Synthesis, cytotoxic, and carbonic anhydrase inhibitory effects of new 2-(3-(4-methoxyphenyl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazole derivatives

Article abstract of DOI:10.1002/jhet.3985

2-(3-[4-Methoxyphenyl]-5-aryl-4,5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazoles (1b-7b) were synthesized for the first time except 1b, and spectral methods such as ¹H NMR, ¹³C NMR and HRMS were utilized to illuminate the chemical structures of the synthesized compounds. Phenyl (1b), 2-methoxyphenyl (2b), 4-methoxyphenyl (3b), 4-methoxy-3-hydroxyphenyl (4b), 2,5-dimethoxyphenyl (5b), 3,4,5-trimethoxyphenyl (6b), or thiophene-2-yl (7b) was used as a aryl part. The inhibitory effects of the compounds were evaluated toward human carbonic anhydrase I and II enzymes (hCA I and hCA II). In vitro cytotoxic effects of the compounds against human oral squamous carcinomas and human normal oral cells were carried out via MTT. The compounds (1b-7b) had Ki values of 36.87 ± 11.62-66.24 ± 2.99 μM (hCA I) and 22.66 ± 1.41-89.95 ± 6.25 μM (hCA II). Compounds 1b (Ki = 36.87 ± 11.62 μM) toward hCA I, 6b (Ki = 22.66 ± 1.41 μM) toward hCA II had significant enzyme inhibitory potency. Compound 6b had the highest tumor selectivity (TS = 29.3) and potency selectivity expression (PSE = 272.3) values. Therefore, compounds 1b and 6b with CAs inhibition effect and compound 6b with the cytotoxicity may be forwarded to further studies as potent compounds.

Full text of DOI:10.1002/jhet.3985

Received: 20 February 2020
DOI: 10.1002/jhet.3985
Revised: 4 March 2020
Accepted: 13 March 2020
A R T I C L E
Synthesis, cytotoxic, and carbonic anhydrase inhibitory
effects of new 2-(3-(4-methoxyphenyl)-5-(aryl)-4,5-
dihydro-1H-pyrazol-1-yl)benzo[d]thiazole derivatives
Mehtap Tugrak1
| Halise Inci Gul1
| Hiroshi Sakagami2 | Ilhami Gulcin3
1Department of Pharmaceutical
Abstract
Chemistry, Faculty of Pharmacy, Ataturk
University, Erzurum, Turkey
2-(3-[4-Methoxyphenyl]-5-aryl-4,5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazoles
(1b-7b) were synthesized for the first time except 1b, and spectral methods
such as 1H NMR, 13C NMR and HRMS were utilized to illuminate
the chemical structures of the synthesized compounds. Phenyl (1b),
2-methoxyphenyl (2b), 4-methoxyphenyl (3b), 4-methoxy-3-hydroxyphenyl
(4b), 2,5-dimethoxyphenyl (5b), 3,4,5-trimethoxyphenyl (6b), or thiophene-2-yl
(7b) was used as a aryl part. The inhibitory effects of the compounds were eval-
uated toward human carbonic anhydrase I and II enzymes (hCA I and hCA
II). In vitro cytotoxic effects of the compounds against human oral squamous
carcinomas and human normal oral cells were carried out via MTT. The
compounds (1b-7b) had Ki values of 36.87 11.62-66.24 2.99 μM (hCA I)
and 22.66 1.41-89.95 6.25 μM (hCA II). Compounds 1b (Ki = 36.87
11.62 μM) toward hCA I, 6b (Ki = 22.66 1.41 μM) toward hCA II had signifi-
cant enzyme inhibitory potency. Compound 6b had the highest tumor selectiv-
ity (TS = 29.3) and potency selectivity expression (PSE = 272.3) values.
Therefore, compounds 1b and 6b with CAs inhibition effect and compound
6b with the cytotoxicity may be forwarded to further studies as potent
compounds.
2Division of Pharmacology, Meikai
University Research Institute of
Odontology, Sakado, Japan
3Faculty of Science, Department of
Chemistry, Ataturk University, Erzurum,
Turkey
Correspondence
Mehtap Tugrak and Halise Inci Gul,
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ataturk University,
Erzurum, Turkey.
Email: mehtaptugrak@hotmail.com
(M. T.) and
Email: incigul@atauni.edu.tr (H. I. G)
Funding information
Ataturk University; TUBITAK, Grant/
Award Number: 117S939
1 | INTRODUCTION
Cancer is a disease defined by rapid and uncontrolled
proliferation of abnormal cells. Globally, cancer is the
Heterocyclic chemistry has great importance especially in
the development of new drug candidates. Compounds
including a pyrazoline scaffold are of great importance
for heterocyclic chemistry and it is available in many bio-
active molecules. Pyrazoline is a partially reduced form
of pyrazole, which has two adjacent nitrogen atoms and
an endocyclic double bond.[1,2]
Pyrazoline has quite different pharmacological
activities such as antimicrobial,[3] anticancer,[4,5] anti-
convulsant,[6] anti-depressant,[7] anti-oxidant,[8] anti-
inflammatory,[9] anti-tubercular,[10] and carbonic
anhydrase (CA, EC4.2.1.1) inhibiting activities.[11,12]
second leading disease that causes death after cardiovas-
cular diseases. Resistance to drugs, lack of selectivity, and
the emergence of side effects in chemotherapeutic agents
are the biggest problems in the treatment of cancers.
Hence, there is a need to enhance new effective and
selective drugs in the treatment of cancer.[13,14]
Oral squamous cell carcinoma (OSCC) is the most
common type of oral cancer.[15] Morbidity and functional
impairments may occur during the treatment of the dis-
ease. Although there are improvements in surgical tech-
niques, post-operative radiation, or chemotherapy, the
diagnosis and prevention of this malignancy has not
J Heterocyclic Chem. 2020;1–7.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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TUGRAK ET AL.
improved considerably.[16] Therefore, early diagnosis of
OSCC is extremely important for the treatment of the
disease.
CA is an enzyme responsible for the hydration of car-
− [5,12,17–19]
bon dioxide (CO2) and dehydration of HCO3 .
Carbonic anhydrases (CAs, E.C.4.2.1.1) are found in pro-
karyotes and eukaryotes. They take part in various physio-
logical and pathological events, for instance, neurological
disorders, bone resorption, pH regulation, gluconeogenesis,
neurogenesis, glaucoma, lipogenesis, cancer, calcification,
osteoporosis, tumorgenicity, and bicarbonate synthesis.
CA I and CA II are cytosolic CA isoenzymes. The most
important function of these isoenzymes is their role in the
respiratory event. Also, CA II plays a role in primary trans-
port of sodium to the eye and helps regulate intraocular
pressure. CA-IX and CA-XII isoenzymes are transmem-
brane proteins and are targeted for treatment in cancer.
Their expression increases in response to hypoxia. They
contribute to tumor development by triggering extracellu-
lar acidification. In addition, patients with Alzheimer's dis-
ease (AD) have been shown to have reduced CA levels in
brain tissue significantly, which is evident that CA iso-
zymes (hCA I, II, IV, and VII) play an important role in
cognitive function.[18–21]
SCHEME 1 The synthetic route for the synthesis of
compounds 1b-7b. i: Hydrazine hydrate (%80), ethanol, reflux,
ii: NaOH (%10), ethanol, rt, iii:Ethanol, glacial acetic acid, reflux.
Ar: Phenyl (1b), 2-methoxyphenyl (2b), 4-methoxyphenyl (3b),
4-methoxy-3-hydroxyphenyl (4b), 2,5-dimethoxyphenyl (5b),
3,4,5-trimethoxyphenyl (6b), thiophene-2-yl (7b)
This study objected to synthesize new 2-(3-(4-methoxy
phenyl)-5-aryl-4,5-dihydro-1H-pyrazol-1-yl) benzo[d]thia-
zole (1b-7b) to investigate their cytotoxic /anticancer
activities against human oral squamous cell lines (Ca9-22,
HSC-2, HSC-3, HSC-4) and normal oral cells (HGF,
HPLF, HPC) and their CA (hCA I and hCA II) inhibition
potency to ascertain their applicability as possible leading
compounds.
Selectivity index (SI) value was calculated via
equation of [Mean D] on normal cells / [CC50] on
OSCC and presented in Table 1. SI value (SI > 1) is con-
sidered as a key parameter to identify tumor-selective
compounds.[20–26] According to Table 1, the SI values of
the compounds toward a specific cell line, were as fol-
lows: [Ca9-22: 2b (6.3), 4b (13.8), 5b (1.2), 6b (28.5), 7b
(2.0)], [HSC-2: 2b (3.9), 4b (16.5), 6b (11.8)], [HSC-3: 2b
(3.5), 4b (19.5), 5b (61.3), 6b (18.6), 7b (3.0)], [HSC-4: 2b
(2.6), 4b (12.1), 5b (14.2), 6b (14.3), 7b (1.2)]. The highest
SI value calculated was 63.1 for the compound 5b toward
HSC-3. Among others, compound 1b did not show
remarkable tumor specificity against cancer cells.
The most important problem in anticancer drugs is
selective cytotoxicity. In this study, tumor selectivity
(TS) was calculated by 2 equations as TS1 and TS2. First TS
calculation was made by dividing the average CC50 value
toward normal cells to the average CC50 value toward can-
cer cell lines (TS1 = Column D/Column B, Table 1). Sec-
ond TS values were also generated by considering the fact
that HGF is the corresponding normal cell of Ca9-22 can-
cer cell line having the same origin (both derived from gin-
gival tissues). Second TS values were generated for a
compound by dividing the CC50 value toward HGF cells
into the CC50 value toward Ca9-22 cell line
(TS2 = Column C/Column A., Table 1). It is clear that
some compounds tested had more selectivity than 5-FU
according to the first type TS1 calculation. The highest TS
value was calculated 16.4 for the compound 4b, which has
2 | RESULTS AND DISCUSSION
The compounds (1b-7b) were synthesized for the first
time except 1b. The synthesis method of the target com-
pounds was shown in Scheme 1. The cytotoxicity proper-
ties of the compounds were given in Table 1 and CAs
inhibitory results are shown in Table 2.
Cytotoxic activities of the compounds were tested via
MTT method.[20–26] The compounds had CC50 value of
3.1 - 400 μM toward OSCC cancer cell lines while the ref-
erence 5-fluorouracil (5-FU) had CC50 value of 7.8 and
261.0 μM. When the compounds cytotoxicities compared
with 5-FU, against Ca9-22 cell line, 2b (1.4 times) and 6b
(2.04 times) showed more cytotoxicity while 2b (10.6
times), 4b (13.5 times) and 6b (10.03 times) were demon-
strated favorable cytotoxicity against HSC-2 cells. Only
compound 5b (2.5 times) was cytotoxic against HSC-3
cells. Besides, compounds did not show any cytotoxicity
against to HSC-4 cells.

TUGRAK ET AL.
3
4-methoxy-3-hydroxyphenyl ring. The bioisosteric modifi-
cation of the compound 1b (phenyl, 0.7) caused to 2 times
increasing of TS value for 7b (thiophene-2-yl, 1.5).
On the other hand, TS2 pointed out that all of the
compounds had less selectivity than 5-FU. In terms of TS,
mono and tri methoxy substitution/s of phenyl ring were
considered a favorable molecular modification. The
compound 2b (2-methoxy phenyl), compound 4b (4-met-
hoxy-3-hydroxy
phenyl)
and
compound
6b
(3,4,5-trimethoxyphenyl) showed the best TS. TS values
were 16.6, 16.8, 29.3 for 2b, 4b, and 6b, respectively.
As another parameter, PSE (Potential Selectivity
Expression) values were calculated by the following
Equations (D/B2) × 100 and (C/A2) × 100. The com-
pounds 4b (with 4-methoxy-3-hydroxy phenyl,
PSE = 70.7) and 6b (with trimethoxyphenyl, PSE = 88)
showed higher PSE values than 5-FU although other
compounds PSE values were much lower than 5-FU
(PSE = 17.1) (Table 1). When the PSE values of the com-
pounds were compared to the non-substituted com-
pound 1b, it was observed that the PSE values of the
substituted compounds increased compared to the non-
substituted compound 1b as follows as compound and
(times of): 2b (362), 4b (242), 5b (5), 6b (906). However,
when non-substituted compound 1b was compared to
compound 7b, which is a bioisoster of compound 1b,
PSE value of compound 7b did not increase. These kinds
of compounds can be considered as the most promising
compounds with good potency and selective cytotoxicity
based on the PSE values.
Based on PSE and TS values, compound 6b
(2-(3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-di-
32#hydro-1H-pyrazol-1-yl)benzo[d]thiazole)
showed
remarkable cytotoxicity toward OSCC.
Carbonic anhydrase (CA) isoenzymes have become
an important target in developing new inhibitor and
activator drugs due to their various pharmacological
effects.[21] In this study, hCA I and hCA II isoenzymes
were used to identify whether the compounds have
CAs inhibitory potency. According to the results
obtained (Table 2), the compounds (1b-7b) had hCA I
inhibitory potency with Ki value of 36.87 11.62 −
66.24 2.99 μM. Among the series, compound 1b
(Ki = 36.87 11.62 μM) showed great inhibition prop-
erties against hCA I when compared with AZA
(30.18 7.77 μM).
Furthermore, compounds (1b-7b) had inhibition
potency against hCA II with Ki values between 22.66 1.41
and 89.95 6.25 μM while AZA had Ki value of 4.41
0.55 μM. Compound 6b, 2-(3-(4-methoxyphenyl)-5-(3,-
4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo
[d]thiazole may be evaluated as the strongest compound
with the lowest Ki value (22.66 1.41 μM) against hCA II.

4
TUGRAK ET AL.
TABLE 2
inhibition results of the compounds
hCA I and hCA II
IC50 (μM)
Ki (μM)
Compounds
hCA I
28.5
46.5
20
r2
hCA II
45.39
29.49
33.16
32.25
37.46
29.24
38.08
8.37
r2
hCA I
hCA II
1b-7b
1b
0.9546
0.9955
0.9498
0.9345
0.9385
0.9674
0.9787
0.9887
0.949
0.943
0.969
0.946
0.947
0.926
0.921
0.983
36.87 11.62
47.06 16.89
45.55 5.37
43.132 4.12
46.56 3.45
46.61 2.15
66.24 2.99
30.18 7.77
57.36 2.39
55.57 3.30
89.95 6.25
25.95 7.46
26.72 8.88
22.66 1.41
36.57 10.48
4.41 0.55
2b
3b
4b
21.3
26.5
27.3
24.3
16.6
5b
6b
7b
AZA*
Note: *Acetazolamide (AZA) was used as a standard inhibitor for both hCA I and hCA II isoenzymes.
The compounds studied here had methoxy substitu-
ent/s (mono, di and, tri methoxy) in different positions of
the phenyl ring. These modifications were provided by
using benzaldehyde and mono, di and tri substituted
methoxy benzaldehydes [2-methoxybenzaldehyde (2b),
4 | MATERIALS AND METHODS
4.1 | Chemistry
The synthesis methods of the compounds were presented
in Scheme 1. During the synthesis studies, to monitor the
reaction and check the purity of the synthesized com-
pounds, thin layer chromatography (TLC) plates
(60 HF254, Merck KGaA) were used. Chloroform: metha-
nol (4.8:0.2) system was used as the mobile phase system.
NMR spectra of the compounds were taken with the Var-
ian Mercury Plus spectrometer (Varian inc., Palo Alto,
California, U.S.). Shimadzu's LCMS-TOF-ESI (Shimadzu,
Kyoto, Japan) device was used for HRMS. Electrothermal
9100/IA9100 instrument (Bibby Scientific Limited, Staf-
fordshire, UK) device was used to determine melting
points.
4-methoxybenzaldehyde
(3b),
4-methoxy-3-hydroxy-
benzaldehyde (4b), 2,5-dimethoxybenzaldehyde (5b) and
3,4,5-trimethoxy benzaldehyde (6b)] to investigate how
methoxy groups affect the bioactivities (Scheme 1).
As a favorable molecular modification, the replace-
ment of hydrogen by di and tri methoxy groups can be
considered for future studies. The compound 4b
(Ki = 43.132 4.12 μM, 4-methoxy-3-hydroxy derivative)
against hCA I and the compound 6b (Ki = 22.66
1.41 μM, trimethoxy derivative) against hCA II were
found promising CAs inhibitors.
It can be expressed here that the substitution of
methoxy groups on phenyl ring affects compounds' CA
inhibitory effects against hCA I/II. So, the different
chemical structure of the compounds which may affect
interaction with the active side may change their
bioactivity.
4.1.1 | Synthesis of 2-hydrazinylbenzo[d]
thiazole (Compound A)
The starting compounds, 2-mercaptobenzothiazole (3.0g)
and hydrazine hydrate (10 mL / 80%), were refluxed for
24 hours by the conventional method in ethanol (20 mL).
At the end of the specified period, the contents of the
flask were kept at room temperature and the separated
product was filtered and dried. Light brown compound A
was used for the next reaction without purification.[22]
3 | CONCLUSION
This research reported synthesis and bioactivities of
the
2-(3-(4-methoxyphenyl)-5-(aryl)-4,5-dihydro-1H-
pyrazol-1-yl)benzo[d]thiazoles. The bioactivity assays
showed that the compounds (2-(3-(4-methoxyphenyl)-
5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1 yl)
benzo [d]thiazole) 6b (PSE = 272.3, TS = 29.3) as cyto-
toxic agent and (2-(3-(4-methoxyphenyl)-5-phenyl-4,-
4.1.2 | General procedure for the
preparation of chalcones, 1a-7a
5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazole) 1b (Ki
=
36.87 11.62 μM, hCA I) and 6b (Ki = 22.66 1.41 μM,
hCA II) as carbonic anhydrase inhibitors can be consid-
ered for further molecular modifications and new
projects.
Starting compounds of the series, which are chalcones,
were synthesized by Claisen-Schmidt condensation.[20–28]
4-Methoxy acetophenone and the appropriate aldehyde
derivative [benzaldehyde (1a), 2-methoxybenzaldehyde

TUGRAK ET AL.
5
(2a),
4-methoxybenzaldehyde
(3a),
3-hydroxy-
ArH, J = 7.7 Hz), 7.08 (t, 1H, ArH, J = 7.7 Hz), 6.93 (d,
3H, ArH, J = 7.3 Hz), 6.85 (t, 1H, ArH, J = 7.5 Hz), 6.03
(dd, 1H, pyrazoline ring, J = 11.7, 5.1 Hz), 3.90 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.16 (dd, 1H, pyrazoline ring,
J = 17.6, 5.1 Hz), (one of the proton peak of the
pyrazoline ring was under methoxy peak). 13C NMR
(100 MHz, CDCl3, δ ppm): 163.6, 161.3, 156.5, 153.7,
153.1, 132.0, 128.98, 128.88, 128.3, 126.5, 125.8, 124.4,
121.7, 120.9, 120.8, 120.1, 114.3, 110.9, 59.6, 55.7, 55.6,
43.1, HRMS (ESI-MS) calc. For C24H21N3O2S [M + H]+
416.1427; found 416.1435.
4-methoxybenzaldehyde (4a), 2,5-dimethoxybenzaldehyde
(5a), 3,4,5-trimethoxybenzaldehyde (6a), thiophene-
2-carboxaldehyde (7a)] were mixed within ethyl alcohol
(6 mL) in 1:1 mol ratio. The mixture was cooled on ice
bath and then NaOH (aqua, 6 mL, 10%) was added drop
by drop to the flask. The mixture was sustained at room
temperature throughout the night. After 24 hours, the
content was taken into the cold water (50 mL). The con-
tent of the flask was acidified with concentrated HCl acid
(pH = 6-7). The collapsed solid was filtered. Water and
ethanol were used to wash the solid compound. After the
drying, the compound was used as a starting material in
the third step.
2-(3,5-bis[4-Methoxyphenyl]-4,5-dihydro-1H-pyrazol-
1-yl)benzo[d]thiazole (3b)
1
A bright yellow solid, yield 6.5%. Mp: 174ꢀC–175ꢀC. H
NMR (400 MHz, CDCl3, δ ppm): 7.71 (d, 2H, ArH,
J = 7.3 Hz), 7.62 (d, 1H, ArH, J = 8.1 Hz), 7.51 (d, 1H,
ArH, J = 8.1 Hz), 7.28–7.21 (m, 3H, ArH), 7.06 (t, 1H,
ArH, J = 7.5 Hz), 6.94 (d, 2H, ArH, J = 7.3 Hz), 6.83 (d,
2H, ArH, J = 7.3 Hz), 5.72 (dd, 1H, pyrazoline ring,
J = 11.7, 4.8 Hz), 3.85 (s, 3H, OCH3), 3.76 (s, 3H, OCH3),
3.26 (dd, 1H, pyrazoline ring, J = 17.2, 5.1 Hz), (one of
the proton peak of the pyrazoline ring was under met-
hoxy peak). 13C NMR (100 MHz, CDCl3, δ ppm): 163.6,
161.4, 159.3, 152.9, 152.7, 133.7, 131.9, 128.3, 127.6, 125.8,
124.2, 121.8, 120.9, 120.2, 114.42, 114.38, 63.3, 55.6, 55.5,
44.1, HRMS (ESI-MS) calc. For C24H21N3O2S [M + H]+
416.1427; found 416.1438.
4.1.3 | Synthesis of the pyrazoline
derivatives, 1b-7b
Synthesis of pyrazoline derivative compounds (1b-7b)
was carried out in acidic medium using a conventional
method with a protic solvent. Briefly, favorable chalcone
derivative (1 mmol) (1a-7a) and 2-hydrazinylbenzo[d]thi-
azole (1.1 mmol, compound A) in ethanol (25 mL) with
acetic acid (0.05 mL) were heated for 19 - 36 hours (for
1b-7b). Then, ethanol was evaporated until half volume
and the flask was left at room temperature. After the col-
lapsed solid was filtered, it was purified by crystallization
from the favorable solvent or solvent mixture (methanol
or methanol-ether).[12,29,30]
5-(1-(Benzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-5-yl)-2-methoxyphenol (4b)
1
2-(3-[4-Methoxyphenyl]-5-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)benzo[d]thiazole (1b)
A cream colour solid, yield 3.3%. Mp: 225ꢀC - 226ꢀC. H
NMR (400 MHz, CDCl3, δ ppm): 9.03 (s, 1H, OH), 7.76–
7.69 (m, 3H, ArH), 7.37 (d, 1H, ArH, J = 7.7 Hz), 7.23–
7.19 (m, 1H, ArH), 7.07 - 7.00 (m, 3H, ArH), 6.83 (d, 1H,
ArH, J = 8.8 Hz), 6.67-6.50 (m, 2H, ArH), 5.61 (dd, 1H,
pyrazoline ring, J = 11.3, 4.4 Hz), 3.95 (dd, 1H,
pyrazoline ring, J = 17.6, 11.7 Hz), 3.78 (s, 3H, OCH3),
3.67 (s, 3H, OCH3), 3.22 (dd, 1H, pyrazoline ring,
J = 17.9, 4.8 Hz). 13C NMR (100 MHz, CDCl3, δ ppm):
163.2, 161.6, 154.5, 152.9, 147.7, 147.3, 134.9, 131.6, 128.9,
126.5, 123.9, 122.3, 121.9, 119.9, 117.2, 115.0, 113.3, 113.0,
63.3, 56.2, 56.0, 44.4, HRMS (ESI-MS) calc. For
C24H21N3O3S [M + H]+ 432.1376; found 432.1363.
A bright green solid, yield 9.3%. Mp: 188ꢀC–190ꢀC; Lit m.
p: 149ꢀC–150ꢀC.[27] 1H NMR (400 MHz, CDCl3, δ ppm):
7.71 (d, 2H, ArH, J = 8.8 Hz), 7.63 (d, 1H, ArH,
J = 7.7 Hz), 7.50 (d, 1H, ArH, J = 8.1 Hz), 7.46–7.22 (m,
6H, ArH), 7.07 (t, 1H, ArH, J = 7.5 Hz), 6.94 (d, 2H, ArH,
J = 8.8 Hz), 5.78 (dd, 1H, pyrazoline ring, J = 12.1,
5.1 Hz), 3.84 (s, 3H, OCH3), 3.27 (dd, 1H, pyrazoline ring,
J = 17.2, 5.1 Hz), (one of the proton peak of the
pyrazoline ring was under methoxy peak). 13C NMR
(100 MHz, CDCl3, δ ppm): 152.9, 152.7, 141.6, 131.9,
129.1, 128.7, 128.4, 127.9, 126.2, 125.8, 124.1, 121.8, 121.4,
120.9, 120.2, 114.4, 63.7, 55.6, 44.1, HRMS (ESI-MS) calc.
For C23H19N3OS [M + H]+ 386.1322; found 386.1336.
2-(5-(2,5-Dimethoxyphenyl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazole (5b)
A cream colour solid, yield 10.7%. Mp: 210ꢀC - 212ꢀC. 1H
NMR (400 MHz, CDCl3, δ ppm): 7.71 - 7.69 (m, 2H,
ArH), 7.64 (d, 1H, ArH, J = 7.7 Hz), 7.51 (d, 1H, ArH,
J = 8.1 Hz), 7.26 - 7.22 (m, 1H, ArH), 7.10 - 7.06 (m, 1H,
ArH), 6.93 - 6.91 (m, 2H, ArH), 6.85 - 6.83 (m, 1H, ArH),
6.75 - 6.72 (m, 2H, ArH), 5.99 (dd, 1H, pyrazoline ring,
2-(5-(2-Methoxyphenyl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazole (2b)
A bright yellow solid, yield 39%. Mp: 199ꢀC - 200ꢀC. H
NMR (400 MHz, CDCl3, δ ppm): 7.71 (d, 2H, ArH,
J = 9.1 Hz), 7.66 (d, 1H, ArH, J = 8.1 Hz), 7.52 (d, 1H,
ArH, J = 7.7 Hz), 7.27 - 7.21 (m, 2H, ArH), 7.15 (d, 1H,
1

6
TUGRAK ET AL.
J = 11.7, 5.1 Hz), 3.87 (s, 3H, OCH3), 3.83 (s, 3H, OCH3),
3.64 (s, 3H, OCH3), 3.16 (dd, 1H, pyrazoline ring,
J = 17.2, 5.1 Hz), (one of the proton peak of the
pyrazoline ring was under methoxy peak). 13C NMR
(100 MHz, CDCl3, δ ppm): 163.7, 161.3, 153.9, 153.6,
153.0, 150.7, 132.0, 130.4, 128.4, 125.7, 124.3, 121.7, 120.9,
120.2, 114.3, 113.0, 112.8, 112.1, 59.5, 56.3, 55.9, 55.6,
43.2, HRMS (ESI-MS) calc. For C25H23N3O3S [M + H]+
446.1533; found 446.1527.
some minor modifications.[23,24,26,28,31,32] The viable cell
numbers were determined by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.
The CC50 values were determined from dose-response
curves. 5-Fluorouracil was used as
compound.
a
reference
4.2.2 | Carbonic anhydrase enzyme assay
2-(3-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)benzo[d]thiazole (6b)
A cream colour solid, yield 7.2%. Mp: 182ꢀC–183ꢀC. H
Purification of cytosolic CA enzymes was carried out
according to the literature.[33] Activities of these enzymes
were designated according to a procedure by Verpoorte
et al[34] at 348 nm. In addition, the amount of protein
was designated at 595 nm.
1
NMR (400 MHz, CDCl3, δ ppm): 7.71 (d, 1H, ArH,
J = 8.8 Hz), 7.64 (d, 1H, ArH, J = 8.1 Hz), 7.53 (d, 1H,
ArH, J = 7.7 Hz), 7.28 - 7.24 (m, 2H, ArH), 7.11–7.07 (m,
1H, ArH), 6.94 (d, 2H, ArH, J = 8.8 Hz), 6.56 (s, 2H,
ArH), 5.69 (dd, 1H, pyrazoline ring, J = 12.1, 5.5 Hz),
3.85 (s, 3H, OCH3), 3.80 (s, 9H, OCH3), 3.28 (dd, 1H,
pyrazoline ring, J = 17.2, 5.1 Hz), (one of the proton peak
of the pyrazoline ring was under methoxy peak). 13C
NMR (100 MHz, CDCl3, δ ppm): 163.8, 161.5, 153.9,
153.8, 152.9, 152.8, 137.3, 131.9, 128.4, 125.9, 124.0, 121.9,
120.9, 120.2, 114.4, 114.1, 64.0, 60.9, 56.4, 56.3, 55.6, 44.2,
HRMS (ESI-MS) calc. For C26H25N3O4S [M + H]+
476.1639; found 476.1635.
According to the Bradford method[35], inhibition
effect of 1b-7b on both hCA enzymes, an activity (%)-[1b-
7b] graph, was drawn. The IC50 values were obtained
from activity (%) vs compounds plots. Ki values were cal-
culated in three different concentrations and Lineweaver-
Burk curves were drawn.[36] Acetazolamide was used as a
reference compound.
ACKNOWLEDGEMENTS
The authors thank TUBITAK for financial support
(Project Number: 117S939) and Ataturk University
(Erzurum, Turkey) for NMR spectra.
2-(3-(4-Methoxyphenyl)-5-(thiophen-2-yl)-4,5-dihydro-
1H-pyrazol-1-yl)benzo[d]thiazole (7b)
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
1
A bright yellow solid, yield 9%. Mp: 167ꢀC–168ꢀC. H
NMR (400 MHz, CDCl3, δ ppm): 7.72 (d, 2H, ArH,
J = 8.8 Hz), 7.65 (d, 1H, ArH, J = 7.7 Hz), 7.59 (d, 1H,
ArH, J = 7.7 Hz), 7.31–7.25 (m, 2H, ArH), 7.18–7.08 (m,
3H, ArH), 6.96–6.91 (m, 2H, ArH), 6.05 (dd, 1H,
pyrazoline ring, J = 11.7, 4.8 Hz), 3.85 (s, 3H, OCH3),
3.44 (dd, 1H, pyrazoline ring, J = 17.6, 4.8 Hz), (one of
the proton peak of the pyrazoline ring was under met-
hoxy peak). 13C NMR (100 MHz, CDCl3, δ ppm): 163.7,
161.5, 152.80, 152.76, 144.0, 132.0, 128.4, 127.1, 125.9,
125.8, 125.1, 123.9, 122.1, 121.0, 120.3, 114.4, 59.5, 55.6,
43.9, HRMS (ESI-MS) calc. For C21H17N3OS2 [M + H]+
392.0886; found 392.0895.
ORCID
Mehtap Tugrak https://orcid.org/0000-0002-6535-6580
Halise Inci Gul https://orcid.org/0000-0001-6164-9602
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How to cite this article: Tugrak M, Gul HI,
Sakagami H, Gulcin I. Synthesis, cytotoxic, and
carbonic anhydrase inhibitory effects of new 2-(3-
(4-methoxyphenyl)-5-(aryl)-4,5-dihydro-1H-
pyrazol-1-yl)benzo[d]thiazole derivatives.
J Heterocyclic Chem. 2020;1–7. https://doi.org/10.
1002/jhet.3985
[25] M. Tugrak, H. I. Gul, H. Sakagami, Lett. Drug Des. Discov.
2015, 12(10), 806.