New selective carbonic anhydrase IX inhibitors: Synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides

Article abstract of DOI:10.1016/j.bmc.2012.10.029

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation

Full text of DOI:10.1016/j.bmc.2012.10.029

Bioorganic & Medicinal Chemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New selective carbonic anhydrase IX inhibitors: Synthesis and
pharmacological evaluation of diarylpyrazole-benzenesulfonamides
Tiphaine Rogez-Florent a,b,c, Samuel Meignan a,d, Catherine Foulon a,c, Perrine Six a,b, Abigaëlle Gros a,d
,
Christine Bal-Mahieu a,d, Claudiu T. Supuran e, Andrea Scozzafava e, Raphaël Frédérick f,
Bernard Masereel f, Patrick Depreux a,b, Amélie Lansiaux a,d, Jean-François Goossens a,c
,
Sébastien Gluszok a,b, Laurence Goossens a,b,
⇑
a Univ. Lille Nord de France, F-59000 Lille, France
b UDSL, EA GRIIOT, ICPAL, F-59000 Lille, France
c UDSL, EA GRIIOT, UFR Pharmacie, F-59000 Lille, France
d INSERM U-837, IRCL, Centre Oscar Lambret, F-59045 Lille, France
e Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Università degli di Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
f Namur Medicine and Drug Innovation Center (NAMEDIC), FUNDP, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic
target since it has been associated with poor prognosis, tumor progression and pH regulation. We report
the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA)
inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study
was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the
active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters
nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyr-
azole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening
has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in
presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an
Keywords:
Carbonic anhydrase
Aryl sulfonamide inhibitors
Pyrazole
Breast cancer
Hypoxia
Doxorubicin
hypoxic environment, expressed in IC50 value, is restored at 20% level with 1 lM CA IX inhibitor.
Ó 2012 Published by Elsevier Ltd.
1. Introduction
and intracellular communication.4 High tumoral carbonic anhy-
drase IX (CA IX) expression has been associated with poor progno-
sis, tumor progression and aggressiveness.5 CA IX is expressed in a
limited number of normal tissues (mainly the gastrointestinal
tract), whereas its overexpression is observed on the cell surface
of a large number of solid tumors, and it is invariably associated
with the hypoxic phenotype, mediated by the transcription factor
hypoxia-inducible factor-1 (HIF-1).6 CA IX has been identified as a
potentially important marker of hypoxia. Furthermore, CA IX
overexpression is often associated with a poor responsiveness to
the classical radio- and chemo-therapies.6,3 Therapeutic inhibition
of CA IX has recently been shown to decrease primary tumor
growth and metastasis in preclinical breast tumor models.5,7 The
overall consequence of CA IX overexpression in tumors is a pH
balance leading to acidification of the extracellular microenviron-
ment of hypoxic tumor (pHe ꢁ 6.8), in contrast to normal tissue
(pHe ꢁ 7.4).8–11 This low pHe is associated with tumorigenic
transformation, chromosomal rearrangements, extracellular ma-
trix breakdown, migration, and invasion.2,5 From a therapeutic
point of view, acidic pHe is also related to a chemoresistance by
Carbonic anhydrases (CAs) are a family of zinc metalloenzymes
that catalyze the rapid reversible hydration of carbon dioxide to
bicarbonate anion and proton (CO2 + H2O M HCO3ꢀ + H+).1 All hu-
man CAs (hCAs) belong to the
a-class and differ widely in their
cellular localizations: CA I, II, III, VII and XIII reside in cytosol,
CA IV, IX, XII and XIV are associated with cell membrane, and
CA VA and CA VB occur in mitochondria, whereas CA VI is secreted
in saliva and milk.2 CA isoenzymes play an important role in
essential cellular processes such as pHe and pHi regulation, secre-
tion of electrolytes, respiration and biosynthetic reactions that re-
ꢀ
quire CO2 and HCO3 as substrates (e.g., lipogenesis, glucogenesis
and ureagenesis).3 CA IX is a dimeric transmembrane glycoprotein
with an extracellular active site and a NH2-terminal proteoglycan-
like region and is almost exclusively associated with tumors. CA
IX is implicated in cell adhesion as well as in acid–base balancing
⇑
Corresponding author. Tel.: +33 (0)3 20 96 47 02; fax: +33 (0)3 20 96 49 06.
E-mail address: laurence.goossens@univ-lille2.fr (L. Goossens).
a
decrease in uptake of weakly basic anticancer drugs as
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.10.029
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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
irinotecan, mitomycin, bleomycin and doxorubicin.12,13 Doxorubi-
cin is one of the most active chemotherapeutic agents for the
treatment of advanced breast cancer and CA IX is described as
being a predictive marker of doxorubicin resistance.14
The acidic extracellular environment can decrease the uptake of
anthracyclins by cells, because these drugs are weak bases, which
ionize at low pH. A recent study demonstrates that CA IX is corre-
lated with worse outcome for early-stage breast cancer patients
treated with doxorubicin.15 Consequently, CA IX has been pro-
posed as a potential therapeutic target.5,7,14 A possible approach
to target CA IX would be via inhibiting its enzymatic activity with
On the other hand a molecular modelisation study was per-
formed on the compound having a 6-MeO-2-naphthyl and a primary
alcohol at position 3 (Fig. 2). This docking show that 6-MeO-2-naph-
thyl fits perfectly into the hydrophobic pocket of CA IX active site,
the sulfonamide interacts with zinc II active site and that hydrogen
bound formed between the primary alcohol and Trp 5. In this study
we designed novel derivatives, 1,5 diarylpyrazole, with different
aromatic groups in 5 position (phenyl, biphenyl, 2-naphthyl, phen-
anthryl. . .), and substituting in the 3 position by donor or acceptor H
bond (primary alcohol) (Fig. 1). Pharmacological evaluations are
carried out using enzymatic activity and cell proliferation studies.
specific pharmacological inhibitors. As all a-CAs, CA IX is inhibited
by several main classes of inhibitors: inorganic anions, sulfona-
mides and their isosteres (sulfamates and sulfamides), phenols,16
coumarins17 and antibodies.18
2. Results and discussion
2.1. Chemistry
Anions and sulfonamides coordinate to the metal ion within the
enzyme cavity in a tetrahedral geometry19 with three anchors. The
best investigated class of inhibitors is the sulfonamide deriva-
tives20 which are characterized by a low nanomolar inhibition con-
stant (Ki). Additional, a selective CA IX inhibition is required for a
cancer therapeutic relevancy without side effects related to the
inhibition of other CA isoforms, especially the ubiquitous CA II en-
zyme. Many low nanomolar sulfonamide CA IX inhibitors have
been identified in the last several years; thus agents, that can inhi-
bit CA IX activity, may have therapeutic value and offer opportuni-
ties for the treatment or prevention of various cancers.
Unfortunately, most of these current inhibitors do not selectively
target CA IX.
The synthetic route to obtain the target derivatives 3a–3o, 3a0
and 3j0 is outlined in Scheme 1. Treatment of suitable commercially
available aryl ketones with sodium ethylate in absolute EtOH, fol-
lowed by addition of diethyl oxalate (or malonate) gave with good
yield (74–82%) the b-diketones 1a–o according to a classical
Claisen procedure. The cyclization of b-diketones 1a–o with
4-aminosulfonylphenylhydrazine hydrochloride, in refluxing EtOH,
gave a mixture of 1,5 and 1,3-diarylpyrazole ethyl esters with a
80:20 ratio in most cases. We were not able to achieve excellent
regioisomeric ratio using modified pH reaction conditions as
described by Singh et al.23 In our case, complex mixtures were
obtained, which complicated the purification. Separation of the
regioisomers was also effected by silica gel chromatography and
followed by a crystallization to afford the pure desired 1,5-diary-
lpyrazole carboxylic ethyl esters 2a–2o. Two 1,3-diaryl compounds
(2a0 and 2j0) were isolated by chromatography (the 1,5 or 1,3-diaryl
structure was established by NOESY experiments). Primary
alcohols 3a–3o, 3a0 and 3j0 were obtained in excellent yields by
reduction of the corresponding ethyl esters 2a–2o, 2a0 and 2j0 with
lithium aluminium hydride in dry THF.
In Scheme 2, the cyclization of b-diketones 1a–1j–1k with 3-
aminosulfonylphenyl hydrazine hydrochloride, in refluxing EtOH,
gave a mixture of 1,5 and 1,3-diarylpyrazole ethyl esters with a
90:10 ratio in most of cases. Separation of the regioisomers was
also performed by crystallization to afford the pure desired 1,5-
diarylpyrazole carboxylic ethyl esters 4a–4j–4k. The confirmation
of the 1,5 structure was performed by NOESY experiments. 1,3-dia-
ryl compounds (4a0–4j0–4k0) were not isolated. Primary alcohols
5a–5j–5k were obtained in excellent yields by reduction of the cor-
responding ethyl esters 4a–4j–4k with lithium aluminium hydride
in dry THF.
It has been shown that celecoxib, a COX-2 sulfonamide contain-
ing compound, was a strong inhibitor of hCA IX (Ki = 16 nM) as well
as hCA II (Ki = 21 nM). Recently, our group described benzylanilino-
sulfonamides as hCA IX inhibitors (Ki = 1.8–27 nM).21 Although
very promising as hCA IX inhibitors, these compounds are not CA
IX selective when compared to the inhibition of the ubiquitous
hCA II (Ki = 17–49 nM). It was therefore proposed to develop aro-
matic molecules that can better fit into the active site of hCA IX
to improve selectivity. Combining the structure of celecoxib and
benzylanilinosulfonamides, series of 1,5-diarylpyroles have been
designed with the aim to obtain selective hCA IX inhibitors. These
compounds have an aromatic ring in position 5 and different
amides in position 3, they show a nanomolar Ki (Ki = 10 nM) but
are not selective for hCA IX versus hCA II (Ki ratio = 5.5).5
According to these results, we proposed a novel design by
changing the heterocycle for a diarylpyrazole structure (Fig. 1).
Various compounds were synthesized to determine structure–
activity relationships. This work has permitted to identify a
pharmacophore with three anchoring points that interact with
the active site of the enzyme. The first is the central heterocycle
ring, as scaffold that can rigidify the structure and so guide
substituent. The second is the aryl sulfonamide that interacts in
the anionic form (SO2NHꢀ) with the zinc, and the last, is a moiety
that will allow hydrogen bound to Trp 5 of the CA IX active site.
Sulfonamides (SO2NH2), as deprotonated state (SO2NHꢀ), bind in
a tetrahedral geometry of the Zn(II) ion: the nitrogen atom of the
sulfonamide moiety coordinated to Zn(II) and an extended net-
work of hydrogen bonds, involving residues Thr199 and Glu106,
also participating to the anchoring of the inhibitor molecule to
the metal ion (Fig. 1).18 The calculated and measured pKa of our
series of compounds (pKa ꢁ 9.3)22 were used to determine the per-
centage of anionic form in relation with the formation of the tetra-
hedral adduct. At physiological pH, our compounds are 99% as
molecular form (SO2NH2) so these values measured in aqueous
solution do not allow to explain the formation of the adduct and
it suppose that the apparent pKa would be acidic shifted in active
site.
The synthetic route to obtain the target derivatives 8a–8j–8k is
outlined in Scheme 3. Aryl ethyl dimethylaminoacrylates 6a–6j–6k
were obtained according to a Claisen condensation, by refluxing
the required ethyl arylacetate and the N,N-dimethylformamide
dimethylacetal. The cyclization of aryl ethyl dimethylaminoacry-
late 6a–6j–6k with 4-aminosulfonylphenyl hydrazine hydrochlo-
ride, in refluxing EtOH, gave compounds 7a–7j–7k. Primary
alcohols 8a–8j–8k were obtained in excellent yields by reduction
of corresponding ethyl esters 7a–7j–7k with lithium aluminium
hydride in dry THF.24
2.2. Carbonic anhydrase inhibition
The inhibitory potency of pyrazole derivatives 3a–o, 3a0, 3j0, 5a–
5j–5k, 8a, 8j, 8k, (Ki, nM) against the three carbonic anhydrase iso-
forms, including physiologically ubiquitous isoforms hCA I, hCA II
and transmembrane tumor-associated isoform hCA IX, was
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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
3
O
OH
CF3
NHR
EtOOC
*
*
N
N
N
NH
O S O
NH2
O S O
NH2
O S O
NH2
celecoxib
1,5-diarylpyrroles
benzylanilinosulfonamide
New 1,5 diaryl pyrazolic derivatives
Interaction with
hydrophobic pocket
Trp 5
H
O
Donor or acceptor
H bond
n = 1 or 2
...
Ar
Tetrahedral adduct
N
N
Rigid scaffold
O
S
Interaction
with Zn2+
HN-
O
His 119
His 94
H
N
His 96
Thr 199
H
O
O-
O
Glu 106
Figure 1. New developed (5-arylpyrazol-1-yl)-benzenesulfonamides from the celecoxib, benzylanilinosulfonamides and 1,5-diarylpyrroles described as hCA inhibitors.
evaluated. The results expressed as Ki (from IC50 values and the
Cheng and Prussof calculations) are summarized in Tables 1 and
2 and reveal significant points. The hCA I/hCAIX and hCA II/hCA
IX Ki ratios are representative of the isozyme selectivity towards
hCA IX. The presence of an aryl group in the position 5 of the pyr-
azole ring, the isomer position, the 1-phenyl substitution by a sul-
fonamide in meta or para were systematically varied. CAs activities
were carried out to determine the compounds inhibitory abilities
to reduce the reaction of hydrogenocarbonate formation and to
gain insight into structure–activity relationships.
naphthyl (3j: Ki = 9.6 nM) and 4-biphenyl (3m: Ki = 8.9 nM). On
the other hand, compounds 3k, 3l and 3n which possess respec-
tively 6-OMe-2-naphthyl, 5-Br-6-OMe-2-naphthyl and 2-phenan-
thryl group are less active inhibitors with Ki values of 264, 388
and 10,232 nM towards hCA II.
As observed in Table 1, important inhibitory activities from 15 to
32 nM were found against hCAIX with compounds 3a–3h, and 3o
possessing a phenyl group at R1. Substituents in para-position of
thephenylringare well toleratedand thebest result is obtainedwith
a
bromine in compound 3d (Ki = 15 nM) compared to 3a
As summarized in Table 1, we observed that all the reported
compounds exhibit a weak inhibitory activity against hCAI with
Ki values from 77 to 19,081 nM, independently of the nature of
the aromatic group R1. These results, in agreement with literature,
show that when compared to AZA, a large number of sulfonamides
containing bulkier scaffolds show a weaker inhibition potential
due to the presence of additional histidine residues (His 200 and
His 67), which delimit a smaller active site in hCA I.24
We observed that most of compounds were considerably more
potent against hCA II than against hCA I (Ki = 8.9–420 nM). On one
hand, a general increase in inhibitory activities against hCA II is ob-
served when the bulkiness of the aromatic group R1 at position 5 of
the pyrazolic ring enhances. Indeed, compounds with a phenyl ring
at R1 such 3a–3h, 3o (Ki = 117–420 nM) are less potent than com-
pounds with bulkier groups such 1-naphthyl (3i: Ki = 46 nM), 2-
(Ki = 27 nM). Among compounds with bulkiest groups, the 2-naph-
thyl (3j–3l), and 4-biphenyl (3m) derivatives, gave the most potent
inhibitors (Ki = 17–30 nM). Moreover, compounds 3i and 3n pos-
sessing 1-naphthyl and 2-phenanthryl group at R1 showed a large
decreaseininhibitionactivityagainsthCAIX(Ki = 254and1857nM).
Most of compounds except 3m are strongly selective towards
hCA IX versus hCA I. Interestingly, the six compounds 3a, 3c, 3d,
3k, 3l and 3o show an important selectivity profile for hCA IX ver-
sus hCA II. Compounds 3a and 3k exert the highest selectivity to-
wards hCA IX versus hCA II (hCA II/hCA IX = 15) and hCA I (hCA
I/hCA IX = 148) and represent the most promising inhibitors. Phar-
macomodulations around these most promising inhibitors (3a, 3j,
3k) were investigated.
The regioisomeric 1,3-diarylpyrazole 3a0 conducts to a loss of
CA IX inhibition (Ki = 157 nM) and a poor selectivity. The regioiso-
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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
OH
N
N
O
SO2NH2
Figure 2. View of the 1-(4-aminosulfonylphenyl)-3-hydroxymethyl-5-(6-methoxynaphtalen-2-yl)-1H-pyrazole (3k), (a) inside the hCA IX active site, (b) inside the hCA II
active site. The zinc ion is represented by a sphere. Pictures made using Pymol.
1,5-diaryl compounds 1,3-diaryl compounds
OEt
R1
3
4
3
n
2
OEt
a
4
O
O
OH
O
b
O
5
5
+
N
N
O
2
R1
CH3
R1
OEt
R1
n
N
N
n
1
1
1a-1n : n = 0
1o : n = 1
O
S
O
O S O
NH2
NH2
2a' : n = 0
2j' : n = 0
2a-2n : n = 0
2o
: n = 1
c
c
R1
3
OH
n
4
4
3
HO
5
5
+
N
N
2
2
R1
N
N
n
1
1
O
S O
O
S O
NH2
NH2
3a' : n = 0
3j' : n = 0
3a-3n : n = 0
3o : n = 1
Scheme 1. aFor R1 see Table 1. bReagents and conditions: (a) (i) EtONa, EtOH, 0 °C, 30 min; (ii) diethyl oxalate or malonate, reflux, 2 h; (b) 4-aminosulfonylphenylhydrazine
hydrochloride, EtOH, reflux, 2 h; (c) LiAlH4, anhydrous THF, 20 °C, 2 h.
meric 1,3-diarylpyrazole 3j0 does not affect the inhibition potential
for hCA II (Ki = 9.1 nM) and hCA IX (Ki = 21 nM) but no selectivity is
observed for hCA IX versus hCA II. Compounds with alcohol group
in position 4 of pyrazole (8a–8j–8k) show good affinity but a de-
crease in selectivity for hCA IX versus hCA II (hCA II/hCA
IX = 0.65). Compounds differing from sulfonamide position in the
phenyl (5a–5j–5k) have an hCA IX activity of same magnitude or-
der with Ki = 17–31 nM, but they are more selective towards hCA
IX versus hCA II, with Ki hCA II/hCA IX = 0.6 (3j) against Ki hCA II/
hCA IX = 26 for 5j.
In our study, the influence of the substitution pattern of pyra-
zole scaffold was examined, as well as the contribution of the sul-
fonamide position on the 1-phenyl. The most promising
compounds show a good affinity for hCA IX (Ki ꢁ 15–30 nM). The
position of primary alcohol group is a crucial factor. In fact, the po-
sition 3 of the alcohol appears to be more favorable for the forma-
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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
1,5-diaryl compounds 1,3-diaryl compounds
5
O
R1
2
OEt
4
3
4
3
a
O
O
OH
b
O
5
5
OEt
2
N
+
N
R1
CH3
R1
R1
N
N
1
1
EtO
O
O
O
1a-1j-1k
S
S
NH2
NH2
O
O
4a'
4a-4j-4k
c
OH
O
3
4
5
2
N
R1
N
1
S
NH2
O
5a-5j-5k
Scheme 2. aFor R1 see Table 1. bReagents and conditions: (a) (i) EtONa, EtOH, 0 °C, 30 min; (ii) diethyl oxalate or malonate, reflux, 2 h; (b) 3-aminosulfonylphenylhydrazine
hydrochloride, EtOH, reflux, 2 h (c) LiAlH4, anhydrous THF, 20 °C, 2 h (for R1 see Table 1).
OH
O
EtO
R1
O
N
R1
O
O
O
O
a
c
b
N
N
EtO
O
N
R1
+
EtO
R1
N
N
S
6a-6j-6k
O
S
O
O
O
NH2
NH2
7a-7j-7k
8a-8j-8k
Scheme 3. Reagents and conditions: (a) reflux, 1 h; (b) 4-aminosulfonylphenylhydrazine hydrochloride, EtOH, reflux, 2 h; (c) LiAlH4, anhydrous THF, 20 °C, 2 h (for R1 see
Table 1).
tion of H-bonded with the Trp 5 NH. The aryl group in position 5 of
pyrazole can be straight but not too bulky (3m) and electronic ef-
fects of its substituents having no influence. The selectivity hCA IX
versus hCA II is one of the main goals of this study. Finally, as ob-
served in the Tables 1 and 2, all the hCA IX affinities are in the same
order range from 20 to 70 nM except for two compounds (3i and
3a0) whereas the variation of hCA II affinities are calculated from
10 to 780 nM. With 3a chosen as reference compound, a greater
hCA II affinity is observed for substituted aryl groups. Interestingly,
the selectivity hCA IX versus hCA II is restored with substituted
naphtyl groups of compounds 3k and 3l. Same conclusions should
be made for compounds with sulfonamide group in meta position
of in the 1-phenyl of pyrazole. In this series, the compound 5j ex-
hibit the greatest selectivity and a Ki value of 19 nM. A molecular
modeling of 5j inserted in the active site of hCA IX is described
in Figure 3.
for a preliminary in vitro study based on the proliferation inhibi-
tion of a breast cancer cell line, the MDA-MB-231 cells in normoxic
or hypoxic conditions.
Anthracyclines, a group of chemotherapeutic agents, known to
target the topoisomerase IIa enzyme, are the most common com-
pounds used worldwide to treat breast cancer. Doxorubicin is the
reference compound used in this study because this weak base is
known to be more ionized in acidic extracellular environment
and a reduced efficacity of the drug is related to the cytosolic mem-
brane uptake decrease.15 The IC50 of doxorubicin has been calcu-
lated from MDA-MB-231 cell proliferation in normoxic and in
hypoxic conditions as reported in the Table 3. A clear reduced effi-
ciency of doxorubicin is observed with a threefold increase of the
IC50 value in normoxic versus in hypoxic conditions (0.12–
0.37 lM).
The selected CA IX inhibitors have been first studied alone in
both culture conditions and all of them revealed weak cytotoxic
2.3. In vitro evaluation in normoxic and hypoxic conditions
activities in both environments (IC50 >50
Table 3, the cytotoxic activity of doxorubicin was evaluated in
the presence of 1 M of CA IX inhibitor, dose for which the com-
pounds present absolutely no cytotoxicity. In the normoxic condi-
lM). As reported in the
The most selective compounds described here as the most po-
tent inhibitors of CA IX, that is, 3d, 3k and 5j, have been selected
l
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6
T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Table 1
Inhibitory potency (Ki, nM), isozyme selectivity ratio data for the pyrazolic derivatives towards isozymes hCA I, hCA II and hCA IX (3a–3o)
OH
OH
N
N
R1
N
S
R1
N
S
O
O
O
O
NH2
NH2
3o
3a-3n
Compounds
R1
Kia (nM)
Ratiob
hCA I
4007
hCA II
hCA IX
27
hCA I/hCA IX
148
hCA II/ hCA IX
15
3a
3o
420
381
351
87
23
13
15
16.6
3.3
3b
43
6.7
3c
3d
3e
3f
1509
19,081
2644
1789
176
117
122
136
22
15
27
20
69
8.0
7.8
4.5
6.8
1272
98
89.5
3g
3h
1710
1168
237
153
26
32
66
9.1
4.8
36.5
3i
5932
46
254
23
0.2
3j
3204
2786
9.6
17
19
188.5
147
0.6
3k
264
13.9
3l
2046
388
30
68
12.9
3m
3n
77
8.9
27
2.9
>5
0.3
>5
>10,000
>10,000
1857
a
Errors in the range 3–5% of reported values from three different assays. (AZA was chosen as the intern control: Ki for hCAI, II and IX are respectively 250, 12, and 25 nM).
The Ki ratios are indicative of isozyme selectivity: a weak selective inhibitor is characterized by a low value ratio.
b
tion, the IC50 values are not significatively differents in the pres-
ence or not of the CA IX inhibitor. However in hypoxic conditions,
for both compounds, 3d and 5j, a significant 20% reduction of the
IC50 value is observed in comparison to that obtained for the doxo-
rubicin alone. For compound 3k, no variation of the doxorubicin
efficiency is observed.
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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
7
Table 2
Inhibitory potency (Ki, nM), isozyme selectivity ratio data for the pyrazolic derivatives towards isozymes hCA I, hCA II and hCA IX (3a0, 3j0–5a, 5j, 5k, 8a, 8b, 8c)
OH
R1
R1
OH
HO
N
N
N
R1
N
N
N
S
O
S
NH2
O
O
S
O
O
O
NH2
NH2
8a-8j-8k
3a'
3j'
5a-5j-5k
Compounds
R1
Kia (nM)
Ratiob
hCA I
hCA II
hCA IX
66
hCA I/ hCA IX
2
hCA II/hCA IX
0.6
8a
8j
134
38
36
2740
49
56
0.7
8k
3a0
3j0
5a
5j
2105
1749
217
31
48
68
11
10
0.65
0.9
0.4
6
142
9.1
157
21
7181
1197
181
490
31
231
63
19
26
5k
2120
782
29
73
27
a
Errors in the range 3–5% of reported values from three different assays. (AZA was chosen as the intern control: Ki for hCAI, II and IX are respectively 250, 12, and 25 nM).
The Ki ratios are indicative of isozyme selectivity: a weak selective inhibitor is characterized by a low value ratio.
b
OH
N
N
SO2NH2
Figure 3. View of the 1-(3-aminosulfonylphenyl)-3-hydroxymethyl-5-(6-methoxynaphtyl)-1H-pyrazole (5j) inside the hCA IX active site. The zinc ion is represented by a
sphere. Pictures made using Pymol.
with the hydrophobic pocket was a phenyl, a 2-naphthyl or a
6-methoxynaphthyl, while hydroxymethyl substituted the
3. Conclusions
a
pyrazole at the position 3. The derivatives 3a, 3d, 3k were the most
potent hCA IX inhibitors with a Ki value of 27, 17 and 19 nM,
respectively (Table 1) and a good selectivity hCA IX versus hCA II.
In this investigation, we designed a series of novel 4-(5-aryl-2-
hydroxymethyl-pyrazole-1-yl)-benzenesulfonamides with the aim
to obtain selective hCA IX inhibitors. The R1-aryl which interacts
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8
T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Table 3
solution (5 mL). The precipitate formed was filtered and washed
with petroleum ether to give 1a as yellow crystal (yield: 62%),
mp 72–74 °C. 1H NMR (CDCl3) d: 15.30 (br s, 1H), 7.96 (m, 2H),
7.58 (m, 1H), 7.47 (m, 2H), 7.06 (s, 1H), 4.35 (q, J = 7.2 Hz, 2H),
1.28 (t, J = 7.2 Hz, 3H). IR (cmꢀ1: neat): 1735, 1600. LC–MS (APCI+)
m/z: 221 (M+H)+.
In vitro cell proliferation of breast cancer MDA-MB-231 cellsa
Doxorubicin IC50
IC50 Doxorubicin without CA IX inhibitor
(lM), in MDA-MB-231 cells in normoxic conditions
0.12 0.02
0.13 0.01
0.10 0.03
0.11 0.02
IC50 Doxorubicin with 3d (1
IC50 Doxorubicin with 3k (1
IC50 Doxorubicin with 5j (1
Doxorubicin IC50
IC50 Doxorubicin without CA IX inhibitor
IC50 Doxorubicin with 3d (1
IC50 Doxorubicin with 3k (1
IC50 Doxorubicin with 5j (1 lM)
l
l
M)
M)
lM)
(lM), in MDA-MB-231 cells in hypoxic conditions
0.37 0.02
0.30 0.01
0.38 0.03
0.29 0.02
4.1.2. 2-Hydroxy-4-oxo-4-pyridin-3-yl-but-2-enoic acid ethyl
ester sodium salt (1b)
l
l
M)
M)
Ethanol (30 mL) was converted to sodium ethoxide by portion
wise addition of sodium (0.92 g, 40 mmol). Then a solution of
diethyl oxalate (5.45 mL; 40 mmol) and 3-acetopyridine (2 mL;
20 mmol) in ethanol was added dropwise at 50 °C. The mixture
was heated at reflux for 3 h. After cooling, the precipitate formed
is filtered and then washed with petroleum ether to give 1b as yel-
low crystal (yield: 78%), mp > 250 °C. 1H NMR (DMSO-d6) d: 8.86
(m, 1H), 8.57 (m, 1H), 8.50 (m, 1H), 8.04 (m, 1H), 6.21 (s, 1/2H),
5.27 (s, 1/2H), 4.07 (m, 2H), 1.22 (m, 3H). IR (cmꢀ1: neat): 1706,
1631. LC–MS (APCI+) m/z: 222 (M+H)+.
a
Results are mean values SD of two independent experiment for n = 6.
The modulation of the position of sulfonamide group in meta of the
1-phenylpyrazole preserved a good affinity for hCA IX and for the
derivatives 5j and 5k increase twofold selectivity hCA IX versus
hCA II. Our compounds present theoretical log P (Molinspiration
Cheminformatics 2012) calculated between 1.8 and 3.9 which sug-
gest that all the compounds cross over the cellular membrane. This
property reinforces the interest to possess selective inhibition of
the hCA IX membrane isoform versus the hCA II cytosolic isoform.
An in vitro antiproliferative screening has been performed on the
breast cancer MDA-MB-231 cell with compounds 3d, 3k and 5j.
4.1.3. 2-Hydroxy-4-oxo-4-p-tolyl-but-2-enoic acid ethyl ester
(1c)
The title compound was prepared from p-methylacetophenone
and diethyl oxalate using the same procedure as for 1a to give 1c as
orange oil (yield: 98%) 1H NMR (DMSO-d6) d: 15.20 (br s, 1H), 7.89
(d, J = 8,2 Hz, 2H), 7.29 (d, J = 9.0 Hz, 2H), 6.99 (s, 1H), 4.39 (q,
J = 7.1 Hz, 2H), 2.43 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). IR (cmꢀ1: neat):
1732, 1607. LC–MS (APCI+) m/z: 235 (M+H)+.
Results have shown that 1 lM 3d or 5j restore 20% level the
cytotoxic efficiency of doxorubicin in a hypoxic environment. The
absence of effect of these compounds in normoxia is consistent
with the fact that CA IX is overexpressed only in hypoxia and
proved their specificity of action in vitro.
4.1.4. 2-Hydroxy-4-oxo-4-p-bromophenyl-but-2-enoic acid
ethyl ester (1d)
The title compound was prepared from p-bromoacetophenone
and diethyl oxalate using the same procedure as for 1a to give
1d as orange oil (yield: 97%). 1H NMR (DMSO-d6) d: 15.40 (br s,
1H), 7.89 (m, 3H), 7.55 (m, 2H), 4.42 (q, J = 7.1 Hz, 2H), 1.47 (t,
J = 7.2 Hz, 3H). IR (cmꢀ1: neat): 1732, 1607. LC–MS (APCI+) m/z:
299 [(M+H)+ for 79Br] and 301 [(M+H) for 81Br].
4. Experimental section
4.1. Chemistry
All commercial reagents and solvents were used without fur-
ther purification. Tetrahydrofuran (THF) was distilled from so-
dium/benzophenone prior to use. All reactions were monitored
by analytical thin-layer chromatography (TLC) on 0.2 mm, Poly-
gram SIL G/UV254 plates (Macherey-Nagel); compounds were
visualized by UV (254 and 366 nm) and/or with iodine. Flash chro-
matography (FC) was performed with silica gel Kieselgel Si 60
0.015–0.040 mm (Macherey-Nagel). Melting points (mp) were
determined with a Büchi 535 capillary melting point apparatus
and remain uncorrected. The structures of each compound were
supported by IR (neat, FT-Brücker Vector 22 instrument) and by
1H NMR at 300 MHz on a Brucker DPX-300 spectrometer. Chemical
shifts (d) are reported in ppm downfield from tetramethylsilane
(TMS), J values are in hertz, and the splitting patterns are designed
as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br s, broad
singlet. The purity of compounds was checked using LC–MS system
Thermo Electron Surveyor MSQ. The mass spectra were operated in
the atmospheric pressure chemical ionization mode (APCI+). Ele-
mental analyses (C, H, N) were performed on a ThermoFinnigan
Flash EA 112 elemental analyser.
4.1.5. 2-Hydroxy-4-oxo-4-p-cyanophenyl-but-2-enoic acid ethyl
ester sodium salt (1e)
The title compound was prepared from 4-cyanoacetophenone
and diethyl oxalate using the same procedure as for 1b to give
1e as beige crystal (yield: 98%), mp 203–205 °C 1H NMR (DMSO-
d6) d: 7.83 (m, 4H), 6.46 (s, 2/3H), 5.30 (s, 1/3H), 4.07 (q,
J = 7.0 Hz, 2H), 1.23 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 2450,
1714, 1626. LC–MS (APCI+) m/z: 246 (M+H)+.
4.1.6. 2-Hydroxy-4-oxo-4-(4-methoxyphenyl)-but-2-enoic acid
ethyl ester (1f)
The title compound was prepared from p-methoxyacetophe-
none and diethyl oxalate using the same procedure as for 1a to give
1f as orange crystal (yield: 63%), mp 57–59 °C 1H NMR (DMSO-d6)
d: 15.2 (br s, 1H), 8.07 (d, J = 9.0 Hz, 2H), 7.10 (d, J = 9.0 Hz, 2H),
7.08 (s, 1H), 4.29 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 1.29 (t,
J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1720. LC–MS (APCI+) m/z: 251
(M+H)+.
4.1.1. 2-Hydroxy-4-oxo-4-phenyl-2-but-2-enoic acid ethyl
ester25 (1a)
4.1.7. 2-Hydroxy-4-oxo-4-(3-methoxyphenyl)-but-2-enoic acid
ethyl ester sodium salt (1g)
Ethanol (200 mL) was converted to sodium ethoxide by portion
wise addition of sodium (6.26 g; 0.27 mol) before a solution of
diethyl oxalate (37 mL; 0.27 mol) and acetophenone (16.36 g;
0.14 mol) in ethanol (100 mL) was added dropwise at 50 °C. The
mixture was heated at reflux for 3 h. After cooling, the precipitate
formed was filtered and then washed with absolute ethanol. The
residue was taken up in water (20 mL) and acidified with 1 N HCl
The title compound was prepared from m-methoxyacetophe-
none and diethyl oxalate using the same procedure as for 1b to
give 1g as yellow crystal (yield: 73%), mp 60–62 °C 1H NMR
(DMSO-d6) d: 7.26 (m, 3H), 6.90 (m, 1H), 6.25 (s, 2/3H), 5.26 (s,
1/3H), 4.01 (m, 2H), 3.76 (s, 3H), 1.26 (t, J = 7.0 Hz, 3H). IR (cmꢀ1
neat): 1690, 1626. LC–MS (APCI+) m/z: 251 (M+H)+.
:
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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
9
4.1.8. 2-Hydroxy-4-oxo-4-(2-methoxyphenyl)-but-2-enoic acid
ethyl ester (1h)
1H), 8.22 (dd, J = 8.8 Hz J = 1.8 Hz, 1H), 7.93 (m, 1H), 7.84 (m,
2H), 7.77 (m, 2H), 7.27 (s, 1H), 4.46 (q, J = 7.2 Hz, 2H), 1.47 (t,
J = 7.2 Hz, 3H). IR (cmꢀ1: neat): 1742, 1633. LC–MS (APCI+) m/z:
321 (M+H)+.
The title compound was prepared from o-methoxyacetophe-
none and diethyl oxalate using the same procedure as for 1a to give
1h as yellow crystal (yield: 79%), mp 79–81 °C 1H NMR (DMSO-d6)
d: 15.30 (br s, 1H), 7.91 (dd, J = 7.9 Hz J = 1.7 Hz, 1H), 7.53 (dt,
J = 7.9 Hz J = 1.7 Hz, 1H), 7.31 (s, 1H), 7.04 (m, 2H), 4.40 (q,
J = 7.1 Hz, 2H), 3.96 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). IR (cmꢀ1: neat):
1741. LC–MS (APCI+) m/z: 251 (M+H)+.
4.1.15. 3-Hydroxy-5-oxo-5-phenyl-pent-3-enoic acid ethyl
ester26 (1o)
A solution of lithium diisopropylamidure was prepared by addi-
tion of diisopropylamine (5 g, 0.05 mol) in n-butyllithium (31 mL,
0.05 mol) at 0 °C. After addition of tetramethylethylenediamine
(2 mL) in ether (50 mL), a mixture of ethyl acetoacetate (2.6 g,
0.02 mol) and ethyl benzoate (3.75 g, 0.025 mol) diluted in ether
was added under an inert atmosphere. After 17 h of reaction, dilute
acetic acid (6 g, 0.1 mol) in ether (75 mL) was added to the reaction
medium before water (50 mL) was added at 0 °C. The ether phase
was dried over MgSO4. After evaporation of the solvent, the residue
was purified by chromatography of silica gel (heptane–ethyl ace-
tate 70:10) to give the yellow oil (yield: 86%). 1H NMR (CDCl3) d:
12.00 (br s, 1H), 7.85 (m, 2H), 7.45 (m, 3H), 6.30 (m, 1H), 4.40
(m, 2H), 3.55 (m, 2H), 1.2 (m, 3H). IR (cmꢀ1: neat): 1739, 1629.
LC–MS (APCI+) m/z: 235 (M+H)+.
4.1.9. 2-Hydroxy-4-oxo-4-(1-naphthyl)-but-2-enoic acid ethyl
ester (1i)
The title compound was prepared from 1-acetylnaphtalene and
diethyl oxalate using the same procedure as for 1a to give 1i as yel-
low crystal (yield: 85%), mp 60–62 °C 1H NMR (DMSO-d6) d: 15.22
(br s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.97 (m,
2H), 7.68 (m, 3H), 7.3 (s, 1H), 4.42 (q, J = 7.3 Hz, 2H), 1.43 (t,
J = 7.3 Hz, 3H). IR (cmꢀ1: neat): 1728, 1610. LC–MS (APCI+) m/z:
271 (M+H)+.
4.1.10. 2-Hydroxy-4-oxo-4-(2-naphthyl)-but-2-enoic acid ethyl
ester (1j)
The title compound was prepared from 2-acetylnaphtalene and
diethyl oxalate using the same procedure as for 1a to give 1j as yel-
low crystal (yield: 74%), mp 54–56 °C 1H NMR (DMSO-d6) d: 15.21
(br s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.97 (m,
2H), 7.68 (m, 3H), 7.3 (s, 1H), 4.42 (q, J = 7.3 Hz, 2H), 1.43 (t,
J = 7.3 Hz, 3H). IR (cmꢀ1: neat): 1728, 1610. LC–MS (APCI+) m/z:
271 (M+H)+.
4.1.16. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-phenyl-
1H-pyrazole (2a) and 1-(4-aminosulfonylphenyl)-5-
ethoxycarbonyl-3-phenyl-1H-pyrazole (2a0)
The diketoester 1a (4 mmol, 0.9 g) was dissolved in ethanol
(25 mL) and a solution of 4-aminosulfonylphenylhydrazine hydro-
chloride (4 mmol, 0.9 g) in ethanol was introduced. The mixture
was refluxed for 4 h. After evaporation under reduced pressure,
the two isomers were separated by chromatography on silica gel
(dichloromethane–ethyl acetate 70:30) to give titles compounds
2a as yellow crystals (yield: 68%) and 2a0 as yellow crystals (yield:
9%).
4.1.11. 2-Hydroxy-4-oxo-4-(6-methoxy-2-naphthyl)-but-2-
enoic acid ethyl ester (1k)
The title compound was prepared from 2-acetyl-6-meth-
oxynaphtalene and diethyl oxalate using the same procedure as
for to 1a to give 1k as yellow crystal (yield: 48%), mp 51–53 °C
1H NMR (DMSO-d6) d: 15.02 (br s, 1H), 8.86 (m, 1H), 7.91 (m,
2H), 7.23 (m, 2H), 7.88 (m, 2H), 4.49 (q, J = 7.2 Hz, 2H), 3.98 (s,
3H), 1.45 (t, J = 7.2 Hz, 3H). IR (cmꢀ1: neat): 1728, 1610. LC–MS
(APCI+) m/z: 301 (M+H)+.
Compound 2a: mp 195–197 °C, 1H NMR (DMSO-d6) d: 7.87 (dd,
J = 8.8 Hz, J = 2.0 Hz, 2H), 7.45 (dd, J = 8.8 Hz, J = 2.0 Hz, 2H), 7.33
(m, 3H), 7.20 (m, 2H), 7.04 (s, 1H), 5.26 (s, 2H), 4.45 (q, J = 7.1 Hz,
2H), 1.43 (t, J = 7.1 H, 3H). IR (cmꢀ1: neat): 1723, 1310, 1150. LC–
MS (APCI+) m/z: 372 (M+H)+.
Compound 2a0: mp 219–221 °C, 1H NMR (DMSO-d6) d: 7.90 (m,
2H), 7.65 (m, 4H), 7.50 (m, 3H), 7.30 (m, 2H), 7.10 (s, 1H), 4.45 (q,
J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1722, 1335,
1164. LC–MS (APCI+) m/z: 372 (M+H)+.
4.1.12. 2-Hydroxy-4-oxo-4-(5-bromo-6-methoxy-2-naphthyl)-
but-2-enoic acid ethyl ester sodium salt (1l)
The title compound was prepared from 2-acetyl-1-bromo-6-
methoxynaphtalene and diethyl oxalate using the same procedure
as for 1b to give 1l as yellow crystal (yield: 97%), mp >250 °C 1H
NMR (DMSO-d6) d: 8.32 (m, 1H), 8.05 (m, 3H), 7.52 (m, 1H), 6.42
(s, 2/3H), 5.46 (s, 1/3H), 4.09 (m, 2H), 3.99 (s, 3H), 1.24 (m, 3H).
IR (cmꢀ1: neat): 1722, 1615. LC–MS (APCI+) m/z: 379 [(M+H)+ for
79Br] and 381 [(M+H) for 81Br].
4.1.17. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(pyridin-3-yl)-1H-pyrazole (2b)
The diketoester 1b (4 mmol, 0.9 g) was dissolved in ethanol
(25 mL), then 4-aminosulfonylphenylhydrazine hydrochloride
(4 mmol, 0.9 g) was introduced. The mixture was refluxed for 4 h.
After cooling to 20 °C the precipitate was filtered under pression
and washed with petroleum ether to give the title compound 2b
as yellow crystals (yield: 89%), mp 137–139 °C. 1H NMR (DMSO-
d6) d: 8.67 (s, 1H), 8.51 (d, J = 4.7 Hz, 1H), 7.93 (s, 1H), 7.80 (d,
J = 8.4 Hz, 1H), 7.58 (d, J = 9.0 Hz, 2H), 7.38 (dd, J = 7.8 Hz J = 4.7 Hz,
1H), 7.18 (d, J = 8.7 Hz, 2H), 7.10 (s, 2H), 4.31 (q, J = 7.0 Hz, 2H),
1.29 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1702, 1310, 1151. LC–MS
(APCI+) m/z: 373 (M+H)+.
4.1.13. 2-Hydroxy-4-oxo-4-(4-biphenylyl)-but-2-enoic acid
ethyl ester (1m)
The title compound was prepared from 4-acetyl-biphenyl and
diethyl oxalate using the same procedure as for 1a to give 1m as
yellow crystal (yield: 78%), mp 109–111 °C 1H NMR (DMSO-d6) d:
15.40 (br s, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H),
7.64 (m, 2H), 7.55 (m, 3H), 7.15 (s, 1H), 4.46 (q, J = 7.2 Hz, 2H),
1.47 (t, J = 7.2 Hz, 3H). IR (cmꢀ1: neat): 1734, 1604. LC–MS (APCI+)
m/z: 297 (M+H)+.
4.1.18. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-p-tolyl-
1H-pyrazole (2c)
The title compound was prepared from diketoester 1c and
4-aminosulfonylphenyl hydrazine hydrochloride using the same
procedure as for 2b to give 2c as white crystal (yield: 78%), mp
224–226 °C. 1H NMR (DMSO-d6) d: 7.86 (d, J = 8.2 Hz, 2H), 7.51
(d, J = 8.2 Hz, 2H), 7.50 (m, 3H), 7.18 (m, 4H), 4.34 (q, J = 7.3 Hz,
2H), 2.31 (s, 3H), 1.29 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1714,
1330, 1159. LC–MS (APCI+) m/z: 386 (M+H)+.
4.1.14. 2-Hydroxy-4-oxo-4-(2-phenantryl)-but-2-enoic acid
ethyl ester (1n)
The title compound was prepared from 2-acetyl-phenanthrene
and diethyl oxalate using the same procedure as for 1a to give
1n as yellow crystal (yield: 82%). 1H NMR (DMSO-d6) d: 15.40 (br
s, 1H), 8.77 (d, J = 8.8 Hz, 1H), 8.70 (m, 1H), 8.55 (d, J = 1.8 Hz,
Please cite this article in press as: Rogez-Florent, T.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.029

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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
4.1.19. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(4-bromophenyl)-1H-pyrazole (2d)
cedure as for 2a to give 2j as yellow crystal (yield: 77%) and 2j0 as
white crystal (yield: 8%).
The title compound was prepared from diketoester 1d and
4-aminosulfonylphenyl hydrazine hydrochloride using the same
procedure as for 2b to give 2d as white crystal (yield: 60%), mp
199–201 °C. 1H NMR (DMSO-d6) d: 7.88 (d, J = 6.8 Hz, 2H), 7.63
(d, J = 8.7 Hz, 2H), 7.52 (m, 4H), 7.26 (d, J = 8.7 Hz, 2H), 7.22 (s,
1H), 4.35 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). IR (cmꢀ1: neat):
1725, 1327, 1158. LC–MS (APCI+) m/z: 450 [(M+H)+ for 79Br] and
452 [(M+H)+ 81Br].
Compound 2j: mp 204–206 °C 1H NMR (DMSO-d6) d: 8.04 (s,
1H), 7.83 (m, 5H), 7.50 (m, 6H), 7.29 (m, 2H), 4.38 (q, J = 7.0 Hz,
2H), 1.35 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1714, 1338, 1169.
LC–MS (APCI+) m/z: 422 (M+H)+.
Compound 2j0: mp 198–201 °C 1H NMR (DMSO-d6) d: 8.00 (s,
1H), 7.58 (m, 5H), 7.53 (m, 5H), 7.30 (m, 3H), 4.48 (q, J = 7.0 Hz,
2H), 1.54 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1722, 1335, 1166.
LC–MS (APCI+) m/z: 422 (M+H)+.
4.1.20. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-p-
cyanophenyl-1H-pyrazole (2e)
4.1.26. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(6-methoxy-2-naphthyl)-1H-pyrazole (2k)
The title compound was prepared from diketoester 1e and
4-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2b to give 2e as white crystal (yield: 77%), mp
226–228 °C. 1H NMR (DMSO-d6) d: 7.88 (m, 4H), 7.50 (m, 6H),
7.22 (s, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). IR
(cmꢀ1: neat): 1712, 1335, 1155. LC–MS (APCI+) m/z: 397 (M+H)+.
The title compound was prepared from diketoester (1k) and
4-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2b to give 2k as white crystal (yield: 79%), mp
99–101 °C. 1H NMR (DMSO-d6) d: 7.71 (m, 2H), 7.66 (m, 3H),
7.54 (m, 2H), 7.19 (m, 2H), 7.12 (m, 4H), 4.47 (q, J = 7.0 Hz, 2H),
3.94 (s, 3H), 1.42 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1726, 1342,
1164. LC–MS (APCI+) m/z: 452 (M+H)+.
4.1.21. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(4-methoxyphenyl)-1H-pyrazole (2f)
4.1.27. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(5-bromo-6-methoxy-2-naphthyl)-1H-pyrazole (2l)
The title compound was prepared from diketoester 1f and
4-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2b to give 2f as white crystal (yield: 74%), mp
205–207 °C. 1H NMR (DMSO-d6) d: 7.86 (d, J = 8.4 Hz, 2H), 7.52
(d, J = 8.4 Hz, 2H), 7.51 (s, 2H), 7.22 (d, J = 8.7 Hz, 2H), 7.07 (s,
1H), 6.95 (d, J = 8.7 Hz, 2H), 4.33 (q, J = 7.0 Hz, 2H), 3.76 (s, 3H),
1.31 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1711, 1339, 1161. LC–MS
(APCI+) m/z: 402 (M+H)+.
The title compound was prepared from diketoester 1l and
4-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2b to give 2l as white crystal (yield: 92%), mp
245–247 °C. 1H NMR (DMSO-d6) d: 8.02 (m, 3H), 7.86 (d,
J = 8.6 Hz, 2H), 7.58 (m, 3H), 7.51 (s, 2H), 7.38 (d, J = 8.6 Hz, 1H),
7.28 (s, 1H), 4.37 (q, J = 7.1 Hz, 2H), 3.34 (s, 3H), 1.34 (t,
J = 7.1 Hz, 3H). IR (cmꢀ1: neat): 1720, 1342, 1165. LC–MS (APCI+)
m/z: 530 [(M+H)+ 79Br] and 532 [(M+H)+ 81Br].
4.1.22. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(3-methoxyphenyl)-1H-pyrazole (2g)
4.1.28. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(4-biphenylyl)-1H-pyrazole (2m)
The title compound was prepared from diketoester 1g and
4-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2b to give 2g as beige crystal (yield: 74%), mp
185–187 °C. 1H NMR (DMSO-d6) d: 7.87 (d, J = 8.4 Hz, 2H), 7.54
(s, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.28 (m, 1H), 6.98 (s, 1H), 6.94 (d,
J = 6.4 Hz, 1H), 6.88 (s, 1H), 6.78 (d, J = 7.8 Hz, 1H), 4.35 (q,
J = 7.1 Hz, 2H), 3.68 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). IR (cmꢀ1: neat):
1712, 1356, 1169. LC–MS (APCI+) m/z: 402 (M+H)+.
The title compound was prepared from diketoester 1m and 4-
aminosulfonyphenylhydrazine hydrochloride using the same pro-
cedure as for 2b to give 2m as white crystal (yield: 60%), mp
214–216 °C. 1H NMR (DMSO-d6) d: 7.92 (d, J = 8.8 Hz, 2H), 7.78
(m, 4H), 7.57 (d, J = 8.8 Hz, 2H), 7.53 (s, 2H), 7.44 (m, 2H), 7.36
(m, 3H), 7.22 (s, 1H), 4.38 (q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.0 Hz,
3H). IR (cmꢀ1: neat): 1717, 1327, 1160. LC–MS (APCI+) m/z: 448
(M+H)+.
4.1.23. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(2-methoxyphenyl)-1H-pyrazole (2h)
4.1.29. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(3-phenantryl)-1H-pyrazole (2n)
The title compound was prepared from diketoester 1h and
4-aminosulfonylphenylhydrazine hydrochloride using the same pro-
cedure as for 2b to give 2h as yellow crystal (yield: 65%), mp 157–
159 °C. 1H NMR (DMSO-d6) d: 7.79 (d, J = 8.4 Hz, 2H), 7.40 (m, 6H),
7.02 (m, 3H), 4.33 (q, J = 7.0 Hz, 2H), 3.32 (s, 3H), 1.31 (t, J = 7.0 Hz,
3H). IR (cmꢀ1: neat): 1699, 1349. LC–MS (APCI+) m/z: 402 (M+H)+.
The title compound was prepared from diketoester 1n and 4-
aminosulfonylphenylhydrazine hydrochloride using the same pro-
cedure as for 2b to give 2n as white crystal (yield: 36%), mp
>250 °C. 1H NMR (DMSO-d6) d: 8.12 (d, J = 1.8 Hz, 1H), 8.04 (m.
1H), 7.95 (m, 4H), 7.75 (m, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.60 (d,
J = 8.8 Hz, 2H), 7.52 (s, 2H), 7.44 (dd, J = 7.0 Hz, J = 1.8 Hz, 1H),
7.34 (s, 1H), 4.37 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). IR
(cmꢀ1: neat): 1714, 1338, 1169. LC–MS (APCI+) m/z: 472 (M+H)+.
4.1.24. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(1-naphthyl)-1H-pyrazole (2i)
The title compound was prepared from diketoester 1i and
4-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2b to give 2i as yellow crystal (yield: 45%), mp
103–105 °C. 1H NMR (DMSO-d6) d: 7.97 (m, 2H), 7.64 (d,
J = 8.5 Hz, 2H), 7.38 (m, 9H), 7.19 (s, 1H), 4.38 (q, J = 7.0 Hz, 2H),
1.34 (t, J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1719, 1340, 1164. LC–MS
(APCI+) m/z: 422 (M+H)+.
4.1.30. 1-(4-Aminosulfonylphenyl)-3-ethylacetate-5-phenyl-1H-
pyrazole (2o)
The diketoester 1o (0.11 g, 0.49 mmol) was dissolved in etha-
nol (15 mL) and a solution of 4-aminosulfonylphenylhydrazine
hydrochloride (0.1 g, 0.49 mmol) in ethanol was introduced. The
mixture was refluxed for 3 h. After cooling, the reaction mixture
was concentrated under reduced pressure and the residue was ta-
ken up into ethylacetate and washed with water (20 mL), and
brine (2 ꢂ 20 mL). The organic layer was dried over MgSO4 and
then concentrated under reduced pressure. The residue was puri-
fied by chromatography of silica gel (dichloromethane–ethyl ace-
4.1.25. 1-(4-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(2-naphthyl)-1H-pyrazole (2j) and 1-(4-aminosulfonylphenyl)-
5-ethoxycarbonyl-3-(2-naphthyl)-1H-pyrazole (2j0)
Title compounds were prepared from diketoester 1j and 4-
aminosulfonylphenylhydrazine hydrochloride using the same pro-
tate 70:30) to give
a yellow crystal (25%), mp 233–235 °C.
Please cite this article in press as: Rogez-Florent, T.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.029

T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
11
1H NMR (DMSO-d6) d: 7.85 (d, J = 8.7 Hz, 2H), 7.46–7.23 (m, 9H),
6.55 (s, 1H), 4.24 (q, J = 7,3 Hz, 2H), 3.8 (s, 2H), 1.32 (t, J = 7,3 Hz,
3H). IR (cmꢀ1: neat): 1736, 1343, 1164. LC–MS (APCI+) m/z: 386
(M+H)+.
4.1.36. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-p-
cyanophenyl-1H-pyrazole (3e)
The title compound was prepared from ester 2e according using
the same procedure as for 3a to give 3e as white crystal (yield:
10%), mp 201–203 °C. 1H NMR (DMSO-d6) d: 7.90 (d, J = 8.5 Hz,
2H), 7.80 (d, J = 8.5 Hz, 2H), 7.45 (s, 2H), 7.40 (m, 4H), 6.80 (s,
1H), 5.30 (t, J = 5.8 Hz, 1H), 4.50 (d, J = 5.8 Hz, 2H). IR (cmꢀ1: neat):
2235, 1344, 1165. LC–MS (APCI+) m/z: 355 (M+H)+. Anal. Calcd for
4.1.31. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-phenyl-
1H-pyrazole (3a)
The ethyl ester (4.8 mmol, 1.4 g) was dissolved in tetrahydrofu-
ran (20 mL) and then a suspension of LiAlH4 (9.6 mmol, 0.37 g) in
tetrahydrofuran (5 mL) was slowly added. After 3 h of reaction at
20 °C, an aqueous solution of HCl 1 N was added dropwise until a
neutral pH. The reaction mixture was concentrated under reduced
pressure the residue was taken into ethyl acetate and washed with
water and brine. The organic layer was dried over MgSO4 and con-
centrated under reduced pressure. The residue was recrystallized
in acetonitrile. The alcohol (3a) give a white crystal (yield: 84%),
mp 186–188 °C 1H NMR (DMSO-d6) d: 7.79 (d, J = 8.4 Hz, 2H),
7.45 (m, 7H), 7.25 (m, 2H), 6.64 (s, 1H), 5.27 (t, J = 5.8 Hz, 1H),
4.76 (s, 2H). IR (cmꢀ1: neat): 3297, 1593. LC–MS (APCI+) m/z: 330
(M+H)+. Anal. Calcd for C16H15N3O3S: C, 58.35; H, 4.59; N, 12.76.
Found: C, 58.42; H, 4.68; N, 12.86.
C17H14N4O3S: C, 57.62; H, 3.98; N, 15.81. Found: C, 57.69; H, 4.31;
N, 15.98.
4.1.37. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(4-methoxyphenyl)-1H-pyrazole (3f)
The title compound was prepared from ester 2f using the same
procedure as for 3a to give 3f as white crystal (yield: 32%), mp
114–116 °C. 1H NMR (DMSO-d6) d: 7.80 (d, J = 8.7 Hz, 2H), 7.44
(s, 2H), 7.41 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 8.7 Hz, 2H), 6.96 (d,
J = 8.7 Hz, 2H), 6.55 (s, 1H), 5.22 (t, J = 5.8 Hz, 1H), 4.50 (d,
J = 5.8 Hz, 2H), 3.76 (s, 3H). IR (cmꢀꢀ1: neat): 1336, 1161. LC–MS
(APCI+) m/z: 360 (M+H)+. Anal. Calcd for C17H17N3O4S: C, 56.81;
H, 4.77; N, 11.69. Found: C, 56.98; H, 4.63; N, 11.78.
4.1.32. 1-(4-Aminosulfonylphenyl)-5-hydroxymethyl-3-phenyl-
1H-pyrazole (3a0)
4.1.38. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(3-methoxyphenyl)-1H-pyrazole (3g)
The title compound was prepared from ester 2a0 using the same
procedure as for 3a to give 3a0 as yellow crystal (yield: 64%), mp
200–202 °C. 1H NMR (DMSO-d6) d: 7.8 (d, J = 8.7 Hz, 2H), 7.44 (m,
7H), 7.24 (m, 2H), 6.64 (s, 1H), 5.27 (t, J = 5.8 Hz, 1H), 4.53 (d,
J = 5.8 Hz, 2H). IR (cmꢀ1: neat): 3336, 1336, 1167. LC–MS (APCI+)
m/z: 330 (M+H)+Å Anal. Calcd for C16H15N3O3S: C, 58.35; H, 4.59;
N, 12.76. Found: C, 58.32; H, 4.74; N, 12.90.
The title compound was prepared from ester 2g using the same
procedure as for 3a to give 3g as white crystal (yield: 59%), mp
177–179 °C 1H NMR (DMSO-d6) d: 7.81 (d, J = 8.7 Hz, 2H), 7.46 (s,
2H), 7.42 (d, J = 8.7 Hz, 2H), 7.30 (t, J = 7.9 Hz, 1H), 6.97 (d,
J = 8.1 Hz, 1H), 6.83 (s, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.66 (s, 1H),
5.27 (t, J = 5.7 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H), 3.69 (s, 3H). IR
(cmꢀ1: neat): 1164. LC–MS (APCI+) m/z: 360 (M+H)+. Anal. Calcd
for C17H17N3O4S: C, 56.81; H, 4.77; N, 11.69. Found: C, 56.68; H,
4.87; N, 11.83.
4.1.33. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
pyridinyl-1H-pyrazole (3b)
The title compound was prepared from ester 2b using the same
procedure as for 3a to give 3b as yellow crystal (yield: 57%), mp
154–156 °C 1H NMR (DMSO-d6) d: 8.55 (s, 1H), 8.46 (s, 1H), 7.82
(d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.45 (m, 5H), 6.77 (s,
1H), 5.29 (t, J = 5.8 Hz, 1H), 4.54 (d, J = 5.8 Hz, 2H). IR (cmꢀ1: neat):
1333, 1158. LC–MS (APCI+) m/z: 331 (M+H)+. Anal. Calcd for
C15H14N4O3S: C, 54.54; H, 4.27; N, 16.96. Found: C, 54.45; H,
4.45; N, 16.90.
4.1.39. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(2-methoxyphenyl)-1H-pyrazole (3h)
The title compound was prepared from ester 2h using the same
procedure as for 3a to give 3h as white crystal (yield: 82%), mp
201–203 °C. 1H NMR (DMSO-d6) d: 7.72 (d, J = 8.7 Hz, 2H), 7.41 (t,
J = 7.9 Hz, 2H), 7.29 (m, 4H), 7.03 (t, J = 7.3 Hz, 1H), 6.97 (d,
J = 8.4 Hz, 1H), 6.49 (s, 1H), 5.22 (t, J = 5.8 Hz, 1H), 4.51 (d,
J = 5.8 Hz, 2H), 3.33 (s, 3H). IR (cmꢀ1: neat): 1327, 1162. LC–MS
(APCI+) m/z: 343 (M+H)+. Anal. Calcd for C17H17N3O4S: C, 56.81;
H, 4.77; N, 11.69. Found: C, 56.67; H, 4.78; N, 11.54.
4.1.34. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-p-tolyl-
1H-pyrazole (3c)
The title compound was prepared from ester 2c using the
same procedure as for 3a to give 3c as white crystal (yield:
73%), mp 166–168 °C. 1H NMR (DMSO-d6) d: 7.80 (d, J = 8.7 Hz,
2H), 7.44 (m, 3H), 7.39 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.0 Hz,
1H), 7.13 (d, J = 8.0 Hz, 2H), 6.58 (s, 1H), 5.24 (t, J = 5.8 Hz, 1H),
4.51 (d, J = 5.8 Hz, 2H) 2.31 (s, 3H). IR (cmꢀ1: neat): 1327,
1162. LC–MS (APCI+) m/z: 344 (M+H)+. Anal. Calcd for
4.1.40. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(1-naphthyl)-1H-pyrazole (3i)
The title compound was prepared from ester 2i using the same
procedure as for 3a to give 3i as yellow crystal (yield: 96%), mp
190–192 °C. 1H NMR (DMSO-d6) d: 8.82 (m, 2H), 8.05 (d,
J = 1.4 Hz, 2H), 8.03 (m, 1H), 7.91 (m, 4H), 7.73 (m, 2H), 6.49 (m,
4H), 6.80 (s, 1H), 5.32 (t, J = 5.7 Hz, 1H). IR (cmꢀ1: neat): 3320,
1339, 1160. LC–MS (APCI+) m/z: 380 (M+H)+. Anal. Calcd for
C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24. Found: C, 59.69; H,
5.08; N, 12.58.
C20H17N3O3S: C, 63.31; H, 4.52; N, 11.07. Found: C, 63.65; H,
4.59; N, 11.48.
4.1.35. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-p-
bromophenyl-1H-pyrazole (3d)
4.1.41. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(2-naphthyl)-1H-pyrazole (3j)
The title compound was prepared from ester 2d using the same
procedure as for 3a to give 3d as white crystal (yield: 62%), mp
203–205 °C 1H NMR (DMSO-d6) d: 7.81 (d, J = 6.8 Hz, 2H), 7.62 (d,
J = 8.7 Hz, 2H), 7.44 (m, 4H), 7.22 (d, J = 8.7 Hz, 2H), 6.67 (s, 1H),
5.27 (t, J = 6.2 Hz, 1H), 4.53 (d, J = 6.2 Hz, 2H). IR (cmꢀ1: neat):
1396, 1159. LC–MS (APCI+) m/z: 408 [(M+H)+ 79Br] and 410
[(M+H)+ 81Br]. Anal. Calcd for C16H14BrN3O3S: C,47.07; H, 3.46; N,
10.29. Found: C, 47.40; H, 3.52; N, 10.34.
The title compound was prepared from ester 2j using the same
procedure as for 3a to give 3j as yellow crystal (yield: 23%), mp
226–228 °C. 1H NMR (DMSO-d6) d: 8.40 (s, 1H), 7.90 (m, 8H),
7.60 (m, 4H), 7.20 (s, 1H), 5.80 (t, J = 5.0 Hz, 1H), 4.80 (d,
J = 6.3 Hz, 2H). IR (cmꢀ1: neat): 3336, 1336, 1167. LC–MS (APCI+)
m/z: 380 (M+H)+. Anal. Calcd for C20H17N3O3S: C, 63.31; H, 4.52;
N, 11.07. Found: C, 63.45; H, 4.65; N, 11.28.
Please cite this article in press as: Rogez-Florent, T.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.029

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T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
4.1.42. 1-(4-Aminosulfonylphenyl)-5-hydroxymethyl-3-(2-
naphthyl)-1H-pyrazole (3j0)
4.1.48. 1-(3-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-phenyl-
1H-pyrazole (4a)
The title compound was prepared from ester 2j0 using the same
procedure as for 3a to give 3j0 as white crystal (yield: 23%), mp
150–152 °C. 1H NMR (DMSO-d6) d: 8.40 (s, 1H), 8.10 (m, 8H),
7.60 (m, 4H), 7.20 (s, 1H), 5.80 (t, J = 5.0 Hz, 1H), 4.80 (d,
J = 6.3 Hz, 2H). IR (cmꢀ1: neat): 3336, 1336, 1167. LC–MSLC–MS
(APCI+) m/z: 380 (M+H)+. Anal. Calcd for C20H17N3O3S: C, 63.31;
H, 4.52; N, 11.07. Found: C, 63.23; H, 4.48; N, 11.38.
The title compound was prepared from diketoester 1a and
3-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2a to give 4a as yellow crystal (yield: 62%), mp
187–189 °C. 1H NMR (DMSO-d6) d: 7.92 (m, 1H), 7.67 (m, 3H),
7.50 (s, 2H), 7.24 (m, 2H), 7.17 (m, 2H), 7.08 (m, 1H), 6.29 (s,
1H), 3.12 (q, J = 7.3 Hz, 2H), 1.26 (t, J = 7.3 Hz, 3H). IR (cmꢀ1: neat):
1714, 1330, 1160. LC–MS (APCI+) m/z: 372 (M+H)+.
4.1.49. 1-(3-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
naphthyl-1H-pyrazole (4j)
4.1.43. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(6-methoxynaphtalen-2-yl)-1H-pyrazole (3k)
The title compound was prepared from diketoester 1j and 3-
aminosulfonylphenylhydrazine hydrochloride using the same pro-
cedure as for 2a to give 4j as yellow crystal yellow crystal (yield:
59%), mp 197–199 °C. 1H NMR (DMSO-d6) d: 7.99 (m, 3H), 7.85
(m, 3H), 7.59 (m, 1H), 7.43 (m, 3H), 7.37 (m, 1H), 7.30 (s, 2H),
6.68 (s, 1H), 4.23 (q, J = 9.0 Hz, 2H), 1.84 (t, J = 9.0 Hz, 3H). IR
(cmꢀ1: neat): 1718, 1280, 1164. LC–MS (APCI+) m/z: 422 (M+H)+.
The title compound was prepared from ester 2k using the same
procedure as for 3a to give 3k as white crystal (yield: 48%), mp
191–193 °C. 1H NMR (DMSO-d6) d: 8.02 (m, 4H), 7.82 (m, 2H),
7.65 (m, 1H), 7.41 (m, 2H), 7.37 (s, 2H), 7.34 (m, 1H), 6.74 (s,
1H), 5.29 (t, J = 5.8 Hz, 1H), 4.59 (d, J = 5.8 Hz, 2H), 4.12 (s, 3H). IR
(cmꢀ1: neat): 1327, 1155. LC–MS (APCI+) m/z: 410 (M+H)+. Anal.
Calcd for C21H19N3O4S: C, 61.60; H, 4.68; N, 10.26. Found: C,
61.65; H, 4.37; N, 10.29.
4.1.50. 1-(3-Aminosulfonylphenyl)-3-ethoxycarbonyl-5-
(6-methoxy-2-naphtalenyl)-1H-pyrazole (4k)
The title compound was prepared from diketoester 1k and
3-aminosulfonylphenylhydrazine hydrochloride using the same
procedure as for 2a to give 4k as yellow crystal (yield: 53%), mp
204–206 °C. 1H NMR (DMSO-d6) d: 7.92 (m, 1H), 7.90 (m, 1H),
7.80 (m, 1H), 7.77 (m, 1H), 7.54 (m, 2H), 7.32 (m, 2H), 7.21 (s,
2H), 7.18 (m, 2H), 6.74 (s, 1H), 4,37 (q, J = 9.1 Hz, 2H), 3.94 (s,
3H), 1.33 (t, J = 9.1 Hz, 3H). IR (cmꢀ1: neat): 1754, 1287, 1162.
LC–MS (APCI+) m/z: 452 (M+H)+.
4.1.44. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(5-bromo-6-methoxynaphtalen-2yl)-1H-pyrazole (3l)
The title compound was prepared from ester 2l using the same
procedure as for 3a to give 3l as white crystal (yield: 61%), mp
>250 °C. 1H NMR (DMSO-d6) d: 7.99 (m, 3H), 7.78 (d, J = 8.7 Hz,
2H), 7.57 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.41 (s, 2H),
7.34 (d, J = 8.7 Hz, 1H), 6.74 (s, 1H), 5.29 (t, J = 5.8 Hz, 1H), 4.55
(d, J = 5.8 Hz, 2H), 4.00 (s, 3H). IR (cmꢀ1: neat): 1342, 1165. LC–
MS (APCI+) m/z: 488 [(M+H)+ 79Br] and 490 [(M+H)+ 81Br)]. Anal.
Calcd for C21H18BrN3O4S: C, 51.65; H, 3.72; N, 16.36. Found: C,
51.43; H, 3.87; N, 16.67.
4.1.51. 1-(3-Aminosulfonylphenyl)-3-hydroxymethyl-5-phenyl-
1H-pyrazole (5a)
The ethyl ester (0.53 mmol, 200 mg) was dissolved in tetrahy-
drofuran (10 mL) and then a suspension of LiAlH4 (2.70 mmol,
102 mg) in tetrahydrofuran (5 mL) was slowly added. After 4 h of
reaction at 20 °C, an aqueous solution of HCl 1 N was added drop-
wise until a neutral pH. The reaction mixture was concentrated un-
der reduced pressure and the residue was taken up into ethyl
acetate and washed with water and brine. The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The
residue was recrystallized in acetonitrile to give 5a as white crystal
(yield: 59%), mp 198–200 °C. 1H NMR (DMSO-d6) d: 7.89 (m, 1H),
7.74 (m, 1H), 7.58 (m, 2H), 7.51 (m, 1H), 7.47 (s, 2H), 7.38 (m,
2H), 7.23 (m, 2H), 6.63 (s, 1H), 5.19 (t, J = 9.1 Hz, 1H), 4.52 (d,
J = 9.1 Hz, 2H). IR (cmꢀ1: neat): 1289, 1164. LC–MS (APCI+) m/z:
330 (M+H)+. Anal. Calcd for C16H15N3O3S: C, 58.35; H, 4.59; N,
12.76. Found: C, 58.23; H, 4.75; N, 12.98.
4.1.45. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
biphenylyl-1H-pyrazole (3m)
The title compound was prepared from ester 2m using the same
procedure as for 3a to give 3m as white crystal (yield: 86%), mp
224–226 °C. 1H NMR (DMSO-d6) d: 7.85 (d, J = 8.4 Hz, 2H), 7.75
(m, 4H), 7.52 (m, 6H), 7.42 (m, 3H), 6.70 (s, 1H), 5.30 (m, 1H),
4.54 (m, 2H). IR (cmꢀ1: neat): 3320, 1339, 1160. LC–MS (APCI+)
m/z: 406 (M+H)+. Anal. Calcd for C22H19N3O3S: C, 65.17; H, 4.72;
N, 10.36. Found: C, 65.45; H, 4.65; N, 10.40.
4.1.46. 1-(4-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(2-phenantryl)-1H-pyrazole (3n)
The title compound was prepared from ester 2n using the same
procedure as for 3a to give 3n as white crystal (yield: 85%), mp
>250 °C. 1H NMR (DMSO-d6) d: 8.82 (m, 2H), 8.05 (d, J = 1.4 Hz,
1H), 8.03 (m, 1H), 7.91 (m, 4H), 7.73 (m, 2H), 6.49 (m, 5H), 6.80
4.1.52. 1-(3-Aminosulfonylphenyl)-3-hydroxymethyl-5-
naphthyl-1H-pyrazole (5j)
(s, 1H), 5.32 (t, J = 5.7 Hz, 1H), 5.30 (d, J = 5.7 Hz, 2H). IR (cmꢀ1
:
The title compound was prepared from ester 4j using the same
procedure as for 5a to give 5j as white crystal (yield: 67%), mp
175–177 °C. 1H NMR (DMSO-d6) d: 7.95 (m, 3H), 7.89 (m, 3H),
7.66 (m, 1H), 7.55 (m, 3H), 7.49 (m, 1H), 7.10 (s, 2H), 6.75 (s,
1H), 5.30 (t, J = 7.1 Hz, 1H), 4.57 (d, J = 7.1 Hz, 2H). IR (cmꢀ1: neat):
1280, 1164. LC–MS (APCI+) m/z: 380 (M+H)+. Anal. Calcd for
neat): 3312, 1335, 1158. LC–MS (APCI+) m/z: 430 (M+H)+. Anal.
Calcd for C24H19N3O3S: C, 67.12; H, 4.46; N, 9.78. Found: C,
67.36; H, 4.56; N, 9.67.
4.1.47. 1-(4-Aminosulfonylphenyl)-3-hydroxyethyl-5-phenyl-
1H-pyrazole (3o)
C
20H17N3O3S: C, 63.31; H, 4.52; N, 11.07. Found: C, 63.23; H,
4.42; N, 11.01.
The title compound was prepared from ester 2o using the same
procedure as for 3a to give 3o as white crystal (yield: 85%), mp
196–198 °C. 1H NMR (DMSO-d6) d: 7.80 (m, 2H), 7.42 (m, 7H),
7.25 (m, 2H), 6.56 (s, 1H), 4.74 (br s, 1H), 3.74 (m, 2H), 2.81 (m,
2H). IR (cmꢀ1: neat): 1736, 1343, 1164. LC–MS (APCI+) m/z: 344
(M+H)+. Anal. Calcd for C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24.
Found: C, 59.34; H, 4.76; N, 12.60.
4.1.53. 1-(3-Aminosulfonylphenyl)-3-hydroxymethyl-5-
(6-methoxy-2-naphthalenyl)-1H-pyrazole (5k)
The title compound was prepared from ester 4k using the same
procedure as for 5a to give 5k as white crystal (yield: 56%), mp
182–184 °C. 1H NMR (DMSO-d6) d: 7.86 (m, 1H), 7.75 (m, 1H),
7.61 (s, 2H), 7.58 (m, 1H), 7.47 (m, 3H), 7.32 (m, 1H), 7.17 (m,
Please cite this article in press as: Rogez-Florent, T.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.029

T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
13
3H), 6.71 (s, 1H), 5.28 (t, J = 9.0 Hz, 1H), 4.59 (d, J = 7.2 Hz, 2H), 3.87
(s, 3H). IR (cmꢀ1: neat): 1287, 1162. LC–MS (APCI+) m/z: 410
(M+H)+. Anal. Calcd for C21H19N3O4S: C, 61.60; H, 4.68; N, 10.26.
Found: C, 61.45; H, 4.57; N, 10.34.
4.1.60. 1-(4-Aminosulfonylphenyl)-4-hydroxymethyl-5-phenyl-
1H-pyrazole (8a)
Ethyl ester 7a (1.3 mmol, 0.500 g) was dissolved in tetrahydro-
furan (25 mL) and then a suspension of LiAlH4 (3.9 mmol, 0.15 g) in
tetrahydrofuran (5 mL) was slowly added. After 2 h of reaction at
20 °C, an aqueous solution of HCl 1 N was added dropwise until a
neutral pH. The reaction mixture was concentrated under reduced
pressure, the residue was taken into ethyl acetate and washed with
water and brine. The organic layer was dried over MgSO4 and con-
centrated under reduced pressure. The residue was recrystallized
in acetonitrile. The alcohol (8a) give a white crystal (yield: 29%),
mp 150–152 °C. 1H NMR (DMSO-d6) d: 7.85 (s, 2H), 7.75 (d,
J = 8.4 Hz, 2H), 7.65 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.26 (m, 5H),
5.10 (br s, 1H), 4.35 (m, 2H). IR (cmꢀ1: neat): 3267, 1598. LC–MS
(APCI+) m/z: 330 (M+H)+. Anal. Calcd for C16H15N3O3S: C, 58.35;
H, 4.59; N, 12.76. Found: C, 58.45; H, 4.82; N, 12.58.
4.1.54. Ethyl 2-benzoyl-3-dimethylaminoacrylate (6a)
The ethyl benzoylacetate (20 mmol, 3.46 mL) and N,N-dimeth-
ylformamide dimethyl acetal was refluxed for 1 h. After evapora-
tion under reduced pressure, the residue was purified by
chromatography on silica gel (diethyl ether) to give title compound
6a as yellow crystals (yield: 40%), mp 154–156 °C. 1H NMR (DMSO-
d6) d: 7.72 (m, 2H), 7.44 (m, 1H), 7.39 (m, 2H), 7.36 (s, 1H), 3.94 (q,
J = 7.0 Hz, 2H), 2.97 (s, 6H), 0.85 (t, J = 6.9 Hz, 3H). IR (cmꢀ1: neat):
1681, 1586. LC–MS (APCI+) m/z: 248 (M+H)+.
4.1.55. Ethyl 2-naphtoyl-3-dimethylaminoacrylate (6j)
The title compound was prepared from ethyl naphtoylacetate
using the same procedure as for 6a to give 6j as yellow crystal
(yield: 58%), mp 145–147 °C. 1H NMR (DMSO-d6) d: 8.10 (m, 2H),
7.90 (m, 1H), 7.78 (m, 2H), 7.35 (m, 2H), 7.29 (s, 1H), 3.80 (q,
J = 6.9 Hz, 2H), 3.10 (s, 6H), 0.90 (t, J = 6.9 Hz, 3H). IR (cmꢀ1: neat):
1687, 1562. LC–MS (APCI+) m/z: 298 (M+H)+.
4.1.61. 1-(4-Aminosulfonylphenyl)-4-hydroxymethyl-5-
(2-naphthyl)-1H-pyrazole (8j)
The title compound was prepared from ester 7j using the same
procedure as for 8a to give 8j as white crystal (yield: 69%), mp
210–212 °C. 1H NMR (DMSO-d6) d: 7.89 (m, 1H), 7.88 (d,
J = 7.2 Hz, 2H), 7.79 (s, 1H), 7.75 (m, 2H), 7.56 (d, J = 7.2 Hz, 2H),
7.40 (m, 2H), 7.53 (s, 2H), 7.19 (m, 2H), 4.36 (br s, 1H), 3.88 (m,
2H). IR (cmꢀ1
:
neat): 3245, 1603. LC–MS (APCI+) m/z: 380
4.1.56. Ethyl 2-methoxynaphtoyl-3-dimethylaminoacrylate (6k)
The title compound was prepared from ethyl methoxynaphtoy-
lacetate using the same procedure as for 6a to give 6k as orange
crystal (yield: 85%), mp 150–152 °C. 1H NMR (DMSO-d6) d: 8.20
(s, 1H), 7.95 (s, 1H), 7.72 (m, 1H), 7.60 (m, 2H), 7.25 (m, 1H),
7.20 (m, 1H), 3.92 (q, J = 6.9 Hz, 2H), 3.31 (s, 3H), 2.75 (s, 6H),
0.78 (t, J = 6.9 Hz, 3H). IR (cmꢀ1: neat): 1692, 1572. LC–MS (APCI+)
m/z: 328 (M+H)+.
(M+H)+. Anal. Calcd for C20H17N3O3S: C, 63.31; H, 4.52; N, 11.07.
Found: C, 63.03; H, 4.69; N, 11.26.
4.1.62. 1-(4-Aminosulfonylphenyl)-4-hydroxymethyl-5-
(6-methoxy-2-naphtalenyl)-1H-pyrazole (8k)
The title compound was prepared from ester 7k using the same
procedure as for 8a to give 5a as white crystal (yield: 63%), mp
221–223 °C. 1H NMR (DMSO-d6) d: 7.99 (s, 1H), 7.94 (m, 2H),
7.92 (d, J = 8.6 Hz, 2H), 7.89 (s, 1H), 7.42 (s, 2H), 7.37 (d,
J = 8.6 Hz, 2H), 7.24 (m, 1H), 7.21 (m, 2H), 5.05 (br s, 1H), 4.40
(m, 2H), 3.40 (s, 3H). IR (cmꢀ1: neat): 3345, 1599. LC–MS (APCI+)
m/z: 410 (M+H)+. Anal. Calcd for C21H19N3O4S: C, 61.60; H, 4.68;
N, 10.26. Found: C, 61.89; H, 4.98; N, 10.69.
4.1.57. 1-(4-Aminosulfonylphenyl)-4-ethoxycarbonyl-5-phenyl-
1H-pyrazole (7a)
The ester 6a (0.8 mmol, 0.92 g) was dissolved in ethanol
(10 mL), then 4-aminosulfonylphenylhydrazine hydrochloride
(0.8 mmol, 0.19 g) was introduced. The mixture was refluxed for
2 h. After cooling to 20 °C the precipitate was filtered and washed
with petroleum ether to give the title compound 7a as yellow crys-
tals (yield: 73%), mp 178–180 °C. 1H NMR (DMSO-d6) d: 7.33 (m,
4H), 8.24 (s, 2H), 7.75 (m, 3H), 7.44 (s, 1H), 7.31 (m, 2H), 4.10 (q,
J = 6.5 Hz, 2H), 1.10 (t, J = 6.5 Hz, 3H). IR (cmꢀ1: neat): 1702,
1310, 1151. LC–MS (APCI+) m/z: 372 (M+H)+.
4.2. Enzymatic evaluation
The CA catalyzed CO2 hydration activity was followed by an
SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument. Phenol red (at a concentration of 0.2 mM) was used
as indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4 (to maintain con-
stant the ionic strength). Saturated CO2 solutions in water at
25 °C were used as substrate. Stock solutions of inhibitor were pre-
pared at a concentration of 10 mM (in DMSO/water 1:1, v/v) and
dilutions up to 0.01 nM were done with the assay buffer men-
tioned above. At least seven different inhibitor concentrations
were used for measuring the inhibition constant. Inhibitor and en-
zyme solutions were preincubated together for 10 min at room
temperature prior assay, in order to allow for the formation of
the E–I complex. Triplicate experiments were done for each inhib-
itor concentration and the values reported throughout the paper
are the mean of such results. KIs were obtained from Linewe-
aver–Burk plots, as reported earlier.27
4.1.58. 1-(4-Aminosulfonylphenyl)-4-ethoxycarbonyl-5-
naphthyl-1H-pyrazole (7j)
The title compound was prepared from ester 6j using the same
procedure as for 7a to give 7j as orange crystal (yield: 74%), mp
145–147 °C. 1H NMR (DMSO-d6) d: 8.29 (m, 1H), 7.93 (d,
J = 8.5 Hz, 2H), 7.90 (s, 1H), 7.82 (m, 2H), 7.62 (d, J = 8.5 Hz, 2H),
7.30 (m, 2H), 7.29 (s, 2H), 7.20 (m, 2H), 4.10 (q, J = 5.5 Hz, 2H),
1.00 (t, J = 5.5 Hz, 3H). IR (cmꢀ1: neat): 1734, 1345, 1158. LC–MS
(APCI+) m/z: 422 (M+H)+.
4.1.59. 1-(4-Aminosulfonylphenyl)-4-ethoxycarbonyl-5-
(6-methoxy-2-naphtalenyl)-1H-pyrazole (7k)
The title compound was prepared from ester 6k using the same
procedure as for 7a to give 7k as orange crystal (yield: 83%), mp
175–177 °C. 1H NMR (DMSO-d6) d: 8.37 (s, 1H), 8.02 (s, 1H), 7.98
(d, J = 8.6 Hz, 2H), 7.92 (m, 2H), 7.65 (s, 2H), 7.44 (d, J = 8.6 Hz,
2H), 7.33 (m, 1H), 7.19 (m, 2H), 4.00 (q, J = 7.3 Hz, 2H), 3.55 (s,
3H), 1.00 (t, J = 7.3 Hz, 3H). IR (cmꢀ1: neat): 1754, 1352, 1171.
LC–MS (APCI+) m/z: 452 (M+H)+.
4.3. In vitro anticancer screening
4.3.1. Cell culture
MDA-MB-231 breast cancer cells were kindly provided by Pro-
fessor X. Le Bourhis (Inserm U908, Lille, France). Cell lines were
maintained as monolayer culture in a humidified incubator at
37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM)
Please cite this article in press as: Rogez-Florent, T.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.029

14
T. Rogez-Florent et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
3. Thiry, A.; Dogne, J. M.; Masereel, B.; Supuran, C. T. Trends Pharmacol. Sci. 2006,
27, 566.
4. Shin, H. J.; Bae Rho, S.; Chul Jung, S.; Han, I. O.; Oh, E. S.; Kim, J. Y. J. Cell Sci.
2010, 124, 1077.
supplemented with 10% fetal calf serum (FCS), 1000 U/ml penicillin
and 100 g/ml streptomycin. All the products for cell culture were
from Life Technologies.
l
5. Lou, Y.; McDonald, P.; Oloumi, A.; Chia, S.; Ostlund, C.; Ahmadi, A.; Kyle, A.;
Ulrich auf dem Keller; Leung, S.; Huntsman, D.; Clarke, B.; Sutherland, B.;
Waterhouse, D.; Bally, M.; Roskelley, C.; Overall, C.; Minchinton, A.; Pacchiano,
F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer
Res. 2011, 71, 3364.
6. Pastorekova, S.; Pastorek, J. Carbonic anhydrase. Its inhibitors and activators; CRC
Press E-publishing Supuran and Scozzafava: Boca, Raton, London, New York,
Washington DC, 2004.
7. Pacchiano, F.; Carta, F.; McDonald, P.; Lou, Y.; Vullo, D.; Scozzafava, A.; Dedhar,
S.; Supuran, C. T. J. Med. Chem. 1896, 2011, 54.
8. Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; Van Zandvoort, M. A.;
Pastorekova, S.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother. Oncol.
2007, 83, 367.
9. Gluszok, S.; Frédérick, R.; Foulon, C.; Klupsch, F.; Supuran, C. T.; Vullo, D.;
Scozzafava, A.; Goossens, J. F.; Masereel, B.; Depreux, P.; Goossens, L. Bioorg.
Med. Chem. 2010, 18, 7392.
10. Neri, D.; Supuran, C. T. Nat. Rev. Drug. Discov. 2011, 10, 767.
11. Swietach, P.; Patiar, S.; Supuran, C. T.; Harris, A.; Vaughan-Jones, R. J. Biol. Chem.
2009, 284, 20299.
12. Betof, A. S.; Rabbani, Z. N.; Hardee, M. E.; Broadwater, G.; Bentley, R. C.; Snyder,
A. S.; Vujaskovic, Z.; Oosterxijk, E.; Harris, L. N.; Horton, J. K.; Dewhirst, M. W.;
Blackwell, K. L. Brit. J. Cancer 2012, 106, 916.
13. Perez-Sayans, M.; Garcia-Garcia, A.; Scozzafava, A.; Supuran, C. T. Curr. Pharm.
Des. 2012, 10, 1407.
4.3.2. Cell growth inhibition assay
Growth inhibition was assessed using MTS assay with CellTiter
96 Aqueous One Solution Cell Proliferation Assay Kit (Promega).
Cells (2 ꢂ 103) were seeded into a 96-well plate in DMEM with
10% FCS and buffered with 22.3 mM bicarbonate,28 and transfered
to hypoxia (1% O2 and 5% CO2 balanced with N2) generated in a H35
hypoxystation (Don Withley). Twenty-four hours later, CA IX
inhibitors (1 lM) were added and cells were treated with increas-
ing concentrations of Doxorubicin (Sigma–Aldrich) and incubated
for 72 h at 37 °C under hypoxia. MTS reagent was then added to
each well and the plate was incubated for 1 h at 37 °C. Absorbance
was measured at 492 nm with a microplate reader. Three indepen-
dent experiments were performed in six replicate wells for each
drug concentration. The IC50 (inhibition concentration 50) value
was defined as the concentration of Doxorubicin needed for 50%
reduction in absorbance and calculated from the survival curves
by SoftMax Pro software (Molecular Devices).
14. Winer, E.; Morrow, M.; Osborne, C. Cancer : Principles and Practice of Oncology,
2001, 1651.
15. Betof, A. S.; Rabbani, Z. N.; Hardee, M. E.; Broadwater, G.; Bentley, R. C.; Snyder,
A. S.; Vujaskovic, Z.; Oosterxijk, E.; Harris, L. N.; Horton, J. K.; Dewhirst, M. W.;
Blackwell, K. L. Brit. J. Cancer 2012, 106, 916.
16. Innocenti, A.; Vullo, D.; Scozzafava, A.; Quinn, R. J.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2008, 18, 1583.
17. Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.;
Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057.
18. Ahlskog, J. K. J.; Schliemann, C.; Marlind, J.; Qureshi, U.; Ammar, A.; Pedley, R.
B.; Neri, D. Brit. J. Cancer 2009, 101, 645.
19. Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 13, 4336.
20. Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.; Scozzafava, A.;
Wouters, B. G.; Lambin, P. Oncology 2009, 92, 423.
21. Thiry, A.; Delayen, A.; Goossens, L.; Houssin, R.; Ledecq, M.; Frankart, A.; Dogné,
J. M.; Wouters, C. T.; Henichart, J. P.; Masereel, B. Eur. J. Med. Chem. 2009, 44,
511.
4.4. Molecular modeling and calculations
The compounds in deprotonated form were built using the
SKETCH module, as implemented in SYBYL (version 8.0),29 and their
geometry was optimized using the MINIMIZE module. The minimi-
zation process uses the POWELL method with the TRIPOS force field
(dielectric constant 1r) to reach
a
final convergence of
0.01 kcal molꢀ1. Docking simulation was then performed into
hCA IX (RCSB Protein Data Bank 3IAI) with the automated GOLD
program.30 The active site was defined including all residues in a
volume of 10 Å around acetazolamide taken as reference.
Acknowledgments
22. http://archemcalc.com/sparc/.
23. Singh, S. K.; Reddy, M. S.; Shivaramakrishna, S.; Kavitha, D.; Vasudev, R.; Moses
Babu, J.; Sivalakshmidevi, A.; Koteswar Rao, Y. Tetrahedron Lett. 2004, 124,
1077.
24. Menozzi, G.; Merello, L.; Fossa, P.; Mosti, L.; Piana, A.; Matioli, F. Il Farmaco
2003, 58, 795.
25. Ferraroni, M.; Tilli, S.; Briganti, F.; Chegwidden, W. R.; Supuran, C. T.; Wiebauer,
K. E.; Tashian, R. E.; Scozzafava, A. Biochemistry 2002, 41, 6237.
26. Pommery, N.; Taverne, T.; Telliez, A.; Goossens, L.; Charlier, C.; Pommery, J.;
Goossens, J. F.; Houssin, R.; Durant, F.; Hénichart, J. P. J. Med. Chem. 2004, 47,
6195.
The authors are grateful to the institutions that support our lab-
oratory: Univ Lille Nord de France and the ‘Ligue Nationale contre
le cancer, Comité du Pas-de Calais’, the PRIM: Pôle de Recherche
Interdisciplinaire du Médicament (Région Nord-Pas-de-Calais,
Ministère délégué à la Recherche et aux Nouvelles Technologies,
European Union (FEDER)). This research was financed (in part) by
an EU FP 7 project (METOXIA) to C.T.S. and A.S.
27. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
28. Svastova, E.; Zilka, N.; Zatovicova, M.; Gibadulinova, A.; Ciampor, F.; Pastorek,
J.; Pastorekova, S. Exp. Cell. Res. 2003, 290, 332.
References and notes
29. Tripos I, SYBYL. 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA.
30. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
1. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
2. De Simone, G.; Supuran, C. T. Biochim. Biophys. Acta 2010, 1804, 404.
Please cite this article in press as: Rogez-Florent, T.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.029