Aryl-4,5-dihydro-1H-pyrazole-1-carboxamide derivatives bearing a sulfonamide moiety show single-digit nanomolar-to-subnanomolar inhibition constants against the tumor-associated human carbonic anhydrases IX and XII

Article abstract of DOI:10.3390/ijms21072621

A series of new 3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide derivatives was designed here, synthesized, and studied for carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory activity against the human (h) isozymes I, II, and VII (cytosolic, off-target isoforms), and IX and XII (anticancer drug targets). Generally, CA I was not effectively inhibited, whereas effective inhibitors were identified against both CAs II (KIs in the range of 5.2-233 nM) and VII (KIs in the range of 2.3-350 nM). Nonetheless, CAs IX and XII were the most susceptible isoforms to this class of inhibitors. In particular, compounds bearing an unsubstituted phenyl ring at the pyrazoline 3 position showed 1.3-1.5 nM KIs against CA IX. In contrast, a subset of derivatives having a 4-halo-phenyl at the same position of the aromatic scaffold even reached subnanomolar KIs against CA XII (0.62-0.99 nM). Docking studies with CA IX and XII were used to shed light on the derivative binding mode driving the preferential inhibition of the tumor-associated CAs. The identified potent and selective CA IX/XII inhibitors are of interest as leads for the development of new anticancer strategies.

Full text of DOI:10.3390/ijms21072621

International Journal of
Molecular Sciences
Article
Aryl-4,5-dihydro-1H-pyrazole-1-carboxamide
Derivatives Bearing a Sulfonamide Moiety Show
Single-digit Nanomolar-to-Subnanomolar Inhibition
Constants against the Tumor-associated Human
Carbonic Anhydrases IX and XII
Priya Hargunani 1, Nikhil Tadge 1, Mariangela Ceruso 2, Janis Leitans 3, Andris Kazaks 3,
Kaspars Tars 3, Paola Gratteri 2, Claudiu T. Supuran 2 , Alessio Nocentini 2,
*
and
Mrunmayee P. Toraskar 1,
*
1
Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai 400 614, India; phargunani@yahoo.in (P.H.);
niktadge@gmail.com (N.T.)
Neurofarba Dept., Section of Pharmaceutical and Nutraceutical Sciences, Universitá degli Studi di Firenze,
Via U. Schiff 6, Sesto Fiorentino, 50019 Florence, Italy; mariangela.ceruso@unifi.it (M.C.);
paola.grateri@unifi.it (P.G.); claudiu.supuran@unifi.it (C.T.S.)
Latvian Biomedical Research and Study Center, Ratsupites 1, LV-1067 Riga, Latvia;
janis.leitans@biomed.lu.lv (J.L.); andris@biomed.lu.lv (A.K.); kaspars@biomed.lu.lv (K.T.)
Correspondence: alessio.nocentini@unifi.it (A.N.); mrunmayeetoraskar@gmail.com (M.P.T.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 March 2020; Accepted: 6 April 2020; Published: 9 April 2020
Abstract: A series of new 3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide
derivatives was designed here, synthesized, and studied for carbonic anhydrase (CAs, EC 4.2.1.1)
inhibitory activity against the human (h) isozymes I, II, and VII (cytosolic, off-target isoforms), and IX
and XII (anticancer drug targets). Generally, CA I was not effectively inhibited, whereas effective
inhibitors were identified against both CAs II (KIs in the range of 5.2–233 nM) and VII (KIs in the
range of 2.3–350 nM). Nonetheless, CAs IX and XII were the most susceptible isoforms to this class of
inhibitors. In particular, compounds bearing an unsubstituted phenyl ring at the pyrazoline 3 position
showed 1.3–1.5 nM KIs against CA IX. In contrast, a subset of derivatives having a 4-halo-phenyl at the
same position of the aromatic scaffold even reached subnanomolar KIs against CA XII (0.62–0.99 nM).
Docking studies with CA IX and XII were used to shed light on the derivative binding mode driving
the preferential inhibition of the tumor-associated CAs. The identified potent and selective CA IX/XII
inhibitors are of interest as leads for the development of new anticancer strategies.
Keywords: sulfonamide; pyrazoline; human carbonic anhydrase; inhibition; selectivity; docking
1. Introduction
Carbon dioxide (CO2) plays a vital role in all life processes. It exists in equilibrium with bicarbonate,
but the interconversion is a slow process [
that catalyze this reversible hydration reaction of carbon dioxide to bicarbonate and protons [1,2
1
]. Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes
]. As a
result, CAs play an important role in respiration, pH and CO2 homeostasis, electrolyte secretion in
a variety of tissues and organs, biosynthetic reactions (such as gluconeogenesis and lipid and urea
synthesis), bone resorption, tumorigenicity, and many other physiological or pathological processes [
Fifteen different -carbonic anhydrase isoforms have been isolated and characterized in humans and
many of them are well-established therapeutic targets to treat a wide range of disorders [ ]. Based
on cellular and sub-cellular location, CAs are classified into four different groups: Cytosolic (CA I,
3].
α
4
Int. J. Mol. Sci. 2020, 21, 2621; doi:10.3390/ijms21072621
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 2621
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II, III, VII, XIII), mitochondrial (CA VA, VB), secretory (CA VI), and membrane-associated (CA IV,
IX, XII, XIV, XV) [5]. The active site of CA is a deep cleft, on one side of which only hydrophobic
residues are present, whereas hydrophilic residues line the opposite side. The Zn2+ ion is coordinated
tetrahedrally by three imidazoles of conserved histidine residues (His94, His96 and His119), and a
water molecule/hydroxide ion is a fourth ligand [
known as the CAs associated with cancers, being expressed in a limited number of normal tissues [5–9
2
]. The membrane-bound CA IX and XII isoforms are
].
Solid tumors usually have a dynamic microenvironment characterized by acidic pH and low levels
of oxygen (i.e., hypoxia). For survival, cancer cells acquire different adaptive features, which are
most probably responsible for the development of invasive and metastatic phenotypes. As a response
to regional hypoxia, cancer cells obtain their need for high levels of ATP synthesis by switching
their metabolism from aerobic respiration to fermentative (anaerobic) glycolysis [10]. CA IX and
XII regulate tumor cell pH by maintaining an intracellular slightly alkaline pH (of 7.2–7.4) while
acidifying the extracellular pH (which arrives at 6.0–6.5), and this is exploited by the cancer cells
for survival and proliferation [6]. The hypoxic conditions in solid tumors induce overexpression
of CA IX and CA XII, which maintain the acidic tumor environment by catalyzing the reversible
hydration of tumor cell-generated CO2 into a bicarbonate anion (HCO3−) and a proton (H+) and then
trap H+ extracellularly to lower the pH [
5,6]. Hence, the overexpression of these isoforms contributes
to the increased acidification of the extracellular hypoxic environment in contrast to normal tissues.
Expression of both membrane-associated CAs is induced by hypoxia in breast tumors and in several
cancer cell lines [7,8]. CA XII expression in breast tumors is indicative of lower grade disease, lower
relapse rates, and better overall patient survival [
associated with poor prognosis [9,10].
9]. In contrast, CA expression in brain tumors is
Although inhibition of hCAs by aromatic and heterocyclic sulfonamides has been largely studied
for 50 years with applications in the treatment of various disorders [ ], these classes of CA inhibitors
(CAIs) remain an attractive chemical family in the design of new inhibitors, as shown by the
2,4
ongoing research in this field [11–
13]. The primary sulfonamide group binds to the Zn2+ ion in
the CA enzyme active site as an anion (SO2NH−) blocking the catalysis [11]. A wealth of recent
studies has shown that the presence of urea as a linker in sulfonamide-containing compounds
is beneficial to generating effective CA inhibitory activity [14,15]. In the present study, novel
3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide derivatives were
designed and synthesized on the basis of sulfonyl semicarbazides and iminoureido CAIs previously
synthesized in our laboratory [16,17]. In vitro inhibition studies were carried out for all newly
synthesized compounds against CA I, II, VII, IX, and XII. The synthesized derivatives were thoroughly
characterized by 1H-NMR, 13C-NMR, IR, and MS, and their purity was checked by HPLC.
2. Results and Discussion
2.1. Chemistry
In the present work, a new series of 3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxamide (
reaction of sulfanilamide with sodium cyanate in ethanol and water, which was further refluxed
with hydrazine hydrate in ethanol to obtain 4-aminosulphonylphenyl semicarbazide . Various
chalcones of the general formula were prepared by Claisen–Schmidt condensation of the appropriate
4-chloro/bromophenyl acetophenone with substituted aromatic/heteroaromatic aldehydes in the
presence of NaOH. Cyclization of semicarbazide with chalcones in the presence of a base afforded the
corresponding 3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide ( 25
(Scheme 1), accomplishing the chemical hybridization of sulfanilamide with pyrazoline. The chemical
structures of titled compounds 25 were established on the basis of spectroscopic analysis. In detail,
compounds 25 were characterized by the presence of the three prominent doublets of doublets, each
having the integration of one proton. Formation of the pyrazoline was confirmed by the presence of a
5–25) is reported. Initially, 4-ureidobenzenesulfonamide 2 was obtained from
3
4
3
5
–
)
5–
5–

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doublet of doublets for CH2 at C4 giving a signal at
δ
3.26–3.13 and
δ
3.93–3.83 ppm, and CH at C5
giving a signal at
δ 5.54–5.48 ppm. The general procedures for the synthesis and spectral data are
described in the Experimental Section.
O
Ar
NH2
SO2
R1
NH2
SO2NH2
SO2
Ar
O
4
H
a
c
b
R1
N
N
N
H
NH2
SO2
HN
NH2
NH2
HN
N
NH2
5-25
O
O
3
1
2
Scheme 1. Synthesis of compounds 5–25. Reagents and conditions: a. NaCNO, glacial AcOH; b.
NH2NH2.H2O, EtOH, reflux, 27 h; c. EtOH, KOH, reflux, 2–3 h, overnight stirring.
2.2. Carbonic Anhydrases Inhibition
Sulfonamides 5–25 were evaluated for their inhibition against the cytosolic CA I, II, and VII
and the membrane-bound CA IX and XII by using a stopped-flow CO2 hydrase assay method [18].
The clinically used acetazolamide (AAZ) was used as standard drug in the kinetic evaluation. The
following SAR can be worked out from the data reported in Table 1.
Table 1. Inhibition data of human carbonic anhydrase (CA) isoforms I, II, VII, IX, and XII with
sulfonamide derivatives 5–25 reported here and the standard sulfonamide inhibitor acetazolamide
(AAZ) by a stopped-flow CO2 hydrase assay.
Ar
H
N
R
N
N
O
SO2NH2
5-25
KI (nM)
CA VII
33.2
7.7
Cmpd
R
Ar
CA I
382
CA II
67.3
8.2
CA IX
1.3
1.4
1.5
1.5
1.4
1.3
1.4
16.5
2.9
11.4
2.7
8
CA XII
7.7
5
6
H
H
C6H5
4-Cl-C6H4
4-F-C6H4
36.2
252
1.2
7
H
58.2
5.2
2.3
6.3
8
H
2-Cl-6-F-C6H3
thiophen-2-yl
anthracene-9-yl
2,4-diCl-C6H3
C6H5
38.3
185
2.4
1.4
9
H
5.2
1.9
2.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
AAZ
H
293
7.1
2.4
6.8
H
39.5
239
5.4
2.5
0.84
9.2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
233
12.3
12.7
71.3
8.8
350
4.5
4-CH3-C6H4
4-Cl-C6H4
4-F-C6H4
66.7
237.9
616.7
296.6
769.6
726
9.2
3.3
1.1
3.2
1.0
2-Cl-C6H4
2,4-diCl-C6H3
thiophen-2-yl
2-Cl-6-F-C6H3
2-OH-C6H4
4-OCH3-C6H4
3,4-diOCH3-C6H3
C6H5
4.3
1.7
13.5
9.2
27.6
2.9
15
1.3
2.5
2.7
76.6
2.8
2.2
1.4
14.3
1.4
25
0.62
6.8
42.7
74.2
367.7
165.9
44.4
45.5
38.2
250
10.8
7.6
2.3
2.1
0.99
0.82
1.5
9.4
2.7
9.3
2.3
6.3
2.9
6.7
4-Cl-C6H4
4-F-C6H4
10.7
5.5
2.7
0.89
2.4
2.6
-
12.1
2.5
5.8

Int. J. Mol. Sci. 2020, 21, 2621
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CA I was the least inhibited isoform by compounds 5–25 with inhibition constants (KIs) ranging
between 36.2 and 769.6 nM. The 4-Br substitution as R (23–25) stood out as the most efficient for
providing <100 nM KIs (i.e., 38.2–45.5 nM), though a comparison can be made on a limited set of Ar
substituents. By contrast, when analog Ar groups are considered, a H atom (5–11) produced better CA
I inhibition than a chlorine atom (12–22).
The most physiologically relevant isoform, CA II, was efficiently inhibited by most derivates with
KIs nearby or even below 10 nM (5.2–13.5 nM), except for 5, 7, 12, and 15. The phenyl derivative 12
induced significantly worse CA II inhibition than most of its relative compounds (KI of 233 nM).
The last tested cytosolic CA, isoform VII, was even more affected than CA II, with the KIs
compacting in a very narrow range spanning between 2.3 and 7.7 nM, comparably to the standard
AAZ (KI of 2.5 nM). Compounds 5 and 17 (KIs of 33.2 and 27.6 nM) reported a 10-fold worse CA VII
inhibition, whereas a drop in efficacy was measured for the 4-Cl derivative 12 (KI of 350 nM), which
also exhibited a reduced action against CA II when compared to the other derivatives.
Undoubtedly, the activity of the tumor-associated CAs IX and XII was the most susceptible to
inhibition by the sulfonamides 5–25 reported here. Most derivatives showed an extremely narrow
range of inhibition of CA IX as KIs spanned in the range 1.3–2.9 nM. Again, compound 12 together
with 14 and 24 exhibited slightly increased KIs (11.4–16.5 nM), whereas the 2-hydroxy derivative 20
turned out as the less active CA IX inhibitor in the study. It should be noted that compounds bearing an
unsubstituted phenyl ring at the pyrazoline 3 position showed the best KIs against CA IX (1.3–1.5 nM).
Even subnanomolar inhibition was achieved against CA XII by some screened compounds (KIs of
0.62–0.99 nM), where another number oddly showed KIs settling around 1 nM. It is interesting to
note that subnanomolar inhibitions are achieved uniquely by derivatives having a 4-halo-phenyl at
the 3 position of the aromatic scaffold. The less active inhibitors 12 and 13 exhibited a KI of 7.7 nM.
Further discussion on the SAR cannot be produced, because of the extremely narrow trend of inhibition
measured for 5–25 against all CAs.
Derivatives 5–25 act as selective inhibitors against the tumor-associated CAs IX and XII over
CA II and mostly CA I, with a selectivity index even reaching four-digit values (e.g., CA I/XII SI of
compound 18).
2.3. Molecular Docking
The binding mode of sulfonamides 5–25 to CA IX (pdb 5FL4) [19] and XII (pdb 1JD0) [8], the
most susceptible isoforms to this class of CAIs, was predicted by docking. The effectiveness of the
adopted computational procedure was testified by a root-mean-square deviation (RMSD) of only 0.49
and 0.35 Å between docked and crystallographic poses of the ligands present in pdb 5FL4 and pdb
1JD0, respectively (Figure 1).
Figure 1. Superimposition of docked (orange and pink) and crystallographic (grey and green) poses of
(
A
) 5-(1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)thiophene-2-sulfonamide bound to CA IX in pdb 5FL4,
and (B) AAZ bound to CA XII in pdb 1JD0.

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The in silico simulation was extended to both enantiomers (R) and (S) for each ligand. Coordination
of the Zn ion from both isozymes occurs by the SO2NH− group of all ligands, which is further involved
in two H-bonds with the OH group and backbone NH of T199 (Figure 2). The aromatic core bearing
the SO2NH2 forms π-alkyl contacts with L198, H94, and V121.
Figure 2. Surface representation of (
) CA XII (pdb 1JD0) bound to (R)-18 (magenta) and (S)-18 (pink). Lipophilic and hydrophilic residues
are colored red and blue, respectively.
A) CA IX (pdb 5FL4) bound to (R)-6 (green) and (S)-6 (cyan) and
(B
Figures 3 and 4 depict the detailed interaction mode of both enantiomers of compound 6 (panel A),
18 (panel B), and 21 (panel C) superimposed within the active site of CA IX and XII, respectively. In
CA IX, the orientations of the phenyl and aryl groups respectively in position 3 and 5 of the pyrazoline
scaffold almost swap from the (R)- to (S)-enantiomer, and vice versa (Figure 3). In fact, the 3-phenyl
portion in the (R)-enantiomer and the 5-aryl moiety in the (S)-enantiomer accommodate in the cleft
region lined by V131, L91, and Q92. Contrariwise, the 3-phenyl portion in the (S)-enantiomer and the
5-aryl moiety in the (R)-enantiomer orient toward the edge between the hydrophilic and lipophilic
regions of CA IX binding clefts (P201, P202, H64, and W5).
Figure 3. Docked binding orientations of (
A
)
(R)-6 (green) and (S)-6 (cyan), (
B) (R)-18 (magenta) and
(S)-18 (pink), and (C) (R)-21 (tan) and (S)-21 (gold) to CA IX (pdb 5FL4). H-bonds are represented as
dashed lines.
Similarly, an almost opposite binding orientation was found for the molecular tail of the (R)- and
(S)-enantiomers of derivative 18 when bound to CA XII (Figure 4B). By contrast, by a 120◦ rotation of
the pyrazoline orientation, the 3-phenyl groups from the two enantiomers of compounds
6 and 21,
as well as their 5-aryl groups, occupy the same area within the active site of CA XII (Figure 4A,C).
It can be speculated that the markedly less lipophilic character of the “hydrophobic region” of the
CA XII active site with respect to CA IX (Figure 2) attracts the most hydrophilic pendant between

Int. J. Mol. Sci. 2020, 21, 2621
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those at the pyrazoline position 3 and 5, in the case where a significant difference among them exists
(e.g., compounds 6 and 21).
It can also be noted that the ureido carbonyl group of the (R)-enantiomers can accept a H-bond
from Q92 side chain CONH2 within both CAs active sites (Figures 3 and 4).
Figure 4. Docked binding orientations of (
A
)
(R)-6 (green) and (S)-6 (cyan), (
B) (R)-18 (magenta) and
(S)-18 (pink), and (C) (R)-21 (tan) and (S)-21 (gold) to CA XII (pdb 1JD0). H-bonds are represented as
dashed lines.
The binding modes depicted in Figures 2–4 are expected to be hindered within the CA II active
site where the mutation of V/A131 to F131 significantly narrows the volume of the cavity. As a result,
the CA II inhibition efficiency of 5–25 drops when compared to the tumor-related CAs, and so that
against CA I does so because of the tight active site of such an isoform.
According to the docking scores computed for both optical isomers of compounds 5–25 reported in
Table 2, the role of eutomers might be ascribed to (S)-enantiomers in the binding to CA IX (GlideScore
–5.86–7.01 kcal/mol) and to (R)-enantiomers in the binding to CA XII (GlideScore –6.48–7.92 kcal/mol).
No further comparison can be made between Glide-predicted scores and KIs determined in vitro. In
fact, the GlideScore function is assumed to distinguish compounds that bind strongly from those that
do not, and compounds 5–25 all act as low nanomolar CA IX and XII inhibitors (and even nanomolar
CAIs against isoforms I and II). The docking scores computed for AAZ when bound within CA IX and
XII active sites are indeed comparable to those of the eutomers of 5–25.
Table 2. Docking score (Glide) of the predicted poses of (R)- and (S)-enantiomers of 5–25 and AAZ
bound to CA IX (pdb 5FL4) and CA XII (pdb 1JD0).
GlideScore (kcal/mol)
GlideScore (kcal/mol)
Cmpd
Cmpd
CA IX
−6.82
−6.54
−6.93
−6.28
−6.34
−7.01
−6.35
−5.86
CA XII
−6.02
−6.28
−5.63
−6.45
−6.63
−6.22
−5.96
−6.29
CA IX
−5.34
−5.67
−5.39
−5.38
−5.94
−5.68
−5.02
−5.49
CA XII
−6.84
−7.01
−6.49
−7.36
−7.57
−6.82
−7.69
−6.57
(S)-5
(S)-6
(R)-5
(R)-6
(R)-7
(R)-8
(R)-9
(R)-10
(R)-11
(R)-12
(S)-7
(S)-8
(S)-9
(S)-10
(S)-11
(S)-12

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Table 2. Cont.
GlideScore (kcal/mol)
CA IX
GlideScore (kcal/mol)
Cmpd
Cmpd
CA XII
−6.07
−6.38
−5.99
−6.23
−6.54
−6.08
−6.63
−6.41
−6.39
−6.28
−6.92
−6.47
−6.19
−7.35
CA IX
−5.79
−5.38
−5.46
−5.82
−5.09
−5.97
−5.01
−5.26
−6.03
−6.05
−5.65
−5.24
−5.37
CA XII
−7.03
−7.51
−7.36
−7.15
−7.64
−7.92
−7.29
−6.86
−7.27
−7.16
−6.48
−7.58
−7.22
(S)-13
(S)-14
(S)-15
(S)-16
(S)- 17
(S)- 18
(S)- 19
(S)- 20
(S)- 21
(S)- 22
(S)- 23
(S)- 24
(S)- 25
AAZ
−6.57
−6.02
−6.83
−6.12
−5.84
−6.14
−6.72
−5.67
−6.35
−6.92
−6.54
−6.24
−6.82
−6.31
(R)-13
(R)-14
(R)-15
(R)-16
(R)- 17
(R)- 18
(R)- 19
(R)- 20
(R)- 21
(R)- 22
(R)- 23
(R)- 24
(R)- 25
3. Conclusions
A series of new 3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide
derivatives was designed here, synthesized, and studied for hCA inhibitory activity against the
cytosolic, off-target isoforms I, II, and VII and the anticancer drug targets IX and XII. While CA I was
not effectively inhibited, effective inhibitors were identified against both CAs II (KIs in the range of
5.2–233 nM) and VII (KIs in the range of 2.3–350 nM). Nonetheless, CAs IX and XII were the most
susceptible isoforms to this class of inhibitors. In particular, compounds bearing an unsubstituted
phenyl ring at the pyrazoline 3 position showed 1.3–1.5 nM KIs against CA IX. By contrast, a subset
of derivatives having a 4-halo-phenyl at the same position of the aromatic scaffold even reached
subnanomolar KIs against CA XII (0.62–0.99 nM). Docking studies with CA IX and XII, performed on
both enantiomers of the ligands, showed the binding mode driving such a preferential inhibition of
the tumor-associated CAs. The identified potent and selective CA IX/XII inhibitors could represent
interesting leads for the development of new anticancer medications.
4. Experimental Section
4.1. Chemistry
All the reagents and solvents were obtained from commercial suppliers and were used as received
unless otherwise indicated. Solvents were dried, wherever necessary, according to standard procedures.
All reactions were performed under N2 atmosphere, unless otherwise indicated. Analytical silica gel
60 F254-coated TLC plates were purchased from Sigma-Aldrich and were visualized with UV light. IR
spectra (ATR) were recorded on a Quest ATR Diamond Accessory (Black) P31482 and Shimadzu 8100
1
infrared spectrophotometer. H-NMR was recorded at 300 MHz in DMSO-d6 solvents using TMS as an
internal reference standard at Sophisticated Analytical Instrument Facility (SAIF) IIT Powai, Mumbai.
Molecular ion peaks of synthesized compounds were recorded using HRLCMS at Laxai-Avanti Life
Sciences Pvt. Ltd. Hyderabad. Melting points were recorded using a Veego® (VMP)-D capillary
melting point apparatus (Veego Instruments Corp. Mumbai, India) and were uncorrected.

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4.1.1. Synthesis of 4-ureidobenzenesulfonamide 2
In a 250 mL beaker, sulfanilamide (0.03 mol) was dissolved in a mixture of glacial HOAc (13 mL)
and hot water (30 mL). A solution of sodium cyanate (0.05 moL) in hot water (25 mL) was added to
the above mixture with continuous stirring. It was allowed to stand for 30 min. The reaction mixture
was then cooled in an ice-bath, vacuum-filtered, dried, and recrystallized from EtOH. Yield: 70%; mp:
175 ◦C; IR (KBr) cm−1: 3462 (N–H str of NH2), 3365 (N–H str), 3227 (aromatic C–H str), 1693 (C=O str),
1533 (aromatic C=C str), 1409, 1321, 1155 (S=O str).
4.1.2. Synthesis of 4-aminosulfonylphenyl Semicarbazide 3
In a 250 mL round bottom flask, equimolar quantities of
1 (0.05 mol) and hydrazine hydrate
(0.05 mol) in EtOH (2.5 mL) were refluxed for 27 h. The progress of the reaction was monitored by
TLC using CHCl3: MeOH (8:2) as the mobile phase. The 2/3rd volume of EtOH was removed under
reduced pressure and the reaction mixture was then poured onto crushed ice. The resultant precipitate
was filtered, washed with H2O, and dried. The crude product was recrystallized from EtOH. Yield:
56%; mp: 207 ◦C; 1H NMR (300 MHz, DMSO-d6)
(
δ = 8.98 (s, 1H, CO-NH), 7.15 (s, 2H, SO2-NH2), 4.40
s, 2H, NH2), 7.60-7.68 (m, 5H, Ar-NH and C–H aromatic); IR (KBr) cm−1: 3365 (N-H str of NH2), 3281
(N-H str), 3074 (aromatic C-H str), 1676 (C=O str), 1521 (aromatic C=C str), 1408, 1301, 1153 (S=O str).
4.1.3. Synthesis of (E)-1-(4-substituted phenyl)-3-substituted Aromatic prop-2-en-1-one 4 [20,21]
Substituted acetophenone (0.01 mol) and various substituted aldehydes (0.01 mol) were mixed in
ethanol (40 mL) in a conical flask placed in an ice bath. To this, 60% NaOH solution (10 mL) was added
dropwise with continuous stirring for 30 min (in ice bath). The mixing was continued for another
2–3 h, maintaining the ice bath. The mixture was kept in a refrigerator overnight. Reaction completion
was confirmed by TLC (hexane:ethyl acetate = 2:1). Then, it was diluted with ice-cold water, filtered,
washed well with cold water, dried in air, and recrystallized from rectified methanol.
4.1.4. General Procedure for Synthesis of
3-phenyl-5-aryl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 5–25 [22]
A mixture of chalcones (E)-1-(substituted phenyl)-3-substituted aromatic prop-2-en-1-one
4
(
0.01 mol) and 4-aminosulfonylphenyl semicarbazide (0.02 mol) in 20 mL ethanol was refluxed for
3
2 h with stirring (heating mantle was used with a temperature of 70–80 ◦C). To this boiling mixture,
alcoholic KOH solution (10 mL, 60%) was added dropwise with continuous stirring for 30 min
(with refluxing at the same temperature: 70–80 ◦C). This mixture was refluxed further for 2 h and
then kept for overnight stirring at room temperature. The reaction was monitored by TLC using
methanol/chloroform (20:80). The resulting solution was poured on slush and the precipitate was
filtered and recrystallized from ethanol. Compounds
general procedure.
5–25 were synthesized according to the above
3,5-Diphenyl-4,5-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 5
Yield = 60%; m.p. 242 ◦C; Purity (HPLC): 95.2%; 1H NMR (300 MHz, DMSO d6)
δ
= 9.46 (s, 1H,
Ar-NH), 7.98–7.24 (m, 13H, Ar-H), 7.20 (S, 2H, SO2-NH2), 3.23–3.16 (dd, 1H, CHHa), 3.96–3.48 (dd, 1H,
CHHb), 5.58–5.53 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6):
= 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; Mass m/z: 421
M + 1)+; IR max (KBr): 3336, 3375 (NH str of NH2), 3279, (NH str), 3063 (Ar C-Hstr), 1674 (C=O str),
δ
(
1585 (C=N str).
5-(4-Chlorophenyl)-3-phenyl-4,5-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 6
Yield = 63%; m.p. 248 ◦C; Purity (HPLC): 88.55%; 1H NMR (300 MHz, DMSO d6)
Ar-NH), 7.98–7.14 (m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.29–3.17 (dd, 1H, CHHa), 3.95–3.84 (dd,
δ = 9.45 (s, 1H,

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1H, CHHb), 5.60–5.54 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6):
δ
= 153.13, 151.43, 142.75, 142.08,
140.61, 137.07, 132.43, 129.94, 129.03, 128.33, 128.01, 127.39, 126.89, 119.0, 60.09, 42.6; Mass m/z: 455
(M + 1)+; IR max (KBr): 3365 (NH str of NH2), 3232, (NH str), 3063 (Ar C-Hstr), 1685 (C=O str), 1587
(C=N str), 790 (C-Cl).
5-(4-Flurophenyl)-3-phenyl-4,5-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 7
Yield = 58%; m.p. 246 ◦C; Purity (HPLC): 100%; 1H NMR (300 MHz, DMSO d6)
Ar-NH), 7.97–7.27 (m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.25–3.17 (dd, 1H, CHHa), 3.95–3.85 (dd,
1H, CHHb), 5.59–5.53 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6):
= 160.61, 152.86, 151.42, 142.78,
δ = 9.46 (s, 1H,
δ
134.32, 131.48, 130.57, 128.98, 128.18, 128.1, 127.39, 126.87, 118.87, 115.91, 60.01, 42.7; Mass m/z: 439
(M + 1)+; IR max (KBr): 3335 (NH str of NH2), 3234, (NH str), 3059 (Ar C-Hstr), 1683 (C=O str), 1585
(C=Nstr), 1411 (Ar-F).
5-(2-Chloro-6-fluorophenyl)-3-phenyl-4, 5-dihydro-pyrazole-1-carboxylic acid
(4-sulfamoyl-phenyl)-amide 8
Yield = 58%, m.p. 244 ◦C, Purity (HPLC): 97.55%; 1H NMR (300 MHz, DMSO d6)
Ar-NH), 7.97–7.23 (m, 11H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.41–3.28 (dd, 1H, CHHa), 3.96–3.86 (dd,
1H, CHHb), 6.04–5.97 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6):
= 152.96, 152.87, 151.36, 142.88,
142.39, 137.74, 134.62, 130.53, 129.12, 127.39, 127.08, 126.88, 126.6, 119.04, 116.08; Mass m/z: 473 (M + 1)+
δ = 9.41 (s, 1H,
δ
;
IR max (KBr): 3389, 3327 (NH str of NH2), 3240, (NH str), 3069 (Ar C-Hstr), 1685 (C=O str), 1585 (C=N
str), 1410 (C-F), 790 (C-Cl).
3-Phenyl-5-thiophene-2-yl-4, 5-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 9
Yield = 56%; m.p. 249 ◦C; Purity (HPLC): 95.23%; 1H NMR (300 MHz, DMSO d6)
Ar-NH), 8.01–6.96 (m, 12H, Ar-H), 7.21 (s, 2H, SO2-NH2), 3.6–3.4 (dd, 1H, CHHa), 4.0–3.88 (dd, 1H,
δ = 9.44 (s, 1H,
CHHb), 6.0–5.9 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6):
δ = 153.04, 151.44, 142.83, 142.58, 137.89,
129.02, 128.63, 128.08, 127.86, 127.17, 126.95, 119.07, 56.24, 42.53; Mass m/z: 427 (M + 1)+; IR max (KBr):
3362, 3327 (NH str of NH2), 3279, (NH str), 3063 (Ar C-Hstr), 1685 (C=O str), 1585 (C=N str), 711 (C-S).
5-Anthracene-9-yl-3-phenyl-4,5-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 10
Yield = 58%; m.p. 252 ◦C; Purity (HPLC): 94.64%; 1H NMR (300 MHz, DMSO d6)
Ar-NH), 8.71-7.38 (m, 18H, Ar-H), 7.14 (s, 2H, SO2-NH2), 3.57–3.47 (dd, 1H, CHHa), 4.22–4.12 (dd, 1H,
CHHb), 7.08–7.01 (dd, 1H, CHHX); 13C NMR (100 MHz, DMSO-d6):
= 153.23, 152.18, 142.9, 140.96,
δ = 9.56 (s, 1H,
δ
137.69, 132.01, 131.54, 130.75, 129.26, 128.39, 127.88, 127.51, 126.97, 126.42, 125.35, 125.21, 124.31, 118.73,
56.71, 42.03; IR max (KBr): 3358, 3294 (NH str of NH2), 3223, (NH str), 3080 (Ar C-Hstr), 1680 (C=O str),
1587 (C=N str).
5-(2,4-Dichlorophenyl)-3-phenyl-4, 5-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 11
Yield = 68%; m.p. 168–170 ◦C; Purity (HPLC): 100%; 1H NMR (300 MHz, DMSO d6)
δ = 9.38 (s, 1H,
Ar-NH), 7.97–7.32 (m, 12H, Ar-H), 7.16 (s, 2H, SO2-NH2), 3.24-3.18 (dd, 1H, CHHa), 3.95–3.88 (dd,
1H, CHHb), 5.75–5.70 (dd, 1H, CHHX); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; IR (ATR) cm−1
:
3396, 3315 (N-H str of NH2), 3240 (N-H str), 3070 (aromatic C-Hstr), 1685 (C=O str), 1589 (C=N str),
1518 (aromatic C=C str), 1491, 1340, 1313, 1234, 1153 (S=O str), 756 (C-S str), 825 (aromatic C-Cl str).
3-(4-Chlorophenyl)-5-phenyl-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 12
Yield = 75%; m.p. 216–218 ◦C; Purity (HPLC): 98.9%; 1H NMR (300 MHz, DMSO-d6)
δ = 9.46
(s, 1H, Ar-NH), 7.98–7.24 (m, 13H, Ar-H), 7.20 (S, 2H, SO2-NH2), 3.23–3.16 (dd, 1H, CHHa), 3.96–3.48
(dd, 1H, CHHb), 5.58–5.53 (dd, 1H, CH);13C NMR (75 MHz, DMSO-d6): 152.8, 151.4, 143.2, 142.9, 142.8,

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137.6, 131.5, 130.5, 128.9, 128.3, 127.6, 127.3, 126.9, 118.8, 60.6, 42.8; MS (m/z): 455 (M + 1)+; IR (ATR)
cm−1: 3406, 3306 (N-H str of NH2), 3234 (N-H str), 3166, 3066, 3032 (aromatic C-Hstr), 1684 (C=O str),
1589 (C=N str), 1518 (aromatic C=C str) 1415, 1340, 1155 (S=O str), 754 (C-S str), 698 (aromatic C-Cl str).
3-(4-Chlorophenyl)-N-(4-sulfamoylphenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 13
Yield = 70%; m.p. 220–221 ◦C; 1H NMR (300 MHz, DMSO d6)
δ = 9.44 (s, 1H, Ar-NH), 7.97–7.19
(m, 12H, Ar-H), 7.14 (s, 2H, SO2-NH2), 2.34(s, 3H, C-H of alkane), 3.26–3.13 (dd, 1H, CHHa), 3.93–3.83
(dd, 1H, CHHb), 5.54–5.48 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; MS (m/z): 469
(M + 1)+; IR (ATR) cm−1: 3342 (N-H str of NH2), 3250 (N-H str), 3198, 3082, 2926 (aromatic C-Hstr),
1680 (C=O str), 1585 (C=N str), 1519 (aromatic C=C str), 1417, 1317, 1234, 1151 (S=O str), 736 (C-S str),
815 (aromatic C-Cl str).
3,5-Bis(4-chlorophenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide 14
Yield = 68%; m.p. 182–183 ◦C; Purity (HPLC): 96.17%; 1H NMR (300 MHz, DMSO d6)
δ = 9.45
(s, 1H, Ar-NH), 7.98–7.14 (m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.29–3.17 (dd, 1H, CHHa), 3.95–3.84
(dd, 1H, CHHb), 5.60–5.54 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; MS (m/z): 489
(M)+; IR (ATR) cm−1: 3354 (N-H str of NH2), 3259 (N-H str), 3097, 2926 (aromatic C-Hstr), 1689
(C=O str), 1587 (C=N str), 1525 (aromatic C=C str), 1406, 1315, 1236, 1136 (S=O str), 732 (C-S str), 817
(aromatic C-Cl str).
3-(4-Chlorophenyl)-5-(4-fluorophenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxamide 15
Yield = 72%; m.p. 198–200 ◦C; 1H NMR (300 MHz, DMSO d6)
δ = 9.46 (s, 1H, Ar-NH), 7.97–7.27
(m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.25–3.17 (dd, 1H, CHHa), 3.95-3.85 (dd, 1H, CHHb), 5.59–5.53
(dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93, 142.83, 137.67, 131.51,
130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; MS (m/z): 473 (M + 1)+; IR (ATR) cm−1
:
3390, 3306 (N-H str of NH2), 3234 (N-H str), 3074, 2939 (aromatic C-Hstr), 1683 (C=O str), 1589 (C=N
str), 1518 (aromatic C=C str), 1413, 1340, 1313, 1219, 1155 (S=O str), 738 (C-S str), 817 (aromatic C-Cl
str), 1087 (C-F str).
5-(2-Chlorophenyl)-3-(4-chlorophenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxamide 16
1
Yield = 65%; m.p. 160–161 ◦C; Purity (HPLC): 100%; H NMR (300 MHz, DMSO d6)
δ = 9.54
(s, 1H, Ar-NH), 7.97–7.32 (m, 12H, Ar-H), 7.21 (s, 2H, SO2-NH2), 3.27–3.10 (dd, 1H, CHHa), 4.04–3.94
(dd, 1H, CHHb), 5.82–5.78 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; MS (m/z): 489
(M)+; IR (ATR) cm−1: 3335 (N-H str of NH2), 3250 (N-H str), 3078, 2935 (aromatic C-H str), 1664
(C=O str), 1587 (C=N str), 1533 (aromatic C=C str), 1421, 1317, 1236, 1149 (S=O str), 734 (C-S str), 833
(aromatic C-Cl str).
3-(4-Chlorophenyl)-5-(2,4-dichlorophenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxamide 17
Yield = 70%; m.p. 180–182 ◦C; Purity (HPLC): 97.26%; 1H NMR (300 MHz, DMSO d6)
δ = 9.53
(s, 1H, Ar-NH), 7.96–7.30 (m, 12H, Ar-H), 7.21 (s, 2H, SO2-NH2), 3.25–3.12 (dd, 1H, CHHa), 4.03–3.46
(dd, 1H, CHHb), 5.81–5.75 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; IR (ATR) cm−1
:
3336 (N-H str of NH2), 3248 (N-H str), 3086, 2928 (aromatic C-H str), 1687 (C=O str), 1589 (C=N str),
1521 (aromatic C=C str), 1404, 1317, 1238, 1153 (S=O str), 732 (C-S str), 817 (aromatic C-Cl str).

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3-(4-Chlorophenyl)-N-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-
1-carboxamide 18
Yield = 75%; m.p. 174–175 ◦C; Purity (HPLC): 93.71%; 1H NMR (300 MHz, DMSO d6)
δ
= 9.44
(s, 1H, Ar-NH), 8.01–6.96 (m, 12H, Ar-H), 7.21 (s, 2H, SO2-NH2), 3.43–3.27 (dd, 1H, CHHa), 3.86–3.88
(dd, 1H, CHHb), 5.90–5.85 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; MS (m/z): 461
(M + 1)+; IR (ATR) cm−1: 3352-3342 (N-H str of NH2), 3223 (N-H str), 3101, 3014, 2914 (aromatic
C-Hstr), 1678 (C=O str), 1587 (C=N str), 1514 (aromatic C=C str), 1404, 1315, 1238, 1153 (S=O str), 689
(C-S str), 825 (aromatic C-Cl str).
5-(2-Chloro-6-fluorophenyl)-3-(4-chlorophenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxamide 19
1
Yield = 70%; m.p. 182–184 ◦C; Purity (HPLC): 100%; H NMR (300 MHz, DMSO d6)
δ = 9.41
(s, 1H, Ar-NH), 7.97–7.23 (m, 11H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.41–3.28 (dd, 1H, CHHa), 3.96–3.86
(dd, 1H, CHHb), 6.04–5.97 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81, 151.48, 143.23, 142.93,
142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63, 42.83; MS (m/z): 507
(M)+; IR (ATR) cm−1: 3387, 3352 (N-H str of NH2), 3252 (N-H str), 3084, 2962 (aromatic C-Hstr), 1678
(C=O str), 1587 (C=N str), 1525 (aromatic C=C str), 1456, 1406, 1317, 1238, 1149 (S=O str), 738 (C-S str),
827 (Aromatic C-Cl str), 1087 (C-F str).
3-(4-Chlorophenyl)-5-(2-hydroxyphenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxamide 20
Yield = 60%; m.p. 182–184 ◦C; Purity (HPLC): 100%; 1H NMR (300 MHz, DMSO d6)
δ = 10.02
(s, 1H, ArOH), 9.21 (s, 1H, Ar-NH), 7.97–7.45 (m, 12H, Ar-H), 7.23 (s, 2H, SO2-NH2), 3.24–3.06 (dd,
1H, CHHa), 3.96-3.72 (dd, 1H, CHHb), 5.66–5.45 (dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6): 152.81,
151.48, 143.23, 142.93, 142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63,
42.83; IR (ATR) cm−1:3217 (O-H str), 3352 (N-H str of NH2), 3282 (N-H str), 2993 (aromatic C-Hstr),
1680 (C=O str), 1585 (C=N str), 1531 (aromatic C=C str), 1460, 1406, 1311 (S=O str), 1236 (aromatic
C–OHstr), 754 (C-S str), 825 (Aromatic C-Cl str).
3-(4-Chlorophenyl)-5-(4-methoxyphenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxamide 21
1
Yield = 80%; m.p. 188–190 ◦C; Purity (HPLC): 100%; H NMR (300 MHz, DMSO d6)
δ = 9.42
(s, 1H, Ar-NH), 7.97–6.88 (m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.30–3.14 (dd, 1H, CHHa), 3.91–3.80
(dd, 1H, CHHb),5.32–5.47 (dd, 1H, CH)3.71 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO-d6): 152.81,
151.48, 143.23, 142.93, 142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83, 60.63,
42.83; IR (ATR) cm−1:3358, 3315 (N-H str of NH2), 3205 (N-H str), 3099, 2966, 2837 (aromatic C-Hstr),
1670 (C=O str), 1587 (C=N str), 1514 (aromatic C=C str), 1406, 1319, 1242, 1155 (S=O str), 727 (C-S str),
825 (aromatic C-Cl str).
3-(4-Chlorophenyl)-5-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxamide 22
Yield = 62%; m.p. 210–211 ◦C; Purity (HPLC): 81.93%; 1H NMR (300 MHz, DMSO d6)
δ = 9.44
(s, 1H, Ar-NH), 7.97–6.72 (m, 11H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.29–3.16 (dd, 1H, CHHa), 3.91–3.78
(dd, 1H, CHHb), 5.52–5.46 (dd, 1H, CH) 3.72, 3.71 (s, 6H, 2OCH3); 13C NMR (75 MHz, DMSO-d6):
152.81, 151.48, 143.23, 142.93, 142.83, 137.67, 131.51, 130.53, 128.97, 128.32, 127.69, 127.36, 126.9, 118.83,
60.63, 42.83; IR (ATR) cm−1: 3352 (N-H str of NH2), 3254 (N-H str), 3093, 3009, 2941, 2837 (aromatic
C-H str), 1681 (C=O str), 1587 (C=N str), 1510 (aromatic C=C str), 1419, 1315, 1261, 1153 (S=O str), 736
(C-S str), 802 (aromatic C-Cl str).

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3-(4-Bromophenyl)-5-phenyl-45-dihydro-pyrazole-1-carboxylic acid (4-sulfamoyl-phenyl)-amide 23
Yield = 60%; m.p. 243 ◦C; 1H NMR (300 MHz, DMSO d6)
= 9.4–9.6 (s, 1H, Ar-NH), 7.98–7.24
δ
(m, 13H, Ar-H), 7.20 (S, 2H, SO2-NH2), 3.23–3.16 (dd, 1H, CHHa), 3.96-3.8 (dd, 1H, CHHb), 5.6–5.53
(dd, 1H, CH); IR max (KBr): 3404, 3363 (NH str of NH2), 3232, (NH str), 3091 (Ar C-Hstr), 1683 (C=O
str), 1587 (C=N str).
3-(4-Bromophenyl)-5-(4-chloro phenyl)-4,5-dihydro-pyrazole-1-carboxylic acid
(4-sulfamoyl-phenyl)-amide 24
Yield = 62%; m.p. 247 ◦C; Purity (HPLC): 96.38%; 1H NMR (300 MHz, DMSO d6)
Ar-NH), 7.98–7.14 (m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.29–3.17 (dd, 1H, CHHa), 3.95–3.84 (dd,
1H, CHHb), 5.60–5.54 (dd, 1H, CH); 13C NMR (100 MHz, DMSO-d6):
= 151.8, 151.42, 142.74, 141.83,
δ = 9.45 (s, 1H,
δ
140.61, 137.77, 132.55, 130.6, 129.18, 128.96, 127.92, 126.87, 118.91, 60.29, 42.46; IR max (KBr): 3362, 3300
(NH str of NH2), 3230, (NH str), 3088 (Ar C-Hstr), 1683 (C=O str), 1585 (C=N str).
3-(4-Bromo phenyl)-5-(4-fluro phenyl)-4, 5-dihydro-pyrazole-1-carboxylic acid
(4-sulfamoyl-phenyl)-amide 25
◦
1
Yield = 59%; m.p. 245 C; H NMR (300 MHz, DMSO d6)
(m, 12H, Ar-H), 7.20 (s, 2H, SO2-NH2), 3.25–3.17 (dd, 1H, CHHa), 3.95–3.85 (dd, 1H, CHHb), 5.59–5.53
(dd, 1H, CH); 13C NMR (75 MHz, DMSO-d6):
= 160.64, 153.29, 151.81, 143.5, 142.76, 137.85, 136.82,
δ = 9.46 (s, 1H, Ar-NH), 7.97–7.27
δ
129.64, 129.18, 128.71, 128.11, 126.85, 118.99, 115.79, 60.22, 42.55; IR max (KBr): 3362, 3294 (NH str of
NH2), 3232, (NH str), 3082 (Ar C-Hstr), 1680 (C=O str), 1587 (C=N str), 1411 (C-F).
4.2. CA Inhibition
An Applied Photophysics stopped-flow instrument was used for assaying the CA catalyzed CO2
hydration activity [18]. Phenol red (at a concentration of 0.2 mM) was used as an indicator, working
at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as a 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 were 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 distilled-deionized water and dilutions up to 0.01 nM were
performed 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 [23,24], and represent the mean from at least three different
determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier [25–27].
4.3. Molecular Docking
The crystal structures of CA IX (pdb 5FL4) [19] and XII (pdb 1JD0) [8] were prepared using the
Protein Preparation Wizard tool implemented in Maestro-Schrödinger suite, assigning bond orders,
adding hydrogens, deleting water molecules, and optimizing H-bonding networks [28]. An energy
minimization protocol with a root-mean-square deviation (RMSD) value of 0.30 was applied using an
Optimized Potentials for Liquid Simulation (OPLS3e) force field. Three-dimensional ligand structures
were prepared by Maestro [28] and evaluated for their ionization states at pH 7.4
±
0.5 with Epik [28].
The OPLS3e force field in Macromodel [28] was used for energy minimization for a maximum number
of 2500 conjugate gradient iterations and setting a convergence criterion of 0.05 kcal mol−1Å−1. The
docking grid was centered on the center of mass of the co-crystallized ligands and Glide was used with
default settings. Ligands were docked with the standard precision mode (SP) of Glide [28] and the best

Int. J. Mol. Sci. 2020, 21, 2621
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5 poses of each molecule were retained as output. The best pose for each compound, evaluated in terms
of coordination, hydrogen bond interactions, and hydrophobic contacts, was refined with Prime [28
with a VSGB solvation model considering the target flexible within 3 Å around the ligand [29–31].
]
Author Contributions: P.H. synthesized and characterized the compounds; N.T. synthesized and characterized
the compounds; M.C. assays the compounds in vitro; J.L. prepared the enzymes used in the kinetic assays; A.K.
prepared the enzymes used in the kinetic assays; K.T. supervised the enzymes preparation; P.G. supervised the
computational study; C.T.S. participated in funding acquisition and supervision; A.N. worked on in silico data,
interpretation, writing, review and editing of the ms; M.P.T. participated in data interpretation, writing, and
funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: MIUR is gratefully acknowledged for the grant PRIN 2017XYBP2R.
Conflicts of Interest: The authors declare no conflict of interest.
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