Synthesis, anticholinesterase activity and molecular modeling studies of novel carvacrol-substituted amide derivatives

Article abstract of DOI:10.1080/07391102.2019.1590243

In the present study, 23 novel carvacrol derivatives involving the amide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and tested in vitro as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhib

Full text of DOI:10.1080/07391102.2019.1590243

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis, Anticholinesterase Activity and
Molecular Modeling Studies of Novel Carvacrol
Substituted Amide Derivatives
Belma Zengin Kurt, Serdar Durdagi, Gulsen Celebi, Ramin Ekhteiari Salmas &
Fatih Sonmez
To cite this article: Belma Zengin Kurt, Serdar Durdagi, Gulsen Celebi, Ramin Ekhteiari Salmas
& Fatih Sonmez (2019): Synthesis, Anticholinesterase Activity and Molecular Modeling Studies of
Novel Carvacrol Substituted Amide Derivatives, Journal of Biomolecular Structure and Dynamics,
DOI: 10.1080/07391102.2019.1590243
To link to this article: https://doi.org/10.1080/07391102.2019.1590243
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Synthesis, Anticholinesterase Activity and Molecular Modeling Studies of
Novel Carvacrol Substituted Amide Derivatives
Belma Zengin Kurt1,*, Serdar Durdagi2,*, Gulsen Celebi3, Ramin Ekhteiari Salmas2, Fatih
Sonmez4
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, 34093, Istanbul,
Turkey
2Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine,
Bahcesehir University, Istanbul, Turkey
3Department of Pharmacology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey;
4Sakarya University of Applied Sciences, Pamukova Vocational High Scholl, 54900, Sakarya, Turkey
Abstract
In the present study, 23 novel carvacrol derivatives involving the amide moiety as a linker
between the alkyl chains and/or the heterocycle nucleus were synthesized and tested in vitro
as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. 2-(5-isopropyl-
2-methylphenoxy)-N-(quinolin-8-yl)acetamide (5v) revealed the highest inhibition properties
against AChE and BuChE with the IC50 values of 1.93 µM and 0.05 µM, respectively. The
blood-brain barrier (BBB) permeability of the potent inhibitor (5v) was also assessed by the
widely used parallel artificial membrane permeability assay (PAMPA-BBB). The results
showed that 5v is capable of crossing the BBB. Pharmacokinetic and toxicity profiles of the
studied molecules predictions were investigated by MetaCore/MetaDrug platform. Bioactive
conformations of the synthesized molecules, their predicted binding energies as well as
structural and dynamical profiles of molecules at the binding pockets of AChE and BuChE
targets were also investigated using different docking algorithms and molecular dynamics
(MD) simulations.
Keywords: Acetylcholinesterase; Alzheimer’s disease; Butyrylcholinesterase; Carvacrol;
Molecular Docking, Molecular Dynamics (MD) Simulations.
Abbreviations: acetylcholinesterase, AChE; blood brain barrier, BBB; butyrylcholinesterase,
BuChE; molecular dynamics (MD); parallel artificial membrane permeability assay, PAMPA.
*Corresponding
BZengin@bezmialem.edu.tr (BZK).
authors;
E-mails:
serdar.durdagi@med.bau.edu.tr
(SD);
1

1. Introduction
Alzheimer's disease (AD) known as the most common cause of dementia, is a deadly and age-
related neurodegenerative disease that affects almost 50 million people in the world and
worsens with severe behavioural and psychiatric symptoms in the long run (Wang et al 2014;
Wu et al 2017). This disease is progressive and irreversible, with the symptoms of memory
loss, a decline in language skills, behavioural disturbances and much other cognitive
impairment (Li et al 2017; Tommonaro et al 2016). The exact etiology of AD remains
unknown, but many factors, such as β-amyloid (Aβ) deposits, tau protein (τ) aggregation, and
oxidative stress, decreased level of acetylcholine (ACh), neuroinflammation, and
dyshomeostasis of biometal are thought to play significant roles in the pathogenesis of the
disease (Li et al 2017; Xia et al 2017; Guzior et al 2015). Although numerous therapeutic
approaches have been reported, only non-competitive N-methyl-D-aspartate (NMDA)
receptor antagonist memantine, and acetylcholinesterase (AChE) inhibitors, donepezil,
rivastigmine, tacrin and galantamine have been approved by the FDA (Saglik et al 2016;
Estrada et al 2016).
Many studies have been carried out in recent years in order to develop new hit molecules
against AD targets in both in silico and experimental studies conducted in academia and
industry. (Iqbal et al 2018; Ambure et al 2018; Ferreira Neto et al 2018; Dutta et al 2018;
Shiri et al 2018). Most of the drugs used in AD are aimed at preventing the decrease of
acetylcholine level. According to the cholinergic hypothesis, the cognitive deficits in AD are
related with correlations deficits such as cholinergic reduced choline acetyltransferase
(ChAT) activity and synaptic acetylcholine synthesis. The main purpose here is to increase
the acetylcholine (ACh) level in the synaptic cleft by inhibition of cholinesterases (Wang et al
2017; Maryamabadi et al 2017; Liu et al 2017) There are two types of cholinesterases in the
body, acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC
3.1.1.8). AChE as a key enzyme target is not only used in enhancing cholinergic transmission
in the synaptic cleft, but also it can be used in reducing the aggregation of amyloid-beta (Aβ)
peptide and in the hydrolysis of ACh which causes neurotoxic fibril formation in AD (Guzior
et al 2015; Kurt et al 2017) BuChE is another enzyme which expressed in selected areas of the
central and peripheral nervous systems, is also capable of hydrolyzing ACh (Eghtedari 2017;
Panek et al 2017). The similarity between the amino acid sequence of AChE and BuChE is
very high (almost 84%), so the answers they provide are matching (Chen et al 2017).
2

Although AChE inhibitors (AChEI) in AD show beneficial palliative properties, cholinergic
drugs have not shown much efficacy to prevent disease progression. As a result, there is
currently no effective therapy to treat, stop, or even slow down the progression of the disease,
and therefore the discovery of effective novel therapeutics is needed (Leon et al 2013).
Carvacrol is the main component of the monoterpenes found as essential oils in Thymus
vulgaris (Majdi et al 2017; Sow et al 2017) Carvacrol is an important small therapeutic
molecule due to their antioxidative, antimicrobial, antitussive and antibacterial properties
(Rodríguez et al 2013; Höferl et al 2009). Apart from these, carvacrol has also been reported
to reveal anti-inflammatory, immunomodulatory and chemopreventive properties (Hussein et
al 2017). Carvacrol demonstrates the potential protective effect of central nervous system
(CNS) diseases through various mechanisms. These properties are the inhibitory effect on
anti-depressant effect, anxiolytic effect and AChE activity (Zhong et al 2013; Dati et al 2017;
Lopez et al 2015). In addition, amidic or imidic substituents are important functional groups
interacting with the unpaired electrons of the N and O atoms as hydrogen bond donors in the
catalytic domain of human AChE (Sonmez et al 2017; Kurt et al 2015; Mohamed et al 2013).
Besides all of these reported evidences, a good penetration across the blood-brain barrier
(BBB) is necessary condition for the central nervous system (CNS) drugs and it is known that
the BBB allows the diffusion of hydrophobic and small polar molecules, whereas it cut down
large or hydrophilic molecules into the blood and cerebrospinal fluid (Pérez-Areales et al
2014). Although many synthetic AChEIs and BuChEIs have been reported in the last decades,
there is still limited study on the natural products and their derivatives having high BBB
permeability as the ChEIs. Therefore, the main goal of this present study is the synthesis of
effective AChEIs and BuChEIs including natural compounds, to investigate their BBB
permeability and also to determine their inhibition mechanism and structure-activity
relationships as a continuation of our interest in AChEIs and BuChEIs.
Based on above consideration, we hypothesized that carvacrol is a small polar natural
compound and can easily penetration across the BBB; however, it has low inhibitory activity
against ChEs, and so the presence of amide moiety could contribute to the inhibitory activity
of carvacrol. Therefore, in the present study, 23 novel carvacrol (5a-w) derivatives substituted
with aliphatic and aromatic amide derivatives were synthesized and their inhibitory effects on
AChE and BuChE were evaluated. Also, the blood-brain barrier permeability of the novel
compounds has been assessed by the widely used parallel artificial membrane permeability
assay (PAMPA-BBB). Moreover, molecular modeling studies were also applied. Molecular
docking studies were carried out to clarify the inhibition mode for the studied compounds at
3

the active sites of the target structures. Binding poses and binding energies of studied
compounds at the binding pockets of AChE and BuChE targets were determined. Predicted
binding energies of these compounds and structural and dynamical profiles of molecules at
the target sites were estimated using different docking algorithms (i.e., induced fit docking
(IFD), quantum polarized ligand docking (QPLD), and GOLD) as well as molecular dynamics
(MD) simulations and post-processing MD analysis. Moreover, the studied compounds were
put through further investigation regarding their pharmacokinetic and toxicity properties using
MetaCore/MetaDrug comprehensive systems biology analysis suite using binary disease and
toxicity QSAR models.
2. Results and Discussion
Although the pathogenesis of AD has not been fully clarified, one of the most important
theories is the decreased levels of acethylcholine and butyrylcholine which is observed in the
brains of patients with AD. Thus, the inhibition of AChE and BuChE enzymes that hydrolyse
ACh and BCh neurotransmitters can be considered as therapeutic approach. For this reason,
many research groups have conducted investigations of the inhibitory activity for these
enzymes involved in AD pathogenesis. (Cavdar et al 2019; Zilbeyaz et al 2018; Pascoini et al
2019; Gao et al 2019). In this study, 23 novel carvacrol derivatives involving the amide
moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized
and tested in vitro as AChE and BuChE inhibitors.
2.1. Chemistry
The synthesis of the target carvacrol derivatives (5a-w) was accomplished in four steps
(Scheme 1). The ethyl 2-(5-isopropyl-2-methylphenoxy)acetate (2) was synthesized from the
carvacrol (1) by a nucleophilic substitution. 2 was hydrolysed with 10% NaOH (aq) to give 2-
(5-isopropyl-2-methylphenoxy) acetic acid (3) and then 3 was chlorinated with SOCl2. In the
last step, 2-(5-isopropyl-2-methylphenoxy)acetyl chloride (4) was reacted with aliphatic or
aromatic amine derivatives. 5a-w were obtained in acceptable yields ranging from 40% to
1
86%. All the novel compounds were characterized by H NMR, 13C NMR, IR, MS and
elemental analyses.
4

Scheme 1. Synthesis of new carvacrol substituted amide derivatives. Reaction conditions: (i)
Ethylbromoacetate, K2CO3, Acetone, reflux, 5h; (ii) 10% NaOH (aq), reflux, 6h; (iii)
SOCl2, 80oC, 2h; (iv) Amine derivatives, Et3N, CH2Cl2, 60oC, overnight.
2.2. Cholinesterase inhibitory activity
The inhibitory activities of the synthesized compounds (5a-w) on AChE and BuChE were
determined by the Ellman’s method (Ellman et al 1961). The IC50 values for AChE and
BuChE inhibitions are summarized in Table 1. The IC50 values against AChE and BuChE
ranged from 1.93 µM to >200 µM and from 0.05 µM to184.50 µM, respectively.
The majority of the synthesized carvacrol substituted amide derivatives revealed significantly
higher inhibition activity than carvacrol against AChE. Similarly, all of the synthesized
compounds have higher BuChE inhibitory activity than carvacrol. Among them, 5v exhibited
the strongest inhibition against AChE with an IC50 value of 1.93 µM, which is 149-fold more
than that of carvacrol (IC50 = 288.26 µM), and it showed similar activity with the
galantamine, used as a standard (IC50 = 2.21 µM), while it represented lesser inhibitory
activity than donepezil (IC50 = 0.03 µM) and tacrine (IC50 = 0.17 µM), which are well-known
as AChE inhibitors. Compound 5v displayed the strongest inhibition against BuChE with an
IC50 value of 0.05 µM, which is 8216-fold more than that of carvacrol (IC50 = 410.79 µM),
372-fold more than that of galantamine (IC50 = 18.60 µM), and 52-fold more than that of
donepezil (IC50 = 2.58 µM), but 2.5-fold less than that of tacrine (IC50 = 0.02 µM).
The following structure-activity relationship (SAR) observations can be drawn from data of
Table 1: (i) In comparison between 5a-e and 5i, involving alkyl or cyclic alkyl group binding
to N- atom, an increase in the C number of alkyl chains and the steric hindrance by binding of
the second alkyl group on the -NH of the amide moiety may lead to a decline in the inhibitory
activity against both AChE and BuChE, compare 5a (having N-methyl group, IC50 = 20.54
5

and 21.87 µM for AChE and BuChE, respectively) with 5b (having N-propyl group, IC50 =
37.51 and 32.65 µM for AChE and BuChE, respectively), 5e (having N-cyclohexyl group,
IC50 = 58.28 and 32.89 µM for AChE and BuChE, respectively) and 5i (having N-dihydro-
indenyl group, IC50 ≥ 200 and 45.04 µM for AChE and BuChE, respectively)) and (compare
5c (having N,N-diethyl group, IC50 = 73.70 and 80.20 µM for AChE and BuChE,
respectively) with 5d (having N,N-diisopropyl group, IC50 = 95.96 and 96.50 µM for AChE
and BuChE, respectively)). (ii) The expansion of the pyrrolidine ring of 5f (IC50 = 144.21
µM) to a piperidine ring (compound 5g: IC50 ≥ 200 µM) and morpholine ring (5h: IC50
=
197.77 µM) decreased the AChE inhibitory activity. These declines in item (i) and (ii) can
simply be explained as increasing steric hindrances decreases H-bonding capability of N-
atoms. (iii) The presence of an ethyleneamine (-HNCH2CH2-) group as a spacer between the
carbonyl moiety and the pyrrolidine or morpholine ring negatively affected the inhibitory
activity against both ChEs (compare 5f (IC50 = 144.21 and 30.31 µM for AChE and BuChE,
respectively) with 5j (IC50 ≥ 200 and 74.40 µM for AChE and BuChE, respectively), and
(compare 5h (IC50 = 197.77 and 9.39 µM for AChE and BuChE, respectively) with 5k (IC50 ≥
200 and 137.38 µM for AChE and BuChE, respectively). (iv) The presence of an amine (-NH)
group between the carbonyl moiety and the piperidine or morpholine ring increased the AChE
inhibitory activity (compare 5g (IC50 ≥ 200 µM) with 5q (IC50 = 66.70 µM), and (compare 5h
(IC50 = 197.77 µM) with 5o (IC50 = 191.56 µM). This enhancement can be linked to the
presence of amine group, increasing the possibility of hydrogen bond formation with the
amino acid residues in active side. (v) The increase in the electron density of the heterocyclic
aromatic moiety (pyridine, pyrimidine and quinoline) and binding amide moiety, enhanced
the inhibitory activity against both AChE and BuChE targets (compare 5t (having pyridine,
IC50 = 92.31 and 184.50 µM for AChE and BuChE, respectively) with 5u (having pyrimidine,
IC50 = 20.74 and 2.82 µM for AChE and BuChE, respectively) and 5v (having quinoline, IC50
= 1.93 and 0.05 µM for AChE and BuChE, respectively). This is an expected effect, because
the increase in electron density of heterocyclic aromatic moiety can lead to forming of -
stacking or -cation interactions with the active site amino acids. When the sizes of the
molecules compared to the approved drugs, they have similar sizes (i.e., while donepezil has
28 non-hydrogen atoms, corresponding number is 25 in 5v), so the ligand efficiency trend can
be also interpreted from the inhibitory activity results of studied molecules.
6

Table 1. In vitro inhibition IC50 values (µM) and selectivity of compounds 5a-w for AChE
and BuChE.
Selectivity
indexb
Compound
Carvacrolc
Structure
AChE (IC50, μM)a
BuChE (IC50, μM)a
288.26±1.112
410.79±1.238
1.43
20.54±0.47
21.87±1.51
1.07
5a
37.51±1.14
73.70±1.47
95.96±1.22
32.65±0.98
80.20±0.98
96.15±1.21
0.87
1.09
1.00
5b
5c
5d
58.28±0.55
37.89±0.68
30.31±0.78
0.65
0.21
5e
5f
144.21±1.22
>200
2.72±0.51
9.39±0.26
<0.01
0.05
5g
5h
197.77±1.61
7

>200
>200
>200
>200
45.04±0.95
74.40±1.42
137.38±1.29
43.16±1.23
<0.23
<0.37
<0.69
<0.22
5i
5j
5k
5l
>200
59.56±1.44
<0.30
5m
116.69±1.01
191.56±1.14
2.68±0.38
0.02
0.73
5n
5o
140.07±1.33
60.11±1.85
66.70±1.08
3.08±0.63
0.05
0.93
5p
5q
61.89±1.27
8

88.68±1.43
88.28±1.28
92.31±1.65
44.05±1.51
30.15±1.66
184.50±1.73
0.50
0.34
2.00
5r
5s
5t
20.74±0.67
1.93±0.32
2.82±0.66
0.05±0.07
0.14
0.03
5u
5v
65.61±1.32
37.40±1.14
0.57
5w
Galantamine
Donepezild
Tacrinee
-
-
-
2.21±0.05
18.60±0.52
2.58±0.65
0.02±0.004
15.37
86.00
0.12
0.03±0.0005
0.17±0.0119
a IC50 values represent the means ± S.E.M. of three parallel measurements (p< 0.05).
b Selectivity index = IC50 (BuChE) / IC50 (AChE).
c From ref. [13]
d From ref. [36]
e From ref. [37]
2.3. Molecular Docking
Studied molecules were docked into the binding pockets of AChE and BuChE targets using
different docking algorithms. Table 2 represents GOLD Fitness and ChemScore results of
studied compounds at the binding cavities of AChE and BuChE. Docking results were
compared with known inhibitors donepezil, galantamine, and tacrine. Docking results show
that while standard inhibitors have better GoldFitness and ChemScore values than studied
molecules at the binding pocket of AChE, corresponding results were similar for BuChE.
9

Docking scores of studied molecules and known inhibitors using other docking programs
were represented and compared to each other with Figure 1. Detailed results were provided at
the Table S1 (Supporting Materials). All used docking programs predicted the donepezil as
tightest binder molecule which fits well with the experimental results. Because of the
limitations in the basis of docking algorithm, it’s not expected to get absolute binding affinity
values obtained from in vitro results. However, when the studied molecules, which are
divided into the weak, moderate and strong binders, it can be seen that experimental results
and especially Glide/IFD results fit well to each other. Thus, for the further analysis (i.e., MD
simulations), initial structures were used from top-docking poses derived from Glide/IFD
algorithm.
Figure 1. Glide/IFD, MOE/IFD, QPLD and Gold (ChemScore) results of studied compounds
at the binding cavities of AChE (top) and BuChE (bottom). Docking scores are in kcal/mol.
2.4. Molecular Dynamics (MD) Simulations
In order to evaluate the dynamics of the selected ligands (5v and 5w) and also fluctuations of
the protein backbones in both ligand-bound and ligand-free (apo) systems, the complexes
obtained from the docking simulations (Figure S1) were submitted into all atom MD
simulations. The results were monitored throughout the MD simulations as the root-mean-
square deviation (RMSD) of the atomic positions respect to the initial conformers (Figure 2).
10

Figure 2. ProFit and LigFit RMSD analysis of compounds 5v and 5w throughout MD
simulations. (ProFit shows the RMSD of a ligand when the protein-ligand complex is first
aligned on the protein backbone of the reference and then the RMSD of the ligand heavy
atoms is measured. If the values observed are significantly larger than the RMSD of the
protein, then it is likely that the ligand has diffused away from its initial binding site. LigFit
shows the RMSD of a ligand that is aligned and measured just on its reference conformation.
This RMSD value measures the internal fluctuations of the ligand atoms.)
Table 2. (top) GOLD Fitness and ChemScore results of studied compounds at the binding
cavities of AChE and BuChE; (bottom) GOLD Fitness and ChemScore results of known
approved compounds at the binding cavities of AChE and BuChE.
Comp.
AChE
BuChE
GoldScore
Fitness
Min.
GoldScore
Fitness
Max.
Average
ChemScore
(kcal/mol)
GoldScore
Fitness
Min.
GoldScore Average
Fitness
Max.
ChemScore
(kcal/mol)
51.55
59.73
53.24
29.68
62.64
52.68
48.53
51.13
65.19
59.22
±2.64
66.26
±2.33
59.98
±2.36
50.76
±7.62
68.61
±1.85
60.82
±2.40
58.30
±3.49
60.53
±3.35
36.45
42.47
39.24
41.93
49.14
42.90
40.79
38.04
44.00
50.25
46.86
51.46
57.04
46.63
47.49
48.21
39.78
±2.10
46.84
±1.59
43.65
±1.87
47.67
±2.42
54.69
±1.93
45.01
±1.00
45.62
±1.98
44.94
±1.57
5a
5b
5c
5d
5e
5f
-10.35
-10.22
-12.51
-10.51
-12.88
-11.52
-12.40
-10.82
-11.19
-10.92
-11.23
-9.57
69.86
64.85
65.99
73.15
65.60
67.21
66.77
-10.92
-9.12
5g
5h
-11.19
-10.08
11

68.53
69.62
65.32
69.73
74.36
66.92
60.73
60.36
59.19
73.17
60.71
63.30
61.23
80.77
79.98
79.95
83.61
88.21
86.40
68.63
73.37
68.98
89.75
76.94
76.56
76.40
73.58
±3.03
74.33
±2.57
74.56
±3.18
77.53
±3.14
81.60
±3.27
78.83
±3.69
64.98
±2.27
68.09
±3.03
68.71
±2.27
84.32
±3.57
69.34
±4.18
70.48
±3.20
70.45
±3.56
48.35
48.05
48.18
50.32
48.89
48.72
44.63
44.87
45.15
54.29
49.15
44.79
43.79
58.01
58.60
58.77
63.31
62.79
62.17
54.11
52.96
57.51
68.73
59.75
55.35
54.09
53.60
±1.95
53.25
±2.55
53.82
±1.88
55.83
±3.08)
56.54
±3.13
54.15
±3.36
49.01
±2.20
49.11
±2.05
51.96
±2.85
61.21
±2.64
56.49
±2.53
51.80
±2.73
50.47
±2.26
5i
-12.81
-11.10
-11.69
-12.95
-12.74
-10.60
-11.47
-11.96
-11.76
-11.26
-13.79
-10.53
-11.37
-13.01
-11.26
-12.95
-10.68
-10.60
-11.07
-11.15
-6.89
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
-6.05
-9.54
-6.34
5u
-10.48
-11.04
-13.36
-6.32
-8.24
-7.57
66.40
66.48
79.73
77.60
72.08
±3.24
72.72
±3.18
52.81
48.98
65.06
60.41
59.36
±3.18
55.47
±3.45
5v
5w
Comp.
AChE
BuChE
GoldScore
Fitness (top)
Average
Score
ChemScore
(top)
GoldScore
Fitness (top)
Average Score
ChemScore
(top)
(kcal/mol)
(kcal/mol)
94.31
84.64
83.70
86.53±5.16
79.96±1.98
82.43±1.15
-14.90
-11.19
-13.80
64.63
51.04
47.65
56.04±2.88
46.57±0.60
47.11±1.79
-7.49
-6.98
-7.07
Donepezil
Galantamine
Tacrine
The calculations were performed based on two different approaches, “ProFit” and “Lig Fit”,
which respectively shows the dynamics of the systems when the trajectory frames were fitted
based on the protein (i.e., translational) and ligand atoms (i.e., rotational), respectively. It
provides better understanding of translational and rotational dynamics of studied ligands at
the binding cavity of targets through the MD simulations. The average values of RMSD were
calculated for the individual systems, helping compare the fluctuations of the systems, as
shown in Figure 2. The results are in consistent with the experimental data. The apo
conformer, which is free of ligand, revealed less structural stability in compression with the
ligand-bound systems.
12

In the case of the ligand-bound systems, the ProFit RMSD average values for the 5v and 5w
were calculated to be 4.84 Å and 5.60 Å, respectively, which demonstrate that compound 5v
seems slightly more stable with lower diffusion than compound 5w. Also, 5v showed smaller
rotational changes in the cavity. The observed data can be linked to strong formed chemical
interactions of 5v within the active site amino acids.
In addition, interactions of studied molecules with the active site residues were monitored
throughout the simulations. (Figures 3 and 4) These contacts between ligand and target
residues were categorized into four different types (i.e., hydrogen bonding, hydrophobic,
ionic, and water-bridge) and represented as stacked bar charts which are normalized over the
course of the trajectories. These figures also assist for monitoring to see the changes of
constructing chemical interactions from beginning of simulations (i.e., input coordinates from
docking) throughout the simulations. Moreover, conformational evolution of rotatable bonds
in the 5v and 5w were analysed throughout the simulation. (Figures 5 and 6) Each rotatable
bond torsion is accompanied by a radial plot and bar plot. While radial plots describe the
change of the dihedral angle during the simulation (i.e., beginning of the simulation is in the
center of the plot and the time evolution is plotted radially outwards), the bar plots summarize
the data on the radial plots by showing the probability density of the torsion. The rotatable
bonds in 5v seems more stable than those in 5w, in which wide spectrum of torsional angles
was appeared – as it is mentioned in the RMSD analysis, the structural stability of the ligand
can due to the interactions establishing in the binding site.
When top-docking poses and ligand-binding pocket residue interactions throughout the MD
simulations were compared, it can be seen that ligands change their initial position (RMSD is
about 5 Å) during the simulations in order to construct more strong new interactions.
It must be noted that, MD simulations of known inhibitor donepezil at the binding pocket of
AChE represents similar crucial residues (i.e., Trp84, Trp279, Tyr334) which is also found
important for inhibitor binding with the studied molecules. (Figure S2, Supporting
Information)
13

Figure 3. (left) A timeline representation of the interactions and contacts of the compound 5v
at the binding pocket of AChE throughout MD simulations. (right) Interaction fractions of
residues are shown by the stacked bar charts which are normalized over the course of the
trajectory frames. Interactions that occur more than 15% of the simulation time throughout
50-ns MD simulations are also shown.
Figure 4. (left) A timeline representation of the interactions and contacts of the compound 5w
at the binding pocket of AChE throughout MD simulations. (right) Interaction fractions of
14

residues are shown by the stacked bar charts which are normalized over the course of the
trajectory frames. Interactions that occur more than 15% of the simulation time throughout
50-ns MD simulations are also shown.
Figure 5. Torsional analysis of 5v throughout MD simulations. (The ligand torsions plot
summarizes the conformational evolution of every rotatable bond in the ligand throughout the
simulation trajectory. The top panel shows the 2D schematic of a ligand with color-coded
rotatable bonds. Each rotatable bond torsion is accompanied by a dial plot and bar plots of the
same color.)
15

Figure 6. Torsional analysis of 5w throughout MD simulations. (The ligand torsions plot
summarizes the conformational evolution of every rotatable bond in the ligand throughout the
simulation trajectory. The top panel shows the 2D schematic of a ligand with color-coded
rotatable bonds. Each rotatable bond torsion is accompanied by a dial plot and bar plots of the
same color.)
2.5. Ligand binding domain analysis
Interestingly, in comparison between 5v (having quinoline) and 5w (having 2-methyl-
quinoline), the presence of methyl group and binding position (5v bound from C8 to amide
moiety, while 5w bound from C4) of heterocyclic ring significantly decreased inhibitory
activity against both ChEs. To understand this decline, the structural and dynamical profiles
of these molecules at the target sites were estimated using docking and MD simulations
methods. According to MD results (Figures 2-6), compound 5v at the AChE revealed high
affinity toward Trp84 and Phe330 by forming mainly - stacking interactions and Gln69,
Ser122, and Gly123 via hydrogen bonding interactions. On the other hand, Tyr121 and
Trp279 at the catalytic anionic site (CAS) of AChE interacted with the quinoline ring of
compound 5w forming of hydrophobic interactions. The hydrophobic interactions were also
observed for Tyr70, Trp84, Leu282 and Tyr334 with compound 5w. Furthermore, Tyr121
16

engaged in a hydrogen bond interaction with the carbonyl oxygen of the compound 5w. Based
on these findings, we can say that compound 5w engaged in the CAS at the vicinity of bottom
of active site, while compound 5v stacked at the PAS at the gorge rim and covered the
entrance of active site.
2.6. MetaCore/MetaDrug Analysis
The studied molecules were investigated for their pharmacokinetic and toxicity properties
using MetaCore/MetaDrug comprehensive systems biology analysis suite by the help of
available absorption, distribution, metabolism, and excretion (ADME), disease and toxicity
QSAR models. Table 3 compares predicted pharmacokinetic profiles of 5v and 5w with
approved drugs rivatigmine, tacrine and donepezil. Results show that both molecules pass the
BBB. The compounds show high lipophilicity, moderate human serum protein binding
profiles and they do not block the human ether a-go-go-related gene (hERG) channel. (Table
3) Toxicity properties of 5v and 5w were studied with 26-different toxicity QSAR models.
(Table 4) Both of the compounds 5v and 5w did not show any high toxicity risks. Slight
toxicity risk appeared only in one property (epididymis) for 5w (the predicted value (0.51)
was very close to cutoff (0.50)). It must be noted that while none of the toxicity QSAR
models show 5v as toxic, toxicity predicted only 1 model for 5w. Corresponding toxicity
results were appeared in 3 models (i.e., cardiotoxicity, genotoxicity, and liver cholestasis) for
rivastigmine, in 19 models for tacrine, and in 4 models for donepezil.
Table 3. Pharmacokinetic predictions of 5v and 5w using MetaCore/MetaDrug. Results are
compared with positive control molecules rivastigmine, tacrine and donepezil.
ADME QSAR and Protein Binding QSAR Models
5v
5w
0.25
3.92
78.50
-0.08
0.46
rivastigmine tacrine donepezil
BBB, log ratio (1)
G-LogP (2)
0.24
3.42
76.97
-0.02
0.99
0.22
1.81
0.40
2.81
67.04
0.04
1.82
0.05
4.10
79.13
0.24
1.03
Prot-bind, % (3)
Prot-bind, Log t (4)
WSol, log mg/L (5)
45.45
-0.12
2.83
hERG-inh, pKi (6)
0.29
0.21
-0.42
-0.82
0.42
(1)Blood brain barrier penetration model. The data is expressed as log values of the ratio of the metabolite
concentrations in brain and plasma. Cutoff is -0.3. Larger values indicate that the metabolite is more likely to
enter the brain. Model description: N=107, R2=0.89, RMSE=0.26. (2)Lipophilicity, log of compound octanol-
water distribution. Cutoffs are -0.4 to 5.6. Values greater than 5.6 correspond to overly hydrophobic compounds.
Model description: N=13474, R2=0.95, RMSE=0.21. (3)Human serum protein binding, %. Cutoff is 50%. A value
of more than 95% is highly bound, less than 50% is a low binding metabolite. Model description: N=265,
R2=0.909, RMSE=10.11. (4)Affinity to human serum albumin, log value of the retention time. Cutoff is 0.
Positive values correspond to higher protein binding, negative values to lower protein binding. The model is
17

based on retention times of compounds assayed by HPLC using an immobilized HSA column. Values are
expressed as log values of the retention time. Model description: N=95, R2=0.904, RMSE=0.2. (5)Water
solubility at 25oC, log mg/L. Cutoffs are from 2 to 4. An acceptable level of solubility is project dependent.
(6)
Model description: N=2871, R2=0.91, RMSE=0.54;
Human hERG (human ether a-go-go-related gene)
channel inhibition, pKi (uM). Cutoff is -1.7. The higher the value, the higher the inhibition activity. Lower
values are preferable. Model description: N=196, R2=0.93, RMSE=0.23.
Table 4. Prediction of toxicity values using MetaCore/MetaDrug. Results are compared with
positive control molecules rivastigmine, tacrine and donepezil.
rivastigmine
tacrine
donepezil
Property
AMES (1)
5v
5w
0.45
0.09
0.08
0.14
0.19
0.04
0.08
0.29
5.33
0.16
0.37
0.17
0.14
0.03
0.27
0.13
0.22
0.03
-0.02
0.09
0.05
0.08
0.30
0.19
0.17
0.27
0.27
0.21
0.28
0.26
5.15
0.51
0.31
0.34
0.22
0.07
0.39
0.22
0.14
0.08
0.21
0.11
0.16
0.24
0.42
0.17
0.33
0.23
0.20
0.22
0.16
0.78
5.29
0.14
0.62
0.27
0.14
0.19
0.56
0.38
0.31
0.35
0.37
0.22
0.33
0.08
0.38
0.46
0.88
0.78
0.77
0.73
0.75
0.83
0.80
0.63
5.11
0.53
0.71
0.75
0.85
0.23
0.61
0.55
0.82
0.87
0.34
0.26
0.87
0.36
0.63
0.15
0.35
0.17
0.08
0.11
0.12
0.02
0.03
0.75
5.21
0.04
0.58
0.14
0.06
0.09
0.62
0.29
0.78
0.54
0.22
0.04
0.38
0.12
0.18
0.37
Anemia (2)
Carcinogenicity (3)
Carcinogenicity Mouse Female (4)
Carcinogenicity Mouse Male (5)
Carcinogenicity Rat Female (6)
Carcinogenicity Rat Male (7)
Cardiotoxicity (8)
Cytotoxicity Model, -log GI50 (M) (9)
Epididymis Toxicity (10)
Genotoxicity (11)
Hepatotoxicity (12)
Kidney Necrosis (13)
Kidney Weight Gain (14)
Liver Cholestasis (15)
Liver Lipid Accumulation (16)
Liver Necrosis (17)
Liver Weight Gain(18)
MRTD (19)
Nasal Pathology (20)
Nephron Injury (21)
Nephrotoxicity (22)
Neurotoxicity (23)
0.21
0.08
0.41
0.10
Pulmonary Toxicity (24)
SkinSens, EC3 (25)
50.30 24.73
0.11 0.23
32.04
0.03
5.66
0.24
21.85
0.04
Testicular Toxicity (26)
1. Potential to be mutagenic (AMES positive), range from 0 to 1. A value of 1 is AMES positive (mutagenic),
and a value of 0 is AMES negative (non-mutagenic). Cutoff is 0.5. Values close to zero are preferable. The
AMES assay is based upon the reversion of mutations in the histidine operon in the bacterium Salmonella
enterica sv Typhimurium.
2. Potential for causing anemia. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic compounds.
Training set consists of chemicals and drugs causing anemia in vivo. Model organisms: human. Model
description: Training set N=324, Test set N=51, Sensitivity= 0.82, Specificity=0.90, Accuracy=0.86, MCC=0.72.
3. Potential for inducing carcinogenicity in rats and mice. Cutoff is 0.5. Values higher than 0.5 indicate
potentially toxic compounds. Training set consists of chemicals and drugs causing carcinogenicity in vivo.
Model organisms: mouse, rat. Model description: Training set N=1210, Test set N=185, Sensitivity= 0.96,
Specificity=0.90, Accuracy=0.93, MCC=0.86.
4. Potential for inducing carcinogenicity in female mice. Cutoff is 0.5. Values higher than 0.5 indicate potentially
toxic compounds. Training set consists of chemicals and drugs causing carcinogenicity in vivo. Model
18

organisms: female mice. Model description: Training set N=640, Test set N=94, Sensitivity= 0.90,
Specificity=0.93, Accuracy=0.92, MCC=0.83.
5. Potential for inducing carcinogenicity in male mice. Cutoff is 0.5. Values higher than 0.5 indicate potentially
toxic compounds. Training set consists of chemicals and drugs causing carcinogenicity in vivo. Model
organisms: mouse male. Model description: Training set N=584, Test set N=93, Sensitivity= 0.91,
Specificity=0.88, Accuracy=0.89, MCC=0.78.
6. Potential for inducing carcinogenicity in female rats. Cutoff is 0.5. Values higher than 0.5 indicate potentially
toxic compounds. Training set consists of chemicals and drugs causing carcinogenicity in vivo. Model
organisms: female rat. Model description: Training set N=667, Test set N=120, Sensitivity= 0.90,
Specificity=0.96, Accuracy=0.93, MCC=0.86.
7. Potential for inducing carcinogenicity in male rats. Cutoff is 0.5. Values higher than 0.5 indicate potentially
toxic compounds. Training set consists of chemicals and drugs causing carcinogenicity in vivo. Model
organisms: male rat. Model description: Training set N=715, Test set N=117, Sensitivity= 0.92,
Specificity=0.88, Accuracy=0.90, MCC=0.79.
8. Potential for inducing cardiotoxicity. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing cardiotoxicity in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=143, Test set N=30, Sensitivity= 0.80, Specificity=1.00,
Accuracy=0.90, MCC=0.82.
9. Growth inhibition of MCF7 cell line (human caucasian breast adenocarcinoma), pGI50. Cutoff is 6. Values
from 6 to 8 correspond to a toxic metabolite, values less than 6 are preferable, values less than 3 are more
preferable and less toxic. Model description: N=1474, R2=0.9, RMSE=0.05.
10. Potential for inducing epididymis toxicity. Training set consists of chemicals and drugs causing epididymis
toxicity in vivo. Model organisms: mouse, rat, human. Cutoff is 0.5. Values higher than 0.5 indicate potentially
toxic compounds. Model description: Training set N=252, Test set N=42, Sensitivity= 0.90, Specificity=0.86,
Accuracy=0.88, MCC=0.76.
11. Potential for inducing genotoxicity. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing genotoxicity in vivo. Model organisms: mouse,
rat. Model description: Training set N=372, Test set N=86, Sensitivity= 0.75, Specificity=0.84, Accuracy=0.79,
MCC=0.59.
12. Potential for inducing hepatotoxicity. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing hepatotoxicity in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=1380, Test set N=231, Sensitivity= 0.73,
Specificity=0.88, Accuracy=0.81, MCC=0.62.
13. Potential for inducing kidney necrosis. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing renal necrosis in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=221, Test set N=42, Sensitivity= 0.96, Specificity=1.00,
Accuracy=0.98, MCC=0.95.
14. Potential for inducing kidney weight gain. Cutoff is 0.5. The values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing kidney weight gain in vivo. Model organisms:
mouse, rat. Model description: Training set N=240, Test set N=49, Sensitivity= 0.95, Specificity=1.00,
Accuracy=0.98, MCC=0.96.
15. Potential for inducing liver cholestasis. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing cholestasis in vivo. Model organisms: mouse,
rat, human. Model description: Training set N=218, Test set N=35, Sensitivity= 0.79, Specificity=0.67,
Accuracy=0.74, MCC=0.46.
16. Potential for inducing liver lipid accumulation. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing lipid accumulation in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=172, Test set N=28, Sensitivity= 0.80, Specificity=0.85,
Accuracy=0.82, MCC=0.64.
17. Potential for inducing liver necrosis. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing hepatic necrosis in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=300, Test set N=57, Sensitivity= 0.91, Specificity=0.91,
Accuracy=0.91, MCC=0.82.
18. Potential for inducing liver weight gain. Cutoff is 0.5. Values higher than 0.5 indicate potential liver weight-
changing compounds. Training set consists of chemicals and drugs causing liver weight gain in vivo. Model
organisms: mouse, rat. Model description: Training set N=292, Test set N=52, Sensitivity= 1.00,
Specificity=1.00, Accuracy=1.00, MCC=1.00.
19. Maximum Recommended Therapeutic Dose, log mg/kg-bm/day, range is from -5 to 3. Cutoff is 0.5.
Chemicals with high log MRTDs can be classified as mildly toxic compounds, chemicals with low log MRTDs
as highly toxic compounds. Model description: N=1209, R2= 0.86, RMSE=0.42.
19

20. Potential for causing nasal pathology. Training set consists of chemicals and drugs causing nasal pathology
in vivo. Model organisms: mouse, rat, human. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Model description: Training set N=246, Test set N=47, Sensitivity= 1.00, Specificity=0.93,
Accuracy=0.96, MCC=0.92.
21. Potential for inducing nephron injury. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing nephron injury in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=598, Test set N=109, Sensitivity= 0.91, Specificity=1.00,
Accuracy=0.96, MCC=0.93.
22. Potential for inducing nephrotoxicity. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Training set consists of chemicals and drugs causing nephrotoxicity in vivo. Model organisms:
mouse, rat, human. Model description: Training set N=847, Test set N=154, Sensitivity= 0.90, Specificity=0.84,
Accuracy=0.87, MCC=0.74.
23. Potential for inducing neurotoxicity. Training set consists of chemicals and drugs causing neurotoxicity in
vivo. Model organisms: mouse, rat, human. Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic
compounds. Model description: Training set N=175, Test set N=34, Sensitivity= 0.94, Specificity=0.94,
Accuracy=0.94, MCC=0.88.
24. Potential for inducing pulmonary toxicity. Training set consists of chemicals and drugs causing pulmonary
toxicity in vivo. Model organisms: mouse, rat, human. Cutoff is 0.5. Values higher than 0.5 indicate potentially
toxic compounds. Model description: Training set N=482, Test set N=87, Sensitivity= 0.89, Specificity=0.88,
Accuracy=0.89, MCC=0.77.
25. Skin sensitization potential expressed as effective concentration 3, EC3 %. Values higher than 10 indicate
weak and moderate sensitizers. Model description: N=89, R2=0.67, RMSE=22.56.
26. It consists of chemicals and drugs causing testicular toxicity in vivo. Model organisms: mouse, rat, human.
Cutoff is 0.5. Values higher than 0.5 indicate potentially toxic compounds. Model description: Training set
N=439, Test set N=88, Sensitivity= 0.81, Specificity=0.85, Accuracy=0.83, MCC=0.66.
2.7. In vitro blood-brain barrier permeability using PAMPA-BBB
A good penetration across the blood-brain barrier is necessary condition for the central
nervous system (CNS) drugs (Pérez-Areales et al, 2014). Brain permeations of 5v, the most
potent compound in this study, and the well-known AChE inhibitors (tacrine, donepezil and
rivastigmine) was determined through the parallel artificial membrane permeation assay
(PAMPA), described by Di et al. (2003). This assay involved measuring the rate at which a
compound passively diffuses across the lipid barrier separating a donor compartment and an
acceptor compartment filled with phosphate buffer (pH 7.4). The concentrations of the
compound in both compartments were then measured to obtain the effective permeability rate
(Pe). It has been established that compounds with the Pe values above 4×10−6 cm s−1 penetrate
into CNS easily (CNS+), while compounds with the Pe value below 2×10−6 cm s−1 do not
(CNS−). In hybrids having the permeability values between these boundary limits, it is
difficult to estimate whether they cross BBB or not (CNS +/-) [32]. 5v (Pe = 4.38×10−6 cm
s−1) showed greater permeability values than that limit, pointing out that these molecules
would cross the BBB by passive diffusion (Table 5). Donepezil (Pe = 6.80×10−6 cm s−1) and
rivastigmine (Pe = 5.78×10−6 cm s−1) showed a higher Pe than 5v, while the Pe of tacrine (Pe =
4.51×10−6 cm s−1) and 5v were fairly close.
20

Table 5. Prediction of blood-brain barrier penetration of drugs expressed as Pe ± SEM (n = 4-
6)
BBB penetration estimation
Compound
Pe (10-6 cm s-1 )
4.38±0.54
CNS (+/-)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
5v
4.51±0.32
6.80±0.66
5.78±0.85
Tacrine
Donepezil
Rivastigmine
‘CNS(+)’ (High BBB permeation predicted; Pe (10-6 cm s-1 )>4.0
‘CNS(-)’ (Low BBB permeation predicted; Pe (10-6 cm s-1 )<2.0
‘CNS(+/-)’ (BBB permeation uncertain; Pe (10-6 cm s-1 ) from 4.0 to 2.0
3. Conclusions
A series of 23 novel carvacrol substituted amide moiety as a linker between the alkyl chains
and/or the heterocycle nucleus was synthesized and their inhibitory activities on AChE and
BuChE were evaluated. Among them, 5v showed the strongest inhibition against AChE and
BuChE with IC50 values of 1.93 µM, which is 149-fold more than that of carvacrol (IC50 =
288.26 µM), and it showed similar activity with the galantamine, used as a standard, (IC50 =
2.21 µM). Compound 5v also exhibited the strongest inhibition against BuChE with an IC50
value of 0.05 µM, which is 8216-fold more than that of carvacrol (IC50 = 410.79 µM), 372-
fold more than that of galantamine (IC50 = 18.60 µM), and 52-fold more than that of
donepezil (IC50 = 2.58 µM). Molecular modeling approaches, including docking and MD
simulation methods suggested that both compounds 5v and 5w were conformationally stable
inside the binding cavity, which is due to the strong polar and non-polar interactions forming
between the ligands and the active site amino acids. The result of parallel artificial membrane
permeability assay indicated that 5v (Pe = 4.38×10−6 cm s−1) showed greater permeability
values than that limit, pointing out that these molecules would cross the BBB by passive
diffusion.
Both docking and MD simulations were also repeated for known AChE and BuChE inhibitors
and results showed similar crucial amino acids both in studied molecules and in known
inhibitors.
Since BBB profiles of are important for CNS drugs, we studied in detail the BBB properties
of synthesized molecules, both MetaCore/MetaDrug and PAMPA-BBB results show that
molecules can permeate the BBB. Thus, with this study, we improved the low inhibitory
21

activity of carvacrol; and derivatives of carvacrol were able to penetrate from BBB.
Moreover, MetaCore/MetaDrug which includes 26-different toxicity QSAR models showed
that studied molecules have less side effect predictions compared to known inhibitors.
Overall these derivatives can be recommended as new chemotypes to develop new ChEs
inhibitors for the treatment of AD disease by suitably modulating the substitution pattern also
in the perspective of multifunctional anti AD agents.
4. Experimental section
4.1. General
All solvents, reagents, and starting materials were obtained from commercial sources unless
otherwise indicated. Melting points were taken on a Barnstead Electrothermal 9200. IR
1
spectra were registered on a Bruker-Alpha infrared spectrometer. H and 13C NMR spectra
were registered on a Varian Infinity Plus spectrometer at 300 and at 75 Hz, respectively. 1H
13
and C chemical shifts are referenced to the internal deuterated solvent. Mass spectra were
obtained using Agilent GC-7890a and Agilent MS-5975c spectrometer. The elemental
analyses were carried out with a Leco CHNS-932 instrument. Spectrophotometric analyses
were performed by a BioTek Power Wave XS (BioTek, USA). The electric eel
acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, 425.84 U/mg, Sigma) and horse serum
butyrylcholinesterase (BuChE, EC 3.1.1.8, 11.4 U/mg, Sigma) were purchased from Sigma
(Steinheim, Germany). The other chemicals and solvents were purchased from Fluka Chemie,
Merck, Alfa Easer and Sigma-Aldrich.
4.2. Synthesis of ethyl 2-(5-isopropyl-2-methylphenoxy)acetate (2)
Potassium carbonate (5.8 mmol) was added to a solution of the carvacrol (5.7 mmol) and
ethyl bromoacetate (5.7 mmol) in acetone (25 mL) and the reaction mixture was refluxed for
5 h. The mixture was filtrated and the solution was concentrated under reduced pressure, dried
1
in vacuo and the solid obtained was crystallized from ethanol [33]. H NMR (CDCl3, 300
MHz) δ/ppm: 1.21 (6H, d, J=6.7 Hz), 1.29 (3H, t, J=7.3 Hz), 2.25 (3H, s), 2.79-2.86 (1H,m),
4.23-4.30 (2H, q), 4.63 (2H, s), 6.57 (1H, s), 6.77 (1H, d, J=7.6 Hz), 7.0 (1H, d, J=7.6 Hz);
13C NMR (CDCl3, 75 MHz) δ/ppm: 14.4, 15.5, 16.0, 24.2, 33.9, 34.2, 61.4, 66.1, 110.0, 113.2,
118.8, 119.5, 124.8, 131.0, 148.0, 153.9, 156.3, 169.5.
4.3. Synthesis of 2-(5-isopropyl-2-methylphenoxy)acetic acid (3)
The crude product 2 was dissolved in aqueous 10% NaOH (25 ml) and the solution was
refluxed for 4 h. The reaction mixture was cooled and acidified with aqueous 6% HCl. The
precipitated white solid was filtered off and subsequently washed with water to give
compound 3 [34]. 1H NMR (CDCl3, 300 MHz) δ/ppm: 1.13 (6H, d, J=7.0 Hz), 2.11 (3H, s),
22

2.73-2.80 (1H,m), 3.34 (1H, s, OH), 4.65 (2H, s), 6.65 (1H, s), 6.7 (1H, d, J=7.6 Hz), 7.0 (1H,
d, J=7.4 Hz) ; 13C NMR (CDCl3, 75 MHz) δ/ppm:
4.4. Synthesis of 2-(5-isopropyl-2-methylphenoxy)acetyl chloride (4)
3 mmol of SOCl2 were added over 1 mmol of 2-(5-isopropyl-2-methylphenoxy)acetic acid (3)
and stirred at room temperature for half an hour. It was then continued for 2 hours at 80oC.
When the gas outlet was completed, the reaction was terminated and cooled. It was
1
crystallized at ether and dried in vacuum. H NMR (CDCl3, 300 MHz) δ/ppm: 1.15 (6H, d,
J=7.0 Hz), 2.13 (3H, s), 2.75-2.84 (1H,m), 4.67 (2H, s), 6.67 (1H, s), 6.71 (1H, d, J=7.6 Hz),
13
7.0 (1H, d, J=7.6 Hz); C NMR (CDCl3, 75 MHz) δ/ppm: 16.3, 24.5, 34.0, 39.3, 41.0, 65.3,
110.2, 118.9, 123.8, 130.9, 147.9, 156.4, 171.1.
4.5. Synthesis of 2-(5-isopropyl-2-methylphenoxy)-N-R-acetamide (5a-w)
Compound 4 (1.0 mmol) and Et3N (1.1 mmol) were dispersed in dry acetonitrile (5 ml). And
then appropriate amine (1.0 mmol) was added to the mixture. The mixture was stirred at 60oC
for overnight. The solvent was evaporated and the residue was washed with water. The
resulting crude product was purified by column chromatography (Hexane:Ethylacetate).
2-(5-isopropyl-2-methylphenoxy)-N-methylacetamide (5a): Brown liquid, 68% yield; IR:
3446, 3324, 2958, 2927, 2870, 1661, 1539, 1511, 1444, 1414, 1248, 1177, 1129, 995, 939,
1
850 cm-1 ; H NMR (CDCl3, 300 MHz) δ/ppm: 1.12 (6H, d, J=6.7 Hz ), 2.14 (3H, s), 2.70-
2.77 (1H, m), 2.81 (3H, d, J= 4.9 Hz), 4.39 (2H, s), 6.53 (1H, s), 6.64 (1H, s, NH), 6.70 (1H,
13
d, J=7.6 Hz), 6.97 (1H, d, J=7.6 Hz) ; C NMR (CDCl3, 75 MHz) δ/ppm: 16.2, 24.3, 26.0,
34.2, 67.7, 110.1, 119.7, 123.9, 131.1, 148.6, 155.6, 169.5. GC-MS (m/z): 221,1 [M+]. Anal.
Calcd. for C13H19NO2: C, 70.56; H, 8.65; N, 6.33; found: C, 70.54; H, 8.61; N, 6.35.
2-(5-isopropyl-2-methylphenoxy)-N-propylacetamide (5b): Yellow liquid, 58% yield; IR:
3428, 3305, 2960, 2930, 2873, 1659, 1614, 1580, 1538, 1512, 1442, 1250, 1177, 1130, 1057,
1
936, 815, 640, 563 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 0.93 (3H, t, J=7.3 Hz), 1.21
(6H, d, J=7.0 Hz ), 1.54-1.61 (2H, q), 2.24 (3H, s), 2.83-2.87 (1H, m), 3.35 (2H, t, J=6.7 Hz),
4.52 (2H, s), 6.65 (1H, s), 6.70 (1H, s, NH), 6.81 (1H, d, J=7.6 Hz), 7.08 (1H, d, J=7.6 Hz) ;
13C NMR (CDCl3, 75 MHz) δ/ppm: 11.5, 16.1, 23.0, 24.2, 34.2, 40.9, 67.8, 110.2, 119.8,
123.9, 131.1, 148.7, 155.6, 168.8. GC-MS (m/z): 249,2 [M+]. Anal. Calcd. for C15H23NO2 C,
72.25; H, 9.30; N, 5.62; found: C, 72.28; H, 9.31; N, 5.63.
N,N-diethyl-2-(5-isopropyl-2-methylphenoxy)acetamide (5c): Yellow liquid, 71% yield; IR:
2960, 2932, 2872, 1760, 1642, 1511, 1459, 1419, 1243, 1178, 1129, 1071, 1036, 813, 642,
1
463 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.12-1.24 (12H, m), 2.23 (3H, s), 2.83-2.88
(1H, m), 3.38-3.48 (4H, m), 4.69 (2H, s), 6.73 (1H, s), 6.76 (1H, d, J=7.3 Hz), 7.06 (1H, d,
23

J=7.6 Hz) ; 13C NMR (CDCl3, 75 MHz) δ/ppm: 13.0, 14.6, 16.1, 24.3, 30.5, 34.2, 40.6, 41.9,
68.3, 109.9, 119.1, 124.2, 130.9, 148.2, 156.4, 167.8. GC-MS (m/z): 263,1 [M+]. Anal. Calcd.
for C16H25NO2:C, 72.96; H, 9.57; N, 5.32; found: C, 72.92; H, 9.58; N, 5.33.
N,N-diisopropyl-2-(5-isopropyl-2-methylphenoxy)acetamide (5d): Yellow solid, 58% yield;
mp.56°C; IR: 2961, 2928, 2871, 1737, 1612, 1586, 1443, 1422, 1342, 1239, 1175, 1127,
1034, 871, 812, 735, 590 cm-1 ; 1H NMR (CDCl3, 300 MHz) δ/ppm: 1.10-1.15 (12H, m), 1.34
(6H, d, J=7.0 Hz ), 2.13 (3H, s), 2.72-2.79 (1H, m), 3.31-3.35 (1H, m), 4.12-4.16 (1H, m),
4.55 (2H, s), 6,66 (1H, d, J=7.9 Hz), 6,67 (1H,s), 6.96 (1H, d, J=7.9 Hz) ; 13C NMR (CDCl3,
75 MHz) δ/ppm: 16.2, 20.5, 21.1, 24.3, 30.5, 34.3, 46.3, 49.0, 69.7, 109.5, 118.9, 123.9,
130.8, 148.3, 156.3, 167.6. GC-MS (m/z): 291,1 [M+]. Anal. Calcd. for C18H29NO2: C, 74.18;
H, 10.03; N, 4.81; found: C, 74.20; H, 10.07; N, 4.80.
N-cyclohexyl-2-(5-isopropyl-2-methylphenoxy)acetamide (5e): Light yellow solid, 62%
yield; mp. 78.3°C; IR: 3292, 2956, 2929, 2852, 1665, 1613, 1578, 1536, 1417, 1277, 1244,
1140, 971, 847, 715, 644, 585 cm-1; 1H NMR (CDCl3, 300 MHz) δ/ppm: 1.20-1.29 (10H, m),
1.32-1.47 (2H,m), 1.59-1.73 (2H,m), 1.90-1.96 (2H, m), 2.24 (3H, s), 2.81-2.90 (1H,m), 3.85-
3.88 (1H, m), 4.49 (2H, s), 6.52-6.53 (1H, s, NH), 6.65 (1H, s), 6.80 (1H, d, J=7.6 Hz), 7.09
(1H, d, J=7.6 Hz) ; 13C NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 24.2, 24.8, 25.6, 33.1, 34.2,
47.8, 67.8, 110.3, 119.8, 123.8, 131.0, 148.7, 155.6, 168.0. GC-MS (m/z): 289,2 [M+]. Anal.
Calcd. for C18H27NO2: C, 74.70; H, 9.40; N, 4.84; found: C, 74.73; H, 9.42; N, 4.82.
2-(5-isopropyl-2-methylphenoxy)-1-(pyrrolidin-1-yl)ethanone (5f): White liquid, 40% yield;
IR: 2957, 2927, 2872, 1641, 1580, 1511, 1448, 1418, 1340, 1288, 1247, 1177, 1131, 1038,
1
993, 858, 813, 716, 642, 522, 463 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.21 (6H, d,
J=7.0 Hz ), 1.81-1.95 (4H, m), 2.22 (3H, s), 2.80-2.87 (1H, m), 3.51 (2H, t, J=6.7 Hz), 3.55
(2H, t, J=6.7 Hz), 4.62 (2H, s), 6,72 (1H, s), 6,76 (1H, d, J=7.9 Hz), 7.05 (1H, d, J=7.9 Hz);
13C NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 24.0, 24.3, 26.5, 34.2, 46.4, 68.8, 109.9, 119.1,
124.2, 130.9, 148.3, 156.3, 167.2. GC-MS (m/z): 261,1 [M+]. Anal. Calcd. for C16H23NO2: C,
73.53; H, 8.87; N, 5.36; found: C, 73.52; H, 8.85; N, 5.38.
2-(5-isopropyl-2-methylphenoxy)-1-(piperidin-1-yl)ethanone (5g): Light brown liquid, 45%
yield; IR: 2970, 2860, 1771, 1642, 1613, 1511, 1445, 1418, 1240, 1176, 1120, 1039, 997,
1
852, 813, 642, 575 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=6.7 Hz), 1.55-
1.64 (6H, m), 2.21 (3H, s), 2.83-2.88 (1H, m), 3.52-3.59 (4H, m), 4.68 (2H, s), 6.73 (1H, s),
13
6.77 (1H, d, J=7.9 Hz), 7.04 (1H, d, J=7.3 Hz) ; C NMR (CDCl3, 75 MHz) δ/ppm: 16.1,
24.3, 24.7, 25.7, 26.7, 34.3, 43.5, 46.8, 68.4, 109.7, 119.0, 124.1, 130.8, 148.3, 156.1, 166.8.
24

GC-MS (m/z): 275,1 [M+]. Anal. Calcd. for C17H25NO2: C, 74.14; H, 9.15; N, 5.09; found: C,
74.16; H, 9.17; N, 5.06.
2-(5-isopropyl-2-methylphenoxy)-1-morpholinoethanone (5h): White solid, 40% yield; mp.
79°C; IR 2979, 2958, 2907, 2879, 1653, 1610, 1577, 1502, 1461, 1433, 1302, 1234, 1176,
1113, 1031, 851, 818, 760, 566 cm-1; 1H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=7.0
Hz ), 2.18 (3H, s), 2.81-2.88 (1H, m), 3.64 (8H, s), 4.68 (2H, s), 6.72 (1H, s), 6.77 (1H, d,
J=7.6 Hz), 7.06 (1H, d, J=7.6 Hz) ; 13C NMR (CDCl3, 75 MHz) δ/ppm: 16.0, 24.3, 34.3, 42.7,
46.3, 67.0, 68.2, 109.6, 119.3, 123.9, 131.0, 148.5, 155.8, 167.1. GC-MS (m/z): 277,1 [M+].
Anal. Calcd. for C16H23NO2: C, 69.29; H, 8.36; N, 5.05; found: C, 69.27; H, 8.34; N, 5.07.
N-(2,3-dihydro-1H-inden-2-yl)-2-(5-isopropyl-2-methylphenoxy)acetamide (5i): Yellow
liquid, 50% yield; IR: 3327, 3068, 3026, 2960, 2930, 2866, 1655, 1540, 1485, 1248, 1128,
1059, 1019, 947, 860, 800, 734, 639, 585, 565, 459 cm-1; 1H NMR (CDCl3, 300 MHz) δ/ppm:
1.21 (6H, d, J=7.0 Hz), 2.09 (3H, s), 2.80-2.87 (1H,m), 2.84 (2H, dd, J=5.2; 15.8 Hz), 3.37
(2H, dd, J=7.3; 16.1 Hz), 4.49 (2H, s), 4.81-4.87 (1H, m), 6.63 (1H, s), 6.79 (1H, d, J=7.6
Hz), 6.80 (1H, s, NH), 7.04 (1H, d, J=7.6 Hz), 7.16-7.26 (4H, m); 13C NMR (CDCl3, 75
MHz) δ/ppm: 15.9, 24.2, 34.2, 40.2, 50.3, 67.9, 110.4, 119.9, 123.9, 124.9, 127.1, 131.0,
131.1, 140.7, 148.7, 155.5, 168.5. GC-MS (m/z): 323,1 [M+]. Anal. Calcd. for C21H25NO2: C,
77.98; H, 7.79; N, 4.33; found: C, 77.96; H, 7.76; N, 4.35.
2-(5-isopropyl-2-methylphenoxy)-N-(2-(pyrrolidin-1-yl)ethyl)acetamide (5j): Brown liquid,
62% yield; IR: 3390, 2958, 2929, 2874, 2797, 1677, 1614, 1582, 1511, 1418, 1344, 1250,
1
1178, 1130, 1055, 994, 877, 814, 640, 573, 422 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm:
1.21 (6H, d, J=6.7 Hz), 1.71-1.76 (4H, m), 2.23 (3H, s), 2.44-2.50 (4H, m), 2.63 (2H, t, J=6.1
Hz), 2.80-2.86 (1H,m), 3.40-3.46 (2H, m), 4.49 (2H, s), 6.63 (1H, s), 6.78 (1H, d, J=7.6 Hz),
7.07 (1H, d, J=7.6 Hz), 7.43 (1H, s, NH) ; 13C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 23.7,
24.3, 34.2, 37.5, 53.8, 54.1, 67.3, 109.7, 119.5, 123.9, 131.0, 148.5, 155.5, 168.6. GC-MS
(m/z): 304,2 [M+]. Anal. Calcd. for C18H28N2O2: C, 71.02; H, 9.27; N, 9.20; found: C, 71.04;
H, 9.25; N, 9.23.
2-(5-isopropyl-2-methylphenoxy)-N-(2-morpholinoethyl)acetamide (5k): Yellow liquid, 66%
yield; IR: 3404, 2958, 2856, 2813, 1677, 1511, 1445, 1418, 1297, 1251, 1178, 1116, 1055,
1
1036, 867, 815, 763, 640, 575, 446 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.13 (6H, d,
J=7.6 Hz), 2.22 (3H, s), 2.32-2.35 (4H, m), 2.43 (2H, t, J=6.1 Hz), 2.73-2.82 (1H,m), 3.33-
3.38 (2H, m), 3.53-3.56 (4H, m), 4.44 (2H, s), 6.55 (1H, s), 6.71 (1H, d, J=7.6 Hz), 7.01 (1H,
13
d, J=7.6 Hz), 7.25 (1H, s, NH) ; C NMR (CDCl3, 75 MHz) δ/ppm: 16.4, 24.3, 34.2, 35.1,
53.4, 56.8, 67.1, 67.1, 109.5, 119.5, 123.6, 131.0, 148.6, 155.4, 168.5. GC-MS (m/z): 320,2
25

[M+]. Anal. Calcd. for C18H28N2O3: C, 67.47; H, 8.81; N, 8.74; found: C, 67.45; H, 8.83; N,
8.76.
N-(2-(cyclohex-1-en-1-yl)ethyl)-2-(5-isopropyl-2-methylphenoxy)acetamide (5l): Brown
liquid, 82% yield; IR: 3417, 3306, 2957, 2925, 2836, 1674, 1525, 1511, 1418, 1248, 1177,
1130, 1055, 814, 639, 555, 448 cm-1 ; 1H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=6.7
Hz), 1.49-1.63 (4H, m), 1.91-2.09 (4H, m), 2.17 (2H, t, J=6.4 Hz), 2.22 (3H, s), 2.81-2.90
(1H,m), 3.40-3.46 (2H, q), 4.49 (2H, s), 5.43 (1H, s), 6.63 (1H, s), 6.66 (1H, s, NH), 6.80 (1H,
d, J=7.6 Hz), 7.1 (1H, d, J=7.6 Hz); 13C NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 22.5, 22.9,
24.3, 25.4, 27.8, 34.3, 36.3, 37.5, 67.3, 109.7, 119.6, 123.7, 124.4, 131.0, 134.4, 148.6, 155.4,
168.5. GC-MS (m/z): 315,2 [M+]. Anal. Calcd. for C20H29NO2 : C, 76.15; H, 9.27; N, 4.44;
found: C, 76.18; H, 9.24; N, 4.46.
N-(3,4-dimethoxyphenethyl)-2-(5-isopropyl-2-methylphenoxy)acetamide (5m): Light brown
liquid, 60% yield; IR: 3416, 3359, 2957, 2932, 2869, 2835, 1673, 1612, 1512, 1441, 1417,
1
1381, 1257, 1177, 1130, 1027, 939, 852, 812, 638, 558 cm-1 ; H NMR (CDCl3, 300 MHz)
δ/ppm: 1.21 (6H, d, J=6.7 Hz), 2.06 (3H, s), 2.80 (2H, t, J=6.7 Hz), 2.84-2.86 (1H,m), 3.60-
3.66 (2H, q), 3.83 (3H, s), 3.85 (3H, s), 4.48 (2H, s), 6.61-6.81 (6H, m), 7.0 (1H, d, J=7.6 Hz)
;
13C NMR (CDCl3, 75 MHz) δ/ppm: 16.0, 24.2, 34.2, 35.3, 40.1, 56.0, 56.1, 67.5, 110.0,
111.6, 111.9, 119.7, 120.8, 123.8, 131.0, 148.0, 148.6, 149.4, 155.4, 168.8. GC-MS (m/z):
371,2 [M+]. Anal. Calcd. for C22H29NO4: C, 71.13; H, 7.87; N, 3.77; found: C, 71.15; H,
7.85; N, 3.80.
N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-(5-isopropyl-2-methylphenoxy)acetamide (5n): Light
yellow solid, 74% yield; mp.66.8°C; IR 3307, 2963, 2930, 2876, 2784, 1649, 1534, 1486,
1
1429, 1375, 1239, 1175, 1126, 1007, 926, 824, 771, 716, 676, 578, 455, 419 cm-1; H NMR
(CDCl3, 300 MHz) δ/ppm: 1.21 (6H, d, J=7.0 Hz), 2.19 (3H, s), 2.80-2.87 (1H,m), 4.45 (2H,
d, J=6.1 Hz), 4.55 (2H, s), 5.93 (2H, s), 6.64 (1H, s), 6.74-6.86 (5H, m, NH), 7.07 (1H, d,
J=7.6 Hz) ; 13C NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 24.2, 34.2, 42.9, 67.9, 101.3, 108.4,
108.5, 110.3, 119.9, 121.0, 124.0, 131.1, 131.9, 147.2, 148.7, 155.6, 168.7. GC-MS (m/z):
342.9 [M+]. Anal. Calcd. for C20H23NO4: C, 70.36; H, 6.79; N, 4.10; found: C, 70.38; H,
6.77; N, 4.12.
2-(5-isopropyl-2-methylphenoxy)-N-morpholinoacetamide (5o): Beige solid, 70% yield;
mp.107 °C; IR: 3199, 3064, 2969, 2948, 2864, 2824, 1737, 1666, 1559, 1513, 1460, 1449,
1
1415, 1242, 1106, 1022, 974, 917, 864, 804, 714, 647, 586, 478 cm-1; H NMR (CDCl3, 300
MHz) δ/ppm: 1.21 (6H, d, J=6.5 Hz), 2.25 (3H, s), 2.81-2.89 (5H,m), 3.83-3.86 (4H, m), 4.55
13
(2H, s), 6.64 (1H, s), 6.83 (1H, d, J=8.4 Hz), 7.1 (1H, d, J=7.6 Hz), 7.32 (1H, s, NH) ; C
26

NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 24.2, 34.2, 56.2, 66.4, 67.7, 110.7, 120.1, 123.8, 131.2,
148.8, 155.4, 166.1. GC-MS (m/z): 292,1 [M+]. Anal. Calcd. for C16H24N2O3: C, 65.73; H,
8.27; N, 9.58; found: C, 65.75; H, 8.29; N, 9.55.
2-(5-isopropyl-2-methylphenoxy)-N-(4-methylpiperazin-1-yl)acetamide (5p): Light brown
solid, 68% yield; mp.103 °C; IR: 3207, 3068, 2955, 2932, 2825, 2795, 2745, 1660, 1613,
1
1561, 1455, 1415, 1241, 1178, 1131, 1064, 1014, 971, 811, 706, 588, 461 cm-1; H NMR
(CDCl3, 300 MHz) δ/ppm: 1.21 (6H, d, J=7.0 Hz), 2.23 (3H, s), 2.32 (3H, s), 2.60-2.63
(4H,m), 2.83-2.88 (5H, m), 4.54 (2H, s), 4.65 (1H, s, NH), 6.63 (1H, s), 6.82 (1H, d, J=7.6
Hz), 7.1 (1H, d, J=7.6 Hz); 13C NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 24.2, 34.2, 45.8, 54.2,
55.7, 67.7, 110.1, 120.1, 123.8, 131.1, 148.7, 155.4, 165.9. GC-MS (m/z): 305,2 [M+]. Anal.
Calcd. for C17H27N3O2: C, 66.85; H, 8.91; N, 13.76; found: C, 66.87; H, 8.90; N, 13.74.
2-(5-isopropyl-2-methylphenoxy)-N-(piperidin-1-yl)acetamide (5q): Yellow solid, 74% yield;
mp. 94°C; IR: 3211, 3066, 2942, 2924, 2864, 2811, 1668, 1557, 1513, 1453, 1414, 1240,
1179, 1140, 1128, 1060, 990, 849, 805, 712, 579, 546, 453, 433 cm-1 ; 1H NMR (CDCl3, 300
MHz) δ/ppm: 1.22 (6H, d, J=7.0 Hz), 1.44-1.48 (2H, m), 1.72-1.77 (4H, m), 2.25 (3H, s),
2.80-2.87 (5H, m), 4.54 (2H, s), 6.64 (1H, s), 6.82 (1H, d, J=7.6 Hz), 7.1 (1H, d, J=7.6 Hz),
7.27 (1H, s, NH); 13C NMR (CDCl3, 75 MHz) δ/ppm: 16.2, 23.3, 24.2, 25.3, 34.2, 57.4, 67.7,
110.0, 120.0, 123.8, 131.1, 148.7, 155.5, 165.7. GC-MS (m/z): 290,2 [M+]. Anal. Calcd. for
C17H26N2O2: C, 70.31; H, 9.02; N, 9.65; found: C, 74.33; H, 9.05; N, 9.62.
N-(3-(dimethylamino)propyl)-2-(5-isopropyl-2-methylphenoxy)acetamide
(5r):
Yellow
liquid, 50% yield; IR: 3337, 2957, 2867, 2818, 2770, 1665, 1529, 1512, 1459, 1418, 1248,
1
1177, 1130, 1056, 994, 852, 814, 641, 581, 461 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm:
1.19 (6H, d, J=6.7 Hz), 1.60-1.68 (2H, m), 2.06 (6H, s), 2.22 (3H, s), 2.28-2.32 (2H, m), 2.80-
2.84 (1H,m), 3.39-3.45 (2H, m), 4.48 (2H, s), 6.60 (1H, s), 6.75 (1H, d, J=7.6 Hz), 7.04 (1H,
13
d, J=7.6 Hz), 7.7 (1H, s, NH, br) ; C NMR (CDCl3, 75 MHz) δ/ppm: 16.1, 24.3, 26.2, 34.2,
39.1, 45.5, 58.8, 67.7, 109.9, 119.5, 123.9, 130.9, 148.5, 155.8, 168.8. GC-MS (m/z): 292,1
[M+]. Anal. Calcd. for C17H28N2O2: C, 69.83; H, 9.65; N, 9.58; found: C, 69.80; H, 9.63; N,
9.59.
1-(4-benzylpiperidin-1-yl)-2-(5-isopropyl-2-methylphenoxy)ethanone (5s): White liquid, 86%
yield; IR: 3060, 3024, 2911, 2853, 2771, 2729, 1646, 1512, 1452, 1417, 1367, 1232, 1136,
1056, 991, 826, 748, 697, 634, 591, 461 cm-1; 1H NMR (CDCl3, 300 MHz) δ/ppm: 1.13-1.19
(2H, m), 1.22 (6H, d, J=7.0 Hz), 1.67-1.78 (3H, m), 2.20 (3H, s), 2.50-2.57 (3H, m), 2.81-
2.88 (1H, m), 2.93-3.03 (1H, m), 4.05 (1H, d, J=13.4 Hz), 4.56 (1H, d, J=11.4 Hz), 4.66
(2H, d, J=4.9 Hz), 6.71 (1H, s), 6.77 (1H, d, J=7.6 Hz), 7.05-7.12 (3H, m), 7.17-7.30 (3H, m);
27

13C NMR (CDCl3, 75 MHz) δ/ppm: 16.2, 24.4, 31.9, 32.9, 34.3, 38.4, 42.8, 43.2, 46.0, 68.4,
109.8, 119.1, 124.1, 126.3, 128.6, 129.3, 130.9, 140.1, 148.4, 156.2, 166.7. GC-MS (m/z):
365,2 [M+]. Anal. Calcd. for C24H31NO2 : C, 78.86; H, 8.55; N, 3.83; found: C, 78.84; H,
8.57; N, 3.85.
2-(5-isopropyl-2-methylphenoxy)-N-(pyridin-2-yl)acetamide (5t): Yellow liquid, 56% yield;
IR: 3394, 2957, 2924, 2867, 1698, 1667, 1577, 1521, 1415, 1307, 1256, 1168, 1134, 1056,
1
775, 678, 571, 514 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=7.0 Hz), 2.35
(3H, s), 2.85-2.89 (1H, m), 4.65 (2H, s), 6.70 (1H, s), 6.83 (1H, d, J=7.6 Hz), 7.07-7.14 (2H,
m), 7.74 (1H, t, J=8.4 Hz), 8.26-8.34 (2H, m), 8.96 (1H, s, NH); 13C NMR (CDCl3, 75 MHz)
δ/ppm: 16.3, 24.3, 34.2, 67.9, 110.4, 114.3, 120.1, 120.5, 124.3, 131.2, 138.6, 148.3, 148.6,
150.6, 155.3, 167.4. GC-MS (m/z): 284,1 [M+]. Anal. Calcd. for C17H20N2O2: C, 71.81; H,
7.09; N, 9.85; found: C, 71.83; H, 7.11; N, 9.83.
5-isopropyl-2-methylbenzyl pyrimidin-2-ylcarbamate (5u): White solid, 60% yield; IR: 3144,
3072, 2958, 2919, 2866, 1705, 1582, 1512, 1451, 1403, 1278, 1255, 1227, 1181, 1133, 1074,
1
848, 811, 637, 536, 414 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=7.1 Hz),
2.35 (3H, s), 2.82-2.89 (1H,m), 4.72 (2H, s), 6.71 (1H, s), 6.83 (1H, d, J=7.7 Hz), 7.09-7.15
13
(2H, m), 8.7 (2H, d, J=5.5 Hz), 9.1 (1H, s, NH) ; C NMR (CDCl3, 75 MHz) δ/ppm: 16.3,
24.3, 34.2, 68.1, 110.3, 117.5, 120.2, 124.1, 131.2, 148.7, 155.2, 157.0, 158.8, 166.8. GC-MS
(m/z): 285,1 [M+]. Anal. Calcd. for C16H19N3O2: C, 67.35; H, 6.71; N, 14.73; found: C,
67.37; H, 6.72; N, 14.70.
2-(5-isopropyl-2-methylphenoxy)-N-(quinolin-8-yl)acetamide (5v): Purple solid, 40% yield;
mp. 127.5 °C; IR: 3323, 3046, 2956, 2886, 2866, 1679, 1578, 1535, 1417, 1257, 1173, 1131,
1057, 996, 939, 827, 808, 750, 640, 572, 465, 422 cm-1; 1H NMR (CDCl3, 300 MHz) δ/ppm:
1.25 (6H, d, J=6.7 Hz), 2.57 (3H, s), 2.87-2.92 (1H,m), 4.74 (2H, s), 6.77 (1H, s), 6.84 (1H,
d, J=7.6 Hz), 7.17 (1H, d, J=7.6 Hz), 7.44-7.49 (1H, m), 7.55-7.57 (2H, m), 8.17 (1H, d,
13
J=8.2 Hz), 8.81-8.83(2H, m), 11.2 (1H, s, NH) ; C NMR (CDCl3, 75 MHz) δ/ppm: 16.2,
24.3, 34.3, 67.7, 109.8, 116.8, 119.6, 121.9, 122.3, 124.4, 127.5, 128.1, 131.1, 134.0, 136.4,
139.0, 148.5, 148.6, 155.5, 167.0. GC-MS (m/z): 334,1 [M+]. Anal. Calcd. for C21H22N2O2:
C, 75.42; H, 6.63; N, 8.38; found: C, 75.46; H, 6.65; N, 8.40.
2-(5-isopropyl-2-methylphenoxy)-N-(2-methylquinolin-4-yl)acetamide (5w): White solid,
56% yield; mp. 111°C; IR: 3402, 2956, 2916, 2868, 1706, 1616, 1532, 1498, 1418, 1250,
1
1178, 1128, 1053, 999, 978, 949, 865, 801, 746, 646, 624, 579, 533, 462 cm-1; H NMR
(CDCl3, 300 MHz) δ/ppm: 1.25 (6H, d, J=7.0 Hz), 2.44 (3H, s), 2.75 (3H, s), 2.85-2.94
(1H,m), 4.75 (2H, s), 6.76 (1H, s), 6.90 (1H, d, J=7.6 Hz), 7.18 (1H, d, J=7.6 Hz), 7.32 (1H, t,
28

13
J=8.2 Hz), 7.67-7.75 (2H, q), 8.0 (1H, d, J=8.5 Hz), 8.3 (1H, s), 9.3 (1H, s, NH) ; C NMR
(CDCl3, 75 MHz) δ/ppm: 16.6, 24.3, 26.0, 34.3, 67.8, 110.2, 111.5, 118.4, 118.5, 120.4,
123.6, 126.1, 129.8, 130.0, 131.4, 139.3, 148.6, 149.0, 154.9, 160.4, 167.3. GC-MS (m/z):
348,1 [M+]. Anal. Calcd. for C22H24N2O2: C, 75.83; H, 6.94; N, 8.04; found: C, 75.85; H,
6.96; N, 8.07.
4.3. Anticholinesterase activity assays
Acetyl- (AChE) and butyryl-cholinesterase (BuChE) inhibitory activities of the synthesized
compounds were determined according to Ellman's method. The IC50 was determined by
constructing an absorbance and/or inhibition (%) curve and examining the effect of five
different concentrations. IC50 values were calculated for a given antagonist by determining the
concentration needed to inhibit half of the maximum biological response of the agonist. The
substrates of the reaction were acetylthiocholine iodide and butyrylthiocholine iodide. 5,5'-
dithio-bis(2-nitrobenzoic) acid (DTNB) was used to measure anticholinesterase activity.
Stock solutions of the compounds and galanthamine in methanol were prepared at a
concentration of 4000 μg/mL. Aliquots of 150 μL of 100 mM phosphate buffer (pH 8.0), 10
μL of sample solution and 20 μL AChE (2.476x10-4 U/μL) (or 3.1813x10-4 U/μL BuChE)
solution were mixed and incubated for 15 min at 25oC. 10 μL of DTNB solution was prepared
by adding 2.0 mL of pH 7.0 and 4.0 mL of pH 8.0 phosphate buffers to a mixture of 1.0 mL
of 16 mg/mL DTNB and 7.5 mg/mL NaHCO3 in pH 7.0 phosphate buffers. The reaction was
initiated by the addition of 10 μL acetylthiocholine iodide (or butyrylthiocholine iodide). In
this method, the activity was measured by following the yellow colour produced as a result of
the thio anion produced by reacting the enzymatic hydrolysis of the substrate with DTNB.
Also, methanol was used as a control solvent. The hydrolysis of the substrates was monitored
using a BioTek Power Wave XS at 412 nm.
4.4. In vitro blood brain barrier permeation assay
The Corning Gentest Pre-coated PAMPA Plate System (Cat. No. 353015) was used to
perform permeability assays for novel compounds (Chen et al 2008). In summary, the 96 well
filter plate, pre-coated with lipids, was used as the permeation acceptor and a matching 96
well receiver plate was used as the permeation donor. Compound solutions were prepared by
diluting 10 mM DMSO stock solutions in PBS (in most cases we used a final concentration of
200 μM). The compound solutions were added to the wells (300 μL/well) of the receiver plate
and PBS was added to the wells (200 μL/well) of the pre-coated filter plate. The filter plate
was then coupled with the receiver plate and the plate assembly was incubated at room
29