Synthesis and biological evaluation of cyclic imides incorporating benzenesulfonamide moieties as carbonic anhydrase I, II, IV and IX inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.01.032

A group of cyclic imides was synthesized by reaction of amino-substituted benzenesulfonamides with a series of acid anhydrides such as succinic, maleic, tetrahydrophthalic, pyrazine-2,3-dicarboxylic acid anhydride, and substituted phthalic anhydrides. The synthesized sulfonamides were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV and IX, involved in a variety of diseases among which glaucoma, retinitis pigmentosa, etc. Some of these sulfonamides showed effective inhibitory action (in the nanomolar range) against the cytosolic isoform hCA II and the transmembrane, tumor-associated one hCA IX, making them interesting candidates for preclinical evaluation in glaucoma or various tumors in which the two enzymes are involved. hCA I and IV were on the other hand less inhibited by these sulfonamides, with inhibition constants in the micromolar range.

Full text of DOI:10.1016/j.bmc.2017.01.032

Accepted Manuscript
Synthesis and biological evaluation of cyclic imides incorporating benzenesul-
fonamide moieties as carbonic anhydrase I, II, IV and IX inhibitors
Alaa A.-M. Abdel-Aziz, Andrea Angeli, Adel S. El-Azab, Mohamed A. Abu
El-Enin, Claudiu T. Supuran
PII:
S0968-0896(16)31418-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.032
BMC 13511
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 December 2016
18 January 2017
19 January 2017
Please cite this article as: Abdel-Aziz, A.A., Angeli, A., El-Azab, A.S., Abu El-Enin, M.A., Supuran, C.T., Synthesis
and biological evaluation of cyclic imides incorporating benzenesulfonamide moieties as carbonic anhydrase I, II,
IV and IX inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.032
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1
Synthesis and biological evaluation of cyclic imides incorporating benzenesulfonamide moieties as
carbonic anhydrase I, II, IV and IX inhibitors
Alaa A.-M. Abdel-Aziza,b*, Andrea Angeli,c Adel S. El-Azaba,d, Mohamed A. Abu El-Enin,b
Claudiu T. Supuran*c
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
cUniversità degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino,
Florence, Italy.
dDepartment of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Abstract. A group of cyclic imides was synthesized by reaction of amino-substituted
benzenesulfonamides with a series of acid anhydrides such as succinic, maleic, tetrahydrophthalic,
pyrazine-2,3-dicarboxylic acid anhydride, and substituted phthalic anhydrides. The synthesized
sulfonamides were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h)
isoforms hCA I, II, IV and IX, involved in a variety of diseases among which glaucoma, retinitis
pigmentosa, etc. Some of these sulfonamides showed effective inhibitory action (in the nanomolar
range) against the cytosolic isoform hCA II and the transmembrane, tumor-associated one hCA IX,
making them interesting candidates for preclinical evaluation in glaucoma or various tumors in which
the two enzymes are involved. hCA I and IV were on the other hand less inhibited by these
sulfonamides, with inhibition constants in the micromolar range.
Keywords: cyclic imide; benzenesulfonamide; carbonic anhydrase; inhibitor
* Corresponding authors. Tel.: 00966-53-5991127; fax: 00966-1-4676220; e-mail: alaa_moenes@yahoo.com (AAM Abdel-Aziz); Tel./Fax.
+39-055-473729, E-mail: claudiu.supuran@unifi.it (CT Supuran).

2
1. Introduction
In previous works from these and other groups,1-11 the preparation and biological activity of cyclic
imides incorporating primary sulfonamide moieties were investigated. Phthalimides were definitely the
privileged such scaffolds, being shown that they may lead to effective inhibitors of the metallo-enzyme
carbonic anhydrase (CA, EC 4.2.1.1).2,8-11 Furthermore, some substituted-phthalimides derived from
aromatic sulfonamides such as sulfanilamide, metanilamide or aminoalkyl-benzenesulfonamides, not
only showed low nanomolar activity as CA inhibitors (CAIs), but some of them were also highly
isoform-selective inhibitors for several CAs involved in relevant pathologies such as glaucoma,12
epilepsy,13 obesity,14 or cancer.15 Indeed, of the 15 different human(h) CA isoforms known to date, many
are involved in such and other pathologies,16 and finding isoform-selective inhibitors is rather
challenging, although many such compounds belonging to various classes of inhibitors were reported by
now to possess such properties.17 Sulfonamides are in clinical use as CAIs for more than 70 years,
primarily as antiglaucoma agents or diuretics.12-17
Continuing our interest in sulfonamide CAIs, in this work we report the synthesis and human (h) hCA
I, II, IV and IX inhibitory activity of cyclic imides obtained from amino-substituted
benzenesulfonamides and a series of acid anhydrides such as succinic, maleic, tetrahydrophthalic,
pyrazine-2,3-dicarboxylic acid anhydride, and substituted phthalic anhydrides.
2. Results and discussion
2.1. Drug design and Chemistry
The lead molecules for designing the CAIs synthetized here were the sulfanilamides incorporating
phthalimides moieties reported earlier by one of these groups.2,9-11 We have shown that reaction of
amino-substituted benzenesulfonamides, such as sulfanilamide, 4-aminomethylbenzenesulfonamide or
4-aminoethylbenzenesulfonamide with phthalic anhydride or substituted-phthalic anhydrides leads to the
corresponding phthalimides derivatives in high yields.2,9-11 Furthermore, the new sulfonamides showed
effective inhibitory action against human CA isoforms I, II, IV and IX.2,9-11

3
In the present work we have included diverse anhydrides for the preparation of the imides, among which
succinic, maleic, tetrahydrophthalic, pyrazine-2,3-dicarboxylic acid anhydride, substituted phthalic
anhydrides and norbornene dicarboxylic anhydride. In this way, a wider range of chemical space is
being explored in order to investigate the effects of the cyclic imide scaffold on the biological activity of
these new sulfonamides.
The preparation of designed imides analogues 1-15 is shown in Schemes 1 and 2. Condensation of acid
anhydrides with 4-(2-aminoethyl)benzenesulfonamide in refluxing acetic acid afforded imides 1-12 in
good yields (Scheme 1).1-6
O
O
O
S
N
H N
2
O
1
O
O
O
S
N
H N
2
2
O
O
O
O
S
O
N
H N
2
NH
2
O
O
O
O
S
3
Acetic acid, Reflux, 12h
NH
2
O
O
O
O
N
Anhydrides
S
N
H N
2
N
4
O
O
O
O
S
N
H N
2
R
O
R= 5; H, 6; 5-Me, 7; 5-tert-But, 8; 5,6-Dichloro,
9; 4,5,6,7-Tetrachloro,10; 4,5,6,7-Tetrabromo,
11; 5-NO , 12; 4-NO
2
2
Scheme 1. Synthesis of the designed cyclic imide derivatives 1-12.

4
NH
2
O
O
S
NH
2
Cl
NH
2
Cl
O
Cl
O
O
S
Cl
NH
2
Sulfonamides
O
Cl
NH
2
Cl
O
O
S
O
H N
2
Acetic acid, Reflux, 6h
Cl
Cl
Cl
Cl
O
Cl
Cl
O
Cl
Cl
O
Cl
Cl
Cl
Cl
NH
2
N
N
N
S
Cl
Cl
Cl
O
NH
O
Cl
Cl
2
Cl
S
O
O
O
O
O
O
S
NH
13
14
15
2
O
Scheme 2. Synthesis of the designed hexachloro norbornene dicarboxylic imides derivatives 13-15.
The remaining compounds, 13-15 (Scheme 2) were prepared in a similar manner, but in this case the
norbornene dicarboxylic anhydride has been employed, leading to the tricyclic sulfonamides 13-15.
Such scaffolds were not employed earlier for obtaining CAIs.
Compounds 1-15 have been characterized in detail by physico-chemical methods which
confirmed their structures (see Experimental for details).
2.2. Carbonic anhydrase inhibition

5
The newly synthesized compounds 1-15 were screened for their CA inhibitory activity by a stopped-
flow CO2 hydrase assay18 against the following isoforms: hCA I, II (cytosolic isoforms involved in
glaucoma13 and possibly other pathologic states)19 and hCA IV (an isoform involved in glaucoma and
other eye diseases, such as retinitis pigmentosa),20 as well as the transmembrane, tumor-associated hCA
IX 15,21 see Table 1.
Table 1: In vitro CA I, II, IV and IX inhibition with compounds 1-15 and acetazolamide as standard, by
a stopped-flow, CO2 hydrase assay.18
KI (nM)*
Compound
hCA I
hCA II
hCA IV
hCA IX
1
2
3
4
5
6
7
8
446
617
49.2
441
491
425
95.7
435
562
5121
865
179
390
201
484
250
73.6
84.5
5.0
9578
4849
973
296
274
296
4188
296
390
459
428
284
2389
1636
2667
13.7
16.9
113.6
25.2
426
9380
5461
2925
9391
9358
9232
8839
8175
8517
83.0
912
49.4
60.5
89.9
79.0
68.1
693
9
10
11
12
13
14
15
AAZ
140
85.4
41.5
54.6
23.2
12.1
6764
74
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5-10
% of the reported values).
The following structure–activity relationships (SARs) are observed and reported for these series of
compounds:
i)
The slow cytosolic isoform hCA I was inhibited by all the investigated compounds with
inhibition constants ranging between 49.2 and 5121 nM. Sulfonamides incorporating

6
tetrahydrophthalimido and tert-butyl phthalimido moietes 3 and 7 showed the best
inhibitiory activity (KI of 49.2 and 95.7 nM, respectively). The other scaffolds were less
potent and inhibited this isoform in the high nanomolar range (KI ranging between 179 and
865 nM). An interesting case was constituted by the isosteric substitution of the chlorine
atoms in the phthalimido compounds 9 with bromine, in 10, which led to a significant
decrease of the inhibitory potency. Considering the monocyclic imides 1, 2 and the bicyclic
scaffold 3-15, the inhibition potency against the hCA I did not vary significantly, except for
compound 3, which was the best inhibitor in the subseries. The linker between the
hexachloronorbornene dicarboxylic scaffold and the sulfonamide part of the molecules
present in 13, 14 and 15 also modulated the inhibition profile. The introduction of a
methylene chain, as in 14, increased the inhibition potency from 390 nM (13) to 201 nM
(14), but a further chain elongation, as in 15, decreased two times the KI for the last
compound.
ii)
hCA II, the dominant physiologic isoform, was inhibited by all compounds in the low to high
nanomolar range, with KIs of 5 – 693 nM. The tetrahydrophthalimido derivative 3 showed a very
good inhibition activity against this isoform, with a KI of 5 nM. The replacement of the
monocyclic imido scaffold with a bicyclic one did not have a significant effect on the inhibition
profiles of these sulfonamides (Table 1). Only in the case of the pyrazine containing sulfonamide
4, a decreased inhibitory potency was observed compared to the other members of the series, this
compound being one of the weakest hCA II inhibitor (KI of 426 nM). The substitution on the
phthalimido scaffold in compounds 5-12 did not lead to significant changes in the inhibition
potency, except for the tetrabromo-substituted compound 10, which was a medium potency –
weak hCA II inhibitor (KI 693 nM). Furthermore, the substitution of the nitro group from
position 5 of compound 11 to position 4 (12), increased nearly two times the potency of the

7
corresponding sulphonamide against this. Unlike the other cytosolic isoform, the introduction of
a linker in the norborneneimido derivatives 13-15 had diverse effects on the inhibition profile: a
decrease of the potency was observed for a methylene linker (13, a KI of 41.5 nM whereas 14
had a KI of 54.6 nM), whereas a further chain elongation, as in 15, led to an increase of the
potency with a KI of 23.2 nM for the last compound.
iii)
The membrane-bound hCA IV was inhibited by most of compounds investigated here in the
micromolar range. The monocyclic imido derivatives 1 and 2 showed KIs in the range of 4.85 –
9.57 µM. The maleimido scaffold present in 3 led to a compound which was two times more
potent than the succinimido analog 1. Also for this isoform, the groups present on the
phthalimido scaffold did not influence significantly the inhibition profile. The compounds
incorporating hexachloronorbornene dicarboxylic scaffolds, 13-15, showed a very interesting
inhibition profile. The linker between the imido scaffold and the sulphonamide head was crucial
for the inhibition potency. A direct correlation between the length of the linker and the inhibition
constant was observed: 13 showed a nanomolar inhibition constant (KI 83 nM), whereas an
increase in the linker length to a methylene or an ethylene group led to a drastic decrease of
potency, to the micromolar range (KI of 912 nM for compound 14 and KI of 6764 nM for 15).
hCA IX, the tumor-associated isoform, was effectively inhibited by many of the compounds
reported here, in high nanomolar range except for compounds 13 and 14 which were active in the
low nanomolar range. The monocyclic imido derivatives 1 and 2 had nearly the same inhibition
profile with a KI of 296 nM and 274 nM, respectively. The phthalimido scaffold present in 5-12
led to high nanomolar hCA IX inhibitors, except for nitro-substituted compounds 11 and 12. For
these compounds the potency has decreased to the micromolar range, with KI of 1636 nM and
2667 nM, respectively. Moreover, the hexachloro-norbornene scaffold peseent in 13-15, led to an
interesting SAR for the inhibition of this isoform. Compounds 13 and 14 incorporating no linker
iv)

8
or a methylene linker, exhibited low nanomolar KIs whereas the derivative with a longer chain
15, as for isoform hCA IV, led to a marked decreased in the inhibition potency (Table 1).
3. Conclusions
We investigated a series of cyclic imides as CA inhibitors, continuing our research in this field of
enzyme modulators. The compounds were synthesized by reaction of amino-substituted
benzenesulfonamides with a series of dicarboxylic acid anhydrides such as succinic, maleic,
tetrahydrophthalic, pyrazine-2,3-dicarboxylic acid anhydride, as well as substituted phthalic anhydrides.
The synthesized sulfonamides were evaluated for the inhibition of isoforms hCA I, II, IV and IX,
involved in a variety of diseases among which glaucoma, retinitis pigmentosa, tumors, etc. They showed
effective inhibitory action against the cytosolic isoform hCA II and the transmembrane, tumor-
associated one hCA IX, making them interesting candidates for preclinical evaluation in glaucoma or
various tumors in which the two enzymes are involved. hCA I and IV were on the other hand less
inhibited by these sulfonamides.
4. Experimental Part
4.1. Chemistry
Melting points (uncorrected) were recorded on Barnstead 9100 Electrothermal melting apparatus. IR
spectra were recorded on a FT-IR Perkin-Elmer spectrometer. 1H NMR and 13C NMR were recorded in
DMSO-d6 on Bruker 500 and 125 MHz instrument, respectively, using TMS as internal standard
(chemical shifts in δppm). Mass spectra were recorded on a Agilent 6320 Ion Trap mass spectrometers.
Elemental analysis was carried out for C, H and N at the Research Centre of College of Pharmacy, King
Saud University and the results are within 0.4% of the theoretical values. Compounds 2, 5, 9, 10 and
11 were prepared according to their reported procedure.7-11

9
4.1.1. General procedure for the synthesis of cyclic imides 1, 3, 4, 6-8, 12.
A mixture of 4-(2-aminoethyl)benzenesulfonamide (0.5g, 2.5 mmol), anhydrous sodium acetate
(0.41g, 5.0 mmol) and an acid anhydride (2.5 mmol) was stirred in glacial acetic acid (15 mL). The
mixture was heated under reflux for 12 h and the reaction was monitored with the help of TLC. After
completion of reaction the solvent was removed under reduced pressure and the crude solid obtained
was washed with water, dried and re-crystallised from an appropriate solvent.
4.1.1.1. 4-(2-(2,5-Dioxopyrrolidin-1-yl)ethyl)benzenesulfonamide (1)
White crystals, M.p 243-245ο, 95% yield (CH3OH); IR (KBr, cm-1) ν: 3496 (NH), 1795, 1725
(C=O), 1382, 1194 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ2.60 (s, 4H), 2.85-2.88 (t, 2H, J
= 7.5 Hz), 3.60-3.63 (t, 2H, J = 7.5 Hz), 7.28 (s, 1H), 7.35-7.37 (d, 2H, J = 8.0 Hz), 7.74-7.76 (d, 2H, J
= 8.0 Hz), 8.22-8.23 (t, 1H, J = 1.5 Hz); 13C NMR (125 MHz, CDCl3/DMSO-d6): δ28.37, 33.15, 39.21,
126.28, 129.47, 142.76, 142.78, 177.74; C12H14N2O4S: m/z (281.99).
4.1.1.2. 4-(2-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindol-2-yl)ethyl)benzenesulfonamide (3)
White crystals, M.p 196-198ο, 91% yield (CH3OH); IR (KBr, cm-1) ν: 3230 (NH), 1767, 1699
(C=O), 1333, 1179 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ2.11-2.15 (m, 2H), 2.34-2.37 (d,
2H, J = 13.5 Hz), 2.83-2.86 (t, 2H, J = 7.5 Hz), 3.04-3.05 (t, 2H, J = 3.0 Hz), 3.61-3.64 (t, 2H, J = 7.0
13
Hz), 5.73-5.77 (m, 2H), 7.21 (s, 2H), 7.27-7.29 (d, 2H, J = 8.0 Hz), 7.72-7.74 (d, 2H, J = 8.5 Hz); C

10
NMR (125 MHz, CDCl3/DMSO-d6): δ25.33, 33.09, 38.86, 42.74, 127.08, 129.33, 142.30, 142.42,
142.78, 179.94; C16H18N2O4S: m/z (333.70).
4.1.1.3. 4-(2-(5,7-Dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)ethyl)benzenesulfonamide (4)
Gray powder, M.p 120-122ο, 76% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3430 (NH), 1782,
1
1707 (C=O), 1318, 1129 (O=S=O); H NMR (500 MHz, DMSO-d6): δ2.76-2.79 (t, 2H, J = 7.0 Hz),
3.27-3.03 (t, 2H, J = 7.0 Hz), 7.38-7.41 (t, 3H, J = 8.0 Hz), 7.74-7.75 (d, 3H, J = 8.5 Hz), 8.01-8.03 (t,
1H, J = 5.0 Hz), 9.11 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 35.28, 38.65, 126.13, 129.54, 142.47,
144.22, 149.01, 169.77; C14H12N4O4S: m/z (331.76).
4.1.1.4. 4-(2-(5-Methyl-1,3-dioxoisoindolin-2-yl)ethyl)benzenesulfonamide (6)
White crystals, Mp 221-222ο, 93% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3466 (NH), 1767,
1722 (C=O), 1300, 1131 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ2.43-2.45 (d, 3H, J = 9.0
Hz), 2.96-2.99 (t, 2H, J = 7.0 Hz), 3.79-3.84 (q, 2H, J = 8.0, 7.0 Hz), 6.97 (s, 2H), 7.26-7.00 (t, 2H, J =
8.5 Hz), 7.46-7.49 (t, 1H, J = 7.5 Hz), 7.53-7.54 (d, 1H, J = 7.5 Hz), 7.60-7.63 (t, 1H, J = 7.5 Hz), 7.71-
7.74 (t, 2H, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3/DMSO-d6): δ22.00, 34.19, 38.62, 123.17, 123.80,
126.30, 129.19, 132.20, 134.76, 142.41, 142.50, 145.43, 167.91, 168.02; C17H16N2O4S: m/z (344.07).
4.1.1.5. 4-(2-(5-(tert-Butyl)-1,3-dioxoisoindolin-2-yl)ethyl)benzenesulfonamide (7)

11
White powder, M.p 160-162ο, 89% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3493, 3095 (NH),
1
1780, 1727 (C=O), 1360, 1236 (O=S=O); H NMR (500 MHz, DMSO-d6): δ1.32 (s, 9H), 2.96-2.99 (t,
2H, J = 7.0 Hz), 3.82-3.85 (t, 2H, J = 7.5 Hz), 6.94 (s, 2H), 7.28-7.30 (d, 2H, J = 8.0 Hz), 7.69-7.70 (t,
13
2H, J = 7.0 Hz), 7.73-7.55 (t, 3H, J = 8.5 Hz); C NMR (125 MHz, DMSO-d6): δ31.17, 34.22, 35.73,
38.61, 120.30, 123.13, 126.32, 129.18, 131.22, 132.07, 142.39, 142.46, 158.56, 167.88, 168.26;
C20H22N2O4S: m/z (387.15).
4.1.1.6. 4-(2-(5,6-Dichloro-1,3-dioxoisoindolin-2-yl)ethyl)benzenesulfonamide (8)
White cystals, Mp 295-296ο, 91% yield (CH3OH); IR (KBr, cm-1) ν: 3469, 3369 (NH), 1727,
1
1670 (C=O), 1366, 1167 (O=S=O); H NMR (500 MHz, DMSO-d6): δ2.99-3.02 (t, 2H, J = 7.5 Hz),
3.83-3.86 (t, 2H, J = 7.5 Hz), 7.31 (s, 2H), 7.40-7.42 (d, 2H, J = 8.0 Hz), 7.71-7.73 (t, 2H, J = 8.5 Hz),
13
8.15-8.16 (t, 2H, J = 8.5 Hz); C NMR (125 MHz, DMSO-d6): δ33.78, 39.34, 125.74, 126.26, 129.67,
131.88, 137.81, 142.80, 166.32; C16H12Cl2N2O4S: m/z (398.60).
4.1.1.7. 4-(2-(4-Nitro-1,3-dioxoisoindolin-2-yl)ethyl)benzenesulfonamide (12)
Yellow powder, Mp 238-240ο, 86% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3485 (NH), 1779,
1
1732 (C=O), 1362, 1228 (O=S=O); H NMR (500 MHz, DMSO-d6): δ2.99-3.02 (t, 2H, J = 6.0 Hz),

12
3.83-3.86 (t, 2H, J = 7.5 Hz), 7.32 (s, 2H), 7.44-7.45 (d, 2H, J = 8.5 Hz), 7.73-7.75 (t, 2H, J = 6.0 Hz),
8.05-8.07 (t, 1H, J = 5.0 Hz), 8.15-8.16 (t, 1H, J = 5.5 Hz), 8.28-8.29 (t, 1H, J = 5.0 Hz); 13C NMR (125
MHz, DMSO-d6): δ33.72, 39.44, 123.44, 126.29, 127.32, 128.80, 129.65, 133.92, 136.70, 142.82,
142.89, 144.76, 163.65, 166.19; C16H13N3O6S: m/z (374.54).
4.1.2. General procedure for the synthesis of cyclic imides 13-15.
A mixture of benzenesulfonamide (2.5 mmol), anhydrous sodium acetate (0.41g, 5.0 mmol) and
4,5,6,7,8,8-hexachloro-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1,3-dione (0.927g, 2.5 mmol)
were stirred in glacial acetic acid (15 mL) and heated under reflux for 6 h. After the evaporation of the
solvent under reduced pressure, the precipitate obtained was washed with water, dried and re-
crystallised from an appropriate solvent.
4.1.2.1. 4-(4,5,6,7,8,8-Hexachloro-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-
yl)benzenesulfonamide (13)
White powder, M.p >350ο, 95% yield (CH3OH); IR (KBr, cm-1) ν: 3370, 3250 (NH), 1769, 1708
(C=O), 1388, 1144 (O=S=O); 1H NMR (500 MHz, DMSO-d6): δ4.28 (s, 2H), 7.34-7.35 (d, 2H, J = 8.5
Hz), 7.52 (s, 2H), 8.00-8.01 (d, 2H, J = 8.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ52.70, 79.44,
104.15, 127.52, 131.12, 134.07, 145.07, 169.85; C15H8Cl6N2O4S: m/z (526.43).
4.1.2.2. 4-((4,5,6,7,8,8-Hexachloro-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-
yl)methyl)benzenesulfonamide (14)
Cl
Cl
O
Cl
Cl
N
Cl
Cl
O
O
S
NH2
O

13
White powder, M.p 201-203ο, 87% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3307, 3229 (NH),
1
1759, 1711 (C=O), 1369, 1180 (O=S=O); H NMR (500 MHz, CDCl3/DMSO-d6): δ3.95 (s, 2H), 4.52
(s, 2H), 6.89 (s, 2H), 7.36-7.38 (d, 2H, J = 8.5 Hz), 7.77-7.78 (d, 2H, J = 8.0 Hz); 13C NMR (125 MHz,
CDCl3/DMSO-d6): δ42.49, 52.09, 79.43, 104.37, 126.39, 129.74, 130.58, 138.23, 143.87, 170.05;
C16H10Cl6N2O4S: m/z (539.50).
4.1.2.3. 4-(2-(4,5,6,7,8,8-Hexachloro-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-
2-yl)ethyl)benzenesulfonamide (15)
White powder, M.p 226-228ο, 83% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3323, 3213 (NH),
1751, 1714 (C=O), 1373, 1165 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ2.71-2.74 (t, 2H, J =
10.0 Hz), 3.55-3.58 (q, 2H, J = 10.0, 5.5 Hz), 4.01-4.02 (d, 2H, J = 3.5 Hz), 7.17 (s, 2H), 7.33-7.34 (t,
13
2H, J = 4.0 Hz), 7.76-7.78 (q, 2H, J = 8.5, 4.5 Hz); C NMR (125 MHz, CDCl3/DMSO-d6): δ33.42,
39.60, 52.17, 79.26, 104.36, 126.41, 129.20, 130.71, 141.39, 143.04, 170.38; C17H12Cl6N2O4S: m/z
(552.19).
4.2. Carbonic anhydrase inhibition
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay
the catalytic/inhibition of various CA isozymes.18 Phenol Red (at a concentration 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

14
inhibitory in the used concentration),22 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 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 7 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 the Cheng-Prusoff equation, as reported earlier,11 and represent the mean from at least three
different determinations. All CA isozymes used here were recombinant proteins obtained as reported
earlier by our group.23-26
Acknowledgments. The authors express their appreciation to the Deanship of Scientific Research at
King Saud University for funding the work through the research group project No. RGP-163.

15
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