N-(2-methyl-indol-1H-5-yl)-1-naphthalenesulfonamide: A novel reversible antimitotic agent inhibiting cancer cell motility

Article abstract of DOI:10.1016/j.bcp.2016.06.016

A series of compounds containing the sulfonamide scaffold were synthesized and screened for their in vitro anticancer activity against a representative panel of human cancer cell lines, leading to the identification of N-(2-methyl-1H-indol-5-yl)-1-naphtha

Full text of DOI:10.1016/j.bcp.2016.06.016

Accepted Manuscript
N-(2-methyl-indol-1H-5-yl)-1-naphthalenesulfonamide: a novel reversible an-
timitotic agent inhibiting cancer cell motility
Clara Aceves-Luquero, Cristina Galiana-Roselló, Guillem Ramis, Ruth
Villalonga-Planells, Enrique García-España, Silvia Fernández de Mattos, Rafael
Pelaez, José M. Llinares, M. Eugenia González-Rosende, Priam Villalonga
PII:
S0006-2952(16)30142-3
http://dx.doi.org/10.1016/j.bcp.2016.06.016
BCP 12576
DOI:
Reference:
Biochemical Pharmacology
To appear in:
Received Date:
Accepted Date:
25 April 2016
23 June 2016
Please cite this article as: C. Aceves-Luquero, C. Galiana-Roselló, G. Ramis, R. Villalonga-Planells, E. García-
España, S.F.d. Mattos, R. Pelaez, J.M. Llinares, M. Eugenia González-Rosende, P. Villalonga, N-(2-methyl-
indol-1H-5-yl)-1-naphthalenesulfonamide: a novel reversible antimitotic agent inhibiting cancer cell motility,
Biochemical Pharmacology (2016), doi: http://dx.doi.org/10.1016/j.bcp.2016.06.016
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1
N-(2-methyl-indol-1H-5-yl)-1-naphthalenesulfonamide: a novel reversible
antimitotic agent inhibiting cancer cell motility
Clara Aceves-Luquero,1,4 Cristina Galiana-Roselló,5,7 Guillem Ramis,1,4Ruth Villalonga-
Planells,3 Enrique García-España,6Silvia Fernández de Mattos,1,2,4Rafael Pelaez,8José
M. Llinares,7M. Eugenia González-Rosende5,§,*and Priam Villalonga1,2,4,§,
*
1Cancer Cell Biology Laboratory, Institut Universitari d’Investigació en Ciències de la
Salut (IUNICS),2Departament de Biologia Fonamental i Ciències de la Salut,
and3Departament de Química, Universitat de les llles Balears, Palma, Illes Balears,
Spain.4Institut d’Investigació Sanitària de Palma (IdISPa), Palma, Illes Balears, Spain.
5Departamento de Farmacia, Universidad CEU-Cardenal Herrera, Moncada, Valencia,
Spain.6Departamento de Química Inorgánica and 7Departamento de Química
Orgánica, ICMoL, Universitat de València, Paterna, Spain. 8Departamento de Química
Farmacéutica, Universidad de Salamanca, Salamanca, Spain.
§Joint senior co-authors.
*Corresponding authors:
Priam Villalonga. Mailing address: Institut Universitari d’Investigació en Ciències de la
Salut (IUNICS), Universitat de les Illes Balears. EdificiCientificotècnic, Ctra.
Valldemossa Km 7,5, 07122, IllesBalears, Spain. Phone: +34 971 173004 Fax: +34
971 259501 E-mail: priam.villalonga@uib.es
M. Eugenia González-Rosende. Mailing address: Departamento de Farmacia,
Universidad CEU Cardenal Herrera, Avda. Seminario s/n, 46113, Moncada, Valencia,
Spain. Phone: +34 961 369 000,Fax: +34961 395 272E-mail: eugenia@uch.ceu.es

2
Abstract
A series of compounds containing the sulfonamide scaffold were synthesized
andscreened for their in vitro anticancer activity against a representative panel of
human cancer cell lines, leading to the identification ofN-(2-methyl-1H-indol-5-yl)-1-
naphthalenesulfonamide (8e) as a compound showing a remarkable activity across the
panel, with IC50 values in the nanomolar-to-low micromolar range. Cell cycle
distribution analysis revealed that 8e promoted a severe G2/M arrest, which was
followed by cellular senescence as indicated by the detection of senescence-
associated β-galactosidase (SA-β−gal) in 8e-treated cells. Prolonged 8e treatment also
led to the onset of apoptosis, in correlation with the detection of increased Caspase 3/7
activities. Despite increasingγ-H2A.X levels, a well-established readout for DNA
double-strand breaks, in vitro DNA binding studies with 8e did not support interaction
with DNA. In agreement with this, 8e failed to activate the cellular DNA damage
checkpoint. Importantly, tubulin staining showed that 8e promoted a severe
disorganization of microtubules and mitotic spindle formation was not detected in 8e-
treated cells. Accordingly, 8e inhibited tubulin polymerization in vitro in a dose-
dependent manner andwas also able to robustly inhibit cancer cell motility. Docking
analysis revealed a compatible interaction with the colchicine-binding site of tubulin.
Remarkably, thesecellular effects were reversible since disruption of treatment resulted
in the reorganization of microtubules, cell cycle re-entry and loss of senescent markers.
Collectively, our data suggest that this compound may be a promising new anticancer
agent capable of both reducing cancer cell growth and motility.
Keywords: Sulfonamides, Cancer, Mitosis, Tubulin, Cell Motility, Apoptosis.

3
1.
Introduction
Despite overwhelming advances in cancer research and clinical oncology, which have
resulted in higher successful outcomes for a number of malignancies, cancer remains
the second leading cause of death in developing as well as developed
countries.Moreover, the global cancer burden is growing at an alarming pace, in 2030
alone, 21.3 million new cancer cases and 13.1 million cancer deaths are expected to
occur, simply due to the growth and aging of the population[1]. Even though
chemotherapy is the mainstay of cancer therapy, the use of available
chemotherapeutics is often limited, mainly due to undesirable side effects caused by
the lack of selectivity of most antitumoral drugs as well as the constant emergence of
drug-resistant and multidrug resistant tumors, which clearly underscores the need of
developing alternative chemotherapeutic agents for more effective cancer treatments
[2]. In this context, research efforts directed at the development of new anti-mitotic
agents is clearly a priority, with the aim to addressing issues such as enhanced tumor
specificity, insensitivity to chemo-resistance and reduced neurotoxicity [3].Anti-mitotic
compounds elicit their antitumor activity by altering tubulin polymerization
and thus microtubule dynamics which impairs the correct assembly of the mitotic
spindle, leading to mitotic arrest. Among these compounds, tubulin-inhibiting agents
binding to the colchicine site on tubulin have been of special interest [4].
Sulfonamides and their derivatives constitute an important class of therapeutic agents
that exhibit a broad spectrum of pharmacological profiles, such as antibacterial,
diuretic, hypoglycaemic, antithyroid, antiviral, antiinflammatory and antiparasitic
activities among others [5]. Particularly, compounds belonging to this structural class
have also been recently reported to show substantial in vitro and in vivo anticancer
activities and some of them are currently undergoing clinical trials [5d,5e,6].
Interestingly, although these novel chemotypesshare
a
common motif of
aromatic/heterocyclic sulfonamides, a varietyof mechanisms have been involved in
their antitumor action, i.e. disruption of microtubule protein assembly [6,7], carbonic
anhydrase inhibition [8], cell cycle arrest [9],NF-κB transcription factorinhibition[10],
Histone Deacetylase inhibition [11],Matrix Metaloproteinases (MMP) inhibition[12],as
well as targeting protein kinase-mediated signaling pathways[13], or mitochondrial
membrane[14].

4
It is also well documented that tubulin may constitute a suitable target for antiprotozoal
compounds [15]. In this context, we have recently reported benzene and
naphthalenesulfonamide derivatives as potent antiparasitic agents targetingβ-tubulin of
Leishmaniainfantum[16]. Hence, based on our previous results on leishmanicidal
tubulin-interacting sulfonamides andthe increasing biological importance of
sulfonamides in the field of anti-tumoral chemotherapy, the potential
antiproliferativeactivity of our in-house sulfonamide library was evaluated against a
large panel of tumor cell lines. In addition, to determine electronic and structural
features, we designed and synthesized a new series of sulfonamides (Figure 1). Here,
we describe the identification of N-(2-methyl-1H-indol-5-yl)-1-naphthalenesulfonamide
(compound 8e) as a sulfonamide with potentialanticancer activity and characterize its
cellular mechanism of action as a novel, and reversible, tubulin polymerization inhibitor
inducing mitotic arrest, senescence and cellular apoptosis. Importantly, we also show
that sulfonamide 8e significantly inhibits cancer cell motility, enhancing the potential
interest of this sulfonamide as a novel anticancer compound.
2. Materials and Methods
2.1. Chemistry
2.1.1. General
All reagents were purchased from Aldrich and used without purification. All experiments
were made under nitrogen atmosphere. Melting points were determined with a Kofler
hot-stage apparatus and are uncorrected. Column chromatography was performed
using silica gel (Merck 60, 70-230 mesh). 1H and 13C NMR spectra were recorded on a
Bruker AC-300 instrument. Chemical shifts (δ values) and coupling constants (J values)
are given in ppm and Hz respectively. HRMS were obtained using a VG Autospec
TRIO 1000 instrument. The ionization mode used in mass spectra was electron impact
(EI), fast atom bombardment (FAB) or Time-of-flight mass spectrometry (TOFMS).
Elemental analysiswereperformedbythe Servicio de Espectroscopía Atómica,
Molecular y Óptica, Universitat de València-SCSIE (Servei Central de Suport a la
Investigació Experimental), València, Spain. The purity of the compounds (≥95%) and
molecular mass were confirmed by elemental microanalysis and HRMS. The analytical
results for C, H, and N were within 0.4 of the theoretical values.
2.1.2. Synthesis and Characterization

5
General procedure for the synthesis of sulfonamides 1-14.To an ice-cooled
solution of the amine (20 mmol) in pyridine (8 mL) was slowly added the corresponding
sulfonyl chloride (30 mmol) in pyridine (6 mL). The mixture was stirred at 0 ºC for 2 h
and allowed to reach room temperature. Water was added (100 mL) and the solid was
collected and recrystallized from MeOH:CH2Cl2.
Compounds 1-4, 5b-d, 6a-c, and 8a-e, were synthesized as previously described [16].
N-(1H-Indazol-5-yl)-4-nitrobenzenesulfonamide (5a)[17].Yield: 73%, mp: 234-236
ºC, 1H NMR (300 MHz, DMSO-d6): δ = 7.08 (d, J = 8.8, 1H), 7.44 (m, 2H), 7.93 (d, J =
8.7, 2H), 8.00 (s, 1H), 8.31 (d, J = 8.7, 2H), 10.5 (brs, 1H, NH), 13C NMR (75 MHz,
DMSO-d6) δ: 111.2 (CH), 114.1 (CH), 122.9 (CH), 123.2 (C), 124.9 (CH), 128.7 (CH),
129.6 (C), 133.9 (CH), 138.2 (C), 145.1 (C), 150.0 (C), HRMS-EI+: m/z calcd for
C13H10N4O4S: 318.0422, found: 318.0429.
1
N-(1H-indol-5-yl)-1-naphthalenesulfonamide (7a). Yield 56%, mp: 107-108 ºC, H
NMR (300 MHz, DMSO-d6) δ = 6.29 (m, 1H), 6.76 (dd, J = 2, J = 8.6, 1H), 7.18 (d, J =
8.6, 1H), 7.21 (d, J = 2, 1H), 7.26 (t, J = 2.7, 1H), 7.51 (t, J = 7.8, 1H), 7.64 (td, J = 1, J
= 8.6, 1H), 7.74 (td, J = 1.4, J = 8.6, 1H), 8.03 (d, J = 7.8, 1H), 8.11 (m, 2H), 8.87 (d, J
= 8.6, 1H), 10.22 (s, 1H, NH), 11.01 (s, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ =
101.4 (CH), 111.9 (CH), 113.6 (CH), 117.0 (CH), 124.7 (CH), 125.0 (CH), 126.6 (CH),
127.2 (CH), 127.9 (C), 128.1 (C), 128.2 (C), 129.0 (CH), 129.3 (CH), 130.0 (CH), 133.9
(C), 134.0 (C), 134.3 (CH), 135.2 (C), HRMS (ES+): m/z (M+H) calcd for C18H15N2O2S:
323.0849 found: 323.0826. Anal. Calcd for C18H14N2O2S: C, 67.06, H, 4.38, N, 8.69, S,
9.94. Found: C, 66.86, H, 4.65, N, 8.48, S, 9.59.
N-(1H-indol-5-yl)-5-(dimethylamino)-1-naphthalenesulfonamide (7b). Yield 74%,
mp 118-121 ºC, 1H NMR (300 MHz, DMSO-d6) δ = 2.49 (s, 6H), 6.25 (m, 1H), 6.73 (dd,
J = 2.1 J = 8.6, 1H), 7.14 (d, J = 8.6, 1H), 7.15 (d, J = 2, 1H), 7.25 (m, 2H), 7.49 (t, J =
7.3, 1H), 7.61 (t, J = 8.6, 1H), 8.05 (dd, J = 1.1, J = 7.3, 1H), 8.38 (d, J = 8.6, 1H), 8.46
(d, J = 8.6, 1H), 10.13 (s, 1H, NH), 10.96 (s, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ
= 45.4 (CH3), 101.3 (CH), 111.9 (CH), 113.2 (CH), 115.5 (CH), 116.7 (CH), 119.4 (CH),
123.8 (CH), 126.6 (CH), 127.9 (C), 128.2 (CH), 129.1 (C), 129.2 (C), 129.6 (C), 129.9
(CH), 130.0 (CH), 133.7 (C), 135.7 (C), 151.7 (C), HRMS (ES+): m/z (M+H) calcd for
C20H20N3O2S: 366.1271 found: 366.1248. Anal. Calcd for C19H18N4O2S: C, 62.28, H,
4.95, N, 15.29, S, 8.75. Found: C, 62.24, H, 5.24, N, 14.71, S, 8.60.

6
N-(2-methyl-1H-indol-5-yl)-8-quinolinesulfonamide(8f). Yield 58%, mp 192-194 ºC,
1H NMR (300 MHz, DMSO-d6) δ = 2.25 (s, 3H), 5.88 (s, 1H), 6.60 (dd, J = 2, J = 8.6,
1H), 6.94 (d, J = 8.6, 1H), 6.98 (d, J = 2, 1H), 7.58 (t, J = 7.7, 1H), 7.74 (dd, J = 4.2, J
=8.4, 1H), 8.2 (d, J = 7.7, 2H), 8.52 (dd, J = 1.7, J = 8.4, 1H), 9.21 (dd, J = 1.7, J = 4.2,
1H), 9.39 (bs, 1H, NH), 10.76 (bs, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ = 13.6
(CH3), 99.3 (CH), 110.6 (CH), 113.0 (CH), 116.1 (CH), 122.9 (CH), 126.0 (CH), 128.4
(C), 128.7 (C), 129.1 (C), 132.0 (CH), 134.0 (C), 134.1 (CH), 135.8 (C), 136.9 (C),
137.4 (CH), 143.4 (C), 151.7 (CH), HRMS (ES+): m/z (M+H) calcd for C18H16N3O2S:
338.0958 found: 338.0934. Anal. Calcd for C18H15N3O2S: C, 64.08, H, 4.48, N, 12.45,
S, 9.50. Found: C, 63.97, H, 4.64, N, 12.01, S, 9.12.
1
N-(2-anthryl)-4-nitrobenzenesulfonamide(9a).Yield 58%, mp 183-187 ºC, H NMR
(300 MHz, DMSO-d6) δ = 7.05 (dd, J = 2, J = 9, 1H), 7.36 (m, 2H), 7.71 (s, 1H), 7.80
(m, 1H), 8.02 (m, 2H), 8.09 (d, J = 9, 2H), 8.35 (d, J = 9, 2H), 8.42 (s, 1H), 8.46 (s, 1H),
13C NMR (75 MHz, DMSO-d6) δ = 115.6 (CH), 123.4 (CH), 125.0 (CH), 125.5 (CH),
125.6 (CH), 126.2 (CH), 126.4 (CH), 126.9 (CH), 127.4 (C), 128.0 (CH), 128.4 (CH),
130.1 (C), 132.0 (CH), 132.3 (C), 134.0 (C), 145.7 (C), 146.4 (C), 150.1 (C), HRMS
(ES+): m/z (M-H) calcd for C20H13N2O4S: 377.0596, found: 377.0593. Anal. Calcd for
C20H14N2O4S: C, 63.48, H, 3.73, N, 7.40, S, 8.47. Found: C, 63.95, H, 4.04, N, 7.45, S,
8.95.
N-(2-anthryl)-4-chlorobenzenesulfonamide (9b). Yield 53%, mp 160-161 ºC, 1H
NMR (300 MHz, DMSO-d6) δ = 7.31 (dd, J = 2.1, J = 9, 1H), 7.47 (m, 2H), 7.61 (d, J =
8.9, 2H), 7.70 (s, 1H), 7.84 (d, J = 8.9, 2H), 8.0 (m, 3H), 8.42 (s, 1H), 8.47 (s, 1H),
10.69 (bs, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ = 115.4 (CH), 121.4 (CH), 125.5
(CH), 125.6 (CH), 126.2 (CH), 126.4 (CH), 128.0 (CH), 128.4 (CH), 128.9 (C), 129.0
(CH), 129.9 (CH), 130.1 (CH), 131.0 (C), 131.5 (C), 132.1 (C), 134.9 (C), 138.3 (C),
138.7 (C), HRMS (ES+): m/z (M+H) calcd for C20H14ClNO2S: 368.0512, found:
368.0511. Anal. Calcd for C20H14ClNO2S: C, 65.30, H, 3.83, N, 3.80, S, 8.71. Found: C,
63.19, H, 3.53, N, 7.45, S, 8.90.
N-(2-anthryl)-benzenesulfonamide(9c). Yield 97%, mp 195-197 ºC (lit [18]184 ºC), 1H
NMR (300 MHz, DMSO-d6) δ = 7.31 (dd, J = 2.0, J = 9, 1H), 7.36 (m, 1H), 7.47 (m,
2H), 7.61 (d, J = 8.7, 2H), 7.70 (s, 1H), 7.85 (d, J = 8.7, 2H), 8.0 (m, 3H), 8.42 (s, 1H),
8.46 (s, 1H), 10.70 (bs, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ = 115.4 (CH), 121.4
(CH), 125.5 (CH), 125.6 (CH), 126.2 (CH), 126.4 (CH), 128.0 (CH), 128.4 (CH), 128.9

7
(C), 129.0 (CH), 129.4 (CH), 129.8 (CH),130.1 (CH), 131.0 (C), 132.1 (C), 134.9 (C),
138.3 (C), 138.7 (C). Anal. Calcd for C20H15NO2S: C, 72.05, H, 4.53, N, 4.20, S, 9.62.
Found: C, 71.77, H, 4.17, N, 3.95, S, 9.44.
1
N-(2-anthryl)-1-naphthalenesulfonamide (9d).Yield 99%, mp 105-109 ºC, H NMR
(300 MHz, DMSO-d6) δ = 7.26 (dd, J = 2.1, J = 9, 1H), 7.43 (m, 2H), 7.64 (t, J = 8, 2H),
7.76 (m, 2H), 7.91 (d, J = 9, 1H), 7.95 (dd, J = 2.4, J = 6.8, 2H), 8.04 (d, J = 7.7, 1H),
8.19 (d, J = 8.3, 1H), 8.32 (s, 1H), 8.36 (d, J = 1.7, 1H), 8.38 (s, 1H), 8.83 (d, J = 8.5,
1H), 13C NMR (75 MHz, DMSO-d6) δ = 113.5 (CH), 120.6 (CH), 124.6 (CH), 124.8
(CH), 125.1 (CH), 125.5 (CH), 126.2 (CH), 126.3 (CH), 127.4 (CH), 127.8 (C), 127.9
(CH), 128.4 (CH), 128.6 (CH), 129.5 (CH), 129.9 (CH), 130.6 (CH), 130.8 (C), 131.4
(C), 132.0 (C), 134.1 (C), 134.6 (C), 134.9 (CH), 135.0 (C), 138.8 (C). HRMS (TOF+):
m/z (M+H) calcd for C24H18NO2S: 384.1053, found: 384.1047. Anal. Calcd for
C24H17NO2S: C, 75.17, H, 4.47, N, 3.65, S, 8.36. Found: C, 71.77, H, 4.17, N, 3.95, S,
9.44.
N-(2,3-dihydro-1H-inden-1-yl)-1-naphthalenesulfonamide (10). Yield 46%, mp 164-
1
165 ºC, H NMR (300 MHz, DMSO-d6) δ = 1.54 (m, 1H), 1.92 (m, 1H), 2.59 (m, 1H),
2.74 (m, 1H), 4.66 (m, 1H), 6.87 (d, J = 7.7, 1H), 7.04 (td, J = 2.9, J = 7.7, 1H), 7.14 (m,
2H), 7.69 (m, 3H), 8.13 (dd, J = 2, J = 7.3, 1H), 8.25 (d, J = 7.3, 1H), 8.26 (d. J = 8.5,
1H), 8.49 (d, J = 9, 1H, NH), 8.73 (d, J = 8.5, 1H), 13C NMR (75 MHz, DMSO-d6) δ =
29.7 (CH2), 33.8 (CH2), 58.2 (CH), 124.3 (CH), 124.8 (CH), 124.9 (CH), 125.3 (CH),
126.6 (CH), 127.2 (CH), 128.0 (CH), 128.1 (CH), 129.1 (CH), 129.3 (CH), 134.2 (CH),
136.8 (C), 142.2 (C), 142.8 (C), 143.0 (C), 143.4 (C), HRMS (ES+): m/z (M+H) calcd for
C19H18NO2S: 324,1053 found: 324.1037.
N-(2,3-dihydro-1H-inden-2-yl)-1-naphthalenesulfonamide(11).Yield 51%, mp 184-
187 ºC, 1H NMR (300 MHz, DMSO-d6) δ = 2.65 (dd, J =7.3, J = 15.8, 2H), 2.83 (dd, J
=7.3, J = 15.8, 2H), 3.91 (sextet, J =7.3, 1H), 7.1 (m, 4H), 7.7 (m, 3H), 8.11 (d, J = 7.7,
1H), 8.21 (dd, J = 1.1, J = 7.7, 1H), 8.25 (d, J = 8.3, 1H), 8.38 (d, J = 7.5, 1H, NH), 8.70
(d, J =8.3, 1H), 13C NMR (75 MHz, DMSO-d6) δ = 37.7 (CH2), 50.18 (CH), 124.6 (CH),
125.0 (CH), 125.1 (CH), 126.8 (CH), 127.2 (CH), 127.9 (C), 128.2 (CH), 129.1 (CH),
129.3 (CH), 134.20 (CH), 134.2 (C), 136.3 (C), 140.6 (C), HRMS (ES+): m/z (M+H)
calcd for C19H18NO2S: 324,1053 found: 324.1032. Anal. Calcd for C19H17NO2S: C,
70.56, H, 5.30, N, 4.33, S, 9.91. Found: C, 70.30, H, 5.09, N, 4.48, S, 9.59.

8
N-(2,3-dihydro-1H-inden-4-yl)-4-nitrobenzenesulfonamide(12a).Yield 70%, mp 175-
1
178 ºC, H NMR (300 MHz, DMSO-d6) δ = 1.82 (quintet, J = 7.5, 2H), 2.57 (t, J = 7.5,
2H), 2.77 (t, J = 7.5, 2H), 6.83 (dd, J = 3.5, J = 5.5, 1H), 7.03 (s, 1H), 7.05 (d, J = 5.5,
1H), 7.92 (d, J = 8.8, 2H), 8.34 (d, J = 8.8, 2H), 10.05 (bs, 1H, NH), 13C NMR (75 MHz,
DMSO-d6) δ = 24.9 (CH2), 30.6 (CH2), 33.0 (CH2), 122.7 (CH), 122.8 (CH), 124.9 (CH),
127.4 (CH), 128.5 (CH), 132.4 (C), 139.5 (C), 146.0 (C), 146.2 (C), 150.1 (C). Anal.
Calcd for C15H14N2O4S: C, 56.59, H, 4.43, N, 8.80, S, 10.07. Found: C, 56.83, H, 4.51,
N, 8.91, S, 9.68.
N-(2,3-dihydro-1H-inden-4-yl)-benzenesulfonamide(12b).Yield 96%, mp 140-143
ºC, 1H NMR (300 MHz, DMSO-d6) δ = 1.77 (quintet, J = 7.5, 2H), 2.50 (t, J = 7.5, 2H),
2.75 (t, J = 7.5, 2H), 6.87 (dd, J = 2.5, J = 6.6, 1H), 7.01 (m, 2H),7.55 (m, 3H), 7.66 (dd,
J = 1.5, J = 7, 2H), 9.67 (bs, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ = 24.9 (CH2),
30.4 (CH2), 33.0 (CH2), 122.2 (CH), 122.5 (CH), 126.9 (CH), 127.2 (CH), 129.5 (CH),
133.0 (CH), 133.1 (C), 139.0 (C), 140.7 (C), 145.6 (C), HRMS (TOF+): m/z (M+H) calcd
for C15H16NO2S: 274.0896, found 274.0902. Anal. Calcd for C15H15NO2S: C, 65.91, H,
5.53, N, 5.12, S, 11.73. Found: C, 65.67, H, 5.57, N, 5.13, S, 11.72.
N-(2,3-dihydro-1H-inden-4-yl)-4-fluorobenzenesulfonamide(12c).Yield 97%, mp
1
115-117 ºC, H NMR (300 MHz, DMSO-d6) δ = 1.80 (quintet, J = 7.5, 2H), 2.52 (t, J =
7.5, 2H), 2.77 (t, J = 7.5, 2H), 6.85 (dd, J = 2.8, J = 6, 1H), 7.02 (m, 2H), 7.40 (t, J =
8.8, 2H), 7.72 (ddd, J = 5.2, J = 8.8, 2H), 9.71 (bs, 1H, NH), 13C NMR (75 MHz, DMSO-
d6) δ = 24.9 (CH2), 30.5 (CH2), 33.0 (CH2), 116.6 (d, JC-F = 22.5, CH), 122.4 (CH),
122.6 (CH), 127.2 (CH), 130.0 (d, JC-F = 9.7, CH), 133.0 (C), 137.1 (d, JC-F = 3, C)
139.2 (C), 145.7 (C), 164.5 (d, JC-F = 247.5, C), HRMS (TOF+): m/z (M+H) calcd for
C15H15FNO2S: 292.0802, found 292.0808.
N-(2,3-dihydro-1H-inden-4-yl)-1-naphthalenesulfonamide (12d). Yield 63%, mp
1
185-186 ºC, H NMR (300 MHz, DMSO-d6) δ = 1.65 (q, J = 7.4, 2H), 2.39 (t, J = 7.4,
2H), 2.66 (t, J = 7.4, 2H), 6.86 (dd, J = 1.8, J = 7.3, 1H), 6.92 (d, J = 7.3, 1H), 6.96 (t, J
= 7.3, 1H), 7.57 (t, J = 8.2, 1H), 7.66 (m, 2H), 8.07 (m, 2H), 8.21 (d, J = 8.2, 1H), 8.70
(m, 1H), 9.98 (bs, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ = 24.7 (CH2), 30.3 (CH2),
32.9 (CH2), 121.6 (CH), 121.8 (CH), 124.8 (CH), 125.0 (CH), 127.1 (CH), 127.2 (C),
127.9 (C), 128.1 (C), 129.3 (CH), 129.6 (C), 133.0 (CH), 134.1 (C), 134.5 (C), 135.7
(CH), 138.3 (CH), 145.7 (CH), HRMS (ES+): m/z (M+H) calcd for C19H18NO2S:

9
324,1053 found: 324.1039. Anal. Calcd for C19H17NO2S: C, 70.56, H, 5.30, N, 4.33, S,
9.91. Found: C, 70.3, H, 5.31, N, 4.46, S, 9.55.
N-(2,3-dihydro-1H-inden-5-yl)-benzenesulfonamide(13a)[17].Yield: 91%, mp: 150-
154 ºC, 1H NMR (300 MHz, DMSO-d6) δ = 1.92 (m, 2H), 2.72 (m, 4H), 6.84 (dd, J = 8.1,
J = 2.1, 1H), 6.94 (s, 1H), 7.02 (d, J = 8.1, 1H), 7.53 (m, 3H), 7.72 (d, J = 7.9, 2H), 10.1
(brs, 1H, NH), 13C NMR (75 MHz, DMSO-d6) δ: 25.4 (CH2), 31.9 (CH2), 32.6 (CH2),
117.1 (CH), 118.9 (CH), 124.9 (CH), 127.0 (CH), 129.5 (CH), 134.0 (CH), 136.0 (C),
139.9 (C), 140.0 (C), 145.4 (C), HRMS-EI+: m/z calcd for C15H15NO2S: 273.0823,
found: 273.0826. Anal. Calcd for C15H15NO2S: C, 65.91, H, 5.53, N, 5.12, S, 11.73.
Found: C, 66.075, H, 5.48, N, 5.15, S, 11.46.
N-(2,3-dihydro-1H-inden-5-yl)-4-fluorobenzenesulfonamide(13b). Yield 87%, mp
134-136 ºC, 1H NMR (300 MHz, DMSO-d6) δ = 1.93 (quintet, J = 7, 2H), 2.73 (m, 4H),
6.82 (dd, J = 2, J = 8, 1H), 6.94 (s, 1H), 7.03 (d, J = 8, 1H), 7.38 (t, J = 8.8, 2H), 7.72
(dd, J = 5.1, J = 8.8, 2H), 13C NMR (75 MHz, DMSO-d6) δ = 25.4 (CH2), 32.0 (CH2),
32.6 (CH2), 116.8 (CH, d, JC-F = 22, CH), 117.4 (CH), 119.2 (CH), 125.0 (CH), 130.0 (d,
JC-F = 9.75, CH), 135.8 (C), 136.3 (d, JC-F = 3, C), 140.3 (C), 145.1 (C), 164.5 (d, JC-F
=
249.7, C), HRMS (ES+): m/z (M+H) calcd for C15H15FNO2S: 292.0808, found: 292.0805.
Anal. Calcd for C15H14FNO2S: C, 61.84, H, 4.84, N, 4.81, S, 11.00. Found: C, 61.72, H,
4.88, N, 4.78, S, 10.95.
N-(2,3-dihydro-1H-inden-5-yl)-1-naphthalenesulfonamide(13c).Yield 88%, mp 112-
1
115 ºC, H NMR (300 MHz, DMSO-d6) δ = 1.86 (quintet, J = 7.5, 2H), 2.65 (t, J = 7.5,
4H), 6.67 (dd, J = 2.1, J = 8, 1H), 6.88 (s, 1H), 6.94 (d, J = 8, 1H), 7.71 (m, 3H), 8.06
(d, J = 8, 1H), 8.16 (m, 2H), 8.74 (d, J = 8, 1H), 10.48 (bs, 1H, NH), 13C NMR (75 MHz,
DMSO-d6) δ = 25.3 (CH2), 31.9 (CH2), 32.6 (CH2), 116.1 (CH), 117.9 (CH), 124.7
(CH),124.8 (CH), 124.9 (CH), 127.3 (CH), 127.9 (C), 128.4 (CH), 129.4 (CH), 130.1
(CH), 134.1 (C), 134.6 (CH), 135.0 (C), 136.0 (C), 139.5 (C), 144.9 (C), HRMS (ES+):
m/z (M+Na) calcd for C19H17NNaO2S: 346.0878 found 346.0883. Anal. Calcd for
C19H17NO2S: C, 70.78, H, 5.00, N, 4.34, S, 9.94. Found: C, 70.40, H, 5.25, N, 4.38, S,
9.69.
N-(5,6,7,8-tetrahydronaphthalen-1-yl)-1-naphthalenesulfonamide(14). Yield 45%,
mp 187-189 ºC, 1H NMR (300 MHz, DMSO-d6) δ = 1.34 (m, 2H), 1.47 (m, 2H), 2.34 (t,
J = 6.2, 2H), 3.38 (t, J = 6.2, 2H), 6.70 (dd, J = 1.5, J = 7.54, 1H), 6.84 (d, J = 7.5, 1H),

10
6.87 (d, J = 7.5, 1H), 7.57 (t, J = 7.7, 1H), 7.65 (m, 2H), 8.05 (m, 2H), 8.20 (d, J = 8.29,
1H), 8.65 (m, 1H), 9.75 (bs, 1H, NH),13C NMR (75 MHz, DMSO-d6) δ = 22.4 (CH2),
22.5 (CH2), 24.6 (CH2), 29.3 (CH2), 123.7 (CH), 124.9 (CH), 125.1 (CH), 125.6 (CH),
127.2 (CH), 127.6 (CH), 127.9 (C), 128.0 (CH), 129.2 (CH), 129.3 (CH), 133.7 (C),
134.1 (C), 134.4 (CH), 134.8 (C), 136.4 (C), 138.3 (C), HRMS (ES+): m/z (M+H) calcd
for C20H20NO2S: 338.1209 found: 338.1202.
2.2. Biological Studies
2.2.1. Cell culture
Jurkat (Acute T-cell Leukemia, a gift from Prof. Joan Gil, IDIBELL, Barcelona), Jeko-1
(Non-Hodgkin Lymphoma, a gift from Beatriz Martínez, CNIO, Madrid), JVM-2, Granta-
549 (Non-Hodgkin Lymphoma, a gift from Dr. DolorsColomer, Hospital Clínic,
Barcelona), and Z-138 (Non-Hodgkin Lymphoma, a gift from Dr. Eva Ortega-Paino,
Lund University, Lund) were maintained in RPMI-1640 with L-Glutamine and HEPES
(Biological Industries Ltd., Kibbutz BeitHaemek, Israel). LN229, U251, U373 (Glioma, a
gift from Dr. Joan Seoane, Institut de RecercaValld’Hebron, Barcelona), SKMG-3
(Glioma, a gift from Dr.HansSkovgaard, Rigshospitalet, Oslo), PC-3 (Prostate Cancer,
a gift from Prof. Anne J. Ridley, King’s College, London), SW480 and SW620
(Colorectal cancer, a gift from Prof. Eric Lam, Imperial College, London) cells were
maintained in DMEM High Glucose (4.5 g/l) with L-glutamine (Invitrogen, Carlsbad,
CA). All cells were grown in a humidified incubator at 37ºC with 5% CO2. RPMI and
DMEM were supplemented with 10% heat inactivated foetal bovine serum and 100
units/ml penicillin/streptomycin (Sigma-Aldrich, St Louis, MO). All cell lines were
subconfluently grown and passaged, routinely tested for mycoplasma contamination
and subjected to frequent morphological tests and growth curve analysis as quality-
control assessments. All cell lines were treated at a prophylactic concentration of 5
µg/ml Plasmocin™ (InvivoGen, San Diego, CA).
2.2.2. Cell Viability Analysis
The number of viable cells in culture was determined based on quantification of ATP,
which signals the presence of metabolically-active cells, using the Cell Titer-
Gloluminiscent assay kit (Promega, Madison, WI). Following the manufacturer’s
instructions, the cells were plated in 96-well plates, treated 24 h later with the
compounds for the indicated times and concentrations, followed by addition of Cell

11
Titer-Glo reagent. Luminiscence was detected using a multi-well Synergy Mx scanning
spectrophotometer (Biotek, Winooski, VT).
2.2.3 Cell Cycle Analysis
Cell cycle analysis was performed using propidium iodide staining. Briefly, cells were
washed in phosphate-buffered saline (PBS) and fixed in 70% ethanol(Sigma-Aldrich, St
Louis, MO). Fixed cells were then washed twice in PBS and stained with50
µg/mlpropidium iodide (Sigma-Aldrich, St Louis, MO) in the presence of 50 µg/ml
RNase A (Sigma-Aldrich, St Louis, MO), then analysed by flow cytometry using a
FACScan (Coulter Epics XL-MSL, Beckman Coulter, Fullerton, CA) and winMDI
software.
2.2.4. Annexin V-FITC / propidium iodide flow cytometric analysis
Analysis of phosphatidylserine externalization in apoptotic cells was determined by an
ApoTargetAnnexin-V-FITC Apoptosis kit (Invitrogen, Carlsbad, CA), according to the
manufacturer’s instructions. 2 x 105 cells were seeded in 6-well plates and treated as
indicated. Cells were then collected and suspended in 100µl of Annexin V-binding
buffer. 5µL of Annexin-V-FITC and 10µL of propidium iodide were added and incubated
15 min at room temperature in the dark. Flow cytometry analysis was carried out using
a FACScan (Coulter Epics XL-MSL, Beckman Coulter, Fullerton, CA) and winMDI
software.
2.2.5. Caspase activity analysis
Enzymatic activity of caspases was determined by measurement of caspases-3 and 7
activity by means of the luminometricCaspase-Glo 3/7 assay (Promega, Madison, WI)
according to the manufacturer’s protocol using a Synergy HT multi-detection microplate
reader (Bio-Tek, Winooski, VT).
2.2.6. γ-H2A.X quantification
DNA damage was assessed monitoring the intensity of γ -H2A.X fluorescence using
flow cytometry. Briefly, trypsinized cells were collected by centrifugation, washed in
PBS and fixed in 3.7% formaldehyde(Sigma-Aldrich, St Louis, MO) for 15 min on ice.
Cells were then permeabilized with 0.2% v/v Triton-X (Sigma-Aldrich, St Louis, MO)for
10 min, and incubated with 1:400 rabbit anti-p-(S139)-H2A.X antibody (Cell Signaling
Technology, Beverly, MA) for 30 min on ice, washed in Triton-X 0.1% in PBS and
incubated with 1:400 anti-rabbit Alexa 555-conjugated antibody (Jackson

12
ImmunoResearch, West Grove, PA) for 20 min on ice and washed. Flow cytometry
analysis was carried out using a FACScan (Coulter Epics XL-MSL, Beckman Coulter,
Fullerton, CA) and Flowing software.
2.2.7. Clonogenic Assays
Cells were seeded in 12-well plates. 24 h later, cells were treated for 3h with 10 µM 8e
or vehicle alone as a control. Cells were then washed with PBS, trypsinized and plated
at low density (3000 cells/60-mm plate). Cells were allowed to divide and form colonies
for 7–10 days. The colonies were fixed and stained with 0.5% (w/v) crystal
violet(Sigma-Aldrich, St. Louis, MO) in 70% ethanol and the number of colonies
counted. All experiments were performed in triplicate.
2.2.8. Gel electrophoresis and immunoblotting
Cells were harvested in a buffer containing 50 mMTris-HCl pH 7.4, 150 mMNaCl, 1 mM
EDTA and 1% (v/v) Triton X-100 plus protease and phosphatase inhibitors(all from
Sigma-Aldrich, St. Louis, MO). Protein content was measured by the Bradford
procedure. Cell lysates were electrophoresed in SDS-polyacrylamide gels. After
electrophoresis the proteins were transferred to Immobilon-P strips (Millipore, Billerica,
MA) for 2 h at 60 V. The sheets were pre-incubated in TBS (20 mMTris-HCl pH 7.5,
150 mMNaCl), 0.05% Tween 20 and 5% defatted milk powder for 1 h at room
temperature and then incubated for 1 h at room temperature in TBS, 0.05% Tween 20,
1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) and 0.5% defatted milk
powder containing the appropriate antibodies: p-Chk1 (Ref. 2348, Cell
SignalingTechnology,Beverly, MA 1:1000) and β-tubulin (T0198, Sigma-Aldrich, St.
Louis, MO, 1:4000). After washing in TBS, 0.05% Tween 20, the sheets were
incubated with a peroxidase-coupled secondary antibody (Dako, Glostrup, Denmark,
1/2000 dilution,) for 1 h at room temperature. After incubation, the sheets were washed
twice in TBS, 0.05% Tween 20 and once in TBS. The peroxidase reaction was
visualized by the enhanced chemiluminiscence detection system (Millipore, Billerica,
MA).
2.2.9. Tubulin staining
Cells grown on coverslips were fixed in ice-cold Methanol (Sigma-Aldrich, St. Louis,
MO)for 2 min, permeabilized in 0.2% (v/v) Triton X-100 for 30 min and blocked by
incubating in 5% BSA for 30 min. Tubulin filaments were visualized by incubating the
fixed cells for 1h at 37°C with anti-β-Tubulin (T0198, Sigma-Aldrich, St. Louis, MO,

13
1:400), washing thrice in PBS and incubating with donkey anti-mouse-Alexa 488
(A21202, Life Technologies, Carlsbad, CA,1:400) in the presence of DAPI (32670,
Sigma-Aldrich, St. Louis,MO, 1:300). Stained cells were analyzed on a Leica TCS SPE
confocal microscope (Leica Microsystems, Wetzlar, Germany).
2.2.10. Senescence Assays
Cells were plated subconfluently and treated for 24h with 10 µM 8e. Cells were then
washed with PBS, fixed in 3% (v/v) formaldehyde in PBS for 10 min at room
temperature, and then incubated with a stain solution containing 1 mg/ml of β-D-
galactoside, for 24 h at 37°C with the Senescence Cells Histochemical Staining Kit
(CS0030, Sigma-Aldrich, St. Louis, MO). Blue-stained cells were counted in at least 10
fields at 10× magnification, and the number of stained cells was expressed as the
percentage of positive cells relative to total cell number.
2.2.11. Cell motility assays
MCF-7 cells and LN229 cells were plated in 6-well plates (4x105 cells/plate), wounded
thrice with a sterile tip and 4 representative images were collected (time 0h). After 16 h,
images of the same regions were collected and the ratio of cell motility in each
experimental condition quantified.
2.2.12 In vitro tubulin polymerization assays
The polymerization of purified monomeric tubulin in vitro was measured using the
tubulin polymerization assay kit (Cytoskeleton Inc, Denver, CO) according to the
manufacturer’s instructions.Briefly, Tubulin (>99% pure) was reconstituted to 3 mg/ml
in ice-cold 80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, 10.2%
glycerol. 100 µl of the reconstituted tubulin was added to each well of a 96-well plate
(pre-warmed at 37°C) and was either untreated or exposed to the indicated compounds
at the specified concentrations, in duplicate. The absorbance at 340nm was measured
every 60 sec for one hour at 37°C using a Synergy HT multi-detection microplate
reader (Bio-Tek, Winooski, VT).
2.2.13 Statistical analysis
The statistical significance of differences was assessed by Student's t test using
GraphPad Prism (GraphPad Software Inc. La Jolla, CA). Statistically significant
differences are indicated by ***p<0.001, **p<0.01 and *p<0.05.

14
2.3. DNA binding studies
Double-stranded ctDNA was purchased from commercial sources (Sigma-Aldrich, St.
Louis, MO) and used without further purification. Polynucleotide was dissolved in
sodium cacodylate buffer, I = 0.05 M (pH = 7.0), additionally sonicated and filtered
through a 0.45 µm filter. DNA concentration per nucleotide was determined by
absorption spectroscopy, using the molar extinction coefficient of 6600 M−1cm−1 at 260
nm. The stock solution of compound 8e was prepared, due the poor solubility in water,
at a concentration of 1 mM in DMSO and diluted into buffer solution at the desired
concentration.
2.3.1. Thermal denaturation experiments
Thermal denaturation experiments were performed in a stoppered quartz cuvettes (1
cm path length) using an Agilent 8453 spectrometer equipped with a Peltier
temperature controller system ( 0.1° C). Thermal melting curves for ctDNA were
determined by following the absorption change at 260 nm in the absence or presence
of 8e as a function of temperature.
The absorbance intensity of wavelength 260 nm was measured over a temperature
range 25-98ºC. The temperature of the samples was raised in 0.5°C increments and
equilibrated for 1 min at each temperature setting. Absorbance of the ligands was
subtracted from every curve, and the absorbance scale was normalized. The Tm values
were taken as the midpoints of the transition curves, determined from the maximum of
the first derivative and checked graphically by the tangent method. ∆Tm values were
calculated subtracting Tm for the free polynucleotide from Tm of the complex ctDNA-8e.
Every ∆Tm value here reported was the average of at least two measurements, the
error in ∆Tm is 0.5 °C.
2.3.2. Fluorimetric experiments
Titration experiments were carried out at room temperature by adding increasing
amounts of ctDNA to a 1 × 10−4 M ligand solution. All solutions were prepared in 50
mM sodium cacodylate buffer (pH 7.0) using doubly distilled water and passed through
a Millipore apparatus. The absorbance of aqueous buffered solution of 8e was
proportional to their concentrations up to 100 µM. Hence, no significant intermolecular
aggregation of the compounds, which would be expected to give rise to hypochromicity
effects, occurred in the concentration range needed for the following spectroscopic
studies. The emission spectra were recorded in the 317-500 nm range with excitation

15
wavelengths of 300 nm. Absorption and volume correction procedures were applied to
the raw fluorescence data.
Ethidium Bromide Displacement Assays were recorded on a spectrofluorimeter in the
−680 nm range
540
with an excitation wavelength of 520 nm. The fluorescence was
normalized by the maximum fluorescence signal when EB was bound to the DNA in the
absence of competition for binding and was corrected for background fluorescence of
free EB in solution.
2.3.3. Circular dichroism experiments
CD spectra were recorded on a JASCO J815 spectropolarimeter between 400 and 200
nm in continuous scanning mode (50 nm/min, 1 nm bandwidth, and 1 s response time).
All of the CD spectra were generated and represented averages of five scans.
Measurements were performed by adding progressively increasing amounts of 8e to
different solutions of ctDNA (1 × 10–5 M) in 50 mMcacodylate buffer (pH 7).
2.4. Docking experiments
The pdbs of tubulin in complex with benzenesulfonamides ABT-751 (3HKC.pdb) and
T138067 (3HKE.pdb), colchicine (3UT5), pyrrolidinedione TN16 (3HKD), two
enantiomericpyridopyrazines (3N2G.pdb and 3N2K.pdb) and podophyllotoxin (1SA1)
were retrieved from the protein data bank [19] and only the chains and ligands
corresponding to one tubulin dimer were used. The ligands were built with Spartan´08,
prepared with AutodockTools[20] and docked with AutoDock 4.2[21] by running 100-
300 times the Lamarckian genetic algorithm (LGA) with a maximum of 2.5 106 energy
evaluations, 150 individuals in population and a maximum of 27000 generations. The
results were analyzed with AutoDockTools and with an in-house developed java-based
software tool [22].
3. Results
Taking into account our previous results on antiparasitic agents targeting L. infantum β-
tubulin and the increasing therapeutic relevance of sulfonamide derivatives as
antitumoral compounds, we decided to assess the potential anticancer activity of our
sulfonamide library in a combined effort performing an array of cell biological and
chemical studies in order to identify and characterize bioactive compounds.

16
3.1. Chemistry
The insertion of the secondary sulfonamide group into the organic scaffolds is
chemically straightforward and different methods have been adopted [23].
Sulfonamides 1-14 were prepared as previously described by condensation of the
corresponding sulfonyl chloride with amine derivatives in pyridine at 0 ºC [16].The
compounds were obtained in good yields and characterized by 1H NMR and 13C NMR
spectroscopy, CHNS elemental analysis and HRMS.
3.2. Biological activities
3.2.1. Antiproliferative activities
All compounds were evaluated for their in vitroantiproliferative activity against a panel
of representative human cancer cell lines, including cells from both solid tumors
(colorectal, brain, prostate) and hematological malignancies (leukemia, lymphoma).
Cells were treated for 48h with 10 µM of the indicated compounds and cell viability was
assessed using a luminescent-based ATP assay. This commonly-used concentration
was selected in our initial screen in order to discard as ineffective any compounds
showing a negative biological effect. These results clearly indicated that one
compound, sulfonamide 8e, showed a significant antiproliferative activity across the
panel (Figure 2A). Similar results were obtained with additional cell lines including
U87MG, U251, U373, Granta-519, JVM-2, Z-138, and SW480 (data not shown).In
agreement with its effects on cell viability, phase-contrast microscopy indicated that
treatment with 8e reduced LN229 and PC-3 cell proliferation and induced
morphological changes consistent with the induction of cell death (Figure 2B). Dose-
response experiments in selected cell lines indicated that sulfonamide 8e was
endowed with
a
pharmacologically-interesting antiproliferative activity in the
nanomolar/low micromolar range, with IC50 values ranging from 91 nM (Jurkat) to 17
µM (PC-3) (Figure 2C). Differences in sensitivity are not likely to be related to
expression of ATP-binding cassette drug transporters, since the IC50 value for U87MG
cells is within the same range as for LN229 cells (Figure 2C), and LN229 but not
U87MG cells have been shown to overexpress P-gp and MRP-1 [24].
Based on the results obtained with 8e, we decided to synthesize an additional series of
compounds including naphthyl groups or related aromatic ringsin order to test
whetherthis functional group could somehow be responsible for the antiproliferative

17
activity induced by 8e. These compounds were tested for their antiproliferative activity
against three representative cancer cell lines: Jeko-1, SKMG-3 and SW620, analyzing
their viability at 48h upon a standard 10 µM treatment. Surprisingly, none of the newly-
synthesized compounds showed significant antiproliferative activity when compared to
8e (Figure 2D). We thus proceeded to further characterize the cellular effects of 8e,
selected as the only active compound within this series of sulfonamides.
3.2.2. Effect on Cell Cycle Progression and Cellular Senescence
Flow cytometry studies upon propidium iodide staining in a number of cell lines treated
with 10 µM 8ewere performed in order to analyze its impact on cell cycle progression.
A 24h treatment with 8e induced a remarkable increase in the G2/M population in both
LN229 and PC-3 cells (Figure 3A), which led to a dramatic G2/M arrest at 48h in all the
cell lines tested (Figure 3B). We next analyzed induction of cellular senescence upon
8e treatment, performing a standard senescence assay based on the quantification of
β-galactosidase positivity[25]. Whereas untreated PC-3 cells were β-gal negative at all
observed time-points, treatment of PC-3 cells with 8e led to a dramatic increase in the
proportion of β-gal positive cells, already detectable at 24h and further increased at 48h
and 72h (Figure 3C). These data indicate that prolonged cell cycle arrest induced by
sulfonamide 8e also leads tothe induction of the cellular senescence program.
3.2.3. Induction of apoptosis
We next assessed whether treatment with 8e was also able to promote cellular death.
For this purpose, we investigated whether treatment with 8e could induce apoptosis in
LN229 and PC-3 cells. Flow cytometric analysis upon double Annexin V /propidium
iodide staining of 8e-treated cells indicated that, in both cell lines, there was an
increase in the early apoptotic (Annexin V-positive) fraction at 24h (Figure 4A). This
was then followed by a dramatic shift of the cellular population to the early apoptotic
quadrant at 72h, with a significant proportion of cells progressing to late apoptosis
(double Annexin V and propidium iodide-positive cells, upper right quadrant) (Figures
4A and 4B).
In order to confirm that cell death in response to 8e was associated with the activation
of caspases, we analyzed caspase activation. For this purpose, we first assessed
caspase 3 cleavage in response to 8e treatment in LN229 and in the highly-sensitive

18
SKMG-3 cell line. As expected, exposure to 8e led to the appearance of the low
molecular bands indicative of cleaved, active caspase-3 (Figure 4C). In correlation with
their higher sensitivity to 8e, SKMG-3 cells showed a more dramatic induction of
caspase-3 cleavage (Figure 4C). In agreement with these observations, treatment with
10 µM 8e for 48hinduced a detectable increase in the activity of the executioner
caspases 3/7 in most cell lines (Figure 4D). Importantly, the levels of caspase
activation inversely correlated with the reduction on cell viability induced by 8e
treatment in those same cell lines (Figure 4D). Taken together, these results indicate
that prolonged treatment with 8e, which rapidly induces a G2/M cell cycle arrest, leads
to the onset of caspase-dependent apoptosis and thus to cell death.
3.2.4. DNA damage analysis
In order to assess whether 8e could induce cellular DNA damage we used flow
cytometry to measure the staining of histone H2A.X phosphorylated at Ser-139 (also
termed γ-H2AX) which is a well-established readout for DNA damage, since H2A.X
becomes rapidly phosphorylated upon DNA double-strand breaks[26].Whereas only a
modest background level of γ-H2AX staining was detected in untreated Z-138 cells,
treatment with 8e induced a clear shift yielding a γ-H2AX-positive population (Figure
5A). Quantification of γ-H2AX staining revealed a 6-fold increase upon 8e treatment in
Z-138 cells, and very similar results were also obtained in both LN229 and SP-53 cells
(Figure 5B).
We next investigated whether 8e could promote the sustained growth-inhibitory effects
elicited by DNA-damaging agents, performing clonogenic assays upon a short
exposure to 8e. To this end, LN229 and PC-3 cells were treated with 10 µM 8e for 3h
and seeded at low density to follow-up colony formation. Surprisingly, the clonogenicity
of 8e-treated cells was very similar to that of untreated cells (Figure 5C), whereas
treatment with a standard genotoxic agent such as etoposide significantly reduced
colony formation (data not shown).
In order to investigate whether 8e was able to promote the activation of the DNA
damage checkpoint, we analyzed the phosphorylation levels of the checkpoint signal-
transducing kinase Chk1. As expected, treatment with etoposide clearly induced a
time-dependent increase in the phosphorylation levels of Chk1. In sharp contrast, Chk1
activation was undetectable upon 8e exposure as indicated by the absence of
phosphorylation induction (Figure 5D). In agreement with these observations, studies

19
aimed at elucidating whether 8e could structurally alter purified DNA in vitro yielded
negative results. Thermal denaturation, fluorescence titration, ethidium bromide
displacement assays and circular dichroism studies, all indicated low affinity binding,
ruling out 8e as an in vitro DNA-damaging compound (data not shown). Altogether,
these observations indicate that 8e does not induce cell cycle arrest secondary to the
activation of the DNA damage checkpoint.
3.2.5. Disruption of microtubuleassembly
Since the cellular effects of 8e are compatible with the response to antimitotic agents,
we next investigated whether 8e could alter the pattern of the microtubule network in a
number of cell lines. In NIH3T3, T98G, LN229 and MCF-7 cells tubulin staining
revealed a well-organized network of filamentous microtubules. In contrast, treatment
with 8e for 24h induced microtubule disassembly, with tubulin staining appearing as a
disorganized meshwork mostly restricted to the perinuclear region (Figure 6A).
Analysis of tubulin staining in synchronized, untreated,NIH3T3 cells indicated the
presence of a well-formed mitotic spindle at ~24h shortly followed by cytokinesis.
Importantly, neither the assembly of the mitotic spindle nor the presence of the
cytokinetic ring was detected in 8e-treated cells (Figure 6B). Interestingly, in correlation
with the effects of other established tubulin binders [27], long term treatment (>48h)
with 8e also induced micronucleation (Figure 6C).We finally investigated whether 8e
could directly influence tubulin polymerization in vitro. To this end, in vitro
polymerization assays were performed to monitor the kinetics of tubulin polymerization
in the absence or presence of 8e. Interestingly, 8e clearly induced a dose-dependent
decrease in tubulin polymerization (Figure 6D).These results strongly indicate that
sulfonamide 8e interferes with tubulin polymerization dynamics, leading to microtubule
cytoskeleton malfunction and mitotic arrest.
3.2.6. Inhibition of cancer cell motility
Since sulfonamide 8e can interfere with tubulin polymerization, we investigated
whether it could also inhibit cancer cell motility. To this end, we performed wound-
healing assays using MCF-7 breast cancer cells and LN229 glioma cells, since they
both are good models for cell motility assays. Both cell lines clearly showed a high rate
of cell motility as indicated by the efficiency of gap closure at 16h. Interestingly,
treatment with either 5 µM or 10 µM 8e strongly reduced cell motility in both cell lines

20
(Figure 6E). These results indicate that 8e, together with its previously described
effects on cell proliferation, senescence and apoptosis, can inhibit cancer cell motility.
3.2.7. Sulfonamide 8e is a reversible antimitotic agent
Since reversibility of antimitotic compounds is an important parameter in terms of
predicting in vivo efficacy and undesirable side effects[28], we investigated the
reversibility of the cellular effects of sulfonamide 8e. We first analyzed whether
disrupting the treatment with 8e would lead to the proper re-assembly of the
microtubule network. Interestingly, 48h after the removal of 8e, cells displayed a
completely reorganized microtubule cytoskeleton (Figure 7A), which is
undistinguishable from that of untreated cells (Figure 6A). In agreement with this, cell
proliferation inhibition was also reverted 48h upon 8e washout, as indicated by the
increase in both LN229 and PC-3 cell number detected in viability assays (Figure 7B).
As expected, proliferation is accompanied byprogressive mitotic exit as indicated by the
increase in the proportion of LN229 cells entering into the G1 phase of the cell cycle
(Figure 7C). Remarkably, even the detection of senescence is reduced in a time-
dependent manner upon 8e removal in PC-3 cells (Figure 7D). Taken together, our
observations clearly indicate that the cellular effects elicited by sulfonamide 8e are fully
reversible.
3.2.8. Docking studies and SAR analysis
With the goal of better understanding the structure–activity relationships, we performed
docking experiments for the synthesized sulfonamides in different crystal structures of
the colchicine site of tubulin in complex with structurally diverse ligands. From among
these, the docking results for 3HKC, the complex of tubulin with the
benzenesulfonamide ABT-751[29]were selected due to its higher structural similarity
with the new compounds here described (Figure 8A). Overall the sulfonamides bind to
the ABT-751 site with the two aromatic rings occupying the sites for the p-
methoxyphenyl ring and the 2,3-diaminopyridine moiety of ABT-751, thus placing the
sulphonamide brigde close to that of ABT-751, while the more deeply buried pocket for
the p-aminophenol is only occupied by ligands with large ring systems, such as 9a-d.
The docked ligands show two different dispositions corresponding to the reversal of the
sulfonamide moiety depending on the geometry of the attached aromatic rings, mainly
to the benzofused ones. When there is only one alpha linked benzofused system (e.g.
the naphthyl group in 8e), it preferentially binds at the 2,3-diaminopyridine pocket, thus

21
allocating the other ring in the p-methoxyphenyl pocket. When two such alpha rings are
present, both orientations can occur. When no alpha ring is present, the
benzofusedsystems still prefer to bind at the 2,3-diaminopyridine pocket, as we can
see for instance for 8b. Therefore, the presence of the 1-naphthyl group causes a
reversal in the binding mode for 8e, which probably explains its different activity profile
(Figure 8B). The same reversal is observed in7a,bbut in these cases, it does not result
in a potency increase. This is probably due to the lack of a substituent to replace the
methoxy group of ABT-751 that is an important pharmacophoric element in the
recognition of colchicine site ligands (hydrophobic site 1 of the pharmacophoric model
of Nguyen et al. [30]) The introduction of additional nitrogen atoms is also detrimental
for the activity, due to unsatisfied hydrogen bonds acceptor potential in the complexes.
4.
Discussion
In this report we have investigated the in vitro anticancer activities of our in-house
panel of sulfonamides, consisting both of novel structures and previously-reported
compounds some of which have shown interesting biological properties as antiparasitic
agents [16]. The analysis of the antiproliferative activity of a series of 24 sulfonamide-
based compounds in a comprehensive panel of representative human cancer cell lines
indicated that only one compound, sulfonamide 8e, had clear antiproliferative
properties. Since this compound was the only one within the series containing a
naphthyl group, we reasoned that this structural motif might be responsible for its
biological activity, and thus synthesized 12 additional sulfonamide compounds
containing naphthyl or related aromatic rings such as quinolyl or dansyl moieties in
order to test whether the naphthalene functional group was the major determinant of
the activity of compound 8e. Surprisingly, however, none of the novel sulfonamides
including naphthalene-related structures showed antiproliferative activity against three
representative cancer cell lines, in contrast with the performance of 8e in these cells.
The observation that only one compound within a series of highly-related structures
promotes significant biological effects suggests that 8eis likely to specifically interfere
with a cellular target.
Interestingly 8e treatment led to a very robust cell cycle arrest and the induction of
cellular senescence, which is a form of long-term and often irreversible proliferative
arrest characterized by a larger and flattened cell shape and the expression of
senescent markers such as β-galactosidase [31]. The observed G2/M arrest indicates

22
that sulfonamide 8e impairs cell proliferation by preventing entry into mitosis or its
completion. These results are compatible with the activation of the checkpoint pathway
that imposes a G2 arrest in order to prevent mitosis entry following the detection of
cellular DNA damage[32], which is normally observed in response to genotoxic agents.
These results are also compatible with the detection of apoptotic cell death upon
prolonged exposure to 8e. In agreement with this, a significant increase in γ-H2AX was
detected in a number of cell lines treated with 8e, thus suggesting the presence of
double-strand breaks in cellular DNA. An established assay to monitor the sustained
antiproliferative effects of genotoxic agents is the analysis of cellularclonogenic
capacity upon a transient exposure to such genotoxic drugs. Interestinlgy these assays
indicated that, whereas a standard genotoxic agent such as etoposide induced a
sustained antiproliferative effect, treatment with 8e was unable to reduce cellular
clonogenicity, thus indicating that 8e does not promote the hallmark response of a DNA
damage-inducing agent. In view of these conflicting observations, we directly monitored
the activation of the DNA damage checkpoint by analyzing the phosphorylation levels
of the checkpoint transducing kinase Chk1, which is rapidly phosphorylated in
response to DNA damage and amplifies the checkpoint signal phosphorylating crucial
downstream mediators [33]. The observation that the genotoxic agent etoposide, but
not 8e, was able to induce an increase in Chk1 phosphorylation indicates that 8e does
not promote the activation of the DNA damage checkpoint. In agreement with these
observations, a number of studies including thermal denaturation, fluorescence
titration, ethidium bromide displacement assays and circular dichroism measurements
indicated low affinity binding of 8e with purified DNA. These observations suggest
that8edoes not function as a genotoxic agent and therefore indicate that its cellular
effects are likely to be unrelated to the observed increase in γ-H2AX. The most
plausible explanation for the detection of γ-H2AX in the absence of other evidences of
DNA damage, such as the observed lack of checkpoint activation, is the so called
“pseudo DNA damage response” that has been reported in senescent cells. This
response is characterized by the detection of γ-H2AX in senescent cells without the
detection of DNA damage by direct measures of DNA fragmentation[34]. Taken
together, our results indicate that despite the detection of γ-H2AX, presumably as a
consequence of senescence induction, 8e does not function as a cellular genotoxic
agent with the ability to induce the DNA damage checkpoint.
The observation that 8e leads to a dramatic increase in G2/M content in the absence of
a detectable measure of checkpoint activation, which would induce a G2 arrest,

23
strongly points to the disruption of mitotic progression as the key mechanism
underlying the activity of 8e. The cellular effects of this compound are compatible with
the hallmark response to other well-established anticancer agents inducing a G2/M-
content increase followed by apoptosis: the mitotic poisons,such astubulin-binding
agents. Due to the crucial role of microtubules for the completion of mitosis, tubulin
heterodimers, the subunits of microtubules, constitute key cellular targets for the
development of new anticancer drugs and thus tubulin inhibitors are currently used for
the treatment of cancer. These inhibitors have antitumor activity by destabilizing tubulin
polymerization, which alters microtubule dynamics and prevents the correct assembly
of the mitotic spindle, leading to mitotic arrest. Several compounds, such as the
taxanes, the vinca alkaloids, and estramustine all prevent cell cycle progression by
inhibiting mitosis[35], and several tubulin binders are clinically-used chemotherapeutic
agents[4].Interestingly, a number of sulfonamides have been recently reported capable
of binding microtubules and exerting antimitotic activity[7]. In agreement with our
previous observations, 8e treatment led to a clear disruption of the microtubule
cytoskeleton in a number of cell lines, with tubulin staining accumulating in the
perinuclear region. Moreover, the formation of the mitotic spindle followed by the
cytokinetic ring detected in synchronized NIH3T3 cells was not observed upon 8e
treatment. Accordingly, in vitro tubulin polymerization assays with purified tubulin
monomers confirmed that 8e is able to inhibit the polymerization of tubulin.
Noteworthy, in correlation with our observations in response to 8e exposure, mitotic
poisons such as paclitaxel[36], discodermolide[37], vincristine and vinblastine[38] also
have been shown to induce cellular senescence and some of these compounds have
also been reported to promote an increase in γ-H2AX[39]. Collectively, our data
strongly indicate that sulfonamide 8e targets tubulin polymerization to elicit its cellular
effects.
The dynamic regulation of the cellular cytoskeleton, including the microtubule network,
is central to many complex cellular processes such as the control of cell shape and
motility.Cell motility is frequently altered in cancer cells, which usually display a highly
motile phenotype that is required to traverse the basal lamina and the endothelial layer
of vessels in the process of localized invasion required for the metastatic dissemination
of tumors [40]. Remarkably, and in agreement with its ability to disassemble the tubulin
cytoskeleton, compound 8e is also able to reduce cancer cell motility as indicated by
our results in wound healing assays performed with MCF-7 and LN229 cells, thus
improving its anticancer potential. Another interesting property displayed by some

24
antimitotic agents is reversibility of action, since compounds that disrupt microtubule
dynamics show a wide degree of reversibility in their ability to block mitosis. The classic
antimitotic compound colchicine, for instance, induces an almost irreversible mitotic
block, whereas structurally-related colcemid exerts reversible effects. This property of
mitotic poisons is not only compound-specific but also difficult to explain in terms of
SAR: even small structural changes in compounds within a given class can lead to
profound differences in mitotic block reversibility[41]. Reversibility is an important
parameter in terms of predicting in vivo efficacy and undesirable side effects, as
illustrated by the fact that slow dissociation from tubulin is thought to be the main
reason behind colchicine’s toxicity, which has led to its failure in the clinic[28]. Taking
this into account, we investigated the cellular response upon 8e removal. Interestingly,
8e washout led to a robust time-dependent reversion of its cellular effects, as shown by
the full assembly of the tubulin cytoskeleton, the restoration of cell proliferation
accompanied by progressive mitotic exit and concomitant G1 increase and, finally, the
reduction of cellular senescence. This latter observation is remarkable since
senescence has been, until recently,often described as an irreversible growth arrest.
However, similar observations showing reversibility in the detection of senescent
markers have also been previously reported, such as in ceramide-induced
senescence[42] or in senescence induced by FGFR3 [43].
We finally show that docking studies and SAR analysis further confirm our
experimental observations and strongly suggest that sulfonamide 8e interacts with the
colchicine site of tubulin. In summary, our data have enabled to identify a
naphtalenesulfonamide, compound 8e, as a potential anticancer agent with
antiproliferative activity within a library of sulfonamide compounds. Exposure to 8e
leads to a dramatic G2/M arrest followed by the induction of apoptosis and the
detection of cellular senescence, and also a reduction of cancer cell motility. These
cellular effects, which are reversible upon removal of the compound, are likely related
to the ability of 8e to inhibit tubulin polymerization and thus mitotic progression, and
therefore allow us to conclude that sulfonamide 8e is a novel reversible antimitotic
compound inhibiting cancer cell motility.
5.
Acknowledgements
This work was supported by the Spanish MINECO and FEDER (Consolider-Ingenio,
Project CSD2010-000652010 and CTQ2013-48917-C3-1-P) and Generalitat

25
Valenciana (PROMETEO II 2015/002).C.G-R. thanks the Ministry of Education of
Spain for a FPU Grant.We are grateful to Joan Seoane (Institut de Recerca Hospital
Valld’Hebron, Barcelona), Hans Skovgaard (Rigshospitalet, Oslo), DolorsColomer
(Hospital Clínic, Barcelona) and Beatriz Martínez (CNIO, Madrid) for cell lines, Manel
Joaquin (Universitat Pompeu Fabra, Barcelona) for advice on γ-H2A.X staining and
Antoni Costa (Universitat de les Illes Balears, Palma) for advice on non-linear
regression analysis and IC50 determinations. Rafael Peláez thanks Consejería de
Educación, Junta de Castilla y León (SA147U13).
6.
Conflict of Interest Statement
The authors declare that there are no conflicts of interest.
7.
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