Synthesis and carbonic anhydrase inhibition of polycyclic imides incorporating N-benzenesulfonamide moieties

Article abstract of DOI:10.1016/j.bmc.2017.07.056

A series of polycyclic imides was prepared by reaction of the benzenesulfonamide with an appropriate polycyclic acid anhydride in refluxing glacial acetic acid. The synthesized mono- and bis-sulfonamides were evaluated as a carbonic anhydrase inhibitors (CA, EC 4.2.1.1), more precisely against the human (h) isoforms hCA I, II, IX and XII, some of which are involved in various pathologies, such as glaucoma, epilepsy and cancer. Several low nanomolar and isoform-selective hCA II, IX and XII inhibitors were detected, and the structure-activity relationship for CA inhibition with this class of compounds is discussed in details. Computational studies allowed us to explain the efficacy and isoform-selective behaviour for some of these enzyme inhiibtors.

Full text of DOI:10.1016/j.bmc.2017.07.056

Accepted Manuscript
Synthesis and carbonic anhydrase inhibition of polycyclic imides incorporating
N-benzenesulfonamide moieties
Andrea Angeli, Alaa A.-M. Abdel-Aziz, Alessio Nocentini, Adel S. El-Azab,
Paola Gratteri, Claudiu T. Supuran
PII:
S0968-0896(17)31256-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.056
BMC 13893
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 June 2017
19 July 2017
28 July 2017
Please cite this article as: Angeli, A., Abdel-Aziz, A.A., Nocentini, A., El-Azab, A.S., Gratteri, P., Supuran, C.T.,
Synthesis and carbonic anhydrase inhibition of polycyclic imides incorporating N-benzenesulfonamide moieties,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.056
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Synthesis and carbonic anhydrase inhibition of polycyclic
imides incorporating N-benzenesulfonamide moieties
Andrea Angelia, Alaa A.-M. Abdel-Azizb,c*, Alessio Nocentini, d Adel S. El-Azabb,e, Paola
Gratteri,d Claudiu T. Supurana*
aUniversità degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino,
Florence, Italy
bDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
cDepartment of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
d Università degli Studi di Firenze, NEUROFARBA Dep., Pharmaceutical and Nutraceutical Section, Laboratory of Molecular Modeling
Cheminformatics & QSAR, via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
e Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Abstract. A series of polycyclic imides was prepared by reaction of the benzenesulfonamide with an appropriate
polycyclic acid anhydride in refluxing glacial acetic acid. The synthesized mono- and bis-sulfonamides were
evaluated as a carbonic anhydrase inhibitors (CA, EC 4.2.1.1), more precisely against the human (h) isoforms
hCA I, II, IX and XII, some of which are involved in various pathologies, such as glaucoma, epilepsy and cancer.
Several low nanomolar and isoform-selective hCA II, IX and XII inhibitors were detected, and the structure-
activity relationship for CA inhibition with this class of compounds is discussed in details. Computational studies
allowed us to explain the efficacy and isoform-selective behaviour for some of these enzyme inhiibtors.
Keywords: polycyclic imides; benzenesulfonamide; carbonic anhydrase; inhibitor.
____
*Corresponding authors. College of Pharmacy, P.O. Box 2457, King Saud University, Riyadh-11451,
Saudi Arabia, E-mail: almoenes@ksu.edu.sa & alaa_moenes@yahoo.com (A. A.-M. Abdel-Aziz) and
NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, E-mail: claudiu.supuran@unifi.it (CT
Supuran).

2
1. ntroduction
Carbonic anhydrases (CA, EC 4.2.1.1) are zinc metalloenzymes present in most living organisms
and encoded by seven unrelated gene families, the α-, β-, γ-, δ-, ζ-, ɳ-, and the recently
discovered θ-CA class 1-3,5. The major function of CA is to rapidly catalyzed the reversible
hydration of carbon dioxide to bicarbonate and protons, assuring thus an optimal pH regulation
system in most living organisms. This simple process is involved in many crucial physiological
and pathological processes1-4. For this reason since the ‘40s, inhibitors of CA (CAIs) started to
play an important role in clinical use as diuretics and antiglaucoma drugs, whereas more recently
sulfonamide CAIs proved to be useful for the management of a variety of disorders, such as
obesity, metastatic tumors, epilepsy and several other neurologic diseases.1–4
However, the principal drawback of this class of valuable drugs is related to their many side
effects, because most mammalian isoforms are rather well inhibited by the first and second-
generation sulfonamide CAIs, which are still nowadays in clinical use.1-4 Thus, many efforts
were registered ultimately to find inhibitors with selectivity for the different CA isoforms or even
CA families. The strategy used here is based on the ‘tail’ approach,6 which consists in attaching
moieties that induce the desired physico-chemical properties and CA inhibitory selectivity
profiles to the molecules of aromatic sulfonamides possessing free amino groups. By modulating
the functionality (tail) appended to the aromatic/heterocyclic ring present in the scaffold of the
CAIs, in order to differently interact with the middle and the rim parts of the active site cavity,
(which is the most variable region among the many CA isozymes), a large number of isoform-
selective CAIs were obtained.1-6 Some of the tails incorporated in sulfonamide CAIs were of the
cyclic imide type, and they led to compounds with interesting inhibitory profiles against CA
isoforms involved in a variety of disorders, such as the cytosolic CA I and II, or the
transmembrane, tumor-associated CA IX and XII.7-10

3
As a continuation our investigation on sulfonamides incorporating cyclic imide
scaffolds,7-10 we report here the synthesis and inhibitory profile against four physiologically
relevant isoforms (the human (h) hCA I, II, IX and XII) of a new series of such derivatives
incorporating polycyclic imide scaffolds.
2. Results and discussion
2.1. Drug Design and Chemistry
Sulfonamide with different cyclic imide scaffolds were reported earlier by our groups7-10 and showed
effective inhibitory action against human CA isoforms I, II, IX and XII. In the present work continued
this study for synthetic CAIs incorporating new imide moieties such as benzoisoquinoline,
dibenzoazepine, dihydropyrroloisoindole and tetrahydrobenzophenanthroline. The rationale of the study
was to use a simple chemistry for obatining sulfonamides incorporating different polycyclic, bulky
imide moieties, in order to investigate whether the incorporation of such polycyclic structures in the
molecule of an inhibitor is compatible with an efficien binding within the enzyme active site, which for
α-CAs is rather deep and large (at least at the entrance and up until the middle of the cavity).1-3 Aromatic
sulfonamides having free amino groups such as sulfanilamide (1), 4-aminomethylbenzenesulfonamide
(2) or 4-aminoethylbenzenesulfonamide (3) were converted to the corresponding polycyclic imides by
reaction with polycyclic acid anhydrides in refluxing acetic acid and in the presence of anhydrous
sodium acetate, afforded these imides in a reasonable yield 10-15 (Scheme 1 and Scheme 2 ).

4
O
N
O
n
NH2
n = 0 (4), 1 (5), 2 (6)
H2NO2S
H2NO2S
1
O
N
O
Acetic acid, Reflux, 12h
NH2
n
O
+
O
NO2
H2NO2S
2
3
O
H2NO2S
n = 0 (7), 1 (8), 2 (9)
Acid anhydrides
NH2
O
N
H2NO2S
n
O
H2NO2S
n = 0 (10), 1 (11), 2 (12)
Scheme 1. Synthesis of the designed polycyclic imide derivatives 4-12.
Scheme 2. Synthesis of the bis-polycyclic imides 15-20.
The chemical structures of the final products 4-12 and 15-20 were assigned on the basis of spectral data
analyses (see Experimental for details).
2.2 Carbonic anhydrase inhibition

5
The newly synthesized compounds 4-20 were screened for their CA inhibitory activity by a stopped-
flow CO2 hydrase assay16 against the following isoforms: hCA I, II (cytosolic isoforms involved in
glaucoma17 and possibly other pathologic states)18 and the transmembrane, tumor-associated hCA IX
and XII (anticancer drug targets) 19,20 see Table 1.
Table 1: In vitro CA I, II, IX and XII inhibition with compounds 4-12, 15-20 and acetazolamide as
standard, by a stopped-flow, CO2 hydrase assay.16
KI (nM)
Compound
hCA I
hCA II
hCA IX
hCA XII
4
1439.5
142.9
143.0
83.9
63.0
286.9
107.9
107.9
217.9
250.6
242.4
93.1
51.7
42.8
42.8
80.5
84.6
46.9
47.2
74.1
90.3
26.0
39.9
84.4
28.3
47.7
19.9
5.7
5
32.7
6
32.7
7
6.7
8
221.8
924.9
781.4
4975.9
7214.0
293.0
177.8
8390.6
548.3
2988.1
3095.2
250
28.1
9
68.6
10
11
12
15
16
17
18
19
20
AAZ
92.4
6512.4
802.0
203.1
295.6
9188.4
97.1
190.5
301.2
250.9
639.4
2050.6
23.9
704.3
177.3
12.1
2782.7
25.5
25.8
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5-10 % of the
reported values).

6
The following structure–activity relationship (SAR) can be drawn from the data of Table 1:
i) hCA I was moderately inhibited by mosst sulfonamides investigated here, except 7 which
showed a reasonable activity (KI of 83.9 nM). The methylene linker between
benzensulfonamide and the isoquinolin scaffold showed a crucial role for the potency of
inhibition of this isoform. Compound 4, without a linker, was 10 times less potent than
compounds 5 and 6 (respectively with one and two carbon atoms). On the contrary, the
methylene spacer for compounds 7-9, with a nitro group in position 5 of the isoquinolin
scaffold, increased the potency of inhibition for this isoform. In addition, the imide scaffolds
also showed an important contribution to the inhibitory profile, with the dibenzoazepine
moiety present in 10-12 and the tetrahydrobenzo phenanthroline one in 18-20 leading to a
decreased inhibition potency against hCA I (Ki 548.3 nM to Ki 3095.2 nM) compared to less
bulky scaffolds (Table 1).
ii) The dominant cytosolic human isoform hCA II, was inhibited in the low - medium
nanomolar range by compounds incorporating the isoquinolin scaffold 4-9 (Ki 6.7 nM to Ki
68.6 nM). The presence of one and two atoms of carbon in the methylene linker, also for this
isoform, slightly increased the inhibition potency for compounds 5, 6 over the ones without a
linker (4). The interesting case for these molecules was the presence of the nitro group in
position 5 of the isoquinolin scaffold (7-9), leading to an increased potency of inhibition with
a further chain elongation. The presence of the methylene spacer in the other scaffolds, such
as the dibenzoazepine, dihydropyrrolo isoindole and tetrahydrobenzo phenanthroline ones
(10-20), decreased the efficacy of inhibition, respectively, compared to derivatives without a
linker. The monosulfonamides (4-10) were generally better hCA II inhibitors compared to
the bis-sulfonamides 11 -20 (Table 1).

7
iii) The transmmebrane, tumor-associated hCA IX was effectively inhibited by some
compounds reported here, such as 18 and 20, which showed inhibition constants comparable
to those of the standard drug acetazolamide, in the range of 23.9 – 25.8 nM. The spacer
between the polycyclic imide scaffold and sulfonamide, proved to have a crucial role for the
inhibition potency of hCA IX. Compounds 18 and 20, the best inhibitors of this isoform, are
bis-sulfonamides and thsy did not inhibit so potently the cytosolic, off-target isoforms hCA I
and II, being thus also isoform-selective CAIs (Table 1). For the other scaffolds present in
the compounds here considered, a methylene chain elongation decreased the efficacy of
inhibition whereas the ethylene chain was usually more effective in generating good
inhibitors (compare for example 18, 19 and 20).
iv) A very interesting inhibition profile with the polyciclilc amides investigated here was
observed for the second transmembrane isoform, hCA XII. Indeed, all of them were effective
inhibitors, with KIs varying between 19.9 and 90.3 nM (Table 1). The best inhibitor was the
bulky bis-sulfonamide 20 (KI of 19.9 nM). However, all polycyclic imide scaffold present in
these compounds are beneficial for obtaining compounds with efficacy and selectivity
against hCA XII.
2.3. Molecular modeling studies
Docking simulations were performed to point out the stereochemical features of the synthesized
compounds involved in the binding with the isoforms II, IX and XII of the human carbonic anhydrase.
Docking followed by the Prime MM-GBSA protocol was applied to a subset of representative
sulfonamides selected according to the following criteria: (i) two unsubstituted benzoisoquinoline
derivatives 4 and 6 as representative tricyclic compounds with an efficacy mainly directed against hCA

8
II and XII; (ii) compound 10, as it showed the strongest inhibitory effectiveness against the varied CA
isoforms among the dibenzoazepine-bearing compounds; (iii) the dihydropyrroloisoindole derivative as
representative for the dual-sulfonamides-endowed compounds (15-20) mainly active towards isoform
XII.
Consistently with the binding modes extensively described in literature, the benzenesulfonamide
moieties of the investigated molecules went in depth into the binding cavity of all the studied isoforms,
establishing two hydrogen bonds with T199 (N-H…OG1; S=O…HN), the negatively charged nitrogen
atom coordinated the Zn2+ and the phenyl ring was stabilized by CH…π interactions with V121 and L198
(L199/hCA IX). Irrespective of the nature of the tricyclic core and the length of the spacer, 4, 6 and 10
oriented the aromatic tails towards the hydrophobic region of the enzymes. As far as the hCA II is
concerned, the aromatic portions of the tricyclic tails of 4, 6 and 10 were engaged in T-shaped π- π
stacking π-alkyl hydrophobic interactions respectively occurring with the side chain ring of F131 and
with I91, P202 and Q92. The poses were further stabilized by the conventional H-bond occurring
between C=O… HE(Q92) and, to less extent, by a weak C=O… C-H hydrogen bond involving Leu98
(Fig.1a). The presence of an ethylenic linker between the benzenesulfonamide and the tricyclic tail
allowed 6 to better accommodate within the hydrophobic area defined by F131, I91, V121, Q92, thus
positively impact on the inhibitory efficacy.
The “mutations” N67/K67 (hCAII/hCAXII) and F131/A131 (hCAII/hCAXII) played an essential
role in the interactions established by the molecular tails of 4, 6 and 10 within hCA XII binding site,
which in turn lacks aromatic residues. Indeed, the docking pointed out some π-cation interactions
between the aromatics and the positively charged NZ nitrogen atom of lysine residue according to the
nature of the scaffold and the presence of the linker (Fig.1c). Furthermore, the tricyclic tail position was
stabilized by Van der Waals interactions occurring with T91, S132 and S135 and by C=O… HE(Q92)
H-bond (compounds 4 and 10). Conversely, the mutations N67/Q71 (hCAII/hCAIX) and F131/V130

9
(hCAII/hCAIX) did not allow analogue stabilization for the tails of 4, 6 within hCA IX binding site,
which again lacked aromatic residues, thus resulting in the decrease of efficacy in the midnanomolar
range. It is worth noting the π-alkyl hydrophobic interactions that the benzoisoquinoline cores formed
with V130, L134 and P202 (Fig.1b), whereas the low nanomolar efficacy of 10 could be the
consequence of the better adaptation of the dibenzoazepine scaffold within the hydrophobic cleft.
With respect to the dual-sulfonamides-endowed derivative 16, the docking findings located the
molecular tail towards the boundary of the catalytic site of hCA XII, in solvent-exposed region which is
endowed with a high variability among CAs (Fig.1d). The wide network of H-bonds the representative
16 formed uniquely in case of isozyme XII, combined with the roomy nature of the binding site, could
explain the hCA XII-selective inhibition of compounds 15-20.
(b)
(a)
(c)
(d)

10
Figure 1. Predicted binding modes of compounds 4 (grey), 6 (aquamarine) and 10 (green) within hCA II
(a), hCA IX (b) and hCA XII (c) active site; (d) predicted binding mode of compounds 16 within hCA
XII. H-bonds, π-π, π-cation interactions are highlighted respectively by yellow, blue and green dashed
lines.
3. Conclusion
A new series of polycyclic amides incorporating N-benzenesulfonamide was prepared and
investigated for the inhibition of the four physiologically relevant hCA isoforms hCA I, hCA II,
hCA IX and hCA XII. These compounds were generally good to moderate inhibitors of hCA II
and hCA XII, and were moderate to poorly effective within the micromolar range for hCA I and
hCA IX. Some of the compounds here considered, were selectivity inhibitors for the
transmembrane isoforms hCA IX and hCA XII over the cytosolic isoforms hCA I hCA II. These
compounds could be a starting point for the development of more selective and potent inhibitors
belonging to the cyclcic imide sulfonamide class.
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were recorded on Barnstead 9100 Electrothermal melting apparatus. IR
1
spectra were recorded on a FT-IR Perkin-Elmer spectrometer. H 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 δ ppm). 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 4 and 5 were
prepared according to their reported procedure.10,12
4.1.1. General procedure for the synthesis of cyclic imides 6-12.

11
A mixture of benzenesulfonamide (5.0 mmol), anhydrous sodium acetate (0.82 g, 10.0 mmol)
and an appropriate acid anhydride (5.0 mmol) in glacial acetic acid (15 mL) was heated under reflux for
12 h. After cooling of the reaction mixture, the precipitate obtained was filtered, washed with water,
dried and re-crystallised from an appropriate solvent.
4.1.1.1. 4-(2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)benzenesulfonamide (6)
Mp 256-258ο, 91% yield (CH3CH2OH); IR (KBr, cm-1) ν: 3503 (NH), 1790, 1728 (C=O), 1376,
1225 (O=S=O); 1H NMR (500 MHz, DMSO-d6): δ 3.00-3.03 (t, 2H, J = 7.5 Hz), 4.24-4.27 (t, 2H, J =
7.5 Hz), 7.34 (s, 2H), 7.47-7.49 (d, 2H, J = 8.0 Hz), 7.77-7.79 (d, 2H, J = 8.0 Hz), 7.82-7.85 (t, 2H, J =
7.5 Hz), 7.87-7.90 (t, 2H, J = 8.0 Hz), 8.48-8.52 (q, 2H, J = 12.0, 7.5 Hz); 13C NMR (125 MHz, DMSO-
d6): δ 33.74, 39.48, 119.38, 122.32, 126.36, 127.65, 127.72, 127.99, 129.61, 130.11, 131.18, 131.70,
131.79, 132.91, 134.82, 135.83, 142.78, 143.44, 161.12, 163.69; Ms, [C20H16N2O4S: M+2, 382].
4.1.1.2. 4-(5-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzenesulfonamide (7)
M.p >350ο, 93% yield (CH3OH); IR (KBr, cm-1) ν: 3466 (NH), 1771, 1709 (C=O), 1398, 1114
(O=S=O); 1H NMR (500 MHz, DMSO-d6): δ 7.54 (s, 2H), 7.65-7.67 (d, 2H, J = 8.5 Hz), 8.01-8.02 (d,
2H, J = 8.5 Hz), 8.07-8.12 (q, 1H, J = 10.0, 7.5 Hz), 8.69-8.70 (d, 1H, J = 6.0 Hz), 8.80-8.82 (d, 1H, J =
8.5 Hz), 8.94 (s, 1H), 9.51 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 123.28, 123.47, 124.92, 126.99,

12
129.75, 130.26, 130.37, 130.39, 131.42, 134.44, 137.01, 139.09, 144.60, 146.23, 162.89, 163.42; Ms,
[C18H11N3O6S: M+1, 398].
4.1.1.3. 4-((5-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)methyl)benzenesulfonamide (8)
M.p 238-240ο, 84% yield (CH3OH); IR (KBr, cm-1) ν: 3468 (NH), 1774, 1703 (C=O), 1397,
1218 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 5.33 (s, 2H), 7.32 (s, 2H), 7.57-7.59 (d, 2H, J
= 8.5 Hz), 7.75-7.77 (t, 2H, J = 5.0, 3.5 Hz), 8.06-8.09 (t, 1H, J = 7.5 Hz), 8.69-8.71 (d, 1H, J = 7.0 Hz),
8.80-8.81 (d, 1H, J = 8.5 Hz), 8.97 (s, 1H), 9.51 (s, 1H); 13C NMR (125 MHz, CDCl3/DMSO-d6):
δ 43.52, 122.99, 123.60, 124.43, 126.24, 128.29, 129.76, 130.22, 130.40, 131.39, 134.66, 137.04,
141.38, 143.39, 146.31, 162.94, 163.41; Ms, [C19H13N3O6S: M+1, 412].
4.1.1.4. 4-(2-(5-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)benzenesulfonamide (9)
M.p 264-266ο, 79% yield (CH3CH2OH); IR (KBr, cm-1) ν: 3046 (NH), 1769, 1709 (C=O), 1392,
1160 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 3.02 (s, 2H), 4.25 (s, 2H), 7.32 (s, 2H), 7.49
(s, 2H), 7.76-7.77 (d, 2H, J = 7.5 Hz), 8.00-8.01 (d, 1H, J = 7.5 Hz), 8.59-8.60 (d, 1H, J = 6.0 Hz), 8.69-
8.70 (d, 1H, J = 7.0 Hz), 8.82-8.84 (t, 1H, J = 8.5, 2.0 Hz), 9.39 (s, 1H); 13C NMR (125 MHz, DMSO-
d6): δ 33.57, 41.40, 123.25, 124.25, 126.38, 129.63, 129.71, 130.19, 131.26, 134.40, 136.81, 142.83,
143.27, 146.22, 162.11, 163.70; Ms, [C20H15N3O6S: M+1, 426].
4.1.1.5. 4-(5,7-dioxo-5,7-dihydro-6H-dibenzo[c,e]azepin-6-yl)benzenesulfonamide (10)

13
M.p 221-223ο, 73% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) ν: 3504 (NH), 1786, 1729 (C=O),
1304, 1114 (O=S=O); 1H NMR (500 MHz, DMSO-d6): δ 7.24 (s, 4H), 7.74-7.76 (t, 8H, J = 9.5, 6.5 Hz),
10.28 (s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 127.40, 127.68, 128.55, 129.54, 138.53, 139.14,
172.41, 172.67; Ms, [C20H14N2O4S: m/z, 378].
4.1.1.6. 4-((5,7-dioxo-5,7-dihydro-6H-dibenzo[c,e]azepin-6-yl)methyl)benzenesulfonamide (11)
M.p 243-244ο, 84% yield (CH3OH); IR (KBr, cm-1) ν: 3448 (NH), 183, 1728 (C=O), 1385, 1128
(O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 4.23 (s, 2H), 6.94-6.95 (d, 3H, J = 7.0 Hz), 7.32-
7.46 (m, 11H); 13C NMR (125 MHz, CDCl3/DMSO-d6): δ 42.23, 126.15, 127.26, 127.47, 127.72,
127.98, 128.67, 129.60, 138.33, 139.20, 172.53, 172.77; Ms, [C21H16N2O4S: M+1, 393].
4.1.1.7. 4-(2-(5,7-dioxo-5,7-dihydro-6H-dibenzo[c,e]azepin-6-yl)ethyl)benzenesulfonamide (12)
M.p 211-213ο, 88% yield (CH3CH2OH/CH2Cl2); IR (KBr, cm-1) ν: 3479 (NH), 1778, 1712
(C=O), 1370, 1120 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 2.88 (s, 2H), 3.26 (s, 2H), 6.94-
6.96 (d, 3H, J = 7.5 Hz), 7.33-7.39 (m, 8H), 7.46-7.47 (d, 3H, J = 7.5 Hz); 13C NMR (125 MHz, DMSO-

14
d6): δ 35.31, 39.47, 126.14, 127.35, 127.65, 128.46, 129.51, 129.55, 138.68, 139.10, 172.31, 172.62; Ms,
[C22H18N2O4S: m/z, 406].
4.1.2. General procedure for the synthesis of cyclic imides 15-20.
A mixture of benzenesulfonamide (5.0 mmol), anhydrous sodium acetate (0.82 g, 10.0 mmol)
and an acid anhydride (2.5 mmol) in glacial acetic acid (15 mL) was heated under reflux for 24 h. After
cooling the reaction mixture, the precipitate obtained was filtered, washed with water, dried and re-
crystallised from an appropriate solvent.
4.1.2.1.
4,4'-(1,3,5,7-tetraoxo-5,7-dihydropyrrolo[3,4-f]isoindole-2,6(1H,3H)-
diyl)dibenzenesulfonamide (15)
M.p >350ο, 70% yield (CH3OH); IR (KBr, cm-1) ν: 3434 (NH), 1779, 1712 (C=O), 1369, 1118
(O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 7.19 (s, 2H), 7.73 (s, 4H), 8.25 (s, 2H), 8.98 (s,
6H); 13C NMR (125 MHz, CDCl3/DMSO-d6): δ 118.85, 127.06, 136.14, 138.24, 138.50, 142.66, 167.92;
Ms, [C22H14N4O8S2: m/z, 526].
4.1.2.2.
4,4'-((1,3,5,7-tetraoxo-5,7-dihydropyrrolo[3,4-f]isoindole-2,6(1H,3H)-
diyl)bis(methylene))dibenzenesulfonamide (16)

15
M.p 182-184ο, 68% yield (CH3CH2OH); IR (KBr, cm-1) ν: 3480 (NH), 1782, 1720 (C=O), 1397,
1121 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 4.31-4.32 (d, 4H, J = 5.5 Hz), 7.32 (s, 4H),
7.41-7.43 (d, 4H, J = 8.0 Hz), 7.76-7.78 (t, 4H, J = 6.5, 2.0 Hz), 8.45 (s, 2H); 13C NMR (125 MHz,
CDCl3/DMSO-d6): δ 42.22, 126.14, 127.99, 143.04, 144.22, 169.83; Ms, [C24H18N4O8S2: m/z, 554].
4.1.2.3. 4,4'-((1,3,5,7-tetraoxo-5,7-dihydropyrrolo[3,4-f]isoindole-2,6(1H,3H)-diyl)bis(ethane-2,1-
diyl))dibenzenesulfonamide (17)
M.p >350ο, 77% yield (CH3OH); IR (KBr, cm-1) ν: 3355, 3180 (NH), 1790, 1725 (C=O), 1372,
1115 (O=S=O); 1H NMR (500 MHz, DMSO-d6): δ 3.02-3.05 (t, 4H, J = 7.0 Hz), 3.88-3.91 (t, 4H, J =
7.0 Hz), 7.31 (s, 2H), 7.42-7.44 (dd, 2H, J = 6.0, 2.0 Hz), 7.71-7.73 (dd, 2H, J = 6.0, 2.5 Hz), 8.15-8.16
(d, 1H, J = 2.5 Hz), 8.97-8.98 (d, 7H, J = 3.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 33.74, 39.45,
126.26, 129.68, 136.20, 137.29, 138.30, 142.85, 166.48, 167.74; Ms, [C26H22N4O8S2: m/z, 582].
4.1.2.4.
4,4'-(1,3,6,8-tetraoxo-1,3,6,8-tetrahydrobenzo[lmn][3,8]phenanthroline-2,7-
diyl)dibenzenesulfonamide (18)
M.p >350ο, 69% yield (CH3OH); IR (KBr, cm-1) ν: 3376, 3185 (NH), 1780, 1729 (C=O), 1377,
1099 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 7.54 (s, 2H), 7.67-7.71 (t, 4H, J = 15.0, 6.5
Hz), 8.02-8.03 (d, 2H, J = 6.0 Hz), 8.18-8.19 (d, 2H, J = 6.0 Hz), 8.56-8.57 (d, 2H, J = 6.5 Hz), 8.71-
13
8.74 (d, 4H, J = 16.5 Hz); C NMR (125 MHz, CDCl3/DMSO-d6): δ 122.00, 125.09, 125.92, 126.99,

16
127.47, 128.85, 129.53, 130.32, 130.95, 132.08, 132.12, 132.25, 144.64, 159.92, 160.25, 160.62, 163.33,
168.77; Ms, [C26H16N4O8S2: M+2, 578].
4.1.2.5.
4,4'-((1,3,6,8-tetraoxo-1,3,6,8-tetrahydrobenzo[lmn][3,8]phenanthroline-2,7-
diyl)bis(methylene))dibenzenesulfonamide (19)
M.p >350ο, 63% yield (CH3CH2OH); IR (KBr, cm-1) ν: 3336, 3108 (NH), 1782, 1709 (C=O),
1372, 1115 (O=S=O); 1H NMR (500 MHz, DMSO-d6): δ 5.34 (s, 4H), 7.32 (s, 4H), 7.56-7.60 (t, 4H, J =
14.0, 8.5 Hz), 7.76-7.78 (d, 4H, J = 8.0 Hz), 8.69-8.71 (t, 4H, J = 5.50 Hz); 13C NMR (125 MHz,
DMSO-d6): δ 43.66, 126.27, 126.86, 128.16, 128.39, 131.19, 141.32, 143.43, 163.24; Ms,
[C28H20N4O8S2: m/z, 604].
4.1.2.6.
4,4'-((1,3,6,8-tetraoxo-1,3,6,8-tetrahydrobenzo[lmn][3,8]phenanthroline-2,7-
diyl)bis(ethane-2,1-diyl))dibenzenesulfonamide (20)
M.p >350ο, 71% yield (CH3OH); IR (KBr, cm-1) ν: 3329, 3099 (NH), 1738, 1712 (C=O), 1353,
1112 (O=S=O); 1H NMR (500 MHz, DMSO-d6): δ 3.04-3.07 (t, 4H, J = 7.5 Hz), 4.30-4.33 (t, 4H, J =
7.5 Hz), 7.30-7.33 (d, 4H, J = 14.5 Hz), 7.47-7.51 (t, 4H, J = 9.5 Hz), 7.74-7.77 (t, 4H, J = 8.5 Hz), 8.70
(s, 4H); Ms, [C30H24N4O8S2: m/z, 632].
4.2. Carbonic anhydrase inhibition

17
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay
the catalytic/inhibition of various CA isozymes.16 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
inhibitory in the used concentration),21 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,22 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
4.3. Molecular modelling studies
The crystal structures of hCA II (5LJT)27 , hCA IX (5FL4 monomer A)28 and hCA XII (1JD0)29 were
prepared for docking calculations using the Protein Preparation Wizard protocol30a in Maestro, deleting
water molecules, using default input parameters and Prime for adding missing side chains or loops. The
prediction of the side chains hetero groups ionization and tautomeric states was performed using Epik30b
.
The ligand 3D structures were generated with Maestro30c, evaluated for their ionization states at pH
7.4 1.0 with Epik30b and geometrically minimized with MacroModel30d (FF OPLS_2005)31 for a

18
maximum number of 2500 conjugate gradient iterations and setting a convergence criterion of 0.05 kcal
mol-1Å-1. Docking studies were carried out with the program Glide.30e Grids for docking were centred in
the centroid of the complexed ligand. The standard precision (SP) mode of the GlideScore function was
applied to evaluate the predicted binding poses. The best three poses for each compound were re-docked
by means of the Prime MM-GBSA protocol30f with a VSGB solvatation model.32 The pictures were
generated with Maestro.30c
Acknowledgments. The authors extend their appreciation to the Deanship of Scientific Research at
King Saud University, Riyadh, Saudi Arabia, for funding the work through the research group projects
No. RG-1435-046 and RGP-163.
References
1. (a) Krishnamurthy, V. M.; Kaufman, G. K.; Urbach, A. M.; Gitlin, I.; Gudiksen, K. L.; Weibel,
D. B.; Whitesides, G. M. Chem. Rev. 2008, 108, 946; (b) Supuran, C. T. Bioorg. Med. Chem.
2013, 21, 1377; (c) Supuran, C. T. Front. Pharmacol. 2011, 2, 34; (d) Supuran, C. T. Expert
Opin. Ther. Pat. 2013, 23, 677; (e) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem.
2015, 30, 325.
2. (a) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (b) Supuran, C. T.; Capasso, C.
Expert Opin. Ther. Targets 2015, 19, 551; (c) Capasso, C.; Supuran, C. T. Expert Opin. Ther.
Pat. 2013, 23, 693; (d) De Simone, G.; Alterio, V.; Supuran, C. T. Expert Opin. Drug Discov.
2013, 8, 793; (e) Supuran, C. T. Expert Rev. Neurother. 2015, 15, 851.
3. (a) Aggarwal, M.; Kondeti, B.; McKenna, R. Expert Opin. Ther. Pat. 2013, 23, 717; (b) Alterio,
V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421;
(c) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (d) Carta, F.; Di Cesare

19
Mannelli, L.; Pinard, M.; Ghelardini, C.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Bioorg.
Med. Chem. 2015,1828, 23.
4. (a) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; (b) Aggarwal, M.; Boone, C. D.;
Kondeti, B.; McKenna, R. J. Enzyme Inhib. Med. Chem. 2013, 28, 267; (c) Winum, J. Y.;
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2015, 30, 321; (d) D’Ambrosio, K.; Carradori, S.;
Monti, S. M.; Buonanno, M.; Secci, D.; Vullo, D.; Supuran, C. T.; De Simone, G. Chem.
Commun. 2015, 302.
5. Kikutani, S; Nakajima, K; Nagasato, C; et al. Proc Natl Acad Sci USA. 2016;113, 9828.
6. a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran, C. T. J.
Med. Chem. 1999, 42, 2641; b) Briganti F, Pierattelli R, Scozzafava A, Supuran CT. Eur J Med
Chem. 1996, 31, 1001.
7. a) Abdel-Aziz, A.A.-M.; Angeli A.; El-Azab A.S.; Abu El-Enin M.A.; Supuran C.T. Bioorg.
Med. Chem. 2017, 25, 1666; (b) Mohamed, M.A.; Abdel-Aziz, A.A.-M.; Sakre, H.M.; El-Azab
A.S.; Silvia Buaf, S.; Supuran C.T. Bioorg. Med. Chem. 2017, 25, 2524; (c) El-Azab A.S.;
Abdel-Aziz, A.A.-M.; Rezk R. Ayyad, R.R.; Ceruso, M.; Supuran C.T. Bioorg. Med. Chem.
2016, 24, 20.
8. . Sethi, K.K.; VermaS.M.; Tanc, M.; Purper, , G.; Calafato, G.; Carta, F.; Supuran, C.T. Bioorg.
Med. Chem. 2014, 22, 1586.
9. Sethi, K.K.; Verma, S.M.; Tanc, M.; Carta, F.; Supuran, C.T. Bioorg. Med. Chem. 2013, 21,
5168.
10. Abdel-Aziz, A.A.-M.; El-Azab, A.S.; Ceruso, M.; Supuran, C.T. Bioorg. Med. Chem Lett. 2014,
24, 5185.
11. Abdel-Aziz, A.A.-M. Eur. J. Med. Chem. 2007, 42, 612.
12. Abdel-Aziz, A.A.-M.; ElTahir, K.E.H.; Asiri, Y.A. Eur. J. Med. Chem. 2011, 46, 1648.

20
13. Alanazi, A.M.; El-Azab, A.S.; Al-Suwaidan, I.A.; ElTahir, K.E.H.; Asiri, Y.A.; Abdel-
Aziz, N.I.; Abdel-Aziz, A.A.-M. Eur. J. Med. Chem. 2015, 92, 115.
14. Al-Suwaidan, I.A.; Alanazi, A.M.; El-Azab, A.S.; Al-Obaid, A.M.; ElTahir, K.E.H.; Maarouf,
A.R.; Abu El-Enin, M.A.; Abdel-Aziz, A.A.-M. Bioorg. Med. Chem Lett. 2013, 23, 2601.
15. Abdel-Aziz, A.A.-M.; El-Azab, A.S.; Attia, S.M.; Al-Obaid, A.M.; Al-Omar, M.A.; El-Subbagh,
H.I. Eur. J. Med. Chem. 2011, 46, 4324.
16. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
17. (a) Carta, F.; Supuran, C.T. Expert Opin. Ther. Pat. 2013, 23, 681; (b) Thiry, A.; Dognè, J. M.;
Supuran, C.T.; Masereel, B. Curr. Pharm. Des. 2008, 14, 661; (c) Winum, J.Y.; Scozzafava, A.;
Montero, J.L.; Supuran, C.T. Med. Res. Rev. 2006, 26, 767; (d) Di Fiore, A.; De Simone, G.;
Alterio, V.; Riccio, V.; Winum, J.Y.; Carta, F.; Supuran, C.T. Org. Biomol. Chem. 2016, 14,
4853.
18. (a) Gao, B.B.; Clermont, A.; Rook, S.; Fonda, S.J.; Srinivasan, V.J.; Wojtkowski, M.; Fujimoto,
J.G.; Avery, R.L.; Arrigg, P.G.; Bursell, S.E.; Aiello, L.P.; Feener, EP. Nat. Med. 2007, 13, 181;
(b) Supuran CT, Scozzafava A, Mastrolorenzo A. Expert Opin Ther Pat 2001, 11, 221; c)
Chohan ZH, Arif M, Akhtar MA, Supuran CT. Bioinorg Chem Appl. 2006, 83131.
19. (a) Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168; (b) Supuran CT. J Enzyme Inhib Med
Chem 2016, 31, 345.
20. (a) Lock, F.E.; McDonald, P.C.; Lou, Y.; Serrano, I.; Chafe, S. C.; Ostlund, C.; Aparicio, S.;
Winum, J.Y.; Supuran, C. T.; Dedhar, S. Oncogene 2013, 32, 5210; (b) Bayram, E.; Senturk, M.;
Kufrevioglu, O.I.; Supuran, C.T. Bioorg. Med. Chem. 2008, 16, 9101; (c) Vullo, D.; Voipio, J.;
Innocenti, A.; Rivera, C.; Ranki, H.; Scozzafava, A.; Kaila, K.; Supuran, C.T. Bioorg. Med.
Chem. Lett. 2005, 15, 971.
21. a) De Simone, G.; Supuran, C.T. J. Inorg. Biochem. 2012, 111, 117; b) Supuran CT. Biochem J.
2016, 473, 2023.

21
22. Sethi, K.K.; Vullo, D.; Verma, S.M.; Tanc, M.; Carta, F.; Supuran, C.T. Bioorg. Med. Chem.
2013, 21, 5973.
23. (a) Korkmaz, N.; Obaidi, O.A.; Senturk, M.; Astley, D.; Ekinci, D.; Supuran, C.T. J. Enzyme
Inhib. Med. Chem. 2015, 30, 75; (b) Akdemir, A; De Monte, C.; Carradori, S.; Supuran, C.T. J.
Enzyme Inhib. Med. Chem. 2015, 30, 114; (c) Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava,
A.; Mastrolorenzo, A.; Supuran, C.T. J. Med. Chem. 2005, 48, 7860; (d) Scozzafava, A.;
Menabuoni, L.; Mincione, F.; Supuran, C.T. J. Med. Chem. 2002, 45, 1466.
24. (a) Sahin, A.; Isik, S.; Arslan, O.; Supuran, C.T.; Ozensoy Guler, O. J. Enzyme Inhib. Med.
Chem. 2015, 30, 224; (b) Bozdag, M.; Isik, S.; Beyaztas, S.; Arslan, O.; Supuran, C.T. J. Enzyme
Inhib. Med. Chem. 2015, 30, 240; (c) De Luca, V.; Del Prete, S.; Supuran, C.T.; Capasso, C. J.
Enzyme Inhib. Med. Chem. 2015, 30, 277; (d) Vullo, D.; Milos, M.; Galic, B.; Scozzafava, A.;
Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2015, 30, 341; (e) Del Prete, S.; Vullo, D.; De Luca,
V.; AlOthman, Z.; Supuran, C.T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2015, 30, 366; (f)
Riafrecha, L.E.; Vullo, D.; Supuran, C.T.; Colinas, P.A. J. Enzyme Inhib. Med. Chem. 2015, 30,
857.
25. (a) Göçer, H.; Akincioğlu, A.S.; Gülçin, G.I.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2015,
30, 316; (b) Ceruso, M.; Bragagni, M.; AlOthman, Z.; Osman, S.M.; Supuran, C.T. J. Enzyme
Inhib. Med. Chem. 2015, 30, 430; (c) Zolfaghari Emameh, R.; Syrjänen, L.; Barker, H.; Supuran,
C.T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2015, 30, 505; (d) Alafeefy, A.M.; Ceruso, M.;
Al-Tamimi, A.M.S.; Del Prete, S.; Supuran, C.T.; C. Capasso, J. Enzyme Inhib. Med. Chem.
2015, 30, 592; (e) Scozzafava, A.; Passaponti, M.; Supuran, C.T.; Gülçin, I. J. Enzyme. Med.
Chem. 2015, 30, 586; (f) Le Darz, A.; Mingot, A.; Bouazza, F.; Castelli, U.; Tanc, M.; Karam,
O.M.; Supuran, C.T.; Thibaudeau, S. J. Enzyme Inhib. Med. Chem. 2015, 30, 737.

22
26. (a) Supuran, C.T.; Barboiu, M.; Luca, C.; Pop, E.; Brewster, M.E.; Dinculescu, A. Eur. J. Med.
Chem. 1996, 31, 597; (b) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.;
Masini, E.; Supuran, C.T. J. Med. Chem. 2012, 55, 1721-1730; (c) Puccetti, L.; Fasolis, G.;
Vullo, D.; Chohan, Z.H.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2005, 15,
3096-3101; (d) Supuran, C.T.; Nicolae, A.; Popescu, A. Eur. J. Med. Chem. 1996, 31, 431.
27. Nocentini, A.; Ferraroni, M.; Carta, F.; Ceruso, M.; Gratteri, P.; Lanzi, C.; Masini, E.; Supuran,
C. T. J. Med. Chem. 2016, 59, 10692-10704.
28. Leitans, J.; Kazaks, A.; Balode, A.; Ivanova, J.; Zalubovskis, R.; Supuran, C.T.; Tars, K. J. Med.
Chem. 2015, 58, 9004-9;
29. Whittington, D.A.; Waheed, A.; Ulmasov, B.; Shah, G.N.; Grubb, J.H.; Sly, W.S.; Christianson,
D.W. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 9545-50.
30. (a) Schrödinger Release 2016-1: Schrödinger Suite 2016-1 Protein Preparation Wizard; Impact,
Schrödinger, LLC, New York, NY, 2016; (b) Schrödinger Release 2016-1:, Epik Schrödinger,
LLC, New York, NY, 2016; (c) Schrödinger Release 2016-1: Maestro, Schrödinger, LLC, New
York, NY, 2016; (d) Schrödinger Release 2016-1: Macromodel, Schrödinger, LLC, New York,
NY, 2016; (e) Schrödinger Release 2016-1: Glide, Schrödinger, LLC, New York, NY, 2016; (f)
Schrödinger Release 2016-1: Prime, Schrödinger, LLC, New York, NY, 2016.
−1666. (b) Jorgensen,
31. (a) Jorgensen, W. L.; Tirado-Rives, J. J. Am. Chem. Soc. 1988, 110, 1657
W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118, 11225
−11236
32. Li, J.; Abel, R.; Zhu, K.; Cao, Y.; Zhao, S.; Friesner, R. A. Proteins, 2011, 79, 2794−2812.

23
Synthesis and carbonic anhydrase inhibition of polycyclic
imides incorporating N-benzenesulfonamide moieties
Andrea Angelia, Alaa A.-M. Abdel-Azizb,c*, Adel S. El-Azabb,d, Claudiu T. Supurana*