Synthesis of tacrine-lophine hybrids via one-pot four component reaction and biological evaluation as acetyl- and butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.01.029

A novel series of tacrine-lophine hybrids was synthesized and tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC₅₀ in the nanomolar concentration scale. The key step is the one-pot four component condensation reaction of 9-aminoalkylamino-1,2,3, 4-tetrahydroacridines, benzil, different substituted aromatic aldehydes and NH₄OAc, using InCl₃ as the best catalyst. Tacrine-lophine hybrids were found to be potent and selective inhibitors of cholinesterases. As an extension of the four component approach to tetrasubstituted imidazoles, a new series of bis-(2,4,5-triphenyl-1H-imidazoles) or bis(n)-lophines was tested against AChE and BuChE.

Full text of DOI:10.1016/j.ejmech.2013.01.029

European Journal of Medicinal Chemistry 62 (2013) 556e563
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of tacrine-lophine hybrids via one-pot four component
reaction and biological evaluation as acetyl- and
butyrylcholinesterase inhibitors
Jessé Sobieski da Costa a, João Paulo Bizarro Lopes a, Dennis Russowskya,
Cesar Liberato Petzhold a, Antonio César de Amorim Borges a, Marco Antonio Ceschi a
Eduardo Konrath b, Cristiane Batassini b, Paula Santana Lunardi b,
Carlos Alberto Saraiva Gonçalves b
,
*
,
a Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Campus do Vale, 91501-970 Porto Alegre e RS, Brazil
b Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600, Prédio Anexo, 90035-003, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of tacrine-lophine hybrids was synthesized and tested for their ability to inhibit ace-
tylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50 in the nanomolar concentration
scale. The key step is the one-pot four component condensation reaction of 9-aminoalkylamino-1,2,3,4-
tetrahydroacridines, benzil, different substituted aromatic aldehydes and NH4OAc, using InCl3 as the best
catalyst. Tacrine-lophine hybrids were found to be potent and selective inhibitors of cholinesterases. As
an extension of the four component approach to tetrasubstituted imidazoles, a new series of bis-(2,4,5-
triphenyl-1H-imidazoles) or bis(n)-lophines was tested against AChE and BuChE.
Received 12 September 2012
Received in revised form
23 January 2013
Accepted 24 January 2013
Available online 4 February 2013
Keywords:
Tacrine
Ó 2013 Elsevier Masson SAS. All rights reserved.
Lophine
Four component reaction
Indium trichloride
AChE inhibitory activity
1. Introduction
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
are enzymes which constitute the group of the cholinesterases. Ace-
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder of the central nervous system (CNS), characterized by
deposits of aberrant proteins namely b-amyloid (Ab) and s-protein,
tylcholinesterase hydrolyses acetylcholine and is mainly associated
withnervesandmuscles,beingtypicallyfoundonthesynapses,while
butyrylcholinesterase hydrolyses butyrylcholine and is synthesized
by the liver, being found in large concentration in serum [8].
The current therapeutic options for the treatment of AD are limi-
ted to four AChE inhibitors [9,10], namely donepezil, rivastigmine,
oxidative stress, loss of synapses and death of cholinergic neurons
[1]. The etiopathogenesis of AD still remains unknown although, in
the last decades, several involved factors have been identified and
found consistent with its onset. Among them is the impairment of
the cholinergic system, as indicated by the presence of altered
cholinergic markers in AD patients, resulting in a pronounced
acetylcholine (ACh) deficiency which translates into a generalized
withdrawal of cholinergic tone [2e4]. Nonetheless, interest in
developing improved ChE inhibitors for treatment of AD has
increased recently, likely due to the body of empirical evidence
showing the benefits of ChE inhibitors in AD patients as well as the
lack of successful alternative approaches [5e7].
galantamine and tacrine, and one N-methyl-D-aspartate receptor
antagonist, memantine [11]. These compounds have been approved
by the U.S. Food and Drug Administration for the treatment of AD.
In healthy brains, AChE hydrolyzes the majority of acetylcholine
while BuChE plays a secondary role. However, as AD progresses, the
activity of AChE decreases, while that of BuChE significantly in-
creases in the hippocampus and temporal cortex [12].
All current kinetic models for AChE propose the existence of at
least two substrate-binding sites; the active site and the peripheral
anionic site (PAS). Inhibitors directed to the active site prevent the
binding of the acetylcholine, or its hydrolysis, by occupying the site
with a high affinity molecule, such as tacrine. The active site is
composed of two subsites. In the catalytic anionic subsite (CAS), it
* Corresponding author. Tel.: þ55 51 33087205; fax: þ55 51 33087304.
E-mail address: mceschi@iq.ufrgs.br (M.A. Ceschi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.029

J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
557
has been proposed that the choline moiety in AChE is stabilized
principally via a cation- interaction with Trp84, and also interacts
with Glu199 and Phe330 [5,13]. A similar cation- interaction oc-
The key step is a one-pot four component synthesis of 1,2,4,5-
tetrasubstituted imidazoles catalyzed by Lewis acid.
p
p
2. Results and discussion
curs in human BuChE, where Trp82 interacts with the product
choline and the substrate butyrylthiocholine [14].
2.1. Chemistry
Tacrine, the first approved drug for AD, is a potent nonselective
inhibitor of both AChE and BuChE. Although this lack of selectivity,
and hepatotoxicity, has reduced its therapeutic use, it has been the
most studied compound and remains a reference structure in the
development of new ChEs inhibitors as potential drugs for AD.
About fifteen years ago, bis(n)-tacrine analogues linked by an
alkylene chain were prepared, and it was proved that these dimeric
molecules of tacrine offered a much stronger potency and selectivity
toward AChE [15]. Bis(7)-tacrine simultaneously binds at both the
CAS and the PAS sites and provides a higher selectivity towards AChE
over BuChE. Since then, most of the dual binding site AChE inhibitors
developed so far contain at least one unit related to tacrine, probably
because of its ease of synthesis and its affinity for both the CAS and
PAS sites of AChE, and an enlarged biological profile [16].
Among achievements in synthesis and pharmacomodulation,
various tacrine-based heterodimers have been designed and stud-
ied. These tacrine heterodimers are usually obtained by connecting
natural or synthetic compounds by a linker of suitable length. Ex-
amples of tacrine-based heterodimer families include the combi-
nation of tacrine with structural units such as tacrine-huperzine A
[17e19], tacrine-donepezil [16,20,21], tacrine-indole [22], tacrinee
4-Oxo-4H-chromene [10] and tacrine-8-hydroxyquinoline [23].
In continuation with our search for new ChE inhibitors based on
tacrine units [24,25] as well as the development of new synthetic
methods using Lewis acids as catalysts [25e27], we describe in the
present study, the synthesis of a novel class of hybrids tacrine-
2,4,5-triphenyl-1H-imidazole (or tacrine-lophine hybrids) as in-
hibitors of AChE and BuChE (Scheme 1). Compounds containing
imidazole moiety have many pharmacological properties and play
important roles in biochemical processes [28,29]. As an extension
of the four component approach to tetrasubstituted imidazoles, we
also synthesized a new series of bis-(2,4,5-triphenyl-1H-imidaz-
oles) or bis(n)-lophines (Scheme 2). These non-based tacrine di-
mers were also subjected to inhibitory essays to AChE and BuChE.
The general synthesis for the novel hybrids tacrine-2,4,5-
triphenylimidazole 2aeg, 3aeg, 4aeg, 5 and 6 is depicted in
Scheme 1. The key step is the one-pot four component condensation
reaction of 9-aminoalkylamino-1,2,3,4-tetrahydroacridines, benzil,
different substituted aromatic aldehydes and NH4OAc using Lewis
acid as catalyst. According to a previously reported protocol, 9-
chloro-1,2,3,4-tetrahydroacridine was first prepared by the cycli-
zation of anthranilic acid with cyclohexanone [30,31]. In order to
introduce the side chain, different alkylenediamines reacted with 9-
chloro-1,2,3,4-tetrahydroacridine to generate the 9-aminoalkylam-
ino-1,2,3,4-tetrahydroacridines in good yields (87e95%) [32].
Despite the use of Lewis acid catalyst on the multicomponent
synthesis of 1,2,4,5-tetrasubstituted imidazoles, Sharma and co-
workers found InCl3.3H2O as a mild and effective catalyst [29].
Thus, in order to evaluate the scope of the multicomponent syn-
thesis of hybrids tacrine-2,4,5-triphenyl-1H-imidazole, we chose
compound 1c as the substrate, the aim being to examine some of
the general features of these reactions, such as Lewis acid catalysts,
stoichiometry, solvent, temperature and product conversion.
As shown in Table 1 (entry 1), we found that the use of InCl3
(15 mol%) under ethanol reflux for 96 h, afforded 2a in quite good
yield (55%). This developed protocol was applied to the synthesis of
tacrine-2,4,5-triphenyl-1H-imidazole 2aeg, 3aeg, 4aeg, 5 and 6
which have a lophine framework substituted at the meta- or para-
position of the 2-phenyl imidazole ring, as shown in Table 2. Next,
bis(n)-lophines 7aee were synthesized by using a similar protocol
as depicted in Scheme 2.
2.2. AChE and BuChE inhibition evaluation
The inhibitory activities against AChE and BuChE of new tacrine-
lophine hybrids and bis(n)lophines, together with that of bis(7)-
Scheme 1. Synthesis of tacrine-lophine hybrids.

558
J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
Scheme 2. Synthesis of bis(n)-lophines.
tacrine taken as reference, are reported in Table 2, and are
expressed as IC50 values. Tacrine-lophine hybrids were found to be
potent inhibitors of cholinesterases with IC50 in the nanomolar
concentration scale. The most active AChE inhibitor in the assayed
series of tacrine-lophines was hybrid 2b (X ¼ 4-Cl) with its IC50 of
5.87 nM (entry 2), bearing an octane chain between the tacrine and
lophine moieties, pointing out that this linker allows optimal
interaction between the aromatic fragments of such an inhibitor
and the CAS and PAS of AChE. Moreover, compound 2b showed very
high selectivity for AChE over BuChE (18.5-fold), comparing to only
2.2-fold BuChE selectivity seen with bis(7)-tacrine (entry 29). From
this dataset with octamethylene linker, compounds 2d (X ¼ 4-F)
and 2e (X ¼ 4-OMe) were active for both AChE and BuChE, com-
pounds 2f (X ¼ 4-CN) and 2g (X ¼ 4-NO2) were active only for
BuChE while 2a (X ¼ H) and 2c (X ¼ 3-Cl) were inactive toward both
enzymes.
In the dataset of tacrine-lophines with the heptane chain,
compound 3f (R ¼ 4-CN) entry 13, showed the best inhibition for
BuChE in the assayed series with IC50 of 7.10 nM and was inactive
toward AChE. It is noteworthy that 3f was an effective inhibitor of
BuChE and exhibited more potent inhibition value compared with
prototype bis(7)-tacrine. With the same chain length, compounds
3a (R ¼ H), 3b (R ¼ 4-Cl), 3d (R ¼ 4-F), 3e (R ¼ 4-OMe), 3g (R ¼ 4-
NO2) were active for both AChE and BuChE, while 3c (X ¼ 3-Cl) was
the only compound inactive for both enzymes in this dataset. In the
case of hexane chain, none of the compounds 4ae4g exhibited
AChE activity while compounds 4ae4g exhibited BuChE inhibitory
IC50 values ranging from 12.44 to 58.00 nM. Comparing products
possessing the same substituents in the 2-phenyl group fragment,
it was possible to detect the effective influence of changes in the
substructure of lophine on the inhibition of ChEs. In the case of
chloride substituent in the para-position of 2-phenyl group (entries
2, 9 and 16) when the length of the linker is made smaller from the
optimum octane chain (entry 2), the ability to inhibit AChE pro-
gressively diminished. On the other hand, for compounds con-
taining the chloride substituent in the para-position (entries 2, 9
and 16), an increased inhibitory activity against BuChE was
observed. Also, hybrids with chloride substituent in the meta-po-
sition (entries 3 and 10) were inactive toward AChE. This result
could be explained as a consequence of the fact that all phenyl rings
bonded to the imidazole core are not coplanar and the dihedral
angles between the phenyl ring planes can be increased with
substituent groups in the meta-position [33]. So, it can be inferred
that the loss of AChE activities with substituent at phenyl meta-
substituted hybrids results from a smaller
pep interaction of
lophine framework with the active sites of the enzyme. On the
other hand, this can explain the improved AChE inhibitor 2b
bearing a phenyl para-substituted which has smaller dihedral an-
gles on the phenyl ring planes of the lophine framework, favoring
binding interactions.
On the bis(n)-lophines dataset, homodimer 7c (entry 26) proved
to be an interesting compound as a non-tacrine dimer, showing
selective AChE inhibitory activity with its IC50 of 42.55 nM.
3. Conclusion
In summary, we have developed a simple and convenient
approach for the synthesis of tacrine-lophine and lophineelophine
dimers via one-pot four component condensation reaction using
InCl3 as catalyst. Several tacrine-lophine hybrids were found to be
potent and selective inhibitors of AChE and BuChE with IC50 in the
nanomolar concentration scale. The non-tacrine bis(8)-lophine (7c)
showed a selective AChE inhibitory activity with its IC50 value of
Table 1
One-pot four component condensation reaction for the synthesis of hybrid 2a under
different conditions.
Entry
Catalyst
(15 mol%)
Solvent
Temp.
(ꢀC)
Reaction
time (h)
Yield (%)a,b
1
2
3
4
5
6
7
8
9
10
InCl3
FeCl3
SnCl2$2H2O
AlCl3
BF3$Et2O
InCl3
InCl3
InCl3
InCl3
InCl3
Ethanol
Ethanol
Ethanol
Ethanol
78
78
78
78
78
120
120
25
78
78
96
96
96
96
96
19
48
180
48
96
55c
45c
41c
44c
33c
3
Ethanol
Free solvent
n-Pentanol
Ethanol
Ethanol
Ethanol
11d
12
31d
35
a
All yields refer to purified product 2a.
The reactions were performed using 1.0 equivalent of 9-octylamino-1,2,3,4-
b
tetrahydroacridine 1c, benzaldehyde, benzil and ammonium acetate.
c
After 48 h, one more equivalent of benzaldehyde, benzil and ammonium acetate
was added.
d
After 24 h, one more equivalent of benzaldehyde, benzil and ammonium acetate
was added.

J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
559
Table 2
Inhibitory activity on AChE and BuChE, and IC50 ratio of the studied compounds.
Entry
Compd
n
X
Yield
(%)a
IC50 (nM)b
AChE
IC50 ratio
BuChE/AChE
BuChE
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
8
8
8
8
8
8
8
7
7
7
7
7
7
7
6
6
6
6
6
6
6
9
10
H
55
57
37
41
74
33
39
54
33
42
52
44
53
44
53
37
29
50
33
53
55
43
33
22
42
37
29
30
n.a.
n.a.
e
4-Cl
3-Cl
4-F
4-OMe
4-CN
4-NO2
H
4-Cl
3-Cl
4-F
4-OMe
4-CN
4-NO2
H
4-Cl
3-Cl
4-F
4-OMe
4-CN
4-NO2
4-Cl
4-Cl
5.87 ꢂ 0.30
108.97 ꢂ 6.41
18.5
e
n.a.
n.a.
25.78 ꢂ 4.66
57.30 ꢂ 4.15
82.34 ꢂ 4.51
30.47 ꢂ 3.65
120.13 ꢂ 29.25
92.42 ꢂ 1.95
37.57 ꢂ 6.24
n.a.
67.31 ꢂ 4.79
61.69 ꢂ 6.71
7.10 ꢂ 2.49
76.89 ꢂ 28.13
58.00 ꢂ 5.37
29.28 ꢂ 2.67
34.75 ꢂ 2.51
55.25 ꢂ 3.66
35.98 ꢂ 3.30
29.56 ꢂ 4.37
12.44 ꢂ 2.84
n.a.
2.2
0.6
e
125.90 ꢂ 15.84
n.a.
n.a.
e
80.65 ꢂ 4.90
1.1
0.6
e
9
59.76 ꢂ 2.09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
n.a.
38.51 ꢂ 3.63
1.7
0.9
e
68.74 ꢂ 7.10
n.a.
72.10 ꢂ 2.18
1.0
e
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
e
e
e
e
e
4g
5
6
7a
7b
7c
7d
7e
e
e
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
9.03 ꢂ 1.67
e
Bis(10)-lophine
Bis(9)-lophine
Bis(8)-lophine
Bis(7)-lophine
Bis(6)-lophine
Bis(7)-tacrine
n.a.
n.a.
e
e
42.55 ꢂ 2.54
e
n.a.
e
n.a.
4.12 ꢂ 0.70
e
2.2
n.a. ¼ not active.
a
All yields refer to purified product.
ꢂ95% Confidence limits.
b
42.55 nM. We can infer that several new hybrids synthesized have
targeted both the catalytic active site and the peripheral anionic
site of AChE.
4.1. Synthesis
4.1.1. General procedure for the preparation of 9-alkylamino-
1,2,3,4-tetrahydroacridines
Intermediates 1aee were prepared according to the previous
literature procedure [32].
4. Experimental protocols
4.1.2. General procedure for the preparation of hybrids 2aeg, 3aeg,
4aeg, 5, 6
All melting points were determined in open glass capillaries
using a Büchi M-565 apparatus. IR spectra were recorded on
a Varian 640-IR spectrometer in KBr disks. 1H NMR and 13C NMR
spectra were recorded in CDCl3 solution on a Varian VNMRS
300 MHz spectrometer. The assignment of chemical shifts is based
on standard NMR experiments (1H; 13C-APT; 1H, 1H-COSY; 1H, 13C-
A mixture of 9-alkylamino-1,2,3,4-tetrahydroacridines 1aee
(0.5 mmol), benzil (0.5 mmol), aldehyde (0.5 mmol), ammonium
acetate (0.5 mmol) and InCl3 (15 mol%) in absolute ethanol (1 mL),
placed in a round bottom flask fitted with a condenser and drying
tube, was stirred at 78 ꢀC. After 48 h, one more equivalent of benzil,
aldehyde and ammonium acetate was added and the mixture was
maintained at 78 ꢀC for an additional 48 h. The solvent was
removed under reduced pressure, and the crude product purified
by column chromatography (eluting with hexane-ethyl acetate-
triethylamine, 60:39:1) to give the desired product.
HMQC). Chemical shifts (
peak of tetramethylsilane (
1H NMR or from the solvent peak of CDCl3
NMR; and multiplicities are given as s (singlet), d (doublet), t
(triplet), qn (quintet), m (multiplet) or br (broad); coupling con-
stants (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 (Man-
d) are given in part per million from the
d
¼ 0.00 ppm) as internal standard in
(d
¼ 77.23 ppm) in 13C
4.1.2.1. 1,2,3,4-Tetrahydro-N-(8-(2,4,5-triphenyl-1H-imidazol-1-yl)
octyl)acridin-9-amine (2a). Intermediate 1c was treated with ben-
zil, benzaldehyde and ammonium acetate according to general
procedure to give the desired product 2a as a yellow solid (55%
yield): m.p. 71e72 ꢀC; IR (KBr) nmax/cmꢁ1: 3346, 3058, 2927, 2854,
chester, UK). Samples were infused from a 100
mL Hamilton sy-
ringe at flow rate range from 5 to 10 L/min, depending on the
m
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 analysed samples and filtered prior to injection. Purifica-
tion by column chromatography was carried out on silica gel 60
(70e230 mesh). Analytical thin layer chromatography (TLC) was
conducted on aluminum plates with 0.2 mm of silica gel 60F-254
(MachereyeNagel).
1579, 1500, 762, 698; 1H NMR (CDCl3)
d 7.97e7.87 (m, 2H), 7.73e
7.66 (m, 2H), 7.58e7.28 (m, 12H), 7.24e7.10 (m, 3H), 4.00e3.80
(br, 1H), 3.87 (t, 2H, J ¼ 7.5 Hz), 3.40 (t, 2H, J ¼ 7.2 Hz), 3.12e3.00
(m, 2H), 2.74e2.64 (m, 2H), 1.98e1.84 (m, 4H), 1.53 (qn, 2H,
J ¼ 7.2 Hz), 1.41e1.12 (m, 4H), 1.12e0.86 (m, 6H); 13C NMR (CDCl3)
d
158.6,150.8,147.8,147.7,137.8,134.7,131.7,131.6,131.1,129.7,129.3,
129.1, 129.0, 128.9, 128.7, 128.3, 128.1, 126.9, 126.3, 123.7, 122.9,

560
J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
120.4, 116.0, 49.6, 44.7, 34.2, 31.8, 30.3, 29.0, 28.6, 26.8, 26.1, 24.9,
4.1.2.6. 4-(1-(8-(1,2,3,4-Tetrahydroacridin-9-ylamino)octyl)-4,5-
diphenyl-1H-imidazol-2-yl)benzonitrile (2f). Intermediate 1c was
treated with benzil, 4-cyanobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 2f as
a yellow solid (33% yield): m.p. 93e94 ꢀC; IR (KBr) nmax/cmꢁ1: 3402,
23.2, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 605.8463, found 605.8474.
4.1.2.2. N-(8-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
octyl)-1,2,3,4-tetrahydroacridin-9-amine (2b). Intermediate 1c was
treated with benzil, 4-chlorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 2b as
a yellow solid (57% yield): m.p. 62e63 ꢀC; IR (KBr) nmax/cmꢁ1: 3367,
3056, 2927, 2225, 1604, 1579, 771, 698; 1H NMR (CDCl3)
d 8.02 (d,
1H, J ¼ 8.7 Hz), 7.96 (d, 1H, J ¼ 8.4 Hz), 7.86 (d, 2H, J ¼ 8.5 Hz), 7.77
(d, 2H, J ¼ 8.5 Hz), 7.62e7.32 (m, 9H), 7.24e7.12 (m, 3H), 3.91 (t, 2H,
J ¼ 7.6 Hz), 3.50 (t, 2H, J ¼ 7.3 Hz), 3.20e3.04 (m, 2H), 2.75e2.60 (m,
2H), 2.00e1.83 (m, 4H), 1.56 (qn, 2H, J ¼ 7.3 Hz), 1.44e1.15 (m, 5H),
3058, 2927, 2854, 1500, 835, 762, 698; 1H NMR (CDCl3)
d 7.97e7.87
(m, 2H), 7.63 (d, 2H, J ¼ 8.4 Hz), 7.58e7.28 (m, 11H), 7.24e7.09 (m,
3H), 4.00e3.75 (br, 1H), 3.85 (t, 2H, J ¼ 7.5 Hz), 3.41 (t, 2H,
J ¼ 7.3 Hz), 3.12e2.98 (m, 2H), 2.76e2.60 (m, 2H), 2.00e1.83 (m,
4H), 1.54 (qn, 2H, J ¼ 7.3 Hz), 1.40e1.12 (m, 4H), 1.12e0.86 (m, 6H);
1.15e0.84 (m, 6H); 13C NMR (CDCl3)
d 157.3, 151.8, 146.0, 145.6,
138.9, 136.1, 134.2, 132.6, 131.1, 131.0, 129.6, 129.4, 129.3, 129.2,
128.4, 126.9, 126.8, 124.1, 123.2, 119.5, 118.8, 115.0, 112.3, 49.4, 45.1,
33.1, 31.8, 30.5, 29.1, 28.7, 26.9, 26.2, 24.7, 23.0, 22.6; HRMS-ESI:
calcd for [M ꢁ H]þ 630.8561, found 630.8572.
13C NMR (CDCl3)
d 158.6, 150.8, 147.7, 146.6, 138.1, 135.0,134.5, 131.5,
131.1, 130.6, 130.2, 130.1, 129.2, 129.0, 128.9, 128.8, 128.4, 128.2,
126.9, 126.5, 123.7, 123.0, 120.4, 116.0, 49.6, 44.8, 34.2, 31.8, 30.4,
29.0, 28.6, 26.9, 26.2, 24.9, 23.2, 23.0; HRMS-ESI: calcd for [M ꢁ H]þ
640.2910, found 640.2910.
4.1.2.7. 1,2,3,4-Tetrahydro-N-(8-(2-(4-nitrophenyl)-4,5-diphenyl-1H-
imidazol-1-yl)octyl)acridin-9-amine (2g). Intermediate 1c was
treated with benzil, 4-nitrobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 2g as
a brown solid (39% yield): m.p. 90e91 ꢀC; IR (KBr) nmax/cmꢁ1: 3422,
4.1.2.3. N-(8-(2-(3-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
octyl)- 1,2,3,4-tetrahydroacridin-9-amine (2c). Intermediate 1c was
treated with benzil, 3-chlorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 2c as
a yellow solid (37% yield): m.p. 43e44 ꢀC; IR (KBr) nmax/cmꢁ1: 3367,
3058, 2929, 2854, 1599, 1519, 1342, 698; 1H NMR (CDCl3)
d 8.35 (d,
2H, J ¼ 7.8 Hz), 8.03 (d, 1H, J ¼ 8.4 Hz), 7.99e7.88 (m, 3H), 7.60e7.30
(m, 9H), 7.25e7.12 (m, 3H), 3.95 (t, 2H, J ¼ 7.6 Hz), 3.51 (t, 2H,
J ¼ 7.2 Hz), 3.14e3.02 (m, 2H), 2.70e2.58 (m, 2H), 1.98e1.80 (m,
4H), 1.58 (qn, 2H, J ¼ 7.2 Hz), 1.44e1.15 (m, 5H), 1.15e0.80 (m, 6H);
3056, 2927, 2854, 1562, 1498, 767, 696; 1H NMR (CDCl3)
d 7.97e7.86
(m, 2H), 7.72 (s,1H), 7.60e7.27 (m,12H), 7.23e7.10 (m, 3H), 4.00e3.80
(br, 1H), 3.87 (t, 2H, J ¼ 7.5 Hz), 3.41 (t, 2H, J ¼ 7.1 Hz), 3.12e3.00 (m,
2H), 2.74e2.62 (m, 2H), 2.00e1.83 (m, 4H), 1.54 (qn, 2H, J ¼ 7.1 Hz),
13C NMR (CDCl3)
d 157.1, 151.9, 147.7, 145.8, 145.2, 139.1, 137.9, 134.2,
131.4, 131.1, 131.0, 129.7, 129.4, 129.2, 128.4, 127.0, 126.9, 124.2, 124.1,
123.3, 119.4, 114.9, 49.4, 45.2, 33.0, 31.7, 30.6, 29.1, 28.7, 26.9, 26.2,
24.7, 23.0, 22.5; HRMS-ESI: calcd for [M ꢁ H]þ 650.8439, found
650.8471.
1.40e1.13 (m, 4H),1.13e0.88 (m, 6H); 13C NMR (CDCl3)
d 158.6, 150.8,
147.7, 146.2, 138.1, 134.7, 134.4, 133.4, 131.4, 131.1, 130.2, 130.0, 129.4,
129.2,129.0,128.9,128.8,128.4,128.2,127.2,126.9,126.5,123.7,122.9,
120.4, 116.0, 49.6, 44.8, 34.2, 31.8, 30.4, 29.0, 28.6, 26.8, 26.1, 24.9,
23.2, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 640.2910, found 640.2930.
4.1.2.8. 1,2,3,4-Tetrahydro-N-(7-(2,4,5-triphenyl-1H-imidazol-1-yl)
heptyl)acridin-9-amine (3a). Intermediate 1b was treated with
benzil, benzaldehyde and ammonium acetate according to general
procedure to give the desired product 3a as a yellow solid (54%
yield): m.p. 61e62 ꢀC; IR (KBr) nmax/cmꢁ1: 3369, 3058, 2929, 2854,
4.1.2.4. N-(8-(2-(4-Fluorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
octyl)-1,2,3,4-tetrahydroacridin-9-amine (2d). Intermediate 1c was
treated with benzil, 4-fluorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 2d as
a yellow solid (41% yield): m.p. 57e58 ꢀC; IR (KBr) nmax/cmꢁ1: 3380,
1579, 1498, 762, 698; 1H NMR (CDCl3)
d 7.94e7.86 (m, 2H), 7.67 (d,
2H, J ¼ 6.9 Hz), 7.59e7.26 (m, 12H), 7.22e7.09 (m, 3H), 3.86 (t, 2H,
J ¼ 7.5 Hz), 3.96e3.76 (br, 1H), 3.42e3.28 (m, 2H), 3.14e3.00 (m,
2H), 2.70e2.56 (m, 2H), 1.98e1.80 (m, 4H), 1.43 (qn, 2H, J ¼ 7.3 Hz),
1.38e1.24 (m, 2H), 1.17e1.02 (m, 2H), 1.01e0.84 (m, 4H); 13C NMR
3056, 2927, 1500, 1224, 843, 763, 698; 1H NMR (CDCl3)
d 7.97e7.86
(m, 2H), 7.72e7.63(m, 2H), 7.58e7.28 (m, 9H), 7.23e7.10 (m, 5H),
3.98e3.78 (br, 1H), 3.84 (t, 2H, J ¼ 7.6 Hz), 3.41 (t, 2H, J ¼ 7.2 Hz),
3.12e2.98 (m, 2H), 2.76e2.64 (m, 2H), 2.00e1.84 (m, 4H), 1.54 (qn,
2H, J ¼ 7.2 Hz), 1.40e1.13 (m, 4H), 1.13e0.88 (m, 6H); 13C NMR
(CDCl3)
d 158.5, 150.6, 147.7, 147.6, 137.7, 134.6, 131.5, 131.0, 129.6,
129.2, 129.1, 128.9, 128.8, 128.6, 128.2, 128.1, 126.8, 126.3, 123.6,
122.8, 120.3, 116.0, 49.4, 44.6, 34.2, 31.5, 30.1, 28.3, 26.5, 26.0, 24.8,
23.1, 22.8; HRMS-ESI: calcd for [M ꢁ H]þ 591.8194, found 591.8218.
(CDCl3)
d
163.2 (d, J ¼ 247.3 Hz), 158.6, 150.8, 147.7, 146.8, 137.8,
134.6,131.5,131.3,131.2,131.1,129.8,129.2,128.9,128.8,128.4,128.2,
127.8 (d, J ¼ 3.3 Hz), 126.9, 126.4, 123.7, 122.9, 120.4, 116.0, 115.8 (d,
J ¼ 21.4 Hz), 49.6, 44.7, 34.2, 31.8, 30.3, 29.0, 28.6, 26.8, 26.2, 24.9,
23.2, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 623.8367, found 623.8380.
4.1.2.9. N-(7-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
heptyl)- 1,2,3,4-tetrahydroacridin-9-amine (3b). Intermediate 1b
was treated with benzil, 4-chlorobenzaldehyde and ammonium
acetate according to general procedure to give the desired product
4.1.2.5. 1,2,3,4-Tetrahydro-N-(8-(2-(4-methoxyphenyl)-4,5-diphenyl-
1H-imidazol-1-yl)octyl)acridin-9-amine (2e). Intermediate 1c was
treated with benzil, 4-methoxybenzaldehyde and ammonium ac-
etate according to general procedure to give the desired product 2e
3b as a yellow solid (33% yield): m.p. 64e65 ꢀC; IR (KBr) nmax/cmꢁ1
:
3367, 3057, 2927, 2854, 1500, 835, 762, 698; 1H NMR (CDCl3)
d
7.94e7.87 (m, 2H), 7.63 (d, 2H, J ¼ 8.7 Hz), 7.58e7.28 (m, 11H),
as a yellow solid (74% yield): m.p. 56e57 ꢀC; IR (KBr) nmax/cmꢁ1
:
7.23e7.10 (m, 3H), 3.86 (t, 2H, J ¼ 7.5 Hz), 3.34 (t, 2H, J ¼ 7.2 Hz),
3.12e3.00 (m, 2H), 2.73e2.60 (m, 2H), 1.98e1.84 (m, 4H), 1.48 (qn,
2H, J ¼ 7.2 Hz), 1.40e1.24 (m, 3H), 1.20e1.05 (m, 2H), 1.04e0.88 (m,
3349, 3056, 2929, 1612, 1250, 1027, 837, 700; 1H NMR (CDCl3)
d
7.98e7.86 (m, 2H), 7.60 (d, 2H, J ¼ 8.7 Hz), 7.58e7.28 (m, 9H),
7.24e7.08 (m, 3H), 6.99 (d, 2H, J ¼ 8.7 Hz), 3.96e3.78 (m, 3H), 3.84
(s, 3H), 3.41 (t, 2H, J ¼ 7.2 Hz), 3.12e3.00 (m, 2H), 2.74e2.64 (m, 2H),
1.98e1.84 (m, 4H), 1.54 (qn, 2H, J ¼ 7.2 Hz), 1.48e1.13 (m, 4H), 1.13e
4H); 13C NMR (CDCl3)
d 158.7, 150.8, 147.7, 146.6, 138.1, 135.0, 134.5,
131.5, 131.1, 130.6, 130.2,130.1, 129.2, 129.1, 129.0, 128.9, 128.8, 128.4,
128.3, 126.9, 126.5, 123.8, 122.9, 120.4, 116.1, 49.5, 44.8, 34.2, 31.7,
30.4, 28.5, 26.7, 26.2, 25.0, 23.2, 23.0; HRMS-ESI: calcd for [M ꢁ H]þ
626.2641, found 626.2669.
0.86 (m, 6H); 13C NMR (CDCl3)
d 160.2, 158.7, 150.9, 147.7, 137.6,
134.8, 131.9, 131.2, 130.7, 129.5, 129.2, 129.0, 128.7, 128.4, 128.2,
126.9, 126.3, 124.1, 123.7, 123.0, 120.4, 116.1, 114.2, 55.5, 49.6, 44.8,
34.3, 31.9, 30.4, 29.1, 28.7, 26.9, 26.2, 25.0, 23.2, 23.0; HRMS-ESI:
calcd for [M ꢁ H]þ 635.8725, found 635.8731.
4.1.2.10. N-(7-(2-(3-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
heptyl)-1,2,3,4-tetrahydroacridin-9-amine (3c). Intermediate 1b

J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
561
was treated with benzil, 3-chlorobenzaldehyde and ammonium
acetate according to general procedure to give the desired product
treated with benzil, 4-nitrobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 3g as
a brown solid (44% yield): m.p. 84e85 ꢀC; IR (KBr) nmax/cmꢁ1: 3411,
3c as a yellow solid (42% yield): m.p. 59e60 ꢀC; IR (KBr) nmax/cmꢁ1
:
3367, 3056, 2927, 2854, 1562, 1498, 767, 696; 1H NMR (CDCl3)
7.95e7.86 (m, 2H), 7.72 (s, 1H), 7.60e7.27 (m, 12H), 7.23e7.10 (m,
3058, 2929, 2856, 1599, 1519, 1342, 696; 1H NMR (CDCl3)
d 8.35 (d,
d
2H, J ¼ 8.4 Hz), 7.86e7.97 (m, 4H), 7.28e7.58 (m, 9H), 7.24e7.12 (m,
3H), 3.94 (t, 2H, J ¼ 7.5 Hz), 3.37 (t, 2H, J ¼ 7.2 Hz), 3.12e2.59 (m,
2H), 2.72e2.60 (m, 2H), 1.98e1.83 (m, 4H), 1.49 (qn, 2H, J ¼ 7.2 Hz),
1.42e1.22 (m, 3H), 1.20e1.08 (m, 2H), 1.08e0.90 (m, 4H); 13C NMR
3H), 4.00e3.78 (br, 1H), 3.87 (t, 2H, J ¼ 7.5 Hz), 3.35 (t, 2H,
J ¼ 7.1 Hz), 3.14e3.00 (m, 2H), 2.76e2.58 (m, 2H), 2.00e1.82 (m,
4H), 1.47 (qn, 2H, J ¼ 7.1 Hz), 1.39e1.24 (m, 2H), 1.20e1.15 (m, 2H),
1.15e0.88 (m, 4H); 13C NMR (CDCl3)
d
158.6, 150.7, 147.7, 146.2,
(CDCl3) d 158.6, 150.9, 147.8, 147.5, 145.2, 139.2, 137.9, 134.1, 131.4,
138.1, 134.7, 134.4, 133.3, 131.3, 131.1, 130.1, 130.0, 129.4, 129.2, 129.0,
128.9, 128.8, 128.4, 128.2, 127.2, 126.9, 126.5, 123.7, 122.9, 120.4,
116.1, 49.5, 44.7, 34.2, 31.7, 30.3, 28.4, 26.6, 26.1, 24.9, 23.2, 22.9;
HRMS-ESI: calcd for [M ꢁ H]þ 626.2641, found 626.2654.
131.1, 131.0, 129.7, 129.4, 129.2, 128.8, 128.6, 128.4, 127.0, 126.9,
124.2, 123.8, 122.9, 120.3, 116.1, 49.5, 45.2, 34.1, 31.7, 30.6, 28.6, 26.7,
26.3, 25.0, 23.2, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 636.8170,
found 636.8161.
4.1.2.11. N-(7-(2-(4-Fluorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
heptyl)-1,2,3,4-tetrahydroacridin-9-amine (3d). Intermediate 1b
was treated with benzil, 4-fluorobenzaldehyde and ammonium
acetate according to general procedure to give the desired product
4.1.2.15. 1,2,3,4-Tetrahydro-N-(6-(2,4,5-triphenyl-1H-imidazol-1-yl)
hexyl)acridin-9-amine (4a). Intermediate 1a was treated with
benzil, benzaldehyde and ammonium acetate according to general
procedure to give the desired product 4a as a yellow solid (53%
yield): m.p. 62e63 ꢀC; IR (KBr) nmax/cmꢁ1: 3369, 3058, 2929, 2856,
3d as a yellow solid (52% yield): m.p. 61e62 ꢀC; IR (KBr) nmax/cmꢁ1
:
3361, 3056, 2929,1500,1224, 843, 761, 698; 1H NMR (CDCl3)
d
7.95e
1579, 1500, 764, 698; 1H NMR (CDCl3)
d
7.91 (d, 1H, J ¼ 8.4 Hz), 7.84
7.86 (m, 2H), 7.70e7.62 (m, 2H), 7.59e7.28 (m, 9H), 7.24e7.09 (m,
5H), 3.96e3.76 (br, 1H), 3.84 (t, 2H, J ¼ 7.5 Hz), 3.42e3.28 (m, 2H),
3.14e2.98 (m, 2H), 2.74e2.58 (m, 2H), 2.00e1.80 (m, 4H), 1.47 (qn,
2H, J ¼ 7.2 Hz), 1.38e1.24 (m, 2H), 1.19e1.05 (m, 2H), 1.05e0.84 (m,
(d, 1H, J ¼ 8.4 Hz), 7.66 (d, 2H, J ¼ 7.3 Hz), 7.59e7.25 (m, 12H), 7.23e
7.08 (m, 3H), 3.86 (t, 2H, J ¼ 7.3 Hz), 3.79 (br,1H), 3.32e3.20 (m, 2H),
3.12e3.00 (m, 2H), 2.68e2.52 (m, 2H), 1.98e1.78 (m, 4H), 1.44e1.20
(m, 4H), 1.10e0.88 (m, 4H); 13C NMR (CDCl3)
d 158.3, 150.6, 147.6,
4H); 13C NMR (CDCl3)
d
163.1 (d, J ¼ 247.2 Hz), 158.6, 150.7, 147.6,
147.4,137.7,134.5,131.4,130.9,129.6,129.1,129.0,128.8,128.7,128.6,
128.2, 128.0, 126.7, 126.2, 123.5, 122.8, 120.2, 115.9, 49.1, 44.4, 34.0,
31.2, 30.0, 25.8, 25.7, 24.7, 23.0, 22.7; HRMS-ESI: calcd for [M ꢁ H]þ
577.7925, found 577.7941.
146.7, 137.8, 134.5, 131.4, 131.2, 131.1, 131.0, 129.7, 129.2, 128.9, 128.8,
128.3, 128.2, 127.8 (d, J ¼ 3.3 Hz), 126.8, 126.4, 123.7, 122.9, 120.3,
116.1, 115.8 (d, J ¼ 22.0 Hz), 49.4, 44.6, 34.2, 31.6, 30.2, 28.4, 26.5,
26.1, 24.9, 23.1, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 609.8098, found
609.8119.
4.1.2.16. N-(6-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
hexyl)- 1,2,3,4-tetrahydroacridin-9-amine (4b). Intermediate 1a was
treated with benzil, 4-chlorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 4b as
a yellow solid (37% yield): m.p. 71e72 ꢀC; IR (KBr) nmax/cmꢁ1: 3377,
4.1.2.12. 1,2,3,4-Tetrahydro-N-(7-(2-(4-methoxyphenyl)-4,5-diphenyl-
1H-imidazol-1-yl)heptyl)acridin-9-amine (3e). Intermediate 1b was
treated with benzil, 4-methoxybenzaldehyde and ammonium ace-
tate according to general procedure to give the desired product 3e as
a yellow solid (44% yield): m.p. 67e68 ꢀC; IR (KBr) nmax/cmꢁ1: 3378,
3056, 2931, 2856, 1500, 835, 761, 698; 1H NMR (CDCl3)
d 7.90 (d, 1H,
J ¼ 8.4 Hz), 7.85 (d, 1H, J ¼ 8.4 Hz), 7.62 (d, 2H, J ¼ 8.4 Hz), 7.56e7.27
(m, 11H), 7.23e7.10 (m, 3H), 3.85 (t, 2H, J ¼ 7.5 Hz), 3.79 (br, 1H),
3.28 (t, 2H, J ¼ 7.2 Hz), 3.10e3.00 (m, 2H), 2.68e2.58 (m, 2H), 1.98e
1.80 (m, 4H),1.44e1.25 (m, 4H),1.10e0.88 (m, 4H); 13C NMR (CDCl3)
3056, 2929, 1500, 1249, 1027, 771, 700; 1H NMR (CDCl3)
d 7.94e7.86
(m, 2H), 7.60 (d, 2H, J ¼ 9.0 Hz), 7.56e7.28 (m, 9H), 7.22e7.08
(m, 3H), 6.98 (d, 2H, J ¼ 9.0 Hz), 3.90e3.78 (m, 3H), 3.82 (s, 3H),
3.35 (t, 2H, J ¼ 7.2 Hz), 3.10e3.00 (m, 2H), 2.70e2.62 (m, 2H), 1.96e
1.84 (m, 4H), 1.47 (qn, 2H, J ¼ 7.2 Hz), 1.31 (qn, 2H, J ¼ 7.2 Hz), 1.17e
d
158.6, 150.7, 147.6, 146.5, 138.1, 135.0, 134.4, 131.4, 131.0, 130.5,
130.1, 130.0, 129.2, 129.0, 128.9, 128.8, 128.4, 128.2, 126.9, 126.5,
123.7, 122.8, 120.4, 116.2, 49.3, 44.7, 34.2, 31.4, 30.3, 26.1, 26.0, 24.9,
23.1, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 612.2373, found 612.2393.
1.04 (m, 2H), 1.04e0.88 (m, 4H); 13C NMR (CDCl3)
d 160.2, 158.7,
150.8, 147.7, 147.6, 137.7, 134.8, 131.8, 131.2, 130.7, 129.5, 129.1, 129.0,
128.7, 128.4, 128.2, 126.9, 126.3, 124.1, 123.7, 122.9, 120.4, 116.1, 114.2,
55.5, 49.5, 44.7, 34.2, 31.7, 30.3, 28.5, 26.6, 26.2, 25.0, 23.2, 23.0;
HRMS-ESI: calcd for [M ꢁ H]þ 621.8457, found 621.8468.
4.1.2.17. N-(6-(2-(3-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
hexyl)- 1,2,3,4-tetrahydroacridin-9-amine (4c). Intermediate 1a was
treated with benzil, 3-chlorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 4c as
a yellow solid (29% yield): m.p. 62e63 ꢀC; IR (KBr) nmax/cmꢁ1: 3361,
4.1.2.13. 4-(1-(7-(1,2,3,4-Tetrahydroacridin-9-ylamino)heptyl)-4,5-
diphenyl-1H-imidazol-2-yl)benzonitrile (3f). Intermediate 1b was
treated with benzil, 4-cyanobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 3f as
a yellow solid (53% yield): m.p. 78e79 ꢀC; IR (KBr) nmax/cmꢁ1: 3400,
3056, 2929, 2856, 1562, 1498, 767, 696; 1H NMR (CDCl3)
d 7.90 (d,
1H, J ¼ 8.4 Hz), 7.86 (d, 1H, J ¼ 8.4 Hz), 7.71 (s, 1H), 7.60e7.27 (m,
12H), 7.23e7.10 (m, 3H), 3.88 (t, 2H, J ¼ 7.5 Hz), 3.79 (br, 1H), 3.28 (t,
2H, J ¼ 7.2 Hz), 3.12e2.98 (m, 2H), 2.72e2.58 (m, 2H), 1.98e1.80 (m,
3056, 2929, 2225,1606,1500, 771, 700; 1H NMR (CDCl3)
d 7.95 (d,1H,
J ¼ 8.7 Hz), 7.91 (d,1H, J ¼ 8.4 Hz), 7.85 (d, 2H, J ¼ 8.5 Hz), 7.77 (d, 2H,
J ¼ 8.5 Hz), 7.59e7.30 (m, 9H), 7.24e7.12 (m, 3H), 4.20e3.80 (br, 1H),
3.91 (t, 2H, J ¼ 7.5 Hz), 3.41 (t, 2H, J ¼ 7.2 Hz), 3.14e3.02 (m, 2H), 2.72e
2.60 (m, 2H),1.98e1.83 (m, 4H),1.50 (qn, 2H, J ¼ 7.2 Hz),1.33 (qn, 2H,
J ¼ 7.5 Hz), 1.20e1.06 (m, 2H), 1.06e0.88 (m, 4H); 13C NMR (CDCl3)
4H), 1.48e1.25 (m, 4H), 1.12e0.91 (m, 4H); 13C NMR (CDCl3)
d 158.6,
150.7, 147.6, 146.2, 138.2, 134.7, 134.4, 133.3, 131.3, 131.0, 130.1, 130.0,
129.3, 129.2, 129.0, 128.9, 128.4, 128.2, 127.2, 126.9, 126.5, 123.7,
122.8, 120.4, 116.2, 49.3, 44.6, 34.2, 31.5, 30.3, 26.1, 26.0, 24.9, 23.1,
22.9; HRMS-ESI: calcd for [M ꢁ H]þ 612.2373, found 612.2372.
d
158.1,151.2,146.9,145.6,138.9,136.0,134.2,132.6,131.1,131.0,129.5,
129.3,129.1, 128.8, 128.3,126.9,126.8, 123.9,123.0,120.0,118.7, 115.7,
112.3, 49.4, 45.1, 33.7, 31.7, 30.5, 28.6, 26.7, 26.2, 24.8, 23.1, 22.8;
HRMS-ESI: calcd for [M ꢁ H]þ 616.8292, found 616.8281.
4.1.2.18. N-(6-(2-(4-Fluorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
hexyl)- 1,2,3,4-tetrahydroacridin-9-amine (4d). Intermediate 1a was
treated with benzil, 4-fluorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 4d as
a yellow solid (50% yield): m.p. 64e65 ꢀC; IR (KBr) nmax/cmꢁ1: 3355,
4.1.2.14. 1,2,3,4-Tetrahydro-N-(7-(2-(4-nitrophenyl)-4,5-diphenyl-
1H-imidazol-1-yl)heptyl)acridin-9-amine (3g). Intermediate 1b was
3056, 2929,1500,1224, 843, 764, 698; 1H NMR (CDCl3)
d 7.89 (d, 1H,

562
J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
J ¼ 8.7 Hz), 7.85 (d, 1H, J ¼ 8.4 Hz), 7.70e7.60 (m, 2H), 7.58e7.27 (m,
9H), 7.23e7.08 (m, 5H), 3.84 (t, 2H, J ¼ 7.5 Hz), 3.77 (br, 1H), 3.38e
3.20 (m, 2H), 3.12e2.98 (m, 2H), 2.68e2.54 (m, 2H), 1.98e1.80 (m,
7.58e7.28 (m, 11H), 7.24e7.09 (m, 3H), 4.00e3.78 (br, 1H), 3.85 (t,
2H, J ¼ 7.5 Hz), 3.44 (t, 2H, J ¼ 7.2 Hz), 3.12e2.98 (m, 2H), 2.76e2.63
(m, 2H), 1.98e1.84 (m, 4H), 1.59 (qn, 2H, J ¼ 7.2 Hz), 1.38e1.20 (m,
4H), 1.44e1.20 (m, 4H), 1.10e0.88 (m, 4H); 13C NMR (CDCl3)
d
163.1
4H), 1.20e0.86 (m, 8H); 13C NMR (CDCl3)
d 158.7, 150.8, 147.7, 146.5,
(d, J ¼ 247.7 Hz), 158.7, 150.6, 147.7, 146.7, 137.9, 134.5, 131.5, 131.2,
131.1, 131.0, 129.7, 129.2, 129.0, 128.8, 128.3, 128.2, 127.8 (d,
J ¼ 3.3 Hz), 126.8, 126.4, 123.7, 122.8, 120.4, 116.2, 115.8 (d,
J ¼ 22.0 Hz), 49.3, 44.6, 34.2, 31.4, 30.3, 26.1, 26.0, 24.9, 23.1, 22.9;
HRMS-ESI: calcd for [M ꢁ H]þ 595.7830, found 595.7809.
138.1, 135.0, 134.5, 131.5, 131.1, 130.5, 130.2, 130.1, 129.2, 129.0, 128.8,
128.3, 128.2, 126.9, 126.5, 123.7, 123.0, 120.4, 116.0, 49.6, 44.9, 34.3,
31.9, 30.4, 29.3, 29.2, 28.6, 27.0, 26.2, 24.9, 23.2, 23.0; HRMS-ESI:
calcd for [M ꢁ H]þ 654.3179, found 654.3195.
4.1.2.23. N-(10-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
decyl)-1,2,3,4-tetrahydroacridin-9-amine (6). Intermediate 1e was
treated with benzil, 4-chlorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 6 as
a yellow solid (33% yield): m.p. 52e53 ꢀC; IR (KBr) nmax/cmꢁ1: 3422,
4.1.2.19. 1,2,3,4-Tetrahydro-N-(6-(2-(4-methoxyphenyl)-4,5-diphenyl-
1H-imidazol-1-yl)hexyl)acridin-9-amine (4e). Intermediate 1a was
treated with benzil, 4-methoxybenzaldehyde and ammonium ace-
tate according to general procedure to give the desired product 4e as
a yellow solid (33% yield): m.p. 68e69 ꢀC; IR (KBr) nmax/cmꢁ1: 3365,
3058, 2925, 2852,1499, 834, 760, 696; 1H NMR (CDCl3)
d 7.94 (d,1H,
3056, 2931,1500,1249,1028, 764, 698; 1H NMR (CDCl3)
d
7.89 (d,1H,
J ¼ 8.7 Hz), 7.90 (d, 1H, J ¼ 8.7 Hz), 7.63 (d, 2H, J ¼ 8.4 Hz), 7.58e7.28
(m, 11H), 7.24e7.09 (m, 3H), 3.92 (br, 1H), 3.85 (t, 2H, J ¼ 7.6 Hz),
3.44 (t, 2H, J ¼ 7.2 Hz), 3.14e2.98 (m, 2H), 2.78e2.60 (m, 2H), 2.00e
1.84 (m, 4H), 1.61 (qn, 2H, J ¼ 7.2 Hz), 1.45e0.86 (m, 14H); 13C NMR
J ¼ 8.7 Hz), 7.85 (d, 1H, J ¼ 8.4 Hz), 7.60 (d, 2H, J ¼ 8.7 Hz), 7.34e7.55
(m, 8H), 7.34e7.26 (m, 1H), 7.21e7.07 (m, 3H), 6.96 (d, 2H,
J ¼ 8.7 Hz), 3.90e3.70 (br, 1H), 3.84 (t, 2H, J ¼ 7.5 Hz), 3.79 (s, 3H),
3.26 (t, 2H, J ¼ 7.2 Hz), 3.10e3.00 (m, 2H), 2.68e2.58 (m, 2H), 1.96e
1.82 (m, 4H), 1.44e1.23 (m, 4H), 1.10e0.88 (m, 4H); 13C NMR (CDCl3)
(CDCl3)
d 158.6, 150.9, 147.7, 146.5, 138.1, 134.9, 134.5, 131.5, 131.1,
130.5, 130.2, 130.1, 129.2, 129.0, 128.9, 128.8, 128.3, 128.2, 126.9,
126.5, 123.7, 123.0, 120.4, 116.0, 49.6, 44.9, 34.2, 31.9, 30.4, 29.4,
29.3, 29.2, 28.7, 27.0, 26.2, 24.9, 23.2, 23.0; HRMS-ESI: calcd for
[M ꢁ H]þ 668.3448, found 668.3459.
d
160.1, 158.6,150.6,147.7, 147.6, 137.6, 134.7, 131.7, 131.0, 130.5,129.4,
129.1, 128.9, 128.6, 128.3, 128.1, 126.8, 126.2, 124.0, 123.6, 122.8,
120.3, 116.1, 114.1, 55.4, 49.3, 44.5, 34.2, 31.4, 30.1, 26.0, 25.9, 24.8,
23.1, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ 607.8188, found 607.8179.
4.1.3. General procedure for the preparation of bis(n)-lophines 7aee
A mixture of benzil (1.0 mmol), diamine (0.5 mmol), benzal-
dehyde (1.0 mmol), ammonium acetate (1.0 mmol) and InCl3
(10 mol%) in absolute ethanol (1 mL), placed in a round bottom
flask fitted with a condenser and drying tube, was stirred under
reflux for 24 h. The solvent was removed under reduced pressure,
and the crude product purified by column chromatography
(eluting with hexane-ethyl acetate-triethylamine, 40:59:1) to
give the desired product.
4.1.2.20. 4-(1-(6-(1,2,3,4-Tetrahydroacridin-9-ylamino)hexyl)-4,5-
diphenyl-1H-imidazol-2-yl)benzonitrile (4f). Intermediate 1a was
treated with benzil, 4-cyanobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 4f as
a yellow solid (53% yield): m.p. 74e75 ꢀC; IR (KBr) nmax/cmꢁ1: 3384,
3056, 2927, 2225, 1604, 1498, 769, 696; 1H NMR (CDCl3)
d 7.90 (d,
1H, J ¼ 8.7 Hz), 7.85 (d, 1H, J ¼ 7.8 Hz), 7.84 (d, 2H, J ¼ 8.5 Hz), 7.75
(d, 2H, J ¼ 8.5 Hz), 7.58e7.28 (m, 9H), 7.24e7.12 (m, 3H), 3.91 (t, 2H,
J ¼ 7.6 Hz), 3.79 (br, 1H), 3.30 (t, 2H, J ¼ 7.3 Hz), 3.12e3.00 (m, 2H),
2.68e2.58 (m, 2H), 1.98e1.82 (m, 4H), 1.48e1.24 (m, 4H), 1.12e0.90
4.1.3.1. 2,4,5-Triphenyl-1-(10-(2,4,5-triphenyl-1H-imidazol-1-yl)
decyl)-1H-imidazole (7a). Following the general procedure, 1,10-
decanodiamine, benzil, benzaldehyde and ammonium acetate
afforded product 7a as a pale yellow solid (22% yield): m.p. 165e
166 ꢀC; IR (KBr) nmax/cmꢁ1: 3059, 2925, 2852, 1601, 1501, 1443,
(m, 4H); 13C NMR (CDCl3)
d 158.7, 150.7, 147.6, 145.6, 138.9, 136.0,
134.1, 132.6, 131.1, 131.0, 129.5, 129.4, 129.2, 129.0, 128.5, 128.3,
126.9, 126.8, 123.8, 122.8, 120.4, 118.7, 116.3, 112.4, 49.3, 45.0, 34.2,
31.5, 30.5, 26.2, 26.1, 25.0, 23.2, 22.9; HRMS-ESI: calcd for [M ꢁ H]þ
602.8023, found 602.8043.
773, 696; 1H NMR (CDCl3)
d
7.71e7.64 (m, 4H), 7.56e7.37 (m, 20H),
7.23e7.09 (m, 6H), 3.84 (t, 4H, J ¼ 7.6 Hz), 1.38e1.22 (m, 4H), 0.96e
0.78 (m, 12H); 13C NMR (CDCl3)
147.8, 137.9, 134.8, 131.8, 131.7,
4.1.2.21. 1,2,3,4-Tetrahydro-N-(6-(2-(4-nitrophenyl)-4,5-diphenyl-
1H-imidazol-1-yl)hexyl)acridin-9-amine (4g). Intermediate 1a was
treated with benzil, 4-nitrobenzaldehyde and ammonium ace-
tate according to general procedure to give the desired product
d
131.2, 129.8, 129.4, 129.2, 129.0, 128.8, 128.2, 127.0, 126.4, 44.9, 30.5,
29.0, 28.6, 26.2; HRMS-ESI: calcd for [M ꢁ H]þ 732.0039, found
732.0035.
4g as a brown solid (55% yield): m.p. 73e74 ꢀC; IR (KBr) nmax
/
cmꢁ1: 3417, 3058, 2929, 2856, 1599, 1519, 1340, 696; 1H NMR
(CDCl3)
4.1.3.2. 2,4,5-Triphenyl-1-(9-(2,4,5-triphenyl-1H-imidazol-1-yl)
nonyl)-1H-imidazole (7b). Following the general procedure, 1,9-
nonanodiamine, benzil, benzaldehyde and ammonium acetate
afforded product 7b as a pale yellow solid (42% yield): m.p. 83e
84 ꢀC; IR (KBr) nmax/cmꢁ1: 3057, 2925, 2852, 1600, 1499, 1442, 773,
d
8.32 (d, 2H, J ¼ 8.7 Hz), 7.98e7.87 (m, 3H), 7.85 (d, 1H,
J ¼ 8.1 Hz), 7.58e7.27 (m, 9H), 7.25e7.12 (m, 3H), 3.94 (t, 2H,
J ¼ 7.5 Hz), 3.73 (br, 1H), 3.29 (t, 2H, J ¼ 7.0 Hz), 3.12e2.98 (m,
2H), 2.72e2.56 (m, 2H), 1.98e1.80 (m, 4H), 1.50e1.28 (m, 4H),
1.14e0.90 (m, 4H); 13C NMR (CDCl3)
d
158.6, 150.7, 147.7, 147.5,
695; 1H NMR (CDCl3)
d
7.70e7.63 (m, 4H), 7.56e7.34 (m, 20H), 7.23e
7.08 (m, 6H), 3.84 (t, 4H, J ¼ 7.3 Hz),1.38e1.18 (m, 4H), 0.92e0.68 (m,
10H); 13C NMR (CDCl3)
147.8, 137.9, 134.7, 131.8, 131.7, 131.2, 129.7,
145.2, 139.1, 137.7, 134.1, 131.4, 131.0, 130.9, 129.6, 129.4, 129.2,
128.9, 128.5, 128.3, 127.0, 126.9, 124.1, 123.8, 122.8, 120.4, 116.2,
49.3, 45.1, 34.1, 31.5, 30.5, 26.2, 26.1, 24.9, 23.1, 22.9; HRMS-ESI:
calcd for [M ꢁ H]þ 622.7901, found 622.7902.
d
129.3, 129.2, 128.9, 128.7, 128.2, 127.0, 126.4, 44.8, 30.4, 28.7, 28.5,
26.2; HRMS-ESI: calcd for [M ꢁ H]þ 717.9770, found 717.9766.
4.1.2.22. N-(9-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)
nonyl)- 1,2,3,4-tetrahydroacridin-9-amine (5). Intermediate 1d was
treated with benzil, 4-chlorobenzaldehyde and ammonium acetate
according to general procedure to give the desired product 5 as
4.1.3.3. 2,4,5-Triphenyl-1-(8-(2,4,5-triphenyl-1H-imidazol-1-yl)
octyl)-1H-imidazole (7c). Following the general procedure, 1,8-
octanodiamine, benzil, benzaldehyde and ammonium acetate affor-
ded product 7c as a white solid (37% yield): m.p. 150e151 ꢀC; IR (KBr)
nmax/cmꢁ1: 3056, 2933, 2856, 1600, 1479, 1442, 773, 696; 1H NMR
a yellow solid (43% yield): m.p. 153e154 ꢀC; IR (KBr) nmax/cmꢁ1
:
3379, 3053, 2931, 2854, 1495, 836, 770, 697; 1H NMR (CDCl3)
(d, 1H, J ¼ 8.4 Hz), 7.89 (d, 1H, J ¼ 8.1 Hz), 7.63 (d, 2H, J ¼ 8.7 Hz),
d
7.93
(CDCl3)
d
7.65 (d, 4H, J ¼ 7.8 Hz), 7.52 (d, 4H, J ¼ 7.8 Hz), 7.48e7.34 (m,
16H), 7.23e7.08 (m, 6H), 3.81 (t, 4H, J ¼ 7.5 Hz), 1.21 (qn, 4H, J ¼ 7.5 Hz),

J.S. da Costa et al. / European Journal of Medicinal Chemistry 62 (2013) 556e563
563
0.81e0.60 (m, 8H); 13C NMR (CDCl3)
d
147.8, 137.8, 134.7, 131.7, 131.6,
Acknowledgements
131.1,129.7,129.3,129.2,128.9,128.7,128.2,126.9,126.4, 44.7, 30.3, 28.2,
26.0; HRMS-ESI: calcd for [M ꢁ H]þ 703.9501, found 703.9473.
The authors wish to thank the following Brazilian agencies for
financial support and fellowships: CNPq, FAPERGS, PROPESQ e UFRGS.
4.1.3.4. 2,4,5-Triphenyl-1-(7-(2,4,5-triphenyl-1H-imidazol-1-yl)hep-
tyl)-1H-imidazole (7d). Following the general procedure, 1,7-
heptanodiamine, benzil, benzaldehyde and ammonium acetate
afforded product 7d aswhite solid (29%yield): m.p. 84e85ꢀC; IR (KBr)
nmax/cmꢁ1: 3058, 2929, 2856, 1600, 1500, 1442, 773, 696; 1H NMR
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.029.
(CDCl3)
7.23e7.08 (m, 6H), 3.77 (t, 4H, J ¼ 7.5 Hz),1.20e1.08(m, 4H), 0.74e0.50
(m, 6H); 13C NMR (CDCl3)
147.7, 137.8, 134.7, 131.7, 131.6, 131.1, 129.7,
d
7.63 (d, 4H, J ¼ 7.2 Hz), 7.54e7.47 (m, 4H), 7.47e7.33 (m,16H),
d
129.3, 129.2, 129.0, 128.7, 128.2, 126.9, 126.4, 44.6, 30.2, 27.7, 25.9;
References
HRMS-ESI: calcd for [M ꢁ H]þ 689.9233, found 689.9232.
[1] M. Goedert, M.G. Spillantini, Science 314 (2006) 777e781.
[2] A.V. Terry Jr., J.J. Buccafusco, J. Pharmacol. Exp. Ther. 306 (2003) 821e827.
[3] R.T. Bartus, Exp. Neurol. 163 (2000) 495e529.
[4] S. Rizzo, A. Bisi, M. Bartolini, F. Mancini, F. Belluti, S. Gobbi, V. Andrisano,
A. Rampa, Eur. J. Med. Chem. 46 (2011) 4336e4343.
[5] M. Kozurkova, S. Hamulakova, Z. Gazova, H. Paulikova, P. Kristian, Pharma-
ceuticals 4 (2011) 382e418.
[6] J.J. Bornstein, T.J. Eckroat, J.L. Houghton, C.K. Jones, K.D. Green, S. Garneau-
Tsodikova, Med. Chem. Commun. 2 (2011) 406e412.
4.1.3.5. 2,4,5-Triphenyl-1-(6-(2,4,5-triphenyl-1H-imidazol-1-yl)hex-
yl)-1H-imidazole (7e). Following the general procedure, 1,6-
hexanodiamine, benzil, benzaldehyde and ammonium acetate
afforded product 7e as a white solid (30% yield): m.p. 166e167 ꢀC;
IR (KBr) nmax/cmꢁ1: 3060, 2937, 2858,1600,1500,1442, 773, 698; 1H
NMR (CDCl3)
16H), 7.23e7.08 (m, 6H), 3.71 (t, 4H, J ¼ 7.5 Hz), 1.14e0.98 (m, 4H),
0.64e0.48 (m, 4H); 13C NMR (CDCl3)
147.7, 137.8, 134.6, 131.6,
d 7.63e7.57 (m, 4H), 7.54e7.48 (m, 4H), 7.45e7.29 (m,
[7] C. Ronco, L. Jean, H. Outaabout, P.Y. Renard, Eur. J. Org. Chem. (2011) 302e310.
[8] R.T. Delfino, T.S. Ribeiro, J.D. Figueroa-Villar, J. Braz. Chem. Soc. 20 (2009)
407e428.
d
[9] D.A. Smith, Am. J. Health-syst. Pharm. 66 (2009) 899e907.
[10] M.I. Fernández-Bachiller, C. Pérez, L. Monjas, J. Rademann, M.I. Rodríguez-
Franco, J. Med. Chem. 55 (2012) 1303e1317.
[11] G.T. Grossberg, V. Pejovic, M.L. Miller, S.M. Graham, Dement. Geriatr. Cogn.
Disord. 27 (2009) 164e172.
131.5, 131.0, 129.6, 129.2, 129.1, 128.9, 128.7, 128.2, 126.9, 126.4, 44.5,
30.0, 25.3; HRMS-ESI: calcd for [M ꢁ H]þ 675.8964, found 675.8976.
4.2. In vitro assay of brain AChE and BuChE activities
[12] E.K. Perry, R.H. Perry, G. Blessed, B.E. Tomlinson, Neuropathol. Appl. Neuro-
biol. 4 (1978) 273e277.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
activities were measured by the colorimetric method of Ellman et al.
[34] with some minor modifications, adapted for determining the
effects of analogs on enzyme activity in supernatants of rat’s brain
homogenates and human plasma, respectively. Adults Wistar male
rats (2e3 months) were supplied by Department of Biochemistry-
UFRGS. Animals were housed in plastic cages and maintained at
22e23 ꢀC under a 12 h light/dark cycle (lights on at 7:00 a.m.) with
free access to food and water. All experiments were following and
approved by the Animal Care and Ethics Committee of the Uni-
versidade Federal do Rio Grande do Sul. For AChE sources, rats were
decapitated, the brain was rapidly dissected on ice into cortex and
homogenized in cold 10 mM TriseHCl buffer, pH 7.2 containing
160 mM sucrose. The homogenates were centrifugated at 10,000ꢃ g
for 10 min at 4 ꢀC; supernatants were used as AChE sources, kept into
aliquots and stored at ꢁ20 ꢀC. For BuChE sources, humanplasmawas
obtained from 5 healthy volunteers (24e60 years old) of both sexes
with written consent and the protocol was approved by the Human
Ethic Committee of the Universidade Federal do Rio Grande do Sul.
The samples were centrifugated at 1000ꢃ g for 15 min at room
temperature and aliquots were stored at ꢁ20 ꢀC.
[13] J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman,
Science 253 (1991) 872e879.
[14] Y. Nicolet, O. Lockridge, P. Masson, J.C. Fontecilla-Camps, F. Nachon, J. Biol.
Chem. 278 (2003) 41141e41147.
[15] Y.P. Pang, P. Quiram, T. Jelacic, F. Hong, S. Brimijoin, J. Biol. Chem. 271 (1996)
23646e23649.
[16] P. Camps, X. Formosa, C. Galdeano, T. Gómez, D. Muñoz-Torrero,
M. Scarpellini, E. Viayna, A. Badia, M.V. Clos, A. Camins, M. Pallàs, M. Bartolini,
F. Mancini, V. Andrisano, J. Estelrich, M. Lizondo, A. Bidon-Chanal, F.J. Luque,
J. Med. Chem. 51 (2008) 3588e3598.
[17] A. Badia, J.E. Baños, P. Camps, J. Contreras, D.M. Görbig, D. Muñoz-Torrero,
M. Simón, N.M. Vivas, Bioorg. Med. Chem. 6 (1998) 427e440.
[18] P. Camps, X. Formosa, C. Galdeano, T. Gómez, D. Muñoz-Torrero, L. Ramírez,
E. Viayna, E. Gómez, N. Isambert, R. Lavilla, A. Badia, M.V. Clos, M. Bartolini,
F. Mancini, V. Andrisano, A. Bidon-Chanal, O. Huertas, T. Dafni, F.J. Luque,
Chem. Biol. Interact. 187 (2010) 411e415.
[19] P. Camps, X. Formosa, C. Galdeano, D. Muñoz-Torrero, L. Ramírez, E. Gómez,
N. Isambert, R. Lavilla, A. Badia, M.V. Clos, M. Bartolini, F. Mancini,
V. Andrisano, M.P. Arce, M.I. Rodríguez-Franco, O. Huertas, T. Dafni, F.J. Luque,
J. Med. Chem. 52 (2009) 5365e5379.
[20] D. Shao, C.Zou, C. Luo,X.Tang, Y.Li, Bioorg. Med. Chem. Lett.14(2004)4639e4642.
[21] D. Alonso, I. Dorronsoro, L. Rubio, P. Muñoz, E. García-Palomero, M. Del Monte,
A. Bidon-Chanal, M. Orozco, F.J. Luque, A. Castro, M. Medina, A. Martínez,
Bioorg. Med. Chem. 13 (2005) 6588e6597.
[22] P. Muñoz-Ruiz, L. Rubio, E. García-Palomero, I. Dorronsoro, M. del Monte-
Millán, R. Valenzuela, P. Usán, C. de Austria, M. Bartolini, V. Andrisano,
A. Bidon-Chanal, M. Orozco, F.J. Luque, M. Medina, A. Martínez, J. Med. Chem.
48 (2005) 7223e7233.
AChE and BuChE enzyme samples, in 20 mM phosphate buffer pH
7.4, were incubated 150 s with acetylthiocholine iodide or butyrylth-
iocholine iodide (0.8 mM), respectively, in the presence of 10 mM 5,50-
dithiobis-(2-nitrobenzoic acid) (DTNB), for color development (all
chemicals from Sigma). Production of the yellow anion of 5-thio-2-
nitrobenzoic acid was measured with a SPECTRAmax 190, 96-well
plate reader, at 415 nm. Protein concentration for both type of samples
were measured by determined by Peterson’s modification of the pro-
cedure of Lowry et al. [35], using bovine serum albumin as standard.
[23] D. Antequera, M. Bolos, C. Spuch, C. Pascual, I. Ferrer, M.I. Fernandez-Bachiller,
M.I. Rodríguez-Franco, E. Carro, Neurobiol. Dis. 46 (2012) 682e691.
[24] D.S. Pisoni, J.S. da Costa, D. Gamba, C.L. Petzhold, A.C.A. Borges, M.A. Ceschi,
P. Lunardi, C.A.S. Gonçalves, Eur. J. Med. Chem. 45 (2010) 526e535.
[25] J.S. da Costa, D.S. Pisoni, C.B. da Silva, C.L. Petzhold, D. Russowsky, M.A. Ceschi,
J. Braz. Chem. Soc. 20 (2009) 1448e1454.
[26] M.V. Marques, M.M. Ruthner, L.A.M. Fontoura, D. Russowsky, J. Braz. Chem.
Soc. 23 (2012) 171e179.
[27] C.S. Schwalm, M.A. Ceschi, D. Russowsky, J. Braz. Chem. Soc. 22 (2011) 623e636.
[28] S. Samai, G.C. Nandi, P. Singh, M.S. Singh, Tetrahedron 65 (2009) 10155e10161.
[29] S. Das Sharma, P. Hazarika, D. Konwar, Tetrahedron Lett. 49 (2008) 2216e2220.
[30] M.K. Hu, C.F. Lu, Tetrahedron Lett. 41 (2000) 1815e1818.
[31] M.K. Hu, J. Pharm. Pharmacol. 53 (2001) 83e88.
4.3. Statistical analysis
[32] W. Luo, Y.P. Li, Y. He, S.L. Huang, J.H. Tan, T.M. Ou, D. Li, L.Q. Gu, Z.S. Huang,
Bioorg. Med. Chem. 19 (2011) 763e770.
[33] D. Yanover, M. Kaftory, Acta Cryst. E65 (2009) o711.
[34] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, Biochem. Pharmacol.
7 (1961) 88e95.
[35] G.L. Peterson, A simplification of the protein assay method of Lowry et al.
which is more generally applicable, Anal. Biochem. 83 (1977) 346e356.
The IC50 values were determined from a plot of activity value of
log (inhibitor) vs. response, which was processed by a software of
GraFit 6.0. All results are calculated as mean ꢂ SD of five inde-
pendent measurements, each performed in triplicate.