In silico studies, nitric oxide, and cholinesterases inhibition activities of pyrazole and pyrazoline analogs of diarylpentanoids

Article abstract of DOI:10.1002/ardp.202000161

A new series of pyrazole, phenylpyrazole, and pyrazoline analogs of diarylpentanoids (excluding compounds 3a, 4a, 5a, and 5b) was pan-assay interference compounds-filtered and synthesized via the reaction of diarylpentanoids with hydrazine monohydrate and phenylhydrazine. Each analog was evaluated for its anti-inflammatory ability via the suppression of nitric oxide (NO) on IFN-γ/LPS-activated RAW264.7 macrophage cells. The compounds were also investigated for their inhibitory capability toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), using a modification of Ellman's spectrophotometric method. The most potent NO inhibitor was found to be phenylpyrazole analog 4c, followed by 4e, when compared with curcumin. In contrast, pyrazole 3a and pyrazoline 5a were found to be the most selective and effective BChE inhibitors over AChE. The data collected from the single-crystal X-ray diffraction analysis of compound 5a were then applied in a docking simulation to determine the potential binding interactions that were responsible for the anti-BChE activity. The results obtained signify the potential of these pyrazole and pyrazoline scaffolds to be developed as therapeutic agents against inflammatory conditions and Alzheimer's disease.

Full text of DOI:10.1002/ardp.202000161

Received: 19 May 2020
Revised: 12 August 2020
Accepted: 14 August 2020
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DOI: 10.1002/ardp.202000161
F U L L P A P E R
In silico studies, nitric oxide, and cholinesterases inhibition
activities of pyrazole and pyrazoline analogs of
diarylpentanoids
Siti Munirah Mohd Faudzi1,2
Faridah Abas2,3
Khozirah Shaari1,2
| S. Wei Leong2
| Lam K. Wai4 | Syahida Ahmad5 | Chau L. Tham6
| Nordin H. Lajis2 | Bohari M. Yamin7
| Faruk A. Auwal1
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1Department of Chemistry, Faculty of Science,
Universiti Putra Malaysia, Serdang, Selangor,
Malaysia
Abstract
A new series of pyrazole, phenylpyrazole, and pyrazoline analogs of diarylpentanoids
(excluding compounds 3a, 4a, 5a, and 5b) was pan‐assay interference compounds‐
filtered and synthesized via the reaction of diarylpentanoids with hydrazine
monohydrate and phenylhydrazine. Each analog was evaluated for its anti‐
inflammatory ability via the suppression of nitric oxide (NO) on IFN‐γ/LPS‐activated
RAW264.7 macrophage cells. The compounds were also investigated for their in-
hibitory capability toward acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE), using a modification of Ellman's spectrophotometric method. The most po-
tent NO inhibitor was found to be phenylpyrazole analog 4c, followed by 4e, when
compared with curcumin. In contrast, pyrazole 3a and pyrazoline 5a were found to
be the most selective and effective BChE inhibitors over AChE. The data collected
from the single‐crystal X‐ray diffraction analysis of compound 5a were then applied
in a docking simulation to determine the potential binding interactions that were
responsible for the anti‐BChE activity. The results obtained signify the potential of
these pyrazole and pyrazoline scaffolds to be developed as therapeutic agents
against inflammatory conditions and Alzheimer's disease.
2Laboratory of Natural Products, Institute of
Bioscience, Universiti Putra Malaysia, Serdang,
Selangor, Malaysia
3Department of Food Science, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia
4Drug and Herbal Research Centre, Faculty of
Pharmacy, Universiti Kebangsaan Malaysia,
Kuala Lumpur, Malaysia
5Department of Biochemistry, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia
6Department of Biomedical Science, Universiti
Putra Malaysia, Serdang, Selangor, Malaysia
7School of Chemical Sciences and Food
Technology, Universiti Kebangsaan Malaysia,
Bangi, Selangor, Malaysia
Correspondence
Siti Munirah Mohd Faudzi, Department of
Chemistry, Faculty of Science, Universiti Putra
Malaysia, 43400 UPM, Serdang,
Selangor, Malaysia.
Email: sitimunirah@upm.edu.my
K E Y W O R D S
Funding information
Universiti Putra Malaysia,
Grant/Award Numbers: 9301700, GP‐IPM/
2016/9480700
AChE and BChE inhibition, diarylpentanoids, nitric oxide inhibition, pyrazole, pyrazoline
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1
INTRODUCTION
and chronic inflammation‐related disorders including cancer, in-
flammatory arthritis, and Alzheimer's disease (AD).[5–8] In addition,
Nitric oxide (NO) is an important cellular messenger in a complex
biological system, which is typically involved in the regulation of the
inflammatory process.[1] This small bioactive molecule also plays pi-
votal roles in intravascular endocrine NO transport, vasodilation, and
cytoprotection.[2–4] However, excessive production of NO may in-
teract with superoxide to generate the cytotoxic species peroxyni-
trite (ONOO–), which is responsible for cell destruction and toxicity,
thus contributing to health risks through the induction of both acute
inflammatory processes are among the pathological features asso-
ciated with the central nervous system (CNS) in AD.[9]
AD, also known as senile dementia, is often characterized by the
presence of amyloid‐β plaques and neurofibrillary tangles in the
brain, which may result in the loss of mental abilities and cognitive
disturbance. The loss of cognitive functions in AD patients is asso-
ciated with a continuous decline of cholinergic neurotransmission in
the cortical or other parts of the brain.[10] Most treatment strategies,
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Arch Pharm. 2020;e2000161.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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to date, are based on the regulation of the cholinergic function, which
could elevate acetylcholine (ACh) levels by inhibiting acet-
ylcholinesterase (AchE) and butyrylcholinesterase (BChE), and there
is evidence that AChE inhibitors also have anti‐inflammatory
potential.[9]
AChE is substrate‐specific in nature and is mainly found in
high concentrations in the brain, whereas BChE is nonspecific and
is widely distributed throughout the body.[11] In a healthy brain,
the AChE enzyme dominantly degrades ACh, whereas BChE only
plays a supportive role. These two enzymes display diverse ki-
netic characteristics, depending on ACh concentrations. At low
ACh concentrations, AChE's activity becomes highly dominant,
whereas BChE is more efficient during hydrolysis at high ACh
concentrations.[11] Some initial studies underrated the im-
portance of BChE in the human brain, owing to its low expres-
sion.[12] Recently, the importance of BChE within the nervous
system in the late stages of AD has been discovered.[13,14] It was
observed that BChE activity progressively increases in patients
with AD, whereas AChE activity remains unchanged. Moreover,
AChE knockout experiments performed on mouse models con-
firmed the role of BChE in maintaining cholinesterasic function,
even in the absence of AChE.[15]
FIGURE 2 The synthesized pyrazoles, phenylpyrazoles, and
pyrazolines (series 3, 4, and 5)
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2
RESULTS AND DISCUSSION
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2.1
Pan‐assay interference compounds (PAINS)
and aggregator identification
Molecules with false‐positive signals in biological assays often cause
substantial problems in biological screenings.[22] Assay artifacts and
interferences might only be detected in the later stages of the
compound development process, leading to a significant loss of time
and resources.[23] Hence, this situation triggered the scientific com-
munity to raise awareness on the importance of assay interference.
Several known assay interference molecules such as colloidal ag-
gregator and chemically reactive compounds under assay conditions
may originate from both synthetic and natural sources.[23,24] One
way to proactively address this problem is by noting or identifying
any PAINS‐containing molecules in the designed compounds before
removing them in the early stages of a drug discovery project. In view
of this, we input all 15 pyrazole, phenylpyrazole, and pyrazoline
analogs of diarylpentanoids into publicly available filter servers, FAF‐
Drugs4 and Aggregator Advisor for PAINS and aggregator identifi-
cation, respectively.[25,26]
The pyrazole and pyrazoline scaffolds (Figure 1) have at-
tracted considerable attention from the research community due
to their wide range of chemical and biological applications.[16]
Pyrazole (1) is a five‐membered aromatic ring, consisting of two
nitrogen atoms in adjacent positions with two conjugated double
bonds. Meanwhile, pyrazoline (2) is a reduction product of pyr-
azole, which comprises only one endocyclic double bond in its
structure. The pyrazole and pyrazoline, together with their de-
rivatives, have been proven to be a great source of medicinal
agents, as they display significant anti‐inflammatory,[17,18] an-
ticancer, antidiabetic, antibacterial, and antifungal properties, as
well as a potent selective activity, for example, acting as a nitric
oxide synthase (NOS) inhibitor.[19–21]
Referring to the PAINS identification results, four compounds
(3c, 4c, 5a, and 5e) were non‐PAINS, whereas the others were in-
termediate PAINS molecules containing halogen (Cl and F) and/or
phenol‐associated fragments, as tabulated in Table 1. Nevertheless,
as the total substitution of the mentioned PAINS fragments was
below the rejection threshold designated by the public filter, and the
phenol and/or halogen alert referred to a stable moiety, the desired
molecules were considered as low‐risk compounds. To summarize, a
compound is categorized as a PAINS molecule if it contains a high‐
risk structural alert and/or exceeds the threshold cut‐off on a num-
ber of undesirable substructures in the same compound.[25,27]
Furthermore, all molecules were predicted to be potential
Therefore, in the context of searching for a new entity for the
treatment of inflammation‐associated disorders and AD, a new
series of 15 pyrazole, phenylpyrazole, and pyrazoline of dia-
rylpentanoid analogs (Figure 2) was synthesized and evaluated
for their NO, AChE, and BChE inhibitory potency. The X‐ray
diffraction (XRD) analysis and molecular docking were then
performed on the most active molecule to further understand the
binding interactions between the ligand and specific active sites
to elicit its biological property.
aggregates, as they were calculated to possess high log
P
(3.1–6.9). It was observed that the bulkier phenylpyrazole series
(4a–e) recorded the higher log P, compared with the other series,
as they contained
a potential chelator moiety, the ortho‐
H
N
H
N
hydroxyphenylhydrazone, and thus may form a more robust
macromolecular aggregate.
N
N
The physicochemical properties of each molecule in series 3, 4,
and 5 were also analyzed in silico through the FAF‐Drugs4 server.
The tabulated results in Table 2 show that most compounds did not
violate Lipinski's rule of five (RO5), excluding 3d and 4a–e, as these
1
2
FIGURE 1 General structures of pyrazole and pyrazoline

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TABLE 1 PAINS analysis of the synthesized compounds
PAINS‐associated
Rejection
threshold
Number of substitutions
in compound
group
Structure
Compound
Phenol
5
3a
3e
4a
5b
1
1
1
1
Halogen
Cl, Br, I
5
3b
3d
4b
4d
4e
1
2
1
2
1
Halogen_F
F
7
5d
1
molecules possess a value of log P exceeding 5. RO5 is a simple rule
applied to evaluate “drug‐likeness” characteristics of compounds
based on several factors, including molecular weight <500 (MW ≤
500), no more than five hydrogen bond donor groups (HBD ≤ 5), no
more than 10 hydrogen bond acceptor groups (HBA ≤ 10), and a
calculated partition coefficient between n‐octanol and water <5 (log
P ≤ 5, log P measure lipophilicity).
blood–brain barrier (BBB) and to exhibit CNS‐related activity, in-
cluding lipophilicity (1.5 ≤ log P ≤ 2.7 and 0 ≤ log D ≤ 3), molecular
weight (MW ≤ 450), hydrogen bonding ([O + N] count ≤ 5, HBA ≤
2.12, and HBD ≤ 1.5), polar surface area (60 Å2 ≤ TPSA ≤ 70 Å2), and
flexibility (NRB ≤ 5).[28]
Lipophilicity was the first identified descriptor that is important
for CNS penetration. According to Fichert et al.[29] as well as Hansch
and Leo,[30] for drug‐like molecules to have a better BBB and in-
testinal penetration, the log P value was suggested to be in the range
of 1.5–2.7, and log D needs to be >0 and <3. However, on the basis of
the results, none of the synthesized molecules possess the predicted
As this study focuses on AD‐related AChE and BChE inhibition,
besides the evaluation of their CNS drug‐likeness, all molecules
should encompass the fundamental physicochemical features of CNS
drugs. These factors are related to their ability to penetrate the
TABLE 2 Physicochemical properties of compounds calculated via FAF‐Drugs4
MW
O + N
count
RO5
Compounds
(g/mol)
262.31
296.75
292.33
331.2
Log P
3.83
4.45
3.8
Log D
4.26
4.79
4.1
TPSA (Å2)
48.91
48.91
58.14
48.58
62.05
38.05
38.05
47.28
37.72
47.28
24.39
44.62
33.62
33.62
61.86
NRB
3
HBD
2
HBA
3
violations
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
3
3
4
3
4
3
3
4
3
4
3
4
4
4
4
0
0
0
1
0
1
1
1
1
1
0
0
0
0
0
3
2
3
4
2
4
5.08
2.89
5.5
4.63
3.4
3
2
3
252.27
338.4
3
2
4
6.04
6.57
5.88
6.4
4
1
3
372.85
368.43
407.29
402.87
248.32
264.32
278.35
296.34
298.4
6.13
5.47
6.76
6.1
4
1
3
5
1
4
4
1
3
6.48
3.8
5
1
4
3.97
3.61
3.94
4.04
4.36
3
1
2
3.48
3.65
3.79
4.2
3
2
3
4
1
3
4
1
3
4
1
3
Abbreviations: HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; MW, molecular weight; NRB, number of rotatable bond; RO5, rule of five;
TPSA, total polar surface area.

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optimal log P and log D values, signifying their low potential for brain
penetration and intestinal permeability.
On the basis of the predicted CNS drug‐likeness, we can con-
clude that compound 3e displayed physicochemical parameters that
were closest to the ideal CNS drug‐like parameters. Through some
slight chemical modifications, the log P and log D of 3e might be
improved to meet the optimum values, and thus may be a potential
compound for the treatment of CNS‐related disorders, including
inflammation‐associated disorders and AD.
Marketed CNS drugs were recorded to have a mean MW value
of 310 g/mol (with a cut‐off at 400) for significant BBB penetration,
which is a reduced MW as compared with other therapeutics.[31] The
MW of potential CNS drug molecules was suggested to be below 450
to facilitate brain penetration, as small molecules may undergo sig-
nificant passive lipid‐mediated transport through the BBB.[32] As
shown, all the compounds' MW were below the 450 g/mol threshold.
Compounds with high hydrogen bond‐forming potential are ob-
served to have a minimal distribution throughout the BBB, as the higher
the hydrogen bonding, the lower is the BBB penetration. This descriptor
could be measured through TPSA, HBD, and HBA counts, or simply by
counting the number of heteroatoms capable of inducing hydrogen
bonding, including oxygen and nitrogen. Marketed CNS drugs, on
average, possess 60–70 Å2 TPSA, heteroatom count (O + N) of 4.32,
HBA of 2.12, and HBD of 1.5 for a significant BBB penetration cap-
ability, indicating the reduced polar properties of CNS drugs, relative to
other medicines.[32–34] As shown in Table 2, only analogs 3e and 5e
were the closest to achieving that ideal CNS drug‐likeness parameter,
with the exception of their HBD count being slightly higher.
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2.2
Chemistry
The new pyrazole, phenylpyrazole, and pyrazoline analogs (series 3, 4,
and 5), excluding the known compounds, 3a, 4a, 5a, and 5b, were syn-
thesized accordingly, as depicted in Scheme 1. Intermediates of dia-
rylpentanoid derivatives, 1,5‐diarylpentene‐1,3‐dienone analogs (1a–e),
were obtained via the Baker–Venkataraman rearrangement, in which
acrylic acid (I) was reacted with selected acetophenone to produce
phenolic ester (II) before being further subjected to the rearrangement
step.[35] Meanwhile, 1,5‐diarylpenta‐2,5‐dien‐1‐one analogs (2a–e) were
prepared via a base‐catalyzed Claisen–Schmidt condensation reaction
between the appropriate cinnamaldehyde (III) and substituted acet-
ophenone.[36] Further reactions of intermediate diarylpentanoids 1a–e
and 2a–e with hydrazine monohydrate and phenylhydrazine in refluxing
acetic acid formed the pyrazole, phenylpyrazole, and pyrazoline analogs
3a–e, 4a–e, and 5a–e, respectively. Three important signals have been
identified as an indication of complete cyclization between dia-
rylpentadienone (2a) with hydrazine hydrate. The presence of a me-
thylene proton, H‐2 (approx. 3.14 and 3.59 ppm), methine moiety, H‐3
Molecular flexibility is another important parameter for CNS‐
related drug‐likeness, which is related to the ease of a molecule
traversing a membrane. It appears that limited flexibility is ad-
vantageous and is very much related to the number of rotatable
bonds (NRBs) in a molecule. Most CNS drugs have an NRB of five or
less,[34] and all the synthesized compounds in this study were ob-
served to meet this ideal parameter.
SCHEME 1 The synthesis of the pyrazole, phenylpyrazole, and pyrazoline analogs 3a–e, 4a–e, and 5a–e, respectively. Reagents and
conditions: (a) substituted acetophenone, POCl3, pyridine, room temperature (RT; overnight); (b) KOH, pyridine, RT (overnight); (c) acetic acid,
hydrazine monohydrate, reflux (2–3 hr); (d) acetic acid, phenylhydrazine, reflux (2 hr); (e) substituted acetophenone, NaOH, EtOH, RT
(overnight)

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at approx. 5.30 ppm, and methyl of N‐acetyl group at 2.43 ppm strongly
approved the conversion of compound 2a to pyrazoline analog 5a.
Another significant feature in the 1H nuclear magnetic resonance
(NMR) spectrum of 5a is the resonance of vinylic protons at 6.22 ppm
(H‐4) and 6.60 ppm (H‐5). The coupling constant (J) values of H‐4 and
H‐5 are in the range of 15.9–16.1 Hz, indicating trans configuration for
the conjugated olefinic system. All compounds were produced in a
significant yield, with the percentage of conversion >75%, except for
compounds 5a and 5b. This lower yield was probably due to the
unsubstituted ring B as compared with p‐methoxy ring B that bears
compounds 5c–e in the same series.
The pyrazoline analogs of 1,5‐diarylpenta‐2,4‐dien‐1‐ones (5a–e) did not
display significant anti‐inflammatory properties, compared with the
pyrazole and phenylpyrazole analogs of series 3 and 4, as the percen-
tages of NO inhibition recorded were below 50%. The inactiveness of
this class of compounds could be caused by the N‐acetylated pyrazoline
scaffold they possess, implying that this structural motive is undesirable
in improving the anti‐inflammatory activity.
However, the pyrazole analogs 3a–e moderately inhibited
nitrite production in macrophage cells, with IC50 values ranging
from 28 to 60 µM. However, it is less promising to have more
than one electron‐withdrawing group (EWG; −Cl) in ring A as the
inhibition activity percentage is lowered by approximately six-
fold, as exhibited by compound 3d, compared with 3b and 3a.
Meanwhile, the replacement of heterocyclic moiety, furanyl on
ring B, further reduced the activity by twofold, as shown by
compound 3e, compared with compound 3a, reflecting that the
heterocyclic scaffold is unfavorable for the targeted NO sup-
pression activity. The most probable reason between these two
moieties that may alter the activity is the aromaticity, which
implies resonance. During the resonance process, the phenyl
group on ring A might not form a charge separation, unlike the
furanyl moiety. It is assumed that this separation of charges that
occurred in furanyl contributed less to the overall aromaticity of
the system, thus decreasing the anti‐inflammatory effect.
Conversely, the phenylpyrazole moiety appeared to be the most
crucial feature for anti‐inflammatory activity, as compound 4c ex-
hibited a better NO inhibitory effect by twofold, compared with the
pyrazole analog 3c. The same pattern could also be observed in
compounds 4b and 4d, compared with their respective pyrazole
compounds 3b and 3d. It is worth noting that the activity was im-
proved with the presence of an electron‐donating group (EDG;
−OCH3) in ring A, ignoring the electron density difference in ring B,
as shown by compounds 4c and 4e with IC50 values of 19.05 0.4
and 17.47 0.1 µM, respectively. In addition, with a disubstituted
EWG on ring A (compound 4d), the NO suppression activity was
lowered by onefold, compared with its monosubstituted analog
(compound 4b), suggesting that a lower electron density will not
improve NO inhibition. The overall results imply that the phe-
nylpyrazole (series 4) is a vital molecular fragment in enhancing NO
inhibition, whereas the pyrazoline fragment (series 5) conversely
tends to weaken the NO inhibition activity.
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2.3
Pharmacology/biology
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2.3.1
NO inhibitory activity
All 15 pyrazole, phenylpyrazole, and phenylpyrazoline analogs were
then screened for their NO suppression activity in IFN‐γ/LPS‐stimulated
RAW264.7 macrophage cells at 50 µM concentration. On the basis of
the preliminary screening, eight compounds (3a–c, 4a–e) significantly
inhibited NO production with the percentage of inhibition >50%
(Figure 3), which indicates that our designed molecules might possess
anti‐inflammatory properties and could be potential candidates for
further investigations. The eight bioactive compounds were further
subjected to IC50 determination, and MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐
2,5‐diphenyltetrazolium bromide) assay was performed to ensure the
NO suppression activity was not caused by the cytotoxicity of
the compounds. The IC50 values (>50% inhibition) were measured in the
presence of serial dilutions of tested compounds, calculated by the
nonlinear regression analysis using GraphPad Prism (version 8.0;
GraphPad Software) and expressed as mean standard error of the
mean (SEM). The results of NO inhibition are tabulated in Table 3.
According to the results, compounds 4c and 4e were observed to
be the most active NO inhibitors, with IC50 values of 19.05 and
17.47 µM, respectively. It was apparent that the IC50 values of series
3 and 4 against NO inhibition were approximately twofold lower than
that of the MTT assay, signifying that the NO suppression activity
was not caused by the cytotoxicity of the tested compounds.
A structure–activity relationship (SAR) study was performed to
correlate the structural features with NO inhibitory effects.
FIGURE 3 NO inhibitory effects of
compounds 3a–e, 4a–e, and 5a–e at a testing
concentration of 50 µM

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2.3.2
AChE and BChE inhibition activity
The AChE and BChE inhibitory activities of the synthesized pyrazole,
phenylpyrazole, and pyrazoline derivatives of diarylpentanoids (series 3,
4, and 5) were evaluated according to Ellman's spectrophotometric
method with slight modifications. The inhibitory evaluation of the
compounds against AChE and BChE enzymes showed that only com-
pounds 3a, 3e, and 5a were found to be active against BChE, with a
strong selective inhibition on BChE over AChE (selective index of 2.3,
>3.3, and >6.6, respectively), whereas all analogs, except 3a and 3b,
poorly inhibited AChE. The IC50 values of most compounds could not be
calculated due to their weak inhibition potency, which did not exceed
50% at the highest concentration (100 µM).
FIGURE 4 Oak ridge thermal ellipsoid diagram of compound 5a
with thermal ellipsoids drawn at 50% probability
The SARs revealed that the pyrazole moiety might play a sup-
portive role as a scaffold for the inhibition of AChE, whereas the
phenylpyrazole and pyrazoline group were not suitable fragments for
the induction of anti‐AChE activity. Apparently, the addition of either
an electron‐donating or electron‐withdrawing atom (−Cl) on the ring
A moiety will only decrease the AChE inhibition, which suggests that
the AChE inhibitory activity was independent of the electron density
of the phenyl ring. The same trend could also be seen in the sup-
pression of the BChE enzyme.
monomer was nonplanar and adopted a trans configuration with re-
spect to the position of the 1‐(4,5‐dihydro‐5‐methyl‐3‐
phenylpyrazolyl‐1‐yl)ethanone and benzene across their C12–C13
double bond of 1.293(3) Å. The crystal system and refinement data,
together with the bond lengths and angles, are tabulated in Sup-
porting Information Materials. The crystallographic data for the
structural analysis were deposited with the Cambridge Crystal-
lographic Data Centre (CCDC) No. 1987366.
It is interesting to highlight that the most hydrophilic pyrazole
compound, 3a, exhibited the highest IC50 value for BChE inhibition,
followed by the slightly hydrophobic N‐acetylated pyrazoline, and
lastly, the most hydrophobic phenylpyrazole moiety recorded the
lowest BChE inhibition capacity. However, the replacement of
the phenyl group (ring B) with a heterocyclic furanyl in 3e lowered
the suppression activity by approximately twofold.
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2.5
Molecular docking
Molecular docking studies were performed to provide possible binding
interactions of the most bioactive compounds 3a and 5a within the
protein crystal structure of human BChE (PDB ID: 4BDS, 2.1 Å re-
solution). All the ligands were removed from the active pocket sites of
the 4BDS, while the obtained single‐crystal XRD atomic coordinates for
compounds 3a and 5a were then employed in the molecular docking
using Discovery Studio® 3.1 (Accelrys, San Diego).
To conclude, the pyrazole and pyrazoline scaffolds might be a
suitable structural motif for the selective inhibition of BChE enzyme
in AD therapy, as shown by compounds 3a, 3e, and 5a, compared
with the phenylpyrazole series. Although these compounds showed a
100 times weaker activity, compared with the standard tacrine, these
analogs still exist as one of the most active compounds in this enzyme
inhibition test and are worth further exploration into their structural
modifications to enhance their potency. Moreover, the addition of
strong EDG and EWG on ring A and/or ring B may further decrease
or deactivate the BChE inhibition effects of the analogs.
Although a new human BChE (hBChE) protein has been published
recently (PDB ID: 6QAE) with an X‐ray resolution of 2.49 Å, 4BDS was
chosen, as it possesses a better X‐ray resolution (2.1 Å) and is within the
acceptable range of X‐ray resolution for molecular docking prediction. It
has been reported that high‐resolution protein structures possess re-
solution values of 0–1 Å and the lower resolution structures possess a
resolution of >3 Å. However, most of the crystallographic‐defined pro-
tein structures are within the range of 1–3 Å. Therefore, the confidence
of molecular docking prediction is higher with a smaller resolution value
of protein.[37,38] The 4BDS protein has been even widely adopted to
study the interaction of bioactive ligands and hBChE by the scientific
community to date.[39–42]
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2.4
XRD
The characterization of compound 5a was conducted by a single‐
crystal structural analysis, with sufficient reflections for a structural
solution with an R1 value of 6%. Figure 4 shows the oak ridge
thermal ellipsoid plots of the crystallized compound. The refrain and
constraint technique was applied in the structural analysis due to the
presence of a crystallized water molecule possessing high thermal
parameters that impeded the analysis. The single‐crystal studies of
compound 5a adopted an orthorhombic system with a space group
Pī, a = 18.118(8) Å, b = 20.572(11) Å, c = 8.790(4) Å, α = β = γ = 90°,
Z = 8, and V = 3,276.3(3) Å3. It was observed that the asymmetric
On the basis of Figure 5a, it is observed that both phenyl rings in
compound 3a were bonded to the cationic π active site of the BChE
protein through π–π stacking and alkyl/π interactions with the indole
group of Trp82 (3.70 and 5.10 Å) and Ala328 with a distance of
5.19 Å. Besides, additional π–π interactions were observed between
a 2‐hydroxyphenyl moiety with residues Phe329 (5.73 Å) and indole
moiety of Trp231 with distances of 5.26 and 5.40 Å, respectively.

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For compound 5a, the existence of an acetyl group on the pyr-
azoline moiety caused a strong hydrogen bonding with Gly116 (2.86 Å),
together with a strong C–H interaction with residues of Gly115 (2.79 Å)
and Ser198 (2.52 Å), to elicit the compound's BChE inhibitory property
(Figure 5b). Moreover, its phenyl rings formed π–π interactions with
Trp82, Trp231, and Phe329 with a distance of 5.86, 5.98, and 5.60 Å,
respectively. Additionally, the 2‐hydroxyl group of ring A formed a
π–cation/anion interaction with residue His438 (2.31 Å).
selective BChE inhibitors. The molecular docking of compounds 3a and
5a with hBChE gave clear interpretations of the detailed interactions at
the active site of hBChE, in which the docking explained the importance
of the N‐acetylated moiety, together with the hydrophobic phenyl rings,
to elicit its BChE inhibition capability. On the basis of the overall results,
the phenylpyrazole skeleton would be a promising structural template
for the development of new anti‐inflammatory agents. Meanwhile, the
pyrazole and pyrazoline scaffolds could be further exploited for the
development of new cholinesterase inhibitors, particularly as a BChE
inhibitor for the treatment of AD. To conclude, we believe that this class
of new derivatives of diarylpentanoids containing N‐acetylated moiety is
promising and deserves further hit‐to‐lead optimization and in‐depth
mechanism studies for the development of novel cholinesterase
inhibitors.
To summarize, the molecular docking results showed that the
BChE inhibition effects of pyrazole compound (3a) were reflected via
π–π and alkyl/π interactions through the hydrophobic features of its
phenyl rings to the catalytic active site (Trp82 and Ala328) of BChE.
The finding also reveals the importance of the N‐acetylated moiety of
pyrazoline (5a) in exhibiting a significant BChE suppression activity,
as it formed crucial C–H/O hydrogen bonds with residues of the
catalytic triad of BChE (His438 and Ser198) and Gly116.
|
4
EXPERIMENTAL
|
|
In silico studies (PAINS, aggregator filtration,
3
CONCLUSION
4.1
and physicochemical properties)
In summary, we reported the in silico studies involving PAINS filtration,
physicochemical properties and molecular docking analyses, synthesis,
as well as biological evaluation of 15 new pyrazole, phenylpyrazole, and
pyrazoline analogs of diarylpentanoids. The results indicate that phe-
nylpyrazole derivatives (series 4) were potent NO inhibitors, whereas
pyrazole and pyrazoline analogs (3a, 3e, and 5a) were potent and
|
4.1.1
In silico PAINS identification and
physicochemical properties' determination
All diarylpentanoid analog chemical structures were drawn using
ChemDraw Ultra 12.0 and converted into the simplified
FIGURE 5 Three‐ and two‐dimensional (2D) diagram of the binding interaction between (a) compound 3a and (b) compound 5a with their
respective active BChE sites. Hydrogen atoms were omitted in the 2D diagram for clarity. The orange line represents the π–π interaction,
whereas the green line indicates hydrogen bonding. Green line, H bond; orange line, π–cation/anion; purple line, π–π stacked/T‐shaped; light
purple, alkyl/π–alkyl with distance in angstroms (Å)

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General procedure for the synthesis of series
3 and series 5
molecular input line entry system (SMILES) format. The SMILES
formula were then converted into Symyx spatial data file (SDF)
file format, as recommended by the FAF‐Drugs4 Bank Formatter
(http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#
forms::Bank-Formatter), before being automatically redirected as
the input file into the FAF‐Drugs4 server (http://mobyle.rpbs.univ-paris-
diderot.fr/cgi-bin/portal.py#forms::FAF-Drugs4) for the filtration of
PAINS with PAINS filters A, B, and C.[25]
4.2.2
The diarylpentanoid analogs were prepared following a previously
described method.[31,32] A mixture of the appropriate diarylpenta-
noids (1a–e, 2a–e; 1 mmol), hydrazine hydrate (10 mmol), and acetic
acid (12 ml) was refluxed for 3 hr. The resultant mixture was then
poured into crushed ice to quench the reaction and stirred for
15 min, followed by an extraction process with ethyl acetate
(3 × 20 ml). The resulting organic layer was collected and washed
with a NaHCO3 solution to remove excess acetic acid, dried over
anhydrous MgSO4, and concentrated under reduced pressure. The
crudes were subjected to purification by column chromatography on
silica gel, using hexane/ethyl acetate as a solvent system. The hy-
drogens and carbons of 1H and 13C NMR spectrum were assigned on
the basis of the numbering system as follows:
|
4.1.2
In silico aggregator identification
The SMILES file containing the synthesized molecules generated
from the in silico PAINS analysis was input into the Aggregator
Advisor server (http://advisor.bkslab.org/) for the identification of
potential aggregators.[26]
2‐(5‐Styryl‐1H‐pyrazol‐3‐yl)‐phenol (3a)
|
4.2
Chemistry
Beige solid (92%), m.p. 114–116°C. 1H NMR (500 MHz, CDCl3) δ:
6.80 (s, 1H, H‐2), 6.94 (dd, J = 7.4 Hz, 1H, H‐3′), 6.98 (d, J = 7.4 Hz, 1H,
H‐5), 7.07 (dd, J = 7.4 Hz, 1H, H‐4″), 7.11 (d, J = 7.4 Hz, 1H, H‐4), 7.25
(td, J = 7.4, 3.7 Hz, 1H, H‐5′), 7.33 (td, 1H, H‐4′), 7.39 (td, J = 7.4,
3.7 Hz, 2H, H‐3″, H‐5″), 7.48 (dd, J = 7.3 Hz, 2H, H‐2″, H‐6″), 7.61 (dd,
J = 7.6, 3.7 Hz, 1H, H‐6′), 9.94 (br s, 1H, −NH), 10.69 (br s, 1H, −OH).
13C NMR (126 MHz, CDCl3) δ: 99.7 (C‐2), 114.4 (C‐3′), 116.5 (C‐5′),
117.1 (C‐1′), 119.5 (C‐4), 126.6 (C‐6′), 126.7 (C‐2″, C‐6″), 128.6
(C‐4″), 128.9 (C‐3″, C‐5″), 129.4 (C‐4′), 132.1 (C‐5), 135.9 (C‐3), 142.1
(C‐1″), 152.7 (C‐1), 155.8 (C‐2′). High‐resolution mass spectrometry
(HRMS) electrospray ionization (ESI): m/z calcd. for C17H14N2O–H–:
261.1045 [M−H]–; found: 261.1048.
|
4.2.1
Materials and instrumentations
All chemicals and reagents were purchased from Merck, Sigma‐
Aldrich, and Thermo Fischer Scientific. All solvents were dried
and distilled before use. The solvents used in the spectroscopic
measurements were of spectroscopic grade. The reaction mix-
tures were extracted with organic solvents, dried over anhydrous
magnesium sulfate, and then concentrated in vacuo. To monitor
the progress of a reaction, thin‐layer chromatography (TLC) was
performed on aluminum TLC sheets of silica gel 60 F254 (layer
thickness, 0.2 mm) from Merck. Column chromatography pur-
ification was performed on silica gel 60 (70–230 mesh ASTM;
Merck). Mass spectra were measured on a GCMS‐QP5050A mass
spectrometer (Shimadzu, Kyoto, Japan). NMR spectra were re-
corded using a Varian 500 MHz NMR spectrometer (Varian Inc.,
Palo Alto, CA), and the melting points were determined using a
Fisher–Johns melting point apparatus.
4‐Chloro‐2‐(5‐styryl‐1H‐pyrazol‐3‐yl)‐phenol (3b)
White solid (98%), m.p. 149–150°C. 1H NMR (500 MHz, CDCl3) δ:
6.78 (s, 1H, H‐2), 6.96 (dd, J = 7.4 Hz, 1H, H‐5), 6.98 (d, J = 7.6, 1.5 Hz,
1H, H‐6′), 7.13 (d, J = 7.4 Hz, 1H, H‐4), 7.18 (dt, J = 7.4 Hz, 1H, H‐4′),
7.34 (td, J = 7.4 Hz, 1H, H‐4″), 7.40 (td, J = 7.4, 3.7 Hz, 2H, H‐3″, H‐5″),
7.50 (dd, J = 7.4, 3.7 Hz, 2H, H‐2″, H‐6″), 7.55 (d, J = 7.4 Hz, 1H, H‐3′),
10.12 (br s, 1H, −NH), 10.78 (br s, 1H, −OH). 13C NMR (126 MHz,
CDCl3) δ: 99.8 (C‐2), 113.4 (C‐3′), 117.7 (C‐4), 118.5 (C‐1), 124.1
(C‐6′), 126.0 (C‐5′), 126.7 (C‐2″, C‐6″), 128.8 (C‐4″), 128.9 (C‐3″,
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Informa-
tion Data.

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C‐5″), 129.1 (C‐4′), 132.7 (C‐5), 135.7 (C‐3), 142.2 (C‐1″), 151.8 (C‐1),
154.6 (C‐2′). HRMS (ESI): m/z calcd. for C17H13ClN2O–H–: 296.0994
[M−H]–; found: 296.0995.
1‐(3‐Phenyl‐5‐styryl‐4,5‐dihydro‐pyrazol‐1‐yl)‐ethanone (5a)
Light yellow solid (29%), m.p. 103–104°C. 1H NMR (500 MHz, CDCl3)
δ: 2.43 (s, 3H, COCH3), 3.14 (dd, J = 17.6, 4.4 Hz, 1H, H‐2trans), 3.56
(dd, J = 17.6, 11.7 Hz, 1H, H‐2cis), 5.30 (dt, J = 11.2, 5.6 Hz, 1H, H‐3),
6.23 (dd, J = 15.9, 7.1 Hz, 1H, H‐4), 6.62 (d, J = 16.1 Hz, 1H, H‐5),
7.19–7.31 (m, 3H, H‐3″, H‐4″, H‐5″), 7.29–7.31 (m, 1H), 7.37
(d, J = 7.8 Hz, 2H, H‐2″, H‐6″), 7.44 (s, 3H, H‐3′, H‐4′, H‐5′), 7.76
(s, 2H, H‐2′, H‐6′). 13C NMR (126 MHz, CDCl3) δ: 22.1 (C‐7), 39.4 (C‐
2), 57.9 (C‐3), 126.5 (C‐1′, C‐2′, C‐6′), 126.6 (C‐2″, C‐6″), 127.1 (C‐4),
127.1 (C‐4), 127.8 (C‐4″), 128.5 (C‐3″, C‐5″), 128.7 (C‐3′, C‐5′), 130.3
(C‐4′), 131.5 (C‐5), 136.2 (C‐1″), 154.2 (C‐1), 169.1 (C‐6). HRMS (ESI):
m/z calcd. for C19H18N2O–H–: 289.1419 [M−H]–; found: 289.1521.
4‐Methoxy‐2‐(5‐styryl‐1H‐pyrazol‐3‐yl)‐phenol (3c)
White solid (85%), m.p. 108–110°C. 1H NMR (500 MHz, CDCl3) δ:
3.83 (s, 3H, –OCH3), 6.75 (s, 1H, H‐2), 6.84 (dd, J = 8.7, 2.9 Hz, 1H,
H‐4′), 6.93 (d, J = 14.8 Hz, 1H, H‐5), 6.99 (d, J = 8.7 Hz, 1H, H‐3′), 7.08
(d, J = 11.0 Hz, 1H, H‐4), 7.15 (dd, J = 3.7 Hz, 1H, H‐6′), 7.32 (td, 1H,
H‐4″), 7.37 (td, J = 7.4 Hz, 2H, H‐3″, H‐5″), 7.54 (dd, J = 7.6 Hz, 2H,
H‐2″, H‐6″), 10.48 (br s, 2H, −NH, −OH). 13C NMR (126 MHz, CDCl3)
δ: 56.0 (−OCH3), 99.7 (C‐2), 111.6 (C‐6′), 114.5 (C‐4′), 115.3 (C‐3′),
116.7 (C‐1′), 117.6 (C‐4), 126.7 (C‐2″, C‐6″), 128.6 (C‐4″), 128.8
(C‐3″, C‐5″), 132.1 (C‐5), 135.9 (C‐3), 142.3 (C‐1″), 149.9 (C‐2′), 152.3
(C‐1), 152.7 (C‐5′). HRMS (ESI): m/z calcd. for C18H16N2O2–H–:
291.1150 [M−H]–; found: 291.1152.
1‐[3‐(2‐Hydroxyphenyl)‐5‐styryl‐4,5‐dihydro‐pyrazol‐1‐yl]‐
ethanone (5b)
Yellow solid (25%), m.p. 137–138°C. 1H NMR (500 MHz, CDCl3) δ:
2.40 (s, 3H, −COCH3), 3.27 (dd, J = 17.6, 4.4 Hz, 1H, H‐2trans), 3.67
(dd, J = 17.6, 11.2 Hz, 1H, H‐2cis), 5.24–5.32 (m, 1H, H‐3), 6.21
(dd, J = 15.6, 7.3 Hz, 1H, H‐4), 6.65 (d, J = 15.6 Hz, 1H, H‐5), 6.97
(t, J = 7.3 Hz, 1H, H‐3′), 7.07 (d, J = 8.3 Hz, 1H, H‐2′), 7.28–7.34
(m, 3H, H‐4′, H‐5′, H‐4″), 7.35–7.41 (m, 4H, H‐2″, H‐3″, H‐5″, H‐6″), 10.24
(s, 1H, −OH). 13C NMR (126 MHz, CDCl3) δ: 22.2 (C‐7), 39.8
(C‐2), 56.6 (C‐3), 115.2 (C‐1′), 117.1 (C‐3′), 119.7 (C‐5′), 126.2 (C‐4),
126.6 (C‐2″, C‐6″), 128.0 (C‐4″), 128.4 (C‐6′), 128.5 (C‐4′), 132.2
(C‐3″, C‐5″), 135.9 (C‐1″), 156.6 (C‐1), 157.6 (C‐2), 168.0 (C‐6). HRMS
(ESI): m/z calcd. for C19H18N2O2–H−: 305.1368 [M−H]−; found: 305.1370.
2,4‐Dichloro‐6‐(5‐styryl‐1H‐pyrazol‐3‐yl)‐phenol (3d)
White solid (94%), m.p. 224–225°C. 1H NMR (500 MHz, acetone‐d6)
δ: 7.22 (d, 2H, H‐2, H‐4), 7.34 (d, J = 7.4 Hz, 1H, H‐5), 7.37 (d,
J = 3.7 Hz, 1H, H‐6′), 7.42 (m, 3H, H‐3″, H‐4″, H‐5″), 7.61 (dd,
J = 7.4 Hz, 2H, H‐2″, H‐6″), 7.74 (d, J = 3.7 Hz, 1H, H‐4′), 11.81 (br s,
1H, −NH), 12.83 (br s, 1H, −OH). 13C NMR (126 MHz, acetone‐d6) δ:
100.2 (C‐2), 114.5 (C‐1), 119.3 (C‐4), 122.0 (C‐1′), 123.4 (C‐6′), 124.7
(C‐4″), 126.7 (C‐2″, C‐6″), 128.0 (C‐5′), 128.3 (C‐4′), 128.5 (C‐5),
128.8 (C‐3″, C‐5″), 132.6 (C‐3), 136.3 (C‐1″), 143.0 (C‐1), 151.0 (C‐2′).
HRMS (ESI): m/z calcd. for C17H12Cl2N2O–H–: 330.0950 [M−H]–;
found: 330.0953.
1‐{5‐[2‐(4‐Methoxyphenyl)‐vinyl]−3‐phenyl‐4,5‐dihydro‐pyrazol‐1‐
yl}‐ethanone (5c)
2‐[5‐(2‐Furan‐2‐yl‐vinyl)−1H‐pyrazol‐3‐yl]‐phenol (3e)
Light yellow solid (82%), m.p. 117–118°C. 1H NMR (500 MHz, CDCl3) δ:
2.43 (s, 3H, −COCH3), 3.13 (dd, J = 17.6, 4.4 Hz, 1H, H‐2trans), 3.4–3.59
(dd, J = 17.6, 11.2 Hz, 1H, H‐2cis), 3.80 (s, 3H, –OCH3), 5.24–5.32 (m, 1H,
H‐3), 6.08 (dd, J = 15.9, 7.1 Hz, 1H, H‐4), 6.56 (d, J = 15.6 Hz, 1H, H‐5),
6.83 (d, J = 8.8 Hz, 2H, H‐2″, H‐6″), 7.30 (d, J = 8.8 Hz, 2H, H‐2′, H‐6′),
7.40–7.48 (m, 3H, H‐3′, H‐4′, H‐5′), 7.76 (d, 2H, H‐2″, H‐6″). 13C NMR
(126 MHz, CDCl3) δ: 22.1 (C‐7), 39.4 (C‐2), 55.2 (−OCH3), 58.0 (C‐3),
113.9 (C‐3″, C‐5″), 124.9 (C‐2′, C‐6′), 126.5 (C‐4), 127.8 (C‐3′, C‐5′),
128.7 (C‐2″, C‐6″), 129.0 (C‐4′), 130.2 (C‐1″), 130.9 (C‐5), 131.5 (C‐1′),
154.1 (C‐1), 159.4 (C‐4″), 169.1 (C‐6). HRMS (ESI): m/z calcd. for
C20H20N2O2–H–: 319.1525 [M−H]–; found: 319.1529.
Brown solid (86%), m.p. 128–129°C. 1H NMR (500 MHz, acetone‐d6)
δ: 6.54 (d, J = 3.7 Hz, 1H, H‐2″), 6.58 (d, J = 3.7 Hz, 1H, H‐3″), 6.93
(m, 2H, H‐4, H‐4″), 7.03 (d, J = 16.6 Hz, 1H, H‐5), 7.08 (s, 1H, H‐2),
7.20 (m, 1H, H‐6′), 7.23 (m, 1H, H‐4′), 7.63 (td, J = 7.4 Hz, 1H, H‐5′),
7.73 (dd, J = 7.4 Hz, 1H, H‐3′), 10.89 (br s, 1H, −NH), 12.56 (br s, 1H,
−OH). 13C NMR (126 MHz, acetone‐d6) δ: 99.3 (C‐2), 110.3 (C‐2″),
112.0 (C‐3″), 113.1 (C‐3′), 116.7 (C‐5′), 116.8 (C‐1′), 119.1 (C‐6′),
119.4 (C‐4′), 126.6 (C‐3), 129.0 (C‐4, C‐5), 143.3 (C‐4″), 152.0 (C‐1),
152.2 (C‐1″), 156.2 (C‐2′). HRMS (ESI): m/z calcd. for
C15H12N2O2–H–: 251.0655 [M−H]–; found: 251.0657.

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1‐{3‐(4‐Fluorophenyl)−5‐[2‐(4‐methoxyphenyl)‐vinyl]−4,5‐dihydro‐
pyrazol‐1‐yl}‐ethanone (5d)
hydrogens and carbons of 1H and 13C NMR spectrum were assigned on
the basis of the numbering system as drawn:
Yellow solid (78%), m.p. 130–131°C. 1H NMR (500 MHz, CDCl3) δ:
2.41 (s, 3H, −COCH3), 3.10 (dd, J = 17.6, 4.4 Hz, 1H, H‐2trans), 3.53
(dd, J = 17.3, 11.0 Hz, 1H, H‐2cis), 3.80 (s, 1H, −OCH3), 5.24–5.33
(m, 1H, H‐3), 6.07 (dd, J = 15.6, 6.8 Hz, 1H, H‐4), 6.55 (d, J = 15.6 Hz,
1H, H‐5), 6.83 (d, J = 8.3 Hz, 2H, H‐3″, H‐5″), 7.13 (t, J = 8.5 Hz, 2H, H‐
3′, H‐5′), 7.30 (d, J = 8.3 Hz, 2H, H‐2″, H‐6″), 7.75 (dd, J = 8.5, 5.6 Hz,
2H, H‐2′, H‐6′). 13C NMR (126 MHz, CDCl3) δ: 22.1 (C‐7), 39.5 (C‐2),
55.3 (−OCH3), 58.1 (C‐3), 113.9 (C‐3″, C‐5″), 115.8 (C‐3′, C‐5′), 124.7
(C‐4), 127.8 (C‐1′), 128.4 (C‐2″, C‐6″), 128.9 (C‐5), 131.0 (C‐2′, C‐6′),
153.1 (C‐1), 159.4 (C‐4″), 163.8 (C‐4″), 169.0 (C‐6). HRMS (ESI): m/z
calcd. for C20H19FN2O2–H−: 337.1431 [M−H]–; found: 337.1434.
2‐(2‐Phenyl‐5‐styryl‐2H‐pyrazol‐3‐yl)‐phenol (4a)
White solid (89%), m.p. 145–146°C. 1H NMR (500 MHz, CDCl3) δ:
5.62 (br s, 1H, −OH), 6.79 (s, 1H, H‐2), 6.85 (td, J = 7.5, 1.1 Hz, 1H,
H‐5′), 6.94 (dd, J = 8.2, 0.9 Hz, 1H, H‐3′), 7.00 (dd, J = 7.6, 1.5 Hz,
1H, H‐4′), 7.17–7.32 (m, 9H, H‐4, H‐5, H‐6′, H‐4″, H‐2‴, H‐3‴,
H‐4‴, H‐5‴, H‐6‴), 7.38 (t, J = 7.6 Hz, 2H, H‐3″, H‐5″), 7.53
(d, J = 7.4 Hz, 1H, H‐2″, H‐6″). 13C NMR (126 MHz, CDCl3) δ:
105.9 (C‐2), 116.2 (C‐3′), 117.1 (C‐5′), 120.1 (C‐1′), 120.6 (C‐4′),
124.1 (C‐2‴, C‐6‴), 126.6 (C‐2″, C‐6″), 127.4 (C‐4‴), 127.9 (C‐3),
128.7 (C‐3″, C‐5″), 128.9 (C‐3‴, C‐5‴), 130.8 (C‐4″), 130.9 (C‐6′),
131.2 (C‐4′), 136.9 (C‐5), 138.7 (C‐1″), 139.6 (C‐1‴), 151.6 (C‐2′),
153.4 (C‐1). HRMS (ESI): m/z calcd. for C23H18N2O–H–: 337.0604
[M−H]–; found: 337.0607.
1‐{5‐[2‐(4‐Methoxyphenyl)‐vinyl]−3‐(5‐methylthiophen‐2‐yl)4,5‐
dihydro‐pyrazol‐1‐yl}‐ethanone (5e)
Yellow solid (76%), m.p. 116–117°C. 1H NMR (500 MHz, CDCl3) δ:
2.37 (s, 3H, −COCH3), 2.53 (s, 3H, −CH3), 3.05 (dd, J = 17.6, 4.4 Hz,
1H, H‐2trans), 3.50 (dd, J = 17.3, 11.0 Hz, 1H, H‐2cis), 3.80 (s, 3H,
−OCH3), 5.20–5.28 (m, 1H, H‐3), 6.04–6.08 (dd, J = 7.3, 6.8 Hz, 1H,
H‐4), 6.54 (d, J = 15.6 Hz, 1H, H‐5), 6.74 (d, 1H, H‐3′), 6.83
(d, J = 8.8 Hz, 2H, H‐3″, H‐5″), 7.04 (d, J = 3.4 Hz, 1H, H‐2′), 7.30
(d, J = 8.3 Hz, 2H, H‐2″, H‐6″). 13C NMR (126 MHz, CDCl3) δ: 15.7
(−CH3), 22.0 (C‐7), 39.9 (C‐2), 55.3 (−OCH3), 58.0 (C‐3), 113.9 (C‐3″,
C‐5″), 124.7 (C‐3′), 125.9 (C‐4), 127.8 (C‐2″, C‐6″), 129.0 (C‐1″),
130.9 (C‐2′), 132.8 (C‐5), 144.0 (C‐4′), 150.1 (C‐1), 159.3 (C‐4″),
168.8 (C‐6). HRMS (ESI): m/z calcd. for C19H20N2O2S–H–: 339.1245
[M−H]–; found: 339.1248.
4‐Chloro‐2‐(2‐phenyl‐5‐styryl‐2H‐pyrazol‐3‐yl)‐phenol (4b)
White solid (92%), m.p. 168–169°C. 1H NMR (500 MHz, acetone‐
d6) δ: 6.86 (s, 1H, H‐2), 6.91 (d, J = 8.4 Hz, 1H, H‐4′), 7.22–7.32
(m, 6H, H‐6′, H‐2‴, H‐3‴, H‐4‴, H‐5‴, H‐6‴), 7.33–7.43 (m, 6H, H‐4, H‐5,
H‐3′, H‐3″, H‐4″, H‐5″), 7.60–7.65 (d, J = 8.4 Hz, 2H, H‐2″, H‐6″), 8.80 (s,
1H, −OH). 13C NMR (126 MHz, acetone‐d6) δ: 106.7 (C‐2), 117.5 (C‐3′),
120.1 (C‐4), 120.7 (C‐1′), 123.5 (C‐2‴, C‐6‴), 123.7 (C‐4‴), 126.4 (C‐2″,
C‐6″), 126.8 (C‐5′), 127.6 (C‐4″), 128.6 (C‐3″, C‐5″), 128.7 (C‐3‴, C‐5‴),
130.0 (C‐6′), 130.5 (C‐4′), 137.3 (C‐5), 139.4 (C‐1″), 140.9 (C‐1‴), 150.8
(C‐2′), 153.8 (C‐1). HRMS (ESI): m/z calcd. for C23H17ClN2O–H–:
372.0214 [M−H]–; found: 372.0215.
4‐Methoxy‐2‐(2‐phenyl‐5‐styryl‐2H‐pyrazol‐3‐yl)‐phenol (4c)
White solid (86%), m.p. 128–129°C. 1H NMR (500 MHz, CDCl3) δ:
3.59 (s, 3H, –OCH3), 5.50 (br s, 1H, –OH), 6.54 (d, J = 2.3 Hz, 1H,
H‐6′), 6.77 (s, 1H, H‐2), 6.80–6.87 (m, 2H, H‐4, H‐4′), 7.16–7.34
(m, 8H, H‐5, H‐3′, H‐4″, H‐2‴, H‐3‴, H‐4‴, H‐5‴, H‐6‴), 7.37 (td,
J = 7.6 Hz, 2H, H‐3″, H‐5″), 7.52 (d, J = 7.6 Hz, 2H, H‐2″, H‐6″). 13C
NMR (126 MHz, CDCl3) δ: 55.7 (−OCH3), 105.9 (C‐2), 115.2
(C‐6′), 117.0 (C‐4′), 117.2 (C‐3′), 117.5 (C‐1′), 120.0 (C‐4), 124.1
(C‐2‴, C‐6‴), 126.6 (C‐2″, C‐6″), 127.4 (C‐4‴), 128.7 (C‐3″, C‐5″),
128.9 (C‐3‴, C‐5‴), 131.2 (C‐4″), 136.9 (C‐5), 139.1 (C‐1″), 139.6
(C‐1‴), 147.4 (C‐2), 151.5 (C‐5′), 153.2 (C‐1). HRMS (ESI): m/z
calcd. for C24H20N2O2–H–: 367.0765 [M−H]–; found: 367.0768.
|
4.2.3
General procedure for the synthesis of
series 4
Diarylpentanoids (2a–e, 0.5 mmol) and phenylhydrazine (1 mmol) were
dissolved in acetic acid and refluxed for 2 hr. The resultant reaction
mixture was then poured into cold water to quench the reaction and
stirred for 30 min, followed by extraction with ethyl acetate. The or-
ganic layer was collected and further washed with NaHCO3 solution to
remove the acetic acid. The resultant organic layer was then dried over
anhydrous magnesium sulfate and concentrated in vacuo. The crude
obtained was purified by gravitational column chromatography. The
2,4‐Dichloro‐6‐(2‐phenyl‐5‐styryl‐2H‐pyrazol‐3‐yl)‐phenol (4d)
White solid (90%), m.p. 154–156°C. 1H NMR (500 MHz, acetone‐
d6) δ: 5.82 (br s, 1H, −OH), 6.77 (s, 1H, H‐2), 7.09 (d, J = 0.6 Hz,
1H, H‐4′), 7.16–7.25 (m, 2H, H‐4, H‐6′), 7.27–7.41 (m, 9H, H‐5,
H‐3″, H‐4″, H‐5″, H‐2‴, H‐3‴, H‐4‴, H‐5‴, H‐6‴), 7.53
(d, J = 7.9 Hz, 2H, H‐2″, H‐6″). 13C NMR (126 MHz, acetone‐d6) δ: 106.7
(C‐2), 120.0 (C‐3′), 120.1 (C‐4), 121.4 (C‐4‴), 123.9 (C‐2‴, C‐6‴), 125.3 (C‐
1′), 126.6 (C‐2″, C‐6″), 127.5 (C‐6′), 127.9 (C‐3), 128.7 (C‐3″, C‐5″), 129.0
(C‐3‴, C‐5‴), 129.4 (C‐4″), 129.6 (C‐5′), 131.1 (C‐4′), 136.9 (C‐5), 137.7
(C‐1″), 139.9 (C‐1‴), 147.9 (C‐2′), 151.3 (C‐1). HRMS (ESI): m/z calcd. for
C23H16Cl2N2O–H–: 406.1150 [M−H]–; found: 406.1154.

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|
AChE and BChE inhibition assays
2‐{5‐[2‐(2‐Chlorophenyl)‐vinyl]−2‐phenyl‐5‐2H‐pyrazol‐3‐yl}‐4‐
methoxyphenol (4e)
4.3.2
Beige solid (97%), m.p. 134–135°C. 1H NMR (500 MHz, CDCl3) δ:
3.60 (s, 3H, −OCH3), 5.42 (br s, 1H, −OH), 6.56 (s, 1H, H‐2),
6.79–6.86 (m, 3H, H‐4, H‐6′, H‐4″), 7.17–7.34 (m, 8H, H‐3″, H‐5″,
H‐6″, H‐2‴, H‐3‴, H‐4‴, H‐5‴, H‐6‴), 7.39 (d, J = 7.9 Hz, 1H, H‐4′),
7.58 (d, J = 16.3 Hz, 1H, H‐5), 7.69 (d, J = 7.6 Hz, 1H, H‐3′). 13C
NMR (126 MHz, CDCl3) δ: 55.7 (–OCH3), 106.1 (C‐2), 115.2
(C‐6′), 117.0 (C‐4′), 117.2 (C‐3′), 117.5 (C‐1′), 122.7 (C‐4), 124.1
(C‐2‴, C‐6‴), 126.5 (C‐4‴), 127.0 (C‐5″), 127.1 (C‐3), 127.4 (C‐6″),
128.8 (C‐3″), 128.9 (C‐3‴, C‐5‴), 129.8 (C‐4″), 133.4 (C‐5), 135.0
(C‐2″), 139.2 (C‐1″), 139.6 (C‐1‴), 147.4 (C‐2′), 151.3 (C‐5′),
153.2 (C‐1). HRMS (ESI): m/z calcd. for C24H19ClN2O2–H–:
402.1201 [M−H]–; found: 402.1203.
AChE and BChE inhibitory activity of pyrazoline analogs of dia-
rylpentanoids was evaluated via Ellman's method with slight mod-
ifications. Electric eel AChE (type V‐S, Sigma‐Aldrich; code: C2888)
and equine serum BChE (Sigma‐Aldrich; code: C7512) were used as
sources of cholinesterases, whereas acetylthiocholine iodide
(Sigma‐Aldrich; [ATCI] code: A5751) and S‐butyrylthiocholine iodide
(Sigma‐Aldrich; [BTCI] code: 20820) were used as the substrates.
The enzymes were prepared in 0.1 M sodium phosphate buffer
(pH 7.4) with a concentration of 0.25 units/ml, whereas the sub-
strates were prepared in water with a concentration of 0.25 mM.
For cholinesterase inhibitory assays, 190 µl of 0.15 mM 5,5‐
dithiobis‐2‐nitrobenzoic acid (Sigma‐Aldrich; code: D21200, 99%) in
sodium phosphate buffer (0.1 M, pH 7.4) was added to a 96‐well
microplate, followed by 20 µl of AChE/BChE solution and 20 µl of
test compounds with different concentrations. The mixture was in-
cubated at 37°C for 30 min. Then, 20 µl of ATCI/BTCI was added to
initiate the enzyme reaction and the reaction mixture was further
incubated for 30 min at 37°C. The absorbance of the end product was
measured at 412 nm using a SpectraMax Plus 384 microplate reader
(Molecular Devices LLC, Sunnyvale, CA). All reactions were carried
out in triplicates. The IC50 values (>50% inhibition) were measured in
the presence of serial dilutions of tested compounds, calculated by
the nonlinear regression analysis using GraphPad Prism (version 8.0;
GraphPad Software) and expressed as mean SEM.[43]
|
4.3
Pharmacological/biological assays
|
4.3.1
Nitric oxide inhibitory assay
Cell culture
The RAW 264.7 murine macrophage cells obtained from American
Type Culture Collection (ATCC, Rockville, MD) were grown in Dul-
becco's modified Eagle's medium (DMEM) containing 10% fetal bo-
vine serum and 1% penicillin/streptomycin in a 95% air and 5% CO2
atmosphere at 37°C.[36]
Nitrite determination (Griess assay)
|
The RAW 264.7 cells at 90–95% confluency were detached and
seeded (50,000 cells/well) into a 96‐well culture plate with 50 μl
of DMEM and incubated for 24 hr. The cells were then stimulated
in 5 mg/ml of lipopolysaccharide (Escherichia coli, serotype
0111:B4) and 1 ng/ml of interferon‐gamma (IFN‐γ) in the pre-
sence or absence of test compounds for 17 hr. The nitrite pro-
duction was then determined by a Griess assay. Briefly, 50 μl of
Griess reagent (1% sulfanilamide and 0.1% N‐(1‐naphthyl)
ethylenediamine dihydrochloride in 2.5% phosphoric acid) was
added to 50 μl of cell culture supernatant. The optical density was
measured at 550 nm with a microplate reader after 5 min of in-
cubation at room temperature.[36]
4.4
X‐ray structure determination
Single‐crystal X‐ray experiment of 5a was performed on a Bruker
D‐QUEST diffractometer using graphite‐monochromated Mo‐Kα ra-
diation (λ = 0.71073 Å). The intensity data was measured at 301ºK by
2θ–ω scan method. Accurate cell parameters and orientation matrix
were determined by the least‐squares fit of 25 reflections. Intensity
data were collected for Lorentz and polarization effects. Empirical
absorption correction was carried out using a multiscan. The struc-
ture was solved by direct methods, and the least‐squares refinement
of the structure was performed by the SHELXL‐97 program. All the
non‐hydrogen atoms were refined anisotropically.[44] The hydrogen
atoms were placed in calculated positions, allowing them to ride on
their parent C atom with Uiso(H) = xUeq(C), whereby x = 1.5 for me-
thyl and 1.2 for non‐methyl groups, except the hydrogen atoms at-
tached to oxygen atoms that were located from Fourier maps and
refined isotropically.
Cell cytotoxicity determination (MTT assay)
To determine that the observed nitric oxide inhibition was not
falsely positive due to cytotoxic effects, a cytotoxicity assay was
also performed following the culture assay. After removal of the
supernatant, 100 µl DMEM and 20 µl of MTT (5 mg/ml) were
added to all the wells. The plate was then incubated in 5% CO2 at
37°C for 4 hr. The supernatants in all wells were discarded, and
the formazan crystals formed were dissolved in dimethyl sulf-
oxide (DMSO) and further incubated for 15 min at room tem-
perature. The absorbance was measured at 570 nm using a
microplate reader.[36]
|
4.5
Molecular docking
All molecular modeling methods were performed using Discovery Stu-
dio 3.1 (Accelrys) on an Intel® (TM)2 Quad CPU Q8200 @2.33 GHz
running under a Windows XP Professional environment.[44]

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ACKNOWLEDGMENTS
[24] J.Bisson, J. B. McAlpine, J. B. Friesen, S. N. Chen, J. Graham, G. F.
Pauli, J. Med. Chem. 2016, 59, 1671.
This study was financially supported by Universiti Putra Malaysia
under the Research University Grant Scheme (RUGS) (9301700) and
Putra grant—Putra Young Initiative (IPM) with sponsorship reference
number GP‐IPM/2016/9480700. The first author gratefully ac-
knowledges support by the Ministry of Education Malaysia for
funding his study under the SLAB scheme.
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ORCID
Siti Munirah Mohd Faudzi
http://orcid.org/0000-0001-8043-798X
https://orcid.org/0000-0003-0021-3092
https://orcid.org/0000-0002-8110-9424
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S. Wei Leong
Faridah Abas
Lam K. Wai
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Rullah, K. W. Lam, M. N. Bahari, S. Ahmad, C. L. Tham, K. Shaari, N. H.
Lajis, Molecules 2014, 19, 16058.
https://orcid.org/0000-0002-4366-6772
https://orcid.org/0000-0002-9734-0372
https://orcid.org/0000-0002-7054-0649
Chau L. Tham
Khozirah Shaari
[36] S. M.Mohd Faudzi, S. W. Leong, F. Abas, M. F. F. Mohd Aluwi, K.
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How to cite this article: Mohd Faudzi SM, Leong SW, Auwal
FA, et al. In silico studies, nitric oxide, and cholinesterases
inhibition activities of pyrazole and pyrazoline analogs of
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https://doi.org/10.1002/ardp.202000161