Synthesis, characterization, molecular docking and biological activities of novel pyrazoline derivatives

Article abstract of DOI:10.1002/ardp.201800359

In this study, synthesis of ethyl 2-((4-bromophenyl)diazenyl)-3-oxo-phenylpropanoate 1 was carried out and a series of new 3H-pyrazol-3-ones (P1–7) were synthesized from 1 as well as various hydrazines. The obtained yields of the synthesized compounds were moderate (40–70%) and these compounds were confirmed by spectral data. These novel pyrazoline derivatives were effective inhibitor compounds of the human carbonic anhydrase I and II isozymes (hCAs I and II) and of the acetylcholinesterase (AChE) enzyme, with K_i values in the range of 17.4–40.7 nM for hCA I, 16.1–55.2 nM for hCA II, and 48.2–84.1 nM for AChE. In silico studies were performed on the compounds inhibiting hCA I, hCA II, and AChE receptors. On the basis of the findings, the inhibition profile of the new pyrazoline compounds at the receptors was determined.

Full text of DOI:10.1002/ardp.201800359

Received: 8 December 2018
Revised: 6 March 2019
Accepted: 17 March 2019
|
|
DOI: 10.1002/ardp.201800359
F U L L P A P E R
Synthesis, characterization, molecular docking and biological
activities of novel pyrazoline derivatives
Fikret Turkan1
İlhami Gulçin4
|
Adnan Cetin2
|
Parham Taslimi3
|
Halide S. Karaman4
|
1Health Services Vocational School, Igdır
University, Igdır, Turkey
Abstract
2Department of Science, Faculty of Education,
In this study, synthesis of ethyl 2‐((4‐bromophenyl)diazenyl)‐3‐oxo‐phenylpropanoate
1 was carried out and a series of new 3H‐pyrazol‐3‐ones (P1–7) were synthesized
from 1 as well as various hydrazines. The obtained yields of the synthesized
compounds were moderate (40–70%) and these compounds were confirmed by
spectral data. These novel pyrazoline derivatives were effective inhibitor compounds
of the human carbonic anhydrase I and II isozymes (hCAs I and II) and of the
acetylcholinesterase (AChE) enzyme, with Ki values in the range of 17.4–40.7 nM for
hCA I, 16.1–55.2 nM for hCA II, and 48.2–84.1 nM for AChE. In silico studies were
performed on the compounds inhibiting hCA I, hCA II, and AChE receptors. On the
basis of the findings, the inhibition profile of the new pyrazoline compounds at the
receptors was determined.
Muş Alparslan University, Muş, Turkey
3Department of Biotechnology, Faculty of
Science, Bartin University, Bartin, Turkey
4Department of Chemistry, Faculty of Science,
Atatürk University, Erzurum, Turkey
Correspondences
Fikret Turkan, Health Services Vocational
School, Igdır University, 76000 Iğdır, Turkey.
Email: fikret.turkan@gmail.com
K E Y W O R D S
acetylcholinesterase, enzyme inhibition, human carbonic anhydrase, in silico study, induced fit
docking, pyrazoline
|
1
INTRODUCTION
also were used as a selective inhibitor against to capable of causing
bacterial cell death and DNA gyrase.[18-20] Some known samples
Heterocyclic chemistry has grown very rapidly particularly in the
development of synthetic methods and investigation of bioactive
properties of the synthesized materials. The compounds having
pyrazoline structure are a significant group in the heterocyclic
chemistry. These compounds are scaffold target compounds in the
field of medicinal and synthetic chemistry.[1,2] These compounds
were used in the development of drug research and agricultural
products.[3-5] In the previous studies, it was indicated that these
compounds have significant pharmacological and biological activ-
ities.[6-8] Many pyrazoline compounds are reported to possess a
broad spectrum of biological activities such as antimicrobial,
antidepressant, neuroprotective, anticonvulsant, anti‐inflammatory,
analgesic, antitubercular, local anesthetic, hypoglycemic, hypoten-
sive, insecticide, herbicide, and molluscicidal.[9-16] In addition, the
antioxidant properties of the newly synthesized pyrazoline group
were also studied.[17] The pyrazoline derivatives were used as
efficient and potent inhibitor of glycogen synthase kinase and they
including the synthetic pyrazoline dipyrone having feature analgesic
are used for preoperative pain.[21] Propifenazon has been developed
for antipyretic anti‐inflammatory drugs.[22] Palerol is used as a novel
spasm‐analgesic in obstetrics and gynecology.[23] Nifenazone is
utilized for treating rheumatic and analgesic[24] and muzolimine is
used for the treatment of hypertension[25] and novel N1‐substituted
3,5‐diphenyl pyrazoline compounds have been synthesized for
evaluation as anti‐Helicobacter pylori agents (Figure 1).[26]
Carbonic anhydrases (CAs, E.C.4.2.1.1) exist in many living
systems and have a diversity of physiological and pathological
procedures including fluid balance, neurological disorders, bone
resorption, pH regulation, glaucoma, carboxylation reactions, cancer,
calcification, osteoporosis, tumorigenicity, and the synthesis of
bicarbonate (HCO−3 ).[27-29] CA isoforms play a significant role in the
physiology of coral calcification. CA enzyme has active confidants in
the brain, gastric mucosa, salivary glands, kidney, nerve myelin
sheath, eye lens, pancreas, prostate, and uterus.[30] Also, CA levels
|
Arch Pharm Chem Life Sci. 2019;e1800359
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
1 of 12
https://doi.org/10.1002/ardp.201800359

2 of 12
TURKAN ET AL.
|
FIGURE 1 The chemical structures of some important pyrazolines
determined by dementia, memory loss and cognitive impairment.[38,39]
Perception is affected in derangements such as AD. A low concentration
of ACh can create several neuropsychiatric perturbations and neurop-
sychological like Parkinson's disease and AD.[40,41]
SCHEME 1 Reaction equation of CA
In this article, we synthesized some new pyrazoline derivatives
(P1–7). In addition, characterized their inhibition confidants
against AChE, hCA I, and hCA II, and evaluated their inhibition
confidants to acetazolamide (AZA) compound, which is a clinical
standard used carbonic anhydrase inhibitor (CAI). On the other
hand, tacrine (TAC) molecule was used a control compound for
AChE enzyme inhibition.
are considerably reduced in the brain tissue cells of patients suffering
from Alzheimer's disease (AD), proving an essential involvement of
various human carbonic anhydrase isozymes (hCA I, II, IV, and VII) in
learning functions and cognition.[31] CA isoforms are biological
catalysts, which convert water (H2O) and carbon dioxide (CO2) to a
proton (H+) and HCO−3 (Scheme 1).[32-34] This reaction defines to the
fastest determined reactions (kcat, 106/s) in the environment and is
required to live in plants and animals.[35]
|
2
RESULTS AND DISCUSSION
Chemistry
Cholinesterases (ChE) catalyze the hydrolysis of acetylcholine (ACh)
into acetic acid and choline, a fundamental mechanism for the restoration
act of the cholinergic neurotransmission.[36,37] These enzymes include
butyrylcholinesterase (E.C.3.1.1.8) and acetylcholinesterase (AChE;
E.C.3.1.1.7) enzymes. ACh molecules are synthesized in presynaptic
terminals from choline and it is necessary for cholinergic neurotransmis-
sion in the peripheral nervous systems and their central. AD is
|
2.1
The synthesis of pyrazoline compounds (P1–7) is shown in Scheme 2.
Initially starting material ethyl 2‐((4‐bromophenyl)diazenyl)‐3‐oxo‐
phenylpropanoate (1) was prepared as result of coupling reaction of
ethyl benzoyl acetate and tetra azotized solution which was obtained
SCHEME 2 Synthesis of the pyrazoline derivatives

TURKAN ET AL.
3 of 12
|
4‐bromoaniline with sodium nitrite solution on the ice bath.[42] 1 was
obtained in good yield 84%.
δ −62.5 ppm signal by 19F‐NMR spectrum. Finally, synthesized pyrazoline
derivatives were confirmed by spectral data in agreement with our
previous findings.[6,43]
The IR spectrum of compound 1 showed absorption bands at
3125 cm‒1 corresponding to ‒NH hydrazide, 1721 cm−1 ester and
1670 cm−1 amide carbonyl groups, respectively. The 1H‐NMR spectrum
revealed the signal (‒NH) protons of hydrazide in the region δ 8.3 ppm,
the signals protons of methylene (‒CH2) in the region δ 3.9 ppm, the
signals protons of methyl (‒CH3) in the region δ 1.2 ppm, the multiplied
signals protons of aromatic ring (‒CH) in the region δ 8.0‒6.5 ppm. The
13C‐NMR spectrum revealed the signals carbonyls of benzoyl and ester in
the region δ 191.5 and 164.4 ppm, respectively. The signal carbonyl of
imine in the region δ 150.7 ppm, the signal of methylene carbon in the
region δ 59.7 ppm, the signal of methyl carbon in the region δ 13.4 ppm.
Then, the pyrazoline derivatives (P1–7) were obtained via cycloconden-
sation the starting material and various hydrazines (hydrazine hydrate,
phenyl hydrazine, 2,5‐dimethylphenyl hydrazine, 3,4‐dimethyl phenyl
hydrazine, 4‐trifluoromethyl phenyl hydrazine, 4‐bromo phenyl hydrazine,
and 2,4‐dinitro phenyl hydrazine, respectively) according to the
literature.[6] The yields of pyrazoline derivatives P1–7 were obtained in
40–70%. The pyrazoline derivatives P1–7 were characterized by 1H,
13C‐NMR, FT‐IR, and mass analysis. The IR spectra of synthesized
pyrazoline derivatives (P1–7) showed corresponding absorption bands
3226–3124 cm−1 –NH imine, 1672–1662 cm−1 pyrazole carbonyl group,
and 3082–3060 cm−1 –CH aromatic vibrations, respectively. The 1H‐
NMR spectra of P1–7 revealed the signal protons of –NH in the region δ
8.3–8.00 ppm, the multiplied signals (–CH) protons of aromatic ring in the
region δ 8.0–6.5 ppm. The 13C‐NMR spectrum revealed the signals
carbonyls of pyrazole in the region δ 163.4–160.4, the signal carbonyl of
imine in the region 150.7 ppm, the signal of methylene carbon in the
region δ 59.7 ppm, the signal of methyl carbon in the region 13.4 ppm, the
signals (C3) and (C4) of pyrazole ring in the region δ 157.1–155.1 and
153.4–152.6 ppm, respectively. Furthermore, the signals protons of
methyl groups for P3 compound were observed in the region δ 2.3 and
1.5 ppm in 1H‐NMR spectrum and its 13C‐NMR spectrum revealed the
signals of methyl carbons were observed in the region
|
2.2
Biological activity
All the synthesized new pyrazoline derivatives (P1–7) were tested to
investigate their inhibitory act toward AChE and the slower hCA I and II
isoenzymes. The chemical structures of new pyrazoline derivatives (P1–
7) are seen in Scheme 2 and their AChE, and CA I, and II isoenzymes
inhibition data are shown in Table 1. The pyrazoline derivatives (P1–7)
showed effective inhibition profiles against the enzymes mentioned
above. The following results are presented in this part.
Special isoforms from the CA families of zinc metalloenzymes have
been dependent on kind of diseases.[44] Classical CAIs, generally
sulfonamide‐based compounds and their features were inquired as
antiepileptic, antiobesity, antiglaucoma, anticancer compounds, and
against antineuropathic pain.[45] The cytosolic enzyme hCA I was
inhibited by the novel pyrazoline derivatives (P1–7), with Ki values
ranging between 17.42 3.61 and 40.73 8.17 nM. In addition, (Z)‐4‐(2‐
(4‐bromophenyl)hydrazineylidene)‐5‐phenyl‐2‐(2,4,6‐trichlorophenyl)‐2,4‐
dihydro‐3H‐pyrazol‐3‐one (P6) and (Z)‐4‐(2‐(4‐bromophenyl)hydraziney-
lidene)‐5‐phenyl,4‐trifluoromethylphenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one
(P5) recorded the most powerful hCA I isoform inhibition properties with
Ki values of 17.42 3.61 and 20.14 5.26 nM, respectively (Figure 2). The
clinically and standard used drug AZA calculated a Ki value of
75.16 14.44 nM. Thus, the evaluated novel pyrazoline derivatives
(P1–7) showed better inhibitory profiles when compared to AZA
molecule (Table 1 and Figure 3).
The importance of isoenzyme and physiologically dominant
cytosolic hCA II enzyme is dependent on different illnesses. The
hCA II was impressively inhibited by the novel pyrazoline derivatives
(P1–7) evaluated here. These molecules were shown to strongly
inhibit hCA II, with Ki values ranging from 16.1 2.8 nM to
55.2 12.5 nM. Ki amounts of new molecules are better than those
of the standard used drug AZA (Ki: 86.5 21.2 nM). All the evaluated
δ
21.5 and 16.6 ppm. Also, for P5 compound was observed at
TABLE 1 HCA I and II and AChE enzyme inhibition values of novel pyrazoline derivatives (P1–7)
IC50 (nM)
hCA I
24.7
30.2
33.7
27.1
17.2
12.7
29.3
73.5
–
Ki (nM)
Com-
pounds
r2
hCA II
32.1
40.1
45.3
35.7
23.6
16.4
33.8
81.5
–
r2
AChE
108.3
122.9
104.9
97.4
r2
hCA I
hCA II
AChE
P1
0.9632
0.9683
0.9974
0.9817
0.9627
0.9889
0.9592
0.9845
–
0.9951
0.9783
0.9910
0.9788
0.9971
0.9814
0.9738
0.9391
–
0.9847
0.9698
0.9810
0.9538
0.9941
0.9473
0.9821
–
30.17 3.8
36.12 5.0
40.73 8.2
28.53 4.9
20.14 5.3
17.42 3.6
32.54 6.9
75.16 14.4
–
40.2 10.2
46.3 8.3
55.2 12.5
41.8 7.3
22.1 3.7
16.1 2.8
30.3 5.9
86.5 21.2
–
77.1 12.5
84.1 17.2
68.3 11.4
58.1 10.8
48.2 9.5
52.7 15.4
64.4 12.6
–
P2
P3
P4
P5
82.3
P6
85.3
P7
100.1
–
AZAa
TACb
145.11
0.9721
89.54 11.1
Note: AChE: acetylcholinesterase; AZA: acetazolamide; hCA I and II: human carbonic anhydrase isoenzymes I and II; TAC: tacrine.
aAZA was used as a standard inhibitor for hCA I and II.
bTAC was used as a standard inhibitor for AChE enzyme.

4 of 12
TURKAN ET AL.
|
12
9
20
15
10
5
hCA II
hCA I
6
3
0
0
-1
1
3
5
7
-1
1
3
5
7
1/[S] (mM)-1
1/[S] (mM)-1
120
90
60
30
0
AChE
-6
-3
0
3
6
9
12
1/[S] (mM)-1
FIGURE 2 Determination of Lineweaver‐Burk graphs for novel pyrazoline derivatives (P1–7) against hCA I and II (P6), and AChE (P5)
enzyme. AChE: acetylcholinesterase; hCA I and II: human carbonic anhydrase isoenzymes I and II
novel pyrazoline derivatives (P1–7) showed potent inhibition against
hCA II, but the compound of (Z)‐4‐(2‐(4‐bromophenyl)hydrazineyli-
dene)‐5‐phenyl‐2‐(2,4,6‐trichlorophenyl)‐2,4‐dihydro‐3H‐pyrazol‐3‐
one (P6) and (Z)‐4‐(2‐(4‐bromophenyl)hydrazineylidene)‐5‐phenyl,4‐
trifluoromethylphenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P5) showed a
significant inhibition profile against hCA II with Ki values of 16.1 2.8
and 22.1 3.7 nM, respectively (Table 1 and Figure 3).
chlorophenyl)‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P6), which showed ex-
cellent inhibition profile against AChE with Ki values of 48.15 9.45
and 52.65 15.38 nM, respectively (Figures 2 and 3).
CA enzymes have seven diverse enzyme families: the α‐, β‐, γ‐, δ‐,
ζ‐, ŋ and θ‐CA. α‐CA enzymes have usually scarcely dimer form and
monomer structures.[49] β‐CAs (in prokaryotes) are tetramers or
octamers, dimer forms, γ‐CAs (in archaea) are trimers, whereas the ζ
and δ‐CAs (in marine diatoms) are less well understood up to now.[50]
All human hCAs have belonged to the α‐class. So far, 16 isoenzymes
have been discovered.[51] All of CA classes are metalloenzymes, which
have Zn2+, Cd2+, or Fe2+ at their enzyme active sites.[52] Various
isozymes of CA have been defined as remedial purpose for different
diseases. Indeed, the plan of isoenzyme‐specific inhibitors is searched
for the development of improved treatments. CAIs were classified into
two principal groups, the metal complexing anions and the unsub-
stituted sulfonamides. Sulfonamide molecules are very momentous
CAIs and sulfamide compounds are effective organic drugs in medical
chemistry.[53] The therapy of AD centralized on AChE inhibitor
(AChEI) compounds, such as TAC, rivastigmine, galantamine, and
donepezil.[54] However, in 2013, it was determined that it caused liver
damage. Therefore, the most important side effect is liver damage
other than those mentioned above.[55] AD is defined as a complex
syndrome where diverse factors are accountable for its etiology like
Recently, the most prescribed ChEIs are donepezil, galantamine,
and rivastigmine. Galantamine and donepezil have brief‐rolling
reversible competitive inhibitors, whereas rivastigmine is actively
reacted by ChE.[46] However, rivastigmine has an equal affinity for
AChE enzyme and that is very momentous drug, which is prescribed
for the therapy of AD.[47] It showed well which phenothiazines were
observed to inhibit ChE, especially AChE.[48] This important enzyme
was very strongly inhibited by novel pyrazoline derivatives (P1–7;
Table 1 and Figure 4). These new molecules had Ki values ranging
from 48.2 9.45 to 84.1 17.2 nM for AChE. Additionally, TAC
compound, used as standard inhibitor, had Ki value of 89.5 11.1 nM
toward AChE. In this study, all the evaluated novel pyrazoline
derivatives (P1–7) showed potent inhibition against AChE enzyme,
but the compound of (Z)‐4‐(2‐(4‐bromophenyl)hydrazineylidene)‐5‐
phenyl,4‐trifluoromethylphenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P5)
and (Z)‐4‐(2‐(4‐bromophenyl)hydrazineylidene)‐5‐phenyl‐2‐(2,4,6‐tri-

TURKAN ET AL.
5 of 12
|
FIGURE 3 (a) IC50 values for novel
pyrazoline derivatives (P1–7) against hCA I
and II, and AChE enzyme. (b) Ki values for
novel pyrazoline derivatives (P1–7) against
hCA I and II and AChE enzyme. AChE:
acetylcholinesterase; hCA I and II: human
carbonic anhydrase isoenzymes I and II
β‐amyloid aggregation, low levels of ACh, and tau protein aggrega-
tion.[56] AChEIs are used in the treatment of multiple neuromuscular
diseases, in addition, were searched for therapy of AD. AChE
accelerates the hydrolysis and involves the regulation of ACh.[57]
Anticonvulsant drugs such as topiramate AZA and zonisamide
were recorded CA inhibitors.[58] CA enzymes are classical which are
partition coefficient, percent human oral absorption, and solvent
accessible surface area (SASA) were screened using QikProp module.
The properties were analyzed through ADME (absorption, distribu-
tion, metabolism, and excretion) risk screening study and Lipinski's
rule of five. The data have been presented in Table 2. Compound
should meet Lipinski's rule of five including MW < 500 kDa, donor
HB < 5, accept HB < 10, and logPw/o < 5 to possess drug‐like
characteristics[61] and also it should meet SASA and percent human
oral absorption values. As shown in Table 2, all synthesized
compounds were consistent with almost all these rules. Lipophilicity
(logPw/o) value of all synthesized compounds was in range from 3.54
to 6.86. Molecular weight and percent oral absorption of all
synthesized compound are within the range, 343.18–522.62 and
58.32–100, respectively. The compounds were found with optimum
number of hydrogen bond donors (0–1) and acceptors (4.5–6.5).
SASA value in Å2 was from 618.24 to 805.58, which were within the
range.
inhibited by different compounds with
a sulfonamide‐based
(SO2NH2) zinc‐binding group or their bioisosteres such as sulfamides
and sulfamates. As CA II enzymes are the most physiologically
plentiful isoforms, which they were mostly recorded as the
predominant off‐purpose isozymes of which inhibitions were
eschewed.[59] Recently, multiple kinds of compounds have been
searched as nonclassical CAIs, including polyamines, coumarins,
phenols, carboxylic acids, fullerenes, and their derivatives.[60]
|
2.3
In silico studies
The primary binding site on receptors is often known from the
structure of a cocrystallized complex. In some instances, however,
The physicochemical properties of the synthesized compounds such
as molecular weight, number of hydrogen bond acceptor and donor,

6 of 12
TURKAN ET AL.
|
FIGURE 4 Two‐dimensional receptor–ligand interaction diagram; (a) P6‐hCA I, (b) P6‐hCA II, (c) P5‐AChE, (d) AZA‐hCA I, (e) AZA‐hCA II,
and (f) TAC‐AChE. AChE: acetylcholinesterase; hCA: human carbonic anhydrase
the location of a binding site for protein–ligand or protein–protein
interactions is not exactly known even though the protein structures
are available. So, we identified likely binding sites of the receptors
with SiteScore and Dscore obtained from SiteMap module. We found
three binding sites for hCA I and II receptors and four binding sites
for AChE receptor. The site with highest SiteScore and Dscore was
considered and chosen as druggable site.
molecular docking simulation studies of the synthesized compounds
using Glide docking and induced fit docking (IFD) methodologies. All
receptors to be used for docking studies were chosen due to their
low resolution. Briefly, all ligands were first docked on the binding
sites of the receptors using the Glide docking methodologies.
Docking and Glide scores of predicted best binding pose of ligands
and standard inhibitors were shown in Table 3. P5, P3 and P2
compounds exhibited higher negative Glide score than other
pyrazoline derivatives docked onto hCA I, hCA II, and AChE,
To identify the binding mechanism of the synthesized pyrazoline
derivatives against hCA I, hCA II, and AChE enzymes. We carried out
TABLE 2 Predicted physicochemical data of the pyrazoline derivatives
Compound
MW
DonorHB
AccptHB
4.5
LogPw/o
3.54
5.33
6.49
5.87
6.86
6.74
3.97
Percent oral absorption
SASA
P1
P2
P3
P4
P5
P6
P7
343.18
419.28
447.33
447.33
487.28
522.62
509.28
1
0
0
0
0
0
0
100
100
100
100
100
100
58.32
618.24
684.61
803.62
731.89
805.58
739.85
743.98
4.5
4.5
4.5
4.5
4.5
6.5
Note: AccptHB: H‐bond acceptors; DonorHB: H‐bond donor; logPw/o: predicted octanol/water partition coefficient; percent oral absorption: predicted
human oral absorption on 0–100% scale; MW: molecular weight; SASA: total solvent accessible surface area.

TURKAN ET AL.
7 of 12
|
respectively. According to in vitro inhibition studies, all pyrazoline
derivatives exhibited potent inhibitory activities against hCA I, hCA
II, and AChE enzyme as in docking studies. After Glide docking
experiments, the best active inhibitor‐compounds for each receptor
were selected for IFD methodology and the compounds were docked
onto the binding sites of the receptors. Two‐dimensional ligand
interaction diagram of best‐posed compounds and reference inhibi-
tors were presented in Figure 4. The compounds formed some
important interactions with binding site residues of receptors. Two‐
dimensional interaction diagrams have shown that the selected
compounds and the reference inhibitors interacted with and are
surrounded by similar residues of receptors.
Therefore, the synthesized pyrazoline derivatives can be considered
as drug‐like molecules. to illustrate theoretically inhibiting mechanism
of potential compounds P1–7, further Glide docking and IFD studies
were performed on hCA I, hCA II, and AChE receptors. In Glide
docking studies, we detected that all compounds exhibited high
affinities on three receptors compared with standard inhibitors for the
receptors. Glide docking suggested that all ligands were active
inhibitor as in the in vitro experiment results. After these findings,
we focused on inhibiting mechanism of most active compounds
determined from the in vitro experiments and observed that best‐
scored poses of the compounds well located in detected binding sites
of the receptors. On the basis of molecular docking analyses, we
detected that Trp5 residue of hCA I, and Zn atom of hCA II are key
amino acid and element for inhibition of these enzymes.
The lowest energy poses for the selected compounds on the
binding site of hCA I, hCA II, and AChE were presented in
Figure 5. The P6 compound well located to the binding sites of
hCA I and II as seen in Figure 5a,b. But P6 compound was not
H‐bonded with any residues in the binding site of hCA I. However,
it formed π–π stacking with the side‐chain atoms of Trp5 and
His64 residues. The compound similarly did not form any
hydrogen bond interactions with the binding site of hCA II. The
compound formed π–π stacking with the side‐chain atoms of Trp5
|
3
CONCLUSION
We synthesized novel 3H‐pyrazol‐3‐one derivatives and their
structures were determined by spectroscopic methods. All of the
synthesized compounds effectively inhibited some metabolic en-
zymes such as AChE, CA I and II, at the nanomolar levels. As we
considered above, novel pyrazoline derivatives (P1–7) can be good
inhibitors, for CAIs and AChEIs.
and Phe131 residues. Moreover, the compound formed
π
interaction between ligands and Zn atom. Of the most active
compound, P5 well located to the binding sites of AChE. The
compound exhibited one hydrogen bond interaction with Asn429
residue of the receptor as seen Figure 5c.
Not only is active against a target but also possess adequate
ADME properties is required for a molecule to be considered as a
drug. Hence, ADME properties are important in drug discovery and
development studies. All the synthesized compounds passed most of
the ADME test. Of the most active compound, P5 and P6 slightly
exceeded MW and logPw/o values. But, all compounds meet optimum
parameters for drug‐likeness according to Qik‐prop predictions.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All the used chemical materials were purchased from Merck (Darmstadt,
Germany) and Sigma‐Aldrich (Steinheim, Germany) companies. The
TABLE 3 Glide and IFD scores (kcal/mol) of the pyrazoline derivatives in the catalytic sites of hCA I, hCA II, and AChE
hCA I
hCA II
AChE
Glide XP
IFD
Glide XP
IFD
Glide XP
IFD
Docking
score
Glide
score
Docking
score
Glide
score
Docking
score
Glide
score
Compounds
Glide score
Glide score
Glide score
P1
−3.251
−4.693
−4.415
−5.201
−5.385
−3.792
−4.172
−4.447
–
−4.157
−4.762
−4.474
−5.258
−5.442
−3.934
−4.432
−5.787
–
–
−3.183
−3.519
−3.558
−4.343
−3.231
−3.701
−3.683
−6.982
–
−4.088
−4.821
−4.950
−4.400
−4.644
−4.617
−4.297
−8.417
–
–
−7.909
−9.915
−8.802
−8.776
−8.076
−7.906
−8.927
–
−8.815
−9.985
−8.862
−8.803
−8.133
−8.048
−9.186
–
–
P2
–
–
–
P3
–
–
–
P4
–
–
–
P5
–
–
−4.679
P6
−5.029
−7.034
–
–
–
–
P7
–
–
–
–
–
–
AZAa
TACb
−10.581
10.582
Note: AChE: acetylcholinesterase; AZA: acetazolamide; hCA: human carbonic anhydrase; TAC: tacrine.
aAZA was used as a standard inhibitor for hCA I and II.
bTAC was used as a standard inhibitor for AChE enzyme.

8 of 12
TURKAN ET AL.
|
FIGURE 5 Best‐scored docking pose of (a) P6, (b) P6, and (c) P5 compounds inside catalytic side of the hCA I, hCA II, and AChE receptors,
respectively. Key amino acid and element were determined in the 3D positions in active sites. Ligands are represented as white bool‐stick,
interacted residues are represented as gray bool‐stick. hCA I, hCA II, and AChE receptors are represented as green, pink and blue cartoon,
respectively. AChE: acetylcholinesterase; hCA: human carbonic anhydrase
structures of obtained compounds were determined by 1H (400 MHz),
13C (100 MHz), and 19F (500 MHz) NMR spectra on a Bruker DRX‐400
high performance digital FT‐NMR spectrometer from Japan. The infrared
spectra were recorded on a PerkinElmer Precisely Spectrum one
spectrometer using pressed disc in the range of 4000–450 cm−1 at
Mus Alparslan University Scientific and Technological Research Central.
The mass spectrum was measured on Thermo Fisher Scientific TSQ‐
Quantum Access LC/MS spectrometers from Japan. Melting points of
these compounds were determined on an Electrothermal Gallenkamp
apparatus and are uncorrected at Mus Alparslan University Scientific and
Technological Research Central. The obtained compounds were done
checking the purity and follow up of the reactions tested in each step by
thin layer chromatography (TLC) SiO2 using a DC Alufolien Kieselgel 60F
254 Merck. These compounds were visualized by Camag TLC devices.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
used for coupling reaction with ethyl benzoylacetate (0.163 g,
1 mmol). The ethyl benzoylacetate was taken in 10 ml methanol.
The solution was added dropwise to the solution of diazonnium salt
over a period 30 min with continuous stirring below 0°C. The
reaction was allowed to cool to ambient temperature. The mixture
was stirred 12 h. The product was washed with water. It was filtered
and dried. Then, it was crystallized from acetone. Yield: 84%. Mp.
136–138°C, FT‐IR (ν, cm−1): 3125, 3065, 2945, 1721, 1670, 1604,
1492, 1444, 1332. 1H‐NMR (400 MHz, DMSO‐d6) δ (ppm): 8.3
(s, 1 H, –NH), 8.0–6.6 (m, 9 H, Ar–H), 3.9 (q, J = 9.6 Hz, 2H, –CH2), 1.2
(t, J = 5.8 Hz, 3H, –CH3). 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm):
191.54, 164.4, 150.7, 143.5, 141.1, 136.2, 132.1, 129.5, 128.7, 128.3,
127.1, 120.6, 110.1, 59.7, 13.4.
|
4.1.2
General procedure for the synthesis of novel
pyrazoline derivatives
Ethyl 2‐((4‐bromophenyl)diazenyl)‐3‐oxo‐phenylpropano-
ate (1)
The starting material 1 (0.5 mmol) and sulfuric acid (0.05 mmol) were
dissolved in ethanol (10 mL). The appropriate hydrazines (0.5 mmol)
were added in the prepared solution and then, it was refluxed for 6 h.
The reaction process was followed by TLC using ethyl acetate/
n‐hexane (1:5) as solvent eluent system. After completion reaction,
the mixture was allowed to cool ambient temperature. The
precipitated product was filtered. The products were crystallized
from toluene.
4‐Bromo aniline (0.172 g, 1 mmol) was dissolved in concentrated
sulfuric acid (1 mmol) and NaNO2 (0.2 g, 3 mmol) was dissolved in
20 ml water. These solutions were kept in ice bath for 10 min below
0°C and then NaNO2 solution was added dropwise to the solution of
4‐bromo aniline over a period of 30 min with constant stirring at 0°C.
The diazonium salt of 4‐bromo aniline generated was immediately

TURKAN ET AL.
9 of 12
|
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐5‐phenyl‐2,4‐dihy-
dro‐3H‐pyrazol‐3‐one (P1)
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐5‐phenyl‐2‐(2,4,6‐
trichlorophenyl)‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P6)
P1 was synthesized according to the general procedure. Yield: 64%.
Mp. 198–200°C; FT‐IR (ν, cm−1): 3184, 3125, 3065, 1670, 1600,
1492–1444, 1332; (+)ESI‐HRMS m/z: calculated for [C15H11BrN4O+H+]:
344.1839; observed 344.1846. 1H‐NMR (400 MHz, DMSO‐d6)
δ (ppm): 8.3 (s, 1H, –NH), 8.0‒6.6 (m, 9H, Ar–H). 13C‐NMR (100 MHz,
DMSO‐d6) δ (ppm): 163.6, 159.6, 157.3, 141.3, 135.5, 132.2, 130.5,
129.8, 128.5, 127.5, 122.2.
P6 was synthesized according to the general procedure. Yield: 40%.
Mp. 232‒234°C; FT‐IR (ν, cm−1): 3124, 3065, 1662, 1604, 1498–
1443, 1317; (+)ESI‐HRMS m/z: calculated for [C21H12BrCl3N4O+H+]:
523.6215; observed 523.6224; 1H‐NMR (400 MHz, DMSO‐d6)
δ
(ppm): 8.0 (s, 1H, –NH), 7.9‐6.5 (m, 11H, Ar–H); 13C‐NMR
(100 MHz, DMSO‐d6) δ (ppm): 160.2, 156.6, 153.4, 146.3, 145.6,
141.6, 141.2, 137.9, 137.9, 134.7, 132.0, 130.8, 130.2, 129.6, 129.2,
128.9, 127.8, 121.7, 120.4, 119.5.
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐2,5‐diphenyl‐2,4‐
dihydro‐3H‐pyrazol‐3‐one (P2)
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐5‐phenyl,2,4‐dini-
trophenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P7)
P2 was synthesized according to the general procedure. Yield: 70%.
Mp. 212–214°C; FT‐IR (ν, cm−1): 3226, 3080, 1666, 1610, 1496–
1442, 1315; (+)ESI‐HRMS m/z: calculated for [C21H15BrN4O+H+]:
420.2775; observed 420.2786; 1H‐NMR (400 MHz, DMSO‐d6)
δ (ppm): 8.3 (s, 1H, –NH), 7.9–6.5 (m, 14 H, Ar–H); 13C‐NMR
(100 MHz, DMSO‐d6) δ (ppm): 161.5, 155.2, 152.8, 143.5, 136.5,
131.3, 130.5, 129.6, 129.0, 128.5, 121.6, 119.7.
P7 was synthesized according to the general procedure. Yield: 62%.
Mp. 219–221°C; FT‐IR (ν, cm−1): 3155, 3066, 1664, 1608, 1497–
1443, 1321; (+)ESI‐HRMS m/z: calculated for [C21H13BrN6O5+H+]:
510.2803; observed 510.2804; 1H‐NMR (400 MHz, DMSO‐d6)
δ (ppm): 8.1 (s, 1H, –NH), 8.0–6.5 (m, 12H, Ar–H); 13C‐NMR
(100 MHz, DMSO‐d6) δ (ppm): 162.5, 156.3, 152.6, 144.1, 141.3,
136.3, 131.8, 130.6, 130.3, 129.8, 129.0, 128.5, 127.5, 126.5, 125.8,
124.7, 124.5, 124.1, 123.1, 122.4, 118.8.
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐5‐phenyl,2,5‐di-
methylphenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P3)
|
P3 was synthesized according to the general procedure. Yield: 56%.
Mp. 216–218°C; FT‐IR (ν, cm−1): 3157, 3082, 1672, 1610, 1498–
1442, 1321; (+)ESI‐HRMS m/z: calculated for [C23H19BrN4O+H+]:
448.3381; observed 448.3386; 1H‐NMR (400 MHz, DMSO‐d6)
δ (ppm): 8.1 (s, 1H, –NH), 7.9–6.6 (m, 12H, Ar–H), 2.3 (s, 3H, CH3),
1.5 (s, 3H, CH3); 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm): 156.3,
152.7, 143.2, 142.3, 139.0, 135.4, 135.1, 131.7, 130.9, 130.5, 129.9,
129.2, 128.2, 127.5, 126.9, 125.1, 124.4, 122.4, 118.4, 21.5, 16.6.
4.2
Biochemical studies
|
4.2.1
hCA isoenzyme purification and inhibition
studies
Both hCA isoenzymes from human erythrocytes were purified via a
simple single‐step by method sepharose‐4B‐L‐tyrosine‐sulfanilamide
affinity gel chromatography.[62] For this purpose, the human erythro-
cyte samples were centrifuged at 13,000 rpm for 25 min. Then, the
solution was filtered to remove precipitate. Both hCA isoenzymes
were isolated from the serum, which its pH was adjusted to 8.7 by
adding solid Tris.[63] The affinity column was equilibrated by buffer
solution (25 mM Tris‐HCl/0.1 M Na2SO4) at the pH 8.7. The serum was
loaded to affinity gel and washed with buffer solution (25 mM Tris‐
HCl/22 mM Na2SO4 at the pH 8.7. The hCA I isoenzyme was eluted by
buffer solution (1.0 M NaCl/0.25 M sodium phosphate at the pH
6.3).[64] On the other hand, hCA II isoenzyme was eluted by another
buffer solution (0.1 M CH3COONa/0.5 M NaClO4 at the pH 5.6). Both
isoenzymes were taken from the column in fractions of 2 ml. All works
were realized at 4°C.[65] The hCA isoenzymes activity were measured
by following the change at absorbance specific (348 nm) of
p‐nitrophenyl acetate (PNA) to p‐nitrophenolate ion over a period of
3 min at room temperature (25°C) using a spectrophotometer (UV‐Vis
spectrophotometer; Thermo Fisher Scientific) according to the method
of Verpoorte et al.[66] There was 0.4 ml of 0.05 M Tris‐SO4 buffer (pH
7.4), 03 ml of 3 mM PNA, 0.2 ml of H2O, and 0.1 ml of enzyme solution
in a test tube content of this reaction.[67]
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐5‐phenyl,3,4‐di-
methylphenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P4)
P4 was synthesized according to the general procedure. Yield: 58%.
Mp. 222–224°C; FT‐IR (ν, cm−1): 3145, 3065, 1662, 1610, 1496–1443,
1318; (+)ESI‐HRMS m/z: calculated for [C23H19BrN4O+H+]:
448.3386; observed 448.3387; 1H‐NMR (400 MHz, DMSO‐d6)
δ (ppm): 8.1 (s, 1H, –NH), 7.9–6.5 (m, 12H, Ar–H) 2.4 (s, 3H, CH3),
1.6 (s, 3H, CH3); 13C‐NMR (100 MHz, DMSO‐d6) δ (ppm): 155.3, 153.1,
145.9, 143.5, 141.6, 140.7, 135.4, 132.4, 132.0, 130.8, 130.2, 130.0,
129.6, 129.2, 127.8, 126.1, 125.1, 124.0, 123.3, 119.4, 23.4, 16.3.
(Z)‐4‐(2‐(4‐Bromophenyl)hydrazineylidene)‐5‐phenyl,4‐tri-
fluoromethylphenyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (P5)
P5 was synthesized according to the general procedure. Yield: 65%.
Mp. 228–230°C; FT‐IR (ν, cm−1): 3184, 3064, 1664, 1598, 1497–
1444, 1321; (+)ESI‐HRMS m/z: calculated for [C22H14BrF3N4O+H+]:
488.2283; observed 488.2292; 1H‐NMR (400 MHz, DMSO‐d6)
δ
(ppm): 8.1 (s, 1H, –NH), 7.8‐6.5 (m, 13H, Ar–H); 13C‐NMR
(100 MHz, DMSO‐d6) δ (ppm): 156.6, 153.7, 146.9, 142.9, 137.2,
136.5, 135.4, 132.0, 131.4, 130.7, 130.2, 129.6, 129.2, 128.5, 126.8,
125.8, 124.7, 124.4, 123.7, 122.7, 122.1, 118.1. 19F NMR (470 MHz,
DMSO‐d6 ppm) δ −62.55.
|
4.2.2
AChE inhibition study
The inhibitory effects of novel pyrazoline derivatives (P1–7) on AChE
activity were performed according to the spectrophotometric

10 of 12
TURKAN ET AL.
|
method of Ellman et al.[68] Acetylthiocholine iodide (AChI) substrate
was utilized for the inhibition act and enzymatic reaction. 5,5′‐Dithio‐
bis(2‐nitro‐benzoic) acid (DTNB) compound was utilized in this study
for the measurement of the AChE activity.[69] Briefly, 750 μl of
sample solution dissolved in deionized water at different concentra-
tions with 100 μl of Tris/HCl buffer (1.0 M, pH 8.0), and 50 μl of AChE
solution were mixed and incubated for 64 min at 21°C.[70] The
reaction was initiated by the addition of 50 μl of AChI. Then, 50 μl of
DTNB (0.5 mM) was added. The hydrolysis of these substrates was
monitored spectrophotometrically by formation of the yellow 5‐thio‐
2‐nitrobenzoate anion as the result of the reaction of thiocholine
with DTNB, released by enzymatic hydrolysis of AChI, with an
minimized structure was the best structure to utilize for molecular
docking. After protein preparation, top‐ranked potential protein binding
sites were identified to determine most suitable binding site of proteins
using SiteMap module in Maestro 11.4.[74]
|
4.3.4
Molecular docking studies
Ligand docking was performed for the prediction of best poses and
binding energies of ligands at binding sites identified by SiteMap
on the receptor. First of all, all ligands were docked on the
receptors using Glide docking module in Maestro 11.4. Briefly, grid
box was generated around the selected residues at the binding site
using the receptor grid generation platform. Then rigid receptor‐
docking simulations were performed using the standard precision
and extra precision options in Maestro 11.4. Following Glide
docking, we performed IFD using IFD module in Maestro 11.4. In
the IFD experiment, three ligands, which exhibited the most
effective inhibition in vitro experiment, were docked on the
receptors. Briefly, centroid of the residues was generated around
the selected residues at the binding site. After that, side chains
were automatically trimmed based on B‐factor; closest residues to
the ligand were refined within 3.4 Å of ligand pose in prime
refinement. Then flexible receptor‐docking simulations were
performed in Maestro 11.4.[74]
absorption maximum at
a
wavelength of 412 nm.[71] For the
computation of Ki values of these compounds, three diverse novel
pyrazoline derivatives (P1–7) concentrations were utilized. One
AChE enzyme unit is the amount of enzyme that hydrolyzes 1.0 mol
of AChI to choline and acetate per minute at pH 8.0 at 37°C,[72] and
Lineweaver‐Burk curves were drawn.[73]
|
4.3
In silico studies
|
4.3.1
Ligand preparation
Molecular docking studies were performed with Small Drug
Discovery Suites package (Schrödinger 2017‐2, LLC). Two‐dimen-
sional structures of the synthesized compounds were sketched and
then converted to 3D structures using LigPrep module in Maestro
11.4. To prepare ligands for docking process, the ligands were set to
the physiological pH and using OPLS‐2005 force field performed
energy minimization. The Epik option was used for keeping ligand in
the correct protonation state.[74]
ACKNOWLEDGEMENTS
This study was supported by Scientific Research Fund of Iğdır
University. Project Number: 2018‐SBE‐A01. A certain part of this
study was carried out in Igdır University Research Laboratory
Practice and Research Center.
|
4.3.2
Prediction of ADME properties
CONFLICT OF INTERESTS
To determine pharmacokinetic properties of all synthesized compounds,
ADME test was performed with QikProp module in Maestro 11.4. The
test provides information about ADME of the synthesized compounds
in comparison to a particular molecule's properties with those of 95% of
known drugs. Prepared ligands were used in the calculation of QikProp.
The drug‐likeness of the compounds were determined with Lipinski's
rule of five (mol_MW, logPo/w, donorHB, and accptHB).[74]
The authors declare that there are no conflict of interests.
ORCID
Fikret Turkan
http://orcid.org/0000-0002-0538-3157
http://orcid.org/0000-0002-3171-0633
Parham Taslimi
REFERENCES
|
4.3.3
Protein processing and binding site
identification
[1] A. Lévai, A. M. S. Silva, J. A. S. Cavaleiro, I. Alkorta, J. Elguero, J. Jekő,
Eur. J. Org. Chem. 2006, 12, 2825.
The 3D crystal structures of hCA I, hCA II, and AChE enzymes were
obtained from RCSB Protein Data Bank (PDB ID: 2FW4, 5AML, and
4TVK, respectively). The 3D crystal structure was repaired and
prepared via protein preparation wizard in Maestro 11.4. First of all,
all water molecules were removed from the crystal structure. Bond
orders and charges were assigned and then all missing hydrogen atoms
were added to protein structure. Amino acids were ionized by setting
physiological pH with the help of Propka software. Finally, restrained
minimization step has also been performed using OPLC force field. This
[2] A. Saikia, M. G. Barthakur, M. Borthakur, C. J. Saikia, U. Bora, R. C.
Boruah, Tetrahedron Lett. 2006, 47, 43.
[3] S. D. Joshi, S. R. Dixit, M. N. Kirankumar, T. M. Aminabhavi, K. V. S. N.
Raju, R. Narayan, K. S. Yang, Eur. J. Org. Chem. 2016, 107, 133.
[4] P. Jeschke, Pest Manage. Sci. 2016, 72, 433.
[5] B. Varghese, S. N. Al‐Busafi, F. O. Suliman, S. M. Z. Al‐Kindy, RSC Adv.
2017, 7, 46999.
[6] A. Çetin, İ. Bildirici, J. Saudi Chem. Soc. 2018, 22(3), 279.
[7] F. Turkan, A. Cetin, P. Taslimi, M. Karaman, İ. Gulçin, Bioorg. Chem.
2019, 86, 420.

TURKAN ET AL.
11 of 12
|
[8] M. S. Shah, S. U. Khan, S. A. Ejaz, S. Afridi, S. U. F. Rizvi, M. Najam‐ul‐
Haq, J. Iqbal, Biochem. Biophys. Res. Commun. 2017, 482, 615.
[9] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, L. Zaprutko, A. Gzella,
R. Lesyk, Eur. J. Org. Chem. 2009, 44, 1396.
[43] W. Zhao, Z. H. Shen, J. H. Xing, G. Yang, T. M. Xu, W. L. Peng, X. H.
Liu, Chem. Pap. 2017, 71(5), 921.
[44] P. Taslimi, İ. Gulci
[45] M. U. Koçyigit, P. Taslimi, F. Gurses, S. Soylu, S. Durna Dastan,
İ. Gulcin, J. Biochem. Mol. Toxicol. 2018, 32, e22031.
[46] Y. Sarı, A. Aktaş, P. Taslimi, Y. Gök, İ. Gulcin, J. Biochem. Mol. Toxicol.
2018, 32(1), e22009.
[47] N. Bulut, U. M. Koçyiğit, I. H. Gecibesler, T. Dastan, H. Karci, P.
Taslimi, S. Durna Dastan, İ. Gulcin, A. Cetin, J. Biochem. Mol. Toxicol.
2018, 32(1), e22006.
̧ n İ, J. Food Biochem. 2018, 42(3), e12516.
[10] E. Nassar, J. Am. Sci. 2010, 6, 463.
̧
[11] M. A. Berghot, E. B. Moawad, Eur. J. Pharm. Sci. 2010, 20(2), 173.
[12] N. D. Amnerkar, K. P. Bhusari, Eur. J. Med. Chem. 2010, 45(1), 149.
[13] A. Sarkar, S. Rakshit, S. C. Bhattacharya, J. Photochem. Photobiol.
2017, A 343, 77.
̧
̧
[14] N. Kahriman, Z. Haşimoğlu, V. Serdaroğlu, F. Ş. Beriş, B. Barut,
N. Yaylı, Arch. Pharm. 2017, 350, 1600285.
[48] G. Gondolova, P. Taslimi, A. Medjidov, V. Farzaliyev, A. Sujayev, H.
Mansura, O. Sahin, B. Yalçın, F. Turkan, I. Gulçin, J. Biochem. Mol.
Toxicol. 2018, 32(9), e22197.
[15] R. Sadashiva, D. Naral, J. Kudva, N. Shivalingegowda, N. K. Lokanath,
K. J. Pampa, Med. Chem. 2017, 26, 1213.
[16] H. Bao, Q. Zhang, Z. Zhu, H. Xu, F. Ding, M. Wang, Z. Yan, Sci. Rep.
2017, 7(1), 9153.
[49] A. Cetin, F. Turkan, P. Taslimi, İ. Gulci
33, e22261.
[50] P. Taslimi, E. Sujayev, F. Turkan, E. Garibov, Z. Huyut, F. Farzaliyev,
S. Mamedova, İ. Gulcin, J. Biochem. Mol. Toxicol. 2018, 32(2), e22019.
̧ n, J. Biochem. Mol. Toxicol. 2018,
[17] N. S. Shringare, V. H. Chavan, S. P. Bhale, B. S. Dongare, B. Y. Mule, D.
N. Kolekar, P. B. Bandgar, Croat. Chem. Acta 2018, 91(3), 357.
[18] I. Khan, M. A. Tantray, M. S. Alam, H. Hamid, Eur. J. Med. Chem. 2017,
125, 464.
̧
[51] İ. Gülçin, A. Scozzafava, C. T. Supuran, H. Akıncıoğlu, Z. Koksal, F.
Turkan, S. Alwasel, J. Enzyme Inhib. Med. Chem. 2016, 31(6), 1095.
[52] İ. Gülçin, A. Scozzafava, C. T. Supuran, Z. Koksal, F. Turkan,
S. Çetinkaya, Z. Bingöl, Z. Huyut, S. H. Alwasel, J. Enzyme Inhib.
Med. Chem. 2016, 31(6), 1698.
[19] M. Arnost, A. Pierce, E. ter Haar, D. Lauffer, J. Madden, K. Tanner, J.
Green, Bioorg. Med. Chem. Lett. 2010, 20(5), 1661.
[20] X. H. Liu, P. Cui, B. A. Song, P. S. Bhadury, H. L. Zhu, S. F. Wang,
Bioorg. Med. Chem. 2008, 16, 4075.
[53] F. Turkan, Z. Huyut, P. Taslimi, İ. Gulci
2018, 32(3), e22041.
[54] P. Taslimi, C. Çağlayan, F. Farzaliyev, O. Nabiyev, A. Sujayev, F. Turkan,
R. Kaya, İ. Gulcin, J. Biochem. Mol. Toxicol. 2018, 32(4), e22042.
[55] P. Taslimi, S. Osmanova, C. Çağlayan, F. Turkan, S. Sardarova,
V. Farzaliyev, A. Sujayev, N. Sadeghian, İ. Gulcin, J. Biochem. Mol.
Toxicol. 2018, 32(9), e22191.
̧ n, J. Biochem. Mol. Toxicol.
[21] B. B. Lorenzetti, S. H. Ferreira, Eur. J. Pharmacol. 1985, 114, 375.
[22] G. Dowling, E. Malone, J. Pharm. Biomed. Anal. 2011, 56, 359.
[23] I. Başar, K. Bircan, C. Taşar, A. Ergen, F. Cakmak, D. Remzi, Int. Urol.
Nephrol. 1991, 23(3), 227.
̧
[24] F. D. Hart, P. L. Boardman, Br. Med. J. 1964, 1(5397), 1553.
[25] R. L. Jayaraj, K. Tamilselvam, T. Manivasagam, N. Elangovan, J. Mol.
Neurosci. 2013, 51, 863.
̧
[56] İ. Gülçin, P. Taslimi, A. Aygün, N. Sadeghiana, E. Bastem, O. I.
Küfrevioglu, F. Türkan, F. Sen, Int. J. Biol. Macromol. 2018, 119, 741.
[57] T. Daştan, U. M. Koçyiğit, S. D. Daştan, C. Kiliçkaya, P. Taslimi, Ç.
[26] F. Chimenti, B. Bizzarri, F. Manna, A. Bolasco, D. Secci, P. Chimenti,
M. I. Brenciaglia, Bioorg. Med. Chem. Lett. 2005, 15(3), 603.
[27] P. Taslimi, İ. Gulcin, B. Ozgeris, S. Goksu, F. Tumer, S. H. Alwasel, C. T.
Supuran, J. Enzyme Inhib. Med. Chem. 2016, 31(1), 152.
[28] H. İ. Gül, K. Kucukoglu, C. Yamali, S. Bilginer, H. Yuca, İ. Ozturk, P.
Taslimi, İ. Gülçin, C. T. Supuran, J. Enzyme Inhib. Med. Chem. 2016,
31(4), 568.
Özge, M. Koparir, A. Çetin, C. Orek, İ. Gulci
2017, 31(11), e21971.
[58] C. Yamali, H. İ. Gül, A. Ece, P. Taslimi, İ. Gulci
2018, 91, 854.
̧
n, J. Biochem. Mol. Toxicol.
̧
n, Chem. Biol. Drug Des.
[59] F. Turkan, A. Cetin, P. Taslimi, I. Gulçin, Arch. Pharm. 2018, 351(10),
[29] A. Behcet, T. Çağlılar, D. Barut Celepci, A. Aktaş, P. Taslimi, Y. Gök,
1800200.
M. Aygün, R. Kaya, İ. Gulci
̧
n, J. Mol. Struct. 2018, 1170, 160.
[60] S. Burmaoglu, A. O. Yilmaz, P. Taslimi, O. Algul, D. Kılıç, İ. Gulci
̧ n,
̈
̈
̈
Arch. Pharm. 2018, 351, e1700314.
[30] P. Taslimi, İ. Gulci̧ n, N. Oztasķ ın, Y. Çetinkaya, S. Goksu, S. H.
Alwasel, C. T. Supuran, J. Enzyme Inhib. Med. Chem. 2016, 31(4), 603.
[31] B. Ozgeriş, S. Göksu, L. Köse Polat, İ. Gülçin, R. E. Salmas, S. Durdagi,
F. Tümer, C. T. Supuran, Bioorg. Med. Chem. 2016, 24(10), 2318.
[32] M. Boztaş, Y. Çetinkaya, M. Topal, İ. Gülçin, A. Menzek, E. Şahin,
M. Tanc, C. T. Supuran, J. Med. Chem. 2015, 58(2), 640.
[61] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug
Deliv. Rev. 2001, 46(1‐3), 3.
[62] E. Garibov, P. Taslimi, A. Sujayev, Z. Bingöl, S. Çetinkaya, İ. Gulcin, S.
Beydemir, V. Farzaliyev, S. H. Alwasel, C. T. Supuran, J. Enzyme Inhib.
Med. Chem. 2016, 31, 1.
[33] A. Yıldırım, U. Atmaca, A. Keskin, M. Topal, M. Çelik, İ. Gülçin, C. T.
Supuran, Bioorg. Med. Chem. 2015, 23(10), 2598.
[63] K. Aksu, B. Özgeris, P. Taslimi, A. Naderi, İ. Gülçin, S. Göksu, Arch.
Pharm. 2016, 349, 944.
[34] M. Rezai, Ç. Bayrak, P. Taslimi, İ. Gulci
̧
n, A. Menzek, Turk. J. Chem.
[64] P. Taslimi, A. Sujayev, S. Mamedova, P. Kalın, İ. Gulcin, N. Sadeghian,
S. Beydemir, İ. Ö. Küfrevioglu, S. H. Alwasel, V. Farzaliyev,
S. Mamedov, J. Enzyme Inhib. Med. Chem. 2017, 32(1), 137.
[65] H. I. Gul, E. Mete, P. Taslimi, I. Gulcin, C. T. Supuran, J. Enzyme Inhib.
Med. Chem. 2017, 32, 189.
2018, 42(3), 808.
[35] Z. Huyut, S. Beydemir, İ. Gülçin, J. Enzyme Inhib. Med. Chem. 2016,
31(6), 1234.
[36] L. Polat Köse, İ. Gulci̧ n, A. C. Gören, J. Namiesnik, A. L. Martinez‐
̈
Ayala, S. Gorinstein, Ind. Crops Prod. 2015, 74, 712.
[37] D. Ozmen Ozgun, C. Yamali, H. İ. Gül, P. Taslimi, İ. Gülçin, T. Yanik,
C. T. Supuran, J. Enzyme Inhib. Med. Chem. 2016, 31(6), 1498.
[38] A. Sujayev, E. Garibov, P. Taslimi, İ. Gülçin, S. Gojayeva, V. Farzaliyev,
S. H. Alwasel, C. T. Supuran, J. Enzyme Inhib. Med. Chem. 2016,
31(6), 1531.
[66] J. A. Verpoorte, S. Mehta, J. T. Edsall, J. Bio.l Chem. 1967, 242, 4221.
[67] H. I. Gul, A. Demirtas, G. Ucar, P. Taslimi, İ. Gülçin, Lett. Drug Des.
Discov. 2017, 14, 573.
[68] G. L. Ellman, K. D. Courtney, V. Andres, R. M. Featherston, Biochem.
Pharmacol. 1961, 7, 88.
[69] Ç. Bayrak, P. Taslimi, İ. Gülçin, A. Menzek, Bioorg. Chem. 2017, 72, 359.
[39] B. Turan, K. Sendil, E. Sengul, M. S. Gultekin, P. Taslimi, İ. Gulcin, C. T.
Supuran, J. Enzyme Inhib. Med. Chem. 2016, 31(S1), 79.
[40] F. Ozbey, P. Taslimi, I. Gulcin, A. Maraş, S. Goksu, C. T. Supuran, J.
Enzyme Inhib. Med. Chem. 2016, 31, 79.
[70] A. Aktas, P. Taslimi, I. Gul
̈
ci̧ n, Y. Gok, Arch. Pharm. 2017, 350,
̈
e1700045.
[71] P. Taslimi, A. Sujayev, E. Garibov, N. Nazarov, Z. Huyut, S. H. Alwasel,
İ. Gülçin, J. Biochem. Mol. Toxicol. 2017, 31, e21897.
[72] N. Öztaskın, P. Taslimi, A. Maras, S. Göksu, İ. Gülçin, Bioorg. Chem. 2017,
74, 104.
[41] M. Işık, S. Beydemir, A. Yılmaz, M. E. Naldan, H. E. Aslan, İ. Gülçin,
Biomed. Pharmacother. 2017, 87, 561.
[42] H. Kurt, A. Çetin, S. Bozarı, J. Inst. Sci. Technol. 2018, 8(2), 223.
[73] H. Lineweaver, D. Burk, J. Am. Chem. Soc. 1934, 56, 658.

12 of 12
TURKAN ET AL.
|
[74] Schrödinger L. Small‐Molecule Drug Discovery Suite 2017, Decem-
ber 2018; https://www.schrodinger.com/sites/default/files/s3/mkt/
Documentation/2016‐4/docs/Documentation.htm#Maestro11_
Handbook.html
How to cite this article: Turkan F, Cetin A, Taslimi P,
Karaman HS, Gulçin İ. Synthesis, characterization, molecular
docking and biological activities of novel pyrazoline
derivatives. Arch Pharm Chem Life Sci. 2019;e1800359
https://doi.org/10.1002/ardp.201800359
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.