Synthesis, cytotoxicity and molecular modelling studies of new phenylcinnamide derivatives as potent inhibitors of cholinesterases

Article abstract of DOI:10.1016/j.ejmech.2014.03.015

The present study reports the synthesis of cinnamide derivatives and their biological activity as inhibitors of both cholinesterases and anticancer agents. Controlled inhibition of brain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may slow neurodegeneration in Alzheimer's diseases (AD). The anticholinesterase activity of phenylcinnamide derivatives was determined against Electric Eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) and some of the compounds appeared as moderately potent inhibitors of EeAChE and hBChE. The compound 3-(2-(Benzyloxy)phenyl)-N- (3,4,5-trimethoxyphenyl)acrylamide (3i) showed maximum activity against EeAChE with an IC₅₀ 0.29 ± 0.21 μM whereas 3-(2-chloro-6- nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3k) was proved to be the most potent inhibitor of hBChE having IC₅₀ 1.18 ± 1.31 μM. To better understand the enzyme-inhibitor interaction of the most active compounds toward cholinesterases, molecular modelling studies were carried out on high-resolution crystallographic structures. The anticancer effects of synthesized compounds were also evaluated against cancer cell line (lung carcinoma). The compounds may be useful leads for the design of a new class of anticancer drugs for the treatment of cancer and cholinesterase inhibitors for Alzheimer's disease (AD).

Full text of DOI:10.1016/j.ejmech.2014.03.015

European Journal of Medicinal Chemistry 78 (2014) 43e53
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity and molecular modelling studies of new
phenylcinnamide derivatives as potent inhibitors of cholinesterases
Aamer Saeed a, Parvez Ali Mahesar a, Sumera Zaib b, Muhammad Siraj Khan b,
,e,
*
Abdul Matin c, Mohammad Shahid d, Jamshed Iqbal b
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
b Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Institute of Biomedical and Genetic Engineering, PO Box 2891, Sector G-9/1, Islamabad, Pakistan
d Department of Bioinformatics, Fraunhofer Institute SCAI, Sankt Augustin, Germany
e Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study reports the synthesis of cinnamide derivatives and their biological activity as in-
hibitors of both cholinesterases and anticancer agents. Controlled inhibition of brain acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) may slow neurodegeneration in Alzheimer’s diseases (AD). The
anticholinesterase activity of phenylcinnamide derivatives was determined against Electric Eel acetyl-
cholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) and some of the compounds
appeared as moderately potent inhibitors of EeAChE and hBChE. The compound 3-(2-(Benzyloxy)
phenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3i) showed maximum activity against EeAChE with an
Received 12 October 2013
Received in revised form
15 February 2014
Accepted 6 March 2014
Available online 7 March 2014
Keywords:
IC50 0.29 ꢀ 0.21
m
M whereas 3-(2-chloro-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3k)
M. To better understand the
Phenylcinnamide derivatives
Alzheimer’s disease
Cholinesterases inhibitors
Cytotoxicity
was proved to be the most potent inhibitor of hBChE having IC50 1.18 ꢀ 1.31
m
enzymeeinhibitor interaction of the most active compounds toward cholinesterases, molecular
modelling studies were carried out on high-resolution crystallographic structures. The anticancer effects
of synthesized compounds were also evaluated against cancer cell line (lung carcinoma). The compounds
may be useful leads for the design of a new class of anticancer drugs for the treatment of cancer and
cholinesterase inhibitors for Alzheimer’s disease (AD).
Molecular docking
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
defined accumulation of amyloid-b peptide plaques at extracellular
level but also characterized with the intracellular neurofibrillary
tangles (NFTs) in the brains of suffering patients. With the pro-
gression of disease, the prominent indications are the continuous
memory loss, confusion, petulance, anger and the lack of vigour in
body to function evenly which eventually become the ultimate
cause of death [4e6]. It is estimated that 24.3 million people were
suspected to have Alzheimer’s disease (AD) in 2005; also the pro-
jected number of individuals with AD at world level in 2020 and in
2040 would be 42.3 and 81.1 million, respectively [7]. In this situ-
ation, global escalating occurrence of AD has compelled researchers
to undertake studies on neurodegenerative disorders (NDDS) [8].
Cholinesterases belong to a family of serine hydrolases that split
acetylcholine into choline and acetic acid, an unavoidable step in
retaining the function of cholinergic neuron [9]. AChE has five
important regions within active site and these are important to
understand substrate and inhibitor binding pattern. These regions
are, 1) catalytic triad residues, 2) acyl pocket, 3) oxyanion hole, 4)
anionic site (AS), 5) and a peripheral anionic site (PAS) [10e15]. The
Acetylcholine (ACh) is a cholinergic neurotransmitter, released
presynaptically by cholinergic terminals, and it interacts with
either nicotinic or muscarinic receptors thereby affecting function
of postsynaptic cells. Signalling action of ACh is terminated by the
action of acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) [1,2]. Both enzymes are widely distributed throughout the
body; however, AChE remains the major cholinesterase within the
human brain. All the brain parts that are innervated by cholinergic
transmission in a normal brain hold AChE and BChE activity [3].
Alzheimer’s disease is a long lasting neurodegenerative disorder
that brings about irreversible memory loss in elderly individuals.
Furthermore, this disease has not only been reported of having
* Corresponding author. Centre for Advanced Drug Research, COMSATS Institute
of Information Technology, Abbottabad 22060, Pakistan.
E-mail addresses: drjamshed@ciit.net.pk, jamshediqb@gmail.com (J. Iqbal).
http://dx.doi.org/10.1016/j.ejmech.2014.03.015
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

44
A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
Table 1
AChE binds to PAS resulting in the formation of amyloid-
plaques and acetylcholinesterase inhibitors (AChEIs) bind to the
PAS to prevent amyloid- peptide plaques formation [16,17].
b peptide
Inhibitory potential of phenylcinnamide derivatives against EeAChE and hBChE.
b
Compounds
EeAChE
IC50 mM)/(%Inhibition)
aSEM (
hBChE
However, such treatment (using AChEIs) only relieves the symp-
toms for short term and do not eradicate the disease eternally [18].
The major role of acetylcholinesterase (AChE) is to catalyze the
hydrolysis of acetylcholine (ACh) in cholinergic synapses and
replacement of AChE function in Alzheimer’s brains [19]. Some of
the important cholinesterase inhibitors which are employed in the
treatment of AD are rivastigmine, galantamine, tacrine, ensaculin
and donepezil. Among these remedies the rivastigmine is broader
in its action as it non-specifically inhibits AChE as well as BChE,
whereas galantamine and donepezil specifically inhibit AChE thus
increasing ACh level at neuronal level and relieving AD symptoms
but they all cannot stop progress of dementia [20]. Various com-
pounds like carbamates (e.g., neostigmine and physostigmine etc),
organophosphates, coumarine and cinnamide derivatives are re-
ported in literature as inhibitors of cholinesterases [18,21,22]. Apart
from anticholinesterase and cytotoxic activity cinnamide de-
rivatives (N-(carboxyaryl)-phenylcinnamide) have shown their
activity against leukotriene B4 (LTB4), which is a metabolic product
of arachidonic acid and a potent inflammatory mediator [23,24].
LTB4 has important role in a range of diseases, like psoriasis, adult
respiratory distress syndrome, inflammatory bowel disease and
rheumatoid arthritis, it makes cinnamide derivatives as potent
therapeutic agents against inflammatory disorders [25e29].
The aim of the present study was to synthesize and investigate
phenylcinnamide derivatives as anticholinesterase as well as anti-
cancer agents.
ꢀ
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
6.52 ꢀ 0.39
4.98 ꢀ 0.27
12.54 ꢀ 0.65
12.61 ꢀ 1.48
6.02 ꢀ 2.95
(33.6)
3.93 ꢀ 1.44
(31.1)
3.85 ꢀ 0.91
(32.8)
(31.1)
(32.6)
(29.2)
1.25 ꢀ 0.08
1.59 ꢀ 1.45
0.29 ꢀ 0.21
(36.5)
(27.2)
(32.1)
(37.4)
(34.2)
1.18 ꢀ 1.31
12.77 ꢀ 1.06
1.41 ꢀ 0.77
(36.4)
(35.4)
(24.8)
(34.6)
(26.2)
(25.7)
3.24 ꢀ 2.16
(31.2)
12.91 ꢀ 0.98
6.22 ꢀ 0.33
4.22 ꢀ 0.03
(30.3)
(31.2)
(36.6)
(41.8)
5.19 ꢀ 0.78
7.65 ꢀ 0.51
(36.2)
(41.2)
(18.21)
1.32 ꢀ 0.15
(33.5)
(34.1)
3.72 ꢀ 0.13
6.69 ꢀ 4.0
22.0 ꢀ 3.01
0.03 ꢀ 0.003
3y
(28.1)
Neostigmine
Donepezil
49.1 ꢀ 6.0
6.37 ꢀ 0.32
a
SEM shows standard error of mean of three experiments.
ambient temperature. All of the synthesized compounds were
characterized with 1H and 13C NMR along with IR (Scheme 1).
2. Results and discussion
2.1. Chemistry
2.2. In vitro inhibition studies of EeAChE and huBChE
We envisioned that DoebnereKnoevenagel condensation was
the best available route to synthesize cinnamic acids derivatives (1)
(aey) [30,31]. It was subsequent one pot activation and coupling
which led to formation of corresponding amides with 2,4,6-
trichloro-1,3,5-triazine [32] that ultimately afforded the desired
products phenylcinnamide (3aey) in a good to excellent yield.
Gratifyingly, we came to know that cyanuric chloride (TCT) was
cost-effective and stable reagent for amide formation, more
importantly it was easily handled and transformation took place at
In vitro inhibitory studies of synthesized phenylcinnamide de-
rivatives were carried out on EeAChE and hBChE. Inhibition potency
of compounds expressed as IC50 values is shown in Table 1. Among
the phenylcinnamide derivatives, 3i showed maximum inhibitory
activity against EeAChE because of its electron donating benzyloxy
(eOCH2Ph) group at ortho position, although the same group
attached to meta and para position showed little activity. However,
when eOCH2CHCH2 group was attached to the same ortho position
then a decrease in the activity of the compound (3m) was observed,
3a :
3b :
3c :
3d :
3e :
3f :
3g :
3h :
3i :
R = 3-OCH2Ph
3n :
3o :
3p :
3q :
3r :
3s :
3t :
R = 3,4-dihydroxy
R = 3-NO2
R = 3-OCH3, 4-OOCCH3
R = 4-NHOOCCH3
R = 3-OCH3, 4-OCH2Ph
R = 3-OCH2Ph, 4-OCH3
R = 4-OCH2CHCH2
R = 3-OOCCH3
R = 4-Cl
R = 3-OCH3
R = 2-OH, 3-OCH3
R = 3-OH, 4-OCH3
R = 2,5-dihydroxy
R = 2,3-dihydroxy
R = 2,4-dihydroxy
R = 2-OH, 4-OCH2Ph
R = 2-OCH2Ph
3u :
3v :
3j :
R = 4-OOCCH3
3w :
3x :
3y :
R = 2-F,6-Cl
R =4-OCH2Ph
R = 3-NO2,4-OCH3
3k :
3l :
3m :
R = 2-Cl, 6-NO2
R = 2-OH, 6-NO2
R = 2-OCH2CHCH2
Scheme 1. Synthesis of substituted phenylcinnamide derivatives.

A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
45
same was the case when eOCH2CHCH2 group was attached to the
2.4. Docking and pose ranking
same para position (3f). In addition, the value of IC50 further
decreased when a eNO2 group was attached at the meta position to
the benzene ring (3o). Introduction of two hydroxy substituents to
benzene ring further decreased inhibition against the EeAChE for
3n, 3t, 3u and 3v. As for as the action of the phenylcinnamide de-
rivatives against the hBChE was concerned, 3k having eCl and e
NO2 groups at ortho positions exhibited maximum inhibitory po-
tency against hBChE. The attachment of the eOOCCH3 group
instead of eCl and eNO2 groups (3g) to the meta position slightly
reduced the inhibitory potency. Electron withdrawing groups like e
Cl and eF simultaneously at ortho positions, as in case of compound
3w also resulted in potent inhibitor of hBChE but relatively less
potent than 3k and 3g.
To investigate possible ligandeAChE interactions, docking
studies were performed to generate binding modes. For acetyl-
cholinesterase, the compounds were docked to TcAChE (Torpedo
californica PDB Code: 3I6Z) and for butyrylcholinesterase a high-
ꢀ
resolution crystal structure of 2.00 A huBChE (human PDB Code:
1P0I) was selected for docking studies. The results of re-docking
experiments showed that the structures were well prepared and
the docking program reproduced the natively bound conformations
ꢀ
of the co-crystallized bound ligands. An RMSD of 1.80 A was ob-
tained for the re-docked ligand (galantamine) of TcAChE that
indicated a good prediction by the docking program. However, the
re-docking experiment of using galantamine as a reference ligand
in huBChE did not produce satisfactory results in terms of RMSD,
however, the orientation of the predicted conformation of the re-
docked structure was flipped at 180ꢁ that resulted in larger RMSD
2.3. Cytotoxicity of 3 (aey) against lung carcinoma (H157) cells
ꢀ
values of 8 A and higher.
In the present study we investigated the toxic effects of these
compounds (in dose dependent manner) against lung carcinoma
(H157) cells through SRB assay in order to assess their behaviours.
The vincristine a standard anticancer drug was used as reference
Similarly, the docking results for these compounds 3 (aey) were
obtained for both enzymes, which showed that there were different
conformations (poses) predicted for each compound. Table 2 shows
the docking scores obtained with LeadIT and the rank number of
best pose obtained with NNScore algorithm for the compounds
screened against both TcAChE and huBChE enzymes. Docking
scores of the compounds range from ꢂ20 to ꢂ39 for both enzymes,
however, filtering the poses for the compounds by NNScore showed
there could be other conformations that might interact favourably
with the enzymes. The docking scores for the first conformation
and the selected conformation ranks are given in Table 2.
for comparison to the test compounds. In the present study, at 1 mM
end concentration in the experiment, 3w and 3y demonstrated
almost 66% cytotoxicity, exhibiting highest antiproliferative activ-
ities. Compounds 3e and 3t were the next most potent compounds
showing about 61% and 60% cytotoxicity respectively at the same
concentration (1
mM). The Fig. 1 reveals the cytotoxic effects of
phenylcinnamide derivatives against lungs cancer cells. Anti-
proliferative activity of the positive control vincristine towards lung
carcinoma (H157) cell line was about 46% at final concentration of
100
mM, whereas the result revealed that these compounds were
2.5. Binding mode analysis
many folds potent in comparison to positive control. Therefore, it is
shown that phenylcinnamide derivatives exhibited more cytotoxic
effects than standard anticancer drug (vincristine) even at lowest
tested concentration.
A common binding mode was observed for the all these com-
pounds docked into both structures of TcAChE and huBChE en-
zymes. The compounds interact in a similar way like the reference
Fig. 1. Antiproliferative activity of the synthesized phenylcinnamide derivatives towards lung carcinoma (H157) cell line at various concentrations. Vincristine was used as standard
drug (100 M).
m

46
A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
Table 2
conformations and is involved in a variety of interactions in which
the interaction with Trp279 is common. The hydrogen bond net-
works formed by most of the compounds include hydrogen
bonding with Tyr70, Tyr121 and Ser200 in TcAChE.
Docking scores and best pose ranks for the compounds screened against TcAChE and
huBChE.
Compound
Docking scores (kcal/mol)
Pose ranks in top 10
TcAChE
huBChE
TcAChE
huBChE
2.6. Molecular dynamics simulations
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
ꢂ27.25
ꢂ34.52
ꢂ31.38
ꢂ28.43
ꢂ28.66
ꢂ26.38
ꢂ34.07
ꢂ29.81
ꢂ30.16
ꢂ31.09
ꢂ36.33
ꢂ27.13
ꢂ26.18
ꢂ35.64
ꢂ33.44
ꢂ25.11
ꢂ26.89
ꢂ29.13
ꢂ27.60
ꢂ28.70
ꢂ30.83
ꢂ29.41
ꢂ25.83
ꢂ27.78
ꢂ33.32
ꢂ35.69
ꢂ25.07
ꢂ39.34
ꢂ25.75
ꢂ31.62
ꢂ22.25
ꢂ28.15
ꢂ26.19
ꢂ25.57
ꢂ27.63
ꢂ29.52
ꢂ21.52
ꢂ28.91
ꢂ30.99
ꢂ39.59
ꢂ20.57
ꢂ25.72
ꢂ27.03
ꢂ26.89
ꢂ28.24
ꢂ28.80
ꢂ26.19
ꢂ24.31
ꢂ28.05
ꢂ32.67
10
6
9
4
9
7
10
3
1
10
7
7
6
6
3
6
2
4
8
2
7
8
8
3
7
6
1
4
8
6
2
3
4
1
2
10
4
In total, 50 individual molecular dynamics simulations were
conducted for all the selected poses of the compounds against both
enzyme structures of TcAChE and huBChE. Each enzymeeligand
complex was parameterized for the high-ranking pose filtered and
selected by the external scoring function NNScore. The pre-
equilibration steps of density and final equilibration results
(Figs. 4 and 5) showed that the complexes were equilibrated well,
and the proteineligand complexes can be further simulated to
obtain enough samples for computing free energy of binding values
as well as determine the stability of the binding modes. Fig. 6 shows
the RMSD deviations of ligand atoms in the production MD runs of
5ns simulation time. The ligand atoms of all compounds do not
show deviations during the simulation time and suggests that the
predicted docked conformations remain stable during the simula-
tion and the selected binding poses are favoured inside the active
site pockets of the enzymes.
3s
3t
1
5
10
10
3
5
3u
3v
3w
3x
3y
10
10
5
7
5
9
4
2.7. Calculations of binding free energy
The binding free energies (DG) were estimated by using Me-
ligand galantamine as shown in Figs. 2 and 3. In both enzymes, the
predicted docked conformations of the compounds adapt similar
orientations and form exactly the same interactions with the active
site residues. As shown in Table 2, the poses with preferred binding
modes that are selected by the external scoring functions occupy
the active site of the enzyme and interact with the same residues in
both enzymes. Therefore, the preferred binding modes selected by
external scoring function were in accordance to the interactions
made by the reference ligand galantamine in the active site of
TcAChE as shown in Figs. 2 and 3. As it appears in Fig. 2, the tri-
methoxybenzoic moiety of the compounds is involved with
hydrogen bond interaction with Ser200, that is anchored between
Trp233 and Trp84 and the benzene ring is making aromatic
stacking interactions with Phe290. Tyr121 is involved in both
hydrogen bonding interactions as well as in aromatic interactions
with the phenyl moiety. The tails of the compounds are positioned
in the opening of the enzyme pocket and adapt flexible
chanics/PoissoneBoltzmann Surface Area (MM/PBSA) method for
all compounds against both TcAChE and huBChE enzymes, the
values are given in Table 3. The estimated
DG values showed that
the activities of compounds predicted against TcAChE are higher as
compared to huBChE enzyme for most of the compounds. However,
some of the compounds have similar binding energies estimated
for both enzymes. The observed correlation of estimated
experimental G is positive (i.e. 0.41) for TcAChE but no correlation
is observed between the experimental and estimated G for huB-
DG and
D
D
ChE (i.e. correlation of 0.02). The reason for observing no correla-
tion in the case of huBChE enzyme may be the structural
differences between the horse BChE and human BChE enzymes.
These results show that MM/PBSA based method to systematically
evaluate free energy of binding for a series of compounds can
predict satisfactorily the relative binding free energies when the
modelling and experimental evaluation is performed on the same
structures.
Fig. 2. Preferred binding modes for the compounds docked into the active site pocket of TcAChE. (Left): Predicted conformations of compounds 3e, 3f, 3g, 3h, 3i and 3j shown in CPK
model. The reference ligand Galantamine (G6X) is shown in green. (Right): Predicted conformations of compounds 3p, 3q, 3r, 3s, 3t inside the active site of TcAChE. Compounds are
shown in CPK model and the reference ligand Galantamine is shown in green colour. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
47
Fig. 3. Interaction diagrams of reference ligand (Galantamine, left) and compound 3f (right). Hydrogen bond interactions are shown with red dotted lines and hydrophobic
interacting residues are shown in the regions surrounding by green lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Fig. 4. Density plots showing density equilibrations for all individual proteineligand complexes. Density plots for all compounds during pre-equilibration step for TcAChE com-
plexes (Left). Density plots for all compounds during pre-equilibration step for huBChE complexes (Right).
Fig. 5. Equilibration plots for all enzymeeligand complexes of TcAChE and huBChE. RMSD deviations in protein backbone atoms show that the complexes start to equilibrate after
2ns of MD simulation.

48
A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
Fig. 6. RMSD deviations of ligand atoms during production MD simulation. RMSD plots of ligand atoms during MD simulation of TcAChE complexes (Left). RMSD plots of ligand
atoms during MD simulation of huBChE complexes (Right).
3. Conclusions
research would help in investigating novel drugs that would act
both as anticancer and anticholinesterases inhibitors.
It is evident from the results of cholinesterase assays, cytotox-
icity assays and molecular docking studies that some of synthesized
cinnamide derivatives are potent inhibitors of cholinesterases as
well as effective cytotoxic agents against cancer cell lines (H157).
The 3i demonstrated the best inhibitory potential against EeAChE
4. Experimental
Butyrylcholinesterase (hBChE) (EC 3.1.1.8, from horse serum), S-
butyrylthiocholine chloride, acetylthiocholine iodide (ATCI),
acetylcholinesterase (EeAChE) (EC 3.1.1.7, type VI-S from electric
eel), 5,50-dithio-bis(2-nitrobenzoic acid) (DTNB), dimethylsulph-
oxide (DMSO) and neostigmine methylsulphate, donepezil hydro-
chloride, chloroform, magnesium sulphate were purchased from
SigmaeAldrich (St. Louis, MO,USA). TriseHCl, MgCl2, ZnCl2, RPMI-
1640, fetal bovine serum, glutamine, pyruvate, penicillin, strepto-
mycin, and HBSS (Hank’s Balanced Salt Solution) were purchased
with the IC50 value of 0.29 ꢀ 0.21
m
M and 3k showed promising
results against hBChE with the IC50 value of 1.18 ꢀ 1.31
mM. Since
phenylcinnamides are reported as effective cytotoxic agents and in
the current study we have also showed their anticholinesterase
activities therefore the compounds explored in the present
from SigmaeAldrich, Steinheim, Germany.
A lung carcinoma
Table 3
(H157) cell lines were obtained from ATCC (ATCC CRL-5802).
Substituted benzaldehydes, malonic acid, pyridine, piperidine,
2,4,6-trichloro-1,3,5 triazine, triethylamine, 3,4,5-trimethoxy
aniline and ethyl acetate were purchased from Alfa Aesar (Karls-
ruhe, Germany).
Experimental and estimated binding free energy values calculated for all com-
pounds simulated with TcAChE and huBChE enzymes. Experimental free energy
values were computed from IC50 values and estimated was calculated by MM/PBSA.
Compound
Experimental
(kcal/mol)
D
G
Estimated
D
G (kcal/mol) by MM/PBSA
TcAChE
huBChE
TcAChE
huBChE
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
ꢂ7.00
ꢂ7.16
ꢂ6.62
ꢂ6.61
ꢂ7.05
ꢂ6.04
ꢂ6.12
ꢂ7.82
ꢂ8.82
ꢂ5.99
ꢂ6.03
ꢂ6.61
ꢂ7.90
ꢂ5.99
ꢂ7.41
ꢂ6.08
ꢂ6.60
ꢂ7.03
ꢂ7.25
ꢂ5.93
ꢂ6.08
ꢂ5.99
ꢂ6.03
ꢂ7.33
ꢂ6.98
ꢂ7.30
ꢂ6.08
ꢂ7.31
ꢂ6.05
ꢂ6.08
ꢂ6.06
ꢂ7.97
ꢂ6.16
ꢂ6.07
ꢂ5.98
ꢂ8.00
ꢂ6.01
ꢂ6.22
ꢂ6.02
ꢂ6.18
ꢂ6.20
ꢂ5.91
ꢂ7.13
ꢂ6.91
ꢂ5.99
ꢂ5.92
ꢂ6.40
ꢂ7.94
ꢂ6.04
ꢂ6.14
ꢂ16.5532
ꢂ9.3075
ꢂ10.9926
ꢂ15.761
ꢂ14.6427
ꢂ9.5405
ꢂ9.2465
ꢂ12.4612
ꢂ22.6499
ꢂ12.8805
ꢂ19.776
ꢂ17.2056
ꢂ12.7105
ꢂ8.8962
ꢂ22.0334
ꢂ15.2873
ꢂ15.1877
ꢂ19.9598
ꢂ16.9283
ꢂ9.624
ꢂ16.206
4.1. General procedure for the synthesis of substituted
phenylcinnamide derivatives 3 (aey)
ꢂ15.9281
ꢂ17.1146
ꢂ13.2968
ꢂ13.1293
ꢂ14.6314
ꢂ9.1486
Substituted cinnamic acids (2aey) were synthesized by Knoe-
venagel condensation of corresponding aldehydes (0.01 mol) and
monoethyl malonate (0.01 mol) according to literature procedure
[30]. A stirred solution of the suitable cinnamic acid (0.01 mol) in
acetonitrile was treated with 2,4,6-trichloro-1,3,5-triazine
(0.01 mol) and Et3N (0.02 mol). A slight exotherm (1e2 ꢁC) was
observed, and most of the solid was dissolved. The reaction
mixture thickened noticeably after 20e40 min. After the mixture
was stirred for further 1 h at ambient temperature, the amine
reagent (0.02 mol) was added, and a second exotherm of 2e4 ꢁC
was noticed. The reaction mixture was stirred at 40 ꢁC and on
completion (TLC examination) was cooled to room temperature
and filtered. The solid was washed with a minimal amount of
solvent; the filtrates were combined and washed with 1 M sodium
hydroxide solution and finally with water. The organic layer was
dried over sodium sulphate, and the solvent was removed by
vaporation under reduced pressure. The residue was dried under
vacuum to yield the amide product and recrystallized from
ꢂ15.1736
ꢂ19.7891
ꢂ12.0348
ꢂ13.7864
ꢂ17.7713
ꢂ9.4361
ꢂ14.5672
ꢂ11.8747
ꢂ13.5464
ꢂ13.5457
ꢂ11.8196
ꢂ11.3597
ꢂ13.9076
ꢂ4.8547
3s
3t
3u
3v
3w
3x
3y
ꢂ15.8722
ꢂ4.5775
ꢂ17.6119
ꢂ11.2139
ꢂ15.8111
ꢂ7.7755
ꢂ13.7757
ꢂ10.0878
ꢂ7.5696

A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
49
ethanol to afford the products (3aey) in yields mentioned against
4.1.6. 3-(4-(Allyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)
each.
acrylamide (3f)
Yield: (71%), Rf: 0.57 (n-hexane:ethyl acetate, 8:2), m.p. 220 ꢁC;
IR: (cmꢂ1) 3250 (NH),3070 (sp2 CH), 1685 (C]O),1428,1608 (C]C),
4.1.1. 3-(3-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3a)
1H NMR (300 MHz, CDCl3):
d
10.5 (s, 1H, NH), 7.65(d, 2H, J ¼ 7.5 Hz),
7.45 (d, 1H, J ¼ 15.0 Hz), 6.70 (d, 2H, J ¼ 8.1 Hz), 6.47 (s, 2H), 6.30 (d,
1H, J ¼ 15.0 Hz), 6.10(d, 1H, m), 5.40 (dd, 1H, J ¼ 8.1 Hz), 6.40 (d, 1H,
J ¼ 5.3 Hz), 4.60 (d, 2H), 4.10 (s, 9H); 13C NMR (75 MHz, CDCl3):
Yield: (75%); Rf: 0.71 (n-hexane:ethyl acetate, 8:2); m.p. 201 ꢁC;
IR: (cmꢂ1) 3250e2400 (COOH), 2971(sp2 CH),1685 (C]O), 1640
(C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.40 (m, 5H,
d165.3, 157.2, 156, 144.2, 137, 133.5, 133, 130.5, 126.5, 120.1, 116.5,
AreH), 7.16 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz AreH), 6.47 (s, 2H), 6.18 (dd,
1H, J ¼ 8.1 Hz, 1.2 Hz), 7.6 (pseudo triplet, 1H, J ¼ 8.1 Hz), 7.45 (d, 1H,
J ¼ 15.6 Hz), 6.30 (d, 1H, J ¼ 15.6 Hz), 5.16 (s, 2H), 4.10 (s, 9H); 13C
113.2, 100, 70.5, 60.2. Anal. Calcd for C21H23NO5: C, 68.28; H, 6.28;
N, 3.79%; Found: C, 68.30; H, 6.25; N, 3.74%.
NMR (75 MHz, CDCl3): d165.3, 160.2, 156, 144.9, 137, 136.9, 135.0,
4.1.7. 3-(3-Oxo-3-(3,4,5-trimethoxyphenylamino)prop-1-enyl)
phenyl acetate (3g)
133, 129.36, 128.43, 127.48, 120.8, 116.5, 113.5, 100, 70.8, 60.2. Anal.
Calcd for C25H25NO5: C, 71.58; H, 6.01; N, 3.34%; Found: C, 71.54; H,
6.02; N, 3.30%.
Yield: (72%), Rf: 0.49 (n-hexane:ethyl acetate, 8:2); m.p. 224 ꢁC;
IR: (cmꢂ1) 3265 (NH), 3171(sp2 CH), 1685 (C]O), 1420, 1594 (C]C),
1H NMR (300 MHz, CDCl3):
d 10.5 (s,1H, NH), 7.6 (pseudo triplet,1H,
J ¼ 8.1 Hz), 7.45 (d, 1H, J ¼ 15.0 Hz), 7.38 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz),
7.28 (s, 1H), 7.18 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47 (s, 2H), 6.30 (d, 1H,
J ¼ 15.0 Hz), 4.10 (s, 9H), 2.10 (s, 3H), 13C NMR (75 MHz, CDCl3):
4.1.2. 2-Methoxy-4-(3-oxo-3-(3,4,5-trimethoxyphenylamino)prop-
1-enyl)phenyl acetate (3b)
Yield: (73%), Rf: 0.66 (n-hexane:ethyl acetate, 8:2), m.p. 215 ꢁC;
IR: (cmꢂ1) 3270 (NH), (COOH), 3071(sp2 CH), 1685(C]O), 1470,
d
165.3, 169.2, 156, 152.3, 144.9, 137, 135.9, 133, 129.2, 128.5, 127.48,
1590 (C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.48 (d,
120.8, 116.5, 113.5, 100, 60.2,20.8. Anal. Calcd for C20H21NO6: C,
64.68; H, 5.70; N, 3.77%; Found: C, 64.63; H, 5.67; N, 3.79%.
1H, J ¼ 15.2 Hz), 7.30(s, H, AreH), 7.26(d, 1H, J ¼ 8.1 Hz), 7.13(d, 1H,
J ¼ 8.1 Hz), 6.47 (s, 2H), 6.40 (d, 1H, J ¼ 15.2 Hz), 4.10 (s, 9H), 3.95 (s,
3H), 2.10 (s, 3H); 13C NMR (75 MHz, CDCl3):
d165.3,169.2,156, 151.9,
4.1.8. 3-(4-(Benzyloxy)-2-hydroxyphenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3h)
144.5, 142.9, 137, 133, 132.0, 129.36, 116.5, 110.9; 100, 60.2, 55.8,
22.5. Anal. Calcd for C21H23NO7: C, 62.84; H, 5.78; N, 3.49%; Found:
C, 62.82; H, 5.77; N, 3.51%.
Yield: (74%), Rf: 0.69 (n-hexane:ethyl acetate, 8: 2), m.p.
2290 ꢁC; IR: (cmꢂ1) 3219 (NH), 3080 (sp2 CH), 1685 (C]O), 1525,
1620 (C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.60 (d,
1H, J ¼ 15.8 Hz), 7.50 (d, 1H, J ¼ 8.5 Hz), 7.40 (m, 5H, AreH), 7.25 (s,
1H), 6.90(d, 1H, J ¼ 8.5 Hz), 6.47 (s, 2H), 6.18 (d, 1H, J ¼ 15.8 Hz),
5.16(s, 2H), 5.0 (s, 1H,OH), 4.10 (s, 9H), 4.00 (s, 3H); 13C NMR
4.1.3. 3-(4-(Acetoxyamino)phenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3c)
Yield: (78%), Rf: 0.71 (n-hexane:ethyl acetate, 8:2), m.p. 210 ꢁC;
(75 MHz, CDCl3):
d 171.5, 162.3, 159.5, 156, 141.9, 137, 136.2, 133,
IR: (cmꢂ1) 3240 (NH), 3106 (sp2 CH),1685 (C]O),1440,1633 (C]C),
130.9, 128.5, 127.6, 127.1, 116.5, 113.5, 103.5, 100, 71.8, 60.2. Anal.
Calcd for C25H25NO6: C, 68.95; H, 5.79; N, 3.22%; Found: C, 68.91; H,
5.77; N, 3.20%.
1H NMR (300 MHz, CDCl3):
d
10.5 (s, 1H, NH), 7.70(d, 2H, J ¼ 8.5 Hz),
7.45 (d, 1H, J ¼ 15.8 Hz), 6.58 (d, 2H, J ¼ 8.5 Hz), 6.47 (s, 2H), 6.40(d,
1H, J ¼ 15.8 Hz) 4.10 (s, 9H), 3.95 (s, 1H, NH), 2.10 (s, 3H), 13C NMR
(75 MHz, CDCl3): d165.3,167.2,156,150.0,144.2,137,133,126.5,120.1,
4.1.9. 3-(2-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3i)
116.5,100, 60.2, 26.2. Anal. Calcd for C20H22N2O6: C, 62.17; H, 5.74; N,
7.25%; Found: C, 62.14; H, 5.70; N, 7.26%.
Yield: (79%), Rf: 0.63 (n-hexane:ethyl acetate, 8:2), m.p. 233 ꢁC;
IR: (cmꢂ1) 3258 (NH), 3031 (sp2 CH),1685 (C]O),1474,1600 (C]C).
1H NMR (300 MHz, CDCl3):
d
10.5 (s,1H, NH), 7.60 (d,1H, J ¼ 15.8 Hz),
7.50(m, 4H), 7.40(m, 5H, AreH), 6.47 (s, 2H), 6.18 (d,1H, J ¼ 15.8 Hz),
5.16(s, 2H), 4.10 (s, 9H); 13C NMR (75 MHz, CDCl3):
165.3,155.2,156,
4.1.4. 3-(4-(Benzyloxy)-3-methoxyphenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3d)
d
Yield:(77%), Rf: 0.70 (n-hexane:ethyl acetate, 8:2), m.p. 205 ꢁC;
144.9, 137, 136.9, 135.0, 133, 129.36, 128.43, 127.1, 120.8, 116.5, 113.5,
100, 70.8, 60.2. Anal. Calcd for C25H25NO5: C, 71.58; H, 6.01; N, 3.34%;
Found: C, 71.54; H, 6.02; N, 3.30%.
IR: (cmꢂ1) 3227 (NH), 3020 (sp2 CH),1685 (C]O),1462,1594 (C]C),
1H NMR (300 MHz, CDCl3):
d
10.5 (s,1H, NH), 7.45(d,1H, J ¼ 15.8 Hz),
7.40(m, 5H, AreH), 7.25 (s, 1H), 7.20(d, 1H, J ¼ 8.5 Hz), 6.80(d, 1H,
J ¼ 8.5 Hz), 6.47 (s, 2H), 6.28(d, 1H, J ¼ 15.8 Hz), 5.16(s, 2H), 4.10 (s,
4.1.10. 4-(3-Oxo-3-(3,4,5-trimethoxyphenylamino)prop-1-enyl)
phenyl acetate (3j)
9H), 4.00 (s, 3H); 13C NMR (75 MHz, CDCl3):
d165.3,156,155.5,150.2,
144.9,137,136.9,133,128.5,127.6,127.3,122.5,116.5,113.5,100, 71.8,
60.2, 55.3. Anal. Calcd for C26H27NO6: C, 69.47; H, 6.05; N, 3.12%;
Found: C, 69.44; H, 6.02; N, 3.09%.
Yield: (78%), Rf: 0.66 (n-hexane:ethyl acetate, 8:2), m.p. 205 ꢁC;
IR: (cmꢂ1) 3297 (NH), 3033 (sp2 CH),1685 (C]O),1500,1632 (C]C).
1H NMR (300 MHz, CDCl3):
d
10.5 (s,1H, NH), 7.70 (d, 2H, J ¼ 8.1 Hz),
7.45 (d,1H, J ¼ 15.0 Hz), 7.30(d, 2H, J ¼ 8.1 Hz), 6.47 (s, 2H), 6.30(d,1H,
4.1.5. 3-(3-(Benzyloxy)-4-methoxyphenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3e)
J ¼ 15.0 Hz), 4.10 (s, 9H), 2.10 (s, 3H); 13C NMR (75 MHz, CDCl3):
d169.2,165.3,160.3,156,144.2,137,133.5,133,130.5,126.5,116.5,100,
Yield: (72%), Rf: 0.45 (n-hexane:ethyl acetate, 8:2), m.p. 208 ꢁC;
IR: (cmꢂ1) 3233 (NH), 3178 (sp2 CH),1685 (C]O),1462,1588 (C]C),
60.2, 20.5. Anal. Calcd for C20H21NO6: C, 64.68; H, 5.70; N, 3.70%;
Found: C, 64.72; H, 5.69; N, 3.67%.
1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.45(d, 1H,
J ¼ 15.8 Hz), 7.40(m, 5H, AreH), 7.25 (s, 1H), 7.20(d, 1H, J ¼ 8.5 Hz),
4.1.11. 3-(2-Chloro-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3k)
6.80(d, 1H, J ¼ 8.5 Hz), 6.47 (s, 2H), 6.28 (d, 1H, J ¼ 15.8 Hz), 5.16 (s,
2H), 4.10 (s, 9H), 4.01 (s, 3H), 13C NMR (75 MHz, CDCl3):
d165.3, 156,
Yield: (75%), Rf: 0.47 (n-hexane:ethyl acetate, 8:2), m.p. 207 ꢁC;
155.5, 150.2, 144.9, 137, 136.9, 133, 128.5, 127.6, 127.3, 122.5, 116.5,
113.5, 100, 71.8, 60.2, 55.3. Anal. Calcd for C26H27NO6: C, 69.47; H,
6.05; N, 3.12%; Found: C, 69.44; H, 6.02; N, 3.09%.
IR: (cmꢂ1) 3287 (NH), 3089 1(sp2 CH), 1685 (C]O), 1450, 1595
(C]C). 1H NMR (300 MHz, CDCl3):
d
10.5 (s, 1H, NH), 8.00 (dd, 2H,
J ¼ 8.1 Hz, 1.2 Hz), 7.55 (d, 1H, J ¼ 15.0 Hz) 7.45 (pseudo triplet, 1H,

50
A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
J ¼ 8.1 Hz), 7.18 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47 (s, 2H), 6.18 (d, 1H,
4.1.17. 3-(4-Methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3q) [33]
J ¼ 15.0 Hz), 4.10 (s, 9H); 13C NMR (75 MHz, CDCl3):
d165.3, 156,
148.2, 145.3, 137, 136.2, 133.5, 133, 130.5, 121.5, 116.5, 100, 60.2.
Anal. Calcd for C18H17ClN2O6: C, 55.04; H, 4.36; N, 7.13%; Found: C,
55.07; H, 4.32; N, 7.14%.
Yield: (72%), Rf: 0.23 (n-hexane:ethyl acetate, 8:2), m.p. 199 ꢁC;
IR: (cmꢂ1) 3282 (NH), 3100 (sp2 CH),1685 (C]O),1510,1634 (C]C).
1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.60 (pseudo triplet,
1H, J ¼ 8.1 Hz), 7.55 (d, 1H, J ¼ 15.0 Hz), 7.30 (s, 1H), 7.25 (dd, 1H,
J ¼ 8.1 Hz, 1.2 Hz), 6.80 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47 (s, 2H), 6.20
(d, 1H, J ¼ 15.0 Hz), 4.10 (s, 9H), 4.1 (s, 3H); 13C NMR (75 MHz,
4.1.12. 3-(2-Hydroxy-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3l)
Yield: (62%), Rf: 0.82 (n-hexane:ethyl acetate, 8:2), m.p. 200 ꢁC;
CDCl3): d165.3, 160.5, 156, 141.2, 137.2, 137, 133, 129.2, 120.3, 116.5,
IR: (cmꢂ1) 3257 (NH), 3142 (sp2 CH),1685 (C]O),1473,1588 (C]C),
113.5, 112.5, 100, 60.2, 58.9. Anal. Calcd for C19H21NO5: C, 66.46; H,
6.16; N, 4.08%; Found: C, 66.43; H, 6.17; N, 4.05%.
1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 8.00 (dd, 2H,
J ¼ 8.1 Hz, 1.2 Hz), 7.55 (d, 1H, J ¼ 15.0 Hz), 7.35 (pseudo triplet, 1H,
J ¼ 8.1 Hz), 7.18 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47 (s, 2H), 6.20(d, 1H,
J ¼ 15.0 Hz), 5.0 (s, 1H,OH), 4.10 (s, 9H); 13C NMR (75 MHz, CDCl3):
4.1.18. 3-(2-Hydroxy-3-methoxyphenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3r)
Yield: (73%), Rf: 0.33 (n-hexane:ethyl acetate, 8:2), m.p, 195 ꢁC:
d
165.3, 159.5, 156, 148.2, 145.3, 137, 136.2, 133, 117.2, 121.5, 116.5,
IR: (cmꢂ1) 3271 (NH), 3030 (sp2 CH),1685 (C]O),1447,1609 (C]C).
100, 60.2. Anal. Calcd for C25H25NO5: C, 57.75; H, 4.85; N, 7.48%;
Found: C, 57.70; H, 4.81; N, 7.46%.
1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.55 (d, 1H,
J ¼ 15.0 Hz), 7.26 (pseudo triplet, 1H, J ¼ 8.1 Hz), 7.15 (dd, 1H,
J ¼ 8.1 Hz, 1.2 Hz), 6.80 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47 (s, 2H), 6.20
(d, 1H, J ¼ 15.0 Hz), 4.10 (s, 9H), 4.1(s, 3H); 13C NMR (75 MHz,
4.1.13. 3-(2-(Allyloxy) phenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3m)
CDCl3): d171.5, 156, 137, 133, 150.5, 148.6, 141.2, 122.2, 120.2, 116.5,
Yield: (77%), Rf: 0.44 (n-hexane:ethyl acetate, 8:2), m.p,
113.5, 112.5, 5100, 60.2,8.9. Anal. Calcd for C19H21NO6: C, 63.50; H,
5.89; N, 3.90%; Found: C, 63.48; H, 5.87; N, 3.88%.
210 ꢁC; IR: (cmꢂ1) 3239 (NH), 3074 (sp2 CH), 1685 (C]O), 1500,
1609 (C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.65(d,
1H, J ¼ 7.5 Hz), 7.45 (d, 1H, J ¼ 15.0 Hz), 6.90 (2H, m), 6.70 (d, 1H,
J ¼ 8.1 Hz), 6.47 (s, 2H), 6.30 (d, 1H, J ¼ 15.0 Hz), 6.10(d, 1H, m),
5.40 (dd, 1H, J ¼ 8.1 Hz), 6.40 (d, 1H, J ¼ 5.3 Hz), 4.60 (d, 2H), 4.10
4.1.19. 3-(3-Hydroxy-4-methoxyphenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3s)
Yellow solid (72%), Rf; 0.37 (n-hexane:ethyl acetate, 8:2), m.p,
(s, 9H); 13C NMR (75 MHz, CDCl3):
d165.3, 157.2, 156, 147.2, 137,
222 ꢁC; IR: (cmꢂ1) 3269 (NH), 3120 (sp2 CH), 1685 (C]O), 1470,
133.5, 133, 130.5, 125.2, 128.9, 120.1, 117.5, 116.5, 114.2, 100, 70.5,
60.2. Anal. Calcd for C21H23NO5: C, 68.28; H, 6.28; N, 3.79%;
Found: C, 68.25; H, 6.27; N, 3.77%.
1630 (C]C). 1H NMR (300 MHz, DMSO):
d 10.5 (s, 1H, NH), 7.45 (d,
1H, J ¼ 15.3 Hz), 7.25 (d, 1H, J ¼ 7.5 Hz), 7.15(s, 1H), 6.65 (d, 1H,
J ¼ 7.5 Hz), 6.47 (s, 2H), 6.30 (d, 1H, J ¼ 15.3 Hz), 5.0 (s, 1H,OH), 4.10
(s, 9H). 13C NMR (75 MHz, DMSO):
d165.3, 156, 148.5, 147.2, 144.3,
4.1.14. 3-(2,3-Dihydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3n)
1137, 133, 28.2, 124.3, 117.2, 116.5, 115.5, 100, 60.2. Anal. Calcd for
19H21NO6: C, 63.50; H, 5.89; N, 3.90%; Found: C, 63.53; H, 5.85; N,
C
Yield: (76%), Rf: 0.76 (n-hexane:ethyl acetate, 8:2), m.p.
3.91%.
212 ꢁC; IR: (cmꢂ1) 3239 (NH), 3050 (sp2 CH), 1685 (C]O), 1467,
1634 (C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.45
4.1.20. 3-(2,6-Dihydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3t)
(d, 1H, J ¼ 15.3 Hz), 7.35(d, 1H, J ¼ 7.5 Hz), 7.15(s, 1H), 6.65(d, 1H,
J ¼ 7.5 Hz), 6.47 (s, 2H), 6.30 (d, 1H, J ¼ 15.3 Hz), 5.0 (s, 1H,OH),
Yield: (75%), Rf: 0.32 (n-hexane:ethyl acetate, 8:2), m.p, 242 ꢁC;
IR: (cmꢂ1) 3259 (NH), 3119 (sp2 CH),1685 (C]O), 1470,1607 (C]C).
4.10 (s, 9H); 13C NMR (75 MHz, CDCl3):
d165.3, 156, 147.5, 145.2,
144.3, 137, 133, 128.2, 124.3, 117.2, 116.5, 115.5, 100, 60.2. Anal.
Calcd for C18H19NO6: C, 62.60; H, 5.55; N, 4.06%; Found: C, 62.63;
H, 5.52; N, 4.00%.
1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.55 (d, 1H,
J ¼ 15.0 Hz), 7.35 (pseudo triplet, 1H, J ¼ 8.1 Hz), 6.47 (s, 2H), 6.18 (d,
2H, J ¼ 8.1 Hz), 6.20 (d, 1H, J ¼ 15.0 Hz), 3.91(s, 6H); 13C NMR
(75 MHz, CDCl3): d165.3, 158.5, 156, 137.2, 137, 133, 129, 116.5, 106.8,
4.1.15. (3-Nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3o)
Yield: (78%), Rf: 0.26 (n-hexane:ethyl acetate, 8:2); m.p.
232 ꢁC; IR: (cmꢂ1) 3222 (NH), 3065 (sp2 CH), 1685 (C]O), 1494,
100, 60.2. Anal. Calcd for C18H19NO6: C, 62.60; H, 5.55; N, 4.06%;
Found: C, 62.62; H, 5.53; N, 4.04%.
1598 (C]C). 1H NMR (300 MHz, Acetone-d6):
d 10.5 (s, 1H, NH),
4.1.21. 3-(2,3-Dihydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3u)
8.30 (s, 1H), 8.00 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 7.70 (pseudo triplet,
1H, J 7.55 (d, 1H, J ¼ 15.0 Hz)), 7.18 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47
(s, 2H), 6.20(d, 1H, J ¼ 15.0 Hz), 4.10 (s, 9H); 13C NMR (75 MHz,
Yield: (79%), Rf: 0.30 (n-hexane:ethyl acetate, 8:2), m.p, 234 ꢁC;
IR: (cmꢂ1) 3218 (NH), 3075 (sp2 CH), 1480, 1610 (C]C); 1H NMR
Acetone-d6): d165.3, 156, 147.5, 141.2, 137.2, 137, 134.5, 133, 129.2,
(300 MHz, CDCl3):
d
10.5 (s, 1H, NH), 7.65 (d, 1H, J ¼ 15.0 Hz), 7.26
124.3, 123.5, 116.5, 100, 60.2. Anal. Calcd for C18H18N2O6: C, 60.33;
H, 5.06; N, 7.82%; Found: C, 60.30; H, 5.07; N, 7.83%.
(pseudo triplet, 1H, J ¼ 8.1 Hz), 7.15 (dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.80
(dd, 1H, J ¼ 8.1 Hz, 1.2 Hz), 6.47 (s, 2H),6.20 (d, 1H, J ¼ 15.0 Hz), 4.10
(s, 9H), 3.95 (s, 6H); 13C NMR (75 MHz, CDCl3):
d165.3, 156, 150.5,
4.1.16. 3-(4-Chlorophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide
(3p)
148.6, 145.5, 141.2, 137, 133, 122.2, 120.2, 116.5, 113.5, 61.3, 100, 60.2,
58.9. Anal. Calcd for: C18H19NO6: C, 62.60; H, 5.55; N, 4.06%; Found:
C, 62.58; H, 5.53; N, 4.05%.
Yield: (75%), Rf: 0.31 (n-hexane:ethyl acetate, 8:2), m.p.
200 ꢁC; IR: (cmꢂ1) 3248 (NH), 3050 (sp2 CH), 1685 (C]O), 1453,
1612 (C]C). 1H NMR (300 MHz, CDCl3):
d
10.5 (s, 1H, NH), 7.60 (d,
4.1.22. 3-(2,4-Dihydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3v)
2H, J ¼ 8.1 Hz),7.41 (d, 1H, J ¼ 15.0 Hz), 7.35 (d, 2H, J ¼ 8.1 Hz),
6.47(s, 2H), 6.30(d, 1H, J ¼ 15.0 Hz), 4.10 (s, 9H). 13C NMR
Yield: (75%); Rf: 0.45 (n-hexane:ethyl acetate, 8:2), m.p; 211 ꢁC,
(75 MHz, CDCl3): d165.3, 156, 144.2, 137, 133.5, 133, 132.5, 129.5,
IR: (cmꢂ1) 3254 (NH), 3133 (sp2 CH),1685 (C]O),1440,1570 (C]C).
128.5, 116.5, 100, 60.2. Anal. Calcd for C18H18ClNO4: C, 62.16; H,
5.22; N, 4.03%; Found: C, 62.18; H, 5.20; N, 4.04%.
1H NMR (300 MHz, CDCl3):
d
10.5 (s, 1H, NH), 7.45 (d, 1H,
J ¼ 15.0 Hz), 7.15(d, 1H, J ¼ 8.1 Hz), 6.80 (d, 1H, J ¼ 8.1 Hz), 6.47 (s,

A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
51
2H), 6.20(d, 1H, J ¼ 15.0 Hz), 6.10 (s, 1H, AreH), 5.12 (s, 2H,OH), 4.10
(s, 9H); 13C NMR (75 MHz, CDCl3):
165.3, 160.5, 158.6, 156, 145.5,
137, 133, 131.2, 115.2, 116.5, 108.5, 103.5, 100, 60.2. Anal. Calcd for
18H19NO6: C, 62.60; H, 5.55; N, 4.06%; Found: C, 62.63; H, 5.52; N,
regression parameters were determined for each curve and the IC50
values were measured. The computer program GraphPad Prism 5.0
(San Diego, CA, USA) was used to analyze the data.
d
C
4.00%.
4.3. Cell lines and cultures
4.1.23. (E)-3-(2-Chloro-6-fluorophenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3w)
Lung carcinoma (H157) cell lines (ATCC CRL-5802) were kept in
RPMI-1640 [having heat-inactivated fetal bovine serum (10%)
glutamine (2 mM), Pyruvate (1 mM), 100 U/mL penicillin and
Yield: (76%); Rf: 0.62 (n-hexane:ethyl acetate, 8:2), m.p.
231 ꢁC; IR: (cmꢂ1) 3209 (NH), 3090 (sp2 CH), 1685 (C]O), 1425,
100
a 5% CO2 incubator at 37 ꢁC [35]. 6-well plates were used for
growing H157 cells by inoculating 104 cells per 100
L per well. For
experiments, H157 were grown in 96-well plates by inoculating
104 cells/100 L/well, and plates were incubated at 37 ꢁC in a 5% CO2
m
g/mL streptomycin] in T-75 cm2 sterile tissue culture flasks in
1525 (C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.55(d,
1H, J ¼ 15.0 Hz), 7.15 (m, 3H AreH), 6.47 (s, 2H), 6.20(d, 1H,
m
J ¼ 15.0 Hz), 4.10 (s, 9H); 13C NMR (75 MHz, CDCl3):
d165.3, 158.5,
156, 147.2, 137, 133, 132.8, 130.5, 125.8, 122.6, 116.5, 115.8, 100,
60.2. Anal. Calcd for C18H17ClFNO4: C, 59.11; H, 4.68, N, 3.83%;
Found: C, 59.13; H, 4.64, N, 3.81%.
m
incubator. Within 24 h, a uniform monolayer was formed which
was used for experiments.
4.1.24. 3-(4-(Benzyloxy)-3-methoxyphenyl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (3x)
4.3.1. Cytotoxicity analysis by sulforhodamine B (SRB) assays
To perform cytotoxicity assay with H157 cells, a previously
described method by Skehan et al. [36] was adopted with some
modifications. Briefly, H157 cells were cultured in different 96 well
plates for 24 h. The compounds in different concentrations (100, 50,
25 and 1 mM) were inoculated in test wells while control and blank
wells were also prepared containing standard drug (VCN) and
culture media with cells respectively. The plates were then incu-
Yield: (77%); Rf: 0.55 (n-hexane:ethyl acetate, 8:2). m.p,
195 ꢁC; IR: (cmꢂ1) 3243 (NH), 3132 (sp2 CH), 1685 (C]O), 1495,
1603 (C]C). 1H NMR (300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 7.60(d,
2H, J ¼ 8.5 Hz), 7.45 (d, 1H, J ¼ 15.8 Hz), 7.40(m, 5H, AreH), 6.80
(d, 2H, J ¼ 8.5 Hz), 6.28 (d, 1H, J ¼ 15.8 Hz), 5.16 (s, 2H), 4.10 (s,
9H); 13C NMR (75 MHz, CDCl3):
d165.3, 160.5, 156, 144.9, 137,
136.9, 133, 130.2, 128.5, 127.6, 127.3, 116.5, 115.5, 100, 70.8, 60.2.
Anal. Calcd for C25H25NO5: C, 71.58; H, 6.01; N, 3.34%; Found: C,
71.56; H, 6.01; N, 3.33%.
bated for 48 h. After that cells were fixed with 50 mL of 50% ice cold
TCA solution at 4 ꢁC for 1 h. The plates were washed 5 times with
PBS and air dried. Fixed cells were further treated with 0.4% w/v
sulforhodamine B dye prepared in 1% acetic acid solution and left at
room temperature for 30 min. After that the plates were rinsed
with 1% acetic acid solution and allowed to dry. In order to solu-
bilize the dye, the dried plates were treated with 10 mM Tris base
solution for 10 min at room temperature. Absorbance was
measured at 490 nm subtracting the background measurement at
630 nm [37].
4.1.25. 3-(4-Methoxy-3-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)
acrylamide (3y)
Yield: (76%); Rf: 0.56 (n-hexane:ethyl acetate, 8:2), m.p. 202 ꢁC;
IR: (cmꢂ1) 3278 (NH), 3121 (sp2 CH), 1421, 1610 (C]C). 1H NMR
(300 MHz, CDCl3):
d 10.5 (s, 1H, NH), 8.15(s, 1H), 8.10(d, 1H,
J ¼ 7.5 Hz), 7.45 (d, 1H, J ¼ 15.3 Hz), 7.30 (d, 1H, J ¼ 7.5 Hz), 6.47 (s,
2H), 6.30 (d, 1H, J ¼ 15.3 Hz), 4.10 (s, 9H), 4.0 (s, 1H,CH3); 13C NMR
(75 MHz, CDCl3): d165.3, 156, 155.3, 149.5, 144.3, 137, 136.2, 133,
130.3, 121.2, 117.2, 116.5, 100, 60.3, 60.2. Anal. Calcd for C19H20N2O7:
C, 58.76; H, 5.19; N, 7.21%; Found: C, 58.74; H, 5.16; N, 7.22%.
4.4. Molecular modelling studies of cholinesterases
4.4.1. Structure selection and preparation
4.2. In vitro inhibitory studies on EeAChE and hBChE
Molecular modelling studies were conducted to observe the
interactions and selectivity of the compounds against both AChE
and BChE enzymes. In order to perform efficient modelling studies,
high-resolution structures of both enzymes were selected from the
Protein DataBank [38] and prepared for the modelling. A high-
Ellman’s method was used to evaluate the inhibitory activities of
phenylcinnamide derivatives against EeAChE and hBChE [34]. The
compounds studied were first dissolved in DMSO (end concentra-
tion of DMSO was 1% in the assay) and studied at a 0.5 mM final
concentration for initial screening. The compounds exhibiting more
than 50% inhibition were further tested by making eight to ten
serial dilutions in assay buffer (50 mM TriseHCl, 0.1 M NaCl and
0.02 M MgCl2, pH 8.0). Composition of the reaction mixture was:
ꢀ
resolution crystal structure of 2.19 A from T. californica (PDB
Code: 3I6Z) was selected for acetylcholinesterase (TcAChE) and a
ꢀ
high-resolution crystal structure of 2.00 A from human (PDB Code:
1P0I) was selected for human butyrylcholinesterase (huBChE). Both
structures have well defined binding sites, as the X-Ray structures
for both enzymes are co-crystallized with bound inhibitors. The
TcAChE was co-crystallized with Galantamine (PDB ID: G6X) and
huBChE was co-crystallized with bound ligands Butanoic acid (PDB
ID: BUA) and Glycerol (PDB ID: GOL) that occupy the binding site of
the enzyme.
Before studying the proteins in molecular modelling experi-
ments, the structures of enzymes and compounds were prepared.
The enzyme structures were protonated with Protonate3D [39]
algorithm implemented in the molecular modelling tool MOE
[40]. Missing atoms of some residues were completed with utility
programs built-in in AmberTools-1.5 [41] package. The structures
were energy minimized using Amber99 force field with all the
heteroatoms and solvent molecules present in the protein. The
backbone atoms were restrained with a small force in order to
avoid collapse of the binding pockets during energy minimization
20 mL assay buffer, 10 mL of test compound and 10 mL of enzymes
(0.5 and 3.4 U/mg of EeAChE or hBChE, respectively). A 10 min pre-
incubation of the reaction mixture at 25 ꢁC was carried out. At the
end of the pre-incubation period, 10 mL of 1 mM acetylthiocholine
iodide or butyrylthiocholine chloride were added to the respective
EeAChE or hBChE enzyme solution to start the enzymatic reactions.
The mixtures were incubated for 15 min at 25 ꢁC. The change in the
absorbance was measured at 405 nm to determine the amount of
the product using
a microplate reader (Bio-Tek ELx800Ô).
Neostigmine and donepezil were used as standard inhibitors. As-
says were performed with a blank containing all of the components
except enzyme or substrate in order to account for a non-enzymatic
reaction. Each concentration was analyzed in triplicate. Enzyme
dilution buffer consisted of 50 mM TriseHCl buffer containing 0.1%
(w/v) bovine serum albumin (BSA) and pH 8.0. The linear

52
A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
calculations. After minimization, the co-crystallized ligands and
solvent molecules were removed.
equilibration at 300 K. The equilibration process was carried out
using Sander program of Amber12 package. Molecular dynamics
simulations were performed by using Particle Mesh Ewald (PME)
MD program of Amber12 package on the equilibrated complexes.
With a step size of 2fs, the production MD simulations were per-
formed for 5ns time scale and atomic coordinates after each 500
step were recorded. The SHAKE algorithm was applied to constrain
4.4.2. Compounds preparation
The 3D structural coordinates of the compounds were generated
for all the compounds using MOE followed by assignment of pro-
tonation and ionization states in physiological pH range by using
the “wash” module. Afterwards, the compounds’ structures were
energy minimized with the MMFF94x force field for the docking
studies. For molecular dynamics simulations, the compound
structures were prepared by using antechamber program present
in AmberTools, to derive AM1-BCC charges and force field param-
eters for the individual compounds.
ꢀ
bonds involving hydrogen atoms with 8 A van der Waals cut off
using the PME method.
4.4.6. Calculation of binding free energy
The binding free energies were estimated by using Mechanics/
PoissoneBoltzmann Surface Area (MM/PBSA) and Molecular Me-
chanics/Generalized Born Surface Area (MM/GBSA) calculations.
MM/PBSA and MM/GBSA based energies were calculated for the
snapshots extracted from the 5ns MD trajectories that included
5000 snapshots recorded at 1ps time interval. The interaction en-
ergies and salvation free energies were computed for the complex,
receptor and ligand at each 5th snapshot in the trajectory to obtain
statistically significant results. In order to correlate the estimated
binding energy values, the experimental IC50 values were first
4.4.3. Docking studies
For the docking studies, the binding sites of both enzymes were
defined by including residues surrounding the co-crystallized
ꢀ
ligand in 7.5 A radius. The default docking and scoring parame-
ters were chosen for the docking calculations and the top 10 docked
conformations based on docking scores were retained for analyzing
the interactions and binding modes. In order to make sure that the
protein structure is prepared well for docking, the co-crystallized
bound ligands were re-docked and the same binding modes were
transformed to
DG by the following formula:
ꢀ
D
G ¼ ꢂRTln Ki
reproduced with an RMSD less than 1.8 A for the 7th ranking
conformation for galantamine re-docked to TcAChE. A re-docking
experiment was also performed with huBChE by first aligning its
X-ray structure to that of TcAChE and then using galantamine as a
reference ligand in huBChE.
where R is gas constant, T is temperature in Kelvin (295 K) and Ki (in
nM) is the experimental affinity value. A comparison of estimated
binding energy values calculated by both MM/PBSA and MM/GBSA
methods with that of experimental
DG values for the compounds
was carried out to observe correlation between the predicted and
experimentally determined values.
4.4.4. Pose ranking
The docked poses of each compound were evaluated for
favourable interactions with the enzymes and pose ranking and
selection for each docked conformation of the compound was
performed by using an external scoring function, NNScore that
evaluates and predicts the most favourable conformation (pose)
among a set of different poses of the same ligand.
Acknowledgement
This work was financially supported by COMSTECHeTWAS and
German-Pakistani Research Collaboration Programme.
4.4.5. Molecular dynamics simulations
Appendix A. Supplementary data
Molecular dynamics (MD) simulations were performed on the
ligand-docked structures of TcAChE and huBChE inhibitor com-
plexes. The aim of MD studies was to evaluate the stability of the
docked binding modes as well as to determine the free energies of
binding for the docked conformations of the compounds. The
protonated enzyme structures were subsequently parameterized
for MD simulations. The force field charges for the protein struc-
tures were derived by using Amber [41] force field ff99SB. The force
field charges and parameters for the compounds were derived
using Antechamber module of Amber. AM1-BCC charges were used
for the ligands calculated by the Antechamber and the parameters
for the ligands were adapted from the general amber force field
(GAFF) library. The docked enzymeeligand complexes were sol-
vated using the LeaP module of Amber by using an octahedron box
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.015.
References
[1] Y. Shen, J. Zhang, R. Sheng, X. Dong, Q. He, B. Yang, Y. Hu, Synthesis and
biological evaluation of novel flavonoid derivatives as dual binding acetyl-
cholinesterase inhibitors, Journal of Enzyme Inhibition and Medicinal Chem-
istry 24 (2009) 372e380.
ꢁ
ꢁ
[2] P. Zelík, A. Lukesová, L.N. Voloshko, D. Stys, J. Kopecký, Screening for acetyl-
cholinesterase inhibitory activity in cyanobacteria of the genus Nostoc, Journal
of Enzyme Inhibition and Medicinal Chemistry 24 (2009) 531e536.
[3] D.H. Small, S. Mok, J.C. Bornstein, Alzheimer’s disease and Abeta toxicity: from
top to bottom, Nature Reviews Neuroscience 2 (2001) 595e598.
[4] G. Waldemar, B. Dubois, M. Emre, J. Georges, I.G. McKeith, M. Rossor,
P. Scheltens, P. Tariska, B. Winblad, Recommendations for the diagnosis and
management of Alzheimer’s disease and other disorders associated with de-
mentia: EFNS guideline, European Journal of Neurology 14 (2007) e1e26.
[5] M.H. Tabert, X. Liu, R.L. Doty, M. Serby, D. Zamora, G.H. Pelton, K. Marder,
M.W. Albers, Y. Stern, D.P. Devanand, A 10-item smell identification scale
related to risk for Alzheimer’s disease, Annals of Neurology 58 (2005)
155e160.
ꢀ
of the TIP3P water model with a distance of 8 A between the edge of
the box and the protein. The solvated complexes were neutralized
by using counter chloride or sodium ions to neutralize the nega-
tively charged proteineligand complexes. Each individual
enzymeeligand solvated complex system was minimized with
2500 steps of conjugate gradient minimization followed by 2500
steps of steepest decent minimization.
After minimization, the proteineligand complex systems were
equilibrated before running the MD simulations to avoid any
instability during the production MD runs. The equilibration steps
included 50ps of gradual heating (from 0 to 300 K temperature)
with NMR option, 50ps of density equilibration with weak re-
straints on the complex atoms followed by 5ns of constant pressure
[6] A.V. Terry, J.J. Buccafusco, The cholinergic hypothesis of age and Alzheimer’s
disease-related cognitive deficits: recent challenges and their implications for
novel drug development, Journal of Pharmacology and Experimental Thera-
peutics 306 (2003) 821e827.
[7] C.P. Ferri, M. Prince, C. Brayne, H. Brodaty, L. Fratiglioni, M. Ganguli, K. Hall,
K. Hasegawa, H. Hendrie, Y. Huang, A. Jorm, C. Mathers, P.R. Menezes,
E. Rimmer, M. Scazufca, Global prevalence of dementia: a Delphi consensus
study, Lancet 366 (2005) 2112e2117.
[8] A.V. Maltsev, S. Bystryak, O.V. Galzitskaya, The role of beta-amyloid peptide in
neurodegenerative diseases, Ageing Research Reviews 10 (2011) 440e452.

A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43e53
53
[9] M. Pohanka, Cholinesterases,
a
target of pharmacology and toxicology,
[27] N. Clapp, M. Henke, R. Hansard, R. Carson, D. Widomski, C. Anglin, R. Walsh,
S. Djuric, D. Fretland, Anti-inflammatory activity of the leukotriene B4 re-
ceptor antagonist, SC-41930, in colitic cotton-top tamarins, Agents and Ac-
tions 41 (1994) C254eC255.
[28] N. Ahmadzadeh, M. Shingu, M. Nobunaga, T. Tawara, Relationship between
leukotriene B4 and immunological parameters in rheumatoid synovial fluids,
Inflammation 15 (1991) 497e503.
[29] A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini,
C. Melchiorre, Multi-target-directed ligands to combat neurodegenerative
diseases, Journal of Medicinal Chemistry 51 (2008) 347e372.
Biomedical Papers 155 (2011) 219e229.
[10] G. Gibney, S. Camp, M. Dionne, K. MacPhee-Quigley, P. Taylor, Mutagenesis of
essential functional residues in acetylcholinesterase, Proceedings of the National
Academy of Sciences of the United States of America 87 (1990) 7546e7550.
[11] M. Ekholm, H. Konschin, Comparative model building of human butyr-
ylcholinesterase, Journal of Molecular Structure: THEOCHEM 467 (1999) 161e172.
[12] A. Ordentlich, D. Barak, C. Kronman, N. Ariel, Y. Segall, B. Velan, A. Shafferman,
Functional characteristics of the oxyanion hole in human acetylcholinesterase,
Journal of Biological Chemistry 273 (1998) 19509e19517.
[13] D.C. Vellom, Z. Radic, Y. Li, N.A. Pickering, S. Camp, P. Taylor, Amino acid
residues controlling acetylcholinesterase and butyrylcholinesterase speci-
ficity, Biochemistry 32 (1993) 12e17.
[30] J. Lu, P.H. Toy, Organocatalytic decarboxylative Doebner-Knoevenagel re-
actions between arylaldehydes and monoethyl malonate mediated by
a
bifunctional polymeric catalyst, Synlett (2011) 1723e1726.
[14] P. Taylor, S. Lappi, Interaction of fluorescence probes with acetylcholines-
terase. Site and specificity of propidium binding, Biochemistry 14 (1975)
1989e1997.
[15] D. Barak, C. Kronman, A. Ordentlich, N. Ariel, A. Bromberg, D. Marcus, A. Lazar,
B. Velan, A. Shafferman, Acetylcholinesterase peripheral anionic site de-
generacy conferred by amino acid arrays sharing a common core, Journal of
Biological Chemistry 269 (1994) 6296e6305.
[31] K. Tanaka, K. Matsuo, A. Nakanishi, T. Hatano, H. Izeki, Y. Ishida, W. Mori,
Syntheses and anti-inflammatory and analgesic activities of hydroxamic acids
and acid hydrazides, Chemical & Pharmaceutical Bulletin (Tokyo) 31 (1983)
2810e2819.
[32] H.L. Rayle, L. Fellmeth, Development of
a process for triazine-promoted
amidation of carboxylic acids, Organic Process Research & Development 3
(1999) 172e176.
[16] M. Bartolini, C. Bertucci, V. Cavrini, V. Andrisano,
b
-Amyloid aggregation
[33] R.S. Dothager, K.S. Putt, B.J. Allen, B.J. Leslie, V. Nesterenko, P.J. Hergenrother,
Synthesis and identification of small molecules that potently induce apoptosis
in melanoma cells through G1 cell cycle arrest, Journal of the American
Chemical Society 127 (2005) 8686e8696.
[34] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, A new and rapid
colorimetric determination of acetylcholinesterase activity, Biochemical
Pharmacology 7 (1961) 88e95.
induced by human acetylcholinesterase: inhibition studies, Biochemical
Pharmacology 65 (2003) 407e416.
[17] N.C. Inestrosa, A. Alvarez, C.A. Perez, R.D. Moreno, M. Vicente, C. Linker,
O.I. Casanueva, C. Soto, J. Garrido, Acetylcholinesterase accelerates assembly
of amyloid-b-peptides into Alzheimer’s fibrils: possible role of the peripheral
site of the enzyme, Neuron 16 (1996) 881e891.
[18] R. Raza, A. Saeed, M. Arif, S. Mahmood, M. Muddassar, A. Raza, J. Iqbal, Syn-
thesis and biological evaluation of 3-thiazolocoumarinyl Schiff-base de-
rivatives as cholinesterase inhibitors, Chemical Biology & Drug Design 80
(2012) 605e615.
[35] K. Araki-Sasaki, S. Aizawa, M. Hiramoto, M. Nakamura, O. Iwase, K. Nakata,
Y. Sasaki, T. Mano, H. Handa, Y. Tano, Substance P-induced cadherin expres-
sion and its signal transduction in a cloned human corneal epithelial cell line,
Journal of Cellular Physiology 182 (2000) 189e195.
[19] E. Giacobini, Cholinesterase inhibitors: new roles and therapeutic alternatives,
Pharmacological Research 50 (2004) 433e440.
[36] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, Journal of the National Cancer Institute
82 (1990) 1107e1112.
[37] G.S. Longo-Sorbello, G. m Say, D. Banerjee, J.R. Bertino, Cytotoxicity and Cell
Growth Assays, in: Cell Biology, Four-volume Set: a Laboratory Handbook, vol.
1, 2005, p. 315.
[38] D.S. Goodsell, Computational docking of biomolecular complexes with Auto-
Dock, Cold Spring Harbor Protocols 2009 (2009) pdb prot5200.
[39] P. Labute, Protonate 3D, 2007. http://www.chemcomp.com/journal/proton.
htm.
[40] MOE (The Molecular Operating Environment) Version 2010.10, Chemical
Computing Group Inc. http://www.chemcomp.com.
[20] M. Weinstock, Selectivity of cholinesterase inhibition: clinical implications for
the treatment of Alzheimer’s disease, CNS drugs 12 (1999) 307e323.
[21] P. Taylor, Cholinergic Agonists, Goodman and Gilman’s the Pharmacological
Basis of Therapeutics, sixth ed., Macmillan, New York, 1980, pp. 91e99.
[22] X. Zhou, X.-B. Wang, T. Wang, L.-Y. Kong, Design, synthesis, and acetylcho-
linesterase inhibitory activity of novel coumarin analogues, Bioorganic &
Medicinal Chemistry 16 (2008) 8011e8021.
[23] P.D. Greenspan, A.J. Main, S.S. Bhagwat, L.I. Barsky, R.A. Doti, A.R. Engle,
L.M. Frey, H. Zhou, K.E. Lipson, M.H. Chin, N-aryl cinnamides: a novel class of
rigid and highly potent leukotriene B4 receptor antagonists, Bioorganic &
Medicinal Chemistry Letters 7 (1997) 949e954.
[24] A. Ford-Hutchinson, Leukotriene B4 in inflammation, Critical Reviews in
Immunology 10 (1990) 1.
[41] D.A. Case, T. Darden, T.E. Cheatham III, C.L. Simmerling, J. Wang, R.E. Duke,
R. Luo, M. Crowley, R.C. Walker, W. Zhang, K.M. Merz, B. Wang, S. Hayik,
A. Roitberg, G. Seabra, I. Kolossvary, K.F. Wong, F. Paesani, J. Vanicek, X. Wu,
S.R. Brozell, T. Steinbrecher, H. Gohlke, L. Yang, C. Tan, J. Mongan, V. Hornak,
G. Cui, D.H. Mathews, M.G. Seetin, C. Sagui, V. Babin, P.A. Kollman, AMBER11,
University of California, San Francisco, 2010.
[25] J. Grabbe, B.M. Czarnetzki, T. Rosenbach, M. Mardin, Identification of
chemotactic lipoxygenase products of arachidonate metabolism in psoriatic
skin, Journal of Investigative Dermatology 82 (1984) 477e479.
[26] K. Yoshimura, S. Nakagawa, S. Koyama, T. Kobayashi, T. Homma, Leukotriene
B4 induces lung injury in the rabbit: role of neutrophils and effect of indo-
methacin, Journal of Applied Physiology 74 (1993) 2174e2179.