Molecular modeling and biological evaluation of 2-N,N- dimethylaminecyclohexyl 1-N′,N′-dimethylcarbamate isomers and their methylsulfate salts as cholinesterases inhibitors

Article abstract of DOI:10.1016/j.molstruc.2010.09.002

This work presents a detailed theoretical and experimental study on the inhibitory properties of 2-N,N-dimethylaminecyclohexyl 1-N′,N′- dimethylcarbamate isomers and their methylsulfate salts against the cholinesterases enzymes. The in vitro inhibition test performed by the Ellman's method showed that the salt form compounds were more active than the neutral ones in cholinesterases inhibition. The trans salt showed good selectivity towards the inhibition of erythrocyte cholinesterase with a maximum limit around 90% and 55% for the plasma cholinesterase inhibition. Molecular modeling, docking and experimental results performed in this study showed to be important initial steps toward the development of a novel pharmaceuticals in the fight against Alzheimer's disease.

Full text of DOI:10.1016/j.molstruc.2010.09.002

Journal of Molecular Structure 983 (2010) 194–199
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Molecular modeling and biological evaluation of 2-N,N-dimethylaminecyclohexyl
1-N0,N0-dimethylcarbamate isomers and their methylsulfate salts as
cholinesterases inhibitors
a
b
b
c
Cleverson C. Bocca a, , Roberto Rittner , Nelci F. Höehr , Glaucia M.S. Pinheiro , Layara A. Abiko ,
⇑
Ernani A. Basso c
a Chemistry Institute, State University of Campinas, POB 6154, 13083-970 Campinas, SP, Brazil
b Department of Clinical Pathology, School of Medical Sciences, State University of Campinas, POB 6111, 13083-887 Campinas, SP, Brazil
c Chemistry Department, University of Maringa, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2010
Received in revised form 1 September 2010
Accepted 1 September 2010
Available online 7 September 2010
This work presents a detailed theoretical and experimental study on the inhibitory properties of 2-N,N-
dimethylaminecyclohexyl 1-N0,N0-dimethylcarbamate isomers and their methyl sulfate salts against the
cholinesterases enzymes. The in vitro inhibition test performed by the Ellman’s method showed that the
salt form compounds were more active than the neutral ones in cholinesterases inhibition. The trans salt
showed good selectivity towards the inhibition of erythrocyte cholinesterase with a maximum limit
around 90% and 55% for the plasma cholinesterase inhibition. Molecular modeling, docking and experi-
mental results performed in this study showed to be important initial steps toward the development
of a novel pharmaceuticals in the fight against Alzheimer’s disease.
Keywords:
Alzheimer’s disease
Carbamates
Molecular modeling
Docking and Ellman’s method
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
inhibition on human health, agrochemistry, and chemical agents
[6]. AChE inhibitors are widely employed both in the symptomatic
Cholinesterases are serine hydrolases that have important roles
in cholinergic neurotransmission and are involved in other nervous
systems functions, and in neurodegenerative diseases [1,2]. They
are divided into two subfamilies according to their substrate and
inhibitor specificities: acetylcholinesterase (AChE; EC 3.1.1.7) and
butyrylcholinesterase (BChE; EC 3.1.1.8).
Human acetylcholinesterase (hAChE) is responsible for the
hydrolysis of acetylcholine released at synaptic cleft and neuro-
muscular junction in response to nerve action potential [3]. Subse-
quent accumulation of acetylcholine at neural synapses and
neuromuscular junctions results in paralysis, seizures, and others
symptoms of cholinergic syndrome [4,5]. It is among the most effi-
cient enzymes, with a turnover number of >104 sꢀ1. Recent re-
search interest regarding this enzyme is not only due to this high
catalytic efficiency, but also due to a broad implication of AChE
treatment of diseases involving acetylcholine depletion, like Alz-
heimer’s disease (AD), glaucoma, and myasthenia gravis, and as
pesticides for agricultural purposes [7].
Butyrylcholinesterase is present in numerous vertebrate tissues
and its physiological role remains unclear [8,9]. Human butyrylch-
olinesterase (hBChE) is toxicologically relevant because it hydro-
lyzes or scavenges a wide range of toxic esters, including heroin,
cocaine, carbamates pesticides, organophosphorus pesticides, and
nerve agents [10].
The X-ray structure of hAChE are characterized by a deep and
narrow gorge which penetrates halfway into the enzyme, with
the catalytic site of about 4 Å from its base [11]. In addition to
the catalytic triad residue Glu334, two other amino acids are lo-
cated near the bottom of the gorge: Glu202, located next to the cat-
alytic Ser203, and Glu450, 9 Å away. These two residues are part of
hydrogen-bond network in the hAChE active center [12].
Alzheimer’s disease has been recognized as one of the most se-
vere conditions affecting the aged and is life-threatening for this
group of people [13]. The disease is characterized by neural loss,
synaptic damage, neutritic and vascular plaques. At the cellular le-
vel, AD is associated mainly with reduces levels of synaptic acetyl-
choline (ACh) [14].
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BChE, butyr-
ylcholinesterase; DTNB, 5,50-dithiobis(2-nitrobenzoic acid); PES, potential energy
surface; CBS, complete basis set; ZPE, zero point energy; LGA, Lamarckian Genetic
Algorithm.
⇑
Corresponding author.
E-mail address: ccbocca@iqm.unicamp.br (C.C. Bocca).
0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2010.09.002

C.C. Bocca et al. / Journal of Molecular Structure 983 (2010) 194–199
195
Increasing the cholinergic neurotransmission capacity consti-
tutes the fundamental mechanism of the drugs utilized for the
treatment of Alzheimer’s disease [15]. It is possible through the
inhibition of the acetylcholinesterase enzyme (AChE), which func-
tions in cholinergic synapses of the central and peripheral nervous
systems, where its principal biological role is termination of im-
pulse transmission by rapid hydrolysis of acetylcholinesterase
[16]. Thus, the anti-cholinesterase agents represent the drugs of
choice for the treatment of Alzheimer’s disease [17].
Cholinesterase inhibitors of the carbamate type have been
known for decades. Towards the end of the 19th century, physo-
stigmine found medicinal use in the treatment of glaucoma. More
recently, physostigmine has been explored for AD treatment [18].
However, the severity of the side effects associated with high doses
of this compound has spurred the search for other carbamate cho-
linesterase inhibitors that are safe and better tolerated [19].
The aim of this study was to synthesize some carbamates (Fig. 1)
structurally analogous to neostigmine, which has known anti-cho-
linesterase activity, and through the in vitro inhibition tests evalu-
ate the potential cholinesterases inhibition. Docking experiments
were performed with the aim to propose a compounds binding
model in the active site of the hAChE enzyme, which is directly in-
volved in AD. We hope through this work, increase the pharmaco-
logical candidates for the disease combat.
2. Experimental section
2.1. Synthesis
It is important to make clear that the neutral compounds pro-
posed in this paper have already been subject of synthesis and
structural characterization studies by our research group [20].
The compounds were synthesized following the route in Fig. 2.
The individual steps are well described in the literature. Cyclohexa-
none was reacted with bromine to obtain 2-bromocyclohexanone
[21], which was placed in an autoclave with N,N-dimethylamine
solution to give 2-N,N-dimethylaminecyclohexanone [22]. Reduc-
tion of this ketone with lithium aluminum hydride [23] gave a mix-
ture of cis and trans (25:75) 2-N,N-dimethylaminecyclohexanol,
which were subsequently reacted with N,N-dimethylcarbamyl chlo-
ride [24] to obtain the cis an trans-2-N,N-dimethylaminocyclohexyl
1-N0,N0-dimethylcarbamate (compounds 1 and 2).
O
O
11
9
N
O
7
N
O
10
N
N
1
6
2
3
8
5
O
4
1
2
N
O
O
O
N
O
N
O
N
N
N
neostigmine
CH3SO4
CH3SO4
1'
2'
Fig. 1. Chemical structures of neostigmine and synthesized compounds, namely cis and trans-2-N,N-dimethylaminecyclohexyl 1-N0,N0-dimethylcarbamate (1 and 2,
respectively) and cis and trans-2-N,N,N-trimethylammoniumcyclohexyl 1-N0,N0-dimethylcarbamate methylsulfate (10 and 20, respectively).
O
O
O
Br
N
Br2
HN(CH3)2, H2O
135ºC/P
0-5 ºC
THF LiAlH4
OH
OH
N
N
(CH3)2SO4
THF, reflux
Naº, THF
1' + 2'
1 + 2
Cl(CO)N(CH3)2
Fig. 2. Synthetic route for obtaining of studied compounds.

196
C.C. Bocca et al. / Journal of Molecular Structure 983 (2010) 194–199
were subsequently reacted with dimethyl sulfate in order to obtain
their salts (compounds 10 and 20).
2.2. Biological assay
Cholinesterases (AChE and BChE) activities were measured by
Ellman et al. [25] modified method [26]. For measurements of ba-
sal levels of human cholinesterases we used a 20 mL of a 4.5 M
phosphate buffer solution pH 7.6 containing 0.1 M DTNB [5,50-
dithiobis(2-nitrobenzoic acid), Sigma–Aldrich Co.] and 20.0 lL of
heparinized fresh blood. 4.00 mL of this solution (erythro-
cyte + plasma) was transferred to a vial and the remainder was
centrifuged at 3000 rpm for the sedimentation of red blood cells
and, 4.00 mL of the supernatant (plasma) was transferred to an-
other vial and both the samples were kept incubated at 30 °C
throughout the procedure. A solution 1.0 M of eserine (Sigma–Al-
drich Co.) was used as control (vial containing starting solution
and eserine). After 10 min incubated, 1.00 mL of 1.5 M acetylthi-
ocholine iodide (Sigma–Aldrich Co.) solution was added to the
samples and after another 10 min incubated 50.0
solution was added to stop the reaction.
lL of eserine
Fig. 3. Illustrative chart summarizing the experimental procedure used to evaluate
the inhibition potential of the synthesized compounds against the cholinesterase.
(A) basal level measurements; (B) carbamate inhibition tests.
The same procedure was used in the enzymes inhibition test.
However 50.0 L of different synthesized compounds concentra-
l
tions were added to start the reaction in order to analyze the inhi-
bition percentage in relation to the control solution (solution
containing eserine).
The isomers were isolated through elution of the reaction prod-
ucts in a silica flash column (acetone/acetate 7:3). The isomers
100
100
cis (1)
trans (2)
(a)
(b)
cis (1)
trans (2)
2
2
y = y0 + (A/w*sqrt(PI/2)) *exp(-2*((x-xc)/w )
y = y0 + (A/w*sqrt(PI/2)) *exp(-2*((x-xc)/w )
y = y0 + (A/w*sqrt(PI/2))*exp(-2*((x-xc)/w )
y = y0 + (A/w*sqrt(PI/2)) *exp(-2*((x-xc)/w )
2
2
80
60
40
20
0
80
60
40
20
0
0
20
40
60
80
0
20
40
60
80
Concentração(mg/mL)
Concentration (mg/mL)
100
80
60
40
20
0
100
80
60
40
20
0
(c)
(d)
cis (1')
trans (2')
cis (1')
trans (2')
2
2
2
y = y0 + (A/w*sqrt(PI/2))*exp(-2*((x-xc)/w )
y = y0 + (A/w*sqrt(PI/2))
y = y0 + (A/w*sqrt(PI/2)) *exp(-2*((x-xc)/w )
y = y0 + (A/w*sqrt(PI/2))
2
*exp(-2*((x-xc)/w )
*exp(-2*((x-xc)/w )
0
20
40
60
80
100
0
20
40
60
80
100
Concentration (mg/mL)
Concentration (mg/mL)
Fig. 4. Effects of synthesized compounds on inhibition of plasma (a and c) and erythrocyte cholinesterase (b and d) from the human fresh blood.

C.C. Bocca et al. / Journal of Molecular Structure 983 (2010) 194–199
197
Table 1
The change in absorbance at 412 nm was recorded through Hp
Agilent-8453 UV–VIS spectrophotometer and the IC50 values were
determined graphically by plotting percent inhibition versus con-
centration of inhibitor.
Relative energies (in kcal/mol) and mole fractions (n) for the conformers of the
studied compounds.
Conformational equilibrium
cis (1)
trans (2)
2.3. Molecular modeling
E1ea–E1ae
1.64
n1ae
0.94
E2aa–E2ee
2.41
n2ee
0.98
cis (10)
trans (20)
2.3.1. Ligand modeling
0
0
0
0
0
0
E1 ea–E1 ae
n1 ae
E2 aa–E2 ee
n2 ee
The molecular modeling calculations were performed with pro-
grams from the GAUSSIAN 03 package [27]. The stable geometries
of all compounds were obtained by calculating the potential en-
ergy surface (PES) through the HF/3-21G level of theory. Then,
the most stable geometries were optimized by density functional
theory (DFT) calculations with the B3LYP hybrid functional, which
consists of the nonlocal exchange functional of Becke’s three-
parameter set [28] and the nonlocal correlation functional of Lee
et al. [29]. Dunning’s basis set (aug-cc-pVDZ) was used to carry
out these calculations. It is defined as a correlation consistent basis
set that contains all the correlating functions which decrease the
correlation energies by similar amounts, as well as all correlation
functions that decrease the energy by large amounts [30]. These
sets are compact, converge systematically to the CBS limit, and
are well defined with respect to the increase in both size and accu-
racy [31]. Stationary points were fully optimized and characterized
by vibrational frequency calculations, which also provided zero-
point vibrational energies (ZPE).
7.3
ꢂ1.0
3.57
ꢂ1.0
(low activity) for the plasma than erythrocyte cholinesterase inhi-
bition the trans salt showed a maximum inhibition around 55%,
while the maximum limit for the erythrocyte cholinesterase inhi-
bition was near 90%.
The cis salt (10) showed no selectivity, inhibiting the BuChE over
95% and AChE above 75% at maximum concentration (IC50 = 20.1
and 21.8 mg/mL, respectively).
The neutral compounds (1 and 2) were not selective and
showed a small inhibition at maximum concentration. Although
these compounds present low values of IC50 (trans-13.7 mg/mL
and cis-16.0 mg/mL) for plasma cholinesterase, the observed inhi-
bition at maximum concentration was around 50%. For erythrocyte
cholinesterase these compounds were less active, providing values
2.3.2. Docking
Docking experiments were performed using Autodock 4.0 [32].
The search method used was a Lamarckian Genetic Algorithm
(LGA) with 100 LGA runs [33]. The number of individuals in each
population was 50. The maximum number of energy evaluations
was set at 2.5 ꢁ 106. Other parameters were left to their respective
default values. During this searching process, the ligands (e.g. sub-
strates) and the Ser203, Glu202, His447 and Trp86 enzyme residues
were regarded as flexible. The grid box was settled at the center of
the flexible residues and the box size was normally set at 60, 60, and
60 Å (x, y, and z) at resolution of 0.375 Å. The complex optimizations
were performed by using the GROMOS96 parameters set imple-
mented in the Swiss-PdbViewer 4.0.1 program [34].
3. Results and discussion
3.1. Biological results
The in vitro inhibition test performed by the Ellman method
was modified in order to evaluate the viability of the studied com-
pounds in the enzymatic inhibition. This procedure can be best
understood through Fig. 3.
In these experiments we can observe the inhibition potential of
each compound against butyrylcholinesterase (found in plasma)
and acetylcholinesterase (found in red blood cells). We will give
greater emphasis to acetylcholinesterase inhibition results, since
this enzyme is the main target in the AD symptoms treatment.
The inhibition results are plotted in the graphs (a–d) demon-
strated in Fig. 4. The IC50 values for each compound were obtained
through the inhibition percentage versus concentration curve. We
carried out a polynomial fit, since the experiment follows the Lam-
bert–Beer law.
Analyzing Fig. 4 we can see clearly that the salt form com-
pounds (4c and 4d) were more active than the neutral ones in cho-
linesterase inhibition (4a and 4b). The trans salt (20) showed good
selectivity towards the inhibition of erythrocyte cholinesterase
(4d) with IC50 = 21.8 mg/mL against IC50 = 47.4 mg/mL for plasma
cholinesterase inhibition (4c). Besides having a higher IC50 value
Fig. 5. Optimized structures for the conformers of studied compounds.

198
C.C. Bocca et al. / Journal of Molecular Structure 983 (2010) 194–199
of IC25 = 15.1 and 18.0 mg/mL for the trans (2) and cis (1) com-
pounds, respectively. At maximum concentration these com-
pounds not reached 50% of the erythrocyte enzymatic inhibition.
the neutral and protonated forms is due to an increase in size that
the additional methyl group provides, causing an increase in repul-
sive interactions (syn – diaxial-1,3).
The preference of ‘‘ae” conformers in 1 and 10 compounds can
be explained based on the experimental proportions of individual
substituents in the cyclohexane ring. The experimental proportions
of the equatorial conformers in these equilibriums are 80% [24,35]
and 93% [36] for –O(C@O)N(CH3)2 and –N(CH3)2, respectively, and
this behavior prevails when the two groups are combined. The car-
bamate group has a large volume but the atoms are relatively dis-
tant from the ring. On the other hand, the entire amine group is
close enough for the ring to cause a larger repulsion. These results
are consistent with previous works published by our research
group about these compounds [20,37].
3.2. Theoretical analysis of the conformational equilibrium
In Table 1 are showed the relative energies and calculated pop-
ulations (ni) for each conformer of studied compounds.
These values were obtained by approximating the Gibbs (
D
G0)
energy difference by the enthalpy difference at 0 K (
D
H0), that in-
cludes electronic, nuclear repulsion and zero-point energies and
using van’t Hoff equation:
D
G0 ¼ ꢀRT ln K
where K (=nax/neq) is the equilibrium constant, R (=1.987 cal
Kꢀ1 molꢀ1) is the gas constant and T (set as 298.15 K) is the absolute
temperature. Fig. 5 presents the optimized geometry structure for
all compounds.
3.3. Enzyme-inhibitor interactions
The docking experiments were performed with the aim of pro-
posing an enzyme-inhibitor interactions model, which can provide
a good approach of what is experimentally observed in this biolog-
ical mechanism. Is important emphasize that we use only the most
stable conformers of each isomer for the docking calculations.
According to Table 2 the compounds in salt form (10 and 20) had
the lowest docking energy for the AChE binding site when com-
pared with neutral ones. The observed energy difference was the
1.6 kcal/mol favoring the 20 compound. This energy difference is
smaller compared with the neutral compounds (4.6 and 3.1 kcal/
mol for 1 and 2 compounds, respectively). The complex optimized
structures showed the same energy order as the docking structures.
The energy difference favoring the compound 20 can be under-
stood on the basis of compounds interactions in the enzyme active
site. Fig. 6 shows the docked structures for all compounds. The
compounds showed the carbamate group oriented toward the
Ser203 residue at the ranging distance from 4.2 to 4.5 Å. This
behavior was expected since this residue is responsible for acetyl-
choline hydrolysis and main target of the inhibitors of AChE.
According to Table 1 all the compounds present ‘‘ae” and ‘‘ee” as
majoritary conformers. These preferences may be explained in
terms of steric repulsion between the individual substituents and
the hydrogens at 1 and 3 arrangements (syn – 1,3-diaxial interac-
tions). When the substituents adopt equatorial positions (ee), these
repulsions are avoided and otherwise (both axial positions (aa)),
these interactions are maximized. We can now understand the
conformation preference in 2 an 20 compounds, where both sub-
stituents occupy equatorial positions (ee) leading the steric repul-
sion minimization. The difference of about 1.0 kcal/mol between
Table 2
Docking relative energies (kcal/mol) and relative complex energy minimization (in
kcal/mol) for the studied compounds.
Energetic parameters
Neutral
Salt
cis (1)
trans (2)
cis (10)
trans (20)
Erel-docking
Erel-complex
4.6
78.5
3.1
47.6
1.6
41.3
0.0
0.0
Fig. 6. Stereoview of the active site gorge (catalytic triad residues are Ser 203, His 447 and Glu 202) of human AChE (Protein Data Base code 1b41) complexed with
synthesized compounds. The inhibitors are depicted as large ball-and-stick model for emphasis, with carbon atoms colored gray, oxygen atoms colored red, and nitrogen
atoms colored blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C.C. Bocca et al. / Journal of Molecular Structure 983 (2010) 194–199
199
Compound 20 presented important interactions, which contributed
to the high stability of this compound in the enzyme active site.
The positive nitrogen atom is stabilized by the Glu202 residue
through a salt bridge formation (2.9 Å), while the nitrogen atom
from carbamate group is involved in a strong hydrogen bond with
hydroxyl group in the Tyr337 residue (2.3 Å). The cyclohexane ring
is stabilized by the van der Waals interaction provided by the
carbon chain from Trp86 residue (4.2 Å).
to CNPq for fellowships (to E.A.B., R.R. and N.F.H.) and scholarships
(to G.M.S.P. and L.A.A.). Toxicology Control Center assistance in the
Ellman’s method experiments is also gratefully acknowledged.
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The in vitro inhibition test performed by the Ellman’s method
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neutral ones in cholinesterase inhibition. The trans salt showed
good selectivity towards the inhibition of erythrocyte cholinester-
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linesterase inhibition. The cis salt showed no selectivity, inhibiting
the BuChE over 95% and AChE above 75% at maximum concentra-
tion. The neutral compounds were not selective and showed a
small inhibition at maximum concentration.
Molecular modeling, docking and experimental results per-
formed in this study showed be important initial steps toward
the development of a novel pharmaceuticals in the fight against
Alzheimer’s disease.
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Acknowledgments
The authors are grateful to FAPESP (Grant 05/59649-0) for
financial support of this work and for a fellowship (to C.C.B.), and