Benzo[b]thiophene-6-carboxamide 1,1-dioxides: Inhibitors of human cancer cell growth at nanomolar concentrations

Article abstract of DOI:10.1016/j.bmc.2010.06.009

Benzo[b]thiophenesulfonamide 1,1-dioxide derivatives (BTS) were described as candidate antineoplastic drugs. In the hope of finding new compounds with improved antitumour activity and reduced toxicity, we have designed and synthesized a small series of benzo[b]thiophene-6-carboxamide 1,1-dioxide derivatives (BTC) structurally related with the best reported BTS. Growth inhibition of HTB-54, CCRF-CEM and HeLa tumour cells lines at nanomolar concentrations was exhibited by some of the BTC. Hydrophobic substituents on the carboxamide group increased cytotoxicity but substitution by a hydroxy group diminished it, thus pointing to the electronic density on benzo[b]thiophene nucleus as a determinant factor. The process of cell death induced by BTC derivatives was further analyzed in CCRF-CEM cells, where these compounds induced apoptosis in a time and dose-dependent manner and cell cycle arrest at S phase. BTC derivatives also induced a significant increase in intracellular ROS levels in this cell line. Previous treatment of the cells with the antioxidant N-acetyl-cysteine abrogated the induction of apoptosis by BTC indicating that ROS generation is a previous event required to trigger the BTC induced apoptotic process.

Full text of DOI:10.1016/j.bmc.2010.06.009

Bioorganic & Medicinal Chemistry 18 (2010) 5701–5707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzo[b]thiophene-6-carboxamide 1,1-dioxides: Inhibitors of human cancer
cell growth at nanomolar concentrations
Aitziber A. Sagardoy a, María J. Gil b, Raquel Villar b, María J. Viñas a, Aranzazu Arrazola a, Ignacio Encío a,
Victor Martinez-Merino b,
*
a Dpto. de Ciencias de la Salud, Universidad Pública de Navarra, Avda. Barañain, 31008 Pamplona, Spain
b Dpto. de Química, Universidad Pública de Navarra, Campus Arrosadía, 31006 Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzo[b]thiophenesulfonamide 1,1-dioxide derivatives (BTS) were described as candidate antineoplastic
drugs. In the hope of finding new compounds with improved antitumour activity and reduced toxicity,
we have designed and synthesized a small series of benzo[b]thiophene-6-carboxamide 1,1-dioxide deriv-
atives (BTC) structurally related with the best reported BTS. Growth inhibition of HTB-54, CCRF-CEM and
HeLa tumour cells lines at nanomolar concentrations was exhibited by some of the BTC. Hydrophobic sub-
stituents on the carboxamide group increased cytotoxicity but substitution by a hydroxy group diminished
it, thus pointing to the electronic density on benzo[b]thiophene nucleus as a determinant factor. The pro-
cess of cell death induced by BTC derivatives was further analyzed in CCRF-CEM cells, where these com-
pounds induced apoptosis in a time and dose-dependent manner and cell cycle arrest at S phase. BTC
derivatives also induced a significant increase in intracellular ROS levels in this cell line. Previous treatment
of the cells with the antioxidant N-acetyl-cysteine abrogated the induction of apoptosis by BTC indicating
that ROS generation is a previous event required to trigger the BTC induced apoptotic process.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 April 2010
Revised 1 June 2010
Accepted 4 June 2010
Available online 9 June 2010
Keywords:
Antitumour agents
Apoptosis
Benzo[b]thiophenecarboxamide 1,1-dioxide
derivatives
Cytotoxicity
Reactive oxygen species (ROS)
1. Introduction
duced in CCRF-CEM cells an accumulation of intracellular reactive
oxygen species (ROS) and, since previous treatment of the cells with
Many efforts have been made to produce new arylsulfon-
amides1–9 with improved antitumour activity and reduced toxicity.
The last and the most promising N-acyl derivatives from Abbott Lab8
as well as other aromatic sulfonamides (ASU, Fig. 1)10 induce apop-
tosis through the bcl-2-dependent pathway. The core of ASU seems
to be related with the arylsulfonylureas (BSU, Fig. 1) previously
reported by us11–13 and others.14,15 Isozyme IX of human carbonic
anhydrase (hCA IX) has emerged as another interesting target for
the design of antitumour arylsulfonamides because it is highly over
expressed in many cancer types while present in few normal tis-
sues.16,17 Within this group of compounds, we have reported the
synthesis and in vitro antitumour activity of several benzo[b]thi-
ophenesulfonamide 1,1-dioxide derivatives (BTS, Fig. 1).2,11,18 These
compounds, that showed a clear correlation between their lipophil-
icity (log P), their cytotoxic activities2 and their ability to inhibit the
tNOX activity of the plasma membrane,19 induced in human leukae-
mia CCRF-CEM cells a typical process of apoptosis that included cell
shrinkage, mitochondrial dysfunction, phosphatidylserine translo-
cation to cell surface, caspase activation, chromatin condensation
and internucleosomal DNA degradation.12,13 BTS derivatives also in-
the antioxidant N-acetyl-cysteine abrogated the BTS induced cyto-
chrome C release, caspase-3 activation and cell death, it has been
proved that ROS are required for the BTS induced apoptotic effect.12
Interestingly, N-substituted with short chains-BTS also showed
some selectivity for the inhibition of the tumour-associated hCA
IX over its cytosolyc counterpart hCA II.16,18 Despite this fact, since
some of the most potent inhibitors among the BTS are not cytotoxic
(R = R1 = H 2,3-dihydro derivative) and the contrary (R = benzyl,
R1 = H),18 BTS cytotoxic activity seems to be unrelated with hCA
inhibition.
Interestingly, benzo[b]thiophene-4-carboxamide 1,1-dioxide
derivatives (hBTC, Fig. 2) have been described in the literature as
preventive for various inflammatory and neoplastic diseases caused
by an abnormal production of interleukin-6 or interleukin-12.20
Moreover, 2-carboxamide analogues of BTS were patented as micro-
bicides in phytosanitary practice.21 Other carboxamide derivatives
of heteroaryl nucleus including thiophene,22,23 furane,22 pyrrole,22
pyrazole,24 thiazole,25,26 pyridine,27 pyridazine,28 pyridoindole29
or thienopyridine30 are suitable for the treatment of cancer because
of their ability to inhibit different kinases. Remarkably, active het-
eroaryl carboxamides usually bring alternate aromatic systems,
hydrogen bond donor/acceptors or lipophilic groups as in KI (Fig. 2).
Since substitution of the sulfonamide group of the BTS deriva-
tives by a carboxamide group could improve their lipophilicity,
* Corresponding author. Tel.: +34 948 169590; fax: +34 948 169606.
E-mail address: merino@unavarra.es (V. Martinez-Merino).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.009

5702
A. A. Sagardoy et al. / Bioorg. Med. Chem. 18 (2010) 5701–5707
X
R1
H
N
H
N
O
H
N
H
N
S
O
O2
NRR
S
S
S
R
NH
S
Ph
S
O
O2
O
O
O2
O
ASU
BSU
BTS
R
NR1R1
Figure 1.
to expand and further optimise our series of BTS derivatives we
decided to test the replacement of the 6-sulfonamide group within
our BTS series2 by a set of alternative carboxamides. Thus, here we
describe the synthesis and biological evaluation of new benzo[b]-
thiophene-6-carboxamide 1,1-dioxide derivatives (BTC, Fig. 2). Ser-
ies of BTC was built from the residues of the more active BTS’s and
some other residues as reference. Substituents at BTC position 3
were not considered because of R1 electron releasing groups in
BTS drove to inactive compounds. Neither the 2,3-dihydro deriva-
tives from BTC were studied because their BTS analogues were not
very cytotoxic. The studied BTC series starts from an unsubstituted
6-carboxamide (4a) as the reference and continues with deriva-
tives including 4-methoxyphenyl (4b) and butyl (4d) groups, the
best cytotoxic N-aryl and N-alkyl residues found within the BTS
series. Phenylethylamines are much less toxic than arylamines31
and so, since we expect a better metabolic profile N-2-(4-methoxy-
phenyl)ethyl BTC (4c) was also included in the series.
to the carboxamide one displayed a stronger toxicity against every
tested cell line than the lead compound 4a. The N-aryl BTC deriv-
ative 4b came up as the most active compound with GI50 values
ranging from 2 to 4 nM against HTB-54, CCRF-CEM and HeLa cells,
and from 47 to 760 nM against HT-29, K-562 and MEL-AC cells.
Compound 4b was also active at the same level of commercial
Doxorubicin (GI50 values of 33 nM in CCRF-CEM, 6 nM in MEL-AC
and 20 nM in K-562 and HeLa cells).2,19 However, cytotoxicity of
the N-aralkyl or N-alkyl BTC derivatives 4c and 4d was similar
against every tested cell line apart from HTB-54. On the other
hand, when cytotoxic activities of BTC and BTS were compared
we found again that the N-substitution modulates activity of the
compounds: the N-aryl derivative was the most active among the
tested compounds in both series (GI50 values of 5b ranging
1–200 nM).2 However, N-substituents from BTC or BTS produce
slightly different effects depending on the tested cell line. Thus,
while 2-(4-methoxyphenyl)ethyl BTC 4c and BTS 5c derivatives
showed similar GI50 values at HTB-54 and MEL-AC cell lines, 5c
was much more cytotoxic against K-562 and CCRF-CEM cells. On
the contrary, cytotoxic activities of N-unsubstituted 4a and 5a
derivatives were within the same order of magnitude at every cell
line tested, except for HTB-54 cells that were more sensitive to 4a.
Moreover, since GI50 of N-benzyl BTS 5e and its N-methylated
derivative 6e compare in most of the tested cell lines, free NH
group seems to play a residual role in the studied activity. Last,
electronic density on the benzo[b]thiophene 1,1-dioxide nucleus
also seems to be important for the cytotoxic activity: as GI50 of
4a, 5a and 7 reflects, carboxamide and sulfonamide, two electron
withdrawing groups, are both more active than hydroxy, an elec-
tron releasing group. These results, that point to the electrophilic
nature of the thiophene 1,1-dioxide moiety as the main cause of
the cytotoxic properties of benzo[b]thiophene 1,1-dioxide deriva-
tives, are well in agreement with the lost of cytotoxicity of the
BTS derivatives when the double bond of the thiophene was hydro-
genated.11,13 Interestingly, the benzo[b]thiophene nucleus of BTC
allows more simple cytotoxic compounds than other reported het-
eroaryl carboxamides. Thus, BTC 4b displays a slightly lower cyto-
toxic activity against K562 cells than the specifically designed
pyridocarboxamide KI (GI50 1.6 nM27). However, compound 4b is
more toxic against HeLa cervix carcinoma cells than reported N-
(4-aryl-2-thiazoly)benzamides or their equivalent pyridocarboxa-
mides34 and, it is also worth noting that 4b inhibits the growth
of both, HTB-54 lung carcinoma and CCRF-CEM lymphocytic
2. Results and discussion
BTC’s 4a–4d were obtained in 35–75% yield from the 6-amin-
obenzo[b]thiophene 1,1-dioxide 1 following typical methods as de-
scribed in Scheme 1. The 6-ciano derivative 2 was prepared by
diazotization and subsequent treatment of the mixture with cu-
prous cyanide at 40 °C. Acid 3 was obtained by refluxing 2 in 37%
hydrochloric acid. The reaction of 3 with thionyl chloride yielded
its acyl chloride, which was then treated with amines and triethyl-
amine at room temperature to give the corresponding amides 4. To
compare, new N-2-(4-methoxyphenyl)ethyl (5c) and N-benzyl-N-
methyl (6e) BTS derivatives and 6-hydroxybenzo[b]thiophene 1,1-
dioxide (7) were also prepared from 1 through known methods.2,32
The growth inhibitory activity of these compounds was tested
in vitro by examining their cytotoxic effects against HeLa (cervix
epitheloid carcinoma), HTB-54 (lung carcinoma), HT-29 (colon car-
cinoma), MEL-AC (melanoma), K-562 (myelocytic leukaemia) and
CCRF-CEM (lymphocytic leukaemia) cells through an MTT-based
colorimetric assay.33 The response parameter GI50, concentration
that reduces by 50% the growth of treated cells, is shown in
Table 1. As can be observed, under the described conditions BTC
derivatives 4a–4d strongly inhibited growth of every cell line
tested, with HeLa, HTB-54 and CCRF-CEM as the most sensitive
ones. As expected, N-substitution modulates BTC cytotoxicity:
compounds 4b, 4c and 4d that carry hydrophobic groups attached
NR1R1
R
O
O
N
O
Ar SO2
N
N
NH
N
N
H
N
H
H
N
F
S
O
S
R
O
O
O
O
O
OH
hBTC
BTC
KI
Figure 2.

A. A. Sagardoy et al. / Bioorg. Med. Chem. 18 (2010) 5701–5707
5703
i, ii
iii
iv, v
H
N
S
S
S
O
S
O
S
O
NH2
CN
COOH
R
O
O
O
O
O
1
O
2
4
3
O
vi
35-75%
vii
a -H
R=
b -C6H4-p-OCH3
c -C2H4C6H4-p-OCH3
d -C4H9
e -CH2C6H5
X
S
S
O
OH
5 X= NH
R
O
O
7
O
6 X= NCH3
O
Scheme 1. Reagents and conditions: (i) NaNO2, HCl (aq); (ii) CuCN (aq), ethyl acetate; (iii) HCl 37%, reflux; (iv) SOCl2 toluene, reflux; (v) RNH2, toluene, (vi) as previously
described,2 (vii) following the general procedure of Ref. 32.
Table 1
Cytotoxic activities (GI50, lM) of BTC 4a–4d, BTS 5a, 5c, 5e, 6e and hydroxy derivative 7 against tumour cell lines
Compound
4a R = –H
4b
HTB-54
0.22
K-562
7.85
MEL-AC
3.52
HT-29
5.19
CCRF-CEM
3.55
HeLa
0.65
0.002
0.64
0.047
2.66
0.76
1.32
0.35
2.08
0.004
0.73
0.003
0.040
R=
OCH3
4c
R=
OCH3
4d
0.09
2.73
1.42
2.32
0.28
0.08
2.21
R=
5a R = –H
5.89a
29.4a
7.14a
8.55a
2.86a
5c
5e
0.62
0.006
0.72a
1.93
0.22
0.005
0.33a
0.12
R=
OCH3
0.70a
0.40a
0.84a
0.010
R=
6e
0.40
7.53
38.1
51.1
2.30
24.0
35.0
2.04
0.63
19.2
0.010
N.D.
R=
7
a
From Ref. 2 N.D.: not determined.
leukaemia cells at levels that compare to those reported for the
better patented benzo[b][1,6]naphthyridine-4-carboxamides against
the related LLTC and P388 cell lines35 and to those reported for the
best ASU against H146 small lung cancer cells (GI50 30 nM,8 Fig. 1).
To further analyze the process of cell death induced by BTC
derivatives, the apoptotic status and cell cycle phase distribution
was determined in leukaemia CCRF-CEM cells. Apoptosis was eval-
uated by measuring the exposure of phosphatidylserine on the cell
ously reported for BTS,12 where induction of a typical apoptotic
process was observed. Cell cycle distribution was determined by
measurement of the DNA content of cells exposed to 2.5 lM of
the corresponding BTC for 24 h using propidium iodide (PI) stain-
ing and flow cytometry. Obtained results are shown in Figure 4.
A significant increase of the hypodipliod subG1 population was
detected for each one of the tested compounds, a result that con-
firms their role as inducers of apoptosis. At the same time a signif-
icant increase of the cell percent in the S phase, as well as a
reduction of the G0/G1 cell population was detected. These data
are consistent with cell cycle arrest at the S phase. Additional work
to unravel the specific regulatory proteins responsible for the cell
cycle arrest is under progress.
Because accumulation of ROS has been proposed to be an essen-
tial event in apoptotic responses induced by some antitumour
drugs,36–41 we next decided to test the effect of BTC on intracellular
ROS level. N-(4-Hydroxyphenyl)retinamide (4-HPR), which has
been reported to induce ROS overproduction on cervical carcinoma
membranes after 12–48 h of treatment with 0–6 lM of the corre-
sponding BTC. Obtained results are shown in Figure 3. As can be
observed, each one of the tested compounds was able to induce
apoptosis in a time and dose-dependent fashion. Moreover, as
aforementioned for the cytotoxic activity, induction of apoptosis
was stronger with compound 4b than with any other tested com-
pound. Thus, after 12 h of treatment with 6 lM 4b the incidence of
apoptotic cells at the culture was 30.4% (vs 2.6% in untreated con-
trol cells), a value that raised to 83% after 48 h of treatment (vs
2.9% in untreated control cells). This result resembles the previ-

5704
A. A. Sagardoy et al. / Bioorg. Med. Chem. 18 (2010) 5701–5707
4 b
3
120.00
100.00
80.00
60.00
40.00
20.00
0.00
*
control
2.5
2
*
*
1
µM
2.5
3.5
µ
M
M
*
µ
1.5
1
4
6
µ
M
M
µ
0.5
0
12h
24h
48h
4 c
control
4-HPR
4b
4c
4d
60.00
50.00
40.00
30.00
20.00
10.00
0.00
control
Figure 5. Incubation of CCRF-CEM cells with BTC induces an increase in ROS
production. Cells were incubated in the presence or absence (control) of 4b, 4c and 4d
(2.5 lM) for 24 h. N-(4-Hydroxyphenyl)retinamide (4-HPR) was used as a positive
control. Reactive oxygen species were measured using CM-H2DFDA. Results are
presented as the fold increased with respect to untreated control cells. Each bar
1
µM
2.5
3.5
µ
M
M
µ
4
6
µ
M
M
*
represents mean SD of two independent experiments (triplicate wells). p <0.05
respect to control cells.
µ
12h
24h
48h
40.00
4 d
*
120.00
100.00
80.00
60.00
40.00
20.00
0.00
control
1 µM
30.00
2.5 µM
3.5 µM
4 µM
#
20.00
6 µM
10.00
0.00
12h
24h
48h
control
control+NAC
4b+NAC
4b
Figure 3. Apoptotic effect of BTC 4b–4d at 12, 24 and 48 h. CCRF-CEM cells were
incubated in the presence of BTC 4b, 4c and 4d at the indicated concentration. The
percentage of apoptotic cells was determined by flow cytometry. Values represent
means SD of five independent experiments (duplicate wells).
Figure 6. NAC prevents the apoptotic process induced by compound 4b. The
percentage of apoptotic cells in the culture was determined by flow cytometry after
24 h in the absence (control) or presence of 2.5 lM 4b, 1 mM N-acetyl-cysteine
(NAC) or both (4b + NAC). Cells were preincubated with NAC for 1 h before 4b
addition. Results are presented as the mean SD of three independent experiments
(triplicate wells). *p <0.001with respect to the control, #p <0.001with respect to 4b.
SubG1 G0/G1
S
*
G2/M
60.00
50.00
40.00
30.00
20.00
10.00
0.00
*
*
*
sis through a mechanism mediated by ROS.36–41 Additional work is
required to find out the origin of BTC-induced ROS. In this sense,
we have previously shown that BTS are able to inhibit the tNOX
activity of the plasma membrane of CCRF-CEM and other tumour
cells.13,19 The putative implication of this enzyme and/or other
ROS-regulating systems of the cell in BTC-induced ROS generation
is an interesting issue to be analyzed in future experiments.
In conclusion, BTC is a novel family of potent cytotoxic com-
pounds that confirms the thiophene 1,1-dioxide nucleus as the true
lead to obtain improved antitumour agents. BTC induce cell cycle
arrest, ROS over generation and apoptosis in leukaemia CCRF-
CEM cells. Additional experiments are now in progress to help clar-
ify their mechanism of action and specificity towards tumour cells.
*
*
*
*
*
control
4b
4c
4d
Figure 4. BTC effect on cell cycle phase distribution. CCRF-CEM cells were treated
with 2.5 M 4b, 4c or 4d for 24 h. After treatment, cells were processed for propidium
iodide staining and cellular DNA content determined by flow cytometry. Data were
obtained from 10,000 events and the percentage of cells in the Sub G1 G0/G1, S and G2/
M phase was determined by EXPO 32 ADC Analysis System. Results are expressed as
l
*
mean SD of six independent experiments (duplicate wells). p <0.05 respect to
control cells.
3. Experimental
cells,42 was used as a positive control. As shown in Figure 5, BTC
derivatives induced a significant increase in intracellular ROS level.
To investigate if the observed accumulation of ROS was necessary
for the apoptotic effect of BTC, induction of apoptosis by 4b was
analyzed in the presence or absence of the antioxidant N-acetyl-
cysteine (NAC), which was added to the culture medium 1 h earlier
than 4b. As shown in Figure 6, the effect of 4b was inhibited by the
previous addition of NAC, indicating that BTC-induced ROS gener-
ation is a previous event required to trigger the apoptotic process.
In this sense, BTC are also similar to previously reported BTS12 and
to other antitumour drugs that have been shown to induce apopto-
Melting points were determined in a Mettler FP82HT-FP80 sys-
tem and are uncorrected. Routine monitoring of reactions was per-
formed using Allegra SilG/UV254 (0.20 mm). All chromatographic
separations were performed using silica gel (Merck 60 230–400
mesh). 1H NMR spectra were recorded on a Varian 200 MHz
spectrometer with TMS as internal standard. Chemical shifts are
reported in ppm and coupling constants in hertz. IR spectra were
recorded on a Nicolet-Avatar 360 FT-IR spectrophotometer. Elemen-
tal analyses were carried out on a Carlo Erba EA1108 elemental
analyser from vacuum-dried samples (over phosphorus pentoxide
at 3–4 mmHg, 6–12 h at about 30–70 °C).

A. A. Sagardoy et al. / Bioorg. Med. Chem. 18 (2010) 5701–5707
5705
3.1. Synthesis of precursors
3.2.2. N-(4-Methoxy)phenylbenzo[b]thiophene-6-carboxamide
1,1-dioxide (4b)
3.1.1. Benzo[b]thiophene-6-carbonitrile 1,1-dioxide (2)
Starting from the acid 3 and p-anisidine, compound 4b was ob-
tained and recrystallized from ethanol (0.25 g, 49.1%): Mp 215–
216 °C; IR (KBr, cmꢀ1): 1252, 1288 (SO2); 1669 (CO); 3381 (NH).
1H NMR (DMSO-d6 d): 10.37 (s, 1H, NH); 8.39 (s, 1H, H-7); 8.24
(d, J4–5 = 7.7 Hz; 1H, H-5); 7.76–7.72 (m, 2H, H-4, H-3); 7.68 (d,
6-Aminobenzo[b]thiophene 1,1-dioxide 12 (10.0 g, 55 mmol)
was dissolved in a mixture of water (100 mL) and HCl (37%,
10 mL, 121 mmol) by heating if necessary. This solution was cooled
at ꢀ10 °C and a solution of NaNO2 (4.0 g, 58 mmol) in water
(15 mL) was added drop wise. The resulting mixture was stirred
at ꢀ5 °C for 30 min to form the diazonium salt. The mixture was
cautiously neutralized by adding dry sodium carbonate with con-
stant stirring, using litmus paper to determine the end-point. A
solution of NaCN (12.4 g, 253 mmol) in water (60 mL) was added
to a solution of CuCl (9.8 g, 99 mmol) in water (60 mL) at T
<20 °C. This mixture was stirred at room temperature for 30 min
and then ethyl acetate (200 mL) was added. To this mixture, the
solution of the neutralized diazonium salt was slowly added with
stirring at 5 °C. Stirring was continued for 1 h at room temperature
and at 40 °C for 30 min. Cold water (500 mL) was added and the
mixture was extracted with ethyl acetate (2 ꢁ 200 mL). Extracts
were washed with NaHCO3 (aq, 3 ꢁ 200 mL, water), dried (MgSO4)
and evaporated at reduced pressure. The solid material was puri-
fied by column chromatography (dichloromethane) to give 6-cya-
J2´ -3 = 9.2 Hz, 2H, H-20, H-60); 7.54 (d, J2–3 = 6.6 Hz, 1H, H-2); 6.95
´
(d, 2H, H-30, H-50); 3.75 (s, 3H, O–CH3). Anal. Calcd (C16H13NO4S):
C, 60.94; H, 4.16; N, 4.44; S, 10.17. Found: C, 60.49; H, 4,41; N,
4.39; S, 10.28.
3.2.3. N-(4-Methoxyphenyl)ethylbenzo[b]thiophene-6-
carboxamide 1,1-dioxide (4c)
Starting from acid 3 and 2-(4-methoxyphenyl)ethylamine, com-
pound 4c was obtained and recrystallized from ethanol (0.18 g,
32.4%): Mp 149–150 °C; IR (KBr, cmꢀ1): 1252, 1288 (SO2); 1669
(CO); 3381 (NH); 1H NMR (DMSO-d6 d): 8.83 (t, JNH–CH2 = 5.5 Hz,
1H, NH); 8.21 (s, 1H, H-7); 8.12 (d, J5–4 = 7.9 Hz, 1H, H-5); 7.71–
7.67 (m, 2H, H-4, H-3); 7.51 (d, J2–3 = 7.0 Hz, 1H, H-2); 7.15 (d,
J2 –3 ,6 –5 = 8.4 Hz, 2H, H-20, H-60); 6.85 (d, 2H, H-30, H-50); 3.71 (s,
3H, –OCH3); 3.51–3.41 (m, 2H, NH–CH2–CH2); 2.79 (t, JCH2–CH2
0
0
0
0
=
nobenzo[b]thiophene 1,1-dioxide (4.79 g, 45.3%): IR (KBr, cmꢀ1
)
7.0 Hz, 2H, –CH2CH2–C6H4). Anal. Calcd (C18H17NO4S): C, 62.96;
H, 4.99; N, 4.08; S, 9.34. Found: C, 62.54; H, 4.82; N, 3.95; S, 9.32.
1151, 1309 (SO2); 2228 (CN); 1H NMR (CDCl3 d): 7.95 (s, 1H,
H-7); 7.86 (d, J4–5 = 7.7 Hz, 1H, H-4); 7.50 (d, 1H, H-5); 7.27 (d,
J3–2 = 7.0 Hz, 1H, H-3); 6.91 (d, 1H, H-2); Anal. Calcd (C9H5NO2S):
C, 56.53; H, 2.64; N, 7.33; S, 16.77. Found: C, 55.99; H, 2.73; N
7.07; S, 16.73.
3.2.4. N-Butylbenzo[b]thiophene-6-carboxamide 1,1-dioxide
(4d)
Starting from the acid 3 and n-butylamine, compound 4d was
obtained and recrystallized from hexane/ethyl acetate (0.23 g,
53.6%): Mp 121–122 °C; IR (KBr, cmꢀ1): 1148, 1304 (SO2); 1637
(CO); 3286 (NH2). 1H NMR (DMSO-d6 d): 8.73 (t, JNH-CH2 = 5.5 Hz,
1H, NH); 8.15 (s, 1H, H-7); 8.15 (d, J5–4 = 7.9 Hz, 1H, H-5); 7.70
(m, 2H, H-4, H3); 7.52 (d, J3–2 = 7.0 Hz, 1H, H-2); 3.27 (m 2H,
NH–CH2); 1.56–1.23 (m, 4H, CH2–CH2–CH3); 0.91 (t, JCH3–
CH2 = 7.3 Hz, 3H, –CH3). Anal. Calcd (C13H15NO3S): C, 58.85; H,
5.70; N, 5.28; S, 12.09. Found: C, 58,57; H, 5.89; N, 5.33; S, 12.02.
3.1.2. Benzo[b]thiophene-6-carboxylic acid 1,1-dioxide (3)
A suspension of benzo[b]thiophene-6-carbonitrile 1,1-dioxide 2
(4.36 g, 23 mmol) in HCl (37%, 25 mL, 0.30 mol) was refluxed for
8 h. The resulting yellow solid was filtered, washed with water
and dried to give the carboxylic acid 3 (3.02 g, 63.0%). IR (KBr,
cmꢀ1): 1159, 1301 (SO2); 1704 (CO); 3468 (OH).
3.2. General procedure for the synthesis of the benzo[b]thio-
phene-6-carboxamide 1,1-dioxide derivatives (4)
3.3. General procedure for the synthesis of the benzo[b]thi-
ophene-6-sulfonamide 1,1-dioxide derivatives (5 and 6)
A solution of the benzo[b]thiophene-6-carboxylic acid 1,1-diox-
ide 3 (0.34 g, 1.6 mmol) was treated with thionyl chloride (0.6 mL,
8.3 mmol) in boiling dry toluene (25 mL) for 30 min. The mixture
was evaporated to afford a crude solid of the corresponding acid
chloride which was identified by IR spectroscopy (1751, C@O). It
was sufficiently pure for the next step. A solution of the crude acid
chloride in dry toluene (20 mL) was added to a mixture of the
appropriate amine (1.60 mmol) and triethylamine (1.60 mmol) in
toluene (5 mL) (or CH2Cl2 for the butyl derivative) at 0 °C with stir-
ring. Stirring was continued for 30–60 min at room temperature.
Solvents were removed under vacuum. The residual material was
dissolved in ethyl acetate (100 mL), washed successively with 5%
HCl (2 ꢁ 50 mL), NaHCO3 (aq 2 ꢁ 50 mL) and water (50 mL), dried
over MgSO4 and evaporated. The solid was purified by crystalliza-
tion. The following products were obtained using the general
procedure.
Compounds 5 and 6 were obtained from 6-aminobenzo[b]thio-
phene 1,1-dioxide 1 and the corresponding amine by the method
previously reported by us.2
3.3.1. N-(4-Methoxyphenyl)ethylbenzo[b]thiophene-6-
sulfonamide 1,1-dioxide (5c)
Starting from 1 (0.33 g, 1.8 mmol) and 2-(4-methoxyphenyl)eth-
ylamine (0.27 g, 1.8 mmol), compound 5c was obtained and recrys-
tallized from 2-propanol (0.25 g, 36.0%): Mp 123–124 °C; IR (KBr)
cmꢀ1: 1141, 1305 (SO2); 1247 (Car–O–C); 3290 (NH). 1H NMR (CDCl3
d): 8.07 (s, 1H, H-7); 7.5 (d, J54 = 8.1 Hz, 1H, H-5); 7.44 (d, 1H, H-4);
´
0
7.25 (d, J32 = 7.0 Hz, 1H, H-3); 6.99 (d, J2 3,65 = 8.8 Hz, 2H, H-2, H-
´
´ ´
´
´
´
6); 6.88 (d, 1H, H-2); 6.79 (d, 2H, H-3, H-5); 4.44 (t, 1H, NH); 3.76
(s, 3H, –OCH3); 3.24 (c, 2H, –NHCH2–); 2.73 (t, J = 7.0 Hz, 2H, CH2–
CH2–C6H4). Anal. Calcd (C17H17NO5S2): C, 53.81; H, 4.52; N, 3.69; S,
16.90. Found: C, 54.20; H, 4.36; N, 3.60; S, 16.71.
3.2.1. Benzo[b]thiophene-6-carboxamide 1,1-dioxide (4a)
Trough a stirred solution of the acid chloride from 3 in dry tol-
uene (20 mL), ammonia gas was passed for 30 min. The solvent was
removed and the yellow solid was recrystallized from ethanol to
give compound 4a (0.25 g, 74.0%): Mp 216–217 °C; IR (KBr, cmꢀ1):
1129.36, 1160, 1290 (SO2); 1676 (CO); 3340, 3299 (NH2). 1H
NMR (CDCl3 d): 7.95 (s, 1H, H-7); 7.86 (d, J4–5 = 7.7 Hz, 1H, H-4);
7.50 (d, 1H, H-5); 7.27 (s, J3–2 = 7.0 Hz, 1H, H-3); 6.91 (s, 1H, H-
2). Anal. Calcd (C9H7NO3S): C, 51.67; H, 3.37; N, 6.69; S, 15.33.
Found: C, 51.59; H, 3.55; N, 6.46; S, 14.89.
3.3.2. N-Benzyl-N-methyl-benzo[b]thiophene-6-sulfonamide
1,1-dioxide (6e)
Starting from 1 (0.33 g, 1.8 mmol) and N-benzylmethylamine
(0.22 g, 1.8 mmol), compound 6e was obtained and recrystallized
from 2-propanol (0.13 g, 20%): Mp 105–106 °C; IR (KBr) cmꢀ1
:
1146, 1302 (SO2). 1H NMR (CDCl3 d): 8.10 (s, 1H, H-7); 8.02 (d,
J54 = 8.1 Hz, 1H, H-5); 7.54 (d, 1H, H-4); 7.32–7.24 (m, 6H, H-3,
C6H5); 6.90 (d, J23 = 7.0 Hz, 1H, H-2); 4.20 (s, 2H, –CH2–) 2.66 (s,

5706
A. A. Sagardoy et al. / Bioorg. Med. Chem. 18 (2010) 5701–5707
3H, –CH3). Anal. Calcd (C16H15NO4S2): C, 55.00; H, 4.33; N, 4.01; S,
18.35. Found: C, 54.99; H, 4.08; N, 3.87; S, 18.22.
Harvested cells (700 g, 6 min, 4 °C) were washed with PBS and
fixed in 70% ethanol at 4 °C 20 min. After fixation cells were recov-
ered by centrifugation, suspended in PBS containing IP (50
lg/ml)
3.3.3. 6-Hydroxybenzo[b]thiophene 1,1-dioxide (7)
and RNAse (200 g/ml) and incubated at 37 °C for 30 min. Cell cy-
l
Compound 7 was obtained following the method for prepara-
tion of phenols from diazonium ions via generation and oxidation
of aryl radicals by copper salts, previously reported.32 6-Amin-
obenzo[b]thiophene 1,1-dióxido 1 (0.31 g, 1.7 mmol) was dis-
solved at ꢀ5 °C in 3 mL of H2SO4 35%. Then a solution of NaNO2
(0.12 g, 1.7 mmol in water 1 mL) was dropwise added with stirring
at ꢀ5 °C. Subsequently a few crystals of urea were added to decom-
pose any excess sodium nitrite and then a solution of Copper(II) ni-
trate hemi(pentahydrate) (0.48 g, 2.1 mmol in water 90 mL) was
dropwise added with stirring at ꢀ5 °C. To the stirred mixture solid
Copper(I) oxide (0.24 g 1.7 mmol) was added and the stirring was
continued for 1 h at ꢀ5 °C. Cold water (50 mL) was added and
the mixture was extracted with ethyl acetate. The extracts were
dried, evaporated and the solid material was purified by column
chromatography (toluene/dioxane 4:3) to give 7 (0.05 g, 16.2%).
IR (HATR) cmꢀ1: 1146, 1288 (SO2); 3394 (OH). 1H NMR (DMSO-
d6 d): 10.61 (s, 1H, OH); 7.50 (d, J32 = 7.0 Hz, 1H, H-3); 7.38 (d,
J45 = 8.1 Hz, 1H, H-4); 7.11 (d, 1H, H-2); 7.1 (s, 1H, H-7); 7.0 (d,
1H, H-5). Ms: 182 [M+], 153, 125, 97, 89.
cle analysis was performed using a COULTER Epics XL flow cytom-
eter (Beckman Coulter, Miami, FL, USA). Cells were excited with an
argon laser emitting at 488 nm and propidium iodide was detected
using 620 nm band pass filter. Cell cycle distribution was deter-
mined by EXPO 32 ADC cell cycle analysis software. A doublet dis-
criminatory gate in a FL3/Aux FL3 plot was established to ensure
only authentic targeted events were permitted for analysis, and
G0/G1, S and G2/M regions were determined manually in a FL3-n
events histogram. Data from 10,000 cells were collected for each
data file.
3.4.5. Measurement of ROS
Cells were grown at a density of 1 ꢁ 106 cells/ml in a six well
plate and cultured in the presence or in the absence of compound
BTC (2.5 lM) for 24 h. Generation or intracellular ROS was exam-
ined using the oxidation-sensitive fluorescent probe CM-H2DCFDA
(Molecular Probes, Eugene, OR, USA). Briefly, the cells (2 ꢁ 106)
were incubated with 1 lM CM-H2DCFDA for 60 min at 37 °C. Sub-
sequently, the cellular fluorescence was analyzed using a Coulter
Epics XL flow cytometer (Beckman Coulter, Miami, Florida, USA).
3.4. Biological evaluation
Acknowledgement
3.4.1. Cells and cell culture
American Type Culture Collection (ATCC, Manassas, VA, USA) or
European Collection of Cell Cultures (ECACC, Porton Down, Salis-
bury, UK) provided human tumour cell lines. Six cell lines were
used: two human leukaemia (K-562 and CCRF-CEM) and four hu-
man solid tumours, one colon carcinoma (HT-29), one lung carci-
noma (HTB54), one cervix epitheloid carcinoma (HeLa) and one
melanoma (MEL-AC). MEL-AC cells were kindly provided by Dr.
Natalia López-Moratalla (Universidad de Navarra, Pamplona,
Spain). Cells were grown in RPMI 1640 medium (Invitrogen, Carls-
A.A.S. and R.V. were fellows from the Universidad Pública de
Navarra.
References and notes
1. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C. T. Curr. Med. Chem. 2003,
10, 925.
2. Villar, R.; Encio, I.; Migliaccio, M.; Gil, M. J.; Martinez-Merino, V. Bioorg. Med.
Chem. 2004, 12, 963.
3. Mader, M. M.; Shih, C.; Considine, E.; De Dios, A.; Grossman, C. S.; Hipskind, P.
A.; Lin, F. S.; Lobb, K. L.; Lopez, B.; Lopez, J. E.; Cabrejas, L. M. M.; Richett, M. E.;
White, W. T.; Cheung, Y. Y.; Huang, Z. P.; Reilly, J. E.; Dinn, S. R. Bioorg. Med.
Chem. Lett. 2005, 15, 617.
4. Lobb, K. L.; Hipskind, P. A.; Aikins, J. A.; Alvarez, E.; Cheung, Y. Y.; Considine, E.
L.; De Dios, A.; Durst, G. L.; Ferritto, R.; Grossman, C. S.; Giera, D. D.; Hollister, B.
A.; Huang, Z. P.; Iversen, P. W.; Law, K. L.; Li, T. C.; Lin, H. S.; Lopez, B.; Lopez, J.
E.; Cabrejas, L. M. M.; McCann, D. J.; Molero, V.; Reilly, J. E.; Richett, M. E.; Shih,
C.; Teicher, B.; Wikel, J. H.; White, W. T.; Mader, M. M. J. Med. Chem. 2004, 47,
5367.
bad, CA) supplemented with 10% faetal calf serum, 2 mM
L
-gluta-
mine, 100 U mlꢀ1 penicillin, 100
10 mM HEPES buffer (pH 7.4).
l
g mlꢀ1 streptomycin and
3.4.2. Cytotoxicity analysis
The cytotoxic effect of each substance was tested at five differ-
ent doses between 0.01 and 100 M. In the case of compound 4b
additional doses between 10ꢀ5 and 10ꢀ2
M were tested. Each sub-
l
5. Bouissane, L.; El Kazzouli, S.; Léonce, S.; Pfeiffer, B.; Rakib, E. M.; Khouili, M.;
Guillaumet, G. Bioorg. Med. Chem. 2006, 14, 1078.
l
stance was initially dissolved in DMSO at a concentration of 0.1 M,
and serial dilutions were prepared using culture medium. The
plates with cells from the different lines, to which media contain-
ing the substance under test were added, were incubated for 72 h
at 37 °C in a humidified atmosphere containing 5% CO2. Cell viabil-
ity was then determined by assaying for the reduction of MTT to
formazan.33
6. Brzozowski, Z.; Saczewski, F.; Slawinski, J.; Bednarski, P. J.; Grünert, R.; Gdaniec,
M. Bioorg. Med. Chem. 2007, 15, 2560.
7. Slawinski, J.; Brzozowski, Z. Eur. J. Med. Chem. 2006, 41, 1180.
8. Park, C. M.; Bruncko, M.; Adickes, J.; Bauch, J.; Ding, H.; Kunzer, A.; Marsh, K. C.;
Nimmer, P.; Shoemaker, A. R.; Song, X.; Tahir, S. K.; Tse, C.; Wang, X. L.; Wendt,
M. D.; Yang, X. F.; Zhang, H. C.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J.
Med. Chem. 2008, 51, 6902.
9. Elmore, S. W.; Bruncko, M.; Park, C. PCT US 2010/0035879, WO 2005/117543.
Abbott Lab., 2010.
10. Mohan, R.; Banerjee, M.; Ray, A.; Manna, T.; Wilson, L.; Owa, T.; Bhattacharyya,
B.; Panda, D. Biochemistry 2006, 45, 5440.
11. Martínez-Merino, V.; Gil, M. J.; Encío, I.; Migliaccio, M.; Arteaga, C. PCT WO
2000/063202, 2000.
12. Alonso, M. M.; Asumendi, A.; Villar, J.; Gil, M. J.; Martinez-Merino, V.; Encio, I.
J.; Migliaccio, M. Oncogene 2003, 22, 3759.
13. Alonso, M. M.; Encio, I.; Martinez-Merino, V.; Gil, M.; Migliaccio, M. Br. J. Cancer
2001, 85, 1400.
14. Morre, D. J.; Wu, L. Y.; Morre, D. M. Biochim. Biophys. Acta 1995, 1240, 11.
15. Howbert, J. J.; Grossman, C. S.; Crowell, T. A.; Rieder, B. J.; Harper, R. W.;
Kramer, K. E.; Tao, E. V.; Aikins, J.; Poore, G. A.; Rinzel, S. M.; Grindey, G. B.;
Shaw, W. N.; Todd, G. C. J. Med. Chem. 1990, 33, 2393.
16. Winum, J. Y.; Rami, M.; Scozzafava, A.; Montero, J. L.; Supuran, C. Med. Res. Rev.
2008, 28, 445.
3.4.3. Assessment of apoptosis
The apoptotic status of the cells was evaluated by measuring
the exposure of phosphatidylserine on the cell membranes using
Annexin V-FITC Kit (BD Pharmingen, San Jose, CA, USA)43 under
the conditions described by the manufacturer. Briefly, after incuba-
tion with and without BTC, cells (5 ꢁ 105) were pelleted and
washed in PBS. Cells were then stained with annexin V-FITC and
propidium iodide for 15 min at 4 °C in the dark and analyzed on
a Coulter Epics XL flow cytometer (Beckman Coulter, Miami, Flor-
ida, USA).
17. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
18. Innocenti, A.; Villar, R.; Martinez-Merino, V.; Gll, M. J.; Scozzafava, A.; Vullo, D.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 4872.
19. Encio, I.; Morre, D. J.; Villar, R.; Gil, M. J.; Martinez-Merino, V. Br. J. Cancer 2005,
92, 690.
3.4.4. Cell cycle analysis
CCRF-CEM cells were cultured in complete medium in six-well
plates (106 cells/ml; 3 ml) for 24 h with o without BTC at 37 °C.

A. A. Sagardoy et al. / Bioorg. Med. Chem. 18 (2010) 5701–5707
5707
20. Kishimoto, T.; Katsube, N.; Konishi, M.; Cono, M. PCT US 2003/0073706, US
6555555, US 6420391, WO 1999/051587, Ono Pharmaceutical Co. Ltd, 2003.
21. Elbe, H.; Tiemann, R.; Dutzmann, S.; Stenzel, K.; Kugler, M.; Wachtler, P. PCT
WO 1998/045283, Bayer Ag, 1998.
22. Seefeld, M. A.; Rouse, M. B.; Heerding, D. A.; Peace, S.; Yamashita, D. S.;
Mcnulty, K. C. PCT WO 2008/098104 Smithkline Beecham Corp., 2008.
23. Graneto, M.; Hanau, C. E.; Perry, T. D. PCT WO 2003/037886, Pharmacia Corp.,
2003.
24. Wickens, P.; Kluender, H. C. E.; Hong, Z.; Kumarasinghe, E. S.; Kreiman, C.;
Zhang, M.; Enyedy, I.; Chuang, C.-Y. PCT WO 2006/133006 Bayer
Pharmaceuticals Corp., 2006.
25. Chidambaram, R.; Derbin, G. M.; Endo, M.; Zh Gao, J.; Lee, T.; Motheram, R.;
Parker, W. L.; Rosso, V. W.; Varia, S. A. PCT WO 2007/035874, Squibb Bristol
Myers Co., 2007.
26. Shipps, G. W. Jr.; Richards, M.; Cheng, C. C.; Huang, X. PCT WO 2009/058729,
Schering Corp., 2009.
27. Smith, R.; Nagarathnam, D. PCT WO 2008/079968, Bayer Pharmaceuticals
Corp., 2008.
28. Liang, C.; Li, Z. PCT WO 2009/154769 Xcovery Inc., 2009.
29. Graham, L.; Isbester, P.; Lapina, O. V.; Mobele, B. I.; Palmer, G. J.; Reineke, K.;
Salsbury, J. S.; Ulysse, L. PCT WO 2009/129191, Takeda Pharmaceutical, 2009.
30. Ginn, J. D.; Sorcek, R. J.; Turner, M. R.; Young, E. R. R. PCT WO 2007/146602,
Boehringer Ingelheim Int., 2007.
31. Safety Data Sheet, <http://www.sigmaaldrich.com/safety-center.html>, 2010.
32. Cohen, T.; Dietz, A. G.; Miser, J. R. J. Org. Chem. 1977, 42, 2053.
33. Mosmann, T. J. Immunol. Met. 1983, 65, 55.
34. Qiu, X. L.; Li, G. D.; Wu, G. K.; Zhu, J. W.; Zhou, L. E.; Chen, P. L.; Chamberlin, A.
R.; Lee, W. H. J. Med. Chem. 2009, 52, 1757.
35. Baguley, B. C.; Deady, L. W.; Denny, W. A.; Rodemann, T.; Rogers, M. L. PCT WO
2003/097642, Univ. Trobe; Auckland Uniservices Ltd, 2003.
36. Gao, N.; Rahmani, M.; Shi, X. L.; Dent, P.; Grant, S. Blood 2006, 107, 241.
37. Gupta, S. Int. J. Oncol. 2003, 22, 15.
38. Matsunaga, N.; Kaku, T.; Itoh, F.; Tanaka, T.; Hara, T.; Miki, H.; Iwasaki, M.;
Aono, T.; Yamaoka, M.; Kusaka, M.; Tasaka, A. Bioorg. Med. Chem. 2004, 12,
2251.
39. Morales, M. C.; Perez-Yarza, G.; Rementeria, N. N.; Boyano, M. D.; Apraiz, A.;
Gomez-Munoz, A.; Perez-Andres, E.; Asumendi, A. Free Radical Res. 2007, 41,
591.
40. Boutonnat, J.; Barbier, M.; Muirhead, K.; Mousseau, M.; Grunwald, D.; Ronot,
X.; Seigneurin, D. Cytometry 2000, 42, 50.
41. Costantini, P.; Jacotot, E.; Decaudin, D.; Kroemer, G. J. Natl. Cancer Inst. 2000, 92,
1042.
42. Oridate, N.; Suzuki, S.; Higuchi, M.; Mitchell, M. F.; Hong, W. K.; Lotan, R. J. Natl.
Cancer Inst. 1997, 89, 1191.
43. Fimognari, C.; Nusse, M.; Cesari, R.; Iori, R.; Cantelli-Forti, G.; Hrelia, P.
Carcinogenesis 2002, 23, 581.