Synthesis, anticholinesterase activity and molecular modeling study of novel carbamate-substituted thymol/carvacrol derivatives

Article abstract of DOI:10.1016/j.bmc.2016.12.037

New thymol and carvacrol derivatives with the carbamate moiety were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. 5-isopropyl-2-methylphenyl(3-fluorophenyl)carbamate (29) was foun

Full text of DOI:10.1016/j.bmc.2016.12.037

Accepted Manuscript
Synthesis, Anticholinesterase Activity and Molecular Modeling Study of Novel
Carbamate-Substituted Thymol/Carvacrol Derivatives
Belma Zengin Kurt, Isil Gazioglu, Aydan Dag, Ramin Ekhteiari Salmas, Gülru
Kayık, Serdar Durdagi, Fatih Sonmez
PII:
S0968-0896(16)31244-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.037
BMC 13464
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
20 November 2016
22 December 2016
Please cite this article as: Zengin Kurt, B., Gazioglu, I., Dag, A., Ekhteiari Salmas, R., Kayık, G., Durdagi, S.,
Sonmez, F., Synthesis, Anticholinesterase Activity and Molecular Modeling Study of Novel Carbamate-Substituted
Thymol/Carvacrol Derivatives, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.12.037
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Synthesis, Anticholinesterase Activity and Molecular Modeling Study of
Novel Carbamate-Substituted Thymol/Carvacrol Derivatives
Belma Zengin Kurt1, Isil Gazioglu2, Aydan Dag1, Ramin Ekhteiari Salmas3, Gülru Kayık4,
Serdar Durdagi3,*, Fatih Sonmez5,*
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, 34093, Istanbul,
Turkey
2Department of Analytical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, 34093, Istanbul,
Turkey
3Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey
4Department of Chemistry, Istanbul Technical University, Istanbul, Turkey
5Pamukova Vocational High School, Sakarya University, 54900, Sakarya, Turkey
Abstract
New thymol and carvacrol derivatives with the carbamate moiety were synthesized and their
inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were
evaluated. 5-isopropyl-2-methylphenyl(3-fluorophenyl)carbamate (29) was found to be the
most potent AChE inhibitor with IC50 values of 2.22 µM, and 5-isopropyl-2-methylphenyl (4-
fluorophenyl)carbamate (30) exhibited the strongest inhibition against BuChE with IC50 value
of 0.02 µM. Additionally, the result of H4IIE hepatoma cell toxicity assay for compounds 18,
20, 29, 30 and 35 showed negligible cell death. Moreover in order to better understand the
inhibitory profiles of these molecules, molecular modeling studies were applied. Binding
poses of studied compounds at the binding pockets of AChE and BuChE targets were
determined. Predicted binding energies of these compounds as well as structural and
dynamical profiles of molecules at the target sites were estimated using induced fit docking
(IFD) algorithms and post-processing molecular dynamics (MD) simulations methods (i.e.,
Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) approaches).
Keywords: Thymol, Carvacrol, Carbamate, Alzheimer’s Disease, Molecular Docking,
Molecular Dynamics (MD) Simulations, MM-PBSA, Induced Fit Docking (IFD)
*Corresponding authors: serdar.durdagi@med.bau.edu.tr (SD); fsonmez@sakarya.edu.tr (FS)
1

1. Introduction
There is growing evidence that Alzheimer’s disease (AD) is a progressive neurodegenerative
disease, which is among the most extensive form of dementia and affects aged people [1]. AD
is related to a selective loss of cholinergic neurons in the brain and reducing acetylcholine
(ACh) levels [2,3]. The cholinergic hypothesis is one of the AD hypothesis. It proposes that
acetylcholinesterase inhibitors (AChEI) could rise the levels of ACh in AD patients by
inhibiting acetylcholinesterase and, therefore, alleviate some symptoms experienced by AD
patients [4,5].
There are two forms of cholinesterases in the body, i.e., acetylcholinesterase (AChE; EC
3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) [6]. AChE is a hydrolase involved in
the termination of impulse transmission at cholinergic synapses owing to hydrolize the
neurotransmitter ACh in the central and peripheral nervous system. AChEI inhibits the
hydrolysis of ACh improving both the level and of duration of neurotransmitter [7,8]. BuChE
is mainly existed in liver, plasma, and muscle tissues. The scientific researches so far have not
been revealed exactly the pharmacological role of BuChE. It is suggested that BuChE may
have a compensatory role in the transition of the hydrolysis of ACh in brain with degenerative
alterations [9,10].
Carbamates have common biological and pharmacological properties [11]. Additionally,
carbamate-class of the AChEI is well known and used in the cure of a various illnesses
involving AD [12]. Carbamates, such as rivastigmine, neostigmine, phenserine and
physostigmine (Figure 1), easily pass the blood-brain barrier to inhibit cholinesterase in the
central nervous system, spearheading advanced cognition in dementia [13-15]. Therefore, the
synthesis of novel carbamate derivatives as cholinesterase inhibitors has been the focus of
attention of scientists both in academia and in industry.
N
H
H
N
O
N+
N
O
N
O
N
O
N
N
O
O
O
O
N
H
N
H
Neostigmine
Rivastigmine
Phenserine
Physostigmine
Figure 1. The structures of well-known carbamate derivatives used as AChE inhibitors.
2

Many plants and their components have a pivotal role in conventional implementation of
medicine to raise the cognitive purpose and reduce other signs of AD [16,17]. Thymol (2-iso-
propyl-5-methylphenol) and its isomer carvacrol (5-iso-propyl-2-methyl-phenol) are among
the main bioactive monoterpenes isolated from many medicinal herbs such as Thymus
vulgaris and Origanum vulgare [18,19]. It has been found that they possess various
pharmalogical and biological properties, such as antioxidant, antiviral, antifungal, antitumor,
and as well as anti-inflammatory and antibacterial activities [20-23].
According to the literature, we hypothesized that the presence of carbamate moiety
contributes to the inhibitory activity of thymol and carvacrol, which are categorized as
natural products. Therefore, in the current study we synthesized novel carbamate derivatives
substituted thymol/carvacrol (1-38) and not only their inhibitory effects on AChE and BuChE
were evaluated, but also the potential toxicity effect on the hepatoma cell line H4IIE was
examined for the most potent compounds 18, 20, 29, 30 and 35. Moreover molecular
modeling studies were also applied. Molecular docking studies were carried out to clarify the
inhibition mode for the active compounds. Binding poses 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 induced fit docking (IFD) algorithms as well as post-processing molecular
dynamics (MD) simulations methods (i.e., Molecular mechanics Poisson–Boltzmann surface
area (MM-PBSA) approaches).
2. Results and discussion
H
OH
O
N
O
R
or
R N C O
R
O
or
OH
O
N
CH2Cl2, Et3N
reflux, 18h
H
Thymol
Carvacrol
1-19
Thymol
derivatives
20-38
Carvacrol
derivatives
Scheme 1. Synthesis of new thymol/carvacrol substituted carbamate derivatives.
2.1. Chemistry
3

The synthetic procedures are shown in Scheme 1. Thymol or carvacrol reacted with various
isocyanate derivatives in CH2Cl2 to get carbamates (1-38) at acceptable yields. All the novel
compounds were characterized by 1H NMR, 13C NMR, IR, MS and elemental analyses.
2.2. Cholinesterase inhibitory activity
The IC50 values for AChE and BuChE inhibitions are given in Table 1. IC50 values against
AChE and BuChE range from 2.22 µM to 419.64 µM and from 0.02 µM to 405.15 µM,
respectively.
Table 1. In vitro inhibition IC50 values (μM) of 1-38 for AChE and BuChE.
Compound
R
AChE(IC50,μM)a BuChE(IC50,μM)a
1
cyclopentyl
419.64±1.825
na*
167.42±1.342
na
2
cyclohexyl
3
cyclohexanemethyl
cycloheptyl
na
36.79±1.023
na
4
na
5
benzyl
111.39±2.778
156.89±2.326
154.32±1.745
na
1.45±0.0421
96.10±1.221
1.65±0.014
59.96±1.542
9.40±0.425
3.85±0.457
405.15±1.452
2.54±0.568
137.41±2.014
107.22±1.785
4.77±0.748
5.02±0.879
3.31±0.564
37.71±1.022
147.85±1.567
1.01±0.035
1.03±0.056
0.30±0.002
112.20±1.125
0.07±0.001
6
naphtyl
7
3-methoxyphenyl
4-methoxyphenyl
p-tolyl
8
O
9
229.19±2.214
88.52±1.253
269.80±1.124
89.53±1.248
229.46±1.560
98.56±1.263
31.61±0.952
197.99±1.542
82.88±1.224
2.50±0.012
R
O
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3-fluorophenyl
4-fluorophenyl
3.4-dichlorophenyl
4-chlorophenyl
4-bromophenyl
4-iodophenyl
phenyl
H
1-19
Thymol
derivatives
3-nitrophenyl
4-nitrophenyl
4-trifluoromethyl phenyl 98.53±1.114
cyclopentyl
cyclohexyl
3.37±0.231
23.05±1.116
8.08±0.523
125.88±1.330
77.81±1.012
H
O
N
R
O
cyclohexanemethyl
cycloheptyl
benzyl
20-38
Carvacrol
derivatives
4

25
naphtyl
127.26±1.254
2.24±0.322
0.51±0.014
1.64±0.235
0.52±0.041
0.31±0.033
1.02±0.011
0.02±0.002
0.41±0.003
0.13±0.002
0.15±0.011
0.21±0.001
0.55±0.021
0.56±0.112
1.05±0.202
1.23±0.052
226.13±1.214
26
3-methoxyphenyl
4-methoxyphenyl
p-tolyl
27
54.46±1.014
6.17±0.652
28
29
3-fluorophenyl
4-fluorophenyl
3.4-dichlorophenyl
4-chlorophenyl
4-bromophenyl
4-iodophenyl
phenyl
2.22±0.221
3.02±0.124
30
31
86.15±1.032
147.62±1.235
104.03±0.425
137.51±1.212
4.01±0.235
32
33
34
35
36
3-nitrophenyl
4-nitrophenyl
5.05±0.231
37
120.93±1.002
38
4-trifluoromethyl phenyl 85.7±1.141
-
-
-
-
458.23±1.745
288.26±1.112
2.05±0.011
Thymol
410.79±1.238
18.13±0.457
1.00±0.251
Carvacrol
Galantamine
Rivastigmine
12.40±1.011
a IC50 values represent the means ± S.E.M. of three parallel measurements (p< 0.05).
*na: not active.
Among the thymol derivatives (1-19), compound 18 exhibited the strongest inhibition against
AChE with an IC50 value of 2.50 µM, which is 183-fold more than that of thymol (IC50 =
458.23 µM). Compound 18 has higher AChE inhibitory activity compared to that of
rivastigmine (IC50 = 12.40 µM), which is well-known carbamate derivatives as AChE
inhibitor, and it has similar inhibition with standard molecule galantamine (IC50 = 2.051 µM).
Furthermore, most of the synthesized thymol-carbamate derivatives exhibited better AChE
inhibition (IC50 = 2.50 µM – 419.64 µM) than thymol, by 1.09-183 fold. Compound 5
exhibited the strongest inhibition against BuChE with an IC50 value of 1.45 µM, which is 156-
fold more than that of thymol (IC50 = 226.13 µM) and 12.5-fold more than that of galantamine
(IC50 = 18.13 µM), but it shows similar activity with the rivastigmine (IC50 = 1.00 µM).
All the synthesized carvacrol-carbamate derivatives (20-38) revealed significantly higher
inhibition activity in compare with carvacrol against both cholinesterases. Among them,
5

compound 29 exhibited the enhanced inhibition against AChE with an IC50 value of 2.22 µM,
which is 130-fold more than that of carvacrol (IC50 = 288.26 µM). The IC50 value of
compound 30 for BuChE is determined to be 0.02 µM, which is 20500-fold more in compare
to that of carvacrol (IC50 = 410.79 µM), 906-fold more than that of galantamine (IC50 = 18.13
µM), and 50-fold more than that of rivastigmine (IC50 = 1.00 µM).
The following results of the structure-activity relationship should be noted regarding the
cholinesterase inhibitory data of Table 1: In short, most of synthesized carvacrol-carbamate
derivatives are more selective than thymol-carbamates for both cholinesterases.
Carvacrol-carbamates
(i) All the carvacrol-carbamates revealed enhanced inhibition against BuChE in compare with
AChE. (ii) Most of aromatic carbamates had higher BuChE inhibitory activity than that of
cyclic aliphatic carbamates. (iii) Moving the methoxy group at the phenyl ring from the meta-
position to the para-position led to a major decline of the inhibitory activity for AChE
(compare 26 (R=3-OMe, IC50 = 2.24 µM) with 27 (R=4-OMe, IC50 = 54.46 µM)). (iv) The
presence of chlorine atom at the meta-position of phenyl ring led to an increase of the
inhibitory activity for AChE (compare 31 (R=3,4-diCl, IC50 = 86.15 µM) with 32 (R=4-Cl,
IC50 = 147.62 µM)). (v) Moving the nitro group at the phenyl ring from the meta-position to
the para-position led to a significant increase of the inhibitory activity for AChE (compare 36
(R=3-NO2, IC50 = 5.05 µM) and 37 (R=4-NO2, IC50 = 120.93 µM)). (iv) Electron-donating
groups (methoxy) at the meta- and para- positions and methyl group at the para- position of
the phenyl ring exhibited higher inhibitory activity compared to electron-withdrawing groups
(nitro and trifluoromethyl) for AChE (compare 26 (R=3-OMe, IC50 = 2.24 µM) with 36 (R=3-
NO2, IC50 = 5.05 µM)); and (compare 27 (R=4-OMe, IC50 = 54.46 µM) and 28 (R=4-Me, IC50
= 6.17 µM) with 37 (R=4-NO2, IC50 = 120.93 µM) and 38 (R=4-CF3, IC50 = 85.7 µM),
respectively).
Thymol-carbamate derivatives
(i) Most of aromatic carbamates are significantly selective for both cholinesterases compared
to that of cyclic aliphatic carbamates. (ii) Moving the fluorine atom and methoxy group at the
phenyl ring from the meta- to the para-position leds to a major decline of the inhibitory
activity for both cholinesterases (compare 10 (R=3-F, IC50 = 88.52 and 3.85 µM for AChE
and BuChE, respectively) with 11 (R=4-F, IC50 = 269.80 and 405.15 µM for AChE and
6

BuChE, respectively) and (compare 7 (R=3-OMe, IC50 = 154.32 and 1.65 µM for AChE and
BuChE, respectively) with 8 (R=4-OMe, IC50 = not active and 59.96 µM for AChE and
BuChE, respectively). (iii) The presence of chlorine atom at the meta-position of phenyl ring
led to an increase of the inhibitory activity for both cholinesterases (compare 12 (R=3,4-diCl,
IC50 = 89.53 and 2.54 µM for AChE and BuChE, respectively) with 13 (R=4-Cl, IC50
=
229.46 and 137.41 µM for AChE and BuChE, respectively). (iv) Moving the nitro group at
the phenyl ring from the meta-position to the para-position led to a major increase of the
inhibitory activity for AChE (compare 17 (R=3-NO2, IC50 = 82.88 µM) and 18 (R=4-NO2,
IC50 = 2.50 µM). The opposite effect was observed for inhibition of BuChE (compare 17
(R=3-NO2, IC50 = 3.31 µM) and 18 (R=4-NO2, IC50 = 37.71 µM)) (v) Electron-donating
groups (methoxy) at the meta- and para- position and methyl group at the para- position of the
phenyl ring exhibited lower inhibitory activity compared to electron-withdrawing groups
(nitro and trifluoromethyl) for AChE (compare 7 (R=3-OMe, IC50 = 154.32 µM) with 17
(R=3-NO2, IC50 = 82.88 µM) and (compare 8 (R=4-OMe, IC50 = not active) and 9 (R=4-Me,
IC50 = 229.19 µM) with 18 (R=4-NO2, IC50 = 2.50 µM) and 19 (R=4-CF3, IC50 = 98.53 µM).
On the contrary, methoxy group at the meta- position and methyl group at the para- position
increased the BuChE inhibitory activity (compare 7 (R=3-OMe, IC50 = 1.65 µM) with 17
(R=3-NO2, IC50 = 3.31 µM) and (compare 9 (R=4-Me, IC50 = 9.40 µM) with 18 (R=4-NO2,
IC50 = 37.71 µM) and 19 (R=4-CF3, IC50 = 147.85 µM)). (vi) The inhibitor activity on both
cholinesterases seems to be strongly dependent on the size and polarizability of the halogen
substituent at the para-position of the phenyl ring (for size and polarizability, I > Br > Cl > F;
for AChE inhibitory activity, 15 (R=4-I, IC50 = 31.61 µM) > 14 (R=4-Br, IC50 = 98.56 µM) >
13 (R=4-Cl, IC50 = 229.46 µM) > 11 (R=4-F, IC50 = 269.80 µM); for BuChE inhibitory
activity, 15 (R=4-I, IC50 = 4.77 µM) > 14 (R=4-Br, IC50 = 107.22 µM) > 13 (R=4-Cl, IC50 =
137.41 µM) > 11 (R=4-F, IC50 = 405.15 µM). This relationship was not observed for
carvacrol-carbamate derivatives.
Carbamates carbamylate the catalytic Serine and the carbamylated Serine is slowly
hydrolysed to reactivate enzyme, so that the inhibition of AChE using carbamates is also
slowly reversible or pseudoirreversible [24]. Several studies have studied that carbamates
such as rivastigmine and physostigmine derivatives form strong hydrophobic interactions with
the aromatic ring and also a hydrogen bond has been established between a carbamate moiety
and the active site of AChE [25,26].
7

It is observed that most of synthesized carvacrol-carbamate derivatives have higher
cholinesterase inhibitory activity compared to thymol-carbamates, because the steric effect of
the isopropyl group on carbamate moiety is smaller than the methyl group. The mesomeric
electron donation of NH- moiety of carbamate increase the electron density of orto/para
position more than that of meta position. On the other hand the substituents (methoxy, fluoro,
and choloro at both thymol and carvacrol derivatives) at meta can enhance electron density at
this position by the mesomeric electron donation. This increasing electron density may affect
inhibition of AChE with contribution to hydrophobic interactions.
2.3. Molecular Docking
Synthesized molecules are docked to the binding sites of AChE and BuChE targets using
flexible docking algorithm, induced fit docking (IFD) approach. The binding energies of
compounds within the catalytic domain of targets were estimated by molecular mechanics
methods, as shown in Table 2. All the synthesized compounds and well-known AChE
inhibitors produced elevated effect against the both proteins. The top IFD scores were
considered for the individual compounds, in which some overestimation respect to the values
obtained by the in vitro tests was observed. Figure 2 shows top-docking poses of compounds
29 and 30 at the binding pockets of AChE and BuChE, respectively. The IFD scores of the
compounds 29 and 30 in complexes with AChE and BuChE calculated were -9.88 kcal/mol
and -9.89 kcal/mol, respectively. This may be due to the use of coordinate files of target
structures (X-ray structures) from human organism in molecular modeling studies. Whereas in
vitro cholinesterase studies do not use tissues from human. Experimental tests show that
compounds 2, 3, 4 and 8 are inactive molecules. However, the IFD calculations were not able
to reproduce these observations. Thus, more sophisticated binding free energy calculations
were considered. For this aim, we performed molecular dynamics (MD) simulations and post-
processing MD analyses for derived trajectory frames. Tight-binding compounds revealed a
significant degree of shape complementary into the active site of the proteins where they were
docked. This can be observed by generating the 3D poses of the MD representative trajectory
frames (i.e., the frame that has the smallest RMSD to the average coordinate) as shown in
Figure 3. Figure 3 shows electron densities around the ligands, the crucial amino acids at the
binding pocket within the 4 Å of the compounds and the explicit hydrogen bonds established
into the cavities were illustrated.
8

Table 2. Predicted binding energies (kcal/mol) of the compounds, calculated by IFD.
Compound
AChE
-9.60
-10.32
-9.70
-10.64
-10.81
-9.82
-9.89
-8.94
-8.09
-9.88
-10.05
-9.89
-9.36
-10.08
-9.74
-9.38
-10.09
-10.12
-9.93
-9.45
-9.60
BuChE
-8.09
-8.86
-7.97
-9.17
-8.44
-9.73
-8.31
-8.25
-8.34
-9.97
-8.87
-9.06
-9.12
-8.52
-8.40
-8.79
-8.58
-8.02
-9.54
-7.79
-8.24
Compound
AChE
-9.81
-9.72
-9.62
-10.90
-10.08
-8.29
-10.59
-9.88
-9.77
-10.05
-9.21
-9.53
-9.14
-9.84
-10.06
-9.20
-9.54
-9.49
-8.75
-12.68
-11.06
BuChE
-8.67
-8.58
-8.83
-10.12
-8.27
-8.17
-9.20
-10.64
-9.89
-9.68
-9.57
-9.64
-9.16
-8.36
-8.60
-7.80
-10.41
-6.19
-6.84
-7.38
-5.72
1
2
22
23
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Thymol
Carvacrol
Galantamine
Rivastigmine
Figure 2. 2D ligand interaction diagrams of top-docking poses of 29 (left) and 30 (right) at
the active sites of AChE and BuChE, respectively.
9

Figure 3. MD representative 3D poses of the compounds 28 (left) and 29 (right) in complexes
with AChE and BuChE, in which the amino acid within 4 Å of the ligands are illustrated and
the explicit hydrogen bonds established between the water molecules and complexes are
inscribed.
2.4. Ligand-Protein Interaction Analysis
Herein, we attempted to understand the structural and dynamical profiles of the compounds of
interest and their intermolecular complex interactions. For this aim, complexes of two
selected active compounds (28 and 29) with AChE and BuChE were submitted to the MD
simulations. The initial configurations of the complexes were retrieved from the docking
simulations and the simulations were started from these modes. Explicit hydrogen bond,
hydrophobic and water mediated interactions, which were established between the ligands and
the active site amino acids along the MD simulations were identified and their interactions
occupancies were plotted in Figure 4. Compound 28 into the AChE revealed high affinity
toward Tyr341 and Trp286 by hydrophobic interactions and Tyr124 and Phe295 via hydrogen
bonding interactions. In addition, Phe295 and Tyr341 formed water-mediated hydrogen bond
interactions with the ligand. In the case of the compound 29 in complex with BuChE, Trp82
was significantly pronounced via hydrophobic interaction. His438 engaged in a hydrogen
bond interaction with the central nitrogen atom of the compound with an occupancy level of
87%. Both Gly116 and Gly197 contributed in water mediated interactions to stabilize the
ligand. This phenomenon can demonstratively show the significant role of the water
molecules in inhibition effect of this compound.
10

Figure 4. Per-residues analysis of the compounds 28 (left) and 29 (right) in complexes with
AChE and BuChE. The analysis was based throughout the 50-ns MD simulations. Hydrogen
bond and π-π stacking interactions are illustrated by red and green lines, respectively.
2.5. Thermodynamic Properties
The free energy is considered to be an important term in thermodynamic properties. This
quantity is mainly expressed as Gibbs free energy. MD trajectory frames were used to carry
out an energetic calculation by Molecular Mechanics Poisson–Boltzmann surface area (MM-
PBSA), which is introduced as a more efficient approach for the thermodynamic assays. The
main subterms of the free energy were estimated for the both complexes, as shown in Table 3.
All the energetic components were determined with errors of less than 1.20 kcal/mol. The
ΔGtotal values for forming of the 28-AChE and 29-BuChE complexes were favourable and
calculated to be -8.75 kcal/mol and -9.24 kcal/mol, respectively. The results were
overestimated respect to the experiments, but sharing a reasonable qualitative agreement with
the in vitro tests. The both systems are entropically unfavourable, which is associated to the
tight bondings and conformational stabilities of the compounds into the hydrophobic-based
11

cavities. The term ΔEvdw which was calculated to be 45.95 kcal/mol and -39.75 kcal/mol for
the compounds 28 and 29, respectively, made a significant contribution to the forming of the
inter- and intra-molecular interactions in the systems. One of the term of the interest to
biologists involves solvation free energy (ΔGsol), by which the free energy change to transfer
the solute from solvent to cavity can be determined. The ΔGsol components were predicted as
38.75 kcal/mol and 33.03 kcal/mol for the compounds 28 and 29, respectively. The values are
positive and involve the entropic penalty, which is associated to the reorientations of the
solvation molecules around the compounds. The solvation energy in turn can be divided into a
polar and non-polar solvation energies. The polar term is an electrostatic component, which is
very important for the charged molecules due to local polarisation of the solvent and modelled
by dielectric constant ε. By contrast, the non-polar solvation energy expresses non-polar
solvation forces, which can be described by non-polar implicit solvent model. The only main
approximation with the solvation energy calculation is the need to implementation of proper
solvation model. In addition to the above components, the calculated enthalpy energy
differences were determined as -15.99 kcal/mol and -12.69 kcal/mol for the compounds 28
and 29, respectively. The energetic calculations conclusively revealed very satisfactory
agreement with the experimental results.
2.6. Conformational Stability
The biophysical and chemical characteristics of a compound depend mainly upon the
conformations, which it adopts. In this section the objective of conformational analysis is to
measure diffuseness and structural stability of the ligands into the active site, in which root-
mean-square deviations (RMSDs) of heavy atoms for the compounds with respect to the their
initial positions as a function of time are calculated as shown in Figure 5. The conformational
analysis are performed by two different modes, in which ‘Fit on Protein’ mode (ProFit)
represents the RMSD of the compound when the system was first aligned with respect to the
Cα atoms. This provides us to monitor the diffusion and movement degrees of the ligands into
the active site. ‘Fit on Ligand’ mode (LigFit) explores the RMSD of the ligand when system
was first aligned on the heavy atoms of the ligand; hence, this result is referred to the
conformational spaces of the ligands. The compounds 28 and 29 were diffusing into binding
cavities of the AChE and BuChE, respectively with a mean RMSD values of 3.62 and 2.43 Å
and falling into different conformations with a mean RMSD of 1.02 and 0.96 Å, respectively.
As the results suggest, both compounds seems to be stabilized into biding pocket, it can be
12

due to the tight intermolecular interactions, form the complexes, as discussed in the previous
section. The results obtained from the LigFit modes obviously gave very satisfactory
agreements with the entropic terms as predicted by MM-PBSA assays. Both compounds
comprise the entropic penalties, connected to the low reorientation and flexibility of the
molecules into the binding pocket.
Table 3. Energetic analysis of the compounds 28 and 29 in complexes with AChE and BuChe,
respectively, as obtained by MM-PBSA calculations.
Energy (kcal/mol)
Complex
28-AChE
29-BuChE
ΔEvdw
-45.95 ± 0.69
-39.75 ± 0.47
ΔEelec
ΔEMM
ΔGPB
ΔGNP
ΔGsolv
-8.78 ± 1.17
-54.74 ± 0.90
42.81 ± 1.10
-4.06 ± 0.02
38.75 ± 1.08
43.03
-5.97 ± 0.38
-45.73 ± 0.42
36.64 ± 0.63
-3.61 ± 0.07
33.03 ± 0.63
30.67
a
ΔGelec(tot)
ΔH
-TΔS
-15.99 ± 0.94
7.24
-12.69 ± 0.67
3.45
ΔGMM-PBSA
-8.75
-9.24
ΔGexperiment (current study)
aΔGelec(tot) = ΔEelec + ΔGPB
-7.14
-8.22
Figure 5. RMSDs of heavy atoms of the compounds 28 (left) and 29 (right) with respect to
their starting positions along the MD simulations.
13

2.7. H4IIE hepdatoma cell toxicity
On basis of the screening results above, the most potent compounds 18, 20, 29, 30, 35 were
selected to further examine the potential toxicity effect on the hepatoma cell line H4IIE. After
exposing the cells to this compound for 24 h, the cell viability was evaluated by the 3-(4,5-
dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) assay. The result indicated that 18, 20,
29, 30, 35 caused negligible cell death and were nontoxic to H4IIE cell at 0.07-10 μM (Figure
6). The result indicated that 18, 20, 29, 30, 35 caused negligible cell death and was non-toxic
to H4IIE cell at 0.07–10 μM (cell viability; for 18; 0.07 μM: 97.8%, 0.15 μM: 57.8%, 0.3 μM:
54.4%, 0.6 μM: 51.7%, 1.25 μM: 47.2%, 2.5 μM: 40%, 5 μM: 32.8%, 10 μM: 26.1%, for 20;
0.07 μM: 94.4%, 0.15 μM: 75.6%, 0.3 μM: 71.1%, 0.6 μM: 63.3%, 1.25 μM: 50.4%, 2.5 μM:
47.8%, 5 μM: 38.9%, 10 μM: 37.4%, for 29; 0.07 μM: 98.1%, 0.15 μM: 90%, 0.3 μM: 74.1%,
0.6 μM: 64.4%, 1.25 μM: 47.8%, 2.5 μM: 45.8%, 5 μM: 37.2%, 10 μM: 28.5% , for 30; 0.07
μM: 92.8%, 0.15 μM: 69.4%, 0.3 μM: 68.9%, 0.6 μM: 65.9%, 1.25 μM: 62.2%, 2.5 μM:
41.6%, 5 μM: 32.8%, 10 μM: 28.5%, for 35; 0.07 μM: 93.8%, 0.15 μM: 87.9%, 0.3 μM: 90%,
0.6 μM: 82.5%, 1.25 μM: 60%, 2.5 μM: 43.3%, 5 μM: 25.8%, 10 μM: 26.%). Increasing
concentrations of the selected compounds from 0.07 μM to 2.5 μM significantly diminished
cell viability%, but it was almost fixed at 26-30% for 5 and 10 μM of concentrations.
Figure 6. H4IIE cell cells were treated with 0.07-10 μM of various compound for 24 h.
14

3. Conclusions
A series of 38 novel carbamate substituted thymol/carvacrol derivatives (1-38) was
synthesized and their inhibitory activities on AChE and BuChE were evaluated. Among the
thymol derivatives (1-19), compound 18 showed the strongest inhibition against AChE with
an IC50 value of 2.50 µM, which is 183-fold active more compared to that of thymol (IC50 =
458.23 µM). Among the carvacrol derivatives, compound 29 exhibited the strongest inhibition
against AChE with an IC50 value of 2.22 µM, which is 130-fold more active compared to that
of carvacrol (IC50 = 288.26 µM). The structure-activity relationship study showed that the
positions, types and bulks of the substituents affected the inhibitions of ChEs differently.
Moreover, the result of H4IIE hepatoma cell toxicity assay indicated that the most potent
compounds 18, 20, 29, 30 and 35 caused negligible cell death and were nontoxic at 0.07-10
μM. Increasing concentrations of the selected compounds from 0.07 μM to 2.5 μM
significantly diminished cell viability%, but it was almost fixed at 26-30% for 5 and 10 μM of
concentrations. 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. Chemistry. General information
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
13
spectra were registered on a Bruker-Alpha infrared spectrometer. H and C 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 MICROMASS Quattro LC-MS-MS 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. General procedures of synthesis and spectral data
15

A mixture of isocyanate derivative (1 mmol) and thymol/carvacrol (1 mmol) in CH2Cl2 (30
mL) containing triethyl amine (0.5 mL) was refluxed for 18 h. The solution was washed with
water and then, organic phase was dried with Na2SO4, filtered, and evaporated. The
precipitated product was recrystallized from ethanol.
2-isopropyl-5-methylphenyl cyclopentylcarbamate (1): Purity: 98.89%, White powder, 57%
yield; mp. 105-107 °C; IR: 3307, 2956, 2868, 1733, 1703, 1622, 1531, 1500, 1225, 1151,
1
1090, 990, 811 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm:1.23 (6H, d, J= 14.0 Hz), 1.28-1.59
(2H, m), 1.61-1.74 (4H, m), 2.0-2.04 (2H, m), 2.3 (3H, s), 3.01-3.10 (1H, m), 4.02-4.09 (1H,
m), 4.98 (1H, s, NH), 6.88 (1H, s), 6.98 (1H, d, J=7.6 Hz), 7.15 (1H, d, J=7.6 Hz ); 13C NMR
(CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 23.8, 27.2, 33.4, 53.2, 123.3, 126.4, 126.8, 136.5, 137.7,
148.3, 154.6. LC-MS (m/z): 284.3 [M+]. Anal. Calcd. for C16H23NO2: C, 73.53; H, 8.87; N,
5.36; found: C, 72.50; H, 8.71; N, 5.44.
2-isopropyl-5-methylphenyl cyclohexylcarbamate (2): Purity: 99.19%, White powder, 61%
yield; mp. 124-126 °C; IR: 3292, 2931, 2854, 1734, 1702, 1533, 1490, 1248, 1154, 1016, 812
1
cm-1; H NMR (CDCl3, 300 MHz) δ/ppm:1.19 (6H, d, J= 7.0 Hz), 1.26-1.35 (2H, m), 1.39-
1.44 (2H, m), 1.54-1.64 (2H, m), 1.72-1.76 (2H,m), 2.01 (2H, d, J=9.0 Hz), 2.3 (3H, s), 3.01-
3.10 (1H, m), 3.55-3.57 (1H, m), 4.90 (1H, d, J= 6.7 Hz, NH), 6.87 (1H, s), 6.98 (1H, d, J=7.6
Hz), 7.15 (1H, d, J=7.6 Hz ); 13C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.2, 25.0, 25.7, 27.5,
33.5, 50.3, 123.3, 126.4, 126.8, 136.5, 137.7, 148.3, 154.2. LC-MS (m/z): 298.4 [M+]. Anal.
Calcd. for C17H25NO2: C, 74.14; H, 9.15; N, 5.09; found: C, 73.65; H, 8.99; N, 4.93.
2-isopropyl-5-methylphenyl (cyclohexylmethyl)carbamate (3): Purity: 99.96%, White
powder, 20% yield; mp. 84-86 °C; IR: 3294, 2924, 2851, 1733, 1703, 1541, 1506, 1231,
1
1154, 1059, 993, 813 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 0.91-1.02 (2H, m), 1.14-
1.32 (2H, m), 1.19 (6H, d, J= 7.0 Hz), 1.49-1.58 (2H, m), 1.68-1.78 (4H, m), 2.3 (3H, s),
3.02-3.19 (3H, m), 5.0 (1H, s, NH), 6.87 (1H, s), 6.98 (1H, d, J=7.9 Hz), 7.15 (1H, d, J=7.9
Hz ); 13C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 26.0, 26.6, 27.2, 30.8, 38.4, 47.7, 123.3,
126.4, 126.8, 136.6, 137.7, 148.3, 155.2. LC-MS (m/z): 312.3 [M+]. Anal. Calcd. for
C18H27NO2: C, 74.70; H, 9.40; N, 4.84; found: C, 74.74; H, 9.41; N, 4.83.
2-isopropyl-5-methylphenyl cycloheptylcarbamate (4): Purity: 99.94%, White powder, 66%
yield; mp. 121-123 °C; IR: 3302, 2923, 2854, 1734, 1703, 1528, 1505, 1449, 1229, 1154,
1
1094, 988, 809 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.19 (6H, d, J= 6.7 Hz), 1.31-1.65
(10H, m), 1.87-2.23 (2H, m), 2.3 (3H, s), 3.01-3.10 (1H, m), 3.75 (1H, s, br), 4.99 (1H, d,
16

J=7.4 Hz), 6.87 (1H, s), 6.97 (1H, d, J=7.6 Hz), 7.15 (1H, d, J=7.9 Hz ); 13C NMR (CDCl3, 75
MHz) δ/ppm: 21.1, 23.3, 24.1, 24.3, 27.2, 28.2, 28.3, 35.4, 35.9, 51.4, 52.6, 123.4, 126.4,
126.8, 136.5, 137.7, 148.3, 154.2. LC-MS (m/z): 312.3 [M+]. Anal. Calcd. for C18H27NO2: C,
74.70; H, 9.40; N, 4.84; found: C, 75.45; H, 9.31; N, 4.79.
2-isopropyl-5-methylphenyl benzylcarbamate (5): Purity: 99.21%, White powder, 71% yield;
1
mp. 85-87 °C; IR: 3302, 3060, 2955, 1735, 1707, 1542, 1504, 1229, 1156, 1024, 815cm-1; H
NMR (DMSO-d6, 300 MHz) δ/ppm: 1.12 (6H, d, J= 6.7 Hz), 2.24 (3H, s), 2.98-3.02 (1H, m),
4.25 (2H, d, J=6.1 Hz), 6.82 (1H, s), 6.97 (1H, d, J=7.9 Hz), 7.16-7.37 (6H, m), 8.31 (1H, t,
J=6.1 Hz ); 13C NMR (DMSO-d6, 75 MHz) δ/ppm: 21.0 23.6, 27.1, 44.6, 123.9, 126.7, 126.9,
127.6, 128.9, 129.0, 136.5, 137.9, 140.1, 148.7, 155.7. LC-MS (m/z): 284,3 [M+]. Anal.
Calcd. for C18H21NO2: C, 76.29; H, 7.47; N, 4.94; found: C, 75.76; H, 7.32; N, 4.84.
2-isopropyl-5-methylphenyl naphthalen-1-ylcarbamate (6): Purity: 99.3%, White powder,
85% yield; mp. 165-166 °C; IR: 3235, 3048, 2958, 1717, 1698, 1541, 1498, 1213, 1150,
1
1024, 988, 815 cm-1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 1.24-1.55 (6H, s, br), 2.25 (3H,
s), 3.17 (1H, m), 6.99-7.05 (2H, m), 7.20-7.24 (1H, m), 7.33 (1H, s, br), 7.46-7.61 (3H, m),
13
7.70 (1H, d, J=7.9 Hz), 7.85-7.98 (3H, m); C NMR (CDCl3, 75 MHz) δ/ppm: 21.1, 23.4,
27.3, 123.2, 126.0, 126.3, 126.6, 127.3, 129.3, 132.4, 134.3, 136.8, 137.7, 148.3. LC-MS
(m/z): 342.2 [M+]. Anal. Calcd. for C21H21NO2: C, 78.97; H, 6.63; N, 4.39; found: C, 78.39;
H, 6.59; N, 4.30.
2-isopropyl-5-methylphenyl (3-methoxyphenyl)carbamate (7): Purity: 99.97%, White powder,
42% yield; mp. 85-86 °C; IR: 3325, 3040, 2963, 1754, 1685, 1599, 1542, 1457, 1219, 1014,
1
814 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=6.7 Hz), 2.30 (3H, s), 3.08-3.13
(1H, m), 3.77 (3H, s), 6.64 (1H, dd, J=2.3, 8.1 Hz), 6.89-6.92 (2H, m), 7.01-7.11 (2H, m),
13
7.14-7.23 (3H, m); C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 27.2, 55.5, 110.0, 123.2,
126.6, 127.4, 130.0, 136.8, 137.7, 139.0, 147.7, 151.2, 160.5. LC-MS (m/z): 349.3 [M+]. Anal.
Calcd. for C18H21NO3: C, 72.22; H, 7.07; N, 4.68; found: C, 72.20; H, 7.09; N, 4.66.
2-isopropyl-5-methylphenyl (4-methoxyphenyl)carbamate (8): Purity: 98.45%, White powder,
60% yield; mp. 91-92 °C; IR: 3330, 3040, 2960, 1733, 1712, 1607, 1542, 1508, 1411, 1212,
1
1020, 812 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.20 (6H, d, J=6.7 Hz), 2.30 (3H, s),
3.09-3.15 (1H, m), 3.76 (3H, s), 6.81-6.86 (2H, m), 6.92 (1H, s), 6.99-7.06 (2H, m), 7.19
13
(1H, d, J=7.9 Hz), 7.33 (2H, d, J=8.4 Hz) ; C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.3,
27.2, 55.7, 114.5, 120.7, 121.6, 123.3, 126.3, 126.6, 127.2, 136.8, 137.8, 147.9, 152.9, 156.3.
17

LC-MS (m/z): 322.2 [M+]. Anal. Calcd. for C18H21NO3: C, 72.22; H, 7.07; N, 4.68; found: C,
71.05; H, 6.85; N, 4.53.
2-isopropyl-5-methylphenyl p-tolylcarbamate (9): Purity: 99.21%, White powder, 66% yield;
mp. 118-119 °C; IR: 3310, 3052, 2960, 1737, 1684, 1540, 1457, 1384, 1237, 1204, 1019, 826
1
cm-1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 1.14 (6H, d, J= 6.7 Hz), 2.23(3H, s), 2.26 (3H,
s), 3.01-3.06 (1H, m), 6.92 (1H, s), 6.95-7.11 (3H, m), 7.21 (1H, d, J=7.9 Hz), 7.33 (1H, d,
J=8.2 Hz ), 7.39 (1H, d, J=8.2 Hz ), 10.14 (1H, s); 13C NMR (CDCl3, 75 MHz) δ/ppm: 21.0,
21.1, 23.0, 23.4, 27.2, 119.0, 120.1, 123.3, 126.6, 127.3, 128.6, 129.5, 130.1, 133.5, 135.2,
136.8, 137.8, 147.9. LC-MS (m/z): 284.3 [M+]. Anal. Calcd. for C18H21NO2: C, 76.29; H,
7.47; N, 4.94; found: C, 75.76; H, 7.32; N, 4.84.
2-isopropyl-5-methylphenyl (3-fluorophenyl)carbamate (10): Purity: 98.66%, White powder,
73% yield; mp. 102-103 °C; IR: 3307, 3040, 2961, 1733, 1714, 1608, 1541, 1266, 1219,
1
1023, 870, 773 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.20 (6H, d, J= 6.7 Hz), 2.30 (3H,
s), 3.04-3.13 (1H, m), 6.79 (1H, t, J=3.2 Hz ), 6.91 (1H, s), 7.00-7.13 (2H, m), 7.16-7.28 (3H,
13
m), 7.35 (1H, d, J=10.8 Hz); C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 27.2, 106.2-
106.5 (d, JC,F=22.4 Hz, CF), 110.6, 114.2-114.4 (d, JC,F=21.8 Hz, CF), 123.4, 126.4, 127.5,
130.5, 136.9, 137.7, 139.2, 147.6, 152.1, 161.8-165.0 (d, JC,F=244.9 Hz, CF). LC-MS (m/z):
310.3 [M+]. Anal. Calcd. for C17H18FNO2: C, 71.06; H, 6.31; N, 4.87; found: C, 69.92; H,
6.12; N, 4.72.
2-isopropyl-5-methylphenyl (4-fluorophenyl)carbamate (11): Purity: 99.90%, White powder,
73% yield; mp. 115-116 °C; IR: 3343, 3060, 2967, 1740, 1717, 1616, 1543, 1436, 1203,
1
1025, 828 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.20 (6H, d, J= 6.7 Hz), 2.31 (3H, s),
3.07-3.12 (1H, m), 6.92 (1H, s ), 6.98-7.04 (3H, m), 7.14-7.21 (2H, m), 7.33-7.41 (2H, m);
13C NMR (CDCl3, 75 MHz) δ/ppm: 21.1, 23.4, 27.3, 115.9-116.2 (d, JC,F=22.6 Hz, CF),
116.6-116.9 (d, JC,F=23.2 Hz, CF), 120.6, 123.2, 126.7, 127.4, 130.5, 133.8, 136.9, 137.7,
147.7, 152.4, 164.7 (s, JC,F=not detected, CF). LC-MS (m/z): 310.2 [M+]. Anal. Calcd. for
C17H18FNO2: C, 71.06; H, 6.31; N, 4.87; found: C, 71.08; H, 6.30; N, 4.87.
2-isopropyl-5-methylphenyl (3,4-dichlorophenyl)carbamate (12): Purity: 99.61%, White
powder, 72% yield; mp. 115-117 °C; IR: 3386, 3060, 2958, 1756, 1716, 1585, 1521, 1474,
1
1382, 1204, 1019, 858, 826 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J= 6.7
Hz), 2.31 (3H, s), 3.04-3.11 (1H, m), 6.92 (1H, s ), 7.04 (1H, d, J= 7.9 Hz ), 7.18-7.25 (2H,
13
m), 7.27-7.33 (2H, m), 7.60 (1H, d, J= 2.1 Hz); C NMR (CDCl3, 75 MHz) δ/ppm: 21.0,
18

23.3, 27.3, 118.3, 120.7, 123.0, 126.8, 127.2, 127.6, 130.7 133.1, 137.0, 137.3, 137.6, 147.5,
152.2. LC-MS (m/z): 360.2 [M+]. Anal. Calcd. for C17H17Cl2NO2: C, 60.37; H, 5.07; N, 4.14;
found: C, 60.35; H, 5.08; N, 4.11.
2-isopropyl-5-methylphenyl (4-chlorophenyl)carbamate (13): Purity: 99.98%, White powder,
81% yield; mp. 133-134 °C; IR: 3310, 3062, 2960, 1738, 1712, 1600, 1536, 1280, 1216,
1
1092, 1012, 827 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.20 (6H, d, J= 6.7 Hz), 2.30 (3H,
s), 3.04-3.11 (1H, m), 6.91 (1H, s ), 7.03 (1H, d, J= 8.2 Hz), 7.14 (1H, s, NH), 7.18-7.24
(3H, m), 7.35 (1H, d, J= 8.4 Hz); 13C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 27.2, 120.1,
123.1, 126.7, 127.5, 129.0, 129.3, 136.4, 136.9, 137.7, 147.7, 152.1. LC-MS (m/z): 344.4
[M+]. Anal. Calcd. for C17H18ClNO2: C, 67.21; H, 5.97; N, 4.61; found: C, 67.20; H, 5.95; N,
4.63.
2-isopropyl-5-methylphenyl (4-bromophenyl)carbamate (14): Purity: 99.91%, Red powder,
76% yield; mp. 172-173 °C; IR: 3334, 3064, 2957, 1750, 1712, 1596, 1537, 1396, 1217,
1
1151, 1025, 825, 814 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.20 (6H, d, J=7.0 Hz), 2.32
(3H, s), 3.04-3.11 (1H, m), 6.91 (1H, s ), 7.04 (2H, d, J= 7.9 Hz), 7.20 (1H, d, J=7.9 Hz),
13
7.33 (2H, d, J=8.7 Hz), 7.42 (2H, d, J= 6.7 Hz); C NMR (CDCl3, 75 MHz) δ/ppm: 21.0,
23.3, 27.2, 120.3, 123.1, 126.7, 127.5, 132.3, 136.9, 137.7, 147.6, 152.0. LC-MS (m/z): 372.3
[M+]. Anal. Calcd. for C17H18BrNO2: C, 58.63; H, 5.21; N, 4.02; found: C, 58.60; H, 5.22; N,
4.04.
2-isopropyl-5-methylphenyl (4-iodophenyl)carbamate (15): Purity: 98.56%, White powder,
74% yield; mp. 181-182 °C; IR: 3304, 3064, 2956, 1733, 1710, 1577, 1532, 1393, 1218,
1
1117, 1025, 824 cm-1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 1.13 (6H, d, J=6.7 Hz), 2.25
(3H, s), 2.98-3.03 (1H, m), 6.93 (1H, s ), 7.02 (2H, d, J= 7.9 Hz), 7.22 (1H, d, J=7.6 Hz),
13
7.33 (2H, d, J=8.7 Hz), 7.64 (2H, d, J= 8.7 Hz), 10.37 (1H, s); C NMR (DMSO-d6, 75
MHz) δ/ppm: 21.0, 23.6, 27.1, 87.0, 117.1, 121.1, 123.8, 127.0, 127.5, 136.8, 137.9, 138.2,
147.9, 152.5. LC-MS (m/z): 418.1 [M+]. Anal. Calcd. for C17H18INO2: C, 51.66; H, 4.59; N,
3.54; found: C, 50.92; H, 4.49; N, 3.46.
2-isopropyl-5-methylphenyl phenylcarbamate (16): Purity: 99.98%, White powder, 54%
yield; mp. 106-107 °C; IR: 3331, 3060, 2963, 1734, 1710, 1602, 1541, 1444, 1215, 1089,
1
1018, 824 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.13 (6H, d, J=7.0 Hz), 2.26 (3H,
s), 3.01-3.05 (1H, m), 6.93 (1H, s ), 7.03 (2H, d, J= 7.3 Hz), 7.23 (1H, d, J=7.6 Hz),
13
7.30(2H, t, J=7.6 Hz), 7.50 (2H, d, J= 7.9 Hz), 10.23 (1H, s); C NMR (DMSO- d6, 75
19

MHz) δ/ppm: 21.0, 23.6, 27.1, 87.0, 118.8, 118.9, 123.5, 123.9, 126.9, 127.3, 129.4, 129.5,
136.7, 138.0, 139.4, 148.1, 152.6. LC-MS (m/z): 292.3 [M+]. Anal. Calcd. for C17H19NO2: C,
75.81; H, 7.11; N, 5.20; found: C, 75.80; H, 7.13; N, 5.21.
2-isopropyl-5-methylphenyl (3-nitrophenyl)carbamate (17): Purity: 99.44%, White powder,
38% yield; mp. 104-106 °C; IR: 3300, 3060, 2962, 1700, 1716, 1532, 1507, 1350, 1224,
1
1089, 816, 720 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.14 (6H, d, J=7.0 Hz), 2.27
(3H, s), 3.01-3.05 (1H, m), 6.97 (1H, s ), 7.05 (1H, d, J= 7.9 Hz), 7.24 (1H, d, J=7.9 Hz),
7.61 (2H, t, J=8.2 Hz), 7.84-7.91 (2H, m), 8.47 (1H, s), 10.77 (1H, s); 13C NMR (DMSO- d6,
75 MHz) δ/ppm: 21.3, 23.3, 26.6, 110.4, 113.1, 116.1, 117.4, 120.2, 125.4, 126.2, 130.8,
131.8, 135.9, 141.3, 148.7, 154.8. LC-MS (m/z): 337.1 [M+]. Anal. Calcd. for C17H18N2O4: C,
64.96; H, 5.77; N, 8.91; found: C, 64.66; H, 5.65; N, 8.83.
2-isopropyl-5-methylphenyl (4-nitrophenyl)carbamate (18): Purity: 99.98%, White powder,
54% yield; mp. 148-149 °C; IR: 3305, 3060, 2962, 1744, 1723, 1596, 1554, 1505, 1410,
1
1213, 1087, 1026, 851, 819 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=6.7
Hz), 2.34 (3H, s), 3.06-3.11 (1H, m), 6.92 (1H, s ), 7.07 (1H, d, J= 7.9 Hz), 7.23 (1H, d,
13
J=7.9 Hz), 7.36 (1H, s, NH), 7.63 (2H, d, J= 9.0 Hz), 8.23 (2H, d, J= 9.0 Hz); C NMR
(CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 27.2, 118.2, 122.9, 125.4, 126.8, 127.8, 137.1, 137.6,
147.3, 151.7. LC-MS (m/z): 381.3 [M+]. Anal. Calcd. for C17H18N2O4: C, 64.96; H, 5.77; N,
8.91; found: C, 64.99; H, 5.75; N, 8.94.
2-isopropyl-5-methylphenyl (4-(trifluoromethyl)phenyl)carbamate (19): Purity: 99.11%,
White powder, 89% yield; mp. 155-156 °C; IR: 3330, 3066, 2960, 1734, 1706, 1614, 1547,
1
1499, 1223, 1025, 828, 755 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.20 (6H, d, J=7.0
Hz), 2.31 (3H, s), 3.04-3.13 (1H, m), 6.92 (1H, s ), 7.05 (1H, d, J= 7.9 Hz), 7.20-7.24 (2H,
m), 7.55 (4H, s, br); 13C NMR (CDCl3, 75 MHz) δ/ppm: 21.0, 23.3, 27.2, 118.4, 123.0, 126.5,
126.6, 126.7, 126.8 (q, JC,F=25.5 Hz, CF3), 127.7, 137.0, 137.7, 140.8, 147.5, 152.0. LC-MS
(m/z): 393.3 [M+]. Anal. Calcd. for C18H18F3NO2: C, 64.09; H, 5.38; N, 4.15; found: C, 63.80;
H, 5.27; N, 4.06.
5-isopropyl-2-methylphenyl cyclopentylcarbamate (20): Purity: 98.48%, White powder, 82%
yield; mp. 99-100 °C; IR: 3309, 3060, 2958, 1731, 1701, 1526, 1500, 1418, 1230, 1167, 1058,
1
992, 788 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=7.0 Hz), 1.42-1.53 (2H,
m), 1.55-1.74 (4H, m), 1.99-2.16 (2H, m), 2.16 (3H, s), 2.82-2.91 (1H, m), 4.02-4.09 (1H,
m), 4.99 (1H, d, J= 5.8 Hz ), 6.92 (1H, s ), 6.97 (1H, d, J= 7.6 Hz), 7.11 (1H, d, J= 7.6 Hz);
20

13C NMR (CDCl3, 75 MHz) δ/ppm: 16.0, 23.8, 24.1, 33.4, 33.8, 53.2, 120.4, 123.8, 127.9,
130.9, 148.0, 149.5, 154.2. LC-MS (m/z): 284.3 [M+]. Anal. Calcd. for C16H23NO2: C, 73.53;
H, 8.87; N, 5.36; found: C, 72.33; H, 8.71; N, 5.20.
5-isopropyl-2-methylphenyl cyclohexylcarbamate (21): Purity: 98.94%, White powder, 68%
yield; mp. 130-131 °C; IR: 3285, 3060, 2961, 2930, 1738, 1703, 1541, 1507, 1319, 1228,
1
1173, 1010, 819 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=6.7 Hz), 1.30-1.44
(4H, m), 1.60-1.65 (2H, m), 1.71-1.76 (2H, m), 2.01 (2H, d, J= 9.9 Hz), 2.16 (3H, s), 2.84-
2.88 (1H, m), 3.55-3.57 (1H, m), 4.93 (1H, d, J= 7.3 Hz ), 6.92 (1H, s ), 6.97 (1H, d, J= 7.6
Hz), 7.11 (1H, d, J= 7.6 Hz); 13C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 24.1, 25.0, 25.7, 33.5,
33.8, 50.3, 120.4, 123.7, 127.9, 130.9, 148.0, 149.5, 153.9. LC-MS (m/z): 276.3 [M+]. Anal.
Calcd. for C17H25NO2: C, 74.14; H, 9.15; N, 5.09; found: C, 74.10; H, 9.16; N, 5.08.
5-isopropyl-2-methylphenyl (cyclohexylmethyl)carbamate (22): Purity: 98.75%, White
powder, 57% yield; mp. 93-95 °C; IR: 3284, 3060, 2959, 2921, 1732, 1703, 1557, 1490,
1
1256, 1231, 1172, 992, 820 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 0.91-1.02 (2H, m),
1.22 (6H, d, J=6.7 Hz), 1.50-1.56 (1H, m), 1.67-1.78 (8H, m), 2.17 (3H, s), 2.82-2.91 (1H,
m), 3.11 (1H, t, J= 6.4 Hz ), 5.0 (1H, s), 6.92 (1H, s ), 6.98 (1H, d, J= 7.6 Hz), 7.11 (1H, d,
J= 7.6 Hz); 13C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 24.1, 26.0, 26.6, 30.8, 33.8, 38.4, 47.7,
120.4, 123.8, 127.9, 130.9, 148.1, 149.5, 154.9. LC-MS (m/z): 312.3 [M+]. Anal. Calcd. for
C18H27NO2: C, 74.70; H, 9.40; N, 4.84; found: C, 73.20; H, 9.21; N, 4.74.
5-isopropyl-2-methylphenyl cycloheptylcarbamate (23): Purity: 99.94%, White powder, 45%
yield; mp. 140-141 °C; IR: 3293, 3060, 2960, 2920, 1736, 1703, 1616, 1540, 1473, 1314,
1
1228, 1172, 1117, 985, 818 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=7.0
Hz), 1.50-1.64 (10H, m), 2.02-2.04 (2H, m), 2.16(3H, s), 2.81-2.90 (1H, m), 3.76-3.77 (1H,
m ), 4.97 (1H, d, J= 7.9 Hz), 6.92 (1H, s ), 6.97(1H, d, J= 7.9 Hz), 7.11 (1H, d, J= 7.6 Hz);
13C NMR (CDCl3, 75 MHz) δ/ppm: 16.0, 24.1, 24.2, 28.2, 33.8, 35.5, 52.6, 120.4, 123.8,
127.9, 130.9, 148.1, 149.5, 153.8. LC-MS (m/z): 312.3 [M+]. Anal. Calcd. for C18H27NO2: C,
74.70; H, 9.40; N, 4.84; found: C, 74.74; H, 9.40; N, 4.80.
5-isopropyl-2-methylphenyl benzylcarbamate (24): Purity: 98.91%, White powder, 82%
yield; mp. 46-47 °C; IR: 3301, 3028, 2961, 1734, 1709, 1541, 1496, 1456, 1267, 1171, 1034,
1
1024, 817 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.22 (6H, d, J=6.7 Hz), 2.17 (3H, s),
2.81-2.91 (1H, m), 4.44 (2H, d, J= 5.8 Hz ), 5.38 (1H, s), 6.94 (1H, s ), 6.98 (1H, d, J= 7.9
13
Hz), 7.11 (1H, d, J= 7.6 Hz), 7.23-7.38 (5H, m); C NMR (CDCl3, 75 MHz) δ/ppm: 16.0,
21

24.2, 33.8, 45.5, 120.4, 124.0, 127.8, 127.9, 128.9, 131.0, 138.5, 148.2, 149.6, 155.0. LC-MS
(m/z): 306.3 [M+]. Anal. Calcd. for C18H21NO2: C, 76.29; H, 7.47; N, 4.94; found: C, 75.76;
H, 7.32; N, 4.83.
5-isopropyl-2-methylphenyl naphthalen-1-ylcarbamate (25): Purity: 98.18%, White powder,
49% yield; mp. 115-116 °C; IR: 3281, 3028, 2954, 1730, 1702, 1536, 1499, 1404, 1239,
1
1224, 1119, 1004, 767 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.17(6H, d, J=6.7 Hz),
2.19 (3H, s), 2.84-2.88 (1H, m), 7.02-7.06 (2H, m ), 7.19 (1H, d, J= 6.1 Hz), 7.48-7.68 (4H,
m), 7.78 (1H, d, J=8.2 Hz), 7.94 (1H, d, J=7.3 Hz), 8.18 (1H, d, J=7.9 Hz), 9.08 (1H, s) ; 13C
NMR (DMSO- d6, 75 MHz) δ/ppm: 16.1, 24.5, 24.7, 33.5, 120.9, 123.3, 124.2, 126.1, 126.3,
126.7, 126.8, 128.2, 128.8, 131.3, 134.0, 134.4, 148.2, 149.9, 153.7. LC-MS (m/z): 342.3
[M+]. Anal. Calcd. for C21H21NO2: C, 78.97; H, 6.63; N, 4.39; found: C, 77.39; H, 6.49; N,
4.30.
5-isopropyl-2-methylphenyl (3-methoxyphenyl)carbamate (26): Purity: 99.99%, White
powder, 58% yield; mp. 51-52 °C; IR: 3308, 3028, 2961, 1735, 1713, 1598, 1540, 1433,
1
1263, 1221, 1034, 823, 767 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23(6H, d, J=6.7 Hz),
2.21 (3H, s), 2.83-2.92 (1H, m), 3.78 (3H, s), 6.64 (1H, d, J= 8.2 Hz), 6.89-7.07 (4H, m),
13
7.10-7.24 (2H, m); C NMR (CDCl3, 75 MHz) δ/ppm: 16.0, 24.1, 33.8, 55.5, 110.0, 120.3,
124.4, 127.9, 130.0, 131.1, 139.0, 148.3, 149.0, 160.5. LC-MS (m/z): 300.3 [M+]. Anal.
Calcd. for C18H21NO3: C, 72.22; H, 7.07; N, 4.68; found: C, 72.25; H, 7.08; N, 4.66.
5-isopropyl-2-methylphenyl (4-methoxyphenyl)carbamate (27): Purity: 99.91%, White
powder, 74% yield; mp. 130-132 °C; IR: 3341, 3028, 2958, 1733, 1713, 1541, 1473, 1245,
1
1216, 1017, 831 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.16 (6H, d, J=6.7 Hz), 2.12
(3H, s), 2.82-2.87 (1H, m), 3.69 (3H, s), 6.89 (2H, d, J= 9.0 Hz), 6.97 (1H, s), 7.02 (1H, d, J=
13
8.7 Hz), 7.16 (1H, d, J= 7.6 Hz), 7.40 (2H, d, J= 8.4 Hz), 10.04 (1H, s) ; C NMR (DMSO-
d6, 75 MHz) δ/ppm: 16.0, 24.5, 33.5, 55.8, 114.6, 114.7, 120.5, 120.9, 124.1, 128.2, 131.3,
132.4, 148.2, 149.6, 152.4, 155.7. LC-MS (m/z): 322.2 [M+]. Anal. Calcd. for C18H21NO3: C,
72.22; H, 7.07; N, 4.68; found: C, 72.20; H, 7.06; N, 4.65.
5-isopropyl-2-methylphenyl p-tolylcarbamate (28): Purity: 99.96%, White powder, 42%
yield; mp. 100-102 °C; IR: 3329, 3040, 2963, 1738, 1715, 1646, 1540, 1456, 1318, 1224,
1
1017, 824 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.16 (6H, d, J=6.7 Hz), 2.12 (3H,
s), 2.23 (3H, s), 2.82-2.86 (1H, m), 6.98-7.18 (6H, m), 7.38 (2H, d, J= 7.9 Hz), 10.13 (1H, s)
13
; C NMR (DMSO- d6, 75 MHz) δ/ppm: 16.0, 21.0, 24.7, 33.5, 118.8, 119.0, 120.9, 124.1,
22

128.1, 129.9, 131.1, 131.3, 132.4, 136.8, 148.2, 149.6, 152.2. LC-MS (m/z): 306.3 [M+]. Anal.
Calcd. for C18H21NO2: C, 76.29; H, 7.47; N, 4.94; found: C, 77.16; H, 7.42; N, 4.82.
5-isopropyl-2-methylphenyl (3-fluorophenyl)carbamate (29): Purity: 97.14%, White powder,
70% yield; mp. 109-111 °C; IR: 3271, 3040, 2960, 1730, 1702, 1652, 1540, 1444, 1223,
1
1205, 1146, 883 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=6.7 Hz), 2.31 (3H,
s), 2.86-2.90 (1H, m), 6.80 (1H, t, J= 8.4 Hz), 6.97-7.31 (6H, m), 7.38 (1H, d, J=9.8 Hz) ;
13C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 24.1, 33.7, 107.1-107.4 (d, JC,F=23.6 Hz, CF),
110.9, 114.8-115.0 (d, JC,F=23.0 Hz, CF), 120.2, 124.5, 127.8, 130.4, 130.5, 131.2, 139.2,
148.4, 148.9, 151.7, 161.8-165.0 (d, JC,F=244.9 Hz, CF). LC-MS (m/z): 310.3 [M+]. Anal.
Calcd. for C17H18FNO2: C, 71.06; H, 6.31; N, 4.87; found: C, 69.21; H, 6.05; N, 4.67.
5-isopropyl-2-methylphenyl (4-fluorophenyl)carbamate (30): Purity: 97.15%, White powder,
82% yield; mp. 121-123 °C; IR: 3301, 3079, 2963, 1736, 1708, 1616, 1550, 1364, 1217,
1
1102, 1019, 878, 747 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=6.7 Hz), 2.21
(3H, s), 2.84-2.93 (1H, m), 6.97 (1H, s, br), 7.00-7.07 (3H, m), 7.16 (1H, d, J=7.6 Hz), 7.40-
7.44 (2H, m) ; 13C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 24.1, 33.7, 115.8-116.1 (d, JC,F=22.7
Hz, CF), 120.2, 124.4, 127.8, 128.0, 131.1, 133.7, 144.2, 148.4, 151.4, 156.3-159.5 (d,
JC,F=245.4 Hz, CF). LC-MS (m/z): 310,2[M+]. Anal. Calcd. for C17H18FNO2: C, 71.06; H,
6.31; N, 4.87; found: C, 71.04; H, 6.30; N, 4.87.
5-isopropyl-2-methylphenyl (3,4-dichlorophenyl)carbamate (31): Purity: 99.98%, White
powder, 79% yield; mp. 123-125 °C; IR: 3308, 3069, 2958, 1731, 1701, 1527, 1505, 1418,
1
1223, 1202, 1115, 992, 817 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.21 (6H, d, J=7.0
Hz), 2.19 (3H, s), 2.82-2.91 (1H, m), 6.95 (1H, s, br), 7.03 (1H, d, J=7.9 Hz), 7.13-7.24 (2H,
13
m), 7.31 (1H, d, J=8.7 Hz), 7.62 (1H, s) ; C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 24.1,
33.8, 118.2, 120.1, 120.6, 124.6, 127.7, 130.8, 131.2, 133.1, 137.3, 148.4, 148.8, 151.7. LC-
MS (m/z): 363.3 [M+]. Anal. Calcd. for C17H17Cl2NO2: C, 60.37; H, 5.07; N, 4.14; found: C,
60.33; H, 5.05; N, 4.15.
5-isopropyl-2-methylphenyl (4-chlorophenyl)carbamate (32): Purity: 99.49%, White powder,
53% yield; mp. 118-120 °C; IR: 3322, 3070, 2963, 1740, 1716, 1600, 1540, 1490, 1399,
1
1217, 1170, 1090, 832, 707 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H, d, J=6.7
Hz), 2.20 (3H, s), 2.83-2.92 (1H, m), 6.96 (1H, s), 7.03 (2H, d, J=7.6 Hz), 7.08 (1H, s, br),
13
7.16 (1H, d, J=7.9 Hz), 7.24-7.29 (2H, m), 7.38 (1H, d, J=7.6 Hz) ; C NMR (CDCl3, 75
MHz) δ/ppm: 16.0, 24.1, 33.8, 120.0, 120.2, 124.5, 127.8, 129.3, 131.2, 136.4, 148.4, 148.9,
23

151.7. LC-MS (m/z): 326.2 [M+]. Anal. Calcd. for C17H18ClNO2: C, 67.21; H, 5.97; N, 4.61;
found: C, 67.10; H, 5.79; N, 4.50.
5-isopropyl-2-methylphenyl (4-bromophenyl)carbamate (33): Purity: 99.91%, White powder,
69% yield; mp. 134-136 °C; IR: 3326, 3079, 2962, 1739, 1716, 1593, 1536, 1488, 1395,
1
1217, 1115, 1020, 828, 702 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.23 (6H, d, J=6.7
Hz), 2.21 (3H, s), 2.84-2.90 (1H, m), 6.96 (2H, d, J=1.7 Hz), 7.03 (1H, d, J=7.9 Hz), 7.15
13
(1H, d, J=7.9 Hz), 7.35 (2H, d, J=8.7 Hz ), 7.42-7.46 (2H, m) ; C NMR (DMSO- d6, 75
MHz) δ/ppm: 16.0, 24.5, 24.7, 33.5, 115.2, 120.8, 124.4, 128.1, 131.4, 132.3, 138.9, 148.3,
149.4, 152.1. LC-MS (m/z): 372.2 [M+]. Anal. Calcd. for C17H18BrNO2: C, 58.63; H, 5.21; N,
4.02; found: C, 58.58; H, 5.24; N, 4.00.
5-isopropyl-2-methylphenyl (4-iodophenyl)carbamate (34): Purity: 97.98%, White powder,
72% yield; mp. 153-155 °C; IR: 3298, 3079, 2961, 1736, 1725, 1577, 1532, 1474, 1394,
1
1216, 1169, 1061, 851, 813 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.16 (6H, d, J=6.7
Hz), 2.11 (3H, s), 2.82-2.86 (1H, m), 6.99-7.04 (2H, m), 7.17 (1H, d, J=7.9 Hz), 7.33 (2H, d,
13
J=8.7 Hz), 7.64 (2H, d, J=7.0 Hz ), 10.36 (1H, s) ; C NMR (DMSO- d6, 75 MHz) δ/ppm:
16.0, 24.5, 33.5, 87.0, 120.8, 121.2, 124.4, 128.1, 131.3, 138.3, 139.3, 148.3, 149.4, 152.1.
LC-MS (m/z): 437.3 [M+]. Anal. Calcd. for C17H18INO2: C, 51.66; H, 4.59; N, 3.54; found: C,
50.62; H, 4.49; N, 3.46.
5-isopropyl-2-methylphenyl phenylcarbamate (35): Purity: 99.94%, White powder, 66%
yield; mp. 136-137 °C; IR: 3282, 3070, 2961, 1732, 1704, 1600, 1540, 1490, 1457, 1220,
1
1014, 820, 750 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.17 (6H, d, J=6.7 Hz), 2.13
(3H, s), 2.83-2.88 (1H, m), 6.99-7.04 (2H, m), 7.16 (1H, d, J=7.6 Hz), 7.28-7.33 (2H, m),
13
7.50-7.53 (2H, m), 10.24 (1H, s) ; C NMR (DMSO- d6, 75 MHz) δ/ppm: 16.0, 24.5, 33.5,
119.0, 120.9, 123.5, 124.2, 128.1, 129.5, 131.3, 139.4, 148.2, 149.6, 152.2. LC-MS (m/z):
292.2 [M+]. Anal. Calcd. for C17H19NO2: C, 75.81; H, 7.11; N, 5.20; found: C, 75.78; H, 7.14;
N, 5.22.
5-isopropyl-2-methylphenyl (3-nitrophenyl)carbamate (36): Purity: 99.93%, White powder,
79% yield; mp. 123-124 °C; IR: 3283, 3070, 2962, 1723, 1702, 1597, 1529, 1351, 1228,
1
1119, 1032, 885, 733 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.16 (6H, d, J=6.7 Hz), 2.15
(3H, s), 2.82-2.86 (1H, m), 7.02-7.04 (2H, m), 7.18 (1H, d, J=8.2 Hz), 7.56-7.61 (1H, m),
7.87 (2H, dd, J=8.4, 2.0 Hz), 8.49 (1H, s), 10.77 (1H, s) ; 13C NMR (CDCl3, 75 MHz) δ/ppm:
15.9, 24.1, 33.7, 113.8, 118.5, 120.1, 124.8, 127.7, 130.1, 131.2, 139.1, 148.5, 148.8, 152.0.
24

LC-MS (m/z): 337.3 [M+]. Anal. Calcd. for C17H18N2O4: C, 64.96; H, 5.77; N, 8.91; found: C,
64.95; H, 5.78; N, 8.90.
5-isopropyl-2-methylphenyl (4-nitrophenyl)carbamate (37): Purity: 99.93%, White powder,
70% yield; mp. 134-135 °C; IR: 3319, 3070, 2964, 1740, 1728, 1593, 1556, 1386, 1217,
1
1119, 1024, 831, 751 cm-1; H NMR (DMSO- d6, 300 MHz) δ/ppm: 1.16 (6H, d, J=6.7 Hz),
2.13 (3H, s), 2.83-2.87 (1H, m), 6.56-6.59 (1H,m), 7.04-7.07 (2H, m), 7.19 (1H, d, J=8.2
Hz), 7.72-7.76 (2H, m), 8.23 (2H, dd, J=7.0, 2.0 Hz), 10.95 (1H, s) ; 13C NMR (DMSO- d6,
75 MHz) δ/ppm: 16.0, 24.4, 33.5, 113.0, 118.6, 120.7, 124.6, 125.8, 127.0, 128.0, 131.4,
142.7, 145.9, 148.4, 149.2, 152.0. LC-MS (m/z): 318.4 [M+]. Anal. Calcd. for C17H18N2O4: C,
64.96; H, 5.77; N, 8.91; found: C, 64.99; H, 5.79; N, 8.90.
5-isopropyl-2-methylphenyl (4-(trifluoromethyl)phenyl)carbamate (38): Purity: 99.85%,
White powder, 71% yield; mp. 125-126 °C; IR: 3329, 3070, 2969, 1746, 1721, 1662, 1542,
1
1409, 1316, 1218, 1112, 1067, 845, 751 cm-1; H NMR (CDCl3, 300 MHz) δ/ppm: 1.23 (6H,
d, J=6.7 Hz), 2.21 (3H, s), 2.86-2.90 (1H, m), 6.98 (1H, s), 7.05 (1H, dd, J=7.9, 1.7 Hz), 7.16
(1H, d, J=7.9 Hz), 7.23 (1H, s), 7.56 (4H, s); 13C NMR (CDCl3, 75 MHz) δ/ppm: 15.9, 24.1,
33.8, 118.4, 120.2, 124.6 (q, JC,F=12.7 Hz, CF3), 126.6, 126.7, 127.8, 131.2, 140.9, 148.5,
148.8, 151.6. LC-MS (m/z): 360.2 [M+]. Anal. Calcd. for C18H18F3NO2: C, 64.09; H, 5.38; N,
4.15; found: C, 64.00; H, 5.45; N, 4.10.
4.3. Anticholinesterase activity assays
Acetyl- (AChE) and butyryl-cholinesterase (BuChE) inhibitory activities of the synthesized
compounds were determined according to Ellman's method [27]. 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
25

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 (7.1 mM) acetylthiocholine iodide (or 0.79 mM 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 cytotoxicity assay
The cytotoxicity effect of test compound on hepatoma cell H4IIE cells was evaluated by MTT
assay according to described methods [28]. Briefly, H4IIE cells were seeded in a flat-
bottomed 96-well plate at a density of 5×104 cells/well in DMEM containing 10% FBS. The
plate was incubated at 37 °C with 5% CO2 for 24 h, and then 10, 20, 29, 30, 35 were prepared
and added to make a final concentration of 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 μM,
respectively, in serum-free DMEM. Cells were further incubated for 24 h at 37 °C with 5%
CO2; then, the medium was replaced with DMEM containing 10% FBS. 10 μL of filter-
sterilized MTT (3-(4,5 dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) solution (5
mg/mL in PBS) was added to each well and further incubated at 37 °C with 5% CO2 for 4 h.
At the end of incubation media was aspirated from the wells and 100 μL of DMSO was added
to dissolve insoluble formosan crystals formed. The absorbance was measured at 570 nm
using a microtiter plate reader. The relative % cell viability was calculated from the following
equation: Relative percent cell viability = (Atest/Acontrol) ×100%. (Atest is the absorbance of
the sample treated cells and a control is the absorbance of the untreated cells. Each
absorbance was taken to be the mean of triplicate measurements.) The cell viability was
represented as a percentage relative to untreated cells as a control.
4.5. Molecular Modeling
4.5.1. Ligand Setup
All the ligands were sketched manually in 2D structures and then minimized by force field
methods (OPLS 2005) [29] in order to obtain the stable states of the structures. The
compounds were ionized at a biological state (pH = 7.4) by Epik [30] module implemented in
the Schrodinger software package. Atomic electro-static potential (ESP) charges of the atoms
were calculated by Austin Model 1 (AM1) [31], which is a semi-empirical method. In order to
26

obtain the lowest energy configurations of the ligands a conformation search assay was used,
in which stereo-isomers of the compounds were also considered.
4.5.2. Protein Preparation
Crystal structures of the human AChE and human BuChE were retrieved from the Protein
Data Bank [32], (PDB codes: 4M0E [33] and 4TPK [34], respectively). The structures were
submitted in protein preparation wizard implemented in the Schrodinger molecular modeling
package. The amino acids with missing atoms in side chains or backbones were repaired,
water molecules that are far from the ligand binding pocket were removed, topology of the
structures were assigned, missing hydrogen atoms were added, protonation state of the
systems was determined at a physiological pH (pH 7.4) by PROPKA software [35], and
finally all the systems were relaxed using the OPLS force field.
4.5.3. Docking Simulations
All the docking simulations were carried out using Induced Fit Docking (IFD) method [36]
implemented into the Schrodinger molecular modeling package. This method of docking
provides us to dock the compounds into the active site, while ligand and the amino acids
within 6 Å of the ligand are kept flexible. This step-wise method is running in several steps as
following: i) all the ligands were docked into the catalytic domain of the target using Glide/SP
[37] and then complexes with the high docking scores were forwarded for next step, ii) amino
acids of the complexes within 6 Å of the docked ligands were minimized by Prime module of
the Maestro. iii) Finally, all the ligands were re-docked into the refined target via Glide/XP
[38] docking method.
4.5.4. Molecular Dynamics (MD) Simulations
Energetic calculations using MD trajectories for the synthesized compounds that predict their
binding affinity into the catalytic domains of the AChE and BuChE proteins was carried out
by Desmond package [39]. The interactions between the atoms were calculated by OPLS
2005 force field. The particle-mesh Ewald method [40] was implemented to calculate the
long-range electrostatic interactions. A cut-off radius of 12.0 Å was used for van der Waals
and Coulombic interactions. Nose−Hoover thermostat [41] and Martyna−Tobias−Klein
protocols [42] were used to adjust the temperature and pressure of the systems at 300 K and
1.01325 bar, respectively. Time step was assigned to be 2.0 fs in all the simulations. All the
atoms were minimized for a maximum of 5000 iterations, and a convergence threshold of 1
27

kcal mol−1 Å−1 was implemented. Then the proteins and water molecules were relaxed using
step-wise methods, in which the systems were gradually equilibrated in a range of 300-700 K.
Finally, 6 x 50-ns MD simulations were carried out for each systems, by which trajectory
frames were output in an interval of 10 fs.
4.5.5. Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) Assays
Binding free energy predictions have been proven feasible approach for studying of living
systems, such as protein-protein and protein-ligand interactions, designing of high potential
drugs [43]. MM−PBSA is a straightforward method that predicts ligand binding free energy in
complex with biological macromolecules [44]. Binding free energies are achieved by
subtracting the individual free energies of the receptor and ligand from the free energy of the
complex structure as given in equation 1:
ΔGbind = Gcomplex – (Gprotein + Gligand
)
(1)
The free energy of ligand binding can be explained as a composition of enthalpy (H) and
entropy (S) terms as shown in equation 2:
ΔGbind = ΔH - TΔS
ΔH at the equation 2 can be obtained as:
ΔH = ΔEMM + ΔGsol
(2)
(3)
where ΔEMM describes the molecular mechanics (MM) interaction energy between the protein
and the ligand, and ΔGsol is the solvation free energy. ΔEMM is expressed by (4):
ΔEMM = ΔEelec + ΔEvdW
(4)
where ΔEelec and ΔEvdW defines electrostatic and van der Waals interaction energies,
respectively. ΔGsol is subdivided into:
ΔGsol = ΔGPB + ΔGNP
(5)
where ΔGPB defines the polar solvation energy calculated by Poisson−Boltzmann (PB)
methods using the PBSA module of AmberTools15 [45]. ΔGNP describes non-polar solvation
energy which is predicted through estimation of the solvent-accessible surface area (SASA) as
shown in equation 6:
ΔGNP = γSASA + β (6)
28

where the surface tension (γ) and the offset (β) have been set the standard values of 0.005420
kcal mol−1 Å−2 and −1.008 kcal mol−1, respectively. ΔGNP was estimated with the linear
combinations of pairwise overlaps (LCPO) method [46]. In SASA calculation, a probe radius
of 1.4 Å has been assigned for the solvent. MMPBSA.py, a Python script of AmberTools15
was employed to carry out the calculations.
Acknowledgments
This work was supported by the Bezmialem Research Fund of the Bezmialem Vakıf
University. Project Number: 12.2015/28.
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