Carbonic anhydrase inhibitors. Design of anticonvulsant sulfonamides incorporating indane moieties

Article abstract of DOI:10.1016/j.bmcl.2004.09.061

A series of aromatic sulfonamides incorporating indane moieties were prepared starting from commercially available 1- and 2-indanamine, and their activity as inhibitors of two carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I and II was studied. The new sulfonamides incorporating acetamido, 4-chloro-benzoyl, valproyl, tetra-, and pentafluorobenzoyl moieties acted as very potent inhibitors of the slow red blood cell isozyme hCA I (K_is in the range of 1.6-8.5 nM), which usually has a lower affinity for such inhibitors, as compared to isozyme II. Some derivatives also showed excellent hCA II inhibitory properties (K_is in the range of 2.3-12 nM), but the anticonvulsant activity of these sulfonamides was rather low as compared to that of other sulfonamide/sulfamate CA inhibitors, such as methazolamide. Furthermore, the 2-amino/acetamido-indane-5-sulfonic acids prepared during this work also showed interesting CA inhibitory properties, with inhibition constants in the range of 43-89 nM against the two isozymes, being among the most potent sulfonic acid CA inhibitors reported so far.

Full text of DOI:10.1016/j.bmcl.2004.09.061

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5781–5786
Carbonic anhydrase inhibitors. Design of anticonvulsant
sulfonamides incorporating indane moieties
b
b
Celine Chazalette,a,b Bernard Masereel,b, Stephanie Rolin, Anne Thiry,
*
´
Andrea Scozzafava,c Alessio Innocentic and Claudiu T. Supuranc,*
a
´ ´ ´
Universite Joseph Fourier de Grenoble, Laboratoire d’Etudes Dynamiques et Structurales de la Selectivite, Bat. C Chimie,
301 rue de la chimie, Domaine Universitaire, 38400 Saint-Martin d’Heres (Grenoble), France
ˆ
`
Universite de Namur, Departement de Pharmacie, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium
b
c
´
´
`
Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
I-50019 Sesto Fiorentino (Firenze), Italy
Received 16 June 2004; revised 3 September 2004; accepted 17 September 2004
Abstract—A series of aromatic sulfonamides incorporating indane moieties were prepared starting from commercially available 1-
and 2-indanamine, and their activity as inhibitors of two carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I and II was studied.
The new sulfonamides incorporating acetamido, 4-chloro-benzoyl, valproyl, tetra-, and pentafluorobenzoyl moieties acted as very
potent inhibitors of the slow red blood cell isozyme hCA I (Kis in the range of 1.6–8.5nM), which usually has a lower affinity for
such inhibitors, as compared to isozyme II. Some derivatives also showed excellent hCA II inhibitory properties (Kis in the range of
2.3–12nM), but the anticonvulsant activity of these sulfonamides was rather low as compared to that of other sulfonamide/sulfa-
mate CA inhibitors, such as methazolamide. Furthermore, the 2-amino/acetamido-indane-5-sulfonic acids prepared during this
work also showed interesting CA inhibitory properties, with inhibition constants in the range of 43–89nM against the two isozymes,
being among the most potent sulfonic acid CA inhibitors reported so far.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
been discovered, with two such drugs—dorzolamide
DZA and brinzolamide BRZ—now used clinically.4,5
All these CAI drugs have been designed by the ring
approach, that is, investigating a very large number of
ring systems incorporating sulfamoyl moieties.6 More
recently, an alternative approach, the ÔtailÕ one,6 has
been reported by our group for the design of antiglau-
coma CAIs with topical activity, but this approach has
then been extended for other applications of these phar-
macological agents.7–10 The tail approach consists of
attaching tails, that is, moieties that will induce the de-
sired physico-chemical properties to aromatic/heterocy-
clic sulfonamide scaffolds possessing derivatizable
amino or hydroxy moieties. By using this approach,
important progress has been achieved for evidencing
inhibitors with higher affinity for a certain isozyme,
although clear-cut isozyme-specific inhibitors are not
available at this moment for the different 14 CA iso-
zymes discovered so far in humans.11 Inhibitors selective
for the membrane associated (CA IV, IX, XII, and XIV)
versus the cytosolic isozymes were recently described,
belonging either to macromolecular compounds or to
Unsubstituted aromatic sulfonamides were known to
inhibit the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1) since the beginning of research in this field.1
Starting with the 1950s, potent CAIs belonging to the
heterocyclic sulfonamide class have been developed that
led to the benzothiadiazine and high-ceiling diuretics, as
well as to the systemic antiglaucoma drugs acetazol-
amide AAZ, methazolamide MZA, ethoxzolamide
EZA, and dichlorophenamide DCP.2,3
Discovering these drugs highly benefited the chemistry
of sulfonamides, as thousands of derivatives belonging
to the heterocyclic, aromatic, and bis-sulfonamide
classes have been synthesized and investigated for their
biological activity.3 In the late 80s and early 90s, the top-
ically effective antiglaucoma CA inhibitors (CAIs) have
*
Corresponding authors. Tel./fax: +32 081724338 (B.M.); tel.: +39
055 457 3005; fax: +39 055 457 3385 (C.T.S.); e-mail addresses:
bernard.masereel@fundp.ac.be; claudiu.supuran@unifi.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.061

5782
C. Chazalette et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5781–5786
Me
N
N
N
N
N
MeCONH
SO2NH2
S
MeCON
SO2NH2
S
EtO
S
SO2NH2
AAZ
SO2NH2
MZA
EZA
NHEt
NHEt
S
SO2NH2
SO2NH2
N
S
MeO(CH2)3
S
Cl
SO2NH2
S
Me
O
O
Cl
O
O
DCP
DZA
BRZ
SO2NH2
H
O
N
S
Cl
N
H
O
IND
the positively charged derivatives (of low molecular
weight).3,12 CAIs possessing relevant antitumor proper-
ties, and thus potent inhibition of the tumor-associated
isozyme CA IX were also discovered, with one such
derivative—indisulam IND—in advanced clinical trials
for the treatment of solid tumors.13 CAIs with good
anticonvulsant activity and several other biomedical
applications have recently been reported by our
groups.14 This field is in constant progress and may lead
to the discovery of highly interesting pharmacological
agents. All the seven drugs in clinical use/trials belong-
ing to the CAIs mentioned above, have been discovered
SO2NH2
SO2NH2
SO2NH2
H2N
O
.H2O
HCl
2
1
H3C
N
H
3
O
SO2NH2
O
SO2NH2
H
N
H
N
H3C
Cl
5
4
O
SO2NH2
H
N
H3C
F
F
O
F
SO2NH2
H
F
F
N
6
CH3
7
O
SO2NH2
F
F
O
F
SO2NH2
H
N
H
Cl
F
N
. H2O
OCH3
9
8
SO3H
O
SO3H
. H2O
H
H2N
N
H3C
11
10
O
H
N
O
H3C
H2N
HCl
12
H3C
N
H
14
13

C. Chazalette et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5781–5786
5783
CH3COO-Na+
(CH3CO)2O
by the ring approach, whereas most of the work from
O
our laboratory was dedicated to finding novel tails that
will induce useful applications to the CAIs that we de-
signed. Here we report sulfonamide derivatives belong-
ing to a ring system that has never been investigated
for its possible applications in the design of CAIs, that
is, the indane ting. Sulfonamides 1–9 and sulfonic acids
10 and 11 were thus prepared and investigated for their
CA inhibitory and anticonvulsant activity.
H2N
HCl
N
H
H3C
position 1 : 13
position 2 : 14
12
+ HClSO3
- H2O
SO2Cl
O
N
H
position 1 : 15
position 2 : 16
H3C
+ NH4OH
- H2O
2. Chemistry
In a preliminary experiment (data not published) we ob-
served that the indanesulfonamide derivative 1 showed
good CA I and II inhibitory properties (see later in the
text). Thus, we decided to investigate some derivatives
incorporating this ring system. For this reason, we
needed to prepare the amino-indanesulfonamide deriva-
tive(s) of 1, which were not described in the literature as
CAIs. Commercially available 1- and 2-aminoindanes 12
were used for this purpose: acetylation of 12 with acetic
anhydride/sodium acetate, led to the acetamido deriva-
tives 13 and 14 (Scheme 1).15 Both of them were then re-
acted with chlorosulfonic acid, leading to the sulfonyl
chlorides 15 and 16, which were converted to the two
isomeric sulfonamides 3 and 4 (the main product of this
reaction is the 5-sulfonyl chloride of type 15 and 16,
respectively).15 The acetamido intermediate 3 was
obtained pure after several recrystallizations from aceto-
nitrile/water (1/1). In the case of the acetamido
intermediate 4, only one compound is formed by treat-
ment of 14 with chlorosulfonic acid, and subsequent
amidation, due to the symmetric nature of the com-
pound. This procedure also allowed us to isolate and
characterize the hydrolysis product of the sulfonylchlo-
ride intermediate 16, the sulfonic acid 10. Its amino
function was then deprotected, leading to the aminosulf-
onic acid 11. Deprotection of the amino moiety of the
key intermediate 4, with concentrated hydrochloric acid,
afforded the aminosulfonamide 2 (as hydrochloride)
which was thereafter used for the preparation of the
derivatives 5–9, by reaction with acyl halides, as previ-
ously reported.7–10,16
SO2NH2
O
position 1 : 3
position 2 : 4
N
H
H3C
SO2NH2
O
SO2NH2
H2O
H
N
HCl(37%)
2 days
H2N
HCl
H3C
4
2
RCOCl
O
SO2NH2
R
HN
5 - 9
Scheme 1.
they seem to be involved in the anticonvulsant activity
shown by many sulfonamide drugs with CA inhibitory
properties.14 The inhibitory activity was evaluated meas-
uring the CO2 hydrase activity of these two isozymes
with the physiological substrate CO2.18,19 The inhibition
data are shown in Table 1.
The following should be noted regarding data of Table 1:
(i) compounds 1–11 investigated here act as quite potent
hCA I inhibitors, a feature which is noteworthy, since
this is an isozyme with a rather reduced affinity for sul-
fonamide inhibitors, as compared to sulfonamide-avid
isozymes such as CA II, IV, VII, or IX among others.3–5
Thus, compounds 1–11 show inhibition constants in
the range of 1.6–215nM against this isozyme, being
much more effective than the clinically used derivatives
acetazolamide and methazolamide, which show inhibi-
tion constants in the range of 780–900nM. Among deriv-
atives 1–11, the sulfonamide 1 and the two sulfonic acids
10 and 11 are moderate hCA I inhibitors (Kis of 43–
215nM), whereas the sulfonamides 2 and 5 already show
an increased affinity (Kis of 33–36nM). But the other sul-
fonamides, that is, 3, 4, 6–9 are indeed very potent hCA I
inhibitors, with Kis in the range of 1.6–8.5nM, being
among the most potent hCA I inhibitors ever reported.
Thus, this ring system incorporating either an 1-aceta-
mido- or 2-alkyl/arylcarboxamido tails, induces very po-
tent hCA I inhibitory properties to the corresponding
sulfonamides, whereas the simple unsubstituted sulfon-
amide 1 and the sulfonic acids are weaker CA I inhibitors.
The nature of the R moieties in derivatives 5–9 was
chosen considering the good activity of compounds
incorporating chloro-benzoyl, valproyl, tetra-, and
pentafluorobenzoyl moieties and other amino-sulfon-
amide ring systems, such as the sulfanilamide, homosulf-
anilamide, or 5-amino-1,3,4-thiadiazole-2-sulfonamide
derivatives, previously reported by this group (Scheme
1).9,14
3. Carbonic anhydrase inhibition and anticonvulsant
activity
All the compounds reported here were assayed for inhi-
bition of two CA isozymes involved in important phys-
iological processes, hCA I and hCA II.17 It is well
known that these isozymes play a crucial role in the
transport of CO2 in a large variety of tissues as well as
for the pH regulation in many organs.2–6 Furthermore,

5784
C. Chazalette et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5781–5786
Table 1. CA inhibition and MES data for compounds 1–11 reported in
the paper, and standard sulfonamide CA inhibitors, acetazolamide
(AAZ), and methazolamide (MZA)
isozyme hCA I, which usually has a lower affinity for
sulfonamide inhibitors, as compared to isozyme II.
Some derivatives also showed excellent hCA II inhibi-
tory properties, but the anticonvulsant activity of these
derivatives was rather low as compared to that of other
sulfonamide/sulfamate CA inhibitors.
Compound
Ki (nM)
hCA IIa
MES-testb % of
protected mice
hCA Ia
AAZ
MZA
900
780
215
33
12
14
52
28
19
NTd
100 (8/8)c
NTd
1
2
0 (0/8)
NTd
References and notes
3
8.5
4
7.12.3
36
1.6
38 (3/8)
38 (3/8)
1. Mann, T.; Keilin, D. Nature 1940, 146, 164–165.
2. Maren, T. H. Physiol. Rev. 1967, 47, 595–781.
3. Supuran, C. T.; Casini, A.; Scozzafava, A. Development
of Sulfonamide Carbonic Anhydrase Inhibitors (CAIs). In
Carbonic Anhydrase—Its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC Press:
Boca Raton (FL), USA, 2004; pp 67–148.
4. Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
2000, 10, 575–600.
5. Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Expert
Opin. Ther. Pat. 2004, 14, 667–702.
6. Supuran, C. T.; Scozzafava, A. Curr. Med. Chem.—Imm.,
Endoc. Metab. Agents 2001, 1, 61–97.
7. Ilies, M. A.; Vullo, D.; Pastorek, J.; Scozzafava, A.; Ilies,
M.; Caproiu, M. T.; Pastorekova, S.; Supuran, C. T.
J. Med. Chem. 2003, 46, 2187–2196.
5
7.5
3.4
31(2.5/8)
13 (1/8)
6
7
4.16.9
3.5
7.2
8
17
38 (3/8)
13 (1/8)
0 (0/8)
0 (0/8)
9
12
65
84
10
11
89
43
a Human, cloned isozyme.
b Determined as described in Ref. 14.
c % of mice protected (n = 8) against seizures induced by a maximal
electroshock (50mA, 0.2s). Drug (30mg/kg) was injected ip 3h prior
to electroshock.
d NT = not tested.
8. Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
5471–5477.
These sulfonic acids are also among the most potent such
nonsulfonamide inhibitors detected so far;2–5 (ii) Against
the major red cell isozyme hCA II, again the lead mole-
cule 1 as well as the sulfonic acids 10 and 11 show weak
inhibitory activity, with Kis in the range of 52–84nM.
The sulfonamides 2, 3, and 8 on the other hand are med-
ium-potency–strong inhibitors, with Kis in the range of
17–28nM, comparable to that of the clinically used sul-
fonamides acetazolamide and methazolamide. Excellent
CA II inhibitory activity was shown by sulfonamides
4–7 and 9, incorporating aliphatic (acetamido and
valproamido) and aromatic (4-chlorobenzamido-,
pentafluorobenzoylamido, and tetrafluorobenzoylamido)
moieties, which showed inhibition constants in the range
of 2.3–12nM. Thus, clearly the indanesulfonamide deriv-
atives with good affinity for hCA II can be designed
rather easy; (iii) several of the new derivatives, more pre-
cisely 4, 5, 7, and 8, showed a moderate anticonvulsant
activity (% of protected mice in the MES test of 31–
38%), which was much inferior to that of metahzol-
amide, which led to a 100% protection in the same
animal model. Compounds 6 and 9 were on the other
hand less effective anticonvulsants in the same model,
whereas the other investigated derivatives were devoid
of this property. Thus, indanesulfonamide derivatives
are not good lead molecules for the design of anticonvul-
sants belonging to the class of the CA inhibitors.
´
9. de Leval, X.; Ilies, M.; Casini, A.; Dogne, J.-M.; Scozzaf-
ava, A.; Masini, E.; Mincione, F.; Starnotti, M.; Supuran,
C. T. J. Med. Chem. 2004, 47, 2796–2804.
10. Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava,
A.; Supuran, C. T. J. Med. Chem. 2004, 47, 1272–1279.
11. Lehtonen, J.; Shen, B.; Vihinen, M.; Casini, A.; Scozzaf-
ava, A.; Supuran, C. T.; Parkkila, A. K.; Saarnio, J.;
Kivela, A. J.; Waheed, A.; Sly, W. S.; Parkkila, S. J. Biol.
Chem. 2004, 279, 2719–2727.
12. Casey, J. R.; Morgan, P. E.; Vullo, D.; Scozzafava, A.;
Mastrolorenzo, A.; Supuran, C. T. J. Med. Chem. 2004,
47, 2337–2347.
13. Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
14. (a) Masereel, B.; Rolin, S.; Abbate, F.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. 2002, 45, 312–320; (b) Ilies,
ˆ
M. A.; Masereel, B.; Rolin, S.; Scozzafava, A.; Campeanu,
G.; Cımpeanu, V.; Supuran, C. T. Bioorg. Med. Chem.
ˆ
2004, 12, 2717–2726.
15. Bondinell, W. E.; Pendleton, R. G. U.S. Patent 4,192,888,
1980.
16. 2-Acetamido-5-sulfonamidoindane 4: 2-aminoindane
hydrochloride (25g), sodium acetate (12g), and acetic
anhydride (200mL) were stirred one night at room
temperature. The evaporation of the mixture under reduce
pressure led to a white powder, which was extracted from
the aqueous phase using chloroform. The chloroform was
washed with HCl 10% and NaHCO3 5%, and dried upon
MgSO4. The evaporation of the solvent yields a white
powder of 14 whose NMR characteristics are given: 1H
NMR (DMSO) d ppm: 1.79 (s, 3H, CH3); 2.71–3.17 (m,
4H, CH2(1) and (3)); 4.42 (m, 1H, CH(2)); 7.13–7.22 (m,
4H, CH(4), (5), (6), and (7)); 8.14 (d, 1H, NH). The
powder of 14 was added to chlorosulfonic acid (18equiv)
at ꢀ80°C. The mixture remained under stirring for 1h at
room temperature. The excess of acid was then hydrolyzed
with ice water. The product was quickly extracted with
ethyl acetate, yielding the chlorosulfonyl compound after
evaporation. This was quickly dissolved in a minimum
4. Conclusion
We report a small library of indane-5-sulfonamide deriv-
atives possessing either 1-acetamido- or 2-alkyl/aryl-
carboxamido moieties in their molecules, that were
chosen in such a way as to incorporate tails leading to
potent CA inhibitory properties. The new sulfonamides
acted as very potent inhibitors of the slow red blood cell

C. Chazalette et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5781–5786
5785
amount of THF and poured in an excess of an aqueous
ammonia solution. After one night stirring at room
temperature, the white precipitate of 4 obtained was
filtered and recrystallized from CH3CN/H2O 1/1. Yield:
with pentane and recrystallized in CH3CN/H2O 1 /1
affording 5 as a white powder. Yield: 33%. Mp: 252°C.
1H NMR (DMSO) d ppm: 2.99–3.05 (m, 2H, CH2(1) or
(3)); 3.29–3.34 (m, 2H, CH2(1) or (3)); 4.74 (m, 1H,
CH(2)); 7.27 (s, 2H, SO2NH2); 7.41(d, 1H, CH(7)); 7.54
(d, 2H, CH(20)); 7.65 (d, 1H, CH(6)); 7.70 (s, 1H, CH(4));
7.89 (d, 2H, CH(30)); 8.79 (d, 1H, NH). MS (electrospray):
MH+ = 251; MNa+ = 373. Analysis: calcd: C 54.78, H
4.31, N 7.99, S 9.14; found: C 54.93, H 4.43, N 7.83, S
9.00.
2-(Valproylcarboxamido-5-sulfonamidoindane 6: Accord-
ing to the procedure used for 5: valproic acid (0.58g) and 2
(1g) afforded 6 as a lightly brown powder. Yield: 45%.
Mp: 116°C. 1H NMR (DMSO) d ppm: 1.18–1.47 (m, 14H,
CH3(CH2)2); 2.12 (m, 1H, CH); 2.81 (m, 2H, CH2(1) or
(3)); 3.22 (m, 2H, CH2(1) or (3)); 4.52 (m, 1H, CH(2));
7.27–7.67 (m, 5H, CH(4), CH(6), CH(7), and SO2NH2);
8.18 (d, 1H, NH). MS (electrospray): MH+ = 339;
MMH+ = 677. Analysis: calcd: C 58.76, H 7.83, N 8.06,
S 9.23; found: C 58.71, H 7.78, N 8.13, S 9.66.
1
45%. Mp: 218–220°C. H NMR (DMSO) d ppm: 1.81 (s,
3H, CH3); 2.80–3.26 (m, 4H, CH2(1) and (3)); 4.49 (m, 1H,
CH(2)); 7.23 (s, 2H, SO2NH2); 7.41(d, 1H, CH(7)); 7.65
(d, 1H, CH(6)); 7.70 (s, 1H, CH(4)); 8.21 (d, 1H, NH). MS
(electrospray): MH+ = 255; MMH+ = 509; MNa+ = 277;
MMNa+ = 531. Analysis: calcd: C 51.95, H 5.55, N 11.02,
S 12.61; found: C 51.85, H 5.68, N 11.06, S 11.59.
Further recrystallization also led to the hydrolysis product
of the chlorosulfonyl intermediate, compound 10. Mp:
1
159–161°C. H NMR (DMSO) d ppm: 1.84 (s, 3H, CH3);
2.72–2.78 (m, 2H, CH2(1) or (3)); 3.11–3.18 (m, 2H,
CH2(1) or (3)); 4.45 (m, 1H, CH(2)); 7.19 (d, 1H, CH(7));
7.45 (d, 1H, CH(6)); 7.48 (s, 1H, CH(4)); 7.62 (s, 1H, OH);
8.42 (d, 1H, NH). MS (electrospray): MH+ = 254. Anal-
ysis: calcd: C 48.34, H 5.53, N 5.12, S 11.73; found: C
48.39, H 6.12, N 5.10, S 11.75.
Compound 10 was stirred under reflux in an excess of HCl
37%. The evaporation of water led to a white powder of
11, which was recrystallized from a mixture EtOH/H2O 1 /
1. Yield: 85%. Mp: 187–188°C. 1H NMR (DMSO) d ppm:
2.87–2.93 (m, 2H, CH2(1) or (3)); 3.23–3.27 (m, 2H,
CH2(1) or (3)); 4.01 (m, 1H, CH(2)); 7.21 (d, 1H, CH(7));
7.46 (d, 1H, CH(6)); 7.50 (s, 1H, CH(4)); 7.99 (s, 3H, NH2
and OH). MS (electrospray): MH+ = 214. Analysis: calcd:
C 50.69, H 5.20, N 6.57, S 15.04; found: C 51.09, H 5.33,
N 6.66, S 15.54.
2-(Pentafluorobenzoyl)amino-5-sulfonamidoindane
7:
according to the procedure used for 5: pentafluorobenzoic
acid (0.58g) and 2 (1g) afforded 7 as a white powder.
1
Yield: 49%. Mp: decomposition around 350°C. H NMR
(DMSO) d ppm: 2.89–2.95 (m, 2H, CH2(1) or (3)); 3.33–
3.42 (m, 2H, CH2(1) or (3)); 4.72 (m, 1H, CH(2)); 7.29 (s,
2H, SO2NH2); 7.45 (d, 1H, CH(7)); 7.65 (d, 1H, CH(6));
7.72 (s, 1H, CH(4)); 9.33 (d, 1H, NH). MS (electrospray):
MH+ = 407; MNH4+ = 424.
2-(20-Methoxy-50-chlorobenzenecarbonyl)amino-5-sulfon-
amidoindane 8: according to the procedure used for 5: 2-
methoxy-5-chlorobenzoic acid (0.75g) and 2 (1g) afforded
2-Amino-5-sulfonamido-indane hydrochloride 2: com-
pound 4 was stirred under reflux in an excess of HCl
37%. The evaporation of water led to a white powder of 2,
which was recrystallized from a mixture EtOH/H2O 1/1.
1
8 as a white powder. Yield: 39%. Mp: 231°C. H NMR
(DMSO) d ppm: 2.90–3.02 (m, 2H, CH2(1) or (3)); 3.28–
3.32 (m, 2H, CH2(1) or (3)); 3.83 (s, 3H, CH3); 4.73 (m,
1H, CH(2)); 7.15 (d, 1H, CH(30)); 7.28 (s, 2H, SO2NH2);
7.41 (d, 1H, CH(7)); 7.50 (dd, 1H, CH(40)); 7.58 (d, 1H,
CH(60)); 7.64 (d, 1H, CH(6)); 7.69 (s, 1H, CH(4)); 8.50 (d,
1H, NH). MS (electrospray): MH+ = 381. Analysis: calcd:
C 53.61, H 4.50, N 7.36, S 8.42; found: C 53.49, H 4.46, N
7.12, S 7.31.
1
Yield: 79%. Mp: decomposition around 350°C. H NMR
(DMSO) d ppm: 3.12 (m, 2H, CH2(1) or (3)); 3.36 (m, 2H,
CH2(1) or (3)); 4.04 (m, 1H, CH(2)); 7.37 (s, 2H,
SO2NH2); 7.46 (d, 1H, CH(7)); 7.68 (d, 1H, CH(6)); 7.74
(s, 1H, CH(4)); 8.63 (s, 3H, NH3+). MS (electrospray):
MH+ = 213. Analysis: calcd: C 40.52, H 5.67, N 10.50, S
12.02; found: C 40.89, H 5.64, N 10.47, S 12.14.
1-Acetamido-5-sulfonamidoindane 3: according to
method described for 4: 1-aminoindane (2.03g), sodium
acetate (1.25g), and acetic anhydride (5mL) led to a white
powder of 13 (yield: 85%) whose NMR characteristics are
given: 1H NMR (DMSO) d ppm: 1.71–1.77 (m, 1H, CH(2)
or (3)); 1.87 (s, 3H, CH3); 2.34–2.38 (m, 1H, CH(2) or (3));
2.78–2.91 (m, 2H, CH(2) or (3)); 5.25 (m, 1H, CH(1));
7.17–7.24 (m, 4H, CH(4), (5), (6), and (7)); 8.21 (d, 1H,
NH). Compound 13 was treated with an excess of
chlorosulfonic acid and the intermediate sulfonyl chloride
was then treated with an aqueous solution of ammonia
yielding 3. Mp: 199–201°C. 1H NMR (DMSO) d ppm:
1.81–1.89 (m, 1H, CH(2) or (3)); 2.41–2.44 (m, 1H, CH(2)
or (3)); 2.80–3.10 (m, 2H, CH(2) or (3)); 5.34 (m, 1H,
CH(1)); 7.33 (s, 2H, SO2NH2); 7.41(d, 1H, CH(7)); 7.66
(s, 1H, CH(6)); 7.68 (s, 1H, CH(4)); 8.37 (d, 1H, NH). MS
(electrospray): MH+ = 255; MMH+ = 509; MNa+ = 277;
MMNa+ = 531; MNH4+ = 272.
2-(40-Chlorobenzenecarbonyl)amino-5-sulfonamidoin-
dane 5: To 40-chlorobenzoic acid (0.63g) was added SOCl2
(10mL). The mixture was stirred under reflux for 2h. The
brown oil obtained after evaporation of the excess of
SOCl2 was poured into 2mL of anhydrous THF and
added to 2 in 2mL anhydrous pyridine at 0°C. The
mixture was stirred for one night at room temperature.
After evaporation of the solvents, an acid solution (3mL
HCl 37% in 50mL H2O) was added and the formed
precipitate was filtered. The solid obtained was washed
2-(20,30,50,60-Fluorobenzenecarbonyl)amino-5-sulfon-
amidoindane 9: according to the procedure used for 5:
2,3,5,6-fluorobenzoic acid (0.78g) and 2 (1g) afforded 9 as
a white powder. Yield: 46%. Mp: 245°C. 1H NMR
(DMSO) d ppm: 2.89–2.95 (m, 2H, CH2(1) or (3)); 3.34–
3.42 (m, 2H, CH2(1) or (3)); 4.73 (m, 1H, CH(2)); 7.30 (s,
2H, SO2NH2); 7.44 (d, 1H, CH(7)); 7.66 (d, 1H, CH(6));
7.72 (s, 1H, CH(4)); 7.80 (m, 1H, CH(ar)); 9.33 (d, 1H,
NH). MS (electrospray): MH+ = 389; MNH4+ = 406.
17. Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21(DE3) from the plasmids
pACA/pCA
I
and pACA/hCA II as previously
described.14 Cell growth conditions were those described
by this group14 and enzymes were purified by affinity
chromatography according to the method of Khalifah
et al.18 Enzyme concentrations were determined spectro-
photometrically at 280nm, using a molar absorptivity of
49mMꢀ1 cmꢀ1 for CA I and 54mMꢀ1 cmꢀ1 for CA II,
respectively, based on Mr = 28.85kDa for CA I and
Mr = 29.30kDa for CA II.
18. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson,
S. M. Biochemistry 1977, 16, 2241–2247.
19. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573. An
SX.18MV-R Applied Photophysics stopped-flow instru-
ment has been used for assaying the CA-catalyzed CO2
hydration activity. Phenol red (at a concentration of
0.2mM) has been used as indicator, working at the
absorbance maximum of 557nm, with 10mM Hepes

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C. Chazalette et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5781–5786
(pH7.5) as buffer, 0.1M Na2SO4 (for maintaining con-
0.01nM were done thereafter with distilled-deionized
water. Inhibitor and enzyme solutions were preincubated
together for 15min at room temperature prior to 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.
stant the ionic strength), following the CA-catalyzed CO2
hydration reaction for a period of 10–100s. Saturated CO2
solutions in water at 20°C were used as substrate. Stock
solutions of inhibitor (1mM) were prepared in distilled-
deionized water with 10–20% (v/v) DMSO (which is not
inhibitory at these concentrations) and dilutions up to