Inhibition of human carbonic anhydrase isozymes I, II, IX and XII with a new series of sulfonamides incorporating aroylhydrazone-, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenylmethylene)-1,3,4-thiadiazol-3(2H)-yl moieties

Article abstract of DOI:10.3109/14756366.2013.877897

A series of benzenesulfonamides incorporating aroylhydrazone, piperidinyl, sulfone, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties was investigated as inhibitors of four α-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h) isoforms hCA I, II (cytosolic, offtarget enzymes) and hCA IX and XII (transmembrane, tumor-associated isoforms). Low nanomolar activity was observed against hCA II (K_Is of 0.56-17.1 nM) with these sulfonamides, whereas the slow cytosolic isoform hCA I was less inhibited by these compounds (K_Is of 86.4 nM-32.8 μM). Most of these sulfonamides significantly inhibited CA IX, with K_Is in the range of 4.5-47.0 nM, although some of the derivatives incorporating bulkier bicyclic moieties, as well as 2-thienyl fragments, showed a weaker activity against this isoform (K_Is in the range 50.1-553 nM). All the investigated compounds also inhibited CA XII with K_Is in the range 0.85-376 nM. The best inhibitors were those incorporating bulky [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl moieties and 1,3,4-thiadiazol-3(2H)-yl groups.

Full text of DOI:10.3109/14756366.2013.877897

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–5
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.877897
!
RESEARCH ARTICLE
Inhibition of human carbonic anhydrase isozymes I, II, IX and XII
with a new series of sulfonamides incorporating aroylhydrazone-,
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenylmethylene)-
1,3,4-thiadiazol-3(2H)-yl moieties
Ahmed M. Alafeefy1, Hatem A. Abdel-Aziz2,3, Daniela Vullo4, Abdul-Malek S. Al-Tamimi1, Amani S. Awaad5,
Menshawy A. Mohamed1,6, Clemente Capasso7, and Claudiu T. Supuran4,8
2
1Department of Pharmaceutical Chemistry, College of Pharmacy, Salman bin Abdulaziz University, Alkharj, Saudi Arabia, Department of
Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 3Department of Applied Organic Chemistry, National
4
Research Center, Dokki, Cairo, Egypt, Laboratorio di Chimica Bioinorganica, Polo Scientifico, Universita degli Studi di Firenze, Sesto Fiorentino,
`
Florence, Italy, 5Chemistry Department, Faculty of Science, King Saud University, Riyadh, Saudi Arabia, 6Department of Organic Chemistry, Faculty
7
of Pharmacy, Al-Azhar University, Cairo, Egypt, Istituto di Biochimica delle Proteine and IBBR – CNR, Napoli, Italy, and Neurofarba Department,
8
`
Section of Pharmaceutical and Nutriceutical Sciences, Universita degli Studi di Firenze, Sesto Fiorentino, Florence, Italy
Abstract
Keywords
A series of benzenesulfonamides incorporating aroylhydrazone, piperidinyl, sulfone, [1,2,4]tria-
zolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties
was investigated as inhibitors of four a-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h)
isoforms hCA I, II (cytosolic, offtarget enzymes) and hCA IX and XII (transmembrane, tumor-
associated isoforms). Low nanomolar activity was observed against hCA II (KIs of 0.56–17.1 nM)
with these sulfonamides, whereas the slow cytosolic isoform hCA I was less inhibited by these
compounds (KIs of 86.4 nM–32.8 mM). Most of these sulfonamides significantly inhibited CA IX,
with KIs in the range of 4.5–47.0 nM, although some of the derivatives incorporating bulkier
bicyclic moieties, as well as 2-thienyl fragments, showed a weaker activity against this isoform
(KIs in the range 50.1–553 nM). All the investigated compounds also inhibited CA XII with KIs
in the range 0.85–376 nM. The best inhibitors were those incorporating bulky [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazinyl moieties and 1,3,4-thiadiazol-3(2H)-yl groups.
Cancer-associated isoform, carbonic
anhydrase, enzyme inhibitor, sulfonamide
History
Received 3 December 2013
Revised 18 December 2013
Accepted 18 December 2013
Published online 25 March 2014
Introduction
Recently, a large of number of sulfonamides and some of their
derivatives started to be obtained which showed a good selectivity
level for the inhibition of various CA isoforms4,15–28, some of
which are cytosolic (e.g. CA I, II, III, VII and XIII, as well as the
acatalytic isoforms CA VIII, X and XI)1,3,6,21, membrane
The primary sulfonamides, RSO2NH2, are the classical inhibitors
of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1)1–4
.
They have been widely used for almost 60 years as diuretic or
systemically acting antiglaucoma drugs, since the introduction of
acetazolamide (AAZ) in clinical use in 19545–9. However, AAZ
and the other clinically used sulfonamides/sulfamate CA inhibi-
tors (CAIs), such as methazolamide and ethoxzolamide1,4,6, target
all mammalian CA isoforms (16 of them are known to date in
vertebrates)1,8–11, and as thus, they show a range of undesired side
effects1,4,6,11,12, motivating the continuous search of novel such
agents with a selective inhibition profile against the desired
associated/transmembrane (CA IV, IX, XII, XIV and XV)1,2,8,25
mitochondrial (CA VA and VB)1,4, 17 or secreted (CA VI)1,4,15,20
,
.
The tumor associated isoform CA IX is highly overexpressed
in many cancer types by the hypoxia inducible factor-1a (HIF-1a)
cascade1–4. Around 70% of the hypoxic tumors overexpress CA
IX and show a bad response to classical chemo- and radio-
therapies2. CA IX was shown to significantly contribute to the
extracellular acidification of the tumor environment, by means of
the protons resulted from the catalysis of carbon dioxide
hydration1–4. This leads to the acquisition of metastasic pheno-
types and chemoresistance towards many anticancer drugs.
Ultimately, targeted structure-based drug design campaigns of
CAIs against this novel target led to a range of interesting
derivatives with a significant activity, selectivity and promising
in vivo action against several types of tumors29,30. CA XII is also a
tumor-associated CA and its inhibition was also shown to lead to
anticancer effects31.
isoform(s)13–20
.
These enzymes are also versatile catalysts of other hydrolytic
reactions except the hydration of CO2 to bicarbonate and protons,
being esterases with a range of carboxylic acid esters, phosphate
and sulfate esters1,3,13,14
.
Address for correspondence: Clemente Capasso, Istituto di Biochimica
delle Proteine and IBBR – CNR, Via P. Castellino 111, 80131 Napoli,
Italy. Tel: +39 081 6132559. E-mail: c.capasso@ibp.cnr.it

2
A. M. Alafeefy et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
Continuing our interest in the design of CA IX and XII Results and discussion
inhibitors of the sulfonamide type, here we report that a series of
benzenesulfonamides incorporating aroylhydrazone, [1,2,4]tria-
Chemistry
zolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)- Sulfonamides investigated here were obtained from oxo-N0-(4-
1,3,4-thiadiazol-3(2H)-yl moieties, reported earlier by us for the sulfamoylphenyl)propanehydrazonoyl chloride (1)26 which was
inhibition of bacterial enzymes from extremophiles26, shows reacted with aroylhydrazines 2 to form the key intermediates of
interesting inhibition profile against the two tumor-associated type 3 (Scheme 1). The 2-(2-arylhydrazono)-N0-(4-sulfamoylphe-
isoforms CA IX and XII.
nyl)propanehydrazonoyl chlorides 3a–d were then reacted with
piperidine in ethanol led to the formation of piperidine derivatives
4a–d or with sodium benzenesulfinate to afford the corresponding
sulfones 5a–d, respectively (Scheme 1)26.
Materials and methods
Chemistry
Alternatively, treatment of 1 with 4-amino-5-(methyl/phenyl)-
Compounds 3–10 investigated here were reported in a previous 4H-1,2,4-triazole-3-thiol 6a, b gave 1,2,4-triazolo[3,4-b]-1,3,4-
study from our group26.
thiadiazines 7a, b (Scheme 2). In another approach, 1 was reacted
with thioanilide derivatives 8a–d in the presence of triethylamine,
which afforded the 1,3,4-thiadiazoles 10a–d, as depicted in
Scheme 2.
All these compounds have been characterized by physico-
chemical and spectral techniques confirming the proposed
structures (see ref.26 for details).
Enzymology
hCA I, II, IX and XII were recombinant enzymes obtained
in-house as described earlier1–7
.
CA catalytic and inhibition assay
An applied photophysics stopped-flow instrument has been used CA inhibition
for assaying the CA catalyzed CO2 hydration activity27. Phenol
The inhibition studies against four mammalian CA isoforms of
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
HEPES (pH 8.4) and 20 mM NaBF4 (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 (10 mM) were
prepared in distilled-deionized water and dilutions up to 0.01 nM
were done thereafter with distilled-deionized water. 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, whereas the kinetic
parameters for the uninhibited enzymes from Lineweaver–Burk
plots, as reported earlier12–17,28, and represent the mean from at
least three different determinations.
this series of benzenesulfonamides containing the interesting tails
of the [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophe-
nyl-methylene)-1,3,4-thiadiazol-3(2H)-yl type (3–10), i.e. hCA I,
II, IX and XII are shown in Table 1.
As seen from data of Table 1, sulfonamides 3a–10d (and
acetazolamide,
5-acetamido-1,3,4-thiadiazole-2-sulfonamide,
AAZ, as standard inhibitor) showed interesting inhibitory proper-
ties against all four investigated CAs. The following structure-
activity relationship (SAR) has been delineated:
(i) The slow cytosolic isoform hCA I was moderately inhibited
by sulfonamides 3a–3d, 4a–4d, 5a–5d and 7a, 7b, with
inhibition constants in the range of 86.4–753 nM, and
weakly inhibited by derivatives 10a–10d (KIs in the range of
6.54–32.8 mM). It is interesting to note that the Schiff’s
bases 3a–3d showed a rather similar behavior as hCA I
inhibitors, with a relatively small variation of the inhibition
constants irrespective of the nature of moiety Ar. The
strongest inhibitor in the subseries was the phenyl derivative
3a, but its substitution with 4-Cl, 4-MeO groups, or its
Cl
H
O
N
N
H2N
S
Me
1
O
O
O
NH2
Ar
Cl
N
H
2
O
H
N
N
N
N
Ar
PhSO2Na
NH
H
H2N
Me
S
3
O
O
O
Ph
N
N
O
O
S
O
H
H
N
N
N
Ar
C6H5
4-ClC6H4
4-CH3OC6H4
2-thienyl
2-5
a
b
c
d
N
N
N
Ar
N
Ar
H
H
H2N
Me
H2N
Me
S
4
S
O
5
O
O
O
Scheme 1. Synthesis of compounds 3a–d, 4a–d and 5a–d.

DOI: 10.3109/14756366.2013.877897
Inhibition of human carbonic anhydrase isozymes I, II, IX and XII
3
Cl
CN
CN
H
H
O
N
R
Ph
NH
HN
N
N
N
R
R
Ph
O
S
H2N
S
Me
SH
8
S
NH2
1
O
O
O
N
N
Me
9
O
SH
N
- PhNH2
NH2
6
O
NH2
S
R
N
N
Me
N
Me
O
N
N
S
H
O
N
N
N
S
NC
10
R
CN
EtOCO
PhCO
R
H2N
S
10
7
a
b
c
O
O
7
a
b
R
Me
Ph
d
NH2SO2C6H4NHCO
Scheme 2. Synthesis of compounds 7a, b and 10a–d.
10, which probably due to their more rigid scaffold (the
thiadiazole ring is directly connected to the benzene one in
these compounds) can be less well-accommodated within
the restricted space of the hCA I active site28.
Table 1. Inhibition data against the human isoforms hCA I, II, IX and XII
with sulfonamides 3–10 by a stopped-flow, CO2 hydrase assay27
.
KI (nM)*
(ii) All new compounds reported here were excellent inhibitors
of the physiologically dominant (in humans) isoform hCA
II. Indeed, only 3a showed an inhibition constant of 17.1 nM
(comparable to that of acetazolamide, 12.1 nM) whereas all
the remaining derivatives were sub-nanomolar or low
nanomolar hCA II inhibitors, with KIs in the range of
0.55–8.6 nM. The SAR is thus almost impossible to
delineate as all these substitution patterns seem to be
highly favorable for obtaining tight-binding hCA II inhibi-
tors. Among the best such compounds were the Schiff’s base
3d (incorporating the thienyl moiety), the piperidine 4b
(incorporating a 4-chlorophenyl moiety), the sulfones 5b
and 5c, incorporating 4-Cl- and 4-MeO-phenyl moieties, the
bulky phenyl-substituted 7b as well as the 1,3,4-thiadiazoles
10c and 10d. All these compounds showed inhibition
constants 51 nM, being among the most effective hCA II
inhibitors reported up until now.
Compound
hCA I
hCA II
hCA IX
hCA XII
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
7a
7b
134
185
180
193
151
132
247
17.1
6.4
3.1
0.94
5.8
0.57
1.1
0.89
8.6
0.97
0.55
4.5
1.0
0.93
4.2
9.3
16.8
7.6
11.7
17.1
38.5
207
15.1
4.6
29.3
376
36.3
32.8
41.2
8.5
4.3
0.85
7.1
3.4
5.2
7.5
5.8
354
63.0
35.1
47.0
553
64.8
85.1
42.7
57.3
52.7
72.5
8.9
91.3
130
367
162
753
86.4
390
16 300
27 500
32 800
6540
10a
10b
10c
10d
AAZ
5.4
4.5
8.8
50.1
25.0
0.91
0.56
12.1
(iii) Against hCA IX the derivatives 3–10 showed with KIs in
the range of 4.5–553 nM. The most ineffective inhibitors of
this isoform were 3d and 4d (with KIs of 354–553 nM),
which both incorporate 2-thienyl fragment at the end of the
bulky tail derivatizing the sulfanilamide scaffold. It is
interesting to note that other chloro derivatives 3 or
piperidines 4, incorporating a different substitution patterns
(e.g. in 3a–3c or 4a–4c) were quite effective (3a and 3c) or
medium potency (3b, 4a–4c) hCA IX inhibitors (the
last group of compounds showed with KIs in the range of
16.8–63.0 nM, Table 1). Interestingly, irrespective of the
substitution pattern, all the sulfones 5 and the [1,2,4]tria-
zolo[3,4-b][1,3,4]thiadiazinyl-substituted derivatives 7 were
medium potency hCA IX inhibitors, with KIs in the range of
42.7–85.1 nM. The same is true for 10d, incorporating two
sulfamoyl functionalities but on a quite bulky scaffold (KI of
50.1 nM), whereas the remaining 2-(cyanophenyl-methy-
lene)-1,3,4-thiadiazol-3(2H)-yl derivatives 10a–10c, which
incorporate more compact functionalities compared to 10d,
were quite efficient hCA IX inhibitors, with KIs in the range
of 4.5–8.9 nM. These compounds are more effective
compared to acetazolamide AAZ as hCA IX inhibitors
(together with 3a and 3c). Thus, SAR for hCA IX inhibition
with compounds 3–10 is much more complicated compared
250
The standard, clinically used sulfonamide acetazolamide (AAZ, 5-
acetamido-1,3,4-thidiazole-2-sulfonamide) is also included for com-
parison reasons.
*Errors were in the range of 10% of the reported values from three
different assays (data not shown).
replacement by a thienyl scaffold, led to a slight diminution
of the inhibitor potency in compounds 3b–3d. For piperi-
dines 4a–4d, the range of inhibition was already higher
compared to derivatives 3, with the thienyl derivative 4d
being the most active in the subseries (KI of 91.3 nM) and
the 4-methoxyphenyl one 4c the least active (KI of 247 nM,
Table 1). Sulfones 5 also showed a significant variation of
the inhibitory power with the nature of the Ar group, with
the best inhibitor being the phenyl derivative 5a (KI of
130 nM) and the least effective one the thienyl derivative 5d
(KI of 753 nM). For compounds 7, the methyl derivative 7a
showed effective hCA I inhibitory properties (this was the
best inhibitor of this isoform among the reported derivatives,
being almost 3-fold more effective than the clinically used
sulfonamide (AAZ) whereas the bulkier Ph derivative 7b
was much less effective as inhibitor of this isoform. Even a
stronger loss of activity was then observed for sulfonamides

4
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to what we observed for the inhibition of the cytosolic
isoforms hCA I and II discussed earlier.
(iv) The second transmembrane isoforms, hCA XII; was also
inhibited by sulfonamides 3–10 investigated here, with KIs
in the range of 0.85–376 nM (Table 1). As for hCA IX
discussed earlier, the least effective hCA XII inhibitors were
the same two compounds (3d and 4d) incorporating the 2-
thienyl tail (KIs of 207–376 nM). Medium potency inhib-
ition, with KIs in the range of 11.7–41.2 nM was observed
for aroylhydrazones 3a–3c, piperidines 4a and 4c as well as
sulfones 5a–5c. The remaining derivatives, i.e. 4b, 5d, 7a, b
and 10a–10d, were highly effective hCA XII inhibitors with
KIs in the range of 0.85–8.5 nM (Table 1). The best and only
subnanomolar hCA XII inhibitor was 7b (KI of 0.85 nM).
Many of the investigated compounds were more effective or
similar to acetazolamide for the inhibition of this isoform.
(v) The inhibition profile of the four subclasses of derivatives
investigated here, which all carry the sulfanilamide head
group, but highly diverse tail moieties, was very different
and specific for each of them. For example derivatives 7
were highly effective as hCA II and XII inhibitors, medium
potency hCA IX inhibitors and rather weak hCA I inhibitors.
Although no highly hCA IX/XII – selective compounds
were detected in this study, the inhibition profiles of these
derivatives are of great interest considering the many
applications that CAIs possess in various pharmacological
fields, for obtaining diuretics32, antiepileptics33, antiobe-
sity34 and antiglaucoma35,36 agents.
8. Supuran CT. Carbonic anhydrase inhibitors. Bioorg Med Chem Lett
2010;20:3467–74.
9. Supuran CT. Carbonic anhydrase inhibitors and activators for novel
therapeutic applications. Future Med Chem 2011;3:1165–80.
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˘
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methanesulfonates are hydrolyzed by the sulfatase carbonic
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14. Cavdar H, Ekinci D, Talaz O, et al. a-Carbonic anhydrases are
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Conclusions
15. Ekinci D, Kurbanoglu NI, Salamcı E, et al. Carbonic anhydrase
inhibitors: inhibition of human and bovine isoenzymes by
benzenesulphonamides, cyclitols and phenolic compounds.
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sulfonamides as potent carbonic anhydrase inhibitors. J Enzyme
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17. Singh S, Supuran CT. QSARs on human carbonic anhydrase VA and
VB inhibitors of some new not yet synthesized, substituted aromatic/
heterocyclic sulphonamides as anti-obesity agent. J Enzyme Inhib
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18. Fabrizi F, Mincione F, Somma T, et al. A new approach to
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19. Sahin H, Can Z, Yildiz O, et al. Inhibition of carbonic anhydrase
isozymes I and II with natural products extracted from plants,
mushrooms and honey. J Enzyme Inhib Med Chem 2012;27:
395–402.
We investigated a series of recently reported benzenesulfona-
mides, incorporating aroylhydrazone, [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thia-
diazol-3(2H)-yl moieties as inhibitors of four a-CAs, the isoforms
hCA I, II (cytosolic, offtarget enzymes) and hCA IX and XII
(transmembrane, tumor-associated isoforms). Low nanomolar
activity was observed against hCA II (KIs of 0.56–17.1 nM)
with these sulfonamides, whereas the slow cytosolic isoform hCA
I was less inhibited by these compounds (KIs of 86.4 nM–
32.8 mM). Most of the sulfonamides investigated here also
significantly inhibited CA IX, with KIs in the range of 4.5–
47.0 nM, although some of the derivatives incorporating bulkier
bicyclic moieties as well as thienyl fragments, showed a weaker
activity against this isoform (KIs in the range 50.1–553 nM). All
the investigated compounds also inhibited CA XII with KIs in the
range 0.85–376 nM. The best inhibitors were those incorporating
bulky [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl moieties and 1,3,4-
thiadiazol-3(2H)-yl groups. Although no hCA IX/XII – selective
compounds were detected in this study, the inhibition profiles of
these derivatives are of great interest considering the many
applications of CAIs for obtaining diuretics, antiepileptics,
antiobesity and antiglaucoma agents.
¨
20. Sentu¨rk M, Ekinci D, Goksu S, Supuran CT. Effects of dopamin-
ergic compounds on carbonic anhydrase isozymes I, II, and VI.
J Enzyme Inhib Med Chem 2012;27:365–9.
¨
¨
21. Bootorabi F, Janis J, Hytonen VP, et al. Acetaldehyde-derived
modifications on cytosolic human carbonic anhydrases. J Enzyme
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22. Chohan ZH, Shad HA, Supuran CT. Synthesis, characterization
and biological studies of sulfonamide Schiff’s bases and
some of their metal derivatives. J Enzyme Inhib Med Chem 2012;
27:58–68.
Declaration of interest
This research was financed by a 7th FP EU grant, METOXIA (to C.T.S.)
and by the National Plan of Science, Technology and Innovation (Grant
No. 10-MED1188-02), King Saud University, Riyadh and by the
Graduate Studies and Scientific Research Agency, Salman bin
Abdulaziz University, Grant No. 2.H.33, Alkharj, Saudi Arabia (to
A.M.A).
23. Ozensoy O, Arslan M, Supuran CT. Carbonic anhydrase inhibitors:
purification and inhibition studies of pigeon (Columba livia
var. domestica) red blood cell carbonic anhydrase with sulfona-
mides. J Enzyme Inhib Med Chem 2011;26:749–53.
24. Sahin H, Aliyazicioglu R, Yildiz O, et al. Honey, pollen, and
propolis extracts show potent inhibitory activity against the zinc
metalloenzyme carbonic anhydrase. J Enzyme Inhib Med Chem
2011;26:440–4.
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