Synthesis, biological evaluation and in silico modelling studies of 1,3,5-trisubstituted pyrazoles carrying benzenesulfonamide as potential anticancer agents and selective cancer-associated hCA IX isoenzyme inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103222

Inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has clinical importance for the treatment of several diseases. They participate in crucial regulatory mechanisms for balancing intracellular and extracellular pH of the cells. Among CA isoforms, selectiv

Full text of DOI:10.1016/j.bioorg.2019.103222

Bioorganic Chemistry 92 (2019) 103222
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, biological evaluation and in silico modelling studies of 1,3,5-
trisubstituted pyrazoles carrying benzenesulfonamide as potential
anticancer agents and selective cancer-associated hCA IX isoenzyme
inhibitors
a
a,⁎
b
c
c
d
Cem Yamali , Halise Inci Gul , Abdulilah Ece , Silvia Bua , Andrea Angeli , Hiroshi Sakagami ,
e
c
Ertan Sahin , Claudiu T. Supuran
b
c
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Biruni University, Istanbul, Turkey
Neurofarba Department, Sezione di Scienza Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
Meikai University Research Institute of Odontology (M-RIO), Sakado, Saitama 350-0283, Japan
d
e
Department of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has clinical importance for the treatment of several diseases.
They participate in crucial regulatory mechanisms for balancing intracellular and extracellular pH of the cells.
Among CA isoforms, selective inhibition of hCA IX has been linked to decreasing of cell growth for both primary
tumors and metastases. The discovery of novel CA inhibitors as anticancer drug candidates is a current topic in
medicinal chemistry. 1,3,5-Trisubstituted pyrazoles carrying benzenesulfonamide were evaluated against phy-
siologically abundant cytosolic hCA I and hCA II and trans-membrane, tumor-associated hCA IX isoforms by a
Pyrazole
Carbonic anhydrase
hCA IX
Cytotoxicity
Anticancer
Sulfonamide
Molecular modelling
stopped-flow CO hydrase method. Their in vitro cytotoxicities were screened against human oral squamous cell
2
carcinoma (OSCC) cell lines (HSC-2) and human mesenchymal normal oral cells (HGF) via 3-(4,5-
Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) test. Compounds 6, 8, 9, 11, and 12 showed low
nanomolar hCA II inhibitory potency with Ki < 10 nM, whereas compounds 9 and 12 displayed Ki < 10 nM
against hCA IX isoenzyme when compared with reference Acetazolamide (AZA). Compound 9, 4-(3-(hy-
drazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benzenesulfonamide, can be considered as the most selec-
tive hCA IX inhibitor over off-target cytosolic isoenzymes hCA I and hCA II with the lowest Ki value of 2.3 nM
and selectivity ratios of 3217 (hCA I/hCA IX) and 3.9 (hCA II/hCA IX). Isoform selectivity profiles were also
discussed using in silico modelling. Cytotoxicity results pointed out that compounds 5 (CC50 = 37.7 μM) and 11
(
CC50 = 58.1 μM) can be considered as lead cytotoxic compounds since they were more cytotoxic than 5-
Fluorouracil (5-FU) and Methotrexate (MTX).
1
. Introduction
Main classes of CA inhibitors (CAIs) are comprised of primary sul-
fonamides and their bioisosteres such as sulfamates, sulfamides, etc.,
+2
Inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has clinical
which bind to the Zn
ion of the enzyme to generate a tetrahedral
importance for the treatment of several diseases such as glaucoma,
epilepsy, obesity, and gastric ulcers [1–3]. More recently, numerous
studies reported that CA inhibition has an important role in cancer-
ogenesis and tumor metastasis [4–7]. CAs are metalloenzymes present
adduct as zinc-binding groups. Furthermore, their scaffolds participate
in other favorable molecular interactions with amino acid residues lo-
cated in the active site of the enzyme [9]. Using of CAIs for medical
applications draws attention due to the large distribution of CA iso-
forms within different tissues as well as on their involvement in many
physiological/pathological conditions [1]. Among these isoenzymes,
the selective inhibition of cancer-associated hCA IX and hCA XII has
in all living organisms which are necessary for CO hydration to bi-
2
carbonate and protons. They participate in crucial regulatory mechan-
isms for balancing intracellular and extracellular pH of all cells [8].
⁎
Corresponding author at: Ataturk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey.
E-mail address: incigul1967@yahoo.com (H.I. Gul).
https://doi.org/10.1016/j.bioorg.2019.103222
Received 1 May 2019; Received in revised form 16 August 2019; Accepted 26 August 2019
Available online 28 August 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
Table 1
chelating scaffolds besides primary sulfonamides were also offered as
remarkable CA inhibitors such as dithiocarbamates, hydroxamates,
phenols, polyamines, coumarins, benzoic acid derivatives, secondary/
tertiary sulfonamides with various chemical differences in their phar-
macophoric moieties [19]. Additionally, many efforts have been also
made to explore chemical modifications of the CAIs structures that
could have a positive affect on lack of selectivity and water solubility
problems. For this purpose, amino acids, peptides or their hybrids for
designing CAIs which could provide a solution to the problems have
applied [17].
CA inhibitory potency of the well-known CAIs in clinical use [16].
Ki
Compound
hCA I (µm)
hCA II (nM)
hCA IX (nM)
hCA XII (nM)
AZA
MZA
EZA
DCP
DZA
BRZ
IND
0.25
0.05
0.025
1.20
50
12
14
8
25
27
34
50
52
37
24
5.7
3.4
22
38
9
50
3.5
3.0
3.4
45
3
Mollica et al. [20] reported the effects of novel probenecid-based
amide derivatives obtained with natural L-amino acids and tertiary
sulfonamide (Probenecid) against hCA I, II, IX, and XII isoforms. Most of
them presented a complete loss of hCA II inhibition (Kis > 10.000 nM)
and strong inhibitory activity against hCA IX and XII in the nanomolar
0.031
15
anticancer effects leading to decreasing of growth for both primary
tumors and metastases [10]. In particular, hCA IX is expressed in a
limited number of normal tissues, while its overexpression is observed
on the cell surface of a large number of solid tumors [10–12]. Previous
studies revealed that CAIX is over expressed in different human cancers
such as breast, kidney, rectum, ovarian, esophagus, cervix, uterine,
lung, and head [13].
range. The transformation of the COOH group into CONH was also
2
interesting, resulting in a remarkably lower affinity against the off-
target hCA II. This study also provides useful information about the
application of the amino acid tail approach to an atypical zinc-chelating
molecule [20].
SLC-0111 and indisulam (E7070) compounds were found promising
CAIs in clinical trials for the treatment of tumors among the wide range
of sulfonamide type compounds evaluated as potential anticancer
agents. Phase I clinical trials of SLC-0111 for tumors overexpressing
hCA IX were completed and phase II clinical trials will be scheduled in
the near future [21–23]. E7070, another primary sulfonamide was de-
veloped as an antineoplastic agent and reached phase II clinical trials,
which have been stopped however in 2014 [24,25].
The selectivity of the clinically used inhibitors or compounds de-
signed for various isoforms is important to target a specific or a certain
disease without undesired side effects [14]. Selective inhibition of hCA
IX isoenzyme is required for the treatment of cancer without side effects
which would result from the inhibition of other widespread hCA iso-
forms, such as the cytosolic hCA II enzyme [1,12,15].
Well-known first and second generation CAIs as clinically used
drugs such as acetazolamide (AZA), methazolamide (MZA), ethoxzo-
lamide (EZA), dichlorophenamide (DCP), dorzolamide (DZA), brinzo-
lamide (BRZ), and indisulam (IND) were presented in Table 1 with their
CA inhibitory results against hCA I, hCA II, hCA IX and hCA XII iso-
enzymes [16]. The standard CA inhibitors given in Table 1 had Ki value
of 24–52 nM against hCA IX while Ki values of the some drugs towards
hCA II is in the range of 3–38 nM [16].
Khloya et al. [26] reported 1,3-diaryl pyrazole bearing sulfonamide
as potent inhibitors of the tumor-associated carbonic anhydrase iso-
forms. Some compounds tested displayed affinity for hCA IX (Ki <
5
1
nM) at low nanomolar while the others had Ki value lower than <
0 nM against hCA XII. The compounds presented in Fig. 1 were re-
ported as potent CA inhibitors with their impressive results towards
hCA I, hCA II, hCA IX, hCA XII in designing novel, potent, and selective
CAIs [26].
Even if popular primary sulfonamides have inhibitory potency
against the target isoenzymes, still they have no desirable selectivity
profile. Thus, the critical issue for designing novel CAIs is to obtain CA
isoenzyme selective candidates against target enzymes for the desired
bioactivity in order to decrease side effects in addition to physico-
chemical problems such as poor water solubility of the sulfonamides
More recently, Pazopanib, a drug carrying a primary sulfonamide
moiety was approved by FDA as an anticancer agent which is a multi-
target tyrosine kinase inhibitor used clinically for the treatment of
different tumors [27–29]. Its bioactivity was also associated with the
inhibition of the carbonic anhydrase enzyme as well as kinase inhibi-
tion potency [30]. Winum et al. [31] reported CA inhibitory potencies
of Pazopanib against the catalytically active several CA isoforms such as
hCA I, hCA II, hCA IX and hCA XII. Some of the results reported in the
literature are in Table 2. The results reveal that Pazopanib showed
excellent CA inhibitory activity towards cancer-related isoenzymes hCA
IX (Ki = 9.1 nM) and hCA XII (Ki = 0.88 nM) [31]. According to these
data, it was estimated that in addition to the tyrosine kinase inhibitor
action, Pazopanib may show its antitumor/antimetastatic effects due to
the potent inhibition of the tumor-associated, hypoxia-inducible en-
zymes hCA IX and hCA XII [31].
[
17].
Despite sulfonamide derivatives are effective CAIs, most of them do
not have isoform selectivity against tumor related isoforms CA IX and
CA XII. Additionally, similarities of the active side of the isoforms
mentioned effect specificity of CAIs. This situation is particularly con-
sidered in the case of CA II isoform which has the broadest distribution
in body cells such as red blood cells. It was also expressed that as many
drugs are delivered systemically and membrane permeable, it is pos-
sible that CA II isoform cannot allow reaching of drugs as CAIs within
cancer cells and also CAIs show limited efficacy and selectivity against
CA IX and CA XII isoforms [18].
A large number of pyrazoles and its derivatives that exhibit
In an attempt to overcome selectivity problems, atypical zinc
Fig. 1. 1,3-Diarylpyrazole derivatives reported in the literature [26] as potent hCA IX and hCA XII inhibitors.
2

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
Table 2
models by affecting cell cycle regulation [36]. Additionally, it was re-
ported that COX-2 promotes tumor-specific angiogenesis, inhibits
apoptosis, and induces proangiogenic factors such as VEGF [37]. Cel-
ecoxib can also be considered as a representative compound of pyrazole
containing diaryl substituted heterocyclic template in pharmaceutical
chemistry. Moreover, Ruxolitinib and Crizotinib carrying pyrazole
moiety are also used as antitumor agents in the clinic for the treatment
of myeloproliferative neoplasm and non-small cell lung carcinoma,
respectively [38,39].
CA inhibitory potency of the anticancer drug Pazopanib [31].
H
3
C
H
3
C
N
CH
N
3
N
N
N
O
N
SO
2 2
NH
CH
3
H C
3
N
S
H
N
N
SO
2 2
NH
H
Pazopanib (Votrient®)
Asetazolamit (AAZ, Diamox®)
Considering the exciting promising literature survey on primary
sulfonamide and pyrazole pharmacophores, and approved drugs car-
rying sulfonamide and pyrazole moieties which are in clinical use for
treatment of cancer, in the present study, it was aimed to synthesize
Ki (nM)
AAZ
Pazopanib
1
,3,5-trisubstituted pyrazoles carrying benzenesulfonamide, evaluation
hCA I
250
12
12.1
32.4
9.1
of their CA inhibition properties towards hCA I, hCA II, and hCA IX
isoenzymes, and also their cytotoxicities against OSCC cell lines (HSC-2
and HGF) to find out potential drug candidate/s. In an attempt to ex-
plore selectivity profiles for hCA IX versus hCA I and II, we also planned
in silico modelling studies for the lead compound of this study. Target
compounds were derivatized by changing aryl parts as phenyl, 4-methyl
phenyl, 4-nitro phenyl and 2-thienyl on the 5th position of pyrazole. In
addition, insertion of ester and hydrazide groups on the 3th position of
the core ring pyrazole were considered as a second modification to
obtain target compounds (Fig. 2).
hCA II
hCA IX
hCA XII
25
5.7
0.88
remarkable pharmacological activities are known as a group of very
active heterocyclic compounds [32,33]. Currently, some of them are in
clinical use such as Celecoxib, Ruxolitinib, and Crizotinib (Fig. 2).
Celecoxib is a COX-2 inhibitor approved by the FDA for the treatment of
rheumatoid arthritis and osteoarthritis [34,35]. According to data re-
ported, the compound also reduces prostate tumors in experimental
Fig. 2. Chemical structure of anticancer drugs and rationally designed template for anticancer drug candidate compounds targeting for CA IX isoenzyme inhibition.
3

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
Scheme 1. Synthetic pathway of the compounds 1,3,5-trisubstituted pyrazoles carrying benzenesulfonamide. (i) NaOEt, EtOH or THF, ice bath, and rt. (ii) 4-
Hydrazinobenzenesulfonamide.HCl, EtOH, reflux. (iii) Hydrazine hydrate, EtOH, reflux. Ar = Phenyl (1–3), 4-Methyl phenyl (4–6), 4-Nitro phenyl (7–9), Thiophen-
-yl (10–12).
1
2
2
. Results and discussion
(hydrazinecarbonyl)-5-aryl-1H-pyrazol-1-yl) benzenesulfonamide deri-
1
vatives (3, 6, 9, 12) was confirmed by their H NMR spectra which
2.1. Chemistry
revealed the disappearance of the proton signals of eOCH
2
CH
3
2
group in
group. A
addition to the appearance of the proton signals of eNHNH
The synthetic route to obtain 1,3,5-trisubstituted pyrazole carrying
signal of the eNHe group was seen in region δ 10.20–9.59 ppm as a
benzenesulfonamide is presented in Scheme 1. Claisen condensation of
the suitable aryl ketones with diethyl oxalate in the presence of sodium
ethoxide gave ethyl 2,4-dioxo-4-arylbutanoates derivatives with the
yield 1 (50%), 4 (54%), 7 (89%), 10 (50%). The cyclization of β-di-
ketones (1, 4, 7, 10) with 4-hydrazinobenzenesulfonamide hydro-
chloride by refluxing in ethanol gave ethyl 5-aryl-1-(4-sulfamoyl-
phenyl)-1H-pyrazole-3-carboxylate derivatives in excellent yield [2
singlet while a signal of eNH group was seen around δ 4.48 ppm as a
2
singlet. The other aromatic protons’ peaks were observed at the ex-
pected values as expected. HRMS results were also confirmed chemical
structure of the compounds in accordance with their proposed chemical
structure in addition to their purity. According to HRMS results, dif-
ferences of mass results between calculated and measured values were
lower than the value of 0.4%. Differences in calculated and measured
values were in the range of 0.0017–0.0008.
(
71%), 5 (91%), 8 (89%), 11 (80%)]. As a final step, 1,5-diarylpyrazole
ethyl esters were then reacted with hydrazine hydrate to afford 4-(3-
The structure of ethyl-5-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxylate (2) was characterized by X-ray diffraction analysis (Fig. 3).
Compound 2 was crystallized as a white block and was solved in the
(
hydrazinecarbonyl)-5-aryl-1H-pyrazol-1-yl) benzenesulfonamide deri-
vatives with the good yield 3 (90%), 6 (51%), 9 (81%), 12 (86%) by
refluxing in ethanol.
monoclinic space group P2 /c with four molecules in the unit cell. The
1
The chemical structure of the compounds was confirmed by NMR
spectra and HRMS analysis (See Supplementary file for spectra). Ac-
structure has phenyl and eOCH
2
CH groups at opposite positions of
3
pyrazole core. Among these, there is a benzenesulfonamide unit. Devia-
tion from planarity of the molecule is due to significant steric effects and
intermolecular interactions. In the pyrazole heterocycle, the bond lengths
of N3eC9 = 1.3334(3) Å and C8eC7 = 1.378(4) Å are in the range of
typical single bond values. The bond lengths between sulphur and oxygen
S1eO1 = 1.427(3) Å and S1eO2 = 1.436(3) Å falls within the double
bond range. The crystal packing shows (Fig. 3) that molecules form
centrosymmetric dimers connected by N1eH⋯O2 [D⋯A = 2.973(3)Å]
hydrogen bonds. These dimeric units form a polymeric structure with
adjacent molecules. The non-covalent CeH⋯O, NeH⋯C, and NeH⋯N
interactions cause the formation of this polymeric form. The π-π stacking
interactions between the delocalized π-electrons of the phenyl and pyr-
azole rings are relatively weak. Distance between rings centroids is in the
range of 3.98–5.72 Å.
1
cording to H NMR data, chemical structures of the 2,4-dioxo-4-ar-
ylbutanoates derivatives (1, 4, 7, 10) were established to exist in an
1
enol form. H NMR spectra of the compounds 1 and 4 showed broad
singlet signal of enol proton at δ 15.30 and 14.80 ppm, except 7 and 10.
On the other hand, a signal belongs to alkene’s proton for the four in-
termediates was shown at δ 7.09–6.91 ppm. Moreover, the spectrum of
the compounds showed a couple of signal of aliphatic protons at δ
4
.39–4.34 ppm (q, eCH
2
e) and δ 1.42–1.39 ppm (t, eCH e).
3
1
The H NMR spectra of ethyl 5-aryl-1-(4-sulfamoylphenyl)-1H-pyr-
azole-3-carboxylate derivatives (2, 5, 8, 11) showed the characteristic
signal of H-4 of the pyrazole ring in the region δ 7.35–7.05 ppm as a
singlet. A signal of sulfonamide group in the compounds also appeared
in the range δ 7.56–7.44 ppm as a singlet or one within the other aro-
matic peaks. In the current study, 1,5-diaryl pyrazoles were designed as
target compounds. That is why the synthetic method mentioned above
carried out to obtain 1,5-diarylpyrazole isomer. In general, the cycli-
zation of β-diketones with hydrazine derivatives may give a mixture of
2.2. Carbonic anhydrase inhibition
In the present study, a small library of 1,5-diarylpyrazole benze-
1
,5- and 1,3-diarylpyrazole esters based on the reaction conditions
nesulfonamides was evaluated against three human carbonic anhy-
drases such as physiologically abundant cytosolic hCA I and hCA II and
trans-membrane, tumor-associated hCA IX isoforms by a stopped-flow,
[
40]. In most cases, 1,5-diarylpyrazole regioisomers were generated by
carrying out the reaction in the presence of the hydrochloride salt of the
hydrazine derivatives in refluxing ethanol with the high yields [41].
In this study, separation of the 1,5-diarylpyrazole isomers as a target
compounds were performed by crystallization to afford pure target
compounds in the high yields as reported above. After purification of
the 1,5-diarylpyrazole isomers, the exact chemical structure of the re-
presentative compound 2 was established by NOESY experiment (See
supplementary file for spectra). The chemical structure of 4-(3-
CO hydrase assay method [42]. CA inhibition data is shown in Table 3.
2
The results are expressed as Ki (inhibition constant, nM) values and the
lowest Ki value indicates the best inhibitory potency of the compound
against the corresponding isoenzyme. Acetazolamide (AZA) was used as
a reference drug. Moreover, the selectivity ratio of the compounds for
the inhibition of tumor-related CA IX over off-target cytosolic iso-
enzymes I and II was summarized in Table 4. The following results of
4

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
Fig. 3. Dimeric structure of molecule 2 with the short-contact interactions.
Table 3
Table 4
Carbonic anhydrase inhibitory potency (Ki, nM) of the 1,3,5-trisubstituted
pyrazoles carrying benzenesulfonamide towards hCA I, hCA II and hCA IX
isoenzymes.
Isoenzyme selectivity ratios of the compounds 1,3,5-trisubstituted pyrazoles
carrying benzenesulfonamide.
Selectivity ratio
R
Compound
hCA I/hCA IX
hCA II/hCA IX
N
O
2
3
5
6
8
9
0.2
0.3
2.9
0.5
0.3
0.4
3.9
0.04
0.9
H NO S
N
2
2
2.2
52.2
11.7
302
3217
0.04
4.6
Ar
1
1
Ki (nM)
12
AZA
9.7
0.5
Compound
Ar
R
hCA I
hCA II
hCA IX
2
3
5
6
8
9
Phenyl
Phenyl
OCH
2
CH
3
3
3
3
47.1
68.4
9176
368.1
6196
7399
8.3
60.9
87.9
86.1
9.7
225.3
30.5
175.8
31.4
20.5
2.3
NHNH
2
times more than AZA (Ki = 25.8 nM) against hCA IX isoenzyme.
iii. When the inhibitory potencies of the 4-(3-(hydrazinecarbonyl)-5-
aryl-1H-pyrazol-1-yl)benzenesulfonamide derivatives (3, 6, 9, 12)
were considered against all isoenzymes, compound 12 against hCA
I (Ki = 44.3 nM) and hCA II (Ki = 8.4 nM) and compound 9 against
hCA IX (Ki = 2.3 nM) were found the best inhibitors. Among this
type of compounds, compound 9 bearing 4-nitrophenyl ring was
found highly effective for inhibiting tumor-associated hCA IX iso-
enzymes with the lowest Ki value of 2.3 nM. So, compound 9
showed approximately 11.2 times superior CA inhibition profile as
compared to the reference AZA (Ki = 25.8 nM).
4-Methylphenyl
4-Methylphenyl
4-Nitrophenyl
4-Nitrophenyl
Thiophen-2-yl
Thiophen-2-yl
OCH
NHNH
OCH CH
NHNH
OCH CH
NHNH
2
CH
2
2
9.1
2
8.9
1
1
2
2
7.4
184.8
1
AZA
2
44.3
8.4
9.7
25.8
250
12.1
the compounds tested against hCA I, hCA II and hCA IX were drawn
from the data are presented in Tables 3 and 4.
iv. When the hydrazide type compounds’ inhibitory potencies were
considered against tumor-associated hCA IX isoenzyme, it can be
said that converting ethyl ester derivatives into hydrazide was
found a useful modification to increase hCA IX inhibiting ability of
the compounds. Interestingly, converting compound 11, ethyl ester
derivative carrying thiophene ring, into hydrazide derivative 12
extremely increased the inhibitory potency more than 19 times
towards hCA IX isoenzyme.
i. The cytosolic hCA I isoenzyme was inhibited by the eight com-
pounds bearing pyrazole-benzenesulfonamide pharmacophores (2,
3
, 5, 6, 8, 9, 11, 12) with the inhibition constant (Ki) ranging be-
tween 8.3−9176 nM (hCA I), 7.4–87.9 nM (hCA II) and
2
2
.3–225.3 nM (hCA IX) while reference drug AZA has Ki values of
50 nM, 12.1 nM and 25.8 nM towards hCA I, hCA II and hCA IX,
respectively.
ii. Among the ethyl 5-aryl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-car-
boxylate derivatives (2, 5, 8, 11), compound 11 carrying thiophene
ring on the 5th position of pyrazole ring effectively inhibited hCA I
isoenzyme with Ki value of 8.3 nM while 11 inhibited hCA I iso-
enzyme with Ki value of 7.4 nM. It showed that 11 was 30 times
effective than AZA against hCA I and also it was 1.6 times more
effective than AZA (Ki = 12.1 nM) against hCA II. On the other
hand, the ester compound 8 was found as a good inhibitor among
others with Ki value of 20.5 nM and its inhibitory potency was 1.3
v. The data in Table 3 leads to the conclusion that 4-nitrophenyl
bearing compounds (8 and 9) and thiophene bearing compounds
(11 and 12) generally have good inhibitory profile against hCA II
and hCA IX isoenzymes with the lowest Ki values among the
compounds tested and also these compounds drawn attraction to
design novel CA inhibitors. The results considered showed that the
nature of the substituents present on the 5th position of pyrazole
scaffold in terms of electron-withdrawing nitro group on phenyl
5

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
ring and bioisosteric replacement of phenyl ring by thiophene in-
ring in 1,5-diarylpyrazole was useful modification to increase CA
activity while the introduction of an electron-releasing group such as
methyl on 3rd position in 1,3-diaryl pyrazole drew attention. In terms
of both inhibition constant and isoenzyme selectivity compounds 4-
(3-(hydrazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benzene-
sulfonamide (9) for 1,5-diarylpyrazole regioisomer and 4-(4-(hy-
drazinecarbonyl)-3-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfonamide
(6b) for 1,3-diarylpyrazole regioisomer can be considered as lead
compounds to design novel and more selective compounds towards
tumor-related hCA IX isoenzyme for further carbonic anhydrase in-
hibitors research and development studies.
fluenced CA inhibition profile positively.
vi. Overexpression of CA IX isoenzyme specifically in cancer cells
makes it a good therapeutic target as a biomarker for cancer. The
most important problem with the CAIs is the lack of selectivity
towards a specific CA isoenzyme such as cancer-related hCA IX and
hCA XII isoenzymes. Non-specific binding of clinically used CA
inhibitors to other isoforms causes undesired side effects. One of
the goals of this study is to obtain selective hCA IX inhibitors over
the two major off-target CA isoenzymes as hCA I and hCA II. One of
the challenges targeting hCA IX for the treatment of tumors is the
structural similarity of hCA IX to hCA II isoenzyme. In fact, most
isoforms of CAs have only a few differences in the active site of
enzyme which makes it difficult for designing inhibitors that would
specifically bind to one isoform over the others. The hCA I/hCA IX
and hCA II/hCA IX ratios reveal that the isoenzyme selectivity of
the compound against hCA IX isoenzyme as shown in Table 4.
When selectivity ratios of the compounds tested were considered,
some compounds make attraction towards cancer-associated hCA
IX isoenzyme. Compounds 5 (5.4 times), 6 (1.2 times), 8 (31.2
times), 9 (331.7 times) were found more selective towards hCA IX
over off-target isoenzyme hCA I while compounds 3 (5.8 times), 9
It can also be expressed here, converting ethyl ester derivatives into
hydrazide was found a useful modification in increasing hCA IX in-
hibiting ability of the compounds. This may result different pharma-
cokinetic and intraction of the compound with targeted enzyme/re-
septors than previously reported compounds. For the future
perspective, novel pyrazole type compounds can be designed with the
hope to find more potent and selective inhibitor by changing sub-
stituent on the 3rd position of the ring by the reaction of eCOOH or
eCONHNH groups with other functional groups. For instance, several
2
pyrazole carboxamide derivatives can be synthesized with primary
amine derivatives such as amino acids to see the effects of the amide
bond on CA inhibitory potency because recently some peptide deriva-
tives were reported as promising CAIs [20].
(
7.8 times), and 12 (1.8) were found more selective towards hCA IX
over off-target isoenzyme hCA II isoenzyme than reference drug
AZA’s isoform selectivity.
vii. On the other hand, we considered comparing CA inhibitory potency
of 1,5-diaryl pyrazole with some 1,3-diaryl pyrazole towards hCA I, h
CAII, and hCA IX isoenzymes to understand which kind of isomers
can be considered for designing of novel and potent CA inhibitors.
Khloya A. et al. reported pyrazole-4-hydrazinocarbonyl derivatives
against hCA I, hCA II, hCA IX and hCA XII [26]. The inhibition data of
compounds 4-(4-(hydrazinecarbonyl)-3-phenyl-1H-pyrazol-1-yl)ben-
zenesulfonamide (6a), 4-(4-(hydrazinecarbonyl)-3-(p-tolyl)-1H-pyr-
azol-1-yl)benzenesulfonamide (6b) and 4-(4-(hydrazinecarbonyl)-3-
2.3. Cytotoxicity
1,3,5-Trisubstituted pyrazoles were screened for their in vitro cyto-
toxicities against human oral squamous cell carcinoma (OSCC) cell lines
(HSC-2) and human mesenchymal normal oral cells (human gingival fi-
broblast, HGF) via 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide (MTT) cytotoxicity method according to literature [5,43–53].
Doxorubicine (DXR), 5-Fluorouracil (5-FU) and Methotrexate (MTX)
which are anticancer drugs in clinical use for several cancer types were
used as reference compounds in the assay. Dose-response of growth in-
hibition induced by 1,3,5-trisubstituted pyrazoles and references com-
pounds are shown in Fig. 4. Compounds 2, 5 and 11, and DXR showed
potent cytotoxicity, killing most of the HSC-2 cells at higher concentra-
tion. On the other hand, compounds 3, 6, 8, 9, 12, 5-FU and MTX showed
rather cytostatic effects. This may explain that 5-FU and MTX are not
used alone, but rather in combination with other anticancer drugs
clinically. The results in Table 6 reveal that 50% cytotoxic concentration
(4-nitrophenyl)-1H-pyrazol-1-yl) benzenesulfonamide (6 g) are pre-
sented in Table 5. Compounds 6a (Ki = 2.8 nM)) and 6b (Ki = 2 nM)
showed remarkably higher CA inhibition profile than AZA against
hCA IX isoenzyme. Compound 6b bearing 4-methyl phenyl was found
more effective with the lowest inhibition constant against hCA IX
isoenzyme except for compound 6 g including 4-nitrophenyl deriva-
tive. On the other hand, according to present results reported by our
group, compound 9 bearing 4-nitrophenyl ring was found highly ef-
fective for inhibiting tumor-associated hCA IX isoenyzme with the
lowest Ki value of 2.3 nM among others. So, compound 9 showed
approximately 11.2 times superior CA inhibition profile as compared
to the reference AZA (Ki = 25.8 nM). It can be concluded that elec-
tron-withdrawing nitro substitution on the 5th position of the phenyl
(CC50
) values of the compounds tested were in the range of
37.7– > 200 μM towards HSC-2 malign cell lines while the compounds
have cytotoxicities between 132 and > 200 μM against to non-malignant
HGF cells. Many compounds tested were found more cytotoxic towards
malign cell line than reference drugs 5-FU and MTX, except DXR.
Compound ethyl 1-(4-sulfamoylphenyl)-5-(p-tolyl)-1H-pyrazole-3-car-
boxylate, 5 (CC50 = 37.7 μM) were found 26.6 and 10.6 times more cy-
totoxic than reference 5-FU and MTX, respectively. And, compound ethyl
Table 5
Carbonic anhydrase activity of the 1,3-diarylpyrazole derivatives towards hCA
I, hCAII, and hCA IX isoenzymes [26].
1
-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxylate, 11
Ar
N
(
CC50 = 58.1 μM) were also found 17.2 and 6.9 times more cytotoxic
H NO S
N
2
2
than 5-FU and MTX, respectively.
NHNH
2
Cytotoxicity of tested compounds is desirable when it is more se-
lective towards cancer cells than normal cells. To clarify tumor se-
lectivity potency of the compounds, tumor selectivity (TS) values were
calculated by dividing the CC50 value towards normal cells into the
CC50 value towards cancer cell lines (Column B/Column A, Table 6).
With regard to this type calculation, compounds 5 (TS = 3.5) and 11
O
Ki (nM)
hCA I
Selectivity
Compound
Ar
hCA II
hCA IX
I/IX
II/IX
(
TS = 2.3) drew attention with the highest TS values among others
when compared with reference drugs 5-FU and MTX. This was reflected
by wider safety margin of 5 and 11 (indicated by red bidirectional
arrow in Fig. 4). Additionally, potency-selectivity expression (PSE)
6
6
6
a
b
g
Phenyl
7.9
9.6
32
9.3
9.2
71
2.8
2.82
4.8
3.32
4.6
4-Methylphenyl
4-Nitro
2.0
49.3
0.65
1.44
2
values were calculated according to equation i.e. (B/A ) × 100 in
AZA
250
12.1
25
10
0.5
Table 6 in an attempt to identify the most promising compounds with
6

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
Fig. 4. Dose-response curves of growth inhibition by 1,3,5-trisubstituted pyrazoles and reference compounds against HSC-2 and HGF cells.
Table 6
properties of the compounds in tissues. In addition, the method used in
cytotoxicity assay, nature of the cell lines used, and mechanism of ac-
tion of the compounds tested can modify the cytotoxicity results.
It is also necessary to test keratinocyte toxicity of newly manu-
facturing compounds, since DXR, which showed the broadest safety
margin (Fig. 4), killed the normal keratinocyte at micromolar con-
centration by inducing apoptosis [50].
Anticancer/cytotoxicity results of the compounds 1,3,5-trisubstituted pyrazoles
carrying benzenesulfonamide towards HSC-2 and HGF cells.
CC50 (μM)
Malignant
HSC-2
Non-malignant
HGF
TS
PSE
2
Oral squamous cell carcinoma (OSCC) is the major malignant tu-
mors of the oral cancers [54]. Despite of improvements in treatment of
cancer, the mortality rate for the cancer is still high. Therefore, new
prognostic molecular parameters/biomarkers are also needed to enable
an individual therapy concept. One of the biomarkers is also reported as
CA IX isoenzymes in OSCC patients [13,55,56] while the potential role
of another tumor related CA XII as a biomarker in OSCC has not been
investigated in detail [57].
(
A)
SD
(B)
SD
(B/A)
(B/A ) × 100
2
3
5
6
8
9
1
1
121.3
> 200
37.7
6.5
0.0
7.2
0.0
0.0
0.0
19.8
2.3
158.3
> 158
132.0
> 200
152.2
> 200
133.0
> 200
1.2
72.5
4.6
1.3
1.1
> < 0.8
3.5
> < 0.4
9.3
> 200
> 200
> 200
58.1
0.0
> < 1
< 0.8
> < 1
2.3
> < 0.5
> 0.4
> < 0.5
3.9
49.9
0.0
1
2
5.3
> 199
0.0
> < 1
> < 0.5
Several studies revealed a significant correlation between CAIX
expression and clinical stage in OSCC [58]. In accordance with the
some cell experiments, the overexpression of CAIX is also associated
with increased cell invasive and metastatic ability [59,60]. Therefore, it
could be considered that CAIX is an indicator of metastasis in OSCC.
Recently, Chien et al. [57], has reported some results in regard to ex-
pression of CA XII in a large collection of OSCC tissue samples. In that
study, expression of CA XII was detected patients with OSCC and CA XII
expression was present in 185/264 patients (70%) cases. According to
the results, the expression of CA XII in OSCC samples can predict the
progression of OSCC and survival of OSCC patients [57].
DXR
-FU
MTX
0.2
0.2
0.0
0.0
> 9.6
0.7
0.0
0.0
> 43.0
> < 1
> < 1
> 19229.7
5
> 1000
> 400
> 1000
> 400
> < 0.1
> < 0.25
Each value represents mean from triplicate assays. SD: Standard deviation. TS:
Tumor selectivity. PSE: Potency Selectivity Expression. Doxorubicin (DXR), 5-
Fluorouracil (5-FU) and Methotrexate (MTX) are reference drugs.
regard to both good potencies and selectively cytotoxic as described in
literature [46,52]. Compounds 5 (PSE = 9.3) and 11 (PSE = 3.9) were
determined as lead compounds of cytotoxicity assay based on the PSE
values.
When CA inhibitory and cytotoxicites of the compounds compared,
the results showed that hydrazide derivative compounds 9 and 12 made
attraction in terms of selectively inhibition of cancer related CA IX
isoenzyme. On the other hand, surprisingly ester derivatives 5 and 11
showed cytotoxic effects on OSCC lines while compounds 9 and 12 have
not shown remarkably cytotoxicity on the same cell lines. Thus, there is
a noncompatible situation between CA IX inhibitory potency and in-
hibition of cell growth of the compounds. It appears possible that the
According to cytotoxicity data, it can be observed that compounds 5
and 11 which are ethyl 1-(4-sulfamoylphenyl)-5-(aryl)-1H-pyrazole-3-
carboxylate derivatives can be considered for further cytotoxicity stu-
dies for detailed results. Hereby, the cytotoxicities of the compounds
tested can be affected by several factors such as chemical structures of
the compounds, physicochemical properties of the compounds which
direct their pharmacological, pharmacokinetics and pharmacodynamics
7

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
compounds tested have different mechanism of action on cancer cells
except CA IX inhibition. Indeed, the presence of CA IX is not required
for killing cancer cells. But, this problematic situation can be clarified
by Western Blot analysis in further studies. Additionally, under ex-
perimental conditions it is also potential that the compounds undergo
chemical inactivation by several chemical reactions and result in
blocking the inhibition of the enzyme targeted, and evaluating these
uncontrolled situations is also difficult in the context of the experi-
mental media. Additionally, sulfonamide derivatives have also different
targets to show its anticancer ability except CA IX isoenzyme. As
mentioned above, CA XII is an another cancer related biomarker in
OSCC lines. This enzyme can be considered as the new target for the
compounds reported here for further studies. In conclusion, to clarify
how the compounds showed their bioactivities in different ways, fur-
ther investigations can be carried out with the next studies.
amino acid residues were observed to be critical in selectivity (Fig. 5).
Interestingly, compound 9 was observed to have an unusual con-
formation. The sulfonamide moiety orients itself towards the active site
gate. Compound 9 has a large size and hence, a steric hindrance in the
binding pocket would show a weaker inhibitory power. At the entrance
of the active site, hCA IX has valine (VAL131) that has a small hydro-
phobic side chain, which results in favourable binding and selectivity
towards this isoform. Although there is phenylalanine (PHE131) at this
position in hCA II, a plausible π-π interaction with compound 9 is ob-
served. This seems to overcome the diminishing effect of steric clash in
the activity.
The amino acid GLN67 in hCA IX has a longer side chain compared
with ASN67 of hCA II that could account a slight increase in activity of
compound 9 towards hCA IX. The same residue is replaced with a larger
amino acid, HIS67 in hCA I which is believed to prevent compound 9 to
orient itself in the active conformation. Threonine 200 in the deep ac-
tive pocket is replaced with histidine 200. Besides being a bulkier
substituent, the interaction of imidazole ring in histidine with the
benzene ring of compound 9 results in conformational changes.
In addition, the hydrophobic valine (VAL62) located close to the
entrance of the active site in hCA I is replaced by a polar asparagine
2.4. In silico modelling
Computational modelling, especially computer aided drug design
approaches provide powerful tools that enlighten potential inhibition
mechanism and help to design more potent drug candidates [61,62].
Although much complicated, isoform selectivity profiles of the studied
ligands could also be investigated with the help of in silico modelling
(
ASN62) in both hCA II and hCA IX. This polar and bulkier residue
causes a steric hindrance and but it also interacts with the polar ends of
propeller shaped compound 9 that induces conformational changes
enhancing binding and inhibition.
[
18,63,64].
In an attempt to explore selectivity profiles for hCA IX versus hCA I
and II, we performed in silico modelling of those three structures.
Amongst all the compounds, compound 9 exhibits a clear selectivity
against hCA IX over hCA II and especially hCA I (Table 4). Subsequent
to the molecular docking of compound 9, the binding sites of hCA I, II
and IX were aligned. After a detailed visual inspection, a number of
3. Conclusion
In the present paper, 1,3,5-trisubstituted pyrazole benzenesulfona-
mides were evaluated against physiologically abundant cytosolic hCA I
Fig. 5. The binding conformation of compound 9 in the active pocket of hCA IX (top), hCA II (lower left) and hCA I (lower right). Only the discussed critical amino
acid residues are shown.
8

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
and hCA II and trans-membrane, tumor-associated hCA IX isoforms in
terms of carbonic anhydrase inhibition potential and their cytotoxicities
were tested against OSCC cell lines. The influence of the substitution
pattern at the 5th position of 1,3,5-trisubstituted pyrazole with several
aryl groups was examined in terms of their effects on bioactivity.
Compounds 6, 8, 9, 11 and 12 showed low nanomolar hCA II inhibitory
potency with Ki < 10 nM while compounds 9 and 12 displayed
Ki < 10 nM against hCA IX isoenzyme. Compound 9;4-(3-(hy-
drazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benzenesulfona-
mide, can be considered as the most selective tumor-associated hCA IX
inhibitor over off-target cytosolic isoenzymes hCA I and hCA II with the
lowest Ki = 2.3 nM values. In addition, its selectivity ratios were 3217
distilled water (50 ml) was added into the reaction mixture and then pH
was adjusted to pH = 3 by adding hydrochloride acid (37%) dropwise.
The aqueous mixture was extracted with ethylacetate (3 × 25 ml). The
combined extracts were dried on dry MgSO , and the content of the
4
mixture was concentrated under vacuum and then kept at room tem-
perature to obtain the final product. The compounds solidified were
filtered, washed with cold ethanol and then dried at room temperature.
Physical and spectral data of the compounds were given as below.
Ethyl 2,4-dioxo-4-phenylbutanoate, 1: Light orange solid, yield
1
50%. Mp = 38–40 °C. H NMR (CDCl
3
, 400 MHz, δ, ppm): 15.30 (bs,
1H, OH), 7.98–7.96 (m, 2H, ArH), 7.60–7.53 (m, 1H, ArH), 7.49–7.46
(m, 2H, ArH), 7.05 (s, 1H, eCOCH]Ce), 4.37 (q, J = 7.1 Hz, 2H,
(
hCA I/hCA IX) and 3.9 (hCA II/hCA IX). It observed that converting
eOCH
2
CH
3
), 1.39 (t, J = 7.1 Hz, 3H, eOCH
2
CH ). HRMS (ESI-MS) m/z
3
+
ethyl ester group into the hydrazide group generally enhanced the in-
hibitory activity of the compounds against hCA IX isoenzyme.
Moreover, nature of the substituents present on the 5th position of
pyrazole scaffold such as electron-withdrawing nitro group on phenyl
ring and bioisosteric replacement of phenyl ring by thiophene affected
positively CA inhibitory potency for the 1,5-diaryl pyrazole benzene-
sulfonamide type compounds. On the other hand, cytotoxicity results
Calculated: 221.0808 C12
H
13
O
4
[M+H] ; Found: 221.0797.
Ethyl 2,4-dioxo-4-(p-tolyl)butanoate, 4: Light orange solid, yield
1
54%. Mp = 109–111 °C. H NMR (CDCl
3
, 400 MHz, δ, ppm): 14.80 (bs,
1H, eOH), 7.89 (d, J = 8.2 Hz, 2H, ArH), 7.29 (d, J = 8.2 Hz, 2H, ArH),
7.05 (s, 1H, eCOCH]Ce), 4.39 (q, J = 7.1 Hz, 2H, eOCH CH ), 2.43
2
3
(s, 3H, ArCH
3
), 1.40 (t, J = 7.1 Hz, 3H, eOCH
2
CH ). HRMS (ESI-MS)
3
m/z Calculated: 257.0784 C13
H
14
O
4
[M+Na]+; Found: 257.0778.
pointed out that compounds
5
(CC50 = 37.7 μM) and 11
(
CC50 = 58.1 μM) can be considered as lead compounds of the cyto-
Synthesis of the ethyl 4-(4-nitrophenyl)-2,4-dioxobutanoate (7,
toxicity study since they were more cytotoxic than reference drugs 5-FU
and MTX. The results obtained from in silico modelling showed that
specific residues within the binding pocket of hCA I, II and IX might
provide the rationale for selective inhibition of hCA IX over I and II. In
the light of the bioactivity results of this current study, using of 4-(3-
Scheme 1) [40,65–69]
Sodium ethoxide (182 mmol) was stirred in ethanol (200 ml) on an
ice bath, after 10 min, diethyl oxalate (91 mmol) was added into the
orange color mixture. Then, 4-nitro acetophenone (91 mmol) was
added slowly into the previous mixture and the color of the main
mixture turned into dark brown. The reaction process was monitored by
TLC using dichloromethane: methanol (4.8:0.2) solvent system. After
20 h, distilled water (100 ml) was added into the reaction mixture and
then pH was adjusted to pH = 2–3 by adding hydrochloride acid (37%)
dropwise. The aqueous mixture was extracted with ethylacetate
(
hydrazinecarbonyl)-5-aryl-1H-pyrazol-1-yl)benzenesulfonamide scaf-
fold and its derivatives could provide a rational approach because of
their promising selectivity and inhibitory potency against cancer-re-
lated hCA IX isoenzyme as anticancer drug candidates for research and
development studies of CAIs.
4
. Material and methods
(3 × 50 ml). The combined extracts were dried on dry Na
2
SO , and the
4
content of the mixture was concentrated under vacuum and then kept at
4.1. Chemistry
room temperature to obtain a crude product. It was crystallized from
ethanol. Ethyl 4-(4-nitrophenyl)-2,4-dioxobutanoate, 7: Light brown
1
Reagents and solvents were purchased from commercial sources and
solid, yield 20%. Mp = 111–113 °C. H NMR (CDCl
3
, 400 MHz, δ, ppm):
used without further purification. Nuclear Magnetic Resonance (NMR)
8.34 (d, J = 8.8 Hz, 2H, ArH), 8.14 (d, J = 8.8 Hz, 2H, ArH), 7.09 (s,
1
spectra ( H NMR) and Nuclear Overhauser Spectroscopy (NOESY)
1H, eCOCH]Ce), 4.34 (q, J = 7.1 Hz, 2H, eOCH CH ), 1.42 (t,
2
3
spectra of the compounds were recorded with 400 MHz Varian
J = 7.1 Hz, 3H, eOCH
2
CH ). HRMS (ESI-MS) m/z Calculated: 266.0659
3
(
Danbury, ABD) spectrometer. Chemical shifts (δ) were reported in
C
12
H12NO
6
[M+H]+; Found: 266.0665.
ppm. CDCl
3
(Merck) and DMSO‑d (Merck) were used as NMR solvent.
6
High-Resolution Mass Spectra (HRMS) of the compounds were taken
using a liquid chromatography ion trap-time of the flight tandem mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray
ionization (ESI) source, operating in both positive and negative ioni-
zation mode. Shimadzu’s LCMS Solution software was used for data
analysis. Melting points were determined using an Electrothermal 9100
instrument (IA9100, Bibby Scientific Limited, Staffordshire, UK) and
are uncorrected. Process of the reaction was monitored by Thin Layer
Chromatography (TLC) using Silicagel HF254 (Merck Art 5715) plate
under UV lamb (254 and 365 nm, Spectroline, Model ENF-240C/FE,
Spectronics Corporation Westbury, New York U.S.A).
Synthesis of ethyl 2,4-dioxo-4-(thiophen-2-yl)butanoate (10,
Scheme 1) [40,65–69]
2-Acetylthiophen (159 mmol) and diethyl oxalate (318 mmol) was
stirred in THF (250 ml) at rt for 10 min. Then, sodium ethoxide
(318 mmol) was added slowly onto the colorless mixture. The reaction
process was monitored by TLC using ethylacetate: hexane (3:2) solvent
system. After 6 h, the content of the reaction was poured into hexane
(250) and the mixture was stirred at room temperature for 15 min.
Then, 1N HCl aqueous solution (250 ml) was added into the mixture
and stirred for 30 min at room temperature. The light brown solid ob-
tained was washed with water then dried. Ethyl 2,4-dioxo-4-(thiophen-
1
Synthesis of the ethyl 2,4-dioxo-4-phenylbutanoate (1) and
2-yl)butanoate, 10: Light brown solid, yield 50%. Mp = 40–42 °C.
H
ethyl 2,4-dioxo-4-(p-tolyl)butanoate (4, Scheme 1) [40,65–69]
NMR (CDCl
.74 (dd, J = 5.0, 1.1 Hz, 1H, ArH), 7.18 (dd, J = 5.0, 4.0 Hz, 1H, ArH),
6.91 (s, 1H, eCOCH]Ce), 4.38 (q, J = 7.2 Hz, 2H, eOCH CH ), 1.40
(t, J = 7.2 Hz, 3H, eOCH CH ). HRMS (ESI-MS) m/z Calculated:
249.0192 C10 S [M+Na] ; Found: 249.0180.
3
, 400 MHz, δ, ppm): 7.84 (dd, J = 4.0, 1.1 Hz, 1H, ArH),
7
Diethyl oxalate (140 mmol for 1; 120 mmol for 4) and acetophenone
2
3
(
140 mmol for 1; 120 mmol for 4) was respectively added into the
2
3
+
freshly prepared sodium ethoxide [(100 ml ethanol and 140 mmol so-
dium for 1); (100 ml ethanol and 120 mmol sodium for 2)] on ice bath.
Then additional 200 ml ethanol was added into the yellow color mix-
ture and the final mixture was stirred at room temperature. The reac-
tion process was monitored by TLC using ethylacetate: hexane (2:3)
solvent system. After the reaction was stopped (20 h for 1; 30 h for 4),
H
O
11 4
Synthesis of ethyl 5-aryl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxylate derivatives (2, 5, 8, 11, Scheme 1) [40,65–69]
A suitable diketoester, ethyl 2,4-dioxo-4-phenylbutanoate (1) and
9

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
ethyl 2,4-dioxo-4-(p-tolyl)butanoate (4), ethyl 4-(4-nitrophenyl)-2,4-
dioxobutanoate (7), ethyl 2,4-dioxo-4-(thiophen-2-yl)butanoate (10)
were used as a starting material with the aim to synthesize compounds
50 ml for 6, 150 ml for 12). Glacial acetic acid (2–4 ml) was added into
the mixture and then stirred at room temparature until whitish pre-
cipitate formed. The solid obtained was filtered, washed with water and
alcohol, then dried and crystallized from ethanol to obtain pure com-
pounds as 3, 6, 9, and 12.
2
, 5, 8, and 11, respectively.
A suitable starting compound [1 (22 mmol), 4 (21.3 mmol), 7
(
(
19 mmol), 10 (65 mmol)] was dissolved in a suitable amount ethanol
50 ml for 1, 100 ml for 4, 200 ml for 7, 150 ml for 10) by heating and
4-(3-(Hydrazinecarbonyl)-5-phenyl-1H-pyrazol-1-yl)benzenesulfo-
1
namide, 3: White solid, yield 90%. Mp = 265–267 °C, 265 °C [68].
H
then 4-hydrazinobenzenesulfonamide hydrochloride (22 mmol for 1,
NMR (DMSO‑d , 400 MHz, δ, ppm): 9.61 (s, 1H, eCONHe), 7.83 (d,
6
2
1.3 mmol for 4, 19 mmol for 7, 65 mmol for 10) was added onto the
J = 8.8 Hz, 2H, ArH), 7.49 (d, J = 8.4 Hz, 2H, ArH), 7.40–7.38 (m, 5H,
mixture. The reaction process was monitored by TLC using di-
chloromethane: methanol (4.8:0.2) solvent system. The orange color
reaction mixture was refluxed for several hours as 6 h for 1, 7 h for 4,
ArH), 7.28–7.26 (m, 2H, ArH), 7.01 (s, 1H, H-4 pyrazole), 4.48 (s, 2H,
eNHNH
2
). HRMS (ESI-MS) m/z Calculated: 358.0968 C16
H
16
N
5
O S [M
3
+
+H] ; Found: 358.0954.
6
h for 7, 3 h for 10. The content of the reaction mixture was con-
4-(3-(Hydrazinecarbonyl)-5-(p-tolyl)-1H-pyrazol-1-yl)benzenesulfo-
1
centrated half of its volume under vacuum and then kept at room
temperature. The formed precipitate was filtered, dried and crystallized
from ethanol. All spectral data confirmed the chemical structure of the
compounds.
namide, 6: White solid, yield 51%. Mp = 193–195 °C.
H NMR
(DMSO‑d
6
, 400 MHz, δ, ppm): 9.59 (s, 1H, eCONHe), 7.82 (d,
NH ), 7.19 (d,
J = 8.4 Hz, 2H, ArH), 7.51–7.47 (m, 4H, ArH, eSO
2
2
J = 8.4 Hz, 2H, ArH), 7.14 (d, J = 8.1 Hz, 2H, ArH), 6.97 (s, 1H, H-4
Ethyl 5-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylate,
pyrazole), 4.48 (s, 2H, eNHNH
2
), 2.29 (s, 3H, ArCH
3
). HRMS (ESI-MS)
1
+
2
: White color solid. Yield 71%. Mp = 192–195 °C, 192–194 °C [70]. H
m/z Calculated: 372.1125 C17
H
18
N
5
O
3
S [M+H] ; Found: 372.1114.
NMR (DMSO‑d
.51–7.44 (m, 4H, ArH, -SO
m, 2H, ArH), 7.14 (s, 1H, H-4 pyrazole), 4.32 (q, J = 7.1 Hz, 2H,
eOCH CH ), 1.30 (t, J = 7.1 Hz, 3H, eOCH CH ). HRMS (ESI-MS) m/z
6
, 400 MHz, δ, ppm): 7.84 (d, J = 8.4 Hz, 2H, ArH),
4-(3-(Hydrazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benze-
1
7
2
NH ), 7.39–7.34 (m, 3H, ArH), 7.29–7.27
2
nesulfonamide, 9: Cream solid, yield % 81. Mp = 305–307 °C. H NMR
(
(DMSO‑d , 400 MHz, δ, ppm): 9.68 (s, 1H, eCONHe), 8.22 (d,
6
2
3
2
3
J = 8.8 Hz, 2H, ArH), 7.85 (d, J = 8.8 Hz, 2H, ArH), 7.84–7.52 (m, 6H,
+
Calculated: 372.1013 C18
H
18
N
3
O
4
S [M+H] ; Found: 372.1001.
ArH, eSO
2
NH
2
), 7.21 (s, 1H, H-4 pyrazole). HRMS (ESI-MS) m/z
+
Ethyl 1-(4-sulfamoylphenyl)-5-(p-tolyl)-1H-pyrazole-3-carboxylate,
Calculated: 403.0819 C16
H
15
6
N O
5
S [M+H] ; Found: 403.0812.
1
5
: Cream color solid, yield 91%. Mp = 227–229 °C, 224–226 °C [40]. H
4-(3-(Hydrazinecarbonyl)-5-(thiophen-2-yl)-1H-pyrazol-1-yl)benze-
1
NMR (DMSO‑d
6
, 400 MHz, δ, ppm): 7.85 (d, J = 8.8 Hz, 2H, ArH),
nesulfonamide, 12: White solid, yield 86%. Mp = 210–212 °C. H NMR
7
.51–7.49 (m, 4H, ArH, eSO
2
NH
2
), 7.20–7.14 (m, 4H, ArH), 7.08 (s,
(DMSO‑d , 400 MHz, δ, ppm): 10.2 (s, 1H, eCONHe), 7.93 (d,
6
1
H, H-4 pyrazole), 4.31 (q, J = 7.0 Hz, 2H, eOCH
2
CH
3
), 2.29 (s, 3H,
J = 8.8 Hz, 2H, ArH), 7.68–7.65 (m, 3H, ArH), 7.54 (s, 2H, eSO
2
NH ),
2
ArCH
3
), 1.30 (t, J = 7.0 Hz, 3H, eOCH
2
CH
3
). HRMS (ESI-MS) m/z
7.19 (s, 1H, H-4 pyrazole), 7.15 (d, J = 3.3 Hz, 1H, ArH), 7.10–7.07 (m,
+
Calculated: 386.1169 C19
H
20
N
3
O
4
S [M+H] ; Found: 386.1161.
1H, ArH). HRMS (ESI-MS) m/z Hesaplanan: 364.0533 C14
H
14
N
5
O
S
3 2
Ethyl 5-(4-nitrophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-car-
[M+H]+; Found: 364.0523.
1
boxylate, 8: Cream color solid, yield 89%. Mp = 194–196 °C. H NMR
(
DMSO‑d , 400 MHz, δ, ppm): 8.22 (d, J = 8.8 Hz, 2H, ArH), 7.86 (d,
6
4.2. Crystallography
J = 8.4 Hz, 2H, ArH), 7.58–7.54 (m, 4H, ArH), 7.51 (s, 2H, eSO
2
NH
), 1.31
). HRMS (ESI-MS) m/z Calculated:
2
),
7
.35 (s, 1H, H-4 pyrazole), 4.34 (q, J = 7.1 Hz, 2H, eOCH
CH
2
CH
3
For the crystal structure determination, single-crystal of compound
was used for the data collection on a four-circle Rigaku R-AXIS
(
t, J = 7.1 Hz, 3H, eOCH
2
3
2
+
4
17.0863 C18
H
17
N
4
O S [M+H] ; Found: 417.0846.
6
RAPID-S diffractometer (equipped with a two-dimensional area IP de-
Ethyl 1-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-car-
tector). Graphite-monochromated Mo-K
α
radiation (λ = 0.71073 Å)
boxylate, 11: Cream solid, yield 80%. Mp = 198–200 °C, 197–199 °C
and oscillation scans technique with Δw = 5° for one image were used
1
[
69]. H NMR (DMSO‑d
6
, 400 MHz, δ, ppm): 7.92 (d, J = 8.8 Hz, 2H,
for data collection. The lattice parameters were determined by the least-
2
2
ArH), 7.67–7.64 (m, 3H, ArH), 7.56 (s, 2H, eSO
pyrazole), 7.16 (dd, J = 3.7, 2.6 Hz, 1H, ArH), 7.08 (dd, J = 5.1,
.7 Hz, 1H, ArH), 4.32 (q, J = 7.0 Hz, 2H, eOCH CH ), 1.29 (t,
J = 7.0 Hz, 3H, eOCH CH ). HRMS (ESI-MS) m/z Calculated: 378.0577
[M+H] ; Found: 378.0561.
2
NH
2
), 7.17 (s, 1H, H-4
squares methods based on all reflections with F > 2σ(F ). Integration
of the intensities, correction for Lorentz and polarization effects and cell
refinement were performed using CrystalClear (Rigaku/MSC Inc.,
3
2
3
2
3
2
005) software (Rigaku/MSC, Inc., 9009 New Trails Drive,
+
C
16
H
16
N
3
O
4
S
2
TheWoodlands, TX 7738.) The structure was solved by direct methods
using SHELXS-97 [71] and non-hydrogen atoms were refined using
anisotropic displacement parameters by a full-matrix least-squares
procedure using the program SHELXL-97. H atoms were positioned
geometrically and refined using a riding model. The final difference
Fourier maps showed no peaks of chemical significance. Crystal data for
Synthesis of 4-(3-(hydrazinecarbonyl)-5-aryl-1H-pyrazol-1-yl)
benzenesulfonamide derivatives (3, 6, 9, 12, Scheme 1)
[
40,65–69]
A suitable pyrazole-3-carboxylate derivatives, ethyl 5-phenyl-1-(4-
compound 2: C H N O S, crystal system, space group: monoclinic,
1
8 17 3 4
sulfamoylphenyl)-1H-pyrazole-3-carboxylate (2), ethyl 1-(4-sulfamoyl-
phenyl)-5-(p-tolyl)-1H-pyrazole-3-carboxylate (5), ethyl 5-(4-ni-
trophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylate (8), ethyl
P2
1
/c; (no:14); unit cell dimensions: a = 7.9535(7), b = 16.5706(17),
c = 14.0579(14) Å, α = 90, β = 93.939(3), γ = 90°; volume; 1848.4(3)
3
3
Å , Z = 4; calculated density: 1.33 g/cm ; absorption coefficient:
−1
1
-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxylate
0.203 mm ; F(0 0 0): 766; θ-range for data collection 2.8–26.4°; re-
2
(
11) were used as a starting material to synthesize 3, 6, 9, and 12, re-
finement method: full matrix least-square on F ; data/parameters:
2
spectively.
2924/236; goodness-of-fit on F : 1.184; final R-indices [I > 2σ(I)]:
An appropriate pyrazole-3-carboxylate derivative [2 (16 mmol), 5
18 mmol), 8 (14 mmol), 11 (27 mmol)] was dissolved in ethanol
R = 0.075, wR = 0.20; largest diff. peak and hole: 0.780 and −0.707
1
2
−
3
(
(
e Å
.
100 ml for 2 and 5) and methanol (200 ml for 8 and 11) by heating.
Crystallographic data that were deposited in CSD under CCDC-
Afterwards, the reaction mixture was refluxed after adding hydrazine
hydrate (99%) (320 mmol for 2, 360 mmol for 5, 280 mmol for 8 and
1912930 registration number contains the supplementary crystal-
lographic data for this structure (compound 2). These data can be ob-
tained free of charge from the Cambridge Crystallographic Data Centre
(CCDC) via www.ccdc.cam.ac.uk/data_request/cif and are available
free of charge upon request to CCDC, 12 Union Road, Cambridge, UK
5
40 mmol for 12). The reaction process was monitored by TLC using
dichloromethane:metanol (4.8:0.2) solvent system. After 4–25 h, the
reaction content was poured into the ice-water (100 ml for 3 and 9,
10

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
(
fax: +441223 336033, e-mail: deposit@ccdc.cam.ac.uk).
supplemented with 10% heat-inactivated FBS, 100 units/ml, penicillin
G and 100 µg/ml streptomycin sulfate under a humidified 5% CO at-
2
4
.3. Computational part
mosphere. HGF cells at 10–18 population doubling levels were used in
the present study.
3
High-resolution protein crystal structures of human CAI (PDB ID:
LXZ, 1.55 Å), CAII (PDB ID: 3HS4, 1.1 Å) and CAIX (PDB ID: 3IAI,
.2 Å) in complexed with a native ligand were downloaded from RCSB
Assay for cytotoxic activity. Cells were inoculated at 2.5 × 10
4
2
cells/0.1 ml in a 96-microwell plate (Becton Dickinson Labware,
Franklin Lakes, NJ, U.S.A.). After 48 h, the medium was replaced with
0.1 ml of fresh medium containing different concentrations of single
test compounds. Cells were incubated further for 48 h and the relative
viable cell number was then determined by the MTT method [43]. The
relative viable cell number was determined by the absorbance of the
cell lysate at 562 nm, using a microplate reader (Sunrise Rainbow RC-R;
TECAN, Männedorf, Switzerland). Control cells were treated with the
same amounts of DMSO and the cell damage induced by DMSO was
subtracted from that induced by test agents. The concentration of
compound that reduced the viable cell number by 50% (CC ) was
Protein Data Bank and prepared using Schrödinger's [72] Protein Pre-
paration Wizard [73,74]. Charges and bond orders were assigned, hy-
drogens were added to the heavy atoms, chain A, native ligands and
Zinc metal were kept. All water molecules and heteroatoms were re-
moved and the final structure were optimised. Finally, in order to avoid
steric clashes between the atoms, minimisation was performed until
heavy atoms were converged to a RMSD of 0.3 Å.
The studied ligands were also prepared by subjecting to LigPrep
[
75] tool in the Schrödinger software. The 3D structures including all
5
0
possible low energy ionisation and tautomeric states within the pH
range of 7.0 ± 2 were generated and then geometrically minimised.
The binding site of hCA isoforms were defined by generating a grid
around the native ligand. The default setting for the grid size was
changed to allow ligands having ≤20 Å size to be docked. Glide XP
determined from the dose-response curve and the mean value of CC
5
0
for each cell type was calculated from triplicate assays as described
before [5,43–48,51–53,81–84]. Calculation of tumor-selectivity index
(TS). TS was calculated using the following equation as TS = CC
5
0
against HGF non-malignant cells/CC against malignant HSC-2 cells
5
0
(
extra precision) [76,77] module was used to dock the compounds into
[(B/A) in Table 6]. We have confirmed that the TS value thus de-
termined reflects the antitumor potential of test samples, although
normal and tumor cells are derived from different tissues (mesenchymal
or epithelial tissues, respectively) [85]. We did not use human normal
oral keratinocytes as controls, since doxorubicin and 5-fluorouracil
showed potent cytotoxicity against these epithelial cells by an as yet
unidentified mechanism.
the active site. The default OPLS3e7 force field was used throughout all
calculations.
4
.4. Bioactivity
4
.4.1. Carbonic anhydrase inhibition
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
Calculation of potency-selectivity expression (PSE). PSE was calcu-
lated using the following equation: PSE = TS/CC against tumor
instrument was used to assay the catalytic/inhibition of various CA
isoenzymes [42]. Phenol Red (at a concentration of 0.2 mM) has been
used as an indicator, working at the absorbance maximum of 557 nm,
5
0
2
cells × [that is, (B/A ) × 100 in Table 6)] [49,52]. Statistical treat-
ment. Experimental data are the mean ± standard deviation (SD). The
statistical differences between control and treated groups were eval-
uated by paired Student's t-test. A value of p < 0.05 was considered to
be significant.
with 10 mM Hepes (pH 7.4) as a buffer, 0.1 M Na
2
SO
4
or NaClO (for
4
maintaining constant the ionic strength; these anions are not inhibitory
in the used concentration), following the CA-catalyzed CO hydration
reaction for a period of 5–10 s. Saturated CO solutions in the water at
5 °C were used as substrate. Stock solutions of inhibitors were pre-
2
2
2
Acknowledgments
pared at a concentration of 10 mM (in DMSO) and further diluted to
appropriate concentrations dilutions with the assay buffer mentioned
above. At least 7 different inhibitor concentrations have been used for
measuring the inhibition constant. Inhibitor and enzyme solutions were
preincubated together for 10 min at room temperature prior to assay, in
order to allow for the formation of the E-I complex. Triplicate experi-
ments were done for each inhibitor concentration, and the values re-
ported throughout the paper are the mean of such results. The inhibi-
tion constants were obtained by nonlinear least-squares methods using
the Cheng-Prusoff equation, as reported earlier and represent the mean
from at least three different determinations. All CA isozymes
used here were recombinant proteins obtained as reported earlier
Dr. Cem Yamali was financially supported by The Scientific and
Technological Research Council of Turkey (TUBITAK), Ankara, Turkey
(Grant Number: 2214-A/1059B141601251) with 2214-A International
Ph.D. Research Scholarship Program for his Ph.D. thesis in 2018.
Authors also thank Dr. Yusuf Ozkay and MSc. Serkan Levent (Anadolu
University, Turkey) for HRMS and Ataturk University, Department of
Chemistry for NMR results.
Declaration of interest
The authors report no conflict of interest and are responsible for the
contents and writing of the paper.
[
4,5,45–47,68,78,79].
4
.4.2. Cytotoxic activity
Appendix A. Supplementary material
The following chemicals and reagents were obtained from the in-
dicated companies: Dulbecco's modified Eagle's medium (DMEM), from
GIBCO BRL, Grand Island, NY, USA; fetal bovine serum (FBS), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxor-
ubicin, dimethyl sulfoxide (DMSO) from Wako Pure Chem. Ind., Osaka,
Japan; 5-fluorouracil (5-FU) from Kyowa (Tokyo, Japan); methotrexate
from Nacalai Tesque, Inc., Kyoto, Japan; Culture plastic dishes and
plates (96-well) were purchased from Becton Dickinson (Franklin
Lakes, NJ, USA).
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103222.
References
[1] A. Nocentini, C.T. Supuran, Carbonic anhydrase inhibitors as antitumor/antimeta-
static agents: a patent review (2008–2018), Expert Opin. Ther. Pat. 28 (2018)
7
29–740.
[
[
[
2] C.T. Supuran, Structure and function of carbonic anhydrases, Biochem. J. 473
(2016) 2023–2032.
Human normal oral mesenchymal cells (human gingival fibroblast,
HGF), established from the first premolar tooth extracted from the
lower jaw of a 12-year-old girl [80], and human oral squamous cell
carcinoma cell line HSC-2 (derived from tongue) purchased from Riken
Cell Bank (Tsukuba, Japan), were cultured at 37 °C in DMEM
3] C.T. Supuran, Carbonic anhydrase inhibitors and their potential in a range of
therapeutic areas, Expert Opin. Ther. Pat. 28 (2018) 709–712.
4] H.I. Gul, C. Yamali, M. Bulbuller, P.B. Kirmizibayrak, M. Gul, A. Angeli, S. Bua,
C.T. Supuran, Anticancer effects of new dibenzenesulfonamides by inducing
apoptosis and autophagy pathways and their carbonic anhydrase inhibitory effects
11

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
on hCA I, hCA II, hCA IX, hCA XII isoenzymes, Bioorg. Chem. 78 (2018) 290–297.
clinical use, potently inhibits several mammalian carbonic anhydrases, Chem.
[
5] H.I. Gul, C. Yamali, H. Sakagami, A. Angeli, J. Leitans, A. Kazaks, K. Tars,
Commun. (Camb.) 48 (2012) 8177–8179.
D.O. Ozgun, C.T. Supuran, New anticancer drug candidates sulfonamides as selec-
tive hCA IX or hCA XII inhibitors, Bioorg. Chem. 77 (2018) 411–419.
6] S.M. Monti, C.T. Supuran, G. De Simone, Anticancer carbonic anhydrase inhibitors:
a patent review (2008–2013), Expert Opin. Ther. Pat. 23 (2013) 737–749.
7] C.T. Supuran, Carbonic anhydrase inhibition and the management of hypoxic tu-
mors, Metabolites 7 (2017) 48–61.
[32] S.G. Kucukguzel, S. Senkardes, Recent advances in bioactive pyrazoles, Eur. J. Med.
Chem. 97 (2015) 786–815.
[
[
[
[33] D. Raffa, B. Maggio, M.V. Raimondi, S. Cascioferro, F. Plescia, G. Cancemi,
G. Daidone, Recent advanced in bioactive systems containing pyrazole fused with a
five membered heterocycle, Eur. J. Med. Chem. 97 (2015) 732–746.
[34] M.M. Goldenberg, Celecoxib, a selective cyclooxygenase-2 inhibitor for the treat-
ment of rheumatoid arthritis and osteoarthritis, Clin. Ther. 21 (1999) 1497–1513
discussion 1427-1498.
8] J. Meehan, C. Ward, A. Turnbull, J. Bukowski-Wills, A.J. Finch, E.J. Jarman,
C. Xintaropoulou, C. Martinez-Perez, M. Gray, M. Pearson, P. Mullen, C.T. Supuran,
F. Carta, D.J. Harrison, I.H. Kunkler, S.P. Langdon, Inhibition of pH regulation as a
therapeutic strategy in hypoxic human breast cancer cells, Oncotarget 8 (2017)
[35] P.L. McCormack, Celecoxib: a review of its use for symptomatic relief in the
treatment of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis, Drugs
71 (2011) 2457–2489.
4
2857–42875.
[
9] F. Carta, C.T. Supuran, A. Scozzafava, Sulfonamides and their isosters as carbonic
anhydrase inhibitors, Future Med. Chem. 6 (2014) 1149–1165.
[36] A.L. Hsu, T.T. Ching, D.S. Wang, X. Song, V.M. Rangnekar, C.S. Chen, The cy-
clooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in
human prostate cancer cells independently of Bcl-2, J. Biol. Chem. 275 (2000)
11397–11403.
[
[
10] Y. Lou, P.C. McDonald, A. Oloumi, S. Chia, C. Ostlund, A. Ahmadi, A. Kyle, U. Auf
dem Keller, S. Leung, D. Huntsman, B. Clarke, B.W. Sutherland, D. Waterhouse,
M. Bally, C. Roskelley, C.M. Overall, A. Minchinton, F. Pacchiano, F. Carta,
A. Scozzafava, N. Touisni, J.Y. Winum, C.T. Supuran, S. Dedhar, Targeting tumor
hypoxia: suppression of breast tumor growth and metastasis by novel carbonic
anhydrase IX inhibitors, Cancer Res. 71 (2011) 3364–3376.
[37] A.T. Koki, J.L. Masferrer, Celecoxib: a specific COX-2 inhibitor with anticancer
properties, Cancer Control. 9 (2002) 28–35.
[38] P.M. Forde, D.S. Ettinger, Targeted therapy for non-small-cell lung cancer: past,
present and future, Expert Rev. Anticancer Ther. 13 (2013) 745–758.
[39] G. Greenfield, S. McPherson, K. Mills, M.F. McMullin, The ruxolitinib effect: un-
derstanding how molecular pathogenesis and epigenetic dysregulation impact
therapeutic efficacy in myeloproliferative neoplasms, J. Transl. Med. 16 (2018)
360.
11] 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 metas-
tasis, J. Med. Chem. 54 (2011) 1896–1902.
[
[
12] P.C. McDonald, J.Y. Winum, C.T. Supuran, S. Dedhar, Recent developments in
targeting carbonic anhydrase IX for cancer therapeutics, Oncotarget 3 (2012)
[40] T. Rogez-Florent, S. Meignan, C. Foulon, P. Six, A. Gros, C. Bal-Mahieu,
C.T. Supuran, A. Scozzafava, R. Frederick, B. Masereel, P. Depreux, A. Lansiaux,
J.F. Goossens, S. Gluszok, L. Goossens, New selective carbonic anhydrase IX in-
hibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzene-
sulfonamides, Bioorg. Med. Chem. 21 (2013) 1451–1464.
8
4–97.
13] J.S. Yang, C.W. Lin, C.Y. Chuang, S.C. Su, S.H. Lin, S.F. Yang, Carbonic anhydrase IX
overexpression regulates the migration and progression in oral squamous cell car-
cinoma, Tumour Biol. 36 (2015) 9517–9524.
[41] M.J. Uddin, B.C. Crews, K. Ghebreselasie, M.N. Tantawy, L.J. Marnett, [I]-Celecoxib
analogues as SPECT tracers of cyclooxygenase-2 in inflammation, ACS Med. Chem.
Lett. 2 (2011) 160–164.
[
[
[
14] E.R. Swenson, Safety of carbonic anhydrase inhibitors, Expert Opin. Drug Saf. 13
(
2014) 459–472.
15] C.T. Supuran, Inhibition of carbonic anhydrase IX as a novel anticancer mechanism,
World J. Clin. Oncol. 3 (2012) 98–103.
[42] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-
flow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246
(1971) 2561–2573.
16] K. D'Ambrosio, R.M. Vitale, J.M. Dogne, B. Masereel, A. Innocenti, A. Scozzafava,
G. De Simone, C.T. Supuran, Carbonic anhydrase inhibitors: bioreductive nitro-
containing sulfonamides with selectivity for targeting the tumor associated isoforms
IX and XII, J. Med. Chem. 51 (2008) 3230–3237.
[43] H. Sakagami, Y. Uesawa, M. Ishihara, H. Kagaya, T. Kanamoto, S. Terakubo,
H. Nakashima, K. Takao, Y. Sugita, Quantitative structure-cytotoxicity relationship
of oleoylamides, Anticancer Res. 35 (2015) 5341–5351.
[
[
17] N. Chiaramonte, M.N. Romanelli, E. Teodori, C.T. Supuran, Amino acids as building
blocks for carbonic anhydrase inhibitors, Metabolites 8 (2018) 36–58.
18] Z.C. Mam, Y. Mboge, Alyssa Wolff, John V. Mathias, Tu. Chingkuang, Kevin
D. Brown, Murat Bozdag, Fabrizio Carta, Claudiu T. Supuran, Robert McKenna,
Susan C. FrostID, Selective inhibition of carbonic anhydrase IX over carbonic an-
hydrase XII in breast cancer cells using benzene sulfonamides: disconnect between
activity and growth inhibition, PLoS ONE 13 (2018) 1–27.
[44] H.I. Gul, M. Tugrak, H. Sakagami, P. Taslimi, I. Gulcin, C.T. Supuran, Synthesis and
bioactivity studies on new 4-(3-(4-Substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-
c]pyrazol-2-yl) benzenesulfonamides, J. Enzyme Inhib. Med. Chem. 31 (2016)
1619–1624.
[45] E. Unluer, H.I. Gul, A. Demirtas, H. Sakagami, N. Umemura, M. Tanc, C. Kazaz,
C.T. Supuran, Synthesis and bioactivity studies of 1-aryl-3-(2-hydroxyethylthio)-1-
propanones, J. Enzyme Inhib. Med. Chem. 31 (2016) 105–109.
[
[
19] C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist? J.
Enzyme Inhib. Med. Chem. 31 (2016) 345–360.
[46] C. Yamali, H.I. Gul, H. Sakagami, C.T. Supuran, Synthesis and bioactivities of ha-
logen bearing phenolic chalcones and their corresponding bis Mannich bases, J.
Enzyme Inhib. Med. Chem. 31 (2016) 125–131.
20] A. Mollica, R. Costante, A. Akdemir, S. Carradori, A. Stefanucci, G. Macedonio,
M. Ceruso, C.T. Supuran, Exploring new Probenecid-based carbonic anhydrase in-
hibitors: synthesis, biological evaluation and docking studies, Bioorg. Med. Chem.
[47] H.I. Gul, E. Mete, S.E. Eren, H. Sakagami, C. Yamali, C.T. Supuran, Designing,
synthesis and bioactivities of 4-[3-(4-hydroxyphenyl)-5-aryl-4,5-dihydro-pyrazol-1-
yl]benzenesulfonamides, J. Enzyme Inhib. Med. Chem. 32 (2017) 169–175.
[48] H.I. Gul, C. Yamali, F. Yesilyurt, H. Sakagami, K. Kucukoglu, I. Gulcin, M. Gul,
C.T. Supuran, Microwave-assisted synthesis and bioevaluation of new sulfonamides,
J. Enzyme Inhib. Med. Chem. 32 (2017) 369–374.
2
3 (2015) 5311–5318.
[
21] F. Carta, D. Vullo, S.M. Osman, Z. AlOthman, C.T. Supuran, Synthesis and carbonic
anhydrase inhibition of a series of SLC-0111 analogs, Bioorg. Med. Chem. 25 (2017)
2
569–2576.
[
[
22] C.T. Supuran, Carbonic anhydrase inhibitors as emerging agents for the treatment
and imaging of hypoxic tumors, Expert Opin. Investig. Drugs. 27 (2018) 963–970.
23] W.M. Eldehna, M.F. Abo-Ashour, E. Berrino, D. Vullo, H.A. Ghabbour, S.T. Al-
Rashood, G.S. Hassan, H.M. Alkahtani, A.A. Almehizia, A. Alharbi, H.A. Abdel-Aziz,
C.T. Supuran, SLC-0111 enaminone analogs, 3/4-(3-aryl-3-oxopropenyl) amino-
benzenesulfonamides, as novel selective subnanomolar inhibitors of the tumor-as-
sociated carbonic anhydrase isoform IX, Bioorg. Chem. 83 (2019) 549–558.
24] C.T. Supuran, Indisulam: an anticancer sulfonamide in clinical development, Expert
Opin. Investig. Drugs 12 (2003) 283–287.
[49] H. Sakagami, Y. Masuda, M. Tomomura, S. Yokose, Y. Uesawa, N. Ikezoe,
D. Asahara, K. Takao, T. Kanamoto, S. Terakubo, H. Kagaya, H. Nakashima,
Y. Sugita, Quantitative structure-cytotoxicity relationship of chalcones, Anticancer
Res. 37 (2017) 1091–1098.
[50] H. Sakagami, N. Okudaira, Y. Masuda, O. Amano, S. Yokose, Y. Kanda, M. Suguro,
T. Natori, H. Oizumi, T. Oizumi, Induction of apoptosis in human oral keratinocyte
by doxorubicin, Anticancer Res. 37 (2017) 1023–1029.
[
[
[51] H.I. Gul, M. Tugrak, M. Gul, H. Sakagami, N. Umemura, B. Anil, Synthesis and
cytotoxicities of new azafluorenones with apoptotic mechanism of action and cell
cycle analysis, Anticancer Agents Med. Chem. 18 (2018) 1770–1778.
[52] C. Yamali, H.I. Gul, G. Gunesacar, H. Sakagami, N. Okudaira, Y. Uesawa, H. Kagaya,
Cytotoxicity, apoptosis, and QSAR studies of phenothiazine derived methoxylated
chalcones as anticancer drug candidates, Med. Chem. Res. 27 (2018) 2366–2378.
[53] D. Ozmen Ozgun, H.I. Gul, C. Yamali, H. Sakagami, I. Gulcin, M. Sukuroglu,
C.T. Supuran, Synthesis and bioactivities of pyrazoline benzensulfonamides as
carbonic anhydrase and acetylcholinesterase inhibitors with low cytotoxicity,
Bioorg. Chem. 84 (2019) 511–517.
25] D.C. Talbot, J. von Pawel, E. Cattell, S.M. Yule, C. Johnston, A.S. Zandvliet,
A.D. Huitema, C.J. Norbury, P. Ellis, L. Bosquee, M. Reck, A randomized phase II
pharmacokinetic and pharmacodynamic study of indisulam as second-line therapy
in patients with advanced non-small cell lung cancer, Clin. Cancer Res. 13 (2007)
1
816–1822.
[
26] P. Khloya, G. Celik, SitaRam, D. Vullo, C.T. Supuran, P.K. Sharma, 4-Functionalized
1
,3-diarylpyrazoles bearing benzenesulfonamide moiety as selective potent in-
hibitors of the tumor associated carbonic anhydrase isoforms IX and XII, Eur. J.
Med. Chem. 76 (2014) 284–290.
[54] S. Listl, L. Jansen, A. Stenzinger, K. Freier, K. Emrich, B. Holleczek, A. Katalinic,
A. Gondos, H. Brenner, G.C.S.W. Group, Survival of patients with oral cavity cancer
in Germany, PLoS ONE 8 (2013) e53415.
[
[
27] R. Rajendra, R.L. Jones, S.M. Pollack, Targeted treatment for advanced soft tissue
sarcoma: profile of pazopanib, Onco. Targets Ther. 6 (2013) 217–222.
28] S.T. Kim, H.L. Jang, S.J. Lee, J. Lee, Y.L. Choi, K.M. Kim, J. Cho, S.H. Park,
Y.S. Park, H.Y. Lim, M. Yashiro, W.K. Kang, J.O. Park, Pazopanib, a novel multi-
targeted kinase inhibitor, shows potent in vitro antitumor activity in gastric cancer
cell lines with FGFR2 amplification, Mol. Cancer Ther. 13 (2014) 2527–2536.
29] A.M. Czarnecka, M. Kawecki, F. Lian, J. Korniluk, C. Szczylik, Feasibility, efficacy
and safety of tyrosine kinase inhibitor treatment in hemodialyzed patients with
renal cell cancer: 10 years of experience, Future Oncol. 11 (2015) 2267–2282.
30] G. Sonpavde, T.E. Hutson, Pazopanib: a novel multitargeted tyrosine kinase in-
hibitor, Curr. Oncol. Rep. 9 (2007) 115–119.
[55] N.T. Brockton, P. Lohavanichbutr, E.K. Enwere, M.P. Upton, E.N. Kornaga,
S.C. Nakoneshny, P. Bose, C. Chen, J.C. Dort, Impact of tumoral carbonic anhydrase
IX and Ki-67 expression on survival in oral squamous cell carcinoma patients,
Oncol. Lett. 14 (2017) 5434–5442.
[
[56] A.W. Eckert, S. Horter, D. Bethmann, J. Kotrba, T. Kaune, S. Rot, M. Bache,
U. Bilkenroth, W. Reich, T. Greither, C. Wickenhauser, D. Vordermark, H. Taubert,
M. Kappler, Investigation of the prognostic role of carbonic anhydrase 9 (CAIX) of
the cellular mRNA/Protein level or soluble CAIX protein in patients with oral
squamous cell carcinoma, Int. J. Mol. Sci. 20 (2019) 375–392.
[
[
31] J.Y. Winum, A. Maresca, F. Carta, A. Scozzafava, C.T. Supuran, Polypharmacology
of sulfonamides: pazopanib, a multitargeted receptor tyrosine kinase inhibitor in
[57] M.H. Chien, T.H. Ying, Y.H. Hsieh, C.H. Lin, C.H. Shih, L.H. Wei, S.F. Yang, Tumor-
associated carbonic anhydrase XII is linked to the growth of primary oral squamous
12

C. Yamali, et al.
Bioorganic Chemistry 92 (2019) 103222
cell carcinoma and its poor prognosis, Oral Oncol. 48 (2012) 417–423.
inhibitors, Bioorg. Med. Chem. Lett. 26 (2016) 406–412.
[
[
58] N.J. Beasley, C.C. Wykoff, P.H. Watson, R. Leek, H. Turley, K. Gatter, J. Pastorek,
[70] D.V.M. Laxmikant, S. Pavase, Kamalkishor G. Baheti, Anti-inflammatory explora-
tion of sulfonamide containing diaryl pyrazoles with promising COX-2 selectivity
and enhanced gastric safety profile, J. Het. Chem. 55 (2018) 913–922.
[71] G.M. Sheldrick, A short history of SHELX, Acta Crystallogr A 64 (2008) 112–122.
[72] Small-Molecule Drug Discovery Suite 2019-2, Schrödinger, LLC, New York, NY,
2019.
G.J. Cox, P. Ratcliffe, A.L. Harris, Carbonic anhydrase IX, an endogenous hypoxia
marker, expression in head and neck squamous cell carcinoma and its relationship
to hypoxia, necrosis, and microvessel density, Cancer Res. 61 (2001) 5262–5267.
59] H.J. Shin, S.B. Rho, D.C. Jung, I.O. Han, E.S. Oh, J.Y. Kim, Carbonic anhydrase IX
(
(
CA9) modulates tumor-associated cell migration and invasion, J. Cell Sci. 124
2011) 1077–1087.
[73] Schrödinger Release 2019-2: Schrödinger Suite 2019-2 Protein Preparation Wizard;
Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York,
NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2019.
[
[
[
60] E. Svastova, S. Pastorekova, Carbonic anhydrase IX: a hypoxia-controlled “catalyst”
of cell migration, Cell. Adh. Migr. 7 (2013) 226–231.
61] C.A. Taft, V.B. Da Silva, C.H. Da Silva, Current topics in computer-aided drug de-
sign, J. Pharm. Sci. 97 (2008) 1089–1098, https://doi.org/10.1002/jps.21293.
62] A. Ece, Towards more effective acetylcholinesterase inhibitors: a comprehensive
modelling study based on human acetylcholinesterase protein–drug complex, J.
Biomol. Struct. Dyn. 11 (2019) 1–8, https://doi.org/10.1080/07391102.2019.
[74] G.M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju, W. Sherman, Protein and li-
gand preparation: parameters, protocols, and influence on virtual screening en-
richments, J. Comput. Aided Mol. Des. 27 (2013) 221–234.
[75] Schrödinger Release 2019-2: LigPrep, Schrödinger, LLC, New York, NY, 2019.
[76] Schrödinger Release 2019-2: Glide, Schrödinger, LLC, New York, NY, 2019.
[77] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood, T.A. Halgren,
P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and scoring in-
corporating a model of hydrophobic enclosure for protein-ligand complexes, J.
Med. Chem. 49 (2006) 6177–6196.
1
583606.
[
[
63] S. Biswas, R. McKenna, C.T. Supuran, Effect of incorporating a thiophene tail in the
scaffold of acetazolamide on the inhibition of human carbonic anhydrase isoforms I,
II, IX and XII, Bioorg. Med. Chem. Lett. 23 (2013) 5646–5649.
64] A. Ece, e-Pharmacophore mapping combined with virtual screening and molecular
docking to identify potent and selective inhibitors of P90 ribosomal S6 kinase
[78] H.I. Gul, Z. Yazici, M. Tanc, C.T. Supuran, Inhibitory effects of benzimidazole
containing new phenolic Mannich bases on human carbonic anhydrase isoforms
hCA I and II, J. Enzyme Inhib. Med. Chem. 31 (2016) 1540–1544.
(
RSK), Turk. J. Pharm. Sci. 13 (2016) 241–248, https://doi.org/10.4274/tjps.
2
8290.
[79] C. Yamali, M. Tugrak, H.I. Gul, M. Tanc, C.T. Supuran, The inhibitory effects of
phenolic Mannich bases on carbonic anhydrase I and II isoenzymes, J. Enzyme
Inhib. Med. Chem. 31 (2016) 1678–1681.
[
65] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter,
M.J. Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier,
S.S. Yu, E.G. AndersonGd, J.N. Burton, S.A. Cogburn, C.M. Gregory, W.E. Koboldt,
K. Perkins, A.W. Seibert, Y.Y. Veenhuizen, P.C. Isakson Zhang, Synthesis and bio-
logical evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors:
identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benze
nesulfonamide (SC-58635, celecoxib), J. Med. Chem. 40 (1997) 1347–1365.
66] R. Di Santo, R. Costi, A. Roux, G. Miele, G.C. Crucitti, A. Iacovo, F. Rosi,
A. Lavecchia, L. Marinelli, C. Di Giovanni, E. Novellino, L. Palmisano, M. Andreotti,
R. Amici, C.M. Galluzzo, L. Nencioni, A.T. Palamara, Y. Pommier, C. Marchand,
Novel quinolinonyl diketo acid derivatives as HIV-1 integrase inhibitors: design,
synthesis, and biological activities, J. Med. Chem. 51 (2008) 4744–4750.
67] R.V. Ragavan, V. Vijayakumar, N.S. Kumari, Synthesis and antimicrobial activities
of novel 1,5-diaryl pyrazoles, Eur. J. Med. Chem. 45 (2010) 1173–1180.
[80] K. Kantoh, M. Ono, Y. Nakamura, Y. Nakamura, K. Hashimoto, H. Sakagami,
H. Wakabayashi, Hormetic and anti-radiation effects of tropolone-related com-
pounds, In Vivo 24 (2010) 843–851.
[81] C. Yamali, H.I. Gul, D. Ozmen Ozgun, H. Sakagam, N. Umemura, C. Kazaz, M. Gul,
Synthesis and cytotoxic activities of difluoro-dimethoxy chalcones, Anticancer
Agents Med. Chem. 17 (2017) 1426–1433.
[
[82] M. Tugrak, C. Yamali, H. Sakagami, H.I. Gul, Synthesis of mono Mannich bases of 2-
(4-hydroxybenzylidene)-2,3-dihydroinden-1-one and evaluation of their cytotoxi-
cities, J. Enzyme Inhib. Med. Chem. 31 (2016) 818–823.
[83] K. Kucukoglu, F. Oral, T. Aydin, C. Yamali, O. Algul, H. Sakagami, I. Gulcin,
C.T. Supuran, H.I. Gul, Synthesis, cytotoxicity and carbonic anhydrase inhibitory
activities of new pyrazolines, J. Enzyme Inhib. Med. Chem. 31 (2016) 20–24.
[84] H. Inci Gul, C. Yamali, A. Tugce Yasa, E. Unluer, H. Sakagami, M. Tanc,
C.T. Supuran, Carbonic anhydrase inhibition and cytotoxicity studies of Mannich
base derivatives of thymol, J. Enzyme Inhib. Med. Chem. 31 (2016) 1375–1380.
[85] R. Suzuki, S. Matsuno, H. Sakagami, Y. Okada, Y. Shirataki, Search of new cytotoxic
[
[
68] H.S. Ibrahim, S.M. Abou-Seri, M. Tanc, M.M. Elaasser, H.A. Abdel-Aziz,
C.T. Supuran, Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-
associated carbonic anhydrase isoforms IX and XII, Eur. J. Med. Chem. 103 (2015)
5
83–593.
1
[
69] K.R.A. Abdellatif, E.K.A. Abdelall, W.A.A. Fadaly, G.M. Kamel, Synthesis, cycloox-
ygenase inhibition, anti-inflammatory evaluation and ulcerogenic liability of new
crude materials against human oral squamous cell carcinoma using H NMR-based
metabolomics, Anticancer Res. 34 (2014) 4117–4120.
1
,3,5-triarylpyrazoline and 1,5-diarylpyrazole derivatives as selective COX-2
13