Concise Synthesis and Biological Evaluation of 2-Aroyl-5-Amino Benzo[ b ]thiophene Derivatives As a Novel Class of Potent Antimitotic Agents

Article abstract of DOI:10.1021/jm4013938

The biological importance of microtubules make them an interesting target for the synthesis of antitumor agents. The 2-(3′,4′,5′- trimethoxybenzoyl)-5-aminobenzo[b]thiophene moiety was identified as a novel scaffold for the preparation of potent inhibitor

Full text of DOI:10.1021/jm4013938

Article
pubs.acs.org/jmc
Concise Synthesis and Biological Evaluation of 2‑Aroyl-5-Amino
Benzo[b]thiophene Derivatives As a Novel Class of Potent
Antimitotic Agents
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Carlota Lopez-Cara,† Delia Preti,†
Mojgan Aghazadeh Tabrizi,† Jan Balzarini,‡ Marcella Bassetto,§ Andrea Brancale,§ Xian-Hua Fu,∥
Yang Gao,∥ Jun Li,∥ Su-Zhan Zhang,∥ Ernest Hamel,⊥ Roberta Bortolozzi,# Giuseppe Basso,#
and Giampietro Viola*,#
†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
̀
di Ferrara, 44121 Ferrara, Italy
‡Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
§School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, U.K.
∥Cancer Institute, Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, The Second
Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province 310009, People’s Republic of China
⊥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick
National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,
United States
#Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Universita
̀
di Padova, 35131 Padova, Italy
S
* Supporting Information
ABSTRACT: The biological importance of microtubules make them an interesting target for the
synthesis of antitumor agents. The 2-(3′,4′,5′-trimethoxybenzoyl)-5-aminobenzo[b]thiophene
moiety was identified as a novel scaffold for the preparation of potent inhibitors of microtubule
polymerization acting through the colchicine site of tubulin. The position of the methoxy group
on the benzo[b]thiophene was important for maximal antiproliferative activity. Structure−activity
relationship analysis established that the best activities were obtained with amino and methoxy
groups placed at the C-5 and C-7 positions, respectively. Compounds 3c−e showed more potent
inhibition of tubulin polymerization than combretastatin A-4 and strong binding to the colchicine
site. These compounds also demonstrated substantial antiproliferative activity, with IC50 values ranging from 2.6 to 18 nM in a
variety of cancer cell lines. Importantly, compound 3c (50 mg/kg), significantly inhibited the growth of the human osteosarcoma
MNNG/HOS xenograft in nude mice.
Among the synthetic small molecules that inhibit tubulin
assembly, we reported a class of 2-(3′,4′,5′-trimethoxybenzoyl)-
3-aminobenzo[b]thiophene methoxy-substituted analogues
with chemical formula 2. The methoxy substitution and
location on the benzene part of the benzo[b]thiophene moiety
plays an important role in affecting tubulin polymerization and
antiproliferative activity, with the most favorable position for
the substituent being C-6 or C-7, while the C-4 and C-5
isomers were inactive.15 While the unsubstituted derivative 2a
was modestly active as an antiproliferative agent, with IC50
values ranging from 0.29 to 1.8 μM against five cancer cell lines,
compound 2b, with a methoxy group at the 7-position, had
potent activity (IC50 = 9.5−33 nM). These compounds
inhibited tubulin assembly and blocked the K562 cell cycle in
the G2/M phase. These encouraging data led us to continue
our research on this series of derivatives.
INTRODUCTION
■
Microtubules are essential in a variety of cellular processes,
including motility and signaling, separation of duplicated
chromosomes during cell division, shape maintenance, and
intracellular transport.1−3 The crucial involvement of micro-
tubules in mitosis makes them an important target for
anticancer drugs.4−6
A large number of chemically diverse substances that
interfere with the formation of the mitotic spindle have been
identified.7,8 The natural stilbene derivative combretastatin A-4
(CA-4, 1a, Chart 1), isolated by Pettit and co-workers from the
bark of the South African tree Combretum caffrum,9 inhibits
microtubule assembly by interacting with tubulin at the
colchicine binding site.10 To improve the water solubility of
CA-4, its disodium phosphate salt (CA-4P, 1b) was
synthesized11 and CA-4P has yielded promising results in
human clinical trials as a potent vascular disrupting agent
(VDA).12−14
Received: September 10, 2013
Published: October 28, 2013
© 2013 American Chemical Society
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1). This finding led us to undertake a more extensive
structure−activity analysis of this scaffold in an effort to further
increase antiproliferative activity, but no additional dramatic
improvements were observed. We next prepared the 2-(3′,4′,5′-
trimethoxybenzoyl)-5-aminobenzo[b]thiophene derivatives
3b−e to explore the effects of the concomitant presence of a
methyl group at the C-3 position or a methoxy moiety at the C-
6 or C-7 positions. To understand the effect of alkyl
substitution on the amino function of compound 3c, N-
methylamino and N,N-dimethylamino substituted analogues 3f
and 3g were synthesized. The importance of the basicity of the
amine function was evaluated by the synthesis of 3h, the N-
acetyl analogue of compound 3c. Compounds 3i−o,
characterized by the presence of a haloacylamino or a α-
bromoacryloylamino moiety at the 5-position of the benzo[b]-
thiophene ring, were synthesized because previous studies had
shown that the electrophilic nature of the haloacetyl moiety
sometimes improved compound activity.16,17 Moreover, the
alkylation of sulfhydryl groups of tubulin by iodoacetamide18
and the disruption of microtubules by p-bromophenacyl
bromide19 have been reported, with significant inhibitory
effects on tubulin assembly. These derivatives were prepared
by taking advantage of the low chemical reactivity as an
alkylating moiety of the α-bromoacryloyl group. Regarding the
chemical reactivity of the α-acrylic moiety, we speculated that
an intracellular biological nucleophile could perform a first-step
Michael attack on the double bond, possibly followed by a
further reaction of the halogen α to the carbonyl, leading to a β
elimination or a second nucleophilic substitution.20 Finally, in
an effort to further confirm whether the 3,4,5-trimethoxyben-
zoyl moiety at the 2-position of the benzo[b]thiophene nucleus
of compound 3c is important for antiproliferative activity, the
3′,4′-dimethoxybenzoyl and 4′-methoxbenzoyl derivatives 3p
and 3q, respectively, were prepared.
Chart 1. Chemical Structures of CA-4, CA-4P, and 2-
Aroylbenzo[b]thiophene Derivatives 2ab and 3a−q
Our first goal in the studies presented here was to determine
how important the amino substituent at the C-3 position was
for activity. We found that simply shifting the amino group of
derivative 2a from the C-3 to the C-5 position of the
benzo[b]thiophene nucleus to furnish derivative 3a resulted in
an 11−67-fold increase in potency (IC50 = 0.78−18 nM, Table
Table 1. In Vitro Growth Inhibitory Activity of Compounds 2a−b, 3a−o, and CA-4 (1a) against a Panel of Murine and Human
Cancer Cells Lines
a
IC50 (nM)
compd
2a
L1210
FM3A/0
Molt4/C8
CEM/0
HeLa
350 32
33 29
1800 300
27 13
290 10
8.5 1.4
18 0.0
310 20
8.9 2.0
11 0.7
nd
nd
2b
3a
0.78 0.05
8.0 2.2
5.0 0.42
2.8 0.2
200 12
13
4
3b
100
8
72
1
100 40
8.0 3.1
3c
12
17
5
1
2.6 0.0
4.1 1.6
3d
19
12
1
7
16
14
1
1
19
14
2
2
16
13 0.0
66
360 40
69
1
3e
15 0.0
190 10
1100 70
340 10
1700 100
2100 200
200 12
250 21
370 10
170 0.0
200 30
2200 400
>10000
3f
67 22
1100 60
340 30
1400 300
1400 200
80 22
74 10
72 14
9
3g
980 60
940 63
3h
98
6
95
8
6
3i
960 82
330 40
260 40
330 30
1600 200
1500 100
100 40
220 20
200 19
700 21
760 71
1600 600
6300 800
1.9 1.6
3j
3k
78
4
72
1
3l
420 10
410 50
360 20
330 10
270 10
280 12
1700 300
6500 600
16 1.4
340 30
310 10
140 70
260 90
1400 300
6100 200
1.9 1.6
3m
3n
3o
75
96
4
9
3p
1900 500
>10000
3q
CA-4 (1a)
2.8 1.1
42 6.0
a
IC50= compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean SE from the dose−response
curves of at least three independent experiments. nd = not determined.
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a
Scheme 1
a
Reagents and conditions: (a) ClC(S)N(CH3)2, DABCO, DMF, 50 °C, 5 h; (b) toluene, reflux; (c) NaOH, EtOH/H2O, 65 °C; (d) 3,4,5-
trimethoxyphenyl-2-bromoethanone for 7a−e, 3,4-dimethoxyphenyl-2-bromoethanone and 4-methoxyphenyl-2-bromoethanone for 7e, K2CO3,
(CH3)2CO, reflux; (e) SnCl2·2H2O, EtOH, reflux.
Acetylating or haloacetylating the 5-amino group of 3a and
3c with acetyl chloride, chloroacetyl chloride, and bromoacetyl
chloride in the presence of pyridine provided the acetamide
(3h), chloroacetamide (3k), and bromoacetamide (3i and 3l)
derivatives, respectively. The iodoacetyl derivatives 3j and 3m
were prepared from the bromoacetyl derivatives 3i and 3l by an
exchange reaction using NaI in DMA. Finally, the α-
bromoacryloylamido derivatives 3n and 3o were obtained by
the coupling of α-bromoacrylic acid with the corresponding 5-
aminobenzo[b]thiophene derivatives 3a and 3c in dry DMF,
using EDCI as the condensing agent (Scheme 2).
CHEMISTRY
■
The 2-(3′,4′,5′-trimethoxybenzoyl)-5-aminobenzo[b]thiophene
derivatives 3a−e were synthesized by the five-step synthesis
shown in Scheme 1. Starting from the variously substituted 5-
nitro salicylaldehydes 4a and 4c or 5-nitro-2-hydroxyacetophe-
nones 4b and 4d−e,21 the reaction with N,N-dimethyl
thiocarbamoyl chloride in N,N-dimethylformamide in the
presence of DABCO yielded the O-arylthiocarbamates 5a−e
in good yields. These latter were submitted to Newman−Kwart
rearrangement22 in a refluxing toluene solution to furnish the
corresponding S-arylthiocarbamates 6a−e. The subsequent
hydrolysis using a 3 M aqueous NaOH solution furnished the
desired 2-thiophenols 7a−e, which were used without further
purification for the next reaction. The 5-nitrobenzo[b]-
thiophene derivatives 8a−e were synthesized by a “one-pot”
cyclization reaction of the thiophenols 7a−e with 2-bromo-1-
(3′,4′,5′-trimethoxyphenyl)ethanone and K2CO3 in refluxing
acetone. The reduction of the nitro group with SnCl2 in
refluxing ethanol furnished the corresponding amino derivatives
3a−e. Compounds 8f and 8g were prepared by the “one-pot”
condensation from 7c and the corresponding α-bromoaceto-
phenone, followed by reduction of the 5-nitro group, to yield
the 5-amino derivatives 3p and 3q, respectively. The C-5 amino
derivative 3c was converted into the N-methylamino and N,N-
dimethylamino derivatives 3f and 3g, respectively, by reaction
with CH3I in the presence of K2CO3 and DMF.
BIOLOGICAL RESULTS AND DISCUSSION
■
In Vitro Antiproliferative Activities. Table 1 summarizes
the growth inhibitory effects of the new 5-substituted
benzo[b]thiophene derivatives 3a−q against two types of
murine cancer cell lines, leukemia L1210 and mammary
carcinoma FM3A cells, as well as three types of human cancer
cell lines, T-lymphoblastoid leukemia (Molt/4 and CEM) and
cervix carcinoma HeLa cells, in comparison with the 3-
aminobenzo[b]thiophenes 2a−b and CA-4 (1a) as reference
compounds. Among the evaluated compounds, derivatives 3a,
3c, 3d, and 3e showed the most potent antiproliferative activity,
with IC50 values of 0.78−18, 2.6−12, 16−19, and 12−15 nM,
respectively. The four compounds were thus more potent than
CA-4 against the FM3A cells, while 3c was also more active
than CA-4 against the Molt/4 cells. Compounds 3a, 3c, 3d, and
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a
Compounds 3f−h and 3i−o were designed to test the effects
of alkylation and haloalkylation, respectively, of the C-5 amino
group, with and without the C-7 methoxy group. These
compounds all had significantly reduced antiproliferative
activity as compared with 3a and 3c−e, although several had
activity comparable with or greater than that of compound 2a.
This group included the more active 3f, 3h, and 3k and the less
active 3l−o. Of these compounds, all except 3n possessed the
C-7 methoxy moiety. Overall, these data support the idea that
the C-5 amino group acts as a hydrogen bond donor rather
than an acceptor.
Scheme 2
Finally, we wished to confirm what we had previously
observed with related compounds, that the trimethoxyphenyl
ring is required for activity.15 We therefore took the 3c scaffold,
with both the C-5 NH2 and C-7 methoxy moieties, and
eliminated either one (3p) or two (3q) methoxy groups on the
trimethoxyphenyl ring. Both compounds had much reduced
antiproliferative activity, as we had observed before.15
Evaluation of Cytotoxicity in Noncancer Cells. To
investigate the antiproliferative activities of these new
derivatives in normal human cells, compound 3c was tested
in vitro against peripheral blood lymphocytes (PBL) isolated
from healthy donors (Table 2) and human umbilical vein
Table 2. Cytotoxicity of 3c in Human Peripheral Blood
Lymphocytes (PBL) and in HUVEC
a
Reagents and conditions: (a) α-bromoacrylic acid, EDCI, DMF, rt;
(b) MeI, K2CO3, DMF; (c) CH3COCl, Py, CH2Cl2, rt; (d)
ClCH2COCl, Py, CH2Cl2, rt; (e) BrCH2COBr, Py, CH2Cl2, rt; (f)
NaI, CH3CON(CH3)2, rt.
a
IC50 (μM)
3c
b
PBLresting
24.7 4.6
20.6 4.8
5.5 1.1
c
PBLPHA
HUVEC
3e were less active than CA-4 against CEM and HeLa cells.
Only 3a was more potent than CA-4 against the L1210 cells.
Moving the amino group from the C-3 to the C-5 position of
the benzo[b]thiophene ring (3a vs 2a, 3c vs 2b) significantly
increased antiproliferative activity, as noted in the Introduction
for 3a vs 2a. Thus, in these two examples, the amino group
located on the benzene portion of the benzo[b]thiophene
nucleus is preferable to its location on the thiophene part of the
molecule. Compound 3a was more active against the two
murine cell lines as compared with the three human cancer cell
lines. In contrast, the activity of 3c was more uniform across the
five cell lines, and, perhaps more importantly, 3c was more
active than 3a in the three human cell lines. This perhaps
resulted from the introduction of a methoxy group at the C-7
position of the benzo[b]thiophene nucleus in 3c as compared
with 3a.
Compound 3b, with a methyl group at the C-3 position of
derivative 3a, showed a reduction in antiproliferative activity by
6−71-fold as compared with 3a, with the greatest difference
being with the FM3A cells. Comparing 3b with 2a, except for
the concomitant introduction of the NH2 moiety at C-5 in the
former compound, it may be that a methyl group at C-3 is
superior to the amino group at this position. Comparing the 3-
methyl-7-methoxy derivative 3e with the 3-unsubstituted
analogue 3c, there was a 1.3−5-fold decrease in antiproliferative
activity in the five cell lines studied. Finally, the cell growth
inhibitory activities of compounds 3d and 3e were very similar,
indicating that placing a methoxy group at the C-6 (3d) or C-7
(3e) position of the 3-methyl benzo[b]thiophene nucleus had
similar effects, while comparing 3d with 3c reinforces the 3b/3a
finding that the C-3 methyl group was deleterious for
antiproliferative activity.
a
Compound concentration required to reduce cell growth by 50%.
b
Values are the mean SEM for three independent experiments. PBL
not stimulated with PHA. PBL stimulated with PHA.
c
endothelial cells (HUVECs). After treatment of both resting
and PHA-stimulated lymphocytes for 72 h, the IC50 values were
over 20 μM, indicating that 3c did not substantially affect the
viability of these cells. Relatively low toxicity (IC50, 5.5 μM)
was observed for 3c with HUVECs, which were 460−2100-fold
less sensitive than the cancer cell lines. Altogether, these data
suggest that 3c has cancer cell selective killing properties.
Inhibition of Tubulin Polymerization and Colchicine
Binding. To investigate whether these 5-aminobenzo[b]-
thiophene derivatives interacted with tubulin, compounds
3a−e were selected to evaluate inhibitory effects on tubulin
polymerization and measure their ability to compete for the
colchicine binding site (Table 3).23 CA-4 and 3-aminobenzo-
[b]thiophene derivatives 2a−b were evaluated as reference
compounds. 5-Aminobenzo[b]thiophenes 3a−e strongly in-
hibited tubulin assembly, and derivatives 3c, 3d, and 3e, with
IC50 values of 0.58, 0.68, and 0.48 μM, respectively, were about
as effective as CA-4 (IC50, 1.2 μM), while 3b had an IC50 value
of 1.3 μM, comparable to that of CA-4. Compounds 2b, 3a, and
2a were progressively less active than CA-4. Thus, the order of
inhibitory effects on tubulin polymerization was 3e = 3c > 3d >
3b = CA-4 > 2b > 3a > 2a.
The results suggest that antiproliferative activity was
correlated with tubulin polymerization inhibition. We observed
a good correlation for compounds 3c, 3d, and 3e, but not for
3a and 3b. Thus, while compounds 3c, 3d, and 3e were the best
inhibitors of tubulin assembly, their inhibitory effects on cancer
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Table 3. Inhibition of Tubulin Polymerization and
Colchicine Binding by Compounds 2a−b, 3a−e, and CA-4
b
colchicine binding
%
SD
a
compd
2a
tubulin assembly IC50 SD (μM) 5 μM drug 1 μM drug
c
4.2 0.7
2.1 0.09
3.2 0.1
39
39
9
1
nd
nd
nd
nd
2b
3a
65 0.2
3b
1.3 0.07
0.52 0.04
0.68 0.06
0.48 0.01
1.2 0.2
78
97
95
95
2
1
1
2
3c
89
3
4
4
1
3d
83
90
88
3e
CA-4 (1a)
99 0.5
a
Inhibition of tubulin polymerization. Tubulin was at 10 μM.
b
Inhibition of [3H]colchicine binding. Tubulin and colchicine were
c
at 1 and 5 μM, respectively. nd: not determined
cell growth were matched by those of derivative 3a, which was
6-fold less active as an assembly inhibitor. However, this
discrepancy in antitubulin versus antiproliferative activity is not
infrequently observed for antimitotic agents. The most
reasonable explanation is that, in the tubulin polymerization
assay, the composition of bovine brain tubulin, in terms of
tubulin isotypes, differs significantly from that of different
human cancer cell lines.24
In the colchicine competition assay, compounds 3a−e
strongly bound to the [3H]colchicine binding domain of
tubulin because 65−98% inhibition occurred with these agents
and colchicine both at 5 μM.25 Compounds 3c−e were the
most active inhibitors of the binding reaction because 83−90%
and 95−97% inhibition occurred with these agent at 1 and 5
μM, respectively. At these concentrations, derivatives 3c−e
were as active as CA-4, which in these experiments inhibited
colchicine binding by 99% and 88% at 5 and 1 μM, respectively.
Inhibition of colchicine binding by compounds 3a and 3b was
lower at 5 μM, with 65% and 78% inhibition occurring with
these agents. These results suggest that the antiproliferative
activity of these compounds resulted from inhibition of tubulin
polymerization by binding at the colchicine site. In general, in
these experiments, inhibition of tubulin assembly correlated
more closely with inhibition of [3H]colchicine binding to
tubulin than did antiproliferative activity.
Molecular Modeling. We performed a series of molecular
docking simulations to investigate the possible binding mode of
this new series of molecules in the colchicine site of β-tubulin.26
The results for the compounds studied (3a−e) show that all
the structures docked with the trimethoxyphenyl ring in
proximity to Cys241 and with the amino group establishing a
hydrogen bond with Thr179 (Figure 1A). Interestingly, the
methoxy group, either in position 6 or 7 of the benzo[b]-
thiophene ring, allows a better occupancy of the binding pocket
for compounds 3c−e by interacting closely with Met259,
stabilizing the overall binding pose for these compounds.
Furthermore, the methyl group present in 3b, 3d, and 3e
establishes a hydrophobic interaction with Leu248 (Figure 1B).
It should also be noted that the described binding mode for this
series of compounds is very similar to the one observed for
colchicine (see Supporting Information). Overall, the in silico
results correlate well with the tubulin polymerization inhibition
and colchicine binding experimental data.
Figure 1. Proposed binding mode for compounds 3a (A) and 3e B),
obtained by molecular docking simulation into the colchicine binding
pocket of β-tubulin.
progression in HeLa and Jurkat cells were determined by flow
cytometry (Figure 2A,B). The compound induced a remarkable
G2/M arrest in a concentration-dependent manner in the cell
lines tested, with an increase in G2/M cells occurring at a
concentration as low as 10 nM, while at higher concentrations
(15−30 nM), more than 80% of the HeLa and 40% of the
Jurkat cells were blocked in G2/M. The increase in G2/M
phase cells was accompanied by a dramatic reduction in S phase
cells in both lines. There was excellent agreement between the
potency in the inhibitory effects on tubulin polymerization by
compound 3c and its remarkable ability to induce a G2/M
arrest.
We further studied the correlation between 3c-induced G2/
M arrest and alterations in expression of proteins that regulate
cell division. The cdc2/cyclin B complex controls both entry
into and exit from mitosis. Phosphorylation of cdc2 on Tyr15
and phosphorylation of cdc25c phosphatase on Ser216
negatively regulate the activation of the cdc2/cyclin B complex.
Thus, dephosphorylation of these proteins is necessary to
activate the cdc2/cyclin B complex and is mediated by the
phosphatase cdc25c, which dephosphorylates cdc2 and
autodephosphorylates itself. Phosphorylation of cdc25c directly
stimulates both its phosphatase and autophosphatase activities,
a condition required to trigger cdc2/cyclin B induction of entry
of cells into mitosis.27−29 Thus, the protein level of cyclin B and
the phosphorylation status of both cdc2 and cdc25c were
examined by Western blot analysis. As shown in Figure 3,
treatment with 3c in HeLa cells at either 10 or 100 nM caused
no significant variations in cyclin B expression after either a 24
or 48 h treatment. In contrast, changes in the phosphorylation
Analysis of Cell Cycle Effects. The effects of a 24 h
treatment with different concentrations of 3c on cell cycle
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appearance of the phosphorylated histone variant H2A.x (γ-
H2A.X). Compound 3c at 100 nM induced a striking increase
in the expression of γ-H2A.X after a 24 h treatment, while after
48 h γ-H2A.X became apparent also at 10 nM. This result
confirms that 3c induced strong DNA damage during mitotic
arrest and that this effect could contribute to its antiproliferative
activity.
Compound 3c Induced Apoptosis. To evaluate the
mode of cell death induced by 3c, a flow cytofluorimetric
analysis was performed using propidium iodide (PI), which
stains DNA and enters only dead cells, and the protein annexin-
V, which selectively binds to apoptotic cells that expose on their
surface the phospholipid phosphatidylserine.31 Thus, this
analysis is able to distinguish between live cells (annexin-V−/
PI−), early apoptotic cells (annexin-V+/PI−), late apoptotic cells
(annexin-V+/PI+), and necrotic cells (annexin-V−/PI+). As
shown in Figure 4A,B, HeLa cells treated with 3c for 24 h
Figure 2. Percentage of cells in each phase of the cell cycle in HeLa
(A) and Jurkat (B) cells, treated with 3c at the indicated
concentrations for 24 h. Cells were fixed and labeled with PI and
analyzed by flow cytometry as described in the Experimental Section.
Data are presented as mean SEM of three independent experiments.
Figure 4. Flow cytometric analysis of apoptotic cells after treatment of
HeLa cells with 3c at the indicated concentrations after incubation for
24 (A) or 48 h (B). The cells were harvested and labeled with annexin-
V-FITC and PI and analyzed by flow cytometry. Data are presented as
mean SEM of three independent experiments.
Figure 3. Effects of 3c on G2/M regulatory proteins and
phosphohistone γH2A.X. HeLa cells were treated for 24 or 48 h
with 3c at the indicated concentrations. The cells were harvested and
lysed for the detection of cyclin B, p-cdc2Y15, cdc25C, and γH2A.X
expression by Western blot analysis. To confirm equal protein loading,
each membrane was stripped and reprobed with an anti-β-actin
antibody.
showed an accumulation of annexin-V positive cells in
comparison with the control cells and the annexin-V positive
cells further increased after 48 h. Apoptotic cells also increased
in a concentration dependent manner, with an increase in the
percentage of apoptotic cells observed at the lowest 3c
concentration examined (15 nM).
Compound 3c Induced Mitochondrial Dysfunction. It
is well-known that, as a result of apoptotic stimuli, alterations of
the mitochondrial transmembrane potential (Δψmt) are often
observed32 and that the mitochondrial apoptotic pathway is
induced following cell treatment with many antimitotic
agents.33 Thus, to analyze the involvement of the mitochon-
dria-initiated intrinsic pathway, we examined the mitochondrial
membrane permeability of HeLa cells treated with 3c. Δψmt
was monitored staining the cells with the fluorescent probe
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocya-
nine (JC-1). As shown in Figure 5A, 3c induced a time and
status of cdc25c were observed after 24 h of treatment along
with a strong reduction in the expression of this protein. At 48
h, disappearance of the cdc25c persisted and the p-cdc-25c
formed at 24 h disappeared as well. We also observed a
dramatic decrease in the expression of the phosphorylated form
of cdc2 (Tyr15). These data, along with the fact that over 80%
of the cells accumulated in the G2/M phase, suggest that 3c-
induced G2/M arrest was not due to defects in G2/M
regulatory proteins but, rather, is closely linked with
acceleration of entry into mitosis.
Prolonged mitotic arrest can lead to DNA damage.30 To
identify DNA damage, we examined treated cells for the
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of certain proteins and small molecules, such as cytochrome c.
Once cytochrome c is released in the cytoplasm, it binds to
procaspase-9, forming the so-called apoptosome, which
subsequently stimulates proteolytic activation of caspase-3.37
As shown in Figure 6, compound 3c induced proteolytic
Figure 6. Effect of 3c on caspase activation and on Bcl-2 and Xiap
expression in HeLa cells. Cells were treated for 24 or 48 h with 3c at
the indicated concentrations. The cells were harvested and lysed for
the detection of cleaved caspase-9, pro-caspase-3, PARP, Bcl-2, and
Xiap expression by Western blot analysis. To confirm equal protein
loading, each membrane was stripped and reprobed with an anti-β-
actin antibody.
Figure 5. (A) Assessment of mitochondrial membrane potential
(Δψmt) after treatment of HeLa cells with 3c. Cells were treated with
the indicated concentrations of 3c for 24 or 48 h and then stained with
the fluorescent probe JC-1. Data are expressed as mean SEM for
three independent experiments. (B) Mitochondrial production of ROS
in HeLa cells following treatment with compound 3c. After 24 or 48 h
incubations with the indicated concentration of 3c, cells were stained
with H2-DCFDA and analyzed by flow cytometry. Data are expressed
as mean SEM of three independent experiments.
cleavage of caspase-9 and caspase-3, in good agreement with
the mitochondrial depolarization described above. The DNA
repair enzyme poly(ADP-ribose) polymerase (PARP) is
cleaved by caspase-3 from its full length 116 kDa form to an
inactive 85 kDa form, and, accordingly, we also observed that
PARP cleavage was detectable at 24 h and at a low
concentration (10 nM) of 3c. Altogether, these results showed
that 3c-induced apoptosis is caspase-dependent.
Effect of 3c on Bcl-2 and XIAP Expression. It is well-
known that antimicrotubule compounds affect signaling
pathways that involve the regulation of the Bcl-2 family of
proteins.33a Bcl-2 is an antiapoptotic member of the Bcl-2
protein family and is one of the main regulators of the
mitochondrial apoptotic pathway. Moreover, it is located in the
outer mitochondrial membrane and protects cells from
apoptosis through the control of mitochondrial permeability
and release of cytochrome c.38 Many microtubule inhibitors,
such as vincristine, colchicine, and paclitaxel, induce the Bcl-2
downregulation that ultimately leads to apoptotic cell death.39
As shown in Figure 6, the expression of Bcl-2 was strongly
reduced in HeLa cells following treatment with 3c at both
concentrations examined (10 and 100 nM).
concentration−dependent increase in the percentage of cells
with depolarized mitochondria, and this effect correlated well
with the induction of apoptosis described above.
It is also well-known that reduction of mitochondrial
membrane potential is associated with mitochondrial produc-
tion of reactive oxygen species (ROS).34 In particular, with
cytochrome c release from mitochondria, a shift from the
normal four-electron reduction of O2 to a one-electron
reduction produces superoxide anion.34a For that reason, we
evaluated the intracellular level of ROS after treatment with 3c
by flow cytometry utilizing the fluorescence indicator 2′,7′-
dichlorodihydrofluorescein diacetate (H2-DCFDA).35 The
results shown in Figure 5B indicated that 3c induced, in a
time and concentration dependent manner, an increase of the
intracellular level of ROS in comparison with untreated cells,
which is in good agreement with the mitochondrial
depolarization described above. Overall, these results suggest
that 3c induced apoptosis follows the mitochondrial pathway.
Compound 3c Induced Caspase Activation. Caspase
activation plays an essential role in the propagation of
apoptosis.36 Caspases are constitutively expressed as pro-
enzymes that can be activated by a proteolytic cleavage at
specific sites. Caspases-2, -8, -9, and -10 are called activator
caspases and usually are the initiators of the apoptotic process.
Furthermore, mitochondrial depolarization results in the efflux
Xiap is a member of the IAP family (inhibitors of apoptosis
protein). In general, the IAPs suppress cell death response
through a direct interaction with caspases, including caspase-3,
caspase-7, and caspase-9, inhibiting their processing and
activation.40 We found (Figure 6) that expression of Xiap was
strongly downregulated after both 24 and 48 h treatments with
3c, even with only 10 nM 3c. Moreover, this effect was
concurrent with activation of the caspases.
In Vivo Antitumor Activity of Compound 3c. The
antitumor activity of 3c was evaluated in vivo using a human
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osteosarcoma xenograft model in Balb/C-nu nude mice. In
preliminary experiments in vitro, we determined that
compound 3c showed potent cytotoxic activity (IC50 = 1.0
0.1 nM) against the human osteosarcoma cell line MNNG/
HOS. Once the MNNG/HOS xenografts reached a size of
∼100 mm3, 12 mice were randomly assigned to one of three
groups. In two of the groups, compound 3c, dissolved in 5%
(v/v) DMSO/olive oil, or reference compound CA-4P (1b),
dissolved in isotonic saline, were injected intraperitoneally at a
dose of 50 mg/kg. Both drugs, as well as the vehicle control,
were administered four times, once every three days. As shown
in Figure 7A, even the first dose of compound 3c induced a
which caused a 32% reduction at day 18. When the observation
time ended, the animals were sacrificed and the tumors excised
and weighed. The results (Figure 7B again showed a statistically
significant reduction in tumor weight in the 3c treated animals.
Although not as great as the tumor weight reduction with 3c, a
statistically significant weight reduction was also observed for
the animals treated with 1b.
No significant weight changes were observed in the treated
animals during the entire treatment period (Figure 7C),
suggesting that 3c induced minimal toxicity.
CONCLUSIONS
■
Wanting to verify that the ortho-relationship between the
3′,4′,5′-trimethoxybenzoyl and amino moieties in derivatives
with general structure 2 was essential for their activity, we
found instead that moving the amino group from the C-3 to the
C-5 position of the benzo[b]thiophene nucleus led to a
consistent increase in antiproliferative activity. Thus, compound
3a, with the amino group at the C-5 position of the
benzo[b]thiophene ring, showed a 16−640-fold increased
improvement in IC50 values over its C-3 amino analogue 2a
in four cancer cell lines. SAR studies with the available
analogues demonstrated, particularly in the three human cancer
cell lines, that antiproliferative activity was enhanced with the
electron-donating methoxy substituent at the C-7 and possibly
at the C-6 position on the benzo[b]thiophene nucleus and was
reduced by the presence of a methyl group at the C-3 position.
The C-3 methyl derivatives 3b and 3e showed reduced
antiproliferative activity compared with their corresponding C-3
unsubstituted counterparts 3a and 3c. However, the available
analogues also suggest that a C-3 methyl group is more
favorable to activity than the original C-3 amino moiety. Four
compounds (3a, 3c, 3d, and 3e) displayed strong antiprolifer-
ative activity with IC50 values lower than 100 nM against the
panel of cancer cell lines. By alkylation of the C-5 amino group,
we confirmed that this moiety is essential for activity and
molecular modeling indicated formation of a stabilizing
hydrogen bond with Thr179 of β-tubulin. We also found that
the 3,4,5-trimethoxybenzoyl substitution at the C-2 position
was essential for potent antiproliferative activity. The molecular
models indicated that this moiety, like the analogous moiety of
CA-4 and colchicine, interacted with Cys241 of β-tubulin.
Compounds 3c−e had activities higher than that of CA-4 as
inhibitors of tubulin polymerization and also potently inhibited
the binding of [3H]colchicine to tubulin, in agreement with the
molecular models. Considering that toxicity is a major factor
that limits the effectiveness of antimitotic agents, the results
obtained with compound 3c on noncancer cells (PBL and
HUVEC) suggest that 3c may not interfere greatly with
microtubule dynamics in normal cells. These cellular studies
seemed to be corroborated by the limited toxicity (minimal
weight loss) we observed in tumor-bearing mice treated with
3c, even as the cancers shrank dramatically. Further cellular
studies showed that the induction of apoptosis by 3c was
associated with dissipation of the mitochondrial transmem-
brane potential, activation of caspase-9 and caspase-3, and other
terminal events of apoptosis such as PARP cleavage. In
conclusion, our results demonstrated that 3c is a very promising
new tubulin binding agent, and its initial strong in vivo
antitumor activity demonstrates that it warrants further
preclinical evaluation.
Figure 7. Inhibition of human xenograft growth in vivo by compound
3c. (A) MNNG/HOS human osteosarcoma tumor-bearing nude mice
were administered the vehicle, as controls, or 50 mg/kg of 3c or CA-
4P (1b). Injections were given intraperitoneally on days 0, 3, 6, and 9
(indicated by arrows). The figure shows the average measured tumor
volumes (A), the weight of the excised tumor at the end of the
observation period (B), and the body weights of the mice recorded on
the indicated days after treatments (C). Data are expressed as mean
SEM of tumor volume, tumor weigh,t and body weight at each time
point for three animals per group. * p < 0.05 and **p < 0.01 vs.
control.
significant reduction in tumor growth, reaching a 62%
reduction by the end of the observation period (18th day), as
compared with administration of vehicle only. As early as the
day after the second dose of 3c, the reduction in tumor growth
was statistically significant, indicating a fast and efficient
delivery of compound to the tumor mass. The effect of 3c on
the reduction in tumor volume was greater than that of 1b,
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= 8.4 Hz, 1H), 8.36 (dd, J = 8.4 and 2.4 Hz, 1H), 8.82 (d; J = 2.8 Hz,
1H), 10.3 (s, 1H). MS (ESI): [M + 1]+ = 255.0.
EXPERIMENTAL SECTION
■
Chemistry. Materials and Methods. All commercially available
solvents and reagents were used without further purification. Melting
points were recorded on a Buchi-Tottoli apparatus and are
uncorrected. All reactions were monitored using TLC on silica gel
F254 plates, and compounds were visualized with aqueous KMnO4.
Flash chromatography was performed using commercial silica gel
(230−400 mesh). 1H NMR data were obtained in CDCl3 or as
specified with a Varian VXR 200 spectrometer or a Varian Mercury
Plus 400 spectrometer. Peak positions are given in ppm (δ) downfield,
and J values are given in hertz. Electron spray ionization (ESI) mass
spectra were obtained on a double-focusing Finnigan MAT 95
spectrometer. The purity of tested compounds was determined by
combustion elemental analyses conducted by the Microanalytical
Laboratory of the Chemistry Department of the University of Ferrara
with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested
compounds yielded data consistent with a purity of at least 95% as
compared with the theoretical values.
General Procedure A for the Synthesis of Compounds 5a−e.
To a solution of 5-nitrosalicylaldehydes 4a and 4c or 5-nitro-2-
hydroxyacetophenones 4b and 4d−e (5 mmol) in DMF (30 mL)
containing DABCO (1.14 g., 10 mmol) was added N,N-dimethylth-
iocarbamoyl chloride (0.94 g., 7.6 mmol) in one portion. The
temperature rose rapidly to 50 °C, and the mixture was stirred at this
temperature for 5 h. After this time, water was added (30 mL) and the
mixture was extracted with CH2Cl2 (3 × 30 mL). The organic phase
was washed with 5% HCl (20 mL), 1 M NaOH (20 mL), and brine
(20 mL), dried over Na2SO4, and evaporated in vacuo. The crude
product was purified by column chromatography on silica gel or
recrystallized from petroleum ether.
O-2-Formyl-4-nitrophenyl-N,N-dimethylcarbamothioate (5a).
Using general procedure A, the crude product purified by
crystallization from petroleum ether yielded 5a as a yellow solid;
yield 91%; mp 81−83 °C. 1H NMR (CDCl3) δ: 3.03 (s, 3H), 3.19 (s,
3H), 7.78 (d, J = 8.4 Hz, 1H), 8.36 (dd, J = 8.4 and 2.6 Hz, 1H), 8.76
(d; J = 2.8 Hz, 1H), 10.3 (s, 1H). MS (ESI): [M + 1]+ = 255.1.
O-2-Acetyl-4-nitrophenyl-N,N-dimethylcarbamothioate (5b).
Using procedure A, the crude product was purified by column
chromatography with EtOAc−petroleum ether 3:7 as eluent to give 5b
as a yellow oil; yield 95%. 1H NMR (CDCl3) δ: 2.61 (s, 3H), 2.87 (s,
3H), 2.95 (s, 3H), 7.26 (d, J = 8.8 Hz, 1H), 8.36 (dd, J = 8.8 and 2.4
Hz, 1H), 8.62 (d; J = 2.4 Hz, 1H). MS (ESI): [M + 1]+ = 269.2.
O-2-Formyl-4-nitro-6-methoxyphenyl-N,N-dimethylcarbamo-
thioate (5c). Using procedure A, the crude product was purified by
crystallization from petroleum ether to give the product 5c as a yellow
solid; yield 91%; mp 129−131 °C. 1H NMR (CDCl3) δ: 3.45 (s, 3H),
3.48 (s, 3H), 3.97 (s, 3H), 8.00. (d, J = 2.4 Hz, 1H), 8.39 (d, J = 2.4
Hz. 1H), 10.4 (s, 1H). MS (ESI): [M + 1] = 285.2.
S-2-Acetyl-4-nitrophenyl-N,N-dimethylcarbamothioate (6b). Fol-
lowing general procedure B, the product 6b purified by crystallization
from petroleum ether was obtained as a red solid; 89% yield; mp 105−
107 °C. 1H NMR (CDCl3) δ: 2.66 (s, 3H), 3.04 (s, 3H), 3.13 (s, 3H),
7.78 (d, J = 8.6 Hz, 1H), 8.27 (dd, J = 8.6 and 2.4 Hz, 1H), 8.44 (d, J =
2.4 Hz, 1H). MS (ESI): [M + 1]+ = 269.2.
S-2-Formyl-4-nitro-6-methoxyphenyl-N,N-dimethylcarbamo-
thioate (6c). Using general procedure B, the product 6c was obtained
by crystallization from petroleum ether as a yellow solid; 95% yield;
1
mp 168−170 °C. H NMR (CDCl3) δ: 3.03 (bs, 3H), 3.23 (bs, 3H),
4.02 (s, 3H), 7.94 (d, J = 2.6 Hz, 1H), 8.44 (d, J = 2.6 Hz, 1H), 10.4
(s, 1H). MS (ESI): [M + 1] = 285.3.
S-2-Acetyl-4-nitro-5-methoxyphenyl-N,N-dimethylcarbamo-
thioate (6d). Using general procedure B, compound 6d purified by
column chromatography (40% ethyl acetate in petroleum ether) was
1
obtained as a brown solid; 46% yield; mp 123−125 °C. H NMR
(CDCl3) δ: 2.62 (s, 3H), 3.05 (s, 3H), 3.14 (s, 3H), 4.03 (s, 3H), 7.53
(s, 1H), 8.27 (s, 1H). MS (ESI): [M + 1] = 299.2.
S-2-Acetyl-4-nitro-6-methoxyphenyl-N,N-dimethylcarbamo-
thioate (6e). Using general procedure B, compound 6e purified by
column chromatography (30% ethyl acetate in petroleum ether) was
1
obtained as a yellow solid; 78% yield; mp 192−194 °C. H NMR
(CDCl3) δ: 2.60 (s, 3H), 3.00 (s, 3H), 3.15 (s, 3H), 3.98 (s, 3H), 7.81
(d, J = 2.2 Hz, 1H), 7.93 (d, J = 2.2 Hz, 1H). MS (ESI): [M + 1] =
299.2.
General Procedure C for the Preparation of Compounds
7a−e. The S-aryl compounds 6a−e (1 mmol) were dissolved in
MeOH (8 mL). A 3 N aqueous solution of NaOH (4 mL) was added,
and the reaction mixtures were refluxed for 5 h. Upon completion, the
mixtures were cooled to 25 °C and acidified to pH 3 by the addition of
10% aqueous HCl. The resulting mixtures were extracted with CH2Cl2
(3 × 10 mL), and the combined organic extracts were washed with
brine, dried over Na2SO4, and evaporated to dryness under reduced
pressure. The residues were purified by column chromatography on
silica gel or by crystallization from petroleum ether.
2-Mercapto-5-nitrobenzaldehyde (7a). Using general procedure
C, the residue was crystallized from petroleum ether to provide 7a as a
1
yellow solid; yield 92%; mp 147−149 °C. H NMR (CDCl3) δ: 6.12
(s, 1H), 7.52 (d, J = 8.6 Hz, 1H), 8.22 (dd, J = 8.6 and 2.4 Hz, 1H),
8.62 (d, J = 2.4 Hz, 1H), 10.1 (br s, 1H). MS (ESI): [M + 1]+ = 184.2.
1-(2-Mercapto-5-nitrophenyl)ethanone (7b). Using general pro-
cedure C, the crude residue was crystallized from petroleum ether to
provide 7b as a red solid; yield 85%; mp 93−95 °C. 1H NMR (CDCl3)
δ: 2.73 (s, 3H), 5.01 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 8.14 (dd, J = 8.6
and 2.6 Hz, 1H), 8.75 (d, J = 2.6 Hz, 1H). MS (ESI): [M + Na]+ =
209.1.
2-Mercapto-3-methoxy-5-nitrobenzaldehyde (7c). Using general
procedure C, the crude residue was crystallized from petroleum ether
to provide 7c as a yellow solid; yield 90%; mp 101−103 °C. 1H NMR
(CDCl3) δ: 4.09 (s, 3H), 6.10 (s, 1H), 7.86 (d, J = 2.4 Hz, 1H,), 8.32
(d, 1H, J = 2.4 Hz), 10.1 (br s, 1H). MS (ESI): [M + 1]+ = 214.1.
1-(2-Mercapto-4-methoxy-5-nitrophenyl)ethanone (7d). Using
general procedure C, the product 7d, purified by column
chromatography on silica gel (30% ethyl acetate in petroleum
ether), was obtained as a yellow solid; yield 68%; mp 101−103 °C.
1H NMR (CDCl3) δ: 2.65 (s, 3H), 4.03 (s, 3H), 5.82 (bs, 1H), 6.92 (s,
1H), 8.54 (s, 1H). MS (ESI): [M + 1]+ = 228.1.
O-2-Acetyl-4-nitro-5-methoxyphenyl-N,N-dimethylcarbamo-
thioate (5d). Using procedure A, the crude product was purified by
crystallization from petroleum ether to give the product 5d as a yellow
solid; yield 83%; mp 122−123 °C. 1H NMR (CDCl3) δ: 2.60 (s, 3H),
3.06 (s, 3H), 3.14 (s, 3H), 4.03 (s, 3H), 7.53 (s, 1H), 8.27 (s, 1H). MS
(ESI): [M + 1] = 299.2.
O-2-Acetyl-4-nitro-6-methoxyphenyl-N,N-dimethylcarbamo-
thioate (5e). Using procedure A, the crude product was purified by
crystallization from petroleum ether to give the product 5e as a yellow
solid; yield 88%; mp 101−103 °C. 1H NMR (CDCl3) δ: 2.60 (s, 3H),
3.42 (s, 3H), 3.45 (s, 3H), 3.94 (s, 3H), 7.92 (d, J = 2.4 Hz, 1H), 8.27
(d, J = 2.4 Hz, 1H). MS (ESI): [M + 1] = 299.3.
General Procedure B for the Preparation of Compounds
6a−e. The N,N-dimethylcarbamothioates 5a−e (5 mmol) were
dissolved in toluene (30 mL), and the mixtures were refluxed for 12
h. The solvent was evaporated in vacuo, and the residues were purified
by column chromatography (petroleum ether−ethyl acetate as eluent)
or by crystallization from petroleum ether.
S-2-Formyl-4-nitrophenyl-N,N-dimethylcarbamothioate (6a).
Using general procedure B, the product 6a was obtained by
crystallization from petroleum ether as a yellow solid; 91% yield; mp
81−83 °C. 1H NMR (CDCl3) δ: 3.06 (s, 3H), 3.19 (s, 3H), 7.76 (d, J
1-(2-Mercapto-3-methoxy-5-nitrophenyl)ethanone (7e). Follow-
ing general procedure C, the product 7e, purified by column
chromatography (40% ethyl acetate in petroleum ether), was obtained
1
as a yellow solid; yield 73%; mp 143−145 °C. H NMR (CDCl3) δ:
2.71 (s, 3H), 4.08 (s, 3H), 5.64 (bs, 1H), 7.83 (d, J = 2.2 Hz, 1H), 8.43
(d, J = 2.2 Hz, 1H). MS (ESI): [M + 1]+ = 228.2.
General Procedure D for the Preparation of Derivatives 8a−
e. To a solution of the substituted 2-mercaptobenzadehyde 7a or 7c or
2-mercaptoacetophenone 7b, 7d, or 7e (1 mmol) in acetone (15 mL)
was added 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone (289 mg, 1
mmol) and anhydrous K2CO3 (276 mg, 2 mmol). The resulting
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mixture was maintained at reflux for 18 h. After this time, acetone was
removed by evaporation, and the residue was taken up in a CH2Cl2
(15 mL) and water (5 mL) mixture. The organic layer was washed
with brine, dried over Na2SO4, and concentrated to dryness under
reduced pressure. The crude residue was purified by flash column
chromatography or by crystallization from petroleum ether.
(5-Nitrobenzo[b]thiophen-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (8a). Using general procedure D, the crude product was
purified by crystallization from petroleum ether to give 8a as a brown
solid; yield 81%; mp 185−187 °C. 1H NMR (CDCl3) δ: 3.92 (s, 6H),
3.96 (s, 3H), 7.18 (s, 2H), 7.20 (s, 1H), 8.03 (d, J = 8.6 Hz, 1H), 8.36
(dd, J = 8.6 and 1.8 Hz, 1H), 8.84 (d, J = 1.8 Hz, 1H). MS (ESI): [M
+ 1] = 374.2.
(3,4,5-Trimethoxyphenyl)(3-methyl-5-nitrobenzo[b]thiophen-2-
yl)methanone (8b). Using general procedure D, the crude residue was
purified by crystallization from petroleum ether to give 8b as a brown
solid; yield 53%; mp 120−122 °C. 1H NMR (CDCl3) δ: 2.64 (s, 3H),
3.92 (s, 6H), 3.94 (s, 3H), 7.18 (s, 2H), 8.00 (d, J = 8.6 Hz, 1H), 8.34
(dd, J = 8.6 and 2.0 Hz, 1H), 8.84 (s, 1H). MS (ESI): [M + 1] = 388.1.
(7-Methoxy-5-nitrobenzo[b]thiophen-2-yl)(3, 4, 5-
trimethoxyphenyl)methanone (8c). Using general procedure D, the
crude product purified by crystallization from petroleum ether
furnished 8c as a brown solid; yield 95%; mp 209−211 °C. 1H
NMR (CDCl3) δ: 3.92 (s, 6H), 3.93 (s, 3H), 4.12 (s, 3H), 7.18 (s,
2H), 7.74 (s, 1H), 8.00 (s, 1H), 8.44 (s, 1H). MS (ESI): [M + 1] =
404.2.
7.12 (d, J = 2.4 Hz, 1H), 7.19 (s, 2H), 7.65 (d, J = 8.6 Hz, 1H), 7.72
(s, 1H). MS (ESI): [M + 1] = 344.1. Anal. (C18H17NO4S) C, H, N.
(3,4,5-Trimethoxyphenyl)(3-methyl-5-aminobenzo[b]thiophen-2-
yl)methanone (3b). Using general procedure E, the crude residue
purified by column chromatography (30% EtOAc in petroleum ether)
1
furnished 3b as a brown solid; yield 67%; mp 165−167 °C. H NMR
(CDCl3) δ: 2.53 (s, 3H), 3.82 (bs, 2H), 3.89 (s, 6H), 3.92 (s, 3H),
6.96 (dd, J = 8.8 and 2.4 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.19 (s,
2H), 7.62 (d, J = 8.6 Hz, 1H). MS (ESI): [M]+ = 358.3. Anal.
(C19H19NO4S) C, H, N.
(7-Methoxy-5-aminobenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (3c). Using general procedure E, the
crude residue purified by column chromatography (40% EtOAc in
petroleum ether) gave 3c as a yellow solid; yield 71%; mp 176−178
1
°C. H NMR (CDCl3) δ: 3.86 (bs, 2H), 3.91 (s, 6H), 3.93 (s, 3H),
3.96 (s, 3H), 6.36 (d, J = 2.0 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H), 7.17
(s, 2H), 7.71 (s, 1H). MS (ESI): [M]+ = 374.2. Anal. (C19H19NO5S)
C, H, N.
(3-Methyl-6-methoxy-5-aminobenzo[b]thiophen-2-yl)(3,4,5-tri-
methoxyphenyl) methanone (3d). Using general procedure E, the
residue purified by column chromatography (50% EtOAc in petroleum
ether) furnished 3d as a yellow solid; yield 60%; mp 193−195 °C. 1H
NMR (CDCl3) δ: 2.54 (s, 3H), 3.80 (bs, 2H), 3.89 (s, 3H), 3.94 (s,
6H), 3.96 (s, 3H), 7.11 (s, 1H), 7.16 (s, 1H), 7.17 (s, 2H). MS (ESI):
[M]+ = 388.2. Anal. (C20H21NO5S) C, H, N.
(3-Methyl-7-methoxy-5-aminobenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (3e). Using general procedure E, the
crude residue was purified by crystallization from petroleum ether to
give 3e as a yellow solid; yield 82%; mp 193−195 °C. 1H NMR
(CDCl3) δ: 2.52 (s, 3H), 3.84 (bs, 2H), 3.89 (s, 6H), 3.93 (s, 3H),
3.95 (s, 3H), 6.36 (d, J = 1.8 Hz, 1H), 6.72 (d, J = 1.8 Hz, 1H), 7.19
(s, 2H). MS (ESI): [M]+ = 388.3. Anal. (C20H21NO5S) C, H, N.
(7-Metho xy-5-aminobenzo[b ]thiophen-2-yl)( 4, 5 -
dimethoxyphenyl)methanone (3p). Using general procedure E, the
residue chromatographed with 40% EtOAc in petroleum ether gave 3p
(3-Methyl-6-methoxy-5-nitrobenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (8d). Using general procedure D, the
crude residue purified by crystallization from petroleum ether yielded
1
8d as a brown solid; yield 86%; mp 198−200 °C. H NMR (CDCl3)
δ: 2.57 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 3.96 (s, 3H), 4.05 (s, 3H),
7.15 (s, 2H), 7.46 (s, 1H), 8.34 (s, 1H). MS (ESI): [M + 1] = 418.1.
(3-Methyl-7-methoxy-5-nitrobenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (8e). Using general procedure D, the
crude residue purified by crystallization from petroleum ether
1
furnished 8e as a white solid; yield 80%; mp 166−168 °C. H NMR
1
as a yellow solid; yield 73%; mp 156−158 °C. H NMR (CDCl3) δ:
(CDCl3) δ: 2.63 (s, 3H), 3.89 (s, 6H), 3.93 (s, 3H), 4.10 (s, 3H), 7.18
(s, 2H), 7.72 (d, J = 1.8 Hz, 1H), 8.42 (d, J = 1.8 Hz, 1H). MS (ESI):
[M + 1] = 418.1.
3.78 (bs, 2H), 3.95 (s, 3H), 3.96 (s, 3H), 3.98 (s, 3H), 6.34 (d, J = 1.8
Hz, 1H), 6.74 (d, J = 1.8 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 7.49 (d, J
= 2.0 Hz, 1H), 7.59 (dd, J = 8.2 and 1.8 Hz, 1H), 7.68 (s, 1H). MS
(ESI): [M]+ = 344.2. Anal. (C18H17NO4S) C, H, N.
Preparation of (7-Methoxy-5-nitrobenzo[b]thiophen-2-yl)-
(4,5-dimethoxyphenyl)methanone (8f). Following general proce-
dure D, using 2-mercaptobenzadehyde 7c and 2-bromo-1-(4,5-
dimethoxyphenyl)ethanone as reagents, the residue was chromato-
graphed with 30% EtOAc in petroleum ether to give 8f as a yellow
solid; yield 73%; mp 167−168 °C. 1H NMR (CDCl3) δ: 3.97 (s, 3H),
3.99 (s, 3H), 4.12 (s, 3H), 6.36 (d, J = 8.2 Hz, 1H), 7.53 (d, J = 8.2
Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.87 (s,
1H), 8.44 (s, 1H). MS (ESI): [M + 1] = 374.2.
Preparation of (7-Methoxy-5-nitrobenzo[b]thiophen-2-yl)(4-
methoxyphenyl)methanone (8g). Following general procedure D,
using 2-mercaptobenzadehyde 7c and 2-bromo-1-(4-methoxyphenyl)-
ethanone as reagents, the residue was chromatographed with 30%
EtOAc in petroleum ether to give 8g as a yellow solid; yield 68%; mp
152−154 °C. 1H NMR (CDCl3) δ: 3.92 (s, 3H), 4.12 (s, 3H), 7.01 (d,
J = 8.8 Hz, 2H), 7.72 (s, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.98 (s, 1H),
8.44 (d, J = 1.8 Hz, 1H). MS (ESI): [M + 1] = 344.1.
(7-Methoxy-5-aminobenzo[b]thiophen-2-yl)(4-methoxyphenyl)-
methanone (3q). Following general procedure E, the residue
chromatographed with 40% EtOAc in petroleum ether furnished 3q
1
as a yellow solid; yield 67%; mp 190−192 °C. H NMR (CDCl3) δ:
3.78 (bs, 2H), 3.90 (s, 3H), 3.96 (s, 3H), 6.34 (d, J = 1.4 Hz, 1H), 6.73
(d, J = 1.4 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 7.65 (s, 1H), 7.92 (d, J =
8.6 Hz, 2H). MS (ESI): [M]+ = 314.1. Anal. (C17H15NO3S) C, H, N.
Synthesis of (5-(Methylamino)-7-methoxybenzo[b]-
thiophen-2-yl)(3,4,5-trimethoxyphenyl)methanone (3f) and
(5-(Dimethylamino)-7-methoxybenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (3g). K2CO3 (83 mg, 0.6 mmol)
was added to a mixture of CH3I (0.058 mL, 0.9 mmol) and 3c (112
mg, 0.3 mmol) in 4 mL of anhydrous DMF. The mixture was stirred at
40 °C for 48 h. After this period, CH3I (0.058 mL, 0.9 mmol) was
added, and after 24 h, the reaction mixture was diluted with cold water
(1 mL) and extracted with CH2Cl2 (3 × 5 mL). The organic extracts
were combined, washed with water (2 mL) and brine, dried over
Na2SO4, and concentrated in vacuo. The crude residue was purified by
flash column chromatography using ethyl acetate−petroleum ether 4:6
(v:v) as eluent to afford 3f and 3g.
General Procedure E for the Synthesis of Compounds 3a−e
and 3p−q. To a suspension of nitro derivatives 8a−g (1 mmol) in
absolute ethanol (10 mL) was added SnCl2 2H2O (5 mmol), and the
stirring mixtures were refluxed for 1 h. After this time, the reactions
were allowed to cool to ambient temperature, treated with 15 mL of
water, and neutralized with NaHCO3. The mixtures were extracted
with EtOAc (3 × 20 mL), and the organic layers were washed with
water and brine, dried over Na2SO4, and concentrated under reduced
pressure to residues that were purified by flash column chromatog-
raphy to furnish 3a−e and 3p−q.
(5-(Methylamino)-7-methoxybenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (3f). Yellow oil; yield 22%. 1H
NMR (CDCl3) δ: 2.89 (s, 3H), 3.91 (s, 3H), 3.93 (s, 6H), 3.96 (s,
3H), 4.04 (bs, 1H), 6.30 (s, 1H), 6.63 (s, 1H), 7.19 (s, 2H), 7.76 (s,
1H). MS (ESI): [M + 1]+ = 388.2. Anal. (C20H21NO5S) C, H, N.
(5-(Dimethylamino)-7-methoxybenzo[b]thiophen-2-yl)-
(3,4,5-trimethoxyphenyl)methanone (3g). Brown oil; 41% yield.
1H NMR (CDCl3) δ: 3.00 (s, 6H), 3.91 (s, 3H), 3.94 (s, 6H), 3.99 (s,
(5-Aminobenzo[b]thiophen-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (3a). Using general procedure E, the residue was
chromatographed with 30% EtOAc in petroleum ether to give 3a as
1
3H), 6.50 (s, 1H), 6.72 (s, 1H), 7.18 (s, 2H), 7.77 (s, 1H). MS (ESI):
a yellow solid; yield 58%; mp 65−67 °C. H NMR (CDCl3) δ: 3.68
(bs, 2H), 3.91 (s, 6H), 3.94 (s, 3H), 6.91 (dd, J = 8.8 and 2.4 Hz, 1H),
[M + 1]+ = 402.4. Anal. (C21H23NO5S) C, H, N.
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N-[7-Methoxy-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]thien-
5-yl]acetamide (3h). To a solution of 3c (373 mg, 1 mmol) and
pyridine (0.24 mL, 3 mmol) in dry CH2Cl2 (5 mL), acetyl chloride
(0.21 mL, 3 mmol) was added at 0 °C. The reaction mixture was
stirred for 2 h at room temperature, diluted with CH2Cl2 (5 mL),
washed with water (4 mL), dried over Na2SO4, and concentrated in
vacuo. The crude residue was purified by column chromatography
(20% petroleum ether in ethyl acetate) to give 3h as a yellow solid;
2H), 7.11 (s, 1H), 7.18 (s, 2H), 7.69 (s, 1H), 7.83 (s, 1H), 7.88 (bs,
1H). MS (ESI): [M]+ = 541.3. Anal. (C21H20INO6S) C, H, N.
General Procedure H for the Synthesis of Compounds 3n
and 3o. To an ice-cooled solution of aminobenzo[b]thiophene 3a or
3c (1 mmol) in dry DMF (5 mL) were added EDCI (383 mg, 2
mmol) and HOBt (270 mg, 2 mmol), followed by α-bromoacrylic acid
(2 mmol, 306 mg). The reaction solution was stirred at ambient
temperature for 18 h and evaporated under reduced pressure. The
crude material was dissolved with a mixture of CH2Cl2 (15 mL) and
water (5 mL), and the organic phase was washed with brine (5 mL),
dried over Na2SO4, and evaporated to dryness in vacuo. The resulting
crude residue was purified by column chromatography on silica gel.
2-Bromo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]thien-5-yl]-
acrylamide (3n). Using general procedure H, the crude residue,
purified by flash chromatography by eluting with a mixture of EtOAc−
petroleum ether 1:1 (v:v), furnished 3n as a brown solid; yield 71%;
1
yield 69%; mp 141−143 °C. H NMR (CDCl3) δ: 2.17 (s, 3H), 3.92
(s, 6H), 3.96 (s, 3H), 4.01 (s, 3H), 7.12 (bs, 1H), 7.18 (s, 2H), 7.27 (s,
1H), 7.69 (s, 1H), 7.84 (s, 1H). MS (ESI): [M]+ = 416.5. Anal.
(C21H21NO6S) C, H, N.
2-Chloro-N-[7-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-
benzo[b]thien-5-yl]acetamide (3k). To a solution of 3c (373 mg, 1
mmol) and pyridine (0.24 mL, 3 mmol) in dry CH2Cl2 (5 mL),
chloroacetyl chloride (0.24 mL, 3 mmol) was added at 0 °C. The
reaction mixture was stirred for 1 h at room temperature, diluted with
CH2Cl2 (10 mL), washed with water (5 mL), dried over Na2SO4, and
concentrated in vacuo. The crude residue was purified by column
chromatography (30% ethyl acetate in petroleum ether) to furnish 3k
as a yellow solid; yield 96%; mp 81−83 °C. 1H NMR (CDCl3) δ: 3.92
(s, 6H), 3.96 (s, 3H), 4.04 (s, 3H), 4.24 (s, 2H), 7.10 (d, J = 1.6 Hz,
1H), 7.19 (s, 2H), 7.84 (d, J = 1.6 Hz, 1H), 7.86 (s, 1H), 8.32 (bs,
1H). MS (ESI): [M + 1]+ = 450.4. Anal. (C21H20ClNO6S) C, H, N.
General Procedure F for the Preparation of Compounds 3i
and 3l. To a solution of aminobenzo[b]thiophene 3a or 3c (1 mmol)
and pyridine (0.24 mL, 3 mmol) in dry CH2Cl2 (5 mL), bromoacetyl
chloride (0.25 mL, 3 mmol) was added at 0 °C. After 3 h at the same
temperature, the reaction mixture was diluted with CH2Cl2 (5 mL),
washed with water (5 mL), and dried (Na2SO4). CH2Cl2 was
concentrated under reduced pressure and the crude residue purified by
flash column chromatography.
1
mp 150−151 °C. H NMR (CDCl3) δ: 3.91 (s, 6H), 3.94 (s, 3H),
6.16 (d, J = 1.6 Hz, 1H), 7.12 (d, J = 1.6 Hz, 1H), 7.14 (s, 1H), 7.18
(s, 2H), 7.42 (dd, J = 8.8 and 1.8 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H),
8.42 (d, J = 1.8 Hz, 1H), 8.54 (bs, 1H). MS (ESI): [M]+ = 476.4, [M +
2]+ = 478.4. Anal. (C21H18BrNO5S) C, H, N.
2-Bromo-N-[7-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]-
thien-5-yl]acrylamide (3o). Using general procedure H, the crude
residue, purified by flash chromatography by eluting with a solution of
EtOAc−petroleum ether 1:1 (v:v), furnished 3o as a yellow solid; yield
1
62%; mp 165−167 °C. H NMR (CDCl3) δ: 3.92 (s, 6H), 3.95 (s,
3H), 4.03 (s, 3H), 6.17 (d, J = 1.6 Hz, 1H), 7.15 (d, J = 1.6 Hz, 1H),
7.16 (s, 1H), 7.18 (s, 2H), 7.73 (s, 1H), 7.85 (s, 1H), 8.49 (bs, 1H).
MS (ESI): [M]+ = 506.4, [M + 2]+ = 508.4. Anal. (C22H20BrNO6S) C,
H, N.
Biology. Antiproliferative Assays. Murine leukemia (L1210),
murine mammary carcinoma (FM3A), human T-lymphoblastoid
(Molt/4 and CEM), and human cervix carcinoma (HeLa) cells were
suspended at (3−5) × 105 cells/mL of culture medium, and 100 μL of
a cell suspension was added to 100 μL of an appropriate dilution of the
test compounds in wells of 96-well microtiter plates. After incubation
at 37 °C for 48 h, cell number was determined using a Coulter counter
as previously described.41 The IC50 was defined as the concentration of
compound that inhibited cell proliferation by 50%.
PBL from healthy donors were obtained by separation on
Lymphoprep (Fresenius KABI Norge AS) gradient. After extensive
washing, cells were resuspended (1.0 × cells/mL) in RPMI-1640 with
10% fetal bovine serum and incubated overnight in a 96-well tissue
culture microtiter plate. For cytotoxicity evaluations of proliferating
PBL cultures, nonadherent cells were resuspended at 5 × 105 cells/mL
in growth medium containing 2.5 μg/mL PHA (Irvine Scientific).
Varying amounts of compounds being evaluated were added to the
cells, and the MTT assay42 was used to assess viability after a 72 h
treatment.
2-Bromo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]thien-5-yl]-
acetamide (3i). Using general procedure F, the crude residue purified
by flash chromatography (60% EtOAc in petroleum ether) furnished
3i as a brown solid; yield 82%; mp 164−166 °C. 1H NMR (CDCl3) δ:
3.92 (s, 6H), 3.96 (s, 3H), 4.07 (s, 2H), 7.18 (s, 2H), 7.42 (dd, J = 8.6
and 2.2 Hz, 1H), 7.83 (s, 1H), 7.88 (t, J = 4.4 Hz, 1H), 8.28 (bs, 1H),
8.34 (d, J = 2.2 Hz, 1H). MS (ESI): [M]+ = 464.4, [M + 2]+ = 466.4.
Anal. (C20H18BrNO5S) C, H, N.
2-Bromo-N-[7-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]-
thien-5-yl]acetamide (3l). Using general procedure F, the crude
residue purified by flash chromatography (50% EtOAc in petroleum
1
ether) furnished 3l as a brown solid; yield 81%; mp 183−185 °C. H
NMR (CDCl3) δ: 3.92 (s, 6H), 3.95 (s, 3H), 4.03 (s, 3H), 4.07 (s,
2H), 7.10 (d, J = 1.6 Hz, 1H), 7.18 (s, 2H), 7.74 (d, J = 1.6 Hz, 1H),
7.85 (s, 1H), 8.26 (bs, 1H). MS (ESI): [M]+ = 494.4, [M + 2]+ =
496.4. Anal. (C21H20BrNO6S) C, H, N.
General Procedure G for the Preparation of Compounds 3j
and 3m. A mixture of bromoacetamido derivatives 3i or 3l (1 mmol)
and NaI (1.5 g., 10 mmol) in N,N-dimethylacetamide (5 mL) was
stirred at ambient temperature for 18 h. DMA was evaporated under
reduced pressure, followed by addition of CH2Cl2 (15 mL) and a
solution of Na2S2O3 (10%, 5 mL). The organic layer was washed with
water (5 mL) and brine (5 mL) and dried over Na2SO4. After
evaporation of the solvent, the residue was purified by chromatography
on silica gel.
2-Iodo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]thien-5-yl]-
acetamide (3j). Using general procedure G, the residue purified by
flash chromatography (50% ethyl acetate in petroleum ether) yielded
3j as a brown solid; 86% yield; mp 172−174 °C. 1H NMR (CDCl3) δ:
3.89 (s, 6H), 3.92 (s, 3H), 3.96 (s, 2H), 7.18 (s, 2H), 7.40 (dd, J = 8.6
and 2.0 Hz, 1H), 7.87 (t, J = 4.4 Hz, 1H), 7.88 (s, 1H), 8.29 (bs, 1H),
8.34 (d, J = 2.0 Hz, 1H). MS (ESI): [M]+ = 511.3. Anal.
(C20H18INO5S) C, H, N.
HUVECs were obtained from human umbilical veins.43 The
adherent cell population was grown in M200 medium containing
Low Serum Growth Supplement (contains fetal bovine serum (FBS),
bFGF, heparin, hEGF, gentamycin, hydrocortisone and amphotericin
B), which was purchased from Life Technologies, Monza, Italy. When
the cells grew to confluence, they were detached from the plate with a
solution containing EDTA and trypsin. Only HUVECs from the first
to the sixth passage were used in viability experiments. In these
experiments, varying concentrations of the compounds of interest were
added, with ability again determined after a 72 h treatment by MTT
assay.42
Tubulin Polymerization Assay. Bovine brain tubulin (10 μM) in
0.8 M glutamate was preincubated with varying concentrations of drug
for 15 min at 30 °C, and then the samples were cooled on ice. After
addition of 0.4 mM GTP, the reaction mixtures were pipetted into 0
°C cuvettes in temperature controlled recording spectrophotometers.
The effect of each agent on tubulin polymerization was followed
turbidimetrically at 350 nm for 20 min at 30 °C. The IC50 was defined
as the compound concentration that inhibited tubulin polymerization
by 50%. Complete experimental details were previously described.23
2-Iodo-N-[7-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]-
thien-5-yl]acetamide (3m). Using general procedure G, the residue
purified by flash chromatography (50% ethyl acetate in petroleum
1
ether 1:1) gave 3m as a brown solid; yield 84%; mp 200−202 °C. H
NMR (CDCl3) δ: 3.89 (s, 6H), 3.91 (s, 3H), 3.95 (s, 3H), 4.01 (s,
9306
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[3H]Colchicine Binding. Each reaction mixture contained 1.0 μM
tubulin, 5.0 μM [3H]colchicine, and 1.0 or 5.0 μM tested compound.
The mixtures were incubated for 10 min at 37 °C. Full experimental
details were described previously.25
intraperitoneally at 0.01 mL/g of weight of the mouse or a total
dose of 50 mg/kg. Administration of vehicle or compound was once
every 3 days for a total of four doses, with the first dose administered
on day 0, the last on day 9. On day 18, all mice were euthanized. The
formula V = (L × W2)/W was used to calculate tumor volume, with V
being volume, L being length, and W being width of the tumor
nodules. L and W were measured using a vernier calliper. Approval of
this study was obtained from the Institutional Animal Ethical
Committee of the Second Affiliate Hospital, School of Medicine,
Zhejiang University, PRC.
Molecular Modeling. Molecular docking simulations were carried
out on a MacPro dual 2.66 GHz Xeon running Ubuntu 12.04, using
the tubulin structure reported by A. Dorleans et al. (PDB code:
3HKC).44 Molecular Operating Environment (MOE) was used to
prepare the protein structure by adding the hydrogen atoms using the
Protonate3D function.45 MOE was also used to build the ligand
structures, which were minimized using the MMFF94x force field until
a RMSD gradient of 0.05 kcal mol−1 Å−1 was reached. The software
package PLANTS was then used to perform the docking simulations.46
Flow Cytometric Analysis of Cell Cycle Distribution. HeLa cells (5
× 105), treated with varying amounts of the desired agents, were
grown for 24 or 48 h. At these times, the cells were harvested by
centrifugation and fixed with 70% (v/v) ethanol that had been chilled
to 0 °C. The cells were suspended in a solution that contained RNase
and 0.1% (v/v) Triton X-100. Their DNA was then stained with PI.
Each sample was processed on a Beckman-Coulter Cytomic FC500
flow cytometer. DNA content was analyzed in a histogram format
using MultiCycle for Windows from Phoenix Flow Systems.
Annexin-V Assay. The appearance of phosphatidylserine on the
surface of apoptotic cells was measured with annexin-V conjugated to
fluorescein isothiocyanate (FITC), and DNA in the cells was measured
with PI. The flow cytometer described above was also used in these
experiments. Staining of the cells prior to analysis was performed as
instructed by Roche Diagnostics, the manufacturer of Annexin-V
Fluos. The excitation wavelength was 488 nm, and emission at 525 nm
was used to quantitate the FTIC and at 585 nm to quantitate the PI.
Assessment of Mitochondrial Changes. The mitochondrial
membrane potential was measured with the fluorescent probe JC-1
(Molecular Probes), as described.42 The generation of ROS was
measured by flow cytometry using H2DCFDA (Molecular Probes), as
previously described.42
ASSOCIATED CONTENT
* Supporting Information
■
S
Proposed binding mode of compound 3c with colchicine in the
β-tubulin binding site. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*For R.R.: phone, 39-(0)532-455303; fax, 39-(0)532-455953;
E-mail, rmr@unife.it.
*For P.G.B.: phone, 39-(0)532-455293; fax, 39-(0)532-455953;
E-mail, pgb@unife.it.
*For G.V.: phone, 39-(0)49-8211451; fax, 39-(0)49-8211462;
E-mail, giampietro.viola1@unipd.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Jan Balzarini is funded by GOA (Krediet no. 10/14) of the KU
Leuven. We gratefully acknowledge Alberto Casolari and
Lizette van Berckelaer for their excellent technical assistance.
Western Blot Analysis. Aliquots of HeLa cell cultures, both control
and containing the desired compounds at the indicated concentration,
were removed at time points as indicated. The cells were collected by
centrifugation and washed twice with phosphate-buffered saline chilled
to 0 °C. The cells were then suspended in a lysis buffer at 0 °C for 30
min. The resulting suspensions were clarified by centrifugation
(15000g, 4 °C, 10 min) and the protein concentrations of the
supernatants determined with the BCA protein kit (Pierce, Italy).
Protein aliquots of 20 μg were subjected to sodium dodecyl sulfate−
polyacrylamide gel electrophoresis using 7.5−15% gradient poly-
acrylamide gels. Proteins were then electroblotted to PVDF Hybond-p
membranes from GE Healthcare. The membranes were then treated
with ECL advance blocking agent from GE Healthcare, using a rotary
shaker at 4 °C, as instructed by the manufacturer. Membranes were
next incubated for 2 h at room temperature with a variety of primary
antibodies from Cell Signaling, Alexis, Upstate, and Sigma-Aldrich, as
indicated in the individual experiments. Finally, membranes were
incubated with peroxidase-labeled secondary antibodies for 60 min. All
membranes were visualized using ECL Advance (GE Healthcare) and
exposed to Hyperfilm MP (GE Healthcare). To ensure equal protein
loading, each membrane was stripped and reprobed with an anti-β-
actin antibody.
Antitumor Activity in V. BALB/c-nu mice (age 4 weeks, sex female,
weight 15−18 g) were purchased from the Shanghai (China) SLAC
Laboratory Animal Co., Ltd. The mice were kept under specific
panthogen-free conditions, with free access to food and water, in the
Laboratory Animal Center of the Zhejiang University of Traditional
Chinese Medicine. HNNG/HOS cells, derived from a human
osteosarcoma, were grown into exponential phase. The cells were
suspended in FBS-free MEM (107 cells/mL), and 0.1 mL was injected
into the hypodermis of the back of each mouse. When each of the
xenografts was about 100 mm3 large, 12 mice were randomly placed in
three groups: vehicle-treated control, CA-4P-treated (compound
dissolved in isotonic saline), and 3c-treated (compound dissolved in
5% (v/v) DMSO in olive oil). Both compounds were given
ABBREVIATIONS USED
■
CA-4, combretastatin A-4; CA-4P, combretastatin A-4 diso-
dium phosphate; DABCO, 1,4-diazabicyclo[2,2,2]octane;
DMF, N,N′-dimethylformamide; DMA, N,N-dimethylaceta-
mide; EDCI, 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride; SAR, structure−activity relation-
ships; PI, propidium iodide; FITC, fluorescein isothiocyanate;
JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcar-
bocyanine; ROS, reactive oxygen species; H2DCFDA, 2,7-
dichlorodihydrofluorescein; PARP, poly-ADP-ribose polymer-
ase
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