Synthesis of novel isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones as selective inhibitors of the tumor-associated carbonic anhydrase IX

Article abstract of DOI:10.1016/j.bioorg.2018.07.027

The synthesis, characterization and biological evaluation of a library of isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones is disclosed. The set of hydroxyiminoethyl aromatic derivatives 10–18 was designed to assess the potentiality as zinc-binder for a feebly studied functional group in the field of carbonic anhydrase (CA, EC 4.2.1.1) inhibition. Analogue phenylphthalimmides were linked to benzenesulfonamide scaffold by hydrazone spacers in the second subset of derivatives 20–28 to further investigate the application of the “tail approach” as tool to afford CA selective inhibition profiles. The compounds were assayed for the inhibition of physiologically relevant isoforms of human carbonic anhydrases (hCA, EC 4.2.1.1), the cytosolic CA I and II, and the membrane-bound CA IV and tumor-associated CA IX. The new zinc-binders, both of the oxime and sulfonamide types, showed a striking selective activity against the target hCA IX over ubiquitous hCA I and II, with diverse inhibitory ranges and ratio differing the two subsets. With CA IX being a strongly current antitumor/antimetastatic drug target, these series of compounds may be of interest for the development of new, both conventional and unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX with minimum ubiquitous CAs-related side effects.

Full text of DOI:10.1016/j.bioorg.2018.07.027

Accepted Manuscript
Synthesis of novel isoindoline-1,3-dione-based oximes and benzenesulfona-
mide hydrazones as selective inhibitors of the tumor-associated carbonic anhy-
drase IX
Alaa A.-M. Abdel-Aziz, Adel S. El-Azab, Mohamed A. Abu El-Enin,
Abdulrahman A. Almehizia, Claudiu T. Supuran, Alessio Nocentini
PII:
S0045-2068(18)30571-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.027
YBIOO 2446
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 June 2018
23 July 2018
23 July 2018
Please cite this article as: A.A. Abdel-Aziz, A.S. El-Azab, M.A. Abu El-Enin, A.A. Almehizia, C.T. Supuran, A.
Nocentini, Synthesis of novel isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones as selective
inhibitors of the tumor-associated carbonic anhydrase IX, Bioorganic Chemistry (2018), doi: https://doi.org/
10.1016/j.bioorg.2018.07.027
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Synthesis of novel isoindoline-1,3-dione-based oximes and benzenesulfonamide
hydrazones as selective inhibitors of the tumor-associated carbonic anhydrase IX.
Alaa A.-M. Abdel-Aziza,b*, Adel S. El-Azaba,c, Mohamed A. Abu El-Eninb,
Abdulrahman A. Almehiziaa, Claudiu T. Supurand, Alessio Nocentinid,*
a Department of Pharmaceutical Chemistrya, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry b, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c Department of Organic Chemistryc, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
d NEUROFARBA Department, University of Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. The synthesis, characterization and biological evaluation of a library of isoindoline-1,3-dione-based
oximes and benzenesulfonamide hydrazones is disclosed. The set of hydroxyiminoethyl aromatic derivatives 10-
18 was designed to assess the potentiality as zinc-binder for a feebly studied functional group in the field of
carbonic anhydrase (CA, EC 4.2.1.1) inhibition. Analogue phenylphthalimmides were linked to
benzenesulfonamide scaffold by hydrazone spacers in the second subset of derivatives 20-28 to further investigate
the application of the “tail approach” as tool to afford CA selective inhibition profiles. The compounds were
assayed for the inhibition of physiologically relevant isoforms of human carbonic anhydrases (hCA, EC 4.2.1.1),
the cytosolic CA I and II, and the membrane-bound CA IV and tumor-associated CA IX. The new zinc-binders,
both of the oxime and sulfonamide types, showed a striking selective activity against the target hCA IX over
ubiquitous hCA I and II, with diverse inhibitory ranges and ratio differing the two subsets. With CA IX being a
strongly current antitumor/antimetastatic drug target, these series of compounds may be of interest for the
development of new, both conventional and unconventional anticancer drugs targeting hypoxia-induced CA
isoforms such as CA IX with minimum ubiquitous CAs-related side effects.
Keywords: Isoindoline-1,3-dione; oxime; hydrazone; benzenesulfonamide; carbonic anhydrase inhibition,
carbonic anhydrase IX, anti-tumor.
* Corresponding author. Tel.: 00966-53-5991127; fax: 00966-1-4676220; e-mail: almoenes@ksu.edu.sa (A.A.-M.
Abdel-Aziz); alessio.nocentini@unifi.it (A. Nocentini)

1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of zinc enzymes which catalyze the reversible
hydration of CO2 into HCO3 and protons by a metal hydroxide nucleophilic mechanism.[1,2] Seven
genetically distinct CA families (α-, β-, γ-, δ-, ζ-. η- and θ-CAs.) were identified to date.[1-3]. The 15
different α-CA isoforms isolated in humans (h) feature catalytic activity, sub-cellular localization and
organ/tissue distribution. Among the catalytically active isoforms, some are cytosolic (CA I, CA II, CA
III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII, CA XIV and CA XV),
two of them are mitochondrial (CA VA and CA VB), and one isozyme is secreted in saliva (CA VI).[2]
Considering the variety of human physiological-pathological processes showing abnormal levels or
activities of these enzymes, CA isozymes are valuable targets for many pharmacological applications
such as antiglaucoma drugs, diuretics, antiobesity, anticonvulsant and/or antitumor agents/diagnostic
tools.[1] When inhibitors targeting hCA I are useful in retinal and cerebral edema, inhibitors targeting
hCA II, IV, XII and XIV are used as diuretics, in the management edema, as antiglaucoma agents,
antiepileptic drugs, and also for the treatment of altitude sickness.[1,2] With CA I and II being
ubiquitous and physiologically relevant isoforms, their inhibition is often off-target.[2] CA IX, a
transmembrane isoform, is involved in tumor growth, metastases formation with its inhibitors having
shown applications as diagnostic tools for imaging hypoxic tumors and as antitumor/antimetastatic
agents.[4-7] In hypoxic tumors (e.g. breast, brain, colorectal, etc.) hCA IX, whose expression is induced
via the hypoxia inducible factor-1α (HIF-1α), maintains the external pH, supporting an acidic
extracellular microenvironment suited for hypoxic tumor cell survival and proliferation, but detrimental
to normal cells.[4-7] The above made hCA IX become an attractive target for the design of
antineoplastic therapies.[1,2] The achievement of selective inhibition of some isoforms over others is a
necessary endeavor to afford tools for the treatments of a plethora of pathologies endowed with
minimum side effects.[1] In this contest many efforts have been made for the development of isoform-

selective CAIs, and some remarkable results have been achieved in the last 15 years since the
introduction of the “tail approach” to most classical sulfonamide-like inhibitors [8-11] and identification
of varied new CA inhibitory chemotypes.[8] Zinc-binding groups diverse from sulfonamides and their
congeners have been discovered, among which carboxilates, hydroxamates, phosphonates, mono- and
dithiocarbammates, xanthates, thioxanthates and boroles.[12-17] The use of heterocyclic tails is on the
other hand the most exploited tools to seek for selectivity with sulfonamides inhibitors against target
hCAs.[18-29] In this context, heterocycles of the isatin, phthalimide or quinoline types are undoubtedly
amongst the most incorporated scaffolds as tail of zinc-binding group bearing molecules since they
possess a variety pharmacological and biological activities, such as excellent anticancer profiles and
inhibition profile for several CA isoforms.[18-29]
Herein synthesis and biological evaluation of novel isoindoline-1,3-dione-based oximes and
benzenesulfonamide hydrazones is proposed to go forward into the design of heterocyclic compounds of
potential pharmacologic interest. A set of hydroxyiminoethyl aromatic derivatives 10-18 was designed
to assess the potentiality as zinc-binder for a feebly studied functional group in the field of carbonic
anhydrase inhibition.[30,31] Analogue scaffolds were linked to benzenesulfonamide cores by hydrazone
spacers 20-28 to further investigate the application of the tail approach as tool to address CA selective
inhibition profiles, with the target tumor-associated hCA IX being screened beside the physiologically
relevant hCA I, II and IV.
2 Results and discussion
2.1. Chemistry
The present work possesses a dual rationale: hydroxyiminoethyl moieties were place onto variously
substituted phthalimmidophenyl scaffolds to explore the potentiality of isoindoline-1,3-dione-based
oximes to inhibit CAs in a selective manner. Equal scaffolds were appended to benzenesulfonamide
through hydrazone linkers. Hydrazone group is a urea-bioisoster, with this latter being the most

commonly incorporated tailing linker in benzenesulfonamides derivatives. A ureido-sulfonamide CA IX
selective inhibitor (SLC-0111) entered in Phase II clinical trials for the treatment of patients with
advanced solid, metastatic tumors over-expressing CA IX.[32,33] Ureido bioisosterism has been
significantly applied to SLC-0111.[2,8,34] Varied substituents at the isoindoline-1,3-dione groups
considered in the present study allowed to study the compounds interactions at the entrance of the CA
active site cavity. Steric and lipophilic contributions of halogens as well as their ability to form
stabilizing interactions, such as halogen bonding, hydrogen bonding and multipolar interactions were
also exploited to seek for isoform-selectivity.[35,36] Since that region is the most diverse one among the
different α-CAs, selective inhibition may be achieved through particular interactions between the tail of
the inhibitor and this region of the cavity.[2]
The synthesis of isoindoline-1,3-dione-based oximes 10-18 and benzenesulfonamide hydrazones 20-28
was performed as highlighted in Schemes 1 and 2, starting from common intermediates 2-(4-
acetylphenyl)-isoindoline-1,3-diones 1-9.
2-(4-Acetylphenyl)-isoindoline-1,3-diones derivatives 1-9 were obtained in 80-95% yield by heating of
4-aminoacetophenone with the corresponding acid anhydride in glacial acetic acid containing anhydrous
sodium acetate [19-29] (Scheme 1). The structures of obtained compounds were confirmed by their
elemental and spectral analyses. The infrared (IR) spectrum of compound 1-9 exhibited characteristic
1
bands at 1683-1730 cm-1 due to the presence of carbonyl (C=O) groups. The H NMR spectrum of
compounds 1-9 was verified by the presence of new signal due to acetyl group (COCH3) at 2.59-2.66
ppm. Additionally, new peaks were observed at 26.68-27.63 ppm due to acetyl group (COCH3), and
162.80-178.78 ppm for carbonyl (C=O) group due to anhydride moiety as well as characteristic peak of
13
carbonyl (COCH3) group at 197.30-197.80 ppm respectively in C NMR spectrum.

Scheme 1. Synthesis of 2-(4-acetylphenyl)isoindoline-1,3-diones (1-9) and 2-(4-(1-(hydroxyimino)ethyl)phenyl)isoindoline-
1,3-diones (10-18)
2-[4-(1-(Hydroxyimino)ethyl)phenyl] derivatives 10-18 were produced in 83-96% yield by stirring of
the isoindoline-1,3-diones 1-9 and hydroxylamine hydrochloride in glacial acetic acid containing
anhydrous sodium acetate (Scheme 1). The structures of new synthesized compounds were confirmed by
their elemental and spectral data. IR spectra of compounds 10-18 were used to verify their structures
through the appearance of characteristic absorption bands due to hydroxyl group of oxime and carbonyl
1
(C=O) groups at 3245-3471 and 1700-1783 cm-1 respectively. H NMR spectra of compounds 10-18
were characterized by the disappearance of acetyl group (COCH3) at 2.59-2.66 ppm and appearance of
new singlet peaks of hydroxyl group (CH3C=N-OH), as well as ethylimine group (CH3C=N-OH) of
oxime moiety at 11.14-11.58 and 1.91-2.41 ppm respectively. Moreover, 13C NMR spectra of
compounds 10-18 showed the disappearance of acetyl (COCH3), and carbonyl (COCH3) groups at

26.68-27.63 and 197.30-197.80 ppm respectively and presence of new peak for (CH3C=N-OH) group at
11.99-12.76 ppm.
Benzenesulfonamides 20-28 were obtained in 84-93% yield by heating of isoindoline-1,3-diones 1-9 and
4-(hydrazinecarbonyl)benzenesulfonamide (19) in methanol containing catalytic amount of acetic acid
(Scheme 2). Compounds 20-28 were confirmed by their elemental and spectral analyses. The IR
spectrum of compounds 20-28 were characterized by the presence of absorption bands at 3513-3260 and
1
1785-1708 cm-1 representing (NH) and (C=O) groups, respectively. The H NMR spectrum of
compounds 20-28 were verified by the disappearance of acetyl group (COCH3) at 2.59-2.66 ppm and
appearance of new singlet peaks of amide group (CH3C=N-NH-CO-R), as well as ethylimine group
(CH3C=N-NH-COR) due to ethylidenehydrazine moiety at 11.84-11.0 and 2.47-2.36 ppm respectively,
in addition to aromatic protons of benzensulfonamide moiety at aromatic region. Furthermore, 13C NMR
spectra of compounds 20-28 showed the disappearance of acetyl (COCH3), and carbonyl (COCH3)
groups at 26.68-27.63 and 197.30-197.80 ppm respectively and presence of new peak for ethylimine
(CH3C=N-NH-COR) group at 15.26-15.21 ppm.
Scheme 2. Synthesis of hydrazones based on isoindoline-1,3-dione incorporating benzenesulfonamide 20-28

2.2. Carbonic anhydrase inhibition
The CA inhibitory properties of oximes 10-18 and sulfonamides 20-28 against human isoforms CA I, II,
IV and IX were measured by a stopped flow CO2 hydrase assay using acetazolamide (AAZ) as standard
inhibitor.[37] Inhibition profiles were displayed in comparison to SLC-0111. The following structure–
activity relationships (SAR) were gathered from the inhibition data reported in Table 1.
Table 1: Inhibition data of human CA isoforms hCA I, II, IV and IX with isoindoline-1,3-dione-based
oximes 10-18, benzenesulfonamide hydrazones 20-28 and the standard sulfonamide inhibitor
acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.[37]
KI (nM)*
Cmpd
R
hCA I
>100000
>100000
>100000
>100000
>100000
>100000
>100000
9670
hCA II
74880
84790
66150
24110
56250
86570
58040
5585
hCA IV
38700
23180
15850
24640
36900
31910
43960
3660
hCA IX
2038
318.9
280.5
1328
318.4
169.3
323.9
240.1
19900
33.9
-
H
10
11
5-CH3
5-C(CH3)3
12
13
5,6-diCl
4,5,6,7-tetraCl
14
15
4,5,6,7-tetraBr
5-COOH
16
17
-
-
>100000
6901
97840
795.2
834.9
632.7
787.3
670.7
707.1
663.9
369.1
539.8
12
46530
2030
18
20
H
5-CH3
9283
6922
4322
4065
31.4
30.3
21
22
5-C(CH3)3
5,6-diCl
8302
5551
1869
2103
83.8
22.9
23
24
4,5,6,7-tetraCl
4,5,6,7-tetraBr
21090
38090
6113
63000
250
3092
4758
17.3
23.1
25
26
5-COOH
406.1
24410
74
22.6
164.3
25
27
28
-
-
-
AAZ
SLC-0111
5080
960
286
45
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported
values).
(i) Oximes 10-18 did not inhibit the cytosolic hCA I up to 100 µM and showed feeble hCA II and IV
inhibition profiles. Compound 17 stood out amongst the others as unique low micromolar hCA I, II and
IV inhibitors, with inhibition constants (KIs) of 9667.5, 5585.4 and 3660.4 nM, respectively. This is

probably due to presence of a carboxylic group on the phthalimide aromatic portion. Carboxy
derivatives have been shown to inhibit several hCAs in a low micromolar range. The other
hydroxyiminoethyl derivatives showed KIs ranging between 16 and 98 µM for the two off-target
isoforms hCA II and IV. It may be observed that CA IX, overexpressed in hypoxic tumors, was
significantly sensitive to inhibition by isoindoline-1,3-dione-featuring oximes with KIs mainly reaching
a medium-low nanomolar range (169.3-2038.4 nM). The most effective CAI 15 bore the fully
chlorinated phthalimmide scaffold. The loss of aromaticity in the isoindoline-1,3-dione core decreased
the inhibition potency of 10 against hCA IX by almost ten-fold with respect to 11 (KIs of 2038.4 vs
318.9 nM). The 5-COOH-bearing derivative 17 was also an effective hCA IX inhibitor showing an
inhibition constant of 240.3 nM. Incorporation of a bulky hexa-halogenated moiety such as in 18 elicited
a remarkable drop of efficacy in inhibiting the tumor-associated isozyme. Most oximes 10-18 showed a
markedly selective action against the hCA IX over the off-targer hCA I and XII (over two orders of
magnitude) with the standard AAZ showing a more potent but promiscuous activity versus the screened
isozymes.
(ii) The inhibition profiles of isoindoline-1,3-diones 20-28 changed dramatically compared to
compounds 10-18 owing to the incorporation of hydrazone benzenesulfonamides in place of
hydroxyiminoethyl moieties, though a striking preferential effectiveness against hCA IX was kept. The
sulfonamides are the most effective zinc-binding group in CA inhibition due to concomitant important
features in terms of ligand-target interactions; the presence of a negatively charged nitrogen which
coordinates the zinc ion and the presence of a hydrogen atom on it which enables the sulfonamide to
form two H-bonds with diverse portion of residue Thr199, present in all catalytically active human
isozymes.[2] As a result, the different CA inhibition profiles flattened out, with hCA II exhibiting the
greater enhancement of inhibition with sulfonamides 20-28 (KIs ranging between 369.1 and 834.9 nM).
The cytosolic hCA I and the membrane-bound hCA IV were moderately inhibited by sulfonamide

reported here, with inhibition constants spanning the low micromolar range between 1869.1 and 9283.3
nM. Inhibition of CA I was more significantly affected by incorporation of bulkier substituents on the
tails with KIs dropping to 21, 38 and 63 µM for 25, 26 and 28 respectively. Compound 27, that bear a 5-
carboxy moiety, arose as the unique sub-micromolar inhibitor of hCA IV. hCA II was inhibited in a
rather narrow nanomolar range by 20-28 (KIs of 539.8-834.9 nM). Again, 27 stood out as the best hCA
II inhibitor. In a similar manner with the first subset of derivatives, namely oximes 10-18, sulfonamides
20-28 showed a selective activity to the tumor-associated hCA IX (KIs of 17.3-164.3 nM), since KIs
ranged 20-fold below those observed against hCA II. In detail, hCA IX was inhibited in the narrow 17.3-
33.9 nM range by all sulfonamide, except 23 and 28, that possess differently bulkier carbon or halogen
groups at the isoindoline-1,3-dione scaffolds and shows KIs of 83.8 and 169.3 nM, respectively.
(iii) Noteworthy, when 20-22 and 24-27 demonstrated the same hCA IX inhibition efficacy of the
standard AAZ, they showed comparable or better IX/II selective efficacy than lead SLC-0111.
Inhibition data in Table 1 highlighted that a hydrazone linker as connection between the
benzenesulfonamide fragment and phthalimmide tails addressed the inhibitory activity versus hCA IX
with respect to hCA I, II or IV. Aromatic oximes of the hydroxyiminoethyl type 10-18 were shown to
act as selective hCA IX zinc-binders, owing to more than two orders of magnitude CA IX/II ratio.
(iv) The present phthalimmidobenzenesulfonamides showed remarkable inhibition profiles in terms of
selective action against CA IX over CA I/II in comparison to a wealth of heterocycle-tailed
benzenesulfonamides previously reported.[18-29] These data make some such derivatives interesting
leads or tools to design novel anti-cancer therapies.
3. Conclusion
The present work reported the synthesis, characterization and biological evaluation of a library of
isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones. The inhibitory profiles of the

hydroxyiminoethyl aromatic derivatives 10-18 allowed to assess the potentiality as zinc-binder for a
rarely studied moiety in the field of carbonic anhydrase inhibition. Isoindoline-1,3-dione hydrazone
benzenesulfonamides 20-28 showed instead the aptitude of hydrazone and phatlimmides to act as the
ureido-bioisoster linker and tailing components of the “tail approach”. The new zinc-binders, both of the
oxime and sulfonamide types, showed a remarkable selective activity against the target hCA IX over
ubiquitous hCA I and II, with diverse inhibitory ranges and ratio differing the two subsets. With CA IX
being a strongly current antitumor/antimetastatic drug target, these series of compounds may be of
interest for the development of new, both conventional and unconventional anticancer drugs targeting
hypoxia-induced CA isoforms such as CA IX.
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were recorded on Barnstead 9100 Electrothermal melting apparatus. IR
1
13
spectra were recorded on a FT-IR Perkin-Elmer spectrometer. H NMR and C NMR were recorded in
DMSO-d6 and CDCl3 on Bruker 500 & 700 and 125 & 176 MHz instrument, respectively, using TMS as
internal standard (chemical shifts in  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 2 and 19 were prepared according to their reported procedure [38].
2.1.1. General procedure for the synthesis of isoindoline-1,3-diones 1-9 (Scheme 1).
A mixture of 4-aminoacetophenone (10.0 mmol), anhydrous sodium acetate (1.38g, 10.0 mmol)
and an appropriate acid anhydride (10.0 mmol) in glacial acetic acid (50 mL) was heated under reflux
for 12 hr. After cooling of the reaction mixture, the precipitate obtained was filtered, washed with water,
dried and re-crystallised from acetic acid.

11
2.1.1.1. 2-(4-Acetylphenyl)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (1)
Yield, 80%; melting point (mp): 270-271 °C; IR (KBr, cm-1) : 1715, 1698 (C=O), 1591 (C=C), 1176
1
(C-C); H NMR (500 MHz, CDCl3): 8.05 (d, 2H, J = 8.5 Hz), 7.42 (d, 2H, J = 8.5 Hz), 6.00 (s, 2H),
3.30 (t, 2H, J= 9.5, 7.5 Hz), 2.73 (d, 2H, J= 14.5 Hz), 2.62 (s, 3H), 2.34 (d, 2H, J= 13.5 Hz);13C NMR
(125 MHz, CDCl3):  23.75, 26.69, 39.33, 126.35, 127.85, 129.08, 136.10, 136.62, 178.78, 197.03;
C16H15NO3: m/z (269).
2.1.1.2. 2-(4-Acetylphenyl)-5-methylisoindoline-1,3-dione (3)
Yield, 93%; melting point (mp): 220-221 °C; 1H NMR (500 MHz, CDCl3):  8.11 (d, 2H, J= 8.5 Hz),
13
7.86 (d, 1H, J= 7.5 Hz), 7.78 (s, 1H), 7.63 (t, 3H, J= 8.5, 7.0 Hz), 2.66 (s, 3H), 2.58 (s, 3H); C NMR
(125 MHz, CDCl3): 22.12, 26.69, 123.88, 124.43, 126.04, 128.97, 129.13, 131.94, 135.31, 135.91,
136.13, 146.18, 166.84, 166.97, 197.11; C17H13NO3: m/z (279).
2.1.1.3. 2-(4-Acetylphenyl)-5-(tert-butyl)isoindoline-1,3-dione (4)
Yield, 82%; melting point (mp): 175-177 °C; IR (KBr, cm-1) : 1710, 1697 (C=O), 1601 (C=C), 1079
1
(C-C); H NMR (500 MHz, CDCl3):  8.10 (d, 2H, J= 8.0 Hz), 8.01 (s, 1H), 7.89 (d, 1H, J= 7.5 Hz),
13
7.84 (t, 1H, J= 6.5, 7.5 Hz), 7.63 (d, 2H, J= 8.5 Hz), 2.65 (s, 3H), 1.42 (s, 9H); C NMR (125 MHz,
CDCl3):26.68, 31.15, 35.89, 121.10, 123.79, 126.07, 128.87, 129.14, 131.78, 135.90, 136.16, 159.52,
166.80, 167.23, 197.08; C20H19NO3: m/z (321).

12
2.1.1.4. 2-(4-Acetylphenyl)-5,6-dichloroisoindoline-1,3-dione (5)
Yield, 88%; melting point (mp): 259-260 °C; IR (KBr, cm-1) : 1708, 1683 (C=O), 1591 (C=C), 1087
(C-C); 1H NMR (700 MHz, DMSO-d6):  8.34 (s, 2H), 8.12 (d, 2H, J= 9.0 Hz), 7.63 (d, 2H, J= 8.5 Hz),
2.64 (s, 3H); 13C NMR (125 MHz, DMSO-d6): 27.34, 126.15, 127.44, 129.34, 132.05, 136.13, 136.50,
138.10, 165.41, 197.80; C16H9Cl2NO3: m/z (334).
2.1.1.5. 2-(4-Acetylphenyl)-4,5,6,7-tetrachloroisoindoline-1,3-dione (6)
Yield, 95%; melting point (mp): 249-250 °C; IR (KBr, cm-1) : 1717, 1700 (C=O), 1598 (C=C), 1114
(C-C); 1H NMR (700 MHz, DMSO-d6):  8.14 (d, 2H, J= 8.5 Hz), 7.61 (d, 2H, J= 8.5 Hz), 2.65 (s, 3H);
13C NMR (176 MHz, DMSO-d6): 27.36, 127.73, 128.82, 128.86, 129.44, 135.66, 136.85, 138.93,
162.81, 197.79; C16H7Cl4NO3: m/z (403).
2.1.1.6. 2-(4-Acetylphenyl)-4,5,6,7-tetrabromoisoindoline-1,3-dione (7)
Yield, 93%; melting point (mp): 290-292 °C; IR (KBr, cm-1) : 1713, 1698 (C=O), 1595 (C=C), 1117
(C-C); 1H NMR (700 MHz, DMSO-d6):  8.13 (d, 2H, J= 8.5 Hz), 7.60 (d, 2H, J= 8.5 Hz), 2.65 (s, 3H);
13C NMR (176 MHz, DMSO-d6): 27.36, 121.35, 127.82, 129.35, 131.52, 136.00, 136.78, 137.19,
163.14, 172.69, 197.80; C16H7Br4NO3: m/z (580).
2.1.1.7. 2-(4-Acetylphenyl)-1,3-dioxoisoindoline-5-carboxylic acid (8)

13
Yield, 82%; melting point (mp): 330-232 °C; IR (KBr, cm-1) : 3050 (br. OH), 1730, 1711, 1643 (C=O),
1594 (C=C), 1093 (C-C); 1H NMR (700 MHz, DMSO-d6):  13.81 (s, 1H), 8.42 (dd, 1H, J= 6.3, 7.7 Hz),
8.31 (s, 1H), 8.13 (d, 2H, J= 8.5 Hz), 8.10 (d, 1H, J= 7.7 Hz), 7.65 (d, 2H, J=8.5 Hz), 2.64 (s, 3H); 13C
NMR (176 MHz, DMSO-d6): 27.32, 123.95, 124.39, 127.45, 129.29, 132.46, 135.29, 136.05, 136.27,
136.40, 137.04, 166.26, 166.39, 166.41, 197.78; C17H11NO5: m/z (309).
2.1.1.8. 2-(4-Acetylphenyl)-4,5,6,7,8,8-hexachloro-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-
1,3(2H)-dione (9)
1
Yield, 89%; melting point (mp): 274-275 °C H NMR (500 MHz, CDCl3/DMSO-d6): 8.06 (d, 2H, J=
13
8.5 Hz), 7.27 (d, 2H, J= 8.5 Hz), 4.18 (s, 2H), 2.59 (s, 3H); C NMR (125 MHz, CDCl3/DMSO-d6):
27.06, 52.68, 79.41, 104.18, 126.98, 129.45, 131.02, 135.20, 137.37, 169.54, 196.88; C17H9Cl6NO3:
m/z (487).
2.1.2. General procedure for the synthesis of oxime based isoindoline-1,3-diones 10-18 (Scheme 1).
A mixture of an appropriate isoindoline-1,3-diones 1-9 (5.0 mmol), anhydrous sodium acetate
(0.69g, 5.0 mmol) and hydroxylamine hydrochloride (0.56 g, 8 mmol) in glacial acetic acid (15 mL) was
stirred at room temperature for 12-24 hr. After cooling the reaction mixture, the precipitate obtained was
filtered, washed with water, dried and re-crystallised from acetic acid.
2.1.2.1.
2-(4-(1-(Hydroxyimino)ethyl)phenyl)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione
(10)

14
Yield, 98%; melting point (mp): 186-170 °C; IR (KBr, cm-1) : 3421 (OH), 1734, 1710 (C=O), 1654
1
(C=N), 1617, 1508 (C=C), 1171 (C-C), 927 (N-O); H NMR (700 MHz, DMSO-d6): 11.32 (s, 1H),
7.75 (s, 2H), 7.23 (s, 2H), 5.95 (s, 2H), 3.31 (s, 2H), 2.48 (d, 2H, J= 14.0 Hz), 2.28 (d, 2H, J= 12.6 Hz),
13
2.17 (s, 3H); C NMR (176 MHz, DMSO-d6): 11.99, 23.73, 39.31, 126.48, 127.18, 128.19, 129.07,
133.04, 137.33, 152.85, 179.66; C16H16N2O3: m/z (284).
2.1.2.2. 2-(4-(1-(hydroxyimino)ethyl)phenyl)isoindoline-1,3-dione (11)
Yield, 94%; melting point (mp): 290-291 °C; IR (KBr, cm-1) : 3245 (OH), 1732, 1706 (C=O), 1685
1
(C=N), 1615, 1528 (C=C), 1268, 1100 (C-C), 819 (N-O); H NMR (700 MHz, DMSO-d6): 11.34 (s,
1H), 7.96 (s, 2H), 7.91 (s, 2H), 7.80 (s, 2H), 7.48 (s, 2H), 2.21 (s, 3H); 13C NMR (176 MHz, DMSO-d6):
12.03, 123.91, 126.44, 127.57, 129.23, 131.98, 132.54, 135.20, 135.32, 137.05, 152.94, 167.37;
C16H12N2O3: m/z (280).
2.1.2.3. 2-(4-(1-(Hydroxyimino)ethyl)phenyl)-5-methylisoindoline-1,3-dione (12)
Yield, 91%; melting point (mp): 300-302 °C;IR (KBr, cm-1) : 3413 (OH), 1768, 1718 (C=O), 1518
(C=C), 1275, 1128 (C-C), 929 (N-O); 1H NMR (700 MHz, DMSO-d6): 11.36 (s, 1H), 7.85 (d, 1H, J=
8.3 Hz), 7.79 (t, 3H, J= 8.8 Hz), 7.70 (d, 1H, J=7.6 Hz), 7.46 (d, 2H, J= 8.5 Hz), 2.52 (s, 3H), 2.20 (s,
3H); 13C NMR (176 MHz, DMSO-d6): 12.04, 21.88, 123.85, 124.31, 126.39, 127.52, 129.35, 132.32,
132.58, 135.58, 136.91, 146.21, 152.91, 167.34, 167.44; C17H14N2O3: m/z (294).
2.1.2.4. 5-(tert-Butyl)-2-(4-(1-(hydroxyimino)ethyl)phenyl)isoindoline-1,3-dione (13)

15
Yield, 86%; melting point (mp): 215-216 °C; IR (KBr, cm-1) : 3471 (OH), 1787, 1716 (C=O), 1678
1
(C=N), 1601, 1512 (C=C), 1268, 1122 (C-C), 829 (N-O); H NMR (700 MHz, DMSO-d6): 11.34 (s,
1H), 7.94 (s, 2H), 7.89 (d, 1H, J= 7.7 Hz), 7.78 (d, 2H, J= 7.7 Hz), 7.45 (d, 2H, J= 7.0 Hz), 2.20 (s, 3H),
13
1.38 (s, 9H); C NMR (176 MHz, DMSO-d6): 12.03, 31.25, 35.99, 120.71, 123.86, 126.43, 127.46,
127.56, 129.48, 132.08, 132.28, 132.64, 137.00, 152.94, 159.01, 167.22, 167.53; C20H20N2O3: m/z (336).
2.1.2.5. 5,6-Dichloro-2-(4-(1-(hydroxyimino)ethyl)phenyl)isoindoline-1,3-dione (14)
Yield, 94%; melting point (mp): 310-312 °C;IR (KBr, cm-1) : 3421 (OH), 1775, 1719 (C=O), 1654
(C=N), 1603, 1517 (C=C), 1219, 1103 (C-C), 829 (N-O); 1H NMR (700 MHz, DMSO-d6): 11.34 (s,
1H), 8.30 (d, 2H, J= 14 Hz), 7.81 (s, 2H), 7.47 (s, 2H), 2.20 (s, 3H); 13C NMR (176 MHz, DMSO-d6):
12.03, 126.03, 126.51, 127.47, 132.06, 132.22, 137.29, 138.01, 152.91, 165.63; C16H10Cl2N2O3: m/z
(349).
2.1.2.6. 4,5,6,7-Tetrachloro-2-(4-(1-(hydroxyimino)ethyl)phenyl)isoindoline-1,3-dione (15)
Yield, 96%; melting point (mp): 316-318 °C;IR (KBr, cm-1) : 3411 (OH), 1734, 1700 (C=O), 1654
(C=N), 1606, 1508 (C=C), 1297, 1099 (C-C), 824 (N-O); 1H NMR (500 MHz, DMSO-d6): 11.58 (s,
1H), 8.01 (d, 2H, J= 8.5 Hz), 7.65 (d, 2H, J= 8.0 Hz), 2.41 (s, 3H); 13C NMR (125 MHz, DMSO-d6):
12.27, 121.50, 126.77, 128.01, 131.77, 137.29, 163.60; C16H8Cl4N2O3: m/z (418).
2.1.2.7. 4,5,6,7-Tetrabromo-2-(4-(1-(hydroxyimino)ethyl)phenyl)isoindoline-1,3-dione (16)

16
Yield, 95%; melting point (mp): 322-324 °C;1H NMR (500 MHz, DMSO-d6): 11.41 (s, 1H), 7.81 (d,
2H, J= 8.5 Hz), 7.45 (d, 2H, J= 8.0 Hz), 2.20 (s, 3H); 13C NMR (125 MHz, DMSO-d6): 11.99, 126.58,
127.67, 128.76, 128.82, 131.79, 137.62, 138.84, 152.82, 162.99, 172.49; C16H8Br4N2O3: m/z (595).
2.1.2.8. 2-(4-(1-(Hydroxyimino)ethyl)phenyl)-1,3-dioxoisoindoline-5-carboxylic acid (17)
Yield, 83%; melting point (mp): 295-296 °C; IR (KBr, cm-1) : 3447 (OH), 1783, 1727 (C=O), 1654
1
(C=N), 1592 (C=C), 1209, 1128 (C-C), 742 (N-O); H NMR (500 MHz, DMSO-d6): 11.36 (s, 1H),
8.43-8.41 (m, 1H), 8.31 (d, 1H, J= 4.5 Hz), 8.13-8.07 (m, 1H), 7.81 (d, 2H, J= 8.5 Hz), 7.49 (d, 2H, J=
8.5 Hz), 2.21 (s, 3H); 13C NMR (125 MHz, DMSO-d6): 12.04, 123.87, 123.96, 124.30, 126.47, 127.53,
129.28, 132.36, 132.49, 135.37, 135.97, 136.94, 137.19, 152.92, 166.28, 166.66; C17H12N2O5: m/z (324).
2.1.2.9. 4,5,6,7,8,8-Hexachloro-2-(4-(-1-(hydroxyimino)ethyl)phenyl)-3a,4,7,7a-tetrahydro-1H-
4,7-methanoisoindole-1,3(2H)-dione (18)
Yield, 91%; melting point (mp): 265-257 °C; 1H NMR (500 MHz, CDCl3/DMSO-d6): 11.14 (s, 1H),
7.71 (d, 2H, J= 9.0 Hz), 7.09 (d, 2H, J= 8.5 Hz), 4.12 (s, 2H), 2.16 (s, 3H); 13C NMR (125 MHz,
CDCl3/DMSO-d6): 12.76, 52.53, 79.41, 104.18, 126.58, 126.60, 130.96, 131.26, 152.42, 169.67;
C17H10Cl6N2O3: m/z (502).
2.1.3. General procedure for the synthesis of hydrazone based isoindoline-1,3-diones 20-28
(Scheme 2).

17
A
mixture of an appropriate isoindoline-1,3-diones 1-9 (5.0 mmol), and 4-
(hydrazinecarbonyl)benzenesulfonamide (19) (1.08 g, 5.0 mmol) in methanol (15 mL) and glacial acetic
acid (0.5 mL) was heated under reflux for 6 hr. After cooling the reaction mixture, the precipitate
obtained was filtered, dried and re-crystallised from methanol.
2.1.3.1. 4-(2-(1-(4-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindol-2-yl)phenyl)ethylidene)hydrazine-
1-carbonyl)benzenesulfonamide (20)
Yield, 90%; melting point (mp): 288-290 °C;IR (KBr, cm-1) : 3369, 3275 (NH), 17 , 1725 (C=O),
1
1655 (C=N), 1593, 1534 (C=C), 1096 (C-C) 1376, 1259 (O=S=O); H NMR (700 MHz, DMSO-d6):
11.19 (s, 0.22H), 11.00 (s, 0.78 H), 8.07 (d, 2H, J= 7.7 Hz), 7.98-7.95 (m, 4H), 7.56 (s, 1.5 H), 7.50 (s,
0.5 H), 7.31 (2, 2H, J= 7.7 Hz), 5.97 (s, 2H), 3.33 (s, 2H), 2.48 (s, 2H), 2.42 (s, 2.25H), 2.36 (s, 0.75H),
2.29 (d, 2H, J= 12.6 Hz); 13C NMR (176 MHz, DMSO-d6): 15.22, 23.74, 39.36, 125.42, 126.05,
127.18, 127.34, 127.47, 128.25, 129.13, 130.42, 133.88, 137.41, 138.24, 146.97, 155.54, 163.61,
179.68; C23H22N4O5S: m/z (466).
2.1.3.2. 4-(2-(1-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)ethylidene)hydrazine-1-
carbonyl)benzenesulfonamide (21)
Yield, 91%; melting point (mp): 350-352 °C; IR (KBr, cm-1) : 3513, 3424, 3137 (NH), 1784, 1725
1
(C=O), 1653 (C=N), 1550 (C=C), 1103 (C-C) 1360, 1266 (O=S=O); H NMR (700 MHz, DMSO-d6):
11.19 (s, 0.25H), 11.09 (s, 0.75H), 8.11 (d, 1.5H, J= 7.7 Hz), 8.07 (d, 1.5H, J= 7.0 Hz), 8.03 (d, 1.5H,
J= 7.7 Hz), 7.97 (d, 2H, J= 7.0 Hz), 7.93-7.92 (t, 3H, J= 4.2 & 3.5 Hz), 7.65 (d, 1.5H, J= 7.7 Hz), 7.57
(s, 3H), 2.45 (s, 2.4 H), 2.40 (s, 0.6 H); 13C NMR (176 MHz, DMSO-d6): 15.24, 123.95, 124.03,

18
126.06, 127.42, 127.53, 129.25, 131.96, 132.00, 135.25, 135.33, 136.26, 136.48, 146.96, 155.73, 163.60,
167.13, 167.36; C23H18N4O5S: m/z (462).
2.1.3.3. 4-(2-(1-(4-(5-Methyl-1,3-dioxoisoindolin-2-yl)phenyl)ethylidene)hydrazine-1-
carbonyl)benzenesulfonamide (22)
Yield, 89%; melting point (mp): 312-313 °C;IR (KBr, cm-1) : 1730, 1717 (C=O), 1640 (C=N), 1598
(C=C), 1114 (C-C) 1354, 1197 (O=S=O); 1H NMR (700 MHz, DMSO-d6): 11.17 (s, 0.25H), 11.01 (s,
0.75 H), 8.10 (d, 0.5H, J= 12.6 Hz), 8.08 (d, 1.5H, J= 9.8 Hz), 8.02 (d, 1.5H, J= 10.5 Hz), 7.96 (d, 2.5H,
J= 11.2 Hz), 7.86 (q, 1H, J= 7.5, 4.0 Hz), 7.80 (s, 1H), 7.72 (d, 1.5H, J= 10.5 Hz), 7.63 (d, 0.5H, J= 11.9
13
Hz), 7.55 (s, 3H), 2.52 (s, 3H),2.44 (s, 3H); C NMR (176 MHz, DMSO-d6):  15.24, 21.90, 123.88,
123.96, 124.33, 124.41, 126.06, 127.42, 129.14, 129.22, 129.34, 132.31, 135.62, 146.26, 146.37, 146.96,
155.71, 167.30, 167.40; C24H20N4O5S: m/z (476).
2.1.3.4. 4-(2-(1-(4-(5-tert-Butyl-1,3-dioxoisoindolin-2-yl)phenyl)ethylidene)hydrazine-1-
carbonyl)benzenesulfonamide (23)
Yield, 86%; melting point (mp): 263-265 °C; IR (KBr, cm-1) : 3260 (NH), 1729, 1708 (C=O), 1599,
1
1503 (C=C), 1088 (C-C) 1380, 1270 (O=S=O); H NMR (700 MHz, DMSO-d6): 11.17 (s, 0.25H),
11.01 (s, 0.75H), 8.12-7.90 (m, 9H), 7.63-7.44 (m, 4H), 2.44 (s, 3H), 1.38 (s, 9H); 13C NMR (176 MHz,
DMSO-d6): 15.26, 31.26, 36.02, 167.50, 120.77, 123.89, 126.06, 127.41, 127.51, 129.13, 129.24,
129.49, 132.11, 132.28, 137.44, 137.86, 146.96, 155.74, 159.00, 163.58, 167.19; C27H26N4O5S: m/z
(518).

19
2.1.3.5. 4-(2-(1-(4-(5,6-Dichloro-1,3-dioxoisoindolin-2-yl)phenyl)ethylidene)hydrazine-1-
carbonyl)benzenesulfonamide (24)
Yield, 92%; melting point (mp): 361-362 °C;IR (KBr, cm-1) : 1725, 1713 (C=O), 1639 (C=N), 1507
(C=C), 1162 (C-C) 1366, 1207 (O=S=O); 1H NMR (700 MHz, DMSO-d6): 11.84 (s, 0.2H), 11.01 (s,
0.8H), 8.32 (d, 2H, J= 10.5 Hz),), 8.12 (d, 0.5H J= 11.9 Hz), 8.07 (d, 1.5H, J= 10.5 Hz), 8.03 (d, 1.5H,
13
J= 11.2 Hz), 7.96 (d, 2H, J= 10.5Hz), 7.62 (d, 0.5H, J= 11.9 Hz), 7.55-7.45 (m, 4H), 2.47 (s, 3H); C
NMR (176 MHz, DMSO-d6): 15.23, 126.05, 126.13, 127.39, 127.49, 129.13, 129.33, 132.01, 132.06,
136.12, 136.48, 138.02, 146.97, 155.57, 163.59, 165.38, 165.59; C23H16Cl2N4O5S: m/z (531).
2.1.3.6. 4-(2-(1-(4-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl)phenyl)ethylidene)hydrazine-1-
carbonyl)benzenesulfonamide (25)
Yield, 94%; melting point (mp): 351-353 °C;1H NMR (700 MHz, DMSO-d6): 11.19 (s, 0.2H), 11.02
(s, 0.8H), 8.05 (d, 3H, J= 10.5 Hz), 7.95 (d, 3H, J= 10.5 Hz), 7.55-7.53 (m, 4H), 2.45 (s, 3H); 13C NMR
(176 MHz, DMSO-d6): 15.21, 125.46, 126.05, 127.16, 127.70, 127.61, 128.79, 128.82, 128.89,
129.13, 129.43, 130.44, 132.66, 137.34, 138.53, 138.86, 146.97, 155.47, 162.99, 163.64;
C23H14Cl4N4O5S: m/z (600).
2.1.3.7. 4-(2-(1-(4-(4,5,6,7-Tetrabromo-1,3-dioxoisoindolin-2-yl)phenyl)ethylidene)hydrazine-1-
carbonyl)benzenesulfonamide (26)

20
Yield, 93%; melting point (mp): 344-345 °C IR (KBr, cm-1) : 3260 (NH), 1728, 1708 (C=O), 1637
1
(C=N), 1548 (C=C), 1090 (C-C) 1327, 1163 (O=S=O); H NMR (700 MHz, DMSO-d6): 11.20 (s,
0.25H), 11.03 (s, 0.75H), 8.13 (d, 0.5H, J= 8.4 Hz), 8.07-8.03 (m, 3H), 7.96-7.89 (m, 3H), 7.60 (d, 0.5H,
J= 8.4 Hz), 7.56-7.52 (m, 3H), 2.45 (s, 3H); 13C NMR (176 MHz, DMSO-d6): 15.23, 121.28, 121.36,
126.05, 126.15, 127.51, 127.71, 127.81, 129.15, 129.35, 131.53, 133.00, 137.09, 138.45, 146.99, 155.50,
163.13, 163.35, 163.63; C23H14Br4N4O5S: m/z (778).
2.1.3.8. 1,3-Dioxo-2-(4-(1-(2-(4-sulfamoylbenzoyl)hydrazono)ethyl)phenyl)isoindoline-5-carboxylic
acid (27)
Yield, 84%; melting point (mp): 358-359 °C;IR (KBr, cm-1) : 2987 (br. OH), 1731, 1711 (C=O), 1640
(C=N), 1547 (C=C), 1097 (C-C) 1388, 1286 (O=S=O); 1H NMR (700 MHz, DMSO-d6): 13.46 (s, 1H),
11.19 (s, 0.2 H), 11.03 (s, 0.8 H), 8.43 (s, 1H), 8.32 (s, 1H), 8.09 (s, 3H), 8.04 (s, 1H), 7.96 (s, 3H), 7.57
(s, 4H), 2.45 (s, 3H); 13C NMR (176 MHz, DMSO-d6): 15.24, 123.89, 124.32, 126.06, 127.45, 129.14,
132.50,133.26, 135.35, 136.00, 136.97, 137.43, 138.08, 146.96, 155.64, 163.61, 166.30, 166.62, 166.63;
C24H18N4O7S: m/z (506).
2.1.3.9. 4-(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)phenyl)ethylidene)hydrazine-1-carbonyl)benzenesulfonamide (28)

21
Yield, 91%; melting point (mp): 338-340 °C;1H NMR (700 MHz, DMSO-d6): 11.19 (s, 0.25H), 11.03
(s, 0.75H), 8.07 (d, 1.5H, J= 7.0 Hz), 8.03 (d, 1.5H, J= 7.0 Hz), 7.96 (d, 2.5H, J= 7.7 Hz), 7.75 (s,
13
0.5H), 7.55 (s, 2H), 7.22 (d, 1.5H, J= 7.0 Hz), 7.11 (s, 0.5H), 4.27 (s, 2H), 2.42 (s, 3H); C NMR (176
MHz, DMSO-d6): 15.23, 52.61, 79.44, 104.20, 126.05, 126.96, 127.99, 129.16, 131.10, 132.29,
137.36, 139.31, 146.99, 155.19, 163.66, 170.06, 172.53; C24H16Cl6N4O5S: m/z (685).
4.2 Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity.[37] Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mMHepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of
the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed
rates were determined in the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (0.1 mM) were prepared in distilled-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 earlier,[39] and represent the mean from at least three different determinations. All
CA isoforms were recombinant ones obtained in-house as reported earlier.[40,41]
Acknowledgments

22
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for
funding the work through the research group project No. RGP-163. Ente Cassa di Risparmio di Firenze,
Italy, is gratefully acknowledged for a grant to A.N (ECR 2016.0774).
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Highlights
 A new chemotype for CA inhibition was identified: isoindoline-1,3-dione-based oximes.
 Isoindoline-1,3-dione benzenesulfonamides were also evaluated as CAIs.
 Remarkable CA IX selectivity was found within both series
 Promising anti-cancer leads/tools were reported.

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