Inhibition of carbonic anhydrase isoforms I, II, IV, VII and XII with carboxylates and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds

Article abstract of DOI:10.1016/j.bmc.2015.11.034

We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthra

Full text of DOI:10.1016/j.bmc.2015.11.034

Accepted Manuscript
Inhibition of carbonic anhydrase isoforms I, II, IV, VII and XII with carboxylates
and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds
Adel S. El-Azab, Alaa A.-M. Abdel-Aziz, Rezk R. Ayyad, Mariangela Ceruso,
Claudiu T. Supuran
PII:
S0968-0896(15)30157-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.11.034
BMC 12682
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 October 2015
21 November 2015
25 November 2015
Please cite this article as: El-Azab, A.S., Abdel-Aziz, A.A., Ayyad, R.R., Ceruso, M., Supuran, C.T., Inhibition of
carbonic anhydrase isoforms I, II, IV, VII and XII with carboxylates and sulfonamides incorporating phthalimide/
phthalic anhydride scaffolds, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.
2
015.11.034
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Inhibition of carbonic anhydrase isoforms I, II, IV, VII and XII with carboxylates and
sulfonamides incorporating phthalimide/phthalic anhydride scaffolds
1
,2
1,3
4
5
Adel S. El-Azab*
,
Alaa A.-M. Abdel-Aziz , Rezk R. Ayyad, Mariangela Ceruso and
5
Claudiu T. Supuran*
1
2
3
4
5
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt.
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.
Università degli Studi di Firenze, Polo Scientifico, Neurofarba Department and Laboratorio di Chimica
Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
Abstract. We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride
and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic
anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthranilic acids and
trimellitic anhydride chloride, followed by reaction with primary amines and were tested for the
inhibition of five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and
XII, some of which are involved in serious pathologies (CA II, IV and XII in glaucoma; CA VII
in epilepsy; CA XII in some solid tumors). The carboxylic acids were generally poor inhibitors
of isoforms hCA I, II and IV but were highly effective, low nanomolar inhibitors of hCA VII and
XII. The sulfonamides inhibited all isoforms significantly, and some of them were sub-
nanomolar hCA VII inhibitors, although their isoform selectivity was lower compared to the
carboxylates. This study proves that carboxylic acids incorporating
a
phthalic
anhydride/phthalimide based scaffold may lead to isoform-selective inhibitors by applying the
tail approach, mostly used up until now for obtaining sulfonamide, sulfamide and sulfamate CA
inhibitors.
Keywords: carbonic anhydrase; sulfonamide; carboxylic acid; isoform-selectivity
_
______
Correspondence authors: Tel: +966 14679096. Fax: +966
1
467-6383; E-mail:
adelazaba@yahoo.com (ASE); Phone:+39-055-4573005; Fax: +39-055-4573385; E-mail:
claudiu.supuran@unifi.it (CTS).
1

1
. Introduction
Primary sulfonamides (RSO
R scaffolds, represent the most investigated class of inhibitors of the metalloenzyme carbonic
2
NH ) incorporating aromatic, heterocyclic, aliphatic or sugar
2
1
-3
anhydrase (CA, EC 4.2.1.1). The reason for this is that CA inhibition with sulfonamides was
already discovered in the '40s, the first such compounds entered in clinical use as diuretics and
antiglaucoma drugs in the '50s, whereas more recently sulfonamide CA inhibitors (CAIs) proved
to be useful for the management of a variety of disorders, such as obesity, epilepsy and other
1
-3
neurologic diseases, solid, metastatic tumors or neuropathic pain. The complex pharmacology
of the CAIs is due to the fact that a large number of human (h) isoforms are known, i.e., 15,
numbered as hCA I - hCA XIV (but there are two V-type isoforms, hCA VA and VB), and
1
-4
because they possess different roles in various pathologies, as the ones mentioned above.
However, the principal drawback of this class of valuable drugs is related to their many side
effects, due to the fact that most mammalian isoforms are rather well inhibited by the first and
1
-3
second generation sulfonamide CAIs, which are still nowadays in clinical use. Thus, many
efforts were registered ultimately for finding alternative chemotypes to the sulfonamides, and the
3
,4
carboxylic acids are among the most investigated such compounds. Indeed, the carboxylates,
(
unlike the sulfonamides which inhibit CAs by coordinating to the Zn(II) ion from the enzyme
1
-3
active site and replacing the hydroxide ion acting as nucleophile in the catalysis) were shown
1
-4
to possess a multitude of inhibition mechanisms. Some of them, similarly to sulfonamides,
directly coordinate to the Zn(II) ion, but other structurally similar carboxylic acids were shown
to anchor to the zinc-coordinated water molecule/hydroxide ion, or occlude the entrance to the
4
-7
active site cavity, or even binding outside the active site. Although at this moment it is
impossible to forecast the mechanism by which a carboxylic acid will inhibit the CAs (except the
2

compounds formed from coumarins, which acting as prodrug CAIs undergo a hydrolysis within
the active site with generation of derivatives of 2-hydroxy-cinnamic acid which all occlude the
5
-7
entrance to the active site) their multiple binding modes to the enzyme certainly assure a
variety of inhibitory profiles against the various CA isoforms, and thus a high level of isoform-
8
selectivity. This is the reason why we continue our earlier investigation on carboxylates versus
sulfonamides as CAIs, reporting here the synthesis and inhibitory profile against five
physiologically relevant isoforms (hCA I, II, IV, VII and XII) of a new series of such derivatives
incorporating phthalimide/phthalic anhydride scaffolds.
2
. Result and discussion
2
.1. Chemistry
The rationale for the drug design of the carboxylate/sulfonamide inhibitors reported here was the
9
following one. The tail approach was reported by one of these groups more than 15 years ago
and allowed for the facile preparation of a large number of potent and isoform-selective classes
9
-13
of sulfonamide inhibitors.
Indeed, an initial drug design strategy based on appending “tails”
(
i.e., various moieties) of different size, shape or nature to pharmacophores incorporating
scaffolds of derivatizable amino-hydroxy-substituted aromatic/heterocyclic sulfonamides has
9
been proposed. Thus, numerous sulfonamide CAIs possessing both high affinity and desired
9
-19
pharmacologic properties were obtained.
This approach, based on an "extension" of the
aromatic/heterocyclic scaffolds through the anchoring tails has been thereafter explored for
sulfamates, sulfamides and dithiocarbamates (as alternative zinc-binding groups (ZBGs) to the
sulfonamide one, but much less to carboxylic acids). Furthermore, CAIs incorporating scaffolds
1
4,15
belonging to the aliphatic or glycosidic chemical spaces were also explored in this way.
The
advantage of the tail approach over other drug design strategies was thereafter explained at the
3

molecular level, after the report of many X-ray crystallographic structures of adducts of various
1
-3,16-20
CA isoforms with such inhibitors.
These studies demonstrated that the active site of most
CA isoenzymes is a rather large conical cavity in which the Zn(II) ion is positioned at its bottom.
The lining of the active site builds two adjacent very diverse halves, one entirely hydrophilic, the
2
,3,20
opposing one completely hydrophobic,
with the highest variability of amino acid residues
between the different isoforms on the edge/entrance of the active site. This is exactly the region
2
0-23
in which the tails of the inhibitors wereobserved,
explaining why these specific interactions
between the inhibitor tail and amino acid residues at the entrance of the active site may lead to
1
-4
compounds showing selectivity for inhibiting isoforms with pharmacological applications.
5
-7
Indeed, most of the isoform-selective inhibitors reported to date (except the coumarins) were
9
,20-23
obtained by the tail approach.
incorporating COOH and SO NH
Here we apply it for the preparation of compounds
moieties as ZBGs, considering the simple chemistry of
2
2
phthalic anhydride/phthalimide (Scheme 1).
Thus, the substituted 2-(1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxamido)benzoic acids (1-5)
were obtained in 90-95% yield by reaction of anthranilic acid derivatives with trimellitic
anhydride chloride in dichloromethane at room temperature. The resulting 2-(1,3-dioxo-1,3-
dihydroisobenzofuran-5-carboxamido)benzoic acid (1) was treated with various primary amines
in acetic acid in the presence of sodium acetate to give the corresponding 2-(1,3-dioxo-2-
substituted phenylisoindoline-5-carboxamido)benzoic acids (6-13) in 76-85% yield (Scheme 1).
4

O
O
Cl
O
O
OH
NH
OH
NH
X
O
OH
O
O
X
+
O
DCM, r.t.
RNH , AcOH,
2
NH2
O
O
CH3COONa,
reflux
O
O
R N
O
O
1
2
3
4
5
-X=H
-X=5-Cl
-H=4,5-di-OCH
-X=5-I
6-R=4-benzenesulfonamide
7-R=4-methylbenzenesulfonamide
8-R=4-ethylbenzenesulfonamide
9-R=phenyl
3
-X=CH3
10-R=benzyl
1
1
1
1-R=phenethyl
2-R=4-benzoic acid
3-R=4-ethylbenzoate
Scheme 1
Scheme 1: Preparation of compounds 1-13.
Thus, the compounds 1-5 as well as 9-13 prepared as reported in Scheme 1, incorporate only
COOH moieties as possible ZBG, whereas 6-8 incorporate both sulfamoyl and carboxylate as
possible ZBGs.
2
.2. CA inhibition
The compounds 1-13 reported here were investigated for their enzyme inhibitory action against
five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and XII (Tables 1
and 2). Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) was used as standard drug
2
4
in the assay.
The following structure-activity relationship (SAR) can be drawn from the inhibition data
shown in Table 1:
(
i) Isoforms hCA I, II (cytosolic) and IV (membrane-bound) were poorly inhibited by the
carboxylates 1-5 (K s in the micromolar range or > 10 µM), in contrast to the first generation
I
5

sulfonamide inhibitor acetazolamide which was effective against all of them with inhibition
constants ranging between 12 and 250 nM (Table 1).
Table 1: Inhibition of human CA isoforms hCA I, II, IV, VII and XII with substituted 2-(1,3-dioxo-1,3-
dihydroisobenzofuran-5-carboxamido)benzoic acid (1-5) and the standard sulfonamide inhibitor
24
acetazolamide (AAZ) by a stopped flow CO hydrase assay.
2
I
K (nM)
Compound
X
hCA I
hCA II
hCA IV
hCA VII
3.9
hCA XII
228.4
>
10000
494.4
1
2
H
5-Cl
4140
3494
4368
>10000
4753
250
>10000
>10000
>10000
>10000
12
>10000
>10000
>10000
526.7
5.1
2.9
3.3
3.5
2.5
43.7
46.5
57.4
40.8
5.7
3
4,5-di-OCH
3
4
5-I
5
5-CH
3
AAZ
-
74
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported
values).
(
ii) On the contrary, isoforms hCA VII (cytosolic) and XII (transmembrane) were effectively
inhibited by these carboxylates, with K s in the range of 2.9 – 5.1 nM against hCA VII, and of
0.8 – 228.4 nM against hCA XII. For hCA VII the SAR is thus quite flat with all compounds
highly effective inhibitors (similar to AAZ), whereas for hCA XII the unsubstituted derivative
at the anthranilic acid fragment of the molecule) such as compound 1 was the least effective
I
4
(
inhibitor, with the derivatives 2-5 incorporating halogens, methoxy or methyl groups showing an
enhanced activity (Table 1).
6

Table 2: Inhibition of human CA isoforms hCA I, II, IV, VII and XII with 2-(1,3-dioxo-2-substituted
phenylisoindoline-5-carboxamido)benzoic acid (6-13) and the standard sulfonamide inhibitor
24
acetazolamide (AAZ) by a stopped flow CO hydrase assay.
2
K
I
(nM)
Compound
R
hCA I
hCAII
hCA IV
184.4
hCA VII hCA XII
O
S NH2
O
33.6
41.3
8.9
0.46
0.35
0.31
4.3
44.4
28.5
4.1
6
O
S NH2
O
10.5
13.8
72.7
495.6
7
8
9
O
46.8
S NH2
O
>
10000
>10000
>10000
>10000
>10000
51.9
2443
7109
3.2
4.4
27.3
48.9
1
1
0
1
3540
366.4
O
>
>
10000
>10000
891.9
14.5
56.4
1
1
2
3
OH
O
10000
250
>10000
12
280.5
74
9.5
2.5
18.5
5.7
O Et
AAZ
-
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported
values).
7

The phthalimides 6-13 were tested for the inhibition of the same isoforms (Table 2). The
following SAR can be observed from data of Table 2:
(
iii) the sulfonamides 6-8 inhibited all five isoforms quite effectively (with few exceptions such
as compounds 6 and 8 against hCA IV), usually in the low nanomolar or subnanomolar (for hCA
VII) range. Thus, for these sulfonamides the tail approach applied here led to effective but rather
non-selective inhibitors.
(
iv) the carboxylates 9-13 on the other hand, similarly to compounds 1-5 discussed earlier show
isoform selectivity. Indeed, these derivatives were poor inhibitors or did not inhibit significantly
hCA I, II and IV (inhibition constants in the micromolar range), but they were highly effective as
hCA VII inhibitors (K
I
s in the range of 3.2 – 14.5 nM) and hCA XII inhibitors (K s in the range
I
of4.1 – 56.4 nM). It is rather clear that the substitution pattern at the R moiety present in the
starting amine is the main factor controlling potency, with the best hCA VII inhibitors
incorporating phenyl, benzyl and phenethyl moieties, whereas the best hCA XII inhibitor
containing ethyl benzoate (13) and benzyl group, 10 (Table 2).
3
. Conclusions
A small panel of carboxylates and sulfonamides incorporating phthalic anhydride and
phthalimide moieties in their structure were prepared by applying the tail approach for
generating chemical diversity in the family of these classes of CAIs. The new derivatives were
obtained from substituted anthranilic acids and trimellitic anhydride chloride, followed by
reaction with primary amines, and were tested for the inhibition of five physiologically relevant
isoforms, hCA I, II, IV, VII and XII. Some of these enzymes are involved in serious pathologies:
CA II, IV and XII in glaucoma; CA VII in epilepsy; CA XII in some solid tumors. The
carboxylic acid derivatives were generally poor inhibitors of isoforms hCA I, II and IV but were
highly effective, low nanomolar inhibitors of hCA VII and XII. The sulfonamides inhibited all
isoforms significantly, and some of them were sub-nanomolar hCAVII inhibitors, although their
isoform selectivity was lower compared to the carboxylates. This study proved that carboxylic
acids incorporating a phthalic anhydride/phthalimide based scaffold may lead to isoform-
8

selective CAIs by applying the tail approach, mostly used up until now for obtaining
sulfonamide, sulfamide and sulfamate inhibitors.
4
. Experimental
4
.1. Chemistry
Melting points were recorded on Barnstead 9100 Electrothermal melting apparatus. IR
-1
spectra (KBr) were recorded on a FT-IR Perkin-Elmer spectrometer ( cm ) at Research Center,
1
13
King Saud University, Saudi Arabia. Nuclear magnetic resonance ( H and C NMR) spectra
were recorded on Bruker 500 MHz spectrometer using DMSO-d as solvents at Research Center,
6
King Saud University, Saudi Arabia. The chemical shifts are expressed in  ppm using TMS as
internal standard. Mass spectra were recorded on an Agilent 6320 Ion Trap mass spectrometers
at Research Center, King Saud University, Saudi Arabia. Elemental analysis was carried out for
C, H and N at the Research Centre of College of Pharmacy, King Saud University, Saudi Arabia
and the results are within ±0.4% of the theoretical values. Solvent evaporation was performed
under reduced pressure using Buchan Rotatory Evaporator. Thin layer chromatography was
performed on precoated (0.25 mm) silica gel GF254 plates (E. Merck, Germany); compounds
were detected with 254 nm UV lamp. Silica gel (60–230 mesh) was employed for routine
column chromatography separations.
Substituted 2-(1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxamido)benzoic acid (1-5)
A mixture of trimellitic anhydride chloride (2.15 g, 0.01 mol) and the appropriate anthranilic
acid (0.01 mol) in dichloromethane (50 ml) was stirred at room temperature for 4-6 h. The
obtained solid was filtered off, washed with dichloromethane and dried.
2
-(1,3-Dioxo-1,3-dihydroisobenzofuran-5-carboxamido)benzoic acid (1)
9

o
-1
Yield 92%, m.p. 285-287 C; IR (KBr, cm ) :3250 (NH), 2855 (OH), 1777, 1755, 1693,
1
(
C=O); H NMR (DMSO-d
6
): 12.35-12.28 (m, 0.67H), 11.60 (m, 0.33H), 8.68-8.58 (m, 1H),
8
7
.29-8.12 (m, 1H), 8.08-8.01 (m, 1H), 8.96-7.80 (m, 1H), 7.87-7.84 (m, 1H); 7.75-7.58 (m, 1H),
+
.41-7.22 (m, 2H). MS: [M 311].
5
-Chloro-2-(1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxamido)benzoic acid (2)
o
-1
1
Yield 95%, m.p. 294-295 C; IR (KBr, cm ) : 3420 (NH), 1783, 1724, 1654 (C=O); H NMR
(
DMSO-d
6
): 9.69-8.65 (m, 1H), 8.53-8.47 (m, 1H), 8.43 (s, 1H), 8.18-8.11 (m, 1H), 8.06-8.00
1
3
(
m, 2H), 7.63-7.59 (m, 1H). C NMR (DMSO-d6): 120.1, 121.1, 123.7, 125.1, 126.9, 128.2,
+
+2
1
29.9, 131.8, 132.5, 137.2, 139.4, 157.5, 162.2, 164.12, 167.88. MS: [M 345 and M 347].
2
-(1,3-Dioxo-1,3-dihydroisobenzofuran-5-carboxamido)-4,5-dimethoxybenzoic acid (3)
o
-1
1
Yield 90%, m.p. 301-302 C; IR (KBr, cm ) : 3245 (NH), 1774, 1776, 1648 (C=O); H NMR
(
DMSO-d
6
): 8.43-8.39 (m, 2H), 8.15-8.13 (m, 1H), 7.56-7.54 (m, 1H), 7.21 (s, 1H), 7.10 (s,
+
1
H), 3.88 (s, 3H), 3.80 (s, 3H). MS: [M 371].
2
-(1,3-Dioxo-1,3-dihydroisobenzofuran-5-carboxamido)-5-iodobenzoic acid (4)
o
-1
1
Yield 94%, m.p. 278-280 C; IR (KBr, cm ) : 3065 (OH), 1783, 1724, 1681 (C=O); H NMR
(
DMSO-d
6
):12.27 (d, 1H, J=22.0 Hz), 8.44-8.38 (m, 2H), 8.34 (s, 1H), 8.27-8.11 (m, 2H),
+
7
.96 (dd, 1H, J=1.5, 2.0 Hz), 7.37 (d, 1H, J= 8.0 Hz). MS: [M 437].
2
-(1,3-Dioxo-1,3-dihydroisobenzofuran-5-carboxamido)-5-methylbenzoic acid (5)
o
-1
1
Yield 91%, m.p. 298-300 C; IR (KBr, cm ) :3142 (OH), 1785, 1718, 1684 (C=O); H NMR
(
DMSO-d ): 12.27 (s, 1H), 8.50 (d, 1H, J=8.5 Hz), 8.44-8.40 (m, 2H), 8.16 (d, 1H, J=8.0 Hz),
6
10

1
3
7
1
1
.60 (d, 1H, J=8.0 Hz), 7.49-7.43 (m, 2H), 2.38 (s, 3H). C NMR (DMSO-d ): 20.7, 118.1,
6
20.9, 124.7, 129.2, 130.8, 131.7, 132.8, 134.0, 134.6, 135.1, 138.4, 140.7, 163.1, 166.5, 167.0,
+
70.4. MS: [M 432].
2
-(1,3-Dioxo-2-substituted phenylisoindoline-5-carboxamido)benzoic acid (6-13)
A mixture of 2-(1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxamido)benzoic acid (1) (612 mg, 2
mmol) and the appropriate amine (0.02 mmol) in glacial acetic acid (10 ml) in the presence of
anhydrous sodium acetate (246 mg, 3 mmol) was heated for 10-12 h. The reaction mixture was
cooled, the solvent was removed under vacuum, and the obtained solid was filtered, washed with
water, dried and recrystallized with dichloromethane-ethanol (1:1).
2
-(1,3-Dioxo-2-(4-sulfamoylphenyl)isoindoline-5-carboxamido)benzoic acid (6)
o
-1
1
Yield 81%, m.p. 313-315 C; IR (KBr, cm ) : 3328, 3245 (NH), 1779, 1661 (C=O); H NMR
DMSO-d ): 12.42 (s, 1H), 8.63 (d, 1H, J=8.5 Hz), 8.48-8.42 (m, 2H), 8.18 (d, 1H, J=8.0 Hz),
.07 (d, 1H, J=1.5 Hz), 8.01 (d, 2H, J=8.5 Hz), 7.72-7.66 (m, 3H), 7.51 (s, 2H), 7.24 (t, 1H,
(
6
8
1
3
J=7.5 Hz). C NMR (DMSO-d
6
): 120.8, 121.8, 124.1, 126.8, 128.0, 131.7, 132.7, 134.3, 134.6,
+
1
34.7, 135.1, 140.5, 140.8, 143.9, 163.3, 166.4, 166.5. MS: [M 465].
2
-(1,3-Dioxo-2-(4-sulfamoylbenzyl)isoindoline-5-carboxamido)benzoic acid (7)
o
-1
1
Yield 83%, m.p. 322-324 C; IR (KBr, cm ) : 3391, 3253 (NH), 1781, 17334, 1654 (C=O); H
NMR (DMSO-d ): 14.20 (s, 1H), 8.65 (d, 1H J=8.0 Hz), 8.42 (d, 1H, J=1.5 Hz), 8.37 (s, 1H),
.09-8.06 (m, 3H), 7.80 (d, 2H, J=8.5 Hz), 7.55-7.52 (m, 3H), 7.36 (s, 2H), 7.18-7.15 (m, 1H),
6
8
4
1
1
3
.88 (s, 2H). C NMR (DMSO-d
6
): 41.2, 119.8, 121.7, 121.8, 123.4, 124.3, 126.4, 128.2,
+
31.7, 132.7, 133.9, 134.5, 140.8, 140.9, 141.0, 143.7, 163.0, 167.4, 167.5, 170.5. MS: [M 479].
11

2
-(1,3-Dioxo-2-(4-sulfamoylphenethyl)isoindoline-5-carboxamido)benzoic acid (8)
o
-1
Yield 85%, m.p.300-301 C; IR (KBr, cm ) : 3358, 3256 (NH), 3042 (OH), 1769, 1718, 1655
1
(
C=O); H NMR (DMSO-d
6
): 12.28 (s, 1H), 8.60 (d, 1H, J=3.5 Hz), 8.34 (d, 1H, J=8.0 Hz),
8
2
7
1
.27 (s, 1H), 8.02 (t, 2H, J=6.5 & 8.0 Hz), 7.74 (d, 2H, J=8.5 Hz), 7.67 (t, 1H, J=8.0), 7.44 (d,
H, J=8.5 Hz), 7.33 (s, 2H), 7.24 (t, 1H, J=7.5 Hz), 3.89 (t, 2H, J=7.0 Hz), 3.03 (t, 2H, J=7.0 &
1
3
.5 Hz). C NMR (DMSO-d ): 33.9, 39.2, 118.0, 120.9, 121.4, 124.1, 124.2, 126.3, 129.7,
6
31.7, 132.6, 133.9, 134.5, 134.7, 140.2, 140.8, 142.8, 142.9, 163.3, 167.3, 167.4, 170.5. MS:
+
[
M 493].
2
-(1,3-Dioxo-2-phenylisoindoline-5-carboxamido)benzoic acid (9)
o
-1
Yield 80%, m.p. 307-309 C; IR (KBr, cm ) :3105 (OH), 3412 (NH), 1780, 1702, 1655 (C=O);
1
H NMR (DMSO-d
6
):, 12.36 (s, 1H), 8.62 (d, 1H, J=8.0 Hz), 8.42 (d, 1H, J=8.0 Hz), 8.38 (s,
1
H), 8.14 (d, 2H, J=8.0 Hz), 8.05 (d, 1H, J=7.5 Hz), 7.67 (t, 1H, J=7.5 & 8.0 Hz), 7.56 (t, 2H,
1
3
J=7.5 & 8.0 Hz), 7.49-7.46 (m, 3H), 7.25 (t, 1H, J=7.5 & 8.0 Hz). C NMR (DMSO-d
6
):
117.8, 120.8, 121.6, 124.0, 124.6, 127.8, 128.7, 129.3, 131.7, 132.2, 132.7, 134.1, 134.6, 134.7,
+
1
40.3, 140.9, 163.3, 166.7, 170.5.MS: [M 386].
2
-(2-Benzyl-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (10)
o
-1
1
Yield 82%, m.p. 270-272 C; IR (KBr, cm ) :3421 (NH), 1771, 1717, 1654, (C=O); H NMR
(
DMSO-d
6
): 12.30 (s, 1H), 8.60 (d, 1H, J=8.5 Hz), 8.35 (d, 1H, J=8.0 Hz), 8.30 (s, 1H), 8.06
(
d, 1H, J=8.0 Hz), 8.03 (d, 1H, J=8.0 Hz), 7.64 (t, 1H, J=8.0 Hz), 7.34-7.20 (m, 6H), 4.80 (s,
1
3
2
1
H). C NMR (DMSO-d
6
): 41.6, 117.8, 120.8, 121.5, 124.0, 124.3, 127.8, 127.9, 129.0, 131.7,
+
32.7, 134.0, 134.6, 134.7, 136.9, 140.2, 140.8, 163.2, 167.4, 170.5. MS: [M 400].
12

2
-(1,3-Dioxo-2-phenethylisoindoline-5-carboxamido)benzoic acid (11)
o
-1
1
Yield 85%, m.p. 255-257 C; IR (KBr, cm ) :3421 (NH), 1793, 1734, 1684 (C=O); H NMR
(
(
(
DMSO-d
6
): 12.28 (s, 1H), 8.60 (d, 1H, J=8.0 Hz), 8.33 (d, 1H, J=8.0 Hz), 8.25 (s, 1H), 8.05
d, 1H, J=8.0 Hz), 8.02 (d, 1H, J=7.5 Hz), 7.66 (t, 1H, J=7.5 & 8.0 Hz), 7.31-7.18 (m, 6H), 7.83
1
3
t, 2H, J=7.5 & 9.0 Hz), 3.83 (t, 2H, J=7.5 Hz), 2.94 (t, 2H, J=7.0 & 7.5 Hz). C NMR (DMSO-
d
1
6
): 34.0, 40.5, 117.9, 120.8, 121.3, 124.1, 126.9, 127.0, 127.8, 128.9, 129.0, 129.2, 131.6,
+
32.6, 133.8, 134.5, 134.7, 138.6, 140.8, 163.3, 167.3, 170.4. MS: [M 414].
2
-(2-(4-Carboxyphenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (12)
o
-1
Yield 76%, m.p. 330-331 C; IR (KBr, cm ) :3032 (OH), 1778, 1734, 1699 (C=O), 3258 (NH);
1
H NMR (DMSO-d
6
):12.44 (s, 1H), 8.62 (d, 1H, J=8.0 Hz), 8.41 (d, 1H, J=7.5 Hz), 8.37 (s,
1
7
1
H), 8.14 (d, 1H, J=8.0 Hz), 8.10 (d, 2H, J=8.0 Hz), 8.05 (d, 1H, J=8.0 Hz), 7.67-7.62 (m, 3H),
1
3
.23 (t, 1H, J=7.5 Hz). C NMR (DMSO-d ): 118.0, 120.7, 121.8, 124.0, 124.7, 127.4, 130.3,
6
30.6, 131.7, 132.6, 134.2, 134.5, 134.6, 136.0, 140.5, 140.8, 163.2, 166.3, 167.2, 170.5. MS:
+
[
M 430].
2
-(2-(4-(Ethoxycarbonyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (13)Yield
o
-1
1
7
9%, m.p. 228-230 C; IR (KBr, cm ) :3420 (NH), 1772, 1700, 1718, 1654 (C=O); H NMR
): 13.01 (s, 1H), 8.64 (d, 1H, J=8.0 Hz), 8.44 (d, 1H, J=78.0Hz), 8.40 (s, 1H), 8.14
d, 1H, J=8.0 Hz), 8.12 (d, 2H, J=8.5 Hz), 8.07 (d, 1H, J=7.5 Hz), 7.71-7.61 (m, 3H), 7.22 (t,
(
DMSO-d
6
(
1
3
1

1
H, J=7.5 Hz), 4.36 (q, 2H, J=7.0 & 7.5 Hz), 1.30 (t, 3H, J=7.0 Hz). C NMR (DMSO-d ):
6
14.6, 61.4, 121.9, 123.8, 124.7, 127.5, 127.8, 129.3, 129.6, 130.1, 130.6, 132.6, 134.0, 134.2,
+
34.4, 136.4, 140.7, 140.9, 163.1, 165.6, 166.3, 170.5. MS: [M 458].
13

4
.2. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
2
4
CO
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20
mM Na SO (for maintaining constant the ionic strength), following the initial rates of the CA-
catalyzed CO hydration reaction for a period of 10-100 s. The CO concentrations ranged from
.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each
2
hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
2
4
2
2
1
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-
deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor
and enzyme solutions were preincubated together for 15 min at room temperature prior to assay,
in order to allow for the formation of the E-I complex. The inhibition constants were obtained by
non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported
2
5-28
earlier,
and represent the mean from at least three different determinations. All CA isofoms
2
5-28
were recombinant ones obtained in-house as reported earlier.
Acknowledgments.The authors extend their appreciation to the Deanship of ScientificResearch
at King Saud University for funding the work through theresearch group project No. RG-1435-
0
46.The authors would like to express their gratitude and thanks to two European Union grants,
Dynano and Metoxia (to CTS).
14

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1
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1
1
1
1
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17

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2
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2
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2
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2
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2
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2
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2
4
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2
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18

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2
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2
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2
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19

Inhibition of carbonic anhydrase isoforms I, II, IV, VII and XII with carboxylates and
sulfonamides incorporating phthalimide/phthalic anhydride scaffolds
1
,2
1,3
4
5
Adel S. El-Azab*
,
Alaa A.-M. Abdel-Aziz , Rezk R. Ayyad, Mariangela Ceruso and
5
Claudiu T. Supuran*
COOH
NH
O
O
R N
O
R = Ph, PhCH
2
, HOOC-C
6
H
4
, H
2
NO
2
S-C
6
H
4
, etc.
K
I
(hCA VII) = 0.31 - 14.5 nM; K
I
(hCA XII) = 4.1 - 56.4 nM
20