Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.3109/14756366.2015.1014477

New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembr

Full text of DOI:10.3109/14756366.2015.1014477

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1014477
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ORIGINAL ARTICLE
Synthesis and inhibition potency of novel ureido benzenesulfonamides
incorporating GABA as tumor-associated carbonic anhydrase IX and XII
inhibitors
Mariangela Ceruso1, Sabrina Antel1, Andrea Scozzafava1, and Claudiu T. Supuran2
1
2
Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Universita degli Studi di Firenze, Sesto Fiorentino (Firenze), Italy and Neurofarba
`
`
Department, Universita degli Studi di Firenze, Sesto Fiorentino (Florence), Italy
Abstract
Keywords
New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido
and the sulfonamide functionalities were synthesized and their inhibition potency determined
against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated
(hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The
majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I
and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmem-
brane tumor-associated isoforms hCA IX and XII, with KIs in nanomolar range. Only one
derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform
over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential
tool to develop new anticancer agents.
Anticancer agents, carbonic anhydrase
inhibitors, GABA linker, transmembrane
isoforms, ureido benzenesulfonamide
History
Received 13 January 2015
Revised 22 January 2015
Accepted 27 January 2015
Published online 20 March 2015
Introduction
the over-expression of hCA IX and XII isoforms, they are
considered as interesting druggable target for imaging and
treatment of hypoxic tumors7,15–21. Therefore, the selective
inhibition of two transmembrane isoforms CA IX and XII over
the ubiquitous and cytosolic ones, CA I/II, represents a promising
approach for the development of an efficient and low-side effect
antitumor therapy.
Sixteen human (h) a-carbonic anhydrase (CA, EC 4.2.1.1)
isoforms have been discovered till now. They belong to a large
family of metalloenzymes, which comprises six genetic families,
i.e. a, b, g, d, z and Z-classes. Carbonic anhydrases are mainly
involved in the reversible hydration reaction of carbon dioxide to
release bicarbonate and proton1–3. Among the various biological
functions, CAs are involved in pH buffering of the intra- and
extra-cellular spaces4–7. Mainly two carbonic anhydrase isoforms
hCA IX8–10 and CA XII11,12, possessing extracellular active site,
are over-expressed in most of hypoxic tumors, such as gliomas/
ependymomas, mesotheliomas, carcinomas of the bladder, cervix,
kidneys, lungs, breast, and other dysplasia resistant to classic
chemo- and radiotherapy7,13,14. Indeed, in response to hypoxia
event, such as the one occuring in the tumor area, the activated
hypoxia inducible factor-1 (HIF-1) causes an over-expression of
hCA IX6, which increases the production of protons in the district
involved. The pH in the tumor area decreases thus from the
characteristic physiological value of 7.4 possessed by the normal
tissue to about 66,7. Since the main reason of this pH imbalance is
The most studied and clinically used pharmacophores as
carbonic anhydrase inhibitor (CAI) are the sulfonamides2,4,5,17
,
which is their mechanism of action within the enzymatic cavity is
well reported with many representatives in clinical use for
decades15,16,22,23
.
However, one of the main side effects of sulfonamide CAIs,
especially those ones belonging to the first generation of such
drugs, i.e. acetazolamide AAZ, is the fact that they are not able to
discriminate different CA isoforms. The most efficient sulfona-
mide CAIs indeed inhibit the majority of the mammalian CA
isoforms, thus leading to a wide range of side effects.
Only recently it has been developed several novel generations
of sulfonamide CAIs, which posses a remarkable selectivity
profile for inhibiting prevalently the two transmembrane hCA IX
and XII isoforms17–20,24. Among such isoform-selective benze-
nesulfonamide CAIs, the ureido-containing inhibitors are very
interesting, as some representatives of this class (among which
compounds A–D) possessed nanomolar activity for the inhibition
of the tumor-associated isoforms hCA IX and XII; whereas
they were much weaker inhibitors of the widespread, cytosolic
off-target isoforms hCA I and II21. The hCA IX inhibition with
these sulfonamides in vitro, in cell cultures, and in animals with
Address for correspondence: Mariangela Ceruso, Dipartimento di
`
Chimica, Laboratorio di Chimica Bioinorganica, Universita degli
Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino
(Firenze), Italy. E-mail: mariangela.ceruso@unifi.it
`
Claudiu T. Supuran, Neurofarba Department, Universita degli Studi di
Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. E-mail:
claudiu.supuran@unifit.it

2
M. Ceruso et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
(B)
(C)
(A)
O
O
O
NH2
NH2
NH2
S
S
S
O
O
O
O
O
O
N
H
N
H
N
H
N
H
N
H
N
H
(D)
O
NH2
S
I
O
N
N
O
N
H
N
H
H3COCHN
SO2NH2
S
AAZ
Chart 1. Structure of compounds A–D and the clinically used sulfonamide acetazolamide AAZ.
transplanted tumors, leads to a return to more normal extracellular
Also, in this new series of GABA containing ureido
pH values, with consequential delay of tumor growth5,6. One of benzenesulfonamide derivatives many low nanomolar hCA IX
these sulfonamide CA IX/XII inhibitors is currently in Phase I or CA XII inhibitors have been identified25.
clinical trials for the treatment of primary tumors/metastases
overexpressing these enzymes6,21
Since, in our previous studies some ureido benzenesulfona-
mide derivatives containing GABA were found highly efficient
.
A large number of ureido-substituted benzenesulfonamide and selective CA IX/XII inhibitors and distinguished as excellent
derivatives have been extensively studied from our group potential anticancer agents, we decided to continue our interest to
showing an interesting and excellent inhibition profile against the same pathway.
the tumor-associated human carbonic anhydrase. Indeed, by
Therefore, in this paper we expanded the synthesis of the
only one-step synthesis occurring as well in high yield derivatives incorporating GABA as a linker between the ureido
between sulfonamide and stoichiometric amount of aryl/alkyl and the benzenesulfonamide moieties. The inhibition potency of
isocyanates a large number of such derivatives were prepared. these novel compounds against that over-expressed in both
Generally, the ureido containing derivatives, among which hypoxic tumors hCA IX and XII isoforms over the physiologically
compounds A–D are considered the most interesting represen- dominant, cytosolic hCA I and II ones have been determined and
tatives of the series (Chart 1), shown to possess an excellent thus compared with the efficiency obtained from similar deriva-
low nanomolar inhibition potency demonstrated by a signifi- tives previously studied25 and their smaller congeners containing
cant regression of the tumor volume in vivo experiments1,2. It sulfanilamide instead21.
has also been shown that depending on the substitution pattern
on the urea side it was possible to obtain a good inhibition Materials and methods
selectivity of the transmembrane CA isoforms over the
cytosolic ones.
Chemistry
X-ray crystallographic studies of adduct formed between Anhydrous solvents and all reagents were purchased from
hCA II enzyme with ureido benzenesulfonamides, such as A–D Sigma-Aldrich (Milan, Italy), Alfa Aesar (Milan, Italy) and
derivatives, pointed out some important features of their Merck (Darmstadt, Germany). All reactions involving air- or
inhibition mechanism. It is clear that the benzenesulfonamide moisture-sensitive compounds were performed under a nitrogen
moiety of various inhibitors belonging to this series was atmosphere using dried glassware and syringes techniques to
entirely superposable in the enzyme active site with the transfer solutions. Nuclear magnetic resonance (1H-NMR and
deprotonated sulfonamide portion (SO2NHꢀ anion) coordinated 13C-NMR) spectra were recorded using a Bruker Advance III
to the Zn (II) ion, whereas the tails (R) of the ureido moiety 400 MHz spectrometer (Flawil, Switzerland) in DMSO-d6.
adopted different orientations based on their specific chemical Chemical shifts are reported in parts per million (ppm) and
nature.
the coupling constants (J) are expressed in Hertz (Hz). Splitting
Therefore, for sulfanilamide derivatives incorporating the patterns are designated as follows: s, singlet; d, doublet; sept,
ureido linker, it has been shown that the tails attached to the septet; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet;
urea portion are located in various hydrophobic pockets of CA II brm, broad multiplet; dd, double of double; td, triplet of double;
catalytic site and interacted with different amino acid residues tt, triplet of triplet; appd, apparent double; appt, apparent triplet.
according to the chemical properties of the R moiety21.
The assignment of exchangeable protons (NH) was confirmed
Considering the inhibition profiles, the selectivity and the by the addition of D2O. Electron ionization mass spectra
mechanism of inhibition of these compounds, it appeared of great (70 eV) were recorded on Hewlett-Packard 5989 Mass
a
interest to further explore these scaffolds for obtaining more Engine Spectrometer (Milan, Italy). Analytical thin-layer chro-
potent CAIs. We extended consequently our earlier investigations matography (TLC) was carried out on Merck silica gel F-254
on ureido benzenesulfonamide CAIs synthesizing and testing the plates.
inhibition potency of a small series of derivatives containing the The synthesis and the full characterization of all compounds
same scaffold but incorporating instead also an elongation investigated here as hCA inhibitors are reported elsewhere,
between the ureido and the sulfonamide moieties, such as a derivatives 6–11 in reference 24 and 12–20 in Supplemental
GABA portion25.
information.

DOI: 10.3109/14756366.2015.1014477
Synthesis and inhibition potency of novel ureido benzenesulfonamides
3
O
Scheme 1. Preparation of sulfonamides 6–20
by reaction of 4-amino-N-(4-sulfamoyl-
benzyl)-butyramide hydrochloride (n ¼ 1)
(4) or 4-amino-N-[2-(4-sulfamoyl-phenyl)-
ethyl]-butyramide hydrochloride (n ¼ 2) (5)
with arylisocyanates A1–8 (R-NCO) in pres-
ence of diisopropylethylamine (DIPEA), in
acetonitrile (dry).
H
N
( )
(i) Boc2O
n
Boc
N
H
H2N
COOH
SO2NH2
(ii)
2: n =1
3: n = 2
SO2NH2
HCl
3M
( )
n =1
n = 2
n
H2N
O
H2N
HCl
( )
n
N
H
4: n =1
5: n = 2
SO2NH2
O
N
R
C
A1-8
O
H
N
H
N
( )
n
R
N
H
O
SO2NH2
6-20
CA inhibition studies
In previous studies carried out by our group, we have
determined the inhibition potency of some ureido sulfonamide
derivatives, such as compounds A–D, which incorporate a
sulfanilamide moiety in one side and an ureido tail in the
other21 (Chart 1). It has been demonstrated that this series of
compounds act as highly effective hCA II inhibitors with
potencies that correlate well with the orientation of the R
moiety present in the ureido tail of the compound.
Similar to the leads A–D just reported, the new compounds
obtained by the above-mentioned reactions showed as well
remarkable properties as CAIs, against the cytosolic ubiquitous
hCA I and CA II isoforms and the tumor-associated hCA IX and
CA XII ones. Indeed, many such compounds were low nanomolar
hCA IX/XII inhibitors whereas their affinity for the cytosolic off-
target isozyme I was not as good, making them moderate tumor
CA-selective inhibitors.
An Applied Photophysics stopped-flow instrument (Oxford, UK)
has been used for assaying the CA catalyzed CO2 hydration
activity26. Phenol red (at a concentration of 0.2 mM) has been
used as an indicator, working at the absorbance maximum of
557 nm, with 10 mM Hepes (pH 7.4), 10 mM Tris.HCl and 0.1 M
NaSO4 (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 mM 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 non-linear least-squares
methods using PRISM 3, whereas the kinetic parameters for the
uninhibited enzymes from Lineweaver–Burk plots, as reported
earlier27–29, and represent the mean from at least three different
determinations. All CAs were recombinant proteins obtained as
As mentioned above all hypoxic tumors overexpressed CA IX
and CA XII isoforms after activation of the hypoxia inducible
factor-1 (HIF-1)6 with a pH imbalance between the tumor tissue
and the normal tissue as main consequence6,8,14. The hCA IX/XII
isoforms inhibition with sulfonamides was recently shown to
reverse the effect of tumor acidification6,8,14, leading to inhibition
of the primary tumor and metastases growth, and CA IX/XII have
been proposed as novel therapeutic antitumor targets1,13
.
Reported here, there are novel ureido-benzenesulfonamide
CAIs obtained considering compounds A–D as leads, some of
which incorporate the same R-ureido linker, where R was phenyl,
benzyl, cyclopentyl and 4-iodophenyl21 together with a GABA
portion connecting the 4-aminomethyl or 4-aminoethylbenzene
sulfonamide and the ureido moieties.
Therefore, the present findings extend our previous stu-
dies21,25, and report here the synthesis of a series of ureido
benzenzesulfonamide derivatives-containing the same ureido
portion of such compounds or new ureido linker.
The reaction of g-Boc-GABA with 4-(aminomethyl)-benzene-
sulfonamide and 4-(2-aminoethyl)-benzenesulfonamide and con-
sequently hydrolysis of the coupling products afforded the key
intermediates as hydrochlorides 4 and 5, which were reacted with
one equivalent of various arylisocyanates, such as 4-methylben-
zyl, 4-iodiophenyl and 4-ethylphenyl isocyanates, as well as 4-
reported earlier by these groups28,29
.
Results and discussion
Chemistry
After protecting the g-amino group of GABA with tert-
butyloxycabonyl (Boc) group via well-known proced-
a
ure25,28,30,31 and consequently coupling of carboxylic group
with 4-methyl or ethyl benzenesulfonamide in the presence of
1-hydroxybenzotriazole30, the g-Boc-GABA-substituted sulfona-
mides 2 and 3 were successfully synthesized (Scheme 1). The
removal of Boc protecting group of derivatives 2 and 3 in acidic
aqueous solution and subsequently treatment of the hydrolyzed
products 4, 5 with various aryl isocyanates A1–8 (R-N¼C¼O)
(Table 1) gave the novel series of ureido benzenesulfonamides
containing a GABA moiety reported here (Scheme 1).

4
M. Ceruso et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Table 1. Isocyanates used for the preparation of compounds 6–20.
CA inhibition
Inhibition data with the new group of ureido benzensulfonamides
6–20 reported here, against the human (h) CA isoforms hCA I, II
(cytosolic) and hCA IX, XII (transmembrane and tumor
associated), as well as the derivatives A–D reported earlier (for
comparison reasons)21 are shown in Table 2. Therefore, the
following structure activity relationship (SAR) can be observed
from data reported in Table 2:
Isocyanates
R
N=C=O
A1
N=C=O
N=C=O
(i) The cytosolic and widespread isoform hCA I was moder-
ately inhibited by almost all the compounds reported here,
with inhibition constants in the range 192.7–373.4 nM.
The best hCA I inhibitors in the series were the phenyl
and the 4-nitrophenyl-ureido derivatives of 4-aminoethyl-
benzensulfonamide (7 and 11), with KIs of 32.2 and
41.8 nM, respectively. The ethylbenzene-ureido derivative
of 4-aminomethylbenzenesulfonamide (10) (KI of 52.6 nM)
was also a very effective hCA I inhibitor, and revealed an
inhibition potency 5.4-fold higher compared to its longer
congener 12 (KI of 286.1 nM). On the other hand, the
remaining substitution patterns R led to compounds with
inhibition constants of the same order of magnitude as the
clinically used drug acetazolamide, AAZ, 5-acetamido-
1,3,4-thiadiazole-2-sulfonamide (Table 2). The low inhib-
ition of this isoform may be considered as a positive
characteristic of this new series of compounds (shared also
with the corresponding 4-substituted-ureido-benzenesulfo-
namide derivatives A, C and D reported earlier)21 since the
ubiquitous isoform hCA I is abundant in red blood cells, it
can be certainly considered an important off-target when
research of antitumor CAIs is involved24.
A2
A3
N=C=O
A4
NO2
N=C=O
A5
A6
I
N=C=O
(ii) Against the physiologically dominant isoform hCA II;
derivatives 6–8, 10, 11, 19 and 20 behaved as very strong
inhibitors with KIs in the range of 4.87–6.9 nM.
N=C=O
The slightly less effective compound was the benzyl ureido
derivative of the ethylbenzensulfonamide 9 with a KI value of
41 nM, whereas the remaining ones such as 9 and 12–18, were all
quite similar and medium potency hCA II inhibitors, with
inhibition constants in a very narrow range 41.0–93.4 nM.
Although these compounds possess a rather wide range of
substitution patterns, SAR is almost impossible to define as all
substituted moieties lead to effective inhibitors of this isoform
(Table 2). Most probably, introduction of various substituents,
such as halogens, methyl, ethyl and nitro group in the aromatic
ring or cyclopenthyl ring, leads to a not significant increase in the
inhibition potency. Interestingly, the new series of ureido
benzenesulfonamides derivatives were very effective hCA II
inhibitors compared to those reported earlier A–D, showing how
an elongation of the linker chain between the ureido and the
sulfonamide moieties can largely modify the inhibition potency of
such compounds.
(iii) Several nanomolar hCA IX inhibitors are reported here,
such as 6, 7, 13, 14, 16, 17 and 20, derivatives with KIs in
the range of 42.2–53.4 nM and thus about two times higher
compared to that one of the clinically used AAZ. Although
many of the highly effective hCA IX inhibitors reported here
have not shown excellent selectivity ratios for inhibiting this
transmembrane isoform over the cytosolic ones, the most
interesting CA IX inhibitor listed here was derivative 12
which has shown selectivity ratios as high as 52.01 against
hCA I and 16.98 against hCA II (Table 1). Indeed, this
derivative resulted to be the best and the most isoform
selective CA IX inhibitor. The least effective hCA IX
inhibitors were derivatives 8, 10, 15, 18 and 19, which
showed KIs in the range of 289–370.4 nM, being thus low
potency inhibitors of this isoform compared to the clinically
A7
A8
CH3
N=C=O
CH3
nitrophenyl one (Table 1) in the presence of N-ethylisopropyla-
mine (Scheme 1). These isocyanates were firstly chosen in such a
way as to contain moieties which were shown earlier to lead to
interesting CA inhibitory compounds in the ureido benzenesulfa-
nilamide derivatives21 for comparison reason.
Reaction of the key intermediate sulfonamide hydrochlorides 4
and 5, possessing an amino group at the GABA moiety with
arylisocyanates A1–8 reported in Table 1 in the molar ratio of 1:1
in the presence of a weak base afforded the formation of a
large series of ureido benzenesulfonamide derivatives (6–20)
(Scheme 1). The equivalent amount of base used in this synthesis
was required to form the nucleophilic free base of the amine
group. Due to its low reactivity, a tertiary amine such as
diisopropylethylamine (DIPEA) is always used as a base in this
case (Scheme 1).
The characterization of new compounds by 1H- and 13C-NMR
as well as by mass spectroscopy, confirmed their chemical
structures (For further details, see Experimental Protocols
reported in reference 24 and Supplemental information).

DOI: 10.3109/14756366.2015.1014477
Synthesis and inhibition potency of novel ureido benzenesulfonamides
5
Table 2. Inhibition data of human CA isoforms hCA I, II, IX and XII with ureido-sulfonamides 6–20
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase
assay26
.
O
H2N
S
O
O
(CH2)n
NH
R
N
H
N
H
O
6-20
KI (nM)*
hCA IX
Compound
n
R
hCA I
hCA II
hCA XII
6y
1
2
1
2
1
2
2
1
2
1
2
1
2
1
2
–
–
–
–
–
C6H5
C6H5
C6H5CH2
C6H5CH2
C6H5(CH2)2
4-NO2C6H4
C6H5(CH2)2
C5H9
373.4
32.2
359
214.4
52.6
41.8
286.1
254.1
240.4
247.9
232.4
203.2
192.7
217.8
213.4
250
6.01
4.87
6.1
41
6.9
6.8
93.4
70.5
60
71.4
57.5
73.5
64.3
6.8
6.3
12
3730
2200
226
2634
46
37.4
6.2
58.3
50.7
43.5
43
10.4
10
28.5
5.4
6
4.9
6.2
6.1
5.1
5.7
67.3
49.5
7.0
7y
41.6
456.6
106.4
300.9
138.7
5.5
42.2
45.3
289
53.4
50.2
328.2
370.4
46.7
25
8y
9y
10y
11y
12
13
14
15
16
17
18
19
20
AAZ
Az
Bz
Cz
Dz
C5H9
4-C2H5Ph
4-C2H5Ph
4-CH3PhCH2
4-CH3PhCH2
4-IC6H4
4-IC6H4
–
C6H5
760
92
470
5500
575
41.4
7.3
C6H5CH2
C5H9
4-IC6H4
24.5
4.3
yFrom reference 24; zFrom reference 23; *Mean from three different assay, by a stopped flow technique
(errors were in the range of 5–10% of the reported values).
used inhibitor AAZ. The remaining derivatives 9 and 11
have showed moderate inhibitory potency against this
isoform (KIs values of 106.4 and 138.7 nM, respectively).
Although some of the compounds reported here contain same
substitution pattern at the R moiety as part of ureido sulfonamides
previously studied possessed, it is interesting to notice that their
shorter congeners containing sulfanilamide B–D are generally
about 10-fold more effective as hCA IX inhibitors compared to
the new derivatives incorporating a GABA moiety in their
structures (8 and 9 towards B; 13 and 14 corresponding to C; 19
and 20 with D). However, only derivatives 6 and 7 showed higher
inhibition potency against hCA IX isoform with KI 10 times lower
compared to their shorter congener A (with KIs of 41.6–46 nM for
compounds 6 and 7 versus 575 nM of derivatives A), revealing to
be more effective hCA IX inhibitors.
However, it is also interesting to observe that among the
ureido-derivatives incorporating a GABA moiety the ones con-
taining 4-aminoethylbenzenesulfonamide are generally stronger
hCA IX inhibitors than the shorter 4-aminomethylbenzenesulfo-
namide congeners of the same series (Table 2).
(iv) Most of the compounds reported here are shown to be very
efficient hCA XII inhibitors possessing inhibition activity at
low nanomolar range in the same order of magnitude as
AAZ (Table 2). Indeed, it is interesting to notice that this
second transmembrane tumor-associated isoform, hCA
XII, was much better inhibited by the new ureido
benzenesulfonamides investigated here, compared to the
other transmembrane tumor-associated isoform, hCA IX.
The best inhibitors were derivatives 7, 12, 13, 15–17, 19
and 20 with KI values in a narrow range between 4.9 and
10.4 nM. The remaining derivatives were slightly less
effective hCA XII inhibitors, with inhibition constants in
the range of 28.5–58.3 nM, which is about 10-fold higher
compared to that of the standard drug AAZ. However, the
elongation of the main skeleton between the ureido linker
and the benzenesulfonamide portion with a GABA moiety
did not significantly improve the inhibition potency of
these compounds compared to their smaller congeners
(such as in the case of derivatives 8 and 9 with B, 13 and
14 with C and 19, 20 with D). Indeed, benzyl ureido
derivatives 8 and 9 have shown extremely similar inhib-
ition potency against hCA XII isoform compared to their
sulfanilamide congener B, revealing KI values of 58.3 and
50.7 nM, respectively, like that one of the compound B
equal to 49.5 nM. The same occurred both for the
cyclopentyl derivatives 13 and 14, which have shown KI
values of 10 and 28.5 nM, respectively, revealing to be
slightly less effective hCA XII inhibitors than their
congener C with KI of 7 nM and for the 4-iodophenyl
derivatives 19 and 20, with inhibition constants of 6.1 and
5.1 nM of the same order of magnitude as compound D
possessing a KI value equal to 4.3 nM.

6
M. Ceruso et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
We can conclude that in this new series of compounds an
elongation of the linker between the ureido and the sulfonamide
moieties with a GABA scaffold, mostly brought an increase of the
inhibition potency against the tumor-associated target isoforms,
decreasing on the other hand their selectivity ratio against the
off-target cytosolic isoforms.
Conclusions
A novel series of ureido benzenesulfonamides incorporating
GABA scaffold and some of them containing same ureido linker
as their sulfanilamide smaller congeners, were explored here as a
new generation of selective tumor-associated hCAIX and XII
Figure 1. Graphical comparison of inhibition constants of the new
ureido-sulfonamides 6–20 reported here and the standard drug AAZ inhibitors. The compounds investigated contain a GABA portion
against hCA I, II, IX and XII.
as linker between the ureido and the benzenesulfonamide
moieties. Most of the new derivatives were medium potency
inhibitors of the ubiquitous cytosolic hCA I isoform and efficient
inhibitors against hCA II, but they showed significant inhibition
potency against both the transmembrane isoforms hCA IX and
XII, with inhibition constants in nanomolar range. The inhibition
profile of these new GABA containing ureidosulfonamides is very
different from the corresponding analogs not incorporating
GABA, which were previously investigated as inhibitors of
these enzymes.
Considering the selectivity ratios of the derivatives reported
here for inhibiting the tumor-associated hCA IX over the
physiologically dominant hCA I and II isoforms, the phenethyl
derivative is the most selective inhibitor, thus representing an
interesting tool for the development of new anticancer agents.
Declaration of interest
Figure 2. Graphical comparison of inhibition constants of the ureido-
sulfonamides A–D previously reported and the standard drug AAZ
against hCA I, II, IX and XII.
Most of the authors declare no financial interest. CTS reports conflict of
interest as author of many patents on CA inhibitors.
This project was funded by a 7th FP EU grant (METOXIA). MC and
CTS also thank the Erasmus project for a mobility grant.
On the other hand, the only phenyl ureido benzenesulfonamide
derivatives 6 and 7 containing GABA were more effective hCA References
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(v) Regarding the selectivity of the new derivatives reported
here against the two transmembrane tumor-associated hCA
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II ones, some interesting results emerged. Firstly, the
selectivity ratio for inhibiting the target isoforms over the
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