Inhibition studies of new ureido-substituted sulfonamides incorporating a GABA moiety against human carbonic anhydrase isoforms I-XIV

Article abstract of DOI:10.1016/j.bmc.2014.10.041

Reaction of γ-Boc-GABA, prepared by protecting the γ-amino moiety of the amino butyric acid with the tert-butyloxycarbonyl (Boc) protecting group, with 4-methyl/ethyl benzenesulfonamide, followed by removal of the Boc protecting group in 3 M HCl afforded the corresponding hydrochlorides, which were further derivatized by reaction with a varying of aryl isocyanates to give a new classes of ureido substituted benzenesulfonamide containing a GABA moiety. Inhibition studies of the human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, CA I-XIV with these new compounds revealed that they possess moderate-weak inhibition potency against hCA III, IV, VA, VI and XIII, rather efficient inhibitory power against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II and VII, as well as of the two tumor-associated isoforms CA IX and XII. The inhibition profile of the new ureido-substituted benzenesulfonamides reported here is thus very different from the corresponding ureido-substituted analogs incorporating sulfanilamide, which were previously investigated as inhibitors of some of these enzymes.

Full text of DOI:10.1016/j.bmc.2014.10.041

Bioorganic & Medicinal Chemistry 22 (2014) 6768–6775
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition studies of new ureido-substituted sulfonamides
incorporating a GABA moiety against human carbonic anhydrase
isoforms I–XIV
b
a
a
a,c,
Mariangela Ceruso a, , Sabrina Antel , Daniela Vullo , Andrea Scozzafava , Claudiu T. Supuran
⇑
⇑
a Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
b Gottfried Wilhelm Leibniz Universität Hannover, Welfengarten 1, D-30167 Hannover, Germany
c Università degli Studi di Firenze, Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Reaction of c-Boc-GABA, prepared by protecting the c-amino moiety of the amino butyric acid with the
Received 6 October 2014
Accepted 30 October 2014
Available online 5 November 2014
tert-butyloxycarbonyl (Boc) protecting group, with 4-methyl/ethyl benzenesulfonamide, followed by
removal of the Boc protecting group in 3 M HCl afforded the corresponding hydrochlorides, which were
further derivatized by reaction with a varying of aryl isocyanates to give a new classes of ureido substi-
tuted benzenesulfonamide containing a GABA moiety. Inhibition studies of the human carbonic anhy-
drase (CA, EC 4.2.1.1) isoforms, CA I–XIV with these new compounds revealed that they possess
moderate–weak inhibition potency against hCA III, IV, VA, VI and XIII, rather efficient inhibitory power
against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II and VII, as well
as of the two tumor-associated isoforms CA IX and XII. The inhibition profile of the new ureido-substi-
tuted benzenesulfonamides reported here is thus very different from the corresponding ureido-substi-
tuted analogs incorporating sulfanilamide, which were previously investigated as inhibitors of some of
these enzymes.
Keywords:
Carbonic anhydrase
Isoform-selective CA inhibitors
Ureido-substituted sulfonamide
GABA
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
of remarkable interest, is ubiquitous metallo-enzymes mainly cat-
alysing the reversible hydration of CO2 to HCOÀ3 and H+3
.
The discovery of isoform-selective CA inhibitors or at least
organ-specific targeting inhibitors would represent one of the most
important aims to be achieved by the research on CAs which only
recently became very intense. In order to obtain new drugs with
less undesired side effects mostly produced by the first generation
of inhibitors and for physiological studies in which specific/selec-
tive inhibitors may constitute valuable tools for understanding
the physiology/physiopathology of hCA isoforms,1,2 new class of
inhibitors containing already known effective moiety as CAs in
their structure have been designed and synthesised.
These novel types of drugs would mainly have the feature of
being isoenzyme selective inhibitors. However, due to the similar-
ity among the related isoforms of the same class of enzyme, the
selectivity of CAs is essential and difficult to achieve during the
design of either potential drugs or pharmacological tools. Indeed,
the family of carbonic anhydrases (CAs, EC 4.2.1.1), which is
involved in a variety of physiological and pathological processes
This class of enzyme is present either in prokaryotes or eukary-
otes and are encoded by six distinct evolutionarily non-related
gene families: alpha, beta, gamma, delta, zeta and eta.4 All human
CAs (hCAs) belong to the alpha-class; up to now, fifteen isozymes
have been recognized, among these only twelve are catalytically
active (CAs I–IV, CAs VA–VB, CAs VI–VII, CA IX and CAs XII–XIV),
whereas the CA-related proteins (CARPs) VIII, X and XI resulted
without any catalytic activity. The different CA isoforms widely
vary in their inhibition, kinetic properties, pattern of expression
in various tissues and cellular localization. Indeed CA I, II, III, VII
and XIII are expressed in the cytosol, CA IV, IX, XII and XIV are asso-
ciated with the cell membrane, CA VA and VB reside in mitochon-
dria, whereas CA VI is secreted in saliva and milk.
Due to the important roles of carbonic anhydrases in a variety of
physiological processes and the association of abnormal levels and/
or activities of these enzymes to different human diseases (such as
glaucoma, osteoporosis, neurological disorders, cancer, etc.), the CA
isozymes have become in the last years an interesting target for the
design of inhibitors or activators with biomedical applications.
Originally CA inhibitors (CAIs) were clinically used mainly as
diuretics, anti-glaucoma and anti-epileptics, while the novel
⇑
Corresponding authors. Tel.: +39 055 4573143 (M.C.), +39 055 4573005 (C.T.S.).
E-mail addresses: mariangela.ceruso@unifi.it (M. Ceruso), claudiu.supuran@
unifi.it (C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2014.10.041
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
6769
generation compounds are undergoing clinical investigation as
anti-obesity or anti-tumor drugs/diagnostic tools.3–10 The sulfona-
mides constitute the major, the most clinically used and most
studied family of CAIs.3,10–13 However, none of the currently CA
inhibitors in clinical use shows selectivity for a specific isozyme.
Thus, the development of isozyme-specific CA inhibitors would
be extremely helpful to discover novel classes of drugs lacking of
a range of undesired side-effects.
of a small series of new six similar ureido-substituted sulfonamide
CAIs containing this time an elongation of their main skeleton such
as a GABA moiety.
2. Results and discussion
2.1. Chemistry
Only recently a large series of 4-RNHCONH-substituted ben-
zenesulfonamide derivatives was synthesised and investigated as
inhibitors of the cytosolic isoform hCA I and II (h = human) and
the two transmembrane tumour-associated isoforms IX and XII
by our group.14
Starting from GABA, by protecting the c-amino moiety with the
tert-butyloxycarbonyl (Boc) protecting group through a procedure
already reported in literature16 to give the Boc-GABA 1, which was
consequently coupled with 4-methyl/ethyl benzenesulfonamide,
the c-Boc-GABA-substituted sulfonamides 2 and 3 were prepared
It has been reported that the inhibition potency of similar sul-
fonamides containing an ureido group changes by varying the ori-
entation of their R-ureido moieties when the inhibitor was bound
within the enzyme active site. Indeed, X-ray crystallography stud-
ies have shown a highly variable orientation of the R-ureido moie-
ties when the inhibitor was bound within the enzyme active site.14
Due to the specific chemical nature of the varying R-ureido linker,
the compounds can adopt different orientations in the X-ray crys-
tal structures. Therefore, a strong correlation has been observed
between the binding pattern, the R group orientation and the CA
II inhibitory properties for this small series of compounds, where
R was 4-fluorophenyl, pentafluorophenyl, 2-isopropylphenyl, 3-
nitrophenyl, and cyclopentyl.14
On the other hand, it has been also reported new sulfonamide
derivatives with high selective inhibition potency against some
CA isoforms over the most common and ubiquitous cytosolic
ones.15 Indeed, new sulfonamide derivatives containing amino acid
or GABA moiety resulted to be effective and selective inhibitors of
the tumor-associated carbonic anhydrase XII.16
(Scheme 1). After removal of the Boc protecting group in 3 M
HCl, the hydrochlorides 4 and 5 were treated with aryl isocyanate
A1–4 (RAN@C@O) (Scheme 1).
It has been showed that the benzenesulfonamides incorporat-
ing 4-substituted ureido moieties act as potent hCA II inhibitors
with potencies that correlate well with the orientation of the R
moiety present in the ureido tail of the compound. Since it has also
been reported earlier that other classes of ureido-substituted ben-
zenesulfonamides showed to be isoform-selective compounds,14
we considered interesting to continue and expand this investiga-
tion by synthesising new ureido-benzene sulfonamides derivatives
containing this time a GABA as main skeleton.
Therefore, we report here the synthesis of a new series of ure-
ido-substituted compounds 6–11 prepared by reaction of 4 or 5
with aryl isocyanates A1–A4 (Scheme 1). The chemical diversity
was generated by varying the nature of the starting isocyanate,
A1–A4 (Table 1).
The difference of CA isoforms is mainly represent by the chang-
ing of their amino acid sequences in the external portion of their
active sites; therefore, compounds with an R group moiety that
can strongly interact with this region will show a better selectivity
profile for the various isoforms.13
Therefore, it appeared of great interest to further explore this
combination of scaffolds for obtaining new class of CAIs. Here
we extend our earlier investigations on 4-ureido-substituted
sulfonamide and GABA containing CAIs and report the synthesis
and inhibition studies against all human (h) isoforms hCA I–XIV,
This hypothesis was confirmed by the work done earlier in our
group,12 indeed it has been reported that some ureido-substituted
O
H2N
O
S
H2N
S
O
O
CH2
NH2
O
n
O
CH2
NH
OH
n
H2N
OH
N
H
a)
O
b)
N
H
O
O
(Boc)2O,
HOBt, DIPEA, EDCI
n = 1 and 2
O
H2O/Dioxane
O
n=1
n=2
2
3
1
c)
3M HCl
O
S
O
H2N
O
H2N
S
O
CH2
NH
CH2
n
NH
O
n
R
H2N
HCl
N
H
N
H
d)
R-N=C=O
DIPEA, r.t.
O
O
n=1
n=2
4
5
6-11
Scheme 1. Preparation of sulfonamides 6–11 by reaction of 4-amino-N-(4-sulfamoyl-benzyl)-butyramide hydrochloride (n = 1) (4) or 4-amino-N-[2-(4-sulfamoyl-phenyl)-
ethyl]-butyramide hydrochloride (n = 2) (5) with isocyanates (R-NCO) in presence of diisopropylethylamine (DIPEA), in acetonitrile (dry).

6770
M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
Table 1
Our intent is thus to discover a new lead compounds which will
Isocyanates used for the preparation of compounds 6–11
show a selectivity against hCA isoforms. The new compounds were
characterized by 1H and 13C NMR as well as by mass spectroscopy,
confirming their structures (see Section 4 for details).
Isocyanates
R
N=C=O
A1
2.2. CA inhibition
The inhibition studies of all mammalian CA isoforms with the
new ureido-substituted benzenesulfonamides derivatives 6–11,
and the clinically used, standard CAIs such as acetazolamide
(AAZ)17 have been hereby reported. The carbonic anhydrase iso-
forms included in the study were cytosolic (hCA I, II, III, VII and
XIII), membrane-bound (CA IV) or transmembrane ones (hCA IX,
XII and XIV), mitochondrial (CA VA) and secreted (CA VI) and all
of them are involved in a host of important physiological and path-
ological functions in vertebrates.3–10
N=C=O
A2
A3
N=C=O
The following structure activity relationship (SAR) has been
obtained from data of Table 2, showing the inhibition of the CA iso-
forms with the new group of ureido-substituted benzenesulfon-
amide 6–11:
N=C=O
A4
(i) The slow cytosolic isoform hCA I was moderately inhibited
by compounds 6, 8 and 9, with inhibition constants in the
range of 214–373 nM (comparable to those of the clinically
used drug acetazolamide, AAZ, 5-acetamido-1,3,4-thiadia-
zole-2-sulfonamide). The best hCA I inhibitors were the
phenyl and 4-nitro substituted phenyl ureido derivatives of
4-aminoethylbenzenesulfonamide (7 and 11), with KIs of
32–41.8 nM, as well as the ethylbenzene derivative of
4-aminomethylbenzenesulfonamide (10) (KI of 52.6 nM).
Although the compounds reported here contain a similar
substitution pattern at the R moiety, it is interesting to
notice that the phenyl 4-aminoethylbenzenesulfonamide
derivate 7 was about 10-fold more effective as an hCA I
inhibitor compared to its shorter congener 6.
(ii) Against the physiologically dominant isoform hCA II;
derivatives 6–11 behaved as very strong inhibitors with KIs
in the range of 4.9–41 nM. The slightly less effective
compound was the benzyl ureido derivative of the ethylben-
zensulfonamide 9 with a KI value of 41 nM, whereas the
remaining ones were all quite similar and highly effective
hCA II inhibitors, with inhibition constants in a very narrow
NO2
sulfonamides show selective inhibition of isoform hCA I over the
dominant one hCA II. Indeed, it has been shown that the benzene-
sulfonamide moiety of these 4-ureido substituted derivatives binds
in the usual manner, coordinating to the zinc ion as SO2NH– group.
Indeed, the phenylsulfamoyl moiety of the inhibitor was highly
imposable between all these adducts occupying the middle of the
active site cavity and binding in the usual way.3,14
However, the different R moieties present in this series of com-
pounds were showed to occupy various subpockets of the enzyme
active site when bound to the external part of the enzyme, and
none of them was imposable with each other. Due to the varying
flexible nature of the R ureido linker connecting the benzenesul-
fonamide of these compounds, a selective inhibition of hCA I iso-
form over the dominant one hCA II was thus demonstrated.
Normally, it is hCA II having higher affinity for sulfonamides com-
pared to hCA I, at least for most of the clinically used derivatives.1–3
Table 2
Inhibition data of human CA isoforms hCA I–XIV with sulfonamides 6–11 reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2
hydrase assay17
O
H2N
O
S
O
(CH2)n
NH
R
N
H
N
H
O
6-11
N°
n
R
KI (nM)*
hCA VI
hCA I
hCA II
hCA III
hCA IV
hCA VA
hCA VII
hCA IX
hCA XII
hCA XIII
hCA XIV
6
7
8
9
10
11
AAZa
1
2
1
2
1
2
—
C6H5
C6H5
373.4
32.2
359
214.4
52.6
41.8
250
6.0
4.9
6.1
41
6.9
6.8
12
>21000
>21000
>21000
>21000
>21000
>21000
20000
233.19
438.95
879.80
779.70
397.89
737.69
74
156.1
346.7
329.8
359.4
141.7
123.8
63
>120
>120
>120
>120
>120
>120
11
50.6
24.0
253.5
28.7
21.9
17.7
2.5
46
37.4
6.2
58.3
50.7
43.5
43
>240
>240
>240
>240
>240
>240
18
238.6
65.8
344.0
70.9
48.2
59.0
41
41.6
456.6
106.4
300.9
138.7
25
C6H5CH2
C6H5CH2
C6H5(CH2)2
4-NO2C6H4
—
5.7
a
From Ref. 1.
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).

M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
6771
range (4.9–6.9 nM). Therefore, SAR is almost impossible to
define as all substitution patterns lead to highly effective
inhibitors of this isoform (Table 2).
acts as a potent inhibitor against hCA XII). Indeed, this tumor asso-
ciated isoform was strongly inhibited by the phenyl ureido-deriv-
ative of the 4-aminoethylbenzenesulfonamide
7 with KI of
(iii) Isoforms hCA III and IV were the ones which were less inhib-
ited by the compounds investigated here. Most probably,
due to the presence of a bulky side chain such as Phe198
in the centre of the active site of this isoform, it is well
known18 that hCA III is relatively insensitive to inhibition
by bulky sulfonamides. Indeed, the residue of phenylalanine
inside the active side of the enzyme may lead to steric
clashes with the scaffold of the molecules. This hypothesis
is also confirmed by the data reported in Table 2, as all the
ureido substituted benzenesulfonamides were inefficient
6.2 nM, although its activity is in the same range as the clinically
used inhibitor AAZ.
(viii) hCA XIII, a cytosolic isoform like hCA I, II, III and VII; was
poorly inhibited by the sulfonamides investigated here. All
the compounds have shown KIs >240 nM, they were about
ten times less active than the standard clinically used drug
acetazolamide (AAZ) (Table 2).
(ix) The last transmembrane isoform, hCA XIV was moderately
inhibited by compounds 6 and 8 (both of them incorporating
methylbenzenesulfonamide moiety), with KIs of 238.6–
344 nM, and better inhibited by the remaining 4 derivatives
(KIs of 48.2–65.9 nM). The best hCA XIV inhibitor was the
ethylbenzene ureido derivative of 4-aminomethylbenzene-
sulfonamide 10 (KI of 48.2 nM), which has anyhow a similar
inhibition activity of acetazolamide.
inhibitors (KI >21 lM). However, it is also interesting to
notice how acetazolamide is also a very weak hCA III inhib-
itor (Table 2) with KI in the same micromolar range.
The membrane-bound isoform hCA IV was also poorly inhibited
by all compounds 6–11 reported here, with KIs in the range 233.2–
879.8 nM which were anyhow about 10 times much weaker com-
pared to AAZ (KI of 74 nM)
3. Conclusions
(iv) The mitochondrial isoform hCA VA was moderate inhibited
by half of the compounds (6, 10 and 11) with KIs values in
the range 123.8–156.1 nM and thus about 2 fold higher than
the standard used drug AAZ, whereas the derivatives 7–9
were ineffective inhibitors with KIs in the range of 329.8–
359.4 nM.
A small series of ureido substituted benzenesulfonamides con-
taining a GABA moiety was synthesized, characterized and investi-
gated for the inhibition of the 13 hCA earlier isolated and cloned
isoforms from different groups, in particular the physiologically
relevant isoforms hCA I and II, and the other cytosolic isoforms
hCA III and XIII, as well as the two tumour-associated isoforms
hCA IX and XII, with some other hCA isoforms involved in various
pathologies, such as obesity (CA VA), epilepsy (CA VII and XIV).
Therefore, the inhibition studies of all the human carbonic
anhydrase (CA, EC 4.2.1.1) isoforms, CA I–XIV with these new com-
pounds revealed that they possess moderate–weak inhibition
potency against hCA III, IV, VA, VI and XIII, rather efficient inhibi-
tory power against hCA I, VI, and IX, and excellent inhibition of
the physiologically relevant hCA I, II and VII, as well as of the
two tumor-associated isoforms CA IX and XII. The inhibition profile
of the new ureido-substituted benzenesulfonamides reported here
is thus very different from the corresponding ureido-substituted
analogs incorporating sulfanilamide, which were previously inves-
tigated as inhibitors of some of these enzymes. Some of these new
compounds may thus represent interesting candidates for the
development of novel antiglaucoma, edema, altitude sickness or
antiepileptic drugs. Since the two transmembrane isoforms hCA
IX and hCA XII were also strongly inhibited by the compounds
reported here with KI in low nanomolar range, this new class of
ureido-substituted sulfonamide hCAIs may be a novel and interest-
ing lead compounds for the development of more potent hypoxia
induced cancer drug therapy.
(v) The secreted isoform (in saliva, milk and tears) hCA VI was
not inhibited significantly by all the sulfonamides reported
here with KIs over 120 mM. Therefore, the SAR is not possi-
ble to be observed as all the series of derivates have shown
the inhibition constants in the same range (Table 2).
(vi) Generally, the sulfonamides investigated here behaved as
moderate potency inhibitors (KIs in the range of 17.7–
50.6 nM) against the cytosolic isoform hCA VII. The best
hCA VII inhibitor was the nitro derivative 11 with KI of
17.7 nM which was anyhow much weaker compared to
AAZ (KIs of 2.5 nM).However, the least effective inhibitor
was the benzyl ureido derivative of the 4-methylbenzene-
sulfonamide 8 (KI of 253.5 nM) whereas for the remaining
compounds 6, 7, 9 and 10 the SAR is again rather flat as most
of them have the inhibition constants in a narrow range
(21.9–50.6 nM). Considering the important difference of
activity between the congeners 8 and 9 (which differ only
by an extra CH2 group in 9) as hCA VII inhibitors—by a factor
of about 10—it is obvious that very small structural differ-
ences in the scaffold of the inhibitor lead to major differ-
ences of activity.
(vii) Although the most efficient two hCA IX inhibitors were the
only phenyl ureido benzenesulfonamide derivatives 6 and
7, they showed to possess inhibition activity at nanomolar
range in the same order of magnitude of AAZ. The remaining
derivatives 8–11 incorporating bulkier substituents at ure-
ido moiety were less effective hCA IX inhibitors, with inhibi-
tion constants in the range of 106.4–456.6 nM. However, It is
also interesting to notice that generally the ureido-deriva-
tives containing 4-aminoethylbenzenesulfonamide are
higher effective hCAIX inhibitors than the shorter 4-amino-
methylbenzenesulfonamide congeners (Table 2).
4. Experimental protocols
4.1. Chemistry
Anhydrous solvents and all reagents were purchased from
Sigma–Aldrich, Alfa Aesar and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to
transfer solutions. Nuclear magnetic resonance (1H NMR, 13C
NMR, DEPT-135, DEPT-90, and HSQC) spectra were recorded using
a Bruker Avance III 400 MHz spectrometer in DMSO-d6. Chemical
shifts are reported in parts per million (ppm) and the coupling con-
stants (J) are expressed in Hertz (Hz). Splitting patterns are desig-
nated as follows: s, singlet; d, doublet; sept, septet; t, triplet; q,
However, the second transmembrane isoform associated with
tumors, hCA XII, was much better inhibited by the new ureido-
substituted benzenesulfonamides investigated here, compared to
hCA IX. All derivatives showed KIs in a narrow range between
37.4–58.3 nM against this isoform (except for derivative 7 which

6772
M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
quadruplet; m, multiplet; br s, broad singlet; br m, broad multi-
plet; dd, double of double; td, triplet of double; tt, triplet of triplet;
appd, apparent double; appt, apparent triplet. The assignment of
exchangeable protons (NH) was confirmed by the addition of
D2O. Electron ionization mass spectra (70 eV) were recorded on a
Hewlett–Packard 5989 Mass Engine Spectrometer. Analytical
thin-layer chromatography (TLC) was carried out on Merck silica
gel F-254 plates.
stirring for 65 h at room temperature. At the end of the reaction
time the solution was acidified to pH 2 using a 3.0 M HCl solution
and extracted with ethyl acetate. The combined organic layers
were washed with 10% Citric acid, NaHCO3 saturated solution,
brine and then dried over anhydrous sodium sulphate, filtered,
and the solvent was removed under reduced pressure to give
1.83 g of the pure product as white crystals.
tert-Butyl-(4-oxo-4((4-sulfamoylbenzyl)amino)butyl)carbamate
2: 75% yield; dH (400 MHz, DMSO-d6): 8.43 (t, 1H, NH, J 6.0,
exchange with D2O), 7.80 (d, 2H, CH, J 8.4), 7.45 (d, 2H, CH, J 8.4),
4.1.1. Synthesis of 4-tert-butoxycarbonylamino-butyric acid 1
O
O
O
pH = 11
H2N
COOH +
O
O
O
O
N
H
1
COOH
1,4-dioxane/H2O (50:50)
A solution
c
-amino butyric acid (1 g, 1.0 equiv) in a 1,4-dioxane/
7.33 (br s, 2H, NH2, exchange with D2O), 6.85 (br t, 1H, NH, J 5.2,
exchange with D2O), 4.35 (d, 2H, CH2, J 6.0), 2.95 (td, 2H, CH2, J
6.5), 2.18 (t, 2H, CH2, J 7.4), 1.66 (tt, 2H, CH2, J 7.3), 1.42 (s, 9H,
3CH3); dC (100 MHz, DMSO-d6): 172.87 (C@O), 156.51 (C@O),
144.73 (qC aromatic), 143.46 (qC aromatic), 128.34 (2 CH aro-
matic), 126.58 (2 CH aromatic), 78.35 (C-Boc), 42.58 (CH2), 33.64
(CH2), 29.19 (CH3-Boc), 26.67 (CH2); m/z (ESI negative), 370.4
[MÀH]À.
water (50:50) mixture (75 ml) was adjusted to pH 11 with 1.0 M
NaOH aqueous solution and cooled down to À10 °C in a salt ice
bath. Di-tert-butyl dicarbonate (2.1 g, 1.0 equiv) was then added
and the pH maintained at 11 if necessary. The reaction mixture
was then stirred overnight at room temperature. At the end of the
reaction time the solution was acidified to pH 1 using a 3.0 M HCl
solution. The solution was then extracted with ethyl acetate. The
combined organic layers were dried over anhydrous sodium sul-
phate, filtered, and the solvent was then removed under reduced
pressure to give 1.44 g of the pure product as colourless oil.
4.1.3. Synthesis of [3-[2-(4-Sulfamoyl-phenyl)-ethylcarbamoyl]-
propyl]-carbamic acid tert-butyl ester 3
H2NO2S
O
O
H
N
THF, HOBt, EDCI
+
O
N
H
COOH
DIPEA, r.t.
O
N
H
O
3
SO2NH2
1
NH2
4-tert-Butoxycarbonylamino-butyric acid 1: 73% yield; dH
(400 MHz, DMSO-d6): 6.84 (br t, 1H, NH, exchange with D2O),
2.95 (td, 2H, CH2, J 6.4), 2.23 (t, 2H, CH2, J 7.2), 1.62 (tt, 2H, CH2, J
7.2), 1.41 (s, 9H, 3CH3); dC (100 MHz, DMSO-d6): 175.14 (COOH),
156.55 (C@O), 78.36 (C-Boc), 40.0 (CH2), 31.9 (CH2), 29.17 (CH3-
Boc), 25.85 (CH2); m/z (ESI negative), 202.2 [MÀH]À.
4-tert-Butoxycarbonylamino-butyric acid 1 (1.44 g, 1.0 equiv)
was suspended in THF (70 ml) and left stirring at room tempera-
ture. 4-(2-Aminoethyl)benzenesulfonamide (1.42 g, 1.0 equiv)
and 1-hydroxybenzotriazole (0.96 g, 1.0 equiv) were then added
at the same temperature followed by the addition dropwise of
diisopropylethylamine (2.43 ml, 2.0 equiv) and dimethylformam-
ide (2 ml) The reaction mixture was then cooled down to 0 °C in
an ice bath. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (1.50 g, 1.1 equiv) was then added and the reaction
4.1.2. Synthesis of tert-butyl-(4-oxo-4((4-sulfamoylbenzyl)-
amino)butyl)carbamate 2
H2NO2S
O
SO2NH2
O
H
N
DCM, HOBt, EDCI
DIPEA, r.t.
.
+
HCl
O
N
H
COOH
O
N
H
O
NH2
1
2
4-tert-Butoxycarbonylamino-butyric acid 1 (1.59 g, 1.0 equiv)
was suspended in dichloromethane (50 ml) and left stirring at
room temperature. 4-Aminomethylbenzenesulfonamide hydro-
chloride (1.74 g, 1.0 equiv) and 1-hydroxybenzotriazole (1.05 g,
1.0 equiv) were then added to the reaction mixture at the same
temperature followed by the additions dropwise of diisopropyleth-
ylamine (4.00 ml, 3.0 equiv) and dimethylformamide (9 ml). The
reaction mixture was then cooled down to 0 °C in an ice bath
and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochlo-
ride (1.65 g, 1.1 equiv) was added to the reaction mixture and left
mixture was stirred for 65 h at room temperature. At the end of the
reaction time the solution was acidified to pH 2 using a 3.0 M HCl
aqueous solution and then extracted with ethyl acetate. The com-
bined organic layers were washed with 10% Citric acid, NaHCO3
saturated solution, brine and then dried over anhydrous sodium
sulphate, filtered, and the solvent was removed under reduced
pressure to give 2.46 g of the pure product as a light brown solid.
[3-[2-(4-Sulfamoyl-phenyl)-ethylcarbamoyl]-propyl]-carbamic
acid tert-butyl ester 3: 91% yield; dH (400 MHz, DMSO-d6): 7.93 (br
t, 1H, NH, J 5.4, exchange with D2O), 7.77 (d, 2H, CH, J 8.4), 7.42 (d,

M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
6773
2H, CH, J 8.4), 7.32 (s, 2H, NH2, exchange with D2O), 6.82 (br t, 1H,
NH, exchange with D2O), 3.32 (td, 2H, CH2, J 6.4), 2.91 (td, 2H, CH2, J
6.4), 2.81 (t, 2H, CH2, J 7.0), 2.06 (t, 2H, CH2, J 7.6), 1.60 (tt, 2H, CH2, J
7.2), 1.41 (s, 9H, 3CH3); dC (100 MHz, DMSO-d6): 172.69 (C@O),
156.49 (C@O), 144.68 (qC aromatic), 142.94 (qC aromatic), 130.00
(2 CH aromatic), 126.58 (2 CH aromatic), 78.35 (C-Boc), 40.67
(CH2), 40.50 (CH2), 35.79 (CH2), 33.77 (CH2), 29.18 (CH3-Boc),
26.69 (CH2); m/z (ESI negative), 384.6 [MÀH]À.
4.2. General procedure for the preparation of compounds 6–11
The 4-amino-N-(4-sulfamoyl-benzyl)-butyramide hydrochlo-
ride 4 or the 4-amino-N-[2-(4-sulfamoyl-phenyl)-ethyl]-butyra-
mide hydrochloride
5 (1.0 equiv) were suspended in dry
acetonitrile (10 ml) and then treated with stoichiometric amount
of isocyanate A1–4 under anhydrous conditions followed by
dropwise addition of diisopropylethylamine (1.05 equiv). The
mixture was stirred at room temperature for 3 h or overnight
under nitrogen flow until completion (TLC monitoring). At the
end of the reaction time the mixture was quenched with slush
4.1.4. Synthesis of 4-amino-N-(4-sulfamoyl-benzyl)-butyramide
hydrochloride 4
SO2NH2
SO2NH2
O
H
H
N
N
3M HCl
H2N
HCl
O
N
H
o.n.
room temperature
O
O
4
2
A solution of tert-butyl-(4-oxo-4((4-sulfamoylbenzyl)amino)butyl)
carbamate 2 (0.54 g, 1.0 equiv) and 3.0 M HCl (15 ml) was stirred
overnight at room temperature. The solution was then dried under
reduced pressure to give a yellowish oil. The oil was then washed
with dichloromethane (twice) and diethylether (twice). After stir-
ring overnight in diethylether the solvent was then removed by
decantation and the powder was dried under reduced pressure to
give 0.49 g of the pure product as a white solid.
(2–3 g) and 3.0 M HCl (5 ml). The formed precipitate was then
filtered off and dried in vacuo or extracted with ethyl acetate,
the combined organic layers dried over anhydrous sodium sul-
phate, filtered and the solvent removed under reduced pressure.
Further purification such as crystallization from water and
washes with diethylether were required to obtain pure products.
All compounds described here were characterised by spectro-
scopic methods.
4-Amino-N-(4-sulfamoyl-benzyl)-butyramide hydrochloride 4:
quantitative yield; dH (400 MHz, DMSO-d6): 8.68 (br t, 1H, NH, J
5.8, exchange with D2O), 8.06 (br s, 3H, NH+3, exchange with
D2O), 7.80 (d, 2H, CH, J 8.4), 7.46 (d, 2H, CH, J 8.4), 7.37 (br s, 2H,
NH2, exchange with D2O), 4.36 (d, 2H, CH2, J 6.0), 2.81 (td, 2H,
CH2, J 6.8), 2.32 (t, 2H, CH2, J 7.2), 1.85 (tt, 2H, CH2, J 7.4); dC
(100 MHz, DMSO-d6): 172.35 (C@O), 144.57 (qC aromatic),
143.53 (qC aromatic), 128.40 (2 CH aromatic), 126.58 (2 CH aro-
matic), 42.62 (CH2), 39.27 (CH2), 32.92 (CH2), 24.08 (CH2); m/z
(ESI positive), 272.3 [MÀCl]+.
O
O
H2N
H2N
S
S
O
O
CH2
NH
CH2
NH
O
n
n
A1-4
R
R-N=C=O
H2N
N
H
N
H
.
MeCN dry
DIPEA, r.t.
HCl
O
O
4
5
n=1
n=2
6-11
4.1.5. Synthesis of 4-amino-N-[2-(4-sulfamoyl-phenyl)-ethyl]-
butyramide hydrochloride 5
O
H
N
H
N
3M HCl
r.t., o.n.
O
N
H
H2N
HCl
O
O
SO2NH2
SO2NH2
5
3
A solution of [3-[2-(4-sulfamoyl-phenyl)-ethylcarbamoyl]-pro-
pyl]-carbamic acid tert-butyl ester 3 (0.5 g, 1.0 equiv) and 3.0 M
HCl (10 ml) was stirred overnight at room temperature. The solu-
tion was then dried under reduced pressure to give a brownish
oil. The oil was then washed with dichloromethane (twice) and
diethylether (three times). After stirring overnight in diethylether
the solvent was removed by decantation and the powder was dried
under reduced pressure to give 0.38 g of the pure product as a light
brown solid.
4-Amino-N-[2-(4-sulfamoyl-phenyl)-ethyl]-butyramide hydrochloride
5: 93% yield; dH (400 MHz, DMSO-d6): 8.14 (br t, 1H, NH, J 5.6,
exchange with D2O), 8.00 (br s, 3H, NH+3, exchange with D2O),
7.78 (d, 2H, CH, J 8.4), 7.43 (d, 2H, CH, J 8.4), 7.35 (br s, 2H, NH2,
exchange with D2O), 3.34 (td, 2H, CH2, J 6.5), 2.83 (t, 2H, CH2, J
7.2), 2.77 (m, 2H, CH2), 2.20 (t, 2H, CH2, J 7.4), 1.79 (tt, 2H, CH2, J
7.4); dC (100 MHz, DMSO-d6): 172.12 (C@O), 144.59 (qC aromatic),
142.97 (qC aromatic), 130.01 (2 CH aromatic), 126.59 (2 CH aro-
matic), 40.62 (CH2), 39.34 (CH2), 35.69 (CH2), 32.97 (CH2), 24.07
(CH2); m/z (ESI positive), 286.5 [M-Cl]+.
4.2.1. Synthesis of 4-(3-phenylureido)-N-(4-sulfamoylbenzyl)-
butanamide 6
SO2NH2
O
H
N
N
H
N
H
O
6
4-(3-Phenylureido)-N-(4-sulfamoylbenzyl)butanamide 6: white
solid; 70% yield; dH (2 atropisomers were detected in 1/0.21 ratio.
Only the major one is reported herein) (400 MHz, DMSO-d6): 8.48
(t, 1H, NH, J 5.8, exchange with D2O), 8.45 (s, 1H, NH, exchange
with D2O), 7.80 (d, 2H, CH, J 8.4), 7.45 (d, 2H, CH, J 8.4), 7.42 (dd, 2H,
CH, J 7.6), 7.34 (s, 2H, NH2, exchange with D2O), 7.24 (d, 2H, CH, J
8.0), 6.91 (dd, H, CH, J 7.2), 6.18 (t, 1H, NH, J 5.6, exchange with
D2O), 4.36 (d, 2H, CH2, J 6.0), 3.13 (td, 2H, CH2, J 6.4), 2.23 (t, 2H,

6774
M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
CH2, J 7.4), 1.73 (tt, 2H, CH2, J 7.2); dC (100 MHz, DMSO-d6): 172.91
(C@O), 156.17 (C@O), 144.73 (qC aromatic), 143.47 (qC aromatic),
141.48 (qC aromatic), 129.52 (2 CH aromatic), 128.37 (2 CH aro-
matic), 126.59 (2 CH aromatic), 121.85 (1 CH aromatic), 118.54
(2 CH aromatic), 42.62 (CH2), 39.69 (CH2), 33.67 (CH2), 27.03
(CH2), m/z (ESI negative), 389.3 [MÀH]À.
4-(3-Benzylureido)-N-(4-sulfamoylphenethyl)butanamide
9:
white solid; 66% yield; dH (2 atropisomers were detected in 1/
0.21 ratio. Only the major one is reported herein) (400 MHz,
DMSO-d6): 7.97 (t, 1H, NH, J 5.4 exchange with D2O), 7.77 (d, 2H,
CH, J 8.4), 7.42 (d, 4H, CH, J 8.4), 7.33 (s, 2H, NH2, exchange with
D2O), 7.27 (m, 5H, CH), 6.34 (t, 1H, NH, J 6.0, exchange with
D2O), 5.98 (t, 1H, NH, J 5.6, exchange with D2O), 4.23 (d, 2H, CH2,
J 6.4), 3.32 (td, 2H, CH2, J 6.6), 3.01 (td, 2H, CH2, J 6.5), 2.81 (t,
2H, CH2, J 7.2), 2.08 (t, 2H, CH2, J 7.6), 1.61 (tt, 2H, CH2, J 7.2); dC
(100 MHz, DMSO-d6): 172.78 (C@O), 159.01 (C@O), 144.68 (qC aro-
matic), 142.91 (qC aromatic), 141.86 (qC aromatic), 130.00 (2 CH
aromatic), 129.09 (2 CH aromatic), 127.90 (2 CH aromatic),
127.42 (2 CH aromatic), 126.56 (1 CH aromatic), 43.81 (CH2),
40.75 (CH2), 39.97 (CH2), 35.78 (CH2), 33.81 (CH2), 27.27 (CH2);
m/z (ESI negative), 417.3 [MÀH]À.
4.2.2. Synthesis of 4-(3-phenylureido)-N-(4-sulfamoylphenethyl)-
butanamide 7
O
H
N
N
H
N
H
O
SO2NH2
4.2.5. Synthesis of 4-(3-phenethylureido)-N-(4-sulfamoylbenzyl)-
7
butanamide 10
4-(3-Phenylureido)-N-(4-sulfamoylphenethyl)butanamide 7: yel-
low solid; 37% yield; dH (400 MHz, DMSO-d6): 8.49 (s, 1H, NH,
exchange with D2O), 7.98 (t, 1H, NH, J 5.4 exchange with D2O),
7.78 (d, 2H, CH, J 8.4), 7.43 (d, 2H, CH, J 8.4), 7.42 (d, 2H, CH, J
8.4), 7.32 (s, 2H, NH2, exchange with D2O), 7.24 (dd, 2H, CH, J
8.0), 6.91 (dd, 1H, CH, J 7.2), 6.20 (t, 1H, NH, J 5.6, exchange with
D2O), 3.33 (td, 2H, CH2, J 6.6), 3.09 (td, 2H, CH2, J 6.4), 2.82 (t, 2H,
CH2, J 7.2), 2.12 (t, 2H, CH2, J 7.4), 1.67 (tt, 2H, CH2, J 7.2); dC
(100 MHz, DMSO-d6): 172.72 (C@O), 156.18 (C@O), 144.69 (qC aro-
matic), 142.93 (qC aromatic), 141.50 (qC aromatic), 130.01 (2 CH
aromatic), 129.52 (2 CH aromatic), 126.58 (2 CH aromatic),
121.82 (1 CH aromatic), 118.50 (2 CH aromatic), 40.67 (CH2),
39.63 (CH2), 35.79 (CH2), 33.79 (CH2), 27.03 (CH2); m/z (ESI posi-
tive), 427.2 [M+Na]+.
SO2NH2
O
H
N
N
H
N
H
O
10
4-(3-Phenethylureido)-N-(4-sulfamoylbenzyl)butanamide
10:
white solid; 58% yield; dH (400 MHz, DMSO-d6): 8.47 (t, 1H, NH, J
5.8, exchange with D2O), 7.80 (d, 2H, CH, J 8.4), 7.45 (d, 2H, CH, J
8.0), 7.34 (s, 2H, NH2, exchange with D2O), 7.31 (d, 2H, CH, J 7.2),
7.23 (m, 3H, CH), 5.95 (t, 1H, NH, J 5.6 exchange with D2O), 5.84
(t, 1H, NH, J 5.6, exchange with D2O), 4.35 (d, 2H, CH2, J 6.0), 3.25
(td, 2H, CH2, J 6.4), 3.01 (td, 2H, CH2, J 6.5), 2.71 (t, 2H, CH2, J
7.2), 2.18 (t, 2H, CH2, J 7.6), 1.65 (tt, 2H, CH2, J 7.2); dC (100 MHz,
DMSO-d6): 173.14 (C@O), 159.05 (C@O), 144.79 (qC aromatic),
143.47 (qC aromatic), 140.70 (qC aromatic), 129.61 (2 CH aro-
matic), 129.26 (2 CH aromatic), 128.40 (2 CH aromatic), 126.63
(1 CH aromatic), 126.35 (2 CH aromatic), 42.64 (CH2), 41.93
(CH2), 39.86 (CH2), 37.12 (CH2), 33.78 (CH2), 27.32 (CH2); m/z (ESI
negative), 417.3 [MÀH]À.
4.2.3. Synthesis of 4-(3-benzylureido)-N-(4-sulfamoylbenzyl)-
butanamide 8
SO2NH2
O
H
N
N
H
N
H
O
4.2.6. Synthesis of 4-(3-(4-nitrophenyl)ureido)-N-(4-sulfamoyl-
phenethyl)butanamide 11
8
4-(3-Benzylureido)-N-(4-sulfamoylbenzyl)butanamide 8: white
solid; 56% yield; dH (400 MHz, DMSO-d6): 8.47 (br t, 1H, NH,
exchange with D2O), 7.80 (d, 2H, CH, J 8.0), 7.44 (d, 2H, CH, J 8.0),
7.34 (s, 2H, NH2, exchange with D2O), 7.28 (m, 5H, CH), 6.34 (br
t, 1H, NH, exchange with D2O), 6.00 (br t, 1H, NH, exchange with
D2O), 4.35 (d, 2H, CH2, J 5.6), 4.23 (d, 2H, CH2, J 5.2), 3.05 (td, 2H,
CH2), 2.19 (t, 2H, CH2, J 7.2), 1.67 (tt, 2H, CH2); dC (100 MHz,
DMSO-d6): 173.19 (C@O), 159.38 (C@O), 144.97 (qC aromatic),
143.50 (qC aromatic), 141.90 (qC aromatic), 129.12 (2 CH aro-
matic), 128.36 (2 CH aromatic), 127.92 (2 CH aromatic), 127.44
(1 CH aromatic), 126.60 (2 CH aromatic), 43.83 (CH2), 42.60
(CH2), 39.96 (CH2), 33.74 (CH2), 27.31 (CH2); m/z (ESI negative),
403.3 [MÀH]À.
O2N
O
H
N
N
H
N
H
O
SO2NH2
11
4-(3-(4-Nitrophenyl)ureido)-N-(4-sulfamoylphenethyl)butana-
mide 11: yellow solid; 36% yield; dH (400 MHz, DMSO-d6): 9.32
(s, 1H, NH, exchange with D2O), 8.17 (d, 2H, CH, J 9.2), 7.99 (t,
1H, NH, J 5.6, exchange with D2O), 7.77 (d, 2H, CH, J 8.0), 7.66
(d, 2H, CH, J 9.2), 7.42 (d, 2H, CH, J 8.0), 7.33 (s, 2H, NH2,
exchange with D2O), 6.50 (t, 1H, NH, J 5.6, exchange with
D2O), 3.32 (td, 2H, CH2, J 6.4), 3.13 (td, 2H, CH2, J 6.5), 2.81 (t,
2H, CH2, J 7.2), 2.12 (t, 2H, CH2, J 7.6), 1.70 (tt, 2H, CH2, J 7.2);
dC (100 MHz, DMSO-d6): 172.67 (C@O), 155.36 (C@O), 148.17
(qC aromatic), 144.67 (qC aromatic), 142.93 (qC aromatic),
141.26 (qC aromatic), 130.01 (2 CH aromatic), 126.57 (2 CH aro-
matic), 126.05 (2 CH aromatic), 117.71 (2 CH aromatic), 40.64
(CH2), 39.71 (CH2), 35.77 (CH2), 33.71 (CH2), 26.72 (CH2); m/z
(ESI negative), 448.3 [MÀH]À.
4.2.4. Synthesis of 4-(3-benzylureido)-N-(4-sulfamoylphenethyl)-
butanamide 9
O
H
N
N
H
N
H
O
SO2NH2
9

M. Ceruso et al. / Bioorg. Med. Chem. 22 (2014) 6768–6775
6775
5. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone,
G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233.
4.3. CA inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity.17
Phenol 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 7.4) 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 earlier1–3 and represent the mean from at least three
different determinations. All CAs were recombinant proteins
obtained as reported earlier by these groups.1,12,13
ˇ
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C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2014, 24, 4006; (b) Ceruso, M.;
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This research was funded by a 7th FP EU Grant (METOXIA). M.C.
also thanks the Erasmus Project for a mobility Grant.
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