Synthesis and biological evaluation of N-(7-indazolyl)benzenesulfonamide derivatives as potent cell cycle inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.09.037

We herein describe a new synthesis of N-(7-indazolyl)benzenesulfonamide derivatives. These compounds were evaluated for their antiproliferative activities toward L1210 murine leukemia cells. One of them, 4-methoxy-N-(3- chloro-7-indazolyl)benzenesulfonamide, was identified as the most potent with an IC₅₀ of 0.44 μM.

Full text of DOI:10.1016/j.bmc.2005.09.037

Bioorganic & Medicinal Chemistry 14 (2006) 1078–1088
Synthesis and biological evaluation of N-(7-indazolyl)benzenesul-
fonamide derivatives as potent cell cycle inhibitors
c
c
b
L. Bouissane,a,b S. El Kazzouli,a,b S. Leonce, B. Pfeiffer, E. M. Rakib,
´
M. Khouilib and G. Guillaumeta,*
aInstitut de Chimie Organique et Analytique, UMR CNRS 6005, Universite´ dÕOrle´ans, BP 6759, 45067 Orle´ans Cedex 2, France
bFaculte´ des Sciences et Techniques de Beni-Mellal, Universite´ Cadi-Ayyad, BP 523, 23000 Beni-Mellal, Morocco
cInstitut de Recherches Servier, Division Recherche Cance´rologie, 125 chemin de ronde, 78290 Croissy sur Seine, France
Received 29 April 2005; revised 30 August 2005; accepted 13 September 2005
Available online 7 November 2005
Abstract—We herein describe a new synthesis of N-(7-indazolyl)benzenesulfonamide derivatives. These compounds were evaluated
for their antiproliferative activities toward L1210 murine leukemia cells. One of them, 4-methoxy-N-(3-chloro-7-indazolyl)benzene-
sulfonamide, was identified as the most potent with an IC50 of 0.44 lM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The sulfonamide derivatives are known for their numer-
ous pharmacological activities, with among others, anti-
CH3O
H2N
H
H
N
N
N
bacterial,
insulin-release
stimulation,
carbonic
S
S
anhydrase inhibition, and diuretic and antithyroid prop-
erties.1 In addition, recent papers have reported novel
sulfonamide drugs with other properties such as endo-
thelin receptor antagonists,2 5-HT6 receptor antago-
nists,3 b3 adrenergic receptor agonists,4 thrombin
inhibitors,5 and matrix metalloproteinase inhibitors.6
E7010 (Fig. 1), which is an original antitumor agent with
a sulfonamide moiety, was found to induce cell cycle ar-
rest in the M phase and apoptosis. This compound,
which inhibits microtubule assembly owing to its revers-
ible binding to the colchicine binding site on tubulin,7
was found to be active in vivo against various rodent tu-
mors and human tumor xenografts.8 E7010 was the first
drug candidate among the antitumor sulfonamides to be
synthesized by Owa and co-workers.9 Various pharma-
comodulations performed by the same team led to the
identification of N-(7-indolyl)benzenesulfonamide deriv-
atives with different effects on cell cycle progression of
P388 murine leukemia cells compared to E7010.10
O
O
O
O
HN
N
HN
Cl
E7010
ER35745
OH
H2NSO2
H
N
S
O
O
HN
E7070
Cl
Figure 1.
Namely, accumulation of cells in the G1 phase was ob-
served in a dose-dependent manner after a 24-h drug
exposure. Growing interest for these G1-targeting sul-
fonamides has resulted in the discovery of N-(3-chloro-
7-indolyl)-1,4-benzenesulfonamide E707011–13 (Fig. 1),
which demonstrated excellent in vivo efficacy against
Keywords: Indazole; Palladium; Sulfonamide; L1210 murine leukemia
cells.
*
Corresponding author. Tel.: +33 2 38 41 70 73; fax: +33 2 38 41 70
78; e-mail: gerald.guillaumet@univ-orleans.fr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.037

L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
1079
various human tumor xenografts, for example, HCT116
colon carcinoma.
Cl
N
N
N
N
N
N
H
H
Studies performed with A549 cell line and its E7070-
resistant subline A549/ER35745 further clarified that
E7070 inhibits pRb phosphorylation, reduces protein
expression of cyclin A, cyclin B1, CDK2, and CDC2,
and represses CDK2 activity with the induction of p53
and p21 proteins, only in the parental A549 cells.14 This
compound also possesses a potent carbonic anhydrase
inhibitory activity which may account for some of its
antitumor properties.15
H
Me
a
NH
N
b
N
O2S
O2S
Me
O2S
OMe
6c
OMe
15
OMe
16
X
X
N
N
N
N
Recently, Owa and co-workers described the synthesis
and antitumor activity of E7070 analogues containing
a 3-pyridinesulfonamide moiety.9,16 ER35745, which is
a 6-amino-3-pyridinesulfonamide derivative, was found
to display significant oral efficacy against the HCT116
human colon carcinoma xenograft in nude mice. Micro-
array-based gene expression analysis has shown that
E7070 and ER-35745 could operate by the same mecha-
nism of action.
a
Me
Me
NH
N
O2S
O2S
Me
OMe
OMe
X= H 13
X= Cl 14
X= H 17
X= Cl 18
Scheme 2. Reagents and conditions: (a) MeI, KOH, acetone, 0 ꢁC, 93–
97%; (b) NaOCl, MeOH, NaOH (2 N), reflux, 98%.
In this paper, we describe the synthesis and in vitro bio-
logical evaluation on the murine L1210 and on the
human DU145, HCT116, and HT29 (for DU145,
HCT116, and HT29, see Section 4) cell lines of
N-(7-indazolyl)benzenesulfonamides.
I
I
I
b
a
N
N
N
N
N
N
2. Chemistry
H
H
NO2
10
NO2
10
NO
2 O
O
Eighteen new benzenesulfonamide derivatives have been
prepared starting from 7-nitroindazole17 (Schemes 1–4).
19
Scheme 3. Reagents and conditions: (a) Boc2O, DMAP, Et3N,
CH2Cl2, 93%; (b) 4-MeOC6H4B(OH)2, Na2CO3, DME, Pd(PPh3)4,
reflux, 95%.
Compounds 6a–d and 7a–d were prepared according to the
general route depicted in Scheme 1 from 7-nitroindazole.
X
N
X
X
d
c
N
N
N
H
N
N
NH
H
H
O2S
X= H
X= C
NO2
NH2
6a, R= H
7a, R= H
6b, R= Me
6c, R= OMe
6d, R= Br
7b, R= Me
7c, R= OMe
7d, R= Br
X= H
X= Cl
X= I
1
2
3
X= H
X= Cl
4
5
a
b
R
6e, R= SO2NH2 7e, R= SO2NH2
X
e
N
N
X
X
Me
NH
c
f
O2S
N
N
N
N
Me
Me
NO2
NH2
OMe
X= H
X= Cl
8
9
X= H 11
X= Cl 12
X= H 13
X= Cl 14
X= I 10
Scheme 1. Reagents and conditions: (a) NaOCl, MeOH, NaOH (2 N), reflux, 98%; (b) I2, KOH, DMF, rt, 95%; (c) H2, Pd/C, MeOH; (d)
arylsulfonyl chlorides, pyridine, rt, 63–95% (yields were calculated after two steps c and d); (e) MeI, KOH, acetone, 0 ꢁC, 93–97%;
(f) 4-methoxybenzenesulfonyl chloride, pyridine, rt, 92–93% (yields were calculated after two steps c and f).

1080
L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
OMe
I
I
I
a
b
N
N
N
+
N
N
N
N
H
H
SO2
NO2
10
NO2
20
NO2
10
N
H
NO2
21
OMe
Me
OMe
c, d
N
N
H
N
NH
N
O2S
H
NO2
22
OMe
Scheme 4. Reagents and conditions: (a) PhSO2Cl, NaOH, PHCH2NEt3Cl, CH2Cl2, 76%; (b) 4-MeOC6H4B(OH)2, Na2CO3, DME, Pd(PPh3)4, reflux,
45% (36% of 10); (c) H2, Pd/C, MeOH; (d) 4-methoxybenzenesulfonyl chloride, pyridine, rt, 33% (yield was calculated after two steps c and d).
Thus, after hydrogenation of 1 and 2 using 10% palladium
on carbon in methanol, the corresponding amines 4 and 5
were immediately condensed with various arylsulfonyl
chlorides in pyridine. Compounds 13 and 14 were synthe-
sized in the same way. Compounds 6e and 7e were ob-
tained by condensation of the corresponding amines
with 4-sulfamoylbenzenesulfonyl chloride using 3 equiva-
lents of pyridine in acetone. The treatment of 7-nitroin-
dazole with sodium hypochlorite in the presence of
sodium hydroxide (2 N) in MeOH at reflux afforded cor-
responding 3-chloro-7-nitroindazole 2. Iodination of 7-
nitroindazole using solid iodine and potassium hydroxide
pellets in DMF gave the 3-iodo-7-nitroindazole 3.
3 could need the protection of N-1, but treatment in the
same conditions (Pd(PPh3)4/Na2CO3/DME at 92 ꢁC)
of 1-N-Boc-3-iodo-7-nitroindazole 19 gave only the
deprotected parent compound 10 (Scheme 3).
Replacement of the Boc protective group with a tosyl al-
lowed the obtaining, in the same conditions, of the cou-
pled product, but in a yield of only 45%. It is
noteworthy that 36% of compound 10 was recovered
(Scheme 4). The coupled product was hydrogenated in
the presence of 10% palladium on carbon in methanol
and then immediately treated with 4-meth-
oxybenzenesulfonyl chloride in pyridine to gave desired
product 22, which was obtained with a relatively poor
yield (33% after two steps).
The 7-nitroindazole derivatives substituted at the 3-posi-
tion were obtained in good yields. 7-Nitroindazole
derivatives 1–3 have been alkylated with methyl iodide
in the presence of potassium hydroxide in acetone.
These reactions gave the corresponding N-alkylated
derivatives 8–10.
An alternative synthetic approach led us to investigate
the reactivity of 3-iodo-1-(4-methoxybenzyl)-7-nitroin-
dazole 23. This compound was obtained by protecting
the NH group with 4-methoxybenzyl chloride in acetone
at 0 ꢁC in the presence of potassium hydroxide. The
compound 24 was subsequently obtained by reaction
of 23 with 4-methoxyphenyl boronic acid under Suzuki
cross-coupling conditions in 80% yield (Scheme 5). After
hydrogenation using 10% palladium on carbon in meth-
anol, the corresponding crude amine was coupled with
4-methoxybenzenesulfonyl chloride in pyridine at rt to
afford in good yield (76% after two steps) the 4-meth-
oxy-N-[1-(4-methoxybenzyl)-3-(4-methoxyphenyl)-7-in-
dazolyl]benzenesulfonamide 25 which, after deprotection
in refluxing TFA,19 gave the desired compound 22.
Alkylation of 22 with methyl iodide in acetone in the
presence of potassium hydroxide at 0 ꢁC provided the
desired compound 26 in good yield.
Reaction of 6c with methyl iodide and potassium hydrox-
ide in acetone gave the compound 15, which was chlori-
nated to give 16. In the same way, N-alkylation of 13
and 14 gave the compounds 17 and 18 (Scheme 2).
Suzuki cross-coupling reactions were used to introduce
aryl groups at the 3-position. Our initial attempt to syn-
thesize 4-methoxy-N-[3-(4-methoxyphenyl)-7-indazol-
yl]benzenesulfonamide 22 was performed under Suzuki
cross-coupling conditions between 3-iodo-7-nitroindaz-
ole 3 and 4-methoxyphenyl boronic acid in the presence
of catalytic amount of tetrakis(triphenylphosphine)pal-
ladium (0). Unfortunately, after 24 h in refluxing
DME, no reaction was observed and we isolated only
3-iodo-7-nitroindazole 3 at the end of the reaction peri-
od in 95% yield. The same result was observed in the
Suzuki cross-coupling reaction of 3-iodoindazoles.18
Apparently, the Suzuki coupling of 3-iodo-7-nitroindazole
Reaction of 3-iodo-1-methyl-7-nitroindazole 10 with 4-
methoxyphenyl boronic acid under Suzuki-type reaction
conditions gave compound 27 in 80% yield (Scheme 6).
After reduction of the nitro by hydrogenation in the

L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
1081
OMe
I
I
a
b
N
N
N
N
N
H
NO2
N
NO2
OMe
NO2
OMe
OMe
24
3
23
OMe
OMe
N
N
e
c, d
N
f
N
N
N
H
NH
NH
Me
N
O2S
O2S
OMe
O2S
Me
25
22
26
OMe
OMe
OMe
Scheme 5. Reagents and conditions: (a) 4-Methoxybenzyl chloride, KOH, acetone, 0 ꢁC, 90%; (b) 4-MeOC6H4B(OH)2, Na2CO3, DME, Pd(PPh3)4,
reflux, 80%; (c) H2, Pd/C, MeOH; (d) 4-methoxybenzenesulfonyl chloride, pyridine, rt, 76% (after two steps c and d); (e) TFA, reflux, 97%; (f) MeI,
KOH, acetone, 0 ꢁC, 95%.
OMe
OMe
I
b, c
a
N
N
N
N
Me
N
N
Me
NO2
10
Me
NO2
NH
O2S
27
28
OMe
Scheme 6. Reagents and conditions: (a) 4-MeOC6H6B(OH)2, Na2CO3, DME, Pd(PPh3)4, reflux, 80%; (b) H2, Pd/C, MeOH; (c) 4-
methoxybenzenesulfonyl chloride, pyridine, rt, 90%.
presence of 10% palladium on carbon in methanol, the
corresponding crude amine was coupled with 4-meth-
oxybenzenesulfonyl chloride in pyridine leading to
4-methoxy-N-[3-(4-methoxyphenyl)-1-methyl-7-indaz-
olyl]benzenesulfonamide 28.
bear relatively small substituents on position 3 of the
indazole (R3 = H or Cl).
Compounds that are not substituted on the aryl sulfonyl
(R1 = H, 6a) or bear an aminosulfonyl group (6e and 7e)
are inactive with IC50 > 50 lM.
3. Pharmacological results
Compounds 22, 26, and 28, with a 4-methoxyphenyl
group on the indazole, were significantly less active than
their analogues substituted with less bulky groups (H or
Cl) with IC50 ranging from 14.2 to 29.1 lM. We can
conclude that both positions 3 of indazole and 4 of the
phenylsulfonamide are sensitive to the sizes of their
substituents.
3.1. Antiproliferative activity on the murine leukemia
L1210 cell line
The antiproliferative properties of various N-(7-indazol-
yl)benzenesulfonamide derivatives on the murine leuke-
mia L1210 cell line are reported in Table 1.
The perturbations of the cell cycles induced by the most
potent compounds were studied on the L1210 cell line
by flow cytometry. Except for 7c, all of these com-
pounds induced a marked accumulation in G2M + 8N
phases of the cell cycle. This observation is typical of
The N-(7-indazolyl)benzene sulfonamides 6b–c, 7c and
13–16 were the most potent with IC50 values ranging
from 0.44 to 1.43 lM. These compounds are substituted
on the phenylsulfonyl moiety (R1 = Me or OMe) and

1082
L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
Table 1. In vitro antiproliferative activity of N-(7-indazolyl)benzenesulfonamide derivatives on the murine L1210 cell line
R3
N
R1
N
R2
N
O
R4
S
O
Compound
R1
R2
R3
R4
IC50 (lM)
Cell cycle analysis
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
13
14
15
16
18
22
26
28
H
Me
H
H
H
>49.9
1.43
0.98
18.3
68.5
51.5
2.52
0.923
25.3
85.5
1.16
1.16
1.18
0.44
1.9
Not tested
H
H
H
93% G2M + 8N at 5 lM
93% G2M + 8N at 2.5 lM
Not tested
OMe
Br
H
H
H
H
H
H
SO2NH2
H
Me
H
H
H
Not tested
Not tested
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
92% G2M + 8N at 5 lM
27% G2M at 1 to 10 lM
Not tested
OMe
Br
H
H
H
H
SO2NH2
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
Not tested
Me
Me
H
H
86% G2M + 8N at 5 lM
88% G2M + 8N at 5 lM
93% G2M + 8N at 5 lM
92% G2M + 8N at 1 lM
90% G2M + 8N at 10 lM
Not tested
Cl
H
H
Me
Me
Me
H
H
Cl
Cl
Me
H
4-MeO-C6H4
4-MeO-C6H4
4-MeO-C6H4
14.2
24.3
29.1
Me
Me
Me
H
Not tested
Not tested
the modification of the cell cycle induced by numerous
tubulin interacting drugs.
There again, compounds 6c, 7c, and 16 were the most
potent with IC50 values ranging from 0.38 to 1.18 lM
on human DU145, HCT116, and HT29 cell lines.
3.2. Antiproliferative activity on human DU145,
HCT116, and HT29 cell lines
In conclusion, we have described here the synthesis and
the antiproliferative activities of new N-(7-indazol-
yl)benzenesulfonamide derivatives. Some of these com-
pounds exhibit significant cytotoxicity against human
(colon and prostate) and murine (leukemia) cell lines.
The most potent compounds induced cell cycle modifi-
cations typical of tubulin interacting agents (tetraploid
cells). Compound 16, 4-methoxy-N-(3-chloro-7-indazol-
yl)benzenesulfonamide, was the most active of the series.
The antiproliferative properties of most of the com-
pounds were also evaluated on human DU145,
HCT116, and HT29 cell lines (results reported in Table 2).
Table 2. In vitro antiproliferative activity (IC50 in lM) of N-(7-
indazolyl)benzenesulfonamide derivatives on the DU145, HCT116,
and HT29 cell lines
4. Experimental
4.1. General
Compound
DU145
HCT116
HT29
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
13
14
15
16
18
22
26
28
NT
NT
1.2
NT
1.2
2.39
1.18
NT
0.6
0.5
Melting points were determined with a Buchi SMP-20
¨
NT
37.2
NT
0.7
NT
78.8
NT
2.1
melting point apparatus and are uncorrected. 1H
NMR and 13C NMR were recorded on a Bruker Avance
DPX250 spectrometer (250.19 MHz 1H, 62.89 MHz
13C) using tetramethylsilane as the internal standard,
multiplicities determined by the DEPT 135 sequence.
Chemical shifts were reported in parts per million
(ppm, d units). Coupling constants were reported in
units of hertz (Hz). Splitting patterns were designated
as s, singlet; d, doublet, and t, triplet. Low-resolution
mass spectra (MS) were recorded on a Perkin-Elmer
SCIEX API 3000 spectrometer. All commercial solvents
were used without further purification. The following
solvents and reagents have been abbreviated: dimethyl-
formamide (DMF), dimethylsulfoxide (DMSO), ethyl
88.25
NT
1.70
0.96
NT
0.6
0.9
NT
0.9
NT
23.5
0.8
7.07
1.98
1.91
3.53
0.91
3.2
0.7
0.8
1.0
1.0
1.2
0.38
2.1
0.40
2.1
30.5
38.4
47.9
11.2
31.2
27.0
10.8
32.5
34.7
NT, Not tested.

L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
1083
acetate (EtOAc), methanol (MeOH), trifluoroacetic acid
(TFA), and ethylene glycol dimethyl ether (DME). Thin
layer chromatography (TLC) was carried out on Merck
silica gel 60F254 precoated plates. Visualization was
made with ultraviolet light.
J = 7.5 Hz), 7.74 (d, 2H, J = 8.5 Hz), 10.12 (s, 1H),
12.71 (s, NH). 13C NMR (DMSO-d6) d 117.7, 118.4,
120.6, 124.5, 126.8, 129.2, 133.1, 133.9, 134.8, 135.1,
139.2. MS m/z = 274 [M+1]+. Anal. Calcd for
C13H11N3O2S: C, 57.13; H, 4.06; N, 15.37; S, 11.73.
Found: C, 57.06; H, 4.21; N, 15.25; S, 11.66.
4-Sulfamoylbenzenesulfonyl chloride was prepared
according to methods described in the literature.20,21
4.2. General procedure for the synthesis of 6b–d
4.1.1. 7-Nitroindazole (1). This compound was prepared
according to the method described in the literature17
(65% yield). Mp 185–186 ꢁC (lit.17 mp 186 ꢁC). 1H
NMR (DMSO-d6) d 7.32 (t, 1H, J = 7.8 Hz), 8.32 (d,
1H, J = 7.8 Hz), 8.35 (d, 1H, J = 7.8 Hz), 8.41 (s, 1H),
13.94 (s, NH). 13C NMR (DMSO-d6) d 120.2, 123.5,
127.1, 129.9, 131.9, 132.1, 135.6. MS m/z = 164.1
[M+1]+.
These products were synthesized as described for 6a by
using the appropriate sulfonyl chlorides.
4.2.1. 4-Methyl-N-(7-indazolyl)benzenesulfonamide (6b).
Obtained as a colorless solid in 93% yield. Mp 207–
208 ꢁC. 1H NMR (DMSO-d6) d 2.31 (s, 3H, CH3),
6.94 (d, 2H, J = 8.1 Hz), 7.30 (d, 2H, J = 7.8 Hz), 7.48
(t, 1H, J = 7.8 Hz), 7.65 (d, 2H, J = 8.1 Hz), 8.04 (s,
1H), 10.04 (s, NH), 12.67 (s, NH). 13C NMR (DMSO-
d6) d 20.9, 113.9, 117.4, 117.9, 120.6, 121.0, 124.6,
126.9, 129.6, 131.2, 136.4, 143.4. MS m/z = 288
[M+1]+. Anal. Calcd for C14H13N3O2S: C, 58.52; H,
4.56; N, 14.62; S, 11.16. Found: C, 58.34; H, 4.45; N,
14.88; S, 11.29.
4.1.2. 3-Chloro-7-nitroindazole (2). To 7-nitroindazole
(1 g, 6.13 mmol) dissolved in 25 ml MeOH was added
2 N aq sodium hydroxide (20 ml) and then sodium
hypochlorite (6 ml, 98.2 mmol) was added to the solu-
tion. The mixture was refluxed for 1 h. After cooling,
the solution was acidified with acetic acid. The precipi-
tated solid was filtered, washed with water, and dried
in vacuo to give a yellow solid (98% yield). Mp 167–
4.2.2.
4-Methoxy-N-(7-indazolyl)benzenesulfonamide
(6c). Obtained as a colorless solid in 92% yield. Mp
230–231 ꢁC. 1H NMR (DMSO-d6) d 3.76 (s, 3H,
OCH3), 6.9 (d, 2H, J = 7.9 Hz), 7.25 (d, 2H,
J = 8.7 Hz), 7.49 (t, 1H, J = 7.9 Hz), 7.59 (d, 2H,
J = 8.7 Hz), 8.04 (s, 1H), 9.97 (s, NH), 12.66 (s, NH).
13C NMR (DMSO-d6) d 55.6, 114.3, 117.4, 117.9,
120.6, 120.9, 124.4, 129.1, 130.7, 133.9, 134.6, 162.3.
MS m/z = 304 [M+1]+. Anal. Calcd for C14H13N3O3S:
C, 55.43; H, 4.32; N, 13.85; S, 10.57. Found: C, 55.56;
H, 4.20; N, 13.62; S, 10.71.
1
168 ꢁC (lit.22 mp 166–168 ꢁC). H NMR (DMSO-d6) d
7.45 (t, 1H, J = 7.8 Hz), 8.20 (d, 1H, J = 7.8 Hz), 8.44
(d, 1H, J = 7.8 Hz), 14.13 (s, NH). 13C NMR (DMSO-
d6) d 121.3, 123.4, 125.1, 127.8, 132.5, 133.2, 134.7.
MS m/z = 198.1 (35Cl) [M+1]+, 201.1 (37Cl) [M+3]+.
4.1.3. 3-Iodo-7-nitroindazole (3). To a solution of 7-
nitroindazole (1 g, 6.13 mmol) in DMF (60 ml) were
added iodine (3.1 g, 12.26 mmol) and potassium hydrox-
ide pellets (1.28 g, 23 mmol) at rt under stirring. After
1 h, the reaction mixture was poured into 10% aq NaH-
SO3 (200 ml) and extracted with Et2O (2 · 150 ml). The
combined organic layers were washed with water and
brine, dried over MgSO4, and the solvent was evaporat-
ed to give a light yellow solid (95% yield). Mp 188–
4.2.3. 4-Bromo-N-(7-indazolyl)benzenesulfonamide (6d).
Obtained as a colorless solid in 71% yield. Mp 224–
225 ꢁC. 1H NMR (DMSO-d6)
d
6.88 (d, 1H,
J = 7.5 Hz), 6.95 (dd, 1H, J = 7.5, 7.7 Hz), 7.54 (d, 1H,
J = 7.7 Hz), 7.65 (d, 2H, J = 8.7 Hz), 7.74 (d, 2H,
J = 8.7 Hz), 8.06 (s, 1H), 10.19 (s, NH), 14.32 (s, NH).
13C NMR (DMSO-d6) d 118.2, 119.2, 120.2, 120.6,
125.4, 126.8, 128.8, 132.3, 133.9, 135.1, 138.5. MS m/
z = 353 [M+1]+ (79Br), 354 [M+3]+ (81Br). Anal. Calcd
for C13H10BrN3O2S: C, 44.33; H, 2.86; Br, 22.69; N,
11.93; S, 9.10. Found: C, 44.20; H, 2.75; Br, 22.85; N,
11.98; S, 9.21.
189 ꢁC. 1H NMR (DMSO-d6)
d
7.44 (t, 1H,
J = 7.8 Hz), 8.00 (d, 1H, J = 7.8 Hz), 8.46 (d, 1H,
J = 7.8 Hz), 14.32 (s, NH). 13C NMR (DMSO-d6) d
96.8, 120.9, 124.6, 129.6, 130.8, 132.1, 132.6. MS
m/z = 290.2 [M+1]+. Anal. Calcd for C7H4IN3O2: C,
29.09; H, 1.39; I, 43.91; N, 14.54. Found: C, 29.20; H,
1.51; I, 43.78; N, 14.50.
4.1.4. N-(7-Indazolyl)benzenesulfonamide (6a). A solu-
tion of 7-nitroindazole (200 mg, 1.23 mmol) in MeOH
(20 ml) was hydrogenated over 10% palladium on car-
bon (20 mg) under H2 at 1 atm overnight. After the cat-
alyst was filtered off, the filtrate was evaporated to give
7-aminoindazole 4. The crude amine was immediately
dissolved in pyridine (5 ml) and then reacted with benze-
nesulfonyl chloride (180 ll, 1.35 mmol) at rt for 8 h.
After the reaction mixture was concentrated in vacuo,
the resulting residue was purified by flash chromatogra-
phy using EtOAc/EP to afford a colorless solid in 95%
4.2.4. N-(7-Indazolyl)-1,4-benzenedisulfonamide (6e). A
solution of 7-nitroindazole (200 mg, 1.23 mmol) in
MeOH (20 ml) was hydrogenated over 10% palladium
on carbon (20 mg) under H2 at 1 atm during 2 h. After
the catalyst was filtered off, the filtrate was evaporated
to give almost pure 7-aminoindazole 4. The crude amine
was dissolved in acetone (5 ml) followed by the immedi-
ate addition of 4-sulfamoylbenzenesulfonyl chloride
(408 mg, 1.67 mmol) and pyridine (300 ll, 3.6 mmol).
The reaction mixture was stirred at rt overnight. After
the reaction mixture was concentrated in vacuo, the
resulting residue was purified by flash chromatography
(eluted with EtOAc/hexane) afforded as a colorless solid
1
yield. Mp 215–216 ꢁC. H NMR (DMSO-d6) d 6.90 (d,
1H, J = 8.5 Hz), 7.04 (dd, 1H, J = 7.5, 7.9 Hz), 7.42 (d,
1H, J = 7.9 Hz), 7.54 (d, 2H, J = 8.5 Hz), 7.62 (d, 1H,
1
in 76% yield. Mp 240–241 ꢁC. H NMR (DMSO-d6) d

1084
L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
6.87 (d, 1H, J = 7.3 Hz), 6.96 (dd, 1H, J = 7.3, 7.5 Hz),
7.53 (d, 1H, J = 7.5 Hz), 7.56 (s, 2H, NH2), 7.90–7.95
(m, 4H), 8.06 (s, 1H), 10.34 (s, NH). 13C NMR
(DMSO-d6) d 118.2, 119.0, 120.2, 120.2, 121.5, 124.5,
126.5, 127.8, 131.7, 142.0, 147.7. MS m/z = 353
[M+1]+. Anal. Calcd for C13H12N4O4S2: C, 44.31; H,
3.43; N, 15.90; S, 18.20. Found: C, 44.50; H, 3.48; N,
15.72; S, 18.04.
9.17; N, 10.87; S, 8.29. Found: C, 40.20; H, 2.31; Br,
20.88; Cl, 9.29; N, 10.66; S, 8.37.
4.3.5. N-(3-Chloro-7-indazolyl)-1,4-benzenedisulfonamide
(7e). This compound was synthesized from 3-chloro-7-
nitroindazole as described for 6e. Chromatography
eluted with EtOAc/hexane afforded a colorless solid
in 72% yield. Mp > 250 ꢁC. 1H NMR (DMSO-d6) d
6.88 (d, 1H, J = 7.5 Hz), 7.06 (dd, 1H, J = 7.5,
7.9 Hz), 7.47 (d, 1H, J = 7.9 Hz), 7.58 (s, 2H, NH2),
7.92–7.97 (m, 4H), 10.42 (s, NH). 13C NMR
(DMSO-d6) d 116.6, 122.1, 122.6, 125.4, 126.6, 127.4,
128.6, 132.5, 136.7, 141.8, 147.8. MS m/z = 387
(35Cl) [M+1]+, 389 (37Cl) [M+3]+. Anal. Calcd for
C13H11ClN4O4S2: C, 40.36; H, 2.87; Cl, 9.16; N,
14.48; S, 16.58. Found: C, 40.51; H, 2.62; Cl, 9.02;
N, 14.73; S, 16.40.
4.3. General procedure for the synthesis of 7a–d
These products were synthesized as described for 6a by
using the appropriate sulfonyl chlorides.
4.3.1. N-(3-Chloro-7-indazolyl)benzenesulfonamide (7a).
Obtained as a colorless solid in 93% yield. Mp 198–
199 ꢁC. 1H NMR (DMSO-d6)
d
6.90 (d, 1H,
J = 7.2 Hz), 7.04 (dd, 1H, J = 7.5, 7.8 Hz), 7.42 (d, 1H,
J = 7.8 Hz), 7.54 (d, 2H, J = 7.2 Hz), 7.60 (d, 1H,
J = 7.5 Hz), 7.74 (d, 2H, J = 7.2 Hz), 10.2 (s, NH),
13.00 (s, NH). 13C NMR (DMSO-d6) d 116.1, 120.5,
121.1, 121.2, 121.7, 126.9, 129.2, 132.5, 133.2, 136.5,
138.9. MS m/z = 308.5 (35Cl) [M+1]+, 310.5 (37Cl)
[M+3]+. Anal. Calcd for C13H10ClN3O2S: C, 50.74; H,
3.28; Cl, 11.52; N, 13.65; S, 10.42. Found: C, 50.96; H,
3.44; Cl, 11.44; N, 13.49; S, 10.41.
4.4. General method for alkylation of 7-nitroindazole
derivatives
To a solution of 7-nitroindazole derivatives (6.13 mmol)
in acetone (15 ml) cooled at 0 ꢁC was added potassium
hydroxide (9.2 mmol). After 15 min at 0 ꢁC, iodome-
thane or 4-methoxybenzyl chloride (6.13 mmol) was
added dropwise. Upon disappearance of the starting
material as indicated by TLC, 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 removed in vacuo to give
the corresponding compounds.
4.3.2. 4-Methyl-N-(3-chloro-7-indazolyl)benzenesulfona-
mide (7b). Obtained as a colorless solid in 88% yield.
1
Mp 176–177 ꢁC. H NMR (DMSO-d6) d 2.49 (s, 3H,
CH3), 6.94 (d, 1H, J = 7.5 Hz), 7.05 (dd, 1H, J = 7.5,
7.8 Hz), 7.30 (d, 2H, J = 8.1 Hz), 7.40 (d, 1H,
J = 7.8 Hz), 7.64 (d, 2H, J = 8.1 Hz), 10.11 (s, NH),
13.01 (s, NH). 13C NMR (DMSO-d6) d 20.9, 115.8,
120.2, 121.1, 121.4, 121.8, 126.9, 129.7, 132.6, 136.1,
136.3, 143.5. MS m/z = 322 (35Cl) [M+1]+, 324 (37Cl)
[M+3]+. Anal. Calcd for C14H12ClN3O2S: C, 52.26; H,
3.76; Cl, 11.02; N, 13.06; S, 9.96. Found: C, 52.40; H,
3.52; Cl, 10.91; N, 13.21; S, 9.83.
4.4.1. 1-Methyl-7-nitroindazole (8). Chromatography
using EtOAc/hexane gave a yellow solid in 93% yield.
1
Mp 99–100 ꢁC (lit.22 mp 98 ꢁC). H NMR (DMSO-d6)
d 4.16 (s, 3H, CH3), 7.40 (t, 1H, J = 7.7 Hz), 7.90 (d,
1H, J = 7.7 Hz), 8.29 (d, 1H, J = 7.7 Hz). 13C NMR
(DMSO-d6) d 40.3, 120.1, 124.5, 125.4, 128.6, 130.4,
134.3, 206.4. MS m/z = 178.1 [M+1]+. Anal. Calcd for
C8H7N3O: C, 54.24; H, 3.98; N, 23.72. Found: C,
54.02; H, 4.17; N, 23.83.
4.3.3. 4-Methoxy-N-(3-chloro-7-indazolyl)benzenesulf-
onamide (7c). Obtained as a colorless solid in 90% yield.
1
Mp 201–202 ꢁC. H NMR (DMSO-d6) d 3.78 (s, 3H,
4.4.2. 3-Chloro-1-methyl-7-nitroindazole (9). This com-
pound was similarly prepared from 3-chloro-7-nitroin-
dazole. Chromatography using EtOAc/EP gave a
yellow solid in 96% yield. Mp 150–151 ꢁC (lit.23 mp
148–150 ꢁC). 1H NMR (DMSO-d6) d 4.11 (s, 3H,
CH3), 7.42 (t, 1H, J = 7.8 Hz), 8.11 (d, 1H,
J = 7.8 Hz), 8.28 (d, 1H, J = 7.8 Hz). 13C NMR
(DMSO-d6) d 40.9, 121.2, 124.6, 126.1, 126.5, 132.0,
132.8, 206.5. MS m/z = 212 (35Cl) [M+1]+, 214 (37Cl)
[M+3]+. Anal. Calcd for C8H6ClN3O: C, 45.41; H,
2.86; Cl, 16.75; N, 19.86. Found: C, 45.55; H, 2.90; Cl,
16.49; N, 19.77.
OCH3), 6.95 (d, 1H, J = 7.5 Hz), 7.01 (dd, 1H, J = 7.5,
7.8 Hz), 7.07 (d, 2H, J = 8.7 Hz), 7.24 (d, 1H,
J = 7.8 Hz), 7.67 (d, 2H, J = 8.7 Hz), 10.02 (s, NH),
13.00 (s, NH). 13C NMR (DMSO-d6) d 55.6, 114.3,
115.7, 120.0, 120.1, 121.5, 121.8, 129.2, 130.5, 132.5,
136.2, 162.6. MS m/z = 338 (35Cl) [M+1]+, 340 (37Cl)
[M+3]+. Anal. Calcd for C14H12ClN3O2S: C, 49.78; H,
3.58; Cl, 10.50; N, 12.44; S, 9.46. Found: C, 49.60; H,
3.61; Cl, 10.77; N, 12.32; S, 9.30.
4.3.4. 4-Bromo-N-(3-chloro-7-indazolyl)benzenesulfona-
mide (7d). Obtained as a colorless solid in 63% yield.
1
Mp 220–221 ꢁC. H NMR (DMSO-d6) d 6.89 (d, 1H,
4.4.3. 3-Iodo-1-methyl-7-nitroindazole (10). This com-
pound was similarly prepared from 3-iodo-7-nitroindaz-
ole. Chromatography using EtOAc/EP gave a yellow
J = 7.5 Hz), 7.08 (dd, 1H, J = 7.5, 8.1 Hz), 7.47 (d, 1H,
J = 8.1 Hz), 7.65 (d, 2H, J = 8.5 Hz), 7.78 (d, 2H,
J = 8.5 Hz), 10.27 (s, NH), 13.10 (s, NH). 13C NMR
(DMSO-d6) d 116.5, 120.8, 121.3, 121.8, 122.5, 127.0,
128.9, 132.3, 132.5, 136.8, 138.2. MS m/z = 387 (35Cl)
[M+1]+, 389 (37Cl) [M+3]+. Anal. Calcd for
C13H9BrClN3O2S: C, 40.38; H, 2.35; Br, 20.67; Cl,
1
solid in 97% yield. Mp 171–172 ꢁC. H NMR (DMSO-
d6) d 4.16 (s, 3H, CH3), 7.40 (t, 1H, J = 7.7 Hz), 7.9
(d, 1H, J = 7.7 Hz), 8.29 (d, 1H, J = 7.7 Hz). 13C
NMR (DMSO-d6) d 40.8, 95.1, 120.8, 125.7, 128.6,
131.4, 132.1, 134.7. MS m/z = 304 [M+1]+. Anal. Calcd

L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
1085
for C7H4IN3O: C, 29.09; H, 1.39; I, 43.91; N, 14.54.
Found: C, 29.20; H, 1.45; I, 43.70; N, 14.37.
onamide as described in general method for alkylation
of 7-nitroindazole derivatives. Chromatography using
EtOAc/hexane as eluent afforded a colorless solid in
97% yield. Mp 164–165 ꢁC. 1H NMR (DMSO-d6) d
3.22 (s, 3H, CH3), 3.88 (s, 3H, CH3), 4.29 (s, 3H,
OCH3), 6.54 (d, 1H, J = 7.5 Hz), 6.97 (dd, 1H, J = 7.5,
7.8 Hz), 7.17 (d, 2H, J = 8.7 Hz), 7.58 (d, 2H,
J = 8.7 Hz), 7.74 (d, 1H, J = 7.8 Hz), 8.11 (s, 1H). 13C
NMR (DMSO-d6) d 38.1, 39.6, 55.7, 114.5, 120.4,
121.4, 124.5, 125.4, 125.8, 127.5, 130.2, 132.5, 136.6,
163.0. MS m/z = 332 [M+1]+. Anal. Calcd for
C16H17N3O3S: C, 57.99; H, 5.17; N, 12.68; S, 9.68.
Found: C, 57.60; H, 5.36; N, 12.45; S, 9.61.
4.4.4. 4-Methoxy-N-(1-methyl-7-indazolyl)benzenesulf-
onamide (13). This compound was synthesized as de-
scribed for 6a as a colorless solid in 92% yield. Mp
163–164 ꢁC. 1H NMR (DMSO-d6) d 3.48 (s, 3H,
CH3), 4.23 (s, 3H, OCH3), 6.43 (d, 1H, J = 7.5 Hz),
6.90 (dd, 1H, J = 7.5, 7.9 Hz), 7.10 (d, 2H, J = 8.5 Hz),
7.59 (d, 2H, J = 8.5 Hz), 7.65 (d, 1H, J = 7.9 Hz), 8.06
(s, 1H), 9.83 (s, NH). 13C NMR (DMSO-d6) d 38.7,
55.7, 114.0, 114.3, 120.2, 120.6, 123.1, 125.4, 127.6,
129.6, 133.7, 137.8, 162.8. MS m/z = 318 [M+1]+. Anal.
Calcd for C15H15N3O3S: C, 56.77; H, 4.76; N, 13.24; S,
10.10. Found: C, 56.58; H, 4.49; N, 13.50; S, 10.25.
4.4.9.
4-Methoxy-N-methyl-N-(3-chloro-1-methyl-7-
indazolyl)benzenesulfonamide (18). This compound was
prepared from 4-methoxy-N-(3-chloro-1-methyl-7-
indazolyl)benzenesulfonamide as described in general
method for alkylation of 7-nitroindazole derivatives.
Chromatography using EtOAc/hexane gave a colorless
4.4.5. 4-Methoxy-N-(3-chloro-1-methyl-7-indazolyl)ben-
zenesulfonamide (14). This compound, synthesized from
12 as described for 6a, was obtained as a colorless solid
in 93% yield. Mp 160–161 ꢁC. H NMR (DMSO-d6) d
3.24 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 6.63 (d, 1H,
J = 7.5 Hz), 6.91 (d, 2H, J = 8.5 Hz), 7.05 (dd, 1H,
J = 7.5, 7.9 Hz), 7.47 (d, 2H, J = 8.5 Hz), 7.60 (d, 1H,
J = 7.9 Hz), 10.49 (s, NH). 13C NMR (DMSO-d6) d
38.6, 55.7, 114.4, 118.5, 121.4, 121.3, 122.5, 124.5,
126.1, 127.0, 130.0, 130.1, 162.9. MS m/z = 352 (35Cl)
[M+1]+, 354 (37Cl) [M+3]+. Anal. Calcd for
C15H14ClN3O3S: C, 51.21; H, 4.01; Cl, 10.08; N,
11.94; S, 9.11. Found: C, 51.00; H, 4.23; Cl, 9.87; N,
11.99; S, 8.93.
1
1
solid in 93% yield. Mp 181–182 ꢁC. H NMR (DMSO-
d6) d 3.21 (s, 3H, CH3), 3.88 (s, 3H, CH3), 4.26 (s, 3H,
OCH3), 6.67 (d, 1H, J = 7.8 Hz), 7.11 (t, 1H,
J = 7.8 Hz), 7.17 (d, 2H, J = 8.7 Hz), 7.56 (d, 2H,
J = 8.7 Hz), 7.65 (d, 1H, J = 7.8 Hz). 13C NMR
(DMSO-d6) d 38.5, 39.4, 55.7, 114.5, 119.5, 121.7,
122.6, 125.4, 126.0, 127.0, 130.2, 130.7, 138.1, 163.1.
MS m/z = 366 (35Cl) [M+1]+, 368 (37Cl) [M+3]+. Anal.
Calcd for C16H16N3O3S: C, 52.53; H, 4.44; Cl, 9.69;
N, 11.49; S, 8.76. Found: C, 52.19; H, 4.69; Cl, 9.55;
N, 11.66; S, 8.51.
4.4.6. 4-Methoxy-N-methyl-N-(7-indazolyl)benzenesulf-
onamide (15). This compound was prepared from
4-methoxy-N-(7-indazolyl)benzenesulfonamide as de-
scribed in general method for alkylation of 7-nitroindaz-
ole derivatives. Chromatography using EtOAc/hexane
as eluent afforded a colorless solid in 95% yield. Mp
165–166 ꢁC. 1H NMR (DMSO-d6) d 3.21 (s, 3H,
CH3), 3.85 (s, 3H, OCH3), 6.67 (d, 1H, J = 7.5 Hz),
6.98 (dd, 1H, J = 7.5, 7.8 Hz), 7.11 (d, 2H, J = 8.7 Hz),
7.53 (d, 2H, J = 8.7 Hz), 7.71 (d, 1H, J = 7.8 Hz), 8.10
(s, 1H), 13.27 (s, NH). 13C NMR (DMSO-d6) d 38.7,
55.7, 114.3, 120.2, 120.4, 123.1, 124.9, 125.4, 127.6,
130.1, 133.7, 137.8, 162.8. MS m/z = 318 [M+1]+. Anal.
Calcd for C15H15N3O3S: C, 56.77; H, 4.76; N, 13.24; S,
10.10. Found: C, 56.86; H, 4.92; N, 13.04; S, 9.91.
4.4.10. 1-N-Boc-3-iodo-7-nitroindazole (19). To a solu-
tion of 3-iodo-7-nitroindazole (1.65 mmol) in CH2Cl2,
were added Et3N (1.82 mmol), DMAP (1.65 mmol),
and di-tert-butyl dicarbonate (1.82 mmol). The reaction
mixture was refluxed with vigorous stirring for 20 h. The
organic solvent was removed under reduced pressure
and the crude product was purified by chromatography
(silica gel, EtOAc/EP) to give compound 19 as a yellow
solid in 93% yield. Mp 108–109 ꢁC. 1H NMR (CDCl3) d
1.64 (s, 9H, 3CH3), 7.48 (dd, 1H, J = 8.1, 7.8 Hz), 7.79
(d, 1H, J = 8.1 Hz), 8.08 (d, 1H, J = 7.8 Hz). 13C
NMR (CDCl3) d 28.4, 88.2, 101.9, 124.3, 126.1, 128.0,
130.9, 133.9, 137.9, 174.8. MS m/z=390 [M+1]+. Anal.
Calcd for C12H12IN3O4: C, 37.04; H, 3.11; I, 32.61; N,
10.80. Found: C, 37.25; H, 3.02; I, 32.77; N, 10.69.
4.4.7. 4-Methoxy-N-methyl-N-(3-chloro-7-indazolyl)ben-
zenesulfonamide (16). Obtained as a colorless solid in
98% yield. Mp 198–199 ꢁC. 1H NMR (DMSO-d6) d
3.20 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 6.77 (d, 1H,
J = 7.2 Hz), 7.07–7.13 (m, 3H), 7.52 (d, 2H,
J = 8.5 Hz), 7.61 (d, 1H, J = 8.3 Hz), 13.56 (s, NH).
13C NMR (DMSO-d6) d 38.6, 55.7, 114.4, 118.5, 121.3,
121.3, 121.4, 124.5, 126.1, 127.1, 130.1, 132.3, 139.1,
162.9. MS m/z = 352 (35Cl) [M+1]+, 354 (37Cl) [M+3]+.
Anal. Calcd for C15H14ClN3O3S: C, 51.21; H, 4.01; Cl,
10.08; N, 11.94; S, 9.11. Found: C, 51.12; H, 4.15; Cl,
10.17; N, 12.20; S, 8.95.
4.4.11. 1-Benzenesulfonyl-3-iodo-7-nitroindazole (20). To
a solution of sodium hydroxide (5.1 mmol) in 10 ml of
CH2Cl2 at 0 ꢁC, 3-iodo-7-nitroindazole (1.65 mmol)
and benzyltriethylammonium chloride (0.04 mmol) were
added. To the mixture, benzenesulfonyl chloride
(2 mmol) was added dropwise. The reaction mixture
was refluxed with vigorous stirring overnight. The
organic solvent was removed under reduced pressure
and the crude product was purified by chromatography
(silica gel, EtOAc/EP) to give compound 20 as a yellow
1
solid in 76% yield. Mp 185–186 ꢁC. H NMR (DMSO-
d6) d 7.47 (d, 2H, J = 7.5 Hz), 7.57 (dd, 1H, J = 8.1,
7.9 Hz), 7.63–7.68 (m, 3H), 8.04 (d, 1H, J = 7.9 Hz),
8.10 (d, 1H, J = 8.1 Hz). 13C NMR (DMSO-d6) d
115.5, 117.1, 127.9, 128.1, 129.5, 130.2, 133.8, 134.7,
4.4.8. 4-Methoxy-N-methyl-N-(1-methyl-7-indazolyl)ben-
zenesulfonamide (17). This compound was prepared
from 4-methoxy-N-methyl-N-(7-indazolyl)benzenesulf-

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L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
139.6, 138.2, 171.7. MS m/z = 444 [M+1]+. Anal. Calcd
for C14H10IN3O4S: C, 37.94; H, 2.27; I, 28.63; N, 9.48.
Found: C, 37.99; H, 2.49; I, 28.41; N, 9.30.
NMR (DMSO-d6) d 55.0, 55.7, 114.1, 121.3, 126.0,
128.1, 128.4, 128.9, 130.4, 132.6, 134.7, 158.6, 206.4.
MS m/z = 410 [M+1]+. Anal. Calcd for C15H12IN3O3:
C, 44.03; H, 2.96; I, 31.01; N, 10.27. Found: C, 43.92;
H, 3.15; I, 31.20; N, 10.11.
4.5. General procedure for Suzuki-type cross-coupling
reaction of 3-iodo-7-nitroindazole derivatives
4.5.4. 1-(4-Methoxybenzyl)-3-(4-methoxyphenyl)-7-nitro-
indazole (24). The title compound was synthesized from
3-iodo-1-(4-methoxybenzyl)-7-nitroindazole. Chroma-
tography using EtOAc/hexane afforded a yellow solid
To a solution of 3-iodo-7-nitroindazole derivatives
(1.65 mmol) and 4-methoxyphenyl boronic acid
(2.47 mmol) in DME (10 ml), sodium carbonate
(4.95 mmol) dissolved in H2O (5 ml) was added followed
by the addition of Pd(PPh3)4 (0.165 mmol). The reaction
mixture was refluxed with vigorous stirring under argon
atmosphere for 2 h. The organic solvent was removed
under reduced pressure and the crude product was puri-
fied by chromatography (silica gel, EtOAc/EP) to give
the corresponding compounds. Spectral data for repre-
sentative compounds were as follows.
1
in 80% yield. Mp 144–145 ꢁC. H NMR (DMSO-d6) d
3.66 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 5.72 (s, 2H,
CH2), 6.80 (d, 2H, J = 8.7 Hz), 6.90 (d, 2H,
J = 8.7 Hz), 7.14 (d, 2H, J = 8.8 Hz), 7.39 (dd, 1H,
J = 7.7, 7.9 Hz), 7.92 (d, 2H, J = 8.8 Hz), 8.14 (d, 1H,
J = 7.7 Hz), 8.45 (d, 1H, J = 7.9 Hz). 13C NMR
(DMSO-d6) d 55.0, 55.3, 55.7, 114.1, 114.6, 120.9,
123.7, 125.4, 128.1, 128.5, 128.7, 129.1, 130.9, 135.1,
144.7, 144.9, 158.6, 159.8. MS m/z = 390 [M+1]+. Anal.
Calcd for C22H19N3O4: C, 67.86; H, 4.92; N, 10.79.
Found: C, 67.63; H, 4.82; N, 10.94.
4.5.1. 3-(4-Methoxyphenyl)-7-nitroindazole (21). Ob-
tained as a colorless solid in 92% yield. Mp 209–
1
210 ꢁC. H NMR (DMSO-d6) d 3.83 (s, 3H, OCH3),
7.12 (d, 2H, J = 8.5 Hz), 7.43 (dd, 1H, J = 7.7, 8.1 Hz),
7.94 (d, 2H, J = 8.5 Hz), 8.41 (d, 1H, J = 7.7 Hz), 8.55
(d, 1H, J = 8.1 Hz). 13C NMR (DMSO-d6) d 55.3,
114.5, 120.7, 123.7, 124.4, 124.6, 128.7, 130.0, 132.1,
133.5, 145.3, 159.7. MS m/z = 270 [M+1]+. Anal. Calcd
for C14H11N3O4: C, 62.45; H, 4.12; N, 15.61. Found: C,
62.31; H, 4.00; N, 15.88.
4.5.5. 4-Methoxy-N-[1-(4-methoxybenzyl)-3-(4-methoxy-
phenyl)-7-indazolyl]benzenesulfonamide (25). The title
compound was synthesized from 3-iodo-1-methyl-7-
nitroindazole as described for 6a as a colorless solid in
76% yield. Mp 132–133 ꢁC. 1H NMR (DMSO-d6) d
3.75 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 5.78 (s, 2H, CH2), 6.72 (d, 1H, J = 7.2 Hz),
6.82 (d, 2H, J = 8.8 Hz), 6.91 (d, 2H, J = 8.8), 6.97 (d,
1H, J = 7.5 Hz), 7.04 (d, 2H, J = 8.8 Hz), 7.13 (d, 2H,
J = 8.8 Hz), 7.62 (d, 2H, J = 8.8 Hz), 7.84–7.89 (m,
3H). 13C NMR (DMSO-d6) d 54.1, 55.4, 55.5, 55.7,
114.3, 114.5, 114.5, 119.2, 121.0, 121.4, 125.1, 125.8,
126.1, 128.1, 129.0, 130.3, 130.5, 130.9, 131.0, 136.9,
159.3, 160.0, 163.4. MS m/z = 530 [M+1]+. Anal. Calcd
for C29H27N3O4S: C, 65.77; H, 5.14; N, 7.93. S, 6.05.
Found: C, 65.44; H, 5.02; N, 8.25. S, 6.19.
4.5.2.
4-Methoxy-N-[3-(4-methoxyphenyl)-7-indazol-
yl]benzenesulfonamide (22). A mixture of 4-methoxy-N-
[1-(4-methoxybenzyl)-3-(4-methoxyphenyl)-7-indazolyl]
benzenesulfonamide (200 mg, 0.37 mmol) and TFA
(5 ml) was refluxed for 6 h. After the reaction mixture
had been concentrated in vacuo to dryness, water was
added to the residue and then extracted with EtOAc.
The organic layer was washed successively with satd
aq NaHCO3, water, and brine, and dried over MgSO4.
The solvent was concentrated in vacuo. The resulting
residue was purified by flash chromatography (eluted
with EtOAc/hexane) to give a colorless solid in 97%
yield. Mp 170–171 ꢁC. 1H NMR (CDCl3) d 3.76 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 6.82 (d, 2H,
J = 9.1 Hz), 7.03 (d, 2H, J = 8.8 Hz), 7.11 (d, 1H,
J = 7.5 Hz), 7.20 (d, 1H, J = 8.1 Hz), 7.70 (dd, 1H,
J = 7.5, 8.1 Hz), 7.74 (d, 2H, J = 9.1 Hz), 7.77 (d, 2H,
J = 8.8 Hz). 13C NMR (CDCl3) d 56.0, 56.2, 114.9,
115.4, 120.0, 122.5, 123.6, 123.9, 124.2, 129.9, 130.1,
130.3, 136.7, 144.8, 161.5, 164.0, 172.4. MS m/z = 410
[M+1]+. Anal. Calcd for C21H19N3O4S: C, 61.60; H,
4.68; N, 10.26; S, 7.83. Found: C, 61.41; H, 4.48; N,
10.56; S, 7.92.
4.5.6. 4-Methoxy-N-methyl-N-[3-(4-methoxyphenyl)-1-
methyl-7-indazolyl]benzenesulfonamide (26). This com-
pound was prepared from 4-methoxy-N-[3-(4-methoxy-
phenyl)-7-indazolyl]benzenesulfonamide as described in
general method for alkylation of 7-nitroindazole deriva-
tives using 2 equivalents of iodomethane. Chromatogra-
phy using EtOAc/hexane gave a colorless solid in 95%
yield. Mp 204–205 ꢁC. 1H NMR (CDCl3) d 3.27 (s,
3H, CH3), 3.87 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 4.47
(s, 3H, OCH3), 6.50 (d, 1H, J = 7.8 Hz), 6.95 (dd, 1H,
J = 7.5, 7.8 Hz), 7.00 (d, 2H, J = 8.7 Hz), 7.04 (d, 2H,
J = 8.7 Hz), 7.65 (d, 2H, J = 8.8 Hz), 7.82 (d, 2H,
J = 8.8 Hz), 7.89 (d, 1H, J = 7.5 Hz). 13C NMR (CDCl3)
d 38.8, 40.2, 55.4, 55.8, 113.2, 114.2, 114.4, 120.3, 121.6,
122.0, 124.5, 126.0, 126.2, 128.2, 129.0, 130.7, 138.6,
159.6, 163.4. MS m/z = 438 [M+1]+. Anal. Calcd for
C23H23N3O4S: C, 63.14; H, 5.30; N, 9.60; S, 7.33.
Found: C, 63.51; H, 5.12; N, 9.87; S, 7.11.
4.5.3. 3-Iodo-1-(4-methoxybenzyl)-7-nitroindazole (23).
This compound was prepared from 3-chloro-7-nitroin-
dazole as described in general method for alkylation of
7-nitroindazole derivatives. Chromatography using
EtOAc/hexane gave a yellow solid in 90% yield. Mp
125–126 ꢁC. 1H NMR (DMSO-d6) d 3.67 (s, 3H,
OCH3), 5.69 (s, 2H, CH2), 6.80 (d, 2H, J = 8.7 Hz),
6.87 (d, 2H, J = 8.7 Hz), 7.40 (dd, 1H, J = 7.8, 8.3 Hz),
7.94 (d, 1H, J = 8.3 Hz), 8.21 (d, 1H, J = 7.8 Hz). 13C
4.5.7.
3-(4-Methoxyphenyl)-1-methyl-7-nitroindazole
(27). The title compound was synthesized from
3-iodo-1-methyl-7-nitroindazole as described in general
procedure for Suzuki-type cross-coupling reaction of
3-iodo-7-nitroindazole derivatives. Chromatography

L. Bouissane et al. / Bioorg. Med. Chem. 14 (2006) 1078–1088
1087
using EtOAc/hexane gave a yellow solid in 80% yield.
Mp 175–176 ꢁC. H NMR (DMSO-d6) d 3.84 (s, 3H,
and incubated in PBS containing 100 lg/ml RNase
and 25 lg/ml propidium iodide for 30 min at 20 ꢁC.
For each sample, 104 cells were analyzed on an Epics
XL/MCL flow cytometer (Beckman Coulter, France).
Results are expressed as the percentage of cells found
in the different phases of the cell cycle.
1
CH3), 4.16 (s, 3H, OCH3), 7.11 (d, 2H, J = 8.7 Hz),
7.38 (dd, 1H, J = 7.7, 7.9 Hz), 7.86 (d, 2H, J = 8.7 Hz),
8.22 (d, 1H, J = 7.7 Hz), 8.41 (d, 1H, J = 7.9 Hz). 13C
NMR (DMSO-d6) d 40.4, 55.3, 114.6, 120.5, 123.9,
124.7, 125.4, 128.4, 128.8, 132.5, 135.0, 143.9, 159.7.
MS m/z = 284 [M+1]+. Anal. Calcd for C15H13N3O3:
C, 63.60; H, 4.63; N, 14.83. Found: C, 63.86; H, 4.41;
N, 15.13.
References and notes
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4.5.8.
4-Methoxy-N-[3-(4-methoxyphenyl)-1-methyl-7-
indazolyl]benzenesulfonamide (28). This compound was
synthesized as described for 6a as a colorless solid in
90% yield. Mp 159–160 ꢁC. 1H NMR (DMSO-d6) d
3.82 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.24 (s, 3H,
CH3), 6.47 (d, 1H, J = 7.3 Hz), 6.97 (dd, 1H, J = 7.3,
8.1 Hz), 7.06 (d, 2H, J = 8.9 Hz), 7.14 (d, 2H,
J = 8.9 Hz), 7.61 (d, 2H, J = 8.7 Hz), 7.83 (d, 2H,
J = 8.5 Hz), 7.89 (d, 1H, J = 8.1 Hz), 9.88 (s, NH). 13C
NMR (DMSO-d6) d 38.6, 55.2, 55.7, 114.3, 114.4,
115.1, 120.0, 120.6, 120.8, 121.2, 123.8, 124.7, 126.1,
128.3, 129.3, 130.5, 137.4, 141.3. MS m/z = 424
[M+1]+. Anal. Calcd for C22H21N3O4S: C, 62.40; H,
5.00; N, 9.92; S, 7.57. Found: C, 62.19; H, 4.82; N,
10.20; S, 7.42.
4.6. Cell culture and cytotoxicity
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4.6.1. Cell lines. The human cell lines, HCT116 colorec-
tal carcinoma, HT29 colorectal adenocarcinoma,
DU145 prostate carcinoma, and the murine L1210 lym-
phocytic leukemia, were obtained from the American
type culture collection (Rockville, MD).
Cells were maintained in RPMI 1640 medium supple-
mented with 10% decomplemented fetal calf serum
(FCS), 2 mM L-glutamine, 100 U/ml penicillin, 100 lg/
ml streptomycin, and 10 mM Hepes, pH 7.4. Cells were
grown at 37 ꢁC in 5% CO2/95% air. All media and sup-
plements were from Life Technologies (Cergy_Pontoise,
France), except for FCS, which was purchased from Sig-
ma Chemical, Co. (St. Louis, MO).
4.6.2. Standard proliferation assay. Cytotoxicity was
measured by the microculture tetrazolium assay as de-
scribed.24 Briefly, adherent cells were seeded in 96-well
microplates and incubated for 2 days. Tested com-
pounds were then added and plates were incubated for
4 doubling times. The nonadherent L1210 cells were
directly incubated for 48 h with the compounds. At
the end of this period, 15 ll of 5 mg/ml 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Sigma) was added to each well and the plates were incu-
bated for 4 h at 37 ꢁC. The medium was aspirated and
the formazan was solubilized by 100 ll DMSO. The
IC50, concentration reducing by 50% the optical density
at 540 nm, was calculated by a linear regression per-
formed on the linear zone of the dose–response curve.
All the measurements were performed in triplicate. For
the cell cycle analysis, (2.5 · 105 cells/ml) were incubated
for 21 h with various concentrations of the compounds.
Cells were then fixed with 70% ethanol (v/v), washed,
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