Sulfonamide/sulfamate switch with a series of piperazinylureido derivatives: Synthesis, kinetic and in silico evaluation as carbonic anhydrase isoforms I, II, IV, and IX inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111896

We report here a thorough structure-activity relationship (SAR) with piperazinylureido sulfamates as inhibitors of human (h) carbonic anhydrase (CA, EC 4.2.1.1). A SAR investigation over the structure of reported anti-cancer zinc-binder CAIs such as SLC-0

Full text of DOI:10.1016/j.ejmech.2019.111896

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Sulfonamide/sulfamate switch with a series of piperazinylureido derivatives:
Synthesis, kinetic and in silico evaluation as carbonic anhydrase isoforms I, II, IV, and
IX inhibitors
Alessio Nocentini, Davide Moi, Alessandro Deplano, Sameh M. Osman, Zeid A.
AlOthman, Gianfranco Balboni, Claudiu T. Supuran, Valentina Onnis
PII:
S0223-5234(19)31048-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111896
EJMECH 111896
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2019
Revised Date: 15 November 2019
Accepted Date: 15 November 2019
Please cite this article as: A. Nocentini, D. Moi, A. Deplano, S.M. Osman, Z.A. AlOthman, G. Balboni,
C.T. Supuran, V. Onnis, Sulfonamide/sulfamate switch with a series of piperazinylureido derivatives:
Synthesis, kinetic and in silico evaluation as carbonic anhydrase isoforms I, II, IV, and IX inhibitors,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111896.
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Sulfonamide/sulfamate switch with a series of piperazinylureido derivatives: synthesis, kinetic
and in silico evaluation as carbonic anhydrase isoforms I, II, IV, and IX inhibitors
c
Alessio Nocentini a,§, Davide Moi b,§, Alessandro Deplano , Sameh M. Osmand, Zeid A.
AlOthmand Gianfranco Balboni b, Claudiu T. Supuran a,*, Valentina Onnis b,*
a Department NEUROFARBA – Pharmaceutical and Nutraceutical Section, University of Firenze, via Ugo Schiff 6, I-50019 Sesto
Fiorentino, Firenze, Italy
b Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University
of Cagliari, via Ospedale 72, I-09124 Cagliari, Italy
c Pharmacelera, Placa Pau Vila, 1, Sector 1, Edificio Palau de Mar, Barcelona 08039, Spain
d Chemistry Department, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
Highlights
* Piperazinylureido aryl sulfamates were designed and synthesized.
* Sulfamates were assayed in vitro as human Carbonic Anhydrase (CA) isoforms inhibitors.
* Synthesized compounds show weak activity against off-target CA I and CA IV isoforms
* Some sulfamates are CA II/IX selective inhibitors representing leads for the development of new
anticancer agents.
* Docking revealed that CA-selective inhibition is related to distinguished interactions.
Keywords: metalloenzyme; inhibitor; bioisoster; antitumor; selectivity.
Graphical abstract

Abstract
We report here a thorough structure-activity relationship (SAR) with piperazinylureido sulfamates
as inhibitors of human (h) carbonic anhydrase (CA, EC 4.2.1.1). A SAR investigation over the
structure of reported anti-cancer zinc-binder CAIs such as SLC-0111 and S4 was carried out by
including the urea outer nitrogen atom into a substituted piperazine ring reducing the linker
flexibility. The derivatives were assessed for the inhibition of CA I, II and IV (off-target isoforms)
and the tumor-associated CA IX (anticancer drug target). CA I and IV were not effectively
inhibited, whereas many low nanomolar inhibitors were evidenced against CA II (KIs in the range
of 1.0-705.5 nM), and IX (KIs in the range of 0.91-155.9 nM). Interestingly, a subset of CA II/IX
selective inhibitors was detected which might represent interesting lead for the development of new
anticancer strategies.
1. Introduction
Carbonic anhydrases (CAs; EC 4.2.1.1) are a superfamily of metalloenzymes that catalyze the CO2
hydration/dehydration reaction, and are classified into eight genetically distinct families, named α-,
β-, γ-, δ-, ζ-, η-, ɵ- and ι-CAs [1,2]. Among α-class enzymes are all human (h) CAs, that are fifteen
different human isoforms identified to date [3]. Human CAs are also sorted into four different
subsets depending on their subcellular localization: CA I, II, III, VII, VIII, X, XI, XIIII are cytosolic
proteins, CA VA and VB are present in the mitochondrial matrix, CA VI is a secreted enzyme, CA
IV is a glycosylphosphatidylinositol (GPI)-anchored protein and CA IX, XII and XIV are trans-
membrane isoforms [1-3]. As these enzymes are disseminated in the human body and are
implicated in a multitude of crucial physiological processes, serious pathological conditions might
occur due to their dysregulated expression and/or abnormal activity [2]. The isoform CA IX is at the
forefront of the research over CAs since its validation as marker of disease progression in many
aggressive hypoxic tumors. Its targeted inhibition is associated with a significant reduction of the
growth of both primary tumors and metastases [4].
CA inhibitors (CAIs) are grouped in different categories according to their inhibition mechanism.
The zinc-binders, among which sulfonamides and their bioisosteres sulfamates, are the most
effective and utilized for drug-design purposes, but lack of isoform specificity [5]. By applying
drug-design methods such as the tail approach, it has been possible to endow derivatives of this
category with a significant degree of isoform selectivity to apply for the treatment of the target
disease reducing side effect related to promiscuous CAs inhibition. In this context, an
ureidobenzenesulfonamide successfully completed Phase I clinical trials for the treatment of
advanced, metastatic hypoxic tumors over-expressing hCA IX, and is currently in Phase Ib/II

clinical trials in a multi-center, open-label study of oral SLC-0111 (Figure 1) in combination with
gemcitabine (administered i.v.) in subjects affected by metastatic pancreatic ductal adenocarcinoma
[6-8]. By switching the zinc-binding group in the series of ureidobenzenesulfonamide to sulfamate,
Gieling et al. described a set of ureidophenyl sulfamates able to inhibit migration and spreading of
tumor cells under oxygen-decreased conditions. In addition, the low dose maintenance therapy with
one such sulfamates (S4, Figure 1) strongly inhibited the development of MDA-MB-231 metastases
in lung with no signs of toxicity [9].
We recently reported a series of piperazinylureidosulfonamides (A, Figure 1) as analogs of SLC-
0111, detecting several low nanomolar inhibitors of CA IX depending on the substitution pattern at
the piperazine ring [2]. The considerable flexibility of the urea linker was constrained by including
the outer nitrogen atom into a substituted piperazine ring to observe the effect on the CA inhibitory
potency.
Therefore, to investigate the impact on CA inhibition of a sulfonamide/sulfamate switch, such as
that producing S4 from SLC-0111, a new series ureidopiperazinylsulfamates (B, Figure 1) was
designed [11]. Based on promising preliminary CA inhibition outcomes with these derivatives (B)
[11], we extend here the series to provide thorough SAR for this series of
piperazinylureidosulfamates (3a-l and 7a-i).
Figure 1. Drug design of ureidobenzenesulfamates 3a-l, 7a-i from sulfonamide of series A based on
S4 development from SLC-0111.
2. Results and discussion
2.1. Chemistry
The synthesis of the new compounds is reported in Schemes 1 and 2. Coupling of 1-substituted
piperazines 2a–l with 4-(phenoxycarbonyl)aminophenylsulfamate 1 in anhydrous DMSO gave the
piperazinylurea derivatives 3a–l in 38-98% yields (Scheme 1).

Scheme 1. General synthetic procedure for sulfamates subsets 3. Reagents and conditions: (i) anhydrous
DMSO, r.t., 24 h.
Enaminones are versatile synthons to yield a variety of heterocycles, and their preparation has been
recognized for some time. The convenient synthesis of β-dimethylaminovinyl ketones from the
reaction of aryl ketones and formamide acetal has already been described [12]. Accordingly,
enaminones 5a-i were prepared from the appropriate acetophenone and N,N-dimethylformamide
dimethyl acetal (DMF-DMA). The pyrimidinyl-piperazines 6a–i were obtained by heterocyclization
of 4-Boc-piperazine-1-carboxamidine (4) with enaminones 5a-i in boiling ethanol in the presence of
sodium methoxide, followed by a trifluoroacetic acid (TFA)-mediated deprotection in
dichloromethane (DCM) solution (Scheme 2). Then piperazinyl urea derivatives 7a-i we prepared
using the same reaction method described for sulfamates 3.
Scheme 2. General synthetic procedure for sulfamates of subset 7. Reagents and conditions: (i) DMF-DMA,
toluene, 1h, reflux then r.t., 24 h; (ii) 30% MeONa in MeOH, EtOH, 8 h, reflux; (iii) CF3COOH, CH2Cl2, r.t.
12 h; iv) compound 1, anhydrous DMSO, r.t., 24 h.
2.2. Carbonic anhydrase inhibition
Derivatives 3a-l and 7a-i were assayed for their inhibition of the target CA IX and off-target
isoforms such as CA I, II and IV by a stopped flow CO2 hydrase assay [13] (details are showed in
supporting information). Acetazolamide (AAZ) was as standard inhibitor. The inhibition profiles
were assembled to those previously reported and are shown in Table 1. The following structure–
activity relationships (SAR) were worked out: the 4-fluorophenyl substituted sulfamate 3e was a
potent CA I inhibitor, with an inhibition constant of 9.4 nM, whereas the replacement of the
fluorine with a chlorine atom led to a reduction in activity (3f and 3g). Likewise, the unsubstituted
compound 3a and methyl substituted compounds 3b and 3c showed weak efficacy against CA I
with inhibition constants (KIs) ranging between 692 and 897 nM.

Table 1. Inhibition data of human CA isoforms I, II, IV and IX with sulfamates 3a-l and 7a-i and
the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
KI (nM)a
CA I
896.8b
692.0
851.5b
63.5b
CA II
71.9b
56.9
15.9b
11.1b
18.2b
16.9b
45.4
11.2b
9.1b
36.2
15.9
21.0
705.5
206.9
58.7
1.1b
CA IV
CA IX
11.1b
29.7
Compound
R/R’
C6H5
2-CH3-C6H4
3-CH3-C6H4
2,3-(CH3)2-C6H3
4-F-C6H4
-
3a
3b
3c
3d
3e
3f
3g
3h
3i
759.3
-
0.91b
32.3b
61.5b
10.4b
90.3
1087.1
890.3
2592.2
1641.6
994.3
1625.6
422.3
4797.5
4189.5
4097.6
1317.3
457.7
-
9.4b
4-Cl-C6H4
581.3b
918.8
88.1b
3,4-Cl2-C6H3
3-OCH3-C6H4
4-OCH3-C6H4
2-methylbenzofuryl
n-octyl
34.1b
114.1b
32.7
31.4
32.2
282.4b
760.3
752.3
680.1
2370.9
4069.9
3472.9
290.9b
7769.1
277.2b
3930.4
5284.0
70.2b
3j
3k
3l
n-decyl
C6H5
3-CH3-C6H4
3-CF3-C6H4
4-CF3-C6H4
3-Br-C6H4
155.9
93.3
7a
7b
7c
7d
7e
7f
7g
7h
7i
27.1
24.3b
30.5
124.2
1.2b
831.3
-
384.1
279.8
-
4-Br-C6H4
113.6b
31.8
4-OCH3-C6H4
3,4,5-(OCH3)3-C6H2
4-benzofuran-2-yl
-
29.1
6.8
24.2
1.0b
6.7b
250
12
74
25
AAZ
aMean from 3 different assays, by a stopped flow technique (errors were in the range of
±
5-10 % of the
reported values). bvalues taken from [11]
On the contrary, the presence of a 2,3-dimethylphenyl group (compound 3d) or 3-methoxyphenyl
group (compound 3h) restored the activity. The displacement of the methoxy group in 4-position
produced about a third reduction in activity (3i, KI of 282 nM). The replacement of the aryl ring
with an heteroaryl as in compound 3j or with alkyl chains as in compound 3k and 3l also afforded
weak inhibitors of this slow cytosolic CA isoform. In sulfamates of subset 7, the presence of the
pyrimidine ring generally afforded poor CA I inhibitors except for compound 7i and in part for the
4-trifluoromethyl and 4-bromine substituted 7d and 7f, displaying about the same potency of AAZ.
Interestingly, the shift of these groups in 3-position to give the analogs 7c and 7e led drop in
activity against CA I isoform maintaining at the same time high potency on tumor expressed CA IX
isoform. As CAI is basically an off- target isoform, its weak inhibition with sulfamates 7c and 7e

reported here might be considered of interest for the development of anticancers based on CA
inhibition.
CA II is the physiologically dominant isoform, being ubiquitous and involved in a variety of
physiologic functions [2]. CA II is considered target for antiglaucoma drugs, but off-target when the
tumor-associated isoform CA IX should be inhibited [1]. The sulfamates of series 3 and 7 behaved
as weak to potent CA II inhibitors depending by the nature and position of substituents on the
piperazine N-4 atom. In the sulfamate subseries 3, the unsubstituted compound 3a showed both the
worse KI against CA II and an approximately 7-fold II/IX selectivity. The 3-methylphenyl
compound 3c showed a very potency to AAZ against CA II and a 16-fold II/IX selectivity. The shift
of the methyl in the 2-position to give the analog 3b produced about a 4-fold reduction of activity
against CA II as compared to compound 3c, while the selectivity toward CAIX was worsened. The
alkyl substituted compound 3l and 4-chlorine derivative 3f showed about the same KIs values
against CA II and CA IX (21.2 and 32.2 nM; 16.9 and 10.4 nM, respectively). The introduction of a
second chlorine atom as in compound 3g reduced the activity against both isoforms (45.4 and 90.3
nM, respectively). In the sulfamate subset 7, the 4-trifluoromethyl and 4-bromine substituted
compounds 7d and 7f showed activity against CA II about 10-fold greater than AAZ and an
approximately 20-fold and 100-fold selectivity for CA IX over CA II. The shift of the bromine from
4- to 3-position to afford compound 7e led to about a 100-fold reduction of CA II inhibition and a
4-fold increase of action against CAIX. Likewise, the 3-trifluoromethylated compound 7c showed
about a 60-fold reduced CAII inhibition as compared with its analog 7d, while the two compounds
showed very similar KIs against CA IX. The presence on the aryl ring of three methoxy groups
(sulfamate 7h, KI of 6.8 nM) led to about a 4-fold increase in activity against CA II as compared
with the 4-methoxy analog 7g. On the contrary, both compounds 7g and 7h showed about the same
KIs against the CA IX (31.8 and 24.2 nM).
All assayed sulfamates of series 3 and 7 showed low inhibitory activity against the membrane-
bound CA IV in comparison to CA II and IX. This low CA IV inhibitory activity might be related to
the position of the enzyme α-helix including residues 126-136. In many CAs, this is located in the
direction of the binding site stabilizing the binding orientations of substituents at N-4 of piperazine
ring. Conversely, in CA IV the helix is replaced by a loop that extends away from the active site
preventing the binding stabilization (Figure 1S).
2.3. Molecular Docking
Selected compounds were docked in the isoforms CA II and CA IX to explore the interactions
behind the CA isoform selectivity showed by some compounds. In this regard, four compounds of

series 7 were selected to evaluate the effect on activity of substituents on 3- and 4-position of the
phenyl ring. Compounds that clearly show a modification on selectivity related to the shift of the
substituent from 3- to 4-position are the trifluoromethylphenyl- (7c, 7d) and bromophenyl
substituted (7e, 7f). These four compounds were docked in CA II (pdb 4g0c) and CA IX (pdb 3iai),
and the obtained poses were analyzed to understand the reason behind this behavior. All compounds
are characterized by the same phenylsulfamate moiety that establishes comparable sets of
interactions in both isoforms. The sulfamate moiety fits deeply into the active site where the
negative charged nitrogen coordinates the zinc. The hydrogen atom bonded to the sulfamate
nitrogen forms a H-bond with the hydroxyl group of Thr199, whereas the amidic hydrogen atom of
the residue was H-bonded to the S=O of the ligand. An important difference highlighted by the
docking was the orientation of the urea oxygen atom (Figure 2).
Figure 2. Focus on the different orientation of the best poses of compounds 7c, 7d, 7e and 7f on
hCAII (green, PDBID: 4g0c) and hCAIX (yellow, PDBID: 3iai).
In CA II, the oxygen atom points towards Asn62, Asn67 and Gln92 establishing at least one H-bond
with this orientation allowing at the same time the generation of another H-bond between the
Thr200 and the oxygen Ph-O-S. Conversely, in CA IX the carbonyl is 90° degrees rotated with the
oxygen pointing in the direction of Thr200 establishing a long-range H-bond (3.6 - 3.8 Å).

For what concerns selectivity and influence for the activity of the position of the substituent on the
phenyl ring, the main differences are on the 4-arylpyrimidine portion of the molecules. The docking
experiment with CA II highlights differences on the stability of poses; in detail the 4-substituted
phenyl compounds 7d and 7f showed lower RMSD between the best scored pose and all the others,
0.472±0.33 Å and 0.825±0.57 Å respectively, compared to the 3-substitutend analogs 7c and 7e
(1.595±0.75 Å and 1.52±1.11 Å, respectively). Analyzing the docking poses, two main aspects can
explain the stability of the 4-substitued compounds. First, the presence in 4-position of the phenyl
ring of halogens allow the establishing of H-bonds and/or van de Waals interaction that stabilize the
tail of the inhibitors (Figure 3A). These kinds of interaction do not appear optimal for the 3-phenyl
substituted analogs. The docking poses of these compounds showed two possible tail positions, one
that generates steric clash and the second that lets the tail of the compounds more exposed to the
solvent than the 4-substituted analogs (Figure 3B).
Figure 3. A) Best scored pose for compound 7d on hCAII (PDBID: 4g0c); B) Two proposed poses
of compound 7e on hCAII (PDBID: 4g0c); in yellow one generating steric clash, in green one with
high solvent exposure.
This could explain the possible reason why compounds 7d and 7f are better CA II inhibitor respect
to their 3-substituted analogs 7c and 7e. About the docking results with CA IX the variability of the
poses RMSD obtained with the CA II disappeared, all compound showed similar RMSD values
(0.67±1.2 Å, 0.69±0.62 Å, 0.41±0.25 Å, 0.52±0.31 Å respectively for 7c, 7d, 7e and 7f) confirming
the flat CA IX inhibitory activity showed by all compounds except 7f. The poses of all four

compounds present similar binding mode. H-Bond between the urea oxygen atom and Thr200
pushes the molecules on the lower portion of the binding site and the presence of smaller residue on
position 131 (a Val instead of a Phe) allows 3-phenylsubstituted compounds 7c and 7e to assume a
similar position to 4-phenylsubstituted analogs 7d and 7f. The minor activity of compound 7f could
be assumed due to bromine atom steric effects.
Conclusions
We report here an extension of a series of piperazinylureido sulfamates as inhibitors of hCA I, II,
IV and IX providing a thorough SAR investigation over the structure of SLC-0111 and S4. The
urea outer nitrogen atom was included into a substituted piperazine ring to explore the effect of a
reduction of the linker flexibility on the CAs inhibition. CA I and IV were not effectively inhibited,
whereas many low nanomolar inhibitors were evidenced against CA II (KIs in the range of 1.0-
705.5 nM), and IX (KIs in the range of 0.91-155.9 nM). A subset of CA II/IX selective inhibitors
were detected which might represent interesting lead for the development of new anticancer
strategies.
4. Experimental section
4.1. Chemistry
All commercially available solvents and reagents were used without further purification. NMR
spectra were recorded on an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). The chemical
shifts (δ) are reported in part per million downfield from tetramethylsilane (TMS), which was used
as internal standard, and the
spectra were recorded in hexadeuteriodimethylsulphoxide (DMSO-d6). Infrared spectra were
recorded on a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol mulls. The main bands
are given in cm-1. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a
double-focusing MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geometry. Melting
points (mp) were determined with a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK)
and are uncorrected. All products reported showed 1H NMR spectra in agreement with the assigned
structures. The purity of the tested compounds was determined by combustion elemental analyses
conducted by the Microanalytical Laboratory of the Chemistry Department of the University of
Ferrara with a MT-5 CHN recorder elemental analyzer (Yanagimoto, Kyoto, Japan) and the values
found were within 0.4% of theoretical values. 4-(Phenoxycarbonyl)aminophenylsulfamate (1) [11]
and 4-Boc-piperazine-1-carboxamidine (4) [14], sulfamates 3a, 3c-f, 3h, 3i, 7d, 7f, and 7i [11], 3k
and 3l [15] were synthesized as previously described.

4.1.1. General procedure for the synthesis of 4-(piperazinocarbonyl)aminosulfamates (3a-l)
A mixture of 4-(phenoxycarbonyl)aminophenylsulfamate 1 (0,31 g, 1 mmol) and substituted 1-
substituted piperazine 2a-l (1 mmol), in anhydrous DMSO (3 mL) was stirred at room temperature
for 24 h. Then, water (10 mL) was added and the mixture was stirred at room temperature until a
solid is formed. The formed solid was filtered off, washed with water, air dried and recrystallized
from EtOH to give ureas 3a-l.
4.1.1.1. 4-(4-(2-Methylphenyl)piperazinocarbonyl)aminophenylsulfamate (3b). Following the
general procedure, the title compound was obtained from 2b in 72% yield, mp 170-171°C. ESIMS
(m/z): 391 (M+H)+. 1H NMR: δ 2.29 (s, 3H, CH3), 2.85 (s, 4H, CH2), 3.60 (s, 4H, CH2), 6.98 (d, J =
7.0 Hz, 1H, Ar), 7.04 (d, J = 8.5 Hz, 1H, Ar), 7.15 (d, J = 8.0 Hz, 2H, Ar), 7.18 (d, J = 7.0 Hz, 2H,
Ar), 7.52 (d, J = 8.0 Hz, 2H, Ar), 7.86 (s, 2H, NH2), 8.70 (s, 1H, NH); IR (Nujol) 3408 (NH), 3329
(NH), 3096 (NH), 1647 (C=O), 1607 (C=C) cm-1. Anal. Calcd for C18H22N4O4S: C, 55.37; H, 5.68;
N,14.35. Found: C, 55.31; H, 5.66; N, 14.29.
4.1.1.2. 4-(4-(3,4-Dichlorophenyl)piperazinocarbonyl)aminophenylsulfamate (3g). Following the
general procedure, the title compound was obtained from 2g in 98% yield, mp 152-154°C. ESIMS
1
(m/z): 447, 445 (M+H)+. H NMR: δ 2.63 (m, 2H, CH2), 2.74 (m, 2H, CH2), 3.23 (m, 2H, CH2),
3.58 (m, 2H, CH2), 6.97 (s, 1H, Ar), 7.14 (d, J = 8.5 Hz, 2H, Ar), 7.20 (m, 2H, Ar), 7.52 (d, J = 8.5
Hz, 2H, Ar), 7.87 (s, 2H, NH2), 8.72 (s, 1H, NH); IR (Nujol) 3390 (NH), 3341 (NH), 3126 (NH),
3032 (NH), 1718 (C=O), 1649 (C=O) cm-1. Anal. Calcd for C17H18Cl2N4O4S: C, 45.85; H, 4.07;
N,12.58. Found: C, 45.91; H, 4.06; N, 12.63.
4.1.1.3. 4-(4-(Benzofuran-2-ylmethyl)piperazine-1-carboxamido)phenyl sulfamate (3j). Following
the general procedure, the title compound was obtained from 2j in 40% yield, mp 152-154°C.
1
ESIMS (m/z): 431 (M+H)+. H NMR: δ 2.54 (s, 2H, CH2), 3.30 (m, 4H, CH2), 3.51 (m, 4H, CH2),
6.81 (s, 1H, Ar), 7.13 (d, J = 9.0 Hz, 1H, Ar), 7.16 (d, J = 9.0 Hz, 1H, Ar), 7.26 (d, J = 9.5 Hz, 1H,
Ar), 7.43 (d, J = 7.0 Hz, 1H, Ar), 7.46 (d, J = 7.0 Hz, 1H, Ar), 7.49 (d, J = 9.5 Hz, 1H, Ar), 7.67 (m,
2H, Ar), 7.88 (s, 2H, NH2), 8.61 (s, 1H, NH); IR (Nujol) 3386 (NH), 1724 (C=O), 1646 (C=O),
1607 (C=C) cm-1. Anal. Calcd for C20H22N4O5S: C, 55.80; H, 5.15; N,13.02. Found: C, 55.74; H,
5.17; N, 13.07.
4.1.2. General procedure for the synthesis of arylpropenones (5a-i)
A mixture of 1-arylethanone (5 mmol) and DMF-DMA (1.79 g, 15 mmol) in anhydrous toluene (10
mL) was refluxed for 1 h, then was allowed to reach the room temperature and stirred for additional
24 h. The mixture was carefully concentrated in vacuum to give the title compounds.

4.1.2.1. (E)-3-(Dimethylamino)-1-(phenyl)prop-2-en-1-one (5a)
Following the general procedure, the title compound was obtained from 1-phenylethanone in 87 %
yield. mp 90-91 °C (n-hexane), lit. [16] 89-90 °C. ESIMS (m/z): 176 (M+H)+. 1H NMR: δ 2.88 (s,
3H, CH3), 2.92 (s, 3H, CH3), 5.77 (d, J = 12.5 Hz, 1H, CH), 7.37-7.74 (m, 5H, Ar), 7.80 (d, J = 12.5
Hz, 1H, CH); IR (Nujol) 3350, 1643 cm-1. Anal. Calcd for C11H13NO: C, 75.40; H, 7.48; N,7.99.
Found: C, 75.34; H, 7.51; N, 7.95.
4.1.2.2. (E)-3-(Dimethylamino)-1-(m-tolyl)prop-2-en-1-one (5b)
Following the general procedure, the title compound was obtained from 1-(m-tolyl)ethanone in 92
% yield. Oil. ESIMS (m/z): 135 (M+H)+. 1H NMR: δ 2.36 (s, 3H, CH3), 2.90 (s, 3H, CH3), 3.12 (s,
3H, CH3), 5.79 (d, J = 12.5 Hz, 1H, CH), 7.29 (m, 2H, Ar), 7.67 (m, 3H, Ar); IR (Nujol) 3348, 1642
cm-1. Anal. Calcd for C12H15NO: C, 76.16; H, 7.99; N,7.40. Found: C, 76.09; H, 8.03; N, 7.44.
4.1.2.3. (E)-3-(Dimethylamino)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5c)
Following the general procedure, the title compound was obtained from 1-(3-
(trifluoromethyl)phenyl)propan-1-one in 80 % yield. mp 44-46 °C Lit. [16] ESIMS (m/z): 244
(M+H)+. 1H NMR: δ 2.92 (s, 3H, CH3), 3.14 (s, 3H CH3), 5.88 (d, J = 12.5 Hz, 1H, CH), 7.43 (d, J
= 7.0 Hz, 1H, Ar), 7.70 (d, J = 8.0 Hz, 1H, Ar), 7.80 (d, J = 12.5 Hz, 1H, CH), 7.88 (d, J = 7.0 Hz,
1H, Ar), 8.04 (s, 1H, Ar); IR (Nujol) 1643, 1594 cm-1. Anal. Calcd for C12H12F3NO: C, 59.26; H,
4.97; N, 5.76. Found: C, 59.32; H, 4.79; N, 5.74.
4.1.2.4. (E)-1-(3-Bromophenyl)-3-(dimethylamino)prop-2-en-1-one (5e)
Following the general procedure, the title compound was obtained from 1-(3-
bromophenyl)ethanone in 90% yield. mp 53-54°C. ESIMS (m/z): 255 (M+H)+. 1H NMR: δ 2.94 (s,
3H, CH3), 3.16 (s, 3H, CH3), 5.82 (d, J = 12.0 Hz, 1H, CH), 7.40 (d, J = 7.5 Hz, 1H, Ar), 7.67 (d, J
= 8.0 Hz, 1H, Ar), 7.72 (d, J = 12.0 Hz, 1H, CH), 7.90 (d, J = 7.5 Hz, 1H, Ar), 8.02 (s, 1H, Ar); IR
(Nujol) 1641, 1573 cm-1. Anal. Calcd for C11H12BrNO: C, 51.99; H, 4.76; N,5.51. Found: C, 52.05;
H, 4.77; N, 5.48.
4.1.2.5. (E)-3-(Dimethylamino)-1-(4-methoxyphenyl)-prop-2-en-1-one (5g)
Following the general procedure, the title compound was obtained from 1-(4-
methoxyphenyl)ethanone in 87% yield. mp 113-115°C. Lit. 89-91 °C [17] ESIMS (m/z): 206
(M+H)+. 1H NMR: δ 2.93 (s, 3H, CH3), 3.15 (s, 3H, CH3), 3.83 (s, 3H, CH3), 5.83 (d, J = 12.0 Hz,
1H, CH), 7.20 (d, J = 8.0 Hz, 2H, Ar), 7.68 (d, J = 12.0 Hz, 1H, CH), 8.12 (d, J = 8.12 Hz, 2H, Ar);
IR (Nujol) 3330, 1642, 1601, 1581 cm-1. Anal. Calcd for C11H15NO2: C, 70.22; H, 7.37; N, 6.82.
Found: C, 70.15; H, 7.40; N, 6.80.
4.1.2.6. (E)-3-(Dimethylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5h)

Following the general procedure, the title compound was obtained from 1-(3,4,5
trimethoxyphenyl)ethanone in 76 % yield. mp 123-125 °C, Lit. 125-127 °C [18] ESIMS (m/z): 206
1
(M+H)+. H NMR: δ 2.94 (s, 3H, CH3), 3.15 (s, 3H, CH3), 3.72 (s, 3H, CH3), 3.83 (s, 6H, CH3),
5.81 (d, J = 12.0 Hz, 1H, CH), 7.18 (s, 2H, Ar), 7.70 (d, J = 12.0 Hz, 1H, CH); IR (Nujol) 1638,
1548 cm-1. Anal. Calcd for C14H19NO4: C, 63.38; H, 7.22; N, 5.28. Found: C, 63.43; H, 7.20; N,
5.31.
4.1.3. General procedure for the synthesis of 1-(4-arylpyrimidin-2-yl)piperazines (6a-i)
A
solution of 1-(aryl)-3-(dimethylamino)prop-2-en-1-one (5a-i) (2 mmol), 4-(tert-
butoxycarbonyl)piperazine-1-carboxamidine (4) (1.02 g, 2.2 mmol) and sodium methylate 30%
MeOH solution (0.8 ml, 4 mmol) in anhydrous EtOH (5 mL) was refluxed 8 h. After cooling to
room temperature, the solvent was removed under reduced pressure. The residue was treated with
ethyl acetate (20 mL) and washed with water (3 x 10 mL) and brine (10 mL). After drying over
sodium sulphate the solvent was removed under reduced pressure. Then the residue was solubilised
in anhydrous dichloromethane (10 mL) and trifluoroacetic acid (5 mL) was added. The mixture was
stirred at room temperature overnight and after evaporation of the solvent, the residue was treated
with isopropyl ether to obtain a solid, that was filtered off and dried. The formed solid was used in
the next step without further purification.
4.1.3.1. 4-Phenyl-2-(piperazin-1-yl)pyrimidine trifluoroacetate (6a)
Following the general procedure, the title compound was obtained from 5a in 42% yield, mp 119-
1
120 °C. ESIMS (m/z): 241 (M+H)+. H NMR: δ 3.22 (s, 4H, CH2), 4.04 (s, 4H, CH2), 7.31 (d, J =
5.0 Hz, 2H, Ar), 7.53 (m, 2H, Ar), 8.15 (d, J = 5.0 Hz, 2H, Ar), 8.49 (d, J = 5.0 Hz, 1H, Ar), 8.93
(s, 1H, NH); IR (Nujol) 3367, 1687 cm-1. Anal. Calcd for C16H17F3N4O2: C, 54.24; H, 4.84; N,
15.81. Found: C, 54.18; H, 4.85; N, 15.84.
4.1.3.2. 2-(Piperazin-1-yl)-4-(m-tolyl)pyrimidine trifluoroacetate (6b)
Following the general procedure, the title compound was obtained from 5b in 58% yield, mp 113-
1
114 °C. ESIMS (m/z): 255 (M+H)+. H NMR: δ 2.09 (s, 1H, NH), 2.41 (s, 3H, CH3), 3.23 (s, 4H,
CH2), 4.05 (s, 4H, CH2), 7.31 (d, J = 5.0 Hz, 1H, Ar), 7.35 (d, J = 7.0 Hz, 1H, Ar), 7.40 (d, J = 7.5
Hz, 1H, Ar), 7.95 (d, J = 7.5 Hz, 2H, Ar), 8.49 (d, J = 5.0 Hz, 1H, Ar), 9.16 (s, 1H, OH); IR (Nujol)
2502,1673 cm-1. Anal. Calcd for C17H19F3N4O2: C, 55.43; H, 5.20; N, 15.21. Found: C, 55.49; H,
5.22; N, 15.17.
4.1.3.3. 2-(Piperazin-1-yl)-4-(3-(trifluoromethyl)phenyl)pyrimidine trifluoroacetate (6c)
Following the general procedure, the title compound was obtained from 5c in 60% yield, mp 105-
1
106 °C. ESIMS (m/z): 309 (M+H)+. H NMR: δ 2.08 (s, 1H, NH), 3.24 (s, 4H, CH2), 4.06 (s, 4H,

CH2), 7.46 (d, J = 5.0 Hz, 1H, Ar), 7.77 (d, J = 7.5 Hz, 1H, Ar), 7.91 (d, J = 7.5 Hz, 1H, Ar), 8.86
(d, J = 8.0 Hz, 2H, Ar), 8.57 (d, J = 5.0 Hz, 1H, Ar), 9.03 (s, 1H, OH); IR (Nujol) 3371, 2514,
1668, 1589 cm-1. Anal. Calcd for C17H16F6N4O2: C, 48.35; H, 3.82; N, 13.27. Found: C, 48.39; H,
3.80; N, 13.24.
4.1.3.4. 4-(3-Bromophenyl)-2-(piperazin-1-yl)pyrimidine trifluoroacetate (6e)
Following the general procedure, the title compound was obtained from 5e in 40% yield, mp 108-
1
109 °C. ESIMS (m/z): 321, 319 (M+H)+. H NMR: δ 2.08 (s, 1H, NH), 3.23 (s, 4H, CH2), 4.05 (s,
4H, CH2), 7.38 (d, J = 5.0 Hz, 1H, Ar), 7.49 (d, J = 7.0 Hz, 1H, Ar), 7.74 (d, J = 7.0 Hz, 1H, Ar),
8.16 (d, J = 6.5 Hz, 1H, Ar), 8.33 (m, 1H, Ar), 8.54 (d, J = 5.0 Hz, 1H, Ar), 9.15 (s, 1H, OH); IR
(Nujol) 3368, 2510, 1671 cm-1. Anal. Calcd for C16H16BrF3N4O2: C, 44.36; H, 3.72; N, 12.93.
Found: C, 44.30; H, 3.74; N, 12.97.
4.1.3.5. 2-(Piperazin-1-yl)-4-(4-methoxyphenyl)pyrimidine trifluoroacetate (6g)
Following the general procedure, the title compound was obtained from 5g in 38% yield, mp > 240
1
°C. ESIMS (m/z): 385 (M+H)+. H NMR: δ 3.68 (s, 4H, CH2), 3.83 (s, 4H, CH2), 4.02 (s, 3H,
OCH3), 7.06 (d, J = 7.0 Hz, 2H, Ar), 7.38 (d, J = 5.0 Hz, 1H, Ar), 7.65 (d, J = 7.0 Hz, 2H, Ar), 8.43
(d, J = 5.0 Hz, 1H, Ar); IR (Nujol) 3320, 1662 cm-1. Anal. Calcd for C17H19F3N4O3: C, 53.12; H,
4.98; N, 14.58. Found: C, 53.18; H, 4.97; N, 14.55.
4.1.3.6. 2-(Piperazin-1-yl)-4-(3,4,5-trimethoxyphenyl)pyrimidine trifluoroacetate (6h)
Following the general procedure, the title compound was obtained from 5h in 36% yield, mp > 240
1
°C. ESIMS (m/z): 331 (M+H)+. H NMR: δ 3.73 (s, 4H, CH2), 3.79 (s, 4H, CH2), 3.88 (s, 9H,
OCH3), 7.41 (d, J = 6.5 Hz, 1H, Ar), 7.44 (s, 2H, Ar), 8.35 (d, J = 6.5 Hz, 1H, Ar); IR (Nujol) 2485,
1662 cm-1. Anal. Calcd for C19H23F3N4O5: C, 51.35; H, 5.22; N, 12.61. Found: C, 51.28; H, 5.25; N,
12.65.
4.1.4.
General
procedure
for
the
synthesis
of
4-(4-(4-aryl)pyrimidin-2-
yl)piperazinocarbonyl)aminophenyl sulfamate (7a-i)
A mixture of 4-(phenoxycarbonyl)aminophenylsulfamate 1 (0,31 g, 1 mmol) and pyrimidines 6a-i
(1 mmol), in anhydrous DMSO (3 mL) was stirred at room temperature for 24 h. Then, water (10
mL) was added and the mixture was stirred at room temperature until a solid is formed. The formed
solid was filtered off, washed with water, air dried and recrystallized from EtOH to give ureas 7a-i.
4.1.4.1. 4-(4-(4-Phenylpyrimidin-2-yl)piperazine-1-carboxamido)phenyl sulfamate (7a)
Following the general procedure, the title compound was obtained from 6a in 25% yield, mp 134-
135 °C. ESIMS (m/z): 455 (M+H)+. 1H NMR: δ 3.55 (m, 4H, CH2), 3.87 (m, 4H, CH2), 6.65 (s, 1H,
CH), 7.22 (m, 4H Ar), 7.25 (d, J = 5.0 Hz, 2H, Ar), 7.39-7.52 (m, 4H, Ar), 8.15 (s, 2H, NH2), 8.47

(s, 1H, NH); IR (Nujol) 3336 (NH), 1701 (C=O), 1634 (C=N), 1567 (C=C) cm-1. Anal. Calcd for
C21H22N6O4S: C, 55.49; H, 4.88; N, 18.49. Found: C, 55.55; H, 4.87; N, 18.52.
4.1.4.2. 4-(4-(4-(m-Tolyl)pyrimidin-2-yl)piperazine-1-carboxamido)phenyl sulfamate (7b)
Following the general procedure, the title compound was obtained from 6b in 31% yield, mp 164-
165 °C. ESIMS (m/z): 469 (M+H)+. 1H NMR: δ 2.42 (s, 3H, CH3), 3.60 (m, 4H, CH2), 3.89 (m, 4H,
CH2), 7.16 (d, J = 6.0 Hz, 1H, Ar), 7.27 (d, J = 6.0 Hz, 1H, Ar), 7.37 (m, 4H, Ar), 7.51 (d, J = 8.0
Hz, 2H Ar), 7.53 (d, J = 8.0 Hz, 2H Ar), 7.88 (s, 2H, NH2), 8.71 (s, 1H, NH); IR (Nujol) 3295
(NH), 1644 (C=O) cm-1. Anal. Calcd for C22H24N6O4S: C, 56.40; H, 5.16; N, 17.94. Found: C,
56.34; H, 5.18; N, 17.97.
4.1.4.3.4-(4-(4-(3-(Trifluoromethyl)phenyl)pyrimidin-2-yl)piperazine-1-carboxamido)phenyl
sulfamate (7c)
Following the general procedure, the title compound was obtained from 6c in 41% yield, mp 174-
1
175 °C. ESIMS (m/z): 523 (M+H)+. H NMR: δ 3.51-3.60 (m, 4H, CH2), 3.90 (m, 4H, CH2), 7.16
(d, J = 7.0 Hz, 2H, Ar), 7.40 (d, J = 5.0 Hz, 1H, Ar), 7.53 (m, 3H, Ar), 7.78 (d, J = 7.2 Hz, 2H, Ar),
7.90 (s, 2H, NH2), 8.44 (s, 1H, Ar), 8.55 (d, J = 5.0 Hz, 1H, Ar), 8.72 (s, 1H, NH); IR (Nujol) 3307
(NH), 1642 (C=O), 1598 (C=N), 1571 (C=C) cm-1. Anal. Calcd for C22H21F3N6O4S: C, 50.57; H,
4.05; N, 16.08. Found: C, 50.63; H, 4.02; N, 16.12.
4.1.4.4. 4-(4-(4-(3-Bromophenyl)pyrimidin-2-yl)piperazine-1-carboxamido)phenyl sulfamate (7e)
Following the general procedure, the title compound was obtained from 6e in 26% yield, mp 163-
1
164 °C. ESIMS (m/z): 533, 535 (M+H)+. H NMR: δ 3.59 (m, 4H, CH2), 3.89 (m, 4H, CH2), 7.17
(m, 2H, Ar), 7.31 (d, J = 5.0 Hz, 1H, Ar), 7.53 (m, 3H, Ar), 7.73 (d, J = 7.5 Hz, 1H, Ar), 7.87 (s,
2H, NH2), 8.16 (d, J = 7.5 Hz, 1H Ar), 8.31 (s, 1H, Ar), 8.52 (d, J = 5.0 Hz, 1H, Ar), 8.72 (s, 1H,
NH); IR (Nujol) 3323 (NH), 1646 (C=O), 1605 (C=N), 1571 (C=C) cm-1. Anal. Calcd for
C22H21BrN6O4S: C, 47.29; H, 3.97; N, 15.76. Found: C, 47.25; H, 3.98; N, 15.80.
4.1.4.5. 4-(4-(4-(4-Methoxyphenyl)pyrimidin-2-yl)piperazine-1-carboxamido)phenyl sulfamate (7g)
Following the general procedure, the title compound was obtained from 6g in 40% yield, mp 93-95
°C. ESIMS (m/z): 485 (M+H)+. 1H NMR: δ 3.59 (s, 4H, CH2), 3.64 (s, 4H, CH2), 3.85 (s, 3H, CH3),
7.07 (d, J = 8.0 Hz, 2H, Ar), 7.19 (m, 4H, Ar), 7.53 (d, J = 8.5 Hz, 2H, Ar), 8.13 (d, J = 8.5 Hz, 2H,
Ar), 7.88 (s, 2H, NH2), 8.73 (s, 1H, NH); IR (Nujol) 3404 (NH), 3317 (NH), 3182 (NH), 1650
(C=O), 1569 (C=N) cm-1. Anal. Calcd for C22H24N6O5S: C, 54.53; H, 4.99; N, 17.34. Found: C,
54.47; H, 5.01; N, 17.38.
4.1.4.6. 4-(4-(4-(3,4,5-Trimethoxyphenyl)pyrimidin-2-yl)piperazine-1-carboxamido)phenyl
sulfamate (7h)

Following the general procedure, the title compound was obtained from 6h in 31% yield, mp 119-
1
120 °C. ESIMS (m/z): 545 (M+H)+. H NMR: δ 3.59 (s, 4H, CH2), 3.74 (s, 4H, CH2), 3.90 (s, 9H,
CH3), 7.16 (d, J = 7.5 Hz, 2H, Ar), 7.29 (m, 1H, Ar), 7.45 (m, 2H, Ar), 7.53 (d, J = 7.5 Hz, 2H, Ar),
7.88 (s, 2H, NH2), 8.45 (s, 1H, Ar), 8.72 (s, 1H, NH); IR (Nujol) 3330 (NH), 3175 (NH), 1632
(C=O), 1552 (C=N) cm-1. Anal. Calcd for C24H28N6O7S: C, 52.93; H, 5.18; N, 15.43. Found: C,
52.97; H, 5.20; N, 15.40.
4.2. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity [13]. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mMHepes (pH 7.5) as buffer, and 20 mM
Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least
six traces of the initial 5–10% of the reaction have been used for determining the initial velocity.
The uncatalyzed rates were determined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in 10% DMSO aqueous solution and
dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions
were preincubated together for 15 min at room temperature prior to assay, in order to allow for the
formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier [19-21] and represent
the mean from at least three different determinations. All CA isofoms were recombinant ones
obtained in-house as reported earlier [22-24].
4.3. Molecular Docking
Molecular Docking simulations were carried out as previously described [25] using rDock [26]. The
crystal structures of CAII (pdb 4G0C) and CAIX (pdb 3IAI) were retrieved from RCSB Protein
Data Bank web server (http://www.rcsb.org/). Proteins preparation was executed using HTMD
(HighThroughput MD) tool [27] to add hydrogens, ionize side chain of amino acids at physiological
pH using propKa, deleting water molecules and co-crystallized small molecules (AAZ included).
3D ligands were prepared using an in-house python script developed using RDKit toolkit [28] and
minimized using MMFF94 forcefield. Considering the high conservation of the binding mode of the
sulfamide moiety of co-crystallized compounds a tethered docking was executed [29] to enforce the
partial binding modes of the sulfamates group. The ‘dock_solv’ rDock protocol was used, this
protocol allows a full docking search but using the desolvation scoring function. Docking grid with

radius of 10.0 Å was centered on the co-crystalized ligand. Number of poses was set to 10. Docking
protocol validation was described in our previous paper [26]. It contemplates the re-docking of the
co-crystallized ligand AAZ and the evaluation of the RMSD with the crystallographic pose.
Acknowledgements
This study was funded by FIR grants from the University of Cagliari, the Italian Ministero
dell’Istruzione, Università e della Ricerca (PRIN 2017, Prot. No. 2010E84AA4_002) (to V.O. and
G.B.) and by the Researchers Supporting Project No. (RSP-2019/1) King Saud University, Riyadh,
Saudi Arabia.
Conflict of interest
The authors declare no conflict of interest.
References
[1] A. Nocentini, C.T. Supuran. Advances in the structural annotation of human carbonic
anhydrases and impact on future drug discovery. Expert Opin. Drug Discov. 14 (2019) 1175-1197.
[2] C.T. Supuran. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators.
Nat. Rev. Drug. Discov. 7 (2008) 168–181.
[3] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone G. Multiple binding
modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different
isoforms? Chem. Rev. 112 (2012) 4421–4468.
[4] A. Nocentini, C.T. Supuran. Carbonic anhydrase inhibitors as antitumor/antimetastatic agents: a
patent review (2008-2018). Expert Opin. Ther. Pat. 28 (2018) 729-740.
[5] C.T. Supuran. How many carbonic anhydrase inhibition mechanisms exist? J. Enzyme Inhib.
Med. Chem. 31 (2016) 345-360.
[6] https://clinicaltrials.gov/ct2/show/NCT02215850 (last updated on May 17, 2016); last accessed
on October 10, 2019.
[7] https://clinicaltrials.gov/ct2/show/NCT03450018 (last updated on March 1, 2018); last accessed
on October 10, 2019.
[8] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar, C.T.
Supuran. Ureido-substituted benzenesulfonamides potently inhibit carbonic anhydrase IX and show
antimetastatic activity in a model of breast cancer metastasis. J. Med. Chem. 54 (2011) 1896-1902.

[9] R.G. Gieling, M. Babur, L. Mamnani, N. Burrows, B.A. Telfer, F. Carta, J.Y. Winum, A.
Scozzafava, C.T. Supuran, K.J. Williams. Antimetastatic effect of sulfamate carbonic anhydrase IX
inhibitors in breast carcinoma xenografts. J. Med. Chem. 14 (2012) 5591-5600.
[10] C. Congiu, V. Onnis, A. Deplano, G. Balboni, N. Dedeoglu, C. T. Supuran. Synthesis of
sulfonamides incorporating piperazinyl-ureido moieties and their carbonic anhydrase I, II, IX and
XII inhibitory activity. Bioorg. Med. Chem. Lett. 25 (2015) 3850-3853.
[11] C. Congiu, V. Onnis, A. Deplano, G. Balboni, M. Ceruso, C.T. Supuran. Synthesis and
carbonic anhydrase I, II, IX and XII inhibitory activity of sulfamates incorporating piperazinyl-
ureido moieties. Bioorg. Med. Chem. 23 (2015) 5619-5625.
[12] (a) S-S. Tseng, J. W. Epstein, H. J. Brabander, G. Francisco. A simple regioselective synthesis
of pyrimido[1,2 a]benzimidazoles. J. Heterocycl. Chem. 24 (1987) 837-843; (b) S. Chimichi, M.
Boccalini, M. M. M. Hassan, G. Viola, F. Dall’Acqua, M. Curini. Synthesis, structural
determination and photo-antiproliferative activity of new 3-pyrazolyl or -isoxazolyl substituted 4-
hydroxy-2(1H)-quinolinones. Tetrahedron 62 (2006) 90-96; (c) C. Reidlinger, R. Dworczak, H.
Junek. Cyanoacetophenone as a Synthon for 1,4,5-Substituted Pyrazoles. Monatsh. Chem. 129
(1998) 1207-1211; (d) F. Al-Omram, A.-Z. A. Elassar, A. A. El-Khair. Synthesis of condensed
heteroaromatics: novel synthesis of aminoquinolizinone derivatives as anti-HIV agents.
Tetrahedron 57 (2001) 10163-10170.
[13] R.G. Khalifah. The carbon dioxide hydration activity of carbonic anhydrase. J. Biol. Chem.
246 (1971) 2561–2573.
[14] D. L. Coffen, M. P. Dillon, A. P. D. W. Ford, Z. Li, T. J. Williams. US Patent 2002, 6355641
B1.
[15] D. Moi, P. A. Foster, L. G. Rimmer, A. Jaffri, A. Deplano, G. Balboni, V. Onnis, B. V.L.
Potter. Synthesis and in vitro evaluation of piperazinyl-ureido sulfamates as steroid sulfatase
inhibitors. Eur. J. Med. Chem. 182 (2019) 111614.
[16] F. A. Rosa, P. Machado, H. G. Bonacorso, N. Zanatta, M. A. P. Martins. Reaction of β-
Dimethylaminovinyl Ketones with Hydroxylamine: a Simple and Useful Method for Synthesis of 3-
and 5-Substituted Isoxazoles. J. Heterocyclic Chem. 45 (2008) 879-885.
[18] A. Balbi, M. Anzaldi, C. Maccio, C. Aiello, M. Mazzei, R. Gangemi, P. Castagnola, M. Miele,
C. Rosano, M. Viale. Synthesis and biological evaluation of novel pyrazole derivatives with
anticancer activity. Eur. J. Med. Chem. 46 (2011) 5293-5309.
[19] L. E. Kiss, H. S. Ferreira, L. Torrao, M. J. Bonifacio, P. N. Palma, P. Soares-da-Silva, D.A.
Learmonth. Discovery of a Long-Acting, Peripherally Selective Inhibitor of Catechol-O-
methyltransferase. J. Med. Chem. 53 (2010) 3396-3411.

[20] D. Vullo, S. Del Prete, A. Nocentini, S.M. Osman, Z. AlOthman, C. Capasso, M. Bozdag, F.
Carta, P. Gratteri, C.T. Supuran, Dithiocarbamates effectively inhibit the b-carbonic anhydrase from
the dandruff-producing fungus Malassezia globose. Bioorg. Med. Chem. 25 (2017) 1260–1265.
[21] Y. Entezari Heravi, S. Bua, A. Nocentini, S. Del Prete, A. A. Saboury, H. Sereshti, C. Capasso,
P. Gratteri, C.T. Supuran. Inhibition of Malassezia globosa carbonic anhydrase with phenols.
Bioorg. Med. Chem. 25 (2017) 2577-2582.
[22] A. Nocentini, A. Bonardi, P. Gratteri, B. Cerra, A. Gioiello, C.T. Supuran. Steroids interfere
with human carbonic anhydrase activity by using alternative binding mechanisms. J. Enzyme Inhib.
Med. Chem. 33 (2018)1453-1459.
[23] A. Nocentini, D. Moi, G. Balboni, V. Onnis, C.T. Supuran. Discovery of thiazolin-4-one-based
aromatic sulfamates as a new class of carbonic anhydrase isoforms I, II, IV, and IX inhibitors.
Bioorg. Chem. 77 (2018) 293-299.
[24] A. Bonardi, A.B. Vermelho, V. da Silva Cardoso, M.C. de Souza Pereira, L.L. da Silva, S.
Selleri, P. Gratteri, C.T. Supuran, A. Nocentini. N-Nitrosulfonamides as Carbonic Anhydrase
Inhibitors: A Promising Chemotype for Targeting Chagas Disease and Leishmaniasis. ACS Med.
Chem. Lett. 10 (2018) 413-418.
[25] A. Nocentini, D. Moi, G. Balboni, S. Salvadori, V. Onnis, C.T. Supuran. Synthesis and
biological evaluation of novel pyrazoline-based aromatic sulfamates with potent carbonic anhydrase
isoforms II, IV and IX inhibitory efficacy. Bioorg. Chem. 77 (2018) 633-639.
[26] D. Moi, A. Nocentini, A. Deplano, G. Balboni, C. T. Supuran, V. Onnis. Structure-activity
relationship with pyrazoline-based aromatic sulfamates as carbonic anhydrase isoforms I, II, IX and
XII inhibitors: synthesis and biological evaluation. Eur. J. Med. Chem. 182 (2019) 111638.
[27] S. Ruiz-Carmona, D. Alvarez-Garcia, N. Foloppe, A.B. Garmendia-Doval, S. Juhos, P.
Schmidtke, X. Barril, R.E. Hubbard, S.D. Morley. rDock: a fast, versatile and open source program
for docking ligands to proteins and nucleic acids. PLoS Comput. Biol. 10 (2014) e1003571.
[28] S. Doerr, T. Giorgino, G. Martínez-Rosell, J.M. Damas, G. De Fabritiis, Highthroughput
automated preparation and simulation of membrane proteins with HTMD. J. Chem. Theory
Comput. 13 (2017) 4003-4011.
[29] RDKit, Cheminformatics and machine learning software. http://www.rdkit. Org (2013)

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: