Cholinesterase inhibitory activities of adamantyl-based derivatives and their molecular docking studies

Article abstract of DOI:10.3390/molecules22061005

Adamantyl-based compounds are clinically important for the treatments of type 2 diabetes and for their antiviral abilities, while many more are under development for other pharmaceutical uses. This study focused on the acetylcholinesterase (AChE) and buty

Full text of DOI:10.3390/molecules22061005

molecules
Article
Cholinesterase Inhibitory Activities of
Adamantyl-Based Derivatives and
Their Molecular Docking Studies
Huey Chong Kwong 1, Siau Hui Mah 2,*, Tze Shyang Chia 3, Ching Kheng Quah 3,
Gin Keat Lim 1 and C. S. Chidan Kumar 4
1
School of Chemical Sciences, Universiti Sains Malaysia, Penang 11800 USM, Malaysia;
stonekg01@gmail.com (H.C.K.); limgk@usm.my (G.K.L.)
School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia
X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, Penang 11800 USM, Malaysia;
2
3
chiatzeshyang@hotmail.com (T.S.C.); ckquah@usm.my (C.K.Q.)
Department of Engineering Chemistry, Vidya Vikas Institute of Engineering & Technology,
4
Visvesvaraya Technological University, Alanahalli, Mysuru 570028, Karnataka, India;
chidankumar@gmail.com
*
Correspondence: SiauHui.Mah@taylors.edu.my; Tel.: +60-3-5629-5000; Fax: +60-3-5629-5001
Academic Editors: Anthony H. Futerman, Yacov Ashani, Gabriel (Gabi) Amitai and Lev Weiner
Received: 23 May 2017; Accepted: 14 June 2017; Published: 17 June 2017
Abstract: Adamantyl-based compounds are clinically important for the treatments of type 2 diabetes
and for their antiviral abilities, while many more are under development for other pharmaceutical
uses. This study focused on the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
inhibitory activities of adamantyl-based ester derivatives with various substituents on the phenyl
ring using Ellman’s colorimetric method. Compound 2e with a 2,4-dichloro electron-withdrawing
substituent on the phenyl ring exhibited the strongest inhibition effect against AChE, with an IC50
value of 77.15
the phenyl ring exhibited good AChE inhibition effects with an ascending order for the substituents:
Cl < NO2 < CH3 < OCH3. Furthermore, compounds with electron-withdrawing groups (Cl and NO2
µM. Overall, the adamantyl-based ester with the mono-substituent at position 3 of
)
substituted at position 3 on their phenyl rings demonstrated stronger AChE inhibition effects,
in comparison to their respective positional isomers. On the other hand, compound 2j with a
3-methoxyphenyl ring showed the highest inhibition effect against BChE, with an IC50 value of
223.30 µM. Molecular docking analyses were conducted for potential AChE and BChE inhibitors,
and the results demonstrated that the peripheral anionic sites of target proteins were predominant
binding sites for these compounds through hydrogen bonds and halogen interactions instead of
hydrophobic interactions in the catalytic active site.
Keywords: adamantane; acetylcholinesterase; butyrylcholinesterase; molecular docking
1. Introduction
Alzheimer’s disease (AD) is a degenerative brain disease resulting in a decline in memory,
language, problem-solving, and cognitive skills, which affects a person’s ability to perform daily
activities. Additionally, AD can be fatal after a prolonged period of struggle and suffering [1].
Although the intrinsic nature of the pathophysiology of AD is still unclear, a “cholinergic hypothesis”
was developed from the study, targeted on people at an advanced age as well as AD patients’
basal forebrain and rostral forebrain cholinergic pathways [
a cholinesterase inhibitor is used to enhance cognitive functions and slow down AD progression,
and involves symptomatic treatment [ ]. There are a limited number of drugs used as cholinesterase
2]. Based on the cholinergic hypothesis,
3
Molecules 2017, 22, 1005; doi:10.3390/molecules22061005
www.mdpi.com/journal/molecules

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inhibitors in AD treatment; examples are physostigmine and galantamine [4], which originate from
plants, in addition to propidium iodide [
synthetic drugs.
5], tacrine [6], donepezil [7], and rivastigmine [8], which are
Adamantane is a bulky diamondoid with a chemical formula of C10H16. Adamantane was first
suggested by H. Decker in 1924, before it was discovered in petroleum by S. landa, V. Machaced,
and M. Mzoured in 1932 using fractional distillation [9]. A decade later, V. Prelog (1941) successfully
synthesized adamantane using Meerwein’s ester with a five-stage procedure [10]. Plukenetione A
is the first natural product bearing an adamantane framework isolated from a plant source, namely
Clusia plukenetii (Guttiferae), in 1966 [11]. It was found to inhibit the enzymatic activities of both
topoisomerase I and DNA polymerase [12]. Furthermore, adamantan-1-amine (or amantadine) was the
first adamantyl-based compound that was established as a treatment for influenza A infections, as well
as to alleviate a number of symptoms of Parkinson’s disease in 1979 [13]. Additionally, the discovery
of (RS)-1-(adamantyl)ethanamine (or rimantadine) gives a more effective prophylaxis and treatment of
influenza A infections with reduced side effects [14]. Nowadays, its derivatives are applied clinically
in the treatment of acne vulgaris [15], for antivirals [16
AD [24].
–20], for type 2 diabetes [21–23], and also for
Inspired by the diverse bioactivities of adamantane-containing molecules, herein the AChE
and BChE inhibitory activities of 18 adamantyl-based ester derivatives with varying substituents
were evaluated using Ellman’s colorimetric test. As reported previously, the structure of the
18 adamantyl-based ester derivatives were confirmed by NMR, FTIR, MS and single-crystal X-ray
diffraction [25]. In this study, we also investigated the in silico binding mode of the proposed
ligands in AChE and BChE enzymes, in comparison with tacrine as a reference, and the molecular
docking procedure was validated using tacrine as the native ligand. There are two major binding
sites in cholinesterase, namely the catalytic anionic site (CAS) and the peripheral anionic site (PAS).
Since the inhibitors of acetylcholine bind to either CAS (tacrine, galantamine, rivastigmine), PAS
(propidium iodide), or to both (donepezil), we examined the interaction of the potent inhibitors
with the whole binding sites (including both CAS and PAS) through molecular docking simulations.
The Genetic Optimization for Ligand Docking (GOLD) methodology was paired with a Piecewise
Linear Potential (PLP) scoring function to generate the best putative pose and conformation for all
subjected inhibitors.
2. Results and Discussion
Eighteen adamantyl-based ester derivatives (2a
confirmed wit H-NMR, FTIR, MS, and single-crystal X-ray diffraction, as reported previously [18].
All compounds 2a were examined for their AChE and BChE inhibitory activities by Ellman’s
–r) were synthesized, and their structures were
–
r
assay [26], and their IC50 values were tabulated in Table 1. Adamantyl-based ester derivatives
with unsubstituted phenyl (2a) and pyridine (2r) rings were synthesized as reference compounds
to accentuate the electronic effects of different substituents attached to the phenyl ring, including
electron-donating (CH3, OCH3 and NH2) and electron-withdrawing (Cl and NO2) groups, upon their
cholinesterase inhibitory activities. Compounds 2a and 2r showed low inhibition effects towards both
AChE and BChE up to a concentration of 1000 µM. Similarly, compounds 2o, 2p and 2q that bore
strong electron-donating (NH2) substituents were inactive against these cholinesterase enzymes.
Table 1. IC50 values of the AChE and BChE inhibitory effects of adamantyl-based compounds.
Compounds
Substituent
AChE IC50 (µM)
BChE IC50 (µM)
2a
2b
2c
2d
2e
benzene
994.26 ± 19.35
360.56 ± 30.05
280.43 ± 17.35
340.52 ± 34.69
77.15 ± 7.86
>1500
>1500
971.50 ± 62.55
1291.99 ± 30.05
306.77 ± 25.15
2-chlorobenzene
3-chlorobenzene
4-chlorobenzene
2,4-dichlorobenzene

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Table 1. Cont.
Compounds
Substituent
AChE IC50 (µM)
BChE IC50 (µM)
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2-methybenzene
3-methybenzene
4-methybenzene
2-methoxybenzene
3-methoxybenzene
4-methoxybenzene
2-nitrobenzene
3-nitrobenzene
4-nitrobenzene
2-aminobenzene
3-aminobenzene
4-aminobenzene
2-pyridine
174.98 ± 25.87
190.99 ± 14.43
>1500
>1500
>1500
>1500
>1500
>1500
166.46 ± 19.58
223.30 ± 17.58
>1500
>1500
689.23 ± 44.48
232.98 ± 25.88
349.47 ± 25.22
>1500
>1500
>1500
776.59 ± 33.63
>1500
>1500
>1500
>1500
>1500
>1500
>1500
Tacrine
0.21 ± 0.04
0.06 ± 0.01
Note: AChE represents Acetylcholinesterase and BChE represents Butyrylcholinesterase.
For AChE inhibition effects, compounds 2f and 2g with moderate electron-donating moiety and
2-methyl and 3-methyl substitutions on their phenyl rings exhibited a moderate effect, with IC50 values
of 174.98 and 190.99
group substituted at position 4 on the phenyl ring, as seen in compound 2h, which was inactive up
to a concentration of 1500 M. Although compounds 2i 2j and 2k also carried electron-donating
methoxy-substituents on their phenyl rings, only 2j with a 3-methoxy substituent showed moderate
inhibition effects towards AChE, with an IC50 value of 166.46 M. Comparing compounds 2f 2g and
µM, respectively. The inhibition effect dropped dramatically with the methyl
µ
,
µ
,
2j, these showed no statistical difference towards their AChE inhibition activities, as shown in Figure 1.
1000
800
600
d
c, d
c, d
400
200
0
b
a,b
a
a
a
2a
2b
2c
2d
2e
2f
2g
2j
2l
2m
2n
Compound
Figure 1. The percentage of AChE inhibition as means
letters are significantly different (p < 0.05).
±
SD (n = 3). Means with different alphabet
In contrast, compounds bearing electron-withdrawing substituents on the phenyl ring, such
as chlorine and nitro groups, showed relatively lower AChE inhibitory activity. As evidence,
compounds 2b
3 and 4, respectively, showed a range of IC50 values from 280 to 360
compounds with a nitro group attached to their phenyl rings, 2m 2n and 2l, fell in the range from 233
to 690 M. By comparing the position of the electron-withdrawing substituent on the phenyl group,
,
2c and 2d, which had chlorine substituents on their phenyl rings at positions 2,
µM. Similarly, IC50 values for the
,
µ
adamantyl compounds with the substituent at position 3 exhibited the strongest AChE inhibition
effect, followed by those with position 4 and position 2 substituents.
Overall, compound 2e, which bore 2,4-dichloro substituents on the phenyl ring, was the most
potent AChE inhibitor among the adamantyl derivatives, with an IC50 value of 77.15
µM. However, this
compound showed weak activities against BChE, with an IC50 value of 306.77 M, which was almost
µ

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5 times lower than that of the AChE inhibition effect. Although compound 2e showed much weaker
effects in the BChE inhibition assay, this compound still existed as one of the most active compounds
in this enzyme inhibition test. Among the derivatives, compound 2j exhibited the strongest inhibition
towards BChE, with an IC50 value of 223.30 µM, followed by 2e.
The results suggest that the substances with mono-substituents at position 3 of the phenyl ring
exhibited profound effects on the AChE inhibition, and these effects increased in the order of the
substituent moieties: Cl < NO2 < CH3 < OCH3. On the other hand, the AChE inhibition effects of
the compounds with mono-substituted electron-withdrawing groups (Cl and NO2) on their phenyl
rings increased in the order of substituents: position 2 < position 4 < position 3. A similar effect of
positional substitution was also observed in 2-(2-(4-benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione
derivatives [27]. Compound 2e, with two chlorine groups substituted at positions 2 and 4, was far more
potent in the AChE inhibition test compared to other mono-chloro-substituted derivatives. In fact, these
compounds exerted less potency toward BChE inhibition, and encompassing an electron-donating
group (methoxyl) at position 3 of the phenyl ring did increase the BChE inhibitory activity effectively.
In other words, the removal of a functional group or the addition of a strong electron-donating group
may decrease or deactivate the cholinesterase inhibition effects of adamantyl-based derivatives.
Molecular docking studies were performed to provide a binding mode of adamantyl-based
derivatives within the cholinesterase enzymes. Differential validation of the docking accuracy was
investigated by docking native co-crystallized ligand (tacrine) into parent enzymes to compare the
conformation of the best-scored pose with the bound ligand in the native crystal (see Figure 2a).
The overall structure of a human’s BChE is similar to a human’s AChE, with the difference of several
aromatic residues lining the gorge of AChE being replaced by hydrophobic aliphatic residue in BChE.
Generally, phenylalanines (Phe) in the acyl binding pocket of AChE are replaced by leucine (Leu)
and valine (Val) in BChE, which allow bulkier substrate moieties to bind more readily to BChE than
to AChE [28]. However, our recent cholinesterase enzymes inhibitory study showed that bulky
adamantyl-based ester derivatives exhibited a greater AChE inhibitory when compared to BChE.
Figure 2. Differential validation in the GOLD package by docking the native ligands of AChE into their
binding sites: (a) The native co-crystallized tacrine represented by green sticks; docked ligands are
shown in the form of balls and sticks, colored by element. Putative binding modes of: (b) compound
2c; (c) compound 2m; (d) compound 2g; (e) compound 2j; and (f) compound 2f.

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In the protein crystal structure of 1ACJ, the inhibitor tacrine was bound to the catalytic active
site of the protein AChE through interactions, involving residues Trp84 and Phe330, as seen in
Figure 2a. Similarly, adamantyl moiety in all potent substances was able to bind at the catalytic active
site via alkyl/ interactions with the residues Trp84 and Phe330. Furthermore, an additional alkyl/
interaction with residue His440 was observed for compounds 2b 2d 2f and 2j. For those compounds
with position 3 substituents on their phenyl rings (2c 2g 2j and 2m), their phenyl rings tended to form
interactions with residues Trp279 and Tyr334 at the peripheral anionic site [29]. For compounds
π/π
π
π
,
,
,
,
π/π
2c and 2m, electron-withdrawing substituents on their phenyl rings failed to bind with other residues
at the catalytic active site, which led to a decrease in enzyme inhibition activities (see Figure 2b,c).
Conversely, the electron-donating substituent present in compounds 2g and 2j managed to form an
extra C–H/
π
interaction with residue Phe330 (Figure 3d,e), and resulted in stronger inhibition effects.
interaction formed with the compounds 2c 2g 2j and 2m, the 2-methyl
Besides the
π/π
,
,
substituent in compound 2f was able to bind with residues Tyr70 and Tyr121 (see Figure 2f) in
the adjacent peripheral anionic site and procure comparable inhibitory activity to compounds 2g and
2j. As shown in Figures 2b and 3a,b, the phenyl ring of mono-chloro-substituted compounds 2b
and 2d was favored to bind with residues Trp279 and Tyr334 at the peripheral anionic site through
interactions. Furthermore, the halogen atoms of the di-chloro-substituted compound 2e were
capable of binding with residues Trp279, Tyr334 and Tyr70 via halogen and halogen/ interactions, as
seen in Figure 4c, therefore exhibiting stronger inhibition effects than those of 2b and 2d by threefold.
, 2c
π/π
π
Figure 3. Putative binding modes in AChE enzymes of: (
and (c) compound 2e.
a) compound 2b; (b) compound 2d;
For the BChE molecular docking study with a protein crystal structure of 4BDS, the inhibitor
tacrine was bound to the catalytic active site of the protein BChE with alkyl/ and interactions,
involving residues Trp82 and Ala328, as seen in Figure 4a. Similar to AChE, adamantyl moiety of the
potent compounds was able to bound at the catalytic active site via alkyl/ interactions with residues
Trp82 and Ala328 in BChE, while additional alkyl/ interactions with residues His438 or Phe329 were
observed for compounds 2e and 2j, respectively. For compound 2e, its di-chloro-substituted phenyl
ring formed and halogen/ interactions with residues Tyr332 and Pro285 in both the peripheral
π
π/π
π
π
π/
π
π
anionic site and the acyl pocket, resulting in an increase of its BChE inhibition effect (see Figure 4b).
In contrast, the 3-methoxy-substituted phenyl ring in compound 2j was able to form intermolecular
C–H/O hydrogen bonds with Pro285 of the acyl pocket and Ser287 of the peripheral anionic site

Molecules 2017, 22, 1005
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(see Figure 4c). The additional C–H/
π
interactions with residue Trp231 led to stronger BChE inhibition
effects compared to 2e.
As a summary, the molecular docking results showed that the compounds with adamantyl moiety
were able to bind with the catalytic active site via alkyl/ interactions (Trp84 and Phe330 in AChE;
π
Trp82 and Ala328 in BChE) by utilizing the hydrophobic feature of the adamantyl ring. The finding
reveals that AChE inhibition effects are attributed to the di-chloro-substituted rings, which are able
to bind with residues Trp279, Tyr334 and Tyr70 at the peripheral anionic site through halogen and
halogen/π interactions. On the other hand, BChE inhibition effects are mainly contributed by C–H/O
hydrogen bonds between the substituted 3-methoxyphenyl ring and the residue in both the peripheral
anionic site and the acyl pocket.
Figure 4. Differential validation in the GOLD package by docking the native ligands of BChE into their
binding sites: (a) The native co-crystallized tacrine is represented by green sticks, while the docked
ligands are shown in the form of balls and sticks, colored by element. Putative binding modes of:
(b) compound 2e in BChE, and (c) active compound 2j in BChE.
3. Materials and Methods
Electric eel acetylcholinesterase (AChE, EC 3.1.1.7), equine serum butyrylcholinesterase (BChE, EC
3.1.1.8), 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB), and sodium phosphate dibasic (Na2HPO4)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). S-butyrylthiocholine chloride (BTCC)
from Sigma-Aldrich (Buchs, Switzerland), acetylthiocholine iodide (ATCI) from Sigma-Aldrich
(Dorset, UK) and sodium phosphate monobasic (NaH2PO4) from Sigma-Aldrich (Munich, Germany)
were purchased. Tacrine hydrochloride from Cayman Chemical Company was used as the standard
drug, and absolute ethanol from Merck Emsure® Germany, was used as the solvent. The reagents
and solvents for the synthesis work were obtained commercially from Sigma-Aldrich Corporation
(St. Louis, MO, USA) and used without any further purification.
3.1. Synthesis and Structural Elucidation of Adamantane
General synthetic routes for 2-(adamantan-1-yl)-2-oxoethyl benzoates, 2a–q, and 2-(adamantan-1-
yl)-2-oxoethyl-2-pyridinecarboxylate, 2r, are shown in Figure 5. Synthesis methods, spectroscopy
analyses, and single-crystal X-ray diffraction studies were described in detail in the previous report [25].

Molecules 2017, 22, 1005
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Figure 5. General reaction scheme for the preparation of 2-(adamantan-1-yl)-2-oxoethyl benzoates,
2a–q, and 2-(adamantan-1-yl)-2-oxoethyl-2-pyridinecarboxylate, 2r.
2-(Adamantan-1-yl)-2-oxoethyl benzoate (2a): Yield: 80%; m.p.: 101–103 ◦C; FT-IR (ATR (solid) cm−1):
2917, 2850 (C–H,
(500 MHz, CDCl3):
ν), 1709 (C=O, ν), 1602, 1413 (Ar, C–H, ν), 1277, 1120 (C–O, ν), 706 (C–H, ω
); 1H-NMR
δ
ppm 8.095–8.081 (d, 2H, J = 7.5 Hz, 15CH, 19CH), 7.579–7.549 (t, 1H, J = 7.5 Hz,
17CH), 7.455–7.424 (t, 2H, J = 7.5 Hz, 16CH, 18CH), 5.096 (s, 2H, 12CH2), 2.079 (br-s, 3H, 2CH, 4CH,
6CH), 1.935–1.930 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.796–1.719 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR
(125 MHz, CDCl3):
δ
ppm 207.16 (C11), 166.04 (C13), 133.19 (C17), 129.87 (C15, C19), 129.58 (C14), 128.37
(C16, C18), 64.94 (C12), 45.39 (C8), 38.02 (C1, C7, C9), 36.47 (C3, C5, C10), 27.79 (C2, C4, C6); GC-MS (EI)
m/z: 298 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 2-chlorobenzoate (2b): Yield: 83%; m.p.: 105–107 ◦C; FT-IR (ATR (solid)
cm−1): 2904, 2850 (C–H,
ν
), 1709 (C=O,
ppm 8.008–7.992 (d, 1H, J = 8.0 Hz, 19CH), 7.468–7.452 (d, 1H,
17CH), 5.115
ν), 1590, 1416 (Ar, C–H, ν), 1247, 1024 (C–O, ν), 742 (C–Cl,
1
ν
); H-NMR (500 MHz, CDCl3):
δ
J = 8.0 Hz
,
16CH), 7.446–7.416 (t, 1H, J = 8.0 Hz, 18CH), 7.344–7.315 (t, 1H, J = 8.0 Hz
,
(
(
s, 2H,
12CH2), 2.085 (br-s, 3H, 2CH, 4CH, 6CH), 1.934–1.929 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.798–1.719
br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 206.98 (C11), 164.89 (C13), 133.98
(C15), 132.84 (C17), 131.95 (C19), 131.03 (C16), 129.35 (C14), 126.62 (C18), 65.18(C12), 45.38 (C8), 37.99
(C1, C7, C9), 36.44 (C3, C5, C10), 27.75 (C2, C4, C6); GC-MS (EI) m/z: 332 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 3-chlorobenzoate (2c): Yield: 73%; m.p.: 130–132 ◦C; FT-IR (ATR (solid)
cm−1): 2911, 2850 (C–H,
(C–Cl,
ν
), 1718 (C=O,
ν), 1571, 1442 (Ar, C–H, ν), 1295, 1253, 1130 (C–O, ν), 745
ν
); 1H-NMR (500 MHz, CDCl3): ppm 8.070 (s, 1H, 15CH), 7.978–7.962 (d, 1H, J = 7.8 Hz, 17CH),
δ
7.554–7.538 (d, 1H, J = 7.8 Hz, 19CH), 7.407–7.376 (t, 1H, J = 7.8 Hz, 18CH), 5.107 (s, 2H, 12CH2), 2.086
(br-s, 3H, 2CH, 4CH, 6CH), 1.928–1.925 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.800–1.719 (br-q, 6H, 3CH2,
5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 206.94 (C11), 164.88 (C13), 134.56 (C16), 133.27 (C17),
131.27 (C14), 129.95 (C18), 129.73 (C15), 128.01 (C19), 65.23(C12), 45.37 (C8), 37.98 (C1, C7, C9), 36.43
(C3, C5, C10), 27.74 (C2, C4, C6); GC-MS (EI) m/z: 332 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 4-chlorobenzoate (2d): Yield: 75%; m.p.: 138–140 ◦C; FT-IR (ATR (solid)
cm−1): 2910, 2851 (C–H, ν), 1723 (C=O,
ν), 1593, 1421 (Ar, C–H, ν), 1269, 1119 (C–O, ν), 752

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(
(
(
C–Cl, ν); 1H-NMR (500 MHz, CDCl3):
d, 2H, J = 8.7 Hz
br-d, 6H, 1CH2, 7CH2, 9CH2), 1.797–1.717 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 8.029–8.011 (d, 2H, J = 8.7 Hz, 16CH, 18CH), 7.429–7.412
2
4
6
,
15CH, 19CH), 5.093 (s, 2H, 12CH2), 2.082 (br-s, 3H, CH, CH, CH), 1.926–1.921
δ
ppm 207.07 (C11), 165.19 (C13), 139.71 (C17), 131.27 (C15, C19), 128.76 (C16, C18), 128.01 (C14), 65.11(C12),
45.37 (C8), 37.99 (C1, C7, C9), 36.43 (C3, C5, C10), 27.75 (C2, C4, C6); GC-MS (EI) m/z: 332 (M+).
◦
2-(Adamantan-1-yl)-2-oxoethyl 2,4-dichlorobenzoate (2e): Yield: 85%; m.p.: 122–124 C; FT-IR (ATR (solid)
cm−1): 2912, 2850 (C–H,
(C–Cl,
ν
), 1711 (C=O,
ν), 1583, 1417 (Ar, C–H, ν), 1244, 1129, 1098, 1023 (C–O, ν), 829
ν
); 1H-NMR (500 MHz, CDCl3): ppm 7.976–7.959 (d, 1H, J = 8.5 Hz, 19CH), 7.484 (s, 1H, 16CH),
δ
7.329–7.312 (d, 1H, J = 8.5 Hz, 18CH), 5.105 (s, 2H, 12CH2), 2.085 (br-s, 3H, 2CH, 4CH, 6CH), 1.925–1.920
(br-d, 6H, 1CH2, 7CH2, 9CH2), 1.799–1.717 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 206.86 (C11), 164.01 (C13), 138.66 (C15), 135.20 (C17), 133.00 (C19), 131.00 (C16), 127.62 (C14),
127.06 (C18), 65.30 (C12), 45.38 (C8), 37.98 (C1, C7, C9), 36.42 (C3, C5, C10), 27.73 (C2, C4, C6); GC-MS
(EI) m/z: 366 (M+).
◦
2-(Adamantan-1-yl)-2-oxoethyl 2-methylbenzoate (2f): Yield: 83%; m.p.: 94–96 C; FT-IR (ATR (solid)
cm−1): 2905, 2851 (C–H,
ν
), 1708 (C=O,
1H-NMR (500 MHz, CDCl3): ppm 8.018–8.002 (d, 1H, J = 8.0 Hz, 19CH), 7.421–7.391 (t, 1H, J = 8.0 Hz
17CH), 7.252–7.235 (m, 2H, 16CH, 18CH), 5.087 (s, 2H, 12CH2), 2.607 (s, 3H, 20CH3), 2.085 (br-s, 3H, 2CH,
ν), 1602, 1415 (Ar, C–H, ν), 1253, 1026 (C–O, ν), 733 (C–H, ω);
δ
,
4CH, CH), 1.940–1.935 (br-d, 6H, CH2, CH2, CH2), 1.799–1.721 (br-q, 6H, CH2, CH2, 10CH2);
6
1
7
9
3
5
13C-NMR (125 MHz, CDCl3):
δ
ppm 207.41 (C11), 166.98 (C13), 140.43 (C15), 132.19 (C17), 131.58 (C16),
130.89 (C19), 129.07 (C14), 125.72 (C18), 64.74 (C12), 45.37 (C8), 38.03 (C1, C7, C9), 36.47 (C3, C5, C10),
27.78 (C2, C4, C6), 21.60 (C20); GC-MS (EI) m/z: 312 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 3-methylbenzoate (2g): Yield: 75%; m.p.: 99–101 ◦C; FT-IR (ATR (solid)
cm−1): 2902, 2848 (C–H,
(C–H,
ν
), 1724 (C=O,
ν
), 1588, 1421, (Ar, C–H,
ν), 1302, 1279, 1195, 1109 (C–O, ν), 740
ω
); 1H-NMR (500 MHz, CDCl3):
7.388–7.372 (d, 1H, J = 7.6 Hz, 17CH), 7.345–7.315 (t, 1H, J = 7.6 Hz, 18CH), 5.093 (s, 2H, 12CH2), 2.397
s, 3H, 20CH3), 2.083 (br-s, 3H, 2CH, 4CH, 6CH), 1.936–1.931 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.798–1.721
δ
ppm 7.909 (s, 1H, 15CH) 7.894–7.879 (d, 2H, J = 7.6 Hz, 19CH),
(
(br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 207.32 (C11), 166.25 (C13), 138.17
(C16), 134.00 (C15), 130.39 (C17), 129.44 (C14), 128.29 (C18), 127.04 (C19), 64.91 (C12), 45.39 (C8), 38.01
(C1, C7, C9), 36.47 (C3, C5, C10), 27.78 (C2, C4, C6), 21.26 (C20); GC-MS (EI) m/z: 312 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 4-methylbenzoate (2h): Yield: 81%; m.p.: 140–142 ◦C; FT-IR (ATR (solid)
cm−1): 2902, 2853 (C–H,
ν
), 1714 (C=O,
ppm 7.984–7.968 (d, 2H, J = 8.1 Hz, 15CH, 19CH), 7.249–7.233
16CH, 18CH), 5.080 (s, 2H, 12CH2), 2.410 (s, 3H, 20CH3), 2.080 (br-s, 3H, 2CH, 4CH,
6CH), 1.934–1.929 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.795–1.718 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR
ν), 1610, 1418, (Ar, C–H, ν), 1258, 1115, (C–O, ν), 747 (C–H,
1
ω
); H-NMR (500 MHz, CDCl3):
δ
(d, 2H, J = 8.1 Hz,
(125 MHz, CDCl3):
δ
ppm 207.37 (C11), 166.12 (C13), 143.93 (C17), 129.92 (C15, C19), 129.10 (C16, C18),
126.80 (C14), 64.82 (C12), 45.39 (C8), 38.01 (C1, C7, C9), 36.47 (C3, C5, C10), 27.79 (C2, C4, C6), 21.70 (C20);
GC-MS (EI) m/z: 312 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 2-methoxybenzoate (2i): Yield: 80%; m.p.: 89–91 ◦C; FT-IR (ATR (solid)
cm−1): 2902, 2851 (C–H,
(
ν
), 1702 (C=O,
C–H, ω); 1H-NMR (500 MHz, CDCl3):
J = 7.8 Hz, 17CH), 7.005–6.972 (t, 2H, J = 7.8 Hz, 16CH, 18CH), 5.071 (s, 2H, 12CH2), 3.907 (s, 3H, 20CH3),
ν
), 1599, 1442 (Ar, C–H,
ν), 1248, 1096, 1020 (C–O, ν), 758
δ
ppm 7.966–7.950 (d, 1H, J = 7.8 Hz, 19CH), 7.500–7.468 (t, 1H
,
2
4
6
1
7
9
2.074 (br-s, 3H, CH, CH, CH), 1.931–1.926 (br-d, 6H, CH2, CH2, CH2), 1.791–1.714 (br-q, 6H,
5
3CH2, CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 207.41 (C11), 165.23 (C13), 159.52 (C15),
133.90 (C17), 132.21 (C19), 120.17 (C18), 119.19 (C14), 111.99 (C16), 64.72 (C12), 56.03 (C20), 45.38 (C8),
38.01 (C1, C7, C9), 36.48 (C3, C5, C10), 27.79 (C2, C4, C6); GC-MS (EI) m/z: 328 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 3-methoxybenzoate (2j): Yield: 74%; m.p.: 157–159 ◦C; FT-IR (ATR (solid)
cm−1): 2926, 2853 (C–H,
ν
), 1711 (C=O,
ν), 1584, 1489 (Ar, C–H, ν), 1288, 1221, 1029 (C–O, ν), 759 (C–H,
1
ω
); H-NMR (500 MHz, CDCl3):
δ
ppm 7.700–7.685 (d, 1H, J = 7.9 Hz, 19CH), 7.598 (s, 1H, 15CH),

Molecules 2017, 22, 1005
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7.365–7.333 (t, 1H, J = 7.9 Hz, 18CH), 7.127–7.110 (d, 1H, J = 7.9 Hz, 17CH), 5.093 (s, 2H, 12CH2), 3.846
s, 3H
20CH3), 2.083 (br-s, 3H, 2CH, 4CH, 6CH), 1.936–1.931 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.798–1.721
(
,
(br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 207.18 (C11), 165.96 (C13), 159.56
(C16), 130.79 (C14), 129.42 (C18), 122.35 (C19), 120.04 (C17), 114.05 (C15), 65.04 (C12), 55.44 (C20), 45.39
(C8), 38.01 (C1, C7, C9), 36.46 (C3, C5, C10), 27.77 (C2, C4, C6); GC-MS (EI) m/z: 328 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 4-methoxybenzoate (2k): Yield: 80%; m.p.: 117–119 ◦C; FT-IR (ATR (solid)
cm−1): 2903, 2851 (C–H,
(C–H,
ν
), 1710 (C=O,
ν), 1605, 1417 (Ar, C–H, ν), 1253, 1167, 1028 (C–O, ν), 767
ω
); 1H-NMR (500 MHz, CDCl3): ppm 8.053–8.035 (d, 2H, J = 9.0 Hz, 15CH, 19CH), 6.932–6.914
δ
(d, 2H, J = 9.0 Hz, 16CH, 18CH), 5.069 (s, 2H, 12CH2), 3.863 (s, 3H, 20CH3), 2.080 (br-s, 3H, 2CH, 4CH,
6CH), 1.933–1.928 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.795–1.718 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR
(125 MHz, CDCl3):
δ
ppm 207.54 (C11), 165.77 (C13), 163.61 (C17), 131.96 (C15, C19), 121.94 (C14), 113.66
(C16, C18), 64.73 (C12), 55.44 (C20), 45.39 (C8), 38.02 (C1, C7, C9), 36.47 (C3, C5, C10), 27.79 (C2, C4, C6);
GC-MS (EI) m/z: 328 (M+).
◦
2-(Adamantan-1-yl)-2-oxoethyl 2-nitrobenzoate (2l): Yield: 70%; m.p.: 118–120 C; FT-IR (ATR (solid)
cm−1): 2919, 2850 (C–H,
1079 (C–O, ), 732 (C–H,
ν
), 1731 (C=O,
ν
), 1579, 1450 (Ar, C–H,
ν), 1535, 1353 (N–O, ν), 1290, 1132,
ν
ω
); 1H-NMR (500 MHz, CDCl3):
δ
ppm 7.970–7.954 (d, 1H, J = 7.7 Hz, 16CH),
7.935–7.920 (d, 1H, J = 7.7 Hz, 19CH), 7.726–7.695 (t, 1H, J = 7.7 Hz, 18CH), 7.663–7.631 (t, 1H, J = 7.7 Hz
,
17CH), 5.123 (s, 2H, 12CH2), 2.088 (br-s, 3H, CH, CH, CH), 1.919 (br-s, 6H, CH2, CH2, CH2),
2
4
6
1
7
9
1.776–1.743 (br-d, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 206.75 (C11), 164.97
(C13), 147.74 (C15), 133.17 (C17), 131.78 (C18), 130.35 (C19), 127.46 (C14), 123.94 (C16), 65.92 (C12), 45.39
(C8), 37.95 (C1, C7, C9), 36.41 (C3, C5, C10), 27.72 (C2, C4, C6); GC-MS (EI) m/z: 343 (M+).
2-(Adamantan-1-yl)-2-oxoethyl 3-nitrobenzoate (2m): Yield: 72%; m.p.: 112–114 ◦C; FT-IR (ATR (solid)
cm−1): 3092 (Ar, C–H,
(N–O,
15CH), 8.449–8.432 (d, 1H, J = 8.0 Hz, 17CH), 8.417–8.402 (d, 1H, J = 8.0 Hz, 19CH), 7.685–7.653 (t, 1H,
J = 8.0 Hz
18CH), 5.167 (s, 2H, 12CH2), 2.101 (br-s, 3H, 2CH, 4CH, 6CH), 1.940 (br-s, 6H, 1CH2, 7CH2,
ν
), 2917, 2851 (C–H,
ν
), 1727 (C=O,
ω
ν
), 1631, 1421 (Ar, C–H,
ν), 1533, 1347
ν
), 1297, 1261, 1131 (C–O, ), 716 (C–H,
ν
); 1H-NMR (500 MHz, CDCl3):
δ
ppm 8.923 (s, 1H,
,
9CH2), 1.788–1.756 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 206.60 (C11),
164.00 (C13), 148.31 (C16), 135.52 (C19), 131.35 (C14), 129.67 (C18), 127.66 (C17), 124.92 (C15), 65.61 (C12),
45.39 (C8), 37.99 (C1, C7, C9), 36.42 (C3, C5, C10), 27.74 (C2, C4, C6); GC-MS (EI) m/z: 343 (M+).
◦
2-(Adamantan-1-yl)-2-oxoethyl 4-nitrobenzoate (2n):Yield: 76%; m.p.: 164–166 C; FT-IR (ATR (solid)
cm−1): 3112 (Ar, C–H,
), 1283, 1118 (C–O,
ν
), 2905, 2854 (C–H,
ν
), 1723 (C=O,
ν), 1606, 1422 (Ar, C–H, ν), 1530, 1344 (N–O,
ν
ν
), 714 (C–H,
ω
); 1H-NMR (500 MHz, CDCl3):
δ ppm 8.310–8.292 (d, 2H, J = 8.8 Hz,
16CH 18CH), 8.265–8.248 (d, 2H, J = 8.8 Hz, 15CH, 19CH), 5.156 (s, 2H, 12CH2), 2.100 (br-s, 3H, 2CH,
4CH, 6CH), 1.936 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.811–1.729 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR
(125 MHz, CDCl3):
δ
ppm 206.61 (C11), 164.21 (C13), 150.72 (C17), 134.97 (C14), 131.00 (C15, C19), 123.56
(C16, C18), 65.61 (C12), 45.40 (C8), 37.99 (C1, C7, C9), 36.41 (C3, C5, C10), 27.73 (C2, C4, C6); GC-MS (EI)
m/z: 343 (M+).
2-(adamantan-1-yl)-2-oxoethyl 2-aminobenzoate (2o): Yield: 76%; m.p.: 167–169 ◦C; FT-IR (ATR (solid)
cm−1): 3497, 3373 (N–H,
1243, 1104 (C–O, ), 746 (C–H,
19CH), 7.287–7.256 (t, 1H, J = 8.3 Hz 17CH), 6.667–6.639 (m, 2H, 16CH, 18CH), 5.647 (br-s, 2H, 1NH2),
ν
), 2902, 2848 (C–H,
ν
), 1695 (C=O,
ν
), 1613, 1418 (Ar, C–H,
ν), 1581 (N–H, δ),
ν
ω
); 1H-NMR (500 MHz, CDCl3):
δ
ppm 7.954–7.940 (d, 1H, J = 8.3 Hz
,
2
4
6
1
7
9
5.068 (s, 2H, 12CH2), 2.083 (br-s, 3H, CH, CH, CH), 1.933–1.928 (br-d, 6H, CH2, CH2, CH2),
1.798–1.721 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 207.64 (C11), 167.29
(C13), 150.52 (C15), 134.38 (C17), 131.63 (C19), 116.67 (C18), 116.41 (C16), 110.34 (C14), 64.46 (C12), 45.38
(C8), 38.05 (C1, C7, C9), 36.47 (C3, C5, C10), 27.79 (C2, C4, C6); GC-MS (EI) m/z: 313 (M+).
2-(adamantan-1-yl)-2-oxoethyl 3-aminobenzoate (2p): Yield: 70%; m.p.: 123–125 ◦C; FT-IR (ATR (solid)
cm−1): 3465, 3347 (N–H,
1248, 1113 (C–O, ), 751 (C–H,
ν
), 2902, 2849 (C–H,
ν
), 1700 (C=O,
ν
), 1631, 1461 (Ar, C–H,
ν), 1603 (N–H, δ),
ν
ω
); 1H-NMR (500 MHz, CDCl3):
δ
ppm 7.502–7.486 (d, 1H, J = 7.8 Hz,

Molecules 2017, 22, 1005
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19CH), 7.415 (s, 1H, 15CH), 7.242–7.211 (t, 1H, J = 7.8 Hz, 18CH), 6.907–6.891 (d, 1H, J = 7.8 Hz, 17CH),
2
4
6
1
7
9
5.074 (s, 2H, 12CH2), 2.080 (br-s, 3H, CH, CH, CH), 1.929–1.923 (br-d, 6H, CH2, CH2, CH2),
1.794–1.717 (br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 207.36 (C11), 166.17
(C13), 145.98 (C16), 130.49 (C14), 129.34 (C18), 120.42 (C19), 119.99 (C17), 116.28 (C15), 64.91 (C12), 45.39
(C8), 38.00 (C1, C7, C9), 36.46 (C3, C5, C10), 27.77 (C2, C4, C6); GC-MS (EI) m/z: 313 (M+).
2-(adamantan-1-yl)-2-oxoethyl 4-aminobenzoate (2q): Yield: 76%; m.p.: 155–157 ◦C; FT-IR (ATR (solid)
cm−1): 3493, 3362 (N–H,
1276, 1118 (C–O, ), 768 (C–H,
15CH, 19CH), 6.648–6.631 (d, 2H, J = 8.8 Hz, 16CH, 18CH), 5.039 (s, 2H, 12CH2), 4.075 (b-s, 2H, 1NH2),
ν
), 2906, 2850 (C–H,
ν
), 1697 (C=O,
ν
), 1619, 1414 (Ar, C–H,
ν), 1600 (N–H, δ),
ν
ω
); 1H-NMR (500 MHz, CDCl3):
δ
ppm 7.907–7.890 (d, 2H, J = 8.8 Hz,
2
4
6
1
7
9
3
2.074 (br-s, 3H, CH, CH, CH), 1.929 (br-s, 6H, CH2, CH2, CH2), 1.789–1.714 (br-q, 6H, CH2,
5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3): ppm 207.88 (C11), 166.07 (C13), 151.12 (C17), 132.06
C15, C19), 118.99 (C14), 113.88 (C16, C18), 64.57 (C12), 45.42 (C8), 38.04 (C1, C7, C9), 36.50 (C3, C5, C10),
27.82 (C2, C4, C6); GC-MS (EI) m/z: 313 (M+).
δ
(
2-(Adamantan-1-yl)-2-oxoethyl picolinate (2r): Yield: 76%; M.P.: 129–131 ◦C; FT-IR (ATR (solid) cm−1):
2903, 2849 (C–H, ), 1707 (C=O, ), 1582, 1419 (Ar, C–H, ), 1304 (C–N, ) 1242, 1128 (C–O, ), 745
(C–H,
); 1H-NMR (500 MHz, CDCl3): ppm 8.787–8.772 (d, 1H, J = 7.6 Hz, 18CH), 8.188–8.172 (d, 1H,
J = 7.6 Hz, 15CH), 7.883–7.852 (t, 1H, J = 7.6 Hz, 16CH), 7.519–7.495 (q, 1H, J = 7.6 Hz, 17CH), 5.203
s, 2H
12CH2), 2.085 (br-s, 3H, 2CH, 4CH, 6CH), 1.934–1.929 (br-d, 6H, 1CH2, 7CH2, 9CH2), 1.798–1.720
ν
ν
ν
ν
ν
ω
δ
(
,
(br-q, 6H, 3CH2, 5CH2, 10CH2); 13C-NMR (125 MHz, CDCl3):
δ
ppm 206.36 (C11), 164.54 (C13), 149.86
(C18), 147.41 (C14), 137.12 (C16), 127.18 (C17), 125.53 (C15), 65.70 (C12), 45.31 (C8), 37.99 (C1, C7, C9),
36.44 (C3, C5, C10), 27.75 (C2, C4, C6); GC-MS (EI) m/z: 299 (M+).
3.2. Cholinesterases Inhibitory Assay
AChE and BChE inhibitory activities of the compounds were determined by Ellman’s assay with
minor modifications [26]. Briefly, 210
96-well microplate, followed by 20 L of the compounds in ethanol and 20
After a pre-incubation of 15 min at room temperature, 10
µL of a 100 mM sodium phosphate buffer (pH 8) was added to a
µ
µ
L of AChE (5.32 × 10−3
U).
µL of 0.50 mM DTNB was added into each
well, followed by 10 µL of 0.71 mM acetylthiocholine iodide as the substrate. The absorbance of each
well was measured 10 times with 1 min intervals using the Epoch2, BioTek instrument microplate
reader (Winooski, VT, USA) at 412 nm. For the BChE inhibition assay, the same procedure as described
above was used, except the enzyme and substrate were replaced with BChE (6.85
×
103 U) from Equine
serum and 0.20 mM butyrylthiocholine chloride, respectively. Tacrine was used as a reference drug.
A series of dilutions made up of 6 concentrations of compounds or the reference drug were tested to
obtain the half-maximal inhibitory concentration (IC50). Each test was carried out in triplicate and the
results were expressed as an average value. The percentage of inhibition was calculated using the
following formula:
S blank − S sample
Percentage of inhibition =
× 100%
(1)
S blank
where
S
blank is slope of the blank reaction (without compounds or a reference standard) and
S
sample
is the slope of the sample reaction (with compounds or a reference drug).
3.3. Statistical Analysis
The results of the cholinesterase inhibitory assay were expressed as means
if p < 0.05 using ANOVA of IBM SPSS Statistics for Windows, Version 23.0 (IBM, New York, NY, USA).
±
SD, and were labeled
3.4. Docking Protocol
Molecular docking was performed for the most potent inhibitors using the Genetic Optimization
for Ligand Docking (GOLD) package 5.4.1 [30–32]. The atomic coordinates of target inhibitors were

Molecules 2017, 22, 1005
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obtained directly from previously reported X-ray crystallography data [10], except for ligand 2m, for
which coordinates were constructed in the ChemBio3D workspace and were energy-minimized by
MM2 force fields. The crystal structures of AChE (PDB ID: 1ACJ [33]) and BChE (PDB ID: 4BDS [34])
were isolated from Pteronarcys californica and Homo sapiens, respectively, with tacrine obtained from the
Protein Data Bank ligand-protein binding space, and the conformational flexibility of the ligand inside
the protein was explored by the Genetic Algorithm (GA). A spherical binding site with a radius of
15 Å was used, which lay around the binding site of the tacrine covering both the catalytic anionic and
peripheral anionic sites. Runs were carried out (100 GA), and the top 100 ranked docking poses were
scored using the Piecewise Linear Potential (PLP) scoring function. Default values were used for all
other parameters. The intermolecular interactions of the best-scored pose of each ligand were analyzed
and illustrated using Discovery Studio 4.5 software (Discovery Studio, v4.5.0.15071; San Diego, CA,
USA, Accelrys Inc.; 2015).
4. Conclusions
Eighteen adamantyl-based ester derivatives were synthesized, and their anti-cholinesterase
activities were assessed using Ellman’s assay. The AChE inhibition effects of the adamantyl-based
ester derivatives increased in the order Cl < NO2 < CH3 < OCH3 for mono-substituents at position
3 of the phenyl ring. Moreover, those derivatives with mono-substituted electron-donating groups
(Cl and NO2) on their phenyl rings showed an increasing inhibition effect in the order: position 2
< position 4 < position 3. As such, compound 2e, which bore a 2,4-dichlorophenyl ring, exhibited
the strongest AChE inhibition effects, while compound 2j, with a 3-methoxyphenyl ring, was the
most potent BChE inhibitor. The in silico study of the adamantyl derivatives demonstrated that the
halogen interaction and hydrogen bond between the compounds and their surrounding residues in
the peripheral anionic site and acyl pocket of target enzymes were predominant when compared to
hydrophobic interactions in the catalytic active site.
Acknowledgments:
The authors acknowledge the Fundamental Research Grant Scheme (FRGS)
(FRGS/1/2015/SG01/TAYLOR/03/1) from the Ministry of Higher Education (MOHE) for financial support.
HCK thank the Malaysian Government for the MyBrain15 (MyPhD) scholarship.
Author Contributions: C.S.C.K. and S.H.M. conceived and designed the experiments; H.C.K. and S.H.M.
performed the experiments; S.H.M., T.S.C. and C.K.Q. analyzed the data; S.H.M., C.K.Q. and G.K.L. contributed
reagents/materials/analysis tools; and H.C.K. and S.H.M. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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