Synthesis and biological evaluation of 2- and 3-aminobenzo[b]thiophene derivatives as antimitotic agents and inhibitors of tubulin polymerization

Article abstract of DOI:10.1021/jm070050f

Two new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene molecular skeleton and its 3-amino positional isomer were synthesized and evaluated for antiproliferative activity, inhibition of tub

Full text of DOI:10.1021/jm070050f

J. Med. Chem. 2007, 50, 2273-2277
2273
Synthesis and Biological Evaluation of 2- and 3-Aminobenzo[b]thiophene Derivatives as
Antimitotic Agents and Inhibitors of Tubulin Polymerization
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Dora Carrion,† Carlota Lopez Cara,† Delia Preti,† Francesca Fruttarolo,†
Maria Giovanna Pavani,† Mojgan Aghazadeh Tabrizi,† Manlio Tolomeo,‡ Stefania Grimaudo,‡ Antonella Di Cristina,‡
Jan Balzarini,# John A. Hadfield,§ Andrea Brancale,| and Ernest Hamel
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, 44100 Ferrara, Italy, Dipartimento di Oncologia, DiVisione di Ematologia e
SerVizio AIDS, Policlinico “P. Giaccone”, UniVersita` di Palermo, Palermo, Italy, Laboratory of Virology and Chemotherapy, Rega Institute for
Medical Research, Minderbroedersstraat 10, B-3000 LeuVen, Belgium, Centre for Molecular Drug Design, Cockroft Building, UniVersity of
Salford, Manchester M5 4W5, U.K., The Welsh School of Pharmacy, Cardiff UniVersity, King Edward VII AVenue, Cardiff CF10 3XF, U.K.,
and Toxicology and Pharmacology Branch, DeVelopmental Therapeutics Program, DiVision of Cancer Treatment and Diagnosis, National
Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702
ReceiVed January 13, 2007
Two new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)-
benzo[b]thiophene molecular skeleton and its 3-amino positional isomer were synthesized and evaluated
for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. Although many
more 3-amino derivatives have been synthesized so far, the most promising compound in this series was
2-amino-6-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene, which inhibits cancer cell growth at
subnanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.
Chart 1. Inhibitors of Tubulin Polymerization
Introduction
There has long been considerable interest in the discovery
and development of novel small molecules able to affect tubulin
polymerization.1 More recently, it has been established that some
tubulin-binding agents selectively target tumor vasculature and
thus can also be considered vascular disrupting agents.2 These
compounds induce morphological changes in the endothelial
cells of the tumor’s blood vessels, resulting in their occlusion
and therefore interruption of blood flow.3
One of the most important tubulin-binding agents is com-
bretastatin A-4 (CA-4, 1; Chart 1). CA-4, isolated from the bark
of the South African tree Combretum caffrum,4 is one of the
well-known natural tubulin-binding molecules affecting micro-
tubule dynamics. CA-4 strongly inhibits the polymerization of
tubulin by binding to the colchicine site.5 Because of its simple
structure, a large number of CA-4 analogues have been
developed and evaluated in SAR studies.6 Among synthetic
small-molecule tubulin inhibitors, replacement of the double
bond of 1 with a carbonyl group furnished a benzophenone-
type CA-4 analogue named phenstatin (2). This compound
demonstrated interesting efficacy in a variety of tumor models
while retaining the characteristics of 1.7 The 2-aminobenzophe-
none derivative 3 also strongly inhibited cancer cell growth and
tubulin polymerization and caused mitotic arrest, as did 2.8
The classical bioisosteric equivalence between benzene and
thiophene prompted us recently to synthesize a series of
thiophene derivatives in which a 2-aminothiophene system
replaced the 2-aminobenzene moiety in the 2-aminophenstatin
analogue 3. Many of the 2-amino-3-(3,4,5-trimethoxybenzoyl)-
5-phenylthiophene molecules with general structure 4 are potent
inhibitors of tubulin polymerization.9 These derivatives are as
effective as their 2-(3,4,5-trimethoxybenzoyl)-3-aminothiophene
isomeric analogues with general structure 5.10
Our preliminary study revealed that a limited series of 2- and
3-aminobenzo[b]thiophenes (6a-c and 7a,d) showed interesting
activities in inhibiting microtubule polymerization and cell
proliferation, with 3-amino-6-methyl-2-(3,4,5-trimethoxyben-
zoyl)benzo[b]thiophene (7d) being the most promising deriva-
* To whom correspondence should be addressed. For R.R.: phone, 39-
(0)532-291290; fax, 39-(0)532-291296; e-mail, rmr@unife.it. For P.G.B.:
phone, 39-(0)532-291293; fax, 39-(0)532-291296; e-mail, baraldi@unife.it.
† Universita` di Ferrara.
‡ Universita` di Palermo.
# Rega Institute for Medical Research.
§ University of Salford.
| Cardiff University.
National Cancer Institute at Frederick.
10.1021/jm070050f CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/10/2007

2274 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Brief Articles
Scheme 1a
Scheme 2a
a Reagents: (a) cyclohexanone, 4-methylcyclohexanone or 4-methoxy-
cyclohexanone, S8, morpholine, EtOH, 70 °C for 1 h, then 18 h at rt; (b)
Ac2O, pyridine, rx; (c) 10% Pd/C moistened with 50% water, heating, 18
h; (d) 1 N NaOH, EtOH, rx, 2 h.
tive in this collection.11 These compounds with general structures
6 and 7 are structurally related to 3, in which the 2-aminophenyl
moiety of this latter derivative was replaced by various 2- or
3-aminobenzo[b]thiophenes. The benzo[b]thiophenes 6 and 7
also represent benzofused analogues of 5-phenylthiophene
derivatives 4 and 5, respectively. Here, we report further work
to improve the potency of this series of tubulin polymerization
inhibitors.
a Reagents: (a) C6H5CH2SH, KOH, DMF; (b) AlCl3, C6H6; (c) NaNO2,
HCl, 0 °C, then KS2COEt, 65 °C; (d) NaOH, 65 °C; (e) 24, K2CO3,
(CH3)2CO, rx, 18 h; (f) MeI, NaH, DMF; (g) Ac2O, Py, rx.
Chemistry
By the synthesis of 7b-j, we focused on effects of electron-
donating methyl and methoxy substituents at positions 4-7 of
the 3-aminobenzo[b]thiophene skeleton. By the synthesis of 7k-
l, we evaluated whether the 3-amino substituent was important
in restricting the conformation of the adjacent trimethoxybenzoyl
moiety through an intramolecular hydrogen bond with the
carbonyl oxygen.
benzo[b]thiophene derivatives 11a-e following aromatization
by heating with 10% Pd/C. These latter compounds were
transformed by ethanolysis into the final products 6a-e.
The 3-amino-2-(3,4,5-trimethoxybenzoyl)benzo[b]thiophenes
7a-j were prepared in excellent yields. Condensation was
followed by intramolecular cyclization when 2-cyanothiophenol
(23a) and methyl/methoxy substituted 2-mercaptobenzonitriles
23b-j were reacted with 1-(3,4,5-trimethoxyphenyl)-2-bromo-
ethanone (24), using potassium carbonate in refluxing acetone
(Scheme 2).
2-Mercaptobenzonitrile (23a)15 and methyl substituted 2-mer-
captobenzonitriles 23b-e were obtained by S-debenzylation of
thioethers 20a-e with aluminum chloride in benzene. These
latter derivatives were obtained in good yields by condensation
of the corresponding 2-nitrobenzonitriles 19a-e with the
potassium salt of phenyl methanethiol in cold aqueous DMF.16
Methoxy substituted 2-mercaptobenzonitriles 23f-j were syn-
thesized by the Leuckart reaction17 starting from the precursor
amines 21f-j. The method involved diazotation of 21f-i with
NaNO2 in a MeOH/H2O mixture of HCl/AcOH, followed by
nucleophilic displacement of the diazonium moiety with potas-
sium O-ethylxanthate to furnish the resulting arylxanthate 22f-
j. The subsequent hydrolysis with NaOH followed by acidifi-
cation furnished the desired 2-mercaptobenzonitriles 23f-j,
which were used without further purification. Compound 7d
was bis-N-methylated with methyl iodide or acetylated with a
mixture of acetic anhydride and pyridine to give 7k or 7l,
respectively.
The benzo[b]thiophene molecular skeleton is the core struc-
ture of a series of inhibitors of microtubule polymerization,
which showed activity comparable with that of CA-4.12 The
most representative compounds in this series are derivatives with
general structure 8, which incorporates the 3-(3,4,5-trimethoxy-
benzoyl)-6-methoxybenzo[b]thiophene ring system. Previous
studies have shown that the 6-methoxy substituent significantly
contributes to maximize activity and mimics the 4-methoxy
group in the B-ring of 1 and aminobenzophenones 2 and 3.8,12
Building upon these observations, as represented by the synthesis
of 6e, we were intrigued with studying the biological effects of
replacing the aryl substituent with an amino group at the
2-position of the benzo[b]thiophene ring of 8a and 8b. Since
the C-6 methoxy group proved to be favorable for bioactivity,
by the synthesis of 6d we evaluated the effect of replacing it
with a weaker electron-releasing methyl group, which had been
well-tolerated in a series of CA-4 analogues published previ-
ously.13 Finally, through the synthesis of 6b and 6c we
investigated the effect on antiproliferative activity of the 3,4,5-
trimethoxybenzoyl group in the 2-aminobenzo[b]thiophene
derivative 6a.
Derivatives 6a-e were synthesized in four steps, as shown
in Scheme 1. The Gewald reaction14 applied to â-ketonitriles
13, 14, and 15 and cyclohexanone, 4-methylcyclohexanone, or
4-methoxycyclohexanone furnished the 2-amino-3-aroyl-4,5,6,7-
tetrahydrobenzo[b]thiophenes 9a-c, 9d, and 9e, respectively.
Acetylation of the amino group, using a mixture of acetic
anhydride and pyridine, yielded 10a-e, which afforded the
Results and Discussion
Table 1 summarizes the antiproliferative effects of benzo[b]-
thiophene derivatives 6 and 7 against a panel of tumor cell lines
with 1 as reference compound. Several derivatives demonstrated
substantial inhibitory effects on the growth of murine leukemia

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2275
Table 1. In Vitro Inhibitory Effects of Compounds 6a-e, 7a-l, and
CA-4 (1) against the Proliferation of Murine Leukemia (L1210), Murine
Mammary Carcinoma (FM3A), and Human T-Lymphocyte (Molt/4 and
CEM) Cells
Table 2. Inhibition of Tubulin Polymerization and Colchicine Binding
by Compounds 6-7a, 6-7d, 6-7e, 7h-j, and CA-4
tubulin assemblya
colchicine bindingb
compd
6a
6d
6e
7a
7d
7e
7h
IC50 ( SD (µM)
% ( SD
IC50 (nM)a
1.9 ( 0.1
0.76 ( 0.1
1.8 ( 0.2
2.4 ( 0.1
1.3 ( 0.2
2.6 ( 0.1
1.3 ( 0.1
2.1 ( 0.3
1.8 ( 0.1
1.2 ( 0.1
25 ( 2
91 ( 2
66 ( 2
15 ( 1
60 ( 4
19 ( 3
60 ( 0.1
39 ( 1
16 ( 2
86 ( 3
compd
L1210
FM3A
Molt4/C8
CEM
6a11
90 ( 3
3700 ( 60
>10000
0.76 ( 0.06
4.4 ( 0.5
350 ( 320
>10000
100 ( 0
4700 ( 250
>10000
73 ( 9
74 ( 15
1700 ( 500
>10000
0.52 ( 0.06
4.5 ( 0.5
310 ( 20
>10000
6b11
6c11
6d
1700 ( 200
9200 ( 200
0.09 ( 0.069 0.69 ( 0.02
6e
4.8 ( 0.7
1800 ( 300
>10000
>10000
71 ( 5
360 ( 60
>10000
>10000
46 ( 12
27 ( 13
380 ( 120
84 ( 10
190 ( 100
42 ( 6
4.1 ( 0.0
290 ( 10
>10000
4200 ( 1700 4600 ( 3400
34 ( 3
83 ( 6
>10000
>10000
10 ( 7
8.5 ( 1.4
87 ( 19
85 ( 1
75 ( 36
1.6 ( 1.4
7i
7j
7a11
7b
CA-4 (1)
7c
9500 ( 1300
58 ( 50
7d11
7e
24 ( 2
98 ( 18
>10000
>10000
7.7 ( 2.9
8.9 ( 2.0
460 ( 440
90 ( 10
100 ( 10
1.9 ( 1.6
a Inhibition of tubulin polymerization. Tubulin was at 10 µM. b Inhibition
of [3H]colchicine binding. Tubulin, colchicine, and tested compound were
at 1, 5, and 1 µM, respectively.
160 ( 10
>10000
7f
7g
7h
7i
7j
7k
7l
>10000
39 ( 16
of substitution in the benzo[b]thiophene ring on antiproliferative
activity. Thus, 7b, with a methyl group at C-4, had no
antiproliferative activity and only marginal improvement oc-
curred with the methyl group at C-5 (7c). Simply moving the
methyl group to C-6 (7d) resulted in a highly active compound,
while moving the methyl to C-7 (7e) resulted in a 2- to 4-fold
loss of activity. With methoxy substituents, the differences
between C-4/5 versus C-6/7 were even more dramatic. Both 7f
and 7g were inactive, while 7h and 7i were the most active
compounds in the 3-amino series. However, a moderate loss of
activity was observed with methoxy substituents at C-6 and C-7
(7j). Activity generally intermediate between the activities of
7d and 7e were observed with 7k and 7l, the N,N-dimethylamino
and N,N-diacetyl derivatives, respectively, of 7d. Thus, the
3-amino hydrogens of 7d cannot be considered essential for its
antiproliferative activity.
33 ( 29
150 ( 30
87 ( 1
150 ( 60
2.8 ( 1.1
CA-4 (1)
a IC50: compound concentration required to inhibit tumor cell prolifera-
tion by 50%. Data are expressed as the mean ( SE from the dose response
curves of at least three independent experiments.
(L1210), murine mammary carcinoma (FM3A), and human
T-lymphoblastoid (Molt/4 and CEM) cells. In general, the
antiproliferative activities of the compounds were greater against
the Molt/4 and CEM cells compared with the two murine lines.
The most active benzo[b]thiophene compound identified in this
study was 6d, which was more cytostatic than 1 in all four cell
lines. Derivative 6d inhibited the growth of L1210, FM3A, Molt/
4, and CEM cancer cell lines with IC50 values of 0.76, 0.09,
0.69, and 0.52 nM, respectively.
Derivatives 6a-c demonstrated that the 3′,4′,5′-trimethoxy-
benzoyl substitution in 6a was crucial for potent cell growth
inhibition. Its substitution with 3,4-dimethoxybenzoyl and
4-methoxybenzoyl moieties (6b and 6c, respectively) led to the
loss of growth inhibition activity. In terms of antiproliferative
activity, 6a and 7a, which were both unsubstituted in the
benzene portion of the benzo[b]thiophene ring, were substan-
tially less active than derivatives with a methyl or methoxy
group at C-6 or C-7.
In the 3-aminobenzo[b]thiophene derivatives, the results
indicated that inhibition of cell growth was strongly dependent
on the position of the methoxy or methyl moiety. A fairly
dramatic difference was observed between C-4/5 and C-6/7
substituted compounds (7b,c and 7f,g versus 7d,e and 7h,i).
Invariably, the greatest activity occurred when the methyl and
methoxy groups were located at the C-6 or C-7 position, the
least when located at the C-4 or C-5 position. A comparison of
substituent effects revealed that the replacement of the methoxy
by a methyl group at the C-6 or C-7 position resulted in a slight
reduction in antiproliferative activity. We observed the opposite
effect in the isomeric 2-aminobenzo[b]thiophene series, where
the substitution of C-6 methoxy with a C-6 methyl (6e and 6d,
respectively) caused a generally modest increase in activity (6-
to 9-fold except in the FM3A cells) and yielded the most active
compound of the whole series. Elimination of the C-6 substituent
(6a) significantly decreased antiproliferative activity in all four
cell lines. Specifically, by comparison of 6e to 8a and 8b, the
2-amino group of 6e is a good surrogate for the substituted
phenyl rings present in the latter compounds.
Finally although only few compounds have been synthesized
in the 2-amino series, thus far all 2-amino-3-(3,4,5-trimethoxy-
benzoyl)benzo[b]thiophenes are significantly more potent than
the isomeric 3-amino compounds (compare 6a,d,e with 7a,d,h,
respectively).
To investigate whether the antiproliferative activities of these
compounds were related to an interaction with the microtubule
system, 6a,d,e and 7a,d,e,h-j were evaluated for their inhibitory
effects on tubulin polymerization and on the binding of [3H]-
colchicine to tubulin (Table 2).18,19 With the exception of 7i,
there was a positive correlation between the inhibition of
tubulin polymerization and antiproliferative activity. The most
potent compound in this series was 6d, with an IC50 of 0.76
µM. This is in agreement with 6d being the compound with
the greatest antiproliferative activity. Compounds 7d and 7h
were as active as CA-4 as inhibitors of tubulin assembly,
although both compounds were less active in their effects on
cell growth..
In the colchicine binding studies, 6d,e and 7d,h potently
inhibited the binding of [3H]colchicine to tubulin, since 60-
91% inhibition occurred with these agents at 1 µM with
colchicine at 5 µM. Only 6d was as potent as CA-4, which in
these experiments inhibited colchicine binding by 86%.
Because molecules exhibiting effects on tubulin assembly
should cause the alteration of cell cycle parameters with
preferential G2-M blockade, flow cytometry analysis was
performed to determine the effect of the most active compounds
on K562 (human chronic myelogenous leukemia) cells. Cells
were cultured for 24 h in the presence of each compound at the
IC50 determined for 24 h of growth (6a, 150 nM; 6d, 2.5 nM;
6e, 20 nM; 7a, 800 nM; 7d, 80 nM; 7e, 250 nM; 7h, 25 nM;
7i, 25 nM; 7j, 950 nM), with CA-4 as reference compound.
The more extensive synthetic work thus far with the 3-amino
derivatives permits us to analyze more extensively the effects

2276 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Brief Articles
cytometry that the most active compounds had cellular effects
typical of agents that bind to tubulin, causing accumulation of
cells in the G2-M phase of the cell cycle. Molecular modeling
studies were also performed, and the proposed binding modes
for 6d and 7d are consistent with the experimental data (see
Supporting Information).
Experimental Section
General Procedure A for Synthesis of 9a-e. A mixture of
aroylacetonitriles 13-15 (5 mmol), cyclohexanone, 4-methylcy-
clohexanone or 4-methoxycyclohexanone (5 mmol), morpholine
(0.44 mL, 5 mmol), and sulfur (164 mg, 5 mmol) was heated at
70 °C for 1 h, then stirred at room temperature for 20 h. The mixture
was evaporated and the residue diluted with dichloromethane
(25 mL). After the mixture was washed with water (10 mL), the
organic layer was dried and filtered and the solvent removed by
evaporation. The crude product was purified by flash column
chromatography and recrystallized from petroleum ether to give
9a-e.
General Procedure B for Synthesis of 10a-e. Pyridine (10
drops) was added to a stirred solution of 9a-e (4 mmol) in Ac2O
(10 mL). The solution was refluxed for 2 h, poured into water, and
extracted with EtOAc (15 mL). The extract was washed succes-
sively with saturated aqueous NaHCO3 (5 mL), water (5 mL), and
brine (5 mL), dried, and concentrated in vacuo. The crude product
was purified by flash column chromatography and recrystallized
from petroleum ether to give 10a-e.
General Procedure C for the Synthesis of 11a-e. A mixture
of 10a-e (2 mmol), 10% Pd/C (50% wet with water, 1.4 g), and
CHCl3 (20 mL) was stirred at room temperature for 10 min,
followed by removal of the solvent by evaporation. The resulting
powder was heated at 130 °C for 20 h, cooled to room temperature,
and extracted with dichloromethane. The insoluble solids were
removed by filtration, and the filtrate was concentrated in vacuo.
The crude product was purified by flash column chromatography
to furnish 11a-e.
General Procedure D for the Synthesis of 6a-e. A mixture
of 11a-e (1 mmol), 1 N aqueous NaOH (1 mL, 1 mmol), and
EtOH (10 mL) was refluxed for 5 h, and the solvent was removed
by evaporation. The residue was triturated with water (5 mL) and
extracted with dichloromethane (3 × 5 mL). The extract was washed
with water to neutral reaction of the aqueous phase and dried. The
residue after evaporation was purified by flash chromatography on
silica gel and crystallized from petroleum ether to give 6a-e.
Figure 1. Effects of 6a,d,e, 7a,d,e,h-j, and CA-4 on DNA content/
cell following treatment of K562 cells for 24 h. The cells were cultured
without compound (control), with 2.5 nM or with compound used at
the concentration leading to 50% cell growth inhibition after 24 h of
treatment. Cell cycle distribution was analyzed by the standard
propidium iodide procedure as described in the Experimental Section
of Supporting Information. Sub-G0-G1 (A), G0-G1, S, and G2-M cells
are indicated in the control panel.
Compounds 6a,d,e and 7d,h-j caused a marked increase in
the percentage of cells blocked in the G2-M phase of the cell
cycle, with a simultaneous decrease of cells in S and G0-G1.
Compounds 7a and 7e were less effective as agents able to
induce cell cycle changes (Figure 1). These data confirm that
this class of molecules acts selectively on the G2-M phase of
the cell cycle, as expected for inhibitors of tubulin assembly
(Table 2).
Supporting Information Available: Detailed biological pro-
tocols, physical and spectroscopic data for 6a-e, 7a-l, 9a-e, 10a-
e, 11a-e, 20a-e, 22f-j, 23a-e, 23f-j; elemental analysis results
of 6a-e and 7a-l; and molecular modeling studies of 6d and 7d.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Conclusion
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