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

Article abstract of DOI:10.1016/j.bioorg.2018.03.027

In this study, new dibenzensulfonamides, 7–9, having the chemical structure 4,4′-(5′-chloro-3′-methyl-5-aryl-3,4-dihydro-1′H,H-[3,4′-bipyrazole]-1′,2-diyl)dibenzenesulfonamide were synthesized in five steps to develop new anticancer drug candidates. Their chemical structures were confirmed by ¹H NMR, ¹³C NMR and HRMS spectra. Cytotoxicities of the dibenzensulfonamides were investigated towards HCC1937, MCF7, HeLa, A549 as tumor cell lines and towards MRC5 and Vero as non-tumor cells. Carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory effects of the dibenzensulfonamides 7–9 were also evaluated on the cytosolic human (h) hCA I and II and the tumor-associated hCA IX and XII isoenzymes. Results indicate that both 7 and 8 induced cleavage of poly (ADP ribose) polymerase (PARP), activation of caspases -3, -7 and -9 which are the hallmarks of apoptosis. Meanwhile both compounds induced autophagy in HCC1937 cells which is shown by enhanced expression of LC3 and decreased level of p62 protein. The compounds tested were also effectively inhibited tumor-associated hCA IX and hCA XII isoenzymes in the range of 20.7–28.1 nM and 4.5–9.3 nM, respectively.

Full text of DOI:10.1016/j.bioorg.2018.03.027

Accepted Manuscript
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
Halise Inci Gul, Cem Yamali, Merve Bulbuller, Petek Ballar Kirmizibayrak,
Mustafa Gul, Andrea Angeli, Silvia Bua, Claudiu T. Supuran
PII:
S0045-2068(18)30155-X
https://doi.org/10.1016/j.bioorg.2018.03.027
YBIOO 2317
DOI:
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
17 February 2018
29 March 2018
29 March 2018
Please cite this article as: H. Inci Gul, C. Yamali, M. Bulbuller, P. Ballar 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, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.03.027
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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
Halise Inci Gula*, Cem Yamalia, Merve Bulbullera, Petek Ballar Kirmizibayrakb, Mustafa
Gulc, Andrea Angelid, Silvia Buad, Claudiu T. Supurand*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University,
Erzurum, Turkey
bDepartment of Biochemistry, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey
cDepartment of Physiology, Faculty of Medicine, Ataturk University, Erzurum, Turkey.
dNeurofarba Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli
Studi di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
*Corresponding authors:
Prof. Halise Inci Gul (H.I. Gul)
Ataturk University
Faculty of Pharmacy
Department of Pharmaceutical Chemistry
25240, Erzurum, TURKEY
Tel: +90 4422315219
Fax: +90 4422315201
E-mail: incigul1967@yahoo.com
Prof. Claudiu T Supuran (C.T. Supuran)
University of Florence
Neurofarba Department
Via Ugo Schiff 6,
Polo Scientifico, 50019-Sesto Fiorentino (Firenze), Italy
Phone: +39-055-4573729/3005
Mobile: +39-349-6639626
E-mail: claudiu.supuran@unifi.it
1

Abstract
In this study, new dibenzensulfonamides, 7-9, having the chemical structure 4,4'-(5'-chloro-3'-
methyl-5-aryl-3,4-dihydro-1'H,H-[3,4'-bipyrazole]-1',2-diyl)dibenzenesulfonamide
were
synthesized in five steps to develop new anticancer drug candidates. Their chemical structures
1
were confirmed by H NMR, 13C NMR and HRMS spectra. Cytotoxicities of the
dibenzensulfonamides were investigated towards HCC1937, MCF7, HeLa, A549 as tumor
cell lines and towards MRC5 and Vero as non-tumor cells. Carbonic anhydrase (CAs, EC
4.2.1.1) inhibitory effects of the dibenzensulfonamides 7-9 were also evaluated on the
cytosolic human (h) hCA I and II and the tumor-associated hCA IX and XII isoenzymes.
Results indicate that both 7 and 8 induced cleavage of poly (ADP ribose) polymerase (PARP),
activation of caspases -3, -7 and -9 which are the hallmarks of apoptosis. Meanwhile both
compounds induced autophagy in HCC1937 cells which is shown by enhanced expression of
LC3 and decreased level of p62 protein. The compounds tested were also effectively inhibited
tumor-associated hCA IX and hCA XII isoenzymes in the range of 20.7-28.1 nM and 4.5-9.3
nM, respectively.
Keywords: Sulfonamide; pyrazole; carbonic anhydrase; anticancer; apoptosis; autophagy
2

1.
Introduction
Cancer is the second leading cause of death in developed countries. According to the
National Cancer Institue’s data, more than 200 drugs have been using for the treatment of
cancer nevertheless there is no cancer treatment that is 100% effective against cancer.1 The
chemotherapy is the most common method for the treatment of different types of cancer but it
fails to cure most cancer patients with advanced disease due to the occurrence of drug
resistance and several side effects such as anemia, appetite loss, diarrhea, hair loss, pain,
sexual and sleep problems.2 Therefore, there is an urgent need to give much attention to
modify drug leads from the point of view of medicinal chemistry and drug design to fulfill
more potent and effective therapies of cancer.
Cancer pathophysiology is characterized by many changes in a cell’s genetic and
metabolic composition that lead to abnormal cell growth and malignancy.3 Apoptosis, the
intrinsic death program in cells, plays a crucial role in regulating tissue homeostasis,
balancing cell death and survival, and regulating cell proliferation. The process of apoptosis is
controlled by a diverse range of cell signals, which may originate either extracellularly or
intracellularly, and is consequently implicated in multiple biochemical events.3 The
extracellular signals that induce apoptosis include toxins, growth factors, hormones, nitric
oxide, or cytokines, and these must either cross the plasma membrane or transduce effective
responses from the mitochondria by inducing apoptotic pathways such as caspase-3 and
caspase-8.4 Many anticancer drugs induce apoptosis by causing DNA damage that enhances
cell death signals. Therefore, the induction of apoptotic cell death is an important mechanism
underlying the therapeutic properties of many anticancer drugs.5
Caspases are well known executioners of apoptosis. Initiator caspases; caspases -8 and
-9, are activated by dimerization and autocatalytic cleavage in presence of apoptotic signals.6
Initiator caspases (caspase 2, 8, 9 and 10) initiate the apoptosis signal by activating the
executioner caspases (caspase 3, 6 and 7) that leads to apoptosis via mass proteolysis.7
Among several substrates of the activated caspases, the DNA damage sensor poly(adenosine
5′-diphosphate-ribose) polymerase 1 (PARP1) is a critical protein, cleavage of which is
widely used apoptotic hallmark. Caspases -3 and -7 are reported to cleave PARP1 into 89-and
24-kDa fragments during drug induced apoptosis.8-10
Like apoptosis, autophagy has been suggested as an important mechanism in
maintaining cellular homeostasis and during development of multicellular organisms.11
Autophagy is a homeostatic, catabolic degradation process by which the cargo is delivered to
3

the lysosome for degradation.11 Autophagy is essential for the removal of damaged or
unwanted organelles and protein aggregates and allows recycling of cellular components to
sustain proper function and metabolism of the cell. Modulating autophagy has recently
emerged as a promising therapeutic approach for certain cancer types.12
Microtubule-associated protein light chain 3 (LC3) widely used for determination of
the status of autophagy. LC3-I is conjugated with the phospholipid phosphatidylethanolamine
(PE) to form LC3-II during autophagosomes formation. Although LC3-II formation is
commonly used to demonstrate autophagy activation, this result may also refer to the
inhibition of autophagic flux.13 Thus, another marker namely p62, a selective substrate of
autophagy, is being used in autophagy studies as the autophagy flux marker.14,15
Carbonic anhydrase (CAs, EC 4.2.1.1), a zinc-containing enzyme, catalyzes the
- 16
reversible reaction between CO2 and HCO3 . CAs are present in many organisms and have
an essential role in pathological and physiological events including pH regulation, bone
resorption, osteoporosis, glaucoma, cancer, and neurological disorders.17 CA IX and XII are
the two CA isoenzymes mainly found in hypoxic tumors, and are atractive targets for cancer
therapy.17,18 Among the 16 CA isoforms known in vertebrates, CA IX, a hypoxia induced,
membrane-tethered enzyme, has been demonstrated to play a role in pH balance, regulation of
cell proliferation, adhesion, malignant cell invasion and cancer stem cell population
regulation.19 Overexpression of CA IX specifically in hypoxic cancer cells makes it a good
therapeutic target as well as a biomarker for cancer.20 CA XII is also a membrane-bonded
cancer-associated CA isoenzyme. It has been suggested that CA XII expression is upregulated
under hypoxic conditions similar to CA IX, but not in all tumor types.21 Sulfonamides bearing
heterocylic and aromatic rings have been reported as attractive scaffolds for the development
of new selective CA inhibitors.18, 22-28
Pyrazole and its derivatives have drawn considerable attention of the medicinal
chemists because of their high therapeutic values.29 Several drugs carring pyrazole ring are
already in the market such as celecoxib (Figure 1), deracoxib, etoricoxib, phenazone,
metamizole, phenylbutazone, sulfinpyrazone, oxyphenbutazone for the treatment of several
diseases.29 Several pyrazole/pyrazoline derivatives have been tested in terms of their
antitumor30,31, cytotoxic18,23,24,27,28, antiangiogenic32, proapoptotic33 properties. They may
exert their anticancer effects through inhibition of different types of enzymes, proteins, and
receptors, which play important roles in cell division such as cyclin dependent kinase
(CDK)34, aurora kinase A=B=C35, heat shock protein (HSP) 9036, epidermal growth factor
receptor (EGFR)37, and tumor growth factor (TGF)-b type.38
4

Several sulfonamide derivatives have recently been reported with antitumor/cytotoxic
activities in vitro and/or in vivo.18,23,24,27,28,39 Therefore, there is much optimism that they may
lead to novel alternative anticancer drugs, devoid of the side effects of the presently available
agents.22,39 Recently several sulfonamide derivatives such as Pazopanib40 (Figure 1), E701041,
and E707042 have been reported as potent anticancer agents and are in advanced clinical trials.
Figure 1. Representative compounds having pyrazole and/or sulfonamide pharmacophore/s
which are in clinical use or clinical trial for the treatment of several diseases
Recently, pyrazole and its derivatives have drawn attention of our research group
because of their impressive cytotoxicities and CA inhibitory effects.18,23-28 In the light of
these informations it was aimed to synthesize sulfonamides having the chemical structure of
4,4'-(5'-chloro-3'-methyl-5-aryl-3,4-dihydro-1'H,H-[3,4'-bipyrazole]-1',2-
diyl)dibenzenesulfonamides, the compounds 7-9 as possible drug candidates and investigate
their cytotoxic/anticancer effects on several cancer and normal cell lines and CA inhibitory
effects on several CA isoenzymes. Finally, it was also aimed to investigate their possible
mechanism of action for their anticancer properties if they are found as cytotoxic.
2.
Results and Discussion
2.1
Chemistry
The synthetic pathway which was presented at Scheme 1 was used for the synthesis of
target compounds 7-9 based on the literature with some modifications.43 The compound 1 was
synthesized by the condensation of 4-hydrazinobenzenesulfonamide hydrochloride with
ethylaceto acetate in ethanol. The peak which was observed at δ 2.11 ppm belonging -CH3
group in 1H NMR confirmed the chemical structure of the compound 1 while -CH2 protons of
the compound 1 hid under DMSO solvent peak. The compound 1 was reactad with
1
DMF/POCl3 under Vilsmeier-Haack condition to obtain the compound 2. In H NMR, peaks
seen at δ 9.89 ppm (aldehyde proton), δ 8.25 ppm (-N=CH- proton), δ 3.14, 2.92, 2.43 ppm (-
CH3 protons) confirmed the chemical structure of 2. By the Vilsmeier-Haack reaction, the
5

amine group on sulfonamide group was reacted with DMF. Dimethylaminomethyl group on
sulfonamide group of the compound 2 was removed by reacting compound 2 with
1
hydrochloric acid in methanol to obtain the compound 3. At the H NMR spectrum of the
compound 3, methyl peaks that belong to DMF moiety were not seen. Then, the compound 3
was reacted with a suitable acetophenone (4-methoxyacetophenone for 4, 2,4-
dimethoxyacetophenone for 5 and 2,4,5-trimethoxyacetophenone for 6) under Claisen-
1
Schmidt condensation condition. Chemical structure of the chalcones were confirmed by H
NMR spectra. Additional methoxy and aryl proton signals in the spectrum confirmed the
chemical structure of the compounds 4-6. The chalcones 4-6 were used to synthesize the
target compounds 7-9 without further purification. At the final step, 4,4'-(5'-chloro-3'-methyl-
5-aryl-3,4-dihydro-1'H,H-[3,4'-bipyrazole]-1',2-diyl)dibenzenesulfonamides,
7-9,
were
obtained by the condensation of chalcones 4-6 with 4-hydrazinobenzenesulfonamide
hydrochloride in ethanol including glacial acetic acid. The chemical structures of the newly
1
synthesized compounds 7-9 were eluciated by H NMR, 13C NMR and HRMS spectra.
Results obtained by NMRs confirmed pyrazoline ring formation. The peak of the methine
proton on fifth position of pyrazoline ring were seen at δ 5.45 ppm as doublet of doublet with
1
coupling constant of 12.5 Hz and 5.5 Hz in H NMR. On the other hand, peaks of the
methylene protons on fourth position of pyrazoline ring were not seen completely since peaks
at issue hidden under the signals of methoxy protons and used NMR solvent signals. 13C
NMR and HRMS results were also confirmed the chemical structure of the compounds 7-9.
2.2
Cytotoxic / Anticancer Activity
The primary goal of this study was to investigate whether the compounds 7-9 had
cytotoxic or anticancer properties. For this aim, cytotoxicities of the compounds 7-9 were
tested on four tumor cell lines as HCC1937, MCF7, HeLa and A549 and on two non-tumor
cells as MRC5 and Vero. Cytotoxicity results presented at Table 1 indicated that the
compound 7 showed selectivity toward HCC1937 with an IC50 value of 22.39 ± 1.76 µM
(Table 1). On the other hand, the compound 8 showed higher potency against breast and lung
cancer cell lines. Both compounds did not affect the viability of Vero cells and had little
cytotoxicity on MRC5 fibroblasts. Interestingly the compound 9 did not show any
cytotoxicity at the same conditions although it has similar chemical structure.
To understand the mechanism underlying the cytotoxic effects of the compounds 7 and
8, we investigated the critical molecular events known to regulate the apoptotic and
autophagic machinery. For this purpose, HCC1937 cell line was used to investigate the
mechanism of actions of the compounds 7 and 8. In order to evaluate the levels of proteins
6

associated with apoptosis and autophagy in HCC1937, the cells were cultured with ½xIC50
and 1xIC50 doses of compounds for 24 h. Then the total protein levels from each treatment for
autophagy and apoptotic associated protein expressions were assayed by Western blotting.
Results are shown in Figure 2A and 2B. Our results indicate that both 7 and 8 induced
cleavage of poly (ADP ribose) polymerase (PARP), activation of caspases -3, -7 and -9 which
are the hallmarks of apoptosis. Moreover these effects found to be in a dose-dependent
manner. Meanwhile both compounds induced autophagy in HCC1937 cells which is shown
by enhanced expression of LC3 and decreased level of p62 protein suggesting augmentation
of autophagic flux.
It is noteworthy that while the compound 7 has very specific cytotoxicity toward
HCC1937, the compound 8 was cytotoxic toward HCC1937, MCF7 and A549. In addition,
both compounds found to have lower cytotoxicity towards nontumoral cell lines. HCC1937
breast cancer cell line has a defective BRCA1 gene (Breast Cancer gene 1). It is defective on
Homologous Recombination, a type of DNA repair mechanism. PARP inhibitors are well
known for their high efficacy in cancer cells with HR deficiency, such as those with BRCA
gene mutations. These cancer cells are very dependent on PARP-mediated repair for survival.
Therefore, the accumulation of single strand breaks in DNA by PARP inhibitor treatment
eventually leads to double strand breaks after encountering by replication forks, which would
not be repaired in the absence of BRCA, resulting in cell death.44,45 On the other hand
compounds 7 and 8 induced apoptosis by the activation of predominant apoptosis executor
caspases at 24-hour treatment at IC50 concentrations of the compounds in question. Similarly,
it was demonstrated that cleaving of apoptotic marker PARP at 89 kDa increased in a
concentration dependent manner in HCC1937 cell line. It was also found that both 7 and 8
increased LC3-II levels but decreased p62 levels; demonstrating their autophagy inducing
effect. In conclusion these findings suggests that compounds 7 and 8 induced cell death via
the activation of both autophagy and apoptosis in HCC1937 cells in a dose-dependent
manner.
The solubility of the compound should be optimum between lipid and water phases for
optimum bioactivity. The indicator of solubility is partition coefficient (P) or its logarithmic
value logP. LogP values of the target compounds (3.24 for 7, 3.11 for 8 and 2.98 for 9) were
calculated by ChemDraw Professional 16.0 Software (PerkinElmer Informatics, Waltham,
MA, USA). When logP values of the compounds considered, it can be said there are
relationship between logP and cytotoxicity. However, it can not be decide exactly whether
cytotoxicity is related to logP value since the number of the compounds is limited.
7

Figure 2A and 2B. The compounds 7 and 8 induced the levels of apoptosis and autophagy
associated proteins. HCC1937 cells were incubated with the compounds at ½xIC50 and 1xIC50
concentrations for 24 hours. The expression levels of apoptosis related proteins (full length
and cleaved PARP1; procaspases 3, 7, 9 and their active cleaved fragments (A) and autophagy
related proteins (LC3 and p62) (B) were determined by immunoblotting. Actin was used as
the loading control.
Table 1: IC50 Values (μM) for compounds 7-9 against different cell lines following 48 h
exposure
IC50 (µM)
Cancer cell lines
HCC1937 MCF7
79.79 ± 5.43 22.39 ± 1.76 47.25 ± 3.23 50.36 ± 3.69
60.6 ± 4.36 16.9 ± 1.53 17.65 ± 2.13 23.1 ± 2.43
Normal cells
HeLa
A549
MRC5
45.13 ± 3.53
48.35 ± 3.08
-
Vero
Compound
-
-
-
7
8
9
-
-
-
-
-
: Compound was not cytotoxic at the condition studied.
8

2.3
CA Inhibitory Effects
The compounds 7-9 were tested for their CA inhibitory properties by stopped-flow,
CO2 hydrase method against four isoforms, the cytosolic human (h) hCA I and II and the
tumor-associated hCA IX and XII. The standard sulfonamide inhibitor acetazolamide (AAZ)
was used as a reference drug. The following structures-activity relationship can be observed
from the inhibition data of Table 2.
The ubiquitous cytosolic hCA I was the least inhibited among the enzymatic isoforms
herein considered, in fact the compounds 7-9 showed KIs spanning between 47.9 and 170.4
nM. It is evidence that the compound 8 which has di-methoxy group showed the best activity
(KI 47.9 nM). Concerning the inhibitory potency of the compound 8, it was found that
positions 2 and 4 on phenyl moiety prove to be crucial for the efficacy against hCA I, in fact
the disubstituted compound 8 (KI 47.9 nM) resulted 3 time more potent, when compared to
the monosubstituted compound 7 (KI 170.4 nM).
The physiologically dominant isoform hCA II was potently inhibited by
dibenzenesulfonamides (7-9) preparated in this study. Substantial differences between the
three derivatives can not be found, considering that the compounds showed similar affinities.
However it could be stressed that the presence of two sulfonamide groups is indisputably the
most responsible for a very strong activity reported here with their nanomolar activity of the
compound 7, 8 and 9 with KI 4.3, 5.5 and 6.4 nM, respectively, for this isoform.
The transmembrane, tumor-associated hCA IX was effectively inhibited by all
compounds herein reported, with KI values comprised between 20.7 and 28.1 nM. As
mentionated above in the other isoform hCA II, the addition of substituents, such as -OCH3
groups, in position 2 and 5 of the phenyl moiety (compared to compound 7 which possessed
only one), improved, even if only slightly, the inhibitory properties of dimethoxy-substituted
compound 8 (KI 25.4 nM) and of trimethoxy-substituted compound 9 (KI 20.7 nM).
The other tumor-associated isoform, hCA XII, was potently inhibited by 7-9 here
synthesized, with KI that spanning between 4.5 and 9.3 nM. It is interesting the case of the
trend a bit different for the compound 9, which was the least active, with a KI 9.3 nM, and
this result is probably attributable to the presence of the three methoxy groups in 2, 4 and 5
positions of the aromatic ring. Contrary to the trend of compounds observed against hCA II
and IX, this last aspect supports the hypothesis, in this case, that substitutions in para and
ortho positions are favored to increase the potency against this isoform.
9

Table 2: Inhibition data of human CA isoforms hCA I, II, IX and XII with the compounds 7-9
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow
CO2 hydrase assay.
KI * (nM)
Compound
hCA I
hCA II
hCA IX
hCA XII
7
170.4
47.9
6.4
5.5
4.3
12.1
28.1
25.4
20.7
25.8
5.1
4.5
9.3
5.7
8
9
110.8
250
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the
reported values).
3.
Conclusions
Newly synthesized dibenzenesulfonamides 7-9 were reported here for the first time
with their cytotoxic/anticancer properties and inhibitory effects on hCA I, II, IX and XII
isoenzymes. The possible mechanism of cytotoxic action of the compounds 7 and 8 may
result from their effects on critical moleculer events known to regulate the apoptosis and
autophagy machinery according to results obtained. The compounds were also effectively
inhibited hCA I, hCA II, hCA IX and hCA XII isoenzymes in nanomolar concentration.
4.
Methods and Materials
Chemistry
4.1
The chemical structures of the final compounds were confirmed by the Nuclear
1
Magnetic Resonance (NMR) spectra H NMR (400 MHz), 13C NMR (100 MHz) (Varian
Mercury Plus spectrometer, Varian inc., Palo Alto, California, U.S.) and HRMS (Shimadzu,
Kyoto, Japan). Chemical shifts (δ) are reported in ppm and coupling constants (J) are
expressed in hertz (Hz). Mass spectra (HRMS) for the compounds were taken using a liquid
chromatography ion trap-time of flight tandem mass spectrometer (Shimadzu, Kyoto, Japan)
equipped with an electrospray ionization (ESI) source, operating in both positive and negative
ionization modes. Shimadzu's LCMS Solution software was used for data analysis. Melting
10

points were determined using an Electrothermal 9100/IA9100 instrument (Bibby Scientific
Limited, Staffordshire, UK) and are uncorrected. Reactions were monitored by Thin Layer
Chromatography (TLC) using silicagel 60 HF254 (Merck KGaA). Dichloromethane:methanol
(4.5:0.5),
petrol
ether:ethylacetate
(1:4),
ethylacetate:methanol
(4.8:0.2),
dichloromethane:methanol (4.8:0.2) solvent mixtures were used as TLC solvent systems.
DMSO-d6 (Merck) was used as a NMR solvent.
4.1.1 Synthesis of 4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide,
Compound 143, Scheme 1
Ethylacetoacetate (17.5 g, 134 mmol) was added onto 4-hydrazinobenzenesulfonamide
hydrochloride (30 g, 134 mmol) in ethanol (100 ml). The mixture was refluxed for 6 hours.
The reaction process was monitored using TLC [Dichloromethane:methanol (4.5:0.5)]. After
stopped the reaction, mixture was kept at room temparature for a day. The solid obtained was
filtered and then washed with cold ethanol for 1. It was crystallized from ethanol. Cream
colour solid. Yield 64 %. Mp= 255-257 °C; 237-238 °C43. 1H NMR (DMSO-d6) δ, ppm: 7.90
(d, J=9 Hz, 2H, ArH), 7.83 (d, J= 9 Hz, 2H, ArH), 7.33 (s, 2H, SO2NH2), 2.11(s, 3H , -CH3)
(-CH2 protons hidden under DMSO peak).
4.1.2 Synthesis
of
N'-((4-(5-chloro-4-formyl-3-methyl-1H-pyrazol-1-
yl)phenyl)sulfonyl)-N,N-dimethylformimidamide, Compound 243, Scheme 1
4-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (20 g, 79 mmol,
compound 1) was dissolved in dimethylformamide (DMF) (200 ml) at room temparature and
then phosphoryl chloride (POCl3) was added slowly into the reaction flask and the mixture
was heated for 23 hours at 60-70 °C. The reaction process was followed by TLC
[Dichloromethane:methanol (4.5:0.5)]. Resulting mixture was poured into the cold water and
the mixture was neutralized with a saturated sodium hydrogen carbonate solution to pH=7.
The mixture was kept at room temparature for several hours. The solid obtained was filtered,
dried, and crystallized from ethanol for 2. Orange colour solid. 44 % Yield. Mp= 172-174 °C;
162-164 °C43. 1H NMR (DMSO-d6) δ, ppm:9.89 (s,1H, aldehyde proton), 8,25 (s,1H, -N=CH
), 7.95 (d, J=8,4 Hz, 2H, ArH), 7.78 (d, J=8,4 Hz, 2H, ArH), 3.14(s, 3H, -CH3), 2.92 (s, 3H , -
CH3), 2,43 (s, 3H, -CH3).
11

4.1.3 Synthesis of 4-(5-chloro-4-formyl-3-methyl-1H-pyrazol-1-yl)benzenesulfonamide,
Compound 343, Scheme 1
The compound N'-((4-(5-chloro-4-formyl-3-methyl-1H-pyrazol-1-yl)phenyl)sulfonyl)-N,N-
dimethylformimidamide (18.7 gr, 57.2 mmol, compound 2) was heated in methanolic HCl
solution (25% HCl in methanol) for 6 hours. The reaction process was followed by TLC
[Dichloromethane:methanol (4.5:0.5)]. After kept at room temperature for a day, the
precipitate formed was filtered, washed with cold water and ethanol for three times for 3.
1
Light brown colour solid. Yield 63%. Mp=199-202 °C; 220-222 °C43. H NMR (DMSO-d6)
δ, ppm: 9,90 (s, 1H, aldehyde proton), 8.00 (d, J=8,8 Hz, 2H, ArH), 7.84 (d, J=8.8 Hz, 2H,
ArH), 7.55 (s, 2H, SO2NH2), 2.44(s, 3H, -CH3).
4.1.4 General synthesis method of 4-(5-chloro-4-(3-(aryl)-3-oxoprop-1-en-1-yl)-3-
methyl-1H-pyrazol-1-yl)benzenesulfonamides, Compound 4-643, Scheme 1
The compound 4-(5-chloro-4-formyl-3-methyl-1H-pyrazol-1-yl)benzenesulfonamide (2 g, 6.7
mmol, compound 3) and suitable acetophenone [4-methoxyacetophenone (1 g, 6.7 mmol) for
4, 2,4-dimethoxyacetophenone (1.2 gr, 6.7 mmol) for 5, 2,4,5-trimethoxyacetophenone (1.5
g, 6.7 mmol) for 6] were added into the cold etanolic solution of KOH (3.7 g, 66.7 mmol, in
ethanol 35 ml) and the mixture was stirred for 30 min at ice cold condition. Then, the reaction
flask was stirred at room temperature for additional 30 hours. The reaction process was
followed by TLC [Petrol ether (40-60°C):ethylacetate (1:4) for 4, ethylacetate: methanol
(4.8:0.2) for 5, dichloromethane:methanol (4.8:0.2) for 6]. After the reaction was stopped, the
contents of the flask was poured into ice cold water and neutralizated with HCl solution (37
%) to pH= 3. The solids obtained were filtered, washed with water, and dried. Chemical
1
structures of the chalcone analogues 4-6 were confirmed according to H NMR spectra
results. The chalcone analogues were used to synthesize the final compounds 7-9 without
further purification.
12

4.1.5 General synthesis method of 4,4'-(5'-chloro-3'-methyl-5-aryl-3,4-dihydro-1'H,H-
[3,4'-bipyrazole]-1',2-diyl)dibenzenesulfonamide, Compound 7-943, Scheme 1
An acidic ethanolic solution [ethanol:glacial acetic acid (15:15)] of a suitable chalcone
analogue 4-6 (1 mmol) and 4-hydrazinobenzenesulfonamide hydrochloride (2.5 mmol) was
refluxed for 25-45 hours. The reaction was monitored using TLC [ethyl acetate:methanol
(4.8:0.2)]. After the reaction was stopped, the mixture was poured onto the cold water (100
ml). The crude compounds were filtered, and crystallized from chloroform-methanol. Spectral
and phycisal data of the dibenzenesulfonamides (the compounds 7, 8, and 9) were presented
below.
4.1.5.1
4,4'-(5'-Chloro-5-(4-methoxyphenyl)-3'-methyl-3,4-dihydro-1'H,2H-[3,4'-
bipyrazole]-1',2-diyl)dibenzenesulfonamide, Compound 743, Scheme 1
Yellow colour solid. Yield 57 %. Mp= 191-193 °C43. 1H NMR (DMSO-d6, 400 MHz, δ, ppm)
7.94 (d, J= 8.8 Hz, 2H), 7.75 (d, J= 6.8 Hz, 2H), 7.72 (d, J= 6.8 Hz, 2H), 7.61 (d, J= 8.8 Hz,
2H), 7.51 (s, 2H), 7.04-6.99 (m, 6H), 5. 45 (dd, J= 12.5, 5.5 Hz, 1H, pyrazoline), 3.77 (s, 3H,
-OCH3), 2.05 (s, 3H, -CH3). Another 2 peaks of protons on pyrazoline ring was hidden under
13
DMSO-d6 peak. C NMR (DMSO-d6, 100 MHz, δ, ppm) 160.1, 150.7, 148.7, 146.6, 143.9,
140.4, 133.4, 128.4, 127.9, 127.6, 125.6, 125.4, 124.7, 118.4, 114.9, 112.2, 55.9, 54.0, 40.5,
13.4. HRMS Calculated [M+H]+ 601.1089; found [M+H]+ 601.1063
4.1.5.2
4,4'-(5'-Chloro-5-(2,4-dimethoxyphenyl)-3'-methyl-3,4-dihydro-1'H,2H-
[3,4'-bipyrazole]-1',2-diyl)dibenzenesulfonamide, Compound 8, Scheme 1
Light green colour solid. Yield 9%. Mp=178-180 °C. 1H NMR (DMSO-d6, 400 MHz, δ, ppm)
7.92 (d, J= 8.4 Hz, 2H), 7.82 (d, J= 8.3 Hz, 1H), 7.54 (d, J=8.4 Hz, 2H), 7.58 (d, J=8.8 Hz,
2H), 7.50 (s, 2H), 7.01 (s, 2H), 6.97 (d, J= 8.8 Hz, 2H), 6.61 (d, J= 8.3, 1.8 Hz, 2H), 5.45 (dd,
J= 12.5, 5.5 Hz, 1H), 3.79 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 2.06 (s, 3H, -CH3). Another 2
peaks of protons on pyrazoline ring was hidden under DMSO-d6 peak. 13C NMR (DMSO-d6,
100 MHz, δ, ppm) 153.6, 152.0, 149.8, 148.7, 146.6, 143.9, 143.6, 140.4, 133.3, 127.9, 127.5,
125.4, 118.7, 112.2, 111.8, 99.2, 56.4, 56.1, 53.7, 40.5, 13.5. HRMS Calculated [M+H]+
631.1195; found [M+H]+ 631.1199.
4.1.5.3
4,4'-(5'-Chloro-3'-methyl-5-(2,4,5-trimethoxyphenyl)-3,4-dihydro-1'H,2H-
[3,4'-bipyrazole]-1',2-diyl)dibenzenesulfonamide, Compound 9 , Scheme 1
13

Yellow colour solid. Yield 12 %. Mp=184-185 °C. 1H NMR (DMSO-d6, 400 MHz, δ, ppm)
7.93 (d, J= 8.8 Hz, 2H), 7.74 (d, J= 8.8 Hz, 2H), 7.58 (d, J= 9.1 Hz, 2H), 7.50 (s, 2H), 7.44 (s,
1H), 7.03- 6.98 (m, 4H), 6.72 ( s, 1H), 5.45 (dd, J= 12.4, 5.9 Hz, 1H), 3.81 (s, 3H, -OCH3),
3.79 (s, 3H, -OCH3), 3.76 (s, 3H, - OCH3), 2.06 (s, 3H, -CH3). Another 2 peaks of protons on
pyrazoline ring was hidden under DMSO-d6 peak. 13C NMR (DMSO-d6, 100 MHz, δ, ppm)
162.5, 159.6, 149.9, 148.7, 146.6, 143.9, 140.4, 133.2, 130.2, 127.9, 127.5, 125.4, 118.6,
113.7, 112.1, 106.9, 99.4, 57.3, 56.9, 56.4, 53.9, 40.5, 13.5. HRMS Calculated [M+H]+
661.1300; found [M+H]+ 661.1324.
14

Reagent and conditions. (i) Ethylacetoacetate, ethanol, reflux (ii) Dimethylformamide (DMF), phosphorus oxychloride (POCl3), heat (iii) HCl
solution (25% in methanol), reflux (iv) KOH solution in ethanol, a suitable acetophenone (4-methoxyacetophenone for 4, 2,4-
dimethoxyacetophenone for 5, 2,4,5-trimethoxyacetophenone for 6) (v) 4-Hydrazinobenzenesulfonamide.HCl, ethanol, glacial acetic acid.
Scheme 1. Synthetic pathway for the compounds 7-9
15

4.2
Biological Activity
4.2.1 Cell Culture
Human breast cancer cell lines HCC1937 and MCF7, human endometrial carcinoma HeLa,
human lung carcinoma A549, human lung fibroblasts MRC5 and African green monkey kidney
cell line Vero were obtained from American Type Culture Collection and maintained as
exponentially growing monolayers by culturing according to the supplier’s instructions.
4.2.2 Cytotoxicity Test
The cytotoxic potencies of test compounds were determined by using WST1 Cell Protliferation
Assay (Roche) according to the manufacturer’s instructions. All tested compounds were
dissolved in DMSO. Cells were seeded into 96-well plate at a density of 5000 cells/well for
HeLa, MCF7, Vero, A549, HCC1937 and 10000 cells per well for MRC5. Next day, cells were
treated with compounds with the final concentrations of 1, 2, 5, 10, 25 μM and incubated for 48
hours at conventional cell culture conditions. DMSO was used negative solvent control. The
ratio of surviving cells after compound treatment was determined by the colorimetric WST-1
assay (Roche) as indicated in the protocol provided by manufacturer. The absorbance was
measured by using Varioscan microplate reader (Thermo) at at 450 nm and a 620-nm reference
filter. To determine the IC50 values, a sigmoid-dose response curve was fitted to the data using
nonlinear regression in GraphPad Prism 5 software.
4.2.3 Immunoblotting
HCC1937 cells, incubated with the respective conditions, were harvested and lysed. An equal
amount of protein was separated by SDS-PAGE (5–15%) and transferred to PVDF membranes.
The blotted membranes were blocked with 5% skim milk for 1 h and then incubated with the
desired primary antibodies at 37°C for 1 h. Mouse monoclonal antibodies used in this study
included anti-actin (Sigma), anti-caspase 9 (Cell Signaling) and anti-cleaved caspase 9 (Cell
Signaling). The rabbit polyclonal antibodies used were anti-PARP-1, anti caspase 3, anti-
caspase 7, anti-LC3 and anti-p62 (Cell Signaling). Then the immunoreactive bands were
visualised by enhanced chemiluminescence using horseradish peroxidase-conjugated IgG
secondary antibodies (Thermo). To quantify equal loading, membrane was reprobed with β-actin
antibody. Chemiluminescence signals were detected using Clarity ECL substrate solution
(BIORAD) by Fusion-FX7 (Vilber Lourmat).
16

4.2.4 Carbonic Anhydrase I, II, IX, and XII Inhibition Studies
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalyzed CO2 hydration reaction. Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH=7.5) as
buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial
rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial 5 –10% of the reaction
have been used for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1
mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together
for 15 min at room temperature prior to assay, in order to allow for the formation of the E –I
complex. The inhibition percantage were obtained by using PRISM 3, as reported earlier, and
represent the mean from at least three different determinations. All CA isoforms were
recombinant ones obtained in-house as reported earlier. 18,23,27,46-50
Acknowledgements
The authors thank to Ataturk University BAP office for the financial supports, Ataturk
University, Faculty of Science, Department of Chemistry for NMRs, and Anadolu University,
Faculty of Pharmacy, Department of Pharmaceutical Chemistry for HRMS analysis.
Declaration of Interest
The authors declare that they have no competing interests.
17

Figure Legends and Tables
Figure 1. Representative compounds having pyrazole and/or sulfonamide pharmacophore/s
which are in clinical use or clinical trial for the treatment of several diseases
Figure 2A and 2B. The compounds 7 and 8 induced the levels of apoptosis and autophagy
associated proteins. HCC1937 cells were incubated with the compounds at ½xIC50 and 1xIC50
concentrations for 24 hours. The expression levels of apoptosis related proteins (full length
and cleaved PARP1; procaspases 3, 7, 9 and their active cleaved fragments (A) and autophagy
related proteins (LC3 and p62) (B) were determined by immunoblotting. Actin was used as
the loading control.
Scheme 1. Synthetic pathway for the compounds 7-9
Table 1: IC50 Values (μM) for compounds 7-9 against different cell lines following 48 h
exposure
Table 2: Inhibition data of human CA isoforms hCA I, II, IX and XII with the compounds 7-9
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow
CO2 hydrase assay.
18

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21

Graphical abstract
22

Highlights
The compounds 7 and 8 induced cleavage of poly (ADP ribose) polymerase (PARP).
The compounds 7 and 8 induced autophagy in HCC1937 cells.
The compounds were effectively inhibited hCA IX and hCA XII isoenzymes.
23