Synthesis and bioactivities of pyrazoline benzensulfonamides as carbonic anhydrase and acetylcholinesterase inhibitors with low cytotoxicity

Article abstract of DOI:10.1016/j.bioorg.2018.12.028

4-(3-Substitutedphenyl-5-polymethoxyphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamides (9–16) were synthesized and their chemical structures were elucidated by ¹H NMR, ¹³C NMR, and HRMS. The compounds designed include pyrazoline

Full text of DOI:10.1016/j.bioorg.2018.12.028

Accepted Manuscript
Synthesis and Bioactivities of Pyrazoline Benzensulfonamides as Carbonic An-
hydrase and Acetylcholinesterase Inhibitors with Low Cytotoxicity
Dilan Ozmen Ozgun, Halise Inci Gul, Cem Yamali, Hiroshi Sakagami, Ilhami
Gulcin, Murat Sukuroglu, Claudiu T. Supuran
PII:
S0045-2068(18)31324-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.028
YBIOO 2692
Reference:
Bioorganic Chemistry
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Accepted Date:
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11 December 2018
19 December 2018
Please cite this article as: D. Ozmen Ozgun, H. Inci Gul, C. Yamali, H. Sakagami, I. Gulcin, M. Sukuroglu, C.T.
Supuran, Synthesis and Bioactivities of Pyrazoline Benzensulfonamides as Carbonic Anhydrase and
Acetylcholinesterase Inhibitors with Low Cytotoxicity, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.12.028
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Synthesis and Bioactivities of Pyrazoline Benzensulfonamides as Carbonic Anhydrase and
Acetylcholinesterase Inhibitors with Low Cytotoxicity
Dilan Ozmen Ozguna, Halise Inci Gulb*, Cem Yamalib, Hiroshi Sakagamic,d, Ilhami Gulcine,f, Murat
Sukuroglug, and Claudiu T. Supuranh
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Agri Ibrahim Cecen University,
Agri, Turkey
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum,
Turkey
cDivision of Pharmacology, Meikai University School of Dentistry, Saitama, Sakado, Japan
dMeikai University Research Institute of Odontology (M-RIO), Meikai University School of
Dentistry, Saitama, Sakado, Japan
eDepartment of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey
fDepartment of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia
gDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara, Turkey
hNeurofarba Department e Laboratorio di Chimica Bioinorganica, Universita Degli Studi di Firenze,
Sesto Fiorentino (Florence), Italy
*Corresponding author:
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
1

Abstract
4-(5-Substitutedphenyl-3-polymethoxyphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamides
13
(9-16) were synthesized and their chemical structures were elucidated by 1H NMR, C NMR, and
HRMS. The compounds designed include pyrazoline and sulfonamide pharmacophores in a single
molecule by hibrit molecule approach which is a useful technique in medicinal chemistry in
designing new compounds with potent activity for the desired several bioactivities. Inhibition
potency of the sulfonamides were evaluated against human CA isoenzymes (hCA I and hCA II) and
acetylcholinesterase (AChE) enzyme and also their cytotoxicities were investigated towards oral
squamous cancer cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) and non-
tumor cells (HGF, HPLF, and HPC). Cytosolic hCA I and hCA II isoenzymes were inhibited by the
sulfonamide derivatives (9-16) and Ki values were found in the range of 27.9±3.2-74.3±28.9 nM
and 27.4±1.4-54.5±11.6 nM, respectively. AChE enzyme was strongly inhibited by the sulfonamide
derivatives with Ki values in the range of 37.7±14.4-89.2±30.2 nM The CC50 values of the
compounds were found between 15-200 µM towards OSCC malign cell lines. Their tumor
selectivities were also calculated with two ways. Compound’s selectivities towards cancer cell line
generally low, except compounds bearing 3,4-dimethoxyphenyl as 14 (TS1=1.3, TS2=1.4) and 10
(TS2=1.4). All sulfonamide derivatives studied here can be considered as good candidates to
develop novel CAs or AChE inhibitor candidates based on the enzyme inhibition potencies with
their low cytotoxicity and tumor selectivity.
Keywords: Sulfonamide; pyrazoline; cytotoxicity; carbonic anhydrase; acetylcholinesterase
2

1. Introduction
Alzheimer’s disease (AD) is an important neurodegenerative disorder of the central nervous
system.1 The total population suffering from AD was reported 44 million in 2015.2 According to
reports, this number is expected to double by 2030. Some pathogenesis hypothesis of AD have been
offered such as amyloid cascade hypothesis, oxidative stress hypothesis, tau protein hypothesis, and
cholinergic hypothesis.3 Among others, the cholinergic hypothesis is a highly approved hypothesis,
which suggests that the low level of acetylcholine in the brain is the major cause leading to learning
and memory dysfunctions.3
Acetylcholinesterase (AChE, E.C. 3.1.1.7) is a membrane-bound enzyme found in many types
of tissues.4 It is responsible for the termination of cholinergic signaling by hydrolyzing
acetylcholine (ACh).5 The primitive approach of AD treatment is reducing ACh breakdown and
therefore increasing ACh concentration to enhance central cholinergic function and provide
symptomatic treatment with AChE inhibitors (AChEIs).6 Clinically, donepezil, rivastigmine,
galantamine, and memantine have been approved for the treatment of AD.7 These drugs are very
important for the palliative treatment of AD but their clinical efficacy is limited due to their poor
selectivity, bioavailability and undesired side effects on peripheral nervous system and liver.3 The
development of novel and target selective AD drugs is an important field of scientific activity, due
to the toxic side effect problems of recent drugs in the market.
Carbonic anhydrases (CAs, EC 4.2.1.1), therapeutically-relevant protein targets which are zinc-
- 8
including metalloenzymes and catalyze the reversible hydration/dehydration of CO2/HCO3 . CA
inhibitors or activators find various applications such as diuretics, antiglaucoma agents,
antiepileptics, antiobesity drugs, antiinfectives, Alzheimer’s disease, and anticancer.9 However,
clinically used CA inhibitors are not selective and inhibit not only the target CA isoforms but also
others causing undesired side effects.10
It is noteworthy to mention here that Celecoxib, a clinically used nonsteroidal antiinflammatory
drug that selectively inhibits COX-2, has sulfonamide and pyrazoline moieties in its chemical
structure.11 It was reported that Celecoxib induced learning and memory deficits may be attributed
to its stimulatory effect on brain beta-amyloid-42 peptide segment level, brain AChE levels, and
oxidative stress.12 Additionally, preclinical studies indicated that Celecoxib demonstrated dose-
dependent chemopreventive activity against colon carcinogenesis13 and it was also reported as a
potent CAs inhibitor.14,15
3

Figure 1. Celecoxib and target compounds designed to be inspired by Celecoxib in this study
Cancer is another mortal public health problem in the world.16 Chemotherapy is one of the
most common cancer therapies which inhibits tumor cell division, angiogenesis or induces cell
death with various chemical agents.16 Many of the anticancer agents in the market have undesired
side effects such as reduced bioavailability, toxicity, and drug-resistance.17 Therefore, the
development of new anticancer agents with better properties is another important field of medicinal
chemistry.
Pyrazoline derivatives have received considerable attention due to their valuable biological
activities such as antimicrobial18, antiinflammatory19, antidepressant20, anticancer21,22, antioxidant23,
AChE inhibitory24, and CA inhibitory22,25-27. Pyrazolines are also well-known pharmacophore to
design novel AChE and selective monoamine oxidase B (MAO-B) inhibitors for the treatment of
AD.28
In recent years, pyrazole, pyrazoline, and sulfonamide derivatives have greatly drawn the
attention of our research group owing to their significant CAs, AChE inhibitory effects and
cytotoxicities.22,24-27,29-31 In the light of the literature survey and our experience, it was aimed to
synthesize pyrazolines having the chemical structure of 4-(5-substitutedphenyl-3-
polymethoxyphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzene sulfonamides (9-16) to investigate their
CA inhibitory effects against hCA I and hCA II isoenzymes, AChE inhibitory effects, and cytotoxic
properties on oral squamous cancer cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, and
4

HSC-4) and non-tumor cells (HGF, HPLF, and HPC) to find out a possible candidate for further
studies.
2. Results and Discussion
4-(5-Substitutedphenyl-3-polymethoxyphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzene
sulfonamides (9-16) were synthesized according to the pathway presented in Scheme 1. Their CAs
and AChE enzyme inhibition results were presented in Table 1. Cytotoxicities of the compounds
against human oral squamous cell carcinoma cell lines and human normal oral cells were shown in
Table 2. Structures of the newly synthesized compounds were confirmed by the spectral techniques
(1H NMR, 13C NMR, and HRMS).
Recently, carbonic anhydrase (CAs) isozymes have become an attractive target for the design of
new inhibitors and/or activators for several bioactivities as mentioned before.9 In this study, the
compounds were tested to see their inhibitory properties against hCA I and hCA II isoenzymes
since sulfonamides bearing compounds are good candidates for CA inhibition (Table 1). According
to the results obtained, physiologically dominant cytosolic hCA I isoenzyme was strongly inhibited
by the compounds (9-16) and Ki values were calculated in the range of 27.9±3.2-74.3±28.9 nM
while the reference drug acetazolamide (AZA) showed inhibition with Ki value of 164.7±14.1 nM
against hCA I isoenzyme. hCA I isoenzyme was effectively inhibited by the compounds 5.9-2.2
times than reference drug. The compound 4-(5-(3,4-dimethoxyphenyl)-3-(4-fluorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl) benzene sulfonamide (12) was the best inhibitor against hCA I isoenzyme
with Ki 27.9±3.2 nM.
Additionally, cytosolic hCA II isoenzyme was effectively inhibited by the compounds (9-16)
with Ki between 27.4±1.4 and 54.5±11.6 nM. The reference drug AZA had Ki value of 132.6±13.1
nM against hCA II isoenzyme. The compound 4-(3-(4-bromophenyl)-5-(3,4-dimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl) benzenesulfonamide (16) was the strongest inhibitor among others with
Ki value as 27.7±1.4 nM against hCA II isoenzyme.
To see whether the compounds bearing pyrazoline and sulfonamide pharmacophores have
AChE inhibitory properties, the compounds were evaluated toward AChE enzyme (Table 1).
According to the results, AChE was significantly inhibited by the sulfonamide-pyrazoline
derivatives (9-16). The Ki values of the compounds were in the range of 37.7±14.4- 89.2±30.2 nM
(Table 1) while the reference compound Tacrine had Ki value of 282.5±55.4 nM. AChE enzyme
was powerfully inhibited by the compounds 5.9-2.2 times than reference drug Tacrine. The
compound
4-(3-(4-bromophenyl)-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
5

benzenesulfonamide, 15, was found the best AChE inhibitor in the series with Ki value of
37.7±14.4 nM.
The compounds studied here had different halogens (fluorine, chlorine, bromine) on the phenyl
ring and also non substituted phenyl ring. These modifications were provided by using
acetophenone and 4-halogenated acetophenones. They had also dimethoxy groups on a different
position of another phenyl ring. This was provided by using different dimethoxy aldehydes in
reactions such as 2,4-dimethoxybenzaldehyde and 3,4-dimethoxybenzaldehyde to investigate how
halogens and polymetoxy groups affect the bioactivities (Scheme 1).
According to inhibition results, replacement of hydrogen by any halogen was found a useful
modification in series. The compound 12 having the structure of 4-(5-(3,4-dimethoxyphenyl)-3-(4-
fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamide was found as the best inhibitor
against hCA I isoenzyme with the lowest Ki value of 27.9 ± 3.2 nM while the compound 16 4-(3-
(4-bromophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamide was
the best inhibitor of the study against hCA II isoenzyme with Ki value of 27.4 ± 1.4 nM. CA
inhibitory potency of the compounds was definitively affected by the position of the methoxy
groups on the phenyl ring, so 3,4-dimethoxylated compounds were found better inhibitors than 2,4-
dimethoxylated compounds against both CA isoenzymes..
On the other hand, for AChE inhibition, the compounds having 3,4-dimethoxyphenyl group had
better AChE inhibitory potency than the compounds bearing 2,4-dimethoxyphenyl, except the
compound 15. Interestingly, the compound 15, 4-(3-(4-bromophenyl)-5-(2,4-dimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl) benzenesulfonamide, was the best AChE inhibitor with the lowest Ki
value as 37.7 ± 14.4 nM among all compounds. In this case, the size of halogen studied and the
positions of methoxy groups were found important in increasing inhibition potency on AChE.
When fluorine and chlorine atoms were compared, the bromine which has bigger atomic diameter
than other halogens (fluorine and chlorine) had more potent inhibition profile on AChE enzyme.
3,4-Dimethoxy substituted compounds had better inhibition profile than 2,4-dimethoxy ones which
may result from different interactions of the compounds with the active side of the enzyme at issue,
except compound 15 carrying 2,4-dimethoxyphenyl group,. Molecular docking studies can be
considered to investigate exact enzyme-ligand interactions of the lead compounds in the further
studies.
6

Table 1. hCA I, hCA II and AChE inhibition results of the compounds 9-16
IC50 (nM)
KI (nM)
hCA II
Compounds
hCA I
45.9
38.4
57.8
34.7
76.9
43.3
46.2
40.8
r2
hCA II
30.1
48.8
34.6
40.5
45.1
49.1
33.0
36.5
r2
AChE
r2
hCA I
53.1±12.9
38.6±0.5
51.7±10.4
27.9±3.2
74.3±28.9
44.1±4.1
59.9±14.1
41.8±17.3
164.7±14.1
-
AChE
89.2±30.2
40.5±2.7
73.6±12.9
38.5±7.1
58.3±3.7
49.2±17.6
37.7±14.4
85.3±13.9
-
0.9784
0.9818
0.9890
0.9931
0.9522
0.9845
0.9772
0.9733
0.9795 225.2 0.9776
0.9528 82.2 0.9842
0.9703 209.9 0.9701
0.9630 86.0 0.9764
0.9911 171.4 0.9695
0.9915 174.9 0.9887
0.9792 100.2 0.9770
0.9840 233.7 0.9878
35.5±7.8
54.5±11.6
39.8±5.6
43.5±4.2
41.2±2.2
36.2±3.6
33.5±10.6
27.4±1.4
132.6±13.1
-
9
10
11
12
13
14
15
16
169.3 0.9873 149.5 0.9924
-
-
Acetazolamide
Tacrine
-
-
-
-
698.6 0.9909
282.5±55.4
Cytotoxic activities of the compounds were tested against cancer cell lines (Ca9-22, HSC-2,
HSC-3, HSC-4) and non-tumour normal cells (HGF, HPLF, HPC).22,26,27,29,31-38 The cytotoxicities
of the compounds were compared with reference drugs 5-Fluorouracil (5-FU) and Melphalan (Table
2). The CC50 values of the compounds were found in the range of 15-200 µM towards OSCC
malign cell lines while the reference drugs 5-FU and Melphalan had CC50 values between 24.4-55.1
µM and 10.3-40.3 µM, respectively. The compounds 9 (2.1;1.5 times), 10 (2.6;1.9 times), 12
(2.3;1.7 times), 13 (2.9;2.1 times), 14 (3.4;2.5 times), 15 (3.1;2.2 times), 16 (3.2;2.4 times) were
found more cytotoxic than the reference drugs 5-FU and Melphalan, respectively against Ca 9-22
cancer cell line, except the compound 11. The compounds had not considerably cytotoxic properties
towards HSC-2 cell line. When the cytotoxicities of the compounds were considered against HSC-3
cell line, the compounds 9 (1.8 times), 11 (1.8), 13 (3.2 times), 14 (2.9 times), 15 (3.6 times), 16 (3
times) were more cytotoxic than 5-FU, except the compounds 10 and 12. On the other hand, the
compounds were less cytotoxic than reference drug Melphalan towards HSC-3 cell line. The
7

compounds 9 (1.1 times), 11 (1.1 times), 12 (1.2 times), 13 (1.7 times), 14 (2.2 times), 15 (2.6
times), 16 (2.3 times) were more cytotoxic than 5-FU towards HSC-4 cell line except the compound
10.
Selective cytotoxicity is the main problem of the anticancer drugs in the market. If the
compound has selectivity towards cancer cells, that compound can be evaluated as a possible
anticancer drug candidate for further experiments. To understand whether the compounds reported
here have selectivity or not, tumor selectivity (TS) values were calculated by two equations.
First TS calculations were made by dividing the average CC50 value towards normal cells to
the average CC50 value towards cancer cell lines (TS1=Column D/Column B, Table 2). Second TS
values were also generated by considering the fact that HGF is the corresponding normal cell of
Ca9-22 cancer cell line having the same origin (both derived from gingival tissues). Second TS
values were generated for a compound by dividing the CC50 value towards HGF cells into the CC50
value towards Ca9-22 cell line (TS2=Column C/Column A, Table 2). When the calculated TS
values were compared with the reference drugs’, it can be obviously said that the compounds tested
had less selectivity than references toward cancer cell lines. Compounds’ selectivities towards
cancer cell line generally low, except compounds bearing 3,4-dimethoxyphenyl as 14 (TS1=1.3,
TS2=1.4) and 10 (TS2=1.4). It can be said that compounds are neither cytotoxic nor tumor
selective. This can be an advantage in possible drug development with different bioactivity rather
than
cytotoxic
activity.
8

Table 2. Cytotoxicities of the compounds 9-16
CC50 (μM)
Human oral squamous carcinoma cell lines
Ca9-22 HSC-2 HSC-3 HSC-4 Mean
Human oral normal cells
SD
HGF HPLF
(C)
HPC
Mean
(D)
SD
TS1
TS2
Compounds
(A)
27
21
65
24
19
16
18
17
(B)
36.0
116.3
45.8
96.5
21.8
20.0
18.3
24.3
(D)/(B) (C)/(A)
41
200
42
41
200
41
35
44
35
33
23
18
15
17
6.6
97.2
13.2
79.3
1.9
31
29
37
25
19
22
18
18
41
112
37
29
28
35
80
21
17
19
19
33.7
56.3
36.3
81.3
21.0
26.7
19.3
19.0
6.4
48.2
1.2
0.9
0.5
0.8
0.8
1.0
1.3
1.1
0.8
1.1
1.4
0.6
1.0
1.0
1.4
1.0
1.1
9
10
11
152
22
177
23
139
23
57.0
2.0
12
13
21
25
3.9
41
12.7
1.5
14
15
20
20
2.4
21
39
24
10.4
20
1.0
16
Positive control
5-Fluorouracil
Melfalan
55.1
40.3
24.4
13.6
72.7
11
38.9
10.3
47.8
18.8
-
-
>200
>200
>200
>200
-
-
>4.2
>9.3
>3.6
>4.9
>200 151.0
>175.5
>175.2
CC50 values refer to the concentrations of the compounds in micromoles which reduce the viable cell number by 50%. Oral squamous cell carcinoma (OSCC) cell lines used are Ca9-
22 (derived from gingiva), HSC-2, HSC-3, HSC-4 (derived from tongue). Normal oral cells used are human gingival fibroblasts (HGF), human periodontal ligament fibroblasts
(HPLF), and human pulp cells (HPC). SD: Standart Deviation. TS: Tumor selectivity.
9

3. Conclusion
In conclusion, pyrazoline-sulfonamide derivatives inhibited three different type of
enzymes such as hCA I, hCA II and AChE at the nanomolar concentrations. All sulfonamide
derivatives studied here may be considered as good candidates to develop novel CA or AChE
inhibitors based on the enzyme inhibition potencies and their low cytotoxicities.
4. Materials and Methods
4.1.Chemistry
Chemical structure of the compounds 4-(5-substitutedphenyl-3-polymethoxyphenyl-4,5-
1
dihydro-1H-pyrazol-1-yl)benzene sulfonamides (9-16) were determined by H NMR (400
MHz) and 13C NMR (100 MHz) spectroscopies using a Varian Mercury Plus spectrometer
(Varian inc., Palo Alto, California, U.S.). Chemical shifts (δ) are reported in ppm and
coupling constants (J) are expressed in hertz (Hz). Mass spectra of the compounds were
undertaken on an HPLC-TOF Waters Micromass LCT Premier XE (Milford, MA, USA) mass
spectrometer using an electrospray ion source (ESI). Melting points were determined using an
Electrothermal 9100/IA9100 instrument (Bibby Scientific Limited, Staffordshire, UK) and are
uncorrected. The reactions were monitored using silica gel HF254-366 Thin Layer
Chromatography (TLC) plates (E. Merck, Germany).
4.1.1. Synthesis of the chalcones, 1-8, Scheme 1
Chalcones were synthesized by Claisen-Schmidt condensation according to literatures.22,24-
27,29-31,39-43 Suitable acetophenone and aldehyde in 1:1 mol ratios were dissolved in ethanol (10
ml). Aqueous NaOH solution (10%, 20 ml) was added into the reaction mixture. Reactions
were monitored by TLC using chloroform: methanol (4.8:0.2) as a solvent system. When at
least one of the starting compounds finished, reactions were stopped. Reaction content was
poured on ice-water and neutralized by HCl (10%). The precipitates obtained were washed
with cold water and ethanol, filtered, and dried. Chalcone derivatives 1-8 were used for the
synthesis of pyrazoline derivatives 9-16 without further purification.
4.1.2. Synthesis of the pyrazoline derivatives, 9-16, Scheme 1
Pyrazolines were synthesized according to literature.22,24-27,29-31,39 A mixture of a suitable
chalcone (1-8) and p-hydrazinobenzenesulfonamide hydrochloride in 1:1 (for 9 and 10) and
1:1.1 (for 11-16) mol ratio in ethanol (25 ml) in the presence of glacial acetic acid (0.05 ml)
was refluxed for 4-19 h [9 (12 h), 10, 11 (17 h), 12 (4 h), 13, 14, 16 (19 h), 15 (7h)].
Reactions were monitored by TLC using chloroform: methanol (4.8:0.2) as a solvent system.
When the reactions were stopped, the precipitate obtained was filtered, dried and
recrystallized from ethanol.
10

Scheme 1. Synthetic pathway of the compounds
Reagents and conditions. i : Etanol, aq. NaOH (20 %), rt. ii : Ethanol, glacial acetic acid, reflux. R1= H ( 1, 2, 9,
10), F (3, 4, 11, 12), Cl (5, 6, 13, 14), Br (7, 8, 15, 16). R2=R4 = OCH3, R3 (H). for 1, 3, 5, 7, 9, 11, 13, 15.
R3=R4=
OCH3, R2 (H) for 2, 4, 6, 8, 10, 12, 14, 16.
4-(5-(2,4-Dimethoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide,
Compound 9
White colour solid, mp 206 °C, yield 35 %. 1H NMR (DMSO-d6, ppm, 400 MHz) δ 7.73 (d, J
= 7.3 Hz, 2H, Ar-H), 7.56 (d, J = 8.8 Hz, 2H, Ar-H), 7.42-7.36 (m, 3H, Ar-H), 7.02 (s, 2H, -
SO2NH2), 6.97 (d, J = 8.4 Hz, 1H, Ar-H), 6.73 (d, J = 8.4 Hz, 1H, Ar-H), 6.59 (d, J = 2.0 Hz,
1H, Ar-H), 6.37 (dd, J = 8.4, 2.0 Hz, 2H, Ar-H), 5.61 (dd, J = 12.0, 4.9 Hz, 1H, H-5
(pyrazoline)), 3.83 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3), 3.01 (dd, J=17.8, 4.9 Hz, 1H, H-4
(pyrazoline)). 13C NMR (DMSO-d6, ppm, 100 MHz) δ 160.8, 157.9, 150.9, 146.5, 133.2,
132.5, 129.9, 129.4, 127.8, 127.4, 126.6, 121.1, 112.3, 105.7, 99.6, 57.7 (C-5 pyrazoline),
56.4 (-OCH3), 55.8 (-OCH3), 42.5 (C-4 pyrazoline). HRMS (ESI-MS) C23H24N3O4S,
Calculated [M+H]+: 438.1488; Found [M+H]+: 438.1472
11

4-(5-(3,4-Dimethoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide,
Compound 10
White colour solid, mp 176 °C, yield 22 %. 1H NMR (DMSO-d6, ppm, 400 MHz) δ 7.75 (d, J
= 7.0 Hz, 2H, Ar-H), 7.56 (d, J = 8.9 Hz, 2H, Ar-H), 7.44-7.37 (m, 3H, Ar-H), 7.06 (d, J = 8.9
Hz, 1H, Ar-H), 7.03 (s, 2H, -SO2NH2), 6.91 (d, J = 1.8 Hz, 1H, Ar-H), 6.84 (d, J = 8.4 Hz,
1H, Ar-H), 6.64 (dd, J = 8.0, 1.8 Hz, 2H, Ar-H), 5.48 (dd, J = 12.1, 5.5 Hz, 1H, H-5
(pyrazoline)), 3.88 (dd, J = 17.6, 12.1 Hz, 1H, H-4 (pyrazoline)),3.68 (s, 3H, -OCH3), 3.58 (s,
3H, -OCH3), 3.14 (dd, J = 17.6, 5.5 Hz, 1H, H-4 (pyrazoline)). 13C NMR (DMSO-d6, ppm,
100 MHz) δ 150.5, 149.7, 148.7, 146.8, 134.5, 133.5, 132.4, 130.0, 129.5, 127.8, 126.7,
118.0, 112.8, 110.3, 62.9 (C-5 pyrazoline), 56.1 (-OCH3), 56.0 (-OCH3), 43.7 (C-4
pyrazoline). HRMS (ESI-MS) C23H24N3O4S, Calculated [M+H]+: 438.1488; Found [M+H]+:
438.1488
4-(5-(2,4-Dimethoxyphenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide, Compound 11
1
White colour solid, mp 188 °C (178 °C lit), yield 48 %. H NMR (DMSO-d6, ppm, 400 MHz)
δ 7.80 (t, J = 6.2 Hz, 2H, Ar-H), 7.57 (d, J = 8.4 Hz, 2H, Ar-H), 7.25 (t, J = 8.1 Hz, 2H, Ar-
H), 7.01 (s, 2H, -SO2NH2), 6.98 (d, J= 7.3 Hz, 2H, Ar-H), 6.75 (d, J = 8.4 Hz, 1H, Ar-H),
6.62 (s, 1H, Ar-H), 6.39 (d, J = 8.4 Hz, 1H, Ar-H) 5.62 (dd, J = 11.5, 4.3 Hz, 1H, H-5
(pyrazoline)), 3.86 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.05 (dd, J = 17.6, 4.3 Hz, 1H, H-4
(pyrazoline)). 13C NMR (DMSO-d6, ppm, 100 MHz) δ 164.5 (d, 1JCF = 246 Hz), 160.8, 157.8,
149.9, 146.5, 133.5, 129.3, 128.9 (d, 3JCF = 8.4 Hz), 127.9, 127.3, 121.1, 116.4 (d, 2JCF = 22.1
Hz), 112.3, 105.7, 99.7, 57.8 (C-5 pyrazoline), 56.5 (-OCH3), 55.9 (-OCH3), 42.6 (C-4
pyrazoline). HRMS (ESI-MS) C23H23N3O4SF, Calculated [M+H]+: 456.1393; Found [M+H]+:
456.1390
4-(5-(3,4-Dimethoxyphenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide, Compound 12
1
Light brown colour solid, mp 201 °C (198 °C lit), yield 32 %. H NMR (DMSO-d6, ppm, 400
MHz) δ 7.82 (dd, J = 5.5, 9.2 Hz, 2H, Ar-H), 7.56 (d, J = 9.2 Hz, 2H, Ar-H), 7.27 (t, J = 9.0
Hz, 2H, Ar-H), 7.06 (d, J = 9.2 Hz, 2H, Ar-H), 7.01 (s, 2H, -SO2NH2), 6.92 (d, J = 1.9 Hz,
1H, Ar-H), 6.85 (d, J = 8.4 Hz, 1H, Ar-H), 6.66 (dd, J = 8.3, 1.9 Hz, 1H, Ar-H), 5.51 (dd, J =
12.0, 5.5 Hz, 1H, H-5 (pyrazoline)), 3.90 (dd, J = 17.6, 12.0 Hz, 1H, H-4 (pyrazoline)), 3.69
13
(s, 3H, -OCH3), 3.67 (s, 3H, -OCH3), 3.18 (dd, J = 17.6, 5.5 Hz, 1H, H-4 (pyrazoline)). C
NMR (DMSO-d6, ppm, 100 MHz) δ 164.6, 149.6, 148.8, 146.8, 134.5, 133.7, 129.2, 128.9 (d,
12

3JCF = 8.3 Hz), 127.8, 118.1,116.6, 116.3, 112.8, 112.7, 110.4, 63.1 (C-5 pyrazoline), 56.1 (-
OCH3), 56.0 (-OCH3), 43.8 (C-4 pyrazoline). HRMS (ESI-MS) C23H23N3O4SF, Calculated
[M+H]+: 456.1393; Found [M+H]+: 456.1390
4-(3-(4-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide, Compund 13
Cream colour solid, mp 217 °C (208 °C lit), yield 57 %. 1H NMR (DMSO-d6, ppm, 400 MHz)
δ 7.74 (d, J = 8.8 Hz, 2H, Ar-H), 7.56 (d, J = 9.0 Hz, 2H, Ar-H), 7.45 (d, J = 8.8 Hz, 2H, Ar-
H),7.02 (s, 2H, -SO2NH2), 6.98 (d, J = 9.0 Hz, 2H, Ar-H), 6.74 (d, J = 8.4 Hz, 1H, Ar-H),
6.59 (d, J = 2.1 Hz, 1H, Ar-H), 6.34 (dd, J = 8.8, 2.6 Hz, 1H, Ar-H), 5.61 (dd, J = 12.0, 5.1
Hz, 1H, H-5 (pyrazoline)), 3.83 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3), 3.01 (dd, J = 17.6, 5.1
Hz, 1H, H-4 (pyrazoline)). 13C NMR (DMSO-d6, ppm, 100 MHz) δ 160.8, 157.9, 149.8,
146.3, 134.3, 133.5, 131.5, 129.4, 128.3, 127.8, 127.5, 120.9, 112.5, 105.7, 99.6, 57.9 (C-5
pyrazoline), 56.4 (-OCH3), 55.8 (-OCH3), 42.3 (C-4 pyrazoline). HRMS (ESI-MS)
C23H23N3O4SCl, Calculated [M+H]+: 472.1098; Found [M+H]+: 472.1096
4-(3-(4-Chlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide, Compound 14
Cream colour solid, mp 182 °C (184 °C lit), yield 68 %. 1H NMR (DMSO-d6, ppm, 400 MHz)
δ 7.76 (d, J = 8.8 Hz, 2H, Ar-H), 7.56 (d, J = 9.1 Hz, 2H, Ar-H), 7.47 (d, J = 8.8 Hz, 2H, Ar-
H), 7.07 (d, J = 9.1 Hz, 2H, Ar-H), 7.03 (s, 2H, -SO2NH2), 6.90 (d, J = 2.1 Hz, 1H, Ar-H),
6.84 (d, J = 8.2 Hz, 1H, Ar-H), 6.64 (dd, J = 8.2, 2.1 Hz, 1H, Ar-H),5.51 (dd, J = 12.0, 5.3
Hz, 1H, H-5 (pyrazoline)), 3.88 (dd, J = 17.8, 12.0 Hz, 1H, H-4 (pyrazoline), 3.68 (s, 3H, -
OCH3), 3.65 (s, 3H, -OCH3), 3.14 (dd, J = 17.8, 5.3 Hz, 1H, H-4 (pyrazoline)). 13C NMR
(DMSO-d6, ppm, 100 MHz) δ 149.7, 149.4, 148.8, 146.6, 134.4, 134.3, 133.8, 131.4, 129.5,
128.4, 127.8, 118.1, 112.9, 112.7, 110.3, 63.2, (C-5 pyrazoline), 56.1 (-OCH3), 56.0 (-OCH3),
43.5 (C-4 pyrazoline). HRMS (ESI-MS) C23H23N3O4SCl, Calculated [M+H]+: 472.1098;
Found [M+H]+: 472.1108
4-(3-(4-Bromophenyl)-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide, Compound 15
Cream colour solid, mp 225 °C, yield 55 %. 1H NMR (DMSO-d6, ppm, 400 MHz) δ 7.69 (d, J
= 8.4 Hz, 2H, Ar-H), 7.61 (d, J = 8.4 Hz, 2H, Ar-H), 7.57 (d, J =8.8 Hz, 2H, Ar-H), 7.01 (d, J
= 6.2 Hz, 2H, Ar-H), 6.98 (s, 2H, -SO2NH2), 6.75 (d, J = 8.4 Hz, 1H, Ar-H), 6.62 (d, J = 2.3
Hz, 1H, Ar-H), 6.39 (dd, J = 8.3, 2.3 Hz, 1H, Ar-H), 5.64 (dd, J = 12.1, 5.0 Hz, 1H, H-5
(pyrazoline)), 3.85 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.04 (dd, J = 17.6, 5.0 Hz, 1H, H-4
(pyrazoline)). 13C NMR (DMSO-d6, ppm, 100 MHz) δ 160.8, 157.8, 149.8, 146.3, 133.7,
13

132.3, 131.9, 128.6, 127.9, 127.4, 122.9, 120.9, 112.5, 105.7, 99.7, 57.9 (C-5 pyrazoline),
56.5 (-OCH3), 55.9 (-OCH3), 42.3 (C-4 pyrazoline). HRMS (ESI-MS) C23H23N3O4SBr,
Calculated [M+H]+: 516.0593; Found [M+H]+: 516.0605.
4-(3-(4-Bromophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide, Compound 16
Light green colour solid, mp 191 °C, yield 38 %. 1H NMR (DMSO-d6, ppm, 400 MHz) δ 7.70
(d, J = 8.4 Hz, 2H, Ar-H), 7.62 (d, J = 8.8 Hz, 2H, Ar-H), 7.57 (d, J = 8.8 Hz, 2H, Ar-H), 7.08
(d, J = 8.8 Hz, 2H, Ar-H), 7.02 (s, 2H, -SO2NH2), 6.91 (d, J = 2.0 Hz, 1H, Ar-H), 6.85 (d, J =
8.4 Hz, 1H, Ar-H), 6.66 (dd, J = 8.0, 2.0 Hz, 1H, Ar-H), 5.54 (dd, J = 12.0, 5.7 Hz, 1H, H-5
(pyrazoline)), 3.90 (dd, J = 17.7, 12.0 Hz, 1H, H-4 (pyrazoline), 3.69 (s, 3H, -OCH3), 3.67 (s,
13
3H, -OCH3), 3.17 (dd, J = 17.7, 5.7 Hz, 1H, H-4 (pyrazoline)). C NMR (DMSO-d6, ppm,
100 MHz) δ 149.8, 149.4, 148.8, 146.6, 134.5, 133.9, 132.4, 131.8, 128.6, 127.8, 123.1,
118.1, 112.9, 112.8, 110.4, 63.2 (C-5 pyrazoline), 56.2 (-OCH3), 56.1 (-OCH3), 43.5 (C-4
pyrazoline). HRMS (ESI-MS) C23H23N3O4SBr, Calculated [M+H]+: 516.0593; Found
[M+H]+: 516.0598
4.2.Biological Activity
4.2.1. Carbonic anhydrase inhibitory assay
The purification of cytosolic CA isoenzymes (CA I and CA II) was previously described
with a simple one-step method by a Sepharose-4B-L tyrosine-sulphanilamide affinity
chromatography.24,26,27,30,31,44-46 The protein quantity in the column effluents was determined
spectrophotometrically at 280 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was applied with a Bio-Rad Mini Gel system Mini-PROTEIN system, Bio-Rad
Laboratories, Inc., China after purification of both CA isoenzymes. Briefly, it was performed
in acrylamide for the running (10%) and the stacking gel (3%) contained SDS (0.1%),
respectively. The increase in absorbance of the reaction medium was spectrophotometrically
recorded at 348 nm. Also, the quantity of protein was determined at 595 nm according to the
method as decribed previously.24,26,27,30,31,44-46 Bovine serum albumin was used as standard
protein. The IC50 values were obtained from activity (%) versus compounds plots. For
calculation of Ki values, three different concentrations were used. The Lineweaver–Burk
curves were drawn and calculations were realized. 24,26,27,30,31,44-46
4.2.2. Cholinesterase inhibitory assay
AChE activities of the compounds were measured by slightly modifying the
spectrophotometric method as decribed previously.24,44-46 Acetylthiocholine iodide (AChI)
was used as a substrate of the reaction. 5,5-dithiobis(2-nitro-benzoic acid) (DTNB) was used
14

for the determination of the AChE activities. Briefly, 100 mL of Tris/HCl buffer (1 M, pH=8)
and 10 mL of sample solution dissolved in ultra pure water at different concentrations and 50
mL AChE solution were mixed, incubated for 10 min at 25 C. Then 50 mL of DTNB (0.5
mM) was added. The reaction was then started by the addition of 50 mL of AChI (10 mM).
The hydrolysis of these substrates was observed spectrophotometrically by the formation of
yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine,
determined by the enzymatic hydrolysis of AChI at a wavelength of 412 nm. For the
determination of inhibition effect of the compounds on AChE, an activity (%)-[Compound]
graph was drawn. The IC50 values were obtained from activity (%) versus compounds plots.
For calculation of Ki values, three different concentrations were used. The Lineweaver–Burk
curves were drawn and calculations were realised.
4.2.3. Cytotoxicity assay
The cytotoxicities of the compounds 9-16 were tested towards human squamous cell
carcinoma cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) and human normal
oral cells (HGF, HPLF and HPC) as described before.22,26,27,29,31-38 All cells were cultured in
DMEM supplemented with 10% fetal bovine serum (FBS). All test samples were
dissolved in dimethylsulfoxide (DMSO). Near confluent cells were incubated in 96-
microwell (Becton Dickinson, Franklin Lakes, NJ, USA) for 48 h with 0.78, 1.56, 3.12, 6.25,
12.5, 25, 50, 100 or 200 µM of each test compound. Any cytotoxicity induced by DMSO
(0.002, 0.004, 0.0078, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5 or 1%) were subtracted from
the corresponding treated groups. The viable cell numbers were determined using the
MTT method. In brief, the treated cells were incubated for another 3 hours in fresh culture
medium containing 0.2 mg/ml MTT. Cells were then lysed with 0.1 ml of DMSO and the
absorbance at 562 nm of the cell lysate was determined using a microplate reader (Sunrise
Rainbow RC-R, TECAN, Männedorf, Switzerland). CC50 (the concentrations of the
compounds in micromoles which reduce the viable cell number by 50%) was determined from
the dose-response curve and the mean value of CC50 for each cell type was calculated
from triplicate assays.
Acknowledgments
Authours thank to Department of Chemistry, Faculty of Science, Ataturk University (Erzurum,
Turkey) for NMR spectra.
15

Declaration of interest
The authors report no conflict of interest and are responsible for the contents and writing of the
paper.
Captions for the scheme, figure, and tables
Figure 1. Celecoxib and target compounds designed to be inspired by Celecoxib in this study
Scheme 1. Synthetic pathway of the compounds
Table 1. hCA I, hCA II and AChE inhibition results of the compounds 9-16
Table 2. Cytotoxicities of the compounds 9-16
16

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19

GRAPHICAL ABSTRACT
20

Highlights
 Sulfonamide derivatives bearing pyrazoline ring can be considered as CAs and AChE
inhibitors.

Ki values were in the range of 27.9 - 74.3 nM against hCAI.
 Ki values were in the range of 27.4 - 54.5 nM against hCAII.
 Ki values were in the range of 37.7 - 89.2 nM against AChE.
 The compounds had low cytotoxicity and selectivity against cancer cell lines.
21