Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: Solution and crystallographic investigations

Article abstract of DOI:10.1016/j.bmc.2010.06.028

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4- yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (K_Is of 73-131 nM), effective hCA II inhibition (K_Is of 9.1-36 nM) and less effective hCA IX and XII inhibition (K_Is of 55-128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with K_Is of 5.9-14.2 nM, although it was less effective as hCA I and II inhibitor (K_Is of 36-120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)- benzenesulfonamide (K_I of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum.

Full text of DOI:10.1016/j.bmc.2010.06.028

Bioorganic & Medicinal Chemistry 18 (2010) 4873–4878
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Coumarinyl-substituted sulfonamides strongly inhibit several human
carbonic anhydrase isoforms: solution and crystallographic investigations q
Jason Wagner a, Balendu Sankara Avvaru a, Arthur H. Robbins a, Andrea Scozzafava b, Claudiu T. Supuran b,
,
*
Robert McKenna a,
*
a Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, FL 32610, USA
b Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic
anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the trans-
membrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acet-
amide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency
inhibition of hCA I (KIs of 73–131 nM), effective hCA II inhibition (KIs of 9.1–36 nM) and less effective hCA
IX and XII inhibition (KIs of 55–128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-
benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane
isoforms, with KIs of 5.9–14.2 nM, although it was less effective as hCA I and II inhibitor (KIs of 36–
120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetam-
ido)-benzenesulfonamide (KI of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc
ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking
with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and
amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct
inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibi-
Received 12 May 2010
Revised 8 June 2010
Accepted 9 June 2010
Available online 15 June 2010
Keywords:
Carbonic anhydrase
Coumarinyl-substituted sulfonamides
X-ray crystallography
Isoformselective inhibitor
Inhibition mechanism
tion profiles against various
a
-CAs found in mammals or parasites, such as Plasmodium falciparum.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
between the two extremes mentioned above (micromolar–nano-
molar range, depending on the isoform and the substitution pat-
tern of the inhibitor scaffold).5,6 There are many X-ray crystal
structures of adducts of all these classes of CAIs with several CA
isoforms (of the 16 presently known in mammals),2,3 which
undoubtedly prove these different binding modes of the inhibitor
to the enzyme.7
Coumarins were recently shown to constitute a novel class of
inhibitors of the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1), whereas their mechanism of action is different from that
of all other known inhibitors.1,2 Indeed most CA inhibitors (CAIs)
investigated to date directly interact with the metal ion (which is
Zn(II) in
a
-CAs) from the enzyme active site, directly coordinating
However, sulfonamides remain the main chemotype of clinically
used CAIs, with many such drugs available to date.3,4,8 Members of
this class include aromatic, heterocyclic or aliphatic primary sulfon-
amides, but most drugs belong to the heterocyclic class.3,8 CAIs are
clinically employed for the management of a variety of disorders
connected to CA disbalances, such as glaucoma;3,8 in the treatment
of edema due to congestive heart failure,3,9 or for drug-induced ede-
ma;3,9 as mountain sickness drugs,9 whereas other agents of this
pharmacological class show applications as anticonvulsants,10,11
antiobesity12 or antitumor drugs/tumor diagnostic agents.3,13 As
there are few isoform-selective inhibitors to date,3 new sulfona-
mides are continuously reported to find derivatives with better inhi-
bition profiles as compared to the promiscuous, first generation
inhibitors such as acetazolamide (5-acetamido-1,3,4-thiadiazole-
2-sulfonamide).3 Recently, we have investigated some coumari-
nyl-substituted sulfonamides as inhibitors of the CA from the
to it (inorganic anions, sulfonamides, and their isosteres, etc.)3,4 or
anchoring to the zinc-bound water molecule/hydroxide ion
through a network of hydrogen bonds (phenols,5 polyamines6)
which stabilize the enzyme–inhibitor adduct. Whereas metal-com-
plexing anions are weak CAIs, with affinities generally in the
millimolar range,7 sulfonamides and their isosteres (sulfamates,
sulfamides, etc.) easily arrive to a low nanomolar inhibition
potency.3,4 Phenols and polyamines have an intermediate potency
q
Coordinates and structure factors have been deposited in the Protein Data Bank
as entry 3ml2.
* Corresponding authors. Tel.: +39 055 457 3005; fax: +39 055 4573385 (C.T.S.);
tel.: +1 352 392 5696; fax: +1 352 392 3422 (R.M.).
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), rmckenna@ufl.edu (R.
McKenna).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.028

4874
J. Wagner et al. / Bioorg. Med. Chem. 18 (2010) 4873–4878
malaria producing protozoa Plasmodium falciparum.14 These com-
pounds incorporate in their molecule both the coumarin ring, found
in the new class of CAIs reported recently,1,2 as well as the classical
benzenesulfonamide moiety normally associated with CA inhibi-
tion. As such coumarinyl-sulfonamides have not been investigated
for the inhibition of thr mammalian CAs with medicinal chemistry
applications, we report here such a study, investigating the inhibi-
tion of four human (h) hCA isoforms with a series of coumarinyl-
substituted benzenesulfonamides. We were also interested to
understand whether the presence of the substituted coumarin ring
in these derivatives will lead to an inhibition mechanism typical of
the coumarins (which are hydrolyzed to 2-hydroxy-cinnamic acids
which subsequently occlude the entrance to the active site)1,2 or
whether the binding to the enzyme occurs as that typical for sulfon-
amides, that is, by coordination to the metal ion from the enzyme ac-
tive site. To understand this, we also report a high resolution X-ray
crystal structure of a coumarinyl-substituted benzenesulfonamide
in adduct with the physiologically dominant isoform hCA II.
obviously their interaction with the enzymes, but these com-
pounds have not yet been tested as inhibitors of mammalian
CAs. Interest in this class of derivatives reemerged recently after
the report that coumarins act as a completely new class of CAIs.1,2
Indeed, the natural product coumarin A1 or the very simple non-
substituted derivative B (and many of its congeners possessing var-
ious substitution patterns at the coumarin ring)2 act as effective
CAIs against many of the mammalian isoforms CA I–CA XV. Fur-
thermore, the real enzyme–inhibitor is constituted by the hydro-
lyzed coumarins, such as compounds A1 and B1, formed from
the original coumarins A and B, respectively (Scheme 1). They have
been evidenced by X-ray crystallography of enzyme–inhibitor ad-
ducts and investigated in detail by kinetic methods.1,2 The 2-hy-
droxy-cinnamic acids thus formed (A1 and B1), bind in an
unprecedented way to the enzyme,1,2 at the entrance of the active
site cavity, plugging the entire entrance to it.
Compounds 1–5 thus incorporate two chemotypes known to
interact in very different ways with the CA active site: (i) the aro-
matic sulfonamide, classical CAI pharmacophore, and (ii) the
substituted coumarin moieties. Both of them lead to low nanomo-
lar and sometimes isoform-selective CAIs.1–4
2. Results and discussion
2.1. Chemistry
2.2. CA inhibition
The ‘tail approach’ has been extensively used to develop sulfon-
amide CAIs possessing a range of desired physico-chemical proper-
ties, and consists in using aromatic/heterocyclic sulfonamide
scaffolds to which tails that will induce water solubility (or other
desired physico-chemical properties, e.g., enhanced liposolubility;
membrane impermeability, etc.) are attached at the amino, hydro-
xy, imino or hydrazino moieties contained in such precursor scaf-
folds.15,16 Some coumarin-substituted sulfonamides of type 1–5
have also been reported by this approach,14 and they contain 7-
methoxy-coumarin-4-yl-acetyl moieties, attached to the classical
scaffolds of the aminobenzenesulfonamides (in para, meta, and
ortho positions, compounds 1–3) or to the 4-hydrazino-benzene-
sulfonamide (4) and 4-amino-6-trifluoromethyl-benzene-1,3-
disulfonamide (5) scaffolds.
Inhibition of four physiologically relevant
a-CA isoforms with
compounds 1–5 and acetazolamide (5-acetamido-1,3,4-thiadia-
zole-2-sulfonamide, AZA, a clinically used drug) is presented in
Table 1.17 hCA I and II (cytosolic, widespread enzymes) as well as
hCA IX and XII (transmembrane, tumor-associated CAs) have been
included in this study due to their relevance as targets/offtargets
when developing CAIs. Indeed, CA II for example is the drug target
for developing antiglaucoma CAIs,3,4 but it is an offtarget when
considering CA IX/XII inhibition.13 In this latter case, only the
transmembrane, tumor-associated isozymes (IX and XII) should
be inhibited, as CA II may have the function of housekeeping en-
zyme, and its inhibition may lead to side effects. Inhibition of the
malaria enzyme pfCA with compounds 1–5 are also shown in Table
1, for comparison reasons, as these compounds were investigated
earlier only for their interaction with this enzyme.14 Thus, we
investigated in detail the inhibition of the coumarinyl-substituted
sulfonamides 1–5 with these four human enzymes, that is, hCA I, II,
IX, and XII.
SO2NH2
SO2NH2
1: R =
2: R =
O
R
N
H
The following structure–activity relationship (SAR) can be
drawn from data of Table 1:
SO2NH2
3: R =
4: R =
(i) Compounds 1–5 were medium potency hCA I inhibitors,
with inhibition constants in the range of 73–131 nM, being
more effective than acetazolamide (KI of 250 nM). The best
hCA I inhibitor was the sulfanilamide derivative 1, whereas
MeO
O
O
O
1-5
N
H
SO2NH2
N
N
CF3
SO2NH2
Me
N
SO2NH2
S
H
OH
OH
5: R =
AZA
CA
H2NO2S
-
COO
r.t, pH 7.4
4-6 h
MeO
O
O
MeO
OH
A
A1
These compounds were prepared from the corresponding ami-
no/hydrazino-aromatic sulfonamides and the 7-methoxy-couma-
rin-4-yl-acetyl chloride,14 by the classical tail approach,15,16 and
-
CA
COO
were investigated for the inhibition of the
a-CA from P. falcipa-
r.t, pH 7.4
4-6 h
O
O
rum.14 Their activity against this enzyme was in the low micromo-
lar range, but they showed no activity in vivo as antimalarials, in
contrast to other types of such derivatives investigated by us.14
The substituted coumarinyl moieties were introduced in the mole-
cules 1–5 in order to change their physico-chemical properties and
OH
B
B1
Scheme 1. Formation of 2-hydroxy-cinnamic acids A1 and B1 by the CA-mediated
hydrolysis of coumarins A and B.

J. Wagner et al. / Bioorg. Med. Chem. 18 (2010) 4873–4878
4875
Table 1
glycerol molecule was also evident, located adjacent to the aro-
matic ring of the benzene sulfonamide. The inhibitor is buried deep
in the active site, with N1 of the sulfonamide bound to the zinc ion,
at a distance of 2.2 Å.
As with all sulfonamide compounds in complex with hCA II, for
example AZA (PDB accession code 3hs4),7c the nitrogen atom of the
sulfonamide moiety is complexed with the active site zinc ion, at a
distance of 2.2 Å. The O2 atom of the sulfonamide accepts a hydro-
gen bond from the main-chain nitrogen of Thr199, at a distance of
2.9 Å. No hydrogen bonds donate to O3 of the sulfonamide, but this
atom is in Van der Waals contact with atoms near the active site
zinc ion (Figs. 2 and 3).
Inhibition of human a-CAs and Plasmodium falciparum enzymes (hCA and pfCA) with
sulfonamides 1–5, by a stopped-flow CO2 hydrase assay.17 Data for AZA are from Ref.
3 whereas data against pfCA from Ref. 14
Inhibitor
KI# (nM)
hCA Ia
hCA IIa
hCA IXb
hCA XIIb
pfCAc
1
2
3
4
5
AZA
73
94
131
91
120
250
9
16
79
12
36
12
87
59
113
91
14
25
63
55
128
78
6
2.5
970
3506
4580
1440
2520
315
#
Errors in the range of 5% of the reported data from three different assays by a
stopped-flow CO2 hydration method.17
a
Cytosolic, recombinant enzyme.
Catalytic domain, recombinant enzyme.
From Ref. 14.
b
c
its meta (2) and ortho (3) isomers were progressively less
inhibitory. The hydrazino derivative 4 was slightly more
active than 2 and 3, whereas the benzene-1,3-disulfonamide
less active compared to 1. Thus, in this case the aromatic sul-
fonamide head strongly influences activity for this small ser-
ies of compounds, as hCA I inhibitors.
(ii) The physiologically dominant isoforms hCA II was highly
inhibited by compounds 1–5 and acetazolamide. Com-
pounds 1, 2, 4 and AZA were very effective CAIs, with KIs
in the range of 9.1–15.8 nM whereas 3 and 5 were less effec-
tive (KIs of 36–79 nM). The best inhibitor was again 1, and
SAR is rather similar to what observed above for the inhibi-
tion of hCA I. The least effective hCA II inhibitor was the
orthanilamide derivative 3.
Figure 1. Stick representation compound 1 bound in the active site of hCA II. Atoms
are colored: zinc, gray; sulfur, orange; oxygen, red; nitrogen, blue; and carbon,
yellow. Solvent are depicted as red spheres. Amino acids are as labeled. The electron
(iii) The tumor-associated isoforms hCA IX and XII were less
prone to be inhibited by these sulfonamides, except com-
pound 5 which behaved as an effective inhibitor of both iso-
forms, with inhibition constants of 5.9–14.2 nM (in the same
range as the clinically used AZA, Table 1). The remaining
derivatives 1–4 were medium potency hCA IX and hCA XII
inhibitors, with KIs in the range of 59–113 nM against the
first enzyme, and of 55–128 nM against the second one. It
is obvious that SAR is different for the inhibition of the trans-
membrane versus the cytosolic isoforms with this small
groups of CAIs. Indeed, the disulfonamide 5 was the best
hCA IX and XII inhibitor, whereas the sulfanilamide 1 was
the best hCA I and II inhibitor detected here.
density is represented by a 2
made using PyMOL.
r-weighted 2Fo À Fc Fourier map (gray mesh). Figure
Table 2
Data and final model statistics of the hCA II–1 adduct
Data-collection statistics
PDB accession number
Temperature (K)
3ML2
100
Wavelength (Å)
1.5418
Space group
P21
Unit-cell parameters (Å, °)
a = 42.14, b = 41.20,
c = 71.58, b = 104.13
19896(1860)*
23.1–1.8 (1.86–1.80)
6.5% (21.6%)
10.6 (3.2)
(iv) As reported erlier,14 compounds 1–5 are less effective inhib-
Total number of measured reflections
Resolution (Å)
Rsym
I/r(I)
itors of an
a
-CA from the malaria parasite P. falciparum, with
M. AZA
inhibition constants in the range of 970 nM–4.578
l
was one of the most effective pfCA inhibitors detected so
far,14 with a KI of 315 nM. However, it has been proven that
it is possible to inhibit the growth of the malaria parasite by
inhibiting this enzyme with potent sulfonamide CAIs.18
Thus, it is possible to develop antimalarials based on the
CAIs, although few nanomolar inhibitors of pfCA were
reported to date.14,18
Completeness (%)
Redundancy
89.0 (89.0)
3.3 (3.5)
Final model statistics
a
Rcryst (%)
18.7
22.2
b,c
Rfree (%)
Residue Nos.
No. of protein atoms (including alternate
conformations)
4–261
2060
No. of drug atoms
27
No. of H2O molecules
185
R.m.s.d. for bond lengths (Å), angles (°)
Ramachandran statistics (%)
Most favored, additionally allowed, and
generously allowed regions
B factors (Å2)
0.018, 1.7
88.0, 11.6, 0.5
2.3. X-ray crystallography
In order to understand why compound 1 is such a potent hCA II
inhibitor, whereas it is less effective against other isoforms, we re-
solved its X-ray crystal structure in complex with this isozyme.
From an initial refinement of the model of hCA II at 1.8 Å resolu-
tion, an |Fo À Fc| electron density omit map was calculated and
revealed density consistent with the entire molecule compound 1
except for the methoxy moiety on the aromatic coumarin ring
( Fig. 1 and Table 2). Electron density consistent with a bound
22.1, 27.2, 28.5, 32.1
Average, main-, side-chain, inhibitor, solvent
*
a
b
c
Values in parenthesis represent highest resolution bin.
Rsym <I>.
|I À <I>|/
Rcryst = ( |Fobs|) Â 100.
|Fo| À |Fc|/
Rfree is calculated in same manner as Rcryst, except that it uses 5% of the
=
R
R
R
R
reflection data omitted from refinement.

4876
J. Wagner et al. / Bioorg. Med. Chem. 18 (2010) 4873–4878
Figure 2. Schematic of hCA II–compound 1 interactions. Hydrophobic contacts are indicated by red hash marks and H-bonds by black dashed lines. Atoms are colored: zinc,
gray; sulfur, yellow; oxygen, red; nitrogen, blue; and carbon, black. Amino acids are as labeled. Figure made using Ligplot.
Figure 3. Overall (a) and zoom-in (b) view of hCA II–sulfonamide 1 adduct, superposed onto acetazolamide (AZA) one (PDB accession code 3hs4).7c Note the two compounds
follow the same trajectory out of the active site with the extended coumarin heterocycle ring at an angle to the benzene ring with an edge-to-face interaction with the side
chain of Phe131. hCA II is depicted as a surface representation (bulk solvent accessible area, light pink; hydrophilic and hydrophobic regions of the active site, blue and
orange, respectively). Compounds 1 and AZA are represented as sticks. Compounds 1 (gray) and AZA (yellow) are represented as sticks. Non-carbon atoms of both compounds
are colored as in Figure 1. Figure made using PyMOL (DeLano Scientific).
The benzene scaffold of compound 1 follows the same trajec-
tory out of the active site, as the thiadiazole ring of acetazolamide
AZA.7c The benzene core of the inhibitor is at van der Waals dis-
tance from the side chain of Leu98, and is sandwiched by the
bound glycerol molecule, coming from the cryoprotectant solution.
Hydrogen bonds from the three hydroxyl groups of the glycerol to
the side chain nitrogen atoms of Gln92, Asn67, and Asn62 were
also observed (Figs. 1 and 2).
The extended coumarin heterocycle ring lies at an angle to the
benzene ring, and is in an edge-to-face interaction with the side
chain of Phe131. C10–C11 and O4 deviate slightly from the plane
of the remaining nine atoms of the heterocyclic coumarin core,
respectively. The torsion angles C9–C10–C11–O4 and C13–O12–
C11–C10 are À9.1° and 7.4°, respectively. O4 interacts via a hydro-
gen bond with a solvent molecule and contacts the CG atom of
Pro202 and the CD1 of Leu204. C10 has a hydrophobic interaction

J. Wagner et al. / Bioorg. Med. Chem. 18 (2010) 4873–4878
4877
a
with the CD atom of Pro202, and C14 contacts the C atom of
4.2. CA inhibition
Gly132. In a packing contact with a symmetry-related molecule,
a
the terminal methyl group C19 is 3.9 Å from the C and N atoms
of Gly235.
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA-catalyzed CO2 hydration activity.17 Phenol
red (at a concentration of 0.2 mM) was used as the indicator, work-
ing at the absorbance maximum of 557 nm, with 20 mM Hepes (pH
7.5) as buffer, and 20 mM Na2SO4 (for maintaining a constant ionic
strength), following the initial rates of the CA-catalyzed CO2 hydra-
tion reaction for a period of 10–100 s. The CO2 concentrations ran-
ged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants (five different substrate con-
centrations were used). For each inhibitor at least six traces of
the initial 5–10% of the reaction have been used for determining
the initial velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed rates. Stock
solutions of inhibitor (0.1 mM) were prepared in distilled-deion-
ized water and dilutions up to 0.01 nM were done thereafter with
distilled-deionized water. Experiments were done using six differ-
Thus, its is obvious from the above data that the coumarin ring
in compounds also incorporating a sulfonamide moiety, such as
derivatives 1–5 investigated here, does not have the same fate as
normal coumarins (without a zinc-binding group of the sulfon-
amide type in their molecule), which inhibit CAs after active site
mediated hydrolysis, as depicted in Scheme 1. On the contrary,
similar to all sulfonamides investigated so far,3–7 the coumarinyl-
substituted derivatives inhibit CAs by coordination of the sulfon-
amide moiety to the metal ion from the enzyme active site, and
participation in hydrophobic and hydrophilic interactions of the
organic scaffold of the inhibitor with amino acid residues known7
to be involved in the binding of CAIs, Thr199, Phe131, Pro201, etc.
The X-ray data presented above also allow us to understand why
compound 1 is a potent hCA II inhibitor and is less effective against
hCA IX, one of the most important drug targets in this family of en-
zymes. Indeed, as shown in Fig. 2, a very important interaction be-
tween this inhibitor and hCA II is the stacking of the coumarin ring
with Phe131, something evidenced earlier for other potent hCA II
inhibitors.11d In hCA IX (PDB accession code 3iai), Phe131 from
hCA II is ‘substituted’ by a valine residue,13c and in hCA XII (PDB
accession code 1jcz) Phe is an alanine residue,13d both of which
are unable to participate in such an edge-to-face stacking interac-
tion. This lack of interaction may thus explain the roughly 10- and
7-times weaker inhibition of hCA IX/hCA XII, respectively, over hCA
II, with compound 1.
ent inhibitor concentrations, varying from 100 lM to 0.1 nM.
Inhibitor and enzyme solutions were preincubated together for
15 min–24 h at room temperature (15 min) or 4 °C (all other incu-
bation times) prior to assay, in order to allow for the formation of
the E–I complex or for the eventual active site mediated hydrolysis
of the inhibitor. Data reported in Table 1 show the inhibition after
15 incubation, as there were no differences of inhibitory power
when the enzyme and inhibitors were kept for longer periods in
incubation.1 The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3, as reported earlier,1,2 and
represent the mean from at least three different determinations.
3. Conclusions
4.3. X-ray crystallography
We investigated here a series of coumarinyl-substituted aro-
matic sulfonamides as inhibitors of four CA isoforms, the cytosolic
hCA I, and II, and the transmembrane, tumor-associated isozymes
hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-
yl-acetamide-tails and benzenesulfonamide and benzene-1,3-
disulfonamide scaffolds showed medium potency inhibition of
hCA I (KIs of 73–131 nM), effective hCA II inhibition (KIs of 9.1–36
nM) and less effective hCA IX and XII inhibition (KIs of 55–128
nM). Only one compound, the derivatized with the coumarinyl tail
4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide, showed
effective inhibition of the transmembrane isozymes, with KIs of
5.9–14.2 nM, although it was less effective as hCA I and II inhibitor
(KIs of 36–120 nM). An X-ray crystal structure of hCA II in complex
The plasmid encoding hCA II was transformed into Escherichia
coli BL21 cells through standard procedures and the transformed
cells were expressed at 37 °C in LB medium containing 100 lg/ml
ampicillin.19 hCA II production was induced by the addition of iso-
propyl thiogalactoside to a final concentration of 1 mM at an
O.D600 of 0.6 AU. The cells were harvested after 4 h of post induc-
tion. The cell pellets were lysed and hCA II was purified through
affinity chromatography using pAMBS resin as has been described
elsewhere.20
Co-crystals of hCA II compound 1 complex were obtained using
the hanging drop vapor diffusion method.21 10
ll drops (0.2 mM
hCA II; 0.4 mM compound 1; 0.8 M sodium citrate; 50 mM Tris–
Cl; pH 8.0) were equilibrated against 1 ml of precipitant solution
(1.6 M sodium citrate; 50 mM Tris–Cl pH 8.0) at room temperature
(ꢀ20 °C).7c Useful crystals were observed 4 days after the crystalli-
zation setup. A crystal was cryoprotected by quick immersion into
25% glycerol precipitant solution and flash-cooled by exposure to a
gaseous stream of nitrogen at 100 K. X-ray diffraction data were
obtained using an R-AXIS IV++ image plate system with OsmicVari-
max optics and a Rigaku RU-H3R Cu rotating anode operating at
50 kV and 22 mA. The detector-crystal distance was set to
80 mm. The oscillation steps were 1° with a 6 min exposure per
image. Indexing, integration, and scaling were performed using
HKL2000.22
with
4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfon-
amide (KI of 9.1 nMagainst this isozyme)showed the intact inhibitor
coordinated to the zinc ion from the enzyme active site by the sul-
fonamide moiety, and participating in a edge-to-face stacking with
Phe131, in addition to other hydrophobic and hydrophilic interac-
tions with water molecules and amino acid residues from the active
site. Thus, sulfonamides incorporating coumarin rings have a dis-
tinct inhibition mechanism compared to the coumarins, and may
lead to compounds with interesting inhibition profiles against vari-
ous enzymes of mammalian or parasitic (e.g., P. falciparum) origin.
4. Experimental
4.1. Chemistry
The crystal structure of hCA II (PDB accession code: 2ili)7c was
used to obtain initial phases using PHENIX.23 The solvent mole-
cules were removed and 5% of the unique reflections were selected
randomly and excluded from the refinement data set for the pur-
pose of Rfree calculations. Coordinates for the inhibitor were gener-
ated using the PRODRG2 website.24 Goemetric restraints for
inhibitor 1 were created using the eLBOW facility within the PHE-
NIX software suite, and planar restraints for C10, C11, O3, and O4
were manually added. All manual rebuilding was done using
Sulfonamides 1–5 have been reported previously14 and were
prepared by the tail approach from the corresponding amino-/
hydrazino-sulfonamide and the acyl chloride.15,16 All reagents
and buffers were the highest grade available, from Sigma–Aldrich,
Milan, Italy.

4878
J. Wagner et al. / Bioorg. Med. Chem. 18 (2010) 4873–4878
Coot25 and the geometry of the final refined model of the complex
was checked using Procheck.26
9. (a) van Patot, M. C.; Leadbetter, G., 3rd; Keyes, L. E.; Maakestad, K. M.; Olson, S.;
Hackett, P. H. High Alt. Med. Biol. 2008, 9, 289; (b) Supuran, C. T. Curr. Pharm.
Des. 2008, 14, 641.
10. (a) De Simone, G.; Vitale, R. M.; Di Fiore, A.; Pedone, C.; Scozzafava, A.;
Montero, J. L.; Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2006, 49, 5544; (b) De
Simone, G.; Di Fiore, A.; Menchise, V.; Pedone, C.; Antel, J.; Casini, A.;
Scozzafava, A.; Wurl, M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15,
2315; (c) Winum, J. Y.; Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.;
Ciattini, S.; Montero, J. L.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49,
7024.
11. (a) Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.;
De Simone, G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329; (b) Casini, A.;
Antel, J.; Abbate, F.; Scozzafava, A.; David, S.; Waldeck, H.; Schafer, S.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2003, 13, 841; (c) Weber, A.; Casini, A.; Heine, A.;
Kuhn, D.; Supuran, C. T.; Scozzafava, A.; Klebe, G. J. Med. Chem. 2004, 47, 550;
(d) Menchise, V.; De Simone, G.; Alterio, V.; Di Fiore, A.; Pedone, C.; Scozzafava,
A.; Supuran, C. T. J. Med. Chem. 2005, 48, 5721.
Acknowledgments
This research was financed in part by a Grant of the 6th Frame-
work Programme of the European Union (DeZnIT project) and by a
7th FP EU project (METOXIA) to A.S. and C.T.S., and in part by an
NIH (GM 25154) and Maren Grant to R.M. R.M. would also like to
thank the Centre of Structure Biology, University of Florida, for
their continued support to help maintain the in-house X-ray
facilities.
12. (a) Supuran, C. T.; Di Fiore, A.; De Simone, G. Expert Opin. Emerg. Drugs. 2008,
13, 383–392; (b) De Simone, G.; Di Fiore, A.; Supuran, C. T. Curr. Pharm. Des.
2008, 14, 655–660.
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