Design, solid-phase synthesis, and biological evaluation of novel 1,5-diarylpyrrole-3-carboxamides as carbonic anhydrase IX inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.09.007

Following previous studies we herein report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a solid-phase strategy involving a PS(HOBt) resin. A mol

Full text of DOI:10.1016/j.bmc.2010.09.007

Bioorganic & Medicinal Chemistry 18 (2010) 7392–7401
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, solid-phase synthesis, and biological evaluation of novel
1,5-diarylpyrrole-3-carboxamides as carbonic anhydrase IX inhibitors
Sébastien Gluszok a, Raphaël Frédérick b, Catherine Foulon c, Frédérique Klupsch a,
d
d
c
Claudiu T. Supuran d, , Daniela Vullo , Andrea Scozzafava , Jean-François Goossens ,
⇑
Bernard Masereel b, Patrick Depreux a, Laurence Goossens a,
⇑
a Univ Lille Nord de France, UDSL, ICPAL, EA 4481, F-59000 Lille, France
b Drug Design and Discovery Center, FUNDP, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium
c Univ Lille Nord de France, UDSL, EA 4481, F-59000 Lille, France
d Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Università degli di Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2010
Revised 31 August 2010
Accepted 2 September 2010
Available online 7 September 2010
Following previous studies we herein report the synthesis and the pharmacological evaluation of a new
class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a
solid-phase strategy involving a PS(HOBt) resin. 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.
This study revealed that the 3-position of the pyrrole was opened to the solvent, so we introduced an
amino side-chain, protonated at physiological pH both to enhance the aqueous solubility and to decrease
the cell membrane penetration. This strategy consisted of preparing membrane-impermeant inhibitors
that may selectively target the tumor-associated hCA IX. Physico-chemical characterizations including
aqueous solubility and lipophilic parameters are described. Pharmacological studies revealed high hCA
IX inhibitory potency in the nanomolar range. Some compounds are selective for hCA IX displaying
hCA I/hCA IX and hCA II/hCA IX ratios higher than 20 and 5, respectively.
Keywords:
Carbonic anhydrase
Sulfonamide
Pyrrole
Molecular modeling
Lipophilicity
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tumors is a pH imbalance leading to the acidification of the
extracellular space of hypoxic tumors (pHe ’ 6), in contrast to nor-
The carbonic anhydrases (CA) are zinc enzymes that catalyze
the reversible hydration of carbon dioxide into a bicarbonate anion
mal tissue (pHe ’ 7.4).7–9 This low pHe is associated with tumori-
genic transformation, chromosomal rearrangements, extracellular
matrix breakdown, migration, and invasion.1–3,6 Acidic pHe is also
related to a chemoresistance by a reduced uptake of weakly basic
anticancer drugs. The tumor-associated CA IX is thus clearly an
attractive target in cancer therapy.10
and a proton (CO2 þ H2O $ HCOꢀ þ Hþ).1 To date, 16 different
3
a-CA isozymes were identified in humans and are distinguished
by their localization and their catalytic activity. Many of these iso-
zymes are involved in numerous physiological processes and path-
ological disorders including edema, glaucoma, obesity, epilepsy.1
Throughout the last three decades, expression of different carbonic
anhydrase isoforms was investigated in various types of cancer
cell.2,3 Functional implication of catalytically active carbonic
anhydrases in cancer was revealed after the identification of two
transmembrane CAs: CA IX, which is almost exclusively associated
with tumors and CA XII, which is expressed in some tumor types.
As CA XII is also found in normal cells, it is a less attractive target
than CA IX in the cancer therapy. CA IX expression is strongly
Recently,11 one of our groups reported that celecoxib 1 (Fig. 1),
a COX-2 sulfonamide containing inhibitor, was a strong inhibitor
of hCA IX (Ki = 16 nM) as well as hCA II (Ki = 21 nM). The crystal
structure of the hCA II-celecoxib adduct demonstrated that this
coxib binds with its sulfonamide toward the zinc ion resulting
in a tetrahedral coordination, as observed for the numerous sul-
fonamides acting as CA inhibitors.12 In a recent work,13 we de-
scribed benzylanilinosulfonamides
2 as very potent hCA IX
inhibitors (Ki = 1.8–27 nM). Although very promising as hCA IX
inhibitors, these compounds revealed to be poorly selective when
compared to the ubiquitous hCA II (Ki = 17–49 nM), and they are
characterized by two chiral centers which are certainly not
advantageous for further drug development.
upregulated by hypoxia, which is
a key feature of many
tumors.4–6 The overall consequence of CA IX overexpression in
With respect to these results, we designed novel constrained
derivatives 3a–3e based on the benzylanilinosulfonamide scaffold,
and including a central pyrrole ring, isoster of the pyrazole present
in celecoxib (Fig. 1).
⇑
Corresponding authors. Tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S);
tel.: +33 (0)3 20 96 47 02; fax: +33 (0)3 20 96 49 06 (L.C.).
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), laurence.goossens@
univ-lille2.fr (L. Goossens).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.007

S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
7393
O
CF3
HOH2C
*
*
OC2H5
NH
N
N
H3C
SO2NH2
SO2NH2
1
2
Position
open to solvent
N
N
CH3
CH3
Hydrophobic
pocket
O
N
N
N ( )n
R
4
3
H
5
2
4a-4i
CH3
N
N ( )n
1
H
R=
3a-3e
N
N ( )n
H
SO2NH2
Interaction with Zn++
5a-5i
N
N
O
N ( )n
H
N CH3
N ( )n
H
n = 2 or 3
Figure 1. Design of 4-(5-aryl-2-methyl-pyrrol-1-yl)-benzenesulfonamides from the coxib scaffold and from the benzylanilinosulfonamides described as hCA IX inhibitors.
As the 3D-coordinates of the catalytic domain of hCA IX
were available,14 the binding conformation of this novel
1-(4-aminosulfonylphenyl)-2-methyl-5-phenyl-1H-pyrrole tem-
plate was obtained by docking in the active site of hCA IX
(Fig. 2). Interestingly, this study revealed that the phenyl ring
in the 5-position of the pyrrole partly fills a hydrophobic pocket
bordered by residues Pro-202, Leu-198, Val-131, and Val-121 in
CA IX. In order to better accommodate this lipophilic pocket, we
suggested the introduction of bulkier hydrophobic moieties
such as a 2-naphthyl (4a–4i) or a 4-biphenylyl (5a–5i) in this
position.
The study also revealed that the 3-position of the pyrrole was
opened to the solvent, and thus adequately oriented for the intro-
duction of an additional residue. For this position, we selected an
amino side-chain, protonated at physiological pH, to decrease the
cell membrane penetration, thus preventing the inhibition of cyto-
solic hCAs I and II which are ubiquitous. On the contrary, the active
site of hCA IX is located on the external side of the cell membrane.
This strategy consisting of preparing membrane-impermeant
inhibitors that may selectively target the tumor-associated hCA
IX was already applied.15 Moreover, the presence of an amino
side-chain in position 3 could improve the drug solubility.
Figure 2. View of the 1-(4-aminosulfonylphenyl)-2-methyl-5-phenyl-1H-pyrrole template inside the hCA IX active site with (a) the lipophilic potential mapped onto the
Connoly surface (brown: high, green: medium, and blue: low lipophilicity) and (b) secondary structure and main active site residues. The zinc ion is represented by a sphere.
Pictures made using (a) Molcad and (b) Pymol, respectively.

7394
S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
between 781 and 2151 nM as observed for the R1-naphthyl mole-
2. Results and discussion
2.1. Chemistry
cules 4a–4i (427–10,000 nM). The 4-biphenylyl derivatives 5a–5i
were also weak hCA I inhibitors with Ki values higher than
1736 nM. This weak inhibitory potency may be explained by the
fact that the hCA I active site is more restricted due to the presence
of a His200 residue.1,20,21
The synthetic pathway to obtain the carboxylic acids 9a–9c is
outlined in Scheme 1. Reaction of ethyl acetoacetate with sodium
All the synthesized molecules were found much more potent
against hCA II (Ki: 9–620 nM) than against hCA I. The phenyl
3a–3e and the 4-biphenylyl 5a–5i derivatives were moderate
hCA II inhibitors with Ki values ranging from 127 to 620 nM. The
best hCA II inhibitors (Ki 6 70 nM) belonged to the R1-naphthyl
series (4a–4e and 4h).
Against the hCA IX isozyme, the newly synthesized molecules
showed a powerful inhibitory potency (Ki: 10–242 nM). Among
the R1-phenyl (Ki: 89–242 nM) and the R1-biphenylyl (Ki:
68–207 nM) derivatives, the nature of the R2-side-chain poorly
influenced the hCA IX inhibitory potency, differently from the
R1-naphthyl compounds (Ki: 10–147 nM). Indeed, the most potent
hCA IX inhibitors are the 2-naphthyl compounds 4a, 4b, 4d, and 4h
(Ki: 10–22 nM) whereas 4c, 4e–4g, and 4i are less active on hCA IX
(Ki: 125–147 nM).
Docking of 4a, the most potent hCA IX inhibitor identified, in-
side the hCA IX binding cleft (Fig. 3) revealed a binding orientation
very similar to the one initially reported for the 1-(4-aminosulfo-
nylphenyl)-2-methyl-5-phenyl-1H-pyrrole template (Fig. 2). In this
orientation, 4a is found to be deeply inserted in the hCA IX cavity,
with its naphthyl group perfectly fitting the hydrophobic pocket.
This might explain, in part, the generally weaker inhibitory po-
tency of the phenyl (3a–3e) or the biphenylyl (5a–5i) that are,
respectively, too small or too bulky to adequately fit this pocket.
As expected, the amino side-chain was found in a position open
to the solvent with the carboxamido function H-bonded to the
Trp-5 NH.
As far as hCA IX selectivity is concerned, all the molecules were
much more potent on hCA IX than hCA I and had a moderate to
high hCA I/hCA IX Ki ratio ranging from 4 to 246. Regarding the
hCA II/hCA IX selectivity all the molecules, except the R1-naphthyl
compounds 4c–4e, were moderately selective inhibitors of hCA IX
(hCA II/hCA IX Ki ratio P1). The R1-naphthyl 4a and the R1-biphe-
nylyl derivatives 5a–5c, 5i were the most selective hCA IX inhibi-
tors characterized by hCA I/hCA IX and hCA II/hCA IX ratios
higher than 20 and 5, respectively. Among these five selective
hCA IX inhibitors, 4a (Ki: 10 nM) was 7- to 11-fold more potent
on hCA IX than the other inhibitors.
hydride in THF, followed by addition of suitable
a-bromomethyl-
ketones 6a–6c gave the 1,4-diketones 7a–7c with a good yield
(61–84%).16 The pyrrole cyclization step was performed following
the usual Paal–Knoor cyclization between the 1,4-diketones
7a–7c and 4-aminobenzenesulfonamide in the presence of a cata-
lytic amount of p-toluenesulfonic acid in refluxing EtOH, to afford
1-[4-(aminosulfonyl)phenyl]-5-aryl-2-methyl-1H-pyrrol-3-carbo-
xylic ethyl esters 8a–8c (yield: 73–83%). The hydrolysis of the ethyl
esters 8a–8c with sodium hydroxide in refluxing EtOH yielded the
corresponding carboxylic acids 9a–9c (yield: 78–94%).
To introduce a large chemical diversity among the 1-[4-(amin-
osulfonyl)phenyl]-5-aryl-2-methyl-1H-pyrrol-3-carboxamides,
a
solid-phase strategy involving a PS(HOBt) resin as coupling agent
was developed on a Quest 205Ò synthesizer (Scheme 2). The choice
of the activating agent is crucial, because we have already
demonstrated that the sulfonamide moiety can react with HBTU
(O-(1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluo-
rophosphate) during the amidification reaction, leading to the
formation of stable tetramethylsulfonylguanidines.17 Another pro-
cedure for loading PS-HOBt with carboxylic acids uses PyBroP as
coupling reagent.18 In our case, the use of diisopropylcarbodiimide
(DIC) and 4-dimethylaminopyridine (DMAP) was preferred: indeed,
it requires fewer equivalents of carboxylic acid because a double
loading of the resin is not required differently from PyBroP.19 This
method is a two steps procedure: (a) formation of resin-bound ac-
tive esters of PS-HOBt using the coupling process with DIC/DMAP
(‘catch’); (b) amide formation is effected by mixing the PS-HOBt es-
ter resin (previously purified by washing sequences) with a mix of
amine (in default) and DIEA in dichloromethane (‘release’). The best
conditions for coupling were 8 h at room temperature.
The obtained amides were easily purified by flash chromatogra-
phy or single crystallization. This pathway afforded the phenyl
3a–3e, the 2-naphthyl 4a–4i and the 4-biphenylyl 5a–5i deriva-
tives with moderate to good yields (30–86%) (Table 1).
2.2. Carbonic anhydrase inhibition
The inhibitory potency (Ki, nM) of all the derivatives was eval-
uated against the tumor-associated hCA IX and against the ubiqui-
tous and physiologically relevant hCA I and hCA II (Table 1). The
isozyme selectivity towards hCA IX was expressed as the hCA I/
hCA IX and hCA II/hCA IX Ki ratios.
From the inhibition data reported in Table 1, we observed that
all the compounds exhibit a weak inhibitory activity against hCA I.
The Ki values of the R1-phenyl derivatives 3a–3e were found
2.3. Lipophilicity and solubility
In drug development, lipophilicity and solubility are important
physico-chemical parameters which strongly influence the bio-
availability. The lipophilicity was theoretically determined with
ACD software,22 and expressed as the logarithm of the distribution
O
O
O
OH
OEt
CH3
R1
O
c
b
OEt
CH3
7a-c
a
Br
R1
CH3
R1
O
N
N
R1
O
6a-c
a : R1 = phenyl
SO2NH2
8a-c
SO2NH2
9a-c
b : R1 = 2-naphthyl
c : R1 = 4-biphenylyl
Scheme 1. Reagents and conditions: (a) (i) NaH, ethyl acetoacetate, anhydrous THF, 0 °C, 30 min; (ii)
aminobenzenesulfonamide, p-toluenesulfonic acid, EtOH, reflux, 24 h; (c) NaOH, EtOH, reflux, 24 h.
a-bromomethylketone 6a–6c, 0 °C, 2 h then rt, 8 h; (b) 4-

S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
7395
O
O
R2
OBt
HOBt
HOBt
R1
CH3
R1
CH3
N
N
9a-c
a
b
"catch"
"release"
SO2NH2
SO2NH2
3a - 3e : R1 = phenyl
4a - 4i : R1 = 2-naphthyl
5a - 5i : R1 = 4-biphenylyl
Scheme 2. Reagents and conditions: (a) PS-HOBt(HL), DIC, DMAP, CH2Cl2/DMF (80/20), rt, 3 h. (b) R-NH2, or N-methylpiperazine, DIEA, CH2Cl2, rt, 8 h.
coefficient (log D) calculated at physiological pH (7.4) (Table 1).
The log D7.4 of all the synthesized pyrroles was ranging from
0.64 to 3.52. The replacement of the R1-phenyl by a 2-naphthyl
4a and 4b were the most potent hCA IX inhibitors with a Ki value
of 10 and 11 nM, respectively (Table 1). They are selective regard-
ing their hCA I inhibitory potency (Ki ratio: 78 and 39, respec-
tively). As far as the hCA II/h CA IX selectivity is concerned, the
derivative 4a is moderately selective for hCA IX (Ki ratio = 5.5),
while 4b is non-selective (Ki ratio = 1.5). Overall 4a seems to
emerge as a lead for further drug development. However, their
capacity to cross the cell membrane needs to be assessed on
in vitro models prior to selecting either.
increased the log D7.4 value (
3a–4a; 3b–4b; 3c–4c; 3d–4e; 3e–4h). A higher log D increase
log D7.4 = +1.42) was observed when the R1-phenyl is replaced
D log D7.4 = +1.12 to +1.19; compare
(D
by 4-biphenylyl moiety (compare 3a–5a; 3b–5b; 3c–5c; 3d–5e;
3e–5h). Surprisingly, when the N,N-dimethylaminoethylamino
side-chain was replaced by the more lipophilic N,N-dimethylami-
nopropylamino moiety, the log D7.4 strongly dropped
(
D
log D7.4 = ꢀ1.1; compare: 3a–3b; 4a–4b; 5a–5b) whatever
4. Experimental section
4.1. Chemistry
the nature of R1. This result was attributed to the increase of the
calculated pKa value (from 8.37 to 9.41, ACD software22) of the
N,N-dimethylamino function. Indeed, the insertion of a supple-
mentary methylene group reduced the impact of the carboxamido
function on the dimethylamino moiety. So, this pKa increase en-
hanced the cationic form of the molecule at pH 7.4, and reduced
its log D7.4, as well as its solubility. Indeed, the theoretical solubil-
ity at pH 7.4 of the N,N-dimethylaminoethylamino derivatives was
13-fold lower than their N,N-dimethylaminopropylamino counter-
parts (compare: 3a–3b; 4a–4b; 5a–5b).
The ACD program calculated the drug solubility at pH 7.4
(Table 1).22 For an identical R2 side-chain, the theoretical solu-
bility at pH 7.4 decreased with the following sequence: phe-
nyl > 2-naphthyl > 4-biphenylyl (compare 3a–4a–5a; 3b–4b–5b;
3c–4c–5c; 3d–4e–5e; 3e–4h–5h). The R2-N,N-dimethylamino-
propylamino compounds (3b, 4b, 5b) were more soluble than
their N,N-dimethylaminoethylamino counterparts (3a, 4a, 5a),
as expected by their log D7.4
All commercial reagents and solvents were used without
further purification. Tetrahydrofuran (THF) was distilled from so-
dium/benzophenone prior to use. All reactions were monitored
by analytical thin-layer chromatography (TLC) on 0.25 mm pre-
coated Kieselgel 60F254 plates (Merck); compounds were visual-
ized by UV (254 and 366 nm) and/or with iodine. Flash
chromatography (FC) was performed with silica gel Kieselgel Si
60 0.040–0.063 mm (Merck). Melting points (Mp) were deter-
mined with a Büchi 535 capillary melting point apparatus and re-
main 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+).
Elemental analyses (C, H, N) were performed on a ThermoFinnigan
Flash EA 112 elemental analyser.
3. Conclusion
Combining the structure of celecoxib 1 and benzylanilinosulf-
onamides 2, we designed a series of novel 4-(5-aryl-2-methyl-pyr-
rol-1-yl)-benzenesulfonamides with the aim to obtain selective
hCA IX inhibitors. The R1-aryl which interacts with the hydropho-
bic pocket was a phenyl, a 2-naphthyl or a 4-biphenylyl, while a
carboxamidoalkylamino R2-side-chain substituted the pyrrole at
the position 3. At physiological pH (7.4), this R2-residue is posi-
tively charged, particularly in the case of the N,N-dimethylamino-
propylamino moiety (calculated pKa = 9.42 for 3b, 4b, and 5b). This
positive charge is reinforced when the extracellular space of tumor
cells is acidified by the overexpression of hCA IX and proton trans-
porters. Even a log D7.4 >1.5–2.0 is an optimal value for membrane
penetration,23 we assumed that this cationic R2-residue hinders
the crossing of the cell membrane, preventing to reach cytosolic
CAs, particularly ubiquitous CA I and CA II; this poor cell penetra-
tion increased the hCA IX selectivity. The R2-naphthyl derivatives
4.1.1. Ethyl 2-acetyl-4-oxo-4-phenylbutanoate (7a)
To a suspension of sodium hydride (16.5 mmol, 0.66 g) in tetra-
hydrofuran (30 mL) cooled at 0 °C was added dropwise a solution
of ethyl acetoacetate (15 mmol, 1.89 mL) in THF (5 mL). The reac-
tion mixture was stirred 30 min at 0 °C before adding a solution
of 2-bromo-1-phenyl-ethan-1-one 6a (16.5 mmol, 3.28 g) in THF
(20 mL). The solution was stirred 2 h at 0 °C, then 8 h at rt. The
reaction was quenched with 1 N HCl solution (30 mL) and ex-
tracted with diethylether (3 ꢁ 50 mL). The organic layers were
combined, dried over MgSO4, filtered and the solvent eliminated
under reduced pressure. The residue was purified by FC (hep-
tane/EtOAc; 85:15) to give 7a as a yellow oil (yield: 84%). 1H
NMR (300 MHz, CDCl3) d: 8.02–7.94 (m, 2H), 7.62–7.55 (m, 1H),

7396
S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
Table 1
Inhibitory potency (Ki, nM), isozyme selectivity ratio data for the R2-phenyl-(3a–3e), the R2-2-naphthyl (4a–4i), and the R2-4-biphenylylpyrazoles (5a–5i) towards hCA I, hCA II,
and hCA IX, and calculated log D7.4 and solubility
O
R2
R1
CH3
N
SO2NH2
Compd
R1
R2
Kia (nM)
Ratiob
hCA II/hCA IX
Calculatedc
Solubility (lM)
hCA I
hCA II
hCA IX
hCA I/hCA II
log D7.4
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
Phenyl
1691
1460
2151
781
391
127
516
286
620
55
242
131
135
89
7
1.6
1
1.76
0.64
1.21
2.10
1.75
2.88
1.82
2.40
2.22
3.29
2.72
2.71
2.94
2.93
3.18
127
1680
114
101
38
NH-(CH2)2
NH-(CH2)3
N
N
Phenyl
11
16
9
Phenyl
N
N
3.8
3
Phenyl
NH-(CH2)2
NH-(CH2 2
N
N
N
N
Phenyl
2131
781
200
10
11
78
39
>75
198
4
3
)
O
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
4-Biphenylyl
5.5
1.5
0.07
0.3
0.1
1.6
1.7
3.2
1.9
5.7
15
NH-(CH2)2
NH-(CH2)3
427
16
11
201
13
N
N
>10,000
5940
479
10
133
30
10
79
NH-(CH2)2
NH-(CH2)2
N
N
9
125
126
147
22
13
2489
1751
5412
1435
1736
196
243
70
20
12
246
11
21
9
NH-(CH2)2
NH-(CH2)3
NH-(CH2)2
NH-(CH2)3
N
N
N
N
N
N
O
4g
4h
4i
12
5
256
475
137
83
6
O
5a
11
NH-(CH2)2
NH-(CH2)3
N
N
5b
5c
5d
5e
5f
4-Biphenylyl
4-Biphenylyl
4-Biphenylyl
4-Biphenylyl
4-Biphenylyl
2169
7085
6799
5853
4424
553
538
604
247
518
111
68
20
5
2.04
2.63
2.45
3.52
2.95
150
10
61
10
7
N
N
104
44
35
21
8
153
167
207
4
NH-(CH2)2
NH-(CH2)2
NH-(CH2)2
NH-(CH2)3
NH-(CH2)2
NH-(CH2)3
N
N
N
N
N
N
1.5
2.5
N
N
O
O
5g
5h
5i
4-Biphenylyl
4-Biphenylyl
4-Biphenylyl
5542
6135
3924
205
352
574
198
187
101
28
33
39
1
2.94
3.17
3.16
9
4
5
2
5.7
a
Errors in the range of 3–5% of the reported values from three different assays. Acetazolamide was chosen as the standard reference drug: Ki for hCA I, II, and IX are 250, 12,
and 25 nM, respectively).
b
The Ki ratios are indicative of isozyme selectivity: a weakly selective inhibitor is characterized by a low ratio value.
Calculated at pH 7.4 by ACD Software.
c
7.50–7.43 (m, 2H), 4.27–4.18 (m, 3H), 3.74 (dd, J = 18.4 Hz, 8.2 Hz,
1H), 3.53 (dd, J = 18.4 Hz, 5.5 Hz, 1H), 2.44 (s, 3H), 1.29 (t, J = 7.2 Hz,
3H). IR (cmꢀ1: neat): 1740, 1716, 1640. LC–MS (APCI+) m/z: 249
(M+H)+.
procedure than that described for ethyl 2-acetyl-4-oxo-4-phenyl-
butanoate 7a. The crude product was purified by FC (toluene/
EtOAc; 9:1) and crystallized from diisopropyl ether to afford 7b
as white crystals (yield: 68%). Mp: 64–66 °C. 1H NMR (300 MHz,
CDCl3) d: 8.49 (s, 1H), 8.05–7.82 (m, 4H), 7.68–7.50 (m, 2H),
4.34–4.20 (m, 3H), 3.85 (dd, J = 18.4 Hz, 8.1 Hz, 1H), 3.65 (dd,
J = 18.4 Hz, 5.9 Hz, 1H), 2.47 (s, 3H), 1.29 (t, J = 7 Hz, 3H). IR
(cmꢀ1: neat): 1736, 1710, 1680. LC–MS (APCI+) m/z: 299 (M+H)+.
4.1.2. Ethyl 2-acetyl-4-(2-naphthyl)-4-oxobutanoate (7b)
The title compound was prepared from 2-bromo-1-(2-naph-
thyl)-ethan-1-one 6b and ethyl acetoacetate according to the same

S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
7397
Figure 3. View of 4a inside the hCA IX active site with (a) the lipophilic potential mapped onto the Connoly surface (brown: high, green: medium, and blue: low lipophilicity)
and (b) the secondary structure and main active site residues. The zinc ion is represented by a sphere. Pictures made using (a) Molcad and (b) Pymol, respectively.
4.1.3. Ethyl 2-acetyl-4-(4-biphenylyl)-4-oxobutanoate (7c)
The title compound was prepared from 2-bromo-1-(4-bipheny-
lyl)-ethan-1-one 6c and ethyl acetoacetate according to the same
procedure than that described for ethyl 2-acetyl-4-oxo-4-phenyl-
butanoate 7a. The crude product was purified by FC (heptan/
EtOAc; 8:2) and crystallized from diisopropyl ether to afford 7c
as white crystals (yield: 61%). Mp: 87–89 °C. 1H NMR (300 MHz,
CDCl3) d: 8.08 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.67–
7.62 (m, 2H), 7.53–7.39 (m, 3H), 4.29–4.22 (m, 3H), 3.77 (dd,
J = 18.4 Hz, 8.3 Hz, 1H), 3.57 (dd, J = 18.4 Hz, 5.5 Hz, 1H), 2.48 (s,
3H), 1.32 (t, J = 7 Hz, 3H). IR (cmꢀ1: neat): 1737, 1713, 1672.
LC–MS (APCI+) m/z: 325 (M+H)+.
crystallized from CH3CN to afford 8c as white crystals (yield:
80%). Mp: 137ꢀ139 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.92 (d,
J = 8.7 Hz, 2H), 7.66–7.59 (m, 2H), 7.57–7.51 (m, 6H), 7.46–7.39
(m, 2H), 7.36–7.30 (m, 1H), 7.14 (d, J = 8.2 Hz, 2H), 6.79 (s, 1H),
4.24 (q, J = 7 Hz, 2H), 2.34 (s, 3H), 1.30 (t, J = 7 Hz, 3H). IR
(cmꢀ1: neat): 1674, 1340, 1236, 1162. LC–MS (APCI+) m/z: 461
(M+H)+.
4.1.7. 1-[4-(Aminosulfonyl)phenyl]-2-methyl-5-phenyl-1H-
pyrrole-3-carboxylic acid (9a)
A solution of the ester 8a (10 mmol, 3.84 g) and sodium hydrox-
yde (50 mmol, 2 g) in 250 mL of aqueous ethyl alcohol (95%) was
refluxed for 24 h. After cooling the reaction mixture was concen-
trated under reduced pressure. The resulting crude residue was di-
luted into water (150 mL) and the pH adjusted to 2 with HCl 1 N.
The mixture was stirred for 1 h at rt. The white precipitate was ex-
tracted with EtOAc (3 ꢁ 400 mL). The combined organic layers
were washed with brine, dried over MgSO4, and the solvent evap-
orated under reduced pressure. The residual solid was crystallized
in CH3CN to afford title compound 9a as white crystals (yield: 78%).
Mp: 223ꢀ226 °C. 1H NMR (300 MHz, DMSO-d6) d: 12.06 (br s, 1H),
7.88 (d, J = 8.2 Hz, 2H), 7.56–7.43 (m, 4H), 7.27–7.12 (m, 3H), 7.08–
7.01 (m, 2H), 6.68 (s, 1H), 2.32 (s, 3H). IR (cmꢀ1: neat): 1655, 1331,
1253, 1161. LC–MS (APCI+) m/z: 357 (M+H)+.
4.1.4. Ethyl 1-[4-(aminosulfonyl)phenyl]-2-methyl-5-phenyl-
1H-pyrrole-3-carboxylate (8a)
A
solution of ethyl 2-acetyl-4-oxo-4-phenylbutanoate 7a
(10 mmol, 2.48 g), 4-aminobenzenesulfonamide (10 mmol, 1.77 g)
and 4-toluenesulfonic acid (0.01 mmol, 1.9 mg), in absolute EtOH
(100 mL) was refluxed for 24 h. After cooling to rt the solvent
was evaporated under reduced pressure and the crude product
was purified by FC (cyclohexane/EtOAc; 6:4) and crystallized from
toluene to give the title compound 8a as yellow crystals (yield:
83%). Mp: 192ꢀ194 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.88 (d,
J = 8.6 Hz, 2H), 7.58–7.46 (m, 4H), 7.26–7.14 (m, 3H), 7.07–7.04
(m, 2H), 6.67 (s, 1H), 4.23 (q, J = 7.0 Hz, 2H), 2.33 (s, 3H), 1.29 (t,
J = 7.0 Hz, 3H). IR (cmꢀ1: neat): 1666, 1342, 1230, 1168. LC–MS
(APCI+) m/z: 385 (M+H)+.
4.1.8. 1-[4-(Aminosulfonyl)phenyl]-2-methyl-5-(2-naphthyl)-
1H-pyrrole-3-carboxylic acid (9b)
The title compound was prepared from carboxylic ethyl ester 8b
according to the same procedure than that described for 9a. The
crude product was crystallized from cyclohexane to afford 9b as
white crystals (yield: 85%). Mp: 235–236 °C. 1H NMR (300 MHz,
DMSO-d6) d: 12.1 (br s, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.83–7.78 (m,
1H), 7.73–7.67 (m, 2H), 7.64 (s, 1H), 7.55–7.49 (m, 4H), 7.47–
7.42 (m, 2H), 7.12 (dd, J = 8.5 Hz, 1.8 Hz, 1H), 6.83 (s, 1H), 2.36 (s,
3H). IR (cmꢀ1: neat): 1649, 1339, 1256, 1166. LC–MS (APCI+) m/z:
407 (M+H)+.
4.1.5. Ethyl 1-[4-(aminosulfonyl)phenyl]-2-methyl-5-(2-naphth
yl)-1H-pyrrole-3-carboxylate (8b)
The title compound was prepared from ethyl 2-acetyl-4-(2-
naphthyl)-4-oxobutanoate 7b and 4-aminobenzenesulfonamide
according to the same procedure than that described for 8a. The
crude product was purified by crystallization (EtOH) to afford 8b
as yellow crystals (yield: 73%). Mp: 195ꢀ197 °C. 1H NMR
(300 MHz, DMSO-d6) d: 7.88 (d, J = 8.6 Hz, 2H), 7.84–7.78 (m,
1H), 7.77–7.67 (m, 3H), 7.57–7.51 (m, 4H), 7.49–7.43 (m, 2H),
7.11 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.86 (s, 1H), 4.26 (q, J = 6.9 Hz,
2H), 2.38 (s, 3H), 1.31 (t, J = 6.9 Hz, 3H). IR (cmꢀ1: neat): 1667,
1337, 1231, 1170. LC–MS (APCI+) m/z: 435 (M+H)+.
4.1.9. 1-[4-(Aminosulfonyl)phenyl]-2-methyl-5-(4-biphenylyl)-
1H-pyrrole-3-carboxylic acid (9c)
The title compound 9c was prepared in 94% yield from carbox-
ylic ethyl ester 8c according to the same procedure than that de-
scribed for 9a to afford 9c (yield: 94%) without further
purification. Mp: 246–248 °C. 1H NMR (300 MHz, DMSO-d6) d:
12.09 (br s, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.64–7.59 (m, 2H), 7.56–
7.50 (m, 6H), 7.46–7.39 (m, 2H), 7.36–7.30 (m, 1H), 7.13 (d,
J = 8.2 Hz, 2H), 6.76 (s, 1H), 2.34 (s, 3H). IR (cmꢀ1: neat): 1651,
1334, 1247, 1171. LC–MS (APCI+) m/z: 433 (M+H)+.
4.1.6. Ethyl 1-[4-(aminosulfonyl)phenyl]-2-methyl-5-(4-
biphenylyl)-1H-pyrrole-3-carboxylate (8c)
The title compound was prepared from ethyl 2-acetyl-4-(4-bi-
phenylyl)-4-oxobutanoate 7c and 4-aminobenzenesulfonamide
according to the same procedure than that described for 8a. The
crude product was purified by FC (cyclohexane/EtOAc; 6:4) and

7398
S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
4.1.10. N-[2-(Dimethylamino)ethyl]-1-[4-(aminosulfonyl)phen-
yl]-2-methyl-5-phenyl-1H-pyrrole-3-carboxamide (3a)
1161. LC–MS (APCI+) m/z: 467 (M+H)+. Anal. Calcd for
25H30N4O3S: C, 64.35; H, 6.48; N, 12.01. Found: C, 64.45; H,
C
DMAP (0.59 mmol, 0.072 g) dissolved in 10 mL of CH2Cl2 was
added to PS-HOBt(HL) resin (0.98 mmol, 1 g) and stirred for
5 min at rt. Then a solution of the carboxylic acid 9a (1.47 mmol,
0.52 g) dissolved in a mixture of CH2Cl2/DMF (80:20) was added
and stirred for 10 min. Finally, N,N0-diisopropylcarbodiimide
(4.41 mmol, 0.68 mL) dissolved in 1 mL of CH2Cl2 was added to
the mixture and stirred at rt for 3 h. The resin was collected by fil-
tration and washed successively with DMF (3 ꢁ 20 mL), CH2Cl2
(3 ꢁ 20 mL), DMF (3 ꢁ 20 mL), and THF (3 ꢁ 20 mL), to afford the
resin-bound-HOBt active ester. A solution of N,N-dimethylethyl-
enediamine (0.78 mmol, 0.087 mL) and N,N-diisopropylethylamine
(0.98 mmol, 0.16 mL) in CH2Cl2 (20 mL) was added to the resin-
bound-HOBt active ester, and stirred at 25 °C for 8 h. At the end
of the reaction, DMF (5 mL) was added and the resin collected by
filtration and washed with a mixture of CH2Cl2/DMF (80/20)
(3 ꢁ 20 mL). The filtrate was then concentrated under reduced
pressure. The crude product was purified by FC (CH2Cl2/MeOH;
6:4) and crystallized from EtOH to afford 3a as white crystals
(yield: 30%). Mp: 248–250 °C. 1H NMR (300 MHz, DMSO-d6) d:
7.87 (d, J = 8.8 Hz, 2H), 7.79 (t, J = 5.7 Hz, 1H), 7.51 (s, 2H), 7.43
(d, J = 8.8 Hz, 2H), 7.26–7.12 (m, 3H), 7.05–7.01 (m, 2H), 6.84 (s,
1H), 3.35–3.26 (m, 2H), 3.37 (t, J = 6.9 Hz, 2H), 2.32 (s, 3H), 2.17
(s, 6H). IR (cmꢀ1: neat): 3069, 1629, 1569, 1536, 1339, 1164. LC–
MS (APCI+) m/z: 427 (M+H)+. Anal. Calcd for C22H26N4O3S: C,
61.95; H, 6.14; N, 13.14. Found: C, 64.65; H, 6.04; N, 12.98.
6.44; N, 12.12.
4.1.14. N-(Morpholino-1-ethyl)-1-[4-(aminosulfonyl)phenyl]-2-
methyl-5-phenyl-1H-pyrrole-3-carboxamide (3e)
The title compound was prepared from the carboxylic ethyl es-
ter 9a and N-(2-aminoethyl)morpholine according to the same
procedure than that described for 3a. The crude product was puri-
fied by FC (CH2Cl2/MeOH; 95:5) and crystallized from EtOH to af-
ford 3e as white crystals (yield: 57%). Mp: 254–256 °C. 1H NMR
(300 MHz, DMSO-d6) d: 7.87 (d, J = 8.7 Hz, 2H), 7.81 (t, J = 5.9 Hz,
1H), 7.51 (s, 2H), 7.44 (d, J = 8.7 Hz, 2H), 7.26–7.13 (m, 3H), 7.06–
7.01 (m, 2H), 6.83 (s, 1H), 3.62–3.56 (m, 4H), 3.39–3.30 (m, 2H),
2.47–2.38 (m, 6H), 2.32 (s, 3H). IR (cmꢀ1: neat): 1625, 1568,
1538, 1312, 1158. LC–MS (APCI+) m/z: 469 (M+H)+. Anal. Calcd
for C24H28N4O4S: C, 61.52; H, 6.02; N, 11.96. Found: C, 61.72; H,
6.23; N, 11.57.
4.1.15. N-[2-(Dimethylamino)ethyl]-1-[4-(aminosulfonyl)phen-
yl]-2-methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4a)
The title compound was prepared from the carboxylic acid 9b
and N,N-dimethylethylenediamine according to the same proce-
dure than that described for 3a. The crude product was purified
by FC (CH2Cl2/MeOH; 8:2) to afford 4a as a white solid (yield:
82%). Mp: 226–227 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.87 (d,
J = 8.8 Hz, 2H), 7.82–7.78 (m, 2H), 7.73–7.68 (m, 2H), 7.59 (d,
J = 1.8 Hz, 1H), 7.52–7.42 (m, 6H), 7.11 (dd, J = 8.6 Hz, 1.8 Hz, 1H),
7.00 (s, 1H), 3.38–3.29 (m, 2H), 2.42 (t, J = 6.9 Hz, 2H), 2.37 (s,
3H), 2.21 (s, 6H). IR (cmꢀ1: neat): 2923, 1626, 1568, 1536, 1332,
1280, 1162. LC–MS (APCI+) m/z: 477 (M+H)+. Anal. Calcd for
4.1.11. N-(Dimethylaminopropyl)-1-[4-(aminosulfonyl)phenyl]-
2-methyl-5-phenyl-1H-pyrrole-3-carboxamide (3b)
The title compound was prepared from the carboxylic ethyl es-
ter 9a and N,N-dimethyl-1,3-propanediamine according to the same
procedure than thatdescribed for 3a. The crudeproduct was purified
by FC (CH2Cl2/MeOH; 6:4) to afford 3b as a white solid (yield: 44%).
Mp: 253–255 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.92 (t, J = 5.5 Hz,
1H), 7.86 (d, J = 8.5 Hz, 2H), 7.51 (s, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.25–
7.12 (m, 3H), 7.06–7.01 (m, 2H), 6.83 (s, 1H), 3.27–3.19 (m, 2H), 2.32
(s, 3H), 2.25 (t, J = 7.2 Hz, 2H), 2.13 (s, 6H), 1.68–1.57 (m, 2H). IR
(cmꢀ1: neat): 1622, 1570, 1543, 1322, 1156. LC–MS (APCI+) m/z:
441 (M+H)+. Anal. Calcd for C23H28N4O3S: C, 62.70; H, 6.41; N,
12.72. Found: C, 62.55; H, 6.34; N, 12.62.
C26H28N4O3S: C, 65.52; H, 5.92; N, 11.76. Found: C, 65.46; H,
6.03; N, 11.86.
4.1.16. N-(Dimethylaminopropyl)-1-[4-(aminosulfonyl)phenyl]-
2-methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4b)
The title compound was prepared from the carboxylic acid 9b
and N,N-dimethyl-1,3-propanediamine according to the same pro-
cedure than that described for 3a. The crude product was crystal-
lized from EtOH to afford 4b as white crystals (yield: 52%). Mp:
228–230 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.96 (t, J = 5.7 Hz,
1H), 7.91–7.84 (m, 2H), 7.82–7.78 (m, 1H), 7.74–7.67 (m, 2H),
7.59 (s, 1H), 7.54–7.42 (m, 6H), 7.11 (dd, J = 8.5 Hz, 1.7 Hz, 1H),
6.99 (s, 1H), 3.28–3.22 (m, 2H), 2.37 (s, 3H), 2.30–2.23 (m, 2H),
2.14 (s, 6H), 1.67–1.58 (m, 2H). IR (cmꢀ1: neat): 1627, 1571,
1539, 1323, 1281, 1158. LC–MS (APCI+) m/z: 491(M+H)+. Anal.
Calcd for C27H30N4O3S: C, 66.10; H, 6.16; N, 11.42. Found: C,
66.32; H, 6.04; N, 11.22.
4.1.12. 4-{2-Methyl-3-[(4-methylpiperazino)carbonyl]-5-phenyl
-1H-pyrrolyl-1}-benzenesulfonamide (3c)
The title compound was prepared from the carboxylic ethyl es-
ter 9a and 1-methylpiperazine according to the same procedure
than that described for 3a. The crude product was purified by FC
(CH2Cl2/MeOH; 9:1) to afford 3c as a white solid (yield: 77%). Mp
>260 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.86 (d, J = 8.8 Hz, 2H),
7.51 (s, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.25–7.12 (m, 3H), 7.08–7.02
(m, 2H), 6.44 (s, 1H), 3.66–3.57 (m, 4H), 2.36–2.31 (m, 4H), 2.20 (s,
3H), 2.10 (s, 3H). IR (cmꢀ1: neat): 1600, 1336, 1255, 1160. LC–MS
(APCI+) m/z: 439 (M+H)+. Anal. Calcd for C23H26N4O3S: C, 62.99; H,
5.98; N, 12.78. Found: C, 63.04; H, 6.10; N, 12.54.
4.1.17. 4-{2-Methyl-3-[(4-methylpiperazino)carbonyl]-5-(2-
naphthyl)-1H-pyrrolyl-1}-benzenesulfonamide (4c)
The title compound was prepared from the carboxylic acid 9b
and 1-methylpiperazine according to the same procedure than that
described for 3a. The crude product was recrystallized from a mix-
ture of CH3CN/EtOH (5:5) to afford 4c as white crystals (yield:
52%). Mp >260 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.91–7.78 (m,
3H), 7.75–7.67 (m, 2H), 7.63 (s, 1H), 7.57–7.32 (m, 6H), 7.18–7.07
(m, 1H), 6.58 (s, 1H), 3.72–3.54 (m, 4H), 2.42–2.29 (m, 4H), 2.21 (s,
3H), 2.14 (s, 3H). IR (cmꢀ1: neat): 3357, 1607, 1333, 1254, 1158.
LC–MS (APCI+) m/z: 489 (M+H)+. Anal. Calcd for C27H28N4O3S: C,
66.37; H, 5.78; N, 11.47. Found: C, 66.42; H, 6.10; N, 11.22.
4.1.13. N-(Piperidino-1-ethyl)-1-[4-(aminosulfonyl)phenyl]-2-
methyl-5-phenyl-1H-pyrrole-3-carboxamide (3d)
The title compound was prepared from the carboxylic ethyl es-
ter 9a and N-(2-aminoethyl)piperidine according to the same pro-
cedure than that described for 3a. The crude product was purified
by FC (CH2Cl2/MeOH; 8:2) and crystallized from EtOH to afford 3d
as a white solid (yield: 45%). Mp: 206–208 °C. 1H NMR (300 MHz,
DMSO-d6) d: 7.87 (d, J = 8.5 Hz, 2H), 7.78 (t, J = 5.5 Hz, 1H), 7.51
(s, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.26–7.12 (m, 3H), 7.06–6.99 (m,
2H), 6.83 (s, 1H), 3.38–3.27 (m, 2H), 2.48–2.32 (m, 6H), 2.28 (s,
3H), 1.51–1.37 (m, 6H). IR (cmꢀ1: neat): 1625, 1569, 1536, 1310,
4.1.18. N-[2-(Pyrrolidinyl-1-ethyl)]-1-[4-(aminosulfonyl)phen-
yl]-2-methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4d)
The title compound was prepared from the carboxylic acid 9b
and N-(2-aminoethyl)pyrrolidine according to the same procedure

S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
7399
than that described for 3a. The crude product was purified by FC
(CH2Cl2/MeOH; 8:2) to afford 4d as a white solid (yield: 60%).
Mp: 148–152 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.96 (t,
J = 5.9 Hz, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.82–7.78 (m, 1H), 7.75–
7.68 (m, 2H), 7.60 (s, 1H), 7.54–7.42 (m, 6H), 7.12 (dd, J = 8.6 Hz,
1.9 Hz, 1H), 7.00 (s, 1H), 3.45–3.3 (m, 2H), 2.68–2.53 (m, 6H),
2.38 (s, 3H), 1.78–1.67 (m, 4H). IR (cmꢀ1: neat): 1626, 1569,
1539, 1336, 1271, 1163. LC–MS (APCI+) m/z: 503 (M+H)+. Anal.
Calcd for C28H30N4O3S: C, 66.91; H, 6.02; N, 11.15. Found: C,
67.01; H, 6.14; N, 11.21.
(M+H)+. Anal. Calcd for C28H30N4O4S: C, 64.85; H, 5.83; N, 10.80.
Found: C, 64.52; H, 5.99; N, 10.62.
4.1.23. N-(3-Morpholinopropyl)-1-[4-(aminosulfonyl)phenyl]-
2-methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4i)
The title compound was prepared from the carboxylic acid 9b
and N-(3-morpholinopropyl)amine according to the same proce-
dure than that described for 3a. The crude product was purified
by FC (CH2Cl2/MeOH; 8:2) and crystallized from EtOH to afford
4i as white crystals (yield: 43%). Mp: 240–242 °C. 1H NMR
(300 MHz, DMSO-d6) d: 7.94 (t, J = 5.5 Hz, 1H), 7.86 (d, J = 8.7 Hz,
2H), 7.83–7.78 (m, 1H), 7.74–7.66 (m, 2H), 7.57 (d, J = 1.6 Hz,
1H), 7.51–7.41 (m, 6H), 7.11 (dd, J = 8.6, 1.6 Hz, 1H), 6.98 (s, 1H),
3.62–3.55 (m, 4H), 3.29–3.22 (m, 2H), 2.39–2.28 (m, 9H), 1.73–
1.62 (m, 2H). IR (cmꢀ1: neat): 1624, 1568, 1542, 1332, 1271,
1158. LC–MS (APCI+) m/z: 533 (M+H)+. Anal. Calcd for
C29H32N4O4S: C, 65.39; H, 6.06; N, 10.52. Found: C, 65.21; H,
6.24; N, 10.32.
4.1.19. N-(Piperidino-1-ethyl)-1-[4-(aminosulfonyl)phenyl]-2-
methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4e)
The title compound was prepared from the carboxylic acid 9b
and N-(2-aminoethyl)piperidine according to the same procedure
than that described for 3a. The crude product was purified by FC
(CH2Cl2/MeOH; 8:2) to afford 4e as a white solid (yield: 86%).
Mp: 245–247 °C. 1H NMR (300 MHz, DMSO-d6) d: 7.87 (d,
J = 8.8 Hz, 2H), 7.83–7.79 (m, 2H), 7.74–7.69 (m, 2H), 7.59 (d,
J = 1.9 Hz, 1H), 7.52–7.42 (m, 6H), 7.10 (dd, J = 8.6 Hz, 1.9 Hz, 1H),
6.99 (s, 1H), 3.39–3.30 (m, 2H), 2.50–2.42 (m, 6H), 2.37 (s, 3H),
1.52–1.47 (m, 6H). IR (cmꢀ1: neat): 2926, 1624, 1570, 1538,
1325, 1281, 1161. LC–MS (APCI+) m/z: 517 (M+H)+. Anal. Calcd
for C29H32N4O3S: C, 67.42; H, 6.24; N, 10.84. Found: C, 67.31; H,
6.31; N, 10.22.
4.1.24. N3-[2-(Dimethylamino)ethyl]-1-[4-(aminosulfonyl)phen
yl]-2-methyl-5-(4-biphenylyl)-1H-3 pyrrolecarboxamide (5a)
The title compound was prepared from carboxylic acid 3c and
N,N-dimethylethylenediamine according to the same procedure
than that described for 3a. The crude product was recrystallized
from EtOH to afford 5a as white crystals (yield: 44%). Mp: 245–
247 °C. 1H NMR (DMSO-d6) d: 7.90 (d, J = 8.4 Hz, 2H), 7.79 (t,
J = 5.8 Hz, 1H), 7.65–7.59 (m, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.51–
7.38 (m, 6H), 7.36–7.29 (m, 1H), 7.11 (d, J = 8.4 Hz, 2H), 6.93 (s,
1H), 3.36–3.28 (m, 2H), 2.38 (t, J = 6.8 Hz, 2H), 2.33 (s, 3H), 2.18
(s, 6H). IR (cmꢀ1: neat): 1634, 1560, 1334, 1171. LC–MS (APCI+)
m/z: 503 (M+H)+. Anal. Calcd for C28H30N4O3S: C, 66.91; H, 6.02;
N, 11.15. Found: C, 66.75; H, 5.96; N, 11.22.
4.1.20. N-[2-(4-Methylpiperazino)ethyl]-1-[4-(aminosulfonyl)-
phenyl]-2-methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4f)
The title compound was prepared from the carboxylic acid 9b
and 2-(4-methylpiperazino)ethylamine according to the same pro-
cedure than that described for 3a. The crude product was purified
by FC (CH2Cl2/MeOH; 8:2) and crystallized from EtOH to afford 4f
as white crystals (yield: 39%). Mp >250 °C. 1H NMR (300 MHz,
DMSO-d6) d: 7.92–7.76 (m, 4H), 7.74–7.65 (m, 2H), 7.58 (d,
J = 1.2 Hz, 1H), 7.53–7.39 (m, 6H), 7.11 (dd, J = 8.4, 1.2 Hz, 1H),
6.98 (s, 1H), 3.37–3.28 (m, 2H), 2.47–2.38 (m, 2H), 2.36–2.27 (m,
11H), 2.13 (s, 3H). IR (cmꢀ1: neat): 1628, 1570, 1538, 1335, 1268,
1163. LC–MS (APCI+) m/z: 532 (M+H)+. Anal. Calcd for C39H33N5O3S:
C, 65.51; H, 6.26; N, 13.17. Found: C, 65.41; H, 6.31; N, 13.52.
4.1.25. N3-[3-(Dimethylamino)propyl]-1-[4-(aminosulfonyl)phe
nyl]-2-methyl-5-(2-naphthyl)-1H-3-pyrrolecarboxamide (5b)
The title compound was prepared from carboxylic acid 3c and
N,N-dimethyl-1,3-propanediamine according to the same proce-
dure than that described for 3a. The crude product was purified
by FC (CH2Cl2/MeOH; 7:3) and recrystallized from EtOH to afford
5b as white crystals (yield: 53%). Mp: 216–218 °C. 1H NMR
(DMSO-d6) d: 8.15 (t, J = 5.3 Hz, 1H), 7.90 (d, J = 7.9 Hz, 2H), 7.68–
7.28 (m, 11H), 7.10 (d, J = 7.9 Hz, 2H), 6.97 (s, 1H), 3.35–3.27 (m,
2H), 3.09–2.97 (m, 2H), 2.72 (s, 6H), 2.34 (s, 3H), 1.94–1.81 (m,
2H). IR (cmꢀ1: neat): 1629, 1554, 1329, 1163. LC–MS (APCI+) m/z:
517 (M+H)+. Anal. Calcd for C29H32N4O3S: C, 67.42; H, 6.24; N,
10.84. Found: C, 67.21; H, 6.27; N, 10.96.
4.1.21. N-[3-(4-Methylpiperazino)propyl]-1-[4-(aminosulfonyl)-
phenyl]-2-methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4g)
The title compound was prepared from the carboxylic acid 9b
and 3-(4-methylpiperazino)propylamine according to the same
procedure than that described for 3a. The crude product was puri-
fied by FC (CH2Cl2/MeOH; 7:3) and crystallized from EtOH to afford
4g as white crystals (yield: 43%). Mp: 239–241 °C. 1H NMR (DMSO-
d6) d: 7.94 (t, J = 5.4 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.83–7.78 (m,
1H), 7.74–7.66 (m, 2H), 7.58 (s, 1H), 7.52–7.41 (m, 6H), 7.10 (dd,
J = 8.4, 1.4 Hz, 1H), 6.98 (s, 1H), 3.28–3.21 (m, 2H), 2.52–2.43 (m,
2H), 2.40–2.26 (m, 11H), 2.11 (s, 3H), 1.73–1.62 (m, 2H). IR
(cmꢀ1: neat): 1601, 1570, 1534, 1330, 1283, 1166. LC–MS (APCI+)
m/z: 546 (M+H)+. Anal. Calcd for C30H35N5O3S: C, 66.03; H, 6.46;
N, 12.83. Found: C, 65.98; H, 6.56; N, 12.42.
4.1.26. 4-[2-Methyl-3-[(4-methylpiperazino)carbonyl]-5-(4-
biphenylyl)-1H-1-pyrrolyl]-1-benzenesulfonamide (5c)
The title compound was prepared from carboxylic acid 3c and
1-methylpiperazine according to the same procedure than that de-
scribed for 3a. The crude product was purified by FC (CH2Cl2/
MeOH; 95:5) and recrystallized from EtOH to afford 5c as white
crystals (yield: 39%). Mp >250 °C. 1H NMR (DMSO-d6) d: 7.89 (d,
J = 8.5 Hz, 2H), 7.61 (d, J = 7.3 Hz, 2H), 7.56–7.47 (m, 6H), 7.45–
7.39 (m, 2H), 7.34–7.29 (m, 1H), 7.13 (d, J = 8.2 Hz, 2H), 6.51 (s,
1H), 3.67–3.56 (m, 4H), 2.38–2.29 (m, 4H), 2.21 (s, 3H), 2.11 (s,
3H). IR (cmꢀ1: neat): 1628, 1569, 1340, 1236, 1162. LC–MS (APCI+)
m/z: 515 (M+H)+. Anal. Calcd for C29H30N4O3S: C, 67.68; H, 5.88; N,
10.89. Found: C, 67.51; H, 5.94; N, 11.01.
4.1.22. N-(2-Morpholinoethyl)-1-[4-(aminosulfonyl)phenyl]-2-
methyl-5-(2-naphthyl)-1H-pyrrole-3-carboxamide (4h)
The title compound was prepared from the carboxylic acid 9b
and N-(2-aminoethyl)morpholine according to the same procedure
than that described for 3a. The crude product was crystallized from
EtOH to afford 4h as white crystals (yield: 86%). Mp: 248–250 °C.
1H NMR (300 MHz, DMSO-d6) d: 7.95–7.86 (m, 3H), 7.83–7.78
(m, 1H), 7.73–7.65 (m, 2H), 7.58 (s, 1H), 7.54–7.42 (m, 6H), 7.12
(dd, J = 8.6 Hz, 1.9 Hz, 1H), 6.99 (s, 1H), 3.64–3.56 (m, 4H), 3.42–
3.31 (m, 2H), 2.48–2.42 (m, 6H), 2.37 (s, 3H). IR (cmꢀ1: neat):
1635, 1570, 1538, 1327, 1271, 1158. LC–MS (APCI+) m/z: 519
4.1.27. N3-(2-Tetrahydro-1H-1-pyrrolylethyl)-1-[4-
(aminosulfonyl)phenyl]-2-methyl-5-(4-biphenylyl)-1H-3-
pyrrolecarboxamide (5d)
The title compound was prepared from carboxylic acid 3c and
N-(2-aminoethyl)piperidine according to the same procedure than

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S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
that described for 3a. The crude product was purified by FC
(CH2Cl2/MeOH; 8:2) and recrystallized from EtOH to afford 5d as
white crystals (yield: 49%). Mp >250 °C. 1H NMR (DMSO-d6) d:
8.32 (t, J = 5.4 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.65–7.59 (m, 2H),
7.55 (d, J = 8.4 Hz, 2H), 7.52–7.38 (m, 6H), 7.36–7.29 (m, 1H),
7.11 (d, J = 8.4 Hz, 2H), 7.06 (s, 1H), 3.65–3.54 (m, 4H), 3.31–3.26
(m, 2H), 3.10–2.94 (m, 2H), 2.35 (s, 3H), 2.05–1.82 (m, 4H). IR
(cmꢀ1: neat): 1636, 1555, 1326, 1163. LC–MS (APCI+) m/z: 529
(M+H)+. Anal. Calcd for C30H32N4O3S: C, 68.16; H, 6.10; N, 10.60.
Found: C, 68.32; H, 5.96; N, 10.56.
3.55 (m, 4H), 3.40–3.31 (m, 2H), 2.47–2.38 (m, 6H), 2.33 (s, 3H).
IR (cmꢀ1: neat): 1622, 1552, 1339, 1236, 1164. LC–MS (APCI+) m/
z: 545 (M+H)+. Anal. Calcd for C30H32N4O4S: C, 66.16; H, 5.92; N,
10.29. Found: C, 66.45; H, 6.04; N, 10.22.
4.1.32. N3-(3-Morpholinopropyl)-1-[4-(aminosulfonyl)phenyl]-
2-methyl-5-(4-biphenylyl)-1H-3-pyrrolecarboxamide (5i)
The title compound was prepared from carboxylic acid 3c and
N-(3-morpholinopropyl)amine according to the same procedure
than that described for 3a. The crude product was recrystallized
from EtOH to afford 5i as white crystals (yield: 36%). Mp >250 °C.
1H NMR (DMSO-d6) d: 7.93–7.87 (m, 3H), 7.65–7.59 (m, 2H), 7.54
(d, J = 8.4 Hz, 2H), 7.51–7.46 (m, 4H), 7.45–7.38 (m, 2H), 7.36–
7.29 (m, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.92 (s, 1H), 3.61–3.55 (m,
4H), 3.29–3.21 (m, 2H), 2.39–2.28 (m, 9H), 1.73–1.61 (m, 2H). IR
(cmꢀ1: neat): 1629, 1552, 1329, 1165. LC–MS (APCI+) m/z: 559
(M+H)+. Anal. Calcd for C31H34N4O4S: C, 66.64; H, 6.13; N, 10.03.
Found: C, 66.73; H, 6.21; N, 10.23.
4.1.28. N3-(2-Piperidinoethyl)-1-[4-(aminosulfonyl)phenyl]-2-
methyl-5-(4-biphenylyl)-1H-3-pyrrolecarboxamide (5e)
The title compound was prepared from carboxylic acid 3c and
N-(2-aminoethyl)morpholine according to the same procedure
than that described for 3a. The crude product was purified by FC
(CH2Cl2/MeOH; 9:1) and recrystallized from EtOH to afford 5e as
white crystals (yield: 62%). Mp: 148–150 °C. 1H NMR (300 MHz,
DMSO-d6) d: 8.38 (t, J = 4.9 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.62
(d, J = 8.2, 2H), 7.56–7.30 (m, 9H), 7.12–7.06 (m, 3H), 3.67–3.58
(m, 2H), 3.21–3.13 (m, 2H), 2.55–2.40 (m, 6H), 2.35 (s, 3H), 1.84–
1.73 (m, 4H). IR (cmꢀ1: neat): 1629, 1556, 1330, 1285, 1162. LC–
MS (APCI+) m/z: 543 (M+H)+. Anal. Calcd for C31H34N4O3S: C,
68.61; H, 6.31; N, 10.32. Found: C, 68.51; H, 6.24; N, 10.12.
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 (for maintaining
constant 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 done with the assay buffer mentioned
above. At least seven different inhibitor concentrations were used
for measuring the inhibition constant. Inhibitor and enzyme solu-
tions 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 inhibitor concentration
and the values reported throughout the paper are the mean of such
results. Kis were obtained from Lineweaver–Burk plots, as reported
earlier.24
4.1.29. N3-[2-(4-Methylpiperazino)ethyl]-1-[4-(aminosulfonyl)-
phenyl]-2-methyl-5-(4-biphenylyl)-1H-3-pyrrolecarboxamide
(5f)
The title compound was prepared from carboxylic acid 3c and
2-(4-methylpiperazino)ethylamine according to the same proce-
dure than that described for 3a. The crude product was purified
by FC (CH2Cl2/MeOH; 8:2) and recrystallized from EtOH to afford
5f as white crystals (yield: 43%). Mp: 235–237 °C. 1H NMR
(DMSO-d6) d: 7.90 (d, J = 8.4 Hz, 2H), 7.79 (t, J = 5.5 Hz, 1H), 7.65–
7.59 (m, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.51–7.46 (m, 4H), 7.44–
7.38 (m, 2H), 7.36–7.29 (m, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.90 (s,
1H), 3.36–3.29 (m, 2H), 2.47–2.39 (m, 7H), 2.35–2.28 (m, 6H),
2.14 (s, 3H). IR (cmꢀ1: neat): 1638, 1554, 1329, 1236, 1171. LC–
MS (APCI+) m/z: 558 (M+H)+. Anal. Calcd for C31H35N5O3S: C,
66.76; H, 6.33; N, 12.56. Found: C, 66.71; H, 5.96; N, 12.51.
4.3. Molecular modeling and calculations
4.1.30. N3-[3-(4-Methylpiperazino)propyl]-1-[4-(aminosulfonyl)
phenyl]-2-methyl-5-(4-biphenylyl)-1H-3-pyrrolecarboxamide
(5g)
The compounds were built using the SKETCH module, as imple-
mented in SYBYL (version 8.0),25 and their geometry was optimized
using the MINIMIZE module. The minimization process uses the
POWELL method with the TRIPOS force field (dielectric constant
1r) to reach a final convergence of 0.01 kcal molꢀ1. Docking simu-
lation was then performed into hCA IX (RCSB Protein Data Bank
3IAI)14 with the automated GOLD program.26 The active site was
defined including all residues in a volume of 10 Å around acetazol-
amide taken as reference.
The title compound was prepared from carboxylic acid 3c and
3-(4-methylpiperazino)propylamine according to the same proce-
dure than that described for 3a. The crude product was purified
by recrystallization from EtOH to afford 5g as white crystals (yield:
63%). Mp >250 °C. 1H NMR (DMSO-d6) d: 7.97–7.84 (m, 3H), 7.68–
7.58 (m, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.49–7.46 (m, 4H), 7.44–7.39
(m, 2H), 7.35–7.29 (m, 1H), 7.11 (d, J = 8.9 Hz, 2H), 6.91 (s, 1H),
3.27–3.21 (m, 2H), 3.36–3.30 (m, 11H), 2.55–2.42 (m, 2H), 2.12
(s, 3H), 1.69–1.60 (m, 2H). IR (cmꢀ1: neat): 1632, 1555, 1330,
1169. LC–MS (APCI+) m/z: 572 (M+H)+. Anal. Calcd for
The theoretical lipophilicity, expressed as log D7.4 calculated at
pH 7.4, and solubility, calculated at the same pH were evaluated by
means of the ACD/Structure Designer program v12.0.22
C32H37N5O3S: C, 67.22; H, 6.52; N, 12.25. Found: C, 67.14; H,
Acknowledgments
6.61; N, 12.06.
The authors are grateful to the institutions that support our
laboratory: Univ Lille Nord de France and the ‘Ligue Nationale con-
tre 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 project was financed by grant
PRIM-CAIRICOL. R.F. is Postdoctoral Researcher from the Belgian
F.R.S.-FNRS. We are grateful to Lionel Pochet for helpful discussion
in solubility evaluation. This research was financed (in part) by an
EU FP 7 project (METOXIA) to C.T.S and A.S.
4.1.31. N3-(2-Morpholinoethyl)-1-[4-(aminosulfonyl)phenyl]-2-
methyl-5-(4-biphenylyl)-1H-3-pyrrolecarboxamide (5h)
The title compound was prepared from carboxylic acid 3c and
N-(2-aminoethyl)morpholine according to the same procedure
than that described for 3a. The crude product was purified by FC
(CH2Cl2/MeOH; 95:5) and recrystallized from EtOH to afford 5h
as white crystals (yield: 57%). Mp >250 °C. 1H NMR (DMSO-d6) d:
7.90 (d, J = 8.5 Hz, 2H), 7.82 (t, J = 5.6 Hz, 1H), 7.62 (d, J = 8.5 Hz,
2H), 7.57–7.29 (m, 9H), 7.10 (d, J = 8.5 Hz, 2H), 6.91 (s, 1H), 3.63–

S. Gluszok et al. / Bioorg. Med. Chem. 18 (2010) 7392–7401
7401
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