Synthesis and biological assessment of diversely substituted furo[2,3-b]quinolin-4-amine and pyrrolo[2,3-b]quinolin-4-amine derivatives, as novel tacrine analogues

Article abstract of DOI:10.1016/j.ejmech.2011.09.038

The synthesis and pharmacological analyses of a number of furo[2,3-b]quinolin-4-amine, and pyrrolo[2,3-b]quinolin-4-amine derivatives are reported. Thus, we synthesized diversely substituted tacrine analogues 1-11 and 12-16 by Friedlaender-type reaction o

Full text of DOI:10.1016/j.ejmech.2011.09.038

European Journal of Medicinal Chemistry 46 (2011) 6119e6130
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and biological assessment of diversely substituted
furo[2,3-b]quinolin-4-amine and pyrrolo[2,3-b]quinolin-4-amine
derivatives, as novel tacrine analoguesq
,
b
,
,
Carla Martins a, M. Carmo Carreiras a , Rafael León c, Cristóbal de los Ríos d e, Manuela Bartolini f,
*
Vincenza Andrisano f, Isabel Iriepa d, Ignacio Moraleda d, Enrique Gálvez d, Manuela García c
**
,
,
g
Javier Egea c, Abdelouhaid Samadi b, Mourad Chioua b, José Marco-Contelles b
,
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
b Laboratorio de Radicales Libres y Química Computacional (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
c Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain
d Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcalá, Ctra. Barcelona, Km. 33.6, 28817 Alcalá de Henares, Spain
e Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, C/Diego de León, 62, 28006 Madrid, Spain
f Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
g Hospital La Paz Health Research Institute-IdiPAZ, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and pharmacological analyses of a number of furo[2,3-b]quinolin-4-amine, and pyrrolo
[2,3-b]quinolin-4-amine derivatives are reported. Thus, we synthesized diversely substituted tacrine
analogues 1e11 and 12e16 by Friedländer-type reaction of readily available o-amino(furano/pyrrolo)
nitriles with suitable and selected cycloalkanones. The biological evaluation of furanotacrines 1e11 and
pyrrolotacrine 13 showed that these are good, in the micromolar range, and highly selective inhibitors of
BuChE. In the furanotacrine group, the most interesting inhibitor was 2-(p-tolyl)-5,6,7,8-tetrahydrofuro
Received 21 March 2011
Received in revised form
20 September 2011
Accepted 21 September 2011
Available online 29 September 2011
[2,3-b]quinolin-4-amine (3) [IC50 (eqBuChE) ¼ 2.9 ꢀ 0.4
m
M; IC50 (hBuChE) ¼ 119 ꢀ 15
mM]. Conversely,
Keywords:
pyrrolotacrines 12 and 14 proved moderately equipotent for both cholinesterases, being 1,2-diphenyl-
5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]quinolin-4-amine (12) the most potent for the inhibition of both
AChE/BuChE inhibitors
Neuroprotection
Alzheimer’s disease
enzymes [IC50 (EeAChE) ¼ 0.61 ꢀ 0.04
m
M; IC50 (eqBuChE) ¼ 0.074 ꢀ 0.009
m
M]. Moreover, pyrrolotacrine
-induced
12, at concentrations as low as 300 nM can afford significant neuroprotective effects against A
b
toxicity. Docking studies show that compounds 3 and 12 bind in the middle of the AChE active site gorge,
but are buried deeper inside BuChE active site gorge, as a consequence of larger BuChE gorge void. All
these data suggest that these new tacrine analogues could be used for the potential treatment of Alz-
heimer’s disease.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
are, in principle, effective in treating complex diseases because of
their ability to interact with multiple targets supposed to be
Alzheimer’s disease (AD) is a multifactorial neurodegenerative
disorder with several target proteins contributing to its aetiology
[1e3]. Hence, drug discovery in AD is gradually moving from the
development of agents able to modulate the biological function of
a single target to the multi-target-directed-ligands (MTDLs), which
responsible for the pathogenesis [1e11]. This approach normally
involves the use of synthetic hybrid compounds, rationally
designed to incorporate into a single framework at least two
different pharmacophoric moieties [12]. The use of multifunctional
drugs should be advantageous over the classical polypharmacy,
since it reduces the risk of drugedrug interactions and simplifies
pharmacokinetic and pharmacodynamic studies, manufacture,
formulation, clinical trial designs, and also the therapeutic regi-
mens, thus resulting in a great patient compliance [2,7,12]. Since
acetylcholinesterase (AChE) binds through its peripheral anionic
q
This manuscript is dedicated to Prof. Christian Bruneau on occasion of his 60th
birthday.
* Corresponding author. Tel.: þ351 96 7070270; fax: þ351 21 7946470.
** Corresponding author. Fax: þ34 91 5644853.
E-mail addresses: mcdamaso@ff.ul.pt, micarreiras@gmail.com (M.C. Carreiras),
iqoc21@iqog.csic.es (J. Marco-Contelles).
site (PAS) to the amyloid
b (Ab) non-amyloidogenic form and acts as
a pathological chaperone inducing a conformational transition
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.038

6120
C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
(AChEI) [15]. Due to its pharmacological nature, cholinesterase
inhibitory therapy may be considered as a simple symptomatic
short-term intervention. However, data emerging from long-term
shows that the effects of AChEI are not necessarily confined to
AChE inhibition; they additionally improve the cognitive symptoms
of AD through regulation of the processing and secretion of APP
[16e27]. Stabilization of the cognitive status of patients treated
with AChEI may be related to the interactions of these drugs with
the amyloid cascade.
Due to the overwhelming evidence regarding the neuro-
protective effects of AChEI, most AD clinicians and researchers
unanimously agree that there is no single treatment or therapeutic
approach that will alleviate this condition, and that a multiple-
functional approach or multipotent drugs will be required [1e12].
Moreover, it is highly conceivable that MTDLs may represent the
future treatment for AD and other similarly complex multifactorial
diseases [28].
Some years ago, we embarked on a long-term research project
aimed at the synthesis of a series of multipotent compounds
designed to target both AChE and neuronal Ca2þ modulation,
showing neuroprotection and antioxidant properties. As a result,
we have synthesized and evaluated a number of hybrid compounds
[29], where the benzene ring in tacrine has been replaced by
a heterocyclic ring. Other tacrine analogues have also been reported
from other laboratories [30e34]. Kirsch and co-workers syn-
thesised a few analogues where the benzene ring in tacrine was
replaced by a thiazole ring [30], a 4-azaisoindole ring [31], a sele-
nophene ring [32], and a thiophene ring [33]. In all these analogues
the cyclohexane-fused ring was maintained, contracted or enlarged
[30e33]. Moreover, in the 4-azaisoindole derivatives [31] it was
replaced by functionalized cyclohexane rings, a nitrogen-containing
six-membered heterocyclic ring, a pyran and a thiopyran ring.
Tzeng and collaborators [34] reported the synthesis of certain
alkoxy, aryloxy, alkylamino or arylamino substituted benzofuro[2,3-
b]quinoline derivatives, all of them are tetracyclic analogues of
tacrine.
Scheme 1. Synthesis of 2-aryl-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-amines (1, 3, 5),
2-aryl-6,7,8,9-tetrahydro-5H-cyclohepta[b]furo[3,2-e]pyridin-4-amines (2, 4, 6), 4-
amino-2-(p-tolyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinoline-3-carbonitrile (7), 4-amino-
2-aryl-5,6,7,8-tetrahydrofuro[2,3-b]quinoline-3-carbonitriles (8, 10), and 4-amino-2-
aryl-6,7,8,9-tetrahydro-5H-cyclohepta[b]furo[3,2-e]pyridine-3-carbonitriles (9, 11).
With these precedents in mind, and with the purpose of
developing AChE and butyrylcholinesterase (BuChE) inhibitors
endowed with neuroprotective properties, we report here the
synthesis and pharmacological activities of fused five-membered
aromatic ring tacrine analogues, such as the furanotacrines 1e11
(Scheme 1) and the pyrrolotacrines 12e16 (Scheme 2), bearing the
furo[2,3-b]quinolin-4-amine (I) or the pyrrolo[2,3-b]quinolin-4-
amine (II) (Chart 1) heterocyclic ring systems.
Scheme 2. Synthesis of 2-aryl-1-phenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]quinolin-
4-amines (12, 14, 16), and 2-aryl-1-phenyl-1,5,6,7,8,9-hexahydrocyclohepta[b]pyrrolo
[3,2-e]pyridin-4-amines (13, 15).
to the amyloidogenic form [13,14] most of the MTDLs are dual-
binding AChE inhibitors endowed with additional properties.
Thus, they may simultaneously alleviate cognitive deficits and
2. Chemistry
behave as disease-modifying agents by inhibiting A
b peptide
aggregation through binding to both catalytic and peripheral sites
of the enzyme [12].
2.1. Furo[2,3-b]quinolin-4-amine derivatives
Besides the pathologic hallmarks of the disease, i.e., neurofi-
brillary tangles and neuritic plaques, AD is characterized chemically
by a consistent deficit in cholinergic transmission, particularly
affecting cholinergic neurons in the basal forebrain. Therefore, the
first clinical relevant approach has been the use of AChE inhibitors
The synthesis of the new furanotacrines 1e11 has been ach-
ieved, as shown in Scheme 1, starting from the readily available,
previously synthesized in our laboratory, 2-amino-5-arylfuran-3-
carbonitriles 17e19 [34] or 2-amino-5-arylfuran-3,4-dicarboni-
triles 20e22 [34], according to typical Friedländer reaction (FR)
Chart 1. General structure and heterocyclic ring systems for the new tacrine analogues: furo[2,3-b]quionlin-4-amine (I) and pyrrolo[2,3-b]quinolin-4-amine (II).

C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
6121
Table 1
Inhibition of EeAChE/hrAChE and eqBuChE/hBuChE by 2-aryl-3a,5,6,7,8,9a-hexahydrofuro[2,3-b]quinolin-4-amine (1e6), 4-amino-2-aryl-5,6,7,8-tetrahydrofuro[2,3-b]quin-
oline-3-carbonitrile (10), and 2-aryl-1-phenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]quinolin-4-amine (12e14) derivatives.a
Compound
Structure
IC50
IC50
Selectivity
IC50
IC50
EeAChE (
mM)
eqBuChe (
m
M)
BuChE/AChE
hrAChE (
m
M)
hBuChE (mM)
1
>100
>100
3.6 ꢀ 0.7
>100
3.78 ꢀ 0.11
8.57 ꢀ 0.40
119 ꢀ 15
2
6 ꢀ 1
12
0.61 ꢀ 0.04
0.074 ꢀ 0.009
0.12
45.5 ꢀ 3.1
13b
>30
1.1 ꢀ 0.2
2.9 ꢀ 0.4
3 (Ar: 40-MeC6H4)
> 100
>100
4b (Ar: 40-MeC6H4)
5 (Ar: 40-ClC6H4)
6b (Ar: 40-ClC6H4)
10b (Ar: 40-ClC6H4)
> 30
>100
>100
>100
13 ꢀ 1
23 ꢀ 2
>100
26.1 ꢀ 1.0
43 ꢀ 3
32.4 ꢀ 0.6
>100
>100
14b (Ar: 40-ClC6H4)
24 ꢀ 2
15 ꢀ 2
0.62
Tacrine
0.027 ꢀ 0.002
0.0052 ꢀ 0.0002
0.193
a
IC50 values are the mean ꢀ SEM of quadruplicates of at least three independent experiments.
b
Measured in presence of bovine serum albumin to improve solubility.

6122
C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
protocols [35]. Under the usual conditions [29], using cyclohex-
anone or cycloheptanone, we isolated the expected target
molecules in good yields. The structures of these compounds are
in very good agreement with their analytical and spectroscopic
data (see Experimental part).
60
50
40
30
20
10
0
Control
100nM
300nM
600nM
1uM
2uM
2.2. Pyrrolo[2,3-b]quinolin-4-amine derivatives
The synthesis of the new pyrrolotacrines 12e16 has been ach-
ieved, as shown in Scheme 2, starting from the readily available,
previously synthesized in our laboratory 2-amino-5-aryl-1-phenyl-
1H-pyrrole-3-carbonitriles 23e25 [35], according to typical FR
protocols [36]. Under the usual conditions [29], using cyclohexa-
none or cycloheptanone, the expected target molecules were iso-
lated in good yields. The structure of these compounds has been
established and assigned by their analytical and spectroscopic data
(see Experimental part).
-4
-2
0
2
4
6
8
10
-1
-1
[AThCh] (mM )
3. Results and discussion
Fig. 1. Steady-state inhibition of AChE hydrolysis of acetylthiocholine (ATCh) by
compound 12. LineweavereBurk reciprocal plots of initial velocity and substrate
concentrations (0e0.8 mM) are presented. Lines were derived from a weighted least-
squares analysis of data.
3.1. Pharmacology
3.1.1. AChE/BuChE inhibitory activity of furo[2,3-b]quinolin-4-
amine and pyrrolo[2,3-b]quinolin-4-amine derivatives
inhibitor 14 shows an equipotent profile on both cholinesterases,
whereas compound 12 is much more potent for the inhibition of
eqBuChE. Very interestingly, compared with 12, pyrrolotacrine 13,
differing just by a cycloheptane instead of cyclohexane ring, is
Furanotacrines 1e11 and pyrrolotacrines 12e16 have been eval-
uated as inhibitors of AChE from Electrophorus electricus (EeAChE)
and BuChE from horse serum (eqBuChE) according to the method
based on Ellman [37]. Next, some selected inhibitors (1, 3, 5 and 10)
were also evaluated as inhibitors of human recombinant AChE
(hrAChE), and human serum BuChE (hBuChE), according to the
method based on Ellman [37].
A
###
100
Unfortunately, compounds 7 and 16 were not soluble, even in
their hydrochloride form. Molecules 8, 9, 11 and 15 were soluble in
DMSO, but proved to be inactive on both cholinesterases. The active
molecules gave the IC50 values shown in Table 1. From these data,
and for the furanotacrines we conclude that: (a) these compounds
are from potent to moderate, in the micromolar range, selective
inhibitors of eqBuChE regarding to EeAChE; (b) the most potent
inhibitor was 2-(p-tolyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-
*
80
60
***
40
20
0
[ ] μM
30
30
3
Basal
3
amine (3) [IC50 (eqBuChE) ¼ 2.9 ꢀ 0.4
mM]; (c) for compounds
bearing the same substituent in the aryl ring at C2, the most potent
inhibitors were those bearing a fused cyclohexane ring, instead of
a fused cycloheptane ring [compare 1 with 2 (Ar ¼ C6H5); 3 with
Compound 12
Tacrine
B
4
(Ar ¼ 40-MeC6H4);
5
with
6
(Ar ¼ 40-ClC6H4)]; (d) for the
compounds bearing the same cycloalkane fused ring system, and
regarding the type of the substituent at C40 in the aromatic ring, the
inhibitory potency followed the order: H ꢁ 40-Me [ Cl [compare 1
with 3 and 5 (cyclohexane)]; or H [ 40-Me [ Cl [compare 2 with
4 and 6 (cycloheptane)]; (e) the incorporation of a nitrile (CN)
group at C3 resulted in a total loss of BuChE inhibitory activity
(compare compound 6 with 11, or see molecules 8, and 9), in most
of the cases, only CN-substituted compound 10 afforded a moderate
100
***
**
**
80
60
40
20
0
###
eqBuChE inhibitory activity (IC50 ¼ 32.4
mM); (f) on going from
eqBuChE to hBuChE, the inhibitory potency remained similar
(compounds 1 and 5) or was significantly decreased (compound 3),
the most potent being 2-phenyl-5,6,7,8-tetrahydrofuro[2,3-b]qui-
[Compound 12] μM
Aβ25-35 10μM
Basal
0.3
1
3
Fig. 2. (A) Cell viability, measured as reduction of MTT, in SH-SY5Y cells exposed for
48 h with 3 or 30 M of compound 12 or tacrine alone. Data correspond to the mean
nolin-4-amine (1) [IC50 (hBuChE) ¼ 3.78 ꢀ 0.11
mM], or was totally
m
lost as in compound 10.
and S.E.M. of 4 different experiments performed in triplicate. *p < 0.05, ***p < 0.001 in
comparison to basal condition in the absence of drug and ###p < 0.001 comparing
From the data in Table 1, and for the pyrrolotacrines, the same
structureeactivity relationships (SARs) could also be determined,
regarding the effect of the size of the fused cycloalkane ring, and
the type of the substituent at the C40 in the aromatic substituent.
The most significant difference with the furanotacrines concerning
the inhibition of AChE/BuChE was that in this group of molecules
(12e16), two of them, 12 and 14, also inhibited EeAChE. Thus,
30
trations of compound 12 in cells exposed to the cytotoxic fragment of
Compound 12 was incubated with the cells 24 h before and during the 24 h exposure
to 10 M Ab25e35. Data correspond to the mean and S.E.M. 4 Different experiments
performed in triplicate. ###p < 0.001, comparing with basal conditions. *p < 0.05,
m
M compound 12 with 30
m
M tacrine. (B) Protective effect of increasing concen-
A
b25e35
.
m
**p < 0.01 and ***p < 0.001 comparing to Ab25e35 e induced toxicity in the absence of
drug.

C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
6123
Fig. 3. Binding mode of compound 12 at the EeAChE gorge predicted by the docking simulation. (a) The compound is represented as stick model and illustrated in green. Relevant
residues are shown as sticks and coloured by atoms. Hydrogen bond is represented as pink dashed lines. (b) Molecular docking model of 12eEeAChE complex: 12 (green) (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
almost inactive. Pyrrolotacrine 12 was also assayed for the inhibi-
tion of hrAChE and hBuChE (Table 1). The results show that this
compound inhibited both enzymes, but 5-fold more selectively for
hBuChE.
Overall, 1,2-diphenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]qui-
nolin-4-amine (12) was the most potent for the inhibition of both
SY5Y cells during 48 h with 3
cause reduction of viability. However, at 30
reduced viability by 28% and tacrine by 50%; this cytotoxic effect
was significantly higher for tacrine in comparison to compound 12
(p < 0.001) (Fig. 2A).
m
M of compound 12 or tacrine did not
mM, compound 12
enzymes [IC50 (EeAChE) ¼ 0.61 ꢀ0.04
m
M; IC50 (eqBuChE) ¼
3.1.4. Neuroprotection against A
In another set of experiments we evaluated the potential
protective effect of compound 12 on A -induced toxicity. Exposure
of SH-SY5Y cells for 24 h to 10 M of Ab25e35 reduced cell viability
b-induced cytotoxicity
0.074 ꢀ 0.009 M].
m
b
3.1.2. Kinetic analysis of AChE inhibition by compound 12
To gain further insight into the mechanism of action on AChE of
this family of compounds, a kinetic study was carried out with the
most promising compound of the series, 12, using EeAChE. Graph-
ical analysis of the reciprocal LineweavereBurk plots (Fig. 1)
showed both increased slopes (decreased Vmax) and intercepts
(higher Km) at increasing concentration of the inhibitor. This
pattern indicates a mixed-type inhibition. Replots of the slope
versus concentration of compound 12 gave an estimate of the
inhibition constant, Ki, of 621 nM.
m
to 36.6 ꢀ 4.5%. Cells that were pre-treated 24 h before with
compound 12 and during the 24 h exposure to the Ab25e35 toxin
showed lower reductions of cell viability, i.e. 11.4 ꢀ 2.2%, 19 ꢀ 5.7%
and 23.7 ꢀ4.3% at 0.3, 1 and 3
mM, respectively. Therefore, these
results indicate that compound 12, at concentrations as low as
300 nM, can afford significant neuroprotective effects against Ab-
induced toxicity (Fig. 2B).
3.1.5. Molecular docking studies
Molecular docking studies with pyrrolotacrine 12 and furanota-
crine 3 were carried out in the active site of EeAChE and eqBuChE.
The 3D structures of both enzymes were employed since ligands
bind to enzyme species differently despite the high homology
sequence among species.
3.1.3. Measurement of cell viability with MTT
The effects of compound 12 on cell viability were analysed, first
at the concentration of 3 mM and after, at a concentration 10 times
higher, 30 mM, since this drug is a tacrine analogue. For compar-
ative purposes we also used tacrine in the experiments. Cell
viability measured as MTT reduction showed that exposure of SH-
The technique blind docking [38,39] was adopted since it might
detect possible binding sites and ligand conformations by
Fig. 4. Binding mode of 3 (violet sticks) at the EeAChE gorge. (a) Relevant residues are shown as sticks and coloured by atoms. Hydrogen bond is represented as pink dashed lines.
(b) Molecular docking model of 3eEeAChE complex (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

6124
C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
enlarge the gorge and enhance access for the ligand to the active
centre.
To account for side chain flexibility during docking, flexible
torsions in the ligand were assigned, and the acyclic dihedral angles
were allowed to rotate freely. In addition, receptor flexibility was
introduced by keeping flexible Trp286, Tyr124, Tyr337 and Tyr72
receptor residues mentioned earlier under the docking simulation,
in which the pose with the lowest docking energy was selected as
the best solution.
Fig. 3 illustrates the most energetic favourable binding mode of
12 at the active site of EeAChE. In the complex, TYR341 was found
to allow a
pep stacking interaction with the pyrrolotacrine moiety
of the ligand. The aromatic nitrogen at the tetrahydroquinoline
ring is hydrogen bonded to the hydroxyl group of TYR124 and the
phenyl group at one-position is involved in hydrophobic contacts
with the central hydrophobic region (TRP86 and ASP74). In this
orientation, the phenyl ring at the two-position is buried inside
the protein gorge and it is stacked between TYR337 and TRP86, as
well as it is involved in hydrophobic contacts with the amino acid
residue of the catalytic triad, HIS447. Besides, the cyclohexane
ring is involved in hydrophobic contacts with TYR341 and
TRP286, the latter residue located in the PAS. These results
suggest that this compound could favourably interact with the
catalytic site and the PAS of AChE, therefore, docking results are in
agreement with the experimental kinetic data, indicating
a mixed-type inhibition.
Fig. 5. Complex of compounds 12 (green) and 3 (violet) and eqBuChE homology build
3D-model (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.).
However, a similar behaviour was not observed for furanota-
crine 3, the best pose accommodates the ligand within the
central region of the active site gorge, although no interactions
with the catalytic triad and the PAS residues were found (Fig. 4).
Conversely to pyrrolotacrine 12, the cyclohexane unit is buried
inside the protein gorge and does not make favourable contacts.
The aromatic nitrogen at the tetrahydroquinoline ring is hydrogen
bonded to the hydroxyl group of TYR337 and the p-methylphenyl
ring is involved in hydrophobic contacts with TYR341 and PHE338.
A close inspection of the 12eAChE and 3eAChE complexes
suggests that critical rearrangements of the ligand are those
induced by the phenyl ring at the one-position. In summary, the
geometric and energetic features of the 12eenzyme complex
contribute to the higher affinity of pyrrolotacrine 12 and make it
a more potent inhibitor than 3.
scanning the entire surface of a protein target so that a location
with the highest binding affinity on the protein may be found. The
program AutoDock Vina [40] was carried out to perform the blind
docking using a single catalytic sub-unit of EeAChE (PDB: 1C2B).
This program was shown to be more accurate compared to
previous versions of AutoDock employed in blind docking studies
[38,39].
Comparative crystallographic analysis of the PAS and the
catalytic ligandeAChE complexes revealed distinct features of the
crystalline PAS and the active site gorge, which may reflect
conformational states of AChE associated with PAS and catalytic
ligands in solution. Although the overall structures are virtually
identical, superimposition revealed significant mobility for the
side chain residues of Trp286, located at the entry of the gorge
(PAS), Tyr124 and Tyr337 in the constricted gorge region and Tyr72
in the gorge path. These residues delineate the shape of the gorge
entry and its lining. In particular, the phenol moieties of Tyr124
and Tyr337, located midway, swing in and out with large devia-
tions of its hydroxyl group. This motion is sufficient by itself to
With respect to BuChE, and in the absence of X-ray structure of
eqBuChE, a homology model was used to rationalize experimental
data. The automated homology-modelling SWISS-MODEL program
performed the modelling of the 3-D structure [41e43]. A putative
three-dimensional structure of eqBuChE was created based on the
Fig. 6. Binding modes of pyrrolotacrine 12 and furanotacrine 3 on eqBuChE homology build 3D-model. (a) Top view of the accessible surface of the active site gorge. (b) Super-
position of 12 (green stick) and 3 (violet stick) with choline (red stick), butyrate (blue stick) and the best pose for tacrine (yellow stick) (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.).

C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
6125
crystal structure of hBuChE (pdb: 2pm8). These two enzymes
5. Experimental part
exhibited 89% sequence identity.
In order to simulate the binding of compounds 3 and 12 to
eqBuChE, the reported position of butanoic acid (PDB ID: 1P0I) and
choline (PDB ID: 1P0M) in hBuChE were used to mimic the inter-
mediate product-bound form during the docking studies due to the
lack of native BuChE-substrate structure. The non-availability of
co-crystallized structures for eqBuChE with tacrine prompted us to
apply the same molecular docking protocol that was adopted for
compounds 3 and 12, in order to explore its proteineligand
interactions and compare with the positions of butanoic acid and
choline. All docking experiments were performed as blind dock-
ings following the same computational protocol adopted in
EeAChE.
The best-ranked docking solutions revealed that BuChE could
effectively accommodate pyrrolotacrine 12 and furanotacrine 3
inside the active site gorge (Fig. 5). The 12-BuChE complex is
stabilized mainly by the hydrophobic interactions with the
amino acid residues of the catalytic active site region of the
enzyme.
Fig. 6 shows the position of 12 and tacrine at the active site of
eqBuChE, and choline and butyrate at the active site of hBuChE. The
phenyl ring at one-position is in the region where tacrine and
choline are bound and the cyclohexane moiety occupies the same
region that butanoic acid does (Fig. 5). In the 3eBuChE complex the
inappropriate position of the ligand compared to the position
occupied by tacrine, choline and compound 12 accounts for
a higher affinity of 12 towards eqBuChE and makes it a more potent
inhibitor than compound 3.
The key to increasing the inhibitory potency against AChE and
BuChE seems to be the phenyl ring at one-position of pyrrolotacrine
12 rather than changing the heteroatom of the five-membered
rings.
5.1. Chemistry
Melting points were determined on a Koffler apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded in CDCl3
or DMSO-d6 at 300, 400 or 500 MHz, and at 75.4, 100.5 or
125.7 MHz, respectively, using solvent peaks [CDCl3: 7.27 (D), 77.2
(C) ppm; D2O: 4.60 ppm and DMSO-d6: 2.49 (D), 40 (C) ppm] as
internal reference. The assignment of chemical shifts is based on
standard NMR experiments (1H, 13C-DEPT, 1H, 1H-COSY, gHSQC,
gHMBC). NMR signals with (*) can be interchanged. Mass spectra
were recorded on a GC/MS spectrometer with an API-ES ionization
source. Elemental analyses were performed on a LECO CHNS-932
apparatus. TLC was performed on silica F254 and detection by UV
light at 254 nm or by charring with either ninhydrin, anisaldehyde,
or phosphomolybdic-H2SO4 dyeing reagents. Anhydrous solvents
were used in all experiments. Column chromatography was per-
formed on silica gel 60 (230 mesh). Reactions under MW irradiation
(250 W) were performed in a CEM Discover systemÔ, equipped
with electromagnetic sample stirrer, an infrared temperature
detector and a pressure sensor. The reaction was performed in
30 mL glass tube equipped with septa.
5.2. General method for the Friedländer reaction
To a suspension of AlCl3 (2.2 equiv) in 1,2-dichloroethane (8 mL
solvent/mmol of aminonitrile), was added cyclohexanone
(2.2 equiv) at rt under nitrogen atmosphere. After ca. 5 min, the
corresponding furo or pyrrolo aminonitrile (1 equiv) was added
and the reaction mixture was refluxed (1e8 d). After the reaction
was complete, a NaOH or NaHCO3 10% aqueous solution was added
dropwise until pH 9. Next, the solvents were evaporated to dryness.
The solid residue was purified by column chromatography, using
hexaneeethyl acetate or hexaneedichloromethane mixtures as
eluent, to afford the corresponding furan or pyrrolotacrines.
4. Conclusions
The synthesis and biologicalevaluation of furanotacrines 1e11, and
pyrrolotacrines 12e14 were reported. These tacrine analogues show
a selective affinity in the micromolar range towards BuChE. In the
furotacrine group, the most attractive inhibitor was 2-(p-tolyl)-
5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-amine (3) [IC50 (eqBuChE) ¼
5.2.1. 2-Phenyl-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-amine (1)
Following the general method for the Friedländer reaction, to
a
solution of AlCl3 (599 mg, 4.5 mmol, 1.7 equiv) in 1,2-
dichloroethane (30 mL) was added 2-amino-5-phenylfuran-3-
carbonitrile (17) [35] (503 mg, 2.73 mmol) under argon atmo-
sphere. After a few minutes, cyclohexanone (425 mL, 4.1 mmol,
1.5 equiv) was added and the reaction mixture was refluxed for
72 h, yielding furanotacrine 1 (618 mg, 86%) as light brown crystals:
2.9 ꢀ 0.4
mM; IC50 (hBuChE) ¼ 119 ꢀ 15
mM]. However, pyrrolotacrines
12 and 14 proved to be moderately equipotent for both cholinester-
ases. Pyrrolotacrine 1,2-diphenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-
b]quinolin-4-amine (12) was the most potent [IC50 (EeAChE) ¼
mp 207e210 ꢂC; IR (KBr)
n 3470, 3326, 3215, 3078, 2935, 2909,
0.61 ꢀ0.04
m
M; IC50 (eqBuChE) ¼ 0.074 ꢀ 0.009
m
M] though it
2860, 1633, 1601, 1469, 1336, 1107 cmꢃ1; 1H NMR (CDCl3, 400 MHz)
doesn’t reach affinities to BuChE comparable to tacrine or physo-
stigmine [46]. However, as a BuChEI it shows higher activity than
phenserine [47] and rivastigmine [3]. Cell viability measured as MTT
reduction showed that exposure of SH-SY5Y cells during 48 h with
d
7.81 (d, J ¼ 7.2 Hz, 2H, H20), 7.41 (t, J ¼ 7.3 Hz, 2H, H30), 7.30 (t,
J ¼ 7.3 Hz,1H, H40), 6.84 (s,1H, H3), 4.36 (br s, 2H, NH2), 2.93 (m, 2H,
H8), 2.51 (m, 2H, H5), 1.89 (m, 4H, H6, H7); 13C NMR (CDCl3,
100.5 MHz)
d 161.1 (C9a), 152.8 (C8a), 151.6 (C2), 145.1 (C4), 130.3
3
m
M of compound 12 or tacrine did not cause reduction of viability.
(C10), 128.7 (C30), 128.1 (C40), 124.4 (C20), 110.5 (C4a), 105.5 (C3a),
96.6 (C3), 33.1 (C8), 23.1 (C5), 22.8 (C7)*, 22.7 (C6)*; MS (EIþ) m/z:
264 [Mþ] (100), 263 (36), 236 (27). Anal. Calcd. for C17H16N2O: C,
77.25; H, 6.10; N, 10.60. Found: C, 77.05; H, 6.15, N, 10.46.
Nevertheless, at 30 M, compound 12 reduced cell viability by 28%
m
while tacrine diminished by 50%. Thus, this cytotoxic effect was
significantly higher for tacrine in comparison to compound 12. Pyr-
rolotacrine 12 also showed that at concentrations as low as 300 nM,
can afford significant neuroprotective effects against A
b
-induced
5.2.2. 2-Phenyl-6,7,8,9-tetrahydro-5H-cyclohepta[b]furo[3,2-e]
pyridin-4-amine (2)
toxicity. The molecular modelling studies provided valuable infor-
mation on how this new molecular scaffold represented by the
compounds containing both pyridine and pyrrole or furan rings
interacts with AChE and BuChE. The presence of a phenyl ring at the
one-position of the pyrrole unit is beneficial for both AChE and BuChE
inhibition. These results suggest pyrrolotacrine 12 may represent
a new lead capable of structural improvement for potential treatment
of AD.
Following the general method for the Friedländer reaction, to
a
solution of AlCl3 (694 mg, 5.21 mmol, 1.9 equiv) in 1,2-
dichloroethane (30 mL) was added 2-amino-5-phenylfuran-3-
carbonitrile (17) [35] (500 mg, 2.71 mmol) under argon atmo-
sphere. After a few minutes, cycloheptanone (577 mL, 4.89 mmol,
1.8 equiv) was added and the reaction mixture was refluxed for
24 h, yielding furanotacrine 2 (659 mg, 87%): mp 216e218 ꢂC; IR

6126
C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
(KBr)
n
3433, 3323, 3250, 3103, 3067, 3032, 2949, 2912, 2846, 1642,
48 h, yielding furanotacrine 5 (510 mg, 69%) as dark yellow crystals:
mp 263e265 ꢂC; IR (KBr)
3451, 3328, 3221, 3091, 2932, 2855, 1633,
1599, 1565, 1471, 1340, 1267, 1186, 1114 cmꢃ1
;
1H NMR (DMSO-d6,
n
400 MHz)
d
7.72 (d, J ¼ 7.2 Hz, 2H, H20), 7.47 (t, J ¼ 7.3 Hz, 2H, H30),
1601, 1555, 1486, 1466, 1339, 1088 cmꢃ1 1H NMR (DMSO-d6,
;
7.38 (s, 1H, H3), 7.34 (t, J ¼ 7.3 Hz, 1H, H40), 6.31 (br s, 2H, NH2), 2.86
400 MHz)
d
7.72 (d, J ¼ 8.4 Hz, 2H, H20), 7.53 (d, J ¼ 8.4 Hz, 2H, H30),
(m, 2H, H9), 2.69 (m, 2H, H5), 1.78 (m, 2H, H7), 1.58 (m, 2H, H8), 1.51
7.39 (s, 1H, H3), 6.34 (br s, 2H, NH2), 2.72 (m, 2H, H8), 2.43 (m, 2H,
(m, 2H, H6); 13C NMR (DMSO-d6, 100.4 MHz)
d
160.2 (C10a), 159.1
H5), 1.75 (m, 4H, H6, H7); 13C NMR (DMSO-d6, 100.5 MHz)
d 160.9
(C9a),149.3 (C2),146.5 (C4),130.2 (C10),129.1 (C30),128.0 (C40),123.6
(C20),114.9 (C4a),105.2 (C3a), 99.8 (C3), 38.5 (C9), 31.9 (C7), 27.4 (C6),
26.5 (C8), 24.6 (C5); MS (EIþ) m/z (%): 278 [M]þ (100), 277 (33), 263
(17), 250 (23), 249 (39), 237 (12), 224 (12). Anal. Calcd. for C18H18N2O:
C, 77.67; H, 6.52; N, 10.06. Found: C, 77.54; H, 6.35; N, 9.92.
(C9a), 152.2 (C8a), 147.9 (C2)*, 147.3 (C4)*, 132.3 (C40), 129.2 (C30),
129.1 (C10), 125.3 (C20), 109.6 (C4a), 104.3 (C3a), 100.3 (C3), 32.9 (C8),
23.1 (C5), 22.7 (C6)**, 22.5 (C7)**; MS (EIþ) m/z (%): 300 [M þ 2]þ (57),
298 [M]þ (100), 283 (10), 270 (37). Anal. Calcd. for C17H15ClN2O: C,
68.34; H, 5.06; N, 9.38. Found: C, 68.21; H, 5.13; N, 9.02.
5.2.3. 2-(p-Tolyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-amine
(3)
5.2.6. 2-(4-Chlorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]furo
[3,2-e]pyridin-4-amine (6)
Following the general method for the Friedländer reaction, to
Following the general procedure for the Friedländer reaction, to
a
solution of AlCl3 (277 mg, 2.08 mmol, 1.7 equiv) in 1,2-
a solution of AlCl3 (635 mg, 4.76 mmol, 2.1 equiv) in 1,2-
dichloroethane (33 mL) was added 2-amino-5-(p-tolyl)furan-3-
carbonitrile (18) [35] (202 mg, 1.02 mmol) under argon atmo-
dichloroethane (30 mL) was added 2-amino-5-(40-chlorophenyl)
furan-3-carbonitrile (19) [35] (504 mg, 2.31 mmol) under argon
sphere. After a few minutes, cyclohexanone (218
mL, 2.10 mmol,
atmosphere. After a few minutes, cycloheptanone (550 mL,
1.8 equiv) was added and the reaction mixture was refluxed for
4.66 mmol, 2.0 equiv) was added and the reaction mixture was
24 h, yielding furanotacrine 3 (220 mg, 78%): mp 256e258 ꢂC; IR
refluxed for 48 h, yielding furanotacrine 6 (563 mg, 78%): mp
(KBr)
n
3481, 3333, 3221, 3068, 3020, 2943, 2861, 1632, 1599, 1503,
271e273 ꢂC; IR (KBr)
n 3469, 3339, 3250, 3221, 2949, 2921, 2843,
1468, 1445, 1336, 1112 cmꢃ1
;
1H NMR (DMSO-d6, 300 MHz)
d
7.63
1637, 1594, 1576, 1482, 1471, 1335, 1091 cmꢃ1 1H NMR (DMSO-d6,
;
(d, J ¼ 6.0 Hz, 2H, H20), 7.29 (d, J ¼ 6.0 Hz, 2H, H30), 7.27 (s, 1H, H3),
400 MHz)
d
7.71 (d, J ¼ 8.4 Hz, 2H, H20), 7.53 (d, J ¼ 8.4 Hz, 2H, H30),
6.01 (br s, 2H, NH2), 2.74 (m, 2H, H8), 2.50 (m, 2H, H5), 2.35 (s, 3H,
7.40 (s, 1H, H3), 6.35 (br s, 2H, NH2), 2.85 (m, 2H, H9), 2.69 (m, 2H,
CH3), 1.79 (m, 4H, H6, H7); 13C NMR (DMSO-d6, 75.4 MHz)
d 161.7
H5), 1.78 (m, 2H, H7), 1.58 (m, 2H, H8), 1.50 (m, 2H, H6); 13C NMR
(C9a), 152.6 (C8a), 150.4 (C2), 147.7 (C4), 138.3 (C40), 130.4 (C30),
128.5 (C10), 124.6 (C20), 110.5 (C4a), 105.5 (C3a), 99.4 (C3), 33.7 (C8),
23.8 (C5), 23.5 (C7)*, 23.3 (C6)*, 21.6 (CH3); MS (EIþ) m/z (%): 278
[M]þ$ (100), 277 (32), 250 (30). Anal. Calcd. for C18H18N2O: C, 77.67;
H, 6.52; N, 10.06. Found: C, 77.54; H, 6.47; N, 9.97.
(DMSO-d6, 100.5 MHz) d 160.9 (C10a), 160.0 (C9a), 148.7 (C2), 147.2
(C4), 132.9 (C40), 129.8 (C30), 129.7 (C10), 125.9 (C20), 115.6 (C4a),
105.8 (C3a), 101.2 (C3), 39.1 (C9), 32.5 (C7), 28.0 (C6), 27.1 (C8), 25.2
(C5); MS (EIþ) m/z (%): 314 [M þ 2]þ (51), 312 [M]þ (100), 311 (26),
297 (15), 285 (15), 284 (20), 283 (31). Anal. Calcd. for C18H17ClN2O:
C, 69.12; H, 5.48; N, 8.96. Found: C, 69.31; H, 5.68; N, 8.56.
5.2.4. 2-(p-Tolyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]furo[3,2-e]
pyridin-4-amine (4)
5.2.7. 4-Amino-2-(p-tolyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinoline-
3-carbonitrile (7)
Following the general method for the Friedländer reaction, to
a
solution of AlCl3 (271 mg, 2.04 mmol, 2.0 equiv) in 1,2-
Following the general procedure for the Friedländer reaction, to
dichloroethane (36 mL) was added 2-amino-5-(p-tolyl)furan-3-
carbonitrile (18) [35] (200 mg, 1.01 mmol) under argon atmo-
sphere. After a few minutes, cycloheptanone (240 mL, 2.04 mmol,
a solution of AlCl3 (714 mg, 5.35 mmol, 4.1 equiv) in 1,2-
dichloroethane (35 mL) was added 2-amino-5-(p-tolyl)furan-3,4-
dicarbonitrile (20) [35] (290 mg, 1.30 mmol) under argon atmo-
2.0 equiv) was added and the reaction mixture was refluxed for
sphere. After a few minutes, cyclohexanone (516 mL, 5.27 mmol,
24 h, yielding furanotacrine 4 (238 mg, 81%): mp 272e274 ꢂC; IR
4.1 equiv) was added and the reaction mixture was refluxed for
(KBr)
n 3460, 3337, 3228, 3022, 2953, 2923, 2847, 1638, 1601, 1503,
48 h, yielding furanotacrine 7 (333 mg, 84%): mp 268e270 ꢂC; IR
1471, 1338 cmꢃ1; 1H NMR (DMSO-d6, 400 MHz)
d
7.57 (d, J ¼ 8.0 Hz,
(KBr) n 3472, 3322, 3203, 3032, 2936, 2855, 2217, 1646, 1599, 1504,
2H, H20), 7.26 (s, 1H, H3), 7.25 (d, J ¼ 8.0 Hz, 2H, H30), 6.23 (br s, 2H,
NH2), 2.81 (m, 2H, H9), 2.65 (m, 2H, H5), 2.29 (s, 3H, CH3), 1.75 (m,
2H, H7), 1.54 (m, 2H, H8), 1.47 (m, 2H, H6); 13C NMR (DMSO-d6,
1467,1361,1328,1141 cmꢃ1; 1H NMR (DMSO-d6, 500 MHz)
d 7.92 (d,
J ¼ 8.2 Hz, 2H, H20), 7.43 (d, J ¼ 8.2 Hz, 2H, H30), 5.77 (br s, 2H, NH2),
2.76 (m, 2H, H8), 2.51 (m, 2H, H5), 2.41 (s, 3H, CH3), 1.81 (m, 2H,
100.5 MHz)
d
160.1 (C10a), 158.8 (C9a), 149.5 (C2), 146.3 (C4), 137.4
H7)*, 1.80 (m, 2H, H6)*; 13C NMR (DMSO-d6, 125.7 MHz)
d 159.8
(C40), 129.6 (C30), 127.5 (C10), 123.6 (C20), 114.8 (C4a), 105.2 (C3a),
98.9 (C3), 38.4 (C9), 31.8 (C7), 27.3 (C6), 26.5 (C8), 24.6 (C5), 20.8
(C9a), 158.0 (C2), 154.9 (C8a), 147.7 (C4), 142.1 (C40), 130.8 (C30),
126.5 (C20),125.6 (C10),116.0 (CN),113.0 (C4a),101.9 (C3a), 84.8 (C3),
33.6 (C8), 23.7 (C5), 23.1 (C7)**, 22.9 (C6)**, 21.9 (CH3); MS (EIþ) m/z
ꢄ
(CH3); MS (EIþ) m/z (%): 292 [M]þ (100), 291 (22), 264 (10), 263
ꢄ
(22). Anal. Calcd. for C19H20N2O: C, 78.05; H, 6.89; N, 9.58. Found: C,
77.78; H, 7.10; N, 9.58. The hydrochloride of furanotacrine 4 was
obtained by treating it with hydrogen chloride gas generated by the
reaction of sulphuric acid with sodium chloride crystals: Anal.
Calcd. for C18H19ClN2O$HCl: C, 68.67, H, 6.08; N, 8.90; Cl, 11.26.
Found: 68.70; H, 6.20; N, 8.45; Cl, 10.84.
(%): 303 [M]þ (100), 302 (34), 288 (10), 275 (32). Anal. Calcd. for
C19H17N3O: C, 75.23; H, 5.65; N, 13.85. Found: C, 74.99; H, 5.46; N,
13.76. The hydrochloride of furanotacrine 7 was obtained by
treating it with hydrogen chloride gas generated by the reaction of
sulphuric acid with sodium chloride crystals: Anal. Calcd. for
C19H18ClN3O$HCl: C, 67.15, H, 5.34; N, 12.37; Cl, 10.43. Found: 66.91;
H, 5.52; N, 12.55; Cl, 10.84.
5.2.5. 2-(4-Chlorophenyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-
amine (5)
5.2.8. 4-Amino-2-(4-methoxyphenyl)-5,6,7,8-tetrahydrofuro[2,3-b]
quinoline-3-carbonitrile (8)
Following the general procedure for the Friedländer reaction, to
a
solution of AlCl3 (693 mg, 5.20 mmol, 2.1 equiv) in 1,2-
Following the general procedure for the Friedländer reaction, to
a solution of AlCl3 (458 mg, 3.43 mmol, 4.0 equiv) in 1,2-
dichloroethane (35 mL) was added 2-amino-5-(40-chlorophenyl)
furan-3-carbonitrile (19) [35] (537 mg, 2.46 mmol) under argon
dichloroethane (38 mL) was added 2-amino-5-(4-methoxyphenyl)
atmosphere. After a few minutes, cyclohexanone (510
mL, 4.92 mmol,
furan-3,4-dicarbonitrile (21) [35] (200 mg, 0.85 mmol) under argon
2.0 equiv) was added and the reaction mixture was refluxed for
atmosphere. After
a
few minutes, cyclohexanone (356 mL,

C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
6127
3.44 mmol, 4.0 equiv) was added and the reaction mixture was
refluxed for 24 h, yielding furanotacrine 8 (241 mg, 89%): mp
2.0 equiv) were added and the reaction mixture was refluxed for
3 d yielding furanotacrine 11 (521 mg, 72%): mp 265e267 ꢂC; IR
267e269 ꢂC; IR (KBr)
n
3477, 3338, 3077, 2923, 2851, 2213, 1634,
(KBr)
n 3479, 3398, 3332, 3217, 2919, 2851, 2219, 1644, 1600, 1487,
1604, 1504, 1468, 1261, 1176, 1108 cmꢃ1
;
1H NMR (DMSO-d6,
1474, 1407 cmꢃ1 1H NMR (DMSO-d6, 500 MHz)
; d 8.02 (d,
300 MHz)
d
8.04 (d, J ¼ 8.9 Hz, 2H, H20), 7.18 (d, J ¼ 8.9 Hz, 2H, H30),
J ¼ 10.0 Hz, 2H, H20), 7.69 (d, J ¼ 10.0 Hz, 2H, H30), 5.88 (br s, 2H,
5.72 (br s, 2H, NH2), 3.91 (s, 3H, OCH3), 2.77 (m, 2H, H8), 2.50 (m, 2H,
NH2), 2.92 (m, 2H, H9), 2.76 (m, 2H, H5), 1.82 (m, 2H, H7), 1.62 (m,
H5), 1.80 (m, 4H, H6, H7); 13C NMR (DMSO-d6, 75.4 MHz)
d
162.4
2H, H8), 1.56 (m, 2H, H6); 13C NMR (DMSO-d6, 125.7 MHz)
d 162.4
(C40), 154.6 (C8a), 147.5 (C4), 132.2 (C2), 128.4 (C20), 120.8 (C10), 116.6
(CN),116.2 (C3a)*,115.9 (C30)*,113.0 (C4a), 83.3 (C3), 56.4 (OCH3), 33.6
(C9a), 159.5 (C10a), 156.6 (C2), 147.2 (C4), 136.5 (C40), 130.4 (C30),
128.2 (C20),127.1 (C10),118.4 (C4a),115.6 (CN),102.9 (C3a), 86.4 (C3),
39.2 (C9), 32.2 (C7), 27.7 (C6), 26.9 (C8), 25.3 (C5); MS (EIþ) m/z (%):
339 [M þ 2]þ (34), 338 (30), 337 [M]þ (100), 336 (26), 322 (16), 310
(15), 309 (23), 308 (35), 283 (11), 139 (7). Anal. Calcd. for
C19H16ClN3O: C, 67.56; H, 4.77; N, 12.44; Cl, 10.50. Found: C, 67.87;
H, 5.01; N, 12.28; Cl, 10.88.
ꢄ
(C8), 23.7 (C5), 23.1 (C7)**, 22.9 (C6)**; MS (EIþ) m/z (%): 319 [M]þ
(100), 318 (23), 304 (16), 291 (17). Anal. Calcd. for C19H17N3O2: C,
71.46; H, 5.37; N, 13.16. Found: C, 71.58; H, 5.49; N, 13.26.
5.2.9. 4-Amino-2-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5H-
cyclohepta[b]furo[3,2-e]pyridine-3-carbonitrile (9)
Following the general procedure for the Friedländer reaction, to
solution of AlCl3 (1.15 g, 8.69 mmol, 4.2 equiv) in 1,2-
5.2.12. 1,2-Diphenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]quinolin-
4-amine (12)
a
dichloroethane (50 mL) was added 2-amino-5-(4-methoxyphenyl)
Following the general procedure for the Friedländer reaction, to
furan-3,4-dicarbonitrile (21) [35] (490 mg, 2.05 mmol) under argon
a solution of AlCl3 (700 mg, 5.25 mmol, 3.0 equiv) in 1,2-
atmosphere. After
8.48 mmol, 4.1 equiv) was added and the reaction mixture was
a
few minutes, cycloheptanone (1 mL,
dichloroethane (50 mL) was added 2-amino-1,5-diphenyl-1H-
pyrrole-3-carbonitrile (23) [35] (450 mg, 1.74 mmol) under argon
refluxed for 24 h, yielding furanotacrine 9 (521 mg, 76%): mp
atmosphere. After a few minutes, cyclohexanone (540 mL,
5.21 mmol, 3.0 equiv) was added and the reaction mixture was
249e251 ꢂC; IR (KBr)
n
3478, 3339, 3222, 2928, 2848, 2219, 1642,
1603, 1507, 1476, 1300, 1262, 1177, 1111 cmꢃ1
;
1H NMR (DMSO-d6,
refluxed for 24 h yielding pyrrolotacrine 12 (405 mg, 69%): mp
500 MHz)
d
7.98 (d, J ¼ 8.7 Hz, 2H, H20), 7.18 (d, J ¼ 8.7 Hz, 2H, H30),
308e310 ꢂC; IR (KBr)
n 3467, 3360, 3061, 2930, 2852, 1633, 1595,
5.79 (br s, 2H, NH2), 3.87 (s, 3H, OCH3), 2.91 (m, 2H, H9), 2.75 (m,
1565, 1534, 1499, 1467, 1451, 1440, 1340 cmꢃ1 1H NMR (DMSO-d6,
;
2H, H5), 1.81 (m, 2H, H7), 1.62 (m, 2H, H8), 1.56 (m, 2H, H6); 13C
400 MHz)
d
7.37 (t, J ¼ 7.8 Hz, 2H, H300), 7.28 (d, J ¼ 7.2 Hz, 2H, H20),
NMR (DMSO-d6, 125.7 MHz)
d
162.4 (C40), 161.6 (C9a), 159.2 (C10a),
7.27 (m, 1H, H400), 7.23 (d, J ¼ 7.8 Hz, 2H, H200), 7.19 (t, J ¼ 7.2 Hz, 2H,
H30), 7.16 (d, J ¼ 7.2 Hz,1H, H40), 6.89 (s,1H, H3), 6.01 (br s, 2H, NH2),
2.62 (m, 2H, H8), 2.48 (m, 2H, H5), 1.73 (m, 4H, H6, H7); 13C NMR
158.4 (C2), 146.9 (C4), 128.4 (C20), 120.8 (C10), 118.3 (C4a), 116.3
(CN), 115.9 (C30), 102.9 (C3a), 83.9 (C3), 56.4 (OCH3), 39.1 (C9), 32.2
(C7), 27.7 (C6), 26.9 (C8), 25.5 (C5); MS (EIþ) m/z (%): 333 [M]þ
(100), 332 (18), 318 (12), 305 (10), 304 (19). Anal. Calcd. for
C20H19N3O2: C, 72.05; H, 5.74; N, 12.60. Found: C, 72.27; H, 6.01; N,
12.58.
(DMSO-d6, 100.5 MHz)
d 151.5 (C8a), 148.9 (C9a), 145.6 (C4), 137.7
(C100), 134.9 (C2), 132.7 (C10), 128.6 (C300), 128.3 (C30)*, 128.2 (C200)*,
127.8 (C20), 126.7 (C400), 126.5 (C40), 106.8 (C4a), 106.2 (C3a), 100.1
(C3), 33.3 (C8), 23.1 (C5), 23.0 (C7), 22.8 (C6); MS (EIþ) m/z (%): 339
[M]þ (75), 338 (100). Anal. Calcd. for C23H21N3: C, 81.38; H, 6.24; N,
12.38. Found: C, 81.07; H, 6.51; N, 12.09.
5.2.10. 4-Amino-2-(4-chlorophenyl)-5,6,7,8-tetrahydrofuro[2,3-b]
quinoline-3-carbonitrile (10)
Following the general procedure for the Friedländer reaction, to
solution of AlCl3 (556 mg, 4.17 mmol, 3.3 equiv) in 1,2-
5.2.13. 1,2-Diphenyl-1,5,6,7,8,9-hexahydrocyclohepta[b]pyrrolo
[3,2-e]pyridin-4-amine (13)
a
dichloroethane (35 mL) was added 2-amino-5-(4-chlorophenyl)
Following the general procedure for the Friedländer reaction, to
furan-3,4-dicarbonitrile (22) [35] (307 mg, 1.26 mmol) under argon
a solution of AlCl3 (730 mg, 5.47 mmol, 3.1 equiv) in 1,2-
atmosphere. After
3.78 mmol, 3.0 equiv) was added and the reaction mixture was
a
few minutes, cyclohexanone (392
m
L,
dichloroethane (36 mL) was added 2-amino-1,5-diphenyl-1H-
pyrrole-3-carbonitrile (23) [35] (450 mg, 1.74 mmol) under argon
refluxed for 5 d, yielding furanotacrine 10 (306 mg, 75%): mp
atmosphere. After a few minutes, cycloheptanone (644 mL,
5.46 mmol, 3.1 equiv) was added and the reaction mixture was
240e242 ꢂC; IR (KBr)
n
3344, 2943, 2845, 2219, 1634, 1605, 1463,
1102 cmꢃ1; 1H NMR (CDCl3, 300 MHz)
d
8.06 (d, J ¼ 8.5 Hz, 2H, H20),
refluxed for 48 h yielding pyrrolotacrine 13 (561 mg, 91%): mp
7.48 (d, J ¼ 8.5 Hz, 2H, H30), 4.84 (br s, 2H, NH2), 2.91 (m, 2H, H8),
205e206 ꢂC; IR (KBr)
n 3432, 3337, 3046, 2909, 2847, 1628, 1594,
2.51 (m, 2H, H5), 1.92 (m, 4H, H6, H7); 13C NMR (CDCl3, 75.4 MHz)
1566, 1532, 1499, 1472, 1452, 1417, 1339, 1203 cmꢃ1 1H NMR
;
d
159.7 (C9a), 158.9 (C2), 156.9 (C8a), 146.3 (C4), 137.4 (C40), 129.9
(DMSO-d6, 400 MHz)
d
7.37 (t, J ¼ 7.4 Hz, 2H, H300), 7.28 (d,
(C30), 127.6 (C20), 126.6 (C10), 115.9 (CN), 112.5 (C4a), 103.5 (C3a),
84.6 (C3), 33.6 (C8), 23.3 (C5), 22.9 (C7)*, 22.8 (C6)*; MS (EIþ) m/z
(%): 325 [M þ 2]þ (37), 324 (35), 323 [M]þ (100), 297 (10), 295 (29),
139 (10). Anal. Calcd. for C18H14ClN3O: C, 66.77; H, 4.36; N 12.98.
Found. C, 67.08; H, 4.35; N, 12.54.
J ¼ 7.2 Hz, 2H, H20), 7.26 (m, 1 H, H400), 7.23 (d, J ¼ 7.4 Hz, 2H, H200),
7.19 (t, J ¼ 7.2 Hz, 2H, H30), 7.14 (d, J ¼ 7.2 Hz, 1H, H40), 6.88 (s, 1H,
H3), 6.02 (br s, 2H, NH2), 2.76 (m, 2H, H9), 2.68 (m, 2H, H5), 1.75 (m,
2H, H7), 1.51 (m, 4H, H6, H8); 13C NMR (DMSO-d6, 75.4 MHz)
d
159.3 (C9a), 149.0 (C10a), 145.6 (C4), 138.3 (C100), 135.6 (C2), 133.4
(C10), 129.2 (C300)*, 129.0 (C200)*, 128.9 (C30), 128.5 (C20), 127.4 (C400),
127.2 (C40), 112.9 (C4a), 107.5 (C3a), 101.1 (C3), 39.3 (C9), 32.7 (C7),
28.6 (C8), 27.6 (C6), 25.5 (C5); MS (EIþ) m/z (%): 353 [M]þ (88), 352
(100), 324 (12). Anal. Calcd. for C24H23N3: C, 81.55; H, 6.56; N, 11.89.
Found: C, 81.36; H, 6.58; N, 11.75.
5.2.11. 4-Amino-2-(4-chlorophenyl)-6,7,8,9-tetrahydro-5H-
cyclohepta[b]furo[3,2-e]pyridine-3-carbonitrile (11)
Following the general procedure for the Friedländer reaction, to
a
solution of AlCl3 (1.14 g, 8.59 mmol, 4.0 equiv) in 1,2-
dichloroethane (20 mL) was added 2-amino-5-(4-chlorophenyl)
furan-3,4-dicarbonitrile (22) [35] (518 mg, 2.13 mmol) under argon
5.2.14. 2-(4-Chlorophenyl)-1-phenyl-5,6,7,8-tetrahydro-1H-
pyrrolo[2,3-b]quinolin-4-amine (14)
atmosphere. After
a few minutes, cycloheptanone (500 mL,
4.24 mmol, 2.0 equiv) was added and the reaction mixture was
Following the general procedure for the Friedländer reaction, to
refluxed. After 48 h, AlCl3 (579 mg, 4.34 mmol, 2.0 equiv), in dry
a
solution of AlCl3 (449 mg, 3.06 mmol, 1.8 equiv) in 1,2-
1,2-dichloroethane (20 mL), cycloheptanone (500 mL, 4.24 mmol,
dichloroethane (45 mL) was added 2-amino-5-(4-chlorophenyl)-

6128
C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
1-phenyl-1H-pyrrole-3-carbonitrile (24) [35] (500 mg, 1.70 mmol)
under argon atmosphere. After a few minutes, cyclohexanone
by treating it with hydrogen chloride gas generated by the reaction
of sulphuric acid with sodium chloride crystals. Anal. Calcd. for
C24H24ClN3O$HCl: C, 73.93, H, 6.20; N, 10.78; Cl, 9.09. Found: 73.85;
H, 6.48; N, 10.49; Cl, 9.21.
(320
was refluxed. After 48 h, AlCl3 (209 mg, 1.57 mmol, 0.9 equiv), in
dry 1,2-dichloroethane (5 mL), and cyclohexanone (200 L,
mL, 3.06 mmol, 1.8 equiv) was added and the reaction mixture
m
1.95 mmol, 1.1 equiv) were added and the reaction refluxed for 6 d
5.3. Pharmacology
yielding pyrrolotacrine 14 (516 mg, 81%): mp 293e295 ꢂC; IR (KBr)
n
: 3470, 3366, 3072, 2937, 2847, 1631, 1594, 1564, 1530, 1499, 1467,
5.3.1. Evaluation of EeAChE and eqBuChE inhibitory activities
To assess the inhibitory activity of both cholinesterases, the
spectrophotometric method of Ellman [37] was followed, using
purified AChE from E. electricus (Type V-S, Sigma), or BuChE from
horse serum (lyophilized powder) (SigmaeAldrich, Madrid, Spain).
The reaction took place in a final volume of 3 mL of a phosphate-
buffered solution (0.1 M) at pH 8, containing 0.035 U of AChE or
0.05 U of BuChE and 0.35 mM of 5,50-dithio-bis(2-nitrobenzoic
acid) (DTNB, SigmaeAldrich, Madrid, Spain). Inhibition curves
were made by pre-incubating this mixture with at least nine
concentrations of each compound for 10 min. A sample with no
compound was always present to determine the 100% of enzyme
activity. After this pre-incubation period, acetylthiocholine iodide
(0.35 mM) or butyrylthiocholine iodide (0.5 mM) (SigmaeAldrich,
Madrid, Spain) were added, allowing 15 min of incubation, where
the DTNB produces the yellow anion 5-thio-2-nitrobenzoic acid
along with the enzymatic degradation of acetylthiocholine iodide
or butyrylthiocholine iodide. Changes in absorbance were detected
at 405 nm in a spectrophotometric plate reader (FluoStar OPTIMA,
BMG Labtech). Compounds inhibiting AChE or BuChE activity
would reduce the colour generation, thus IC50 values were calcu-
lated as the concentration of compound that produces 50% of the
AChE activity inhibition. Data are expressed as means ꢀ SEM of at
least three different experiments in quadruplicate.
1440, 1339 cmꢃ1; 1H NMR (CDCl3, 400 MHz)
d
7.37 (t, J ¼ 7.8 Hz, 2H,
H300), 7.28 (d, J ¼ 7.8 Hz, 3H, H200, H400), 7.19 (d, J ¼ 6.6 Hz, 2H, H20),
7.13 (d, J ¼ 6.6 Hz, 2H, H30), 6.53 (s, 1H, H3), 4.31 (br s, 2H, NH2), 2.87
(m, 2H, H8), 2.58 (m, 2H, H5), 1.87 (m, 4H, H6, H7); 13C NMR (CDCl3,
100.4 MHz)
d
153.2 (C8a), 149.0 (C9a), 143.7 (C4), 137.2 (C100), 136.0
(C10), 132.9 (C40), 131.4 (C2), 129.7 (C30), 128.7 (C300), 128.4 (C200),
128.3 (C20), 126.7 (C400), 108.2 (C4a), 107.0 (C3a), 98.0 (C3), 33.8 (C8),
29.6 (C5), 23.2 (C7)*, 23.1 (C6)*; MS (EIþ) m/z (%): 375 [M þ 2]þ (27),
374 (49), 373 [M]þ (79), 372 (100). Anal. Calcd. for C23H20ClN3: C,
73.89; H, 5.39; N, 11.24. Found: C, 73.92; H, 5.87; N, 10.87.
5.2.15. 2-(4-Chlorophenyl)-1-phenyl-1,5,6,7,8,9-hexahydrocyclohe
pta[b]pyrrolo[3,2-e]pyridin-4-amine (15)
Following the general procedure for the Friedländer reaction, to
solution of AlCl3 (548 mg, 4.11 mmol, 2.0 equiv) in 1,2-
a
dichloroethane (30 mL) was added 2-amino-5-(4-chlorophenyl)-
1-phenyl-1H-pyrrole-3-carbonitrile (24) [35] (593 mg, 2.02 mmol)
under argon atmosphere. After a few minutes, cycloheptanone
(476
was refluxed for 48 h, yielding pyrrolotacrine 15 (517 mg, 73%): mp
274e276 ꢂC; IR (KBr)
: 3430, 2918, 2848, 1630, 1593, 1566, 1530,
1451, 1334 cmꢃ1; 1H NMR (DMSO-d6, 500 MHz)
mL, 4.04 mmol, 2.0 equiv) was added and the reaction mixture
n
d
7.41 (t, J ¼ 5.0 Hz,
2H, H300), 7.32 (t, J ¼ 5.0 Hz, 2H, H20, H400), 7.24 (d, J ¼ 5.0 Hz, 2H,
H200), 7.18 (d, J ¼ 5.0 Hz, 2H, H30), 6.94 (s, 1H, H3), 5.88 (br s, 2H,
NH2), 2.81 (m, 2H, H9), 2.72 (m, 2H, H5), 1.78 (m, 2H, H7), 1.56 (m,
5.3.2. Evaluation of hAChE and hBuChE inhibitory activities
The evaluation of the inhibitory activity against human
recombinant AChE (Sigma, Italy) and BuChE from human serum
4H, H6, H8); 13C NMR (DMSO-d6, 125 MHz)
d 159.8 (C9a), 149.1
(C10a), 145.8 (C4), 138.4 (C100), 134.8 (C10), 132.6 (C40)*, 132.4 (C2)*,
130.3 (C30), 129.4 (C300), 129.1 (C20)**, 129.1 (C200)**, 127.5 (C400), 113.3
(C4a), 107.9 (C3a), 101.7 (C3), 39.7 (C9), 32.6 (C7), 28.6 (C8), 27.6
(C6), 25.7 (C5); MS (EIþ) m/z (%): 389 [M þ 2]þ (31), 388 (50), 387
[M]þ (91), 386 (100), 358 (15). Anal. Calcd. for C24H22ClN3: C, 74.31;
H, 5.72; N, 10.83; Cl, 9.14. Found: C, 74.12; H, 5.93; N, 11.03; Cl, 9.44.
(Sigma, Italy) was carried out with 0.02 U/mL of enzyme, 550
substrate (acetylthiocholine and butyrylthiocholine, respectively),
340
M 5,50-dithio-bis(2-nitrobenzoic acid) and 0.1 M potassium
mM of
m
phosphate buffer pH 8 as assay medium, in a final reaction volume
of 1 mL. The method of Ellman [37] was followed to evaluate
activity and calculate inhibition.
5.2.16. 1-Phenyl-2-(p-tolyl)-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]
quinolin-4-amine (16)
5.3.3. Kinetic analysis of the AChE inhibition by compound 12
To obtain estimates of the mechanism of action of compound 12,
reciprocal plots of 1/V versus 1/[S] were constructed at different
concentrations of the substrate acetylthiocholine (0.1e0.8 mM) by
using Ellman’s method [37]. Experiments were performed in
Following the general procedure for the Friedländer reaction, to
a
solution of AlCl3 (306 mg, 2.30 mmol, 2.1 equiv) in 1,2-
dichloroethane (20 mL) was added 2-amino-1-phenyl-5-(p-tolyl)-
1H-pyrrole-3-carbonitrile (25) (302 mg, 1.10 mmol) under argon
atmosphere. After
a
few minutes, cyclohexanone (231
mL,
a transparent 48-well plate containing each well 400
mL of the
2.23 mmol, 2.1 equiv) was added and the reaction mixture was
DTNB 0.875 mM solution in PBS 0.1 M, 1 L of DMSO (control) or
m
refluxed. After 72 h, AlCl3 (228 mg, 1.71 mmol, 1.5 equiv), in dry 1,2-
inhibitor solution to give the desired final concentration. Final
dichloroethane (10 mL), and cyclohexanone (171
were added and the reaction was refluxed for 4 d yielding pyrro-
lotacrine 16 [35] (285 mg, 73%): mp 305e306 ꢂC; IR (KBr)
3469,
3364, 3067, 3014, 2940, 2849, 1632, 1595, 1563, 1539, 1500, 1467,
m
L, 1.5 mmol)
volume (1 mL) was reached by adding PBS (pH 8). Reaction was
initiated by adding 14 m
L of AChE at 30 ꢂC to give a final concen-
n
tration of 0.05 U/mL. Progress curves were monitored at 412 nm
over 1.33 min in a fluorescence plate reader Fluostar Optima (BMG-
technologies, Germany). Progress curves were characterized by
a linear steady-state turnover of the substrate and values of a linear
regression were fitted according to LineweavereBurk replots using
Origin software. The plots were assessed by a weighted least square
analysis. Determination of Michaelis constant for the substrate
ATCh was done at 6 different concentrations (0e0.8 mM) to give
1441, 1338 cmꢃ1; 1H NMR (DMSO-d6, 300 MHz)
d
7.38 (t, J ¼ 9.0 Hz,
2H, H300), 7.31 (d, J ¼ 9.0 Hz, 1H, H400), 7.22 (d, J ¼ 9.0 Hz, 2H, H200),
7.07 (br s, 4H, H20, H30), 6.84 (s, 1H, H3), 5.75 (br s, 2H, NH2), 2.66
(m, 2H, H8), 2.50 (m, 2H, H5), 2.26 (s, 3H, CH3), 1.78 (m, 4H, H6, H7);
13C NMR (DMSO-d6, 75.4 MHz)
d 152.2 (C8a), 149.8 (C9a), 146.2
(C4), 138.8 (C100), 136.9 (C40), 136.3 (C2), 130.9 (C10), 129.6 (C300),
129.3 (C200), 129.2 (C30), 128.7 (C20), 127.2 (C400), 107.9 (C4a), 107.3
(C3a), 100.3 (C3), 34.1 (C8), 23.9 (C5), 23.8 (C7), 23.7 (C6), 21.4
(CH3); MS (EIþ) m/z (%): 353 [M]þ (100), 352 (98), 176 (11). Anal.
Calcd. for C24H23N3: C, 81.55; H, 6.56; N, 11.89. Found: C, 81.28; H,
6.67; N, 11.59. The hydrochloride of pyrrolotacrine 16 was obtained
a value of KM ¼ (0.4 ꢀ 0.1) mM, and Vmax ¼ (0.41 ꢀ0.07) minꢃ1
.
Slopes of the reciprocal plots were then plotted against the
concentration of 12 (range 0e2 mM) to evaluate Ki data. Data
analysis was performed with Origin Pro 8.0 software (Origin
Lab Corp.).

C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
6129
5.3.4. Cell culture of the human neuroblastoma cell line SH-SY5Y
SH-SY5Y cells were maintained in a 1:1 mixture of F-12 nutrient
mixture (Ham12) (SigmaeAldrich, Spain) and Eagle’s minimum
essential medium (EMEM) supplemented with 15 nonessential
amino acids, 1 mM sodium pyruvate, 10% heat-inactivated foetal
Docking calculations were performed with the program Auto-
Dock Vina [40]. AUTODOCKTOOLS (ADT) (version 1.5.4) was used to
add hydrogens and partial charges for proteins and ligands using
Gasteiger charges. Flexible torsions in the ligands were assigned
with the AUTOTORS module, and the acyclic dihedral angles were
allowed to rotate freely.
bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL strepto-
mycin (reagents from Invitrogen, Spain). Cultures were seeded into
flasks containing supplemented medium and maintained at 37 ꢂC
in a humidified atmosphere of 5% CO2 and 95% air. For assays, SH-
SY5Y cells were sub-cultured in 48-well plates at a seeding
density of 1 ꢅ105 cells per well. Cells were treated with the tested
compound before confluence in F12/EMEM with 1% FBS. All the
cells used in this study were used at a low passage number (<13).
Because Vina uses rectangular boxes for the binding site, the box
centre was defined and the docking box was displayed using ADT.
For EeAChE (PDB: 1C2B) the docking procedure was applied to the
whole protein target, without imposing the binding site (“blind
ꢀ
docking”). A grid box of 60 ꢅ 60 ꢅ 72 with grid points separated 1 A,
was positioned at the middle of the protein (x ¼ 21.5911;
y ¼ 87.752; z ¼ 23.591). Default parameters were used except
num_modes, which was set to 40.
5.3.5. Incubation of drugs
To compare the effects of compound 12 with tacrine on cell
viability, SH-SY5Y cells were incubated for 48 h with 3 and 30 mM of
compound 12, and thereafter, cell viability was measured as MTT
reduction.
5.4.2. Molecular docking of compounds 3 and 12 into eqBuChE
The eqBuChE model has been retrieved from The SWISS-MODEL
Repository. This is a database of annotated three-dimensional
comparative protein structure models generated by the fully
automated homology-modelling pipeline SWISS-MODEL. A puta-
tive three-dimensional structure of eqBuChE has been created
based on the crystal structure of hBuChE (pdb: 2pm8). These two
enzymes exhibited 89% sequence identity. Proper bonds, bond
orders, hybridization and charges were assigned using protein
model tool in Discovery Studio, version 2.1, software package.
CHARMm force field was applied using the receptoreligand inter-
actions tool in Discovery Studio, version 2.1, software package.
Docking calculations were performed following the same
protocol described before for EeAChE. All dockings were performed
To assess the neuroprotective effect of compound 12 against
Ab
25e35 e induced toxicity, SH-SY5Y cells were pre-incubated with
compound 12 at the concentrations of 0.3, 1, and 3 M for 24 h.
Then, cells were co-incubated for another 24 h period with
compound 12 in the presence of 10 M Ab25e35. At the end of the
m
m
experiment, cell viability was assessed using the MTT method.
5.3.6. Measurement of cell viability with MTT
Cell viability, virtually the mitochondrial activity of living cells,
was measured by quantitative colorimetric assay with MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma-
eAldrich, Spain), as described previously. MTT was added to all
wells (final concentration 0.5 mg/mL) and allowed to incubate in
the dark at 37 ꢂC for 2 h. The tetrazolium ring of MTT can be cleaved
by mitochondrial reductases in order to produce a precipitated
formazan derivative. After this 2 h period, the formazan produced
ꢀ
as blind dockings where a cube of 75 A with grid points separated
ꢀ
1 A was positioned at the middle of the protein (x ¼ 29.885;
y ¼ ꢃ54.992; z ¼ 58.141). Default parameters were used except
num_modes, which was set to 40.
The lowest docking-energy conformation was considered as the
most stable orientation. Finally, the docking results generated were
directly loaded into Discovery Studio, version 2.1.
was dissolved by adding 200 mL of DMSO, resulting in a coloured
compound whose optical density was measured in an ELISA reader
at 540 nm. All MTT assays were performed in triplicate. Absorbance
values obtained in control cells untreated with the toxic were
considered as 100% viability.
Acknowledgements
Carla Martins thanks Fundação para a Ciência e Tecnologia do
Ministério da Ciência, Tecnologia e Ensino Superior of Portugal for
her Ph
D grant SFRH/BD/17577/2004. JMC thanks MICINN
5.4. Molecular docking
(SAF2006-08764-C02-01; SAF2009-07271), Comunidad de Madrid
(S/SAL-0275-2006), and CSIC-GRICES (2007PT-13) for financial
support.
MG and JE thank the Spanish Ministry of Science and Innovation
Ref. SAF2009-12150 and the Spanish Ministry of Health (Instituto
de Salud Carlos III) RETICS-RD06/0026 to MGL. CM thanks COST
Action D34 for her STSM, which made possible this collaborative
work.
Compounds 3 and 12 were assembled within Discovery Studio,
version 2.1, software package, using standard bond lengths and
bond angles without protonation at the pyridine ring. With the
CHARMm force field [44] and partial atomic charges, the molecular
geometry of these compounds was energy-minimized using the
adopted-based NewtoneRaphson algorithm. Structures were
considered fully optimized when the energy changes between
interactions were less than 0.01 kcal/mol [45].
References
5.4.1. Molecular docking into EeAChE
The coordinates of EeAChE (PDB ID: 1C2B) were obtained from
the Protein Data Bank (PDB). For docking studies, initial protein was
prepared by removing all water molecules, heteroatoms, any co-
crystallized solvent and the ligand. It is well known that PDB files
often have poor or missing assignments of explicit hydrogens, and
the PDB file format cannot accommodate bond order information.
Therefore, proper bonds, bond orders, hybridization and charges
were assigned using protein model tool in Discovery Studio, version
2.1, software package. CHARMm force field was applied using the
receptoreligand interactions tool in Discovery Studio, version 2.1,
software package.
[1] M.L. Bolognesi, A. Cavalli, L. Valgimigli, M. Bartolini, V. Andrisano,
M. Recanatini, C. Melchiorre, J. Med. Chem. 50 (2007) 6446e6449.
[2] M. Rosini, E. Simoni, M. Bartolini, A. Cavalli, L. Ceccarini, N. Pascu,
D.W. McClymont, A. Tarozzi, M.L. Bolognesi, A. Minarini, V. Tumiatti,
V. Andrisano, I.R. Mellor, C. Melchiorre, J. Med. Chem. 51 (2008) 4381e4384.
[3] S. Rizzo, C. Rivière, L. Piazzi, A. Bisi, S. Gobbi, M. Bartolini, V. Andrisano,
F. Morroni, A. Tarozzi, J.P. Monti, A. Rampa, J. Med. Chem. 51 (2008)
2883e2886.
[4] R. Morphy, Z. Rankovic, J. Med. Chem. 48 (2005) 6523e6543.
[5] M.B.H. Youdim, J.J. Buccafusco, Trends Pharmacol. Sci. 26 (2005) 27e35.
[6] L. Piazzi, A. Cavalli, F. Colizzi, F. Belluti, M. Bartolini, F. Mancini, M. Recanatini,
V. Andrisano, A. Rampa, Bioorg. Med. Chem. Lett. 18 (2008) 423e426.
[7] A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini,
C. Melchiorre, J. Med. Chem. 51 (2008) 347e372.

6130
C. Martins et al. / European Journal of Medicinal Chemistry 46 (2011) 6119e6130
[8] L. Fang, D. Appenroth, M. Decker, M. Kiehntopf, A. Lupp, S. Peng, C. Fleck,
Y. Zhang, J. Lehmann, J. Med. Chem. 51 (2008) 7666e7669.
M.G. López, A.G. García, M.C. Carreiras, M. Villarroya, J. Med. Chem. 52 (2009)
2724e2732 and references cited therein.
[9] M. Decker, Mini-Rev. Med. Chem. 7 (2007) 221e229.
[30] (“Thiazolotacrines”) D. Thomae, E. Perspicace, S. Hesse, G. Kirsch, P. Seck,
Tetrahedron 64 (2008) 9309e9314.
[31] ("Pyrrolotacrines”) H. Bekolo, G. Kirsch, Can. J. Chem. 85 (2007) 1e6.
[32] (“Selenophenetacrines”) D. Thomae, G. Kirsch, P. Seck, Synthesis (2008)
1600e1606.
[10] M.L. Bolognesi, R. Banzi, M. Bartolini, A. Cavalli, A. Tarozzi, V. Andrisano,
A. Minarini, M. Rosini, V. Tumiatti, C. Bergamini, R. Fato, G. Lenaz, P. Hrelia,
A. Cattaneo, M. Recanatini, C. Melchiorre, J. Med. Chem. 50 (2007) 4882e4897.
[11] A. Cavalli, M.L. Bolognesi, S. Capsoni, V. Andrisano, M. Bartolini, E. Margotti,
A. Cattaneo, M. Recanatini, C. Melchiorre, Angew. Chem., Int. Ed. 46 (2007)
3689e3692.
[33] (“Thiophenetacrines”) D. Thomae, G. Kirsch, P. Seck, Synthesis (2007)
1027e1032.
[12] D. Muñoz-Torrero, Curr. Med. Chem. 15 (2008) 2433e2455.
[13] N.C. Inestrosa, A. Alvarez, F. Calderón, Mol. Psychiatry 1 (1996) 359e361.
[14] M. Bartolini, C. Bertucci, V. Cavrini, V. Andrisano, Biochem. Pharmacol. 65
(2003) 407e416.
[15] M. Racchi, E. Porrello, C. Lanni, S.C. Lenzken, M. Mazzucchelli, S. Govoni, Aging
Clin. Exp. Res. 18 (2006) 149e152.
[34] (“Benzofuranotacrines”) C.-L. Yang, C.-H. Tseng, Y.-L. Chen, C.-M. Lu, C.-L. Kao,
M.-H. Wu, C.-C. Tzeng, Eur. J. Med. Chem. 45 (2010) 602e607.
[35] C. Martins, A. Eleuterio, M.C. Carreiras, A. Samadi, E. Soriano, R. León,
J.M. Marco-Contelles, Mol. Divers. 13 (2009) 459e468.
[36] J. Marco-Contelles, E.M. Pérez-Mayoral, A. Samadi, M.C. Carreiras, E. Soriano,
Chem. Rev. 109 (2009) 2652e2671.
[16] E. Giacobini, F. Mori, C.C. Lai, Ann. N.Y. Acad. Sci. 777 (1996) 393e398.
[17] E. Giacobini, Ann. N.Y. Acad. Sci. 920 (2000) 321e327.
[18] E. Giacobini, CNS Drugs 15 (2001) 85e91.
[19] M. Pakaski, P. Kasa, Curr. Drug Targets CNS Neurol. Disord. 2 (2003) 163e171.
[20] M. Racchi, M. Mazzucchelli, S.C. Lenzken, E. Porrello, C. Lanni, S. Govoni, Chem.
Biol. Interact. 157e158 (2005) 335e338.
[21] F. Mori, L. Chen-Ching, F. Fusi, E. Giacobini, Neuroreport 6 (1995) 633e636.
[22] E. Giacobini, Neurochem. Res. 25 (2000) 1185e1190.
[23] M. Zimmermann, B. Borroni, F. Cattabeni, A. Padovani, M. Di Luca, Neurobiol.
Dis. 19 (2005) 237e242.
[37] G.L. Ellman, K.D. Courtney, B.J. Andres, R.M. Featherstone, Biochem. Pharma-
col. 7 (1961) 88e90.
[38] C. Hetenyi, D. Van der Spoel, Protein Sci. 20 (2011) 880e893.
[39] C. Hetenyi, D. Van der Spoel, FEBS Lett. 580 (2006) 1447e1450.
[40] O. Trott, A.J. Olson, J. Comput. Chem. 31 (2010) 455e461.
[41] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, Bioinformatics 22 (2006)
195e201.
[42] F. Kiefer, K. Arnold, M. Künzli, L. Bordoli, T. Schwede, Nucleic Acids Res. 37
(2009) D387eD392.
[43] M.C. Peitsch, Bio/Technology 13 (1995) 658e660.
[44] B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan,
M. Karplus, M. Charmm, J. Comput. Chem. 4 (1983) 187e217.
[45] A. Morreale, F. Maseras, I. Iriepa, E. Galvez, J. Mol. Graph. Model. 21 (2002)
111e118.
[46] M.L. Bolognesi, V. Andrisano, M. Bartolini, A. Cavalli, A. Minarini,
M. Recanatini, M. Rosini, V. Tumiati, C. Melchiorre, J. Med. Chem. 60 (2005)
465e473.
[24] H.C. Liu, C.W. Chi, S.Y. Ko, H.C. Wang, C.J. Hong, K.N. Lin, P.N. Wang, T.Y. Liu,
Dement. Geriatr. Cogn. Disord. 19 (2005) 345e348.
[25] M. Pakaski, J. Kálmán, Neurochem. Int. 53 (2008) 103e111.
[26] R. Cacabelos, Neuropsychiatr. Dis. Treat 3 (2007) 303e333.
[27] A. Castro, A. Martinez, Curr. Pharm. Des. 12 (2006) 4377e4387.
[28] M.L. Bolognesi, M. Rosini, V. Andrisano, M. Bartolini, A. Minarini, V. Tumiatti,
C. Melchiorre, Curr. Pharm. Des. 15 (2009) 601e613.
[29] J. Marco-Contelles, R. León, C. de Los Ríos, A. Samadi, M. Bartolini,
V. Andrisano, O. Huertas, X. Barril, F.J. Luque, M.I. Rodríguez-Franco, B. López,
[47] Q.-S. Yu, H.W. Holloway, T. Utsuki, A. Brossi, N.H. Greig, J. Med. Chem. 42
(1999) 1855e1861.