Substituted 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b] thiophene derivatives as potent tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.05.068

The central role of microtubules in cell division and mitosis makes them a particularly important target for anticancer agents. On our early publication, we found that a series of 2-(3′,4′,5′-trimethoxybenzoyl)-3- aminobenzo[b]thiophenes exhibited strong antiproliferative activity in the submicromolar range and significantly arrested cells in the G2-M phase of the cell cycle and induced apoptosis. In order to investigate the importance of the amino group at the 3-position of the benzo[b]thiophene skeleton, the corresponding 3-unsubstituted and methyl derivatives were prepared. A novel series of inhibitors of tubulin polymerization, based on the 2-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene molecular skeleton with a methoxy substituent at the C-4, C-5, C-6 or C-7 position on the benzene ring, was evaluated for antiproliferative activity against a panel of five cancer cell lines, for inhibition of tubulin polymerization and for cell cycle effects. Replacing the methyl group at the C-3 position resulted in increased activity compared with the corresponding 3-unsubstituted counterpart. The structure-activity relationship established that the best activities were obtained with the methoxy group placed at the C-4, C-6 or C-7 position. Most of these compounds exhibited good growth inhibition activity and arrest K562 cells in the G2-M phase via microtubule depolymerization.

Full text of DOI:10.1016/j.bmc.2010.05.068

Bioorganic & Medicinal Chemistry 18 (2010) 5114–5122
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Substituted 2-(30,40,50-trimethoxybenzoyl)-benzo[b]thiophene derivatives
as potent tubulin polymerization inhibitors
a,
a
a
b
Romeo Romagnoli a, , Pier Giovanni Baraldi , Maria Dora Carrion , Olga Cruz-Lopez , Manlio Tolomeo ,
Stefania Grimaudo b, Antonietta Di Cristina b, Maria Rosaria Pipitone b, Jan Balzarini c, Andrea Brancale d,
Ernest Hamel e
*
*
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
b Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
c Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
d The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, UK
e Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, Maryland 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The central role of microtubules in cell division and mitosis makes them a particularly important target
for anticancer agents. On our early publication, we found that a series of 2-(30,40,50-trimethoxybenzoyl)-
3-aminobenzo[b]thiophenes exhibited strong antiproliferative activity in the submicromolar range and
significantly arrested cells in the G2–M phase of the cell cycle and induced apoptosis.
Received 3 April 2010
Revised 25 May 2010
Accepted 25 May 2010
Available online 1 June 2010
In order to investigate the importance of the amino group at the 3-position of the benzo[b]thiophene
skeleton, the corresponding 3-unsubstituted and methyl derivatives were prepared. A novel series of
inhibitors of tubulin polymerization, based on the 2-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene
molecular skeleton with a methoxy substituent at the C-4, C-5, C-6 or C-7 position on the benzene ring,
was evaluated for antiproliferative activity against a panel of five cancer cell lines, for inhibition of tubu-
lin polymerization and for cell cycle effects. Replacing the methyl group at the C-3 position resulted in
increased activity compared with the corresponding 3-unsubstituted counterpart. The structure–activity
relationship established that the best activities were obtained with the methoxy group placed at the C-4,
C-6 or C-7 position. Most of these compounds exhibited good growth inhibition activity and arrest K562
cells in the G2–M phase via microtubule depolymerization.
Keywords:
Antiproliferative agents
Tubulin polymerization inhibitors
Microtubules
Antitumor
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
to the colchicine site.5 The activity and structural simplicity of
CA-4 have stimulated extensive studies to examine the struc-
Besides being critical for cell architecture, the microtubule
system of eukaryotic cells is essential for cell division, since micro-
tubules are key components of the mitotic spindle.1 Microtubules
also play a crucial role in a wide number of essential cellular pro-
cesses, such as maintenance of cellular shape, regulation of motility,
cell signaling, secretion and intracellular transport.2 In recent years,
there has been an intense effort to identify and develop chemically
diverse substances, many of which are derived from natural prod-
ucts, that inhibit tubulin assembly for treatment of cancer.3
Among the microtubule depolymerizing agents, combretastatin
A-4 (CA4, 1; Chart 1) is one of the more studied compounds. CA4,
isolated from the bark of the South African tree Combretum caff-
rum,4 strongly inhibits the polymerization of tubulin by binding
ture–activity relationship (SAR) of this compound and its
analogues.6
Among the synthetic inhibitors of tubulin polymerization, there
are limited examples of small molecules based on the benzo[b]thio-
phene molecular skeleton as the core structure. Pinney and co-
workers described a series of tubulin binding agents with general
structure 2, which incorporates the 3-(30,40,50-trimethoxybenzoyl)-
6-methoxy benzo[b]thiophene ring system.7 Previous studies have
shown that the 6-methoxy substituent significantly contributes to
maximize the activity of these compounds, presumably as a mimic
of the 4-methoxy group in the B-ring of CA-4.6
We have recently reported the biological evaluation of a series
of derivatives with general structure 3, based on the 2-(30,40,50-
trimethoxybenzoyl)-3-aminobenzo[b]thiophene molecular skele-
ton. These compounds inhibited the growth of cancer cell lines
and of tubulin polymerization by binding to the colchicine site of
tubulin. As expected, these compounds caused cells to arrest in
* Corresponding authors. Tel.: +39 (0)532 455303; fax: +39 (0)532 455953 (R.R.);
tel.: +39 (0)532 291293; fax: 39 (0)532 455953 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.068

R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
5115
(4a and 4f) or a methoxy group at each of the four possible posi-
tions on the benzene ring (compounds 4b–e and 4g–j). Keeping
the C-6 methoxy group intact, compound 4k was prepared with
the aim of evaluating the effect on biological activity of one addi-
tional methoxy substituent at the C-5 position. Finally, the 6-meth-
oxy position (corresponding to the 4-methoxy group in the B-ring
of CA4) of 4j was further studied by synthesizing the correspond-
ing 6-ethoxy (4l) and 6-fluorine (4m) analogues.
MeO
MeO
OMe
O
MeO
MeO
A
B
OMe
R
OH
S
OMe
H3CO
OMe
2
Combretastatin A-4 (CA-4), 1
R=H, 2a
R=OH, 2b
2. Chemistry
The 2-(30,40,50-trimethoxybenzoyl)benzo[b]thiophene deriva-
tives 4a–m were synthesized by the four step synthesis shown in
Scheme 1. Starting from the variously substituted salicylaldehydes
5b–e and 5k or 2-hydroxyacetophenones 5f–j and 5l–m, the
reaction with N,N-dimethyl thiocarbamoyl chloride in N,N-dimeth-
ylformamide (DMF) in the presence of DABCO yielded the O-aryl-
thiocarbamates 6b–m in good yields. These latter were
submitted to Newman–Kwart rearrangement9 by heating under
microwave irradiation (MW) in the absence of solvent, to furnish
the corresponding S-arylthiocarbamates 7b–m. The subsequent
hydrolysis using a 3 M aqueous sodium hydroxide solution fur-
nished the desired 2-thiophenols 8b–m, which were used without
further purification for the next reaction. 2-Mercaptobenzaldehyde
8a was obtained by S-debenzylation of 2-benzylthiobenzaldehyde
with aluminum chloride. This latter compound was obtained by
condensation of 2-nitrobenzaldehyde 5a with the potassium salt
of phenyl methanethiol in cold aqueous DMF.10
OMe
R1
N
OMe
R4
R3
R1
OMe
OMe
OMe
OMe
R5
R6
R2
S
S
O
O
R7
4a-m
3
R1=H, R2=4-OCH3, 3a
R1=H, R2=5-OCH3, 3b
R1=H, R2=6-OCH3, 3c
R1=H, R2=7-OCH3, 3d
R1=CH3, R2=6-OCH3, 3e
R3-7=H, 4a
R4=OMe, R3, 5-7=H, 4b
R5=OMe, R3-4, 6-7=H, 4c
R6=OMe, R3-5, 7=H, 4d
R7=OMe, R3-6=H, 4e
R3=Me, R4-7=H, 4f
R3=Me, R4=OMe, R5-7=H, 4g
R3=Me, R5=OMe, R4, 6-7=H, 4h
R3=Me, R6=OMe, R4-5, 7=H, 4i
R3=Me, R7=OMe, R4-6=H, 4j
Finally, the 2-(30,40,50-trimethoxybenzoyl)benzo[b]thiophene
derivatives 4a–m were synthesized by a ‘one-step’ condensation,
followed by intramolecular cyclization, of 8a–m with 2-bromo-1-
(3,4,5-trimethoxyphenyl)ethanone using anhydrous potassium
carbonate in refluxing acetone.
R
5, 6=OMe, R3-4, 7=H, 4k
R3=Me, R6=OEt, R4-5, 7=H, 4l
R3=Me, R6=F, R4-5, 7=H, 4m
Chart 1. Inhibitors of tubulin polymerization.
the G2/M phase of the cell cycle.8 Potent activity appears to be
highly dependent upon the presence and position of a fourth meth-
oxy substituent on benzo[b]thiophene system. A fairly dramatic
difference was observed between C-4/5 and C-6/7-substituted
compounds (3a–b and 3c–d, respectively). The greatest activity oc-
curred when the methoxy group was located at the C-6 or C-7 po-
sition, the least when located at the C-4 or C-5 position (IC50
3. Biological results and discussion
Table 1 summarizes the growth inhibitory effects of 2-(30,40,50-
trimethoxy)benzo[b]thiophene derivatives 4a–m against murine
leukemia (L1210), murine mammary carcinoma (FM3A), human
T-lymphoblastoid (Molt/4 and CEM) and human cervix carcinoma
(HeLa) cells, with 3-amino-2-(30,40,50-trimethoxy)benzo[b]thio-
phene counterparts 3a–d and CA4 (1) as reference compounds.
Five compounds (4b, 4e, 4g, 4i and 4j) displayed strong growth
inhibitory activity, with IC50 values lower than 100 nM against
all 5 cell lines. In addition, 4f, 4l and 4m yielded IC50 values below
100 nM in one to three cell lines. The 3-methyl-4-methoxy deriv-
ative 4g possessed the highest potency, inhibiting the growth of
L1210, FM3A, Molt/4, CEM and HeLa cells with IC50 values of 19,
23, 18, 19 and 16 nM, respectively. These values were similar to
those obtained with CA4 against FM3A and Molt4 cells, while 4g
was 5–10-fold less active than CA-4 against L1210, CEM and HeLa
cells. Compound 4g also showed at least 100-fold greater activity
than its 4-methoxy-3-amino-2-(30,40,50-trimethoxy)benzo[b]thio-
phene counterpart 3a.
Comparing the 3-unsubstituted derivatives 4a–e with their 3-
methyl congeners 4f–j, the introduction of a methyl at the C-3 po-
sition of the benzo[b]thiophene system generally increased anti-
proliferative activity against all five cell lines. The methoxy
substitution and location on the benzene part of benzo[b]thio-
phene moiety plays an important role in affecting antiproliferative
activity. In both these series, the methoxy group located at the C-4
or C-7 position enhanced activity, while the C-5 and C-6 substitu-
ents were detrimental.
>10 lM). The slight reduction in potency of the 3-dimethylamino
derivative 3e, which did not differ significantly from that of the
3-amino counterpart 3c, allowed us to verify that an intramolecu-
lar hydrogen bond between the unsubstituted 3-amino group and
the carbonyl oxygen of the 2-trimethoxybenzoyl moiety is not re-
quired for activity. These encouraging results prompted us to study
this class of compounds in more detail. We herein describe the
synthesis and SAR of additional 2-(30,40,50-trimethoxybenzoyl)-
benzo[b]thiophene derivatives in continuation of our search for
new potent antitubulin agents.
In the present investigation, the 30,40,50-trimethoxyphenyl of the
2-benzoyl moiety was kept unchanged because it is the character-
istic structural requirement for activity in numerous inhibitors of
tubulin polymerization, such as colchicine, CA4 and podophyllo-
toxin.6 In the current studies we examined the importance of the
3-position of the benzo[b]thiophene skeleton by studying the ef-
fects of replacing the amino or dimethylamino substituents of
compounds with general structure 3 with a hydrogen or a methyl
group, to furnish derivatives with general structure 4. The SAR was
elucidated with electron-donating methoxy substitution at the C-4,
C-5, C-6 or C-7 position on the benzo[b]thiophene ring.
In the series of compounds 4a–j, the 3-amino group of the
benzo[b]thiophene system was replaced by hydrogen (4a–e) or
methyl (4f–j). These molecules possessed either no substituent
In the series of 3-unsubstituted derivatives 4b–e, there was an
evident difference in activity between the C-4/7 and the C-5/6

5116
R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
R4
O
R4
O
R4
O
R5
R6
R5
R6
R5
R6
b
R3
OH
a
R3
R3
OC(S)N(CH3)2
SC(O)N(CH3)2
R7
R7
R7
5b-m
6b-m
7b-m
c
R4
O
OMe
R4
R3
d
R5
OMe
OMe
R5
R6
R3
SH
R6
S
R7
O
R7
8a-m
4a-m
R3-7=H, 4-5a, 8a
e,f
R4=OMe, R3, 5-7=H, 4-8b
R5=OMe, R3-4, 6-7=H, 4-8c
R6=OMe, R3-5, 7=H, 4-8d
R7=OMe, R3-6=H, 4-8e
R3=Me, R4-7=H, 4-8f
R4
O
R5
R6
R3
NO2
R3=Me, R4=OMe, R5-7=H, 4-8g
R3=Me, R5=OMe, R4, 6-7=H, 4-8h
R3=Me, R6=OMe, R4-5, 7=H, 4-8
R3=Me, R7=OMe, R4-6=H, 4-8j
R5, 6=OMe, R3-4, 7=H, 4-8k
R7
5a
R3=Me, R6=OEt, R4-5, 7=H, 4-8l
R3=Me, R6=F, R4-5, 7=H, 4-8m
Scheme 1. Reagents and conditions. (a) ClC(S)N(CH3)2, Dabco, DMF, 50 °C, 5 h; (b) no solvent, heating of fused substrates, microwaves; (c) NaOH, EtOH/H2O, 65 °C; (d) (3,4,5-
trimethoxyphenyl)-2-bromo-ethanone, K2CO3, (CH3)2CO, rx; (e) C6H5CH2SH, KOH, DMF; (f) AlCl3, C6H6.
A different effect was observed in the series of 3-methyl substi-
tuted derivatives 4g–j, in which the greatest activity occurred
when the methoxy group was located at the C-4, C-6 or C-7 posi-
tion, the least when it was located at the C-5 position. The C-3
methyl, C-6 methoxy derivative (4i) was selected as a scaffold for
further modifications, by replacing the methoxy group with either
an ethoxy group (4l) or an electron-withdrawing fluorine atom
(4m). Both these changes resulted in a decrease in antiproliferative
activity against all cell lines relative to 4i, but overall compounds 4l
and 4m had very similar activity despite the significant difference
in the chemical properties of the two C-6 substituents.
The marked improvement in activity of 4g relative to 4-meth-
oxy-3-amino-2-(30,40,50-trimethoxybenzoyl)benzo[b]thiophene
derivative 3a derives from the substitution of an amino with a
methyl group at the C-3 position of the 2-(30,40,50-trimeth-
oxy)benzo[b]thiophene core.
Table 1
In vitro inhibitory effects of compounds 3a–d, 4a–m and CA4 against the proliferation
of murine leukemia (L1210), murine mammary carcinoma (FM3A), human
T-lymphocyte (Molt/4 and CEM) and human cervix carcinoma (HeLa) cells
a
Compound
IC50 (nM)
Molt4/C8
L1210
FM3A/0
CEM/0
Hela
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
>10,000
>10,000
39 16
33 29
490 40
87
580 15
1200 200 1500
84 85
140 20
19
600 10
30
25
1100 60
100
>10,000
>10,000
46 12
27 13
860 16
>10,000
>10,000
10
>10,000
>10,000
7.7 2.9 n.d.
8.9 2.0 n.d.
n.d.
n.d.
7
8.5 1.4
500 31
510 17
83
790 18 2100 300 3000 100
1100 100
75
900 23
590 32
24
99
16
460 10
18
21
220 16
140 24
110 13
1.9 1.6
5
99
0
75
6
9
5
0
4
550 14
1700 40
5
27
93
18
430 10
18
28
440 14
56
94
2
5
0
46
94
19
500 50
19
18
350 15
56
110 10
1.9 1.6
7
5
1
1
1
1
190 30
23
550 10
2
7
Comparing the 4-methoxy derivatives 3a, 4b and 4g, the order
of activity for the substituent at the 3-position of the benzo[b]thi-
ophene moiety was methyl (4g) > hydrogen (4b) ꢀ amino (3a).
With these three compounds, IC50 values were 16–23 nM for 4g
and 75–99 nM for 4b, while for 3a all IC50’s were greater than
2
4
45
35
2
4
0
2
1
4
2
1
980 53
130 10
0
2
8
3
4m
CA4
130 20
2.8 1.1
140
9
42 6.0
16 1.4
10 lM. Comparing the C-6 and C-7 methoxy derivatives 3cd and
n.d. = not determined.
4ij, replacement of the 3-amino group with a methyl resulted in
minimal loss of activity. Thus, with a C-4 methoxy group, the C-3
substituent had a much greater impact on antiproliferative activity
than was the case with either a C-6 or C-7 methoxy substituent.
With the latter compounds, in which the benzo[b]thiophene meth-
oxy substituent is more distant from the trimethoxybenzene ring,
the ortho relationship between the 30,40,50-trimethoxybenzoyl moi-
ety and the C-3 amino group does not play an important role in
activity. Thus the 3-amino function can be removed and replaced
with a hydrogen or a methyl without significant change in activity.
Because 3c and 3d inhibited tubulin assembly,8 we next inves-
tigated whether the antiproliferative activities of the new
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.
methoxy derivatives. Our findings showed that the methoxy group
located at the C-4 or at the C-7 position (4b and 4e, respectively)
resulted, on average, in antiproliferative activity, 19-fold greater
than with the methoxy group at the C-5 or C-6 position (4b and
4e vs 4c and 4d). The C-5/6 dimethoxy derivative 4k had activity
on average about twice that of either 4c or 4d, and 4k was partic-
ularly more active than either monosubstituted compound in the
CEM/0 and HeLa cells.

R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
5117
compounds were related to an interaction with the microtubule
system. Compounds 4b, 4e–g, 4i–j and 4lm were evaluated for
employed by our research group to determine the alteration of cell
cycle parameters following exposure to antitumour compounds.13
Cells were cultured for 24 h in the presence of each compound at
the concentration able to inhibit cell growth 100% after 24 h. Anal-
ysis of sub-G0–G1 (apoptotic peak, A), G0–G1, S, and G2–M peaks
revealed that the compounds caused somewhat different effects
on cell cycle distribution (Table 3). After a 24 h treatment, all
tested compounds caused a massive accumulation of cells in the
G2–M phase (51–82%) relative to the untreated control (15.1%),
with a simultaneous decrease of cells in the S and G0–G1. With
compound 4e, the accumulation of cells in G2–M phase was
accompanied by the appearance of a significant sub-G0–G1 peak
(A = 17.7%) due to apoptosis.
A set of molecular docking calculations was also performed to
investigate the possible binding mode of this series of compounds.
All the compounds reported in Table 1 were docked in the colchi-
cine binding site of tubulin. The majority of the compounds
showed a binding pose similar to the one observed with the co-
crystallized DAMA-colchicine, with the trimethoxyphenyl ring
placed in proximity of Cys241 (residue numbering derived from
the crystal structure used). In addition, the benzo[b]thiophene ring
is placed deep in the pocket, interacting with tubulin through a
series of hydrophobic contacts (Fig. 1). In addition, the benzo[b]thi-
ophene ring is placed deep in the pocket, interacting with tubulin
through a series of hydrophobic contacts (Fig. 1). Compounds 3a–b,
4c–d, 4h and 4l either did not dock with the trimethoxyphenyl in
contact with Cys241 or they docked outside the binding pocket,
hence their results were not further analyzed. For compounds
their in vitro inhibition of the polymerization of 10 lM tubulin
and for their inhibitory effects on the binding of [3H]colchicine to
tubulin (in the latter assay, tubulin was examined at a concentra-
tion of 1 lM, while colchicine was at 5 l
M).11,12 For comparison,
derivatives 3cd and CA4 were examined in contemporaneous
experiments as a reference compounds (Table 2). All tested com-
pounds strongly inhibited tubulin assembly and derivatives 4e,
4f, 4g and 4m, with IC50 values of 0.58–0.71
ulin activity about 1.5–2 times greater than that of CA4 (IC50
1.0 M), while 4b, 4i, 4j and 4l had IC50 values of 0.92–1.2 M,
lM, exhibited antitub-
,
l
l
essentially equivalent to that of CA4 and 3c. Comparing the 7-
methoxy benzo[b]thiophene derivatives 3d, 4e and 4j, the 3-amino
compound 3d was less effective than 3-unsubstituted and 3-
methyl analogues 4e and 4j, respectively, as an inhibitor of tubulin
polymerization.
Compounds 4f and 4m were almost 1.5-fold more active than
CA4 as inhibitors of tubulin assembly, although both compounds
were less active in their effects on cell growth.
In the colchicine studies, all compounds were active inhibitors
of the binding reaction. Compound 4g, the agent with the greatest
antiproliferative activity, was the most active inhibitor of the bind-
ing of [3H]colchicine to tubulin, since 85% and 93% inhibition oc-
curred with this agent at 1 and 5 lM, respectively. Compound 4g
was as active as CA4, which in these experiments inhibited colchi-
cine binding by 88% and 99%. Although 4e was almost twice as ac-
tive as 4i as an inhibitor of tubulin polymerization, these two
compounds showed similar activity as inhibitors of colchicine
binding.
Table 3
While this group of compounds were all highly potent in the
biological assays (inhibition of cell growth, tubulin assembly and
colchicine binding), correlations between the three assay types
were imperfect. Thus, while compound 4e was the best inhibitor
of tubulin assembly, its effect on colchicine binding was matched
by compounds 4i and 4j, both of which were almost half as active
as assembly inhibitors. Compound 4e was, in general, somewhat
less effective as an inhibitor of cell growth than the other two com-
pounds 4i and 4j.
Because molecules exhibiting effects on tubulin assembly
should cause alteration of cell cycle parameters, with preferential
G2–M blockade, flow cytometry analysis was performed to deter-
mine the effect of compounds 4b, 4e–g, 4i–j and 4l–m on K562
(human chronic myelogenous leukemia) cells, which are usually
Effects of compounds 4b, 4e–g, 4i–j and 4l–m on K562 cell cycle progression
a
b
Compound
IC50 (nM)
IC100 (nM)
Cell cycle distribution (%)
Sub-G1c
G0G1
S
G2/M
Control
4b
4e
4f
4g
4i
4j
4l
4m
3.76
8.28
17.7
6.00
7.92
7.80
9.46
11.2
10.5
34.6
2.13
14.8
3.72
3.74
11.7
4.19
10.8
6.16
50.4
18.9
26.8
13.8
16.9
22.1
15.2
38.1
17.1
15.1
78.9
58.3
82.5
79.4
66.2
80.6
51.2
76.7
220 50
800
150
500
85
25
75
95
2
180 20
38
6
42
47
7
0.1
5
8
230
400
220 40
a
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.
b
Compound concentration required to inhibit tumor cell proliferation by 100%.
Percentage of the cell population with hypodiploid DNA content peak (apop-
c
Table 2
totic cells).
Inhibition of tubulin polymerization and colchicine binding by
compounds 3c–d, 4b, 4e–g, 4i–j, 4l–m and CA4
Compound Tubulin assemblya
Colchicine
IC50 SD (
l
M)
bindingb
%
SD
1
l
M
5
lM
drug
drug
3c
1.3 0.1
2.1 0.3
60 0.1 n.d.
3d
4b
4e
4f
4g
4i
4j
4l
4m
CA4 (1)
39
49
1
1
n.d.
81
1.2 0.07
0.58 0.02
0.63 0.1
0.67 0.1
0.92 0.05
0.95 0.06
0.93 0.01
0.71 0.1
1.0 0.09
4
60 0.3 88 0.5
51
85
66
54
n.d.
48
88
2
7
4
3
88
93
92 0.3
87
75
80
99
4
5
1
3
1
2
2
1
n.d.: not determined.
a
Inhibition of tubulin polymerization. Tubulin was at 10
l
l
M.
M,
b
Inhibition of [3H]colchicine binding. Tubulin was at 1
colchicine was at 5 lM.
Figure 1. Presumptive binding mode of 4g. DAMA-colchicine in blue.

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R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
3c–d, the benzo[b]thiophene was placed with the amino group ori-
ented on the same side of the carbonyl linker, allowing a possible
intramolecular hydrogen bond (see Fig. 1 in Supplementary data).
In the case of methyl substituted analogues (4f–j), it is often the
sulfur atom that is oriented on the same side of the carbonyl group,
allowing, in the case of compound 4g, the positioning of the meth-
oxy substituent in position 4 on the heterocyclic ring to overlap
well with the corresponding moiety on ring C of the colchicine ana-
logue (Fig. 1). In particular, the methoxy group establishes a non-
polar interaction deep in the biding pocket, in contact with
Val181, which appear to be important for biological activity. Com-
pound 4j showed a similar orientation of the heterocyclic ring to
the one observed for 3c–d and also in this case, the methoxy sub-
stituent is in contact with Val181. It should also be noted that a
similar binding pose was observed for the series of benzo[b]furan
derivatives we have recently reported.14
acetophenone (5j), 2-hydroxy-4-ethoxyacetophenone (5l), 2-hy-
droxy-4-fluoroacetophenone (5m) are commercially available
and were used as received. For the preparation of 2-hydroxy-4,5-
dimethoxybenzaldehyde (5k) see: Foyer, R.; Rene, L.; Cavier, R.;
Lemoine, J. Eur. J. Med. Chem. 1977, 12, 455.
1H NMR spectra were recorded on a Bruker AC 200 spectrome-
ter. Chemical shifts (d) are given in ppm upfield from tetramethyl-
silane as internal standard, and the spectra were recorded in
appropriate deuterated solvents, as indicated. Melting points
(mp) were determined on a Buchi-Tottoli apparatus and are uncor-
rected. All products reported showed 1H NMR spectra in agreement
with the assigned structures. Elemental analyses were conducted
by the Microanalytical Laboratory of the Chemistry Department
of the University of Ferrara. Mass spectra were obtained by electro-
spray ionization (ESI) in positive mode using an ESI Micromass
ZMD 2000 mass spectrometer. All reactions were carried out under
an inert atmosphere of dry nitrogen, unless otherwise described.
Standard syringe techniques were applied for transferring dry sol-
vents. Reaction courses and product mixtures were routinely mon-
itored by TLC on silica gel (precoated F254 Merck plates) and
visualized with aqueous KMnO4. Flash chromatography was per-
formed using 230–400 mesh silica gel and the indicated solvent
system. Organic solutions were dried over anhydrous Na2SO4. Cal-
cium chloride was used in the distillation of DMF, and the distilled
solvent was stored over molecular sieves (3 Å). The MW reaction
was performed in a focused MW oven (Discover, CEM, 2450 MHz).
4. Conclusions
In continuing experiments to optimize the 2-(30,40,50-trim-
ethoxybenzoyl)-3-aminobenzo[b]thiophene core structure, we
developed a new series of compounds with general structure 4,
in which the 3-amino moiety was replaced with a hydrogen or
methyl group. The results suggest that the hydrophobic methyl
group is a good surrogate for the 3-amino moiety and resulted in
an improvement in antiproliferative activity. Maximal activity
was dependent on the location of the methoxy substituent on
the benzene part of benzo[b]thiophene moiety. The SAR study
showed that a single methoxy group located at the C-4 or C-7 posi-
tion resulted in the best activity, while activity was decreased
when the methoxy group was located at the C-5 position.
In the series of derivatives without a substituent at C-3, the C-5
and C-6 methoxy derivatives were significantly less active than C-4
and C-7 positional isomers. In the 3-methyl series, compounds
with the methoxy group at the C-4, C-6 and C-7 position exhibited
about 20-fold greater activity than the C-5 methoxy isomer.
The most active compound in this series was the 2-(30,40,50-
trimethoxybenzoyl)-3-methyl-4-methoxybenzo[b]thiophene deriv-
ative 4g, with IC50 antiproliferative values of 16–23 nM. Compound
4g also showed at least 100-fold greater activity than its 4-meth-
oxy-3-amino-2-(30,40,50-trimethoxy)benzo[b]thiophene counter-
5.2. General procedure (A) for the synthesis of compounds 6b–m
To a solution of phenol 5b–m (5 mmol) in 8 mL of DMF contain-
ing DABCO (1.68 g, 15 mmol) was added N,N-dimethylthiocarbam-
yl chloride (1.85 g, 15 mmol) in one portion. The temperature rose
rapidly to 50 °C, and the mixture was held at this temperature for
5 h. After this time, the mixture was poured into water (30 mL),
and the product was extracted with dichloromethane (3 ꢁ
30 mL). The combined organic phases were washed with 5% HCl
(20 mL), 0.1 M NaOH (20 mL) and brine (20 mL), dried over Na2SO4
and concentrated under reduced pressure. The residue was puri-
fied by flash column chromatography on silica gel.
5.2.1. O-2-Formyl-3-methoxyphenyl N,N-dimethylcarb-
amothioate (6b)
part 3a. Compound 4g was
polymerization, with an IC50 of 0.67
a
potent inhibitor of tubulin
M, and it arrested K562 can-
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 6b as a white solid, yield: 79%, mp 119–121 °C.
1H NMR (CDCl3) d: 3.43 (s, 3H), 3.48 (s, 3H), 3.87 (s, 3H), 7.19 (d,
J = 8.0 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 10.1
(s, 1H).
l
cer cells in the G2–M phase of the cell cycle. Several additional
compounds (4e, 4f, 4g and 4m) were more potent than CA4 as
inhibitors of tubulin assembly. Nevertheless, except for 4g, all
members of the series were less active than CA4 as inhibitors of
the binding of [3H]colchicine to tubulin. As a consequence of their
interactions with tubulin, all compounds examined caused cell cy-
cle arrest in the G2–M phase, with an increase from the control va-
lue of 15% to 51–82% of cells in G2–M. This is well supported by the
molecular modeling studies, where it was observed that the meth-
oxy group in these positions could occupy an hydrophobic region
deep in the binding pocket.
5.2.2. O-2-Formyl-4-methoxyphenyl N,N-dimethylcarb-
amothioate (6c)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 6c as a white solid, yield: 92%, mp 87–89 °C.
1H NMR (CDCl3) d: 3.41 (s, 3H), 3.47 (s, 3H), 3.86 (s, 3H), 7.03 (d,
J = 8.8 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 10.0 (s, 1H).
5.2.3. O-2-Formyl-5-methoxyphenyl N,N-dimethylcarb-
amothioate (6d)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 6d as a yellow solid, yield: 72%, mp 91–93 °C.
1H NMR (CDCl3) d: 3.41 (s, 3H), 3.47 (s, 3H), 3.88 (s, 3H), 6.63 (s,
1H), 6.91 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 9.91 (s, 1H).
5. Experimental
5.1. Chemistry
5.1.1. Materials and methods
2-Nitrobenzaldehyde (5a), 2-hydroxy-6-methoxybenzaldehyde
(5b), 2-hydroxy-5-methoxybenzaldehyde (5c), 2-hydroxy-4-me-
thoxybenzaldehyde (5d), 2-hydroxy-3z-methoxybenzaldehyde
(5e), 1-(2-hydroxyphenyl)ethanone (5f), 2-hydroxy-6-methoxy-
acetophenone (5g), 2-hydroxy-5-methoxyacetophenone (5h),
2-hydroxy-4-methoxyacetophenone (5i), 2-hydroxy-3-methoxy-
5.2.4. O-2-Formyl-6-methoxyphenyl N,N-dimethylcarb-
amothioate (6e)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 6e as a white solid, yield: 72%, mp 142–144 °C.

R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
5119
1H NMR (CDCl3) d: 3.40 (s, 3H), 3.46 (s, 3H), 3.94 (s, 3H), 6.70 (d,
J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 8.2 Hz, 1H), 10.3
(s, 1H).
conditions described for each compound. The progress of the reac-
tion was followed by thin-layer chromatography. The reaction
mixture was then cooled and purified by flash column chromatog-
raphy on silica gel.
5.2.5. O-2-Acetylphenyl N,N-dimethylcarbamothioate (6f)
The residue was chromatographed with EtOAc–petroleum ether
1:9 as eluent to give 6f as a white solid, yield: 95%, mp 77–79 °C. 1H
NMR (CDCl3) d: 2.66 (s, 3H), 3.36 (s, 3H), 3.45 (s, 3H), 7.07 (d,
J = 8.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.80
(d, J = 8.2 Hz, 1H).
5.3.1. S-2-Formyl-3-methoxyphenyl N,N-dimethylcarb-
amothioate (7b)
MW irradiation conditions: power (P): 200 W, temperature (T):
210 °C, ramp time (RT): 2 min; time: 2 min. The residue was chro-
matographed with EtOAc–petroleum ether 3:7 as eluent to give 7b
as a yellow oil, yield: 60 %. 1H NMR (CDCl3) d: 3.01 (s, 3H), 3.13 (s,
3H), 3.90 (s, 3H), 7.02 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H),
7.47 (t, J = 8.4 Hz, 1H), 10.5 (s, 1H).
5.2.6. O-2-Acetyl-3-methoxyphenyl N,N-dimethylcarb-
amothioate (6g)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 6g as a white solid, yield: 79%, mp 89–92 °C.
1H NMR (CDCl3) d: 2.53 (s, 3H), 3.29 (s, 3H), 3.39 (s, 3H), 3.86 (s,
3H), 6.68 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.35 (d,
J = 8.4 Hz, 1H).
5.3.2. S-2-Formyl-4-methoxyphenyl N,N-dimethylcarb-
amothioate (7c)
MW conditions: P: 280 W, T: 240 °C, RT: 2 min; time: 1 min. The
residue was chromatographed with EtOAc–petroleum ether 2:8 as
eluent to give 7c as a yellow oil, yield: 48%. 1H NMR (CDCl3) d: 3.03
(s, 3H), 3.16 (s, 3H), 3.87 (s, 3H), 7.12 (d, J = 8.2 Hz, 1H), 7.46 (d,
J = 8.2 Hz, 1H), 7.52 (s, 1H), 10.3 (s, 1H).
5.2.7. O-2-Acetyl-4-methoxyphenyl N,N-dimethylcarb-
amothioate (6h)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 6h as a white solid, yield: 94%, mp 83–85 °C.
1H NMR (CDCl3) d: 2.52 (s, 3H), 3.38 (s, 3H), 3.45 (s, 3H), 3.84 (s,
3H), 7.01 (s, 1H), 7.04 (m, 1H), 7.29 (m, 1H).
5.3.3. S-2-Formyl-5-methoxyphenyl N,N-dimethylcarb-
amothioate (7d)
MW conditions: P: 280 W, T: 170 °C, RT: 2 min; time: 10 min.
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 7d as a white solid, yield: 92%, mp 81–
83 °C. 1H NMR (CDCl3) d: 3.02 (s, 3H), 3.17 (s, 3H), 3.87 (s, 3H),
7.07 (d, J = 8.4 Hz, 1H), 7.08 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 10.25
(s, 1H).
5.2.8. O-2-Acetyl-5-methoxyphenyl N,N-dimethylcarb-
amothioate (6i)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 6i as colourless oil, yield: 95%. 1H NMR (CDCl3)
d: 1.42 (t, J = 7.0 Hz, 3H), 2.49 (s, 3H), 3.39 (s, 3H), 3.46 (s, 3H), 4.04
(q, J = 7.0 Hz, 2H), 6.56 (d, J = 2.6 Hz, 1H), 6.84 (dd, J = 2.6 and
8.8 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H).
5.3.4. S-2-Formyl-6-methoxyphenyl N,N-dimethylcarb-
amothioate (7e)
5.2.9. O-2-Acetyl-6-methoxyphenyl N,N-dimethylcarb-
amothioate (6j)
MW conditions: P: 200 W, T: 210 °C, RT: 2 min; time: 2 min. The
residue was chromatographed with EtOAc–petroleum ether 3:7 as
eluent to give 7e as a yellow oil, yield: 67 %. 1H NMR (CDCl3) d: 3.01
(s, 3H), 3.21 (s, 3H), 4.31 (s, 3H), 7.18 (d, J = 8.0 Hz, 1H), 7.52 (t,
J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 10.4 (s, 1H).
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 6j as a white solid, yield: 78%, mp 144–146 °C.
1H NMR (CDCl3) d: 2.55 (s, 3H), 3.40 (s, 3H), 3.46 (s, 3H), 3.84 (s,
3H), 7.12 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 8.4 Hz, 1H), 7.36 (d,
J = 8.4 Hz, 1H).
5.3.5. S-2-Acetylphenyl N,N-dimethylcarbamothioate (7f)
MW conditions: P: 200 W, T: 210 °C, RT: 2 min; time: 3 min. The
residue was chromatographed with EtOAc–petroleum ether 2:8 as
eluent to give 7f as a brown oil, yield: 61%. 1H NMR (CDCl3) d: 2.57
(s, 3H), 3.04 (s, 3H), 3.07 (s, 3H), 7.45 (m, 2H), 7.59 (m, 2H).
5.2.10. O-2-Formyl-4,5-dimethoxyphenyl N,N-dimethylcarb-
amothioate (6k)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 6k as a white solid, yield: 69%, mp 172–
174 °C. 1H NMR (CDCl3) d: 3.42 (s, 3H), 3.48 (s, 3H), 3.94 (s, 6H),
6.61 (s, 1H), 7.36 (s, 1H), 9.94 (s, 1H).
5.3.6. S-2-Acetyl-3-methoxyphenyl N,N-dimethylcarb-
amothioate (7g)
MW conditions: P: 200 W, T: 210 °C, RT: 2 min; time: 5 min. The
residue was chromatographed with EtOAc–petroleum ether 4:6 as
eluent to give 7g as a brown oil, yield: 82%. 1H NMR (CDCl3) d: 2.46
(s, 3H), 2.99 (s, 3H), 3.02 (s, 3H), 3.79 (s, 3H), 6.92 (d, J = 8.4 Hz, 1H),
7.11 (d, J = 8.4 Hz, 1H), 7.27 (t, J = 8.4 Hz, 1H).
5.2.11. O-2-Acetyl-5-ethoxyphenyl N,N-dimethylcarb-
amothioate (6l)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 6l as a white solid, yield: 81%, mp 84–86 °C. 1H
NMR (CDCl3) d: 2.48 (s, 3H), 3.38 (s, 3H), 3.45 (s, 3H), 3.84 (s, 3H),
6.58 (d, J = 2.8 Hz, 1H), 6.84 (dd, J = 2.4 and 8.8 Hz, 1H), 7.80 (d,
J = 8.8 Hz, 1H).
5.3.7. S-2-Acetyl-4-methoxyphenyl N,N-dimethylcarb-
amothioate (7h)
MW conditions: P: 280 W, T: 240 °C, RT: 2 min; time: 1 min. The
residue was chromatographed with EtOAc–petroleum ether 4:6 as
eluent to give 7h as a brown oil, yield: 53%. 1H NMR (CDCl3) d: 2.56
(s, 3H), 3.04 (s, 3H), 3.10 (s, 3H), 3.83 (s, 3H), 6.95 (d, J = 8.6 Hz, 1H),
6.99 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H).
5.2.12. O-2-Acetyl-5-fluorophenyl N,N-dimethylcarbamothioate
(6m)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 6m as a colourless oil, yield: 90%. 1H NMR
(CDCl3) d: 2.52 (s, 3H), 3.39 (s, 3H), 3.45 (s, 3H), 6.85 (dd, J = 2.8
and 7.0 Hz, 1H), 7.03 (m, 1H), 7.82 (dd, J = 6.4 and 8.8 Hz, 1H).
5.3.8. S-2-Acetyl-5-methoxyphenyl N,N-dimethylcarb-
amothioate (7i)
5.3. General procedure (B) for the synthesis of compounds 7b-m
MW conditions: P: 200 W, T: 210 °C, RT: 2 min; time: 4 min.
The residue was chromatographed with EtOAc–petroleum ether
4:6 as eluent to give 7i as a brown oil, yield: 51%. 1H NMR
The N,N-dimethylcarbamothioate 6b–m (0.5 mmol) was placed
in a 10 mL closed vial and irradiated in a focused MW oven at the

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R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
(CDCl3) d: 2.56 (s, 3H), 3.04 (s, 3H), 3.09 (s, 3H), 3.84 (s, 3H),
6.89 (d, J = 2.8 and 8.8 Hz, 1H), 7.14 (d, J = 2.6 Hz, 1H), 7.65 (d,
J = 8.6 Hz, 1H).
5.6. General procedure (C) for the synthesis of 2-thiophenols
8b–m
The S-aryl compound 7b–m (3 mmol) was dissolved in MeOH
(10 mL). A 3 N aqueous solution of NaOH (12 mL) was added, and
the reaction mixture was heated to reflux for 2 h. The reaction mix-
ture was cooled to 25 °C and acidified to pH 5 by the addition of
10% aqueous HCl. The resulting mixture was extracted with EtOAc
(3 ꢁ 15 mL), and the combined organic extracts were washed with
brine, dried over Na2SO4 and concentrated in vacuo to furnish the
corresponding 2-mercaptoarylaldehyde, which was used without
further purification for the next reaction.
5.3.9. S-2-Acetyl-6-methoxyphenyl N,N-dimethylcarb-
amothioate (7j)
MW conditions: P: 240 W, T: 210 °C, RT: 2 min; time: 2 min. The
residue was chromatographed with EtOAc–petroleum ether 2:8 as
eluent to give 7j as a brown oil, yield: 69 %. 1H NMR (CDCl3) d: 2.58
(s, 3H), 2.96 (s, 3H), 3.24 (s, 3H), 4.24 (s, 3H), 7.12 (d, J = 8.0 Hz, 1H),
7.48 (t, J = 8.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H).
5.3.10. S-2-Formyl-4,5-dimethoxyphenyl N,N-dimethylcarb-
amothioate (7k)
5.7. General procedure (D) for the synthesis of 2-(30,40,50-trim-
ethoxybenzoyl) benzo[b]thiophenes 4a–m
MW conditions: P: 300 W, T: 240 °C, RT: 2 min; time: 4 min. The
residue was chromatographed with EtOAc–petroleum ether 1:1 as
eluent to give 7k as a yellow oil, yield: 55%. 1H NMR (CDCl3) d: 3.03
(s, 3H), 3.16 (s, 3H), 3.96 (s, 6H), 6.99 (s, 1H), 7.54 (s, 1H), 10.3 (s,
1H).
To a solution of the appropriate substituted 2-mercaptobenzade-
hyde 8b–e and 8k or 2-mercaptoacetophenone 8f–j and 8lm
(1 mmol) in dry acetone (15 mL) was added 2-bromo-1-(3,4,5-tri-
methoxyphenyl)ethanone (289 mg, 1 mmol) and anhydrous potas-
sium carbonate (276 mg, 2 mmol) while stirring, and the reaction
mixture was refluxed for 18 h. After cooling, the reaction mixture
wasevaporated, andtheresiduewasdissolvedinamixtureofdichlo-
romethane (15 mL) and water (5 mL). The organic layer was washed
with brine, dried and concentrated under reduced pressure to obtain
a residue, which was purified by flash column chromatography.
5.3.11. S-2-Acetyl-5-ethoxyphenyl N,N-dimethylcarbamothioate
(7l)
MW conditions: P: 280 W, T: 220 °C, RT: 2 min; time: 2 min. The
residue was chromatographed with EtOAc–petroleum ether 2:8 as
eluent to give 7l as a brown oil, yield: 53%. 1H NMR (CDCl3) d: 1.41
(t, J = 7.0 Hz, 3H), 2.56 (s, 3H), 3.04 (s, 3H), 3.09 (s, 3H), 4.09 (q,
J = 7.0 Hz, 2H), 6.87 (d, J = 2.8 and 8.6 Hz, 1H), 7.14 (d, J = 2.6 Hz,
1H), 7.65 (d, J = 8.6 Hz, 1H).
5.7.1. (Benzo[b]thiophen-2-yl)(3,4,5-trimethoxyphenyl)
methanone (4a)
The residue was chromatographed with EtOAc–petroleum ether
1.5:8.5 as eluent to give 4a as a white solid, yield: 96%, mp 113–
115 °C. 1H NMR (CDCl3) d: 3.93 (s, 6H), 3.96 (s, 3H), 7.19 (s, 2H),
7.42 (t, J = 8.0 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.92 (m, 3H). Anal.
Calcd for C18H16O4S: C, 65.84; H, 4.91. Found: C, 65.72; H, 4.78.
5.3.12. S-2-Acetyl-5-fluorophenyl N,N-dimethylcarbamothioate
(7m)
MW conditions: P: 240 W, T: 210 °C, RT: 1 min; time: 4 min.
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 7m as a brown oil, yield: 78%. 1H NMR
(CDCl3) d: 2.58 (s, 3H), 3.04 (s, 3H), 3.08 (s, 3H), 7.12 (m, 1H),
7.36 (dd, J = 2.6 and 8.8 Hz, 1H), 7.64 (dd, J = 5.6 and 8.6 Hz,
1H).
5.7.2. (4-Methoxybenzo[b]thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4b)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 4b as a white solid, yield: 55%, mp 163–165 °C.
1H NMR (CDCl3) d: 3.92 (s, 3H), 3.93 (s, 6H), 3.96 (s, 3H), 6.77 (dd,
J = 7.6 and 1.0 Hz, 1H), 7.19 (s, 2H), 7.44 (m, 2H), 8.07 (s, 1H). Anal.
Calcd for C19H18O5S: C, 63.76; H, 5.06. Found: C, 63.56; H, 4.98.
5.4. Synthesis of 2-(benzylthio) benzaldehyde
To a cold solution (ice bath) containing 2-nitrobenzaldehyde
(3.0 g, 20 mmol) and benzylmercaptan (2.48 g, 20 mmol) in
20 mL of DMF was added dropwise a solution of KOH (2 g,
36 mmol) in 5 mL of water. The mixture was stirred at 4 °C for
2 h and then poured into ice-water. The solid was collected,
washed with cold water (50 mL), dried in vacuo over P2O5 and
recrystallised from petroleum ether. Yield: 76%, brown oil. 1H
NMR (CDCl3) d: 4.13 (s, 2H), 7.28 (m, 6H), 7.33 (t, J = 8.0 Hz, 1H),
7.49 (m, 1H), 7.82 (d, J = 4.4 Hz, 1H), 10.2 (s, 1H).
5.7.3. (5-Methoxybenzo[b]thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4c)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 4c as a yellow oil, yield: 78%. 1H NMR (CDCl3)
d: 3.88 (s, 3H), 3.92 (s, 6H), 3.96 (s, 3H), 7.12 (m, 2H), 7.33 (s, 2H),
7.49 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H). Anal. Calcd for C19H18O5S: C,
63.76; H, 5.06. Found: C, 63.61; H, 4.89.
5.7.4. (6-Methoxybenzo[b]thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4d)
5.5. Synthesis of 2-mercaptobenzaldehyde (8a)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 4d as a white solid, yield: 63%, mp 114–
116 °C. 1H NMR (CDCl3) d: 3.92 (s, 3H), 3.93 (s, 6H), 3.95 (s, 3H),
7.03 (dd, J = 8.8 and 2.4 Hz, 1H), 7.16 (s, 2H), 7.34 (d, J = 2.0 Hz,
1H), 7.76 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 1.0 Hz, 1H). Anal. Calcd
for C19H18O5S: C, 63.76; H, 5.06. Found: C, 63.62; H, 4.78.
A solution of 2-benzylthiobenzaldehyde (4.56 g, 20 mmol) in
dry benzene (40 mL) was added dropwise over 30 min to a suspen-
sion of finely powdered anhydrous aluminium chloride (4.5 g,
33 mmol) in dry benzene (40 mL), and the mixture was stirred un-
der nitrogen at room temperature for 48 h. The green-brown mix-
ture was decomposed by the cautious addition of ice-water
(100 mL), and the organic layer was separated and washed succes-
sively with water (50 mL) and 5% aqueous sodium hydroxide
(2 ꢁ 50 mL). The alkaline extracts were acidified (pH 2) and ex-
tracted with dichloromethane (3 ꢁ 50 mL). The organic extracts
were washed with brine, dried and evaporated to yield 2-merca-
ptobenzaldehyde as a brown oil, yield: 24%. 1H NMR (CDCl3) d:
7.31 (m, 2H), 7.33 (t, J = 8.0 Hz, 1H), 7.82 (d, J = 4.4 Hz, 1H), 10.1
(s, 1H), 10.4 (s, 1H).
5.7.5. (7-Methoxybenzo[b]thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4e)
The residue was chromatographed with EtOAc–petroleum ether
2:8 as eluent to give 4e as a yellow solid, yield: 68%, mp 105–
107 °C. 1H NMR (CDCl3) d: 3.92 (s, 3H), 3.96 (s, 6H), 4.03 (s, 3H),
6.89 (d, J = 7.2 Hz, 1H), 7.20 (s, 2H), 7.38 (t, J = 8.0 Hz, 1H), 7.52
(d, J = 8.0 Hz, 1H), 7.92 (s, 1H). Anal. Calcd for C19H18O5S: C,
63.76; H, 5.06. Found: C, 63.61; H, 4.92.

R. Romagnoli et al. / Bioorg. Med. Chem. 18 (2010) 5114–5122
5121
5.7.6. (3,4,5-Trimethoxyphenyl)(3-methylbenzo[b]thiophen-2-
yl)methanone (4f)
(dd, J = 5.6 and 8.6 Hz, 1H). Anal. Calcd for C19H17FO4S: C, 63.32;
H, 4.75. Found: C, 63.11; H, 4.56.
The residue was chromatographed with EtOAc–petroleum ether
1:9 as eluent to give 4f as a white solid, yield: 56%, mp 124–126 °C.
1H NMR (CDCl3) d: 2.61 (s, 3H), 3.89 (s, 6H), 3.95 (s, 3H), 7.20 (s,
2H), 7.43 (m, 2H), 7.90 (m, 2H). Anal. Calcd for C19H18O4S: C,
66.65; H, 5.30. Found: C, 66.51; H, 5.18.
5.8. Cell growth inhibitory activity
Murine leukemia L1210, murine mammary carcinoma FM3A
and human T-lymphocyte Molt 4 and CEM and human cervix car-
cinoma (HeLa) cells were suspended at 300,000–500,000 cells/mL
5.7.7. (4-Methoxy-4-methylbenzo[b]thiophen-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4g)
The residue was chromatographed with EtOAc–petroleum ether
1:9 as eluent to give 4g as a white solid, yield: 60%, mp 160–162 °C.
1H NMR (CDCl3) d: 2.73 (s, 3H), 3.89 (s, 6H), 3.94 (s, 6H), 6.73 (dd,
J = 7.6 and 1.0 Hz, 1H), 7.18 (s, 2H), 7.38 (m, 2H). Anal. Calcd for
of culture medium, and 100
lL of a cell suspension was added to
100 L of an appropriate dilution of the test compounds in wells
l
of 96-well microtiter plates. After incubation at 37 °C for two days,
cell number was determined using a Coulter counter. The IC50 va-
lue was defined as the compound concentration required to inhibit
cell proliferation by 50%.
C20H20O5S: C, 64.50; H, 5.41. Found: C, 64.38; H, 5.23.
5.9. Effects on tubulin polymerization and on colchicine
binding to tubulin
5.7.8. (5-Methoxy-3-methylbenzo[b]thiophen-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4h)
The residue was chromatographed with EtOAc–petroleum ether
1:9 as eluent to give 4h as a white solid yield: 52%, mp 132–133 °C.
1H NMR (CDCl3) d: 2.58 (s, 3H), 3.89 (s, 3H), 3.92 (s, 3H), 3.94 (s,
3H), 3.96 (s, 3H), 7.11 (d, J = 8.8 Hz, 1H), 7.20 (s, 2H), 7.24 (s, 1H),
7.72 (d, J = 8.8 Hz, 1H). Anal. Calcd for C20H20O5S: C, 64.50; H,
5.41. Found: C, 64.32; H, 5.21.
Bovine brain tubulin was purified as described previously.15 To
evaluate the effect of the compounds on tubulin assembly in vitro,11
varying concentrations were preincubated with 10 lM tubulin in
glutamate buffer at 30 °C and then cooled to 0 °C. After addition of
GTP, the mixtures were transferred to 0 °C cuvettes in a recording
spectrophotometer and warmed to 30 °C, and the assembly of tubu-
lin was observed turbidimetrically. The IC50 value was defined as the
compound concentration that inhibited the extent of assembly by
50% after a 20 min incubation. The ability of the test compounds
to inhibit colchicine binding to tubulin was measured as de-
5.7.9. (6-Methoxy-3-methylbenzo[b]thiophen-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4i)
The residue was chromatographed with EtOAc–petroleum ether
1.5:8.5 as eluent to give 4i as a white solid, yield: 60%, mp 130–
132 °C. 1H NMR (CDCl3) d: 2.59 (s, 3H), 3.89 (s, 3H), 3.91 (s, 3H),
3.92 (s, 3H), 3.94 (s, 3H), 7.11 (d, J = 2.8 and 8.8 Hz, 1H), 7.17 (s,
2H), 7.39 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H). Anal. Calcd for
scribed,15 except that the reaction mixtures contained 1
M [3H]colchicine and 1
M or 5 M test compound.
lM tubulin,
5
l
l
l
5.10. Flow cytometric analysis of cell cycle distribution
For details, see Ref. 8a.
C
20H20O5S: C, 64.50; H, 5.41. Found: C, 64.33; H, 5.19.
5.7.10. (7-Methoxy-3-methylbenzo[b]thiophen-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4j)
5.11. Molecular modeling
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 4j as a yellow solid, yield: 71%, mp 112–
114 °C. 1H NMR (CDCl3) d: 2.43 (s, 3H), 3.89 (s, 3H), 3.94 (s, 6H),
3.98 (s, 3H), 6.87 (d, J = 7.2 Hz, 1H), 7.12 (s, 2H), 7.42 (t,
J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H). Anal. Calcd for C20H20O5S:
C, 64.50; H, 5.41. Found: C, 64.32; H, 5.20.
All molecular modeling studies were performed on a MacPro
dual 2.66 GHz Xeon running Ubuntu 8. The tubulin structure was
downloaded from the PDB data bank (http://www. rcsb.org/-PDB
code: 1SA0).16 Hydrogen atoms were added to the protein, using
Molecular Operating Environment (MOE) 2009.10,17 and mini-
mized keeping all the heavy atoms fixed until a RMSD gradient
of 0.05 kcal molꢂ1 Åꢂ1 was reached. Ligand structures were built
with MOE and minimized using the MMFF94x forcefield until a
RMSD gradient of 0.05 kcal molꢂ1 Åꢂ1 was reached. The docking
simulations were performed using FlexX18 with the MOE interface.
5.7.11. (5,6-Dimethoxybenzo[b]thiophen-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4k)
The residue was chromatographed with EtOAc–petroleum ether
4:6 as eluent to give 4k as a yellow solid, yield: 62%, mp 153–
155 °C. 1H NMR (CDCl3) d: 3.93 (s, 6H), 3.95 (s, 6H), 4.00 (s, 3H),
7.17 (s, 2H), 7.26(s, 1H), 7.30 (s, 1H), 7.80 (s, 1H). Anal. Calcd for
Acknowledgments
C20H20O6S: C, 61.84; H, 5.19. Found: C, 61.62; H, 5.02.
Financial support was provided by GOA (Krediet no. 10/014) of
the K. U. Leuven. The authors would like to thank Mrs. Lizette van
Berckelaer, Dr. Alberto Casolari and Elisa Durini for technical
assistance.
5.7.12. (6-Ethoxy-3-methylbenzo[b]thiophen-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4l)
The residue was chromatographed with EtOAc–petroleum ether
3:7 as eluent to give 4l as a red solid, yield: 64%, mp 157–158 °C. 1H
NMR (CDCl3) d: 1.50 (t, J = 7.2 Hz, 3H), 2.58 (s, 3H), 3.89 (s, 6H),
3.94 (s, 3H), 4.14 (t, J = 6.8 Hz, 2H), 7.10 (d, J = 2.8 and 8.6 Hz,
1H), 7.16 (s, 2H), 7.29 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H). Anal. Calcd
for C21H22O5S: C, 65.27; H, 5.74. Found: C, 65.01; H, 5.62.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.068.
5.7.13. (6-Fluoro-3-methylbenzo[b]thiophen-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4m)
References and notes
The residue was chromatographed with EtOAc–petroleum ether
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132 °C. 1H NMR (CDCl3) d: 2.60 (s, 3H), 3.90 (s, 6H), 3.96 (s, 3H),
7.16 (s, 2H), 7.23 (m, 1H), 7.52 (dd, J = 2.6 and 8.8 Hz, 1H), 7.84
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