New series of sulfonamides containing amino acid moiety act as effective and selective inhibitors of tumor-associated carbonic anhydrase XII

Article abstract of DOI:10.3109/14756366.2014.942659

New benzenesulfonamides incorporating water solubilizing moieties were synthesized using N-α-acetyl-l-lysine or γ-aminobutyric acid as scaffolds followed by the conversion of their terminal amino group to the guanidine one. Their inhibition activity was a

Full text of DOI:10.3109/14756366.2014.942659

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–5
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.942659
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ORIGINAL ARTICLE
New series of sulfonamides containing amino acid moiety act as
effective and selective inhibitors of tumor-associated carbonic
anhydrase XII
Mariangela Ceruso1, Marco Bragagni2, Zeid AlOthman3, Sameh M. Osman3, and Claudiu T. Supuran2
2
1Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences,
3
Universita degli Studi di Firenze, Sesto Fiorentino (Florence), Italy, and Department of Chemistry, College of Science, King Saud University, Riyadh,
`
Saudi Arabia
Abstract
Keywords
New benzenesulfonamides incorporating water solubilizing moieties were synthesized using
N-a-acetyl-L-lysine or g-aminobutyric acid as scaffolds followed by the conversion of their
terminal amino group to the guanidine one. Their inhibition activity was assessed by
determining their KIs values against the human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms
hCA I, II, IX and XII. Some of these compounds were medium potency inhibitors of the cytosolic
(CA I, II) and transmembrane (CA IX) isoforms and highly effective, nanomolar inhibitors of
the second transmembrane isoform hCA XII. Some of these sulfonamides possessing
good selectivity inhibition for the tumor-associated CA XII isoform over the cytosolic
and physiologically dominant isoforms CA I and II may be used as tools to develop new
anticancer agents.
Amino acid, carbonic anhydrase inhibitor,
sulfonamide, tumor-associated CA XII
History
Received 26 June 2014
Revised 3 July 2014
Accepted 4 July 2014
Published online 30 July 2014
Introduction
a (ERa), a hormone-regulated transcription factor belonging to
the superfamily of nuclear receptors, which is expressed in about
70% of breast cancers12. CA XII has also been identified in brain
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that
catalyze a simple and physiologically relevant reaction in all life
kingdoms: the carbon dioxide hydration to bicarbonate and
protons1–7. In humans, 15 CA isoforms belonging to the a-CA
family are known to be involved in many physiologic processes.
Due to the fact that different isoforms are involved in diverse
physiologic or pathologic processes1,2, this class of enzyme
constitutes drug targets for diuretics, antiglaucoma agents,
tumors, such as astrocytoma and glioblastoma13–16
.
The main consequence of an overexpression of hypoxia-induced
CA IX and CA XII isoforms is a pH imbalance, with most of
hypoxic tumors having lower pH value (about 6–6.5) compared
with that characteristic of normal tissue equal to 7.417–19
.
Therefore, hypoxia-induced CA IX and CA XII overexpression
can be considered as the major tumor pH-regulating enzymes, and
their inhibition may represent a promising target for antitumor
antiepileptics, antiobesity and antitumor drugs1–7
.
It has been found that isoforms CA IX and XII are
transmembrane proteins characterized by an N-terminal extracel-
lular domain, a putative transmembrane a-helix and an intramo-
lecular C-terminal domain1,8. Both of them are overexpressed in a
large number of human cancers, such as glioblastoma9, colorec-
tal10 and breast cancer11 as markers of hypoxia and thus used in
therapy1,8,13–16
.
Although all a-CAs, including CA IX and XII, are inhibited by
several main classes of inhibitors, such as the inorganic anions20,
the phenols21 and the coumarins22 the sulfonamides and their
bioisosteres (sulfamates, sulfamides, etc.) are still the most
investigated CAIs18,19,23. Many low nanomolar sulfonamide CA
IX17,18 or CA XII24 inhibitors have been identified in the last few
years. It has been recently shown that in vitro, in cell cultures and
in animals with transplanted tumors, CA IX inhibition with
sulfonamides led to a return to more normal extracellular pH
values, with consequential delay of tumor growth5,6. Furthermore,
in mice suffering from T lymphoma, it was observed that the
inhibition of the overexpressed CA XII by sulfonamides decreased
cell proliferation and induced apoptosis16.
clinical settings1,2,8
.
Although it has been demonstrated that the overexpression of
CA IX can be initiated by the activation of the hypoxia-inducible
factor-1 (HIF-1) cascade6, the molecular mechanisms at the basis
of the induction of CA XII are not so well understood yet4.
However, CA XII is known to be involved in breast cancer and
recently it has been shown to be regulated by the estrogen receptor
In this article we continue our interest in sulfonamide CAIs as
potential anticancer agents, and report the synthesis of benzene-
sulfonamides incorporating amino acid scaffolds and guanidine
moieties. These new compounds were investigated for the
inhibition of the overexpressed in hypoxic tumors human CA
(hCA) IX and XII isoforms over the physiologically dominant,
Address for correspondence: Mariangela Ceruso, Dipartimento di
`
Chimica, Laboratorio di Chimica Bioinorganica, Universita degli Studi
di Firenze, Via della Lastruccia 3, I 50019 Sesto Fiorentino (Firenze),
Italy. E-mail: mariangela.ceruso@unifi.it

2
M. Ceruso et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
cytosolic ones hCA I and II, in order to develop possibly isoform- compounds incorporating 4-aminoethyl/methyl-benzenesulfona-
selective CA IX/XII inhibitors as anticancer agents5.
mide as well as metanilamide/sulfanilamide as heads for binding
to the zinc ion from the enzyme active site (Schemes 1 and 2, and
Chart 1).
Materials and methods
The first group of compounds were obtained starting from N-
a-acetyl-L-lysine 1, followed by the protection of the "-amino
Chemistry
Anhydrous solvents and all reagents were purchased from group with the Boc moiety, and the coupling with benzenesulfo-
Aldrich, Merck or Carlo Erba. All reactions involving air- or namides 2 or 3 to give the "-Boc-a-acetyl-protected-lysyl
moisture-sensitive compounds were performed under nitrogen sulfonamides 4 and 5 (Scheme 1). After removing of the Boc
atmosphere using oven-dried glassware and syringes to transfer (tert-butyloxycarbonyl) protecting group in 3 M HCL, the hydro-
solutions. Nuclear magnetic resonance (1H-NMR and NOE- chlorides 6, 7 were treated with N,N0-di-Boc-N00-trifluoro-
difference) spectra were determined in CDCl3 and DMSO-d6 on a methane-sulfonylguanidine 8 for converting the terminal amino
Varian XL-400 (400 MHz) spectrometer (Germany). Chemical moiety to the di-Boc-protected guanidines 9 and 10. Deprotection
shifts (d scale) are reported in parts per million downfield from of the guanidine moiety in the presence of acid led to the
tetramethylsilane used as an internal standard. The assignment of guanidine-substituted sulfonamides 11 and 12 (Scheme 1).
exchangeable protons (OH and NH) was confirmed by the
The remaining derivatives were synthesized using a similar
addition of D2O. Electron ionization mass spectra (70 eV) were approach. In this case GABA 13 was used as starting material
recorded on a Hewlett-Packard 5989 Mass Engine Spectrometer following by similar derivatization reactions, compounds 14–21
(Germany). Analytical thin-layer chromatography was performed incorporating guanidine–GABA moieties were thus obtained
on Merck silica gel F-254 plates (Darmstadt, Germany). For flash (Scheme 2). Furthermore, several metanilamide (22, 23), sulf-
chromatography Merck Silica gel 60 was used with a particle size anilamide (24) and 4-carboxy-benzenesulfonamide (25) deriva-
0.040–0.063 mm (230–400 mesh ASTM). The synthesis and the tives incorporating lysyl- or GABA moieties were also obtained
full characterization of the compounds investigated here (4–25) as by following the same synthetic procedure (Chart 1).
hCA inhibitors are reported elsewhere25.
CA inhibition
CA inhibition studies
Inhibition data with the new group of sulfonamides 4–25 reported
An applied photophysics stopped-flow instrument has been used
here, against hCA isoforms hCA I, II (cytosolic) and hCA IX, XII
for assaying the CA catalyzed CO2 hydration activity26. Phenol
(transmembrane and tumor associated) are shown in Table 1.
red (at a concentration of 0.2 mM) has been used as indicator,
Therefore, the following structure–activity relationship can be
working at the absorbance maximum of 557 nm, with 20 mM
observed from data reported in mentioned table:
HEPES (pH 7.4) and 20 mM NaBF4 (for maintaining constant the
(i) The new sulfonamides reported here were generally inef-
ionic strength), following the initial rates of the CA-catalyzed
fective or medium potency inhibitors against the cytosolic
CO2 hydration reaction for a period of 10–100 s. The CO2
slow isoform hCA I, with KIs values ranging between 88.9
concentrations ranged from 1.7 to 17 mM for the determination of
and 6030 nM. However, only two sulfonamides, 14 and 23,
the kinetic parameters and inhibition constants. For each inhibitor,
both containing Boc–GABA moieties, showed to be
at least six traces of the initial 5–10% of the reaction have been
effective inhibitors against this isoform, with KIs of 19.8–
used for determining the initial velocity. The uncatalyzed rates
22.1 nM. For all substitution patterns, generally the
were determined in the same manner and subtracted from the total
4-aminoethyl-benzenesulfonamides were more effective
observed rates. Stock solutions of inhibitor (10 mM) were
hCA
I inhibitors compared with the corresponding
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 RT prior to assay, in order to allow for the formation of
the E-I complex. The inhibition constants were obtained by
nonlinear least-squares method using PRISM 3, and the Cheng–
Prusoff equation, as reported earlier27,28, and represent the mean
from at least three different determinations. All CAs were
recombinant proteins obtained as reported earlier by these
4-aminomethylbenzenesulfonamide derivates (Table 1).
(ii) The physiologically dominant human isoform hCA II was
effectively inhibited by derivatives 14 and 23–25. Indeed,
these compounds showed KIs in the range of 4.4–29.4 nM,
of the same order of magnitude as acetazolamide (AAZ; KIs
of 12 nM). These compounds again incorporate the Boc-
GABA moieties, except 25 which is a derivative of
4-carboxybenzenesulfonamide to which the derivatization
was achieved at the a-amino moiety, whereas the "-amino
one was protected by the benzyloxycarbonyl group. Some of
the derivatives reported here, such as 4, 5, 12, 15, 16, 18 and
19, were medium potency hCA II inhibitors (KIs in the range
of 44.0–91.9 nM). The remaining compounds showed to be
ineffective inhibitors against hCA II, with KIs in the range of
384–5100 nM (Table 1).
groups27,28
.
Results and discussion
Chemistry
Sulfonamides derivates are known to be highly effective CAIs1–3
.
However, their low water solubility represents one of the main (iii) The transmembrane isoform, hCA IX was poorly inhibited
weak points of this class of pharmacological agents29. This is
probably due to the fact that the highly polar sulfamoyl moiety
generally is attached to aromatic/heterocyclic rings and no water
hydrophilic moieties are present in the molecule. Therefore, in
this work we considered to add amino acyl moieties as tails to the
scaffolds of aromatic sulfonamides, in order to increase the water
solubility of such compounds30. We then used the tail approach
for our drug design employing N-a-acetyl-L-lysine or g-amino-
butyric acid (GABA) as scaffolds, and the conversion of their
terminal amino group to the guanidine one, for synthesizing
by most of the derivatives reported here. Generally the
GABA derivatives were better hCA IX inhibitors compared
with the N-a-acetyl-L-lysines possessing similar sulfona-
mide and protecting group scaffolds (Table 1). Indeed, the
compounds 4, 5, 7, 9–12 and 22 have shown high KIs values
in the range of 1373–2474 nM (except for the compound 16
with KI of 1015 nM, which contains a GABA moiety).
Although this tumor-associated isoform, hCA IX, was
slightly better inhibited by the derivatives 14, 17, 18, 20,
21 and 23 containing a GABA moiety (KIs of 517.8–

DOI: 10.3109/14756366.2014.942659
Sulfonamide CAIs act as anticancer agents
3
(i) Boc O
2
COOH
NHAc
( )n
H N
2
CONH
BocHN
SO NH
2
2
SO NH
NHAc
2
2
(ii)
1
4: n =1
5: n = 2
( )n
H N
2
2: n =1
3: n = 2
HCl
3M
( )n
CONH
H N
2
SO NH
NHAc
2
2
HCl
6: n =1
7: n = 2
BocNH
NHBoc
N
O
O
S
CF
3
8
BocNH
NBoc
( )n
H N
NH
2
CONH
NHAc
( )n
HCl
6M
HN
CONH
NHAc
HN
SO NH
2
2
SO NH
2
2
9: n =1
10: n = 2
11: n =1
12: n = 2
Scheme 1. Synthesis of N-a-acetyl-L-lysine sulfonamides 4–12.
( )n
(i) Boc O
2
COOH
CONH
H N
2
BocHN
2
SO NH
2
SO NH
2
2
13
(ii)
14: n =1
15: n = 2
( )n
H N
2
HCl
3M
2: n =1
3: n = 2
( )n
CONH
H N
2
SO NH
2
2
HCl
16: n =1
17: n = 2
BocNH
NHBoc
O
N
O
S
CF
3
8
H N
NH
2
BocNH
HN
NBoc
( )n
( )n
CONH
HCl
CONH
HN
6M
2
SO NH
2
SO NH
2
2
20: n =1
21: n = 2
18: n =1
19: n = 2
Scheme 2. Synthesis of GABA-containing sulfonamides 14–21.

4
M. Ceruso et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
Chart 1. Structure of compounds 22–25 and
the clinically used sulfonamide acetazola-
mide AAZ.
SO NH
2
2
CONH
NHAc
SO NH
2
2
CONH
BocHN
BocHN
23
22
Ph
O
O
COOMe
NH
HN
CONH
SO NH
2
2
BocHN
24
O
25
N
N
SO NH
2
2
AcNH
SO NH
2
2
S
AAZ
14, 18, 20, 24 and 25 derivatives with KIs in the range of
4.8–12.3 nM. Although many of the highly effective hCA
XII inhibitors reported here have not possessed excellent
selectivity ratios for inhibiting this transmenbrane isoform
over the cytosolic ones, the most interesting CA XII
inhibitor listed here was derivate 20 which has shown
selectivity ratios as high as 139 against hCA I and 67 against
hCA II (Table 1). The remaining derivatives 10, 15, 16, 19
and 23 have showed moderate inhibitory potency against
this isoform (KIs in the range of 26.5–69 nM).
Table 1. Inhibition data against human isoforms hCA I, II (cytosolic) and
IX, XII (transmembrane and tumor-associated) of sulfonamides 4–25
reported here and AAZ (as standard inhibitor) by a stopped-flow CO2
hydrase assay26
.
KI (nM)*
hCA IX
Compound
hCA I
hCA II
CA XII
4
5
6
7
484
430
2330
626
2310
519
3150
289
19.8
253
2270
517
193
71.2
75.9
563
473
384
452
824
44.0
9.8
91.9
83.5
197
83.1
71.3
470
847
5100
5.9
1718.2
2130
876.4
2242
2474
1373
2216
1795
517.8
65.7
1015
751.4
540.9
95.7
862.2
737.3
1825
648.6
67.5
150.7
157.7
1356
201.2
126.2
31.6
849.7
142.2
12.1
69.0
59.0
145.6
12.3
53.6
7.0
The least effective hCA XII inhibitors were derivatives 4, 5, 7,
9, 12, 17, 21 and 23, which showed KIs in the range of 126.2–
413.7 nM, being thus low potency inhibitors of this isoform
compared with the clinically used inhibitor AAZ.
9
10
11
12
14
15
16
17
18
19
20
21
22
23
24
25
AAZ
Conclusions
Two series of benzenesulfonamides incorporating amino acid
scaffolds (GABA or the N-a-acetyl-L-lysine) were explored here
as a new generation of selective tumor-associated hCA XII
inhibitors. The compounds investigated contain guanidine groups
at the terminal amino moiety which confer them higher water
solubility. Most of the new compounds were medium potency or
ineffective as inhibitors of the human CAs, hCA I, II and IX, but
they showed significant inhibition potency against the transmem-
brane isoform hCA XII, with efficacy in nanomolar range.
Therefore, considering the selectivity ratios of some of these
267
972
2120
6030
22.1
151.3
88.9
250
246.8
413.7
26.5
6.5
4.8
5.7
29.4
4.4
12
47.7
25
*Errors in the range of 10% of the reported value (from three different derivatives for inhibiting the tumor-associated hCA XII over the
assays).
physiologically dominant hCA I and II isoforms, these com-
pounds may constitute interesting tools for the development of
new anticancer agents.
862.2 nM) and derivatives 6 which contain N-a-acetyl-L-
Lysine (KI of 876.4 nM), the most efficient hCA IX
inhibitors were 15, 19 and 24, which showed inhibition
constants between 65.7 and 95.7 nM. The best activity is
Declaration of interest
C.T.S. reports conflict of interest as author of many patents on CA
inhibitors. The authors alone are responsible for the content and writing
correlated with the presence of derivatized GABA moieties
and 4-amino-ethylbenzenesulfonamide (15 and 19) and
of this article. This project was funded by a 7th FP EU grant
(METOXIA).
4-aminobenzenesulfonamide (24) head group. However,
the best hCA IX inhibitor was compound 25, (containing a
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