Combretastatin Aꢀ4
Benzenesulfonates
Docking
DAPI staining
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Introduction
A steadily increasing focus has been drawn to the treatment of cancers along with
the development of human health care, in that cancers and tumors are one of the most
intimidating threats to the public health. Tumor cells differentiate from normal cells
enormously in the propagation rate, to inhibit the cell division, therefore, is one of the
fundamental methods to treat cancers. In the process of cell division, microtubule
plays an essential role; as well as in the process like maintenance of cell shape,
regulation of motility, cell signaling, intracellular transport, and segregation of
chromosomes1, 2, which hence makes it a significant target for the antiꢀtumor drug
design. Some natural products, which are divided into two categories in general, are
reported to demonstrate potent effects on interfering dynamics of microtubules:
stabilizing agents versus destabilizing agents. Colchicine (Figure 1a) with its
analogues, combretastatins, vinca alkaloids3 function as destabilizing agents which
inhibit microtubulin polymerization; taxanes, contrarily function as stabilizing
microtubules by enhancing assembly. Since the past decades, some natural products
were extracted and reported with the ability to combine to the colchicineꢀbinding site
on microtubules, among which is Combretastatin Aꢀ4(CAꢀ4, Figure 1b) that derived
from the South African tree Combretum caffrum. It demonstrated a potent antimitotic
activity, also presented a vascular interruptive activity, and furthermore, the generally
agreeable way that CAꢀ4 causes mitosis arrest is effects on mitotic phases G2/M
phase4, 5. The StructureꢀActivity Researches (SARs) of CAꢀ4 had been focused on the
modulations on Aꢀring, olfenic bridge and Bꢀring5ꢀ8, thereby a conclusion was drawn
that it is remarkably useful to maintain the 3, 4, 5ꢀtrimethoxyl moiety in Aꢀring, a cisꢀ
alkenyl configuration in the bridge, and the hydroxyl group on Bꢀring so as to keep a
relatively considerable activity. According to Stephen L9, etc., cisꢀalkene can be
substituted by the sulfonates, also partially attributed to the cost of starting materials
and the difficulty in the reactions, the Aꢀring with the bridge was modulated to 3,4ꢀ
dimethoxyl bezensulfonate in our research.
On the other hand, according to past reports10, 11, chalcones demonstrated an
antiꢀproliferative effect on cancer cells. Kerr D J12 reported the chalcone analogues of
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CAꢀ4, which is a tough evidence of the chalcones’ antitumor activities. Besides, lots
of reports have proved that the chalcone analogues of CAꢀ4 with potent biological
activities10ꢀ13. Some work has been done by our group to optimize the CAꢀ4 analogues
and the methods of relevant biological assays and molecular imitations were still used
in this research14, 15
.
Hence, based on the Twinꢀdrug Principle, we designed
a
series of
benzenesulfonates derivatives 1bꢀ19b joint with chalcone parts so as to magnify the
inhibitory activities towards the cancer cells. The structures of the compounds 1bꢀ19b
(Table 1) were tested with imitative docking with the microtubule crystal (PDB code:
1SA0), with several compounds showing profoundly high absolute values of
interaction energy. Our present research mainly focused on the derivatives’ chemical
and biological characteristics in regard of their basic structures as in the Xꢀray single
crystal diffraction results; their inhibitory competency on several tumor cell lines
evaluated from their IC50 values, effects on the apoptosis and cell cycle. An
immuneꢀfluorescent staining assay virtually showed the ability on mitotic arrests on
the human breast cancer cell, in the regard of morphological variations between the
living carcinoma cells and mitotic ones of MCFꢀ7 cell line.
Results and Discussion
2.1 Chemistry
The synthetic route for the 3, 4,ꢀdimethoxylbenzenesulfonates derivatives 1bꢀ19b is
outlined in Scheme 1. The intermediate chalcones (1aꢀ19a) were prepared with the
reported methodologies and protocol16 as in the ClaisenꢀSchmidt Condensation of
substituted acetophenones with salicylaldehyde 1aꢀ17a, and substituted
salicylaldehydes with acetophenone 18a, 19a with the presence of hydroxide sodium
aqueous solution; yielding rates were in a range of 70%ꢀ90%. Then the chalcones
1aꢀ19a were treated with 3,4,ꢀdimethoxylbenzenesulfonates under the sulfonation
condition reported17 to obtain the targeting products 1bꢀ19b, which were reported
originally from our research. All the targeting products were purified with column
chromatography before recrystallization in the 25 °C to 30 °C in ethanol/ ethyl acetate
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solutions. All of the synthetic compounds gave satisfactory spectroscopic analysis,
which were in full accordance with their assigned structures. Specifically, the Xꢀray
crystallography of compound 10b justified its structure (Figure 8, also see
experimental parts 4.4).
2.2 Biological Evaluation
In the regard of antiꢀproliferative activities, the compounds were evaluated via
MTT assay against MCFꢀ7 (human breast carcinoma cell line), HepG2 (human liver
carcinoma cell line), HCT116 (human colorectal carcinoma cell line), and Hela cells.
Meanwhile the compounds were also evaluated toxicity towards normal cells 293T
(human embryonic kidney cells) via MTT assay, whose materials and measurements
are shown in experimental part 4.5. The results are summarized in Table 2, that the
tested sulfonate derivatives exhibit appreciable antiꢀproliferative effects on the cell
lines. IC50 values of these compounds against four carcinoma cell lines are distributed
in a range of 70ꢀ190 nM; the inhibitory activities against the distinctive cells tended
likewise.
Besides the antiꢀproliferative activities against carcinoma cells as described above,
the compound 10b was evaluated for its tubulin polymerization inhibitive activity
versus positive control CAꢀ4 (Table 3). The results showed that compound 10b could
bind to colchicineꢀbinding site similarly as CAꢀ4 did.
However, after a careful scrutiny towards the last row in Table 2, will it be not
difficult to summarize that all the targeting products exhibited more potent toxic
damages against 293T cell line, which made it a further focus to optimize this series
upon lessening the toxicity and heightening the selectivity towards carcinoma cells.
Compared from the results shown in Table 2, the positive control colchicine
effected on MCFꢀ7 cell line with a very high activity (IC50 = 150.4 nM) comparing to
those of HepG2 (IC50 = 123.9 nM), HCTꢀ116 (IC50 = 125.4 nM), and Hela cells (IC50
= 131.4 nM), however, most compounds evaluated for the IC50 values manifested a
relatively smaller value than that of colchicine’s. Among them is the compound 10b,
which is one of the most potent agents against MCFꢀ7 (IC50 = 79.2 nM) and likewise
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against other cell lines (79.2 nM, 81.34 nM, 86.8 nM, for HepG2, HCTꢀ116, and Hela
cells, respectively). Compound 10b hence was selected to be evaluated the effects on
apoptosis and mitotic arrests.
The structureꢀactivity relationships (SARs) are depicted combining the data from
Table 1 and Table 2. First, comparing the IC50 values of compound 17b, 18b, 19b
with those of 1b to 16b, substitutes on R1ꢀR6 are better stratagems than substitutes on
R7ꢀR9. Second, there is a general pattern for electronꢀdonating groups and
electronꢀwithdrawing groups on R1ꢀR6. From each row, IC50 values of compound 2b
overweighed those of compound 4b; methyl, methoxy, and ethoxy groups on paraꢀ,
metaꢀ, orthoꢀ substitute, gave better inhibitory activity than nitro and halogen
substituted compounds. Last, the compounds with substitution at the para were of
better activity than those with substitution at the ortho and meta position. For example,
IC50 value of 2b (paraꢀmethoxy substituted) against MCFꢀ7 cells (83.1 nM) is smaller
than that of 12b (metaꢀmethoxy substituted, 107.4 nM). Similarly, compound 4b
(paraꢀnitro substituted) has a better activity against MCFꢀ7 cells (IC50 = 110.3 nM)
than 9b (orthoꢀnitro substituted, IC50 = 113.4 nM).
The death pattern of MCFꢀ7 cells treated with compound 10b (200 nM) was
depicted in the Figure 2. The morphological changes between MCFꢀ7 cells without or
with the treatment of compound 10b are manifest. DAPI can partially penetrate into
the nucleus to stain nuclear DNA; thereby a relatively less intensive blue fluorescence
will be captured if the nucleus is intact. While the apoptotic cells show intensive blue
fluorescence of DAPI by staining of DAPI more, in that the permeability of nuclear
membrane of apoptotic cells changes. Except for the increasing fluorescent intensity,
the cytological morphology differs in apoptotic cells and normal cells. Compared to
Figure 2a, the apoptotic cells in Figure 2b showed not only a much higher fluorescent
intensity, but also the nucleus were irregularly edged with the chromatins unevenly
stained. It is the typical characteristics of the apoptotic cells. Furthermore, a flow
cytometry was used to further analyze the apoptosis. The MCFꢀ7 cells were cross
stained with Annexin V/PI18, and then the results were shown in Figure 3. Annexin V
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binds with phosphatidylserine (PS) at the outer plasma membranes, which refined
only to apoptotic cells. Therefore, the apoptotic cells are distinguished from intact
ones, which are the (FITC labelled) annexin Vꢀnegative ones. Besides, the double
labelling with propidium iodide (PI) can further quantitatively discriminates the
damaged cells and necrotic ones. To conclude, the results from Figure 3 quantitatively
shows intact cells (FITCꢀ/PIꢀ, Q4), apoptotic cells (FITC+/PIꢀ, Q3), necrotic cells
(FITC+/PI+, Q2), and damaged ones (FITC+/PI+, Q1). It is conspicuous that along
the concentration of compound 10b added up from 0 to 200 nM, the percentage of
apoptotic cells and necrotic ones gave a relative increase as well. One thing should be
noted that the necrotic cells added at the meantime, which mainly due to a long time
treatment with compound 10b as in the 24 hours incubation.
To determine the effects of the derivatives upon mitotic arrest and the effects on
cell cycle concerning concentration, particularly, at nanomole level, a flow
cytometry19 was carried out, whose results were shown in Figure 4 and Figure 5. After
24 hours of treatment of compound 10b as in the concentration order from 0 to 200
nM in the culture of MCFꢀ7 cells, it can be evidenced that along with the
concentration added yet still at a nanomole level, the percentage of cells arrested at
the G2/M phases gave a positively related increase. Usually, the optimal ratio of CV
of G2/M phase to G1 phase is expected less than 119, which showed a consistence in
Figure 4a, vehicle. When the concentration of compound 10b added to 200 nM in
Figure 4d, the ratio of DNA concentrations of GG2/M phase to G1 phase was nearly
2.
2.3 Molecular Docking
In the regard of the interaction of the derivatives and the microtubules, a Docking
molecular modeling was used. Raimond B.G. Ravelli20 gave a detailed description of
mictotubulin colchicineꢀbinding site in 2004, which was justified by a crystal
structure of DAMAꢀcolchicineꢀ microtubule complex (PDB code: 1SA0). The
colchicineꢀbinding site was mostly buried in the intermediate domain of β subunit,
while surrounded by strands S8 and S9, loop T7 and helices H7 and H8. Buriedꢀin
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structure on the active site making it less likely for the compounds to fit deep in the
domain, so a movement of T7 loop and H8 helix facilitates.
The results obtained from docking imitation were presented in Figure 6 and Figure
7. Researches have proved that methoxyl group on the Aꢀring on colchicine can be
linked with cys24121. Our products, on the contrary, interacted with microtubules in a
distinct way. The compound 6b was fit in the colchicineꢀbinding site with the lowest
absolute value of interaction energy (Figure 6a, and Figure 6b). A strong piꢀpi bond
stabilizes the interaction between the LYS352 amino acid residue and the conjugated
system, also a hydrogen bond happens accessorily between ASN101 and the methoxyl
group. As the surface model of microtubuleꢀligand interaction in Figure 7, it reveals
that the basic structure could be embedded into the active site, which makes it of more
likelihood to be onꢀtarget. The binding mode of the analogues and the colchicine site
predicted in the computational docking could be regarded as a consultation, we
assumed that compound 10b with the ethoxy group presented higher activity partially
attributed to the stability of phenol ring on chalcone part that the ethoxy group helped
with donating electrons, besides, ethoxy group inserted in the residueꢀslots near the
active site.
2.4 Western blot analysis
To test the ability of series to combine with colchicineꢀbinding site, causing the
mitotic arrest, a western blotting was conducted on MCFꢀ7 cells after 10 hours
treatment of compound 10b (200 nM) and positive control Colchicine (200 nM). As
were shown from Figure 9, after 10h treatment of colchicine and 10b, histone
remained similar amounts, Pꢀhistone, on the contrary, has given a rise. As was
reported before22, 23, the phosphorylated histone is a significant marker of mitosis, the
increases of Pꢀhistone, specifically the one treated with 10b, has steadily proved that
the compound 10b had effects on mitosis, even in a more favorable way, as
colchicine.
Conclusion
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In this research, a series of benzenesulfonate derivatives have been synthesized and
evaluated for their antiꢀproliferative activity (IC50 values) against MCFꢀ7, Hela,
HCTꢀ116, Hep G2 carcinoma cell lines, together with toxicity towards 293T cells as
well. The derivatives exhibited potent inhibitory activity at a nanomolar level. Among
the series, compound 10b acted out ubiquitous strong effects on those four carcinoma
cells (approximately 80 nM in a common place) and, a relatively low toxicity towards
normal cells. Therefore, a flow cytometric research regarding the effects on apoptosis
and cell cycle was committed upon compound 10b at a soundly low concentration.
With the auxiliary support from fluorescent PI staining assay, we justified how 10b
induced a mitotic arrest and apoptosis in the end. Then a molecular docking evidenced
our prediction that the derivatives could interact with microtubules intimately; by the
establishment of a piꢀpi bond and two strong hydrogen bonds. Also, it was expected to
fit in the embedded active site on the microtubules. The research somehow revealed
the prospectively optimizational orientation for this series of compounds, which is
also the future focus for this kind of designing upon the similar structure, namely,
enhancement of the selectivity towards the mutant cells instead of making effects on
both normal cells and carcinoma cells.
Experimental Section
4.1 Materials and Methodologies
All of the synthesized compounds were assessed by thin layer chromatography
1
(TLC), proton nuclear magnetic resonance (1H NMR). H NMR spectra were
measured on a Bruker AVꢀ300, AVꢀ400 or AVꢀ500 spectrometer with
tetramethylsilane (TMS) as an internal reference (δ= 0) at 25 °C. Chemical shifts are
reported in parts per million (ppm) using the residual solvent line as internal standard.
Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. Analytic
thinꢀlayer chromatography (TLC) was performed on the glassꢀbacked silica gel sheets
(silica gel 60 Å GF254). All compounds were detected using UV light (254 nm or 365
nm).
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4.2 General protocols for synthesis of chalcones 1a-19a
Procedures for the synthesis of 1a-19a (except for 3a, 4a, 9a, 14a):
Substituted acetophenones (5 mmol) and salicylaldehyde (5 mmol) were mixed in
methanol (40 mL) in an ice bath. Then NaOH aqueous solution (40%, 10 mL) was
added dropwise to the mixture maintaining in the ice bath. Then the stirring continued
in the room temperature overnight. Then the mixture was washed by cold distilled
water, neutralized with HCl solution (1 M), during which a precipitation appeared.
After filtration, the residue was recrystallized in ethanol and estate ether at the room
temperature then washed with water, to obtain the purified products.
Procedures for the synthesis of 3a, 4a, 9a and 14a:
To the mixture of substituted acetophenones (5 mmol) and salicylaldehyde (5 mmol)
in methanol (40 mL), KOH (2 e.q., 10 mmol) was added and continuously stirred for
30min. Then the mixture was removed to room temperature, reflux at 80 °C for 24
hours. Then the mixture was washed and neutralized with HCl diluted solution (1 M),
during which a precipitation appeared. After filtration, the residue was recrystallized
in ethanol and acetate ether at the room temperature, and then washed with water to
obtain the products.
4.3. General protocols for synthesis of benzenesulfonate derivatives 1b-19b
To the mixture of compound 3ꢀ(2ꢀhydroxyphenyl)ꢀ1ꢀpꢀtolylpropꢀ2ꢀenꢀ1ꢀone (1a) (2
mmol) and triethylamine (2 mL) in dichloromethane (20 mL) add 3,4ꢀ
dimethoxybenzeneꢀ1ꢀsulfonyl chloride (2 mmol) dropwise for over 20 min, stirring
continuously in room temperature for two hours. The reaction mixture was washed
with saluted brine and acetate ether (100 mL). The organic layer was dried with
anhydrous sodium sulfate and the solvent was removed to obtain a rough solid
product. The product then was purified with column chromatography, with
developing solvents ethyl acetate (20 mL) in petroleum ether (400 mL). The purified
product was obtained after the recrystallization in ethyl acetate and ethanol.
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4.3.1(E)-2-(3-Oxo-3-p-tolylprop-1-enyl)phenyl-3,4-dimethoxybenzenesulfonate
(1b)
1
m.p. 179ꢀ182 °C. H NMR (400 MHz, CDCl3) δ: 7.48 (dd, J = 7.7, 1.2 Hz, 1H),
7.36 ~ 7.27 (m, 3H), 7.25 (d, J = 6.4 Hz, 1H), 7.17 (ddd, J = 18.5, 9.9, 5.1 Hz, 5H),
7.01 (d, J = 2.1 Hz, 1H), 6.84 (d, J = 16.1 Hz, 1H), 6.67 (d, J = 8.6 Hz, 1H), 3.69 (d, J
= 11.9 Hz, 6H), 2.30 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 188.45 (s), 163.29 (s),
153.37 (s), 151.80 (s), 141.22 (s), 137.53 (s), 128.15 (d, J = 9.6 Hz), 127.89 (s),
127.65 (d, J = 11.9 Hz), 127.35 (s), 125.44 (s), 124.99 (s), 123.76 (s), 120.45 (s),
118.97 (s), 113.39 (s), 113.17 (s), 110.98 (s), 110.51 (s), 56.69 (d, J = 11.3 Hz), 21.53
(s). IR (KBr, ν, cm–1): 3412, 3299, 3060, 2945, 1709, 1650, 1547, 1508, 1479, 1373,
1281, 1192, 1141, 1028, 968, 930, 897, 828, 749, 697, 495. MS (ESI) m/z: 438.1 (M+),
calculated MS 438.49.
4.3.2(E)-2-(3-(4-Methoxyphenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzene
sulfonate (2b)
m.p. 180ꢀ183 °C. 1H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 8.6 Hz, 2H), 7.64 ~
7.52 (m, 2H), 7.45 ~ 7.28 (m, 4H), 7.11 (s, 1H), 6.97 (d, J = 8.5 Hz, 3H), 6.74 (d, J =
8.5 Hz, 1H), 3.89 (s, 3H), 3.77 (s, 3H), 3.68 (s, 3H). 13C NMR (101 MHz, CDCl3) δ:
188.12 (s), 165.75 (s), 156.37(s), 156.26 (s), 140.05 (s), 130.95 (s), 130.88 (s), 129.74
(s), 129.66 (s), 127.89 (s), 125.37 (s), 125.28 (s), 123.78 (s), 123.64 (s), 121.86 (s),
114.67 (s), 114.59 (s), 112.64 (s), 112.38 (s), 110.56 (d, J = 11.7 Hz), 56.68 (d, J =
9.3 Hz), 54.37 (s). IR (KBr, ν, cm–1): 3413, 3297, 3063, 2941, 1708, 1651, 1549, 1507,
1478, 1369, 1280, 1194, 1141, 1029, 966, 932, 898, 829, 750, 698, 496. MS (ESI) m/z:
454.1 (M+), calculated MS 454.11.
4.3.3(E)-2-(3-(4-Bromophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzenes
ulfonate (3b)
m.p. 155ꢀ157 °C. 1H NMR (500 MHz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.72 ~
7.57 (m, 4H), 7.57 ~7.27 (m, 5H), 7.19 (dd, J = 24.5, 8.7 Hz, 2H), 6.76 (d, J = 8.5 Hz,
1H), 3.80 (s, 3H), 3.73 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 189.16 (s), 153.27 (s),
149.79 (s), 148.36 (s), 139.87 (s), 136.69(s), 130.71 (s), 130.54(s), 129.85 (d, J = 6.6
Hz), 128.24 (s), 127.76 (s), 124.38 (s), 123.98 (s), 123.59 (s), 122.29 (s), 120.59 (s),
114.35 (s), 112.57 (s), 112.35 (s), 110.78 (s), 56.98 (d, J = 10.8 Hz). IR (KBr, ν, cm–1):
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3414, 3300, 3061, 2948, 1710, 1649, 1546, 1505, 1480, 1374, 1283, 1191, 1143, 1030,
969, 931, 898, 826, 750, 698, 496. MS (ESI) m/z: 502.1 (M+), calculated MS 502.01.
4.3.4(E)-2-(3-(4-Nnitrophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzenesu
lfonate (4b)
1
m.p. 145ꢀ147 °C. H NMR (400 MHz, CDCl3) δ 7.62 (dd, J = 7.8, 1.5 Hz, 1H),
7.49 ~ 7.28 (m, 9H), 7.18 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 16.1 Hz, 1H), 6.83 (d, J =
8.6 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 187.37 (s),
151.88 (s), 151.32 (d, J = 7.5Hz), 149.63 (s), 143.27 (s), 140.59 (s), 130.76 (s),
130.37 (s), 128.79 (s), 127.66 (s), 124.35 (s), 124.09 (s), 121.68 (s), 120.92 (s),
120.65 (s), 114.37 (s), 112.57(s), 88.93 (s), 88.05 (s), 87.88 (s), 54.06 (d, J = 12.1 Hz).
IR (KBr, ν, cm–1): 3411, 3296, 3062, 2947, 1706, 1651, 1548, 1506, 1480, 1371, 1283,
1195, 1146, 1024, 969, 932, 898, 829, 751, 698, 496. MS (ESI) m/z: 469.1 (M+),
calculated MS 469.08.
4.3.5(E)-2-(3-(4-Chlorophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzenes
ulfonate (5b)
1
m.p. 155 ꢀ 158 °C. H NMR (400 MHz, CDCl3) δ 7.92 – 7.85 (m, 2H), 7.60 (dd, J
= 8.7, 7.4 Hz, 2H), 7.52 ~ 7.32 (m, 6H), 7.17 (dd, J = 16.1, 9.0 Hz, 2H), 6.76 (d, J =
8.6 Hz, 1H), 3.81 (s, 3H), 3.72 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 187.05 (s),
152.85 (s), 151.62 (s), 148.96 (s), 140.83 (s), 138.27 (s), 135.72 (s), 128.77 (d, J = 6.2
Hz), 126.88 (s), 126.29 (d, J = 7.3 Hz), 125.75 (s), 124.79 (s), 124.64 (s), 121.39 (s),
120.54 (s), 115.79 (s), 115.03 (s), 99.07(s), 98.65(s), 56.97(d, J = 10.85 Hz). IR (KBr,
ν, cm–1): 3415, 3300, 3064, 2942, 1711, 1653, 1546, 1512, 1483, 1370, 1283, 1190,
1139, 1031, 968, 933, 894, 829, 752, 698, 491. MS (ESI) m/z: 458.1 (M+), calculated
MS 458.06.
4.3.6(E)-2-(3-(3,4-Dichlorophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenze
nesulfonate (6b)
m.p. 180 ꢀ 182 °C. 1H NMR (400 MHz, CDCl3) δ 8.04 (t, J = 1.7 Hz, 1H), 7.85 (d,
J = 7.8 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.61 (dd, J = 8.6, 7.3 Hz, 2H), 7.44 (dd, J =
8.1, 1.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.35 (ddd, J = 8.6, 5.3, 2.5 Hz, 2H), 7.16
(dd, J = 8.9, 6.8 Hz, 2H), 6.76 (d, J = 8.6 Hz, 1H), 3.81 (s, 3H), 3.74 (s, 3H). 13C
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NMR (100 MHz, CDCl3) δ: 188.02(s), 153.57 (s), 150.73 (s), 148.59 (s), 139.62 (s),
137.37 (s), 136.93 (s), 136.01 (s), 135.49 (s), 135.27 (s), 133.38 (s), 131.89 (s),
129.18 (s), 121.07 (s), 117.27 (s), 117.08 (s), 114.71 (s), 110.34 (s), 78.53 (s), 77.65
(s), 77.39 (s), 56.29 (d, J = 9,7 Hz). IR (KBr, ν, cm–1): 3410, 3296, 3064, 2942, 1714,
1653, 1542, 1512, 14783, 1375, 1279, 1190, 1138, 1029, 966, 935, 892, 829, 750, 694,
498. MS (ESI) m/z: 492.0 (M+), calculated MS 492.02.
4.3.7(E)-2-(3-(3,4-Dimethylphenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenze
nesulfonate (7b)
1
m.p. 210ꢀ213 °C. H NMR (400 MHz, CDCl3) δ 7.79 ~ 7.29 (m, 9H), 7.22 (d, J =
11.6 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H), 6.74 (d, J = 8.6 Hz, 1H), 3.77 (s, 3H), 3.69 (s,
13
3H), 2.35 (s, 6H). C NMR (100 MHz, CDCl3) δ: 188.32 (s), 151.76 (s), 150.37 (s),
148.41 (s), 140.23 (s), 140.11 (s), 135.71 (s), 134.28 (s), 130.94 (s), 130.81 (s),
128.59 (s), 128.17 (s), 127.25 (s), 121.68 (s), 120.37 (d, J = 4.6 Hz), 118.83 (s),
113.78 (s), 112.49 (s), 112.05 (s), 110.57 (s), 56.72 (d, J = 6.7 Hz), 18.18 (s), 18.07
(s). IR (KBr, ν, cm–1): 3415, 3296, 3066, 2938, 1712, 1655, 1553, 1512, 1483, 1375,
1287, 1196, 1144, 1030, 969, 933, 898, 825, 736, 693, 499. MS (ESI) m/z: 452.1 (M+),
calculated MS 452.13.
4.3.8(E)-2-(3-Oxo-3-o-tolylprop-1-enyl)phenyl-3,4-dimethoxybenzenesulfonate
(8b)
1
m.p. 170ꢀ172 °C. H NMR (400 MHz, CDCl3) δ 7.61 (dd, J = 7.7, 1.2 Hz, 1H),
7.48 ~ 7.37 (m, 4H), 7.36 – 7.26 (m, 5H), 7.15 (d, J = 2.1 Hz, 1H), 6.97 (d, J = 16.1
Hz, 1H), 6.80 (d, J = 8.6 Hz, 1H), 3.82 (d, J = 11.9 Hz, 6H), 2.44 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ: 189.87 (s), 151.27 (s), 150.28 (s), 148.33 (s), 142.29 (s), 138.87
(s), 133.79 (s), 133.24 (s), 130.64 (s), 130.07 (s), 128.53 (s), 126.81 (s), 125.59 (s),
124.76 (s), 124.27 (s), 124.06 (s), 121.08 (s), 120.54(s), 114.07 (d, J = 6.9 Hz),
110.58 (s), 56.73 (d, J = 11.3 Hz), 18.11(s). IR (KBr, ν, cm–1): 3414, 3301, 3063,
2947, 1711, 1652, 1548, 1510, 1483, 1376, 1283, 1194, 1143, 1026, 969, 936, 894,
826, 751, 696, 496. MS (ESI) m/z: 438.1 (M+), calculated MS 438.11.
4.3.9(E)-2-(3-(2-Nitrophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzenesul
fonate (9b)
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1
m.p. 197ꢀ198 °C. H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 7.6 Hz, 1H), 7.54 ~
7.34 (m, 6H), 7.24 (dd, J = 8.5, 2.2 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.99 ~ 6.90 (m,
2H), 6.62 (d, J = 8.6 Hz, 1H), 6.34 (s, 1H), 3.69 (s, 3H), 3.60 (s, 3H). 13C NMR (100
MHz, CDCl3) δ: 187.35 (s), 153.27 (s), 151.78 (s), 151.39 (s), 150.27 (s), 140.26 (s),
136.19 (s), 136.08 (s), 130.79 (s), 130.63 (s), 127.57 (s), 127.28 (s), 123.68 (s),
123.21 (s), 122.54 (s), 120.77 (s), 120.32 (s), 114.67 (s), 114.29 (s), 110.58 (s),
110.23 (s), 56.91 (d, J = 12.8 Hz). IR (KBr, ν, cm–1): 3416, 3298, 3068, 2942, 1714,
1658, 1546, 1506, 1484, 1378, 1290, 1185, 1144, 1035, 968, 934, 896, 822, 754, 695,
494. MS (ESI) m/z: 469.1 (M+), calculated MS 469.08.
4.3.10(E)-2-(3-(4-Ethoxyphenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzene
sulfonate (10b)
m.p. 145ꢀ147 °C. 1H NMR (400 MHz, CDCl3) δ 8.04 ~ 7.93 (m, 2H), 7.63 (dd, J =
15.1, 11.7 Hz, 2H), 7.51 ~ 7.47 (m, 2H), 7.42 (dt, J = 7.0, 3.5 Hz, 2H), 7.31 (d, J =
6.3 Hz, 1H), 7.16 (d, J = 2.2 Hz, 1H), 7.06 ~ 6.96 (m, 2H), 6.79 (d, J = 8.6 Hz, 1H),
4.18 (q, J = 7.0 Hz, 2H), 3.83 (s, 3H), 3.73 (s, 3H), 1.51 (t, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 187.64 (s), 163.02 (s), 153.98 (s), 149.19 (s), 148.39 (s), 136.45
(s), 131.16 (s), 130.83 (s), 130.49 (s), 129.37 (s), 127.79 (s), 127.43 (s), 125.72 (s),
124.29 (s), 123.62 (s), 123.08 (s), 114.34 (s), 110.46 (d, J = 7.9 Hz), 77.39 (s), 77.08
(s), 76.76 (s), 56.09 (d, J = 16.6 Hz), 14.74 (s). IR (KBr, ν, cm–1): 3414, 3302, 3062,
2948, 1712, 1656, 1549, 1512, 1482, 1376, 1283, 1195, 1143, 1032, 972, 935, 899,
825, 753, 699, 496. MS (ESI) m/z: 468.1 (M+), calculated MS 468.12.
4.3.11(E)-2-(3-(4-Fluorophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzenes
ulfonate (11b)
1
m.p. 165ꢀ167 °C. H NMR (400 MHz, CDCl3) δ 8.01 – 7.94 (m, 2H), 7.65 – 7.55
(m, 2H), 7.50 – 7.28 (m, 4H), 7.23 – 7.11 (m, 4H), 6.76 (d, J = 8.6 Hz, 1H), 3.80 (s,
3H), 3.71 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 189.12 (s), 165.31 (s), 151.27 (s),
149.91 (d, J = 4.8 Hz), 149.63 (s), 140.51 (s), 131.78 (d, J = 6.2 Hz), 128.76 (s),
128.37 (s), 127.61 (s), 127.59 (s), 123.07 (s), 120.87 (s), 116.20 (s), 115.88 (s),
115.76 (s), 115.23 (d, J = 12.7 Hz), 113.82 (s), 57.29 (s). IR (KBr, ν, cm–1): 3414,
3294, 3066, 2948, 1713, 1653, 1549, 1503, 1483, 1376, 1285, 1196, 1145, 1022, 968,
933, 894, 822, 756, 699, 498. MS (ESI) m/z: 442.1 (M+), calculated MS 442.09.
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4.3.12(E)-2-(3-(3-Methoxyphenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenze
nesulfonate (12b)
1
m.p. 160ꢀ162 °C. H NMR (400 MHz, CDCl3) δ 7.60 (dd, J = 11.3, 9.4 Hz, 2H),
7.51 ~ 7.29 (m, 7H), 7.25 – 7.17 (m, 1H), 7.17 ~ 7.08 (m, 2H), 6.74 (d, J = 8.6 Hz,
13
1H), 3.89 (s, 3H), 3.78 (s, 3H), 3.70 (s, 3H). C NMR (100 MHz, CDCl3) δ: 187.79
(s), 156.74 (s), 151.57 (s), 149.94 (s), 149.71(s), 140.23 (s), 133.25 (s), 132.74 (s),
130.57 (s), 130.24 (s), 123.71 (s), 123.38(s), 120.89 (s), 117.58 (s), 117.43 (s), 110.44
(d, J = 9.2 Hz), 107.53 (s), 107.23 (s), 106.83 (s), 104.83 (s), 56.12 (d, J = 8.9 Hz),
55.92 (s). IR (KBr, ν, cm–1): 3414, 3302, 3062, 2945, 1712, 1655, 1548, 1512, 1482,
1378, 1288, 1196, 1139, 1032, 970, 933, 898, 834, 752, 698, 496. MS (ESI) m/z:
454.1 (M+), calculated MS 454.11.
4.3.13(E)-2-(3-(2-Chlorophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzene
sulfonate (13b)
m.p. 176ꢀ178°C. 1H NMR (400 MHz, CDCl3) δ 7.62 (dd, J = 7.8, 1.5 Hz, 1H), 7.49
~ 7.28 (m, 9H), 7.18 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 16.1 Hz, 1H), 6.83 (d, J = 8.6 Hz,
1H), 3.88 (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 189.74(s), 152.07 (s),
150.39 (s), 150.29 (s), 143.37 (s), 136.92 (s), 136.58 (s), 130.06 (s), 129.91 (s),
129.27 (s), 128.35(s), 127.33 (s), 127.18 (s), 126.64 (s), 123.28 (s), 123.17(s), 118.15
(s), 117.83(s), 112.89 (s), 110.27 (s), 110.16 (s), 56.64 (d, J = 12.4 Hz). IR (KBr, ν,
cm–1): 3416, 3296, 3066, 2942, 1713, 1655, 1544, 1510, 1482, 1376, 1280, 1188,
1139, 1026, 963, 928, 893, 824, 744, 695, 493. MS (ESI) m/z: 458.1 (M+), calculated
MS 458.06.
4.3.14(E)-2-(3-(3-Bromophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzene
sulfonate (14b)
m.p. 181ꢀ184 °C. 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 2.0 Hz, 1H), 7.77 (dd,
J = 8.3, 2.0 Hz, 1H), 7.62 (ddd, J = 17.7, 9.8, 6.3 Hz, 3H), 7.47 ~ 7.44 (m, 1H), 7.39
(dd, J = 8.2, 1.1 Hz, 1H), 7.37 ~ 7.32 (m, 2H), 7.17 (dd, J = 11.2, 9.0 Hz, 2H), 3.83 (s,
13
3H), 3.77 (s, 3H). C NMR (100 MHz, CDCl3) δ: 188.15 (s), 153.09 (s), 151.76 (s),
151.54 (s), 140.38 (s), 140.12 (s), 136.28 (s), 129.75 (s), 129.63 (s), 127.18 (s),
127.01(s), 123.29 (s), 122.98 (s), 122.14 (s), 117.15 (d, J = 5.6 Hz), 117.09 (s),
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110.87 (s), 110.66 (s), 108.57 (s), 104.38 (s), 57.26 (s), 57.19 (s). IR (KBr, ν, cm–1):
3416, 3300, 3068, 2942, 1714, 1656, 1552, 1513, 1482, 1376, 1285, 1194, 1145, 1032,
962, 935, 892, 825, 744, 698, 499. MS (ESI) m/z: 502.1 (M+), calculated MS 502.01.
4.3.15(E)-2-(3-(3-Fluorophenyl)-3-oxoprop-1-enyl)phenyl-3,4-dimethoxybenzenes
ulfonate (15b)
m.p. 167ꢀ168 °C. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 7.8 Hz, 1H), 7.65 ~
7.58 (m, 3H), 7.46 (ddt, J = 13.4, 8.1, 6.2 Hz, 3H), 7.33 (ddd, J = 10.2, 8.5, 2.1 Hz,
3H), 7.21 ~ 7.14 (m, 2H), 6.77 (d, J = 8.6 Hz, 1H), 3.80 (s, 3H), 3.73 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ: 189.12 (s), 167.53 (s), 152.79 (s), 149.81 (s), 149.62 (s),
142.17 (s), 140.87 (s), 131.95 (s), 130.27 (s), 129.85 (s), 124.35 (s), 124.21 (s),122.35
(s), 121.28 (s), 120.84 (s), 120.77 (s), 114.64 (d, J = 10.3 Hz), 112.88 (s), 112.53 (s),
112.09 (s), 56.87 (d, J = 13.9 Hz). IR (KBr, ν, cm–1): 3409, 3295, 3065, 2940, 1713,
1656, 1543, 1511, 1482, 1370, 1279, 1190, 1139, 1026, 964, 932, 894, 832, 752, 688,
496. MS (ESI) m/z: 442.1 (M+), calculated MS 442.08.
4.3.16(E)-2-(3-(2,4-Dimethylphenyl)-3-oxoprop-1-enyl)-phenyl3,4-dimethoxybenz
ene-sulfonate (16b)
m.p. 160ꢀ163 °C. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 7.8 Hz, 1H), 7.65 ~
7.58 (m, 3H), 7.46 (ddt, J = 13.4, 8.1, 6.2 Hz, 3H), 7.33 (ddd, J = 10.2, 8.5, 2.1 Hz,
3H), 7.21 ~ 7.14 (m, 2H), 6.77 (d, J = 8.6 Hz, 1H), 3.80 (s, 3H), 3.73 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ: 191.76 (s), 152.06 (s), 151.87 (s), 151.64 (s), 142.39 (s),
141.82 (s), 141.08 (s), 132.79 (s), 132.15 (s), 131.28 (s), 129.51 (s), 129.27 (s),
124.28 (s), 123.89 (s), 118.54 (s), 118.33 (s), 114.88 (s), 114.29 (s), 113.07 (s), 89.39
(s), 88.76 (s), 56.25 (d, J = 11.4 Hz), 20.05 (s), 19.58 (s). IR (KBr, ν, cm–1): 3414,
3296, 3058, 2940, 1706, 1648, 1546, 1503, 1482, 1368, 1279, 1190, 1138, 1022, 966,
936, 894, 822, 749, 694, 492. MS (ESI) m/z: 452.1 (M+), calculated MS 452.13.
4.3.17(E)-2-(3-Oxo-3-phenylprop-1-enyl)phenyl-3,4-dimethoxybenzenesulfonate
(17b)
m.p. 144ꢀ146 °C. 1H NMR (400 MHz, CDCl3) δ 8.00 ~ 7.89 (m, 2H), 7.58 (dd, J =
7.8, 4.8 Hz, 2H), 7.56 ~ 7.46 (m, 4H), 7.35 (ddd, J = 8.5, 4.6, 2.2 Hz, 3H), 7.25 ~ 7.14
(m, 2H), 6.74 (d, J = 8.6 Hz, 1H), 3.83 (s, 3H), 3.67 (s, 3H). 13C NMR (100 MHz,
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CDCl3) δ: 188.29 (s), 152.15 (s), 149.87 (s), 149.56 (s), 140.28 (s), 136.36 (s), 135.71
(s), 132.28 (s), 131.99 (s), 131.83 (s), 128.21 (d, J = 12.2 Hz), 127.62 (s), 123.88 (s),
123.57 (s), 120.20 (s), 118.58 (s), 115.75 (s), 115.52 (s), 114.31 (s), 110.89 (s), 56.05
(d, J = 6.9 Hz). IR (KBr, ν, cm–1): 3416, 3295, 3066, 2940, 1712, 1656, 1543, 1512,
1482, 1370, 1286, 1190, 1132, 1033, 969, 936, 899, 824, 752, 696, 493. MS (ESI) m/z:
424.1 (M+), calculated MS 424.10.
4.3.18(E)-4-Chloro-2-(3-oxo-3-phenylprop-1-enyl)phenyl-3,4-dimethoxybenzenes
ulfonate (18b)
1
m.p. 169ꢀ171 °C. H NMR (500 MHz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.66 ~
7.61 (m, 3H), 7.48 ~ 7.30 (m, 5H), 7.22 ~ 7.14 (m, 2H), 6.76 (d, J = 8.5 Hz, 1H), 3.80
(s, 3H), 3.73 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 187.58 (s), 151.87 (s), 149.47
(s), 149.03 (s), 146.37 (s), 140.27 (s), 136.81 (s), 135.12 (s), 130.59 (s), 129.27 (s),
129.03 (s), 128.14 (s), 126.19 (s), 125.83 (s), 125.35 (s), 124.12 (d, J = 5.3 Hz),
120.09 (s), 115.58 (s), 115.12 (s), 112.39 (s), 56.91 (s), 55.87 (s). IR (KBr, ν, cm–1):
3416, 3305, 3058, 2944, 1713, 1654, 1541, 1504, 1482, 1376, 1278, 1188, 1138, 1033,
966, 928, 896, 826, 752, 694, 491. MS (ESI) m/z: 458.1 (M+), calculated MS 458.06.
4.3.19(E)-2,4-Dichloro-6-(3-oxo-3-phenylprop-1-enyl)phenyl-3,4-dimethoxybenze
nesulfonate (19b)
m.p. 170ꢀ173 °C. 1H NMR (400 MHz, CDCl3) δ 7.98 ~ 7.90 (m, 2H), 7.53 (dt, J =
15.0, 7.7 Hz, 4H), 7.42 ~ 7.28 (m, 3H), 7.17 (dd, J = 26.8, 9.0 Hz, 2H), 6.74 (d, J =
8.6 Hz, 1H), 3.80 (s, 3H), 3.68 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 187.32 (s),
153.45 (s), 153.37 (s), 149.27 (s), 142.07 (s), 135.87 (s), 133.76 (s), 130.84 (s),
128.71 (s), 128.67 (s), 128.39 (d, J = 4.8 Hz), 124.12 (s), 124.07 (s), 122.29 (s),
121.73 (s), 121.01 (s), 119.83 (s), 115.35 (s), 115.27 (s), 110.79 (s), 55.28(s), 54.02
(s). IR (KBr, ν, cm–1): 3410, 3296, 3062, 2940, 1703, 1644, 1542, 1500, 1472, 1370,
1279, 1189, 1138, 1024, 960, 933, 890, 822, 752, 695, 491. MS (ESI) m/z: 492.0 (M+),
calculated MS 492.02.
4.4 Single crystal structure determination
The crystal structure determination of compound 10b was carried out on a Nonius
CAD4 diffractometer equipped with graphitemonochromated MoKα (λ 0.7103 Å)
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radiation (Figure 8). The structure was solved by direct methods and refined on F2 by
fullꢀmatrix leastꢀsquares methods using SHELXꢀ9724. All nonꢀhydrogen atoms were
refined anisotropically. All hydrogen atoms were placed in the calculated positions
and were assigned fixed isotropic thermal parameters at 1.2 times the equivalent
isotropic U of the atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen atoms were included in
the structureꢀfactors calculations. The crystal data, data collection, and refinement
parameter for the compound 10b are listed in Table 4.
4.5 Antiproliferation assay and tubulin polymerization inhibition
The antiproliferative activity of the prepared compounds against MCFꢀ7, Hela, Hep
G2, HCTꢀ116 carcinoma cell lines were evaluated as described elsewhere with some
modifications. Target tumor cell lines were grown to log phase in RPMI 1640
medium supplemented with 10% fetal bovine serum. After diluting to 2 ×104 cells
mLꢀ1 with the complete medium, 100 ꢁL of the obtained cell suspension was added to
each well of 96ꢀwell culture plates. The subsequent incubation was permitted at 37 °C,
5% CO2 atmosphere for 24 h before the cytotoxicity assessments. Tested samples at
preꢀset concentrations were added to six wells with colchicine coassayed as positive
references. After 48 h exposure period, 40 µL of PBS containing 2.5 mg/mL of MTT
(3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetꢀrazolium bromide) was added to each
well. Four hours later, 100 µL extraction solution (10% SDS ꢀ 5% isobutyl alcohol ꢀ
0.01 M HCl) was added. After an overnight incubation at 37 °C, the optical density
was measured at a wavelength of 570 nm on an ELISA microplate reader. In all
experiments three replicate wells were used for each drug concentration. Each assay
was carried out for at least three times. The results were summarized in Table 2.
The effects on tubulin polymerization of compound 10b and positive control CAꢀ4
were evaluated as IC50 values showed in Table 3. In this research the methodologies
were as reported previously in our lab14. Concentrations of gradients were
preꢀincubated with purified bovine brain tubulin (10 ꢁM) in glutamate buffer in 30 °C.
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Then they were cooled to 0 °C, after the addition of GTP, and transportation in
spectrophotometer in 30 °C, the assembly of tubulin was observed turbidimetrically
for 50% after 30min incubation.
4.6 Apoptosis morphological assessment by fluorescent DAPI staining
After MCFꢀ7 cells were grown on coverglassꢀbottom concofal dish and incubated
with compound 10b at gradient concentrations of 0, 50 nM, 100 nM and 200 nM in
DMEM media for 24 hours at 37 °C15. The media was removed and cells were
washed with 4,6ꢀDiaminidoꢀ2ꢀphenylindole staining solution (DAPI, 10 ꢁg/mL)
staining solution three times. Then, cells were added DAPI staining solution 500
μL
for each dish for 15 min. Before scanned under microscopy, the staining solution was
removed and the dishes were washed with methanol for three times, then glycerin was
added on the dishes for imaging. All imaging experiments were performed on a fixed
cell DSU spinning confocal microscope (Olympus). Excitation and emission
monitored used Alexa Fluor 568 filters. Imaging performed using X40 objective, and
captured using Slidebook software. The result was shown in Figure. 2.
4.7 Flow cytometry on apoptosis and cell cycle
Cells (1.3 × 105 cells/mL) were cultured in the presence or not of benzenesulfonate
derivatives at 0, 50nM, 100nM, 200nM. After treatment for 24hours, cells were
washed and fixed in PBS/ethanol (30/70). For cytofluorometric examination, cells
(104 cells/mL) were incubated for 30 min in PBS/ Triton X100, 0.2% /EDTA (1 mM),
and propidium iodide (PI) (50 ꢁg/mL) in PBS supplemented by RNase (0.5 mg/mL).
The number of cells in different phases of the cell cycle was determined, and the
percentage of apoptotic cells was quantified. Analyses were accomplished by a FACS
Calibur (Becton Dickinson, Le Pont de Claix, France). Cell Quest software was used
for data acquisition and analysis. The results were presented as in Figure 3.
Approximately 105 cells/well were plated in 6ꢀwell plates and allowed to adhere.
After 12 hours, the medium was replaced with fresh culture medium containing
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compounds 10b at final concentrations of 25 nM, 50 nM, 100 nM and 200 nM.
Nontreated wells received an equivalent volume of ethanol (<0.1%). After 36 h, Cells
in the supernatant and adherent cells were collected using 0.25% Trypsin, 0.02%
EDTA. Cells were washed with PBS and were fixed in 70% ethanol, centrifuged for 1
min at 3000 g at 4 °C, washed once with PBS buffer, treated with 1 mg/mL
ribonuclease (Sigma Chemical Co.) for 15 min at 37 °C and stained with 50 mg/mL
propidium iodide (Sigma Chemical Co.) for 30 min at room temperature. Flow
cytometry analyses were performed on a Becton Dickinson FaCSꢀCalibur using the
Becton Dickinson Cell Quest program. The results were summarized in Figure 4 and
Figure 5.
4.8 Western Blot analysis
MCFꢀ7 cells were harvested at the indicated times, and cell pellets were frozen on
dry ice before lysis. Cell pellets were lysed (25 mM HEPES, 0.1% TritonꢀX100, 300
mM NaCl, 20 mM βꢀglycerophosphate, 0.5 mM DTT, 1.0 mM EDTA, 1.5 mM
MgCl2, pH 7.5, 0.2 mM sodium orthovanadate, and protease inhibitors). Then 100 ꢁg
of each lysate was resolved by 10% SDSꢀpolyacrylamide gel electrophoresis. The
resolved proteins were transferred onto nitrocellulose filter paper and hybridized to
the following antibodies: pꢀHistone H3 (Santa Cruz), histone H3 (Sigma,
H0164ꢀ200UL). Quantitation of the bands was performed using Odyssey software.
4.9 Molecular docking
The threeꢀdimensional Xꢀray structure of microtubules crystal structure (PDB code:
1SA0) was chosen as the template for the modeling study of compound 6b bound to
colchicineꢀbinding site. 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 CDOCKER protocol within Discovery Studio 3.5.
For ligand preparation, the 3D structure of 6b was generated and minimized using
Discovery Studio 3.5. For protein preparation, the hydrogen atoms were added, and
the water and impurities were removed. The molecular docking was performed by
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inserting compound 6b into the colchicineꢀbinding site on microtubules on the
β
domain. Types of interactions of the docked protein with ligandꢀbased pharmacophore
model were analyzed after the end of molecular docking.
Acknowledgement
This work was supported by the Jiangsu National Science Foundation (NO.
BK2009239) and the Fundamental Research Fund for the Central Universities (NO.
1092020804).
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1. C. Dumontet and M. A. Jordan, NAT REV DRUG DISCOV, 2010, 9, 790ꢀ803.
2. J. Zhou and P. Giannakakou, Current Medicinal ChemistryꢀAntiꢀCancer Agents, 2005, 5, 65ꢀ71.
3. J. G. White, The American journal of pathology, 1968, 53, 281.
4. T. L. Nguyen, C. McGrath, A. R. Hermone, J. C. Burnett, D. W. Zaharevitz, B. W. Day, P. Wipf,
E. Hamel and R. Gussio, J MED CHEM, 2005, 48, 6107ꢀ6116.
5. C. M. Lin, H. H. Ho, G. R. Pettit and E. Hamel, BIOCHEMISTRYꢀUS, 1989, 28, 6984ꢀ6991.
6. D. Simoni, R. Romagnoli, R. Baruchello, R. Rondanin, G. Grisolia, M. Eleopra, M. Rizzi, M.
Tolomeo, G. Giannini and D. Alloatti, J MED CHEM, 2008, 51, 6211ꢀ6215.
7. D. J. Edwards, J. A. Hadfield, T. W. Wallace and S. Ducki, ORG BIOMOL CHEM, 2011, 9,
219ꢀ231.
8. A. Kamal, A. Viswanath, M. J. Ramaiah, J. Murty, F. Sultana, G. Ramakrishna, J. R. Tamboli, S.
Pushpavalli, C. Kishor and A. Addlagatta, MEDCHEMCOMM, 2012, 3, 1386ꢀ1392.
9. S. L. Gwaltney, H. M. Imade, K. J. Barr, Q. Li, L. Gehrke, R. B. Credo, R. B. Warner, J. Y. Lee,
P. Kovar and J. Wang, BIOORG MED CHEM LETT, 2001, 11, 871ꢀ874.
10. Y. Kong, K. Wang, M. C. Edler, E. Hamel, S. L. Mooberry, M. A. Paige and M. L. Brown,
BIOORGAN MED CHEM, 2010, 18, 971ꢀ977.
11. K. V. Sashidhara, A. Kumar, M. Kumar, J. Sarkar and S. Sinha, BIOORG MED CHEM LETT,
2010, 20, 7205ꢀ7211.
12. D. J. Kerr, E. Hamel, M. K. Jung and B. L. Flynn, BIOORGAN MED CHEM, 2007, 15,
3290ꢀ3298.
13. A. Boumendjel, J. Boccard, P. Carrupt, E. Nicolle, M. Blanc, A. Geze, L. Choisnard, D.
Wouessidjewe, E. Matera and C. Dumontet, J MED CHEM, 2008, 51, 2307ꢀ2310.
14. Y. Hu, X. Lu, K. Chen, R. Yan, Q. Li and H. Zhu, BIOORGAN MED CHEM, 2012, 20, 903ꢀ909.
15. Y. Qian, H. Zhang, P. Lv and H. Zhu, BIOORGAN MED CHEM, 2010, 18, 8218ꢀ8225.
16. O. Mazimba, I. B. Masesane and R. R. Majinda, TETRAHEDRON LETT, 2011, 52, 6716ꢀ6718.
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277ꢀ282.
18. Z. Junnian, X. Shuliang and C. Jiachun, SHANGHAI J. OF IMMUNOLOGY, 1999.
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NATURE, 2004, 428, 198ꢀ202.
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22. Y. Wei, L. Yu, J. Bowen, M. A. Gorovsky and C. D. Allis, CELL, 1999, 97, 99ꢀ109.
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Figure Captions
Table 1. Structures of compounds 1b ꢀ 19b
Table 2. Proliferation inhibitory activities of compounds 1b -19b and colchicine
against cancer cells (IC50 values) and cytotoxicity towards normal cells (CC50 values)
Table 3. The inhibition of tubulin polymerization (IC50 values)
Table 4. The crystal data, data collection, and refinement parameter for the compound
10b
Figure 1. The structures of Colchicine and Combretastatin A4
Figure 2. The morphological changes of DAPI stained MCFꢀ7 human breast
cancinoma cells after treatment with compound 10b (200 nM). Observed under
coꢀfocal microscope
Figure 3. Compound 10b induced apoptosis in MCFꢀ7 cells with the concentration of
0 (A), 50 nM (B), 100 nM (C), 200 nM (D). MCFꢀ7 cells were treated with for 24 h.
Figure 4. Effects of 10b on the cell cycle distribution in MCFꢀ7 cells
Figure 5. Percentages of MCFꢀ7 cells in each mitotic phase after treatment with
compound 10b at the concentrations of 0, 50 nM, 100 nM, 200 nM
Figure 6. Binding mode of compound 6b with microtubule (PDB code: 1SA0)
Figure 7. The docking surface of compound 6b with microtubule (1SA0) in colchicine
site
Figure 8. The crystal structure of compound 10b
Figure 9. The Western blot analysis of phosphorylated histone (Pꢀhistone) and histone
on MCFꢀ7 cells, treated with colchicine (200 nM) and compound 10b (200 nM)
Scheme 1. The synthesis of compounds 1bꢀ19b
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Table 1. Structures of compounds 1b-19b
R1
H
R2
H
R3
CH3
OCH3
Br
NO2
Cl
R4
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R6
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
R7
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
R9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1b
H
H
H
2b
H
H
H
3b
H
H
H
4b
H
H
H
5b
H
H
Cl
Cl
CH3
H
6b
H
H
CH3
H
7b
CH3
NO2
H
H
8b
9b
H
H
H
H
OEt
F
H
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
H
H
H
H
OCH3
H
H
H
Cl
H
H
H
Br
F
H
H
H
H
H
CH3
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
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Table 2 Proliferation inhibitory activities of compounds 1b-19b and colchicine
against cancer cells and cytotoxicity towards normal cells.
IC50 (nM)a
HepG2d
94.8
CC50 (nM)b
293Tg
MCFꢀ7c
94.6
HCTꢀ116e
102.7
106.6
138.2
122.2
121.9
155.4
92.1
Helaf
98.4
573.6
460.6
360
1b
83.1
82.8
99.5
2b
123.9
110.3
116.8
122.3
91.7
123.8
114.6
120.4
137.9
97.3
129.4
125.2
118.3
143.5
96.0
3b
4b
387.4
572.3
506.6
415.2
574.3
435.7
734.8
435.7
403
5b
6b
7b
99.7
96.3
95.5
100.3
100.3
86.8
8b
113.4
79.2
97.0
107.6
81.34
127.6
98.6
9b
79.2
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
colchicine
111.3
107.4
101.0
144.5
133.9
90.1
123.6
102.7
131.1
157.4
137.1
85.8
113.1
106
98.4
104.2
137.9
135.8
90.1
420
144.0
138.2
86.4
541
442.2
434.6
614
151.4
149.2
175.3
150.4
128.6
116.7
201.9
123.9
189.2
124.9
184.8
125.4
115.7
164.8
182.4
131.4
841.3
442.7
797.5
a IC50 values and CC50 values were averaged values determined by at least two
independent experiments. Variation was generally 5%.
b Minimum cytotoxic concentration required to cause a microscopically detectable
alteration of normal cell morphology.
c Inhibition of MCFꢀ7 cells’ growth.
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d Inhibition of HepG2 cells’ growth.
e Inhibition of HCTꢀ116 cells’ growth.
f Inhibition of Hela cells’ growth.
g Cytotoxicity of 293T cells’ growth.
Table 3 Tubulin polymerization inhibition of Compound 10b and positive control
CAꢀ4.
IC50 values (ꢁM) a
Tubulin polymerization inhibition
CAꢀ4
3.4
Compound 10b
12.0
a The inhibition of tubulin polymerization, N=3, variation generally 5%. Tubulin (10
ꢁM) incubated in glutamate buffer. The IC50 values were defined after 30min
incubation.
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Table 4 The crystal data, data collection, and refinement parameter for the compound
10b.
compound
Empirical formula
Molecular Weight
Temperature (K)
Radiation
10b
C25H24O7S
468.52
273(2)
MoꢀKα(0.7103Å)
Triclinic
Pꢀ1
Crystal system
Space group
a (Å)
8.5318(5)
10.1614(7)
14.1611(10)
70.470(2)
88.224(2)
87.397(2)
1155.71(13)
19
b (Å)
c (Å)
α (°)
β
(°)
γ
(°)
V (Å3)
Z
Dc (g cm–3)
1.667
(mm–1) absort.coeff
0.948
ꢀ
F (000)
589
θ rang (deg)
Reflections collected
Indep. reflns
2.17ꢀ 27.56
12303(Rint= 0.0270)
5177
4025
Refns obs. [I > 2σ(I)]
Data/restr./paras
5177/0/301
1.053
Goodnessꢀofꢀfit on F2
R1, wR2(all data)
0.0622/0.1219
0.0444/0.0106
R1, wR2 [I > 2σ(I)]
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Figure 1 The structures of Colchicine and Combretastatin A 4.
Figure 2 The morphological changes of DAPI stained MCFꢀ7 human breast
cancinoma cells after treatment with compound 10b (200 nM). Observed under
coꢀfocal microscope. (a). MCFꢀ7 cells treated without compound 10b. (b). MCFꢀ7
cells treated with the compound at 200 nM.
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Figure 3 Compound 10b induced apoptosis in MCFꢀ7 cells with the concentration of
0 (A), 50 nM (B), 100 nM (C), 200 nM (D). MCFꢀ7 cells were treated with for 24 h.
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