Synthesis and cytotoxic activity of lipophilic sulphonamide derivatives of the benzo[b]thiophene 1,1-dioxide

Article abstract of DOI:10.1016/j.bmc.2003.12.012

In the search of new compounds with antineoplastic activity, we have analysed the effect of several structural modifications on the nucleus 6-benzo[b]thiophenesulphonamide 1,1-dioxide on its cytotoxic activity on tumour cells. Lipophilic substituents on t

Full text of DOI:10.1016/j.bmc.2003.12.012

Bioorganic & Medicinal Chemistry 12 (2004) 963–968
Synthesis and cytotoxic activity of lipophilic sulphonamide
derivatives of the benzo[b]thiophene 1,1-dioxide
R. Villar,a I. Encio,b,* M. Migliaccio,b M. J. Gila and V. Martinez-Merinoa,*
a
´
´
Dpto. de Quımica, Universidad Pu´blica de Navarra, Campus Arrosadıa, 31006 Pamplona, Spain
bDpto. de Ciencias de la Salud, Universidad Pu´blica de Navarra, Avda. Baran˜ain, 31008 Pamplona, Spain
Received 22 October 2003; accepted 12 December 2003
Abstract—In the search of new compounds with antineoplastic activity, we have analysed the effect of several structural modifi-
cations on the nucleus 6-benzo[b]thiophenesulphonamide 1,1-dioxide on its cytotoxic activity on tumour cells. Lipophilic sub-
stituents on the sulphonamide group significantly increased the cytoxic activity measured using a panel of human tumour cell lines.
Only slight variations on cytotoxicity were obtained when the sulphonamide group occupied the position 5 of the system. The most
active compound was the N-4-methoxyphenyl derivative 15, which showed GI50 values of 1–9 nM against HT-29, CCRF-CEM, K-
562 and MEL-AC cells and of 200 nM against HTB-54 cells. Free access to the 3-position of the heterocyclic system seems to be
required to obtain cytotoxic derivatives. Derivative 15 was also active at the same level of commercial Doxorubicine against cul-
tured normal human lung fibroblasts.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
developed from antineoplastic diarylsulphonylureas4 by
a 3D-QSAR model.5,6 Despite of the interesting char-
acteristics described above, cytotoxicity of the lead
compound 1 (Fig. 1) against tumour cells was observed
at doses moderately high (GI50>2 mM). Therefore, we
decided to look for new derivatives with stronger anti-
neoplastic activity.
A series of benzo[b]thiophenesulphonamide 1,1-dioxide
derivatives (BTS) were recently reported as a new class
of potential antineoplastic agents.1 These compounds
showed a clear correlation between their cytotoxic
activities and their ability to inhibit an specific NADH
oxidase located at the membrane of leukaemia CCRF-
CEM cells.2 Moreover, unsubstituted BTS and their
corresponding sulphonylurea derivatives induced an
apoptotic process in CCRF-CEM cells that is mediated
by the generation of reactive oxygen species (ROS), and
includes typical apoptotic features such as cell shrink-
age, PS translocation to the cell surface, mitochondrial
dysfunction, caspase activation, chromatin condensa-
tion and internucleosomal DNA degradation.3 Thus,
these compounds may exert its effect by affecting the
activity of enzymes related with the control of cellular
ROS levels. Up to now, unsubstituted BTS showed bet-
ter properties as antitumour agents than the corres-
ponding benzo[b]thiophenesulphonylureas, which were
Figure 1.
Some enzymes related with ROS control such as the
glutathione reductase and the glutathione S-transferase,
present a hydrophobic pocket near their active site.7,8
Thus, new benzothiophene 1,1-dioxide derivatives 6–20
were designed carrying hydrophobic substituents of dif-
ferent length and grade of flexibility on the sulphona-
mide group. Moreover, some of them possess groups
with capacity to form hydrogen bonds. Additionally, to
analyse different distances between polar groups, the
sulphonamide group was inserted at the positions 5 and
6 of the benzo[b]thiophene nucleus (Scheme 1). Since
the reaction of the thiol groups at the position 3 of the
system was previously pointed out2 as a determinant
Keywords: Benzo[b]thiophenesulphonamide 1,1-dioxide derivatives;
Cytotoxic activity; Synthesis; Structure–activity relationships;
Lipophilicity.
* Corresponding author. Tel.: +34-948-169590; fax: +34-948-169606;
e-mail: merino@unavarra.es
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.012

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R. Villar et al. / Bioorg. Med. Chem. 12 (2004) 963–968
factor in the cytotoxic activity of this kind of com-
pounds,2 the series also explores substituents that would
hinder such reaction. Our results demonstrate that some
of the synthesised derivatives of the benzo[b]thiophene
1,1-dioxide presented a strong, highly increased cyto-
toxic activity on human colon carcinoma (HT-29),
lung carcinoma (HTB-54), lymphocytic leukaemia
(CCRF-CEM), myelocytic leukaemia (K-562) and mel-
anoma (MEL-AC) cell lines.
The amino derivatives 4 and 5 were transformed into
the corresponding chlorosulphonyl derivatives using the
Meerwein’s method11 (treatment of diazonium salts
with sulphonyl chloride in the presence of cuprous
chloride), and then treated with either ammonia or
amines to give the 6-benzo[b]thiophenesulphonamide
1,1-dioxide derivatives 6–17 (12-95% yield) or the
5-benzo[b]thiophenesulphonamide 1,1-dioxide deriv-
atives 18–20 (30–72% yield) respectively. Spectroscopic
properties of all compounds were in agreement with
their structures.
2. Chemistry
BTS derivatives 6–20 were prepared as shown in
Scheme 1. The oxidation of benzo[b]thiophene 2 (R2=H,
CH3) with 30% hydrogen peroxide, and the subsequent
nitration of the 1,1-dioxide intermediate by the Chal-
lenger’s method9 using nitric acid (d=1.52 g/mL) gave
the corresponding 6-nitrobenzo[b]thiophene 1,1-dioxides
(60% yield). The latest compounds were reduced to 6-
aminobenzo[b]thiophene 1,1-dioxide 4 (R2=H, CH3)
with iron-ammonium chloride (87% yield). The 5-amino
isomer 5 was prepared from the ethyl 5-nitro-2-
benzo[b]thiophenecarboxylate 3, which was obtained
following the method described by Rossi and Trave.10
Compound 3 was hydrolysed and then decarboxylated
by heating in cooper/quinoline under nitrogen atmos-
phere to give the 5-nitrobenzo[b]thiophene (90% yield),
which was then oxidised to its 1,1-dioxide by using 3-
chloroperoxybenzoic acid, and the nitro group selec-
tively reduced with iron-ammonium chloride to give the
5-aminobenzo[b]thiophene 1,1-dioxide 5 (52% yield).
3. Results and discussion
Cytotoxic activity of the lead compound 1 and sulpho-
namides 6–20 against five human tumour cell lines are
displayed in Table 1. All compounds were tested in the
range of 0.01-100 mM, or lower when GI50 was inferior
to 10 nM.12 As shown in Table 1, though CCRF-CEM
and MEL-AC cells were generally more sensitive and
K-562 more resistant to the cytotoxic effect, growth of
every cell line was clearly affected by all the tested com-
pounds and it is worth noting that all compounds were
more cytotoxic against each one of the tested cell lines
than the lead compound 1. Figure 2 shows exemplary
curves with the original data from which the GI50 values
for compounds 7, 10, 13 and 15 in K-562 and HT-29
cells were calculated.
The influence of the sulphonamide position on cyto-
toxicity seems to be weak. When 5 and 6 BTS’s were
Scheme 1. (i) Acetic acid, H2O2 30% (v/v), reflux, 30 min; (ii) nitric acid 100%; (iii) NaOH, Ethyl alcohol/water 50%; Cu/Quinolin 200 ꢀC; (iv)
MCPBA; (v) Fe/NH4Cl, ethanol/H2O 50% (v/v); (vi) NaNO2, HCl (ac); SO2/CuCl, Acetic acid; (vii) NH2R, dioxane.
Table 1. Cytotoxic activities (GI50, mM inhibition of cell growth) against tumoural cell lines and lipophilicity (logP) of BTS
Compd
R1
R2
HT-29
HTB-54
CCRF-CEM
K-562
MEL-AC
LogP
1
6
7
8
H
C2H5
C4H9
CH2C3H5
C6H13
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
8.55
2.80
0.60
5.89
0.06
0.79
2.86
2.11
0.08
0.214.12.00
3.3
0.28
3.30
3.13
2.70
4
29.417.1
6.57
0.810.73
ꢁ0.03
1.25
2.31
1.70
3.37
2.49
0.77
0.33
1.91
1.80
2.34
3.16
0.49
2.31
2.49
2.34
3.00
a
0.51N.D.
6
9
1.46
0.80
5.87
0.47
1.51
2.79
0.007
0.29
N.D.a
0.72
2.50
1.38
0.90
1.92
0.20
N.D.a
2.48
0.710.39
4.86
20.68
2.39
2.09
0.06
10
11
12
13
14
15
16
17
18
19
20
CH2C6H5
C2H4OCH3
C4H8OH
C3H6COOC2H5
C4H8COOC2H5
p-C6H4OCH3
m-C6H4Cl
H
5.49
4.46
2.89
1.12
0.72
0.005
0.210.46 8
0.0010.009
0.1
>100
>100
>100
>100
>100
0.78
0.27
0.44
C4H9
CH2C6H5
m-C6H4OCH3
2.84
2.38
4.06
0.49
0.14
0.57
2.78
1.58
0.23
2.67
1.26
1.41
H
H
a Not determined.

R. Villar et al. / Bioorg. Med. Chem. 12 (2004) 963–968
965
Figure 3. BTS’s Cytotoxicities (pGI50=ꢁlogGI50) against MEL-AC
(*) and K-562 (&) cells vs logP.
(q2) increased from 0.11 to 0.62 for the linear relation of
K562 pGI50 vs logP, and from 0.51to 0.81for the linear
relation of MEL-AC pGI50 vs logP.
BTS 15 was as active as commercial Doxorubicine both
against CCRF-CEM and MEL-AC tumour cells
(GI50=33 nM and 6 nM respectively), and normal
human lung fibroblasts growing in culture (GI50=8 nM
for 15 and 6 nM for Doxorubicine).
In conclusion, BTS are a novel class of potential anti-
tumour agents that includes compounds as cytotoxic as
some commercial drugs. BTS 15 could be a new lead
compound where it should be possible to introduce
structural modifications to gain specificity towards
tumour cells.
Figure 2. Determination of the cytotoxic effect of BTS 7, 10, 13 and 15
on K-562 and HT-29 cells. Cells were incubated in the presence of
every compound at the indicated concentration for 72 h. Cytotoxicity
was then determined by a colorimetric microassay based on the use of
MTT12. Values represent meansꢂSD derived from 3 independent
experiments each performed in quadruplicate.
compared (7 vs 18, 10 vs 19 and, in less degree 16 vs 20),
their cytotoxic activities were within the same order of
magnitude, except for the butyl derivative against
CCRF-CEM cells (Table 1).
4. Experimental
4.1. General
On the other hand, the only presence of a methyl group
at position 3 of the heterocyclic system, as in compound
17, abolished the cytotoxic activity against all tested cell
lines. This fact could be related with a steric hinder on
3-position of BTS towards the thiol reactivity at the
active site of a protein involved in the ROS response.
The lack of cytotoxicity of the 2,3-dihydro-6-benzo[b]-
tiophenesulphonamide 1,1-dioxide,2 a compound with
low reactivity at position 3 with thiol groups, further
supports this idea. To confirm this hypothesis, addi-
tional studies about the mode of action of BTS’s are
under progress.
Melting points were determined in
a
Mettler
FP82HT+FP80 apparatus and are uncorrected. Rou-
tine monitoring of reactions was performed using Alu-
gram SilG/UV254 (0.20 mm). All chromatographic
separations were performed using silica gel (Merck 60
1
230–400 mesh). H NMR spectra were recorded on a
Varian 200 MHz spectrometer with TMS as internal
standard. Chemical shifts are reported in ppm and cou-
pling constants in Hz. IR spectra of precursors were
recorded on an Avatar 360 FT-IR spectrophotometer.
Elemental 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).
In some cases, a clear correlation could be observed
between the lipophilicity (logP)13 and the cytotoxic
activity of the active compounds (pGI50 in Fig. 3). The
observed correlation most probably reflects the interac-
tion with a target molecule rather than a transport phe-
nomenon. However, since the 4-methoxyphenyl
derivative 15 was strongly active, other properties than
lipophilicity should be involved in the cytotoxicity of
these compounds, as it has been pointed above. When
the compound 15 was excluded from the statistical
analysis, leave-one-out square correlation coefficient
4.1.1. General procedure for the synthesis of the
benzo[b]thiophenesulphonamide 1,1-dioxide derivatives
(6–20). To a stirred solution of the corresponding ami-
nobenzo[b]thiophene 1,1-dioxide 4, 5 (1.84 mmol) in
hydrochloric acid (37% v/v, 6 mL) and glacial acetic
acid (3 mL) at ꢁ10 ꢀC, a solution of 0.13 g (1.9 mmol)
of NaNO2 in 1mL of water was drop wise added, and
the stirring was continued at room temperature for 1h.
That mixture was cooled at 0 ꢀC and then drop wise

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R. Villar et al. / Bioorg. Med. Chem. 12 (2004) 963–968
added to a stirred mixture of 0.043 g (0.43 mmol) of
CuCl in glacial acetic acid (3 mL) at 0 ꢀC previously
saturated with SO2. The resulting mixture was further-
more stirred at room temperature for 1h. Cold water
and ice (250 g) were added and the obtained solid was
collected and dried to give the corresponding chloro-
sulphonyl derivative which was used without further
purification. To a solution of the above chlorosulphonyl
derivative (1.84 mmol) in dioxane (5 mL), a solution of
the appropriate amine (1.84 mmol) and triethylamine
(1.84 mmol) in dioxane (5 mL) was added with stirring.
(Compound 17 was prepared by passing ammonia gas
through a stirred solution of the 6-chlorosulphonyl
derivative). Stirring was continued for 30–60 min at
room temperature. Solvents were removed in vacuum.
The residual material was dissolved in dichloromethane
(100 mL), washed successively with 5% HCl (2ꢃ50 mL)
and saturated aqueous NaHCO3 (2ꢃ50 mL), dried over
MgSO4 and evaporated. The solid was purified by crys-
tallization or by chromatography.
4.1.5. N-hexyl-6-benzo[b]thiophenesulphonamide 1,1-di-
oxide (9). Starting from 6-chlorosulfonylbenzo[b]thio-
phenesulphonamide 1,1-dioxide and hexylamine,
compound 9 was obtained and recrystallised from iso-
propanol (30%): Mp 102–103 ꢀC; IR (HATR, cmꢁ1):
1145, 1310 (SO2), 3257 (NH); 1H NMR (CDCl3 d): 8.15
(s, 1H, H-7), 8.05 (d, J54=8.06 Hz, 1H, H-5), 7.50 (d,
1H, H-4), 7.27 (d, J32=6.96 Hz, 1H, H-3), 6.88 (d,
1H, H-2), 4.49 (s, 1H, NH), 2.96 (m, 2H, NH–CH2–),
1.45 (m, 2H, NH–CH2–CH2–), 1.22 (m, 6H,
ꢁCH2 ꢁCH2 ꢁCH2–CH3), 0.84 (t, J=6.22 Hz, 3H,
–CH3). Anal. calcd (C14H19NO4S2): C, 51.04; H, 5.81;
N, 4.25; S, 19.47. Found: C, 51.20; H, 6.16; N, 4.26; S,
19.73.
4.1.6. N-benzyl-6-benzo[b]thiophenesulphonamide 1,1-di-
oxide (10). Starting from 6-chlorosulfonylbenzo[b]thio-
phenesulphonamide 1,1-dioxide and benzylamine,
compound 10 was obtained and recrystallised from iso-
propanol (58,1%): Mp 120 ꢀC; IR (KBr, cmꢁ1): 1322,
1
1151 (SO2), 3275 (NH); H NMR (CDCl3 d): 8.10 (s,
In this manner, the following products were obtained.
1H, H-7), 7.99 (d, J54=8.06 Hz, 1H, H-5), 7.43 (d, 1H,
H-4), 7.33–7.15 (m, 6H, H-3, C6H5), 6.87 (d, 1H, H-2),
5.01(t, J=5.86 Hz, 1H, NH), 4.19 (d, 2H, –CH2–
C6H5). Anal. calcd (C15H13NO4S2): C, 53.72; H, 3.91;
N, 4.18; S, 19.12. Found: C, 53.22; H, 3.94; N, 4.17; S,
18.41.
4.1.2. N-ethyl-6-benzo[b]thiophenesulphonamide 1,1-dioxide
(6). Starting from 6-chlorosulfonylbenzo[b]thiophene-
sulphonamide 1,1-dioxide and ethylamine hydrochlo-
ride, compound 6 was obtained and recrystallised from
ethanol (36%): Mp 162.5–163.5 ꢀC; IR (HATR cmꢁ1
)
1149, 1300 (SO2), 3281(NH). 1H NMR (CDCl3 d): 8.14
(s, 1H, H-7), 8.05 (d, J54=7.69 Hz, 1H, H-5), 7.51 (d,
1H, H-4), 7.27 (d, J32=6.96 Hz, 1H, H-3), 6.89 (d, 1H,
H-2), 4.55 (t, 1H, NH), 3.05 (m, 2H, NH–CH2–); 1.14
(t, 3H, CH3). Anal. calcd (C10H11NO4S2): C, 43.94; H,
4.06; N, 5.12; S, 23.46. Found: C, 44.01; H, 4.27; N,
5.04; S, 24.11.
4.1.7. N-(2-metoxy)ethyl-6-benzo[b]thiophenesulphonamide
1,1-dioxide (11). Starting from 6-chlorosulfonyl-
benzo[b]thiophenesulphonamide 1,1-dioxide and 2-
methoxyethylamine, compound 11 was obtained and
recrystallised from isopropanol (25,4%): Mp 126–
128 ꢀC; IR (KBr, cmꢁ1): 1151, 1301 (SO2), 3299 (NH).
1H NMR (CDCl3 d): 8.16 (s, 1H, H-7), 8.06 (d,
J54=7.69 Hz, 1H, H-5), 7.50 (d, 1H, H-4), 7.26 (d,
J32=6.96 Hz, 1H, H-3), 6.88 (d, 1H, H-2), 4.94 (t,
J=5.86 Hz, 1H, NH), 3.42 (t, J=5.13, 2H, –CH2O–),
3.21(s, 3H, –CH 3), 3.17 (m, 2H, NH–CH2–CH2). Anal.
calcd (C11H13NO5S2): C, 43.55; H, 4.32; N, 4.62; S,
21.14. Found: C, 43.78; H, 4.39; N 4.59; S, 21.31.
4.1.3. N-butyl-6-benzo[b]thiophenesulphonamide 1,1-di-
oxide (7). Starting from 6-chlorosulfonylbenzo[b]thio-
phenesulphonamide 1,1-dioxide and butylamine, com-
pound 7 was obtained and recrystallised from isopropanol
(71%): Mp 117 ꢀC; IR (HATR cmꢁ1): 1154, 1316 (SO2),
3306 (NH); 1H NMR (CDCl3 d): 8.19 (s, 1H, H-7), 8.09
(d, J54=7.69 Hz, 1H, H-5), 7.55 (d, 1H, H-4) 7.32 (d,
J32=6.96 Hz, 1H, H-3), 6.93 (d, 1H, H-2), 4.52 (t,
J=5.86 Hz, 1H, NH), 3.03 (m, 2H, NH–CH2–), 1.59–
1.22 (m, 4H, –CH2–CH2–CH3), 0.91(t, J=7.33 Hz,
3H, –CH3). Anal. calcd (C12H15NO4S2): C, 47.82; H,
5.02; N, 4.65; S, 21.28. Found: C, 48.29; H, 5.34; N,
4.63; S, 21.29; MS: 301, 258, 229, 165.
4.1.8. N-(4-hydroxy)butyl-6-benzo[b]thiophenesulphon-
amide 1,1-dioxide (12). Starting from 6-chlorosulfonyl-
benzo[b]thiophenesulphonamide 1,1-dioxide and 4-
amino-1-butanol, compound 12 was obtained and and
purified by column chromatography (ethyl acetate)
(44,6%): Mp 115.7–116.5 ꢀC;. IR (KBr, cmꢁ1): 1146,
1310 (SO2), 3200–3600 (bs, NH, OH); 1H NMR (CDCl3
d): 8.12 (s, 1H, H-7), 8.01 (d, J54=8.06 Hz, 1, H-5), 7.46
(d, 1H, H-4), 7.23 (d, J32=6.96 Hz, 1H, H-3), 6.84 (d,
1H, H-2), 5.48 (t, J=5.86 Hz, 1H, NH), 3.58 (t, J=5.86,
2H, –CH2–OH), 2.97 (m, 2H, NHCH2–), 1.90 (s, 1H,
OH), 1.55 (m, 4H, –CH2CH2–CH2OH). Anal. calcd
(C12H15NO5S2 ): C, 45.41; H, 4.76; N, 4.41; S, 20.21.
Found: C, 45.33; H, 4.74; N 4.30; S, 21.31.
4.1.4. N-cyclopropylmethyl-6-benzo[b]thiophenesulphon-
amide 1,1-dioxide (8). Starting from 6-chloro-
sulfonylbenzo[b]thiophenesulphonamide 1,1-dioxide and
aminomethylciclopropane hydrochloride, compound 8
was obtained and recrystallised from isopropanol (31%):
Mp 161–162 ꢀC; IR (KBr, cmꢁ1): 1146, 1311 (SO2), 3287
1
(NH); H NMR (CDCl3 d): 8.16 (s, 1H, H-7), 8.06 (d,
J54=7.69 Hz, 1H, H-5), 7.50 (d, 1H, H-4), 7.27 (d,
J32=6.96 Hz, 1H, H-3), 6.88 (d, 1H, H-2), 4.69 (t,
J=5.86 Hz, 1H, –NH), 2.88 (m, 2H, –CH2–), 0.93–0.82
(m, 1H), 0.54–0.45 (m, 2H), 0.16–0.89 (m, 2H). Anal.
calcd (C12H13NO4S2): C, 48.14; H, 4.38; N, 4.68; S,
21.42. Found: C, 47.93; H, 4.14; N, 4.58; S, 20.18.
4.1.9. Ethyl 4-(6-benzo[b]thiophenesulphonamido 1,1-di-
oxide)butyrate (13). Starting from 6-chlorosulfonyl-
benzo[b]thiophenesulphonamide 1,1-dioxide and ethyl
4-aminobutyrate hydrochloride, compound 13 was
obtained and and purified by column chromatography
(hexane 2:3 ethyl acetate) (95%): Mp 61 ꢀC; IR (KBr,

R. Villar et al. / Bioorg. Med. Chem. 12 (2004) 963–968
967
cmꢁ1): 1300, 1169 (SO2), 1706 (COOEt), 3244 (NH); 1H
NMR (CDCl3 d): 8.18 (s, 1H, H-7), 8.08 (d, J54=7.69
Hz, 1H, H-5), 7.54 (d, 1H, H-4), 7.31 (d, J32=6.96 Hz,
1H, H-3), 6.93 (d, 1H, H-2), 5.28 (t, J=5.86 Hz, 1H, –
NH–), 4.14 (q, J=6.96 Hz, 2H, OCH2CH3), 3.07 (m,
2H, –NH–CH2–), 2.40 (t, J=6.96 Hz, 2H, –CH2COO),
1.60 (m, 2H, –CH2–CH2–CO), 1.27 (t, 3H, –CH3). Anal.
calcd (C14H17NO6S2): C, 46.78; H, 4.77; N, 3.90; S,
17.84. Found: C, 46.87; H, 4.92; N, 3.88; S, 17.84.
4.1.14. N-butyl-5-benzo[b]thiophenesulphonamide 1,1-di-
oxide (18). Starting from 5-chlorosulfonylbenzo[b]thio-
phenesulphonamide 1,1-dioxide and butylamine,
compound 18 was obtained and recrystallised from iso-
propanol (65%): Mp 147 ꢀC; (KBr, cmꢁ1): 1163, 1311
(SO2), 3307 (NH). 1H NMR (CDCl3 d): 8.06 (d,
J67=8.06, 1H, H-6), 7.89 (s, 1H, H-4), 7.88 (d, 1H, H-
7), 7.31(d, J32=6.96, 1H, H-3), 6.49 (d, 1H, H-2), 4.59
(t, J=5.86, 1H, –NH), 3.02 (m, 2H, –NH–CH2–), 1.58–
1.21 (m, 4H, –CH2 ꢁCH2–CH3), 0.90 (t, J=7.33, 3H,
–CH3). Anal. calcd (C12H15NO4S2): C, 47.82; H, 5.02;
N, 4.65; S, 21.38. Found: C, 47.72; H, 5.13; N 4.58; S,
20.54; MS: 301, 284, 258, 229, 165.
4.1.10. Ethyl 5-(6-benzo[b]thiophenesulphonamido 1,1-di-
oxide)valerate (14). Starting from 6-chlorosulfonyl-
benzo[b]thiophenesulphonamide 1,1-dioxide and ethyl
5-aminovalerate hydrochloride, compound 14 was
obtained and purified by column chromatography
(hexane 4:7 ethyl acetate) (11,7%): Mp 77–79 ꢀC; IR
(KBr, cmꢁ1): 1147, 1308 (SO2), 1721 (COOEt), 3293
4.1.15. N-benzyl-5-benzo[b]thiophenesulphonamide 1,1-
dioxide (19). Starting from 5-chlorosulfonylbenzo[b]-
thiophenesulphonamide 1,1-dioxide and benzylamine,
compound 19 was obtained and recrystallised from iso-
propanol (71%): Mp 183.7 ꢀC; (KBr, cmꢁ1): 1149, 1303
(SO2), 3258 (NH). 1H NMR (DMSO-d6 d): 8.51(t,
J=6.23, 1H, NH), 8.04 (d, J67=8.42, 1H, H-6), 7.97–
7.93 (m, 2H, H-7, H-4), 7.71(d, J32=6.96, 1H, H-3),
7.55 (d, 1H, H-2), 7.22 (s, 5H, –C6H5), 4.08 (d, 2H,
–CH2). Anal. calcd (C15H13NO4S2): C, 53.72; H, 3.91;
N, 4.18; S, 19.12. Found: C, 53.71; H, 3.91; N, 4.16; S,
18.39.
1
(NH); H NMR (CDCl3 d): 8.16 (s, 1H, H-7), 8.07 (d,
J54=7.69 Hz, 1H, H-5), 7.54 (d, 1H, H-4), 7.32 (d,
J32=6.96 Hz, 1H, H-3), 6.99 (d, 1H, H-2), 5.38 (t,
J=6.23, 1H, NH), 4.11 (q, J=6.96 Hz, 2H, CH2–CH3),
2.98 (m, 2H, –NH–CH2), 2.29 (t, J=6.23 Hz,
2H, –CH2–COO–), 1.62 (m, 4H, –CH2–CH2), 1.26 (t,
3H, –CH3). Anal. calcd (C15H19NO6S2): C, 48.24; H,
5.13, N, 3.75; S, 17.17. Found: C, 48.19; H, 5.59; N,
3.75; S, 17.09.
4.1.11. N-(4-methoxy)phenyl-6-benzo[b]thiophenesulphon-
amide 1,1-dioxide (15). Starting from 6-chlorosulfonyl-
benzo[b]thiophenesulphonamide 1,1-dioxide and p-
anisidine, compound 15 was obtained and recrystallised
from isopropanol (20%): Mp 160–161 ꢀC; (KBr, cmꢁ1):
1146, 1308 (SO2), 3258 (NH). 1H NMR (CDCl3 d): 8.01
(s, 1H, H-7), 7.77 (d, J54=8.06 Hz, 1H, H-5), 7.38 (d,
1H, H-4), 7.22 (d, J32=6.96 Hz, 1H, H-3), 7.00–6.76 (m,
5H, H-2, C6H4), 6.35 (s, 1H, –NH–), 3.76 (s, 3H, O–
CH3). Anal. calcd (C15H13NO5S2): C, 51.27; H, 3.73; N,
3.99; S, 18.25. Found: C, 51.48; H, 3.97; N, 3.96; S,
18.61.
4.1.16. N-(3-methoxy)phenyl-5-benzo[b]thiophenesulpho-
namide 1,1-dioxide (20). Starting from 5-chloro-
sulfonylbenzo[b]thiophenesulphonamide 1,1-dioxide and
m-anisidine, compound 20 was obtained and recrcrys-
tallized from water (28,3%): Mp 151 ꢀC; (KBr, cmꢁ1):
1
1162, 1304 (SO2), 1611 (OCH3), 3248 (NH); H NMR
(DMSO-d6 d): 10.59 (s, 1H, NH), 8.1–7.9 (m, 3H, H-7,
H-4), (d, J67=7.69, 1H, H-6), 7.98 (d, 1H,), 7.92 (s, 1H,
H-4), 7.76 (d, J32=6.69, 1H, H-3), 7.54 (d, 1H, H-2),
7.14 (m, 1H, H-50), 6.69–6.63 (m, 3H, H-20, H-40, H-60),
3.67 (s, 3H, OCH3). Anal. calcd (C15H13NO5S2): C,
51.27; H, 3.73; N, 3.99; S, 18.25. Found: C, 51.05; H,
3.65; N, 3.95; S, 18.73.
4.1.12. N-(3-chloro)phenyl-6-benzo[b]thiophenesulphon-
amide 1,1-dioxide (16). Starting from 6-chlorosulfonyl-
benzo[b]thiophenesulphonamide 1,1-dioxide and 3-
chloroaniline, compound 16 was obtained and recrys-
tallised from toluene (19%): Mp 152–153 ꢀC; (KBr,
cmꢁ1): 1143, 1297 (SO2), 3256 (NH). 1H NMR (DMSO-
d6 d): 10.76 (s, 1H, NH), 8.11 (s, 1H, H-7), 8.04 (d,
J54=7.69 Hz, 1H, H-5), 7.76 (d, 1H, H-4), 7.71 (d,
J32=6.96 Hz, 1H, H-3), 7.48 (d, 1H, H-2), 7.40–7.00 (m,
4H, H-20, H-40, H-50, H-60). Anal. calcd
(C14H10NO4S2Cl): C, 47.26; H,2.83, N, 3.94; S, 18.02.
Found: C, 47.24; H, 2.76; N, 3.79; S, 18.66.
4.2. Biological methods
European Collection of Cell Cultures (ECACC) or
American Type Culture Collection (ATCC) provided
human tumour cell lines. Five cell lines were used:
two human leukaemia (K-562 and CCRF-CEM) and
three human solid tumours, one colon carcinoma (HT-
29), one lung carcinoma (HTB54) and one melanoma
(MEL-AC). MEL-AC cells were kindly provided by Dr.
Natalia Lopez-Moratalla (Universidad de Navarra,
Pamplona, Spain). Human lung fibroblasts were kindly
provided by Dr. Markus Nabholzs (ISREC, Epalinges,
Switzerland). Cells were grown in RPMI 1640 medium
(Life Technologies) supplemented with 10% foetal calf
serum, 2 mM l-glutamine, 100 units/mL penicillin, 100
mg/mL streptomycin and 10 mM HEPES buffer
(pH=7.4). The cytotoxic effect of each substance was
tested at five different doses between 0.01and 100 mM.
Each substance was initially dissolved in DMSO at a
concentration of 0.1M, and serial dilutions were pre-
pared using culture medium. The plates with cells from
the different lines, to which medium containing the
4.1.13. 3-methyl-6-benzo[b]thiophenesulphonamide 1,1-di-
oxide (17). Through a stirred solution of the 3-methyl-6-
chlorosulfonylbenzo[b]thiophenesulphonamide 1,1-dioxide
(1.84 mmol) in dioxane (15 mL) ammonia gas was passed
for 10 min. After removal the solvent under vacuum,
the solid was recrystallised from water to give 0.10 g of
17 (31%). Mp 212–213 ꢀC; IR (KBr, cmꢁ1): 1168, 1280
1
(SO2) 3275, 3369 (NH2); H NMR (DMSO-d6 d): 8.15
(s, 1H, H-7), 8.13 (d, 1H, H-5), 7.85 (d, 1H, H-4), 7.67
(s, 2H, –NH2), 7.35 (s, 1H, H-2), 2.31 (s, 3H, –CH3).

968
R. Villar et al. / Bioorg. Med. Chem. 12 (2004) 963–968
substance under test were added, were incubated for 3
days at 37 ꢀC in a humidified atmosphere containing 5%
CO2. Cytotoxicity was then determined by a colori-
metric microassay based on the use of MTT.12 Results
are expressed as GI50, concentration that reduced by
50% the growth of treated cells with respect to
untreated controls.
J.; Poore, G. A.; Rinzel, S. M. J. Med. Chem. 1990, 33,
2393.
5. Martınez-Merino, V.; Martınez-Gonzalez, A.; Gonzalez,
A.; Gil, M. J. In QSAR and Molecular Modelling: Con-
cepts, Computational Tools and Biological Applications.
Proceedings of the 10th European Symposium on Structure-
Activity Relationships: QSAR and Molecular Modelling,
Barcelona 1994. Edited by Prous Science Publishers,
Barcelona 1995, 478–80.
6. Gil, M. J.; Manu, M. A.; Arteaga, C.; Migliaccio, M.;
Encıo, I.; Gonzalez, A.; Martınez-Merino, V. Bioorg.
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Acknowledgements
7. Karplus, P. A.; Schulz, G. E. J. Mol. Biol. 1989, 210,
163.
R.V. is indebted to the Navarra Government for a grant.
8. Chern, M. K.; Wu, T. C.; Hsieh, C. H.; Chou, C. C.; Liu,
L. F.; Kuan, I. C.; Yeh, Y. H.; Hsiao, C. D.; Ming, F.;
Tam, M. F. J. Mol. Biol. 2000, 300, 1257.
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