Synthesis, antiproliferative activities and in vitro biological evaluation of novel benzofuransulfonamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.07.007

In a cell-based screen of novel antiproliferative agents, the hit compound 1a, which bears a benzofuransulfonamide scaffold, exhibited broad-spectrum antiproliferative activities against a panel of tumor cell lines. The promising in vitro antiproliferative activity and structural novelty of 1a prompted us to investigate the synthesis of five analogs of 1a and test their antiproliferative activities. The most potent analogue, 1h, exhibited enhanced antiproliferative activities compared with the parent 1a, and exhibited an IC₅₀ value against NCI-H460 cells of 4.13 μM compared with 4.52 μM for the positive control cisplatin. Flow cytometric analysis revealed that 1h induces significant levels of apoptosis in NCI-H460 cells in vitro at low micromolar concentrations. These results suggest that 1a and analogs based on its benzofuransulfonamide scaffold may constitute a novel class of antiproliferative agents, which deserve further study.

Full text of DOI:10.1016/j.bmcl.2011.07.007

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5389–5392
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, antiproliferative activities and in vitro biological evaluation
of novel benzofuransulfonamide derivatives
Li Yang b, , Hua Lei a, , Cheng-Gen Mi a, Huan Liu b, Tian Zhou b, Ying-Lan Zhao b,
Xiao-Yun Lai a, Zi-Cheng Li a, Hang Song a, Wen-Cai Huang a,
⇑
a Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, China
b State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medicinal School, Sichuan University, Chengdu 610041, Sichuan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 March 2011
Revised 1 July 2011
Accepted 5 July 2011
Available online 13 July 2011
In a cell-based screen of novel antiproliferative agents, the hit compound 1a, which bears a benzofuran-
sulfonamide scaffold, exhibited broad-spectrum antiproliferative activities against a panel of tumor cell
lines. The promising in vitro antiproliferative activity and structural novelty of 1a prompted us to inves-
tigate the synthesis of five analogs of 1a and test their antiproliferative activities. The most potent ana-
logue, 1h, exhibited enhanced antiproliferative activities compared with the parent 1a, and exhibited an
IC50 value against NCI-H460 cells of 4.13 lM compared with 4.52 lM for the positive control cisplatin.
Keywords:
Flow cytometric analysis revealed that 1h induces significant levels of apoptosis in NCI-H460 cells
in vitro at low micromolar concentrations. These results suggest that 1a and analogs based on its ben-
zofuransulfonamide scaffold may constitute a novel class of antiproliferative agents, which deserve fur-
ther study.
Benzofuransulfonamide
Antiproliferative activity
Apoptosis
Ó 2011 Elsevier Ltd. All rights reserved.
Cancer is the second most important cause of death in the world
and its incidence and resultant mortality continues to increase rap-
idly.1 Chemotherapy with various antiproliferative agents is one of
the most commonly used clinical treatment options, but side effects
and multidrug resistance (MDR) make current treatment regimens
far from satisfactory.2 The development of novel chemotherapeutic
agents with excellent antiproliferative activity and high therapeutic
indexes therefore remains an important target.
Our research group is interested in the design, screening, syn-
thesis and biological evaluation of novel antiproliferative agents.
Recently, a series of promising hit compounds, including 1a, 1b,3
and 1c,4 have been found in a cell-based screen of antiproliferative
agents (Fig. 1). Among them, compound 1a, bearing a benzenesul-
fonamide substituted benzofuran scaffold, exhibited broad-
spectrum activity to inhibit the proliferation of a panel of human
tumor cell lines in vitro (see Table 1).
1a was first reported by our group.11 Given the promising
in vitro activity and structural novelty of 1a, we examined its anti-
proliferative activity further. Herein, we report the synthesis of 1a
and its analogs, preliminary structure–activity relationships (SARs)
and biological evaluation of this novel class of antiproliferative
agents.
No report on the synthesis of compound 1a had been published
when we tackled its synthesis, and the synthetic route shown in
Scheme 1 was designed and explored. The starting material
4-aminoanisole, 2, was acetylated with acetic anhydride in dichlo-
romethane to give 3. Friedel–Crafts acylation of acetamide 3,12 fol-
lowed by condensation with bromoacetophenone accomplished
the annulation of benzofuran to yield 1d. Hydrolysis of the amide
group of 1d with hydrochloric acid gave the key intermediate 5.
Then, 1a was obtained by sulfonylation of
5 with p-fluor-
obenzenesulfonyl chloride in dichloromethane with pyridine as
the base.
Benzofuran derivatives, either natural or synthetic, demonstrate
a variety of biological activities.5 They have been reported to
exhibit antiproliferative activity via different mechanisms, such
as inhibition of tubulin polymerization6, inhibition of histone
deacetylase enzymes7, inhibition of angiogenesis8 and induction
of apoptosis9, among others.10
Recently, sulfonamides have been reported to show substantial
antiproliferative activity both in vitro and in vivo.13 To investigate
whether the aryl sulfonamide moiety in 1a is necessary for its anti-
proliferative activity, analogs 1d and 1e, which have different acyl
groups at the 5-position, were also synthesized. As shown in
Scheme 1, 1d was an intermediate in the synthesis of 1a, and 1e
could be conveniently obtained by the reaction of 5 with p-fluor-
obenzoyl chloride.
To the best of our knowledge, the antiproliferative activity of
benzenesulfonamide substituted benzofuran derivatives such as
Efforts were also made to incorporate different groups such as
an ester group at the 2-position, to investigate the influence of sub-
stituents at this position on antiproliferative activity. The target
⇑
Corresponding author. Tel./fax: +86 28 85405221.
E-mail address: hwc@scu.edu.cn (W.-C. Huang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.007

5390
L. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5389–5392
Figure 1. Hit compounds found in a cell-based screening of antiproliferative agents.
o-Hydroxyacetophenone was condensed with bromoacetophe-
Table 1
none in the presence of potassium carbonate in DMF, or reacted
with ethyl bromoacetate under solvent-free conditions to generate
annulated product 9. This was chlorosulfonated with chlorosul-
fonic acid in dichloromethane to afford 10. Then, 1g and 1h were
generated by the sulfonation of p-fluoroaniline with 10, using pyr-
idine as the base, in dichloromethane.
The antiproliferative activities of compounds 1a, and 1d–h against various cancer cell
lines
a
Compound
IC50
NCI-H460
(
l
M)
HepG2
HeLa
PC-3
1a
1d
24.1 3.3
>40
34.8 2.7
>40
10.6 0.7
>40
38.2 3.4
>40
The regioselectivity of the chlorosulfonation of benzofuran 9 is
likely to be directed by higher electron density at positions 5 and 6,
compared to that at positions 4 and 7. It could be seen by 1H NMR
of 10 (R2 = COOEt) that an AB system of aromatic hydrogens was
present, with a coupling constant of 6.6 Hz. The same coupling
constant was observed in compounds 1g and 1h. The chlorosulfo-
nyl group must therefore have been introduced at the 5- or 6-posi-
tion, but the exact substitution position remained unconfirmed.
Although methyl 3-methylbenzofuran-2-carboxylate has been
reported to be chlorosulfonated to give methyl 5-chlorosulfonyl-
3-methylbenzofuran-2-carboxylate,15 we do not believe that the
regioselectivity of the chlorosulfonylation can be unambiguously
assigned by 1H NMR alone, as provided in the literature. As 1h dis-
played the most potent antiproliferative activity of the compounds
synthesized in the MTT assay, a single crystal of 1h was cultivated
and X-ray single crystal diffraction was carried out.16 The result
(Fig. 2) unambiguously established the position of the substituted
side chain as the 5-position of benzofuran, therefore revealing that
the chlorosulfonation of benzofuran 9 takes place at the 5-position.
Furthermore, the X-ray single crystal diffraction result may also be
helpful to determine the regioselectivity of electrophilic substitu-
tion on the benzofuran ring with substituents at the 2- and
3-positions.
1e
1f
1g
1h
31.2 2.1
5.68 0.28
32.2 2.9
7.53 0.38
16.3 0.6
15.6 1.7
8.89 0.51
22.6 1.6
4.13 0.27
4.52 0.27
18.8 1.4
12.2 1.3
15.4 0.9
9.37 0.44
3.40 0.14
13.5 0.6
15.9 1.3
22.1 1.5
8.56 0.53
0.25 0.02
Cisplatin
a
The cytotoxic effect of compounds on various cancer cell lines were determined
using the MTT assay.17 The results were expressed as the IC50, and were the means
calculated from three independent experiments.
compound 1f was successfully synthesized using 4 as the starting
material (Scheme 2).
As shown in Scheme 2, the solvent-free reaction of 4 and ethyl
bromoacetate in the presence of potassium carbonate14 gave 6,
which was hydrolyzed to afford 7. Target compound 1f was then
prepared by the sulfonylation of 7 with p-fluorobenzenesulfonyl
chloride in dichloromethane with pyridine as the base.
Retro-inverso modification is a useful tool for analog design and
drug development, and it prompted us to target 1g and 1h, which
have a p-fluorophenylaminosulfonyl group instead of the p-fluor-
ophenylsulfonylamino group at the 5-position found in 1a and
1f. The synthesis of 1g and 1h was achieved by the acylation of
p-fluoroaniline with substituted benzofuransulfonyl chloride, as
illustrated in Scheme 3.
Scheme 1. Synthetic route adopted to compounds 1a, 1d, and 1e. Reagents and conditions: (a) Ac2O, CH2Cl2, rt, 2 h, 92%; (b) AcCl, AlCl3, CH2Cl2, reflux, 4.5 h, 82%; (c)
C6H5COCH2Br, K2CO3, DMF, 120 °C, 1 h, 72%; (d) 3 M HCl, EtOH, 90 °C, 4.5 h, 77%; (e) p-FC6H4SO2Cl or p-FC6H4COCl, Py, CH2Cl2, rt, 0.5 h, 70% for 1a and 68% for 1e.
Scheme 2. Synthetic route to compound 1f. Reagents and conditions: (a) BrCH2COOEt, K2CO3, N2, 80 °C for 0.5 h then 160 °C for 1.5 h, 30%; (b) 3 M HCl, EtOH, 90 °C, 4.5 h,
72%; (c) p-FPhSO2Cl, Py, CH2Cl2, rt, 0.5 h, 54%.

L. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5389–5392
5391
Scheme 3. Synthetic route to compounds 1g and 1h. Reagents and conditions: (a) C6H5COCH2Br, K2CO3, DMF, 120 °C, 1 h, 76%/BrCH2COOEt, K2CO3, N2, 80 °C for 1 h then
160 °C for 2.5 h, 45%; (b) ClSO3H, CH2Cl2, 1 h, 41%/39%; (c) p-FC6H4NH2, Py, CH2Cl2, 77%/72%.
Figure 2. Ortep diagram of the crystal structure of compound 1h, with displacement ellipsoids drawn at the 50% probability level.
The antiproliferative activities of compounds 1a, and 1d–h were
examined in four cancer cell lines: the human hepatoma HepG2
cell line, large cell lung cancer cell line NCI-H460 (H460), cervical
cancer cell line HeLa, and prostate cancer cell line PC-3, using an
MTT assay. Cisplatin was used as a positive control. The experi-
mental data are summarized in Table 1.
benzofuransulfonamide scaffold may constitute a novel class of
antiproliferative agents, which deserve further study.
To further disclose the antiproliferative mechanism of 1h, flow
cytometric analysis was performed to identify and measure the
apoptotic cells after Annexin V/propidium iodide (PI) staining.18
The data are shown in Figure 3.
As can be seen from Table 1, the hit compound 1a exhibited
broad-spectrum activity, inhibiting the proliferation of all four hu-
man tumor cell lines tested in vitro. Introduction of a benzamide
substituent at the 5-position on the benzofuran ring (1e) was well
tolerated, whereas introduction of an acetamide group (1d) led to
almost total loss of activity. Replacement of the benzoyl group at
the 2-position with an ester group yielded 1f, which exhibited sig-
nificantly enhanced activities, except against HeLa cells. To our de-
light, retro–inverso modification of the sulfonamide (1g and 1h)
does not compromise activity. The most potent analogue, 1h,
exhibited enhanced antiproliferative activities, and in particular,
As can be seen in Figure 3, a 48 h exposure of NCI-H460 cells to
compound 1h showed Annexin-V positivity (the lower right quad-
rant and the upper right quadrant which contains early and late
apoptotic cells, respectively) that was much higher than untreated
cells (b: 33.88% and c: 41.09% vs a: 3.82%). These results suggest
that compound 1h induces significant levels of apoptosis in H460
cells in vitro at low micromolar concentrations.
In conclusion, compound 1a bearing a novel benzofuransulfona-
mide scaffold exhibited broad-spectrum antiproliferative activities
against a panel of tumor cell lines in a cell-based screen of novel
antiproliferative agents. In addition, five analogs of 1a were de-
signed and synthesized. The most potent analogue, 1h, exhibited
enhanced antiproliferative activities, with an IC50 value against
its IC50 value against NCI-H460 cells was 4.13
an IC50 of 4.52 M for cisplatin, the positive control. These results
suggest that the target compound 1a and its analogs based on the
lM compared with
l
NCI-H460 cells of 4.13 lM, compared with an IC50 of 4.52 lM for
Figure 3. Bivariate annexin V/PI analysis of NCI-H460 cells after 48 h culture in the absence (a: control) or presence of 1h (b: 2.5 lM and c: 10.0 lM).

5392
L. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5389–5392
8. Chen, Y.; Chen, S.; Lu, X.; Cheng, H.; Ou, Y.; Cheng, H.; Zhou, G. C. Bioorg. Med.
Chem. Lett. 2009, 19, 1851.
cisplatin, the positive control. Flow cytometric analysis revealed
that compound 1h induces significant levels of apoptosis in NCI-
H460 cells in vitro at low micromolar concentrations. Further SARs
and mechanistic studies of these benzofuransulfonamide-based
antiproliferative agents are currently in progress.
9. Simoni, D.; Romagnoli, R.; Baruchello, R.; Rondanin, R.; Rizzi, M.; Pavani, M. G.;
Alloatti, D.; Giannini, G.; Marcellini, M.; Riccioni, T.; Castorina, M.; Guglielmi,
M. B.; Bucci, F.; Carminati, P.; Pisano, C. J. Med. Chem. 2006, 49, 3143.
10. Hayakawa, I.; Shiota, R.; Agatsuma, T.; Furukawa, H.; Naruto, S.; Sugano, Y.
Bioorg. Med. Chem. Lett. 2004, 14, 455.
11. To the best of our knowledge, there is no synthetic report of compound 1a,
which was synthesized by ourselves for an anticancer project of our lab.
12. Hu, L. X.; Li, Z. R.; Jiang, J. D.; Boykin, D. W. Anti-Cancer Agents Med. Chem. 2008,
8, 739.
13. Bennett, C. J.; Caldwell, S. T.; McPhail, D. B.; Morrice, P. C.; Duthie, G. G.;
Hartley, R. C. Bioorg. Med. Chem. 2004, 14, 2079.
14. Suzuki, T.; Tanemura, K.; Horaguchi, T.; Shimizu, T.; Sakakibara, T. J. Heteroyclic
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15. Termin, A. P.; Martinborough, E.; Zimmermann, N.; Cohen, C. J.; Gutierrez, C. D.
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Acknowledgment
We thank the Analytical and Testing Center of Sichuan Univer-
sity for NMR and MS measurements.
Supplementary data
16. X-ray crystal structure analysis for compound (1h). Formula C18H16FNO5S, yellow
crystal. The crystal is of monoclinic, space group P21/n with a = 12.2470(5) Å,
b = 10.2822(4) Å, c = 14.9826(6) Å, b = 112.116(5)°, V = 1747.89(11) Å3, Z = 4,
Supplementary data (the list of experimental details and spec-
troscopic characterization of compounds 1a, 1d–h and their inter-
mediates) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2011.07.007.
Dc = 1.434 g/cm3, F(0 0 0) = 784, T = 293(2) K,
l
= 0.225 mmÀ1, R = 0.0666 and
(I0). Crystallographic data
wR = 0.1176 for 5070 observed reflections with I > 2
r
of 1h have been deposited with the Cambridge Crystallographic Data Center as
supplementary publication numbers CCDC 814683. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK (fax: +44 (0) 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
17. Briefly, cells (2000/well) were seeded in 96-well plates and cultured for 24 h,
followed by treatment with the compounds for 48 h. Ten microliters of 10 mg/
mL MTT was added per well and incubated for another 2.5 h at 37 °C. Then the
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