O-Benzenedisulfonimido-sulfonamides are potent inhibitors of the tumor-associated carbonic anhydrase isoforms CA IX and CA XII

Article abstract of DOI:10.1016/j.bmc.2012.12.037

By using phthalimido-substituted aromatic sufonamides as lead molecules, a series of new sulfonamides incorporating ortho-benzenedisulfonimide moieties have been synthesized and tested against the human (h) cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA I and hCA II and the transmembrane, tumor-associated isozymes hCA IX and hCA XII. All these compounds showed K _i values lower than 100 nM and many of them showed better K _is than the reference compound acetazolamide, a clinically used sulfonamide. The tumor-associated isozymes were better inhibited than the cytosolic ones. A molecular docking within the active site of some CA isoforms, such as hCA I, explained these findings, as the benzenedisulfonimide moiety makes favorable interactions (hydrogen bonds) with amino acid residues involved in binding of inhibitors, such as Gln92, His67, and His64.

Full text of DOI:10.1016/j.bmc.2012.12.037

Bioorganic & Medicinal Chemistry 21 (2013) 1386–1391
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
o-Benzenedisulfonimido–sulfonamides are potent inhibitors
of the tumor-associated carbonic anhydrase isoforms
CA IX and CA XII
Özlen Güzel-Akdemir a, Atilla Akdemir b, , Semra Isik c,d, Daniela Vullo d, Claudiu T. Supuran e,
⇑
⇑
a Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
b Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi 34093, Fatih, Istanbul, Turkey
c Balikesir University, Faculty of Art and Sciences, Balıkesir, Turkey
d Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
e Universita degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff, 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:
Available online 4 January 2013
By using phthalimido-substituted aromatic sufonamides as lead molecules, a series of new sulfonamides
incorporating ortho-benzenedisulfonimide moieties have been synthesized and tested against the human
(h) cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA I and hCA II and the transmembrane,
tumor-associated isozymes hCA IX and hCA XII. All these compounds showed Ki values lower than
100 nM and many of them showed better Kis than the reference compound acetazolamide, a clinically
used sulfonamide. The tumor-associated isozymes were better inhibited than the cytosolic ones. A
molecular docking within the active site of some CA isoforms, such as hCA I, explained these findings,
as the benzenedisulfonimide moiety makes favorable interactions (hydrogen bonds) with amino acid res-
idues involved in binding of inhibitors, such as Gln92, His67, and His64.
Keywords:
Carbonic anhydrase
Cytosolic isoforms I and II
Tumor-associated isoforms IX and XII
o-Benzenedisulfonimide
Sulfonamide
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Sulfonamides constitute the main class of CA inhibitors
(CAIs)1–4 and many different families of such compounds have
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of
metalloenzymes that mainly catalyze the interconversion between
CO2 and bicarbonate (HCO3À).1–4 There are 16 mammalian CA iso-
been reported to possess significant inhibitory power against many
isoforms.5–10
Among the interesting classes of such derivatives investigated
earlier11 were the phthalimido sulfonamides of types A, which
showed interesting CA I, II and IV inhibitory power and were easily
obtained by reaction of amino-containing aromatic/heterocyclic
sulfonamides with phthalic anhydride.
forms that belong to the a
-class enzymes which all have a Zn2+ ion
in the active site. Even though, these CA isozymes share similarity
in structure and active site composition, there are differences in
the overall structure, expression and their putative role in
(patho)physiology.1–4
The human CA isoforms I and II (hCA I and hCA II, respectively)
are cytosolic enzymes that are widespread throughout the human
body. They are drug targets for clinically used diuretics, antiglauco-
ma drugs and anticonvulsants.1–4
In contract, the isoforms IX and XII (hCA IX and hCA XII, respec-
tively) are transmembrane enzymes with the active site on the
extracellular side of the cell membrane. These enzymes are mainly
found in hypoxic tumors.1,3 They are validated drug targets for the
design anticancer agents specific for solid, hypoxic tumors.1
O
N
O
S
NH2
O
O
In our current work, continuing our interest in sulfonamides as
CAIS, and considering compounds A as lead mlecules, we have syn-
thesized six new compounds by reaction of o-benzenedisulfonyl
chloride with amino-sulfonamides, obtaining the disulfonamido
analogs of compounds A. We were interested to see whether
substituting the phthalimido moiety with the o-benzenedisulfo-
nimido one has important consequences for the interaction of
these sulfonamides with the various CA isoforms of medicinal
chemistry interest, such as CA I, II, IX and XII.
⇑
Corresponding authors. Tel.: +90 212 523 2288x1142; fax: +90 212 621 7578
(A.A.); tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: aakdemir@bezmialem.edu.tr (A. Akdemir), claudiu.supuran@
unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.037

Ö. Güzel-Akdemir et al. / Bioorg. Med. Chem. 21 (2013) 1386–1391
1387
between the benzenesulfonamide head and the ortho-benzenedi-
sulfonamide scaffold of compounds 3–8 reported here, as shown
in Figure 1.
2. Results and discussion
2.1. Chemistry
2.2. Carbonic anhydrase inhibition
The rationale of the present drug design study was to replace
the CO moieties from the phthalimido sulfonamides A reported
earlier11 by the SO2 ones. In fact the presence of four oxygen in
the disulfonamides reported here compared to the two ones pres-
ent in the phthalimides reported earlier, may lead to a supplemen-
tary stabilization of the enzyme-inhibitor adduct and thus to better
CAIs. The simple chemistry outlined below was employed to pre-
pare the new compounds reported here.
Reaction of o-benzenedisulfonyl chloride 1 with aminosulfona-
mides 2a–f, led to the formation of o-benzenedisulfonimides 3–8
(Scheme 1). The new compounds 3–8 were characterized by spec-
troscopic and analytic methods which confirmed their structures
(see Section 4).
The newly synthesized sulfonamides 3–8 as well as the refer-
ence compound AZA have been tested for their enzyme inhibition
activities against human CA isoforms hCA I, hCA II, hCA IX and hCA
XII (Table 1). All tested compounds have Ki values in the lower nM
region (Ki <100 nM, see Table 1). Interestingly, the overall differ-
ence between the lowest and highest Ki values are only 13-fold
(i.e., Ki value of compound 8 for hCA I = 7.2 nM; Ki value of com-
pound 3 for hCA II = 95.6 nM; Table 1).
The hCA I enzyme was inhibited well by all tested compounds
(Table 1) and the worst inhibition was observed for compounds 3
and 5 (Ki = 51.4 and 48.5 nM, respectively). Compound 4, which
has a sulfonamide group on the meta positon (Fig. 1), showed a
slight decrease in Ki value compared to compounds 3 and 5. The
We have included variously substituted benzenesulfonamides
( ortho, meta and para such derivatives) and different spacers
H
O
O
2N
O
H2
O
N
O
O
O
S
S
O
S
S
Cl
Cl
N
H2
N
+
+
S
S
O
O
O
O
2a
2b
1
1
3
4
O
NH2
O
O
NH2
O
O
O
O
S
S
O
S
Cl
Cl
S
H2
N
N
S
S
O
O
O
O
O
O
O
O
O
O
O
O
S
S
Cl
Cl
H2
N
S
NH2
+
N
S
NH2
n
n
S
S
O
Y
O
Y
O
O
1
2c: n=0, Y=H
2d: n=0, Y=Cl
2e: n=1, Y=H
2f: n=2, Y=H
5: n=0, Y=H
6: n=0, Y=Cl
7: n=1, Y=H
8: n=2, Y=H
Scheme 1. Preparation of o-benzenedisulfonimide derivatives. Reagents and conditions: Et3N, CH2Cl2, reflux.
H2
O
O
N
O
O
NH2
O
O
S
O
S
O
O
Cl
O
O
O
S
S
O
O
O
O
S
S
N
O
N
N
S
NH2
N
S
NH2
S
S
S
S
O
O
O
O
O
O
O
3
4
6
5
O
O
O
O
S
S
N
O
N
O
N
N
O
O
S
S
NH2
S
S
NH2
N
H
S
O
O
O
O
O
AZA
8
7
S
O
O
NH2
Figure 1. The chemical structures of sulfonamides 3–8 and the reference compound acetazolamide (AZA).

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Ö. Güzel-Akdemir et al. / Bioorg. Med. Chem. 21 (2013) 1386–1391
Table 1
nM and 5, Ki = 82.5 nM). However, adding a chlorine group to
compound 5 (to yield compound 6) decreased the Ki value approx-
imately 5-fold (Table 1). This difference was only approximately
1.4-fold for the human hCA I enzyme.
Growing the spacer length of compound 5 with a methylene be-
tween the phenyl and benzenedisulfonamide moieties increased
the Ki value (Table 1). In contrast, addition of an ethylene group
to compound 3 did not increase the Ki value (Table 1).
Finally, the compounds have been tested for the inhibition of the
second the human, tumor-associated enzyme hCA XII (Table 1). The
inhibition data of compounds 3–6 show that the para position
(compound 5) is the best location for the sulfonamide substituent
compared to the ortho and meta positions (Table 1). Adding a
chlorine substituent on compound 5 to yield compound 6 did not
decrease the Ki value. In addition, increasing the size of the spacer
between the phenyl and benzenedisulfonamide groups increased
the Ki value (Table 1).
In summary, all four human CA enzymes respond differently to
compounds 3–8 in enzyme inhibition assays. The Ki values of all
compounds are in the lower nanomolar range. The lowest Ki value
is observed for compound 8, which inhibits hCA I with a Ki value of
7.2 nM. This compound shows a 3.8-fold higher Ki value for hCA II,
a 12-fold higher Ki value for hCA IX and a 4.4-fold higher Ki value
for hCA-XII compared to hCA I.
Inhibition of human CA isoforms hCA I, hCA II, hCA IX and hCA XII with sulfonamides
3–8
Compounds
Kia (nM)
hCA IIb
hCA Ib
hCA IXb
hCA XIIb
3
4
5
6
7
8
AZA
51.4
31.2
48.5
35.8
26.5
4
3
5
4
3
95.6
61.7
54.8
61.7
28.2
27.1
12.1
8
5
6
5
3
1
1
66.9
32.2
82.5
17.1
49.8
86.0
25.4
7
2
6
2
4
7
3
50.5
71.5
19.0
34.9
31.4
32.0
4
5
2
3
1
3
7.2 0.6
250.0 15
5.6 0.2
AZA has been included as a reference compound.
a
Mean standard error, from three different assays, by a CO2 hydration stopped-
flow assay.
b
Human, recombinant isozymes, pH 7.5, 20 mM TrisÁHCl buffer.
latter two compounds have the sulfonamide group on the ortho
and para positions respectively. The addition of a chlorine atom
to the ortho position of compound 5 (to yield compound 6) again
decreased the Ki value (Table 1). Even though the differences in
Ki value between compounds 3–6 were rather small, the data
might suggest that either a sulfonamide group or a chlorine group
on the meta position is well tolerated.
Compounds 7 and 8 having methylene and ethylene groups,
respectively, as spacers between the phenyl and benzenedisulfona-
mide groups (Fig. 1). In contrast, compound 5 had a direct bond
between the two moieties (Fig. 1). Increasing the size of this spacer
led to a decrease in the Ki value of the compounds (compound 5:
Ki = 48.5 nM; compound 7: Ki = 26.5 nM; compound 8: Ki = 7.2
nM). This difference in inhibition is almost 7-fold between com-
pounds 5 and 8 (Table 1).
The human hCA II enzyme showed a different inhibition profile
for the tested compounds. The Ki values for compounds 3–6 show
that the sulfonamide and chlorine substituents in the ortho posi-
tion (compound 3 and 6) and a sulfonamide substituent in the meta
position (compound 4) were less well tolerated compared to a sul-
fonamide substituent in the para position (compound 5; Table 1).
The addition of either a methyl spacer (compound 7) or an ethyl-
ene spacer (compound 8) between the phenyl and benzenedisulf-
onamide moieties of compound 3 decreased the Ki value (Table
1). However, the Ki values of compounds 7 and 8 were similar to
each other and no difference was observed related to the spacer
length (compound 7: Ki = 28.2 nM; compound 8: Ki = 27.1 nM).
Some of the new compounds reported here, tested on the
tumor-associated hCA IX showed similar enzyme inhibition than
the reference compound AZA (Table 1). This enzyme showed a
similar inhibition profile for compounds 3–6 as hCA I, but with lar-
ger changes in the Ki values (Table 1). The sulfonamide group was
best tolerated on the meta position (compound 4, Ki = 32.2 nM)
compared to the ortho and para positions (compounds 3, Ki = 66.9
2.3. Docking studies
The difference between the lowest and highest measured Ki val-
ues per enzyme for compounds 3–8 are relatively small, that is,
approximately 7-fold for hCA I, 3.5-fold for hCA II, 5-fold for hCA
IX and 3.7-fold for hCA XII (Table 1). Nevertheless, docking studies
have been performed to rationalize the enzyme inhibition data of
compounds 3–8. To this end, the GOLD Suite docking programme12
was used with the GoldScore scoring function. The sulfonamide
substituent on the phenyl moiety was assigned a negative charge
(–SO2NHÀ) to facilitate its interaction with the Zn2+ of the enzyme
active site.
The crystal structure of hCA I in complex with topiramate (PDB:
3LXE; 1.90 Å) has been used as a template in our docking studies for
hCA I. The docking results suggests that compound 4 can relatively
easy interact with the Zn2+ ion through its meta-sulfonamide sub-
stitution on the phenyl moiety. This phenyl group of the ligand also
forms hydrophobic interactions with His200. In addition, the ben-
zodithiazole group of compound 4 forms hydrophobic interactions
with Trp5 and His64. Compounds 3 and 5 have slightly more diffi-
culties in forming these interactions with the hCA I active site com-
pared to compound 4, but the difference in docking score is small.
Adding a methyl group between the benzodithiazole group and
the phenyl moiety (compound 7), positions one of the sulfoxide
functionalities of the benzodithiazole group near Gln92 and hydro-
A
B
Thr199
Gln92
Thr199
Gln92
2+
2+
Zn
Zn
Trp5
Trp5
His67
His67
His64
His64
Figure 2. The docked poses of compounds 7 (panel A, green) and 8 (panel B, light blue) in the active site of hCA I. The docked pose of compound 5 (shown in black) is shown in
both panels. Hydrogen bonds are depicted in red dashed lines.

Ö. Güzel-Akdemir et al. / Bioorg. Med. Chem. 21 (2013) 1386–1391
1389
gen bonds are formed (Fig. 2A). An ethyl group as a spacer (com-
pound 8) places the sulfoxide groups near His67 and Gln92 and
two hydrogen bonds are formed (Fig. 2B). This is consistent with
the fact that the lowest Ki value is observed for compound 8 on
hCA I (Table 1).
The docked poses of compounds 4 and 5 in hCA II form hydro-
gen bonds with Asn62 and Asn67. Asn62 is also present in hCA IX
and hCA XII, but not in hCA I (Val62, Table 2). Asn67 (hCA II) is the
counterpart of His67 (hCA I), Gln67 (hCA IX) and Lys67 (hCA XII).
It should be noted that differences in ligand recognition and
binding are not only dependent on the residues that directly line
the binding pocket but also on more distant residues. In addition,
charged residues that are not in direct contact with the ligand, such
as Arg60 (hCA IX), Lys67 (hCA XII) and Glu69 (hCA II), can also
influence binding processes.
For the hCA II enzyme, the crystal structure of hCA II in complex
with
2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide
was used (PDB: 3B4F; 1.89 Å). Compounds 4 and 5 form hydrogen
bonds with the side chains of Asn62 and Asn67. These interactions
are not present in the binding interactions of compound 3 and
hence the Ki value is higher (Table 1). Adding a chlorine substituent
to compound 5 does not influence the binding mode, because the
active site is large enough to accommodate the chlorine atom.
Increasing the spacer size of compound 5 with either a methyl
or an ethyl group places the benzodithiazole groups of compounds
7 and 8 close to the side chain of Gln92 and hydrogen bonds are
formed with both ligands.
Docking studies for the hCA IX enzyme were performed using the
3IAI crystal structure (2.20 Å). No clear indications have been found
for the difference in Ki value between compounds 3–8. Most of the
compounds seem to interact with Asn62 and Gln92. Increasing the
spacer size allows for additional interactions with Trp5 and His64.
The final docking was performed using the hCA XII structure in
complex with acetazolamide (PDB: 1JD0; 1.50 Å). Again, no clear
indications have been found for the measured Ki values of com-
pounds 3–8. It seems however that compound 5 could form hydro-
gen bonding interactions between its sulfoxide group and the side
chain of Gln92.
3. Conclusions
In summary, six structurally new sulfonamides have been syn-
thesized and tested against the cytosolic hCA I and hCA II isozymes
and the tumor-associated hCA IX and hCA XII isozymes. All mea-
sured Ki values were lower than 100 nM for all four hCA isozymes.
Several of the new sulfonamides showed better enzyme inhibition
than the reference sulfonamide in clinical use acetazolamide.
4. Materials and methods
4.1. Chemistry
Melting points were estimated with a Buchi 540 melting point
apparatus in open capillaries and are uncorrected. Elemental
analyses were performed on a Thermo Finnigan Flash EA 1112 ele-
mental analyzer. IR spectra were recorded on KBr discs, using a
Perkin–Elmer Model 1600 FT-IR spectrometer. 1H NMR and
HSQC-2D spectra were obtained on Bruker Avance DPX 400 and
Varian UNITY INOVA 500 spectrophotometers using DMSO-d6.
Mass spectra were determined on a Mass-AGILENT 1100 MSD
instruments. All chemicals and solvents were purchased from
Merck–Schuchardt.
2.4. Structural comparisons of CA binding pockets
In addition to the docking studies, we compared the overall
backbone structures and the binding pockets of the four enzymes
(Table 2) using the MOE software package (version 2011.10, CCG,
Montreal, Canada). The four crystal structures superpose well on
their backbone Ca-atoms (RMSD: 1.560 Å for 260 residues), espe-
4.2. General procedure for the preparation of o-
cially near the binding pocket (RMSD: 0.637 Å for 24 residues;
pocket is defined as all residues within 4.5 Å of the ligand in
3LXE and their counterparts in the other structures). Nevertheless,
some differences in the backbone orientations of the four enzyme
structures are present near the conserved residues Trp5 and His64.
Gln92 of hCA I is conserved in all four crystal structures (Table
2). Even though the conformation of the Gln92 side chain is differ-
ent in the four structures (RMSD: 0.438 Å for all Gln92 heavy
atoms), this residue is able to present its side chain nitrogen or
oxygen atom to ligands for hydrogen bonding. Therefore, this res-
idue is involved in most docking poses that are obtained for the
four enzyme structures.
benzenedisulfonimide derivatives (3–8)13
To
a refluxing solution containing 10 mmol of o-benze-
nedisulfonyl chloride 1, in 20 mL of dry CH2Cl2 was added a solu-
tion of 11 mmol of substituted benzenesulfonamide 2 and 1.0 mL
of Et3N in 25 mL of CH2Cl2 over a period of 60 min. The resulting
mixture was stirred overnight at room temperature. The reaction
mixture was then washed with 0.1 M aqueous HCl, 5% NaHCO3,
water and finally dried over anhydrous MgSO4. Filtration and evap-
oration of the solvent under reduced pressure gave the crude prod-
uct as a solid material. Recrystallization from ethanol.
His67 of hCA I is also involved in hydrogen bonding in the dock-
ing poses. This residue is not conserved amongst the four enzyme
structures and its counterparts in the other enzymes differ with re-
spect to their shape, size and charge (Table 2).
His200 of hCA I forms hydrophobic interactions with the phenyl
group of the ligands. In hCA II, hCA IX and hCA XII this residue is
Thr200.
4.2.1. 2-(1,1,3,3-Tetraoxido-1,3,2-benzodithiazol-2-
yl)benzenesulfonamide (3)
Mp 251–252 °C; IR(KBr) (m
, cmÀ1), 3236, 3352 (NH), 1172, 1354
(SO2); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 7.40 (2H, s, SO2NH2),
7.44 (1H, dd, J = 8.79, 1.46 Hz, phenyl C3–H), 7.75 (1H, td, J = 7.80,
1.95 Hz, phenyl C5–H), 7.82 (1H, td, J = 8.05, 1.46 Hz, phenyl C4–
H), 8.12 (1H, dd, J = 7.56, 1.46 Hz, phenyl C6–H), 8.14 (2H, dd,
J = 5.86, 2.93 Hz, benzothiazole C5,6–H), 8.47 (2H, dd, J = 5.37,
2.93 Hz, benzothiazole C4,7–H). Anal. Calcd for C12H10N2O6S3
(374.413): C, 38.49; H, 2.69; N, 7.48; S, 25.69. Found: C, 38.37; H,
2.68; N, 7.50; S, 25.42.
Table 2
Differences and similarities in the active sites of the four enzymes
hCA I
hCA II
hCA IX
hCA XII
Trp5
Trp5
Trp5
Trp5
Ile60
Leu60
Asn62
His64
Asn67
Glu69
Gln92
Thr200
Arg60
Asn62
His64
Gln67
Thr69
Gln92
Thr200
Thr60
Asn62
His64
Lys67
Asn69
Gln92
Thr200
Val62
His64
His67
Asn69
Gln92
His200
4.2.2. 3-(1,1,3,3-Tetraoxido-1,3,2-benzodithiazol-2-
yl)benzenesulfonamide (4)
Mp 212–214 °C; IR(KBr) (m
, cmÀ1), 3286, 3371 (NH), 1165, 1352
(SO2); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 7.69 (2H, s, SO2NH2),
7.83 (1H, dd, J = 7.56, 1.47 Hz, phenyl C4–H), 7.92 (1H, t, J = 7.81 Hz,

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Ö. Güzel-Akdemir et al. / Bioorg. Med. Chem. 21 (2013) 1386–1391
phenyl C5–H), 8.00 (1H, t, J = 1.95 Hz, phenyl C2–H), 8.17 (1H, d,
J = 7.81 Hz, phenyl C6–H), 8.22 (2H, dd, J = 5.86, 3.42 Hz, benzothi-
azole C5,6–H), 8.61 (2H, dd, J = 5.86, 3.42 Hz, benzothiazole C4,7–H).
Anal. Calcd for C12H10N2O6S3 (374.413): C, 38.49; H, 2.69; N, 7.48;
S, 25.69. Found: C, 38.83; H, 2.79; N, 7.40; S, 25.91.
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 have been used for
measuring the inhibition constant. Inhibitor and enzyme solutions
were preincubated together for 10 min 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. The inhibition constants were obtained by non-linear least-
squares methods using PRISM 3, as reported earlier,16,17 and repre-
sent the mean from at least three different determinations. Human
CA isozymes were prepared in recombinant form as reported ear-
lier by our groups.18,19
4.2.3. 4-(1,1,3,3-Tetraoxido-1,3,2-benzodithiazol-2-
yl)benzenesulfonamide (5)
Mp 234–236 °C; IR(KBr) (m
, cmÀ1), 3280, 3373 (NH), 1161, 1363
(SO2); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 7.60 (2H, s, SO2NH2),
7.81 (2H, d, J = 8.78 Hz, phenyl C3,5–H), 8.11 (2H, d, J = 8.30 Hz, phe-
nyl C2,6–H), 8.22 (2H, dd, J = 5.85, 2.93 Hz, benzothiazole C5,6–H),
8.60 (2H, dd, J = 5.86, 3.42 Hz, benzothiazole C4,7–H). ESI (À)m/z
(%): 373 (MHÀ, 19). Anal. Calcd for C12H10N2O6S3 (374.413): C,
38.49; H, 2.69; N, 7.48; S, 25.69. Found: C, 38.32; H, 2.78; N,
7.43; S, 25.76.
4.4. Ligand preparation
Compounds 3–8 and the reference ligand AZA were built and
converted to three-dimensional structures using the MOE software
package (version 2011.10, Chemical Computing Group, Montreal,
Canada). The sulfonamide substituent of the phenyl group was as-
signed a negative charge (–SO2NHÀ) because it was expected to
interact with the Zn2+ ion of the CA active sites. Subsequently, par-
tial atomic charges were calculated and the molecules were en-
ergy-minimized according to a steepest-descent protocol using
the MMFF94x force field in MOE. The ligands were saved as a mul-
ti-mol2 file.
4.2.4. 3-Chloro-4-(1,1,3,3-tetraoxido-1,3,2-benzodithiazol-2-
yl)benzenesulfonamide (6)
Mp 211–212 °C; IR(KBr) (m
, cmÀ1), 3248, 3325 (NH), 1176, 1359
(SO2); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 7.71 (2H, s, SO2NH2),
7.91 (1H, d, J = 8.30 Hz, phenyl C5–H), 8.02 (1H, dd, J = 8.30,
2.44 Hz, phenyl C6–H), 8.18 (1H, d, J = 2.44 Hz, phenyl C2–H), 8.22
(2H, dd, J = 5.85, 2.93 Hz, benzothiazole C5,6–H), 8.61 (2H, dd,
J = 5.86, 2.93 Hz, benzothiazole
C4,7–H). Anal. Calcd for
C12H9ClN2O6S3 (408.858): C, 35.25; H, 2.22; N, 6.85; S, 23.53.
Found: C, 35.02; H, 2.22; N, 6.47; S, 23.21.
4.5. Template preparation
4.2.5. 4-[(1,1,3,3-Tetraoxido-1,3,2-benzodithiazol-2-
yl)methyl]benzenesulfonamide (7)
Crystal structures of hCA I (PDB: 3LXE; 1.90 Å),20 hCA II (PDB:
3B4F; 1.89 Å),19 hCA IX (PDB: 3IAI; 2.20 Å)21 and hCA XII (PDB:
1JD0; 1.50 Å)22 were obtained from the protein databank. Only 1
protein chain (chain A) and its corresponding Zn2+ ion and ligand
(CA inhibitor) was retained per PDB file and all other protein
chains, ions, buffer molecules and water molecules were deleted.
Hydrogen atoms and charges were added to the protein using
the protonate 3D tool of the MOE software package (version
2011.10, CCG, Montreal, Canada) and a steepest descent energy
minimization was applied (MMFF94x forcefield). All proteins were
Mp 154–155 °C; IR(KBr) (m
, cmÀ1), 3271, 3367 (NH), 1176, 1355
(SO2); 1H NMR (DMSO-d6, 500 MHz) d (ppm): 5.03 (2H, s, N-CH2–),
7.37 (2H, s, SO2NH2), 7.63 (2H, d, J = 7.81 Hz, phenyl C3,5–H), 7.85
(2H, d, J = 7.81 Hz, phenyl C2,6–H), 8.16 (2H, dd, J = 5.86, 3.42 Hz,
benzothiazole C5,6–H), 8.48 (2H, dd, J = 5.85, 3.41 Hz, benzothiazole
C4,7–H). Anal. Calcd for C13H12N2O6S3 (388.439): C, 40.20; H, 3.11;
N, 7.21; S, 24.76. Found: C, 40.35; H, 2.99; N, 7.24; S, 24.98.
4.2.6. 4-[2-(1,1,3,3-Tetraoxido-1,3,2-benzodithiazol-2-
yl)ethyl]benzenesulfonamide (8)
superposed on the hCA-I structure using their Ca-atoms (RMSD:
1.411 Å) and the proteins were saved as mol2-files.
Mp 193–194 °C; IR(KBr) (
m
, cmÀ1), 3253, 3388 (NH), 1168, 1348
(SO2); 1H NMR (DMSO-d6, 500 MHz)
d
(ppm): 3.18 (2H, t,
J = 6.83 Hz, CH2), 3.98 (2H, t, J = 6.83 Hz, N-CH2), 7.29 (2H, s,
SO2NH2), 7.51 (2H, d, J = 8.29 Hz, phenyl C3,5–H), 7.75 (2H, d,
J = 8.29 Hz, phenyl C2,6–H), 8.13 (2H, dd, J = 5.86, 2.93 Hz, benzothi-
azole C5,6–H), 8.42 (2H, dd, J = 5.85, 2.93 Hz, benzothiazole C4,7–H);
13C NMR (HSQC-2D, DMSO-d6, 125 MHz) d (ppm): 34.66 (–CH2-Ph),
43.47 (N-CH2–), 123.60 (benzothiazole C4,7), 126.43 (phenyl C2,6),
130.20 (phenyl C3,5), 137.08 (benzothiazole C5,6), 134.58 (phenyl
C1), 141.93 (benzothiazole C3a,7a), 143.38 (phenyl C4). ESI (À) m/z
(%): 401 (MH–, 100). Anal. Calcd for C14H14N2O6S3 (402.46): C,
41.78; H, 3.51; N, 6.96; S, 23.90. Found: C, 41.95; H, 3.42; N,
6.81; S, 23.52.
4.6. Docking studies
Compounds 3–8 and the reference ligand AZA were docked into
the protein models of hCA I, hCA II, hCA IX and hCA XII using the
GOLD Suite docking package (version 5.1, CCDC, Cambridge,
UK)12 and the GoldScore scoring function (25 docking per ligand).
The binding pocket was defined as all residues within 12 Å of the
central carbon atom of the CA inhibitor topiramate (atom CAL of
topiramate in complex with hCA I; PDB: 3LXE). No restrictions
were applied for the ligand conformations and binding poses dur-
ing the docking procedure except for the requirement to form
hydrogen bonding with Thr199 (or its counterparts in other CA
isozymes).
4.3. CA inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of vari-
ous CA isozymes as reported by Khalifah.14 Phenol red (at a con-
centration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nM, with 10 mM Hepes (pH 7.4) as
buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic
strength; these anions are not inhibitory in the used concentra-
tion),15 following the CA-catalyzed CO2 hydration reaction for a
period of 5–10 s. Saturated CO2 solutions in water at 25 °C were
used as substrate. Stock solutions of inhibitors were prepared at
Acknowledgments
This project was in part financed by the Istanbul University
Scientific Research Projects Department under Project Numbers
ACIP-26528 and BYP-17334 and by an FP7 EU Project (Metoxia).
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