Synthesis, antiproliferative and apoptotic activities of N-(6(4)-indazolyl)-benzenesulfonamide derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.09.013

Recently, it has been reported that compounds bearing a sulfonamide moiety possess many types of biological activities, including anticancer activity. The present work reports the synthesis and antiproliferative evaluation of some N-(6(4)-indazolyl)benzenesulfonamides and 7-ethoxy-N-(6(4)-indazolyl) benzenesulfonamides. All compounds were evaluated for their in vitro antiproliferative activity against three tumor cell lines: A2780 (human ovarian carcinoma) A549 (human lung adenocarcinoma) and P388 (murine leukemia). The results indicated that sulfonamides 2c, 3c, 6d, 8, 13, 3b and 16 were endowed with a pharmacologically interesting antiproliferative activity with compounds 2c and 3c showing the lower IC₅₀ (from 0.50 ± 0.09 to 1.83 ± 0.52 μM and from 0.58 ± 0.17 to 5.83 ± 1.83 μM, respectively). Moreover, these indazoles were able to trigger apoptosis through the upregulation of the typical apoptosis markers p53 and bax. As regard to the hypothetic targets of these compounds, a preliminary docking analysis showed that all compounds seemed to interact with β-tubulin, in particular compound 3b that showed the lower Ki. The cytofluorimetric analysis of the cell cycle phases indicates that all compounds, when administered at their IC ₇₅, caused a block in the G2/M phase of the cell cycle with the generation of subpopulations of cells with a number of chromosome >4n. When the IC₅₀s were applied we observed a prevalent block in the G0/G1 phase except for compounds 16 and 8 where a partial G2/M block was present with a concomitant decrease of cells in the G0/G1 and S phases of the cell cycle. Altogether these results suggest a possible, but not exclusive, interaction with microtubules.

Full text of DOI:10.1016/j.ejmech.2012.09.013

European Journal of Medicinal Chemistry 57 (2012) 240e249
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative and apoptotic activities of N-(6(4)-indazolyl)-
benzenesulfonamide derivatives as potential anticancer agents
,
a
a
Najat Abbassi a, Hakima Chicha a, El Mostapha Rakib a , Abdellah Hannioui , Mdaghri Alaoui ,
**
Abdelouahed Hajjaji a, Detlef Geffken b, Cinzia Aiello c, Rosaria Gangemi c, Camillo Rosano d,
Maurizio Viale c
,
*
a Laboratoire de Chimie Organique et Analytiques, Faculté des Sciences et Techniques, Université Sultan Moulay Slimane, B.P. 523, Béni-Mellal, Morocco
b Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Hamburg, Hamburg, Germany
c IRCCS Azienda Ospedaliera Universitaria San Martino e IST Istituto Nazionale per la Ricerca sul Cancro, U.O.C. Terapia Immunologica, L.go R. Benzi 10, 16132 Genova, Italy
d IRCCS Azienda Ospedaliera Universitaria San Martino e IST Istituto Nazionale per la Ricerca sul Cancro, U.O.S. Biopolimeri e Proteomica, L.go R. Benzi 10, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, it has been reported that compounds bearing a sulfonamide moiety possess many types of
biological activities, including anticancer activity. The present work reports the synthesis and anti-
proliferative evaluation of some N-(6(4)-indazolyl)benzenesulfonamides and 7-ethoxy-N-(6(4)-inda-
zolyl)benzenesulfonamides.
All compounds were evaluated for their in vitro antiproliferative activity against three tumor cell lines:
A2780 (human ovarian carcinoma) A549 (human lung adenocarcinoma) and P388 (murine leukemia).
The results indicated that sulfonamides 2c, 3c, 6d, 8, 13, 3b and 16 were endowed with a pharmaco-
logically interesting antiproliferative activity with compounds 2c and 3c showing the lower IC50 (from
Received 25 June 2012
Received in revised form
6 September 2012
Accepted 7 September 2012
Available online 17 September 2012
Keywords:
Nitroindazoles
N-(6(4)-Indazolyl)benzenesulfonamides
Antiproliferative
Apoptosis
0.50 ꢀ 0.09 to 1.83 ꢀ 0.52
m
M and from 0.58 ꢀ 0.17 to 5.83 ꢀ 1.83
mM, respectively). Moreover, these
indazoles were able to trigger apoptosis through the upregulation of the typical apoptosis markers p53
and bax.
Cell cycle arrest
As regard to the hypothetic targets of these compounds, a preliminary docking analysis showed that all
compounds seemed to interact with b-tubulin, in particular compound 3b that showed the lower Ki. The
cytofluorimetric analysis of the cell cycle phases indicates that all compounds, when administered at
their IC75, caused a block in the G2/M phase of the cell cycle with the generation of subpopulations of
cells with a number of chromosome >4n. When the IC50s were applied we observed a prevalent block in
the G0/G1 phase except for compounds 16 and 8 where a partial G2/M block was present with
a concomitant decrease of cells in the G0/G1 and S phases of the cell cycle. Altogether these results
suggest a possible, but not exclusive, interaction with microtubules.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
substantial anticancer activities in vitro and/or in vivo [8e12], and
our research group has reported the synthesis of some new N-(7-
Indazoles posses many types of biological activities [1,2] and
representatives of this class of pharmacological agents are widely
used as antibacterial [3], anti-inflammatory [4], anti-HIV [5],
antiprotozoal [6] and antimalarial [7]. Recently, a host of struc-
turally novel indazole derivatives have been reported to show
indazolyl)benzenesulfonamide derivatives of type A (Fig. 1).
Some of these compounds exhibited significant cytotoxicity
against human (colon and prostate) and murine (leukemia) cell
lines [13]. The perturbations of the cell cycles induced by the
most potent compounds were studied on the L1210 cell line by
flow cytometry. All of these compounds induced
a marked
accumulation in G2/M þ 8N phases of the cell cycle. This
observation is typical of the modification of the cell cycle
induced by numerous tubulin interacting drugs. Compound B, 4-
methoxy-N-(3-chloro-7-indazolyl)benzenesulfonamide, was the
most active of the series.
* Corresponding author. Tel.: þ39 0105737320; fax: þ39 0105737373.
** Corresponding author. Tel.: þ212 523485112; fax: þ212 523485201.
E-mail addresses: elmostapha1@ymail.com (E.M. Rakib), maurizio.viale@istge.it
(M. Viale).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.013

N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
241
ethoxy group in indazole. As an extension of these results, we
continued our studies on reactions of the reduction of aromatic
nitro compounds with anhydrous SnCl2 in alcohol. Here, we report
the reduction of 6-nitroindazole derivatives by anhydrous SnCl2
in ethanol. Thus, compounds (9a,b) were reduced as previously
described, and subsequent coupling was achieved with 4-
methylbenzenesulfonyl chloride. We noticed that (9a,b) afforded
only N-(3-halogeno-6-indazolyl)-benzenesulfonamides (10a,b) and
(11a,b) (Scheme 4). No trace of nucleophilic substitution of ethoxy
group was observed.
Fig. 1. Cytotoxic activity of N-(7-indazolyl)-benzenesulfonamides against human
(colon and prostate) and murine (leukemia) cell lines.
When we investigated the same conditions to 2-allyl-6-
nitroindazole 12 we obtained a mixture of two products, 6-
aminoindazole and 7-ethoxy-6-aminoindazole, which were
immediately coupled by 4-methylbenzenesulfonamide chloride in
pyridine (Scheme 5). In this case, it is clear that SNH is improved by
N-alkylation of the N-2 position.
In continuation of our efforts in searching for novel effective
anticancer agents, we synthesized a series of N-(6(4)-indazolyl)ben-
zenesulfonamide derivatives and evaluated their antiproliferative
and apoptotic potential. Docking and cytometric analyses were also
performed in order to identify the possible molecular target of these
compounds.
The structure of compound 14 was unambiguously proven by
single crystal X-ray analysis [18] (Fig. 3).
Interestingly, the reduction of 7-nitroindazole 10 as previously
described in our work [14] delivered a mixture of sulfonamide 16
with ethoxy group at position 4 and compound 17 devoid of ethoxy
group (Scheme 6).
2. Chemistry
Previously, we described the syntheses of various N-(alkoxy-1H-
indazol-4(7)-yl)benzenesulfonamide derivatives [14,15] by reduc-
tion of nitroindazole derivatives with SnCl2 in different alcohols
followed by coupling the corresponding amine by benzenesulfo-
namide. This method was employed for the synthesis of N-(1H-
indazol-4-yl)-benzenesulfonamides (2aec) and N-(7-ethoxy-1H-
indazol-4-yl)-benzenesulfonamides (3aec) starting from 3-
halogeno-4-nitroindazoles (1aec) (Scheme 1). We observed a new
kind of transformation of 4-nitroindazoles under the action of
stannous chloride in ethanol leading to 7-ethoxy-4-aminoindazoles
accompanied by the desired amine.
3. Biological results
3.1. Inhibition of cell proliferation and triggering of apoptosis by
indazole compounds
The analysis of the inhibition of cell proliferation obtained by
the treatment for 72 h with our indazole compounds showed that
at least 7 molecules had a significant inhibiting activity, here
Similarly, we prepared the sulfonamides (6d,e) and (7d,e) by
reduction of N-alkyl-4-nitroindazoles (4d,e) with SnCl2 in ethanol
followed by coupling of the corresponding amine with 4-
methylbenzenesulfonamide (Scheme 2). Compounds 4 and 5 have
been synthesized starting from alkylation of 4-nitroindazole 1a
according to a previously described procedure [16].
The structural assignments of the sulfonamides (6d,e) and
(7d,e) are based on a full characterization by 1H NMR and 13C NMR
spectra. The structure of the N-[7-ethoxy-1-(prop-2-en-1-yl)-1H-
indazol-4-yl]-4-methylbenzenesulfonamide 7e, was established by
X-ray crystallography [17] (Fig. 2).
When we applied the same reduction conditions as previously
described for 2-methyl-4-nitroindazole 5d, we obtained only the
N-(7-ethoxy-2-methyl-4-indazolyl)-4-methylbenzenesulfonamide
8 functionalized in position 7 in good yield (Scheme 3). This result
indicates that alkylation of 4-nitroindazole at position 2 leads
selectively to 7-ethoxyindazole derivatives with good yield.
Obviously, the position of nitro group of indazole plays an
important role for the orientation of the nucleophilic substitution of
defined with an IC50 lower than the arbitrary value of 30
three cell lines used for the assays. In particular, 2c and 3c showed
the lower IC50s (from 0.50 ꢀ 0.09 to 1.83 ꢀ 0.52 M and from
0.58 ꢀ 0.17 to 5.83 ꢀ 1.83 M, respectively), demonstrating their
greater activity compared to the other active compounds (from
3.40 ꢀ 1.43 to 6.88 ꢀ 2.10 M, from 4.41 ꢀ 0.52 to 11.9 ꢀ 0.5 M,
M, from 4.19 ꢀ 1.70 to 14.4 ꢀ 3.4, and
mM in all
m
m
m
m
from 5.21 ꢀ1.40 to 17.5 ꢀ 4.0
m
from 5.70 ꢀ 0.29 to 25.1 ꢀ 8.6
mM for 6d, 13, 3b, 8, and 16,
respectively) (Table 1).
Considering the activation of the apoptotic death, in general, the
most active compounds in terms of inhibition of cell proliferation
(i.e. 2c and 3c) were also those displaying the higher activity in
terms of apoptosis (Table 2). An interesting result is that, on
average, while the sensitivity of the human lung A549 cell line to
the antiproliferative activity of the active compounds was lower
than that of human ovarian A2780 cancer cells (Table 1), in terms of
induction of apoptosis and at equitoxic concentrations these cells
seem to be in general (in particular for 2c, 3c, 13 and 6d) more
sensitive than A2780 cells (p < 0.05, Table 2).
Scheme 1.

242
N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
Scheme 2.
3.2. Western blot analysis of p53 and bax apoptosis markers
structures. To verify this hypothesis we repeated the experiments
using a higher compound concentration (IC75) observing that all
compounds in these culture conditions caused a typical and marked
block of cells in the G2/M phase of the cell cycle and a simultaneous
decrease of cells in the S and G0/G1 phases. Moreover, all
compounds generated a cellular subpopulations with a number of
chromosomes >4n.
Our compounds 3b, 2c, 3c, 13, 6d, and 16, all able to induce
apoptosis, were also analyzed by western blot for the activation/
upregulation of some apoptosis markers such as p53 and bax. To
this end we studied the marker levels on A2780 cells after 24, 48
and 72 h incubation with their specific IC50s.
Our results show (Fig. 4) that all tested compounds were able to
upregulate both p53 and bax apoptosis markers, though in
a different manner. In fact, p53 was greatly upregulated already
after 24 h incubation by all active compounds, while bax upregu-
lation generally appeared after 48 h incubation and was much less
evident than that of p53 or, in case of compound 6d, absent.
Nevertheless, on the whole these data suggest a p53-dependent
pathway of apoptosis induction.
3.4. Docking analysis
The docking procedure described below allowed us to identify
the colchicine site as the binding pocket for the considered mole-
cules. Particularly, all the ligand tested “in silico” adopted a bound
conformation similar to that of colchicine where the ligand is mostly
buried in the intermediate domain of the
strands S8, S9, loop T7, and -helices H7, H8. The ligands tested also
interact with loop T5 of the neighboring -subunit. Such a location
b subunit, boxed in by b-
a
a
3.3. Cell cycle analysis
explains how the binding of a colchicine molecule to tubulin
prevents the curved complex from adopting a straight structure
[19]. Among the molecules tested in silico, 3b was identified as the
best tubulin ligand displaying a calculated Ki of about 7.1 ꢁ 10ꢂ7 M
and a favorable clustering. Fig. 5 shows the superposition of mole-
cule 3b, positioned according to the docking simulation results, to
colchicine, as revealed by X-ray crystallography. Compound 2c was
shown to be an ideal candidate but with a slightly lower calculated
Ki (3.2 ꢁ 10ꢂ6 M), the other candidates showed lower ranking scores
and thus were considered as less good ligands.
To evaluate the modifications of the cell cycle phases due to the
exposure to our indazole active compounds A2780 cells were
analyzed after treatment by flow cytometry and propidium iodide
staining. As shown in Table 3, our compounds 3b, 3c, 6d and 13 were
able to cause a block of cells in the G0/G1 phase of the cell cycle at
their IC50s, with a concomitant decrease of cells in the S phase.
While we did not observe the presence of polyploid/aneuploid cells,
a partial block of cells in the G2/M phase, when we used compounds
16 and 8, suggested a possible mechanism of action starting from
a direct interaction of these compounds with the microtubular
4. Discussion and conclusion
Sulfonamides represent an important group of compounds
endowed with different pharmacological activities such as diuretic,
Fig. 2. ORTEP drawing of 7e, showing the molecular numbering scheme. Ellipsoids are
drawn at the 30% probability level.
Scheme 3.

N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
243
Scheme 4.
antibiotic, anti-hypertensive and hypoglycaemic. Obviously, the
sulfonamide group represents a key structural motif for the inter-
action with many cell targets. In particular, in the last 15 years this
motif has been considered as starting point for the synthesis of new
anticancer compounds such as the chloroquinoxaline sulfonamide
[20] or the diarylsulfonylureas [21] or again the sulfonamide E-
7010, ER-34410 and E-7070 [22,23], some of which also entered the
clinical phase of study [24].
On this base and taken into account our previously described
synthesis of N-(7-indazolyl)benzenesulfonamide derivatives of type
A [13], we have developed a method for the synthesis of a series of N-
(6(4)-indazolyl)benzenesulfonamides and N-(7-ethoxy-6(4)-inda-
zolyl)benzenesulfonamides and described their evaluation as anti-
cancer agents. Our methodology for the synthesis of sulfonamides is
based on the reduction of the nitro group of 6(4)-nitroindazoles
using anhydrous stannous chloride in ethanol followed by coupling
of the corresponding amine by benzenesulfonamides.
indicates that our indazoles may interact, although with significant
differences, with the binding site of colchicine, a well-known
tubulin interacting agent, disturbing the normal curvature of the
tubulin complex and complicating the physiological growth of
microtubules during polymerization with the subsequent stop or
alteration of mitosis. This binding site was similar to that of the
already mentioned antimitotic sulfonamides E7010 and ER-34410
and suggest that this kind of chemical structure has a particular
binding “preference” for this molecular site.
Always as regard tothetarget, in a previouspaper we showed that
most of our previously synthesized N-(7-indazolyl)-benzenesulfo-
namide derivatives caused a deep perturbation of the cell cycles with
a marked accumulation of cells in the G/M þ 8N phases [13]. These
results suggested the possibility that this different class of indazoles
could have a mechanism of action starting from an initial binding to
the cell microtubular system. On the basis of this hypothesis and to
verify the hypothesis suggested by docking analysis we thus tried to
evaluate by cytometry whether a similar molecular target could
justify the activity of our new indazole derivatives.
Once analyzed for their ability to cause modifications of the cell
cycle phases, all active compounds showed to cause a block in the
G2/M phase of the cell cycle, and to generate subpopulations with
a number of chromosome >4n. This effect clearly appeared at the
higher concentration used for the treatment (IC75) and, in analogy
with the N-(7-indazolyl)-benzenesulfonamide derivatives, strongly
suggests a possible interaction of indazoles with tubulin or with
other macromolecules involved in the mitosis events such as the
The sulfonamides 2c, 3c, 6d, 13, 3b, 8, and 16 exhibited high or
very high activity against all the tumor cell lines investigated.
Compounds 2c and 3c were the most potent of all derivatives tested
with IC50s near 1 mM in one or more cell lines. A similar result was
found also considering their apoptotic activity, in fact active
compounds were all able to trigger apoptosis, in particular
compounds 2c and 3c, and to upregulate the p53 and bax markers,
thus suggesting the mitochondrial way for the activation of this
important mechanism of tumor cells killing.
As regard to the structure, our data demonstrate that the bio-
logical activity depends mainly on the presence of a methoxy group
in the benzene ring of the sulfonamide moiety associated with and
a chlorine atom in position 3 of the indazole. This important role is
suggested by the fact that the antiproliferative activity of the two
molecules 2c and 3c, which are characterized by the presence of
these residues, is significantly higher compared to that of the other
active compounds. This finding is not surprising since other two
classical antimitotic sulfonamides (E-7010 and ER-34410) were
both characterized by a para-methoxy group linked to the benze-
nesulfonamide moiety [22e24]. On the other hand our docking
aurora kinases or the g-tubulin, whose inhibition blocks the normal
progression of the phases of mitosis with a subsequent poly-
ploidization effect [25,26].
A G2/M block was also present, although to a smaller extent, at
a lower applied concentration (IC50) for compound 16 and 8, and
was linked to a decrease of cells in the G0/G1 or S phases. In the
same culture conditions all the other compounds showed a typical
block in the G0/G1 phase with a concomitant decrease of cells in
the following S phase. As known cell cycle progression and
apoptosis are the result of the interaction of a great number of
biomolecular factors, whose study was outside our present aims;
their definition in our culture conditions could help to better
understand the partially different behavior of our compounds and
to eventually enhance their efficacy in vitro.
analysis identifies as the better ligand for the b-tubulin binding site
the molecule 3b with a Ki of 7.1 ꢁ 10ꢂ7 M, and this molecule was,
together with 16, the less active compound among the active ones.
This introduces the possibility that our compounds may bind to
other possible molecular targets, probably more essential for cell
survival. This results from the fact that 3b, as already mentioned,
does not carry a methoxy and/or a chlorine groups that seem to be
linked to the higher activity of 2c and 3c. Our docking analysis also
In conclusion, in this paper we have described the synthesis, the
antiproliferative and apoptotic activities of new N-(6(4)-indazolyl)-
benzenesulfonamides and N-(7-ethoxy-6(4)-indazolyl)-benzene-
sulfonamides.Someselected compounds exerted a pharmacologically
Scheme 5.

244
N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
Table 1
Concentrations of indazole compounds inhibiting 50% proliferation (IC50) in A2780,
A549 and P388 cells.
Compounds
Cell lines
A2780
A549
P388
2a
3a
2b
3b
2c
3c
6d
7e
>100a
>100
58.5 ꢀ 3.4
82.4 ꢀ 19.5
17.5 ꢀ 4.0
1.83 ꢀ 0.52
5.83 ꢀ 1.83
6.88 ꢀ 2.1
100.4 ꢀ 2.7
14.4 ꢀ 3.4
103.6 ꢀ 13.0
>100
85.5 ꢀ 5.5
43.5 ꢀ 5.1
53.9 ꢀ 2.4
5.21 ꢀ 1.40
0.50 ꢀ 0.09
0.58 ꢀ 0.17
3.40 ꢀ 1.43
51.8 ꢀ 2.1
4.19 ꢀ 1.70
51.3 ꢀ 1.2
71.3 ꢀ 5.7
66.6 ꢀ 4.9
4.41 ꢀ 0.52
68.9 ꢀ 1.4
5.70 ꢀ 0.29
55.1 ꢀ 2.8
70.9 ꢀ 4.01
10.5 ꢀ 2.12
0.86 ꢀ 0.22
1.23 ꢀ 0.36
6.62 ꢀ 1.56
61.6 ꢀ 1.8
8.34 ꢀ 1.53
74.7 ꢀ 12.8
86.6 ꢀ 6.7
101.4 ꢀ 3.9
4.89 ꢀ 1.51
>100
8
10b
11a
11b
13
14
16
>100
Fig. 3. ORTEP drawing of 14, showing the molecular numbering scheme. Ellipsoids are
drawn at the 30% probability level.
11.9 ꢀ 0.5
>100
8.00 ꢀ 1.86
25.1 ꢀ 8.6
a
interesting antiproliferative/apoptotic activity against human and
murine cell lines. Although with different characteristics all
compounds induced a block of cells in the G2/M phase of the cell cycle
and generate cells with an abnormal number of chromosomes,
phenomena that are typical, although not exclusively, of tubulin
interacting agents. These findings together with docking results
suggest that our indazoles may represent good lead compounds for
further studies aimed at the synthesis of new and more active anti-
tumor agents.
Data represent the means ꢀ SD of 3e9 experiments.
basic (pH 7e8) by addition of 5% aqueous potassium bicarbonate
before being extracted with EtOAc. The organic phase was washed
with brine and dried over magnesium sulfate. The solvent was
removed to afford the amine, which was immediately dissolved in
pyridine (5 mL) and then reacted with 4-methylbenzenesulfonyl
chloride (0.26 g, 1.25 mmol) at room temperature for 24 h. After the
reactionmixturewasconcentrated invacuo, theresultingresiduewas
purified by flash chromatography (eluted with EtOAc/hexane 1/9).
5. Experimental
5.1.1.1. N-(1H-4-Indazolyl)-4-methylbenzenesulfonamide
Yield: 35%; mp: 170e172 ꢃC; IR (KBr, cmꢂ1): 3340, 3235 (NH), 1595
(CN), 1335, 1160 (SO2); 1H NMR (DMSO-d6):
2.27 (s, 3H, CH3), 6.92
(2a).
5.1. Chemistry
d
Melting points were determined using a Büchi-Tottoli apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded in
CDCl3 or DMSO-d6 and solution (unless otherwise specified) with
TMS as an internal reference using a Bruker AC 300 (1H) or 75 MHz
(dd, 1H, J ¼ 2.1 Hz and 6.1 Hz), 7.16 (d, 2H, J ¼ 6.2 Hz), 7.28 (d, 2H,
J ¼ 8.3 Hz), 7.68 (d, 2H, J ¼ 8.3 Hz), 8.22 (s, 1H), 10.51 (s, 1H, NH),
13.05 (s, 1H, NH); 13C NMR (DMSO-d6):
d 21.3 (CH3), 106.7 (CH),
111.0 (CH), 117.3 (C), 126.9 (2CH), 127.2 (CH), 130.1 (2CH), 130.5 (C),
132.4 (CH), 137.3 (C), 141.4 (C), 143.7 (C); MS m/z ¼ 288 [M þ 1]þ.
Anal. Calcd. for C14H13N3O2S: C, 58.52; H, 5.56; N, 14.62. Found: C,
58.76; H, 5.42; N, 14.77.
(
13C) instruments. Chemical shifts are given in
d parts per million
(ppm). Multiplicities of 13C NMR resources were assigned by dis-
tortionless enhancement by polarization transfer (DEPT) experi-
ments. Low-resolution mass spectra (MS) were recorded on
a PerkineElmer Sciex API 3000 spectrometer. Column chromatog-
raphy was carried out on SiO2 (silica gel 60 Merck 0.063e
0.200 mm). Thin-layer chromatography (TLC) was carried out on
SiO2 (silica gel 60, F 254 Merck 0.063e0.200 mm), and the spots
were located with UV light (254 nm). Commercial reagents were
used without further purification unless stated.
5.1.1.2. N-(7-Ethoxy-1H-4-indazolyl)-4-methylbenzenesulfonamide
(3a). Yield: 33%, mp: 210e212 ꢃC; IR (KBr, cmꢂ1): 3340, 3220 (NH),
1585 (CN), 1338, 1165 (SO2); 1H NMR (DMSO-d6):
d 1.37 (t, 3H, CH3,
J ¼ 6.9 Hz), 2.28 (s, 3H, CH3), 4.12 (q, 2H, OCH2, J ¼ 6.9 Hz), 6.64 (d,
1H, J ¼ 8.1 Hz), 6.72 (d, 1H, J ¼ 8.1 Hz), 7.25 (d, 2H, J ¼ 8.1 Hz), 7.58
(d, 2H, J ¼ 8.1 Hz), 7.97 (s, 1H), 10.06 (s, 1H, NH), 13.21 (s, 1H, NH);
13C NMR (DMSO-d6):
d 15.1 (CH3), 21.3 (CH3), 64.2 (CH2O), 106.1
5.1.1. Synthesis of N-(1H-indazol-4-yl)-4-methylbenzenesulfonamides
(2aec) and N-(ethoxy-1H-indazol-4-yl)-4-methylbenzenesulfonamides
(3aec)
A mixture of 2-alkyl-4-nitroindazole (3aeb) (1.22 mmol) and
anhydrous SnCl2 (1.1 g, 6.1 mmol) in 25 mL of absolute EtOH was
heated at 60 ꢃC. After reduction, the starting material has disappeared
and the solution is allowed to cool down. The pH was made slightly
(CH), 114.9 (CH), 120.6 (C), 122.3 (C), 127.2 (2CH), 129.9 (2CH), 132.7
(CH), 132.9 (C), 137.4 (C), 142.4 (C), 143.4 (C); MS m/z ¼ 332
[M þ 1]þ. Anal. Calcd. for C16H17N3O3S: C, 57.99; H, 5.17; N, 12.68.
Found: C, 57.86; H, 5.28; N, 12.57.
5.1.1.3. N-(3-Bromo-1H-4-indazolyl)-4-methylbenzenesulfonamide
(2b). Yield: 25%; mp: 150e152 ꢃC; IR (KBr, cmꢂ1): 3335, 3225 (NH),
Scheme 6.

N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
245
Table 2
Triggering of apoptosis by indazole compounds, as evaluated by nuclear morphological analysis after DAPI staining.
Drug concentration Anticancer compounds
3b 13
2c
3c
6d
16
8
A2780
A549
P388
CTR
2.8 ꢀ 1.0a
3.2 ꢀ 2
1.8 ꢀ 1.3
2.2 ꢀ 1.2
1.5 ꢀ 1.4
1.8 ꢀ 1.6
3.0 ꢀ 1.0
b
IC90
IC50
CTR
IC90
IC50
CTR
IC90
IC50
34.3 ꢀ 3.4
12.8 ꢀ 3.5
1.8 ꢀ 0.5
32.3 ꢀ 9.4
2.8 ꢀ 1.0
2.5 ꢀ 1.9
87.5 ꢀ 6.4
9.8 ꢀ 3.5
66.5 ꢀ 15
19.3 ꢀ 6.5
1.6 ꢀ 0.5
46.6 ꢀ 10.1
23.6 ꢀ 4.4
2.0 ꢀ 0.8
36.8 ꢀ 5.9
23.3 ꢀ 5.3
1.7 ꢀ 0.5
24.0 ꢀ 6.6
7.3 ꢀ 2.5
27.0 ꢀ 6.2
12.5 ꢀ 2.1
0.9 ꢀ 1.2
51.5 ꢀ 10.8
33.5 ꢀ 6.5
3.0 ꢀ 2.6
1.0 ꢀ 1.3
29.2 ꢀ 8.9
51.2 ꢀ 11.2
1.5 ꢀ 0.5
70.8 ꢀ 12.3
34.3 ꢀ 7.5
2.5 ꢀ 2.0
64.5 ꢀ 7.0
34.8 ꢀ 9.7
1.5 ꢀ 1.0
41.2 ꢀ 5.8
28.0 ꢀ 4.6
1.5 ꢀ 0.6
24.9 ꢀ 6.7
18.8 ꢀ 7.7
1.8 ꢀ 1.0
54.3 ꢀ 1.5
22.3 ꢀ 4.0
1.5 ꢀ 1.3
68.7 ꢀ 9.4
30.0 ꢀ 12.3
74.0 ꢀ 12.5
11.5 ꢀ 5.6
98.0 ꢀ 1.1
47.3 ꢀ 10.5
91.3 ꢀ 3.9
37.3 ꢀ 11.8
56.0 ꢀ 12.2
19.0 ꢀ 6.7
94.3 ꢀ 4.9
10.3 ꢀ 3.4
a
The means ꢀ SD (4e6 data) express the percentage of apoptotic cells.
b
IC90 ranged from 0.97 mM to 957 mM. When IC90s were higher than 200 mM (A2780, 13, 16, 8, 6d; A549, 13, 8, 6d) this concentration was used to treat cells.
1595 (CN), 1340, 1156 (SO2); 1H NMR (DMSO-d6):
d
2.36 (s, 3H, CH3),
cmꢂ1): 3345, 3231 (NH), 1600 (CN), 1338, 1162 (SO2); 1H NMR
(DMSO-d6):
6.57 (d, 1H, J ¼ 7.4 Hz), 7.25 (t, 1H, J ¼ 7.4 Hz), 7.35 (d, 2H, J ¼ 8.1 Hz),
d
1.39 (t, 3H, CH3, J ¼ 7.0 Hz), 2.36 (s, 3H, CH3), 4.15 (q,
7.41 (d, 1H, J ¼ 7.3 Hz), 7.63 (d, 2H, J ¼ 8.1 Hz), 9.69 (s, 1H, NH), 13.50
2H, OCH2, J ¼ 7.0 Hz), 6.24 (d, 1H, J ¼ 8.1 Hz), 6.38 (d, 1H, J ¼ 8.1 Hz),
7.35 (d, 2H, J ¼ 8.0 Hz), 7.57 (d, 2H, J ¼ 8.0 Hz), 9.49 (s, 1H, NH),13.69
(s, 1H, NH); 13C NMR (DMSO-d6):
d 21.4 (CH3), 109.8 (CH), 117.9 (C),
118.3 (CH), 118.7 (C), 127.4 (2CH), 127.9 (CH), 129.3 (C), 130.0 (2CH),
(s, 1H, NH); 13C NMR (DMSO-d6):
d 15.0 (CH3), 21.4 (CH3), 64.5
138.1 (C), 142.8 (C), 143.5 (C); MS m/z ¼ 367 (79Br) [M þ 1]þ, 369
(CH2O), 106.6 (CH), 118.0 (C), 119.2 (C), 120.8 (C), 121.0 (CH), 127.4
(2CH), 130.0 (2CH), 134.4 (C), 138.4 (C), 142.1 (C), 143.5 (C); MS m/
z ¼ 411 (79Br) [M þ 1]þ, 413 (81Br) [M þ 3]þ. Anal. Calcd. for
(
81Br) [M þ 3]þ. Anal. Calcd. for C14H12BrN3O2S: C, 45.91; H, 3.30; N,
11.47. Found: C, 45.78; H, 3.44; N, 11.32.
C16H16BrN3O3S: C, 46.84; H, 3.93; N, 10.24. Found: C, 46.96; H, 3.82;
5.1.1.4. N-(3-Bromo-7-ethoxy-1H-4-indazolyl)-4-methyl-
benzenesulfonamide (3b). Yield: 30%; mp: 118e120 ꢃC; IR (KBr,
N, 10.37.
5.1.1.5. N-(3-Chloro-1H-4-indazolyl)-4-methoxy-benzenesulfona-
mide (2c). Yield: 27%; mp: 56e58 ꢃC; IR (KBr, cmꢂ1): 3340, 3250
(NH), 1590 (CN), 1325, 1162 (SO2); 1H NMR (CDCl3):
d 3.77 (s, 3H,
CH3O), 6.78 (d, 1H, J ¼ 7.8 Hz), 7.14 (t, 1H, J ¼ 7.8 Hz), 6.72 (d, 2H,
J ¼ 8.4 Hz), 7.42 (d, 1H, J ¼ 7.8 Hz), 7.60 (d, 2H, J ¼ 8.4 Hz), 10.09 (s,
1H, NH), 13.25 (s, 1H, NH); 13C NMR (CDCl3):
d 55.5 (CH3O), 105.8
(CH), 110.2 (CH), 112.1 (C), 114.0 (2CH), 126.7 (CH), 129.4 (2CH),
130.4 (C), 131.6 (C), 135.4 (C), 142.6 (C), 163.4 (C); MS m/z ¼ 338
(
79Cl) [M þ 1]þ, 339 (37Cl) [M þ 3]þ. Anal. Calcd. for C14H12ClN3O3S:
C, 49.78; H, 3.58; N, 12.44. Found: C, 49.92; H, 3.49; N, 12.57.
5.1.1.6. N-(3-Chloro -7-ethoxy-1H-4-indazolyl)-4-methoxy-benzene-
sulfonamide (3c). Yield: 40%; mp: 68e70 ꢃC; IR (KBr, cmꢂ1): 3338,
3260 (NH), 1580 (CN), 1345, 1150 (SO2); 1H NMR (CDCl3):
d 1.47 (t,
3H, CH3, J ¼ 7.0 Hz), 3.77 (s, 3H, CH3O), 4.16 (q, 2H, OCH2, J ¼ 7.0 Hz),
6.66 (d, 1H, J ¼ 8.1 Hz), 6.79 (d, 2H, J ¼ 8.6 Hz), 7.22 (d, 1H,
J ¼ 8.1 Hz), 7.64 (d, 2H, J ¼ 8.6 Hz), 9.85 (s, 1H, NH), 13.1 (s, 1H, NH);
13C NMR (CDCl3):
d 14.7 (CH3), 55.5 (CH3O), 64.3 (CH2O), 106.5 (C),
107.2 (CH), 112.1 (C), 114.0 (2CH), 116.5 (CH), 121.4 (C), 129.4 (2CH),
130.5 (C), 134.2 (C), 142.2 (C), 163.1 (C); MS m/z ¼ 382 (35Cl)
[M þ 1]þ, 384 (37Cl) [M þ 3]þ. Anal. Calcd. for C16H16ClN3O4S: C,
50.33; H, 4.22; N, 11.00. Found: C, 50.48; H, 4.11; N, 11.07.
5.1.2. Preparation of N-alkylated indazoles 4 and 5
To a solution of 4-nitroindazole 1a (6.13 mmol) in THF (30 mL)
cooled at 0 ꢃC was added K2CO3 (9.2 mmol). After 15 min at 0 ꢃC,
allyl bromide/methyl iodide (6.13 mmol) was added dropwise. The
solution was stirred for 16e20 h and the resulting mixture was
evaporated. The crude material was dissolved with EtOAc (50 mL),
washed with water and brine, dried over MgSO4 and the solvent
was evaporated in vacuo. The resulting residue was purified by
column chromatography (EtOAc/hexane 3/7).
Fig. 4. Representative western blot analysis of p53 and bax after exposure of A2780 cells
to compounds 3b, 2c, 3c, 13, 6d, and 16 for 24, 48 and 72 h. Our molecules were
administered at their specific IC50s, as detected in MTT assays. Densitometric normali-
5.1.2.1. 1-Methyl-4-nitro-1H-indazole (4d). Yield: 62%; mp
123e125 ꢃC. 1H NMR (CDCl3):
d
4.17 (s, 3H, NCH3), 7.50 (t, 1H,
J ¼ 8.0 Hz), 7.76 (d, 1H, J ¼ 8.5 Hz), 8.13 (d, 1H, J ¼ 8.0 Hz), 8.58 (s,
1H). 13C NMR (CDCl3):
36.1 (NCH3), 116.1 (CH-5), 117.0 (C-3a),
118.2 (CH-7), 125.4 (CH-6), 132.6 (CH-3), 140.5, 141.5 (C-4, C-7a).
zation was performed using the expression signal obtained by an anti-
clonal antibody. Prestained molecular markers were always included as references. Fifty
g of protein extract from control and treated A2780 cells were seeded in each lane.
b-actin mono-
d
m

246
N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
Table 3
Percentage of A2780 cells in the different cell cycle phases after treatment for 72 h with the IC50 and IC75 of indazole compounds.
Cell cycle phases
IC50
CTR
3b
2c
3c
16
6d
13
8
G0/G1
S
G2/M
51.0 ꢀ 6.2a
25.9 ꢀ 4.7
23.1 ꢀ 2.5
61.5 ꢀ 3.3
14.2 ꢀ 2.9
24.3 ꢀ 3.4
56.1 ꢀ 4.1
18.0 ꢀ 3.7
25.9 ꢀ 1.6
62.7 ꢀ 0.7
10.4 ꢀ 4.6
26.9 ꢀ 4.6
53.7 ꢀ 1.9
12.6 ꢀ 2.6
33.7 ꢀ 3.2
65.0 ꢀ 4.2
11.3 ꢀ 5.3
23.7 ꢀ 1.9
66.0 ꢀ 3.8
9.8 ꢀ 0.9
24.2 ꢀ 4.1
43.5 ꢀ 2.9
27.3 ꢀ 5.0
29.3 ꢀ 2.1
b
Cell cycle phases
IC75
CTR
3b
2c
3c
16
6d
13
8
G0/G1
S
G2/M
Cells with >4n
51.0 ꢀ 6.2
25.9 ꢀ 4.7
23.1 ꢀ 2.5
e
10.2 ꢀ 1.1
10.3 ꢀ 1.0
71.5 ꢀ 2.1
8.1 ꢀ 1
9.4 ꢀ 0.4
7.8 ꢀ 0.8
70.1 ꢀ 0.2
12.8 ꢀ 0.1
10.8 ꢀ 1.8
8.4 ꢀ 6.9
69.6 ꢀ 3.7
11.3 ꢀ 1.4
8.9 ꢀ 0.5
8.2 ꢀ 3.5
71.4 ꢀ 1.5
11.6 ꢀ 1.6
9.1 ꢀ 1.0
15.0 ꢀ 2.1
69.2 ꢀ 1.1
6.8 ꢀ 0.9
10.8 ꢀ 1.7
5.5 ꢀ 0.4
72.7 ꢀ 1.4
11.1 ꢀ 0.6
9.9 ꢀ 2.3
13.7 ꢀ 3.1
64.5 ꢀ 1.1
12.0 ꢀ 0.4
a
The means ꢀ SD (3e4 data) express the percentage of cells in the various phases of the cell cycle.
b
The IC75s ranged from 6.8 mM to 102.3 mM.
5.1.2.2. 2-Methyl-4-nitro-2H-indazole (5d). Yield: 34%; mp 75e77 ꢃC.
d6): d 21.4 (CH3), 35.9 (NCH3), 106.2 (CH), 110.9 (CH), 117.7 (C), 127.0
1H NMR (CDCl3):
d
4.31 (s, 3H, NCH3), 7.40 (t,1H, J ¼ 8.0 Hz), 8.06 (d,1H,
(CH),127.2 (2CH),130.1 (2CH),130.6 (C),131.2 (CH),137.2 (C),141.0 (C),
143.8 (C). MS: m/z 302 [M þ 1]þ. Anal. Calcd. for C15H15N3O2S: C,
59.78; H, 5.02; N, 13.94. Found: C, 59.92; H, 5.14; N, 13.81.
J ¼ 8.2 Hz), 8.16 (d, 1H, J ¼ 8.0 Hz), 8.54 (s, 1H). 13C NMR (CDCl3):
d 40.9
(NCH3), 120.6 (CH-5), 115.1 (C-3a), 124.4 (CH-7), 125.3, 125.8 (CH-6,
CH-3), 140.5 (C-4), 149.9 (C-7a).
5.1.3.2. N-(1-Methyl-7-ethoxy-2H-indazol-4-yl)-4-methyl-
5.1.2.3. 1-Allyl-4-nitro-1H-indazole (4e). Yield: 54; mp 63e65 ꢃC.
benzenesulfonamide (7d). Yield: 14%, mp 190e192 ꢃC. 1H NMR
1H NMR (CDCl3):
d
5.07e5.10 (m, 2H, NCH2), 5.15e5.26 (m, 2H, ]
(DMSO-d6):
d
1.33 (t, 3H, CH3, J ¼ 7.2 Hz), 2.21 (s, 3H, CH3), 3.90 (s,
CH2), 5.95e6.08 (m, 1H, ]CH), 7.44 (t, 1H, J ¼ 7.8 Hz), 7.75 (d, 1H,
3H, NCH3), 4.10 (q, 2H, CH2O, J ¼ 7.2 Hz), 6.45 (d,1H, H-6, J ¼ 8.0 Hz),
6.60 (d, 1H, H-5, J ¼ 8.0 Hz), 7.15 (d, 2H, ArH, J ¼ 7.8 Hz), 7.45 (d, 2H,
ArH, J ¼ 7.8 Hz), 8.14 (s, 1H, H-3), 10.34 (s, 1H, NH). 13C NMR (DMSO-
J ¼ 8.4 Hz), 8.04 (d, 1H, J ¼ 7.8 Hz), 8.51 (s, 1H). 13C NMR (CDCl3):
d
52.2 (NCH2), 116.5 (CH-5), 117.1 (C-3a), 118.2 (CH-7), 118.5 (]CH2),
125.4 (CH-6), 132.0 (]CH), 132.8 (CH-3), 140.6, 141.0 (C-4, C-7a).
d6): d 15.1 (CH3), 21.5 (CH3), 36.1 (NCH3), 63.0 (CH2O), 104.5 (CH),
116.8 (CH), 117.4 (C), 127.2 (2CH), 129.8 (2CH), 130.6 (C), 132.0 (CH),
136.9 (C), 142.5 (C), 143.1 (C), 148.2 (C). MS: m/z 346 [M þ 1]þ. Anal.
Calcd. for C17H19N3O3S: C, 59.11; H, 5.54; N, 12.16. Found: C, 59.24;
H, 5.40; N, 12.25.
5.1.2.4. 2-Allyl-4-nitro-2H-indazole (5e). Yield: 30; mp 68e70 ꢃC.
1H NMR (CDCl3):
d 5.10e5.13 (m, 2H, NCH2), 5.35e5.42 (m, 2H, ]
CH2), 6.09e6.22 (m, 1H, ]CH), 7.37 (t, 1H, J ¼ 7.8 Hz), 8.07
(d, 1H, J ¼ 8.4 Hz), 8.15 (d, 1H, J ¼ 7.8 Hz), 8.56 (s, 1H). 13C NMR
(CDCl3):
d
56.7 (NCH2), 115.0 (C-3a), 120.4 (]CH2), 120.6 (CH-5),
5.1.3.3. N-(1-Allyl-2H-indazol-4-yl)-4-methylbenzenesulfonamide
124.2 (CH-3), 124.4 (CH-7), 126.0 (CH-6), 131.4 (]CH), 140.6 (C-4),
(6e). Yield: 68%, mp 124e126 ꢃC. 1H NMR (CDCl3):
d 2.34 (s, 3H,
149.9 (C-7a).
CH3), 4.86e4.92 (m, 2H, NCH2), 5.30e5.33 (m, 2H, ]CH2), 5.97e
6.07 (m, 1H, ]CH), 6.97 (d, 1H, J ¼ 8.1 Hz), 7.18e7.21 (m, 3H),
7.43 (d, 1H, J ¼ 8.0 Hz), 7.55 (d, 2H, J ¼ 8.4 Hz), 7.99 (s, 1H, H-3), 8.38
5.1.3. Synthesis of sulfonamides (6d,e) and (7d,e)
These compounds were prepared from 1-alkyl-4-nitroindazole
(4d,e) by using the same procedure applied to 3-halogeno-4-
nitroindazoles (1aec).
(s, 1H, NH). 13C NMR (CDCl3):
d 21.5 (CH3), 53.2 (NCH2), 113.5 (CH),
117.1 (]CH2),121.0 (C),127.1 (CH),127.4 (2CH),128.0 (CH),128.3 (C),
129.7 (2CH), 130.9 (CH), 133.8 (CH), 135.8 (C), 136.4 (C), 144.3 (C).
MS: m/z 328 [M þ 1]þ. Anal. Calcd. for C17H17N3O2S: C, 62.37; H,
5.23; N, 12.83. Found: C, 62.48; H, 5.30; N, 12.72.
5.1.3.1. N-(1-Methyl-2H-indazol-4-yl)-4-methylbenzenesulfonamide
(6d). Yield: 75%, mp 204e206 ꢃC. 1H NMR (DMSO-d6):
d 2.26 (s, 3H,
CH3), 3.92(s, 3H, NCH3), 6.90(d,1H, J ¼ 7.2 Hz), 7.20e7.28 (m, 4H), 7.65
5.1.3.4. N-(1-Allyl-7-ethoxy-2H-indazol-4-yl)-4-methylbenzen-
(d, 2H, J ¼ 7.4 Hz), 8.15 (s,1H, H-3),10.52 (s,1H, NH).13C NMR (DMSO-
esulfonamide (7e). Yield: 18%, mp 133e135 ꢃC. 1H NMR (CDCl3):
d
1.47 (t, 3H, CH3, J ¼ 7.0 Hz), 2.33 (s, 3H, CH3), 4.11 (q, 2H, CH2O,
J ¼ 7.0 Hz), 4.94e5.10 (m, 2H, NCH2), 5.18e5.23 (m, 2H, ]CH2),
5.94e6.05 (m, 1H, ]CH), 6.54 (d, 1H, H-6, J ¼ 8.1 Hz), 6.84 (d, 1H, H-
5, J ¼ 8.1 Hz), 7.14 (d, 2H, J ¼ 7.8 Hz), 7.61 (d, 2H, J ¼ 7.8 Hz), 7.78 (s,
1H, H-3), 8.84 (s, 1H, NH). 13C NMR (CDCl3):
d 14.7 (CH3), 21.5 (CH3),
53.8 (NCH2), 64.2 (CH2O), 106.3 (CH), 117.0 (]CH2), 117.5 (CH), 121.1
(C), 122.3 (C), 127.3 (2CH), 129.5 (2CH), 130.6 (CH), 131.4 (C), 134.1
(CH), 136.2 (C), 143.7 (C), 144.2 (C). MS: m/z 372 [M þ 1]þ. Anal.
Calcd. for C19H21N3O3S: C, 61.44; H, 5.70; N, 11.31. Found: C, 61.56;
H, 5.52; N, 11.46.
5.1.4. N-(2-Methyl-7-ethoxy-2H-indazol-4-yl)-4-
methylbenzenesulfonamide (8)
This compound was prepared from 2-Methyl-4-nitro-2H-inda-
zole (5d) by using the same procedure applied to 3-halogeno-4-
nitroindazoles (1aec).
Yield: 72%, mp 66e68 ꢃC. 1H NMR (CDCl3):
d 1.45 (t, 3H, CH3,
Fig. 5. Representation of the interaction between compound 3b (violet) and colchicine
J ¼ 7.2 Hz), 2.32 (s, 3H, CH3), 4.00 (s, 3H, NCH3), 4.15 (q, 2H, CH2O,
(yellow) and
b-tubulin. (For interpretation of the references to colour in this figure
J ¼ 7.2 Hz), 6.34 (d, 1H, H-6, J ¼ 8.0 Hz), 6.56 (d, 1H, H-5, J ¼ 8.0 Hz),
legend, the reader is referred to the web version of this article.)

N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
247
7.12 (d, 2H, J ¼ 7.8 Hz), 7.58 (d, 2H, J ¼ 7.8 Hz), 8.38 (s, 1H, H-3), 10.14
(s, 1H, NH). 13C NMR (CDCl3):
14.6 (CH3), 21.3 (CH3), 40.4 (NCH3),
for C17H17N3O2S: C, 62.37; H, 5.23; N, 12.83. Found: C, 62.50; H,
5.20; N, 12.68.
d
63.9 (CH2O), 103.1 (CH), 117.8 (CH), 120.3 (C), 124.1 (CH), 127.3
(2CH), 129.5 (2CH), 130.6 (C), 135.6 (C), 142.2 (C), 143.7 (C), 147.9 (C).
MS: m/z 346 [M þ 1]þ. Anal. Calcd. for C17H19N3O3S: C, 59.11; H,
5.54; N, 12.16. Found: C, 59.01; H, 5.65; N, 12.20.
5.1.6.2. N-(2-Allyl-7-ethoxy-2H-indazol-6-yl)-4-methylbenzene-
sulfonamide (14). Yield: 36%, mp 108e110 ꢃC. 1H NMR (CDCl3):
d
1.22 (t, 3H, CH3, J ¼ 7.0 Hz), 2.31 (s, 3H, CH3), 4.26 (q, 2H, CH2O,
J ¼ 7.0 Hz), 4.98e5.06 (m, 2H, NCH2), 5.30e5.35 (m, 2H, ]CH2),
6.04e6.13 (m, 1H, ]CH), 7.11 (d, 2H, J ¼ 8.4 Hz), 7.27 (d, 1H, H-6,
J ¼ 8.1 Hz), 7.45 (d, 1H, H-5, J ¼ 8.1 Hz), 7.61 (d, 2H, J ¼ 8.4 Hz), 7.88
5.1.5. Synthesis of compounds (10a,b) and (11a,b)
These compounds were prepared from 3-halogeno-6-
nitroindazoles (9a,b) by using the same procedure applied to 3-
halogeno-4-nitroindazoles (1aec).
(s, 1H, H-3), 9.05 (s, 1H, NH). 13C NMR (CDCl3):
d 15.5 (CH3), 21.5
(CH3), 56.1 (NCH2), 68.6 (CH2O), 114.6 (CH), 118.5 (CH), 120.2 (]
CH2), 122.2 (C), 123.5 (CH), 124.0 (C), 127.1 (2CH), 129.4 (2CH), 131.7
(CH), 136.1 (C), 137.4 (C), 141.3 (C), 143.7 (C). MS: m/z 372 [M þ 1]þ.
Anal. Calcd. for C19H21N3O3S: C, 61.44; H, 5.70; N, 11.31. Found: C,
61.62; H, 5.60; N, 11.38.
5.1.5.1. 4-Methyl-N-[1-(toluene-4-sulfonyl)-1H-indazol-6-yl]-benze-
nesulfonamide (10a). Yield: 27%. Mp 140e142 ꢃC. 1H NMR (CDCl3):
d
2.36 (s, 3H, CH3), 2.38 (s, 3H, CH3), 7.20e7.28 (m, 4H), 7.49e7.68
(m, 2H), 7.79e7.86 (m, 3H), 8.05 (d, 2H), 8.41 (s, 1H, H-3), 9.03 (s,
1H, NH). 13C NMR (CDCl3):
21.6 (CH3), 21.7 (CH3), 103.3 (CH), 117.8
d
5.1.7. Synthesis of sulfonamides (16) and (17)
(CH), 122.2 (CH), 122.5 (C), 127.2 (2CH), 127.6 (2CH), 129.8 (2CH),
129.9 (2CH),134.3 (C),135.9 (C),138.5 (C),140.7 (C),141.0 (CH),144.2
(C), 145.5 (C). MS: m/z 442 [M þ 1]þ. Anal. Calcd. for C21H19N3O4S2:
C, 57.13; H, 4.34; N, 9.52. Found: C, 57.21; H, 3.20; N, 9.68.
These compounds were prepared from 7-nitroindazole (15).
5.1.7.1. N-(4-ethoxy-1H-7-indazolyl)-4-methylbenzenesulfonamide
(16). Yield: 36%, mp: 170e172 ꢃC; IR (KBr, cmꢂ1): 3335, 3240 (NH),
1580 (CN), 1335, 1160 (SO2); 1H NMR (CDCl3):
d 1.45 (t, 3H, CH3,
5.1.5.2. N-(1H-Indazol-6-yl)-4-methylbenzenesulfonamide
(11a).
J ¼ 7.2 Hz), 2.30 (s, 3H, CH3), 4.08 (q, 2H, OCH2, J ¼ 7.2 Hz), 6.24 (d,
Yield: 60%. Mp 166e168 ꢃC. 1H NMR (CDCl3):
d
2.27 (s, 3H, CH3),
1H, J ¼ 8.1 Hz), 6.81 (d,1H, J ¼ 8.1 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz), 7.60 (d,
6.90 (dd, 1H, J ¼ 8.6 Hz, 1.4 Hz), 7.24 (t, 1H, J ¼ 8.6 Hz), 7.29 (d, 2H,
J ¼ 8.0 Hz), 7.58 (dd, 1H, J ¼ 8.6 Hz, 1.4 Hz), 7.65 (d, 2H, J ¼ 8.0 Hz),
7.93 (s, 1H, H-3), 10.37 (s, 1H, NH), 12.87 (s, 1H, NH). 13C NMR
2H, J ¼ 8.4 Hz), 8.15 (s, 1H), 10.15 (s, 1H, NH), 13.35 (s, 1H, NH). 13
C
NMR (CDCl3): d 14.7 (CH3), 21.5 (CH3), 64.0 (CH2O), 100.9 (CH), 112.6
(C), 116.1 (C), 120.8 (CH), 124.3 (C), 127.4 (2CH), 129.6 (2CH), 133.6
(CH), 135.4 (C), 138.9 (C), 144.0 (C); MS m/z ¼ 332 [M þ 1]þ. Anal.
Calcd. for C16H17N3O3S: C, 57.99; H, 5.17; N, 12.68. Found: C, 58.05;
H, 5.12; N, 12.76.
(CDCl3):
d 21.4 (CH3), 100.2 (CH), 115.2 (CH), 120.2 (C), 121.7 (CH),
127.1 (2CH), 130.1 (2CH), 133.8 (CH), 136.5 (C), 137.0 (C), 140.6 (C),
143.7 (C). MS: m/z 288 [M þ 1]þ. Anal. Calcd. for C14H13N3O2S: C,
58.52; H, 4.56; N, 14.62. Found: C, 58.71; H, 4.42; N, 14.68.
5.1.7.2. N-(1H-7-Indazolyl)-4-methylbenzenesulfonamide
Yield: 35%; mp: 202e204 ꢃC; IR (KBr, cmꢂ1): 3340, 3235 (NH), 1595
(CN), 1335, 1160 (SO2); 1H NMR (CDCl3):
2.28 (s, 3H, CH3), 6.99 (t,
(17).
5.1.5.3. 4-Methyl-N-[1-(toluene-4-sulfonyl)-3-bromo-1H-indazol-6-
yl]-benzenesulfonamide (10b). Yield: 40%; mp 180e182 ꢃC. 1H NMR
d
(DMSO-d6):
d
2.32 (s, 3H, CH3), 2.33 (s, 3H, CH3), 7.21 (d, 1H,
1H, J ¼ 7.8 Hz), 7.04 (d,1H, J ¼ 7.8 Hz), 7.14 (d, 2H, J ¼ 8.4 Hz), 7.48 (d,
J ¼ 8.4 Hz), 7.35 (t, 1H, J ¼ 8.4 Hz), 7.50 (d, 1H, J ¼ 8.4 Hz), 7.61 (d, 2H,
1H, J ¼ 7.8 Hz), 7.66 (d, 2H, J ¼ 8.3 Hz), 8.18 (s, 1H), 10.58 (s, 1H, NH),
J ¼ 8.1 Hz), 7.73 (d, 2H, J ¼ 8.1 Hz),10.85 (s,1H, NH). 13C NMR (DMSO-
13.93 (s, 1H, NH); 13C NMR (CDCl3):
d 21.3 (CH3), 118.7 (CH), 122.0
d6):
d
21.4 (CH3), 21.6 (CH3), 101.9 (CH), 118.6 (CH), 121.8 (C), 122.4
(CH), 116.1 (C), 124.2 (C), 125.3 (CH), 127.5 (2CH), 129.8 (2CH), 131.7
(CH), 135.4 (C), 141.4 (C), 143.8 (C); MS m/z ¼ 288 [M þ 1]þ. Anal.
Calcd. for C14H13N3O2S: C, 58.52; H, 5.56; N, 14.62. Found: C, 58.65;
H, 5.51; N, 14.68.
(CH), 127.3 (2CH), 127.4 (2CH), 130.3 (2CH), 130.8 (2CH), 131.6 (C),
133.4 (C),136.8(C),141.5(C),141.8(C),144.3 (C),146.8(C). MS: m/z 521
(
79Br) [M þ 1]þ, 523 (81Br) [M þ 3]þ. Anal. Calcd. for C21H18BrN3O4S2:
C, 48.47; H, 3.49; N, 8.07. Found: C, 48.64; H, 3.34; N, 8.18.
5.2. Biology
5.1.5.4. N-(3-Bromo-1H-indazol-6-yl)-4-methylbenzenesulfonamide
(11b). Yield: 32%. Mp 218e220 ꢃC. 1H NMR (DMSO-d6):
d
2.27 (s,
5.2.1. Chemicals
3H, CH3), 6.97 (d, 1H, J ¼ 8.7 Hz), 7.25 (t, 1H, J ¼ 8.7 Hz), 7.36 (d, 2H,
Because of the low water solubility of our indazole compounds
they were firstly dissolved in 100% dimethylsulfoxide (DMSO) to
100 mM concentration and subsequently diluted in fetal calf serum,
the final DMSO concentrations on cells being 0.6% for 6d and 7e and
0.2% for all other compounds.
J ¼ 8.1 Hz), 7.40 (d, 1H, J ¼ 8.7 Hz), 7.63 (d, 2H, J ¼ 8.1 Hz), 10.46 (s,
1H, NH), 13.14 (s, 1H, NH). 13C NMR (DMSO-d6):
d 21.4 (CH3), 100.2
(CH), 116.1 (CH), 119.4 (C), 120.6 (CH), 120.8 (C), 127.1 (2CH), 130.2
(2CH), 136.9 (C), 138.1 (C), 141.8 (C), 143.9 (C). MS m/z ¼ 367 (79Br)
[M þ 1]þ, 369 (81Br) [M þ 3]þ. Anal. Calcd. for C14H12BrN3O2S: C,
45.91; H, 3.30; N, 11.47. Found: C, 45.82; H, 3.38; N, 11.38.
5.2.2. 3-(2,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay
5.1.6. Synthesis of sulfonamides (13) and (14)
A2780 (human ovarian carcinoma) A549 (human lung adeno-
carcinoma) and P388 (murine leukemia) cells were plated at
opportune densities/well (range: 900e1800/well) in order to
maintain an exponential growth into 96-well microtiter plates. They
were then centrifuged at 275 g for 2 min and maintained for about
6e8 h before treatment. Our indazole compounds were added in
duplicate at the appropriate 10ꢁ concentrations for a minimum of
5 concentrations (1:10 fold serial dilutions) reaching a final
These compounds were prepared from 2-allyl-6-nitroindazole (12).
5.1.6.1. N-(2-Allyl-2H-indazol-6-yl)-4-methylbenzenesulfonamide
(13). Yield: 54%, mp 138e140 ꢃC. 1H NMR (DMSO-d6):
d 2.28 (s, 3H,
CH3), 4.94e4.98 (m, 2H, NCH2), 5.13e5.21 (m, 2H, ]CH2), 5.99e
6.08 (m, 1H, ]CH), 6.82 (t, 1H, J ¼ 8.0 Hz), 7.18 (d, 1H, J ¼ 8.2 Hz),
7.29 (d, 2H, J ¼ 8.4 Hz), 5.54 (d, 1H, J ¼ 8.0 Hz), 7.63 (d, 2H,
J ¼ 8.4 Hz), 8.21 (s, 1H, H-3), 10.19 (s, 1H, NH). 13C NMR (CDCl3):
volume of 200 mL. Opportune controls were always made. The MTT
d
21.4 (CH3), 55.5 (NCH2), 105.8 (CH), 117.3 (CH), 118.9 (]CH2), 119.1
assay was performed as described elsewhere [27,28]. IC50s were
calculated by the analysis of single concentrationeresponse curves.
Each final mean ꢀ SD was calculated from 3 to 9 experiments.
(C), 122.0 (CH), 124.3 (CH), 127.2 (2CH), 130.1 (2CH), 135.9 (C),
137.1 (C), 143.6 (C), 148.4 (C). MS: m/z 328 [M þ 1]þ. Anal. Calcd.

248
N. Abbassi et al. / European Journal of Medicinal Chemistry 57 (2012) 240e249
5.2.3. Visualization of apoptotic cells by 40-6-diamidino-2-
phenylindole (DAPI) staining
Cells were plated in 1 mL at different densities/well (range:
5000e10000/well) into 24-well microtiter plates for about 6e8 h,
then all active indazole compounds were added in order to
obtain their specific IC50 and IC90, as determined with the MTT
assay. After 3 days, floating and adherent cells were harvested and
performed by sorting the different complexes with respect to the
predicted binding energy. A cluster analysis based on root mean
square deviation values, with reference to the starting geometry,
was subsequently performed and the lowest energy conformation
of the more populated cluster was considered as the most prom-
ising bioactive candidate. When clusters of molecules were almost
equipopulated, and their energy distribution is spread, the corre-
sponding moiety was not considered a good ligand. In a second
phase, we validated the correct positioning of the ligands within
the active site cleft by using a second docking program, GOLD
(CCDC, Cambridge, UK). The results obtained by GOLD confirmed
what already achieved with Autodock.
washed twice with cold PBS, fixed with 100
m
L of 70% ethanol in PBS
and maintained at 4 ꢃC until analysis. Five
m
L of a solution of 10 g/
m
mL DAPI in water were added to the samples just before exami-
nation at the microscope, and the percentage of apoptotic
segmented nuclei/cells stained with the fluorescent dye evaluated.
5.2.4. Western blot analysis
5.2.7. Statistical analysis
Analysis by western blots of p53, bax, and
b
-actin was carried on
The Student’s t test was used for the statistical analysis of data.
A2780 after treatment with the IC50s of the active compounds 3b, 2c,
3c, 13, 16, and 6d, as determined by the MTT assay. At 24, 48 and
72 h culture aliquots of treated and untreated cells were harvested,
washed twice with normal saline and treated at 4 ꢃC for 30 min with
a lysis buffer containing 1% Triton X-100, 0.15 M NaCl and 10 mM Tris
(pH 7.4), 2 mg/mL aprotinin and 50 mg/mL phenylmethylsulphonyl
fluoride. The protein concentration was determined by the Bradford
Acknowledgements
The synthetic work was supported by a grant of the University of
Sultan Moulay Slimane, Béni-Mellal and the National Centre for
Scientific and Technical Research (CNRST), Rabat, Morocco.
method (Sigma Chemical Co., St. Louis, MO, USA). Fifty mg of protein
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