Indanesulfonamides as carbonic anhydrase inhibitors. Toward structure-based design of selective inhibitors of the tumor-associated isozyme CA IX

Article abstract of DOI:10.1021/jm0600287

Carbonic anhydrases are ubiquitous metalloenzymes which are involved in fundamental processes (i.e., acid-base regulation, respiration, calcification, etc.). The carbonic anhydrase isozyme IX becomes an interesting pharmacological target due to its overexpression in cancer and its absence in normal tissue. Therefore, several indanesulfonamides were synthesized and tested for their inhibition both against the human CA IX and against two other biologically relevant isozymes (CA I and II). Structure-activity relationships are discussed and point out different compounds for its selectivity and activity against CA IX. To establish preliminary hypothesis for the design of new isozyme-selective CA IX inhibitors, we conducted molecular modeling. We describe here the first human CA IX model built by homology with another CA isozyme already crystallized. Docking studies were performed to explore the binding mode of our indanesulfonamide derivatives.

Full text of DOI:10.1021/jm0600287

J. Med. Chem. 2006, 49, 2743-2749
2743
Indanesulfonamides as Carbonic Anhydrase Inhibitors. Toward Structure-Based Design of
Selective Inhibitors of the Tumor-Associated Isozyme CA IX
Anne Thiry,† Marie Ledecq,† Alessandro Cecchi,‡ Jean-Michel Dogne´,† Johan Wouters,† Claudiu T. Supuran,‡ and
Bernard Masereel†,*
Drug Design and DiscoVery Center, UniVersity of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium, and Polo Scientifico,
Laboratorio di Chimica Bioinorganica, Rm. 188, UniVersita` degli Studi di Firenze, Via della Lastruccia 3,
50019 Sesto Fiorentino, Florence, Italy
ReceiVed January 10, 2006
Carbonic anhydrases are ubiquitous metalloenzymes which are involved in fundamental processes (i.e.,
acid-base regulation, respiration, calcification, etc.). The carbonic anhydrase isozyme IX becomes an
interesting pharmacological target due to its overexpression in cancer and its absence in normal tissue.
Therefore, several indanesulfonamides were synthesized and tested for their inhibition both against the human
CA IX and against two other biologically relevant isozymes (CA I and II). Structure-activity relationships
are discussed and point out different compounds for its selectivity and activity against CA IX. To establish
preliminary hypothesis for the design of new isozyme-selective CA IX inhibitors, we conducted molecular
modeling. We describe here the first human CA IX model built by homology with another CA isozyme
already crystallized. Docking studies were performed to explore the binding mode of our indanesulfonamide
derivatives.
Introduction
activation of CA9 gene by the hypoxia-inducible factor-1 (HIF-
1).22 Moreover, hypoxia stimulates CA IX to decrease the pHe
(by an yet unknown mechanism of action) proving that the
expression levels and the catalytic activity of CA IX are
dependent on the availability of oxygen within the tumor.23
CA IX is clearly implicated in the acidification of the pHe as
shown by Svastova et al.23 Teicher and collaborators also
demonstrated that acetazolamide (AZA) decreased the tumor
growth in vivo and enhanced the action of some chemothera-
peutic agents, such as cisplatin, melphalan, and PtCl4, when
used in combination therapy.24 Several CA IX-selective sul-
fonamide inhibitors were able to reduce the extracellular
acidification of Madin-Darby canine kidney (MDCK)-CA IX
cells in hypoxia but not in normoxia.23 Moreover, a decrease
of the pHe reduces the cytotoxicity of weakly basic chemo-
therapeutics drugs such as paclitaxel, mitoxantrone, and topo-
tecan.25 Taken together, these data increase the interest for
selective CA IX inhibitors in cancer therapy. Such compounds
may prevent the decrease of pHe and may be used in combina-
tions with other antitumor drugs to increase the efficacy or the
uptake of weakly basic drugs.15,24 Thus, the aim of the present
study is the design, synthesis, and in vitro pharmacological
evaluation of original indanesulfonamides as selective CA IX
inhibitors.
At least 13 enzymatically active R-carbonic anhydrase (CA,
EC 4.2.1.1) isoforms have been discovered in higher verte-
brates.1-4 These metalloenzymes are involved in the catalysis
of an important physiological reaction: the hydration of CO2
to bicarbonate and a proton (CO2 + H2O T HCO3- + H+). As
a consequence, they regulate the pH, the secretion of electrolytes,
and the respiration by their presence throughout neural pathways
controlling ventilation.5-7 CAs also play a major role in
biosynthetic pathways which require CO2/bicarbonate as sub-
strate such as gluconeogenesis, lipogenesis, ureagenesis, and
pyrimidines synthesis.8 Other roles for these enzymes were
highlighted, such as calcification and bone resorption.9 The
discovery that CA IX, a transmembrane tumor-associated
protein,10 was prevalently expressed in several human cancer
cells and not in their normal counterparts11 suggests a role for
some CAs in oncogenesis.8 Several studies showed a clear-cut
relationship between high CA IX expression levels in tumors
and a poor prognosis.12,13 CA IX also acts on cells adhesion
and differentiation by its N-terminal proteoglycan related-region
which is absent in other transmembrane CA isozymes, such as
CA XII and CA XIV.14
Tumor cells have a lower extracellular pH (pHe) than normal
cells.15 An acidic pHe contributes to increase tumor progression
by promoting the action of growth factors16,17 and proteases18
and an increased rate of mutation.19 Recently CO2 and lactic
acid were demonstrated as a significant source of acidity in
tumors, pointing out the implication of CA in tumor progres-
sion.20 The expression of CA IX is both regulated by the von
Hippel-Lindau (VHL) tumor suppressor protein and by hypoxia
present in some tumors. Thus, the inactivation of the VHL factor
gene enhances the expression of CA IX,21 whereas hypoxia
induces the expression of CA IX through a direct transcriptional
A series of indanesulfonamide derivatives was synthesized
and tested for their inhibitory activity against CA IX, and against
CA I and II, two other physiologically relevant CA isozymes.
To better understand the structure-activity relationships (SAR)
of this series, we present here the homology modeling of the
isozyme IX based on CA XIV. This is the first CA IX model
described and used to perform docking studies and to establish
preliminary hypothesis for the design of new compounds.
Results and Discussion
Chemistry. To date, X-ray crystallographic structures are not
available for CA IX thus preventing a structure-based design
of selective CA IX inhibitors. The indanesulfonamide template
was first described by our group as a potent inhibitor against
* Corresponding author. Phone, +32 81 72 43 38. Fax, +32 81 72 42
99. E-mail: bernard.masereel@fundp.ac.be.
† University of Namur.
‡ Universita` degli Studi di Firenze.
10.1021/jm0600287 CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/01/2006

2744 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Thiry et al.
Chart 1
Figure 1. Structural elements of CA inhibitors in the CA enzymatic
active site. A sulfonamide attached to the scaffold coordinates the zinc
ion and interacts with threonine by H bond. The side chain might
possess a hydrophilic link able to interact with the hydrophilic part of
the active site and a hydrophobic moiety which can interact in the
hydrophobic pocket.
Scheme 1. Synthesis of Indanesulfonamides from 1- or
2-Aminoindane
Table 1. Inhibition Data of Indanesulfonamide Derivatives and
Standard Inhibitor Acetazolamide
KI (nM)
KI ratiosa
KI(hCAI)/
KI(hCAIX)
KI(hCAII)/
KI(hCAIX)
compound
hCA I
hCA II
hCA IX
CA I and II.26 On the basis of these preliminary results, we
designed indanesulfonamide derivatives based on the recently
described “tail approach”.3,27 A general pharmacophore was
drawn (Figure 1) from the analysis of CAs active site and from
the structure of inhibitors described in the literature.3 Figure 1
shows the structural elements required for the inhibition of CAs.
A side chain which can interact with the hydrophobic and
hydrophilic parts of the CA active site can substitute an aromatic
or heterocyclic sulfonamide scaffold. Therefore, different
hydrophobic side chains were incorporated in the indanesulfona-
mide scaffold with an amide linker which can interact with the
hydrophilic part of the active site (Figure 1). The selected
hydrophobic side chains (methyl, valproyl, cyclohexyl, and
pentafluorophenyl) substitute the indane moiety in positions 1
or 2. Such hydrophobic chains incorporated in other scaffolds
have already demonstrated a powerful CA inhibition.3,28,29
Racemic indanesulfonamides were synthesized following the
Scheme 1. Moreover, investigation of the elongation of the side
chain with a n-ethyl, n-propyl, n-butyl, and n-nonyl was
performed in position 2 of the indane ring.
CA Inhibition. The inhibition activities of indanesulfonamide
derivatives were evaluated against hCA I, II, and IX and are
reported in Table 1. Acetazolamide was used as a standard
inhibitor for comparison. As seen from Table 1, nearly all the
derivatives show a high inhibitory potency against CA IX and
CA II and a weak inhibition against CA I. The KI ratios of CA
I over CA IX reveal that compounds 5, 12b, 11c, 12f, and 12g
are selective against CA IX while the KI ratios of CA II over
CA IX point out that compounds 12b, 11c, 12f, and 12g
preferentially inhibit CA IX. Among these selective CA IX
inhibitors, 11c is also characterized by a potent inhibitory
potency against this isozyme.
AZA
5
6
900
56500
2510
810
4130
880
3560
770
600
12
186
1.0
1.2
0.9
32
118
490
6.7
10
25
193
290
37.5
282
108
30
3.5
33
2015
2320
3.4
36
292
8.6
22
15
0.48
0.964
0.0034
0.032
0.003
0.30
3.9
140
0.20
11a
12a
11b
12b
11c
12c
12d
12e
12f
12g
8.1
118
220
18.2
1.7
0.35
257
1440
3440
825
875
0.0050
0.020
2.2
46
7.5
128
5330
3.7
35
a The KI ratios are indicative of the inhibition selectivity. A weak selective
inhibitor is characterized by low KI ratios.
the side chain on CA inhibition: a methyl provides a Ki of 290
nM (6). An additional carbon (12d, n-ethyl side chain) increases
the Ki value (2015 nM), and the n-propyl side chain (12e) leads
to a higher Ki value (2320 nM). Surprisingly, the inhibition
becomes more efficient with a side chain containing four carbons
(12f, n-butyl moiety) with a KI of 3.4 nM. The n-nonyl moiety
(12g) provides a comparable inhibition. We can conclude that
the optimum length of the side chain for the design of new
inhibitors seems to be about four carbons. (ii) Influence of the
substitution pattern of the indanesulfonamide scaffold on CA
inhibition revealed the following: a methyl group substituted
in position 1 (5) or 2 (6) leads to the same range of inhibitory
potency. With other substituents, some differences can be
highlighted between the corresponding regioisomers (compare
11a-c and 12a-c). On one hand, position 1 seems to be
favorable for a valproyl (11a vs 12a) and a pentafluorophenyl
group (11c vs 12c). On the other hand, a cyclohexyl ring leads
to a better inhibition constant when it is present in position 2
(12b).
The analysis of the Ki values obtained for CA IX can be
summarized in two points. (i) The influence of the length of

Indanesulfonamides as Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2745
Table 2. Selection of Template for CA IX. BLASTp Determination for
the Identity Percentage of the Best Ranked Crystallized Proteins
protein
PDB code
identity percentage
E value
mCA XIV
hCA XII
hCA II
1RJ532
1JCZ45
1CA248
38
36
33
2 × 10-52
2 × 10-49
5 × 10-32
We can conclude that sulfonamides 11c, 12f, and 12g are
potent CA IX inhibitors with a Ki value in the range of 3.4-
3.7 nM. They are characterized by a good selectivity toward
CA IX in comparison to CA I and CA II. In comparison with
the reference drug AZA, these three drugs are more potent and
selective for hCA IX than AZA.
Molecular Modeling
In an attempt to rationalize the high inhibitory activity and
selectivity of compound 11c, we conducted molecular modeling
studies of the enzyme/inhibitor interaction. Similar studies were
performed on the less active and less selective regioisomer 12c.
Due to the lack of experimental 3D data for CA IX, the first
step was to build a model of this isozyme. The modeled active
site was then used to investigate the interactions between CA
IX and both regioisomers of 11c and 12c. We explored through
docking studies, the binding mode of these two inhibitors.
1. Comparative Modeling. Three-dimensional (3D) protein
structure is an important tool to highlight the function of a
protein and the interaction with ligands. Homology (comparative
modeling) is currently the most accurate method to predict the
3D structure of proteins when no experimental structure is
available.30 The modeling process generally consists of four
steps: (1) databank search to identify the template, (2) target-
template alignment, (3) model building and optimization, and
(4) quality assessment of the resulting structure. To identify the
template, the protein-protein BLAST algorithm31 was per-
formed on the sequence of hCA IX. The identity percentage
between hCA IX and several templates have been determined.
Three CA isozymes with a 3D available structure provide the
best ranking score (Table 2). Among them, murine CA XIV
(mCA XIV, available on the Protein Data Bank of Brookaven,
PDB, www.rcsb.org/pdb, PDB code 1RJ5)32 provides the best
identity percentage (38%) and was chosen as template to build
the CA IX model. The target-template alignment step is
generally accepted as the most critical step in homology
modeling.33 Therefore, to improve the accuracy of this essential
stage, a “consensus” alignment (Table 3) between the target
and the chosen template was used with the aid of the automated
Figure 2. The final 3D model of hCA IX where R-helixes are red and
â-sheets are yellow. The active site is delimited by three histidines
which coordinate the zinc ion.
program of homology modeling ESyPred3D.30 All the key
regions corresponding to the amino acids of the active site and
the metal binding site were correctly aligned.
On the basis of this “consensus” alignment, a 3D model was
built with the MODELLER program34 (Figure 2). This final
model was then evaluated against the Ramachandran plot35
provided by the PROCHECK program.36 Only two amino acids,
Thr361 and Ser368 were found in the disallowed region of the
Ramachandran plot. They are located in loops far from the active
site and would probably not modify its topology. It is generally
considered that if at least 90% of the amino acids are in the
allowed region the quality of the model can be evaluated as
good.36 In this case, the model contains 98.4% of the amino
acid in the allowed region and only 0.5% in the disallowed
region. These results showed that our model is reliable for
performing further docking studies.
2. CA IX Active Site Characterization. Superimposition
of the 3D structure of hCA II available from the PDB (PDB
code 1A42)37 and hCA IX model is represented in Figure 3.
Both active sites form a cone where the summit consists of the
zinc ion coordinated to three histidine residues. Hydrophobic
and hydrophilic pockets are also present. Amino acids of the
active site1 are highly conserved (Figure 3). Nevertheless, we
can note three differences in the amino acid sequence of the
active site of hCA IX as compared to hCA II: Thr205, Val262,
Table 3. Sequences Alignments Generated by ESyPred3D for Human CA IX (hCA IX) and the Template Murine CA XIV (mCA XIV)

2746 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Thiry et al.
correctly. A hydrogen bond is established between the sulfona-
mide nitrogen atom and the hydroxyl moiety of Thr332. One
of the oxygen atoms of the sulfonamide moiety is H-bonded
with the backbone amide of Thr332. The hydrophobic side chain
of the inhibitors interacts with the active site residues following
two orientations. In the first, the pentafluorophenyl moiety of
S-11c, R-11c, and S-12c forms a 90° dihedral angle with the
indane moiety. It lies nearby residues Leu331, Ala337, Leu266.
In the second, the pentafluorophenyl moiety of compound R-11c
and the indane moiety are coplanar. In this case, the pentafluo-
rophenyl moiety is surrounded by Asp263, Gln203, and Thr205.
The carbonyl oxygen of R-11c makes an H bond with the
backbone amide nitrogen of Gln203. This interaction could
direct the hydrophobic substituent toward the three “specific”
residues of the CA IX active site. Therefore, we can hypothesize
that regarding the activity and selectivity of both racemics 11c
in comparison with their regioisomers 12c, derivative R-11c
could provide the best activity and selectivity against CA IX.
4. Design of New Selective CA IX Inhibitors. The develop-
ment of selective CA IX inhibitors will be interesting either to
avoid adverse effects due to the inhibition of other CAs and
also to study the exact physiological role of this isozyme.27 This
goal is difficult to achieve due to the high similarity of the CA
isozyme’s active site. Two approaches can be planned. Recently,
the incorporation of a positively charged moiety which confers
membrane-impermeability properties to the molecule has been
developed.40 Such compounds show improved selectivity for
the targeted CA IX because they are confined in the extracellular
compartment.23 They can consequently inhibit only membrane-
associated CA isozyme possessing an extracellular catalytic
domain like CA IX but not the cytosolic CA isozymes (CA
II).41 On the other hand, it is possible to target specifically the
nonconserved residues of hCA IX active site. Three amino acids
have been pointed out in our study: Val262, Asp263, and
Thr205 (which correspond respectively to the Phe129, Gly130,
and Glu68 residues of hCA II). In fact, several substitutions
could be envisaged. (i) The presence of the Asp263 residue in
hCA IX could be interesting for the design of new inhibitors.
Indeed, the incorporation of a positively charged side chain
could form an ionic interaction with this moiety and would
enhance the selectivity. Moreover, it is interesting to note that
such compound will not enter into the cell where CA I and CA
II are located. (ii) Bulky groups should be chosen to avoid the
interaction with the phenyl moiety of Phe129 present in CA II.
(iii) Neutral groups which can form H bond with the hCA IX
Thr205 could also enhance the selectivity. (iv) The amide group
present in our molecules should be maintained to allow the
formation of an additional H bond with hCA IX Glu203. Such
interaction could compel the inhibitor to interact with specific
residues of hCA IX.
Figure 3. Superimposition of CA II and hCA IX active sites. The
conserved active site residues are colored by atom; differences between
amino acids of hCA IX and CA II are colored respectively in blue and
in red.
Figure 4. Interaction modes of 11c and 12c with hCA IX model after
refinement of the best ranked docked solution. Specific hCA IX residues
are colored in blue. Compound S-11c is represented in cyan, R-11c in
orange, S-12c in dark blue, and R-12c in yellow. H bond are indicated
in green dotted.
and Asp263 replace Glu68, Phe129, and Gly130 of hCAII,
respectively. These differences are associated with a modifica-
tion of some physicochemical properties. Moreover, those amino
acids are known to be involved in the binding of inhibitors.38
Therefore, these features are relevant for the design of selective
CA IX inhibitors.
Conclusion
3. Docking. The docking program GOLD39 (Genetic Opti-
mization for Ligand Docking) was used to simulate the
interaction between the inhibitors and the active site of CA IX
model. Docking studies were carried out in order to understand
why the incorporated pentafluorophenyl in position 1 on indane
(11c) enhances by 10 fold the inhibitory constant in comparison
with its regioisomer (12c). For each compound, R and S
enantiomers were considered. All the compounds are anchored
into the active site by the zinc complexation of the sulfonamide
moiety (Figure 4). Indeed, sulfonamides are known to bind to
the zinc ion in a tetrahedral adduct by substituting the nonprotein
zinc ligand.1 The docking studies modeled this major interaction
The synthesis and the biological evaluation of racemic
indanesulfonamide derivatives on hCA I, II, and IX are reported
in this paper. Two indanesulfonamides derivatives, the 1-pen-
tafluorophenylamidoindane-5-sulfonamide (11c) and the 2-non-
ylamidoindane-5-sulfonamide (12g), possess an interesting
profile characterized by a high inhibitory potency and selectivity
against hCA IX. In addition, we have performed homology
modeling and docking studies to rationalize the observed
activities and to design new selective inhibitors. Since the 3D
structure of CA IX is not yet available, we have built a model
for hCA IX by homology with a similar isozyme already
crystallized. Alignment of sequences provides mCA XIV as the

Indanesulfonamides as Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2747
ethyl acetate and ice to hydrolyze the excess of acid. The organic
layer was isolated and rapidly evaporated under reduced pressure.
The chlorosulfonyl compound was dissolved in THF and poured
into an excess of an aqueous ammonia solution. The corresponding
sulfonamide was crystallized from ethanol/water (1/1).
1-Acetamido-5-sulfonamidoindane (5). The title compound
(585 mg, 3.34 mmol) reacted according to the general procedure
previously described. Yield: 55.4%; Mp: 202.8-204.6 °C; Anal.
(C11H14N2O3S) C, H, N, S.
2-acetamido-5-sulfonamidoindane (6). The title compound
(1.46 g, 8.36 mmol) reacted according to the general procedure
previously described. Yield: 35%; Mp: 217.8-221.6 °C; Anal.
(C11H14N2O3S) H, N; C: calcd, 51.95; found, 51.06; S: calcd,
12.61; found, 11.96.
1-valproylamido-5-sulfonamidoindane (11a). The title com-
pound (650 mg, 2.51 mmol) reacted according to the general
procedure previously described. Yield: 30,6%; Mp: 205.9-207.2
°C; Anal. (C17H26N2O3S) C, H, N, S.
1-cyclohexylamido-5-sulfonamidoindane (11b). The title com-
pound (407 mg, 1.67 mmol) reacted according to the general
procedure previously described. Yield: 46,5%; Mp: 198.9-202.1
°C; Anal. (C16H22N2O3S) C, H; N: calcd, 8.69; found, 8.14; S:
calcd, 9.95; found, 9.14.
best template for homology constructing. Our model was
evaluated, and its quality can be considered as good. Docking
studies were then performed in this model with the most active
and selective inhibitors against CA IX, compound 11c. The
corresponding regioisomer (12c) which was less active and less
selective was also docked to understand the SAR. Enantiomers
R and S of 11c and 12c were considered. One binding mode
was observed for both enantiomers of 12c, but two different
binding modes are reported for the enantiomers of 11c.
Compound R-11c established an additional H-bond with Gln203
and could place its the side chain toward the specific residue
of the CA IX active site. Separating and determining the activity
of each enantiomer will give supplementary information on these
docking studies. Moreover comparison of the ubiquitous
isozyme hCA II and the tumor associated CA IX active site
have pointed out the hCA IX nonconserved active site residues
Asp263 and Thr205. These residues could be specifically
targeted for the design of new selective compounds.
We intend to test compound 11c on MDCK cells in order to
evaluate its ability to reduce the medium acidification.23 Then
we will also determine if 11c can modify the cellular uptake of
anticancer drugs and modulate the response of tumor cells to
conventional chemo- and radiotherapy.15,42
1-pentafluorophenylamido-5-sulfonamidoindane (11c). The
title compound (821 mg, 2.51 mmol) reacted according to the
general procedure previously described. Yield: 56.5%; Mp: 241.5-
243.9 °C; Anal. (C16H11F5N2O3S) H, N, S; C: calcd, 47.29; found,
48.56.
Experimental Section
General. Reagents and solvents are supplied by Acros Organics,
Sigma-Aldrich, or Merck Eurolab. They are used without purifica-
tion. Reactions are followed by TLC performed on a silica gel
Merck 5544, and spots are revealed under 254 nm UV illumination.
Melting points were performed on a Tottoli-Bu¨chi Melting Point
B540 in open capillary tubes and are uncorrected. 1H NMR spectra
were recorded on a JEOL JNM EX 400 at room temperature using
DMSO-d6 as solvent and tetramethylsilane as internal standard.
Chemical shifts are expressed in δ (ppm) and coupling constants
(J) in hertz. The abbreviations s ) singulet, d ) doublet, dd)
doublet of doublet, t ) triplet, m ) massif, m ) multiplet, q )
quadruplet, br ) broad were used throughout. Electron ionization
mass spectra were recorded on an Agilent 1100 Series MSD trap
SL. Elemental analyses (C, H, N, S) were performed on a
ThermoFinnigan Flash EA 112 elemental analyzer.
Synthesis of Indanesulfonamides. General Procedure. Com-
pounds 3 and 4 were prepared by reacting 3 g of 1- or 2-aminoin-
dane (1 or 2) (22.5 mmol) with 1.85 g of sodium acetate (22.5
mmol) and 30 mL of acetic anhydride. The mixture was stirred at
room temperature for 2 h and then concentrated under reduced
pressure. The residue was extracted three times by chloroform (3
× 50 mL). The organic layers were combined, washed with water,
with HCl 1% (p/v), with Na2CO3 5% (p/v), and with brine solution,
and dried with MgSO4.
Compounds 9a-c and 10a-g: 2 g of 1- or 2-aminoindane (1
or 2) (11.8 mmol) was dissolved in chloroform (15 mL). A small
excess of the corresponding acid chloride (8a-g) (14.2 mmol) was
added. If the acyl chloride is not commercially available, it was
prepared from 2 g of the corresponding carboxylic acid (7a, 13.9
mmol) by refluxing with an excess of thionyl chloride (20 mL) for
1 h. The excess of SOCl2 was evaporated under reduced pressure.
At the end of the reaction between the acid chloride and the 1- or
2-aminoindane monitered by TLC, the residue was extracted three
times by ether or chloroform (3×50 mL). The organic layers were
combined and washed several times with water, HCl 1% (p/v), Na2-
CO3 5% (p/v), and brine solution. The organic extract was then
dried upon MgSO4 and evaporated under reduced pressure. The
residue was purified by crystallization from ether/pentane or ethyl
acetate/pentane.
2-valproylamido-5-sulfonamidoindane (12a). The title com-
pound (650 mg, 2.51 mmol) reacted according to the general
procedure previously described. Yield: 40%; Mp: 101.2-105.6
°C.
2-cyclohexylamido-5-sulfonamidoindane (12b). The title com-
pound (611 mg, 2.51 mmol) reacted according to the general
procedure previously described. Yield: 47%; Mp: 180-182.1 °C;
Anal. (C16H22N2O3S) H, N; C: calcd, 59.60; found, 59.14; S: calcd,
9.95; found, 9.36.
2-pentafluorophenylamido-5-sulfonamidoindane (12c). The
title compound (1.09 g, 3.34 mmol) reacted according to the general
procedure previously described. Yield: 16,6%, Mp: 174.7-177.1
°C; Anal. (C16H11F5N2O3S) C, H, N, S.
2-ethylamido-5-sulfonamidoindane (12d). The title compound
(633 mg, 3.34 mmol) reacted according to the general procedure
previously described. Yield: 51,3%; Mp: 179.1-181.0 °C; Anal.
(C12H16N2O3S) C, H, N, S.
2-propylamido-5-sulfonamidoindane (12e). The title compound
(510 mg, 2.51 mmol) reacted according to the general procedure
previously described. Yield: 23.2%; Mp: 188.5-190.7 °C; Anal.
(C13H18N2O3S) H, N, S; C: calcd, 55.30; found, 54.56.
2-butylamido-5-sulfonamidoindane (12f). The title compound
(545 mg, 2.51 mmol) reacted according to the general procedure
previously described. Yield: 21.8%, Mp: 131.5-133.2 °C; Anal.
(C14H20N2O3S) H, N; C: calcd, 56.73; found, 56.15; S: calcd,
10.82; found, 10,03.
2-nonylamido-5-sulfonamidoindane (12g). The title compound
(921 mg, 2.51 mmol) reacted according to the general procedure
previously described. Yield: 49%; Mp: 132.8-134.9 °C; Anal.
(C19H30N2O3S) H, N, S; C: calcd, 62.26; found, 62.71.
CA Inhibition. CA I and II were supplied by Sigma-Aldrich.
The recombinant CA IX enzyme was obtained as reported earlier
by Vullo et al.43 An SX.18MV-R Applied Photophysics stopped-
flow instrument has been for assaying the CA CO2 hydration activity
assays.44 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), following the CA-catalyzed CO2
hydration reaction for a period of 10-100 s. Satured CO2 solution
in water at 20 °C was used as substrate. Stock solutions of inhibitor
(1 mM) were prepared in distilled-deionized water with 10-20%
(v/v) DMSO (which do not influence the measure at these
concentrations), and dilutions up to 0.1 nM were done thereafter
Compounds 5, 6, 11a-c, and 12a-g: The substituted indane
(1 equiv) was added to chlorosulfonic acid (18 equiv) at -60 °C
under argon. The mixture remains under stirring for 2 h at room
temperature. The mixture was carefully poured into a mixture of

2748 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Thiry et al.
with distilled-deionized water. To allow for the formation of the
E-I complex, inhibitor and enzyme solutions were preincubated
during 15 min at room temperature prior to assay. Enzyme
concentrations were 1.0 µM for CA I and II and 0.1 µM for CA
IX. Each experiment was done in triplicate. The values reported
throughout the paper were calculated as described in the literature.44
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Acknowledgment. A. Thiry is very indebted to the “Fonds
pour la Formation a` la Recherche dans l′Industrie et dans
l′Agriculture (FRIA)” for the award of a research fellowship.
This work was funded by a FNRS grant (Belgian National Fund
for Scientific Research). This work was also partly supported
by an EU grant of the sixth framework program (EUROXY
project) to C.T.S.
Supporting Information Available: Ramachandran map of the
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