2-Benzoyl-6-benzylidenecyclohexanone analogs as potent dual inhibitors of acetylcholinesterase and butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2016.06.016

In the present study, a series of 2-benzoyl-6-benzylidenecyclohexanone analogs have been synthesized and evaluated for their anti-cholinesterase activity. Among the forty-one analogs, four compounds (38, 39, 40 and 41) have been identified as lead compounds due to their highest inhibition on both AChE and BChE activities. Compounds 39 and 40 in particular exhibited highest inhibition on both AChE and BChE with IC₅₀values of 1.6?μM and 0.6?μM, respectively. Further structure–activity relationship study suggested that presence of a long-chain heterocyclic in one of the rings played a critical role in the dual enzymes’ inhibition. The Lineweaver–Burk plots and docking results suggest that both compounds could simultaneously bind to the PAS and CAS regions of the enzyme. ADMET analysis further confirmed the therapeutic potential of both compounds based upon their high BBB-penetrating. Thus, 2-benzoyl-6-benzylidenecyclohexanone containing long-chain heterocyclic amine analogs represent a new class of cholinesterase inhibitor, which deserve further investigation for their development into therapeutic agents for cognitive diseases such as Alzheimer.

Full text of DOI:10.1016/j.bmc.2016.06.016

Accepted Manuscript
2-Benzoyl-6-benzylidenecyclohexanone analogs as potent dual inhibitors of
acetylcholinesterase and butyrylcholinesterase
Sze Wei Leong, Faridah Abas, Kok Wai Lam, Khozirah Shaari, Nordin H. Lajis
PII:
S0968-0896(16)30439-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.016
BMC 13072
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 March 2016
26 May 2016
9 June 2016
Please cite this article as: Leong, S.W., Abas, F., Lam, K.W., Shaari, K., Lajis, N.H., 2-Benzoyl-6-
benzylidenecyclohexanone analogs as potent dual inhibitors of acetylcholinesterase and butyrylcholinesterase,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.016
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2
-Benzoyl-6-benzylidenecyclohexanone analogs as potent dual inhibitors of acetylcholinesterase and
butyrylcholinesterase
a
a,b*
c
a,d
a,
Sze Wei Leong , Faridah Abas , Kok Wai Lam , Khozirah Shaari , Nordin H. Lajis *
a
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang,
Selangor, Malaysia.
b
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400
UPM, Serdang, Selangor, Malaysia.
c
Drug and Herbal Research Centre Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda
Abd. Aziz, 50300 Kuala Lumpur, Malaysia.
d
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor,
Malaysia.
*Corresponding authors
Tel +603 89471491; Fax: +603 89423552
E-mail addresses: faridah_abas@upm.edu.my (Abas. F), nordinlajis@gmail.com (NHLajis)
1

Abstract
In the present study, a series of 2-benzoyl-6-benzylidenecyclohexanone analogs have been synthesized and
evaluated for their anti-cholinesterase activity. Among the forty-one analogs, four compounds (38, 39, 40 and
4
1) have been identified as lead compounds due to their highest inhibition on both AChE and BChE activities.
Compounds 39 and 40 in particular exhibited highest inhibition on both AChE and BChE with IC50 values of
.6 µM and 0.6 µM, respectively. Further structure-activity relationship study suggested that presence of a
1
long-chain heterocyclic in one of the rings played a critical role in the dual enzymes’ inhibition. The
Lineweaver-Burk plots and docking results suggest that both compounds could simultaneously bind to the PAS
and CAS regions of the enzyme. ADMET analysis further confirmed the therapeutic potential of both
compounds based upon their high BBB-penetrating. Thus, 2-benzoyl-6-benzylidenecyclohexanone containing
long-chain heterocyclic amine analogs represent a new class of cholinesterase inhibitor, which deserve further
investigation for their development into therapeutic agents for cognitive diseases such as Alzheimer.
Keywords:
Acetylcholinesterase; Butyrylcholinesterase; 2-Benzoyl-6-benzylidenecyclohexanone; Kinetic studies;
Molecular docking
1
.
Introduction
Acetylcholinesterase and butyrylcholinesterase are important enzymes that catalyze the degradation of
1
-3
acetylcholine, an important neurotransmitter involved in memory and cognition. These enzymes have been
closely associated to Alzheimer’s disease (AD), a progressive and irreversible neurodegenerative brain
disorder characterized by permanent memory loss, cognitive impairment, disorientation, confusion and
4
language deficits. It is the most common type of age-related dementia which affects more than 46 million
5
elderly globally and the number is expected to be triple by the year 2050. Cholinesterase enzymes have also
6
,
been found to promote the aggregation of neurotoxic β-amyloid which responsible for neuronal cell apoptosis.
7
Therefore, targeting AChE and BChE may be one of the most promising approaches in treating AD.
To date, several cholinesterase inhibitors such as tacrine, donepezil, rivastigmine and galantamine have
been approved for the treatments of AD. Although they offered some improvement in AD, several adverse
8
-10
effects including nausea, diarrhea, dizziness and vomiting, have also been observed.
These unpleasant side
effects may not only affect the patients’ health but also reduce their quality of life. New safer cholinesterase
inhibitors with minimal side effects are therefore urgently warranted.
Curcumin is a well-known chemical constituent abundantly found in Curcuma longa, which has been
extensively studied for centuries due to its valuable medicinal properties, especially the anti-oxidant and anti-
1
1-14
inflammatory activities.
Recent studies showed that curcumin could be an ideal agent for treating AD upon
its excellent safety profile and distinctive interference on several AD-related pathological pathways including
1
5-19
acetylcholine degradation, β-amyloid aggregation, tau protein accumulation and neuronal cell destruction.
However, the therapeutic potential of curcumin is constrained by its poor absorbability and stability, resulting
2
0
in low oral bioavailability, which therefore diminishes its usefulness in clinical trials.
Diarylpentanoids, a bioactive group of compounds derived from curcumin has received increasing attention
for its multiple medicinal properties. Diarylpentanoids have been found to exhibit excellent anti-oxidant and
anti-inflammatory properties based on their distinctive suppressing effects on numerous free radicals and pro-
inflammatory cytokines such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, superoxide radical,
2
1-24
hydroxyl radical, tumor necrosis factor alpha (TNF-α), and interleukins.
Recent studies showed that
diarylpentanoids could be the most impactful candidate to substitute curcumin as therapeutic agent for AD due
to its better stability and anti-Alzheimer properties including anti-cholinesterase and anti-β-amyloid
2
5, 26
aggregation activities.
On the basis of this, we therefore further derivatize and investigate the anti-
2

cholinesterase potential of our novel cyclohexanone containing diarylpentenedione series, which has also been
2
7
shown to exhibit anti-inflammatory properties with excellent metabolic stability.
2
.
Results and discussion
2
.1. Chemistry
Synthesis of target compounds was performed according to the reaction sequence outlined in Scheme 1 and
2
.
x
Scheme 1. General synthetic steps for compounds 1-37
x
Reagents and conditions: (a) pyrrolidine, p-toluene-sulphonic acid, toluene, reflux (2h); (b) benzoic anhydride, RT (24h); (c)
2 2 4
H O, reflux (0.5h); (h) benzaldehyde, acetic acid, H SO , RT (overnight).
As shown in Scheme 1, cyclohexanone was first reacted with pyrrolidine to prepare the enamine
intermediate I. The reaction was carried out using Dean-Stark trap to remove water by product in order to
prevent the hydrolysis of the desired enamine. Enamine I was then reacted with a benzoic anhydride to afford
II, the key intermediate in the synthesis of the target compounds. The formation of diketone intermediate II
was confirmed by the detection of a triplet at 4.38 ppm in the proton NMR spectra (data not shown). The
purified intermediate II was further reacted with commercially available aromatic aldehydes to achieve
compounds 1-37 with appreciable yield, ranging from 35-70 %.
x
Scheme 2. General synthetic steps for compounds 38-41
x
Reagents and conditions: (d) 1,2-dibromoethane, K
8h); (f) acetic acid, H SO , RT (overnight).
2 3 2 3
CO , DMF, 80ºC (6h); (e) pyrrolidine or piperidine, K CO , DMF, reflux
(
2
4
3

To prepare compounds 38-41 (Scheme 2), hydroxylated benzaldehydes were first reacted with 1,2-
dibromoethane to form alkoxylated benzaldehyde III. The reactions were carried out in DMF with the
presence of K CO . Excellent yield (>95%) was obtained for both the 3’- and 4’-hydroxybenzaldehydes after
2 3
six hours of reaction time. The product III was then reacted with appropriate heterocyclic amines to form
aminated aldehyde IV. Finally, aminated aldehydes were further reacted with previously synthesized diketone
intermediate II to afford the target compounds 38-41. All synthesized compounds were purified by column
1
13
chromatography and characterized by H-NMR, C-NMR, and high-resolution electron impact-mass
spectrometry. All purified compounds were of 95–99% purity based on their respective HPLC profiles (refer to
Supplementary data).
2
.2. In-vitro AChE and BChE inhibitory activity
The in-vitro AChE and BChE inhibitory activities of the compounds were determined by Ellman's method
and the preliminary screening results for synthesized compounds are presented in Figure 1.
A
B
Figure 1. AChE (A) and BChE (B) inhibitory effects of compounds 1-41 at a testing concentration of 10 µM.
As depicted in Figure 1, twenty-five compounds were found to inhibit AChE activity as compared to only
seven compounds that showed reasonable inhibition on BChE activity. These results suggested that
cyclohexanone containing diarylpentenedione scaffold is generally more selective towards AChE. The IC50
values of the compounds are listed in Table 1.
Table 1. AChE and BChE inhibitory activities of compounds 1-41.
Compounds
Ar (Ring B)
Phenyl
AChE IC50 (µM) BChE IC50 (µM)
1
2
3
4
5
6
7
8
9
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
3,4-difluorophenyl
2-bromophenyl
4-bromophenyl
3,4-dibromophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
6.6 ± 0.5
9.7 ± 1.1
6.6 ± 0.3
8.7 ± 0.8
8.4 ± 0.6
8.7 ± 0.7
9.0 ± 0.4
8.2 ± 0.9
ND
1
1
0
1
7.6 ± 0.4
4

1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2,3-dichlorophenyl
2,4-dichlorophenyl
3,4-dichlorophenyl
3-methylphenyl
4-methylphenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2,3-dimethoxyphenyl
2,5-dimethoxyphenyl
3,4-dimethoxyphenyl
2,3,4-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3-hydroxyphenyl
5.9 ± 0.6
5.4 ± 0.2
9.8 ± 0.2
8.6 ± 0.6
8.5 ± 0.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
7.4 ± 0.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4-hydroxyphenyl
3,4-dihydroxyphenyl
3-hydroxy-4-methoxyphenyl
4-hydroxy-3-methoxyphenyl
3-chloro-4-hydroxyphenyl
3-bromo-4-hydroxyphenyl
naphthalen-1-yl
6.5 ± 0.6
ND
ND
10.6 ± 0.3
4.7 ± 0.2
ND
7.6 ± 0.1
8.3 ± 1.5
5.1 ± 0.3
5.9 ± 0.5
7.8 ± 0.7
8.4 ± 0.7
3.1 ± 0.4
1.6 ± 0.2
3.5 ± 0.3
2.0 ± 0.3
0.095 ± 0.011
naphthalen-2-yl
thiophen-2-yl
5-methylthiophen-2-yl
3-benzylphenyl
ND
ND
ND
ND
4-benzylphenyl
ND
3-(2-(piperidin-1-yl)ethoxy)phenyl
4-(2-(piperidin-1-yl)ethoxy)phenyl
3-(2-(pyrrolidin-1-yl)ethoxy)phenyl
4-(2-(pyrrolidin-1-yl)ethoxy)phenyl
-
1.4 ± 0.4
2.7 ± 0.6
0.6 ± 0.1
2.3 ± 0.2
0.010 ± 0.004
Tacrine
ND = Not determine
Based on the IC50 values of diarylpentenedione analogs against AChE, four compounds were found to
exhibit significant anti-AChE activity with IC50 values ranging from 1.6 to 3.5 µM. Compound 39 was
identified as the most potent due to its strong inhibition profile with IC50 value of 1.6 µM. The overall results
suggested that heterocyclic amine is particularly important for AChE inhibitory activity as all compounds
bearing pyrrolidine or piperidine fragments exhibited strong AChE inhibition. With respect to the substitution
pattern of heterocyclic amines on aryl ring, para- substitution is preferable since compounds 39 and 41
exhibited approximately 2-fold higher inhibition than their respective meta-aminated analogs, 38 and 40. On
the other hand, the presence of thiophene ring in place of aryl (ring B) was found to contribute moderately to
AChE inhibition on the basis that compounds 34 and 35 achieved the IC50 values of 5.1 and 5.9 µM,
respectively. In contrast, presence of any halogen group in aryl ring B was not favorable since analogs 2-14
generally displayed weaker inhibition. Besides, methoxy and hydroxy groups (17-31) are considered to be
undesirable due to their poor AChE inhibition.
The BChE was strongly inhibited by compounds 38-41 with the IC50 values ranging from 0.6 to 2.7 µM.
Compound 40 demonstrated the strongest inhibitory activity with IC50 value of 0.6 µM. This observation
suggested that heterocyclic amine is not only important for AChE inhibition but is also critical for BChE
inhibitory effect. Unlike the SAR trend exhibited by AChE, the presence of heterocyclic amine at meta-
position is more preferable for BChE inhibition activity, as shown by analogs 38 and 40, which were 2- to 4-
fold more active than the para-aminated analogs 39 and 41. In addition, pyrrolidine fragment appeared to be a
better choice than piperidine, on account that compound 40 exhibited 2.3-fold better activity than 38. Apart
from heterocyclic amine, the only other contributing factor for BChE inhibitory activity was the hydroxyl
5

group. This is clearly implied based on the stronger BChE inhibition of compounds 25-31 (Figure 1) at 10 µM
testing concentration. However, only three compounds, 27, 30 and 31 achieved greater than 50% BChE
inhibition. Further IC50 determination showed that they are moderate BChE inhibitors with IC50 values of 6.5,
1
0.6 and 4.7 µM, respectively.
Interestingly, it was reported that BChE activity is dramatically increased when AChE is inhibited, which
2
8
implied that dual inhibitors are a better choice for treating Alzheimer’s disease. Thus, compounds 38-41 with
strong inhibitory effects on both AChE and BChE activity could be potential candidates, which deserve further
investigation towards a new anti-Alzheimer agent.
2
.3. Kinetic analysis of AChE and BChE inhibition
To determine the inhibition type and inhibition constant, K of the compounds, enzyme kinetic studies were
i
carried out on compounds 39 and 40 using AChE and BChE. The analysis was carried out by investigating the
effects of inhibitors’ concentration on enzyme’s activity at different substrate concentrations. The data resulted
from cholinesterase inhibition was further analyzed with Lineweaver-Burk method followed by dissociating
constant (Ki) calculation from Michaellis-menten using GraphPad prism 5. The calculated results suggest that
both compounds exhibited mixed-type inhibition with K values of 1.9 and 0.2 µM, respectively. The dual
i
character (competitive and uncompetitive) of compounds 39 and 40 suggests that both of them could
simultaneously bind to catalytically active site (CAS) and peripheral anionic site (PAS) of the respective
enzymes. The Lineweaver-Burk double reciprocal plots of compounds 39 and 40 are depicted in Figures 2A
and 2B, respectively.
A
B
Figure 2. Lineweaver–Burk double reciprocal plots resulting from substrate-velocity curves of AChE activity of compound 39
A) and BuChE activity of compound 40 (B).
(
2
.4.Molecular modeling
In order to gain functional and structural insights into the binding mode of compounds 39 and 40, flexible
docking was performed using Discovery Studio 3.1. The docking parameters were first validated by re-docking
of co-crystallized ligands into the active site of the enzymes. The parameters are considered successful if the
Root Mean Square Deviation (RMSD) values of the re-docked ligands are less than 1.5 Å, in comparison with
29
their original conformation in the crystal structure. As depicted in Figure 3, both the re-docked donepezil
and tacrine (red) were found to bind in a similar manner as their respective crystallographic conformations
(
blue), indicating that the selected docking parameters are acceptable. The RMSD values for donepezil and
tacrine conformations are 1.06 Å and 0.89 Å, respectively.
6

B
A
Figure 3. Overlay of re-docked (red) and crystallographic (blue) conformations of donepezil in AChE (A) and tacrine in BChE
(B), respectively.
The optimized parameters were then used for the docking of compounds 39 and 40 in AChE and BChE
active sites, respectively. Based on the docking result (Figure 4), compound 39 was found to simultaneously
interact with both the PAS and CAS of the AChE. This observation further supported the mixed type inhibition
mechanism of compound 39, which has been suggested in our previous kinetic study.
Figure 4. Binding interactions of compound 39 with the active site residues of TcAChE receptor.
The piperidine moiety of compound 39 showed a hydrophobic interaction with Trp-279 residue in PAS
(
Figure 4). This interaction is particularly important as several studies have reported the promoting role of
30, 31
Trp-279 in β-amyloid deposition.
Apart from Trp-279, Tyr-334 residue of PAS also interacted with the
alkoxylated phenyl ring of compound 39 through a π-π stacking. Besides, a similar interaction was also been
observed in CAS in which the alkoxylated phenyl ring stacked against Phe-330 residue with a distance of 5.48
Å. It is worth noting that the cyclohexyl ring of the respective compound displayed a hydrophobic contact with
Phe-331 residue located in the middle of the active site. Interestingly, the benzoyl moiety of compound 39 was
found to bind to the oxyanion site and catalytic triad of TcAChE with multiple interactions. The carbonyl
group of benzoyl fragment forms two hydrogen bonds with the hydroxyl group of Ser-200 and Glu-199
residues in the oxyanion site. Meanwhile, the phenyl ring of the same fragment interacts with Gly-118 residue
of catalytic triad by an amide-π interaction.
7

Figure 5. Binding interactions of compound 40 with the active site residues of hBChE receptor.
Similar to compound 39, compound 40 was also found to be a mixed type inhibitor based upon its double
reciprocal plots in kinetic study and dual binding character in molecular docking. As presented in Figure 5,
compound 40 binds to the PAS of BChE by hydrogen bonding interaction with both Asp-70 and Ser-79
residues. Meanwhile, the cyclohexyl moiety of compound 40 is aligned with the choline binding site and
simultaneously interacts with Trp-82 and Ala-328 residues through hydrophobic bonds. The Trp-82 interaction
is further enhanced by an additional π-π stacking system between the alkoxylated phenyl moiety of compound
4
0 and the indole ring of the respective residue. On the other hand, compound 40 also displayed hydrogen
bonding with catalytic triad through the interaction of oxygen atom of the alkoxylated phenyl ring on both the
His-438 and Ser-198 residues. Interestingly, the pyrrolidine fragment was found to fit into the acyl binding
pocket by interacting with both the Trp-231 and Leu-286 residues through hydrophobic contacts, resulting in
the binding enhancement of compound 40 on BChE. The flexible docking results of compounds 39 and 40 are
summarized in Table 2.
8

Table 2. Data resulted from the flexible docking of compounds 39 and 40 in active site gorge of
TcAChE and hBChE receptors.
Amino
acid
residue
Bonding
distance
Interacting
site
Compound Structure
Enzyme
Bond type
(Å)
Hydrogen
bonding
Ser-286
2.77, 2.91
PAS
Trp-279
Try-334
Phe-330
Phe-331
Hydrophobic
π- π stacking
π- π stacking
Hydrophobic
4.69
4.17
5.48
5.28
Anionic site
TcAChE
Catalytic
triad
Amide - π
stacking
Gly-118
Ser-200
Glu-199
Asp-70
Ser-79
3.87
2.22
Hydrogen
bonding
Oxyanion
site
Hydrogen
bonding
2.20
Hydrogen
bonding
2.60
PAS
Hydrogen
bonding
2.62, 2.58
Hydrophonic;
π- π stacking
4.17, 5.35;
4.93
Trp-82
Ala-328
His-438
Cation – π
site
Hydrophobic
4.79
2.02
hBChE
Hydrogen
bonding
Catalytic
triad
Hydrogen
bonding
Ser-198
2.77
Leu-286
Trp-231
Hydrophobic
Hydrophobic
4.96
Acyl
binding site
4.84, 4.73
9

2.5. ADMET analysis
Apart from strong cholinesterase inhibitory activity, the most fundamental condition for a promising
cholinesterase inhibitor is to exhibit high blood brain barrier (BBB) penetration. Therefore, ADMET analysis
was carried out, specifically to predict the BBB penetration of compounds 39 and 40. The ADMET plot of
compounds 39 and 40 is presented in Figure 6.
Figure 6. ADMET plot of compounds 39 and 40
Compounds 39 and 40 are within the ellipses of 95 and 99% blood brain barrier (BBB) confidence region,
indicating that both compounds could be highly BBB-penetrating agents (Figure 6). This observation further
supports the promising potential of compounds 39 and 40 as cholinesterase inhibitors. Apart from BBB-
penetration prediction, ADMET analysis was also used to predict their aqueous solubility (AS), human
intestinal absorption (HIA), cytochrome P450 2D6 (CYP2D6) inhibition, plasma protein binding (PPB), and
hepatotoxicity (HT). Based on the results in Table 3, both compounds are non-hepatotoxic with good human
intestinal absorption. This implies that they have huge potential to serve as oral drugs. Poor water solubility of
compounds 39 and 40 is the only possible drawback in developing the practical use of them. However, the
presence of nitrogen atom in both compounds could allow the formation of salt which therefore improve their
3
2
solubility. On the other hand, these compounds were also found to bind to plasma protein with no interaction
with cytochrome enzymes, indicating that higher dose may be required to achieve therapeutic concentration in
treatments.
Table 3. Results of ADMET predictions on six important parameters.
Compounds AS HIA BBB CYP2D6
PPB
HT
3
4
9
0
Low Good High Non-inhibit Bound Non-hepatotoxin
Low Good High Non-inhibit Bound Non-hepatotoxin
Notes: AS = Aqueous Solubility; HIA = Human Intestinal Absorption; BBB = Blood Brain Barrier;
CYP2D6 = cytochrome P450 2D6; PPB = Plasma Protein Binding; HT = Hepatotoxicity.
1
0

3
.
Conclusion
In summary, forty-one analogs of cyclohexanone containing diarylpentenedione have been synthesized and
evaluated for their AChE and BChE inhibitory activities. Compounds 39 and 40 appeared to be the most active
analogs based on the determined IC50 values for both AChE and BChE inhibition, respectively. Further kinetic
analyses and docking studies revealed that these compounds exhibited mixed-type inhibition. The overall
findings suggested that the diarylpentenedione core structure is selective towards AChE inhibition, while the
long chain heterocyclic moieties are critical for both AChE and BChE inhibitory activities. ADMET analysis
further confirmed the potential development of both compounds on the basis of their high blood brain barrier
penetration. As a conclusion, we believe that diarylpentenedione containing a long chain heterocyclic amines
is a promising class, which deserve further investigation for their development of novel cholinesterase
inhibitors.
4
. Experimental Section
4
.1. Chemistry
Starting materials and chemical reagents were purchased from Sigma-Aldrich and Merck, and were used
without purification. Solvents, which were purchased from common commercial suppliers, were dried and
distilled before use. The chemical reactions were routinely checked on 0.20 mm Merck TLC plate silica gel 60
F254 in every reaction step. Purification procedures were conducted using column chromatography on Merck
silica gel 60 (mesh 70-230). Melting points were determined using Fisher-Johns melting point apparatus and
were uncorrected. Mass spectra were measured by GCMS-QP5050A (Shimadzu) Mass Spectrometer. High-
resolution electron ionization-mass spectrometry (HREI-MS) was determined using a DFS high resolution
GC/MS (Thermo Scientific, San Jose, CA, USA). Nuclear Magnetic Resonance Spectra were recorded on
Varian 500 MHz NMR Spectrometer.
4
.1.1. General procedure for the synthesis of I and II
A toluene solution (100 mL) containing cyclohexanone (20 mmol), pyrrolidine (20 mmol) and a catalytic
amount of p-toluenesulphonic acid (0.1 g) was heated under Dean-Stark condition for 2 hours to obtain I. Then,
the reaction mixture was cooled to room temperature and further stirred with 20 mmol of benzoic anhydride
for 24 hours. Upon completion, the reaction mixture was added with 10 mL of distilled water and refluxed for
30 minutes. The resulting reaction mixture was extracted thrice with 100 mL HCl (3M) and once with 20 mL
water. The toluene layer was dried over anhydrous magnesium sulphate and concentrated in vacuo to give a
crude product of 2-benzoylcyclohexanone (II). The resulting crude product was purified by column
chromatography.
4
.1.2. General procedure for the synthesis of compounds 1-37
One millimole of 2-benzoylcyclohenxanone (II) was reacted with an equivalent amount of appropriate
aromatic aldehyde in acetic acid (30 mL) in the presence of sulfuric acid as catalyst. The reaction mixture was
stirred at room temperature for overnight. Upon completion, the reaction mixture was poured into 100 mL of
distilled water and stirred for 10 minutes, followed by extraction with 100 mL of ethyl acetate. The organic
layer was then washed with 10% sodium bicarbonate solution and dried over anhydrous magnesium sulphate.
The further evaporation in vacuo gave crude of targeted compounds 1-37. The resulting crude product was
purified by column chromatography.
2
-benzoyl-6-benzylidenecyclohexen-1-ol (1). Yellow; m.p.: 126-127ºC; Mass calculated: 290.1307; Mass
1
found: 290.1324. H NMR (500 MHz, CDCl
3
) δ: 1.68 (quin, J=6.04 Hz, 2 H) 2.50 - 2.57 (m, 2 H) 2.75 - 2.82
(
m, 2 H) 7.29 - 7.35 (m, 1 H) 7.38 - 7.49 (m, 7 H) 7.59 (dd, J=7.86, 1.46 Hz, 2 H) 7.78 (s, 1 H) 16.78 (s, 1
1
3
H). C NMR (125 MHz, CDCl
3
) δ: 23.6, 27.2, 27.6, 108.4, 127.6, 128.1, 128.2, 128.4, 130.1, 130.6, 132.6,
1
33.4, 136.3, 138.3, 176.3, 195.0
1
1

2
-benzoyl-6-(2-fluorobenzylidene)cyclohexen-1-ol (2). Yellow; m.p.: 124-125ºC; Mass calculated: 308.1213;
1
Mass found: 308.1218. H NMR (500 MHz, CDCl
3
) δ: 1.67 (m, 2 H) 2.54 (t, J=6.12 Hz, 2 H) 2.62 - 2.68 (m, 2
H) 7.08 - 7.13 (m, 1 H) 7.14 - 7.19 (m, 1 H) 7.28 - 7.34 (m, 1 H) 7.36 - 7.41 (m, 1 H) 7.41 - 7.50 (m, 3 H) 7.58
1
3
(
d, J=6.99 Hz, 2 H) 7.78 (br. s., 1 H) 16.56 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.5, 27.3, 27.5, 108.5,
1
15.8, 123.7, 124.2, 125.8, 127.5, 128.1, 129.9, 130.6, 134.6, 138.4, 161.6, 174.8, 196.5
2
-benzoyl-6-(3-fluorobenzylidene)cyclohexen-1-ol (3). Yellow; m.p.: 121-122ºC; Mass calculated: 308.1213;
1
Mass found: 308.1228. H NMR (500 MHz, CDCl
3
) δ: 1.68 (quin, J=6.12 Hz, 2 H) 2.51 - 2.58 (m, 2 H) 2.73 -
2
.79 (m, 2 H) 7.02 (td, J=8.45, 1.75 Hz, 1 H) 7.14 (d, J=10.19 Hz, 1 H) 7.21 (d, J=7.57 Hz, 1 H) 7.36 (td,
1
3
J=8.01, 6.12 Hz, 1 H) 7.41 - 7.51 (m, 3 H) 7.55 - 7.61 (m, 2 H) 7.70 (s, 1 H) 16.64 (s, 1 H). C NMR (125
MHz, CDCl ) δ: 23.5, 27.2, 27.5, 108.7, 115.0, 116.5, 125.9, 127.6, 128.2, 129.9, 130.7, 131.8, 133.6, 138.2,
3
1
38.5, 163.6, 175.3, 196.1
2
-benzoyl-6-(4-fluorobenzylidene)cyclohexen-1-ol (4). Yellow; m.p.: 127-128ºC; Mass calculated: 308.1213;
1
Mass found: 308.1218. H NMR (500 MHz, CDCl
H) 7.05 - 7.12 (m, 2 H) 7.39 - 7.50 (m, 5 H) 7.55 - 7.59 (m, 2 H) 7.71 (s, 1 H) 16.74 (s, 1 H). C NMR (125
MHz, CDCl ) δ: 23.5, 27.1, 27.5, 108.3, 115.5, 127.6, 128.1, 130.6, 131.9, 132.1, 132.3, 132.4, 138.2, 163.4,
3
) δ: 1.68 (m, 2 H) 2.54 (t, J=6.12 Hz, 2 H) 2.71 - 2.76 (m, 2
1
3
3
1
76.1, 195.4
2
3
2
-benzoyl-6-(3,4-difluorobenzylidene)cyclohexen-1-ol (5). Yellow; m.p.: 97-98ºC; Mass calculated:
1
26.1118; Mass found: 326.1123. H NMR (500 MHz, CDCl ) δ: 1.69 (quin, J=6.12 Hz, 2 H) 2.51 - 2.57 (m,
3
H) 2.68 - 2.76 (m, 2 H) 7.16 (d, J=8.45 Hz, 1 H) 7.25 (d, J=8.45 Hz, 1 H) 7.41 - 7.50 (m, 4 H) 7.58 (d,
13
J=8.25 Hz, 2 H) 7.63 (s, 1 H) 16.62 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.4, 27.1, 27.4, 108.7, 117.2,
1
18.4, 126.5, 126.6, 127.6, 128.1, 130.7, 130.8, 133.3, 138.2, 147.1, 148.3, 175.2, 196.0
2
-benzoyl-6-(2-bromobenzylidene)cyclohexen-1-ol (6). Yellow; m.p.: 107-109ºC; Mass calculated: 368.0412;
1
Mass found: 368.0427. H NMR (500 MHz, CDCl
3
) δ: 1.64 - 1.70 (m, 2 H) 2.54 (t, J=6.12 Hz, 2 H) 2.57 -
2
.62 (m, 2 H) 7.13 - 7.21 (m, 1 H) 7.33 (d, J=4.66 Hz, 2 H) 7.39 - 7.50 (m, 3 H) 7.57 (d, J=6.99 Hz, 2 H) 7.64
13
(
d, J=8.15 Hz, 1 H) 7.78 (s, 1 H) 16.54 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.6, 27.2, 27.3, 108.6, 124.9,
1
26.8, 127.5, 128.1, 129.3, 130.5, 130.6, 132.4, 132.9, 133.9, 136.6, 138.4, 174.6, 196.7
2
-benzoyl-6-(4-bromobenzylidene)cyclohexen-1-ol (7). Yellow; m.p.: 140-141ºC; Mass calculated: 368.0412;
1
Mass found: 368.0417. H NMR (500 MHz, CDCl
3
) δ: 1.68 (dt, J=12.23, 6.12 Hz, 2 H) 2.54 (t, J=5.82 Hz, 2
H) 2.70 - 2.75 (m, 2 H) 7.30 (d, J=8.15 Hz, 2 H) 7.41 - 7.49 (m, 3 H) 7.52 (d, J=8.74 Hz, 2 H) 7.57 (d, J=6.99
13
3
Hz, 2 H) 7.67 (s, 1 H) 16.65 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.5, 27.1, 27.5, 108.5, 122.3, 127.6,
1
28.1, 130.6, 131.5, 131.5, 131.9, 13325, 135.2, 138.2, 175.5, 195.9
2
-benzoyl-6-(3,4-dibromobenzylidene)cyclohexen-1-ol (8). Yellow; m.p.: 125-126ºC; Mass calculated:
1
445.9517; Mass found: 445.9561. H NMR (500 MHz, CDCl ) δ: 1.68 (dt, J=12.23, 6.12 Hz, 2 H) 2.54 (t,
3
J=6.12 Hz, 2 H) 2.70 (t, J=5.82 Hz, 2 H) 7.21 (d, J=8.15 Hz, 1 H) 7.42 - 7.50 (m, 3 H) 7.55 - 7.60 (m, 3 H)
1
3
7
1
3
.63 (d, J=8.74 Hz, 1 H) 7.68 (s, 1 H) 16.54 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.4, 27.1, 27.4, 108.8,
24.3, 124.8, 127.6, 128.1, 129.8, 130.2, 130.8, 133.5, 134.3, 134.6, 137.1, 138.2, 174.6, 196.5
2
-benzoyl-6-(2-chlorobenzylidene)cyclohexen-1-ol (9). Yellow; m.p.: 92-94ºC; Mass calculated: 324.0917;
1
Mass found: 324.0932. H NMR (CDCl
3
) δ: 1.67 (quin, J=6.12 Hz, 2 H) 2.54 (t, J=5.97 Hz, 2 H) 2.59 - 2.64
(
m, 2 H) 7.23 - 7.30 (m, 2 H) 7.35 (dd, J=6.99, 2.04 Hz, 1 H) 7.41 - 7.50 (m, 4 H) 7.58 (dd, J=8.15, 1.46 Hz, 2
13
H) 7.85 (s, 1 H) 16.56 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.6, 27.3, 27.4, 108.7, 126.2, 127.5, 128.1,
1
29.2, 129.7, 130.1, 130.5, 130.7, 134.2, 134.7, 134.8, 138.4, 174.7, 196.7
2
-benzoyl-6-(3-chlorobenzylidene)cyclohexen-1-ol (10). Yellow; m.p.: 97-99ºC; Mass calculated: 324.0917;
1
Mass found: 324.0928. H NMR (500 MHz, CDCl
3
) δ: 1.68 (quin, J=6.04 Hz, 2 H) 2.54 (t, J=5.97 Hz, 2 H)
2
1
1
.71 - 2.78 (m, 2 H) 7.27 - 7.35 (m, 3 H) 7.40 - 7.51 (m, 4 H) 7.58 (dd, J=8.01, 1.31 Hz, 2 H) 7.67 (s, 1 H)
1
3
6.62 (s, 1 H). C NMR (125 MHz, CDCl
3 3
CDCl ) δ: 23.5, 27.2, 27.5, 108.7, 127.6, 128.0, 128.2, 128.2,
29.6, 129.6, 130.7, 131.5, 133.8, 134.3, 138.14, 138.2, 175.2, 196.2
1
2

2
3
2
-benzoyl-6-(4-chlorobenzylidene)cyclohexen-1-ol (11). Yellow; m.p.: 129-130ºC; Mass calculated:
1
3
24.0917; Mass found: 324.0925. H NMR (500 MHz, CDCl ) δ: 1.68 (quin, J=6.12 Hz, 2 H) 2.50 - 2.57 (m,
H) 2.70 - 2.76 (m, 2 H) 7.37 (s, 4 H) 7.42 - 7.50 (m, 3 H) 7.58 (dd, J=8.01, 1.31 Hz, 2 H) 7.69 (s, 1 H) 16.69
13
(s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.5, 27.2, 27.5, 108.6, 127.6, 128.1, 128.6, 130.7, 131.3, 131.9,
1
33.1, 134.0, 134.8, 138.2, 175.6, 195.8
2
3
-benzoyl-6-(2,3-dichlorobenzylidene)cyclohexen-1-ol (12). Yellow; m.p.: 131-132ºC; Mass calculated:
1
3
58.0527; Mass found: 358.0545. H NMR (500 MHz, CDCl ) δ: 1.66 (dt, J=12.23, 6.12 Hz, 2 H) 2.51 - 2.62
1
3
(
m, 4 H) 7.19 - 7.25 (m, 2 H) 7.41 - 7.50 (m, 4 H) 7.57 (d, J=6.99 Hz, 2 H) 7.79 (s, 1 H) 16.47 (s, 1 H). C
3
NMR (125 MHz, CDCl ) δ: 23.5, 27.2, 27.3, 108.8, 126.7, 127.5, 128.1, 128.6, 129.7, 129.8, 130.7, 132.6,
1
33.5, 134.8, 137.1, 138.3, 174.0, 197.1
2
-benzoyl-6-(2,4-dichlorobenzylidene)cyclohexen-1-ol (13). Yellow; m.p.: 135-136ºC; Mass calculated:
1
358.0527; Mass found: 358.0537. H NMR (500 MHz, CDCl ) δ: 1.67 (dt, J=12.23, 6.12 Hz, 2 H) 2.54 (t,
3
J=5.82 Hz, 2 H) 2.56 - 2.61 (m, 2 H) 7.24 - 7.30 (m, 2 H) 7.41 - 7.50 (m, 4 H) 7.57 (d, J=6.99 Hz, 2 H) 7.76 (s,
1
3
1
1
3
H) 16.48 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.5, 27.3, 27.3, 108.7, 126.6, 127.5, 128.1, 128.8, 129.6,
30.7, 131.2, 133.3, 134.2, 134.7, 135.4, 138.3, 174.1, 197.0
2
3
-benzoyl-6-(3,4-dichlorobenzylidene)cyclohexen-1-ol (14). Yellow; m.p.: 133-135ºC; Mass calculated:
1
58.0527; Mass found: 358.0544. H NMR (500 MHz, CDCl
3
) δ: 1.68 (dt, J=12.23, 6.12 Hz, 2 H) 2.53 - 2.56
(
(
m, 2 H) 2.69 - 2.73 (m, 2 H) 7.25 (dt, J=7.3, 2.3 Hz, 1 H) 7.42 - 7.49 (m, 4 H) 7.52 (d, J=2.33 Hz, 1 H) 7.57
13
d, J=6.99 Hz, 2 H) 7.61 (s, 1 H) 16.56 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.4, 27.1, 27.5, 108.8, 127.6,
1
28.1, 129.2, 130.3, 130.3, 130.8, 131.4, 132.0, 132.5, 134.1, 136.3, 138.2, 174.7, 196.4
2
-benzoyl-6-(3-methylbenzylidene)cyclohexen-1-ol (15). Yellow; m.p.: 84-86ºC; Mass calculated: 304.1463;
1
Mass found: 304.1470. H NMR (500 MHz, CDCl
3
) δ: 1.67 (dt, J=12.23, 6.12 Hz, 2 H) 2.54 (t, J=5.82 Hz, 2
H) 2.76 - 2.80 (m, 2 H) 7.14 (d, J=6.99 Hz, 1 H) 7.25 - 7.31 (m, 3 H) 7.41 - 7.49 (m, 3 H) 7.58 (dd, J=6.99,
13
1
3
.16 Hz, 2 H) 7.75 (s, 1 H) 16.79 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 21.5, 23.6, 27.2, 27.6, 108.3, 127.1,
1
27.6, 128.1, 128.2, 129.0, 130.6, 130.8, 132.3, 133.6, 136.2, 137.9, 138.3, 176.4, 195.4,
2
3
2
-benzoyl-6-(4-methylbenzylidene)cyclohexen-1-ol (16). Yellow; m.p.: 121-122ºC; Mass calculated:
1
04.1463; Mass found: 304.1468. H NMR (500 MHz, CDCl ) δ: 1.67 (quin, J=6.12 Hz, 2 H) 2.51 - 2.56 (m,
3
H) 2.76 - 2.80 (m, 2 H) 7.22 (d, J=8.15 Hz, 2 H) 7.37 (d, J=8.15 Hz, 2 H) 7.41 - 7.50 (m, 3 H) 7.58 (d,
1
3
J=6.99 Hz, 2 H) 7.75 (s, 1 H) 16.84 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 21.4, 23.6, 27.2, 27.7, 108.2,
1
27.6, 128.1, 129.1, 130.2, 130.5, 131.7, 133.4, 133.5, 138.3, 138.4, 176.8, 195.0,
2
-benzoyl-6-(2-methoxybenzylidene)cyclohexen-1-ol (17). Yellow; m.p.: 127-128ºC; Mass calculated:
1
3
320.1412; Mass found: 320.1422. H NMR (500 MHz, CDCl ) δ: 1.66 (dt, J=11.43, 5.79 Hz, 2 H) 2.53 (t,
J=5.68 Hz, 2 H) 2.70 (t, J=5.82 Hz, 2 H) 3.88 (s, 3 H) 6.93 (d, J=8.15 Hz, 1 H) 6.98 (t, J=7.43 Hz, 1 H) 7.29 -
1
3
7
.37 (m, 2 H) 7.41 - 7.48 (m, 3 H) 7.54 - 7.61 (m, 2 H) 7.96 (s, 1 H) 16.77 (s, 1 H). C NMR (125 MHz,
CDCl ) δ: 23.7, 27.4, 27.6, 55.5, 108.1, 110.6, 119.9, 125.3, 127.6, 128.1, 129.2, 129.7, 130.2, 130.1, 132.4,
3
1
38.5, 158.0, 176.4, 195.5
2
3
2
7
-benzoyl-6-(3-methoxybenzylidene)cyclohexen-1-ol (18) . Yellow; m.p.: 95-97ºC; Mass calculated:
1
20.1412 ; Mass found: 320.1424. H NMR (500 MHz, CDCl
3
) δ: 1.67 (quin, J=6.12 Hz, 2 H) 2.51 - 2.56 (m,
H) 2.75 - 2.81 (m, 2 H) 3.84 (s, 3 H) 6.88 (dd, J=8.15, 2.04 Hz, 1 H) 6.99 (s, 1 H) 7.05 (d, J=7.57 Hz, 1 H)
13
.32 (t, J=7.86 Hz, 1 H) 7.41 - 7.50 (m, 3 H) 7.58 (dd, J=8.01, 1.60 Hz, 2 H) 7.74 (s, 1 H) 16.75 (s, 1 H). C
) δ: 23.6, 27.2, 27.6, 55.3, 108.5, 113.8, 115.5, 122.6, 127.6, 128.1, 129.3, 130.6,
32.8, 133.2, 137.7, 138.3, 159.5, 176.2, 195.5
NMR (125 MHz, CDCl
1
3
2
-benzoyl-6-(4-methoxybenzylidene)cyclohexen-1-ol (19). Yellow; m.p.: 120-121ºC; Mass calculated:
1
3
320.1412; Mass found: 320.1421. H NMR (500 MHz, CDCl ) δ: 1.68 (quin, J=6.04 Hz, 2 H) 2.54 (t, J=5.97
Hz, 2 H) 2.78 (t, J=5.39 Hz, 2 H) 3.85 (s, 3 H) 6.94 (d, J=8.74 Hz, 2 H) 7.40 - 7.47 (m, 5 H) 7.58 (dd, J=7.72,
1
3
1
.60 Hz, 2 H) 7.74 (s, 1 H) 16.93 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ : 23.6, 27.1, 27.7, 55.4, 108.0, 113.9,
1
27.7, 128.1, 129.0, 130.5, 130.6, 131.9, 133.4, 138.3, 159.7, 177.5, 194.3
1
3

2
-benzoyl-6-(2,3-dimethoxybenzylidene)cyclohexen-1-ol (20). Yellow; m.p.: 69-71ºC; Mass calculated:
1
3
350.1518; Mass found: 350.1535. H NMR (500 MHz, CDCl ) δ: 1.65 (dt, J=11.87, 6.15 Hz, 2 H) 2.53 (t,
J=5.82 Hz, 2 H) 2.66 (t, J=5.39 Hz, 2 H) 3.84 (s, 3 H) 3.89 (s, 3 H) 6.93 (dd, J=16.02, 7.86 Hz, 2 H) 7.04 -
1
3
7
.09 (m, 1 H) 7.41 - 7.49 (m, 3 H) 7.56 - 7.59 (m, 2 H) 7.89 (s, 1 H) 16.69 (s, 1 H). C NMR (125 MHz,
CDCl ) δ: 23.6, 27.3, 27.6, 55.9, 61.1, 108.2, 112.4, 122.1, 123.4, 127.5, 128.1, 128.9, 130.5, 130.7, 133.5,
3
1
38.4, 148.0, 152.8, 175.9, 195.9
2
-benzoyl-6-(2,5-dimethoxybenzylidene)cyclohexen-1-ol (21). Yellow; m.p.: 128-129ºC; Mass calculated:
1
350.1518; Mass found: 350.1545. H NMR (500 MHz, CDCl
3
) δ: 1.66 (quin, J=6.04 Hz, 2 H) 2.53 (t, J=5.82
Hz, 2 H) 2.71 (t, J=5.39 Hz, 2 H) 3.80 (s, 3 H) 3.84 (s, 3 H) 6.85 (d, J=1.46 Hz, 2 H) 6.91 (s, 1 H) 7.40 - 7.50
1
3
(
m, 3 H) 7.55 - 7.59 (m, 2 H) 7.91 (s, 1 H) 16.71 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.6, 27.3, 27.6,
5
1
5.8, 56.1, 108.2, 111.5, 114.0, 116.4, 126.1, 127.6, 128.1, 128.9, 130.5, 132.7, 138.5, 152.4, 152.9, 176.1,
95.6
2
-benzoyl-6-(3,4-dimethoxybenzylidene)cyclohexen-1-ol (22). Yellow; m.p.: 152-153ºC; Mass calculated:
1
350.1518; Mass found: 350.1542 H NMR (500 MHz, CDCl
3
) δ: 1.69 (quin, J=6.04 Hz, 2 H) 2.52 - 2.57 (m, 2
H) 2.76 - 2.84 (m, 2 H) 3.92 (s, 3 H) 3.92 (s, 3 H) 6.91 (d, J=8.45 Hz, 1 H) 7.00 (d, J=1.75 Hz, 1 H) 7.09 (dd,
13
J=8.30, 1.60 Hz, 1 H) 7.41 - 7.49 (m, 3 H) 7.58 (dd, J=7.72, 1.60 Hz, 2 H) 7.72 (s, 1 H) 16.89 (s, 1 H).
NMR (125 MHz, CDCl ) δ: 23.6, 27.1, 27.7, 55.9, 56.0, 108.1, 110.9, 113.5, 123.5, 127.7, 128.1, 129.3, 130.5,
30.8, 133.5, 138.3, 148.7, 149.3, 177.2, 194.4
C
3
1
2
3
2
7
5
1
-benzoyl-6-(2,3,4,-trimethoxybenzylidene)cyclohexen-1-ol (23). Yellow; m.p.: 140-141ºC; Mass calculated:
1
80.1624; Mass found: 380.1643. H NMR (500 MHz, CDCl ) δ: 1.63 - 1.69 (m, 2 H) 2.53 (t, J=5.82 Hz, 2 H)
3
.69 (t, J=5.24 Hz, 2 H) 3.90 (s, 6 H) 3.91 (s, 3 H) 6.69 (d, J=8.74 Hz, 1 H) 7.09 (d, J=8.74 Hz, 1 H) 7.40 -
13
3
.48 (m, 3 H) 7.55 - 7.59 (m, 2 H) 7.88 (s, 1 H) 16.80 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.7, 27.3, 27.7,
6.1, 61.0, 61.5, 106.7, 107.9, 123.3, 125.0, 127.6, 128.1, 128.7, 130.4, 131.8, 138.5, 142.3, 153.1, 154.1,
76.8, 195.1
2
3
2
1
1
-benzoyl-6-(3,4,5-trimethoxybenzylidene)cyclohexen-1-ol (24). Yellow; m.p.: 150-151ºC; Mass calculated:
1
80.1624; Mass found: 380.1631. H NMR (500 MHz, CDCl
3
) δ: 1.69 (t, J=5.68 Hz, 2 H) 2.54 (t, J=5.97 Hz,
H) 2.80 (t, J=5.24 Hz, 2 H) 3.89 (s, 6 H) 3.89 (s, 3 H) 6.69 (s, 2 H) 7.42 - 7.48 (m, 3 H) 7.58 (dd, J=7.86,
1
3
3
.46 Hz, 2 H) 7.69 (s, 1 H) 16.79 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.6, 27.1, 27.7, 56.2, 56.2, 61.0,
07.6, 108.3, 127.6, 128.1, 130.6, 131.8, 131.9, 131.9, 133.5, 138.2, 138.4, 153.0, 176.4, 195.2
2
-benzoyl-6-(3-hydroxybenzylidene)cyclohexen-1-ol (25). Yellow; m.p.: 120-121ºC; Mass calculated:
1
306.1256; Mass found: 306.1258. H NMR (500 MHz, CDCl
3
) δ: 1.66 (quin, J=6.04 Hz, 2 H) 2.53 (t, J=5.97
Hz, 2 H) 2.76 (t, J=5.53 Hz, 2 H) 5.39 (br. s., 1 H) 6.80 (dd, J=8.01, 2.18 Hz, 1 H) 6.91 (s, 1 H) 7.02 (d,
J=7.86 Hz, 1 H) 7.22 - 7.29 (m, 1 H) 7.40 - 7.50 (m, 3 H) 7.58 (d, J=7.28 Hz, 2 H) 7.70 (s, 1 H) 16.71 (s, 1 H).
1
3
3
C NMR (125 MHz, CDCl ) δ: 23.5, 27.2, 27.6, 108.6, 115.4, 116.7, 122.7, 127.6, 128.2, 129.5, 130.7, 132.8,
1
33.1, 137.8, 138.2, 155.5, 176.2, 195.8
2
-benzoyl-6-(4-hydroxybenzylidene)cyclohexen-1-ol (26). Yellow; m.p.: 158-160ºC; Mass calculated:
1
306.1256; Mass found: 306.1268. H NMR (500 MHz, CDCl ) δ: 1.68 (quin, J=5.97 Hz, 2 H) 2.53 (t, J=5.82
3
Hz, 2 H) 2.77 (t, J=5.53 Hz, 2 H) 6.87 (d, J=8.45 Hz, 2 H) 7.38 (d, J=8.15 Hz, 2 H) 7.41 - 7.49 (m, 3 H) 7.57
13
(
3
d, J=7.28 Hz, 2 H) 7.71 (s, 1 H) 16.87 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.6, 27.1, 27.7, 108.1, 115.4,
1
27.6, 128.1, 129.1, 130.5, 130.6, 132.1, 133.4, 138.2, 155.8, 177.5, 194.4
2
-benzoyl-6-(3,4-dihydroxybenzylidene)cyclohexen-1-ol (27). Yellow; m.p.: 212-213ºC; Mass calculated:
1
322.1205; Mass found: 322.1215. H NMR (500 MHz, acetone) δ: 1.67 (quin, J=5.61 Hz, 2 H) 2.54 (t, J=5.24
Hz, 2 H) 2.79 (t, J=5.97 Hz, 2 H) 6.87 - 7.00 (m, 2 H) 7.09 (s, 1 H) 7.44 - 7.56 (m, 3 H) 7.58 - 7.68 (m, 3 H)
1
3
8
1
.22 (br. s., 2 H) 17.12 (s, 1 H). C NMR (125 MHz, acetone) δ: 23.4, 26.7, 27.5, 107.8, 115.4, 117.2, 123.4,
27.6, 128.1, 128.3, 129.9, 130.5, 133.7, 138.2, 144.9, 146.1, 177.8, 193.8
1
4

2
-benzoyl-6-(3-hydroxyl-4-methoxybenzylidene)cyclohexen-1-ol (28). Yellow; m.p.: 152-153ºC; Mass
1
calculated: 336.1362; Mass found: 336.1375. H NMR (500 MHz, CDCl
2.56 (m, 2 H) 2.77 - 2.81 (m, 2 H) 3.92 (s, 3 H) 5.83 (s, 1 H) 6.95 (d, J=8.44 Hz, 1 H) 6.98 (d, J=2.04 Hz, 1
H) 7.06 (dd, J=8.15, 2.04 Hz, 1 H) 7.42 - 7.47 (m, 3 H) 7.58 (dd, J=7.86, 1.75 Hz, 2 H) 7.71 (s, 1 H) 16.92 (s,
3
) δ: 1.69 (quin, J=6.12 Hz, 2 H) 2.50
-
13
1
1
H). C NMR (125 MHz, CDCl ) δ: 23.6, 27.1, 27.7, 56.00, 108.1, 113.0, 114.4, 124.1, 127.7, 128.1, 128.8,
3
30.5, 130.6, 133.7, 138.2, 146.1, 146.3, 177.4, 194.3
2
-benzoyl-6-(4-hydroxyl-3-methoxybenzylidene)cyclohexen-1-ol (29). Yellow; m.p.: 153-154ºC; Mass
1
calculated: 336.1362; Mass found: 336.1382. H NMR (500 MHz, CDCl
3
) δ: 1.67 (quin, J=5.97 Hz, 2 H) 2.53
(
t, J=5.82 Hz, 2 H) 2.78 (t, J=5.39 Hz, 2 H) 3.93 (s, 3 H) 5.67 (s, 1 H) 6.89 (d, J=8.44 Hz, 1 H) 7.00 (d,
13
J=8.45 Hz, 1 H) 7.10 (s, 1 H) 7.39 - 7.49 (m, 3 H) 7.58 (d, J=6.70 Hz, 2 H) 7.68 (s, 1 H) 16.88 (s, 1 H).
NMR (125 MHz, CDCl ) δ: 23.5, 27.1, 27.7, 56.0, 108.1, 110.4, 115.9, 123.4, 127.7, 128.1, 129.8, 130.5,
31.0, 133.4, 138.3, 145.3, 146.8, 177.3, 194.4
C
3
1
2
-benzoyl-6-(3-chloro-4-hydroxybenzylidene)cyclohexen-1-ol (30). Yellow; m.p.: 164-165ºC; Mass
1
calculated: 340.0866; Mass found: 340.0878. H NMR (500 MHz, CDCl
3
) δ: 1.69 (quin, J=6.04 Hz, 2 H) 2.51
-
2.56 (m, 2 H) 2.69 - 2.78 (m, 2 H) 5.74 (s, 1 H) 7.05 (d, J=8.44 Hz, 1 H) 7.30 (dd, J=8.59, 1.89 Hz, 1 H)
.42 - 7.50 (m, 4 H) 7.56 - 7.60 (m, 2 H) 7.63 (s, 1 H) 16.75 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.5,
3
7.1, 27.6, 108.4, 116.2, 120.0, 127.6, 128.1, 130.1, 130.5, 130.6, 130.7, 131.7, 131.8, 138.2, 151.3, 176.3,
95.2
13
7
2
1
2
-benzoyl-6-(3-bromo-4-hydroxybenzylidene)cyclohexen-1-ol (31). Yellow; m.p.: 172-173ºC; Mass
1
calculated: 384.0361; Mass found: 384.0379. H NMR (500 MHz, CDCl ) δ: 1.69 (quin, J=6.04 Hz, 2 H) 2.50
3
-
2.57 (m, 2 H) 2.71 - 2.78 (m, 2 H) 5.70 (br. s., 1 H) 7.05 (d, J=8.45 Hz, 1 H) 7.34 (dd, J=8.45, 2.04 Hz, 1 H)
.41 - 7.50 (m, 3 H) 7.55 - 7.61 (m, 3 H) 7.63 (s, 1 H) 16.74 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.5,
3
7.1, 27.6, 108.3, 110.3, 116.0, 127.6, 128.1, 130.5, 130.6, 131.4, 131.5, 131.9, 133.6, 138.2, 152.2, 176.2,
95.2
13
7
2
1
2
-benzoyl-6-(1-naphthalenylmethylene)cyclohexen-1-ol (32). Yellow; m.p.: 109-110ºC; Mass calculated:
1
340.1463; Mass found: 340.147. H NMR (500 MHz, CDCl
3
) δ: 1.64 (quin, J=5.97 Hz, 2 H) 2.56 (t, J=5.82
Hz, 2 H) 2.63 (t, J=5.39 Hz, 2 H) 7.43 - 7.56 (m, 7 H) 7.59 - 7.64 (m, 2 H) 7.84 (d, J=8.15 Hz, 1 H) 7.87 -
1
3
7
1
1
3
.91 (m, 1 H) 8.03 - 8.08 (m, 1 H) 8.31 (s, 1 H) 16.74 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.7, 27.5, 27.6,
08.4, 124.9, 125.1, 126.1, 126.4, 126.9, 127.6, 128.2, 128.5, 128.5, 130.6, 131.4, 131.9, 133.5, 133.5, 134.4,
38.5, 175.5, 196.4
2
-benzoyl-6-(2-naphthalenylmethylene)cyclohexen-1-ol (33). Yellow; m.p.: 114-115ºC; Mass calculated:
1
340.1463; Mass found: 340.1488. H NMR (500 MHz, CDCl
3
) δ:1.71 (quin, J=5.82 Hz, 2 H) 2.57 (t, J=5.68
Hz, 2 H) 2.89 (t, J=5.53 Hz, 2 H) 7.42 - 7.53 (m, 5 H) 7.55 - 7.64 (m, 3 H) 7.81 - 7.89 (m, 3 H) 7.93 (br. s., 2
13
H) 16.80 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.6, 27.2, 27.7, 108.5, 126.4, 126.7, 127.6, 127.6, 127.9,
3
1
28.1, 128.3, 129.7, 130.6, 132.8, 132.9, 133.2, 133.4, 133.9, 138.3, 176.2, 195.5
2
2
-benzoyl-6-(thien-2-ylmethylene)cyclohexen-1-ol (34). Orange; m.p.: 110-111ºC; Mass calculated:
1
96.0871; Mass found: 296.0896. H NMR (500 MHz, CDCl ) δ: 1.77 (dt, J=12.09, 6.19 Hz, 2 H) 2.52 - 2.58
3
(
7
m, 2 H) 2.75 - 2.82 (m, 2 H) 7.14 (dd, J=4.95, 3.79 Hz, 1 H) 7.33 (d, J=3.49 Hz, 1 H) 7.43 - 7.48 (m, 3 H)
1
3
.51 (d, J=4.95 Hz, 1 H) 7.58 (dd, J=7.86, 1.46 Hz, 2 H) 7.95 (s, 1 H) 16.87 (s, 1 H). C NMR (125 MHz,
) δ: 23.0, 26.8, 27.7, 108.4, 126.5, 127.6, 127.7, 128.1, 129.3, 129.4, 130.5, 131.9, 138.1, 139.9, 177.3,
CDCl
3
1
93.8
2
3
5
-benzoyl-6-(5-methyl-2-thienylmethylene)cyclohexen-1-ol (35). Yellow; m.p.: 100-102ºC; Mass calculated:
1
10.1028; Mass found: 310.1035. H NMR (500 MHz, CDCl ) δ: 1.75 (quin, J=6.19 Hz, 2 H) 2.49 - 2.58 (m,
3
H) 2.75 (t, J=5.39 Hz, 2 H) 6.79 - 6.82 (m, 1 H) 7.14 (d, J=3.49 Hz, 1 H) 7.39 - 7.48 (m, 3 H) 7.57 (dd,
1
3
J=7.86, 1.46 Hz, 2 H) 7.86 (s, 1 H) 16.96 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 15.7, 23.0, 26.8, 27.6,
1
08.1, 126.1, 127.2, 127.8, 128.0, 128.1, 130.4, 132.5, 138.0, 138.1, 144.9, 178.0, 193.0
1
5

2
-benzoyl-6-(3-benzyloxybenzylidene)cyclohexen-1-ol (36). Yellow; m.p.: 97-98ºC; Mass calculated:
1
3
396.1725; Mass found: 396.1735. H NMR (500 MHz, CDCl ) δ: 1.64 (dt, J=12.23, 6.12 Hz, 2 H) 2.53 (t,
J=6.12 Hz, 2 H) 2.68 - 2.72 (m, 2 H) 5.11 (s, 2 H) 6.95 (dd, J=7.57, 1.16 Hz, 1 H) 7.02 - 7.07 (m, 2 H) 7.29 -
13
7
.36 (m, 2 H) 7.37 - 7.50 (m, 7 H) 7.58 (d, J=7.57 Hz, 2 H) 7.72 (s, 1 H) 16.74 (s, 1 H). C NMR (125 MHz,
CDCl ) δ: 23.5, 27.2, 27.6, 70.1, 108.5, 114.8, 116.3, 123.0, 127.4, 127.6, 128.0, 128.1, 128.6, 129.3, 130.6,
3
1
32.8, 133.1, 136.8, 137.6, 158.6, 176.1, 195.6
2
3
2
-benzoyl-6-(4-benzyloxybenzylidene)cyclohexen-1-ol (37). Yellow; m.p.: 133-135ºC; Mass calculated:
1
96.1725; Mass found: 396.1738. H NMR (500 MHz, CDCl
3
) δ: 1.68 (quin, J=6.12 Hz, 2 H) 2.52 - 2.55 (m,
H) 2.75 - 2.80 (m, 2 H) 5.11 (s, 2 H) 7.01 (d, J=8.74 Hz, 2 H) 7.32 - 7.37 (m, 1 H) 7.38 - 7.48 (m, 9 H) 7.58
13
(
dd, J=6.99, 1.16 Hz, 2 H) 7.73 (s, 1 H) 16.91 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.6, 27.1, 27.7, 70.0,
1
14.8, 127.5, 127.7, 128.1, 128.1, 128.7, 129.2, 130.5, 130.6, 131.9, 133.3, 136.6, 138.2, 158.8, 177.4, 194.4
4
.1.3. General procedure for the synthesis of III
A DMF solution (100 mL) containing hydroxylated benzaldehyde (20 mmol), 1,2-dibromoethane (60 mmol)
and anhydrous potassium carbonate (40 mmol) was heat at 70-80˚C for 6 hours to obtain III. Upon completion,
the reaction mixture was poured into 200 mL of distilled water and stirred for 10 minutes, followed by
extraction with 100 mL of ethyl acetate. The organic layer was then washed twice with 100 mL of distilled
water and dried over anhydrous magnesium sulphate. Further evaporation in vacuo gave crude product of the
targeted intermediate (III). The resulting crude product was purified by column chromatography.
4
.1.4. General procedure for the synthesis of IV
A DMF solution (50 mL) containing intermediate III (2 mmol), appropriate secondary amine (3 mmol) and
anhydrous potassium carbonate (4 mmol) was heated under reflux for 8 hours to obtain IV. Upon completion,
the reaction mixture was poured into 200 mL of distilled water and stirred for 10 minutes, followed by
extraction with 100 mL of ethyl acetate. The organic layer was then washed twice with 100 mL of distilled
water and dried over anhydrous magnesium sulphate. Further evaporation in vacuo gave crude product of the
targeted intermediate (IV). The resulting crude product was purified by column chromatography.
4
.1.5. General procedure for the synthesis of compounds 38-41
One milimole of 2-benzoylcyclohenxanone (II) was reacted with equivalent amount of aminated
benzaldehyde in acetic acid (30 mL), in the presence of sulfuric acid as catalyst. The reaction mixture was
stirred at room temperature for overnight. Upon completion, the reaction mixture was poured into 100 mL of
distilled water and stirred for 10 minutes, followed by extraction with 100 mL of ethyl acetate. The organic
layer was then washed with 10% sodium bicarbonate solution and dried over anhydrous magnesium sulphate.
Further evaporation in vacuo gave crude product of the targeted compounds 38-41. The resulting crude
product was purified by column chromatography.
2
-benzoyl-6-(3-(2-(piperidin-1-yl)ethoxy)benzylidene)cyclohexen-1-ol (38). Yellow; m.p.: - ; Mass
1
calculated: 417.2304; Mass found: 417.2344. H NMR (500 MHz, CDCl
3
) δ: 1.41 - 1.51 (m, 2 H) 1.57 - 1.71
(
m, 6 H) 2.50 - 2.58 (m, 6 H) 2.74 - 2.84 (m, 4 H) 4.14 (td, J=6.26, 1.46 Hz, 2 H) 6.88 (d, J=8.15 Hz, 1 H)
6
.98 (s, 1 H) 7.03 (d, J=7.57 Hz, 1 H) 7.28 - 7.33 (m, 1 H) 7.41 - 7.49 (m, 3 H) 7.58 (d, J=7.57 Hz, 2 H) 7.72
1
3
(s, 1 H) 16.71 (s, 1 H). C NMR (125 MHz, CDCl
3
) δ: 23.6, 24.2, 25.9, 27.2, 27.6, 55.0, 57.9, 66.0, 108.41,
1
14.5, 116.1, 122.7, 127.1, 127.6, 128.2, 129.3, 130.6, 132.7, 133.2, 137.6, 138.3, 176.1, 195.5
2
-benzoyl-6-(4-(2-(piperidin-1-yl)ethoxy)benzylidene)cyclohexen-1-ol (39). Yellow; m.p.: 102-104ºC; Mass
1
calculated: 417.2304; Mass found: 417.2321. H NMR (500 MHz, CDCl
3
) δ: 1.42 - 1.49 (m, 2 H) 1.62 (quin,
J=5.68 Hz, 4 H) 1.67 (dt, J=12.23, 6.12 Hz, 2 H) 2.48 - 2.58 (m, 6 H) 2.73 - 2.84 (m, 4 H) 4.11 - 4.18 (m, 2 H)
13
6
.93 (d, J=8.74 Hz, 2 H) 7.38 - 7.49 (m, 5 H) 7.57 (dd, J=7.57, 1.16 Hz, 2 H) 7.72 (s, 1 H) 16.91 (s, 1 H).
C
NMR (125 MHz, CDCl ) δ: 23.6, 24.2, 25.9, 27.1, 27.7, 55.0, 57.8, 66.0, 108.0, 114.5, 127.7, 128.1, 129.0,
3
1
30.5, 130.5, 131.9, 133.4, 138.2, 158.9, 177.5, 194.3
1
6

2
-benzoyl-6-(3-(2-(pyrrolidin-1-yl)ethoxy)benzylidene)cyclohexen-1-ol (40). Yellow; m.p.: 78-80ºC; Mass
1
calculated: 403.2147; Mass found: 403.2161. H NMR (500 MHz, CDCl
m, 4 H) 2.53 (t, J=5.82 Hz, 2 H) 2.65 (t, J=6.41 Hz, 4 H) 2.73 - 2.80 (m, 2 H) 2.93 (t, J=6.12 Hz, 2 H) 4.14 (t,
J=6.12 Hz, 2 H) 6.89 (dd, J=8.15, 2.91 Hz, 1 H) 6.99 (s, 1 H) 7.03 (d, J=7.57 Hz, 1 H) 7.30 (t, J=7.86 Hz, 1
3
) δ: 1.59 - 1.70 (m, 2 H) 1.80 - 1.85
(
1
3
H) 7.39 - 7.49 (m, 3 H) 7.57 (d, J=7.57 Hz, 2 H) 7.71 (s, 1 H) 16.71 (s, 1 H). C NMR (125 MHz, CDCl ) δ:
3
2
1
3.4, 23.5, 27.2, 27.6, 54.7, 55.0, 67.1, 107.9, 114.5, 116.1, 122.7, 127.6, 127.8, 128.1, 129.2, 130.5, 132.7,
33.2, 137.6, 176.1, 193.2, 195.5
2
-benzoyl-6-(4-(2-(pyrrolidin-1-yl)ethoxy)benzylidene)cyclohexen-1-ol (41). Yellow; m.p.: 99-101ºC; Mass
1
calculated: 403.2147; Mass found: 403.2171. H NMR (500 MHz, CDCl
3
) δ: 1.67 (quin, J=6.12 Hz, 2 H) 1.78
-
(
1
1.86 (m, 4 H) 2.51 - 2.55 (m, 2 H) 2.64 (t, J=6.41 Hz, 4 H) 2.74 - 2.80 (m, 2 H) 2.92 (t, J=5.82 Hz, 2 H) 4.15
t, J=6.12 Hz, 2 H) 6.95 (d, J=8.74 Hz, 2 H) 7.39 - 7.47 (m, 5 H) 7.57 (dd, J=7.86, 1.46 Hz, 2 H) 7.72 (s, 1 H)
6.91 (s, 1 H). C NMR (125 MHz, CDCl ) δ: 23.5, 23.6, 27.1, 27.7, 54.7, 55.0, 67.1, 108.00, 114.5, 127.7,
3
13
1
28.1, 130.5, 130.5, 131.9, 133.4, 138.2, 158.9, 177.5, 194.3
4
.2. In-vitro AChE and BuChE inhibition assay
AChE and BChE inhibitory activity of the compounds were evaluated using Ellman’s microplate assay
following the conditions described by A. Basiri et al. with slight modifications. Electric eel AChE (type V-S,
Sigma Chemical Co. code: C2888) and equine serum BChE (Sigma Chemical Co. code: C7512) were used as
the sources of cholinesterases, while acetylthiocholine iodide (Sigma Chemical Co. [ATCI] code: A5751) and
S-butyrylthiocholine iodide (Sigma Chemical Co. [BTCI] code: 20820) were used as the sources of substrates.
The enzymes were prepared in 0.1M sodium phosphate buffer (pH 7.4) with the concentration of 0.25
units/mL while the substrates were prepared in water with the concentration of 0.25 mM.
For cholinesterase inhibitory assays, 190 µL of 0.15 mM of 5,5-dithiobis-2-nitrobenzoic acid (DTNB)
(Sigma Chemical Co. Code: D21200, 99%) in sodium phosphate buffer ( 0.1M, pH 7.4) was first added to 96-
well microplate followed by the addition of 20 µL of AChE/BChE solution and 20 µL of test compounds. The
mixture was incubated at 37 ºC for 15 minutes. Then, 20 µL of ATCI/BTCI was added to initiate the enzyme
reaction and the reaction mixture was further incubated for 30 minutes at 37 ºC. The absorbance of the colored
end-product was measured at 412 nm using SpectraMax Plus 384 Microplate Reader (Molecular Devices LLC,
Sunnyvale, CA, USA). All reactions were carried out in triplicate. The IC50 values were calculated in µM
using graph Pad software.
4
.3. Kinetic studies of AChE and BChE inhibition
The kinetic studies were performed as described in the above Section (4.2.) using different concentrations
of substrate and compounds. Compound 39 with concentrations of 5, 2.5, 1.25 and 0 µM were used for AChE
kinetic analysis while compound 40 with concentrations of 0.625, 0.313, 0.157 and 0 µM were used for BChE
kinetic analysis. Meanwhile, the concentrations of substrate for both assays were set at 0.2, 0.3, 0.4 and 0.5
mM. Lineweaver-Burk plots were derived from the resulting data and the Ki values of both compounds were
calculated using GraphPad Prism 5.
4
.4. Molecular modeling
Molecular docking studies were carried out using Discovery Studio 3.1 (Accelrys, San Diego, USA) on an
Intel® (TM)2 Quad CPU Q8200 @2.33 GHz running under a Windows XP Professional environment.
4
.4.1. Receptors preparation
The crystal structure of AChE from Torpedo californica (TcAChE; Code ID: 1EVE) and BChE from Homo
sapiens (hBChE; Code ID: 4BDS) were obtained from the Protein Data Bank. Then, all water molecules and
co-crystalized ligands were removed followed by protein preparation protocol with CHARMm force field. The
1
7

crystal structures of TcAChE and hBChE were used for docking studies due to their sequential similarity with
EeAChE and equine serum BChE, respectively.
4
.4.2. Ligands preparation
Compounds 39 and 40, as well as co-crystallized ligands (donepezil and tacrine) were drawn with
ChemDraw Ultra 12.0.Then, the structures were imported to the Discovery Studio 3.1 followed by ligands
preparation protocol with the default setting, as recommended by Accelrys. The prepared ligands were then
subjected to ligands minimization with CHARMm force field before being used for docking analyses.
4
.4.3. Flexible docking
Minimized co-crystallized ligands were re-docked into their respective enzymes with several sets of
amino acids as flexible residues. The top ranked conformations resulted from the docking experiment
were compared to their original crystallographic confirmation in terms of RMSD. The parameters with
lowest RMSD values were selected for the flexible docking of compounds 39 and 40. The flexible
docking results were analyzed using Discovery Studio Visualizer v4.1.0.14169 (Accelrys, San Diego,
USA).
4.5. ADMET analysis
Compounds 39 and 40 were selected for Discovery Studio 3.1 ADMET analysis based on their best
performance in cholinesterase inhibitory activity. The ADMET analysis performed was on their aqueous
solubility (AS), human intestinal absorption (HIA), blood brain barrier (BBB), cytochrome P450 2D6
(CYP2D6), plasma protein binding (PPB), and hepatotoxicity (HT) descriptors.
Acknowledgments
The authors thank the Ministry of Education (MOE) of Malaysia and Universiti Putra Malaysia for
financial support under the project ERGS/1/11/STG/UPM/01/24. The first author acknowledges the support
from the Malaysian Ministry of Science, Technology and Innovation (MOSTI) for a scholarship under the
National Science Foundation (NSF).
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