Synthesis, biological evaluation, and docking studies of some 5-chloro-2(3H)-benzoxazolone Mannich bases derivatives as cholinesterase inhibitors

Article abstract of DOI:10.1002/ardp.201700273

A series of N-substituted-5-chloro-2(3H)-benzoxazolone derivatives were synthesized and evaluated for their acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) inhibitory, and antioxidant activities. The structures of the title compounds were confirmed by spectral and elemental analyses. The cholinesterase (ChE) inhibitory activity studies were carried out using Ellman's colorimetric method. The free radical scavenging activity was also determined by in vitro ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The biological activity results revealed that all of the title compounds displayed higher AChE inhibitory activity than the reference compound, rivastigmine, and were selective for AChE. Among the tested compounds, compound 7 exhibited the highest inhibition against AChE (IC₅₀ = 7.53 ± 0.17 μM), while compound 11 was found to be the most active compound against BuChE (IC₅₀ = 17.50 ± 0.29 μM). The molecular docking study of compound 7 showed that this compound can interact with the catalytic active site (CAS) of AChE and also has potential metal chelating ability and a proper log P value. On the other hand, compound 2 bearing a methyl substituent at the ortho position on the phenyl ring showed better radical scavenging activity (IC₅₀ = 1.04 ± 0.04 mM) than Trolox (IC₅₀ = 1.50 ± 0.05 mM).

Full text of DOI:10.1002/ardp.201700273

Received: 22 August 2017
Revised: 19 February 2018
Accepted: 20 February 2018
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DOI: 10.1002/ardp.201700273
FULL PAPER
Synthesis, biological evaluation, and docking studies of some
5-chloro-2(3H)-benzoxazolone Mannich bases derivatives as
cholinesterase inhibitors
Sirin Uysal1
Sulunay Parlar1
Ayse H. Tarikogullari1
Fadime Aydin Kose2
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Vildan Alptuzun1
Zeynep Soyer1
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1 Faculty of Pharmacy, Department of
Abstract
Pharmaceutical Chemistry, Ege University,
Izmir, Turkey
A series of N-substituted-5-chloro-2(3H)-benzoxazolone derivatives were synthe-
sized and evaluated for their acetylcholinesterase (AChE), butyrylcholinesterase
(BuChE) inhibitory, and antioxidant activities. The structures of the title compounds
were confirmed by spectral and elemental analyses. The cholinesterase (ChE)
inhibitory activity studies were carried out using Ellman's colorimetric method. The
free radical scavenging activity was also determined by in vitro ABTS (2,2-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The biological activity
results revealed that all of the title compounds displayed higher AChE inhibitory
activity than the reference compound, rivastigmine, and were selective for AChE.
Among the tested compounds, compound 7 exhibited the highest inhibition against
AChE (IC50 = 7.53 0.17 μM), while compound 11 was found to be the most active
compound against BuChE (IC50 = 17.50 0.29 μM). The molecular docking study of
compound 7 showed that this compound can interact with the catalytic active site
(CAS) of AChE and also has potential metal chelating ability and a proper log P value.
On the other hand, compound 2 bearing a methyl substituent at the ortho position
on the phenyl ring showed better radical scavenging activity (IC50 = 1.04 0.04 mM)
than Trolox (IC50 = 1.50 0.05 mM).
2 Faculty of Pharmacy, Department of
Biochemistry, Ege University, Izmir, Turkey
Correspondence
Dr. Zeynep Soyer, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Ege
University, 35100 Bornova, İzmir, Turkey.
Email: zeynep.soyer@ege.edu.tr
K E Y W O R D S
2(3H)-benzoxazolone, antioxidant activity, cholinesterase inhibitors, Mannich, molecular
docking
role in the pathophysiology of AD.[4–7] The cholinergic hypothesis was
the first theory proposed to explain AD. According to this hypothesis,
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1
INTRODUCTION
Alzheimer's disease (AD) is a chronic progressive neurodegenerative
disorder characterized by memory loss, language skill and intellectual
ability degression in the elderly population.[1–3] Although the etiology
of AD is not fully understood, several factors such as cholinergic
dysfunction, τ protein aggregation, amyloid-β-deposits, dysregulation
of biometals and oxidative stress are considered to play an important
the main cause of the loss of cognitive functions on AD patients is a
continuous decline of the cholinergic transmission in specific regions
of the brain. After the cholinergic neurotransmission mediated by
acetylcholine (ACh) is accomplished, ACh is rapidly hydrolysed by
acetylcholinesterase (AChE). The decrease of acetylcholine levels in
brain was thought as the widely approved reason of AD. Currently, the
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Arch Pharm Chem Life Sci. 2018;e1700273.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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most efficacious approach for AD is considered to increase cholinergic
neurotransmission in the brain by lowering ACh hydrolysis.[8,9]
2
RESULTS AND DISCUSSION
High levels and dysregulation of biometal ions, such as Cu2+, Zn2+
,
2.1 Chemistry
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and Fe2+ are closely implicated in the pathogenesis of AD. Compounds
with metal-chelating effects might provide an additional therapeutic
strategy for the treatment of AD. Moreover, recent studies have
demonstrated that oxidative stress plays a key role in AD onset and
progression.[10]
In this study, twelve N-substituted-5-chloro-2(3H)-benzoxazolone
derivatives have been synthesized to evaluate antioxidant and
anticholinesterase activities. The target compounds were prepared
by a two steps synthesis procedure (Scheme 1). In the first step, 5-
chloro-2(3H)-benzoxazolone was prepared according to the reported
procedure. In the second step, this intermediate was reacted with
formaldehyde and various amines or imidazole heterocycle in
methanol by Mannich reaction to furnish corresponding Mannich
bases of 5-chloro-2(3H)-benzoxazolone derivatives.
Therefore, designing cholinesterase inhibitors endowed with
antioxidant and biometal-chelating properties may be an effective
strategy for medicinal chemists.
To date, only four acetylcholinesterase inhibitors, i.e., tacrine,
donepezil, galantamine, and rivastigmine, have been approved by FDA
(Food and Drug Administration) (Figure 1). These agents are important
drugs for palliative treatment of AD, but their clinical efficacy is limited
due to their poor selectivity, bioavailability and adverse side effects.
Thus, there is an urgent need for developing new effective,
multifunctional anti-AD drugs.[11–16]
All of the title compounds were reported for the first time in this
study except for compounds 1[26,27] and 4 (341018-25-5 CAS Registry
Number).
The structures of these target compounds were confirmed by IR,
1H NMR, APCI-MS spectral and elemental analysis. Details of synthetic
procedure and structural characterization are described in the
Experimental section.
2(3H)-Benzoxazolone derivatives are important compounds in
drug discovery research due to their various bioactivities such as
anticancer,[17] analgesic,[18] anti-inflammatory,[19] anticonvul-
sant,[20] antimalarial,[21] anti-nociceptive,[22] antioxidant,[23] and
human leukocyte myeloperoxidase chlorinating inhibitor activity.[24]
Additionally, some benzoxazole derivatives have been reported as
β-amyloid imaging agents of AD patients.[25] In our previous study,
we designed and synthesized some N-substituted-5-chloro-2(3H)-
benzoxazolone derivatives as AChE inhibitors. The preliminary
results have demonstrated that N-substituted-5-chloro-2(3H)-
benzoxazolone template has acetylcholinesterase inhibitory
activity.[26]
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2.2 Biological activity
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2.2.1 Cholinesterase inhibitory activity
The cholinesterase inhibitory activities of the target compounds 1–12
were evaluated against AChE and BuChE enzymes in comparison to
rivastigmine as the reference compound. The IC50 values of the
compounds are listed in Table 1. According to the AChEI activity
results, all tested compounds showed higher AChEI activity with
IC50 = 7.53–9.51 μM values than the reference compound rivastig-
mine (IC50 = 10.87 0.24 μM). Compounds 2, 3, 5, 6, 7, 10 and 12
showed similar activity potency with IC50 = 7.53–7.76 μM values.
Among them, compound 7 bearing hydroxyl substituent at ortho
position on N-phenyl ring exhibited relatively higher AChE inhibitory
activity. The introduction of mono- and disubstituents to ortho position
on N-phenyl ring slightly increased AChEI activity, except for
compounds 4, 8 and 9. In case of ortho alkyl substituted derivatives,
compound 2 with methyl substituent showed better activity profile
than those with ethyl and isopropyl substituents (compound 8 and 9,
respectively). Interestingly, introducing a second methyl group on
ortho position of N-phenyl ring did not make a noticeable change in the
AChEI activity. In terms of halogen substituent on the N-phenyl ring, a
decrease on the activity was observed for chlorine atom, whereas an
increase was discovered for iodide substituent. On the other hand, the
replacement of N-phenyl with imidazole ring led to an increase in the
inhibitory activity, whereas a decrease in activity was observed for the
replacement of N-phenyl group with N-benzyl moiety. In terms of
BuChEI activity, the tested compounds exhibited from weak to
moderate inhibitory potency. Interestingly, compound 11 with N-
benzyl group, which have less activity potency than other compounds
against AChE enzyme, is the most active compound with
IC50 = 17.50 0.29 μM value against BuChE enzyme. In case of the
As an ongoing research program on the synthesis of more
effective cholinesterase inhibitors, we synthesized some 5-chloro-
2(3H)-benzoxazolone Mannich bases and evaluated their cholinester-
ase inhibitory and antioxidant activities and performed their docking
studies.
FIGURE 1 Chemical structures of FDA approved AChE inhibitors.

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SCHEME 1 Synthesis of the title compounds
derivatives bearing N-phenyl group, the introduction of halogens and
hydroxyl substituent increased BuChEI activity.
blood-brain-barrier (BBB). Compounds possessing moderate log P
(lower than 4) values often exhibit the highest brain penetration.[28]
The lipophilicities (log P values) of the synthesized compounds were
calculated using MOE 2011.10 (Molecular Operating Environment
It is a well-known fact that lipophilicity (log P) is an important
physicochemical parameter to evaluate or predict the ability to cross
TABLE 1 AChE and BuChE inhibitory activities, ABTS radical scavenging capacities and log P values of the title compounds
IC50 SEM (µM)a
IC50 SEM (mM)a
Comp. No.
AChE
BuChE
ABTS assay
TEACb
0.41
1.44
0.64
–
log Pc
3.73
4.04
3.74
4.38
4.33
3.64
3.43
4.29
4.85
4.35
3.67
1.48
–
8.12 0.12
7.76 0.22
7.65 0.21
8.45 0.56
7.70 0.32
7.74 0.25
7.53 0.17
9.19 0.22
9.23 0.28
7.67 0.14
9.51 0.40
7.70 0.32
10.87 0.24
–
57.67 6.36
>100
3.70 0.04
1.04 0.04
79.67 1.64
44.58 3.65
34.37 0.27
60.12 0.87
31.58 4.63
67.03 2.14
92.22 8.80
73.20 3.69
17.50 0.29
90.13 2.49
5.13 0.18
–
2.35 0.08
–
–
–
–
–
4.65 0.26
0.32
0.60
–
2.50 0.13
–
2.41 0.13
0.62
–
–
–
–
–
–
1.50 0.05
1
–
aIC50 = 50% Inhibitory concentration. The data are means standard error of the main of triplicate independent experiments. The IC50 values were calculated
by using GraphPad 5 software.
bTEAC = Trolox equivalent antioxidant capacity. The data are expressed as mM of Trolox equiv/mM of tested compound.
cLog P values were calculated by using MOE 2014.09.01.

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Chemical Computing Group). The results showed that the log P values
of the compounds ranged from 1.48 to 4.85, which indicated that
compounds have proper log P values and can be considered as optimal
to cross the BBB (Table 1).
which demonstrated the production of the corresponding complex via
metal chelation (Figure 2). The chelating ability of compound 7 may be
related to the presence of the hydroxyl group at ortho position on N-
phenyl ring. However, with the addition of FeSO4 or ZnCl2, there was
no significant change.
As a result, it can be speculated that the nature of the substituents
at the ortho position on the phenyl ring influences the cholinesterase
inhibitor activity profile and potency in this class of compounds. These
results indicated that N-substituted-5-chloro-2(3H)-benzoxazolone
scaffold is generally more selective toward AChE enzyme.
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2.3 Molecular docking study
Docking studies were performed to investigate the possible binding
mode of the most active compounds of N-substituted-5-chloro-2(3H)-
benzoxazolone derivatives in AChE and BuChE active site (compound
7 and compound 11). The three-dimensional structure of Torpedo
californica AChE (TcAChE) was selected (PDB code: 1EVE) and
homology model BuChE was used for docking studies.
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2.2.2 Antioxidant activity
The antioxidant activity profile of the synthesized compounds was
determined by ABTS free radical scavenger assay.[29] Trolox, a water-
soluble vitamin E analog, was used as a reference standard. Preliminary
antioxidant activity screening results are given in Table 1.
As shown in Table 1, six derivatives were found to possess potent
ABTS radical scavenging capacities with IC50 values in the range of
1.04–4.65 mM. Among them, compound 2 bearing methyl substituent
at ortho position on the phenyl ring showed higher radical scavenging
activity (IC50 = 1.04 0.04 mM; 1.44 Trolox equivalents) than Trolox
(IC50 = 1.50 0.05 mM).
Compound 7 exhibited two binding interactions with TcAChE as
shown in Figure 3. The carbonyl group of 2(3H)-benzoxazolone core
interacts with Gly118 and Gly119 via two hydrogen bonds. Gly118
and Gly119 residues are located in the oxyanion hole of AChE and
oxyanion hole is in the catalytic active site (CAS) of AChE. Also, phenyl
ring of 2(3H)-benzoxazolone is oriented towards Phe330 that is also in
CAS so we can propose that compound 7 can interact with catalytic
active site (Figure 3).
Moreover, binding interactions of the most active compounds
(compounds 2, 3, 5, 6, 10 and 12) in TcAChE were studied. Similar to
compound 7-TcAChE complex, the carbonyl group of the 2(3H)-
benzoxazolone scaffold of compounds 2, 3, 5, and 6 were oriented
towards Gly118 and the side chain towards Gly117. Proposed binding
modes of the most active compounds and their docking scores are
given in Figure 4 and Table 2. Exceptionally, compounds 10 and 12
showed different binding interactions as given in Figure 4. The phenyl
side chain of compound 10 makes π–π interaction with Phe330 and
the 2(3H)-benzoxazolone scaffold is oriented toward Trp84 which is
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2.2.3 Metal-chelating study
A UV-Vis spectroscopy assay was performed to evaluate the ability of
most active compound 7 in the series to chelate bio-metals such as
Cu+2, Fe+2, and Zn+2. For this purpose, the absorption spectra of
compound 7 (400 μM) alone or in the presence of CuSO4, FeSO4 or
ZnCl2 (200 μM) solutions in ethanol were recorded for 30 min. It can be
seen that maximum absorption increased dramatically were detected
at 214 nm after the addition of CuSO4 to the solution of compound 7,
FIGURE 2 (a) UV spectrum of compound 7 (40 μM). (b) Spectrum of a mixture of compound 7 (40 μM) and FeSO4 (40 μM). (c) Spectrum of
a mixture of compound 7 (40 μM) and CuSO4 (40 μM). (d) Spectrum of a mixture of compound 7 (40 μM) and ZnCl2 (40 μM)

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FIGURE 4 Proposed binding mode for compounds 2, 3, 5, 6, 10,
and 12 superimposed with the binding mode shown by compound 7
inside TcAChE crystal structure (1EVE.pdb). Compound 7 is showed
as green sticks, while compounds 2, 3, 5, 6, 10, and 12 are showed
as pink, cyan, coral, yellow, green, dark brown, and gray,
respectively. The most involved residues are colored as light brown
sticks
FIGURE 3 Proposed binding mode for compound 7 inside AChE
(PDB code: 1EVE). Compound 7 is represented as green stick in
AChE. The most involved residues are named and represented as
brown sticks for AChE. Hydrogen bond interactions are specified as
blue dashed lines
inhibition, respectively. Furthermore, docking studies revealed that
compound 7 against AChE interacted with CAS of the enzyme.
The cholinesterase inhibitory activity results suggested that the N-
substituted-5-chloro-2(3H)-benzoxazolone core structure is generally
more selective against AChE. These series of compounds might
contribute to a better understanding of the structural features
required for strong inhibition of the main AChE enzyme.
located in the peripheral anionic site (PAS). The orientation difference
of compound 10 may be due to the steric effect of the dimethyl
substituents at ortho positions on N-phenyl ring. Compound 12 also
had different binding interactions. Imidazole oriented towards Glu199
and the 2(3H)-benzoxazolone ring system had a π–π interaction with
Phe330. This binding interaction difference is expected due to the
change of the phenyl ring to imidazole.
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EXPERIMENTAL
Regarding the docking study on BuChE, compound 11 has one
major interaction with model BuChE. The nitrogen atom of the amine
group makes a hydrogen bond with Trp82 side chain which is located
at the anionic substrate binding site of the catalytic active site
(Figure 5).
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4.1 Chemistry
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4.1.1 General
All chemicals, reagents, and solvents were high-grade commercial
products and used without further purification. Reactions were
checked by thin-layer chromatography (TLC) on precoated silica gel
aluminum plates (Kieselgel 60, F254, E. Merck, Germany), spots were
visualized by UV at 254 nm. Melting points were determined using a
Stuart SMP30 (Staffordshire, ST15 OSA, United Kingdom) melting
point apparatus and are not corrected. IR spectra of the compounds
were recorded on a Perkin Elmer 100 FT-IR (ATR) spectrophotometer
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CONCLUSION
In this study, twelve N-substituted-5-chloro-2(3H)-benzoxazolone
derivatives have been synthesized and evaluated for their cholines-
terase inhibitory and antioxidant acitivities. Compounds 7 and 11
appeared to be the most active derivatives for AChE and BuChE

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TABLE 2 Docking score results for the most active compounds
inside TcAChE according to the proposed binding mode using
GoldScore scoring functions
(multiplet). Mass spectra (APCI-MS) were measured on a Thermo MSQ
Plus LC/MS (Thermoscientific Inc., San Jose, CA). Elemental analyses
(C, H, N) were performed by Leco TruSpec Micro (Leco, St. Joseph, MI).
The analytical results for the elements were within 0.4% of the
theoretical values. Microwave irradiation synthesis of the compound
1a was conducted on Milestone MicroSYNTH (Milestone S.r.l.,
Sorisole, Italy) microwave apparatus.
Compound No.
GoldScore
49.252
53.38
AChE IC50 SEM (μM)
7.76 0.22
2
3
7.65 0.21
5
53.607
50.053
49.496
54.312
42.268
7.70 0.32
Compound 1a and the final compounds 1–12 were prepared
according to the reported procedure.[26] The InChI codes of the
investigated compounds as well as some biological activity data are
provided as Supporting Information.
6
7.74 0.25
7
7.53 0.17
10
12
7.67 0.14
7.70 0.3
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4.1.2 Synthesis of compound 1a
(Perkin Elmer Inc., MA). The 1H NMR spectra were recorded on a
Varian AS 400 Mercury Plus NMR (Varian Inc., Palo Alto, CA, USA) and
Agilent 600 MHz PremiumCOMPACT NMR (Agilent Tech., USA)
spectrometers using CDCl3 and DMSO-d6 as solvents. Chemical shifts
were reported in parts per million (δ) with TMS as an internal standard.
J values were given in Hz. Abbreviations for 1H NMR data quoted are
as follows: s (singlet); bs (broad singlet) d, (doublet); t, (triplet); m,
2-Amino-4-chlorophenol (10 mmol), urea (50 mmol) and 37% HCl
(2.5 mL) were irradiated (300 W, 140°C) for 15 min in a microwave
reactor. After completion of reaction by monitoring with TLC, water
(10 mL) was added to the reaction mixture and stirred at room
temperature for 1 h. The resulting precipitate was filtered and washed
with water. After drying, the crude product was crystallized from
ethanol:water (1:1) to yield 5-chloro-2(3H)-benzoxazolone (1a, 40%).
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4.1.3 General procedure for the synthesis of the
final compounds 1–12
The title compounds were prepared by Mannich reaction conditions.
For this purpose, 5-chloro-2(3H)-benzoxazolone (2 mmol) was dis-
solved in methanol (10 mL). Appropriate amines or imidazole (2 mmol)
and 37% formalin (2.5 mmol) were added to this solution. The mixture
was stirred vigorously at room temperature until solid product was
precipitated. The resulting precipitate was filtered and washed with
cold methanol. The crude product was crystallized from ethanol to
yield the target compounds.
5-Chloro-3-((phenylamino)methyl)benzo[d]oxazol-2(3H)-one (1)
Yield 84%; mp 190°C; IR υmax (FT/ATR): 3398, 3066, 2161, 2032,
;
1750, 1604, 1479 cm−1 1H NMR (CDCl3): δ 7.19–7.25 (2H, m, Ar-H),
7.10–7.11 (1H, m, Ar-H), 7.07–7.08 (2H, m, Ar-H), 6.81–6.85 (3H, m,
Ar-H), 5.30 (2H, d, J = 7.0 Hz, CH2), 4.64 (1H, t, J = 6.8 Hz, NH) ppm;
Anal. calcd. for C14H11ClN2O2: C, 61.21; H, 4.04; N, 10.20. Found: C,
61.60; H, 4.02; N, 10.24; MS (APCI) m/z (%): 274 (1) [M]+, 106 (100), 94
(10).
5-Chloro-3-((o-tolylamino)methyl)benzo[d]oxazol-2(3H)-one (2)
Yield 31%; mp 137°C; IR υmax (FT/ATR): 3432, 2975, 2159, 2031,
1775, 1754 1604, 1590, 1478 cm−1; 1H NMR (DMSO-d6): δ 7.90 (1H,
d, J = 2.4 Hz, Ar-H), 7.30 (1H, d, J = 8.4 Hz, Ar-H), 7.13 (1H, dd J = 8.4;
1.8 Hz, Ar-H), 6.95–7.00 (2H, m, Ar-H), 6.80 (1H, d, J = 7.6 Hz, Ar-H),
6.55 (1H, t, J = 7.6 Hz, Ar-H), 6.32 (1H, t, J = 6.4 Hz, NH), 5.27 (2H, d,
J = 6.4 Hz, CH2), 2.09 (3H, s, CH3) ppm; Anal. calcd. for C15H13ClN2O2:
C, 62.40; H, 4.54; N, 9.70. Found: C, 62.45; H, 4.64; N, 10.10; MS
(APCI) m/z (%): 288 (1) [M]+, 132 (68), 118 (11), 106 (44), 94 (50), 93
(88), 91 (100), 65 (50), 42 (14).
FIGURE 5 Proposed binding mode for compound 11 inside
model BuChE. Compound 11 is represented as pink sticks. The most
involved residues are named and represented as gray sticks for
BuChE. Hydrogen bond interactions are specified as blue dashed
lines

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5-Chloro-3-(((2-methoxyphenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (3)
5-Chloro-3-(((2-ethylphenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (8)
Yield 34%; mp 124°C; IR υmax (FT/ATR): 3408, 2993, 2968, 2159,
Yield 10%; mp 122°C; IR υmax (FT/ATR): 3389, 2962, 1772, 1751,
1606, 1588, 1478 cm−1 1H NMR (CDCl3): δ 7.13–7.16 (2H, m, Ar-H),
2032, 1766, 1601, 1482 cm−1
;
1H NMR (DMSO-d6): δ 7.92 (1H, d,
;
J = 2.3 Hz, Ar-H), 7.33 (1H, d, J = 8.3 Hz, Ar-H), 7.15 (1H, dd, J = 8.3;
2.0 Hz, Ar-H), 6.84 (2H, m, Ar-H), 6.76 (1H, t, J = 7.6 Hz, Ar-H), 6.62
(1H, t, J = 7.6 Hz, Ar-H), 6.57 (1H, t, J = 7.2 Hz, NH), 5.26 (2H, d,
J = 7.2 Hz, CH2), 3.79 (3H, s, OCH3) ppm; Anal. calcd. for
7.04–7.09 (3H, m, Ar-H), 6.95 (1H, d, J = 7.2 Hz, Ar-H), 6.79–6.83
(1H, m, Ar-H), 5.36 (2H, d, J = 7.2 Hz, CH2), 4.66 (1H, t, J = 7.2 Hz, NH),
2.52 (2H, q, J = 7.6 Hz, CH2CH3), 1.22 (3H, t, J = 7.6 Hz, CH2CH3) ppm;
Anal. calcd. for C16H15ClN2O2: C, 63.47; H, 4.99; N, 9.25. Found: C,
63.75; H, 5.25; N, 9.40; MS (APCI) m/z (%): 135 (24), 118 (100), 106
(18), 94 (32), 93 (26), 91 (12), 56 (17), 42 (23).
C15H13ClN2O3: C, 59.12; H, 4.30; N, 9.19. Found: C, 59.45; H, 4.36;
N, 9.20; MS (APCI) m/z (%): 161 (13), 150 (6), 120 (44), 109 (100), 81
(10), 80 (48), 65 (8), 42 (7).
5-Chloro-3-(((2-isopropylphenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (9)
5-Chloro-3-(((2-chlorophenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (4)
Yield 28%; mp 136°C; IR υmax (FT/ATR): 3454, 3423, 3064, 2955,
1775, 1752, 1603, 1480 cm−1 1H NMR (CDCl3): δ 7.13–7.18 (3H, m,
;
Yield 11%; mp 131°C; IR υmax (FT/ATR): 3379, 2939, 2161, 1772,
;
1595, 1483 cm−1 1H NMR (CDCl3): δ 7.25 (1H, d, J = 8.4 Hz, Ar-H),
Ar-H), 7.05–7.08 (2H, m, Ar-H), 6.97–6.99 (1H, m, Ar-H), 6.85–6.87
(1H, m, Ar-H), 5.39 (2H, d, J = 4.8 Hz, CH2), 4.83 (1H, t, J = 4.4 Hz, NH),
2.89–2.96 (1H, m, CH), 1.25 (6H, d, J = 4.4 Hz, 2xCH3) ppm; Anal. calcd.
for C17H17ClN2O2. 0.1 CH4O: C, 64.19; H, 5.48; N, 8.75. Found: C,
64.18; H, 5.87; N, 8.61; MS (APCI) m/z (%): 317 (1) [M+H]+, 132 (29),
118 (15), 106 (15), 94 (12), 91 (100), 65 (3), 42 (3).
7.16–7.20 (2H, m, Ar-H), 7.07–7.11 (3H, m, Ar-H), 6.75-6-77 (1H, m,
Ar-H), 5.38 (2H, d, J = 4.4 Hz, CH2), 5.28 (1H, t, J = 4.4 Hz, NH) ppm;
Anal. calcd. for C14H10Cl2N2O2: C, 54.39; H, 3.26; N, 9.06. Found: C,
54.55; H, 3.12; N, 9.12; MS (APCI) m/z (%): 142 (4), 140 (12), 135 (24),
118 (40), 110 (52), 94 (16), 81 (15), 56 (100), 42 (24).
5-Chloro-3-(((2-iodophenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (5)
5-Chloro-3-(((2,6-dimethylphenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (10)
Yield 8%; mp 121°C; IR υmax (FT/ATR): 3381, 2975, 2160, 2040, 1753,
;
1608, 1480 cm−1 1H NMR (CDCl3): δ 7.65 (1H, d, J = 8.0 Hz, Ar-H),
Yield 20%; mp 133°C; IR υmax (FT/ATR): 3375, 2960, 2867, 1796,
;
1775, 1752, 1609, 1479 cm−1 1H NMR (CDCl3): δ 7.02 (1H, d,
7.24–7.27 (1H, m, Ar-H), 7.17 (1H, d, J = 1.2 Hz, Ar-H), 7.09–7.11
(2H, m, Ar-H), 7.01 (1H, d, J = 8.0 Hz, Ar-H), 6.56 (1H, t, J = 8.0 Hz, Ar-
H), 5.36 (2H, d, J = 6.8 Hz, CH2), 5.12 (1H, bs, NH) ppm; Anal. calcd. for
J = 7.6 Hz, Ar-H), 6.96–6.98 (3H, m, Ar-H), 6.90–6.94 (2H, m, Ar-H),
5.06 (2H, d, J = 7.6 Hz, CH2), 3.99 (1H, bs, NH), 2.22 (6H, s, 2xCH3)
ppm; Anal. calcd. for C16H15ClN2O2: C, 63.47; H, 4.99; N, 9.25.
Found: C, 63.70; H, 5.05; N, 9.37; MS (APCI) m/z (%): 303 (1) [M+H]+,
146 (18), 144 (46), 135 (30), 118 (100), 105 (62), 94 (62), 91 (12), 56
(14), 42 (22).
C14H10ClN2O2I. 1.1 C2H6O: C, 43.12; H, 3.71; N, 6.21. Found: C,
43.52; H, 3.47; N, 5.98; N, 7.56; MS (APCI) m/z (%): 145 (32), 134 (10),
118 (18), 105 (26), 93 (100), 78 (28), 56 (20), 42 (68).
5-Chloro-3-(((2-nitrophenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (6)
3-((Benzylamino)methyl)-5-chlorobenzo[d]oxazol-2(3H)-one
(11)
Yield 23%; mp 145°C; IR υmax (FT/ATR): 3382, 2978, 1774, 1611,
1480 cm−1; 1H NMR (CDCl3): δ 8.38–8.43 (1H, m, Ar-H), 8.20 (2H, dd,
J = 8.7; 1.6 Hz, Ar-H), 7.51 (1H, td, J = 8.4; 1.6 Hz, Ar-H), 7.99 (1H, d,
J = 8.0 Hz, Ar-H), 6.77–6.81 (2H, m, Ar-H), 6.01–6.10 (1H, bs, NH),
5.13 (1H, t, J = 5.6 Hz, CH2) ppm; Anal. calcd. for C14H10ClN3O4. 0.5
H2O: C, 51.15; H, 3.37; N, 12.78. Found: C, 50.76; H, 3.12; N, 12.84;
MS (APCI) m/z (%): 319 (1) [M]+, 84 (38), 83 (34), 56 (100), 42 (98).
Yield 28%; mp 177°C; IR υmax (FT/ATR): 3067, 3028, 2869, 1798,
;
1776, 1610, 1482 cm−1 1H NMR (CDCl3): δ 7.29–7.33 (5H, m, Ar-H),
7.03–7.04 (2H, m, Ar-H), 6.54–6.61 (1H, m, Ar-H), 4.84 (2H, s,
─CH2NH─), 3.91 (3H, s, CH2-Phenyl and NH) ppm; Anal. calcd. for
C15H13ClN2O2: C, 62.40; H, 4.54; N, 9.70. Found: C, 62.35; H, 4.18; N,
9.30; MS (APCI) m/z (%): 132 (64), 106 (68), 91 (100), 67 (18), 65 (68),
42 (21).
5-Chloro-3-(((2-hydroxyphenyl)amino)methyl)benzo[d]oxazol-
2(3H)-one (7)
3-((1H-Imidazol-1-yl)methyl)-5-chlorobenzo[d]oxazol-2(3H)-one
(12)
Yield 8%; mp 167°C; IR υmax (FT/ATR): 3338, 2975, 2159, 2031, 1747,
1607, 1480 cm−1 1H NMR (CDCl3): δ 7.09–7.11 (3H, m, Ar-H), 6.97
;
Yield 18%; mp 161°C; IR υmax (FT/ATR): 3391, 2082, 1754, 1607,
;
1471 cm−1 1H NMR (CDCl3): δ 7.20–7.21 (1H, m, Ar-H), 7.13–7.14
(1H, d, J = 8.0 Hz, Ar-H), 6.83–6.87 (1H, m, Ar-H), 6.73–6.74 (2H, m,
Ar-H), 5.31 (2H, d, J = 7.2 Hz, CH2), 4.95 (1H, bs, NH) ppm; Anal. calcd.
for C14H11ClN2O3. 0.3 H2O: C, 56.79; H, 3.95; N, 9.46. Found: C,
56.39; H, 3.58; N, 9.85; MS (APCI) m/z (%): 289 (1) [M–H]+, 106 (58), 94
(30), 65 (22), 56 (100), 42 (56).
(2H, m, Ar-H), 7.09–7.12 (3H, m, Ar-H), 5.37 (2H, s, CH2) ppm; Anal.
calcd. for C11H8ClN3O2. 0.25 H2O: C, 51.98; H, 3.37; N, 16.53. Found:
C, 51.60; H, 3.22; N, 16.36; MS (APCI) m/z (%): 251 (1) [M+2H]+, 84
(28), 80 (74), 42 (100).

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UYSAL ET AL.
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|
4.2 Biological activity assays
curve and their antioxidant properties were expressed as mM Trolox
equivalent.
|
4.2.1 Cholinesterase inhibitor activity assay
|
AChE (E.C.3.1.1.7., Type VI-S, from electric eel) and BuChE (E.-
C.3.1.1.8., from equine serum) were purchased from Sigma–Aldrich
(Steinheim, Germany). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB,
Ellman's reagent), acetylthiocholine iodide (ATC) and butyrylthiocho-
line iodide (BTC) used as substrates were obtained from Fluka. Buffer
compounds (potassium dihydrogen phosphate, potassium hydroxide)
and sodium hydrogen carbonate were purchased from Merck.
Spectrophotometric measurements were performed on a Shimadzu
160-A UV-Vis spectrophotometer.
4.2.3 Metal-chelating study
The chelating studies were performed in ethanol at 25°C using UV-vis
spectrophotometer (a Thermoscientific Evaluation Array, Thermo-
scientific Inc.) with wavelength ranging from 200 to 400 nm. CuSO4,
FeSO4, or ZnCl2 solutions were prepared in 200 μM using ethanol.
Compound 7 was dissolved in DMSO and then diluted with ethanol to
400 μM concentration. To a mixture of 100 μL compound 7 and
700 μL ethanol, 200 μL CuSO4 (FeSO4 or ZnCl2) solution was added.
The solution was incubated at room temperature for 30 min and then
the absorption spectra were recorded at room temperature. The
control was prepared by mixing 100 μL compound 7 solution and
900 μL ethanol. The blank was 4 μL DMSO in 996 μL ethanol. All
absorption spectra subtracted the blank absorption spectra.[32]
The inhibitory effects of the synthesized compounds on AChE and
BuChE were evaluated using a slightly modified colorimetric method of
Ellman et al., with rivastigmine as the reference compound.[30,31] Prior
to use, all solutions were adjusted to 20°C. Enzyme solution (100 μL)
and inhibitor solution (100 μL) were added into a cuvette containing
the phosphate buffer (3.0 mL, 0.1 M; pH 8.0). After 5 min incubation,
required aliquots of the DTNB solution (100 μL) and of the ATC/BTC
(20 μL) were added. After rapid and immediate mixing the absorption
was measured at 412 nm by UV spectroscopy. As a reference, an
identical solution of the enzyme without the inhibitor is processed
following the same protocol. The blank reading contained 3.0 mL
buffer, 200 μL water, 100 μL DTNB, and 20 μL substrate. The enzyme
activity was determined in the presence of at least five different
concentrations of an inhibitor. Each concentration was assayed in
triplicate. The samples were investigated immediately after prepara-
tion. The AChE/BuChE inhibitory activities of the title compounds are
summarized in Table 1.
|
4.3 Molecular modeling
|
4.3.1 Homology modeling
From the lack of eqBuChE X-ray crystal structure a homology model
was initially generated. The model was obtained through SWISS-
MODEL server using a default automatized pipeline.[33,34] The three-
dimensional geometries of eqBuChE were obtained after primary
sequences alignment with huBuChE X-ray crystal structure (2PM8.
pdb) that shows a primary sequence identity of 89.79% with eqBuChE.
|
4.3.2 Molecular docking studies
The crystal structures of donepezil in complex with AChE (PDB code
1EVE resolved at 2.5 Å) were taken from the Protein Data Bank.
Heteroatoms and water molecules in the PDB file were removed and
hydrogen atoms were added to the protein by using MOE 2016.08.[35]
Prior to the docking calculations, energy minimization using the
AMBER99 force field was performed on TcAChE enzyme and model
BuChE. Compounds 2, 3, 5, 6, 7, 10, 11, and 12 were built and
protonated using the protonate 3D protocol and energy minimized
using the MMFF94 force field via MOE 2016.08. Docking of the
ligands was carried out using the GOLD 5.2.1 program with default
settings.[36,37] A sphere of 20 Å around the carbonyl group of Glu199
was defined as the binding site for the ligand docking in TcAChE for
compounds 2, 3, 5, 6, 7, 10 and 11 and 250 confirmations was allowed.
For model BuChE-compound 11 docking, a sphere of 20 Å around the
imidazole group of His438 was selected as binding site and for every
ligand 250 poses were retrained. The GoldScore and ChemScore
standard precision (sp) were calculated and analyzed. The
putative binding modes were carried out through visual inspection
(Figures 3–5). Since ChemScore was not able to find a reasonable
binding mode for all the compounds present in the data set, only the
GoldScore values for the docked compounds according the putative
binding mode are reported (Table 2).
|
4.2.2 Antioxidant activity assay
Trolox equivalent antioxidant capacity of compounds was deter-
mined by an improved assay based on the decolorization of the
radical monocation of [2,2′-azinobis-(3-ethylbenzothiazoline-6-sul-
fonic acid)] (ABTS.+) applied to a 96-well microplate assay. Trolox
was used as an antioxidant standard.[29]
A total of 2.45 mM
potassium persulphate (Sigma) was mixed with 7 mM ABTS (Sigma)
in order to produce an ABTS.+ radical. This radical was left in the dark
at room temperature for 16 h to reach stable absorbance at 734 nm.
Compounds were solved in methanol to a final concentration 1 mg/
1 mL. Methanol used as a blank. The ABTS+ radical was diluted in
5 mM phosphate buffer until it had an absorbance of 0.70 0.02 at
734 nm. Then, 200 μL of this diluted solution was taken and mixed
with 2 μL of a various concentrations of compounds (1–1000 μM).
The absorbance was measured spectrophotometrically at 734 nm at
30°C in Thermo Varioscan Flash microplate reader for 1–6 min after
the initial mixing. Decrease of the absorption was used to calculate
the IC50 values. All measurements were carried out in triplicate.The
standard curve of the Trolox was obtained using a Trolox standard
solution at various concentrations (0.25–2.5 mM) in methanol. The
absorbances of the samples were compared to that of the standard

UYSAL ET AL.
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ACKNOWLEDGMENTS
[20] H. Ucar, K. Van Derpoorten, S. Cacciaguerra, S. Spampinato, J. P.
Stables, P. Depovere, M. Isa, B. Masereel, J. Delarge, J. H. Poupaert, J.
Med. Chem. 1998, 41, 1138.
The authors would like to thank the Pharmaceutical Sciences Research
Centre (FABAL) at Ege University Faculty of Pharmacy for spectral and
elemental analyses of the compounds.
[21] M. Courtois, Z. Mincheva, F. Andreu, M. Rideau, M. C. Viaud-
Massuard, J. Enzyme Inhib. Med. Chem 2004, 19, 559.
[22] T. Onkol, S. Ito, E. Yildirim,K. Erol, M. F. Sahin,Arch. Pharm. 2001,334, 17.
[23] Z. Soyer, S. Uysal, F. A. Kose, A. Pabuccuoglu, Asian J. Chem. 2014, 26,
6642.
CONFLICTS OF INTERESTS
[24] Z. Soyer, M. Bas, A. Pabuccuoglu, V. Pabuccuoglu, Arch. Pharm. 2005,
338, 405.
The authors declare no conflicts of interests.
[25] B. M. Swahn, D. Wensbo, J. Sandell, D. Sohn, C. Slivo, D. Pyring, J.
Malmström, E. Arzel, M. Vallin, M. Bergh, F. Jeppsson, A. E. Johnson, A.
Juréus, J. Neelissen, S. Svensson, Bioorg. Med. Chem. Lett. 2010, 20, 1976.
[26] Z. Soyer, S. Parlar, V. Alptuzun, Marmara Pharm. J. 2013, 17, 15.
[27] R. S. Varma, W. L. Nobles, J. Pharm. Sci. 1968, 57, 39.
[28] R. N. Waterhouse, Mol. Imaging Biol. 2003, 5, 376.
[29] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-
Evans, Free Radical Biol. Med. 1999, 26, 1231.
ORCID
Zeynep Soyer
http://orcid.org/0000-0002-7426-0345
[30] P. Kapková, N. Stiefl, U. Sürig, B. Engels, K. Baumann, U. Holzgrabe,
Arch. Pharm. 2003, 336, 523.
REFERENCES
[31] G. L. Ellman, K. D. Courtney, V. Andres, R. M. Feather-Stone, Biochem.
Pharmacol 1961, 7, 88.
[1] H. W. Querfurth, F. M. La Ferla, N. Engl. J. Med. 2010, 362, 329.
[2] M. Goedertand, M. G. Spillantini, Science 2006, 314, 777.
[3] K. Blennow, M. J. de Leon, H. Zetterberg, Lancet 2006, 368, 387.
[4] E. L. Konrath, C. S. Passos, L. C. Klein-Júnior, A. T. Henriques, J. Pharm.
Pharmacol. 2013, 65, 1701.
[32] F. Mao, L. Huang, Z. Luo, A. Liu, C. Lu, Z. Xie, X. Li, Bioorg. Med. Chem.
2012, 20, 5884.
[33] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, Bioinformatics 2006, 22, 195.
[34] P. Benkert, M. Biasini, T. Schwede, Bioinformatics 2011, 27, 343.
[35] Molecular Operating Environment (MOE), Chemical Computing
Group Inc. 1010 Sherbrooke Street West, Suite 91, Montreal H3A
2R7, Canada, 2016.08.
[5] P. Davies, A. J. F. Maloney, Lancet 1976, 308, 1403.
[6] J. Hardy, D. Allsop, Trends Pharmacol. Sci. 1991, 12, 383.
[7] M. A. Ansari, S. W. Scheff, J. Neuropathol. Exp. Neurol. 2010, 69, 155.
[8] R. T. Bartus, R. L. Dean, B. Beer, A. S. Lippa, Science 1982, 217, 408.
[9] N. Toda, T. Kaneko, H. Kogen, Chem. Pharm. Bull. 2010, 58, 273.
[10] A. I. Bush, R. E. Tanzi, Neurotherapeutics 2008, 5, 421.
[11] S. Shaikh, A. Verma, S. Siddiqui, S. S Ahmad, S. M. Rizvi, S. Shakil, D.
Biswas, D. Singh, M. H. Siddiqui, S. Shakil, S. Tabrez, M. A. Kamal, CNS
Neurol. Disord.: Drug Targets 2014, 13, 391.
[36] G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J. Mol. Biol. 1997,
267, 727.
[37] R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T.
Mainz, M. P. Repasky, E. H. Knoll, M. Shelley, J. K. Perry, D. E. Shaw, P.
Francis, P. S. Shenkin, J. Med. Chem. 2004, 47, 1739.
[12] M. Bajda, A. Więckowska, M. Hebda, N. Guzior, C. A. Sotriffer, B.
Malawska, Int. J. Mol. Sci. 2013, 14, 5608.
SUPPORTING INFORMATION
[13] M. Pohanka, Int. J. Mol. Sci. 2014, 15, 9809.
Additional Supporting Information may be found online in the
supporting information tab for this article.
[14] R. Farina, L. Pisani, M. Catto, O. Nicolotti, D. Gadaleta, N. Denora, R.
Soto-Otero, E. Mendez-Alvarez, C. S. Passos, G. Muncipinto, C. D.
Altomare, A. Nurisso, P. A. Carrupt, A. Carotti, J. Med. Chem. 2015, 58,
5561.
[15] M. A. Khan, S. Fazal-ur-Rehman, A. Hameed, S. Kousar, K. Dalvandi, S.
Yousuf, M. I. Choudharya, F. Z. Basha, RSC Adv. 2015, 5, 59240.
[16] W. Xu, X. B. Wang, Z. M. Wang, J. J. Wu, F. Li, J. Wang, L. Y. Kong, Med.
Chem. Comm. 2016, 7, 990.
How to cite this article: Uysal S, Parlar S, Tarikogullari AH,
Aydin Kose F, Alptuzun V, Soyer Z. Synthesis, biological
evaluation, and docking studies of some 5-chloro-2(3H)-
benzoxazolone Mannich bases derivatives as cholinesterase
inhibitors. Arch Pharm Chem Life Sci. 2018;1–9.
[17] Y. Ivanova, G. Momekov, O. Petrov, M. Karaivanova, V. Kalcheva,
Eur. J. Med. Chem 2007, 42, 1382.
[18] S. Unlu, T. Onkol, Y. Dundar, B. Okcelik, E. Kupeli, E. Yesilada, N.
Noyanalpan, M. F. Sahin, Arch. Pharm. 2003, 336, 353.
[19] M. Koksal, N. Gokhan, E. Kupeli, E. Yesilada, H. Erdogan, Arch. Pharm.
2005, 338, 117.
https://doi.org/10.1002/ardp.201700273