Molecular modeling and anticholinesterasic activity of novel 2-arylaminocyclohexyl N,N-dimethylcarbamates

Article abstract of DOI:10.5935/0103-5053.20130225

This work reports a detailed theoretical and experimental study of the novel isomer series cis- and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates as potential inhibitors of cholinesterases. In vitro inhibition assay by Ellman's method with human bloo

Full text of DOI:10.5935/0103-5053.20130225

http://dx.doi.org/10.5935/0103-5053.20130225
J. Braz. Chem. Soc., Vol. 24, No. 11, 1798-1807, 2013.
Printed in Brazil - ©2013 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Article
A
Molecular Modeling and Anticholinesterasic Activity of
Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
a
a
b
b
Mariane C. Bagatin, Augusto A. Cândido, Glaucia M. S. Pinheiro, Nelci F. Höehr,
c
c
a
,a
Miguel Machinski Jr., Simone A. G. Mossini, Ernani A. Basso and Gisele F. Gauze*
a
Departamento de Química, Universidade Estadual de Maringá,
Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
b
Departamento de Patologia Clínica, Faculdade de Ciências Médicas,
Universidade Estadual de Campinas, CP 6111, 13083-887 Campinas-SP, Brazil
c
Departamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá,
Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
O presente trabalho reporta um estudo teórico e experimental detalhado das séries inéditas
de isômeros cis- e trans-N,N-dimetilcarbamatos de cicloexila 2-arilaminossubstituídos como
potenciais inibidores de colinesterases. Os testes de inibição in vitro, realizados através do método
de Ellman em amostras de sangue humano, mostraram que os novos carbamatos apresentaram
boa seletividade frente à inibição da enzima butirilcolinesterase (BuChE), com um máximo de
-1
inibição de 90% e IC50 de 6 e 8 mmol L para os compostos mais ativos da série. Os estudos de
modelagem molecular apontaram significantes diferenças entre as orientações destes compostos
nos sítios ativos das enzimas BuChE e acetilcolinesterase (AChE). Os resultados mostraram que
os compostos interagem de forma mais efetiva com o sítio ativo da enzima BuChE, pois o grupo
carbamato está próximo aos resíduos chave da tríade catalítica.
This work reports a detailed theoretical and experimental study of the novel isomer series
cis- and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates as potential inhibitors of
cholinesterases. In vitro inhibition assay by Ellman’s method with human blood samples showed
that the new carbamates are selective to the inhibition of enzyme butyrylcholinesterase (BuChE)
-1
with maximum inhibition of 90% and IC50 of 6 and 8 mmol L for the more actives compounds
of the series. Molecular modeling studies point to significant differences for the conformations
of the compounds in the active sites of enzymes BuChE and acetylcholinesterase (AChE). The
results show that the compounds interact more effectively with the active site of enzyme BuChE
since the carbamate group is close to the key residues of the catalytic triad.
Keywords: carbamate derivatives, cholinesterase inhibitors, molecular docking, Alzheimer’s
disease
Introduction
Despite the numerous studies done, the causes of
AD are not fully elucidated and no cure has been found
yet. Nevertheless, its damages and progression can be
minimized. The most efficient therapy for the symptomatic
treatment of AD is based on the cholinergic hypothesis
in which the cognitive deficiency is a consequence of an
acetylcholine (ACh) deficiency with a resulting decrease
Alzheimer’s disease (AD) is a neurodegenerative
disease characterized by progressive loss of memory and
other functions. It was first described by Alois Alzheimer
1
in 1906. This type of mental deterioration is the most
common in individuals aged over 60 and its incidence
has increased as the world population has grown older.
Currently, it is estimated that 26 million people suffer from
3-6
in cholinergic neurotransmission.
In physiological conditions, the activity of
neurotransmitter ACh is known to be related mainly
to acetylcholinesterase (AChE). However, substantive
evidence points to the importance of the function of
2
AD in the world.
*
e-mail: gfgbandoch@uem.br

Vol. 24, No. 11, 2013
Bagatin et al.
1799
7
-10
butyrylcholinesterase (BuChE) in the cholinergic system.
The cyclohexene oxide aminolysis was performed
without a catalyst and with water as a solvent. Bonollo et al.
25
Recent studies have shown that due to a progressive
decrease in the AChE activity in certain regions of the
brain in AD, the BuChE activity increases, suggesting
that BuChE may act as a compensatory mechanism in the
demonstrated that the reaction efficiency depends on a
rigorous pH control. The ideal reactional condition is
attained when 1.0 mmol cyclohexene oxide is mixed with
1.05 mmol amine in 2.0 mL deionized water; the resulting
pH of 8.30 varies little during the reaction.
4
,11
12,13
hydrolysis of ACh.
Due to the cholinergic hypothesis and numerous
reports on the multiple functions of cholinesterases on the
pathogenesis and evolution of AD, AChE and BuChE are
interesting targets for the development of new anti-AD
The trans-2-arylaminocyclohexanols 1a-1c were
carbamoylated with metallic sodium for the preparation
of the corresponding alkoxide and later reacted with
dimethylcarbamoyl chloride. However, the joint analysis
of the nuclear magnetic resonance (NMR) and gas
chromatography-mass spectrometry (GC-MS) data in
the preparation of derivative 2c showed the formation
of a mixture of compounds, 25% of the defluorinated
compound 2a and 75% of the desired product 2c. Attempts
to separate these compounds by column chromatography
were unsuccessful. As an alternative, metallic sodium
was substituted with sodium hydride in the synthesis of
compound 2c, which was obtained as a single product.
The mechanism for hydrodefluorination was reported
1
4-19
drugs.
The action of carbamates as cholinesterase inhibitors
has been known for decades and several studies have been
conducted in search of anticholinesterastic carbamates that
20-24
are active, also safe and better tolerated.
Rivastigmine,
a drug from the carbamate class, is one of four drugs that
act as cholinesterase inhibitors currently approved by
ANVISA, the Brazilian Health Surveillance Agency, for
the treatment of AD.
This study describes the synthesis and evaluation of the
in vitro activity of two novel series of potential cholinesterase
inhibitors (Figure 1). Computational calculations were
performed to determine the conformational preference
of the isomers and molecular docking was performed
for the proposal of a binding model for the compounds
synthesized to the active site of enzymesAChE and BuChE.
We hope this study may contribute to increase the number
of therapeutic agents against AD.
27,28
previously
as a sequence of steps, beginning with the
electron transfer from metal to the substrate and ending with
the formation of the hydrodefluorination product. Therefore,
the product 2c was successfully obtained when the metallic
sodium was substituted by sodium hydride since in this case
it is not possible the occurrence of electron transfer.
The cis carbamates were synthesized in four steps.
First, cyclohexanone was reacted with bromide to produce
2
9
2
-bromocyclohexanone with a yield of 70%. Next,
racemic 2-arylaminocyclohexanones 3a-3c were prepared
with 30% yield by substitution of 2-bromocyclohexanone
3
0
with appropriate arylamines, using quinoline as a
catalyst and ethoxyethanol as a solvent. In the third
step, ketones 3a-3c were stereoselectively reduced in
3
1
the presence of N-selectride, giving the respective
cis-2-arylaminocyclohexanols 4a-4c in moderate yields,
36-50%. Temperature control (-78 C) was very important
Figure 1. Structures of the two novel series of carbamate derivatives
cis- and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates.
o
in this step to ensure that only the cis isomer was obtained.
2
6
Results and Discussion
Finally, compounds 4a-4c were carbamoylated under
the same conditions used in the synthesis of the trans
carbamates to obtain compounds 5a-5c.
Synthesis
New cis- and trans-carbamate derivatives were
synthesized via the route outlined in Scheme 1. trans
Carbamates were synthesized in two steps. First,
cyclohexene oxide was submitted to aminolysis with
Biological activity
Inhibitory potencies of the novel synthesized carbamates
against cholinesterases from fresh human blood were
evaluated by Ellman’s modified spectroscopic method.
This test is based on the reaction of 5,5’-dithiobis-
(2-nitrobenzoic acid), known as DTNB or as Ellman’s
reagent, with the sulfhydryl group of acetylthiocholine
2
5
32
arylamines to obtain trans-2-arylaminocyclohexanols
a-1c with yields from 50 to 80%. Next, the alcohols 1a-1c
1
26
were carbamoylated with dimethylcarbamoyl chloride to
give carbamates 2a-2c with yields ranging from 35 to 50%.

1
800
Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
o
Scheme 1. Synthesis route for isomers cis and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates. Reagents and conditions: (a) arylamine, H O, 60 C,
2
o
o
o
4
(
8 h; (b) Na /THF, 80 C, 8 h; N,N-dimethylcarbamoyl chloride, reflux, 16 h; (c) NaH/THF, 80 C, 8 h; N,N-dimethylcarbamoyl chloride, reflux, 16 h;
o
o
d) Br , H O, 0-5 C; (e) Na CO , quinoline, methoxyethanol, reflux, 2 h; and (f) N-selectride, THF, -78 C, 4 h.
2
2
2
3
or butyrylthiocholine, which results in the formation of a
yellow-colored product, 2-nitro-5-thiobenzoic acid (TNB).
This product has maximum absorbance at 412 nm, thus the
lower the measured absorbance, the greater the activity
of the compounds tested, since the enzyme is inhibited.
Since this method requires water soluble compounds,
chlorohydrates 2a’-2c’ and 5a’-5c’ were prepared.
for any evaluated concentration (Figures 2a and 2c).
The cis and trans salts produced maximum activity
-
1
inhibition for BuChE, around 90% at 0.2 mol L .
Compounds 5a’ and 2b’ were the most active, with IC
values of 6.0 and 8.0 mmol L , respectively.
5
0
-1
Relative to the acetylcholinesterase inhibition
(Figures 2b and 2d), compounds 2a’-2c’ and 5b’-5c’
followed the same tendency and did not inhibit 50% of the
enzyme activity in any of the investigated concentrations.
Only compound 5a’ inhibited AChE significantly, with a
The assays were performed at five concentrations
-
1
(
0.01, 0.025, 0.05, 0.1 and 0.2 mol L ) of the potential
®
inhibitors. Exelon , a drug that contains the active
principle rivastigmine, was used as a standard. In these
experiments, the inhibition potential of each compound
against butyrylcholinesterase (found in plasma) and
acetylcholinesterase (found in red blood cells) was
assessed.
-1
maximum inhibition of 79% at 0.2 mol L .
Likewise, the investigated compounds had good
selectivity in relation to the BuChE inhibition. Derivative 2b’
was the most active selective compound, with an IC of
5
0
-1
8.0 mmol L .Among the tested compounds, only derivative
The inhibition results are shown in Figure 2a-2d. The
IC50 (the testing compound concentration that inhibits
the hydrolysis of substrates by 50%) values (Table 1) of
each compound were obtained through the plot of percent
inhibition versus compound concentration.
5a’ was active against both enzymes, with an IC of
5
0
-1
6.0 mmol L against plasma cholinesterase (BuChE) and
-1
59.2 mmol L against erythrocyte cholinesterase (AChE).
The analysis of the data shows that the cis-trans
isomerism does not influence the activity of the compounds
significantly. However, the presence of donor or acceptor
substituents in the para position of the aromatic ring may
The analysis of Figure 2 shows that all compounds
were more active in the butirylcholinesterase inhibition

Vol. 24, No. 11, 2013
Bagatin et al.
1801
-1
Figure 2. Concentration effects (0.01, 0.025, 0.05, 0.1 and 0.2 mol L ) of 2a’-2c’ and 5a’-5c’ on the BuChE (a and c) and AChE (b and d) activities from
fresh human blood.
Table 1. IC values of carbamate chlorohydrates and reference compound,
rivastigmine, as inhibitors of cholinesterases
carbamate isomers and their methyl sulfate salts, and found
50
-1
an IC of 59 mmol L for the most active compound of the
50
series against BuChE. Comparatively, all compounds tested
a
a
BuChE IC
(
/
AChE IC50
/
5
0
Compound
in this work (2a’-2c’ and 5a’-5c’) were more active against
-
1
-1
mmol L )
(mmol L )
-1
BuChE, with IC values ranging from 6 to 29.8 mmol L .
50
2
2
2
5
5
5
a’
b’
c’
a’
b’
c’
17.9
8.0
n.a.
n.a.
n.a.
59.2
n.a.
n.a.
1.29
These data demonstrate that the presence of the arylamine
group potentialized the activity of these carbamates in the
inhibition of plasma cholinesterase.
29.8
6.0
17.0
17.2
0.031
Conformational equilibrium analysis
Rivastigmine
The molecular modeling calculations were performed
3
3
a
with Gaussian 03 package programs. To investigate
the cis and trans isomer preferential conformations and
the influence of the substituents on the conformational
equilibrium, surface energy potential calculations were
performed at HF/6-31G level. The most stable structures
were then optimized at B3LYP/6-311++G(d,p) level, and
the obtained energy values were used to determine the
contribution of each conformer to the equilibrium. Table 2
shows the relative energies and calculated populations of
each conformer of the studied compounds. Figure 3 presents
the optimized structure for the cis and trans carbamate
derivates of aniline (2a and 5a). Similar conformations
were obtained for the other compounds.
IC values are representative of three independent experiments performed
in triplicate, standard deviations within 10% of mean values reported for
each compound. n.a.: not active (IC50 > 100 mmol L ).
50
-
1
either increase or decrease the activity of the compounds,
depending on the series that is analyzed. For the trans series
compounds 2a’-2c’), the presence of the methoxyl group
in 2b’ potentialized the anticholinesterasic activity and was
approximately two times more active than derivative 2a’.
For the cis series, the presence of activator (5b’) and
deactivator (5c’) substituents in the aromatic ring
significantly reduced the activity of the compounds in
relation to the non-substituted derivative 5a’.
(
2
3
Bocca et al. studied the inhibitory properties of
-N,N-dimethylaminecyclohexyl 1-N’,N’-dimethyl-
According to Table 2, all trans isomers presented ee
as major conformers. This preference can be attributed to
2

1
802
Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
Table 2. Relative energy (E ) and population for conformers of isomers cis- and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates
rel
trans
cis
-
1
-1
Erel / (kcal mol )
Erel / (kcal mol )
Compound
ee / %
Compound
ae / %
ee
0
aa
ae1
0
ae2
0.19
0.27
0.03
ea1
1.13
1.17
0.86
ea2
1.90
1.94
1.89
2
2
2
a
b
c
98.6
97.2
98.2
2.52
2.10
2.37
5a
5b
5c
90.1
90.2
87.6
0
0
0
0
Figure 3. Structures optimized at B3LYP/6-311++G(d,p) for trans- and cis-2-arylaminocyclohexyl N,N-dimethylcarbamates.

Vol. 24, No. 11, 2013
Bagatin et al.
1803
steric repulsion between the individual substituents and
the hydrogens in 1 and 3 arrangements (syn, 1,3-diaxial
interactions). When the substituents adopted equatorial
positions (ee), these repulsions were avoided and otherwise,
when the substituents adopted axial positions (aa), these
interactions were maximized.
conformation, the atoms of the carbamate group (despite
its volume being greater than that of the arylamine group)
are relatively distant from the cyclohexane ring, which
decreases the syn 1,3-diaxial interactions. In contrast, in the
ea conformation, the arylamine group is close to the ring,
causing a greater repulsion between the hydrogen in the
amine group and the 1,3-diaxial hydrogens. These results
are consistent with previous studies published on these
These results agree with the coupling constant values
3
1
3
JHH obtained from the H NMR spectra, with J greater
HH
34,35
than 9.3 Hz for hydrogens H-1 and H-2, indicating that they
are in axial position and the substituents (carbamate and
arylamine) in equatorial position.
compounds.
The effect of hyperconjugative interactions on
conformers ae and ea was assessed by means of NBO
analysis, using B3LYP/6-311++G(d,p) theory level.
Table 3 shows the main interactions involving the
substituent groups. When the carbamate group is in axial
position (ae), a stabilizing orbital interaction occurs between
σC2-H2’ → σ*C1-O, which has considerably more energy than
the interaction between σC1-H1’ → σ*C2-N, which occurs when
the arylamine group is in axial position (ea). This occurs
because antibonding orbital σ*C1-O is a better electron
acceptor than antibonding orbital σ*C2-N. The sum of the
main hyperconjugative interactions of the substituent groups
shows that they are more effective in the ae conformation,
For isomers cis-2-arylaminocyclohexyl N,N-dimethyl-
carbamates, two conformations are possible, one with the
carbamate group in axial position and the arylamine group
in equatorial position (ae), and another with the carbamate
group in equatorial position and the arylamine group in
axial position (ea). Each of the conformations had two
minimum energy structures, represented by ae1, ae2,
ea1 and ea2 (Figure 3). The analysis of the data in Table 2
shows that the conformers with the carbamate group in the
axial position (ae) have lower energy than the conformers
with the arylamine group in axial position (ea). These
3
-1
results agree with the values of the coupling constant J
with a difference of about 1.50 kcal mol in relation to the
HH
1
obtained from the H NMR spectra. In this case, values
ea conformation. The conformational preference of the cis
isomers can be assigned to both steric and electronic effects.
The molecular modeling calculations were also applied
to the protonated form of the carbamates. The differences
of energy between the conformers (E -E and E -E ) were
3
of J over 9.3 Hz were observed only for hydrogen H2,
HH
indicating that it is in axial position and, consequently,
the arylamine group is in equatorial position.
The preference of conformer ae can also be explained by
the steric repulsion between the individual substituents and
the hydrogens in 1 and 3 arrangements. In the ae
ee
ea
ae
ea
-1
higher than 5.0 kcal mol , so ee and ae are the dominant
conformers in the equilibrium.
Table 3. Hyperconjugative interactions obtained through NBO analysis for derivatives cis-2-arylaminocyclohexyl N,N-dimethylcarbamates
-1
E / (kcal mol )
5b
Conformer
ae
NBO donor
NBO acceptor
5
a
5c
σC2-H2’
σC6-H6’
σC3-C4
σC1-C6
σ*C1-O
σ*C1-O
σ*C2-N
σ*C2-N
5.90
4.91
2.71
2.56
16.08
3.98
4.27
3.04
3.29
14.58
5.87
5.88
4.91
2.71
2.57
16.07
3.98
4.29
3.03
3.29
14.59
4.92
2.68
2.55
Total
ea
16.02
3.95
σC1-H1’
σC3-H3’
σC5-C6
σC2-C3
σ*C2-N
σ*C2-N
σ*C1-O
σ*C1-O
4.21
3.06
3.33
Total
14.55

1
804
Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
Enzyme-inhibitor interactions
anionic subsite, Gly118. There is a hydrogen bond between
Hsd440 and the protonated amino group. Residues Trp84,
Tyr334 and Tyr121 contribute to the stabilization through
van der Waals interactions. The hydrolysis mechanism
of acetylcholine involves mainly the catalytic triad
residues (Ser200, His440 and Glu327). Upon analysis of
the enzyme-substrate complex, one can observe that the
carbamate group is distant from the catalytic triad residues,
decreasing the possibility of the enzyme inhibition.
Figure 4b shows the ligand complexed with enzyme
BuChE. In this case, one can observe that the carbamate
group is close to the catalytic triad residues of the enzyme
(Ser198, Hsd438 and Glu325), enabling the nucleophilic
attack of the oxygen of Ser198 to the carbonyl of the
carbamate group. It can also be observed that the arylamine
group is stabilized by hydrogen bonds with Glu197,
Gly116 and Ser198, and those residues Tyr128, Trp82 and
Tyr440 contribute to stabilize the ligand through van der
Waals interactions.
Molecular docking calculations were performed using
the AutoDock 4.2.3 program implemented at the interface
3
6
PyRx 0.9 to assess the enzyme-inhibitor interactions and
propose a bonding model based on the experimental results.
All the experimentally tested compounds were docked to
both enzymes (AChE and BuChE) and their interactions
were analyzed. Due to the similarity of the obtained results,
the most active selective compound 2b’ was chosen as
model in the present discussion. It is important to point
out that only the most stable conformer of each isomer was
used in the docking calculations.
Figure 4a shows the trans-2-(4-methoxyphenylamino)
cyclohexyl N,N-dimethylcarbamate hydrochloride 2b’
complexed with enzyme AChE. One can observe that the
ligand is stabilized by residues of the catalytic anionic
site Glu199 and Phe330 and by one of the residues of the
In general, the molecular docking results show that
the assessed compounds are located in the active site of
both enzymes, but in butyrylcholinesterase, the carbamate
group is close to the catalytic triad residues, whereas in
acetylcholinesterase this group is farther away. These
results agree with the experimental data, which showed that
the tested compounds are selective inhibitors of enzyme
butyrylcholinesterase.
Conclusions
New selective inhibitors of enzyme BuChE may serve
as biological investigative tools of its role in AD and
its treatment. This study resulted in a series of new
cholinesterase inhibitors, selective BuChE inhibitors
(2a’-2c’, 5b’and 5c’), and one non-selectiveAChE/BuChE
inhibitor (5a’). The in vitro inhibition test by Ellman’s
modified method showed that the cis-trans isomerism does
not influence the activity of the compounds significantly,
but that the presence of donor and acceptor substituents in
the para position of the aromatic ring may either increase
or decrease the activity of the compounds, depending on
the series being analyzed.
According to the DFT (density functional theory)
calculations, the trans carbamates presented diequatorial
substitution as major conformers, which can be attributed to
steric repulsion between the substituents and the hydrogens
in 1 and 3 arrangements (syn 1,3-diaxial interactions). For
the cis isomers, the most stable conformer is the one with
the carbamate group in axial position and the arylamine
group in equatorial position. In this case, the conformational
preference is explained both by steric and electronic effects.
Figure 4. Active sites of enzymes (a) AChE and (b) BuChE complexed
with trans-2-(4-methoxyphenylamino)cyclohexyl N,N-dimethylcarbamate
hydrochloride 2b’.

Vol. 24, No. 11, 2013
Bagatin et al.
1805
Molecular modeling studies attributed the selectivity
of the compounds toward BuChE to the distance between
the carbamoyl group and the catalytic site of the enzyme.
In the BuChE, the carbamoyl group is oriented towards
the catalytic triad, creating import interactions between
the triad and the active site. In contrast, in AChE, the long
distance between the carbamoyl group and the catalytic triad
of enzyme makes difficult any interaction between them.
Overall, these results indicate that the novel carbamate
derivatives are interesting structures for the development
of selective and more potent BuChE inhibitors.
To determine the total amount of cholinesterases, 3.0 mL
of solution (erythrocyte + plasma) were transferred to
vials and 40 μL of tested compounds were preincubated
with the enzymes for 10 min at 30 ºC before starting the
reaction by adding of the substrate (acetylthiocholine
-
1
iodide). Next, 50 μL of 10 mmol L DTNB (Ellman’s
reagent, Sigma-Aldrich Co.) solution and 20 μL of
-1
75 mmol L acetylthiocholine iodide (Sigma-Aldrich Co.)
solution were added. Enzyme activity was determined
by measuring the absorbance at 412 nm for 5 min with a
Shimadzu UV-1061PC apparatus.An inhibitor-free sample
was used (100% enzyme activity) as a reference. Each
compound was assayed at five concentrations in triplicate.
The reaction rates were compared and the percent inhibition
due to the presence of the tested compounds was calculated.
The IC50 values were determined by spectrophotometric
measurement of the effect of increasing compound
concentrations on the enzyme activity.
Experimental
General experimental procedures
NMR spectra were acquired in a Varian Mercury
1
Plus BB apparatus operating at 300.059 MHz for H and
1
3
7
2
5.457 MHz for C. The spectra were recorded in
To determine the plasma cholinesterase (BuChE),
heparinized fresh blood was centrifuged at 2000 rpm for
10 min for the sedimentation of red blood cells, and 20 μL
of the supernatant (plasma) were added to 12 mL of a
-3
0 mg cm solutions of CDCl , with a probe temperature
3
of ca. 300 K and TMS (tetramethylsilane) as reference.
Melting points were determined with a Micro-Química
apparatus model MQAPF-301 and are uncorrected.
-1
100 mmol L phosphate buffer solution at pH 8.0.Amounts
of 3.0 mL of this solution were transferred to vials and
40 μL of test compounds were preincubated with the
HRMS analyses were performed for new 2-arylamino-
cyclohexyl N,N-dimethylcarbamates. The compounds
were dissolved in a solution of 50% (v/v) chromatographic
grade acetonitrile (Tedia, Fairfield, OH, USA), 50% (v/v)
deionized water and 0.1% formic acid. The solutions were
infused directly and individually into the ESI (electrospray
ionization) source by means of a syringe pump (Harvard
-
1
enzymes for 10 min at 30 ºC. Next, 25 μL of 10 mmol L
-1
DTNB solution and 20 μL of 75 mmol L acetylthiocholine
iodide solution were added. Enzyme activity was measured
as for total cholinesterase.
−1
Apparatus) at a flow rate of 10 μL min . ESI(+)-MS and
tandem ESI(+)-MS/MS were acquired using a hybrid high-
Ligand modeling
-1
resolution and high accuracy (5 μL L ) MicrOTOF-Q II
mass spectrometer (Bruker Daltonics) under the following
conditions: capillary and cone voltages were set to +3500 and
The molecular modeling calculations were performed
33
with Gaussian 03 package programs. Energy potential
surfaces were obtained for all the compounds at theory
level HF/6-31G to determine the position of lowest
energy adopted by the groups in space. The lowest
energy structures were optimized at a high level of theory
[B3LYP/6-311++G(d,p)] and characterized as absolute
minima by vibrational frequency calculations. NBO
calculations (version 5.0) were performed to evaluate
the hyperconjugative interactions using theory level
B3LYP/6-311++G(d,p).
+
40 V, respectively, with a de-solvation temperature of
o
200 C. For data acquisition and processing, Micro-TOF
software(BrukerDaltonics)wasused.Thedatawerecollected
in the m/z range of 50-400 at the speed of two scans per s,
providing the resolution of 50,000 (FWHM) at m/z 200.
Procedures for the synthesis and purification of all
compounds are described in the Supplementary Information
section.
Cholinesterase inhibition bioassay
Molecular docking
The inhibitory activity of cholinesterases (AChE and
BuChE) was evaluated in fresh human blood by Ellman’s
Molecular docking studies were performed using the
AutoDock 4.2.3 program implemented at the interface
PyRx 0.9. The compounds under study were docked to
1GQR crystallographic structures, AChE complexed with
rivastigmine, and 1POM, BuChE complexed with choline
32
36
modified spectrophotometric method. For measurement,
-
1
1
0 mL of a 100 mmol L phosphate buffer solution
pH 8.0 and 10 μL of heparinized fresh blood were used.

1
806
Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
ions, selected from the Protein Data Bank (PDB). For each
PDB file, molecules of water and other ligands (except the
main ligands rivastigmine and choline ion) were removed.
Redocking calculations were performed to validate the
parameters that had been chosen. The compound structures
2. Oehlrich, D.; Berthelot, D. J. C.; Gijsen, H. J. M.; J. Med. Chem.
2011, 54, 669.
3. Martorana, A.; Esposito, Z.; Koch, G.; CNS Neurosc. Ther.
2010, 16, 235.
4. Lane, R. M.;Potkin, S. G.;Enz,A.;Int. J. Neuropsychopharmacol.
2006, 9, 101.
33
were drawn and optimized with the Gaussian 09 program
as described in the Ligand Modeling section.
5. Benzi, G.; Moretti, A.; Eur. J. Pharmacol. 1998, 346, 1.
6. Hufani, M.; Filocamo, L.; Lappa, S.; Maggi, A.; Drug Future
1997, 22, 397.
The docking calculations for the enzymes and compounds
were performed by building a 50 × 50 × 50 Å box centered
at coordinates x = 8.058, y = 64.887 and z = 61.330 for
AChE and x = 132.478, y = 114.974 and z = 38.835 for
BuChE with a grid space of 0.375 Å. The genetic algorithm
7. Tasso, B.; Catto, M.; Nicolotti, O.; Novelli, F.; Tonelli, M.;
Giangreco, I.; Pisani, L.; Sparatore, A.; Boido, V.; Carotti, A.;
Sparatore, F.; Eur. J. Med. Chem. 2011, 46, 2170.
8. Freitas, H. F.; Paz, O. S.; Castilho, M. S.; Quim. Nova 2009,
32, 2114.
(GA) was used as a standard protocol of 50 poses obtained for
the ligand, an initial population of 150 random individuals,
5
a maximum number of 2.5 × 10 energy evaluations and
9. Sekutor, M.; Majerski, K. M.; Hrenar, T.; Tomic, S.; Primozic, I.;
Bioorg. Chem. 2012, 41, 28.
4
a maximum of 2.7 × 10 generations. The docked results
within an RMSD (root mean square deviation) of 2.0Å were
clustered and the final results of each ligand were selected
considering both the embedded empirical binding free energy
evaluation and the clustering analysis.
10. Wieckowska, A.; Badja, M.; Guzior, N.; Malawska, B.; Eur. J.
Med. Chem. 2010, 45, 5602.
11. Darreh-shori, T.; Brimijoin, S.; Kadir, A.; Almkvist, O.;
Nordberg, A.; Neurobiol. Dis. 2006, 24, 326.
12. Darvesh, S.; Hopkins, D. A.; Geula, C.; Neuroscience 2003, 4,
131.
The best results were submitted to energy minimization
37
with the NAMD2 program. The force field adopted for
proteins was CHARMM C35b2-C36a2, and for the ligands,
they were generated in the same format by the SwissParam
13. Mesulam, M. M.; Guillozet,A.; Shaw, P.; Levey,A.; Duysen, G.;
Lockridge, O.; Neuroscience 2002, 110, 627.
14. Gong, C.; Liu, F.; Grundke-Iqbal, I.; Iqbal, K.; J. Biomed.
Biotechnol. 2006, 1, 11.
3
8
server. Energy minimization was simulated with the
complexes immersed in a box with water measuring at least
+
1
0 Å from the outermost surface of the protein. Either Na
15. Martorana, A.; Esposito, Z.; Koch, G.; CNS Neurosc. Ther.
2010, 16, 235.
–
or Cl counter ion was added in appropriate amounts to
neutralize the charges of the system. The temperature and
the pressure were adjusted to 300 K and 1 atm.After energy
minimization, the protein-ligand complexes were redocked
using the same docking parameters, which gave a maximum
RMSD of 1.5 Å.
16. Lane, R. M.;Potkin, S. G.;Enz,A.;Int. J. Neuropsychopharmacol.
2006, 9, 101.
17. Hufani, M.; Filocamo, L.; Lappa, S.; Maggi, A.; Drug Future
1997, 22, 397.
18. Nascimento, E. C. M.; Martins, J. B. L.; Santos, M. L.;
Gargano, R.; Chem. Phys. Lett. 2008, 458, 285.
19. Kryger, G.; Silman, I.; Sussman, J. L.; J. Physiol. 1998, 92,
Acknowledgments
1
91.
We would like to thank the FAPESP for scholarship
G. M. S. P.), the CNPQ for scholarship (M. C. B.) and
fellowships (E. A. B and N. F. H.) and financial support.
We also thank Sidney Moura for the HRMS analyses.
20. Mustazza, C.; Borioni,A.; Giudice, M. R.; Gatta, F.; Ferretti, R.;
Meneguz, A.; Volpe, M. T.; Lorenzini, P.; Eur. J. Med. Chem.
2002, 37, 91.
(
21. Bolognesi, M. L.; Bartolini, M.; Cavalli, A.; Andrisano, V.;
Rosini, M.; Minarini, A.; Melchiorre, C.; J. Med. Chem. 2004,
47, 5945.
Supplementary Information
2
2. Lin, M. C.; Hwang, M. T.; Chang, H. G.; Lin, C. S.; Lin, G.;
Experimental details and supplementary data are
available free of charge at http://jbcs.sbq.org.br as PDF file.
J. Biochem. Mol. Toxicol. 2007, 21, 348.
23. Bocca, C. C.; Rittner, R.; Höehr, N. F.; Pinheiro, G. M. S.;
Abiko, L. A.; Basso, E. A.; J. Mol. Struct. 2010, 983, 194.
24. Anand, P.; Singh, B.; Med. Chem. Res. 2012, 22, 1648.
References
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5. Bonollo, S.; Fringuelli, F.; Pizzo, F.; Vaccaro, L.; Green Chem.
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Villarroya, M.; Romero, A.; J. Med. Chem. 2010, 53, 5129.
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Submitted: May 27, 2013
Published online: September 13, 2013
FAPESP has sponsored the publication of this article.

J. Braz. Chem. Soc., Vol. 24, No. 11, S1-S8, 2013.
Printed in Brazil - ©2013 Sociedade Brasileira de Química
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Supplementary Information
SI
Molecular Modeling and Anticholinesterasic Activity of
Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
a
a
b
b
Mariane C. Bagatin, Augusto A. Cândido, Glaucia M. S. Pinheiro, Nelci F. Höehr,
c
c
a
,a
Miguel Machinski Jr., Simone A. G. Mossini, Ernani A. Basso and Gisele F. Gauze*
a
Departamento de Química, Universidade Estadual de Maringá,
Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
b
Departamento de Patologia Clínica, Faculdade de Ciências Médicas,
Universidade Estadual de Campinas, CP 6111, 13083-887 Campinas-SP, Brazil
c
Departamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá,
Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
1
2
(A) Preparation of trans-2-arylaminocyclohexanols 1a-1c
(B) Preparation of 2-bromocyclohexanone
Cyclohexanone (19 mL, 180 mmol) and water (70 mL)
were placed in a three-necked flask equipped with a stirrer
and a dropping funnel. Bromine (9.5 mL, 180 mmol)
was added dropwise to the stirred heterogeneous mixture
In a two-necked flask equipped with a magnetic stirrer,
1 mmol of appropriate arylamine (4.65 mL of aniline,
.3 g of p-anisidine or 4.8 mL of p-fluoraniline) and
o
5
6
during 1 h, and a water-ice bath (0-5 C) was employed to
cool the reaction. When addition was completed, stirring
was continued until the reaction mixture was colorless
(30-60 min). The mixture was extracted with ethyl ether
(3 × 30 mL), the combined organic layers were dried over
anhydrous Na SO . The filtrate was concentrated under
cyclohexene oxide (49 mmol) were consecutively added
in water (100 mL) and the resulting mixture was left under
o
vigorous stirring at 60 C for 48 h. The mixture was basified
-1
with 5 mol L NaOH until pH 10 and extracted with ethyl
acetate (3 × 30 mL). The combined organic layers were
dried over anhydrous Na SO . The solvent was evaporated
2
4
o
reduced pressure (73 C, 2 mmHg) to give the product
(70% yield).
2
4
and the product pure was isolated.
3
trans-2-Phenylaminocyclohexanol 1a: white solid;
(C) Preparation of 2-arylaminocyclohexanones 3a-3c
o
mp 58-59 C; 50% yield.
trans-2-(4-Methoxyphenylamino)cyclohexanol 1b:
o
brown solid; mp 67-68 C; 80% yield.
trans-2-(4-Fluoropheyilamino)cyclohexanol 1c: white
o
solid; mp 94-95 C; 50% yield.
0
.2 mol of the appropriate arylamine (18.2 mL of
aniline, 24.6 g of p-anisidine or 19.0 mL of p-fluoroaniline),
-bromocyclohexanone (23 mL, 0.2 mmol), quinoline
2.4 mL, 0.02 mmol), 0.3 mol sodium carbonate (31.8 g,
0.3 mmol) and 150 mL of 2-methoxyethanol were
2
(
*e-mail: gfgbandoch@uem.br

S2
Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
added to a dry flask and the resulting reaction mixture
was heated to reflux for 2 h. The reaction mixture was
cooled to room temperature. The solid was removed by
filtration and washed with chloroform. The filtrate was
concentrated under reduced pressure to give crude product.
Pure racemic α-arylaminocycloalkanone was obtained by
recrystallization from anhydrous methanol.
o
2
-(Phenylamino)cyclohexanone 3a: white solid;
mixture was heated to 80 C for 8 h. After this time,
dimethylcarbamyl chloride (3.1 mL, 34 mmol) was added
and the reaction was heated to reflux for 15 h.
o
mp 81-82 C; 30% yield.
2
-(4-Methoxyphenylamino)cyclohexanone 3b: brown
o
solid; mp 91-93 C; 30% yield.
The reaction mixture was cooled to room temperature,
added cold solution of sodium bicarbonate 1% (50 mL),
and extracted with ethyl ether (3 × 30 mL) and cold water
2
-(4-Fluorophenylamino)cyclohexanone 3c: white solid;
o
mp 75-76 C; 30% yield.
(
2 × 20 mL). The organic layers were dried over anhydrous
4
(
D) Preparation of cis-2-arylaminocyclohexanols 4a-4c
Na SO . The solvent was evaporated and the crude product
2
4
was obtained. Compounds 2a and 5a were purified washing
the obtained solid repeatedly with cold hexane. Pure
compounds 2b and 5b were obtained by silica gel column
chromatography (hexane/ether 8:2).
trans-2-(Phenylamino)cyclohexyl N,N-dimethylcarbamate
2a
4.55 mmol of the appropriate 2-arylaminocyclohexanone
(
0.9 g of 3a, 1.0 g of 3b and 1.0 g of 3c) were dissolved
in dried THF (tetrahydrofuran, 25 mL) in a round-bottom
flask under nitrogen atmosphere and magnetic stirring.
After lowering the temperature to -78 °C, N-selectride
(
9.1 mL, 9.1 mmol) was added, and the reactor was kept
under stirring for 4 h. The reaction mixture was allowed to
attain room temperature, after which it was hydrolyzed with
water (2.0 mL) and ethanol (5.0 mL). The organoborane
1
White solid; mp 72.9-73.5 °C; 50% yield; H NMR
(300 MHz, CDCl ) d 1.16-1.30 (m, 1H, H 6), 1.32-1.42
3
a
-1
was oxidized with 6.0 mol L NaOH (4.0 mL) and 30%
H O (5.0 mL). The aqueous phase was than saturated with
(m, 2H, H 4 and H 5), 1.40-1.52 (m, 1H, H 3), 1.68-1.91
a a a
(m, 2H, H 4 and H 5), 1.94-2.06 (m, 1H, H 3), 2.10-2.22
2
2
e
e
e
CaCO and extracted with ethyl ether. The two organic
(m, 1H, H 6), 2.67 (s, 1H, CH ), 2.81 (s, 1H, CH ), 3.32
e 3 3
3
portions were joined, dried with MgSO and carried to a
rotary evaporator where the solvent was removed.
(ddd, 1H, J 9.3, 9.3, 4.2 Hz, H2), 4.64 (ddd, 1H, J 9.3, 9.3,
4.2 Hz, H1), 6.54-6.64 (m, 2H, H2’ and H6’), 6.58-6.66
(m, 1H, H4’), 7.12 (ddd, 2H, J 8.7, 7.2, 4.2 Hz, H3’ and
4
cis-2-Phenylaminocyclohexanol 4a: white solid;
o
13
mp 70-72 C; 35% yield.
H5’); C NMR (75 MHz, CDCl ) d 24.3 (C5), 24.4 (C4),
3
cis-2-(4-Methoxyphenylamino)cyclohexanol 4b: brown
solid; mp 51-52 C; 45% yield.
31.7 (C3), 32.2 (C6), 35.9 (CH ), 36.5 (CH ), 57.2 (C2),
3
3
o
76.8 (C1), 113.1 (C2’ and C6’), 116.9 (C4’), 129.2 (C3’
and C5’), 148.2 (C1’), 157.0 (C=O); ESI-HRMS calcd.
cis-2-(4-Fluorophenylamino)cyclohexanol 4c: white
o
+
solid; mp 73-74 C; 50% yield.
for C H N O ([M + H] ): 263.1760; found: 263.1837.
15
23
2
2
(
E) Preparation of new cis- and trans-2-arylaminocyclohexyl
trans-2-(4-Methoxyphenylamino)cyclohexyl N,N-dimethyl-
carbamate 2b
5
N,N-dimethylcarbamates 2a, 2b, 5a and 5b
2
3 mmol of the appropriate 2-arylaminocyclohexanol
(
4.4 g of 1a or 1b, 5.1 g of 4a or 4b) were dissolved in
dried THF (30 mL) in a round-bottom flask under nitrogen
atmosphere and magnetic stirring. Following metalic
sodium (1.0 g, 45 mmol) addition, the resulting reaction

Vol. 24, No. 11, 2013
Bagatin et al.
S3
1
Brown oil; 50% yield; H NMR (300 MHz, CDCl ) d
2H, J 9.3 Hz, H3’ and H5’), 6.75 (d, 2H, J 9.0 Hz, H2’
3
13
1
1
1
1
.17-1.28 (m, 1H, H 6), 1.28-1.39 (m, 2H, H 4 and H 5),
and H6’); C NMR (75 MHz, CDCl ) d 20.8 (C5), 23.9
a
a
a
3
.38-1.50 (m, 1H, H 3), 1.60-1.76 (m, 2H, H 4 and H 5),
(C4), 28.4 (C3), 29.5 (C6), 36.0 (CH ), 36.4 (CH ), 54.7
a
e
e
3
3
.94-2.04 (m, 1H, H 3), 2.07-2.18 (m, 1H, H 6), 2.67 (s,
(C2), 55.8 (OCH ), 73.0 (C1), 115.0 (C2’ and C6’), 115.1
e
e
3
H, CH ), 2.82 (s, 1H, CH ), 3.28 (ddd, 1H, J 9.3, 9.3, 4.2
(C3’ and C5’), 141.3 (C1’), 152.1 (C4’), 156.1 (C=O);
3
3
+
Hz, H2), 3.72 (s, 1H, OCH ), 4.62 (ddd, 1H, J 9.3, 9.3,
ESI-HRMS calcd. for C H N O ([M + H] ): 293.1865;
3
16 25
2
3
4
2
.2 Hz, H1), 6.57 (d, 2H, J 9.0 Hz, H2’ and H6’), 6.73 (d,
found: 293.1944.
13
H, J 9.0 Hz, H3’ and H5’); C NMR (75 MHz, CDCl )
3
d 24.2 (C5), 24.4 (C4), 31.5 (C3), 32.3 (C6), 35.8 (CH3),
6.4 (CH ), 55.9 (OCH ), 58.1 (C2), 76.9 (C1), 114.5 (C2’
(F) Preparation of cis- and trans-2-(4-fluorophenylamino)
cyclohexyl N,N-dimethylcarbamate 2c and 5c
3
3
3
and C6’), 114.8 (C3’ and C5’), 142.5 (C1’), 151.8 (C4’),
+
1
56.9 (C=O); ESI-HRMS calcd. for C H N O ([M + H] ):
16
25
2
3
2
93.1865; found: 293.1946.
cis-2-(Phenylamino)cyclohexyl N,N-dimethylcarbamate 5a
12 mmol of the appropriate 2-arylaminocyclohexanol
were dissolved in dried THF (30 mL) in a round-bottom flask
under nitrogen atmosphere and magnetic stirring. Following,
sodium hydride (0.6 g, 24 mmol) was added and the resulting
o
reaction mixture was heated to 80 C for 8 h.After this time,
1
White solid; mp 68.1-70.0 °C; 39% yield; H NMR
dimethylcarbamyl chloride (3.1 mL, 34 mmol) was added
and the reaction was heated to reflux for 15 h. The reaction
mixture was cooled to room temperature, added cold solution
of sodium bicarbonate 1% (50 mL) and extracted with ethyl
ether (3 × 30 mL) and cold water (2 × 20 mL). The organic
layers were dried over anhydrous Na SO . The solvent
(
300 MHz, CDCl ) d 1.34-1.44 (m, 1H, H 4), 1.44-1.58 (m,
3
a
2
1
1
H, H 5 and H 5), 1.50-1.62 (m, 1H, H 6), 1.52-1.66 (m,
a
e
a
H, H 3), 1.69-1.77 (m, 1H, H 4), 1.85-1.94 (m, 1H, H 3),
a
e
e
.94-2.06 (m, 1H, H 6), 2.91 (s, 2H, CH ), 3.50 (ddd, 1H,
e
3
J 9.9, 3.3, 3.3 Hz, H2), 5.00-5.06 (m, 1H, H1), 6.55-6.64
2
4
(
2
m, 2H, H2’ and H6’), 6.62-6.70 (m, 1H, H4’), 7,14 (ddd,
H J 6.9, 6.9, 1.8 Hz, H3’ and H5’); C NMR (75 MHz,
was evaporated and the crude product was obtained. Pure
compounds 2b and 5b were obtained by silica gel column
chromatography (hexane/ether 8:2).
13
CDCl ) d 21.0 (C5), 23.9 (C4), 28.3 (C3), 29.6 (C6), 36.1
3
(
CH ), 36.6 (CH ), 53.6 (C2), 73.3 (C1), 113.4 (C2’ and
3 3
C6’), 117.4 (C4’), 129.4 (C3’and C5’), 147.3 (C1’), 156.4
trans-2-(4-Fluorophenylamino)cyclohexyl N,N-dimethyl-
carbamate 2c
+
(
C=O); ESI-HRMS calcd. for C H N O ([M + H] ):
1
5
23
2
2
2
63.1760; found: 263.1842.
cis-2-(4-Methoxyphenylamino)cyclohexyl N,N-dimethyl-
carbamate 5b
1
White solid; mp 54.5-56.4 °C; 35% yield; H NMR
(
300 MHz, CDCl ) d 1.19-1.29 (m, 1H, H 6), 1.29-1.40 (m,
3
a
2
H, H 4 and H 5), 1.41-1.52 (m, 1H, H 3), 1.67-1.81 (m,
a a a
2
H, H 4 and H 5), 1.96-2.05 (m, 1H, H 3), 2.09-2.19 (m,
e
e
e
1
Brown oil; 23% yield; H NMR (300 MHz, CDCl )
1H, H 6), 2.67 (s, 1H, CH ), 2.82 (s, 1H, CH ), 3.25 (ddd,
e 3 3
3
d 1.28-1.42 (m, 1H, H 4), 1.34-1.42 (m, 1H, H 5), 1.44-1.56
1H, J 9.6, 9.6, 3.9 Hz, H2), 4.63 (ddd, 1H, J 9.3; 9.3; 4.0 Hz,
a
a
(
(
m, 2H, H 5 and H 6), 1.47-1.62 (m, 1H, H 3), 1.64-1.76
H1), 6.49-6.56 (m, 2H, H2’ and H6’), 6.79-6.87 (m, 2H,
e
a
a
13
m, 1H, H 4), 1.80-1.92 (m, 1H, H 3), 1.92-2.06 (m, 1H,
H3’and H5’); C NMR (75 MHz, CDCl ) d 24.3 (C5), 24.5
e
e
3
H 6), 2.90 (s, 2H, CH ), 3.38 (ddd, 1H, J 9.6, 3.3, 3.3 Hz,
(C4), 31.7 (C3), 32.3 (C6), 35.9 (CH ), 36.5 (CH ), 58.0
e
3
3
3
H2), 3.72 (s, 1H, OCH ), 4.96-5.04 (m, 1H, H1), 6.57 (d,
(C2), 76.9 (C1), 114.0 (C2’and C6’), 115.5 (C3’and C5’),
3

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Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
1
44.6 (C1’), 155.6 (C4’), 156.9 (C=O); ESI-HRMS calcd.
6.44-6.52 (m, 2H, H2’ and H6’), 6.76-6.87 (m, 2H, H3’
+
13
for C H FN O ([M + H] ): 281.1665; found: 281.1741.
and H5’); C NMR (75 MHz, CDCl ) d 20.7 (C5), 23.8
1
5
22
2
2
3
(
C4), 28.1 (C3), 29.4 (C6), 35.8 (CH ), 36.3 (CH ), 54.2
3 3
cis-2-(4-Fluorophenylamino)cyclohexyl N,N-dimethyl-
carbamate 5c
(C2), 72.7 (C1), 114.2 (C2’and C6’), 115.5 (C3’and C5’),
143.5 (C1’), 155.6 (C4’), 156.0 (C=O); ESI-HRMS calcd.
+
for C H FN O ([M + H] ): 281.1665; found: 281.1847.
15
22
2
2
References
1
. Bonollo, S.; Fringuelli, F.; Pizzo, F.; Vaccaro, L.; Green Chem.
006, 8, 960.
. Allinger, J.; Allinger, N. L.; Tetrahedron 1958, 2, 64.
2
2
o
1
White solid; mp 66.4-67.5 C; 45% yield; H NMR
3. Liu, S.; Xie, J.; Wang, L.; Zhou, Q.; Angew. Chem., Int. Ed.
2007, 46, 7506.
(
(
(
300 MHz, CDCl ) d 1.30-1.40 (m, 1H, H 4), 1.42-1.52
3
a
m, 2H, H 4 and H 5), 1.46-1.55 (m, 1H, H 6), 1.46-1.58
4. Basso, E. A.; Abiko, L. A.; Gauze, G. F.; Pontes, R. M.; J. Org.
Chem. 2011, 76, 145.
e
a
a
m, 1H, H 3), 1.64-1.74 (m, 1H, H 5), 1.78-1.88 (m, 1H,
a
e
H 3), 1.90-2.02 (m, 1H, H 6), 2.88 (s, 2H, CH ), 3.38
5. Bernard, C.; Maurette, M. T.; Latters, A.; Bull. Soc. Chem. Fr.
1976, 1, 145.
e
e
3
(
ddd, 1H, J 9.6;3.3;3.3 Hz, H2), 4.94-5.02 (m, 1H, H1),
(
G) NMR spectra for new cis- and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates
1
13
Figure S1. H NMR (CDCl , 300 MHz) and C NMR (CDCl , 75 MHz) spectra of trans-2-(phenylamino)cyclohexyl N,N-dimethylcarbamate 2a.
3
3

Vol. 24, No. 11, 2013
Bagatin et al.
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1
13
Figure S2. H NMR (CDCl , 300 MHz) and C NMR (CDCl , 75 MHz) spectra of trans-2-(4-methoxyphenylamino)cyclohexyl N,N-dimethylcarbamate 2b.
3
3
1
13
Figure S3. H NMR (CDCl , 300 MHz) and C NMR (CDCl , 75 MHz) spectra of trans-2-(4-fluorophenylamino)cyclohexyl N,N-dimethylcarbamate 2c.
3
3

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Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
1
13
Figure S4. H NMR (CDCl , 300 MHz) and C NMR (CDCl , 75 MHz) spectra of cis-2-(phenylamino)cyclohexyl N,N-dimethylcarbamate 5a.
3
3
1
13
Figure S5. H NMR (CDCl , 300 MHz) and C NMR (CDCl , 75 MHz) spectra of cis-2-(4-methoxyphenylamino)cyclohexyl N,N-dimethylcarbamate 5b.
3
3

Vol. 24, No. 11, 2013
Bagatin et al.
S7
1
13
Figure S6. H NMR (CDCl , 300 MHz) and C NMR (CDCl , 75 MHz) spectra of cis-2-(4-fluorophenylamino)cyclohexyl N,N-dimethylcarbamate 5c.
3
3
(H) ESI-HRMS spectra for new cis- and trans-2-arylaminocyclohexyl N,N-dimethylcarbamates
Figure S7. ESI-HRMS spectra of trans-2-(phenylamino)cyclohexyl N,N-dimethylcarbamate 2a.
Figure S8. ESI-HRMS spectra of trans-2-(4-methoxyphenylamino)cyclohexyl N,N-dimethylcarbamate 2b.

S8
Molecular Modeling and Anticholinesterasic Activity of Novel 2-Arylaminocyclohexyl N,N-Dimethylcarbamates
J. Braz. Chem. Soc.
Figure S9. ESI-HRMS spectra of trans-2-(4-fluorophenylamino)cyclohexyl N,N-dimethylcarbamate 2c.
Figure S10. ESI-HRMS spectra of cis-2-(phenylamino)cyclohexyl N,N-dimethylcarbamate 5a.
Figure S11. ESI-HRMS spectra of cis-2-(4-methoxyphenylamino)cyclohexyl N,N-dimethylcarbamate 5b.
Figure S12. ESI-HRMS spectra of cis-2-(4-fluorophenylamino)cyclohexyl N,N-dimethylcarbamate 5c.