Synthesis, biological evaluation and molecular docking of novel pyrazole derivatives as potent carbonic anhydrase and acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.02.013

A series of substituted pyrazole compounds (1–8 and 9a, b) were synthesized and their structure was characterized by IR, NMR, and Mass analysis. These obtained novel pyrazole derivatives (1–8 and 9a, b) were emerged as effective inhibitors of the cytosoli

Full text of DOI:10.1016/j.bioorg.2019.02.013

Accepted Manuscript
Synthesis, Biological Evaluation and Molecular Docking of Novel Pyrazole
Derivatives as Potent Carbonic Anhydrase and Acetylcholinesterase Inhibitors
Fikret Turkan, Adnan Cetin, Parham Taslimi, Muhammet Karaman, İlhami
Gulçin
PII:
S0045-2068(18)31420-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.013
YBIOO 2788
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
5 December 2018
2 February 2019
4 February 2019
Please cite this article as: F. Turkan, A. Cetin, P. Taslimi, M. Karaman, I. Gulçin, Synthesis, Biological Evaluation
and Molecular Docking of Novel Pyrazole Derivatives as Potent Carbonic Anhydrase and Acetylcholinesterase
Inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.013
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Synthesis, Biological Evaluation and Molecular Docking of Novel Pyrazole
Derivatives as Potent Carbonic Anhydrase and Acetylcholinesterase Inhibitors
Fikret Turkana, Adnan Cetinb, Parham Taslimic*, Muhammet Karamand, İlhami Gulçinc
aHealth Services Vocational School, Igdır University, 76000, Igdır, Turkey
bDepartment of Science, Faculty of Education, Muş Alparslan University, 49250, Muş, Turkey
cDepartment of Chemistry, Faculty of Science, Atatürk University, 25240, Erzurum, Turkey
dDepartment of Molecular Biology and Genetics, Faculty of Arts and Science, Kilis 7 Aralik
University, 79000, Kilis, Turkey

ABSTRACT
A series of substituted pyrazole compounds (1-8 and 9a, b) were synthesized and their structure
was characterized by IR, NMR, and Mass analysis. These obtained novel pyrazole derivatives (1-
8 and 9a, b) were emerged as effective inhibitors of the cytosolic carbonic anhydrase I and II
isoforms (hCA I and II) and acetylcholinesterase (AChE) enzymes with Ki values in the range of
1.03±0.23-22.65±5.36 µM for hCA I, 1.82±0.30-27.94±4.74 µM for hCA II, and 48.94±9.63-
116.05±14.95 µM for AChE, respectively. Docking studies were performed for the most active
compounds, 2 and 5, and binding mode between the compounds and the receptors were
determined.
Keywords: Substituted pyrazole; acetylcholinesterase; carbonic anhydrase; enzyme inhibition
Running Head: Biological effects of substituted pyrazole derivatives
2

1. INTRODUCTION
Pyrazoles are important class of heterocyclic compounds, which have rapidly increased in
importance due to their wide range of properties. Their biologic activities, have guided
researchers towards the synthesis of substituted pyrazole derivatives using existing methods [1].
Substituted pyrazole compounds are significant target molecules for organic synthesis chemists in
terms of potential applications such as medicine [2], pharmacology [3], agriculture [4] and
material science [5]. Some known applications are antibacterial [6], antifungal [7], antibiotic [8],
pesticide [9], and sensors [10]. Moreover, some molecules having pyrazole structure exhibited
excellent properties such as antiviral [11], antioxidant [12], anti-inflammatory [13], antitumor
[14] and antidepressant [15] activities. On the other hand, pyrazole-containing compounds are
widely commercialized such as Tartrazine [16], Fomepizole [17], Ceftolozane [18], Celebrex
[19], Rimonabant [20] and Viagra [21]. Furthermore, substituted pyrazole molecules were used to
ligand as a catalyst in the cross coupling reaction [22] and also was used α-Helix mimetic as
inhibitors of protein-protein interactions [23]. The cyclocondensation of hydrazines with 1,3-
dicarbonyl compounds, α,β-unsaturated aldehydes ketones and 2,3-furandiones generally were
used for synthesis of the pyrazoles [6,24]. The aim of this study is to synthesize and characterize
novel substituted pyrazole-based molecules. The structure of the synthesized pyrazoles was
confirmed using spectroscopic analysis.
Dementia disease is a main health concern in old people worldwide, which can affect
multitude aspects of a functioning and person’s life. Indeed, there is no cure for most kinds of
dementia disease, but various drugs are utilized in its office [25,26]. The acetylcholinesterase
inhibitors (AChEI) were developed as an effect of the cholinergic assumption of cognitive
reduction [27]. Indeed, the effectiveness of these treatments has been investigated in a great
number of randomized controlled tests among global, functional, neuropsychiatric domains, and
cognitive [28]. AChEI compounds are demonstrated for the symptomatic treatment of mild-to-
moderate Alzheimer’s disease (AD). They inhibit AChE, which is accountable for the separation
of acetylcholine (ACh), a neurotransmitter molecule that associated with memory function [29,
30].
Two genetically distinguished human carbonic anhydrase (hCA) isoforms designated
hCA I, and II isoenzymes are present in RBC cells. Both isoforms exhibit 60% sequence
homology, several particular activities and several immunologic specificities [31]. While, CA II
3

isoform is expressed in an extensive diversity of tissues and cells, CA I isoform has a more
restricted cellular expression. Also, hCA II isoform can produce NO molecule, owing to the
structural similarity between nitrite and bicarbonate [32]. The hCA II isoform is a zinc ions
-
(Zn2+) and catalysis reversible interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 ).
It is found in platelets, vascular smooth muscle, and erythrocytes. Also, hCA II isoenzyme
contributes to vascular adjustment and could be a source of good hCA II-mediated generation of
-
nitric oxide (NO) from nitrite [33]. Since nitrite exhibits similar to sp2 geometry as HCO3 , it is
hypothesized that hCA II isoform acts as a nitrous anhydrase factor, wherein nitrite binds hCA II
-
active site and is dehydrated, analogously to HCO3 [34,35]. CA isoforms are zinc-containing
-
metallocatalists that perform the reversible hydration of CO2 to HCO3 and a H+. This simple
reaction is essential for many physiological mechanisms including electrolyte secretion,
respiration, pH regulation, bone resorption, tumorigenesis, biosynthetic reactions, and
-
calcification, which require HCO3 as a substrate. The hCA isoenzymes are interesting
therapeutic targets where their inhibition could be utilized to treat a range of disorders including
anemia, oxidative stress, glaucoma, edema, cancer, epilepsy, osteoporosis, obesity, and sterility
[36-39].
In this study, we have performed a facile synthesis of well-defined novel pyrazole
derivatives (1-8 and 9a, b) .We also investigated their inhibition potential towards hCA I, and II
isoenzymes as well as towards AChE inhibition.
2. RESULTS AND DISCUSSION
2.1. Chemistry
The starting material pyrazole-3,4-dicarboxylic acid (1) was previously synthesized by our
research group [40]. Pyrazole-3,4-dicarbonyl chloride was prepared via heating compound 1 with
excess thionyl chloride. The novel pyrazole derivatives; pyrazole-3,4-dicarboxyamide, pyrazole-
3,4-dicarbonitrile,
pyrazole-3,4-dicarboxylate,
pyrazolo[3,4-d]pyridazine-4,7-dione,
pyrazolo[3,4-d]pyridazine-4,7-diamine and furo[3,4-c]pyrazole-4,6-dione were prepared by
reactions of carboxylic acid and/or chloride with various O- and/or N-nucleophiles in Scheme1.
Scheme 1
All new compounds were confirmed by spectroscopic methods. Pyrazole-3,4-
dicarboxyamide 3 was synthesized through reaction of 2 and ammonia in xylene for 5 hours. Its
4

yield was 84%. The structure of 3 was clarified by 13C NMR spectroscopy in which characteristic
pyrazolo-carbodiamide (C=O) peaks were observed at δ 167.2 and 160.6 ppm. Pyrazolo-
carbonitrile 4 was obtained by treating pyrazolo-carbodiamide with a mixture of DMF/SOCl2 at
cold for 2 hours. The characteristic FT-IR signal of the nitrile group(s) bands in 4 were observed
13
at 2194, 2186 cm-1. Also, characteristic C-NMR peaks for 4 were seen at δ 114.8, 111.2 ppm.
The pyrazole-3,4-dicarboxylate 5 was easily synthesized using Schotten-Baumann method. The
structure of 5 was seen dicarbonyl carbon signals at δ 159.9 and 156.0 ppm in 13C-NMR
spectroscopy and the characteristic FT-IR absorption of the ester structure at 1721 cm-1 was
observed. Pyrazolo[4,3-d]pyridazine-4,7-dione 6 was obtained from cyclization reaction of 5 and
anhydrous hydrazine (Scheme 1). Groups of amine as a nucleophile were attacked to ester
groups. The related product was occurred at 62% yield. The structure of 6 was clarified 13C NMR
spectroscopy in which characteristic hydrazide (C=O) peaks were seen at δ 168.9 and 160.2 ppm
1
and -NH peaks were observed at δ 8.5 and 8.3 ppm in H NMR spectroscopy. Pyrazolo[4,3-
d]pyridazine-4,7-diamine 7 was synthesized from cyclization reaction of pyrazole-3,4-
1
dicarbonitrile 5 and anhydrous hydrazine. Compound 7 was clarified H NMR spectroscopies in
which characteristic amine peaks were observed at δ 4.4 and 4.1 ppm. The furo[3,4-c]pyrazole-
4,6-dione furo[3,4-c]pyrazole-4,6-dione 8 was obtained with dehydration of pyrazole-3,4-
dicarboxylic acid 1 and DMF/SOCl2 in cold medium. Characteristic 13C NMR signals of the -
C=O carbonyl groups in 8 were observed at δ 167.5 and 159.9 ppm. Also, yield of 8 was occurred
at 68% (see experimental). 9a,b were synthesized with refluxing of corresponding compound 8
and ammonia with ethyl amine as nucleophiles in ethanol for two days (Scheme 1). Theirs yields
13
were obtained at 65%. C-NMR spectrum revealed the signals of -C=O carbonyl groups in the
range of δ 167.9 and 161.2 ppm. All synthesized compounds 1-8 and 9a,b were confirmed by
spectroscopic methods which wasagreement with our previous findings and literature reports [6].
2.2. Biochemical Results
In this study, we obtained the effects of novel pyrazole compounds (1-8 and 9a, b) derivatives
against hCA I, hCA II, and AChE enzymes. Inhibition act of the CA isoforms has pharmacologic
applications in several fields, such as antiglaucoma, diuretics, antiobesity, anticonvulsant, and
anticancer agents/diagnostic tools, also, it can be emerging for designing anti-infective, i.e.,
antibacterial, antifungal, and antiprotozoan factors with a new process of action [41]. For a long
time it has been considered that the pharmacologic effects of CA activation or inhibition are
5

mostly due to effects on pH regulation in tissues or cells where the enzymes are present. We
report the inhibition effects of these derivatives on the activity of hCA I, hCA II AChE enzymes
under in vitro conditions. The following results are presented in Table 1:
Abnormal levels of CA I enzyme in the blood are used as a marker for hemolytic anemia
[42]. The slow cytosolic isoform hCA I was inhibited by the investigated novel pyrazole
compounds (1-8 and 9a, b) derivatives, with Ki values ranging between 1.03±0.23 and
22.65±5.36 nM. Furthermore, 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarbonyl
dichloride (2) and 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylic acid (1) which
demonstrated the most powerful hCA I isoenzyme inhibition properties with Ki values of
1.03±0.23 and 4.88±0.83 µM. In addition, it is known that the molecules, that had affinity against
CA isoenzymes. The standard and clinically used drug acetazolamide (AZA) demonstrated a Ki
value of 28.11±10.33 µM (Table 1). Thus, the investigated compounds showed better inhibitory
profiles compared to AZA, a clinically used CA inhibitor.
In addition, CA II is often associated with several diseases such as glaucoma,
osteoporosis, and renal tubular acidosis. The hCA II was also efficiently inhibited by the novel
pyrazole compounds (1-8 and 9a, b) derivatives investigated here. These compounds appeared to
strongly inhibit hCA II, with Ki values ranging from 1.82±0.30 nM to 27.94±4.74 µM. These
values are better than those of the clinically used drugs acetazolamide (Ki of 34.12±8.23 µM). All
the investigated these novel pyrazole compounds (1-8 and 9a, b) derivatives demonstrated
marked inhibition against hCA II, but the compounds of 1-(3,4-dimethylphenyl)-5-phenyl-1H-
pyrazole-3,4-dicarbonyl dichloride (2) and 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-
dicarboxylic acid (1) showed excellent inhibitions profile against cytosolic hCA II with Ki values
of 1.82±0.30 and 6.95±1.01 µM, respectively (Table 1).
Anti-cholinesterases or AChEIs inhibit cholinesterase, increasing the length and level of
ACh action. A diversity of usages of AChEI compounds is popular in agriculture and medicine
[43,44]. AChEI molecules traditionally utilized for medical aims include carbamates and
organophosphates. In diseases such as AD and myasthenia gravis, in which cholinergic function
is defective, organophosphates/carbamates provide therapeutic options due to high effectiveness
as AChEI compounds [45]. These inhibitor compounds are also utilized as pesticides for the
deletion of insects that pose a threat to agriculture, gardening, and public health [46]. The
inhibitory effects of the synthesized novel pyrazole compounds (1-8 and 9a, b) derivatives on
6

AChE are shown in Table 1. The AChE inhibition profiles of the compounds evaluated here were
quite interesting. Overall, the novel pyrazole compounds (1-8 and 9a, b) derivatives had excellent
inhibitory activity with Ki values ranging from 48.94±9.63 µM to 116.05±14.95 µM.
Furthermore, tacrine, used as a standard AChE inhibitor in this study, demonstrated Ki value of
125.02±18.63 µM toward AChE. As these results show, the inhibition of AChE of novel pyrazole
compounds (1-8 and 9a, b) derivatives is much better than standard drug. The compounds of 1-
(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylate (5) and 1-(3,4-dimethylphenyl)-5-
phenyl-1H-pyrazole-3,4-dicarbonitrile (4) showed excellent inhibitions profile against AChE
with Ki values of 48.94±9.63 and 55.93±10.42 µM, respectively (Table 1).
2.3 Docking studies
We performed docking studies to identify the best pose of the most active compounds that
interact with the receptors and possible inhibition mechanism after in vitro experimental results.
We firstly identified the catalytic site on hCAI, hCAII, and AChE receptors. The catalytic site
was generated using SiteMap module and Dscore was calculated 1.061, 0.945, and 1.113 for
hCAI, hCAII, and AChE receptors, respectively. The catalytic sites were considered as
druggablesite. Following catalytic site generating process, we assessed the correct binding mode
between the receptor and co-crystallized ligand with re-docking procedure performing an
effective docking process. Docking validation provides useful in the assessment of docking
method. The accuracy of docking process has been shown that co-crystallized and docked ligands
are closely docked into the receptors as seen in Figure 1.
Figure 1
Most active inhibitors and reference inhibitors were docked into catalytic site of all
receptors with both Glide docking process and IFD process. AZA was used as a reference
inhibitor for hCAI and hCAII receptors and TAC was used as a reference inhibitor for AChE
receptor. IFD process against the catalytic site of the receptors resulted in multiple docking poses
for each syntheses compound. Pose viewer module was used analyzing the interactions between
best-scored compounds and the receptors. The results of Glide docking and IFD of the best-
scoring compounds and reference inhibitors are presented in Table 2. The results clearly shown
that most of active compounds possessed high binding affinity against hCAI, hCAII, and AChE
receptors.
7

Table 2
We focus on explanation for interactions and binding mechanisms between most active
compounds and the receptors. So we considered IFD score, hydrogen bonds and non-bonded
interactions for docking results analysis. We firstly analyzed 2D interaction diagram of most
active compounds and reference inhibitors on the receptors. According to 2D interaction diagram,
compound 2formed π-π interaction with His200 residue as seen Figure 2-a. The complexed
compound displayed interactions with 35 residues of hCAI. Superimposed pose of compounds 2
has shown that the compound well placed into the catalytic site of hCAI as seen in Figure 3-a. In
detailed binding mode of the compound has also shown that formed halogen bonds with His122,
Ala142, and Thr199 residues and aromatic hydrogen bond with His94 and Pro202 residues. We
considered as key residues His94, Thr199, and His200residues of hCA I, because previously
syntheses inhibitors and reference inhibitor interacted with these residues [47-49].
Figure 2
2D interaction diagram of compound 2 there was no any hydrogen bound or non-bonded
interactions with residues of hCAII, but the compound was located into the cavity creating from
hydrophobic, polar and metal residues. The residues were reasonably similar with residues of
hCAII which surrounding AZA as seen Figures 2b and 2d. Furthermore, the residues in binding
pocket of compound 2are closely similar to residues which surrounding sulfonamide derivatives
[50] and EMAC1011g compound [51]. The complexed compound displayed interactions
withjust25 residues of hCA II, on the contrary, hCA I. In Figure 3b, it can be seen that the
compound formed two halogen bonds with Asn62 and Asn67 residues. According to these
results, we observed that the chloride group of compound 2has been contributed to inhibition of
hCA I, and hCA II enzymes.
The compound 5 formed π-π interaction with Trp286 and Phe297 residues and also
hydrogen bonds with Tyr72 and Phe295 residues by courtesy of water molecules in crystal
structure. The aromatic ring of TAC formed π-π interaction with Trp286 residue as compound 5
as seen Figures 2c and 2f. In previous studies shown that Tyr72 and Trp286 residues play a key
role on inhibition of AChE enzyme [52,53]. The complexed compound displayed interactions
with 27 residues of AChE. Superimposed pose of compounds 5 has shown that the compound
well placed into the catalytic site of AChE as seen in Figure 3c. This figure also shown that these
compounds interacted with the key residues into the catalytic site of the receptors.
8

Figure 3
3. CONCLUSION
We synthesized novel substituted pyrazole derivatives, which have two or three important rings.
Binding free energy of most active compounds was calculated with induced fit docking process.
After docking process, inhibition mechanism of most active compounds is identified by detecting
binding mode of the compounds in the catalytic site of hCA I, hCA II and AChE receptors. Key
residues, which are responsible from inhibition each receptor was detected. These compounds
were investigated for their hCAs I and II isoforms, and AChE inhibition properties. These
molecules have shown inhibition against those enzymes at nanomolar concentration level and
also, the docking results revealed that all compounds inhibited hCAI, hCA II, and AChE enzymes
through interactions including hydrophobic interactions, electrostatic interactions, and H-bonds.
Inhibition of AChE and hCA I, and II isoforms can have a significant role in discovery and drug
design as well as in toxicology and medicine. Additionally, these molecules are drug candidates
as anticholinergic and antiepileptic.
4. EXPERIMENTALS
4.1. Materials and Equipment
All the used chemical materials were purchased from Merck and Sigma Aldrich companies. 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 60 F 254 Merck.
1
13
Camag TLC device was used to visualize in reactions conditions. H and C NMR spectra on a
Bruker DRX-400 high performance digital FT-NMR spectrometer were used to characterizations
of chemical structures. The infrared spectra were recorded on a Perkin Elmer Precisely Spectrum
one spectrometer using pressed disc in the range of 4.000-450 cm-1. The mass spectrum was
measured on Thermo Scientific TSQ-Quantum Access LC/MS spectrometer. Melting points of
synthesized compounds were performed on an Electrothermal Gallenkamp apparatus and are
uncorrected.
4.2. 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylic acid (1)
To synthesize compound 1, procedure existed in literature was followed [40].
9

4.3. 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarbonyl dichloride(2)
To synthesize compound 2, procedure existed in literature was followed [40].
4.4. 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxamide (3)
Pyrazole-3,4-dicarbonyl dichloride 2 (0.373 g, 1 mmol) was dissolved in xylene (10 mL).
Ammonia as nucleophile (1 mmol) was added to solution. The reaction mixed was refluxed in
xylene for 5 hours. After evaporation, the oily residue was treated with dry ether and the formed
o
crude product was crystallized from ethanol. Yield: 84%. mp 211-213 C; FT-IR (ν, cm-1): 3256,
3210, 3062, 2936, 1662, 1654, 1598, 1520, 1498, 1443, 1315; 1H-NMR (400 MHz, CDCl3) 
13
(ppm): 7.7-6.5 (m, 8H, Arom.), 5.5 (s, 2H), 5.0 (s, 2H), 2.0 (s, 3H), 1.6 (s, 3H), C-NMR (100
MHz, CDCl3)  (ppm): 167.2, 160.6, 144.5, 141.4, 140.4, 135.5, 131.0, 130.7, 130.0, 129.6,
128.5, 127.5, 126.8, 125.1, 121.6, 120.2, 111.9, 21.1, 17.9. (+)ESI-HRMS m/z: calculated for
[C19H18N4O2 + H+]: 335.3782; observed 335.3789.
4.5. 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarbonitrile (4)
Pyrazole-3,4-dicarboxamide 3 (0.167 g, 0.5 mmol) was added in xylene (5 mL). SOCl2 (0.146
o
mL, 2 mmol) and DMF (2 mL) were added to solution in 0 C. Reaction mixed was stirred at
room temperature for ten hours. The ice water was added to the mixture. Precipitated solid was
filtered, dried. It was purified from ethanol. Yield: 42%. mp 198 oC. FT-IR (ν, cm-1): 3064, 2932,
2194, 2186, 1597, 1442, 1315; 1H-NMR (400 MHz, CDCl3)  (ppm): 7.9-6.6 (m, 8H, Arom.), 2.0
13
(s, 3H), 1.7 (s, 3H); C-NMR (100 MHz, CDCl3)  (ppm): 145.9, 136.2, 134.1, 130.7, 129.6,
128.9, 127.1, 121.6,120.6, 115.0, 114.8, 111.2, 21.8, 16.5. (+)ESI-HRMS m/z: calculated for
[C19H14N4 + H+]: 240.3426; observed 240.3431.
4.6. Diethyl 1-(3,4-dimethylphenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylate (5)
Pyrazole-3,4-dicarbonyl dichloride 2 (0.373 g, 1 mmol) was dissolved ethanol (15 mL). The
prepared solution was heated with a catalytic amount of pyridine for five hours [54]. It was
cooled to room temperature. It was acidified via hydrochloric acid (12%). Precipitated solid was
o
filtered, dried. It was crystallized from ethanol. Yield: 85%. mp 124 C. FT-IR (ν, cm-1): 3062,
1
2954, 1721, 1592, 1521, 1490, 1442, 1315; H-NMR (400 MHz, CDCl3)  (ppm): 7.9-6.5 (m,
10

8H, Arom.), 3.7 (m, 2H), 3.4 (m, 2H), 2.0 (s, 3H), 1.6 (s, 3H), 1.3 (t, J=5.2 Hz 3H), 1.2 (t, J=5.2
Hz, 3H); 13C-NMR (100 MHz, CDCl3)  (ppm): 159.9, 156.0, 144.9, 140.1, 135.5, 134.1, 133.1,
132.0, 130.0, 128.6, 127.5, 126.1, 125.1, 124.0, 123.3, 110.8, 41.3, 40.2, 21.1, 17.6, 12.4, 11.7.
(+)ESI-HRMS m/z: calculated for [C23H24N2O4 + H+]: 393.4551; observed 393.4556.
4.7. 2-(3,4-dimethylphenyl)-3-phenyl-5,6-dihydro-2H-pyrazolo[3,4-d]pyridazine-4,7-dione (6)
Pyrazole-3,4-dicarboxylate 5 (0.196 g, 0.5 mmol) was added in anhydrous toluene (10 mL).
Anhydrous hydrazine (0.016 g, 0.5 mmol) was added to solution. The reaction mixture was
refluxed for five hours. Precipitated solid was filtered. It was crystallized from hexane. Yield:
o
62%. mp 264-266 C. FT-IR (ν, cm-1): 3226, 3064, 2956,1652, 1650, 1596, 1528, 1496, 1447,
1328; 1H-NMR (400 MHz, DMSO)  (ppm): 8.5, 8.3 (s, 2H), 7.8-6.5 (m, 8H, Arom.), 2.2 (s, 3H),
1.6 (s, 3H); 13C-NMR (100 MHz, DMSO)  (ppm): 168.9, 160.2, 145.2, 143.2, 140.1, 139.0,
133.1, 131.7, 130.7, 129.6, 128.2, 127.5, 126.4, 125.8, 125.1, 119.1, 111.9, 21.1, 16.5. (+)ESI-
HRMS m/z: calculated for [C19H16N4O2 + H+]: 333.3635; observed 336.3641.
4.8. 2-(3,4-dimethylphenyl)-3-phenyl-2H-pyrazolo[3,4-d]pyridazine-4,7-diamine (7)
Pyrazole-3,4-dicarbonitrile 4 (0.15 g, 0.5 mmol) was added dry ethanol (10 mL). Anhydrous
hydrazine (0.016 g, 0.5 mmol) was added to solution. The reaction mixture was refluxed for five
hours. Precipitated solid was filtered and it was washed with diethyl ether. The product was
crystallized by ethanol. Yield: 72 %. mp >300 oC. FT-IR (ν, cm-1): 3264, 3258, 3062, 2936, 1597,
1
1521, 1496, 1442, 1321; H-NMR (400 MHz, CDCl3)  (ppm): 8.0-6.8 (m, 8H, Arom.), 4.4 (s,
2H), 4.1 (s, 2H), 2.3 (s, 3H), 1.8 (s, 2H); 13C-NMR(100 MHz, CDCl3)  (ppm): 164.7, 159.2,
145.2, 141.4, 141.1, 137.2, 134.1, 131.7, 131.4, 130.0, 129.3, 128.9, 128.6, 127.1, 123.7, 22.1,
16.5. (+)ESI-HRMS m/z: calculated for [C19H18N6 + H+]: 331.4042; observed 331.4047.
4.9. 2-(3,4-dimethylphenyl)-3-phenyl-2H-furo[3,4-c]pyrazole-4,6-dione (8)
Pyrazole-3,4-dicarboxylic acid 1 (0.17 g, 0.5 mmol) was added in DMF (0.2 mL) and SOCl2 (0.7
mL). The solution was mixed at 0 °C for four hours. Then, it was stirred to room temperature for
twelve hours. The ice water was added to the mixture. The product was crystallized by ethanol.
Yield: 68%. mp 181-183oC.FT-IR (ν, cm-1):3062, 2928, 1676, 1596, 1524, 1498, 1442, 1316;1H-
NMR (400 MHz, CDCl3)  (ppm): 8.0-6.5 (m, 8H, Arom.), 2.2 (s, 3H), 1.6 (s, 3H);13C-NMR
11

(100 MHz, CDCl3)  (ppm): 167.5, 159.9, 146.6, 141.1, 136.1, 131.7, 130.3, 129.3, 128.9, 127.8,
127.1, 126.1, 124.4, 123.7, 121.3, 119.5, 110.8, 20.8, 16.2. (+)ESI-HRMS m/z: calculated for
[C19H14N2O3 + H+]: 319.3326; observed 319.3329.
4.10. 2-(3,4-dimethylphenyl)-3-phenylpyrrolo[3,4-c]pyrazole-4,6(2H,5H)-dione (9a)
Pyrazole-4,6-dione 8 was (0.16 g, 0.5 mmol) was dissolved ethanol (20 mL). Ammonia as
nucleophile (0.009 g, 0.5 mmol) was added to solution. Reaction was refluxed for two days. It
was cooled room temperature. The solvent was evaporated. The residue compound was washed
with diethyl ether. Precipitated solid was filtered, dried. It was crystallized by ethanol. Yield:
65%. mp 215-217oC. FT-IR (ν, cm-1):3274, 3068, 2927, 1658, 1589, 1517, 1496, 1442, 1313; 1H-
NMR (400 MHz, CDCl3)  (ppm): 8.3 (s, 1H), 7.9-6.5 (m, 8H, Arom.), 2.1 (s, 3H), 1.5 (s,
3H);13C-NMR (100 MHz, CDCl3)  (ppm): 167.9, 161.2, 144.2, 138.3, 134.8, 134.5, 132.4,
131.0, 130.7, 129.3, 128.9, 128.2, 126.4, 125.4, 124.0, 122.7, 110.1, 23.9, 17.9. (+)ESI-HRMS
m/z: calculated for [C19H15N3O2 + H+]: 318.3421; observed 318.3425.
4.11. 2-(3,4-dimethylphenyl)-5-ethyl-3-phenylpyrrolo[3,4-c]pyrazole-4,6(2H,5H)-dione (9b)
Pyrazole-4,6-dione 8 was (0.16 g, 0.5 mmol) was dissolved ethanol (20 mL). Ethylamine as
nucleophile (0.023 g, 0.05 mmol) was added to solution. Reaction was refluxed for two days. It
was cooled room temperature. The solvent was evaporated. The residue compound was washed
with diethyl ether. Precipitated solid was filtered, dried. It was crystallized by ethanol. Yield: 65
%. mp 224 oC. FT-IR (ν, cm-1): 3310, 3064, 2926, 1662, 1594, 1524, 1498, 1444, 1321; 1H-NMR
(400 MHz, CDCl3)  (ppm): 8.0-6.5 (m, 8H), 3.0 (m, 2H, Arom.), 2.1 (s, 3H), 1.7 (s, 3H), 1.1 (m,
3H); 13C-NMR (100 MHz, CDCl3)  (ppm): 163.7, 161.2, 143.5, 140.1, 137.2, 133.8, 131.7,
130.3, 130.0, 129.6, 128.9, 128.5, 127.5, 126.8, 125.8, 124.4, 109.0, 33.6, 23.5, 19.3, 11.4.
(+)ESI-HRMS m/z: calculated for [C21H19N3O2 + H+]: 346.3986; observed 349.8689.
4.12. Biochemical Studies
4.12.1. hCA isoenzymes purification and inhibition studies
In this paper, both CA isozymes were purified by Sepharose-4B-L-tyrosine sulfanilamide as
affinity chromatography in a single phase [55]. The column chemical material of affinity
12

chromatography containing Sepharose-4B-L-Tyrosine-sulfanilamide was created comprising to a
prior procedure [56,57]. The protein molecules flow in the column eluates was
spectrophotometrically obtained at 280 nm as illustrated formerly. CA isozymes’ inhibitory
activity evaluation of novel pyrazole (1-8 and 9a, b) derivatives was obtained using the
spectrophotometric style of Verpoorte et al. [58] as explained in former studies [59-61]. In this
study, changes in absorbance were recorded during 3 min at 348 nm using p-nitrophenylacetate
was utilized as a substrate which converted by both isoenzymes to the p-nitrophenolate ion
molecule [52,63].
4.12.2. AChE inhibition study
The inhibitory effects of novel pyrazole (1-8 and 9a, b) derivatives on AChE activity were
performed according to the spectrophotometric method of Ellman et al. [64] and described
previously [65,66]. 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. Briefly, 750 μL of sample solution
dissolved in deionized water at different concentrations 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 21oC [67]. Then
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 absorption maximum at a wavelength of 412
nm [68].
4.13. Docking studies
The x-ray crystal structures of hCA I, hCAII, and AChE (PDB code: 4WR7, 5AML, and 4M0E,
respectively) were taken in pdb format from RCSB Protein Data Bank with resolution of 1.5 Å,
1.36 Å, and 2 Å, respectively. Then, the crystal structures of the receptors were used for
molecular docking studies. Molecular docking studies were performed using Small Drug
Discovery Suites package (Schrödinger 2017-2, LLC, USA). The receptors preparation was
performed according to our previous study. Briefly, the 3D crystal structures were repaired and
prepared via protein preparation wizard in Maestro 11.4. Bond order and charges were assigned
13

and then all missing hydrogen atoms were added to protein structure. Missing side chains were
filled using Prime module of the program. Amino acids were ionized by setting physiological pH
with the help of Propka software. Water molecules that were formed less than 3 contacts with the
protein or ligand were removed. Then, energy minimization has also been performed using OPLC
force field. Single binding site of the prepared receptors was identified using SiteMap module in
Maestro 11.4.3D structures of synthesized compounds were produced with Maestro 11.4 by
sketching 2D structures. 3D structure of ligands was created using LigPrep module in Maestro
11.4. In order to obtain correct molecular geometries and protonation state at pH 7.0 ± 2.0, Epik
module and OPLS-2005 force field were used.
Molecular docking was performed to identify binding affinity and possible interactions
between synthesized compounds and receptors. Briefly, selecting crystallized inhibitors at the
binding site using the Receptor Grid Generation platform before the docking process generated
grid box. Docking calculations were set as Extra Precision (XP) by keeping the ligand flexible.
After the docking process, the types of interactions and interacted residues for best-scored
compound results were analyzed. In order to identification accuracy of the docking process,
docking validation was performed with re-docking procedure by extracting inhibitor complexed
in the crystal structure of the receptor, before docking of synthesized compounds. Following
Glide docking, induced fit docking (IFD) studies were performed 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 in the catalytic 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
[69].
Acknowledgements
The authors are grateful to Dr. Halide Sedef Karaman for providing her technical guidance while
processing docking study of this article and for supporting small drug discovery suite software.
Conflict of Interest
The authors declare that they have no conflict of interest.
14

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enzymes. Chemical Biology and Drug Design, 91(4) (2018) 854-866.
[68] A. Behcet, T. Çağlılar, D. Barut Celepci, A. Aktaş, P. Taslimi, Y. Gök, M. Aygün, R.
Kaya, İ. ul in, Synthesis, characterization and crystal structure of 2-(4-
22

hydroxyphenyl)ethyl and 2-(4-nitrophenyl)ethyl substituted benzimidazolium bromide
salts: their inhibitory properties against carbonic anhydrase and acetylcholinesterase.
Journal of Molecular Structure, 1170 (2018) 160-169.
[69] L. Schrödinger, Drug Discov. Suite, (2017).
23

Scheme1. Synthesis of 1-8 and 9a-b derivatives
24

Table 1. Human carbonic anhydrases I, and II isoenzymes (hCA I, hCA II), and
acetylcholinesterase (AChE) enzymes inhibition values of substituted pyrazole compounds (1-8
and 9a, b)
IC50 (µM)
Ki (µM)
hCA II
Compounds
hCA I
3.54
r2
hCA II
r2
AChE
85.94
r2
hCA I
AChE
0.9704
0.9935
5.52
1.36
0.9683
0.9052
0.9723
0.9593
4.88±0.83
1.03±0.23
6.95±1.01
1.82±0.30
65.36±11.47
79.74±21.15
1
2
0.83
103.65
13.93
9.26
0.9465
0.9401
0.9880
0.9366
0.9835
0.9535
0.9905
0.9693
18.63
14.28
7.88
0.9693
0.9890
0.9424
0.9882
0.9758
0.9395
0.9692
0.9355
145.26
64.04
60.26
74.98
86.05
100.82
93.72
78.03
0.9205
0.9793
0.9688
0.9816
0.9911
0.9582
0.9105
0.9924
16.46±2.66
12.54±1.46
6.58±0.95
22.65±5.36
8.03±1.83
6.95±1.25
13.77±2.80
18.83±4.05
18.02±2.63
17.73±3.74
8.93±1.72
116.05±14.95
55.93±10.42
48.94±9.63
3
4
5.04
5
18.25
6.66
24.44
9.02
27.94±4.74
11.62±2.41
14.88±3.60
14.88±2.52
24.80±5.04
62.04±15.04
66.04±14.72
84.05±16.03
80.52±25.88
72.07±18.72
6
7
6.01
10.21
14.05
21.58
8
10.15
16.22
9a
9b
21.13
-
0.9665
-
28.55
-
0.9789
-
-
-
28.11±10.33
-
34.12±8.23
-
-
AZA*
166.74
0.9498
125.02±18.63
TAC**
*Acetazolamide (AZA) was used as a standard inhibitor for both human carbonic anhydrases I,
and II isoenzymes (hCA I, hCA II).
**Tacrine (TAC) was used as a standard inhibitor for acetylcholinesterase (AChE) enzyme.
25

Table 2. Glide and IFD scores (kcal/mol) of the pyrazole derivatives in the catalytic sites of hCA
I, and hCA II, and AChE
hCA I
hCA II
AChE
Compounds
Glide Score IFD Score Glide Score IFD Score Glide Score
IFD Score
-3.597
-6.776
-4.896
-5.774
-
-
2
-
-8.331
-
-
-9.016
-
-
-8.178
-
-
-9.560
-
-6.749
-
-9.534
-
5
AZA*
TAC**
-8.978
-9.579
*Acetazolamide (AZA) was used as a standard inhibitor for human carbonic anhydrase isoenzymes I, and
II (hCA I, and II).
** Tacrine (TAC) was used as a standard inhibitor for acetylcholinesterase (AChE) enzyme.
26

FIGURE LEGENDS
Figure 1. Docking validation. Receptors are depicted in the ribbon model which hCA I with red
color, hCA II with blue color, and AChE with purple color. The poses of co-crystallized
ligands are represented in grey color ball and stick modeling while that of docked
ligands is represented in green color ball and stick modeling
Figure 2. 2D receptor–ligand interaction profile; (a) 2-hCA I, (b) 2-hCA II, (c) 5-AChE, (d)
AZA-hCA I, (e) AZA-hCA II, and (f) TAC-AChE
Figure 3. The lowest energy conformation of compounds into hCAI, hCA II, and AChE
receptors. (a) Best-pose of 2 into catalytic active site of hCAI, (b) Best-pose of 2 into
catalytic active site of hCA II, and (c) Best-pose of 5 into catalytic active site of AChE.
Receptors are depicted in the ribbon model which hCAI with red color, hCA II with blue
color, and AChE with purple color. Catalytic active sites are represented as solid
surface. Compounds are represented in ball and stick modeling which green color carbon
and residues are represented in ball and stick modeling which grey color carbon
27

a
b
c
Figure 1
28

c
b
a
d
e
f
Figure 2
29