Chiral bistacrine analogues: Synthesis, cholinesterase inhibitory activity and a molecular modeling approach

Article abstract of DOI:10.21577/0103-5053.20170074

Cholinesterase enzymes are important targets for the therapy of Alzheimer’s disease. Tacrine-based dual binding site cholinesterases inhibitors are potential disease-modifying anti-Alzheimer drug candidates. In the present work, we described the synthesis of a series of chiral homo- and heterodimers of bis(7)-tacrine connected by a heptylene chain as a spacer with the methyl substituent at the C-3 position of the alicyclic region of tacrine nucleus and/or a chlorine atom attached to the C-6. Friedl?nder cyclocondensation between (R) or (S) 3-methylcyclohexanone prepared from monoterpene pulegone and o-aminobenzoic acids in the presence of POCl₃ afford 9-chloroacridines as intermediates, which were used to the synthesis of homo- and heterodimers. All compounds demonstrated to be potent inhibitors of acetylcholinesterase (AChE) at low nanomolar concentration and showed selectivity for AChE over butyrylcholinesterase (BuChE). Furthermore, the affinity difference between enantiomeric bis(7)-tacrine analogues series indicated some degree of stereoselectivity in the active site of AChE for chiral bis-cognitin compounds.

Full text of DOI:10.21577/0103-5053.20170074

http://dx.doi.org/10.21577/0103-5053.20170074
J. Braz. Chem. Soc., Vol. 28, No. 11, 2218-2228, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Chiral Bistacrine Analogues: Synthesis, Cholinesterase Inhibitory Activity and a
Molecular Modeling Approach
João P. B. Lopes,a Jessie S. da Costa,a Marco A. Ceschi,*,a Carlos A. S. Gonçalves,b
Eduardo L. Konrath,c Ana L. M. Karl,d Isabella A. Guedesd and Laurent E. Dardenne*,d
aInstituto de Química, Universidade Federal do Rio Grande do Sul,
Av. Bento Gonçalves, 9500, Campus do Vale, 91501-970 Porto Alegre-RS, Brazil
bDepartamento de Bioquímica, Universidade Federal do Rio Grande do Sul,
Rua Ramiro Barcelos, 2600, Prédio Anexo, 90035-003 Porto Alegre-RS, Brazil
cFaculdade de Farmácia, Universidade Federal do Rio Grande do Sul,
Av. Ipiranga, 2752, Santana, 90610-000 Porto Alegre-RS, Brazil
dLaboratório Nacional de Computação Científica,
Av. Getulio Vargas, 333, 25651-075 Petrópolis-RJ, Brazil
Cholinesterase enzymes are important targets for the therapy of Alzheimer’s disease.
Tacrine-based dual binding site cholinesterases inhibitors are potential disease-modifying
anti-Alzheimer drug candidates. In the present work, we described the synthesis of a series of
chiral homo- and heterodimers of bis(7)-tacrine connected by a heptylene chain as a spacer with
the methyl substituent at the C-3 position of the alicyclic region of tacrine nucleus and/or a chlorine
atom attached to the C-6. Friedländer cyclocondensation between (R) or (S) 3-methylcyclohexanone
prepared from monoterpene pulegone and o-aminobenzoic acids in the presence of POCl3 afford
9-chloroacridines as intermediates, which were used to the synthesis of homo- and heterodimers.All
compounds demonstrated to be potent inhibitors of acetylcholinesterase (AChE) at low nanomolar
concentration and showed selectivity forAChE over butyrylcholinesterase (BuChE). Furthermore,
the affinity difference between enantiomeric bis(7)-tacrine analogues series indicated some degree
of stereoselectivity in the active site of AChE for chiral bis-cognitin compounds.
Keywords: bistacrine, chiral, cholinesterases, synthesis, molecular docking
Introduction
of ACh emerge as a symptomatic treatment to relieve
these symptoms.4-6 The current therapeutic options for
AD are limited to three inhibitors of acetylcholinesterase
(AChEI):4-8 donepezil (Aricept®), rivastigmine (Exelon®)
and galantamine (Razadyne®, Reminyl®). In addition,
the N-methyl-D-aspartate (NMDA) receptor antagonist
memantine (Namenda®) is also used.9-11 However, none of
these therapeutic options represent a real cure.
The cholinesterase enzymes acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE) are enzymes
which are found in the central nervous system (CNS) and
catalyze the hydrolysis of ACh efficiently but at different
rates.9-12 However, as AD progresses, the activity of AChE
decreases, while that of BuChE significantly increases and
may even surpass the AChE activity.13,14
Alzheimer’s disease (AD) is one of the leading causes of
death among elderly people in the World, and its treatment
remains a challenge for the pharmaceutical community.
Nearly a million new cases per year it is expected to
emerge by 2050.1,2 The proposed cholinergic hypothesis
of cognitive impairment has been accepted for decades to
explain AD and is characterized by the loss of cholinergic
basal forebrain, and their projections to the cerebral
cortices.2,3 According to this hypothesis, the memory
and cognitive decline well-marked in AD result from a
deficit of the important neurotransmitter acetylcholine
(ACh). In this context, the inhibition of cholinesterase
enzymes (ChEs) that are responsible for the hydrolyses
The acetylcholine binding site of AChE is located
at the base of a deep hydrophobic channel measuring
*e-mail: mceschi@iq.ufrgs.br; dardenne@lncc.br

Vol. 28, No. 11, 2017
Lopes et al.
2219
approximately 20 Å in length. It is formed by a catalytic
anionic site (CAS) composed by the catalytic triad Ser200,
His440 and Glu327 and the anionic subsite which is
defined by Trp84, Tyr130, Tyr330, and Phe331 amino acid
residues.15 At the entrance of the gorge, there is a specific
peripheral anionic site (PAS) that exhibits an important
conformational flexibility. It is composed of a set of aromatic
residues Tyr70, Tyr121, Trp279, Tyr334 and the negatively
charged Asp72.16 Furthermore, a line of recent evidences
suggests that BuChE plays a key role in the etiology and
progression of neurodegenerative diseases such asAD.17-20
It is suggested that BuChE is a key player in brain areas
that influence the aggregation of neuritic Aβ plaques due
to the correlation between BuChE polymorphisms and
the progression of cognitive impairment in dementia with
Lewy bodies (DLB) and AD.21-23 It is also believed that
BuChE is particularly important in individuals with more
severe dementia, since its activity is increased with disease
development. Therefore, compounds that can interact
specifically with PAS or CAS residues are important in ChE
inhibition and may help in prevention of Aβ aggregation
facilitated by AChE.24-26
hydrophobic interactions with the targeted enzymes.3
Computational studies performed about twenty years ago
by Pang et al.14 tested the strategy of a double interaction
of an inhibitor with the enzyme.38 The authors found that
methylene chain spacers joining two units of tacrine moiety
allowed a double interaction of the compound with the
enzyme by binding simultaneously to the CAS and PAS.14,38-40
These results lead the authors to perform the synthesis
of alkylene-linked bis-tacrine compounds (Figure 1).40
Among the compounds obtained, the heptylene-linked
bis-tacrine (2), also called bis(7)-cognitin or bis(7)-tacrine
was found to be almost fifteen hundred times more potent
againstAChE than tacrine. These findings were ratified with
crystallographic studies.41 In the binding mode observed for
the dimer, a THA component is located at the CAS region,
close to the enzyme catalytic triad, while the other THA
component binds to PAS at the entrance of the catalytic
gorge.42 After Pang et al.14 first report, several examples of
homo- or hetero-dimeric cholinesterase inhibitors (ChEIs),
containing units of tacrine linked by an oligomethylene
chain, have appeared in the literature.43
Tacrine (1, THA) or 9-amino-1,2,3,4-tetrahydroacridine
(Figure 1), sold under the name of Cognex, was the
first approved drug introduced in AD therapy. THA is a
reversible inhibitor and known as a classical cholinesterase
inhibitor (ChEI) pharmacophore.27-30 However, its extensive
use was limited since it showed various side effects and
toxicity. Tacrine showed other pharmacological activities
such as the blockage of potassium channels and inhibition
of the neuronal uptake of noradrenaline, dopamine, and
serotonin.31,32 Therefore, many efforts have been made for the
synthesis of new tacrine analogues, with only a few dealing
with the synthesis of chiral THA derivatives, although
they showed important pharmacological activities.30,33-35 In
Figure 1, we showed the regions of the structure of THA that
can be modified by bioisosterism strategy.3 The quinoline
nucleus present in (a) is the pharmacophore group in THA.
This aromatic region usually interact with AChE binding
site through cation-, ion-dipole- and hydrogen bonding
interactions. The pyridin-4-amine nucleus, interrelated by
the resonance of two aromatic rings, confers higher acidity
to the primary amine (b). The amine becomes a hydrogen
bond donor instead of a hydrogen bond acceptor since its
non-bonding electron pair is involved in the resonance
of the aromatic system. According to experimental and
theoretical studies, the pyridine nitrogen (region (c)) is
protonated under physiological conditions (pKa = 9.836
experimentally measured and pKa = 9.83 1.47 according to
the Epik37 prediction), conferring to tacrine some basicity. The
alicyclic region (d) has low polarity and is able to establish
Figure 1. Structures of tacrine, bis(7)-tacrine and chiral dimers.
In June 2016, Schmidt et al.44 synthesized mono- and
dimeric tacrine derivatives with different substitution
patterns of tacrine moiety and linker lengths combining a
set of substituents at aromatic region (a) and the alicyclic
region (d). Dimers, especially, were found to have an
antiplasmodial activity in the nanomolar concentration
range and so regarded as new potent antimalarials.
We have previously described the synthesis of a series
of chiral terpenic tacrine analogues as AChEIs using chiral

2220
Chiral Bistacrine Analogues
J. Braz. Chem. Soc.
ketonessynthesizedfromnaturalmonoterpenesinanoptically
active form.30 Within the studied series, (3R)-9-amino-
3-methyl-1,2,3,4-tetrahydroacridine was obtained in a
completely regioselective manner from a cyclodehydration
reaction with (R)-(+)-3-methylcyclohexanone and
anthranilonitrile in the presence of BF3.Et2O as catalyst.45
Despite the plethora of synthetic and natural ChEIs,
different extents of chiral selectivity have been observed
for reversible, irreversible or pseudo-irreversible
inhibitors.10,46-48 For instance, several studies of structural
elements of ChEs are being gradually unraveled by X-ray
crystallography, kinetic experiments and computational
techniques.10 However, the lack of chiral prototypes as
ChEIs in both enantiomeric forms is limited and demand
further studies.
Friedländercyclocondensationwith3-methylcyclohexanone
(4) and o-aminobenzoic acids 5 in the presence of POCl3 to
afford 9-chloroacridines (6a-6c) regioselectively in good
yields 60-80% (Scheme 1).
Next, the nucleophilic aromatic substitution reaction
between 9-chloroacridines (6a-6c) and 1,7-diaminoheptane
afforded the chiral homodimers (3a-3b) 63-81% as shown
in Scheme 2. The synthesis of chiral heterodimers required
the preparation of precursor 9-(1,7-diaminoheptyl)-
1,2,3,4-tetraydroacridine (7) containing tacrine nucleus and
the amino group separated by spacer chain, for subsequent
SNAr reaction with the 9-chloroacridine 6a. The reaction
was performed according to a previously reported protocol
affording (3c-3d) in good yields 44-80% as depicted in
Table 1.
As part of our search of novel ChEIs based on tacrine
units.30,49-51 we describe in the present study the synthesis of a
series of chiral homo- and heterodimers of bis(7)-tacrine (3,
Figure 1) with the methyl substituent at C-3 position
(alicyclic region (d)) and/or chlorine atom in the C-6 position
(aromatic region (a)) connected by a heptylene chain as
spacer. The compounds were tested as inhibitors of AChE
and BuChE, which were evaluated by molecular docking.
The inhibitory activities against AChE and BuChE of
new chiral bis(7)-tacrine dimers are reported in Table 1, and
are expressed as IC50 values. Bis(7)-tacrine was synthesized
according to literature protocol and taken as a control
compound in the set of experiments.40,50
The inhibitory activities against AChE and BuChE
of new chiral bis(7)-tacrine dimers, together with that of
bis(7)-tacrine taken as a reference compound in the set of
experiments, are reported in Table 1, and are expressed as
IC50 values.
Results and Discussion
Both enantiomeric bis(7)-tacrine analogues series were
found to be potent inhibitors of cholinesterases with IC50
in the nanomolar concentration scale. The homodimer
(R,R)-3b (X = Cl) was the most active AChE inhibitor in
the homodimers series and the most active of all compounds
was the heterodimer (R)-3d (X = Cl) with IC50 of 4.73 nM
(entry 3) and 2.80 nM (entry 7), respectively, both of them
more active than bis(7)-tacrine (entry 9). The selectivity for
AChE was observed for all compounds, and some dimers
(R,R)-3b and (R)-3c did not inhibit BuChE enzyme. When
comparing the homodimers and heterodimers containing
The chiral 3-methylcyclohexanones were obtained from
natural monoterpene pulegone, commercially available in
(R)-(+)- and (S)-(−) enantiomers. Due to cost consideration,
we first adapted all reaction protocol in a small scale using
the readily and abundantly available (R)-(+)-pulegone.
Pulegone retro-aldol reaction afforded (R)-(+)- and (S)-(−)
enantiomers in 70% yield and the observed optical activities
were +13°and −15°, respectively.
In order to synthesize the bis(7)-tacrine homodimers
(3a-3b) and heterodimers (3c-3d) we performed the
Scheme 1. Synthesis of 9-chloro- and 9-(1,7-diaminoheptyl)-acridine derivatives, 6 and 7.

Vol. 28, No. 11, 2017
Lopes et al.
2221
Scheme 2. Synthesis of chiral bis(7)-tacrine homodimers (3a-3b) and heterodimers (3c-3d).
Table 1. Inhibitory activity on AChE and BuChE, and IC50 ratio of chiral bis(7)-tacrine dimers
IC50c / nM
entry
Compound
X
Yielda / %
[α]D20 b
IC50 ratio BuChE/AChE
AChE
BuChE
80.03 8.46
128.06 5.31
n.a.
1
2
3
4
5
6
7
8
9
(R,R)-3a
(S,S)-3a
(R,R)-3b
(S,S)-3b
(R)-3c
H
H
73
63
81
69
80
68
44
46
−
+18° (c 1.50)
–17° (c 1.54)
+14° (c 0.57)
–12° (c 0.59)
+5° (c 0.74)
–4° (c 0.73)
+10° (c 1.12)
–4° (c 0.85)
−
11.21 3.41
7.64 0.45
4.73 0.67
16.08 1.85
76.23 15.16
13.73 1.12
2.80 0.92
9.97 1.01
5.33 0.48
7.14
16.76
−
Cl
Cl
H
114.59 2.85
n.a.
7.13
−
(S)-3c
H
38.03 3.49
44.82 3.06
92.43 7.07
38.30 10.93
2.77
16.0
9.27
7.18
(R)-3d
Cl
Cl
−
(S)-3d
bis(7)-tacrine
aAll yields refer to purified product; boptical rotations were obtained in CH2Cl2; c 95% confidence limits. n.a.: not active.
the H and Cl substituent it is observed that the presence of
chlorine atom increased the inhibition of AChE activity,
except for the (S,S)-3b homodimer. On the other hand,
this substitution affected the inhibition of BuChE in
different ways, decreasing the activity of (R)-series of
homodimers and (S)-series heterodimers, but increasing
the activity of (R)-series of heterodimers, and did not affect
significantly the (S)-series of homodimers. Regarding the
stereoselectivity, it was observed that the AChE showed a
preference for (R)-series of homo and heterodimers with
chlorine atom in the tacrine moiety (entries 3 and 7) and
for the (S)-series of dimers without this chlorine atom
(entries 2 and 6). The affinity difference between the
stereoisomers indicates some degree of stereoselectivity in
the active site of AChE for chiral bis-cognitin compounds.
However, in the same series, BuChE showed a strong
stereoselectivity for (S,S)-3b (with chlorine) and (S)-3c.
According to the binding affinity predictions (i.e.,
GlideScore, DockTScore), all compounds were predicted
to be highly potent inhibitors of AChE in the range of
low nanomolar concentration (Table 2) obtaining only
small differences between the predicted affinities. The
most potent inhibitor (i.e., (R)-3d) obtained the best score
according to the DockTScore predictions. The lowest
energy binding mode of all inhibitors were predicted with
the 2CKM conformation of AChE (residue numbers of
Tetronace californica AChE are cited in plain text with
the corresponding numbers of Torpedo californica AChE
in parenthesis and in italics), with an overall binding
mode similar to the co-crystallized inhibitor bis(7)-tacrine
(Figure 2). The main characteristic of this binding mode is
the parallel packing of a tacrine moiety with Trp276 (279)
and Tyr67 (70) from PAS and the conserved interactions
with CAS.

2222
Chiral Bistacrine Analogues
J. Braz. Chem. Soc.
Table 2. Results of docking and binding affinity prediction according to the
GlideScore and DockTScore, and IC50 ratio of chiral bis(7)-tacrine dimers
His437 (440) main chain, (ii) quinoline system sandwiched
between Trp81 (84) and Phe327 (330) interacting through
pi-stacking interactions, (iii) terpene ring oriented to
CAS. The chlorine atom in the 6-position is predicted to
interact with the allosteric hydrophobic site formed by
Trp429 (432), Met433 (436) and Ile436 (439), while the
methyl group is oriented to CAS. The main difference
between R and S configurations is that the former provides
the methyl group near Glu196 (199) and the conserved
water HOH2062, while the last allows the methyl group
to be located near Ser197 (200) on a larger cavity more
exposed to the solvent. Despite the differences of the binding
modes, it is not clear the influence of such interactions for
the binding affinities observed for R and S compounds.
In the case of heterodimers (Figure 4), the tacrine
moiety with substituents exhibited a preference for the
bottom of the gorge, performing the same interactions
observed for tacrine with CAS and previously described.
According to the docking results, it is possible to
conclude that: (i) the presence of a chlorine atom increases
binding affinity due to the hydrophobic interactions with
CAS and a possible halogen bond with Glu275 (278) in
PAS, (ii) for homodimers with the presence of chlorine,
the methyl group in PAS is predicted to be exposed to
the solvent and probably contributes negatively for the
binding affinity. Despite the small differences, the predicted
affinities provided by DockTScore (Table 2) follow
qualitatively the experimental results when comparing
R × S and Cl × H compounds. Furthermore, it is probable
Compound
(R,R)-3a
(S,S)-3a
(R,R)-3b
(S,S)-3b
(R)-3c-7
(S)-3c-6
(R)-3d
X
H
AChEa / nM
11.21 3.41
7.64 0.45
4.73 0.67
16.08 1.85
76.23 15.16
13.73 1.12
2.80 0.92
9.97 1.01
5.33 0.48
GlideScoreb
–14.554
–14.994
–14.549
–14.467
–14.291
–14.521
–14.526
–14.817
–15.333
DockTScoreb
–14.030
–14.076
–14.207
–14.123
–13.867
–13.834
–14.223
–14.076
–13.907
H
Cl
Cl
H
H
Cl
Cl
−
(S)-3d
Bis(7)-tacrine
a
95% confidence limits; bpredicted binding affinities given in kcal mol-1.
The overall binding modes of the compounds are very
similar, interacting with both CAS and PAS (Figures 2-4).
In the case of homodimers (Figure 3), one tacrine moiety
is packed between Trp276 (279) and Tyr67 (70) from
PAS through pi-stacking interactions (Figure 3A). For the
compounds where the chlorine atom is present, the halogen
is oriented toward Glu275 (278), probably interacting
through a halogen bond.At the opposite side of tacrine, the
methyl group is totally exposed to the solvent, which might
decrease the binding affinity. The other tacrine moiety
interacts in the bottom of the gorge (Figure 3B) performing
the conserved interactions observed for tacrine: (i) hydrogen
bond between charged nitrogen from pyridine with the
Figure 2. Overall binding modes predicted for (A) the heterodimer (R)-3d and (B) the homodimer (R,R)-3b, superposed with the experimental conformation
of bis(7)-tacrine (colored green) observed in the 2CKM complex. Structural waters are represented as red spheres.

Vol. 28, No. 11, 2017
Lopes et al.
2223
Figure 3. Predicted binding modes of the homodimers (R,R)-3b (yellow) and (S,S)-3b (blue) with the 2CKM conformation ofAChE (Tetronace californica
AChE numbering). The chlorine atoms are highlighted with dashed green line. Structural waters are represented as red spheres.
that there is a cooperativity effect between the chlorine
atom and the methyl group that we were incapable to
explore through docking experiments, requiring more
sophisticated studies such as molecular dynamics and
quantum calculations for further analyses.
overall binding modes of the compounds are very similar,
interacting with both CAS and PAS of AChE. Despite the
differences of the binding modes observed for the R and S
configurations, it is not clear the influence of the resulting
interactions for the binding affinities observed for R and S
compounds.
Conclusions
Experimental
In summary, we have synthesized a new series of chiral
bis(7)-tacrines and tested its ability to inhibit cholinesterase
enzymes. Both enantiomeric bis(7)-tacrine analogues series
were found to be potent inhibitors of AChE and BuChE
with IC50 in the nanomolar concentration scale, according
to the AChE binding affinity predictions (i.e., GlideScore,
DockTScore), possessing only small differences between
the predicted affinities for all tested compounds. Regarding
the stereoselectivity, it was observed that theAChE showed
a preference for (R)-series of homo and heterodimers with
chlorine atom in the tacrine moiety and for the (S)-series of
dimers without this chlorine atom. The affinity difference
between the stereoisomers indicates some degree of
stereoselectivity in the active site of AChE for chiral
bis-cognitin compounds. In a docking approach, the
Chemistry
All melting points were determined in open glass
capillaries using a Büchi M-565 apparatus. Infrared (IR)
spectra were recorded on a Varian 640-IR spectrometer
in KBr disks. H and 13C nuclear magnetic resonance
1
(NMR) spectra were recorded in CDCl3 solution on a
VarianVNMRS 300 MHz spectrometer. The assignment of
chemical shifts is based on standard NMR experiments (1H;
13C-APT (attached proton test); 1H,1H-COSY (correlation
spectroscopy); H,13C-HMQC (heteronuclear multiple
1
quantum correlation)). Chemical shifts (d) are given in part
per million from the peak of tetramethylsilane (d 0.00 ppm)
as internal standard in 1H NMR or from the solvent peak

2224
Chiral Bistacrine Analogues
J. Braz. Chem. Soc.
Figure 4. Predicted binding modes of the heterodimers (R)-3d (orange) and (S)-3d (purple) with the 2CKM conformation of AChE (Tetronace californica
AChE numbering). The chlorine atom is highlighted with dashed green line. Structural waters are represented as red spheres.
13
of CDCl3 (d 77.00 ppm) in C NMR; and multiplicities
are given as s (singlet), d (doublet), dd (double doublet),
t (triplet), q (quartet), m (multiplet) or br (broad); coupling
constants (J) are given in Hz. High resolution mass
spectrometry with eletrospray ionization (HRMS-ESI)
data on the positive mode was collected on a Micromass
Q-Tof instrument from Waters (Manchester, UK). Samples
were infused from a 100 mL Hamilton syringe at flow rate
range from 5 to 10 mL min-1, depending on the sample. The
instrument settings were the following: capillary voltage
3000 V, cone voltage 33 V, extraction cone voltage 2.5 V,
desolvation gas temperature 100 °C. Nitrogen was used as
the desolvation gas. Methanol (Tedia, HPLC grade) was
used as solvent for the analyzed samples and filtered prior to
injection. Optical rotations were measured in a PerkinElmer
341 polarimeter with a 0.1 dm cell at a temperature of
20 °C, sample were dissolved in dichloromethane and the
concentrations are expressed in g per 100 mL of solvent.
Purification by column chromatography was carried out
on silica gel 60 (70-230 mesh). Analytical thin layer
chromatography (TLC) was conducted on aluminium
plates with 0.2 mm of silica gel 60F-254 (Macherey-
Nagel). (R)- and (S)-3-methylciclohexanone (4),45
chloroacridines (6a-6c)34,44 and compound 752 were
synthesized according to the previous literature procedure.
1
Specific rotation, melting point, H and 13C NMR data for
compounds (R)-6b and (S)-6b34
(R)-6b [α]D20 +64° (c 0.44; 20 °C, CHCl3), m.p. 58 °C;
20
(S)-6b [α]D −62° (c 0.45; 20 °C, CHCl3), m.p. 58 °C;
1H NMR (300 MHz, CDCl3) d 1.14 (d, 3H, J 6.6 Hz),
1.44-1.53 (m, 1H), 1.95-2.06 (m, 2H), 2.66-2.73 (m, 1H),
2.82-2.90 (m, 1H), 3.15-3.23 (m, 2H), 7.51 (ddd, 1H, J 8.4,
6.8 and 1.2 Hz), 7.64 (ddd, 1H, J 8.4, 6.8 and 1.5 Hz), 7.96
(d, 1H, J 8.4 Hz), 8.16 (d, 1H, J 8.4 Hz); 13C NMR (75 MHz,
CDCl3) d 21.5, 27.1, 28.9, 30.6, 42.5, 123.6, 125.4, 128.3,
128.7, 129.2, 141.3, 146.8, 159.3.
1
Specific rotation, melting point, H and 13C NMR data for
compounds (R)-6c and (S)-6c44
(R)-6c [α]D20 +60°(c 1.03; 20 °C, CH2Cl2); m.p. 88 °C;
20
(S)-6c [α]D −59° (c 0.99; 20 °C, CH2Cl2); m.p. 88 °C;
1H NMR (300 MHz, CDCl3) d 1.14 (d, 3H, J 6.0 Hz),
1.45-1.56 (m, 1H), 1.95-2.09 (m, 2H), 2.63-2.72 (m, 1H),
2.79-2.90 (m, 1H), 3.12-3.22 (m, 2H), 7.45 (dd, 1H, J 9.0
and 3.0 Hz), 7.96 (d, 1H, J 3.0 Hz), 8.06 (d, 1H, J 9.0 Hz);

Vol. 28, No. 11, 2017
Lopes et al.
2225
13C NMR (75 MHz, CDCl3) d 21.4, 27.0, 28.8, 30.5, 42.3,
123.8, 125.1, 127.3, 127.6, 128.6, 135.1, 141.2, 147.0, 160.7.
(300 MHz, CDCl3) d 1.11 (d, 6H, J 6.6 Hz), 1.33-1.52 (m,
10H), 1.58-1.70 (m, 4H), 1.85-2.08 (m, 4H), 2.50-2.82 (m,
6H), 3.03-3.17 (m, 2H), 3.47 (t, 4H, J 7.2 Hz), 3.89-4.04
(sl, 2H), 7.21-7.30 (m, 2H), 7.81-7.97 (m, 4H); 13C NMR
(75 MHz, CDCl3) d 21.4, 24.1, 26.7, 28.7, 29.0, 30.8, 31.6,
42.2, 49.4, 115.0, 118.2, 124.1, 124.6, 127.4, 133.9, 148.0,
Procedure for the preparation of chiral homodimers:
(3R)-N-(7-((R)-1,2,3,4-tetrahydro-3-methylacridin-
9-ylamino)heptyl)-1,2,3,4-tetrahydro-3-methylacridin-
9-amine ((R)-3a) and (3S)-N-(7-((S)-1,2,3,4-tetrahydro-
3-methylacridin-9-ylamino)heptyl)-1,2,3,4-tetrahydro-
3-methylacridin-9-amine ((S)-3a)
20
150.6, 159.2; (R)-3b [α]D20 +14°(c 0.57), (S)-3b [α]D −12°
(c 0.59 CH2Cl2), HRMS-ESI calcd. for [M − H]+: 589.2859,
found: 589.2866.
1,7-Diaminoheptane (0.15 mmol), (R)- or (S)-6b
(0.3 mmol) and sodium iodide (0.02 mmol) were dissolved
in 1.0 mL of n-pentanol and the mixture was maintained
at 160 °C with stirring during 48 hours. After this time,
the solvent was removed in vacuum and the mixture was
diluted in 10 mL of CH2Cl2. The organic layer was washed
with NaOH 10% and NaCl saturated aqueous solution and
dried by anhydrous Na2SO4. Dichloromethane was removed
under reduced pressure and the crude product was purified
by column chromatography on silica gel.
General procedure for the preparation of chiral heterodimers
3c-3d
Compound 7 (0.20 mmol) and 6b or 6c (0.30 mmol)
were dissolved in 1.0 mL of n-pentanol and the mixture was
maintained at 160 °C with stirring during 48 hours under
inert atmosphere. After this time, the solvent was removed
in vacuum and the mixture was diluted in 10 mL of CH2Cl2.
The organic layer was washed with NaOH 10% and NaCl
saturated aqueous solution and dried by anhydrous Na2SO4.
Dichloromethane was removed under reduced pressure and
the crude product was purified by column chromatography
(eluting with hexane-ethyl acetate-triethylamine, 90:9:1,
gradient until 50:49:1) to give the desired product.
After column chromatography (eluting with hexane-
ethyl acetate-triethylamine, 90:9:1, gradient until 50:49:1),
(R)-3a and (S)-3a were obtained as a yellow solid in 73%
yield, m.p. 78-80 °C; IR (KBr) νmax / cm-1 3410, 2922,
2856, 1615, 1581, 1561, 1499, 1418, 1357, 760; 1H NMR
(300 MHz, CDCl3) d 1.12 (d, 6H, J 6.0 Hz), 1.32-1.42 (m,
6H), 1.43-1.55 (m, 2H), 1.58-1.70 (m, 4H), 1.92-2.09 (m,
4H), 2.58-2.72 (m, 4H), 2.75-2.86 (m, 2H), 3.09-3.20 (m,
2H), 3.40-3.55 (m, 4H), 3.92 (s, 2H), 7.33 (ddd, 1H, J 8.4,
7.7 and 1.2 Hz), 7.54 (ddd, 1H, J 8.4, 7.5 and 1.2 Hz),
7.87-7.98 (m, 4H); 13C NMR (75 MHz, CDCl3) d 21.5,
24.3, 26.8, 28.8, 29.0, 30.9, 31.6, 42.4, 49.3, 115.2, 120.1,
122.8, 123.5, 128.1, 128.7, 147.6, 150.5, 158.3; (R)-3a
N-(7-((R)-1,2,3,4-Tetrahydro-3-methylacridin-9-ylamino)
heptyl)-5,6,7,8-tetrahydroacridin-9-amine ((R)-3c) and
N-(7-((S)-1,2,3,4-tetrahydro-3-methylacridin-9-ylamino)
heptyl)-5,6,7,8-tetrahydroacridin-9-amine ((S)-3c)
The heterodimers (R)- and (S)-3c were obtained
according to general procedure to give a yellow solid (80%
yield); m.p. 50-52 °C; IR (KBr) νmax / cm-1 3411, 2391,
2853, 1615, 1580, 1561, 1498, 760; 1H NMR (300 MHz,
CDCl3) d 1.11 (d, 3H, J 6.3 Hz), 1.23-1.76 (m, 10H),
1.82-2.17 (m, 6H), 2.52-2.90 (m, 5H), 3.03-3.23 (m, 4H),
3.43-3.62 (s, 4H), 4.02-4.18 (sl, 2H), 7.30-7.42 (m, 2H),
7.48-7.63 (m, 2H), 7.88-8.17 (m, 4H); 13C NMR (75 MHz,
CDCl3) d 21.4, 21.5, 22.5, 22.9, 23.1, 24.2, 24.6, 26.8, 28.5,
28.7, 29.0, 29.6, 30.4, 30.8, 31.6, 33.5, 41.8, 49.3, 114.8,
115.4, 119.7, 122.8, 122.9, 123.7, 124.2, 128.0, 128.6,
129.3, 146.7, 146.8, 151.0, 151.1, 157.6, 157.8; (R)-3c
20
[α]D20 +18° (c 1.50), (S)-3a [α]D −17° (c 1.54), HRMS-ESI
calcd. for [M − H]+: 521.3639, found: 521.3631.
Procedure for the preparation of chiral homodimers:
(6R)-N-(7-((R)-3-chloro-5,6,7,8-tetrahydro-6-methylacridin-
9-ylamino)heptyl)-3-chloro-5,6,7,8-tetrahydro-
6-methylacridin-9-amine ((R)-3b), and (6S)-N-(7-((S)-
3-chloro-5,6,7,8-tetrahydro-6-methylacridin-9-ylamino)
heptyl)-3-chloro-5,6,7,8-tetrahydro-6-methylacridin-
9-amine, ((S)-3b)
20
[α]D20 +5° (c 0.74), (S)-3c [α]D −4° (c 0.73), HRMS-ESI
calcd. for [M − H]+: 507.3482, found: 507.3672.
1,7-Diaminoheptane (0.15 mmol), (R)- or (S)-6c
(0.3 mmol) and sodium iodide (0.02 mmol) were dissolved
in 1.0 mL of n-pentanol and the mixture was maintained at
160 °C with stirring during 48 hours. After this time, the
reaction was treated and purified as 3a procedure.
N-(7-((R)-3-Chloro-5,6,7,8-tetrahydro-6-methylacridin-
9-ylamino)heptyl)-5,6,7,8-tetrahydroacridin-9-amine
((R)-3d) and N-(7-((S)-3-chloro-5,6,7,8-tetrahydro-
6-methylacridin-9-ylamino)heptyl)-5,6,7,8-tetrahydroacridin-
9-amine ((S)-3d)
Yellow solid (81% yield); m.p. 54-56 °C; IR (KBr)
max / cm-1 3411, 2924, 2854, 1607, 1489, 1419; 1H NMR
The heterodimers (R)- and (S)-3d were obtained
according to general procedure to give a yellow solid
ν

2226
Chiral Bistacrine Analogues
J. Braz. Chem. Soc.
(46% yield); m.p. 58-59 °C; IR (KBr) νmax / cm-1 3412,
2449, 2854, 1657, 1495, 1419, 1353, 760; 1H NMR
(300 MHz, CDCl3) d 1.10 (d, 3H, J 6.6 Hz), 1.27-1.47
(m, 7H), 1.49-1.67 (m, 4H), 1.81-2.04 (m, 6H), 2.49-2.78
(m, 5H), 3.01-3.17 (m, 3H), 3.37-3.57 (m, 4H), 3.81-4.07
(sl, 2H), 7.18-7.38 (m, 2H), 7.43-7.58 (m, 1H), 7.78-8.01
moieties donates a hydrogen bond to the carbonyl oxygen
of the catalytic His440 main chain.55 An optimization of
the hydrogen bond network between protein and inhibitor
were performed to adjust the orientation of the hydrogen
atoms and all hydrogen atoms were submitted to an energy
minimization process keeping the heavy atoms fixed.
Ensemble docking experiments were performed with
the molecular docking program Glide with SP (standard
precision) mode (Glide, version 6.2, Schrödinger, LLC,
New York, NY, 2014).60-62 Best pose of each ligand was
selected according to the Glide Emodel. Comparison
between different isomers and inhibitors were carried out
according to the score provided by the DockTScore.54,63 The
ligands co-crystallized were redocked into their respective
AChE conformation to validate the docking protocol.
13
(m, 4H); C NMR (75 MHz, CDCl3) d 21.6, 22.9, 23.2,
24.3, 24.9, 26.9, 28.9, 29.2, 31.0, 34.1, 42.6, 49.5, 49.6,
115.2, 116.0, 118.5, 120.3, 122.9, 123.7, 124.2, 124.7,
127.8, 128.4, 128.8, 134.0, 147.5, 148.5, 150.7, 150.8,
20
158.5, 159.5; (R)-3d [α]D20 +10° (c 1.12), (S)-3d [α]D
−4° (c 0.85); HRMS-ESI calcd. for [M − H]+: 541.3093,
found: 541.3107.
Molecular modeling studies of AChE inhibitors
Currently, there are different three-dimensional
structures of the enzyme acetylcholinesterase (AChE)
available in the Protein Data Bank complexed to a wide
variety of inhibitors.53 Due to significant conformational
changes observed on the peripheral anionic site (PAS),16
we selected five representative conformations of AChE
to conduct the ensemble docking experiments. This
methodology consists in performing the docking of small
ligands in each representative conformation of the receptor
aiming to implicitly include the protein flexibility.54 Selected
structures in this work were 1ZGC (Torpedo californica),55
2CKM (Torpedo californica),41 1Q83 (Mus musculus),56
1J07 (Mus musculus),57 and 4EY7 (Homo sapiens).58 The
inhibitors from the representative conformations of AChE
(i) interact with the CAS, (ii) interact with the PAS, and
(iii) are similar to the tacrine inhibitor. The exception is the
1J07, which is complexed with an inhibitor that interacts
exclusively with PAS. Five molecules of structural water,
preserved in the structures of AChE, were used for the
ensemble docking studies.
Isomers, protonation states and tautomers of the ligands
were predicted with LigPrep/Epik tool (LigPrep, version 2.9,
Schrödinger, LLC, New York, NY, 2014).37 Conformations
of AChE were prepared with Protein Preparation Wizard
tool (Schrödinger Suite 2014-1 Protein Preparation Wizard;
Impact version 6.2, Schrödinger, LLC, New York, NY,
2014). The protonation states of the amino acid residues
were predicted using PROPKA with pH = 7, however,
respecting the protonation state of the active site residues
already described in the literature:59 Glu202 and Glu327
negatively charged and neutral His440:ND1. Tacrine moiety
of all inhibitors were predicted to be protonated according
to Epik, obtaining a predicted pKa of 9.83 1.47 for the
tacrine molecule, which corresponds to the experimental
value of 9.8.36 This protonation state is essential since tacrine
Molecular modeling of BuChE
We were not capable to obtain good binding modes of
these compounds with BuChE. This fact is probably due to
the smaller size of the original inhibitors complexed with
the BuChE, when compared with the larger compounds
tested in this work, leading to a conformation of the binding
site with not enough space for accommodating such large
inhibitors (unfortunately there is no experimental structure
available of BuChE complexed with bis-tacrine analogues).
Microplate assay for AChE and BuChE activities
AChE and BuChE inhibitory activities were measured
using a 96-well microplate reader by the colorimetric
Ellman’s method.64 Both enzymes hydrolyze the
substrate acetyl and butyrylcholinesterase resulting in the
product thiocholine, which reacts with Ellman’s reagent
(DTNB), producing the yellow anion 2-nitrobenzoate-
5-mercaptothiocholine, detected at 405 nm. The enzymes
sources used in the present study were rat brain homogenates
(AChE) and human plasma (BuChE).All experiments were
conducted after approval by the Animal Care and Ethics
Committee of the Universidade Federal do Rio Grande do
Sul. In order to obtain the AChE source, Wistar male rats
(3 months) were decapitated, brains were quickly dissected
on ice into cortex and homogenized in cold 10 mM
Tris-HCl buffer, pH 7.2 containing 160 mM sucrose. The
homogenates were centrifuged at 10,000 × g for 10 min at
4 °C and the supernatants were used as enzyme sources. For
BuChE sources, human plasma was obtained from a female
healthy volunteer with written consent and the protocol
was approved by the Human Ethic Committee of the
Universidade Federal do Rio Grande do Sul. Samples were
centrifuged at 1,000 × g for 15 min at room temperature.

Vol. 28, No. 11, 2017
Lopes et al.
2227
The samples of both enzymes were stored at −20 °C. In the
96-well plates, the test compounds were incubated during
30 min in various concentrations at 25 °C in the presence
of 10 mM Ellman’s reagent (5,5’-dithiobis-(2-nitrobenzoic
acid)) and AChE and BuChE samples in 20 mM phosphate
buffer pH 7.4. Then, the substrates acetyltiocholine iodide
or S-butyrylthiocholine iodide (0.8 mM), respectively,
were added, and the absorbance was measured every 30 s
for 180 s at 405 nm. Protein concentration for both type of
enzymes were measured by Peterson’s65 modification of the
procedure of Lowry et al.,66 using bovine serum albumin as
standard. The concentration of each compound to produce
50% of enzyme activity inhibition (IC50) was estimated by
nonlinear regression analysis of the response-concentration
(log) curve, using the GraFit 7 program package (GraFit 7
Software; Horley, UK). Results are expressed as the
mean SEM (standard error of the mean) of at least three
different experiments performed in triplicate.
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Supplementary Information
Supplementary data (1H and 13C NMR spectra) are
available free of charge at http://jbcs.sbq.org.br as PDF file.
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Acknowledgments
We would like to thank the following Brazilian agencies
for financial support and fellowships: CNPq, FAPERGS,
CAPES, PROPESQ-UFRGS, LNCC.
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