Comprehensive study on potent and selective carbonic anhydrase inhibitors: Synthesis, bioactivities and molecular modelling studies of 4-(3-(2-arylidenehydrazine-1-carbonyl)-5-(thiophen-2-yl)-1H-pyrazole-1-yl) benzenesulfonamides

Article abstract of DOI:10.1016/j.ejmech.2021.113351

In this research, rational design, synthesis, carbonic anhydrases (CAs) inhibitory effects, and cytotoxicities of the 4-(3-(2-arylidenehydrazine-1-carbonyl)-5-(thiophen-2-yl)-1H-pyrazole-1-yl)benzenesulfonamides 1–20 were reported. Compound 18 (Ki = 7.0 nM) was approximately 127 times more selective cancer-associated hCA IX inhibitor over hCA I, while compound 17 (Ki = 10.6 nM) was 47 times more selective inhibitor of hCA XI over hCA II compared to the acetazolamide. Compounds 11 (CC₅₀ = 5.2 μM) and 20 (CC₅₀ = 1.6 μM) showed comparative tumor-specificity (TS= > 38.5; >128.2) with doxorubicin (TS > 43.0) towards HSC-2 cancer cell line. Western blot analysis demonstrated that 11 induced slightly apoptosis whereas 20 did not induce detectable apoptosis. A preliminary analysis showed that some correlation of tumor-specificity of 1–20 with the chemical descriptors that reflect hydrophobic volume, dipole moment, lowest hydrophilic energy, and topological structure. Molecular docking simulations were applied to the synthesized ligands to elucidate the predicted binding mode and selectivity profiles towards hCA I, hCA II, and hCA IX.

Full text of DOI:10.1016/j.ejmech.2021.113351

European Journal of Medicinal Chemistry 217 (2021) 113351
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Comprehensive study on potent and selective carbonic anhydrase
inhibitors: Synthesis, bioactivities and molecular modelling studies of
4-(3-(2-arylidenehydrazine-1-carbonyl)-5-(thiophen-2-yl)-1H-
pyrazole-1-yl) benzenesulfonamides
,
Cem Yamali a b, Hiroshi Sakagami c, Yoshihiro Uesawa d, Kota Kurosaki d, Keitaro Satoh e,
Yoshiko Masuda f, Satoshi Yokose g, Abdulilah Ece h, Silvia Bua i, Andrea Angeli i,
,
*
Claudiu T. Supuran i, Halise Inci Gul a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey
b Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Cukurova University, Adana, Turkey
c Research Institute of Odontology (M-RIO), Meikai University, Saitama, Japan
d Department of Medical Molecular Informatics, Meiji Pharmaceutical University, Tokyo, Japan
e Division of Pharmacology, Meikai University School of Dentistry, Saitama, Japan
f Department of Operative Dentistry, Matsumoto Dental University, Nagano, Japan
g Division of Endodontics and Operative Dentistry, Meikai University School of Dentistry, Sakado, Saitama, Japan
h Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Biruni University, Istanbul, Turkey
i Neurofarba Department, Sezione di Scienza Farmaceutiche e Nutraceutiche, Universita Degli Studi di Firenze, Via U. Schiff 6, 50019, Sesto Fiorentino,
Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this research, rational design, synthesis, carbonic anhydrases (CAs) inhibitory effects, and cytotoxic-
ities of the 4-(3-(2-arylidenehydrazine-1-carbonyl)-5-(thiophen-2-yl)-1H-pyrazole-1-yl)benzenesulfo-
namides 1e20 were reported. Compound 18 (Ki ¼ 7.0 nM) was approximately 127 times more selective
cancer-associated hCA IX inhibitor over hCA I, while compound 17 (Ki ¼ 10.6 nM) was 47 times more
Received 11 January 2021
Received in revised form
21 February 2021
Accepted 27 February 2021
Available online 6 March 2021
selective inhibitor of hCA XI over hCA II compared to the acetazolamide. Compounds 11 (CC50 ¼ 5.2
mM)
and 20 (CC50 ¼ 1.6 M) showed comparative tumor-specificity (TS¼ > 38.5; >128.2) with doxorubicin
m
(TS > 43.0) towards HSC-2 cancer cell line. Western blot analysis demonstrated that 11 induced slightly
apoptosis whereas 20 did not induce detectable apoptosis. A preliminary analysis showed that some
correlation of tumor-specificity of 1e20 with the chemical descriptors that reflect hydrophobic volume,
dipole moment, lowest hydrophilic energy, and topological structure. Molecular docking simulations
were applied to the synthesized ligands to elucidate the predicted binding mode and selectivity profiles
towards hCA I, hCA II, and hCA IX.
Keywords:
Carbonic anhydrase
hCA IX
Anticancer
OSCC
Apoptosis
Pyrazole
© 2021 Elsevier Masson SAS. All rights reserved.
Benzenesulfonamide
Hydrazone
Docking
1. Introduction
the clinic also have severe side effects [2]. Therefore, there is an
ongoing need to develop novel anticancer agents with fewer side
Despite the valuable progress in the survival of cancer patients,
cancer is still one of the deathful and rising public health concerns.
In 2018, nearly 10 million patients died due to cancer, and it is also
predicted that new cancer cases will be around 29e37 million by
2040 [1]. Many of the chemotherapeutic anticancer drugs used in
effects that may act by different mechanisms of actions and to find
out new cancer biomarkers or pathways that have a crucial role
throughout the cancer process.
As an attractive superfamily of enzymes in many organisms,
carbonic anhydrases (CAs, EC 4.2.1.1) generally include a tightly
bound Zn2þ ion that interacts with water and histidine molecules at
the active site of the enzyme. The function of Zn2þ ion is to bind and
activate water molecule to catalyze the reversible hydration reac-
tion of carbon dioxide into bicarbonate and proton ions as in
* Corresponding author.
E-mail address: incigul1967@yahoo.com (H.I. Gul).
https://doi.org/10.1016/j.ejmech.2021.113351
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
equation CO2 þ H2O $ HCO3ꢀ þ Hþ by a metal hydroxide nucleo-
philic mechanism. CAs isoenzymes are significant cellular targets
for many pathological processes and pharmacological applications
such as anticonvulsants, diuretics, antiglaucoma, anticancer agents,
and diagnostic tools for imaging hypoxic tumors [3].
individual residues of the active site and also tail part of the com-
pound provides isoenzyme specificity over the off-target isoenzymes
[8,25]. Hydrazone groupasa bioisostericgroup of ureaisreportedasa
tailing linker in several benzenesulfonamide derivatives, which is the
most effective class of CA inhibitors [19]. The hydrazone linker can
also improve the flexibility of the compound to improve the selec-
tivity of the compounds towards hCA IX by leading to favorable in-
teractions with the specific residues of the enzyme [25].
In the members of the superfamily a-CAs in mammals, which
differ by features, cellular distribution, expression levels, and
response to different classes of inhibitors, especially the hypoxia-
induced transmembrane CA IX isoenzyme, along with the trans-
membrane CA XII isoenzyme, has been approved as promising
cancer biomarkers as key pH regulators at the cancer process [4].
The lack of oxygen called hypoxia regulates the expression of
several enzymes or transporters by the hypoxia-inducible factor 1
(HIF1) cascade [5,6]. Since hypoxia-inducible CA IX is an effective
isoenzyme for the conversion of CO2 in bicarbonate and proton
ions, it also contributes to the acidification of the tumor environ-
ment [6]. Because approximately 70% of the hypoxic tumors over-
express CA IX isoenzyme, the cancer cells show unfavorable
response to the classical chemotherapies and radiotherapies due to
increased resistance or several undesired pathways [7]. Whereas
cancer-associated CAs are expressed in a limited amount in normal
tissues, they are upregulated or overexpressed in tumor cells such
as colon, pancreas, breast, brain, head and neck cancers, squamous/
basal cell carcinomas [8,9]. Besides, there are numerous studies on
the role of CA IX in oral oncogenesis as a predictor for oral squa-
mous cell carcinomas (OSCC) [10,11]. Hypoxia is also known as one
of the important symptoms in OSCC, and novel molecular hall-
marks are needed to understand the process of OSCC [12]. Even
different results are reported, some studies found out a relationship
between the expression level of CA IX in OSCC as a prognostic
biomarker [11,13,14]. According to recent studies reported by our
research group, several CA IX and/or CA XII inhibitors showed
promising in vitro cytotoxicities in OSCC cell lines [15e18].
On the other hand, off-target CA I and CA II isoenzymes, which
express in red blood cells, cause problematic issues since CA IX and
CA XII isoenzymes have approximately 34% similarity with CA II
isoenzyme. Therefore, CA I and CA II isoenzymes may reduce the
bioavailability of the non-selective CA IX and CA XII inhibitors
[19e21]. The properties of the most studied CA I, CA II, and CA IX
isoenzymes were given in Table 1 [22]. Based on the findings, the
main drawback for developing anticancer drug candidates target-
ing cancer-associated CAs is known as selective inhibition [21].
The well-known selective anticancer drug candidate is the sul-
fonamide derivative SLC- 0111 (Fig. 1), an effective inhibitor of CA IX
and CA XII, which was forwarded in further clinical studies [23,24].
SLC-0111, which has ureido linker between the zinc-binding group
(ZBG) benzenesulfonamide and the tail of the inhibitor, was designed
usingthewell-knowntailapproach. Thislinker moietyledtoenhance
the flexibility of the tail part of the compound to interact with the
Reported CAs inhibitors having a similar chemical structure to the
presentstudywereshowninFig.1. Inpreviousstudiesreportedbyour
group, pyrazoline-benzenesulfonamides including thiophen ring 1
were declared as a selective inhibitor of cancer-related hCA XII
isoenzymewiththeselectivityvaluesofhCAI/hCAXII¼1250 and hCA
II/hCA XII ¼ 224 (Fig. 1) [17]. In another study, some 1,3,5-
trisubstituted pyrazole-benzenesulfonamides incorporating hydra-
zine moiety were also reported as selective hCA IX inhibitors. In that
study, the compound 2 having thiophen ring showed moderate
selectivity towards hCA IX with the selectivity values of hCA I/hCA
IX ¼ 4.6 and hCA II/hCA IX ¼ 0.9 (Fig. 1) [18]. Sharma V et al. [26]
reported the inhibition potential of the compound 3, tri-
fluoromethylhydrazone-carbonyl-1,2,3-triazole containing benze-
nesulfonamide (Fig. 1) against tumor-associated hCA IX with the
value of Ki ¼ 1.4 nM. Allam HA et al. [20] reported hydrazone de-
rivatives incorporatingbenzenesulfonamidemoiety, 4 (Fig.1), asCAIs,
in the range of 3.1e23.7 nM towards hCA IX. Yazıcı-Demir K et al. [27]
reported indole-based sulfonamide derivatives 5 (Fig. 1) having
hydrazone moiety as selective CAIs with Ki value of 9.2e476.1 nM
(hCA IX) and 1.4e219.7 nM (hCA XII). Based on the studies, it can be
expressed that using heterocyclic rings and/or hydrazone moiety
incorporating benzenesulfonamide can be considered as one of the
modifications to increase the isoenzyme selectivity.
Numerous studies and our previous experiences pointed out
that heterocyclic ring bearing sulfonamides/benzenesulfonamides
are an attractive group of compounds with their significant CAs
inhibitory activity profiles and anticancer effects as mentioned
above. As a part of our ongoing studies to develop potent and se-
lective enzyme inhibitors targeting the cancer-related hCA IX
isoenzyme, herein, we designed and synthesized a class of hydra-
zone derivatives incorporating pyrazole-benzenesulfonamide
(Scheme 1) to evaluate in vitro biological effects towards hCA I,
hCA II, hCA IX, and OSCC cell lines. Furthermore, mechanism of
action studies, molecular docking, and QSAR studies were applied.
2. Results and discussion
2.1. Chemistry
A wide range of bioactivities of hydrazones makes them a popular
compound class in drug design and development studies as well as in
Table 1
The properties of the most studied human CAs [22].
Organ/Tissue
Distribution
Subcellular Localization
Catalytic Activity (CO2 Hydration)
low
Affinity for Sulfonamides
Target Diseases
CAI
erythrocytes
gastrointestinal
tract eye
cytosol
medium
retinal cerebral edema
CA II
erythrocytes eye
gastrointestinal
tract bone
cytosol
high
very high
glaucoma edema epilepsy altitude sickness
osteoclasts kidney,
lung, testis, brain
CA IX
tumors
transmembrane
high
high
cancer
gastrointestinal
mucosa
2

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
Fig. 1. Chemical structure of the potent CAs inhibitors and target compounds 1e20.
synthetic chemistry [28,29]. The synthesis pathway of the hydrazone
derivatives of pyrazole-benzenesulfonamide is presented in Scheme
1. Synthesis of the starting compound 4-(3-(hydrazinecarbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide (I3) and its
structural analysis were reported in our previous studies [18,30]. In
this study, twenty compounds were synthesized and their structures
were confirmed by 1H NMR, 13C NMR, and HRMS. Spectras can be
found in the supplementary file. According to NMRs data, a proton
signal of the functional eN]CH- group was seen in the range of
2.2. Carbonic anhydrase inhibition
In this study, in vitro enzyme inhibitory effects of the com-
pounds were examined towards cytosolic (human) hCA I and hCA II,
and tumor-associated transmembrane hCA IX isoenzymes by a
stopped-flow, CO2 hydrase assay according to previous studies
[15e18,31e34]. Inhibition constants (Ki) and selectivity ratios
calculated are shown in Table 2. The structure-activity relationship
(SAR) analysis was deduced from Table 2. The cytosolic hCA I and
hCA II were inhibited by most of the compounds in the high
nanomolar range with the inhibition constant (Ki) values ranging
from 652 to >10000 nM (hCAI) and 8.9->10000 nM (hCA II) while
the reference drug AZA’s Ki values were 250 and 12.1 nM, respec-
tively. Compounds generally showed weak activity towards hCA I
and hCA II, except compound 19 (Ki ¼ 8.9 nM, hCA II) compared to
AZA. Since compound 19 having furan ring was found 7-times more
potent than its phenyl analog 1 towards hCA II, it can be stated here
bioisosteric replacement remarkably increased inhibitory potency
against hCA II. On the other hand, the substitution of halogens such
as fluorine and chlorine generally led to increased activity towards
hCA II, except bromine. Electron-releasing methoxy groups
dramatically decreased inhibitory potency. Dimethoxy and trime-
thoxy bearing compounds reduced activity in the range of 13e161
times compared to phenyl analog 1. The data showed that poly-
d
8.83e8.35 ppm while another proton belongs to eCONHe moiety
was observed at 12.15e11.49 ppm as a singlet. eNH2 protons
belonging sulfonamide group also was generally seen as a singlet in
therangeof 7.56e7.52ppm.Asignalthatbelongstotheprotonatthe
fourth position of the pyrazole ring was seen in the field of
7.29e7.18 ppm in singlet appearance, except the compounds 4-
d
d
d
methyl and 4-fluoro derivatives. Methoxylated and methylated
compounds had also signals for aliphatic protons and carbons in the
expected areas of the spectra. As a general fact, 4-fluorinated com-
pound 4 gave additional peaks due to strong 19Fe13C spin-spin
1
coupling. Therefore, coupling constants JCF
¼
247.9 Hz,
2JCF ¼ 22.1 Hz, and 3JCF ¼ 9.2 Hz were recorded for the 4-fluorinated
compound 4. HRMS results also confirmed the chemical structure
of the compounds by the calculated and measured m/z values of the
compounds.
3

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
Scheme 1. Synthetic pathway of the target sulfonamides 1e20.
Conditions and reagents: (i) NaOEt, diethyloxalate, EtOH, rt. (ii) 4-hydrazinobenzensulfonamide hydrochloride, EtOH, reflux. (iii) Hydrazine hydrate (99%), MeOH, reflux, (iv)
Aldehyde derivative, EtOH, gl.HAc, reflux. Ar: Phenyl (1), 4-methylphenyl (2), 4-methoxyphenyl (3), 4-fluorophenyl (4), 4-chlorophenyl (5), 4-bromophenyl (6), 4-nitrophenyl (7), 4-
trifluoromethylphenyl (8), 2,3-dimetoxyphenyl (9), 2,4-dimetoxyphenyl (10), 2,5-dimetoxyphenyl (11), 2,3,4-trimetoxyphenyl (12), 3,4,5-trimetoxyphenyl (13), 4-dimethylami-
nophenyl (14), 4-hydroxy-3-methoxyphenyl (15), 3-hydroxy-4-methoxyphenyl (16), 4-hydroxyphenyl (17), thiophene-2-yl (18), furan-2-yl (19), pyridine-4-yl (20).
Table 2
hCA I, hCA II, and hCA IX inhibition effects of the compounds 1e20.
Comp.
Ar
Ki (nM)
hCA I
Selectivity ratio
hCA I/hCA IX
hCA II
hCA IX
hCA II/hCA IX
1
2
3
4
5
6
7
8
Phenyl
4-Methylphenyl
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
7188.8
8662.2
10000
5541.7
7178.9
7034.3
6612.1
5517.6
6456.7
>10000
>10000
>10000
>10000
7702.9
>10000
8175.8
8470.5
8623.8
652
61.9
20
359.4
14.7
6.5
350.7
1071.5
4.8
472.3
318.9
3.6
>6.5
>39.0
>6.7
>5.6
574.8
>5.0
3.1
1.5
3.7
2.6
6.1
5.9
3.2
2.4
870.3
5660.7
41.5
40.8
8710.5
44.3
588.6
1534.3
15.8
6.7
1467.1
14
4-Bromophenyl
4-Nitrophenyl
4-Trifluoromethylphenyl
2,3-Dimetoxyphenyl
2,4-Dimetoxyphenyl
2,5-Dimetoxyphenyl
2,3,4-Trimetoxyphenyl
3,4,5-Trimetoxyphenyl
4-Dimethylaminophenyl
4-Hydroxy-3-methoxyphenyl
3-Hydroxy-4-methoxyphenyl
4-Hydroxyphenyl
Thiophene-2-yl
42.3
17.3
9
798.6
6504.9
4446.8
>10000
>10000
25.5
>10000
53
246.6
58.7
1787.9
1544.9
256.4
1486.5
1778.8
13.4
1993.7
8.1
10.6
0.4
4.2
10
11
12
13
14
15
16
17
18
19
20
17.3
>6.7
>5.6
1.9
>5.0
6.5
23.3
8.4
0.5
1009.4
799.1
1232.0
38.8
7.0
16.8
789.3
Furan-2-yl
Pyridine-4-yl
8.9
8151.4
9650.2
12.2
10.3
Acetazolamide, AZA
250
12.1
25.8
9.7
0.5
methoxylation of the compounds was not a favorable modification
to obtain hCA II inhibitor. As a result, compound 19 (Ki ¼ 8.9 nM)
that is the most potent hCA II inhibitor can be considered for
glaucoma and epilepsy studies since hCA II has a role in these
diseases.
considerably inhibited by the compounds with Ki values in the
range of 6.7e1787.9 nM. The following compounds were found as
the most effective hCA IX inhibitors compared to AZA
(Ki ¼ 25.8 nM): 1, 4, 5, 7, 8, 14, 16, 17, 18, and 19.
The drawback of finding novel CAIs is mainly the lack of selec-
tivity for a specific CA isoenzyme. Therefore, the selectivity ratios
The transmembrane cancer-associated hCA IX isoenzyme was
4

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
calculated are given in Table 2. The results showed that some of the
compounds had high selectivity in the inhibition of hCA IX over hCA
I and hCA II. The following compounds can be considered as the
most selective hCA IX inhibitors over hCA I isoenzyme (assessed as
the ratio of hCA I/hCA IX): 1 (359.4), 4 (350.7), 5 (1071.5), 7 (472.3),
8 (318.9), 14 (574.8), 16 (1009.4), 17 (799.1), 18 (1232). Among the
compounds, 5 having 4-chlorophenyl and 18 having thiophen ring
were approximately 110 and 127 times more selective than AZA
(hCA I/hCA IX ¼ 9.7). On the other hand, all of the compounds
selectively inhibited hCA IX over hCA II, except compounds 9 and
19. The most selective compounds (assessed by the ratio of hCA II/
hCA IX) were 11 (17.3), 17 (23.3), and 20 (10.3). Compound 17
bearing 4-hydroxyphenyl showed 47 times more selective inhibi-
tion of hCA IX over hCA II compared to AZA.
According to the SAR analysis, substitution of electron-
withdrawing groups such as fluorine, chlorine or nitro and bio-
isosteric ring replacements such as thiophen or furan usually
increased the enzyme inhibitory potencies of the compounds,
while mono or poly methoxylation of the compounds generally
decreased activity compared to phenyl derivative towards hCA IX.
Substitution of chlorine atom instead of fluorine or bromine led to
increasing inhibition potency against hCA IX. The differences may
be a result of the atom radius that prevents it from approaching the
enzyme. On the other hand, when compounds 3 having methoxy
and 17 having hydroxy were compared, the data showed that the
hydroxy group increased activity around 145 times towards hCA IX.
When vanillin (15) and its structural isomer isovanillin (16) de-
rivatives were compared, the results also indicated that the hy-
droxy group at the third position of the phenyl ring increased
activity 246 times towards hCA IX. Furthermore, compound 15
having 4-hydroxy-3-methoxyphenyl showed 188 times less activity
than 17 having 4-hydroxyphenyl. This data also indicated that the
methoxy group at the third position of phenyl ring was not favor-
able. It can be concluded that the methoxy group made additional
hydrogen bond interactions with the hydroxy group, and also the
hydroxy group was hindered from interacting with the critical
protein residues of the target enzyme. Moreover, thiophen, and
furan rings considerably increased inhibitory potency towards hCA
XI while pyridine decreased potency compared to phenyl analog 1.
According to the previous study [18], starting compound 4-(3-
(hydrazinecarbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benze-
nesulfonamide showed moderate selectivity towards hCA IX with
the selectivity values of hCA I/hCA IX ¼ 4.6 and hCA II/hCA IX ¼ 0.9.
Converting hydrazine into the hydrazone led to enhanced selec-
tivity for the most of the compounds studied towards hCA IX. Thus,
the hydrazone linker was found a useful modification to obtain
more powerful and selective hCA IX inhibitors.
highest cytotoxicity against HSC-2 cells (CC50 ¼ 1.6
by 17 (2.0 M) > 11 (5.2 M) > 12 (18.0 M) > 8 (19.5
(24.3 M) > 2 (31.4 M). All of the synthesized compounds showed
higher cytotoxicity than references 5-FU (CC50 > 1000 M) and
MTX (CC50 > 400 M). All of these compounds except doxorubicin
m
M), followed
m
m
m
mM) > 4
m
m
m
m
did not reduce the viability of tumor cells completely (Fig. 2).
Tumor selectivity (TS) values were calculated by dividing the
CC50 for HSC-2 by CC50 for HGF (B/A in Table 3). Compound 20
showed the highest TS value (>128.2), followed by doxorubicin
(>43.0) > 11 (>38.5) > 12 (>11.1) > 8 (>10.3) > 4 (7.2) > 2 (>6.4) > 16
(>6.0) > 18 (>5.5) [ 5-FU, methotrexate (><1) (highlighted in
Fig. 2). Therefore, compound 20, having a pyridine ring, was the
most selective one compared to the three of the reference drugs.
Further, potency-selectivity expression (PSE) values were
calculated by dividing TS by CC50 for HSC-2 and then mulptiplied by
100 [ (B/A2)x100 in Table 3], in an attempt to find out the most
favorable anticancer compounds having both potency and selec-
tivity. Doxorubicin showed the highest PSE value (>19229.7), fol-
lowed by 20 (>8218.3) > 11 (>744.2) > 17 (171.1) > 12 (>61.7) > 8
(>52.8) > 4 (29.6) > 2 (>20.2) > 16 (>18.3) > 18 (>15.0) > >
methotrexate (><0.25) > 5-FU (><0.1). The compounds 11 with
_
thiophen ring, [CC50 (5.2
pyridine ring, [CC50 (1.6
m
m
M), TS (>38.5), PSI (>744.2)] and 20 with
_
M), TS (>128.2), PSI (>8218.3)] made great
attraction according to their TS and PSE values.
Western blot analysis was carried out to clarify the mechanism
of action of the compounds. The results showed that representative
compound 11 induced only minor apoptosis (caspase activation
assessed by the production of cleaved poly (ADP-ribose) polymer-
ase (PARP) and cleaved caspase-3). On the other hand, compound
20 did not induce apoptosis (Fig. 4). The data showed that apoptotic
cell death was not the main pathway for these compounds, most
possibly causing other types of cell death or growth arrest in a
certain phase of the cell cycle.
The following SAR analysis was inferred from the Ki, TS, and PSE
values of the compounds compared to unsubstituted phenyl de-
rivative 1. Methoxylation of the compounds generally decreased
cytotoxicity, except 11 having 2,5-dimetoxyphenyl and 12 having
2,3,4-trimetoxyphenyl. Compound 11 was one of the promising
cytotoxic agents with high cytotoxicity and selectivity. In halogen
derivatives, fluorinated compounds 4 and 8 showed approximately
2 and 3 times elevated cytotoxicity, while the compounds having
bromine (6) and chlorine (5) were the weakest compounds towards
HSC-2. Compound 16 carrying isovanillin was more cytotoxic and
selective agent than its structural isomer vanillin derivative 15 in a
similar manner with their effects on hCA IX. Converting the phenyl
group into phenol dramatically increased cytotoxicity around 27
times. Further, phenol derivative was also more cytotoxic than
vanillin and isovanillin derivatives. The data proved that free
phenol function is one of the suitable modifications to increase
cytotoxicity as similar as its hCA IX effects. Bioisosteric replacement
is generally caused by increasing cytotoxicity. Thiophen and pyri-
dine rings were more favorable than the furan ring compared to
phenyl derivative. According to cytotoxicity results, the most active
compound was 20 having a pyridine ring. The pyridine ring led to
enhancing cytotoxicity 33 times compared to phenyl derivative 1.
Also, its PSE value was 1157 times higher than that of 1.
When hCA IX inhibitory effects of the drug and/or drug candi-
dates in phase trials as Pazopanib (Ki ¼ 9.1 nM, hCA IX) [35],
indisulam (Ki ¼ 24 nM, hCA IX) [35] and SLC-0111 (Ki ¼ 45 nM, hCA
IX) [24] were compared with our significant results, compounds 5
(6.7 nM), 18 (7.0 nM), 16 (8.1 nM) and 17 (10.6 nM) with the lowest
Ki values can be taken into account as potent, selective and novel
hCA IX inhibitors for further in vivo experiments.
2.3. Cytotoxicities towards oral squamous cell carcinomas
Moreover, a preliminary QSAR analysis was made to understand
correlations between tumor-specificity of compounds and several
chemical descriptors. The graphs showed that there were some
correlations with several chemical descriptors for vsurf_D7 (hy-
drophobic volume) (r2 ¼ 0.271, p ¼ 0.019), dipoleX (The x compo-
nent of the dipole moment) (r2 ¼ 0.241, p ¼ 0.028), vsurf_D8
(hydrophobic volume) (r2 ¼ 0.221, p ¼ 0.037), vsurf_D6 (hydro-
phobic volume) (r2 ¼ 0.217, p ¼ 0.039), vsurf_EWmin1 (lowest
Invitro cytotoxicities of the compounds,1e20towardshuman oral
squamous cell carcinoma (OSCC) cell line (HSC-2) and human
mesenchymal normal oral cell (human gingival fibroblast, HGF) were
investigatedaccordingtoourpreviousstudies[15,17,18,30,33,36e40].
The 50% cytotoxic concentration (CC50, expressed as mM) of com-
pounds1e20isdeterminedfromthe dose-response curve(Fig. 2)and
listed in Table 3.
According to the cytotoxicity assay, compound 20 showed the
hydrophilic energy) (r2
¼
0.212,
p
¼
0.041) and ATSC8c
5

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
Fig. 2. Dose-response curve for cytotoxicity induced by cytotoxicity of the compounds 1e20 towards HSC-2 and HGF cells. Cells were treated for 48 h without (control) with the
indicated concentration of samples, and viable cell number was determined by MTT methods. Each value represents the mean S.D. of the triplicate assay.
6

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
Table 3
Cytotoxicity of the compounds 1e20 towards HSC-2 and HGF cells.
Code
Ar
CC50
(mM)
Malignant
Non-malignant
TS
PSE
HSC-2
(A)
SD
HGF
SD
(B)
(B/A)
(B/A2) ꢁ 100
1
2
3
4
5
6
7
8
Phenyl
4-Methylphenyl
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
53.0
31.4
>158
24.3
>200
>200
85.1
19.5
>200
>200
5.2
18.0
>181
>200
35.1
33.1
2.0
10.0
15.5
72.2
7.0
0.0
0.0
9.2
1.1
0.0
0.0
>200
>200
>200
174.7
>200
>200
>200
>200
>200
>200
>199
>200
>200
>200
23.1
0.0
0.0
0.0
32.5
0.0
0.0
0.0
0.0
0.0
0.0
2.3
0.0
0.0
0.0
23.4
0.0
4.9
0.0
82.9
0.0
>3.8
>6.4
><1.3
7.2
>7.1
>20.2
><0.8
29.6
><0.5
><0.5
>2.8
>52.8
><0.5
><0.5
>744.2
>61.7
><0.6
><0.5
1.9
>18.3
171.1
>15.0
2.8
><1
><1
>2.3
>10.3
><1
><1
>38.5
>11.1
><1.1
><1
0.7
4-Bromophenyl
4-Nitrophenyl
4-Trifluoromethylphenyl
2,3-Dimetoxyphenyl
2,4-Dimetoxyphenyl
2,5-Dimetoxyphenyl
2,3,4-Trimetoxyphenyl
3,4,5-Trimetoxyphenyl
4-Dimethylaminophenyl
4-Hydroxy-3-methoxyphenyl
3-Hydroxy-4-methoxyphenyl
4-Hydroxyphenyl
Thiophene-2-yl
9
10
11
12
13
14
15
16
17
18
19
20
4.3
6.1
32.9
0.0
22.0
15.0
0.4
35.4
27.4
0.0
>200
7.0
>200
145.3
>200
>6.0
3.5
>5.5
2.0
36.5
72.1
1.6
Furan-2-yl
Pyridine-4-yl
>128.2
>8218.3
Doxorubicin
5-Fluorouracil
Methotrexate
0.2
>1000
>400
0.2
0.0
0.0
>9.6
>1000
>400
0.7
0.0
0.0
>43.0
><1
><1
>19229.7
><0.1
><0.25
Each value represents the mean from triplicate assays. SD: Standard deviation. TS: Tumor selectivity. PSE: Potency Selectivity Expression.
Fig. 3. Top six chemical descriptors that showed correlation with tumor specificity of twenty 4-(3-(2-arylidenehydrazine-1-carbonyl)-5-(thiophen-2-yl)-1H-pyrazole-1-yl)
benzenesulfonamides.
(autocorrelation of topological structure weighted by gasteiger
charge) (r2 ¼ 0.199, p ¼ 0.048) (Fig. 3).
7

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
a similar binding orientation with AZA by occupying the binding
pocket where sulfonamide ring dwells deeply towards gatekeeper
residues and Zn2þ ion (Fig. 6). It makes favorable hydrogen-
bonding interactions with Thr200 and Gln92 and also a
p-p
interaction is observed between compound 19 and His94 which
also plays a critical role in inhibiting hCA II. The furan moiety ori-
ents toward the hydrophobic parts of the external part of the cavity.
Both thiophene and furan rings seem to have favorable interactions
with hydrophobic amino acid residues of hCA II (Pro201-202 and
Leu57, Phe70, respectively). We believe that these are key in-
teractions to stabilize 19-hCA II complex.
On the other hand, the most active compound against hCA IX,
compound 5, was docked in a specific shaped conformation with
sulfonamide ring pointing toward the top of the active site (Fig. 7).
However, it seems to bind to the Zn2þ ion through the carbonyl
oxygen to the coordination site of metal ion. The chlorophenyl,
thiophene, and also sulfonamide aromatic rings point toward the
hydrophobic region of the active site (Pro201, 202, Leu91, 123, 131,
135, 141, Val121, 131). While being in an unusual confirmation,
compound 5 keeps known critical interactions with the binding site
Fig. 4. Western blot analysis for induction of apoptosis by the representative com-
pounds 11 and 20 in HSC-2 cells. The images (long and short exposure) and repeated
experiments to check the reproducibility are shown in Supplementary Fig. S1.
residues: a hydrogen bond with Thr200 and
tion with His94.
p-p stacking interac-
It can be expressed here that some of the compounds tested
acted as both selective CA IX inhibitors and potent cytotoxic agents.
They may show their cytotoxic effects by the inhibition of CA IX
isoenzyme as it was mentioned in one of the cancer biomarkers in
OSCC, supported by some correlation as shown in Fig. 5. Since this
finding is preliminary, further study is needed to confirm the
generalization of this finding.
When it comes to the selectivity, compound 17 shows a high
selectivity for hCA IX over hCA II. Fig. 8 clearly shows that 17 has
different conformations in hCA II and hCA IX binding pockets. The
compound 17 is largely stabilized by positioning sulfonamide
moiety to the bottom of the binding pocket where it coordinates
with Zn metal, makes a hydrogen bond with the gatekeeper res-
idue Thr199 and forms two hydrogen bonds with the neighboring
amino acid residue Thr200. Another critical hydrogen bond is
observed with Gln92 and also Ala129 which is located at the top of
the binding site. Although several interactions are observed be-
tween 17 and hCA II, the two hydrogen bonds with Thr200 are
absent in 17-hCA II predicted complex. Instead, a steric clash is
observed between this critical amino acid residue (located next to
the gatekeeper) and phenol ring of 17. The sulphonamide ring
remains up against the external binding cavity in hCA II. The small
hydrophobic residue Val131 at the entrance of the pocket in hCA
IX is replaced with a larger residue phenylalanine (Phe131) in hCA
II which will make it difficult for large ligands to enter into the
active site.
2.4. Molecular docking studies
In order to enlighten inhibition mechanisms of drug candidates
with target macromolecules, we often refer to computer-aided
drug design techniques that use several approaches involving
quantum mechanical calculations or molecular mechanics mainly
used in molecular docking simulations [41e43]. In our previous
studies, we used several molecular modelling approaches to
simulate both biological properties and potential inhibition path-
ways of novel compounds against a number of therapeutic targets
and obtained successful findings [44e48]. Hence, to help us un-
derstand and assess the inhibition mechanism and selectivity
profiles of the ligands reported here, we have performed molecular
docking studies.
When binding modes of 18-hCA I is compared with that of hCA
IX where a remarkable selectivity is observed, the gatekeeper
Compound 19, the most active compound against hCA II, adapts
Fig. 5. Correlation of hCAII/hCAIX and TS (A) and PSE (B). The data of hCAII/hCAIX (Table 2) and data of TS or PSE (Table 3) were plotted in logarithmic scale. Red symbols indicates
the top six compound with the highest hCAII/hCAIX.
8

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
Fig. 6. Calculated 3D binding mode of 19 with hCA II (For the sake of clarity, only discussed residues are shown. Amino acid residues are coloured by properties. Hydrogen bonds:
yellow dashed lines;
p-
p
interactions: cyan dashed lines; hydrophobic: green; polar: cyan; negatively charged: red).
Fig. 7. The docked pose of 5 in the binding pocket of hCA IX (See Fig. 6 for colouring).
residue Thr199 seems to be of significant importance. As shown in
Fig. 9, compound 18 interacts with this residue in hCA IX complex
by forming two hydrogen bonds while only one hydrogen bond is
observed in hCA I binding pocket. It is also noteworthy to mention
that the coordination bond distance is significantly shorter be-
tween 18 and zinc ion in hCA IX (2.17 vs. 2.60 Å). Those key
differences could help to fit 18 in the binding cavity of hCA IX
stronger. Although several interactions can be observed in 18-hCA I
complex at a first glimpse, after visual inspection, some steric
clashes are observed between 18 and hCA I of which His200
(Thr200 in hCAIX) and His94 are critical residues in enhancing the
activities. Hence, unfavorable close contacts with these residues
9

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
Fig. 8. Comparison of 3D binding mode of 17-hCA II (left) and 17-hCA IX (right) complexes (The models are shown in slightly different orientations for clarity. See Fig. 6 for
colouring).
Fig. 9. Comparison of 3D binding mode of 18-hCA I (left) and 18-hCA IX (right) complexes (The models are shown in slightly different orientations for clarity. See Fig. 6 for
colouring).
_
appear to not only be detrimental to the activity but it also might
block large ligand entry to the active site.
CC50 (5.2
(1.6
m
M), TS (>38.5), PSI (>744.2) values and 20 with CC50
_
m
M), TS (>128.2), PSI (>8218.3) values made great attraction
according to their TS and PSE values for further investigation.
Western blot analysis demonstrated that 11 induced much weaker
apoptosis whereas 20 did not induce detectable apoptosis. Enzyme
inhibitory potency and cytotoxicities of the compounds were
significantly affected by molecular modifications. Besides, molec-
ular modelling analysis helped us to enlighten binding mechanism
and selectivity profiles of the ligands which could potentially be
considered as lead compounds for further optimization to design
more potent and selective hCAs inhibitors.
3. Conclusion
In conclusion, compounds 17 (hCA II/hCA IX ¼ 23.3), a 4-
hydroxyphenyl derivative, and 18 (hCA I/hCA IX ¼ 1232.0), a thio-
phen derivative, displayed selective inhibitory potency towards
cancer-associated hCA IX over hCA I or hCA II whereas compound 5
(Ki ¼ 6.7 nM), a 4-chlorophenyl derivative, had the lowest Ki values
against hCA IX. While compounds 17 (CC50 ¼ 2
(CC50 ¼ 1.6
toxicity against HSC-2 cells, compounds 11 having thiophen with
mM) and 20
mM) with pyridine moiety showed the highest cyto-
10

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
4. Material and methods
White powder. Yield 31%. Mp ¼ 257e259 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d 11.76 (s, 1H, -CONH), 8.47 (s, 1H, eN]CH-), 7.94
4.1. Chemistry
(d, J ¼ 8.4 Hz, 2H, ArH), 7.70 (d, J ¼ 7.7 Hz, 2H, ArH), 7.66 (d,
J ¼ 4.8 Hz, 1H, ArH), 7.57 (d, J ¼ 8.1 Hz, 2H, ArH), 7.53 (s, 2H,
-SO2NH2), 7.27e7.24 (m, 3H, ArH, H-4 pyrazole), 7.18 (d, J ¼ 3.7 Hz,
1H, ArH), 7.10e7.08 (m, 1H, ArH), 2.33 (s, 3H, eCH3). 13C NMR
Nuclear Magnetic Resonance (NMR) spectra (1H NMR and 13C
NMR) were recorded with a 400 MHz Varian (Danbury, ABD)
spectrometer. DMSO‑d6 (Merck) was used as a solvent. High-
Resolution Mass Spectra (HRMS) was recorded taken using a
liquid chromatography ion trap-time of the flight tandem mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with an electro-
spray ionization (ESI) source, operating in both positive and nega-
tive ionization mode. Shimadzu’s LCMS Solution software was used
for data analysis. Melting points were determined using an Elec-
trothermal 9100 instrument (IA9100, Bibby Scientific Limited,
Staffordshire, UK) and are uncorrected. The 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 West-
bury, New York U.S.A).
(DMSO‑d6, 100 MHz, ppm)
d 157.8, 148.9, 147.3, 145.4, 141.9, 140.6,
139.4, 132.3, 130.1, 129.6, 129.5, 128.6, 127.8, 127.5, 109.1, 21.7. HRMS
(ESI-MS) m/z Calc.: 466.1002
466.0983
C
22H20N5O3S2 [MþH]þ; Found:
4.1.1.3. 4-(3-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
3.
White powder. Yield 68%. Mp ¼ 242e244 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d 11.69 (s, 1H, -CONH), 8.44 (s, 1H, eN]CH-), 7.94
(d, J ¼ 8.4 Hz, 2H, ArH), 7.70 (d, J ¼ 7.7 Hz, 2H, ArH), 7.66 (d, J ¼ 5.1,
1.1 Hz, 1H, ArH), 7.63 (d, J ¼ 8.9 Hz, 2H, ArH), 7.53 (s, 2H, -SO2NH2),
7.24 (s, 1H, H-4 pyrazole), 7.17 (dd, J ¼ 3.6, 1.1 Hz, 1H, ArH), 7.09 (dd,
J ¼ 5.1, 3.6 Hz, 1H, ArH), 7.00 (d, J ¼ 8.9 Hz, 2H, ArH), 3.79 (s, 3H,
eOCH3). 13C NMR (DMSO‑d6, 100 MHz, ppm)
d 161.6, 157.7, 148.8,
4.1.1. General synthesis method for 4-(3-(2-arylidenhydrazine-1-
carbonyl)-5-(aryl)-1H-pyrazole-1-yl) benzenesulfonamides 1e20,
Scheme 1
147.4,145.3,141.9,139.4,129.7,129.6,129.4,128.6,127.8,127.6,127.5,
115.0, 109.1, 56.0. HRMS (ESI-MS) m/z Calc.: 482.0951 C22H20N5O4S2
[MþH]þ; Found: 482.0971
4-(3-(Hydrazinecarbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-
yl)benzenesulfonamide (I3) was synthesized according to our
previous studies [18,30,49]. Briefly, 2,4-dioxo-4-(thiophen-2-yl)
butanoate (I1) was synthesized by Claisen condensation reaction of
the 2-acetyl thiophene with diethyloxalate in the presence of so-
dium ethoxide. Ethyl 1-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-
1H-pyrazole-3-carboxylate (I2) was obtained by the cyclization of
I1 and 4-hydrazinobenzenesulfonamide hydrochloride by refluxing
in ethanol. 4-(3-(Hydrazinecarbonyl)-5-(thiophen-2-yl)-1H-pyr-
azol-1-yl)benzenesulfonamide (I3) was synthesized by the reflux-
ing of I2 and hydrazine hydrate (99%) in ethanol. Details of the
synthesis part, 1H NMR, 13C NMR, and HRMS belonging in-
termediates (I1, I2 and I3) were reported in our previous study (18).
Compound I3 was heated in ethyl alcohol: glacial acetic acid
(15 ml:0.25 ml) mixture by heating. Then, a suitable aromatic
aldehyde derivative was added to the mixture in 1:1 mole ratio.
Progress of the reaction was followed by thin-layer chromatog-
raphy (TLC). Dichloromethane: methanol 4.8:0.2 was used as a
mobile phase. The reactions were stopped after a suitable time
(varies from 3 h to 24 h). After the reaction solvent is distilled under
vacuum up to its half volume, the solid raw product obtained was
filtered, and washed with water and then ethanol. The target
compounds 1e20 were purified by crystallization using methanol/
DMF/water (1), methanol (2), ethanol/DMF (3 and 4), methanol/
DMF/water (5e20). Chemical structures of the compounds were
enlightened by spectral analysis techniques such as 1H NMR, 13C
NMR, and HRMS.
4.1.1.4. 4-(3-(2-(4-Fluorobenzylidene)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
4.
White powder. Yield 77%. Mp ¼ 312e314 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d 11.85 (s, 1H, -CONH), 8.51 (s, 1H, eN]CH-), 7.94
(d, J ¼ 7.7 Hz, 2H, ArH), 7.76e7.70 (m, 4H, ArH), 7.66 (d, J ¼ 5.0 Hz,
1H, ArH), 7.53 (s, 2H, -SO2NH2), 7.30e7.25 (m, 3H, ArH, H-4 pyr-
azole), 7.18 (d, J ¼ 3.7 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.0, 3.7 Hz, 1H, ArH).
13C NMR (DMSO‑d6, 100 MHz, ppm)
d
163.8 (1JCF ¼ 247.9 Hz), 157.9,
147.8, 147.2, 145.4, 141.8, 139.5, 131.6, 130.0 (3JCF ¼ 9.2 Hz), 129.6,
129.5, 128.6, 127.8, 127.5, 116.6 (2JCF ¼ 22.1 Hz), 109.1. HRMS (ESI-
MS) m/z Calc.: 470.0751 C21H17N5O3FS2 [MþH]þ; Found: 470.0744
4.1.1.5. 4-(3-(2-(4-Chlorobenzylidene)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
5.
White powder. Yield 48%. Mp ¼ 275e277 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d 11.94 (s, 1H, -CONH), 8.49 (s, 1H, eN]CH-), 7.94
(d, J ¼ 8.4 Hz, 2H, ArH), 7.72e7.69 (m, 4H, ArH), 7.65 (d, J ¼ 5.1 Hz,
1H, ArH), 7.55 (s, 2H, -SO2NH2), 7.50 (d, J ¼ 8.4 Hz, 2H, ArH), 7.26 (s,
1H, H-4 pyrazole), 7.18 (d, J ¼ 3.7 Hz, 1H, ArH), 7.08 (dd, J ¼ 5.1,
3.7 Hz, 1H, ArH). 13C NMR (DMSO‑d6, 100 MHz, ppm)
d 157.9, 147.5,
147.1, 145.4, 141.8, 139.5, 135.2, 133.9, 129.7, 129.6, 129.5, 129.4,
128.6, 127.9, 127.5, 109.1, 94.6. HRMS (ESI-MS) m/z Calc.: 486.0456
C
21H17N5O3S2Cl [MþH]þ; Found: 486.0444
4.1.1.6. 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
6.
White powder. Yield 69%. Mp ¼ 278e280 ꢂC. 1H NMR (DMSO‑d6,
4.1.1.1. 4-(3-(2-(Benzylidene)hydrazine-1-carbonyl)-5-(thiophene-2-
400 MHz, ppm) d 11.91 (s, 1H, -CONH), 8.49 (s, 1H, eN]CH-), 7.94
yl)-1H-pyrazole-1- yl)benzenesulfonamide, 1. White powder. Yield
(d, J ¼ 8.8 Hz, 2H, ArH), 7.70 (d, J ¼ 8.8 Hz, 2H, ArH), 7.67e7.64 (m,
5H, ArH), 7.53 (s, 2H, -SO2NH2), 7.26 (s, 1H, H-4 pyrazole), 7.18 (dd,
J ¼ 3.7, 1.1 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.1, 3.7 Hz, 1H, ArH). 13C NMR
23%. Mp ¼ 322e324 ꢂC. 1H NMR (DMSO‑d6, 400 MHz, ppm)
d 11.83
(s, 1H, -CONH), 8.52 (s, 1H, eN]CH-), 7.94 (d, J ¼ 8.8 Hz, 2H, ArH),
7.71 (d, J ¼ 8.8 Hz, 2H, ArH), 7.68e7.65 (m, 3H, ArH), 7.53 (s, 2H,
-SO2NH2), 7.45e7.42 (m, 3H, ArH), 7.18 (s, 1H, H-4 pyrazole), 7.17 (d,
J ¼ 1.0 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.1, 3.7 Hz, 1H, ArH). 13C NMR
(DMSO‑d6, 100 MHz, ppm)
d 157.9, 147.6, 147.2, 145.4, 141.8, 139.5,
134.3,132.6,129.6,129.5,128.6,127.8,127.5,124.0,109.1. HRMS (ESI-
MS) m/z Calc.: 529.9951 C21H17N5O3S2Br [MþH]þ; Found: 529.9944
(DMSO‑d6, 100 MHz, ppm)
d 157.9, 148.9, 147.2, 145.4, 141.9, 139.5,
135.0, 130.8, 129.6, 129.5, 129.4, 128.6, 127.9, 127.8, 127.5, 109.1.
HRMS (ESI-MS) m/z Calc.: 452.0846 C21H18N5O3S2 [MþH]þ; Found:
452.0831
4.1.1.7. 4-(3-(2-(4-Nitrobenzylidene)hydrazine-1-carbonyl)-5-(thio-
phene-2-yl)-1H-pyrazole-1-yl) benzenesulfonamide, 7. Yellow pow-
der. Yield 48%. Mp ¼ 307e309 ꢂC. . 1H NMR (DMSO‑d6, 400 MHz,
ppm)
d 12.15 (s, 1H, -CONH), 8.62 (s, 1H, eN]CH-), 8.28 (d,
4.1.1.2. 4-(3-(2-(4-Methylbenzylidene)hydrazine-1-carbonyl)-5-
J ¼ 9.1 Hz, 2H, ArH), 7.97e7.94 (m, 4H, ArH), 7.72 (d, J ¼ 8.4 Hz, 2H,
(thiophene-2-yl)-1H-pyrazole-1-
yl)benzenesulfonamide,
2.
ArH), 7.66 (dd, J ¼ 5.1, 1.1 Hz, 1H, ArH), 7.54 (s, 2H, -SO2NH2), 7.28 (s,
11

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
1H, H-4 pyrazole), 7.19 (dd, J ¼ 3.5, 1.1 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.1,
(dd, J ¼ 5.0, 1.2 Hz, 1H, ArH), 7.61 (d, J ¼ 9.2 Hz, 1H, ArH), 7.55 (s, 2H,
-SO2NH2), 7.25 (s, 1H, H-4 pyrazole), 7.17 (dd, J ¼ 3.7, 1.2 Hz, 1H,
ArH), 7.09 (dd, J ¼ 5.0, 3.7 Hz, 1H, ArH), 6.92 (d, J ¼ 9.2 Hz, 1H, ArH),
3.5 Hz, 1H, ArH). 13C NMR (DMSO‑d6, 100 MHz, ppm)
d 158.1, 148.6,
146.9, 146.4, 145.5, 141.8, 141.3, 139.6, 129.7, 129.5, 128.7, 128.6,
127.9, 127.5, 124.8, 109.2. HRMS (ESI-MS) m/z Calc.: 497.0696
3.83 (s, 3H, eOCH3), 3.81 (s, 3H, eOCH3), 3.75 (s, 3H, eOCH3). 13
C
C
21H17N6O5S2 [MþH]þ; Found: 497.0687
NMR (DMSO‑d6, 100 MHz, ppm) d 157.6, 155.8, 153.4, 147.4, 145.3,
144.6, 142.3, 141.9, 139.3, 129.7, 129.6, 129.4, 128.6, 127.8, 127.5,
121.3, 121.2, 109.4, 109.1, 62.6, 61.2, 56.7. HRMS (ESI-MS) m/z Calc.:
542.1163 C24H24N5O6S2 [MþH]þ; Found: 542.1150
4.1.1.8. 4-(3-(2-(4-Trifluoromethylbenzylidene)hydrazine-1-
carbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
8. White powder. Yield 63%. Mp ¼ 259e261 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d
12.04 (s, 1H, -CONH), 8.59 (s, 1H, eN]CH-), 7.95
4.1.1.13. 4-(3-(2-(3,4,5-Trimethoxybenzylidene)hydrazine-1-
carbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
13. White powder. Yield 71%. Mp ¼ 312e314 ꢂC. 1H NMR (DMSO‑d6,
(d, J ¼ 8.8 Hz, 2H, ArH), 7.90 (d, J ¼ 8.1 Hz, 2H, ArH), 7.79 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.71 (d, J ¼ 8.4 Hz, 2H, ArH), 7.66 (dd, J ¼ 5.0,
1.2 Hz, 1H, ArH), 7.53 (s, 2H, -SO2NH2), 7.28 (s, 1H, H-4 pyrazole),
7.19 (dd, J ¼ 3.6, 1.2 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.0, 3.6 Hz, 1H, ArH).
400 MHz, ppm)
d 11.84 (s, 1H, -CONH), 8.42 (s, 1H, eN]CH-), 7.94
(d, J ¼ 8.8 Hz, 2H, ArH), 7.70 (d, J ¼ 8.8 Hz, 2H, ArH), 7.66 (dd, J ¼ 5.0,
1.1 Hz, 1H, ArH), 7.55 (s, 2H, -SO2NH2), 7.25 (s, 1H, H-4 pyrazole),
7.18 (dd, J ¼ 3.7, 1.1 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.0, 3.7 Hz, 1H, ArH),
6.96 (s, 2H, ArH), 3.82 (s, 6H, 2x-OCH3), 3.68 (s, 3H, eOCH3). 13C
13C NMR (DMSO‑d6, 100 MHz, ppm)
d 158.1, 147.1, 147.0, 145.4, 141.8,
139.6, 139.0, 130.8, 129.7, 129.6, 129.5, 128.6, 128.4, 127.9, 127.5,
126.5, 109.1. HRMS (ESI-MS) m/z Calc.: 520.0719 C22H17N5O3F3S2
[MþH]þ; Found: 520.0707
NMR (DMSO‑d6, 100 MHz, ppm)
d 157.8, 153.9, 148.9, 147.3, 145.4,
141.8, 139.9, 139.4, 130.5, 129.6, 129.5, 128.6, 127.8, 127.5, 109.1,
4.1.1.9. 4-(3-(2-(2,3-Dimethoxybenzylidene)hydrazine-1-carbonyl)-
104.9, 60.8 (2C), 56.6. HRMS (ESI-MS) m/z Calc.: 542.1163
C
5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
9.
24H24N5O6S2 [MþH]þ;Found: 542.1141
White powder. Yield 82%. Mp ¼ 245e247 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d
11.94 (s, 1H, -CONH), 8.81 (s, 1H, eN]CH-), 7.95
4.1.1.14. 4-(3-(2-(4-Dimethylaminobenzylidene)hydrazine-1-
carbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
14. Light green powder. Yield 64%. Mp ¼ 252e254 ꢂC. 1H NMR
(d, J ¼ 8.8 Hz, 2H, ArH), 7.71 (d, J ¼ 8.8 Hz, 2H, ArH), 7.65 (dd, J ¼ 5.1,
1.3 Hz, 1H, ArH), 7.56 (s, 2H, -SO2NH2), 7.46 (dd, J ¼ 7.1, 2.4 Hz, 1H,
ArH), 7.26 (s, 1H, H-4 pyrazole), 7.18 (dd, J ¼ 3.5, 1.3 Hz, 1H, ArH),
(DMSO‑d6, 400 MHz, ppm) d 11.49 (s, 1H, -CONH), 8.35 (s, 1H, eN]
7.12e7.08 (m, 3H, ArH), 3.81 (s, 3H, eOCH3), 3.75 (s, 3H, eOCH3). 13
C
CH-), 7.94 (d, J ¼ 8.4 Hz, 2H, ArH), 7.69 (d, J ¼ 8.8 Hz, 2H, ArH), 7.65
(dd, J ¼ 5.1,1.4 Hz,1H, ArH), 7.53 (s, 2H, -SO2NH2), 7.49 (d, J ¼ 8.8 Hz,
2H, ArH), 7.23 (s,1H, H-4 pyrazole), 7.16 (dd, J ¼ 3.5,1.4 Hz,1H, ArH),
7.09 (dd, J ¼ 5.1, 3.5 Hz, 1H, ArH), 6.73 (d, J ¼ 8.8 Hz, 2H, ArH), 2.96
NMR (DMSO‑d6, 100 MHz, ppm)
d 157.8, 153.4, 148.8, 147.2, 145.3,
144.6, 141.9, 139.4, 129.7, 129.6, 129.5, 128.6, 127.8, 127.5, 125.1, 117.7,
114.9, 109.1, 61.9, 56.4. HRMS (ESI-MS) m/z Calc.: 512.1057
C
23H22N5O5S2 [MþH]þ; Found: 512.1055
(s, 6H, eN(CH3)2). 13C NMR (DMSO‑d6,100 MHz, ppm)
d 157.4,152.2,
149.7, 147.6, 145.3, 141.9, 139.3, 129.7, 129.5, 129.4, 129.2, 128.6,
4.1.1.10. 4-(3-(2-(2,4-Dimethoxybenzylidene)hydrazine-1-carbonyl)-
127.7, 122.3, 111.5, 109.0. HRMS (ESI-MS) m/z Calc.: 495.1268
C
5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
10.
23H23N6O3S2 [MþH]þ; Found: 495.1256
Cream powder. Yield 78%. Mp ¼ 182e184 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d
11.75 (s, 1H, -CONH), 8.75 (s, 1H, eN]CH-), 7.94
4.1.1.15. 4-(3-(2-(4-Hydroxy-3-methoxybenzylidene)hydrazine-1-
carbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
(d, J ¼ 8.8 Hz, 2H, ArH), 7.79 (d, J ¼ 9.2 Hz, 1H, ArH), 7.69 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.66 (dd, J ¼ 5.0, 1.2 Hz, 1H, ArH), 7.55 (s, 2H,
-SO2NH2), 7.23 (s, 1H, H-4 pyrazole), 7.16 (dd, J ¼ 3.7, 1.2 Hz, 1H,
ArH), 7.09 (dd, J ¼ 5.0, 3.7 Hz, 1H, ArH), 6.61 (d, J ¼ 6.6 Hz, 2H, ArH),
3.83 (s, 3H, eOCH3), 3.80 (s, 3H, eOCH3). 13C NMR (DMSO‑d6,
15. White powder. Yield 64%. Mp
¼
303e305 ꢂC. 1H NMR
(DMSO‑d6, 400 MHz, ppm) 11.64 (s, 1H, -CONH), 9.27 (s, 1H, eOH),
d
8.35 (s, 1H, eN]CH-), 7.94 (d, J ¼ 8.8 Hz, 2H, ArH), 7.70 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.66 (dd, J ¼ 5.1, 1.1 Hz, 1H, ArH), 7.53 (s, 2H,
-SO2NH2), 7.24 (d, J ¼ 1.8 Hz, 1H, ArH), 7.23 (s, 1H, H-4 pyrazole),
7.17 (dd, J ¼ 3.7, 1.1 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.1, 3.7 Hz, 1H, ArH),
6.99 (d, J ¼ 1.8 Hz, 1H, ArH), 6.95 (d, J ¼ 8.4 Hz, 1H, ArH), 3.79 (s, 3H,
100 MHz, ppm) d 163.1, 159.9, 157.6, 147.4, 145.2, 144.5, 141.9, 139.2,
129.7, 129.6, 129.4, 128.6, 127.5, 127.4, 115.9, 109.1, 107.0, 98.9, 56.4,
56.1. HRMS (ESI-MS) m/z Calc.: 512.1057 C23H22N5O5S2 [MþH]þ;
Found: 512.1042
eOCH3). 13C NMR (DMSO‑d6, 100 MHz, ppm)
d 157.6, 150.5, 149.0,
147.6, 147.4, 145.3,141.9, 139.4,129.7, 129.6,129.4,128.6, 127.8, 127.7,
127.5, 120.9, 113.0, 112.6, 109.1, 56.3. HRMS (ESI-MS) m/z Calc.:
498.0900 C22H20N5O5S2 [MþH]þ; Found: 498.0888
4.1.1.11. 4-(3-(2-(2,5-Dimethoxybenzylidene)hydrazine-1-carbonyl)-
5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
11.
Cream powder. Yield 84%. Mp ¼ 199e202 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d
11.92 (s, 1H, -CONH), 8.83 (s, 1H, eN]CH-), 7.94
4.1.1.16. 4-(3-(2-(3-Hydroxy-4-methoxybenzylidene)hydrazine-1-
carbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
(d, J ¼ 8.4 Hz, 2H, ArH), 7.70 (d, J ¼ 8.4 Hz, 2H, ArH), 7.66 (dd, J ¼ 5.0,
1.3 Hz, 1H, ArH), 7.55 (s, 2H, -SO2NH2), 7.36 (d, J ¼ 3.0 Hz, 1H, ArH),
7.25 (s, 1H, H-4 pyrazole), 7.16 (dd, J ¼ 3.5, 1.3 Hz, 1H, ArH), 7.09 (dd,
J ¼ 5.0, 3.5 Hz, 1H, ArH), 7.04e6.99 (m, 2H, ArH), 3.78 (s, 3H,
eOCH3), 3.75 (s, 3H, eOCH3). 13C NMR (DMSO‑d6, 100 MHz, ppm)
16. White powder. Yield 59%. Mp
¼
293e294 ꢂC. 1H NMR
(DMSO‑d6, 400 MHz, ppm) 11.62 (s, 1H, -CONH), 9.51 (s, 1H, eOH),
d
8.38 (s, 1H, eN]CH-), 7.94 (d, J ¼ 8.6 Hz, 2H, ArH), 7.69 (d,
J ¼ 8.6 Hz, 2H, ArH), 7.55 (dd, J ¼ 5.1, 1.1 Hz, 1H, ArH), 7.52 (s, 2H,
-SO2NH2), 7.27 (d, J ¼ 1.7 Hz,1H, ArH), 7.23 (s,1H, H-4 pyrazole), 7.17
(dd, J ¼ 3.7, 1.1 Hz, 1H, ArH), 7.08 (dd, J ¼ 5.1, 3.7 Hz, 1H, ArH), 7.03
(dd, J ¼ 8.1, 1.7 Hz, 1H, ArH), 6.82 (d, J ¼ 8.1 Hz, 1H, ArH), 3.82 (s, 3H,
d
157.8, 153.9, 153.1, 147.2, 145.2, 144.3, 141.9, 139.3, 129.7, 129.6,
129.5, 128.6, 127.6, 127.5, 123.7, 118.4, 114.2, 109.8, 109.2, 56.9, 56.2.
HRMS (ESI-MS) m/z Calc.: 512.1057 C23H22N5O5S2 [MþH]þ; Found:
512.1055
eOCH3). 13C NMR (DMSO‑d6, 100 MHz, ppm)
d 157.6, 149.7, 149.5,
148.7, 147.4, 145.3, 141.9, 139.4, 129.7, 129.6, 129.4, 128.6, 127.8,
127.5, 126.4, 122.9, 116.1, 109.6, 56.3. HRMS (ESI-MS) m/z Calc.:
498.0900 C22H20N5O5S2 [MþH]þ; Found: 498.0881
4.1.1.12. 4-(3-(2-(2,3,4-Trimethoxybenzylidene)hydrazine-1-
carbonyl)-5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
12. White powder. Yield 77%. Mp
(DMSO‑d6, 400 MHz, ppm) 11.83 (s, 1H, -CONH), 8.70 (s, 1H, eN]
CH-), 7.94 (d, J ¼ 8.8 Hz, 2H, ArH), 7.71 (d, J ¼ 8.4 Hz, 2H, ArH), 7.65
¼
248e250 ꢂC. 1H NMR
d
4.1.1.17. 4-(3-(2-(4-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-
yl)benzenesulfonamide,
17.
12

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
White powder. Yield 71%. Mp ¼ >350 ꢂC. 1H NMR (DMSO‑d6,
number was determined using a microplate reader (Sunrise
€
400 MHz, ppm)
d
11.61 (s, 1H, -CONH), 9.89 (s, 1H, eOH), 8.39 (s, 1H,
Rainbow RC-R; TECAN, Mannedorf, Switzerland). The concentra-
eN]CH-), 7.94 (d, J ¼ 8.4 Hz, 2H, ArH), 7.70 (d, J ¼ 8.8 Hz, 2H, ArH),
7.64 (dd, J ¼ 5.0, 1.1 Hz, 1H, ArH), 7.53e7.50 (m, 4H, ArH, -SO2NH2),
7.23 (s, 1H, H-4 pyrazole), 7.16 (dd, J ¼ 3.6, 1.1 Hz, 1H, ArH), 7.08 (dd,
J ¼ 5.0, 3.6 Hz, 1H, ArH), 6.81 (d, J ¼ 8.8 Hz, 2H, ArH). 13C NMR
tion of compound that reduced the viable cell number by 50%
(CC50) was determined from the dose-response curve, and the
mean value of CC50 for each cell type was calculated from triplicate
assays as described before. Tumor-selectivity index (TS) was
calculated using the following equation as TS ¼ CC50 against HGF
non-malignant cells/CC50 against malignant HSC-2 cells [(B/A)].
Potency-selectivity expression (PSE) was calculated using the
following equation: PSE ¼ [TS/(CC50 against tumor cells)] ꢁ 100
[that is, (B/A2) ꢁ 100]. Experimental data are the mean standard
deviation (SD). The statistical differences between control and
treated groups were evaluated by paired Student’s t-test. A value of
p < 0.05 was considered to be significant.
(DMSO‑d6, 100 MHz, ppm)
d 160.1, 157.6, 149.2, 147.5, 145.3, 141.9,
139.3, 129.7, 129.6, 129.4, 128.6, 127.8, 127.5, 125.9, 116.4, 109.0.
HRMS (ESI-MS) m/z Calc.: 468.0795 C21H18N5O4S2 [MþH]þ; Found:
468.0775
4.1.1.18. 4-(3-(2-(Thiophene-2-yl-methylen)hydrazine-1-carbonyl)-
5-(thiophene-2-yl)-1H-pyrazole-1-yl)benzenesulfonamide,
18.
White powder. Yield 77%. Mp ¼ 314e315 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d 11.86 (s, 1H, -CONH), 8.69 (s, 1H, eN]CH-), 7.94
(d, J ¼ 8.4 Hz, 2H, ArH), 7.70 (d, J ¼ 8.4 Hz, 2H, ArH), 7.66e7.64 (m,
2H, ArH), 7.55 (s, 2H, -SO2NH2), 7.39 (dd, J ¼ 3.3, 1.1 Hz, 1H, ArH),
7.23 (s,1H, H-4 pyrazole), 7.17 (dd, J ¼ 3.3,1.1 Hz,1H, ArH), 7.13e7.07
4.4. Western blot assay
4.4.1. Reagents
(m, 2H, ArH). 13C NMR (DMSO‑d6, 100 MHz, ppm)
d
157.7, 147.2,
RIPA buffer, a protease inhibitor cocktail was purchased from
ATTO (Tokyo, Japan). Skim milk was purchased from Morinaga-
Nyugyo (Tokyo, Japan). Quick Start Bradford protein assay kit was
purchased from Bio-Rad Laboratories (Hercules, CA). Apoptosis
Western Blot Cocktail kit was purchased from Abcam (Cambridge,
UK). Amersham ECL Select was purchased from Cytiva (Tokyo,
Japan).
145.4, 143.9, 141.8, 139.8, 139.5, 131.6, 129.7, 129.6, 129.5, 128.6,
127.9, 127.5, 109.1. HRMS (ESI-MS) m/z Calc.: 458.0410
C
19H16N5O3S3 [MþH]þ; Found: 458.0389
4.1.1.19. 4-(3-(2-(Furan-2-yl-methylen)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-
yl)benzenesulfonamide,
19.
Cream powder. Yield 52%. Mp ¼ 291e292 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d
11.86 (s, 1H, -CONH), 8.39 (s, 1H, eN]CH-), 7.94
4.4.2. Preparation of cell lysate
(dd, J ¼ 8.5, 2.0 Hz, 2H, ArH), 7.83 (s, 1H ArH), 7.71 (dd, J ¼ 8.5,
2.0 Hz, 2H, ArH), 7.65 (dd, J ¼ 4.0, 1.1 Hz, 1H, ArH), 7.55 (s, 2H,
-SO2NH2), 7.25 (d, J ¼ 2.0 Hz 1H, H-4 pyrazole), 7.18 (dd, J ¼ 3.5,
1.3 Hz, 1H, ArH), 7.08 (dd, J ¼ 4.8, 3.5 Hz, 1H, ArH), 6.88 (dd,
J ¼ 3.2 Hz, 1H, ArH), 6.61 (dd, J ¼ 3.2, 1.5 Hz, 1H, ArH). 13C NMR
The incubation medium was removed to new microcentrifuge
tubes from the cultured dish where cells are cultured. Then the
tubes were spun at 10,000 g for 3 min. Precipitations were washed
by ice-cold PBS twice and collected as floating cells. Adherent cells
in the dishes, where the medium had been removed, were washed
by ice-cold PBS twice. Next, the cells were incubated with RIPA
buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate (SDS), 5 mM sodium deoxycholate, and 20 mM HEPES)
containing protease inhibitor and phosphatase inhibitor cocktails
in the dishes on the ice for 15 min. And then the cells were scraped
with a scraper. The cell suspensions in RIPA buffer were transferred
to the tubes where the floating cells have been collected. Both
adherent and floating cells were incubated on the ice for 15 min.
And then the tubes were centrifuged at 14,000ꢁg for 10 min. Su-
pernatants were collected as the cell lysate. Protein concentrations
were determined with a Quick Start Bradford protein assay kit. The
cell lysates were used for western blotting analysis.
(DMSO‑d6, 100 MHz, ppm)
d 157.8, 150.2, 147.2, 145.9, 145.4, 141.8,
139.5, 138.6, 129.6, 129.5, 128.6, 127.9, 127.5, 114.1, 112.9, 109.0.
HRMS (ESI-MS) m/z Calc.: 442.0638 C19H16N5O4S2 [MþH]þ; Found:
442.0638
4.1.1.20. 4-(3-(2-(Pyridine-4-yl-methylen)hydrazine-1-carbonyl)-5-
(thiophene-2-yl)-1H-pyrazole-1-
yl)benzenesulfonamide,
20.
White powder. Yield 64%. Mp ¼ 310e312 ꢂC. 1H NMR (DMSO‑d6,
400 MHz, ppm)
d
12.15 (s, 1H, CONH), 8.63 (d, J ¼ 4.6 Hz, 2H, ArH),
8.50 (s, 1H, -N]CH-), 7.95 (d, J ¼ 8.4 Hz, 2H, ArH), 7.72 (d, J ¼ 8.4 Hz,
2H, ArH), 7.66 (dd, J ¼ 5.1, 1.1 Hz, 1H, ArH), 7.62 (d, J ¼ 4.6 Hz, 2H,
ArH), 7.55 (s, 2H, SO2NH2), 7.29 (s, 1H, H-4 pyrazole), 7.19 (dd,
J ¼ 3.7 Hz, 1H, ArH), 7.09 (dd, J ¼ 5.1, 3.7 Hz, 1H, ArH). 13C NMR
(DMSO‑d6, 100 MHz, ppm)
d
158.1, 150.9, 146.9, 146.5, 145.5, 142.1,
4.4.3. Western blot analysis
141.8, 139.6, 129.7, 129.5, 128.6, 127.9, 127.5, 121.7, 109.2. HRMS (ESI-
All protein samples of cell lysates (10 mg) were separated by
MS) m/z Calc.: 453.0798 C20H17N6O3S2 [MþH]þ; Found: 453.0786
SDS-PAGE using a Mini-Protean 3 Cell system (Bio-Rad Labora-
tories). After electrophoresis, the separated proteins were trans-
ferred onto a PVDF filter using a Trans-Blot Turbo System (Bio-Rad
Laboratories). The blots were blocked at room temperature for
50 min in skim milk and then probed for 120 min with a primary
antibody cocktail (1:250) from Apoptosis Western Blot Cocktail kit.
The blots were washed three times with Tris-buffered saline (pH
7.6) containing 0.05% Tween 20 and then probed for 90 min with
horseradish peroxidase-conjugated secondary antibody cocktail
(1:100) from the kit. Immunoreactivities were detected using
Amersham ECL Select. Images were acquired using ChemiDoc MP
System (Bio-Rad Laboratories) and Image Lab 4.1 software (Bio-Rad
Laboratories). The exposure time was 0.275 or 10. 692 s.
4.2. Carbonic anhydrase inhibition
To assay inhibition effects of the compounds towards hCA I, hCA
II, and hCA IX isoenzymes of CAs, an SX.18 MV-R applied photo-
physics (Oxford, UK) stopped-flow CO2 hydrase assay was carried
out according to previous studies [15e18,31e34].
4.3. Cytotoxic activity and tumor-specificity
Human normal oral mesenchymal cells (human gingival fibro-
blast, HGF), established from the first premolar tooth extracted
from the lower jaw of a 12-year-old girl and human oral squamous
cell carcinoma cell line HSC-2 (derived from the tongue) purchased
from Riken Cell Bank (Tsukuba, Japan). The relative viable cell
number was then determined by the MTT assay according to our
previous studies [15,17,18,30,33,36e40]. The relative viable cell
4.5. QSAR analysis
We used the negative log CC50 (pCC50) values for the comparison
of cytotoxicity between compounds. The mean pCC50 values for
13

C. Yamali, H. Sakagami, Y. Uesawa et al.
European Journal of Medicinal Chemistry 217 (2021) 113351
M.A. Mohamed, C. Capasso, C.T. Supuran, Inhibition of human carbonic
normal cells and tumor cell lines were defined as N and T,
respectively. The difference (TeN) was used as a tumor-selectivity
index [50]. Out of 2180 descriptors [354 Molecular Operating
Environment (MOE) descriptors (version 2019.0101, Chemical
Computing Group Inc., Quebec, Canada), 1826 Mordred descriptors
[51]], descriptors with duplicate names, descriptors of the absolute
value of 1 as internal correlation, missing descriptors, and de-
scriptors that contain outliers based on Q1/4e3.0IQR ~ Q3/
4 þ 3.0IQR were removed. The remaining 1427 descriptors were
used for the analysis. The CC50 values were expressed as
mean S.D. of triplicate assays. Statistical analysis was done with
JMP Pro15.0.0 (SAS Institute Inc. NC, USA). The significance level
was set at p < 0.05.
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169e175.
4.6. Molecular docking
€
The in silico studies have been conducted using Schrodinger
software [52]. Initially, all compounds including standard AZA were
€
prepared using LigPrep tool [53] embedded in Schrodinger suite
[52]. This tool generates all possible tautomer and ionization states
using default conditions at pH 7.0 2.0. Besides, several possible
stereoisomers for each ligand while preserving the specified chiral-
ities were also formed. The default force field used in all calculations
was optimized potential liquid simulations 3e (OPLS3e) [54].
Our previously generated models [18] were used for docking of
the ligands in the binding site of target enzymes which were high-
resolution protein crystal structures of hCA I (2NMX; 1.55 Å), hCA II
(3HS4; 1.10 Å), and hCA IX (3IAI; 2.20 Å). Glide extra precision
(Glide XP) [55] was used for molecular docking.
Declaration of competing interest
[16] 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 on hCA I, hCA II, hCA IX, hCA XII isoenzymes, Bioorg. Chem. 78 (2018)
290e297.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[17] H.I. Gul, C. Yamali, H. Sakagami, A. Angeli, J. Leitans, A. Kazaks, K. Tars,
D.O. Ozgun, C.T. Supuran, New anticancer drug candidates sulfonamides as
selective hCA IX or hCA XII inhibitors, Bioorg. Chem. 77 (2018) 411e419.
[18] C. Yamali, H.I. Gul, A. Ece, S. Bua, A. Angeli, H. Sakagami, E. Sahin, C.T. Supuran,
Synthesis, biological evaluation and in silico modelling studies of 1,3,5-
trisubstituted pyrazoles carrying benzenesulfonamide as potential anti-
cancer agents and selective cancer-associated hCA IX isoenzyme inhibitors,
Bioorg. Chem. 92 (2019) 103222.
[19] A.A. Abdel-Aziz, A.S. El-Azab, M.A. Abu El-Enin, A.A. Almehizia, C.T. Supuran,
A. Nocentini, Synthesis of novel isoindoline-1,3-dione-based oximes and
benzenesulfonamide hydrazones as selective inhibitors of the tumor-
associated carbonic anhydrase IX, Bioorg. Chem. 80 (2018) 706e713.
[20] H.A. Allam, S.H. Fahim, F.A.-A. M, A. Nocentini, M.E. Elbakry,
M.A. Abdelrahman, W.M. Eldehna, H.S. Ibrahim, C.T. Supuran, Application of
hydrazino and hydrazido linkers to connect benzenesulfonamides with hy-
drophilic/phobic tails for targeting the middle region of human carbonic
anhydrases active site: selective inhibitors of hCA IX, Eur. J. Med. Chem. 179
(2019) 547e556.
Acknowledgments
Dr. Cem Yamali was supported by the Scientific and Technological
Research Council of Turkey, Turkey (TUBITAK) under 2214-A Inter-
national Ph.D. Research Scholarship Program (1059B141601251-
2214-A) and Ataturk University BAP office, Turkey (PRJ2017/10).
Authors gratefully thank Serkan Levent (Anadolu University, Turkey)
for HRMS analysis and Ataturk University, Department of Chemistry,
Turkey for NMR analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113351.
[21] S. Singh, C.L. Lomelino, M.Y. Mboge, S.C. Frost, R. McKenna, Cancer drug
development of carbonic anhydrase inhibitors beyond the active site, Mole-
cules 23 (2018).
[22] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Multiple
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hydrazidoureido linker to benzenesulfonamide compounds: synthesis,
in vitro and in silico evaluation of potent carbonic anhydrase II, IX and XII
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