Synthesis, biological assessment, and molecular modeling of racemic 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amines as potential drugs for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2012.06.038

The synthesis, pharmacological analysis and molecular modeling of the readily available racemic tacrine analogs 21-30, bearing the 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine heterocyclic ring system (II), prepared by Friedlaender reaction of 2-amino-4-aryl-4H-benzo[h]chromene-3-carbonitriles (11-20) with cyclohexanone, are described in this paper. Molecules 21-30 are potent and selective inhibitors of hAChE, in the low micromolar range, one of the most potent inhibitors, 4-(8-amino-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-7-yl) -2-methoxyphenol (25), showing a IC₅₀ (hAChE) = 0.33 ± 0.04 μM. Kinetic studies of compound 25 proved that this compound is a mixed type inhibitor for EeAChE (K_i = 81 nM). Accordingly, molecular modeling of inhibitor 25 showed that both enantiomers have two major predicted binding modes at the active and at the peripheral anionic sites of AChE. Inhibitor 25 has an excellent antioxidant profile as determined in the ORAC experiment (1.47 ± 0.10 Trolox equiv). Inhibitors 26-28 and 30 are permeable to BBB as determined in the PAMPA assay. Compared to tacrine, selected compounds 26-28 and 30 showed less hepatic toxicity in HepG2 cells. Moreover, cell viability-related studies in cortical neurons in primary cultures show that compounds 26-28 and 30 (0.1-50 μM) have significant neuroprotective effects against mitochondrial chain blockers-induced cell death, and, unlike tacrine, are not neurotoxic at concentrations lower than 50 μM. It is worth highlighting that compound 27 has the best neuroprotective properties out of all assayed compounds and shows no neurotoxicity. To sum up, these tacrine analogs can be considered as attractive multipotent therapeutic molecules on pharmacological receptors playing key roles in the progress of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2012.06.038

European Journal of Medicinal Chemistry 54 (2012) 750e763
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological assessment, and molecular modeling of racemic 7-aryl-
9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amines
as potential drugs for the treatment of Alzheimer’s disease
,
b,
Emna Maalej a, Fakher Chabchoub a , María Jesús Oset-Gasque c, Mario Esquivias-Pérez b,
María P. González b, Leticia Monjas d, Concepción Pérez d, Cristóbal de los Ríos e,
María Isabel Rodríguez-Franco d, Isabel Iriepa f, Ignacio Moraleda f, Mourad Chioua g,
**
Alejandro Romero h, José Marco-Contelles g , Abdelouahid Samadi
,
g
*
a Laboratoire de Chimie Appliquée, Hétérocycles, Corps Gras et Polymères Faculté des Sciences de Sfax, Université de Sfax, 3018 Sfax, Tunisia
b Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), Madrid, Spain
c Instituto Universitario de Investigación en Neuroquímica (IUIN), Universidad Complutense de Madrid (UCM), 28040 Madrid, Spain
d Instituto de Química Médica (IQM-CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
e Instituto Teófilo Hernando, Fundación de Investigación Biómedica, Hospital Universitario de la Princesa, C/ Diego de León, 62, E-28029 Madrid, Spain
f Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcalá, Ctra. Barcelona, Km. 33.5, 28817 Alcalá de Henares, Spain
g Laboratorio de Química Médica y Computacional (IQOG, CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain
h Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 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, pharmacological analysis and molecular modeling of the readily available racemic tacrine
analogs 21e30, bearing the 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-
amine heterocyclic ring system (II), prepared by Friedländer reaction of 2-amino-4-aryl-4H-benzo[h]
chromene-3-carbonitriles (11e20) with cyclohexanone, are described in this paper. Molecules 21e30 are
potent and selective inhibitors of hAChE, in the low micromolar range, one of the most potent inhibitors,
4-(8-amino-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-7-yl)-2-methoxyphenol (25),
Received 29 May 2012
Received in revised form
11 June 2012
Accepted 13 June 2012
Available online 28 June 2012
showing a IC50 (hAChE) ¼ 0.33 ꢀ 0.04
mM. Kinetic studies of compound 25 proved that this compound is
Keywords:
a mixed type inhibitor for EeAChE (Ki ¼ 81 nM). Accordingly, molecular modeling of inhibitor 25 showed
that both enantiomers have two major predicted binding modes at the active and at the peripheral
anionic sites of AChE. Inhibitor 25 has an excellent antioxidant profile as determined in the ORAC
experiment (1.47 ꢀ 0.10 Trolox equiv). Inhibitors 26e28 and 30 are permeable to BBB as determined in
the PAMPA assay. Compared to tacrine, selected compounds 26e28 and 30 showed less hepatic toxicity
in HepG2 cells. Moreover, cell viability-related studies in cortical neurons in primary cultures show that
7-Aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amines
hAChE
hBuChE
Kinetic analysis
Inhibition mechanism
Antioxidant
ORAC
Neuroprotection
Toxicity
compounds 26e28 and 30 (0.1e50
chain blockers-induced cell death, and, unlike tacrine, are not neurotoxic at concentrations lower than
50 M. It is worth highlighting that compound 27 has the best neuroprotective properties out of all
mM) have significant neuroprotective effects against mitochondrial
m
assayed compounds and shows no neurotoxicity. To sum up, these tacrine analogs can be considered as
attractive multipotent therapeutic molecules on pharmacological receptors playing key roles in the
progress of Alzheimer’s disease.
Molecular modeling
Alzheimer’s disease
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
other cognitive impairments [1]. Although the etiology of AD is still
poorly understood, several factors such as amyloid-b (Ab) deposits
Alzheimer’s disease (AD) is an age-related neurodegenerative
[2], -protein aggregation, and deficits of acetylcholine (ACh) [3] are
s
process characterized by a progressive loss of memory, along with
thought to play significant roles in the pathology of the disease [4].
The cholinergic theory [1] suggests that the selective loss of
cholinergic neurons in AD results in low levels of ACh in specific
regions of the brain that mediate learning and memory functions
[5]. Consequently, acetylcholinesterase (AChE) inhibitors such as
tacrine, donepezil, rivastigmine, and galanthamine, are known to
* Corresponding author. Fax: þ34 91 5644853.
** Corresponding author. Fax: þ216 74676606.
E-mail addresses: fakher.chabchoub@yahoo.fr (F. Chabchoub), iqoc21@
iqog.csic.es (J. Marco-Contelles).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.038

E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
751
improve AD symptoms by inhibiting AChE, i.e. the enzyme
responsible for the hydrolysis of ACh, thereby rising the levels of
ACh in the synaptic cleft. As a result, they have been approved for
commercial use [6]. Recently, a renewed interest for AChE inhibi-
tors has been stimulated by the potential role of AChE in acceler-
ating the formation of amyloid fibrils in the brain and forming
a potent Ca2þantagonist that permeates the blood brain barrier
(BBB), and displays neuroprotective and antioxidant properties
[26].
In this context, with these precedents from our laboratory in
mind, and in order to validate the proposed binding mode [25,26]
and modify the structure of the previous active compounds A, B
and RL2/101, looking for more equipotent AChE vs BuChE inhibi-
tors with higher antioxidant and neuroprotective and lower
toxicity properties, we have recently reported the synthesis, the
biological evaluation, and molecular modeling of a number of
stable complexes with Ab [7]. This role involves the peripheral
anionic binding site (PAS) of AChE [8]. The multifactorial nature of
AD supports new therapeutic strategies [9e14], such as NO-donor-
tacrine-hybrids [15], dual inhibitors of MAO/AChE [16,17], serotonin
transporters [18], ChE inhibitors with antioxidant and neuro-
protective properties [19], gallamineetacrine hybrids binding at
racemic
14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno
[2,3-b]quinolin-13-amines of type (I) (Chart 2) [27]. During the
course of this project, Li [28] and Perumal [29] communicated the
synthesis of compounds of type I, but no biological activity has
been described for them. From our work [27], compound 4-(13-
amino-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]qui-
nolin-14-yl)phenol (A4) (Chart 2) emerged as a selective, potent
and mixed type EeAChE inhibitor (IC50 ¼ 7 ꢀ 2 nM; Ki of 5.00 nM),
4-fold more active than tacrine; however, compound A4 was
unable to displace propidium iodide, suggesting that this inhibitor
does not strongly bind to the PAS of AChE, an observation
confirmed later by docking, molecular dynamics simulations, and
MM-GBSA calculations [27].
ChE/M2 receptors [20], and AChE plus
regulators [21]. On the other hand, Ca2þ overload is the main factor
that augments formation [22], and favors mitochondrial
s-hyperphosphorylation
Ab
disruption, which leads to the activation of the apoptotic cascade
and cell death [23]. Nimodipine, a neuronal L-type Ca2þ channel
blocker, protects neurons from death evoked by focal cerebral
ischemia [24].
In this context, some years ago we embarked on a long-term
research project aimed at the synthesis of a series of multipotent
compounds designed to target AChE inhibition and neuronal Ca2þ
modulation [25]. We have therefore synthesized and evaluated
a
number of hybrid derivatives such as ethyl 5-amino-4-(4-
Based on these results, we have carried out the synthesis and
pharmacological evaluation of a number of related, racemic,
isomeric tacrine analogs bearing the 7-aryl-9,10,11,12-tetrahydro-
7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine structure (II)
(Chart 2). We hypothesized that by changing the location of the
fused benzene ring A [compare compounds of type I with those
of type II (Chart 2)] consequences in the AChE binding should
arise and, consequently, in the inhibition power of these new
inhibitors.
Accordingly, in this work we report the synthesis [30], studies
on the inhibition of hAChE/hBuChE, the antioxidant activities
determined using the ORAC method, and the BBB of compounds
21e30. In addition, we have deepened into the hepatotoxicity and
neuroprotective properties of selected derivatives 26e28 and 30
(Scheme 1). Overall, from the ChE inhibition studies, confirmed by
fluorophenyl)-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyrid-
ine-3-carboxylate (A) and ethyl 5-amino-4-(4-methoxyphenyl)-2-
methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylate
(B), which combine the tetrahydroaminoquinoline moiety present
in tacrine (Chart 1) with a pyridine or a 4H-pyran ring system,
a substitution pattern similar to that found in the isosteric 1,4-
dihydropyridines [25]. Compounds A and B were less potent as
AChEIs than tacrine, but they blocked voltage-dependent Ca2þ
channels (38% and 50% inhibition, respectively) [25].
More recently, we have reported the chemistry and pharma-
cology of RL2/101 (Chart 1), a potent and selective AChE vs BuChE
mixed-type inhibitor, which binds preferentially at the PAS of
AChE, interfering with the pro-aggregation A
thus being a mild inhibitor of A self aggregation, as well as
b effect of hAChE, and
b
Chart 1. Structure of tacrine, ethyl 5-amino-4-(4-fluorophenyl) 2-methyl-6,7,8,9-
tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate (A) [25], ethyl 5-amino-4-(4-
methyoxyphenyl)-2-methyl-6,7,8,9-tetrahydro 4H-pyrano[2,3-b]quinoline-3-carboxylate
(B) [25], and ethyl 5-amino-4-(4-methoxphenyl)-2-methyl-1,4,6,7,8,9-hexahydrobenzo
[b][1,8] naphthridine-3-carboxylate (RL2/101) [26].
Chart 2. Structures of the previously investigated tacrine analogs l, and the most
potent AChE inhibitor in this series (A4) [27], the target 7-aryl-9,10,11,12-tetrahydro-
7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amines (ll), and compound 25, described in
this work.

752
E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
Scheme 1. Synthesis of 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine (21e30).
molecular modeling analysis, along with other pharmacological
results, we have identified 4-(8-amino-9,10,11,12-tetrahydro-7H-
benzo[7,8]chromeno[2,3-b]quinolin-7-yl)-2-methoxyphenol (25)
(Chart 2) with an attractive multipotent profile for the potential
treatment of AD.
substituents in benzene ring. First of all, compounds 21e30 are
potent, in the low micromolar range, and selective inhibitors of
hAChE. The most potent hAChE inhibitor was 7-phenyl-
9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-
amine (21) (IC50 ¼ 0.30 ꢀ 0.04
mM), the parent compound
without any substituent in the benzene ring, followed by prod-
ucts 25, 26 and 22, bearing electron-donating groups such 40-
OH,30-OMe (25), 40-OMe,20-Me (26) or 40-Me (22), respectively,
2. Results and discussion
showing IC50 values in the 0.33e0.40
mM range. Very interest-
2.1. Chemistry
ingly, monosubstituted inhibitors, bearing electron-donating
groups, such as 20-OMe (24), or 40-OH (23), or a trisubstituted
compound bearing electron-donating groups such as 30,50-di-
OMe,40-OMe (27) were 11e26 fold less potent than inhibitor 21,
the most active in this series. Similarly, compounds 28e30
bearing electron-withdrawing groups such as 40-NO2 (28), and
20,6- (29) or 30,40-di-Cl (30) were also 13e19 fold less potent
than inhibitor 21, but still selective hAChE inhibitors; however,
note also that these inhibitors (28e30) are more
potent than compound 23, bearing a free hydroxyl group at
C40. Very surprisingly, only 7-(2-methoxyphenyl)-9,10,11,12-
tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine (24)
The synthesis of 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amines of type II (Chart 2) has been
carried as shown in Scheme 1. Starting from the readily available
2-arylidenemalononitriles 1e10, the reaction with 1-naphtho
provided, as described, racemic compounds 11e20 in very good
yields [31,32]. Compounds 14 [33] and 20 [34] (Scheme 1) have
been described in a non easily available literature source and in
a patent, respectively, but we have described them here from the
corresponding 2-arylidenemalononitriles
4 [35] and 10 [36].
Intermediates 16 and 19 are new, and have been prepared here
for the first time from precursor 6 [37] and 9 [38], respectively
(Scheme 1).
showed
(IC50 ¼ 9.37 ꢀ 0.3
a
modest, but significant inhibition of hBuChE
M) (Table 1). To sum up, the selective hAChE
m
Friedländer reaction [39] of 3-amino-1-aryl-1H-benzo[f]chro-
mene-2-carbonitriles 11e20 with cyclohexanone, under the usual
experimental conditions [20,27], gave, as expected, racemic
compounds 21e30 (Scheme 1) from low to good chemical yields.
These new 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno
[2,3-b]quinolin-8-amines derivatives included compounds bearing
one or more electron donating (withdrawing) groups at different
positions in the benzene ring at C7 in order to understand the effect
of the steric and/or electronic capacities of these groups on their
enzymatic binding and other physicochemical properties.
All new compounds showed analytical and spectroscopic data in
good agreement with their structures (see Experimental Part).
inhibition by 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chro-
meno[2,3-b]quinolin-8-amines is clearly favored by electron-
donating substituents in the benzene ring at C7, but the type,
Table 1
Inhibition (IC50, mM) of human Acetyl- and Butyrylcholinesterase (hAChE and hBu-
ChE), and Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents) by
racemic compounds 21e30.a
Compound
hAChE
hBuChE
ORAC
21
22
23
24
25
26
27
28
0.30 ꢀ 0.04
0.40 ꢀ 0.02
7.83 ꢀ 0.8
4.53 ꢀ 0.1
0.33 ꢀ 0.04
0.37 ꢀ 0.06
3.51 ꢀ 0.3
5.74 ꢀ 0.2
4.01 ꢀ 0.2
5.07 ꢀ 0.4
0.35 ꢀ 0.01
>10
>10
>10
<0.01
<0.01
<0.01
<0.01
1.5 ꢀ 0.1
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
2.2. Pharmacology
9.37 ꢀ 0.3
>10
>10
>10
>10
>10
>10
0.04 ꢀ 0.002
2.2.1. In vitro evaluation of the inhibition of human cholinesterases
Compounds 21e30 were evaluated as inhibitors of hAChE and
hBuChE, according to Ellman’s protocol [40]. Tacrine was also
evaluated for comparative purposes and the IC50 values are shown
in Table 1.
29
30
Tacrine
a
From these data several conclusions can be withdrawn con-
cerning the structureeactivity relationships, and the effect of the
Values are expressed as mean ꢀ standard error of the mean of atleast three
different experiments.

E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
753
number and position of the electron-donating groups also made
a different influence in the inhibitory ability.
pointing out that these molecules would cross the BBB by passive
diffusion (Table 2).
Next, and based on these results, we selected the potent and
selective hAChE inhibitor 4-(8-amino-9,10,11,12-tetrahydro-7H-
benzo[7,8]chromeno[2,3-b]quinolin-7-yl)-2-methoxyphenol (25)
2.2.4. Antioxidant evaluation of compounds 21e30 (ORAC method)
The antioxidant activities of compounds 21e30 were evaluated
by following the well-established ORAC-FL method (Oxygen
Radical Absorbance Capacity by Fluorescence) [52,53] that was
recently applied by us to other compounds [42,43,54]. Peroxyl
radicals were thermally generated from 2,2-azobis-(amidinopro-
pane) dihydrochloride and reacted with fluorescein to form
nonfluorescent products at 520 nm. The antioxidant capacity of
tacrine analogs 21e30 was determined by their competition with
fluorescein in the radical capture, using a fluorescence microplate
reader. Trolox, a vitamin E analog, was used as a standard and the
(IC50 (hAChE) ¼ 0.33 ꢀ 0.04
mM) for the kinetic analysis in order to
investigate the type of inhibition, prior to carrying out the corre-
sponding molecular modeling of the new AChE inhibitors of type II
(Chart 2).
2.2.2. Kinetic study of the AChE inhibition by compound 25
To gain further insight into the mechanism of action on AChE of
this family of compounds, a kinetic study was carried out on
compound 25 using AChE from Electrophorus electricus, as
a cheaper and more readily available source of enzyme. Graphical
analysis of the reciprocal Lineweaver Burk 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 25 gave an estimate of the inhibition constant, Ki of
81 nM.
results were expressed as trolox equivalents (mmol of trolox/mmol
of tested compound), in a relative scale where ORAC (trolox) ¼ 1.
Melatonin was also tested giving an ORAC value of 2.3 that fully
agreed with the value previously described by Sofic et al. (2.0
of trolox/ mol of melatonin) [55], pointing out the reliability of our
experiments. At the maximum concentration used (10 M) tacrine
mmol
m
m
and the majority of tested compounds showed negligible radical-
capture ability. Only compound 25, bearing 40-OH,30-OMe substit-
uents in the benzene ring, showed an interesting antioxidant
capacity that was 1.5-fold more potent than the vitamin E analog
(Table 1).
As the target cholinesterase inhibitors 7-aryl-9,10,11,12-tetrahydro-
7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amines 21e30 have been
designed as tacrine analogs in order to decrease its well-known
hepatotoxicity [56], but retaining the useful cholinergic properties,
we decided to undertake a the appropriate evaluation for the toxicity,
and neuroprotective profile on the permeable inhibitors 26e28,
and 30.
2.2.3. In vitro evaluation of the bloodebrain barrier permeability
To explore whether these tacrine analogs would be able to
penetrate into the brain, we used a parallel artificial membrane
permeation assay for bloodebrain barrier (PAMPA-BBB). This
simple and rapid model, described by Di et al. [41] and successfully
applied by us to different compounds [42e51] has the advantage of
predicting passive BBB permeation with high success. Due to
unexpected solubility problems under the experimental conditions
of the PAMPA-BBB assay, only ChE inhibitors 26e28 and 30 could be
analyzed. The in vitro permeabilities (Pe) of these tacrine analogs
and 15 commercial drugs through a lipid extract of porcine brain
were determined using a mixture of PBS:EtOH (70:30). Assay
validation was made by comparing the experimental permeability
with the reported values for commercial drugs, which gave a good
lineal correlation, Pe (exptl) ¼ 1.24Pe(bibl) þ 1.98 (R2 ¼ 0.93). From
this equation, and taking into account the limits established by Di
et al. for BBB permeation [41], we found that molecules with
a permeability > 7.0 10ꢁ6 cm sꢁ1 would be able to cross the BBB by
passive permeation.
2.2.5. In vitro toxicity of compounds 26e28 and 30 in HepG2 cells
The possible toxicity effects of compounds 26e28, and 30 in the
human hepatocellular carcinoma HepG2 cells has been measured
with MTT assay [57].
The evaluation results, using tacrine [58] as
a reference
compound, are summarized in Table 3. After 24 h incubation with
compounds 26e28 and 30, at increasing concentrations
(1e300
observed in the cell viability. In contrast, cell viability was decreased
only at the highest concentration (100 and 300 M) in cells treated
mM), a decrease concentration dependent pattern was
All tested compounds showed permeability values over the
above limit, as the known CNS drugs used in the assay validation,
m
with compound 28, and no significant variations on cell viability
with compound 27.
In all, and based on the data obtained, compounds 26e28 and
30 exhibited less hepatotoxicity than tacrine.
140
120
100
80
Control
10nM
30nM
100nM
300nM
3uM
2.2.6. Neuroprotective capacities of compounds 26e28, and 30
against mitochondrial electron transport chain complexes I and V
blockers
The neuroprotective effects of compounds 26, 27, 28 and 30
were evaluated on primary neuronal cultures from cerebral cortex,
cultured for 6e9d and subjected to oligomycin A-rotenone treat-
ments. The mixture of oligomycin-A plus rotenone (Olig/Rot)
60
40
20
Table 2
Permeability results from the PAMPA-BBB assay (Pe, 10ꢁ6 cm sꢁ1).a
0
-4
-2
0
2
4
6
8
10
Compd
PAMPA-BBB
Prediction
-1
-1
(mM )
26
27
28
30
14.6 ꢀ 0.3
23.4 ꢀ 0.5
16.1 ꢀ 0.5
12.3 ꢀ 0.5
cnsþ
cnsþ
cnsþ
cnsþ
[ATCh]
Fig. 1. Steady-state inhibition of EeAChE hydrolysis of acetylthiocholine (ATCh) by
inhibitor 25. LineweavereBurk reciprocal plots of initial velocity and substrate
concentrations (0.1e1 mM) are presented. Lines were derived from a weighted least-
squares analysis of data.
a
Results are the mean of three independent experiments (n ¼ 3) ꢀ SD. PBS/EtOH
(70:30) was used as solvent.

754
E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
Table 3
In vitro toxicity of compounds 26e28 and 30 in HepG2 cells.
Viability (%) HepG2 cells
Compounds
1
m
M
3
m
M
10
m
M
30
m
M
100
m
M
300 mM
26
27
28
30
97.8 ꢀ 1.02ns
96.5 ꢀ 0.67ns
95 ꢀ 1.87ns
84.2 ꢀ 0.23***
95.6 ꢀ 1.43ns
93 ꢀ 2.42ns
86.2 ꢀ 4.01*
90 ꢀ 2.95ns
85.3 ꢀ 2.62***
96.4 ꢀ 1.76ns
92.6 ꢀ 3.37ns
79.9 ꢀ 3.14***
88.7 ꢀ 3.42ns
76.9 ꢀ 0.75***
96.6 ꢀ 1.59ns
90.3 ꢀ 1.11ns
68.1 ꢀ 1.30***
81.6 ꢀ 4.88*
78.9 ꢀ 1.69***
94.5 ꢀ 1.08ns
85.0 ꢀ 2.72**
63.4 ꢀ 2.20***
64.3 ꢀ 4.54***
74.8 ꢀ 0.99***
93.5 ꢀ 1.28ns
72.1 ꢀ 1.78***
61 ꢀ 1.49***
40 ꢀ 2.20***
97.5 ꢀ 1.88ns
Tacrine
93.4 ꢀ 4.69ns
Cell viability was measured as MTT reduction and data were normalized as % control. Data are expressed as the means ꢀ s.e.m. of quadruplicate of at least three different
ns
cultures. All compounds were assayed at increasing concentrations (1e300
m
M). ***P ꢃ 0.001, **P ꢃ 0.01, *P ꢃ 0.05 and not significant, with respect to control group.
Comparisons between drugs and control group were performed by one-way ANOVA followed by the NewmaneKeuls post-hoc test.
blocks mitochondrial electron transport chain complexes
I
EC50’s for neuroprotective effects of different compounds are
shown in Fig. 3. The lowest EC50 was obtained for tacrine, followed
by compounds 28, 30 and 27. Compound 26 was the least efficient
neuroprotector against mitochondrial blockers-induced neuronal
death.
and V, respectively, inducing cell death by oxidative stress in
a concentration-dependent manner [59e63]. Cell viability assay
by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide inner salt (XTT) determination was performed to
evaluate the potential neuroprotective effect of these compounds
Because the neuroprotective effect of some compounds
against Olig/Rot. Compounds 26e28 and 30 (ranging from 0.1
m
M to
decreases at concentrations ꢂ50
mM, the possible basal neurotox-
icity of these compounds was tested.
100 M concentrations) were added 15 min before than Olig/Rot to
m
test their potential neuroprotective effect. Tacrine, a well-known
anticholinesterasic agent [58], was used as the reference
compound to assess its neuroprotective features on neuronal
cultures. Results from Table 4 show that 24 h-exposure of neuronal
cultures to Olig/Rot induced a significant decrease in cell viability
(63.15 ꢀ 2.75%; mean ꢀ SEM; n ¼ 15, p < 0.001 versus 100% control,
by one way ANOVA test), which was partially reversed after 24 h
incubation by tacrine and by the tested tacrine analogs, compounds
Data from Fig. 4 indicate that in general the tacrine analogs at
concentrations lower than 50
mM are not neurotoxic. However, at
100 M concentration, compounds 26, 28 and 30 produced
m
a decrease of about 40% in cell viability. The most neurotoxic
compound was tacrine, which showed neurotoxic effects ranking
from 25 to 60% of decrease in cell viability at concentrations
between 25 and 100 mM. Thus, synthetic tacrine analogs trigger
lower neurotoxicity than anticholinesterasic tacrine.
26e28 and 30, in
dependent manner (P < 0.001, one way ANOVA test). However,
with the exception of compound 27 at 25 and 50 M concentra-
tions, drug treatments were not able to reach the 24 h control value
(100%) (Table 4).
a
statistically significant concentration-
Finally, a modeling study was carried out through docking
simulations for the purpose of gaining insights as to the nature and
spatial location of the key interactions of the racemic tacrine analog
25, by analysis of the AChE modulation affinity of enantiomers (R)-
and (S)-25. As in previous studies [16,26], we have chosen the 3D
structure of the enzyme species for our kinetic studies.
m
When the neuroprotective effect induced by compounds 26e28
and 30 was compared with those obtained with tacrine, these
tacrine analogs provided, in general, a very similar neuroprotective
effect profile to that of tacrine (Figs. 2 and 3). Very interestingly,
however, compound 27 showed a significantly higher neuro-
protective effect than tacrine (128.39 ꢀ 6.59 and 119.72 ꢀ 22.93) at
2.2.7. Molecular modeling of compound 25
As the kinetic inhibition studies have been carried out on
EeAChE (see section 2.2.2.), ligand docking studies were performed
with Autodock Vina [64] using a single catalytic sub-unit of EeAChE
(PDB: 1C2B). The docking procedure was applied to the whole
protein target (“blind docking”). To account for side chain flexibility
25 and 50 mM concentrations of compound 27, respectively versus
64.34 ꢀ 17.60 at 25
mM tacrine (P < 0.001; one way ANOVA test).
Table 4
Effect of tacrine and tacrine analogs (compounds 26e28 and 30) on the decrease in cell viability induced by mitochondrial blockers on primary cortical neuronal cultures.
Drug concentration (
m
M)
(Cell viability, %)
Tacrine
26
27
28
30
Control
Olig/Rot
0.01
0.1
1
2.5
5
10
25
50
100.00 ꢀ 0.94***
39.36 0.10ooo
37.65 ꢀ 0.29ooo
48.14 ꢀ 3.74ooo
58.18 ꢀ 6.75ooo
53.01 ꢀ 3.32ooo
62.90 ꢀ 2.81*ooo
65.95 ꢀ 11.19*ooo
65.65 ꢀ 14.41*ooo
46.62 ꢀ 5.62ooo
40.24 ꢀ 5.29ooo
100.00 ꢀ 2.50***
34.96 0.15ooo
e
100.00 ꢀ 2.75***
35.07 0.12ooo
e
37.29 ꢀ 4.80ooo
42.06 ꢀ 4.55ooo
e
72.37 ꢀ 4.32***oo
74.92 ꢀ 3.28***oo
84.19 ꢀ 10.72***
99.46 ꢀ 11.94***
62.75 ꢀ 0.73**ooo
100.00 ꢀ 2.81***
37.35 0.18ooo
e
100.00 ꢀ 0.72***
37.50 0.12ooo
e
34.38 ꢀ 0.12ooo
40.39 ꢀ 3.08ooo
e
63.12 ꢀ 2.25***ooo
68.42 ꢀ 3.74***ooo
74.27 ꢀ 5.29***ooo
49.37 ꢀ 5.67*ooo
55.45 ꢀ 0.73***ooo
40.72 ꢀ 8.22ooo
41.74 ꢀ 6.45ooo
50.80 ꢀ 8.23ooo
60.07 ꢀ 9.63**ooo
64.88 ꢀ 5.75**ooo
72.16 ꢀ 6.47***oo
90.18 ꢀ 1.04***
e
43.67 ꢀ 1.70ooo
52.99 ꢀ 3.65*ooo
55.86 ꢀ 5.53***ooo
61.44 ꢀ 5.10***ooo
74.80 ꢀ 3.68***ooo
76.84 ꢀ 3.07***ooo
64.20 ꢀ 3.00***ooo
74.69 ꢀ 0.73***ooo
100
Data show the percentage of cell viability versus control (C), obtained after 24 h treatment of cortical neuronal cultures with 10
mM olygomicin/30
m
M Rotenone (Olig/Rot), in
the absence or presence of indicated concentrations of tacrine and tacrine analogs (compounds 26e28 and 30). The treatment with Olig/Rot induced a loss in cell viability of
63.15 ꢀ 2.75% (n ¼ 15; P < 0.001, ANOVA test) when compared with cells without treatment (Control). Cell viability corresponding to control cells was considered as 100%. The
values represent the mean ꢀ SEM of three to four independent experiments, each one performed by triplicate, performed in different cell batches. Statistics compares (a) The
increase in cell viability produced by indicated compounds compared with Olig/Rot in the absence of drugs at *P < 0.05, **P < 0.01, ***P < 0.001 or (b) The difference in cell
viability in the presence of drugs versus the control at ooP < 0.01 and oooP < 0.001 by using one-way ANOVA followed by HolmeSidak’s post hoc test, when analysis of variance
was significant.

E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
755
Fig. 2. Neuroprotective effects of tacrine and tacrine analogs (compounds 26e28 and 30) on primary neuronal cultures exposed to OligomycineRotenone insult. Bar charts show
the percentage of neuroprotection by compounds 26e28 and 30 (AeD) and tacrine (E) at indicated concentrations after a 24 h treatment with 10 M olygomicin/30 M Rotenone.
m
m
The treatment with these respiratory chain blockers induced a loss in cell viability of 63.15 ꢀ 2.75% (n ¼ 15; P < 0.001, ANOVA test) when compared with cells without treatment
(control). Cell viability corresponding to control cells was considered as 100%. Neuroprotection was defined as the percentage to reach the control value, defining as 100% neu-
roprotection the difference in cell viability between control values and Olig/Rot values. Data represent the mean ꢀ SEM of three to four independent experiments, each one
performed by triplicate, in different cell batches. Statistics compares the neuroprotective effects of indicated compounds against 0% neuroprotection in the absence of drugs.
*P < 0.05, **P < 0.01, ***P < 0.001 versus 0% neuroprotection by one-way analysis of variance followed by HolmeSidak’s post hoc test, when analysis of variance was significant.
during docking, flexible torsions in the ligands were assigned, and
the acyclic dihedral angles were allowed to rotate freely.
Remarkably, an accurate prediction of receptoreligand complexes
was obtained by allowing flexibility in the residues Trp286, Tyr124,
Tyr337 and Tyr72 [26]. This is the set of residues that were
prescribed flexibility in our earlier works [16,26,65]. However, due
to the large size of both enantiomers, this motion by itself is not
sufficient to enlarge the gorge to enhance ligand access to the active
center. Therefore, Asp74, Thr75, Trp86, and Tyr341 receptor
Protein conformational flexibility is an important aspect of
ligand binding and the conformational rearrangements can lead to
the binding of structurally diverse ligands; thus, the incorporation
of protein structural flexibility into the ligand generation procedure
has been commonly used in our AChE studies [16,26,65].
Fig. 3. Neuroprotective effects of tacrine and tacrine synthetic analogs (compounds 26e28 and 30) on primary neuronal cultures exposed to OligomycineRotenone insult.
Doseeresponse curves showing the percentage of neuroprotection by compounds 26e28 and 30 and tacrine at indicated concentrations against a 24 h treatment with 10
olygomicin/20 M Rotenone. Fitting of the concentrationeresponse curves for estimation of EC50 values was made by weighted nonlinear regression of minimum squares, using
m
M
m
logistic curves. The data are the means ꢀ SEM of three determinations, each one performed by triplicate (n ¼ 9). Data analysis was performed with SigmaPlot v.11 software. The
statistics point out the accuracy of the graph fitting at *P > 0.05, **P < 0.01 and ***P < 0.001 (One way ANOVA test).

756
E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
Fig. 4. Neurotoxic effects of tacrine and tacrine synthetic analogs (compounds 26e28 and 30) on primary cortical neuronal cultures. Bar chart show the percentage of cell viability
in the presence or absence (control; C) of indicated concentrations of tacrine analogs 26e28 and 30 (AeD) and tacrine (E). Cell viability corresponding to control cells was
considered as 100%. The values represent the mean ꢀ SEM of three independent experiments, each one performed by triplicate in different cell batches. Statistics show neurotoxic
effects of these drugs versus controls. *P < 0.05, **P < 0.01, ***P < 0.001 versus 100% cell viability (one-way ANOVA test).
residues were also selected to be flexible during docking simula-
tion. These eight residues delineate the shape of the gorge entry
and lining, as their motion may significantly enlarge the gorge
mouth to facilitate ligand access to the catalytic site.
The docking results reveal that both enantiomers have two
major predicted binding modes at the active site of the enzyme. As
shown in Fig. 5, the two most favored binding modes are presented
along with the residues surrounding (R)-25. Mode I places the (R)-
enantiomer deep in the binding pocket interacting with the cata-
lytic triad residue Ser203. Mode II accommodates this enantiomer
in the PAS and stacks against Trp286 and Tyr341 residues.
A close up of Mode I in the vicinity of the catalytic triad, His447,
Ser203, Glu334, uncovers a likely critical interaction where the
methoxy group is doubly hydrogen bonded to the Ser203 side chain
and backbone of Gly122, whereas the amino group can establish
a hydrogen bond with the side chain of Tyr337. Besides, (R)-25
interacts with Tyr341 forming a face-to-face
the benzochromene moiety.
pep interaction with
A close examination into the first shell of residues surrounding (R)-
25 in Mode II reveals a hydrogen bond interaction between the
hydroxyl substituent of the phenyl ring and the hydroxyl group of
Thr75; the amino group is involved in hydrogen bond interactions
with the carboxylate group of Asp74 and with the hydroxyl group of
the Tyr124; the benzochromene moiety interacts with Trp286 and
tetrahydroquinoline moiety with Tyr341, by means of a pep stacking.
For enantiomer (S)-25, two binding modes were also predicted
(Fig. 6). Mode I, the most energetically favored binding mode,
places the ligand in the PAS with the tetrahydroquinolin moiety
stacking between Trp286 and Tyr341 residues and the benzo-
chromene moiety extended to the central region of the active site
gorge; in this orientation, the nitrogen of the tetrahydroquinolin
moiety establishes hydrogen bond interaction with the hydroxyl
group of Tyr124; however, no interactions with the catalytic triad
residues have been found. Mode II places the ligand in the opening
of the PAS and the benzochromene moiety stacks against Trp286
and Tyr341. Besides, the hydroxyl group of Thr75 establishes
hydrogen bond interactions with the two oxygen atoms of both
methoxy and hydroxyl groups.
Fig. 5. Two major binding modes of (R)-25 at the active site of AChE: Mode I (a): Binding
affinity ¼ ꢁ13.3 kcal molꢁ1; Mode II (b): binding affinity ¼ ꢁ12.5 kcal molꢁ1. The
compound is rendered as sticks and illustrated in blue. The side chains conformations of
the mobile residues are illustrated in the same color as the ligand. Different subsites of
the active site were colored: catalytic triad (CT) in green, oxyanion hole (OH) in pink,
anionic subsite (AS) in orange, acyl binding pocket (ABP) in yellow, aside from Trp86 and
peripheral anionic subsite (PAS) in blue. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)

E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
757
Fig. 8. Overlay of binding Mode I of (R)-25 (blue) and (S)-25 (red). (For interpretation
of the references to color in this figure legend, the reader is referred to the web version
of this article.)
enantiomers share the position of benzene ring B from which the
molecules are arranged as mirror images (Fig. 8).
The molecular modeling results are in good agreement with the
experimental observations in the sense that (S)-enantiomer inter-
acts at the peripheral binding site whereas (R)-enantiomer inter-
acts at both catalytic and peripheral binding sites, giving rise to an
interaction pattern that could explain the mixed-type inhibitory
properties of the racemic specie.
3. Conclusions
In this work, we have described the synthesis, pharmacological
analysis and molecular modeling of tacrine analogs 21e30, bearing
the
7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]
quinolin-8-amine skeleton, prepared by Friedländer reaction of 2-
amino-4-aryl-4H-benzo[h]chromene-3-carbonitriles (11e20) with
cyclohexanone, as potential drugs for the treatment of AD. The
biological evaluation showed that these molecules 21e30 were
good and selective hAChE inhibitors, in the micromolar range, one
of the most potent being 4-(8-amino-9,10,11,12-tetrahydro-7H-
benzo[7,8]chromeno[2,3-b]quinolin-7-yl)-2-methoxyphenol (25)
Fig. 6. Two major binding modes of (S)-25 at the active site of AChE: Mode I (a): Binding
affinity ¼ ꢁ13.3 kcal molꢁ1; Mode II (b): binding affinity ¼ ꢁ11.4 kcal molꢁ1. The
compound is rendered as sticks and illustrated in red. The side chains conformations of
the mobile residues are illustrated in the same color as the ligand. Different subsites of
the active site were colored: catalytic triad (CT) in green, oxyanion hole (OH) in pink,
anionic subsite (AS) in orange, acyl binding pocket (ABP) in yellow except Trp86 and
peripheral anionic subsite (PAS) in blue. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
[IC50 (hAChE) ¼ 0.33 ꢀ 0.04
mM]. Kinetic studies on compound 25
showed that this compound is a mixed type inhibitor for EeAChE
(Ki ¼ 81 nM). In addition, compound 25 has an excellent antioxi-
dant profile as determined in the ORAC experiment (1.47 ꢀ 0.10
Trolox equiv.). The results are in agreement with the observations
in molecular docking studies. In EeAChE both enantiomers were
found to adopt different orientations inside the active-site gorge.
Both compounds have two major favored predicted binding modes,
(R)-25 enantiomer can interact directly with one residue of the
catalytic triad and it can also interact with the enzyme in the PAS,
(S)-25 enantiomer can interact with the enzyme in the opening of
the PAS and it can also occupy a deeper position in the active-site
gorge interacting with the subsite halfway down the gorge and in
the mouth of the aromatic gorge (PAS). Finally, compounds 21e30
showed a good permeability profile as determined in the PAMPA
assay. Very interestingly, and compared to tacrine, compounds
26e28 and 30 showed less toxicity in HepG2 cells. In addition, cell
viability-related studies (XTT assay) in cortical neurons in primary
Interestingly, the examination of the overlay of Mode II of (R)-25
with Mode II of (S)-25 reveals similarities between these docking
events. They share the position of the central cores of the ligands
(methoxyphenol-pyran moieties) and this implies that naphtha-
lene moiety of each enantiomer occupies a similar position as the
tetrahydroquinoline moiety of the other (Fig. 7). Lastly, we exam-
ined overlay of Mode I of (R)-25 with Mode I of (S)-25. Both
cultures show that compounds 26e28 and 30 (0.1e50 mM) have
significant neuroprotective effects against mitochondrial chain
blockers-induced cell death, and unlike tacrine, are not neurotoxic
at concentrations lower than 50 mM. It is worth highlighting that
compound 27, in spite of the fact that it is neither the most efficient
neuroprotector nor the most efficient anticholinesterasic agent, it is
the compound that showed the best neuroprotective properties at
high concentrations out of all assayed compounds, exhibiting not
Fig. 7. Overlay of binding Mode II of (R)-25 (blue) and (S)-25 (red). (For interpretation
of the references to color in this figure legend, the reader is referred to the web version
of this article.)

758
E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
only no neurotoxicity but even a weak neuroprotective effect at
basal levels. Therefore, taking into account these observations,
tacrine 27 could be proposed as a good neuroprotective and anti-
oxidant drug with a consistent therapeutic potential as a neuro-
protector in neurodegenerative diseases, such as Alzheimer
disease; nonetheless, other action mechanisms, aside from that of
AChE inhibitor, could be considered.
To sum up, 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chro-
meno[2,3-b]quinolin-8-amines can be considered as attractive,
non-toxic, neuroprotective, multipotent therapeutic molecules for
the potential treatment of AD. In particular, efforts are now
addressed to preparing and evaluating enantiomerically pure
compounds, such as inhibitors 25 and 27, in order to refine and gain
a deeper insight into the pharmacological properties of this family
of cholinergic derivatives.
1H, H10), 7.85 (d, J ¼ 7.8 Hz, 1 H, H7), 7.60e7.58 (m, 1 H, H9),
7.55e7.53 (m, 2 H, H6, H8), 7.08 (d, J ¼ 8.3 Hz, 1 H, H5), 7.00 (br s,
2 H, NH2), 6.92 (d, J ¼ 8.8 Hz, 1 H, H60), 6.56 (d, J ¼ 2.4 Hz, 1 H, H30),
6.44 (dd, J ¼ 8.8, 2.4 Hz, 1 H, H50), 5.14 (s, 1 H, H4), 3.77 (s, 3 H,
OCH3), 3.71 (s, 3 H, OCH3); 13C NMR (125 MHz, DMSO)
d 160.6 (C2),
159.4 (C20)*, 157.3 (C40)*, 142.8 (C10b), 132.5 (C10b), 129.6 (C60),
127.5 (C7), 126.4 (2 C, C9, C6*), 125.8 (C5), 125.7, 123.6 (C8)*, 122.6,
120.6 (C10), 120.5, 118.3 (CN), 105.4 (C50), 98.6 (C30), 55.7 [C(20)
OCH3)]*, 55.5 (C3), 55.1 [C(40)OCH3)]*, 33.9 (C4); MS (70 eV) m/z:
358 (Mþ, 95), 357 (Mþ ꢁ 1, 53), 343 (28), 341 (23), 261 (22), 221
(100); MS(þ)ESI: m/z: 381 ([M þ Na]þ, 15), 359 ([M þ H]þ, 100).
Anal. Calcd. for C22H18N2O3: C, 73.73; H, 5.06; N, 7.82. Found: C,
74.01; H, 5.33; N, 7.92.
4.4. 2-Amino-4-(2,6-dichlorophenyl)-4H-benzo[h]chromene-3-
carbonitrile (19)
4. Experimental part
Reaction of a solution of 2-(2,6-dichlorobenzylidene)malono-
nitrile 9 [38] (1.0 g, 4.4 mmol) in ethanol with 1-naphthol (630 mg,
4.4 mmol) ans a few drops of piperidine, after 24 h, heating under
4.1. General methods
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, 100 or 125 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).
Mass spectra were recorded on a GC/MS spectrometer with an API-
ES ionization source. Elemental analyses were performed at CNQO
(CSIC, Spain). 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).
reflux, product 19 (1.3 g, 81%) was isolated: mp 264e5 ꢄC; IR (KBr)
n
3478, 3329, 3193, 2197, 1663, 1602, 1575, 1404, 1376, 1102 cmꢁ1; 1H
NMR (500 MHz, DMSO)
d
8.21 (dd, J ¼ 8.3 Hz, 1H, H10), 7.87 (d,
J ¼ 8.3 Hz, 1 H, H7), 7.64e7.55 (m, 4 H, H40, H6, H8, H9), 7.37e7.32
(m, 2 H, H30, H50), 7.21 (br s, 2 H, NH2), 6.85 (d, J ¼ 8.3 Hz, 1 H, H5),
5.96 (s, 1 H, H4); 13C NMR (125 MHz, DMSO)
d 160.7 (C2), 143.5
(C10b), 137.1 (C10), 135.5 (C60)***, 134.8 (C20)***, 132.9 (C6a), 130.8
(C50)*, 129.9 (C30)*, 128.6 (C40)**, 127.6 (C7), 126.8 (C9)**, 126.7 (C8)
**, 124.5 (C5), 123.9 (C6), 122.3 (10a), 120.6 (C10), 119.8 (CN), 114.0
(C4a), 52.1 (C3), 36.9 (C4); MS (70 eV) m/z: 366/368/370 (Mþ, 13/8/
2), 331/333 (Mþ ꢁ 35, 7/2), 221 (100); MS(þ)ESI: m/z: 389/391
([M þ Na]þ, 21), 367/369 (M þ H]þ, 100). Anal. Calcd. for
C20H12Cl2N2O: C, 65.41; H, 3.29; N, 7.63; Cl, 19.31. Found: C, 65.44;
H, 3.31; N, 7.68; C, 19.06.
4.2. 2-Amino-4-(2-methoxyphenyl)-4H-benzo[h]chromene-3-
4.5. 2-Amino-4-(3,4-dichlorophenyl)-4H-benzo[h]chromene-3-
carbonitrile (14) [33]
carbonitrile (20) [34]
Reaction of a solution of 2-(2-methoxybenzylidene)malononi-
trile 4 [35] (1.0 g, 5 mmol) in ethanol with 1-naphthol (720 mg,
5 mmol) and a few drops of piperidine, after 24 h, heating under
reflux, product 14 [33] (1.42 g, 80%) was isolated: mp 204 ꢄC; IR
Reaction of a solution of 2-(3,4-dichlorobenzylidene)malono-
nitrile 10 [36] (1.3 g, 5.8 mmol) in ethanol with 1-naphthol (840 mg,
5.8 mmol) and a few drops of piperidine, after 24 h, heating under
reflux, product 20 (890 mg, 42%) was isolated: mp 256 ꢄC; IR (KBr)
n
(KBr)
n
3467, 3395, 3357, 2186, 1658, 1377 cmꢁ1; 1H NMR (500 MHz,
3464, 3325, 3196, 2195, 1665, 1601, 1412, 1191 cmꢁ1 1H NMR
;
DMSO)
d
8.22 (dd, J ¼ 8.1, 1.0 Hz, 1H, H10), 7.85 (d, J ¼ 7.3 Hz, 1 H,
(500 MHz, DMSO)
d
8.23 (dd, J ¼ 8.3 Hz, 1H, H10), 7.87 (d, J ¼ 8.1 Hz,
H7), 7.62e7.59 (m, 1 H, H9), 7.56e7.52 (m, 2 H, H6, H8), 7.19 (m, 1 H,
H50)*, 7.10 (d, J ¼ 8.8 Hz, 1 H, H5), 7.06 (br s, 2 H, NH2), 7.04e7.00 (m,
2 H, H30, H60), 6.87 (m, 1 H, H40)*, 5.24 (s, 1 H, H4), 3.79 (s, 3 H,
1 H, H7), 7.64e7.55 (m, 4 H, H40, H6, H8, H9, H50), 7.53 (d, J ¼ 2.2 Hz,
1 H, H20), 7.25 (br s, 2 H, NH2), 7.20 (dd, J ¼ 8.3, 2.2 Hz, 1 H, H60), 7.10
(d, J ¼ 8.3 Hz, 1 H, H5), 4.99 (s, 1 H, H4); 13C NMR (125 MHz, DMSO)
OCH3); 13C NMR (125 MHz, DMSO)
d
161.2 (C2), 156.8 (C20), 143.4
d
160.2 (C2), 146.7 (C10b), 142.7 (C10), 132.8 (C30)*, 131.1 (C40)*, 131.0
(C10b), 133.8 (C10), 133.0 (C10b), 129.4 (C60), 128.6 (C50), 128.0 (C7),
127.0 (2 C, C9, C6*), 126.9 (C5),126.2, 124.2 (C8*), 123.1,121.3 (C40)**,
121.0 (C10)**, 118.5 (CN), 112.0 (C30), 56.1 (OCH3)*, 55.7 (C3)*, 34.8
(C4); MS (70 eV) m/z: 328 (Mþ, 73), 327 (Mþ ꢁ 1, 47), 313 (20), 311
(19), 297 (57), 221 (100); MS(þ)ESI: m/z: 351 ([M þ Na]þ, 17), 329
(M þ H]þ, 100). Anal. Calcd. for C21H16N2O2: C, 76.81; H, 4.91; N,
8.53. Found: C, 77.02; H, 4.80; N, 8.53.
(C6a), 129.6 (C20), 129.5 (C50), 128.2 (C60), 127.7 (C7), 126.9 (C8)**,
126.7(C9)**, 125.9 (C5), 124.1 (C6)**, 122.7 (10a), 120.7 (C10)*, 120.2
(CN)*, 116.7 (C4a), 55.2 (C3), 39.6 (C4); MS (70 eV) m/z: 366/368/
370 (Mþ, 10/7/1), 221 (100); MS(þ)ESI: m/z: 389/391 ([M þ Na]þ,
75), 367/369 ([M þ H]þ, 100). Anal. Calcd. for C20H12Cl2N2O: C,
65.41; H, 3.29; N, 7.63; Cl, 19.31. Found: C, 65.58; H, 3.23; N, 7.69; C,
19.48.
4.3. 2-Amino-4-(2,4-dimethoxyphenyl)-4H-benzo[h]chromene-3-
carbonitrile (16)
4.6. General method for the synthesis of 7-phenyl-9,10,11,12-
tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine
(21e30)
Reaction of a solution of 2-(2,4-dimethoxybenzylidene)malo-
nonitrile 6 [37] (2.0 g, 9 mmol) in ethanol with 1-naphthol (1.02 g,
9 mmol) and a few drops of piperidine, after 24 h, heating under
The 2-amino-4-phenyl-4H-benzo[h]chromene-3-carbonitrile
(1.0 equiv) was dissolved in distilled 1,4-dioxane, and to this solu-
tion, AlCl3 (1.5 equiv) and cyclohexanone (1.5 equiv) were added.
The reaction mixture was heated at 100 ꢄC during the time indi-
cated for each compound. When the reaction was complete (TLC
reflux, product 16 (2.10 g, 63%) was isolated: mp 214 ꢄC; IR (KBr)
n
3423, 3331, 3208, 2199, 1667, 1611, 1586, 1574, 1502, 1409, 1337,
1206, 1114 cmꢁ1; 1H NMR (500 MHz, DMSO)
d
8.21 (dd, J ¼ 8.3 Hz,

E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
759
analysis, CHCl3/EtOH, 10/1, v/v), the reaction mixture was diluted
with a solution of chloroform/water (1/1), and treated with an
aqueous solution of sodium hydroxide (10%) until pH 11e12. After
stirring for 30 min, the mixture was extracted with CHCl3, dried
over anhydrous sodium sulfate, filtered and the solvent was evap-
orated. The resultant solid was purified by column chromatography
using methanol/dichloromethane mixtures (1e15%) as eluent to
give pure compound.
(d, J ¼ 8.8 Hz, 1 H, H6), 7.12 (d, J ¼ 8.3 Hz, 2 H, H20, H60), 6.59 (d, 2 H,
H30, H50), 5.53 (br s, 2 H, NH2), 5.32 (s, 1 H, H7), 2.64 (m, 2 H, 2H12),
2.38e2.34 (m, 1 H, H9A), 2.25e2.22 (m, 1 H, H9B), 1.72 (m, 4 H,
2H10, 2H11); 13C NMR (125 MHz, DMSO-d6)
d 155.9 (C13a), 155.0
(C40),152.2 (C8), 151.4 (C12a),144.6 (C14a),142.5 (C10), 1v/v35.8C10),
132.5 (C4a), 128.4 (2C, C20, C60), 127.5 (C4), 126.5 (C6), 126.3 (C2)*,
126.2 (C5)*, 123.5 (C14b), 122.6 (C3), 121.0 (C1), 119.8 (C6a), 115. 2
(2 C, C20, C60),112.4 (C8a), 98.6 (C7a), 38.6 (C7), 32.0 (C12), 23.0 (C9),
22.3, 22.1 (C10, C11); MS (70 eV) m/z: 394 (Mþ, 25), 393 (5), 301
(100); MS (API-ESþ) m/z: 395 ([M þ H]þ, 100). Anal. Calcd for
C26H22N2O2: C, 79.16; H, 5.62; N, 7.10. Found: C, 79.05; H, 5.37; N,
7.24.
4.6.1. 7-Phenyl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-
b]quinolin-8-amine (21)
Following the general method, reaction of compound 11 [31]
(1.0 g, 3.35 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(492 mg, 5.02 mmol) and AlCl3 (670 mg, 5.02 mmol), after 2 h,
4.6.4. 7-(2-Methoxyphenyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amine (24)
product 21 (950 mg, 75%) was obtained: mp 262 ꢄC; IR (KBr)
n
3465,
3406, 3055, 2929, 2857, 1639, 1608, 1586, 1569, 1447, 1431, 1395,
1377, 1262, 1103 cmꢁ1 1H NMR (500 MHz, DMSO-d6)
8.30
Following the general method, reaction of compound 14 [33]
(600 mg, 1.82 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(267 mg, 2.73 mmol) and AlCl3 (364 mg, 2.73 mmol), after 4 h,
;
d
(d, J ¼ 8.3 Hz, 1 H, H1), 7.84 (d, J ¼ 7.9 Hz, 1 H, H4), 7.59 (tm,
J ¼ 8.3 Hz,1 H, H2), 7.54e7.50 (m, 2 H, H3, H5), 7.34 (dd, J ¼ 6.9,1.3 z,
1 H, 2 H, H20, H60), 7.27 (d, J ¼ 8.8 Hz, 1 H, H6), 7.20 (tm, J ¼ 7.8 Hz,
2H, H30, H50), 7.09 (tm, J ¼ 7.4 Hz,1 H, H40), 5.58 (br s, 2 H, NH2), 5.47
(s, 1 H, H7), 2.63 (m, 2 H, 2H12), 2.34 (m, 1 H, H9A), 2.24 (m, 1 H,
H9B), 1.71 (m, 2 H, 2H10, 2H11); 13C NMR (125 MHz, DMSO-d6)
product 24 (200 mg, 27%) was obtained: mp > 260 ꢄC; IR (KBr)
n
3422, 3187, 2930, 1643, 1606, 1595, 1570, 1445, 1436, 1380, 1282,
1245, 1115 cmꢁ1; 1H NMR (500 MHz, DMSO-d6)
d
8.31 (d, J ¼ 8.3 Hz,
1 H, H1), 7.83 (d, J ¼ 7.8 Hz, 1H4), 7.59 (dt, J ¼ 8.3, 1.4 Hz, 1 H, H2),
7.54e7.49 (m, 2 H, H3, H5), 7.14e7.10 (m, 2 H, H6, H50), 7.03
(d, J ¼ 7.8 Hz, 1 H, H60), 6.94 (dd, J ¼ 7.4, 1.4 Hz, 1 H, H30), 6.77 (td,
J ¼ 7.3, 1.0 Hz, 1 H, H40), 5.65 (s, 1 H, H7), 5.34 (br s, 2 H, NH2), 3.91
(s, 3 H, OCH3), 2.62 (m, 2 H, H12), 2.34e2.30 (m, 1 H, H9A), 2.23
(m,1 H, H9B),1.70 (m, 4 H, 2H10, 2H11); 13C NMR (125 MHz, DMSO-
d
155.1 (C13a), 152.5 (C8), 151.4 (C12a), 145.4 (C14a), 144.8 (C10),
132.6 (C4a), 128.5 (2C, C30, C50), 127.6 (C4), 127.4 (2C, C20, C60), 126.5,
126.45, 126.40, 126.3 (4 C, C2, C3, C6, C40), 123.5 (C14b), 122.7 (C5),
121.0 (C1), 119.3 (C6a), 112.4 (C8a), 98.2 (C 7a), 39.5 (C7), 32.0 (C12),
23.0 (C9), 22.3, 22.1 (C10, C11); MS (70 eV) m/z: 378 (Mþ, 26), 301
(100); MS (API-ESþ) m/z: 401 ([M þ Na]þ, 3), 379 ([M þ H]þ, 100);
HRMS (EI): Calcd. for C26H22N2O: 318.1732. Found: 378.1745. Anal.
Calcd. for C26H22N2O: C, 82.51; H, 5.86; N, 7.40. Found: C, 82.50; H,
5.77; N, 7.39.
d6)
d
155.0 (C13a)*, 154.9 (C8)*, 152.3 (C20)**, 151.2 (C12a)**, 145.1
(C14a), 133.1 (C4a), 132.7 (C14b), 129.7 (C30), 128.0 (C10), 127.5 (C4),
126.4 (C50), 126.3 (C6), 126.2 (C5*), 123.4 (C2), 122.6 (C3)*, 121.5
(C40), 121.0 (C1), 118.6 (C8a), 112.2 (C6a), 111.4 (C60), 98.2 (C7a), 56.0
(OCH3), 32.5 (C7), 31.9 (C12), 22.8 (C9), 22.3, 22.09 (C10, C11); MS
(70 eV) m/z: 408 (Mþ, 35), 391 (2), 377 (8), 301 (100); MS (API-ESþ)
m/z: 431 ([M þ Na]þ, 7), 409 (M þ H]þ, 100). Anal. Calcd for
C27H24N2O2: C, 79.39; H, 5.92; N, 6.86. Found: C, 79.62, H, 5.84; N,
6.73.
4.6.2. 7-(p-Tolyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno
[2,3-b]quinolin-8-amine (22)
Following the general method, reaction of compound 12 [32]
(1.0 g, 3.2 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(470 mg, 4.8 mmol) and AlCl3 (640 mg, 4.8 mmol) after 2 h, product
4.6.5. 4-(8-Amino-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno
[2,3-b]quinolin-7-yl)-2-methoxyphenol (25)
22 (817 mg, 65%) was obtained: mp 264 ꢄC; IR (KBr)
n
3450, 3048,
2928, 2861, 1607, 1586, 1568, 1508, 1446, 1431, 1395, 1375, 1264,
1110 cmꢁ1; 1H NMR (500 MHz, DMSO-d6)
Following the general method, reaction of compound 15 [31]
(1.5 g, 4.36 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(640 mg, 6.54 mmol) and AlCl3 (872 mg, 6.54 mmol), after 2 h,
d
8.30 (d, J ¼ 8.3 Hz, 1 H,
H1), 7.83 (d, J ¼ 8.3 Hz, 1 H, H4), 7.58 (td, J ¼ 8.3, 1.0 Hz, 1 H, H2),
7.53e7.50 (m, 2 H, H3, H5), 7.25 (d, J ¼ 8.8 Hz, 1 H, H6), 7.21
(d, J ¼ 7.8 Hz, 2 H, H20, H60)*, 7.00 (d, 2 H, H30, H50)*, 5.53 (br s, 2 H,
NH2), 5.41 (s, 1 H, H7), 2.63 (m, 2 H, 2H12), 2.34 (m, 1 H, H9A), 2.24
(m, 1 H, H9B), 2.15 (s, 3 H, CH3), 1.71 (m, 4 H, 2H10, 2H11); 13C NMR
product 25 (525 mg, 26%) was obtained: mp > 260 ꢄC; IR (KBr)
n
3478, 3369, 2935, 1641, 1607, 1589, 151ꢄ, 1453, 1431, 1380, 1279,
1264, 1112 cmꢁ1; 1H NMR (500 MHz, DMSO-d6)
d
8.79 (s, 1 H, OH),
8.30 (d, J ¼ 8.5 Hz, 1 H, H1), 7.84 (d, J ¼ 8.3 Hz, 1H4), 7.58 (dt, J ¼ 8.5,
1.4 Hz, 1 H, H2), 7.55e7.50 (m, 2 H, H3, H5), 7.29 (d, J ¼ 8.3 Hz, 1 H,
H6), 7.12 (d, J ¼ 1.9 Hz, 1 H, H20), 6.59e6.54 (m, 2 H, H50, H60), 5.52
(br s, 2 H, NH2), 5.30 (s, 1 H, H7), 3.70 (s, 3 H, OCH3), 2.62 (m, 2 H,
H12), 2.34 (m, 1 H, H9A), 2.26 (m, 1 H, H9B), 1.73 (m, 4 H, 2H10,
(125 MHz, DMSO-d6)
d 155.0 (C13a), 152.4 (C8), 151.4 (C12a), 144.7
(C14a),142.5 (C40),135.6 (C10),132.6 (C4a),129.0 (2 C, C30, C50), 127.5
(C4), 127.3 (2 C, C20, C60), 126.4 (C6), 126.3 (2 C, C2, C5), 123.5 (C14b),
122.6 (C3), 121.0 (C1), 119.4 (C6a), 112.4 (C8a), 98.3 (C7a), 39.0 (C7),
32.0 (C12), 23.0 (C9), 22.3, 22.1 (C10, C11), 20.5 (CH3); MS (70 eV) m/
z: 392 (Mþ, 36), 391 (6), 301 (100); MS (API-ESþ) m/z: 315
([M þ Na]þ, 10), 393 ([M þ H]þ, 100); HRMS (EI): Calcd. for
C27H24N2O: 392.1889. Found: 392.1906. Anal. Calcd for C27H24N2O:
C, 82.62; H, 6.16; N, 7.14. Found. C, 82.90; H, 6.10; N, 7.05.
2H11); 13C NMR (125 MHz, DMSO-d6)
d 155.0 (C13a), 152.2 (C8),
151.5 (C12a), 147.2 (C14a), 145.1 (C30), 144.5 (C40), 136.5 (C10), 132.5
(C4a), 127.5 (C4), 126.5 (C6), 126.27 (C14b), 126.21 (C2), 123.5 (C5),
122.5 (C3), 121.0 (C1), 119. 8 (C60), 119.6 (C6a), 115.7 (C50), 112.3
(C8a), 112.2 (C20), 98.5 (C7a), 55.6 (OCH3), 39.0 (C7), 32.0 (C12), 22.9
(C9), 22.4, 22.1 (C10, C11); MS (70 eV) m/z: 424 (Mþ, 19), 423 (3),
301 (100); MS (API-ESþ) m/z: 447 ([M þ Na]þ, 2), 425 ([M þ H]þ,
100); HRMS (EI): Calcd. for C27H24N2O3: 424.1787. Found: 424.1798.
Anal. Calcd. for C27H24N2O3: C, 76.39; H, 5.70; N, 6.60. Found: C,
76.10; H, 5.73; N, 6.69.
4.6.3. 4-(8-Amino-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno
[2,3-b]quinolin-7-yl)phenol (23)
Following the general method, reaction of compound 13 [31]
(600 mg, 2 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(294 mg, 3 mmol) and AlCl3 (400 mg, 3 mmol), after 3 h, product 23
(187 mg, 24%) was obtained as an amorphous solid: IR (KBr)
2931, 1620, 1511, 1453, 1377, 1262, 1172 cmꢁ1 1H NMR (500 MHz,
DMSO-d6)
9.22 (s, 1 H, OH), 8.29 (d, J ¼ 8.3 Hz, 1 H, H1), 7.84
(d, J ¼ 7.9 Hz, 1 H, H4), 7.60e7.50 (m, 3 H, H2, H3, H5), 7.25
n
3485,
4.6.6. 7-(2,4-Dimethoxyphenyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amine (26)
Following the general method, reaction of compound 16 (1.0 g,
2.79 mmol) in 1,4-dioxane (30 mL) with cyclohexanone (409 mg,
;
d

760
E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
4.18 mmol) and AlCl3 (557 mg, 4.18 mmol), after 1 h, product 26
(550 mg, 45%) was obtained: mp > 260 ꢄC; IR (KBr)
3496, 3403,
2933, 2853, 1621, 1606, 1585, 1569, 1501, 1443, 1396, 1378, 1298,
1263, 1206, 1115 cmꢁ1 1H NMR (500 MHz, DMSO-d6)
8.31
4.6.9. 7-(2,6-Dichlorophenyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amine (29)
n
Following the General Method, reaction of compound 19
(600 mg, 1.63 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(239 mg, 2.44 mmol) and AlCl3 (325 mg, 2.44 mmol), after 3 h,
;
d
(d, J ¼ 7.4 Hz, 1 H, H1), 7.83 (d, J ¼ 8.3 Hz, 1H, H4), 7.60 (m 1 H, H2),
7.52 (m, 1 H, H3), 7.50 (d, J ¼ 8.3 Hz, 1 H, H5), 7.13 (d, 1 H, H6), 5.54
(s, 1 H, H7), 5.31 (br s, 2 H, NH2), 3.90 (s, 3 H, OCH3), 3.66 (s, 3 H,
OCH3), 2.63 (m, 2 H, H12), 2.31 (m, 1 H, H9A), 2.25 (m, 1 H, H9B),
product 29 (300 mg, 41%) was obtained: mp > 260 ꢄC; IR (KBr)
n
3437, 3316, 3197, 2933, 2860, 1643, 1606, 1592, 1570, 1448, 1434,
1379, 1264, 1194, 1116 cmꢁ1 1H NMR (500 MHz, DMSO-d6)
;
d
8.31
1.71 (m, 4 H, 2H10, 2H11); 13C NMR (125 MHz, DMSO-d6)
d
159.6
(d, J ¼ 8.3 Hz, 1 H, H1), 7.84 (d, J ¼ 7.8 Hz, 1H, H4), 7.67 (dd,
J ¼ 7.8, 1.0 Hz, 1 H, H30)*, 7.61 (m, 1 H, H2), 7.56 (m, 1 H, H3), 7.51
(d, J ¼ 8.8, 1.0 Hz, 1 H, H5), 7.33 (t, J ¼ 7.8 Hz, 1 H, H40), 7.28 (d,
J ¼ 6.9 Hz, 1 H, H50)*, 7.00 (d, J ¼ 8.8, 1.0 Hz, 1 H, H6), 6.28 (s, 1 H,
H7), 4.95 (br s, 2 H, NH2), 2.64 (m, 2 H, H12), 2.35e2.31 (m, 1 H,
H9A), 2.25e2.21 (m, 1 H, H9B), 1.73 (m, 4 H, 2H10, 2H11); 13C
(C20)*, 156.4 (C13a), 155.3 (C40)*, 152.6 (C8a), 151.6 (C12a)**, 145.4
(C14a)**, 133.1 (C14b), 130.7 (C60), 128.0 (C10), 127.0 (C4), 126.7 (2 C,
C3, C6)**, 125.9 (C2)**, 123.8 (6a), 123.0 (C5), 121.4 (C1), 119.3 (C7a),
112.6 (C8a), 106.8 (C50), 98.8 (C30), 56.5, 55.6 (2 C, 2ꢅ OCH3), 32.5
(C7), 32.4 (C12), 23.4 (C9), 22.8, 22.5 (C10, C11); MS (70 eV) m/z:
438 (Mþ, 40), 437 (10), 423 (3), 407 (18), 391 (5), 301 (100); MS
(API-ESþ) m/z: 461 ([M þ Na]þ, 8), 439 (M þ H]þ, 100). Anal. Calcd
for C28H26N2O3: C, 76.69; H, 5.98; N, 6.39. Found: C, 76.94; H, 5.99;
N, 6.27.
NMR (125 MHz, DMSO-d6) d 155.2 (C13a), 152.9 (C8), 151.1 (C12a),
145.6 (C14a), 137.1 (C10), 135.5 (C20), 134.0 (C60), 133.0 (C4a), 131.4
(C50)**, 130.0 (C40), 128.7 (C30)**, 127.5 (C4), 126.7 (C2)*, 126.4
(C3)*, 125.0 (C6), 122.9 (C14b), 122.6 (C5), 121.1 (C1), 113.5 (C6a)
***, 112.7 (C8a)***, 95.1 (C7a), 36.7 (C7), 31.8 (C12), 22.8 (C9),
22.2, 22.0 (C10, C11); MS (70 eV) m/z: 446/448/450 (Mþ, 14/10/2),
301 (100); MS (API-ESþ) m/z: 469/471/473 ([M þ Na]þ, 3/2/1),
447/449/451 (M þ H]þ, 100/84/17). Anal. Calcd. for C26H20Cl2N2O:
C, 69.81; H, 4.51; N, 6.26, Cl, 15.85. Found: C, 70.10; H, 4.62; N,
6.14.
4.6.7. 7-(3,4,5-Trimethoxyphenyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amine (27)
Following the general method, reaction of compound 17 [31]
(1.0 g, 2.57 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(377 mg, 3.85 mmol) and AlCl3 (513 mg, 3.85 mmol), after 1 h,
product 27 (520 mg, 43%) was obtained: mp 168 ꢄC; IR (KBr)
n
3477,
3428, 2936, 1643, 1604, 1592, 1506, 1448, 1378, 1132, 1110 cmꢁ1; 1H
NMR (500 MHz, DMSO-d6)
4.6.10. 7-(3,4-Dichlorophenyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amine (30). Following the general
method, reaction of compound 20 [34] (787 mg, 2.14 mmol) in 1,4-
dioxane (30 mL) with cyclohexanone (314 mg, 3.21 mmol) and
AlCl3 (428 mg, 3.21 mmol), after 2 h, product 30 (666 mg, 70%) was
d
8.32 (d, J ¼ 8.4 Hz, 1 H, H1), 7.86
(d, J ¼ 7.8 Hz, 1 H, H4), 7.61e7.55 (m, 3 H, H2, H3, H5), 7.32
(d, J ¼ 8.3 Hz, 1 H, H6), 6.69 (s, 2 H, H20, H60), 5.57 (br s, 2 H, NH2),
5.36 (s, 1 H, H7), 3.63 (s, 3 H, OCH3), 3.54 (s, 6 H, 2ꢅ OCH3), 2.64
(m, 2 H, H12), 2.35 (m, 1 H, H9A), 2.28 (m, 1 H, H9B), 1.73 (m, 4 H,
obtained: mp > 260 ꢄC; IR (KBr)
1606, 1594, 1568, 1449, 1397, 1376, 1264, 1111 cmꢁ1
(500 MHz, DMSO-d6)
n
3478, 3410, 2930, 2859, 1624,
2H10, 2H11); 13C NMR (125 MHz, DMSO-d6)
d
155.0 (C13a), 152.8
;
1H NMR
(2 C, C30, C50),152.5 (C8),151.5 (C12a),144.8 (C14a),141.1 (C10),136.2
(C40), 132.7 (C4a), 127.6 (C4), 126.44 (C6), 126.40 (C3), 126.2 (C2),
123.5 (C14b), 122.7 (C5), 121.1 (C1), 119.0 (C6a), 112.3 (C8a), 105.0
(2 C, C20, C60), 98.0 (C7a), 59.8 (OCH3), 55.8 (2 C, 2ꢅ OCH3), 32.0
(C7), 30.6 (C12), 23.0 (C9), 22.3, 22.1 (C10, C11); MS (70 eV) m/z: 468
(Mþ, 31), 301 (100); MS (API-ESþ) m/z: 491 ([M þ Na]þ, 2), 469
(M þ H]þ, 100); HRMS (EI): Calcd for C29H28N2O4: 468.2049. Found:
C, 468.2051. Calcd for C29H28N2O4: C, 74.34; H, 6.20; N, 5.98. Found.
C, 74.08, H, 5.99; N, 6.12.
d
8.31 (d, J ¼ 8.3 Hz, 1 H, H1), 7.86 (d,
J ¼ 7.9 Hz, 1 H, H4), 7.74 (d, J ¼ 2.0 Hz, 1 H, H20), 7.61 (m, 1 H, H2),
7.58 (d, J ¼ 8.3 Hz, 1 H, H5), 7.55 (m, 1 H, H3), 7.46 (d, J ¼ 8.3 Hz, 1 H,
H50), 7.28 (d, J ¼ 8.3 Hz, 1 H, H6), 7.18 (dd, 1 H, H60), 2.64 (m, 2 H,
H12), 2.35e2.31 (m,1 H, H9A), 2.25e2.21 (m,1 H, H9B),1.73 (m, 4 H,
2H10, 2H11); 13C NMR (125 MHz, DMSO-d6)
d 154.9 (C13a), 152.9
(C8), 151.5 (C12a), 146.2 (C14a), 145.0 (C10), 132.8 (C4a), 131.0 (C30),
130.8 (C40), 129.24 (C50), 129.21 (C20), 127.8 (C60), 127.6 (C4), 126.6
(C3), 126.4 (C2), 126.1 (C6), 123.4 (C14b), 123.1 (C5), 121.0 (C1), 118.2
(C6a), 112.6 (C8a), 97.3 (C7a), 38.4 (C7), 32.0 (C12), 23.0 (C9), 22.3,
22.0 (C10, C11); MS (70 eV) m/z: 446/448/450 (Mþ, 20/13/2),
301(100); MS (API-ESþ) m/z: 447/451 (M þ H]þ, 100, 80, 30) HRMS
(EI): Calcd. for C26H20Cl2N2O: 446.0953. Found: C, 446.0963. Anal.
Calcd. for C26H20Cl2N2O: C, 69.81; H, 4.51; Cl, 15.85; N, 6.26. Found:
C, 69.78; H, 4.29; Cl, 15.72; N, 5.98.
4.6.8. 7-(4-Nitrophenyl)-9,10,11,12-tetrahydro-7H-benzo[7,8]
chromeno[2,3-b]quinolin-8-amine (28)
Following the general method, reaction of compound 18 [32]
(600 mg, 1.75 mmol) in 1,4-dioxane (30 mL) with cyclohexanone
(256 mg, 2.62 mmol) and AlCl3 (349 mg, 2.62 mmol), after 1 h,
product 28 (410 mg, 56%) was obtained: mp > 260 ꢄC; IR (KBr)
n
3461, 3401, 2931, 1724, 1607, 1568, 1519, 1448, 1396, 1375, 1345,
4.7. Pharmacology
1265, 1109 cmꢁ1 1H NMR (500 MHz, DMSO-d6)
;
d
8.31
(d, J ¼ 8.3 Hz, 1 H, H1), 8.09 (dt, J ¼ 9.3, 1.0 Hz, 1 H, 2 H, H30, H50),
7.86 (d, J ¼ 8.3 Hz, 1 H, H4), 7.68 (dt, J ¼ 8.9, 1.4 Hz, 1 H, 2 H, H20,
H60), 7.62e7.54 (m, 3 H, H2, H5, H3), 7.27 (d, J ¼ 8.4 Hz, 1 H, H6),
5.73 (br s, 2 H, NH2), 5.71 (s, 1 H, H7), 2.64 (m, 2 H, H12), 2.35 (m,
1 H, H9A), 2.28 (m, 1 H, H9B), 1.71 (m, 4 H, 2H10, 2H11); 13C NMR
4.7.1. Inhibition experiments of AChE and BuChE
To assess the inhibitory activity of the compounds toward AChE
or BuChE, we followed the spectrophotometric method of Ellman
[40], using human AChE, human erythrocytes (buffered aqueous
solution) or human BuChE (Sigma Aldrich, Madrid, Spain). The
reaction took place in a final volume of 3 mL of a phosphate-
buffered solution (0.1 M) at pH ¼ 8, containing 0.035 U of AChE
or 0.05 U of BuChE and 0.35 mM of 5,50-dithiobis-2-nitrobenzoic
acid (DTNB, Sigma Aldrich, Madrid, Spain). Inhibition curves were
made by pre-incubating this mixture with at least nine concen-
(125 MHz, DMSO-d6)
d 155.0 (C13a), 153.0 (C8), 152.7 (C12a), 151.6
(C14a), 146.1 (C40), 145.1 (C10), 132.8 (C4a), 128.6 (2 C, C20, C60),
127.6 (C4), 126.6 (C3), 126.4 (C2), 126.0 (C6), 123.8 (2 C, C30, C50),
123.5 (C14b), 123.1 (C5), 121.0 (C1), 117.9 (C6a), 112.6 (C8a), 97.2
(C7a), 38.9 (C7), 32.0 (C12), 23.0 (C9), 22.3, 22.0 (C10, C11); MS
(70 eV) m/z: 423 (Mþ, 17), 301 (100); MS (API-ESþ) m/z: 446
([M þ Na]þ, 1), 424 (M þ H]þ, 100); HRMS (EI): Calcd for
C26H21N3O3: 423.1597. Found: C, 423.1583. Anal. Calcd for
C26H21N3O3: C, 73.74; H, 5.00; N, 9.92. Found: C, 73.45; H, 4,78; N,
9.65.
trations of each compound (10ꢁ3e10
mM) 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 more

E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
761
of incubation, where the DTNB produces the yellow anion 5-thio-2-
nitrobenzoic acid along with the enzymatic degradation of ace-
tylthiocholine 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 color generation; then,
IC50 values were calculated as the concentration of compound that
produces 50% AChE activity inhibition. Data are expressed as
reader (BMG Labtechnologies GmbH, Offenburg, Germany) with
485-P excitation and 520-P emission filters. The equipment was
controlled by Fluorostar Galaxy software (version 4.11-0) for fluo-
rescence measurement. 2,20-Azobis-(amidinopropane) dihydro-
chloride (AAPH), (ꢀ)-6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid (Trolox) and fluorescein (FL) were purchased from
SigmaeAldrich. The reaction was carried out in 75 mM phosphate
buffer (pH 7.4) and the final reaction mixture was 200
mL. Antiox-
means
quadruplicate.
ꢀ
s.e.m. of atleast three different experiments in
idant (20 L) and FL (120 L; 70 mM, final concentration) solutions
m
m
were placed in a black 96-well microplate (96F untreat, NuncÔ).
The mixture was pre-incubated for 15 min at 37 ꢄC and then, AAPH
4.7.2. Kinetic analysis of the AChE inhibition
solution (60 mL, 12 mM, final concentration) was added rapidly
To obtain estimates of the inhibition constant Ki, 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 [40]. Experiments were performed in a transparent
48-well plate in a final volume of 1 mL of a phosphate-buffered
using a multichannel pipette. The microplate was immediately
placed in the reader and the fluorescence recorded every minute
for 80 min. The microplate was automatically shaken prior to each
reading. Samples were measured at eight different concentrations
(0.1e1
sample solution and eight calibration solutions using Trolox
(1e8 M) were also carried out in each assay. All the reaction
mM). A blank (FL þ AAPH in phosphate buffer) instead of the
solution (0.1 M) at pH 8, containing each well 400
0.875 mM DTNB solution, 1 L DMSO (control) or inhibitor solution
to give desired final concentration, and 14 L of EeAChE (3.6 U/mL)
mL of
m
m
m
mixtures were prepared in duplicate, and at least three indepen-
dent assays were performed for each sample. Raw data were
exported from the Fluostar Galaxy Software to an Excel sheet for
further calculations. Antioxidant curves (fluorescence vs time) were
first normalized to the curve of the blank corresponding to the
same assay, and the area under the fluorescence decay curve (AUC)
was calculated. The net AUC corresponding to a sample was
calculated by subtracting the AUC corresponding to the blank.
Regression equations between net AUC and antioxidant concen-
tration were calculated for all the samples. ORAC-FL values were
expressed as Trolox equivalents by using the standard curve
calculated for each assay, where the ORAC-FL value of Trolox was
taken as 1.
(Type V-S, SigmaeAldrich, Madrid, Spain) at 30 ꢄC to give a final
concentration of 0.05 U/mL. The Reaction was initiated by adding
acetylthiocholine at the concentrations proposed (0.1e0.8 mM).
Progress curves were monitored at 410 nm over 2 min in a fluo-
rescence plate-reader Fluostar Optima (BMG-technologies,
Germany) absorbance ready. 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 the Michaelis constant for the substrate
acetylthiocholine was done at five different concentrations
(0.1e0.8 mM) to give KM ¼ 0.38 mM and Vmax ¼ 0.31 minꢁ1. Slopes
of the reciprocal plots were then plotted against the concentration
of 25 (range 0e3
mM), to evaluate Ki data. Data analysis was per-
4.7.5. Culture of HepG2 cells and treatment
formed with Origin Pro 7.5 software (Origin Lab Corp.).
The human hepatoma cell line HepG2were cultured in Eagle’s
minimum essential medium (EMEM) supplemented with 15 non
essential amino acids, 1 mM sodium pyruvate, 10% heat-inactivated
4.7.3. In vitro bloodebrain barrier permeability assay
Prediction of the brain penetration was evaluated using
a parallel artificial membrane permeation assay (PAMPA), in
a similar manner as described previously [41]. Commercial drugs,
phosphate buffer saline solution at pH 7.4 (PBS), and dodecane
were purchased from Sigma, Aldrich, Across and Fluka. Millex filter
fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL
streptomycin (reagents from Invitrogen, Madrid, Spain). Cultures
were seeded into flasks containing supplemented medium and
maintained at 37 ꢄC in a humidified atmosphere of 5% CO2 and 95%
air. For assays, HepG2 cells were subcultured in 48-well plates at
a seeding density of 1ꢅ105 cells per well. When the HepG2 cells
reached 80% confluence, the medium was replaced with fresh
units (PVDF membrane, diameter 25 mm, pore size 0.45 mM) were
acquired form Millipore. The porcine brain lipid (PBL) was obtained
from Avanti Polar Lipids. The donor microplate was a 96-well filter
medium containing 1e300 mM compounds or 0.1% DMSO as
plate (PVDF membrane, pore size 0.45
microplate was an indented 96-well plate, both from Millipore. The
acceptor 96-well microplate was filled with 180 L of PBS:ethanol
(70:30) and the filter surface of the donor microplate was
m
M) and the acceptor
a vehicle control.
m
4.7.6. MTT assay and cell viability
Cell viability, specifically the mitochondrial activity of living cells,
was measured by quantitative colorimetric assay with 3-[4,5 dime-
thylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) (Sigma
impregnated with 4 mL of porcine brain lipid (PBL) in dodecane
(20 mg mLꢁ1). Compounds were dissolved in PBS:ethanol (70:30)
at 1 mg mLꢁ1, filtered through a Millex filter, and then added to the
Aldrich), as described previously [57]. Briefly, 50 mL of the MTT
donor wells (180
m
L). The donor filter plate was carefully put onto
labelingreagent, at a finalconcentrationof 0.5mg/mL, were added to
each well at the end of the incubation period and the plate was
placed within a humidified incubator at 37 ꢄC with 5% CO2 and 95%
air (v/v) for an additional 2 h period. Then, the insoluble formazan
was dissolved with dimethylsulfoxide. Colorimetric determination
of MTTreductionwas measured at 540 nm. Control cells treated with
EMEM were taken as 100% viability.
the acceptor plate to form a sandwich, which was left undisturbed
for 120 min at 25 ꢄC. After incubation, the donor plate was carefully
removed and the concentration of compounds in the acceptor wells
was determined by UV spectroscopy. Every sample was analyzed at
five wavelengths, in four wells and at least in three independent
runs, and the results are given as the mean ꢀ standard deviation. In
each experiment, 20 quality control standards of known BBB
permeability were included so as to validate the analysis set.
4.7.7. Neuroprotection assay: quantification of neuronal cell
viability by the XTT test
4.7.4. Oxygen Radical Absorbance Capacity assay
The ORAC-FL method of Ou et al. [52], partially modified by
Dávalos et al. [53] was followed, using a Polarstar Galaxy plate
4.7.7.1. Primary neuronal cultures. Primary neuronal cultures from
rat cerebral cortex were prepared as previously described [60] with
minor modifications. All procedures associated with animal

762
E. Maalej et al. / European Journal of Medicinal Chemistry 54 (2012) 750e763
experiments were approved by the Ethics Committee of Uni-
versidad Complutense de Madrid (UCM), Madrid (Spain). Cell
suspensions from cerebral cortex were prepared from 19-day-old
Wistar rat embryos. Living cells in cell suspension were counted by
trypan blue exclusion method. Cells were seeded on plastic 48 well
Docking calculations were performed with the program Autodock
Vina [64]. AutoDockTools (ADT; version 1.5.4) was used to add
hydrogens and partial charges for proteins and ligands using Gas-
teiger charges. Flexible torsions in the ligands were assigned with
the AutoTors module, and the acyclic dihedral angles were allowed
to rotate freely. Trp286, Tyr124, Tyr337, Tyr72, Asp74, Thr75, Trp86,
and Tyr341 receptor residues were selected to keep flexible during
docking simulation using the AutoTors module. Because VINA uses
rectangular boxes for the binding site, the box center was defined
and the docking box was displayed using ADT. The docking proce-
dure was applied to whole protein target, without imposing the
binding site (“blind docking”). A grid box of 60 ꢅ 60 ꢅ 72 with grid
points separated 1 Å, 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. The AutoDock Vina docking
procedure used was previously validated [26].
multidishes, precoated with 0.05 mg/mL poly D-lysine at a density
of 2 ꢅ 105 cells/well, and were kept at 37 ꢄC in a 5.2% CO2 atmo-
sphere in high glucose Dulbecco’s medium supplemented with 15%
heat-inactivated (56 ꢄC for 30 min) fetal calf serum. After 48 h,
cultured cells were placed and maintained in a serum-free medium
(Dulbecco’s: Ham’s F12, 1:1 [vol/vol] containing 3.15 mg/mL
glucose, 2.5 mM Glutamax, and 0.5 mM sodium pyruvate (DMEM/
F-12, GlutaMAXÔ; GIBCO, Life Technologies, Madrid (Spain))), 1%
AntibioticeAntimycotic (Gibco; Life Technologies, Madrid, Spain) at
100 units of penicillin, 100
mg of streptomycin, and 0.25 mg of
amphotericin B final concentrations, and supplemented with 1%
B27 medium (Gibco; Life Technologies, Madrid (Spain)). Six- to nine
day cultures were used in the experiments.
Acknowledgments
4.7.7.2. Assessment of cell viability after exposure of cell cultures
to OligomycineRotenone treatments. To investigate the neuro-
protective effect of tacrine and analogous compounds 26e30, several
A. Samadi thanks CSIC for a I3P-post-doc contract. M. Chioua
thanks ISCIII for a “Sara Borrell” contract. J. Marco-Contelles thanks
MICINN (SAF2006-08764-C02-01; SAF2009-07271). JMC (Spain)
and Fakher Chabchoub (Tunisia) thank AECID Ministerio de Asuntos
Exteriores (Spain) for a grant “Acción Integrada (A1/035457/11)”
with Tunisia. F.C. acknowledges the Ministry of Higher Education,
Scientific Research and Technology in Tunisia for financial support.
M. J. Oset-Gasque thanks MICINN (SAF2010-20337) and UCM-
Santander (GR35/10-B) for financial support. M. I. Rodríguez-Franco
thanks MICINN (SAF2006-01249 and SAF2009-13015-C02-01). L.
Monjas thanks the fellowship from Ministerio de Educación y
Ciencia (MEC, Spain) for Master Studies. C. de los Ríos thanks ISCIII
for a “Miguel Servet” contract (CP10/00531, ISCIII), and financial
support (Fundación CIEN and “Miguel Servet Program”). JMC thanks
Javier Morón-Oset for reading and improving the manuscript.
concentrations of these compounds between 0.01 and 100
used. Neuroprotection was assayed by measuring the increase in cell
viability after 24 h treatment with a mixture of 30 M rotenone and
10 M oligomycin-A, which induced neuronal cell death, as described
mM were
m
m
[59,61]. The mixture of rotenone plus oligomycin-A blocks mito-
chondrial electron transport chain complexes I and V, respectively,
inducing cell death by oxidative stress.
Cell viability, specifically the mitochondrial activity of living
cells, was measured by quantitative colorimetric assay with
the mitochondrial probe XTT [2,3-bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] (Roche,
Madrid, Spain), based on the ability of living metabolically active
cells to reduce the yellow tetrazolium salt (XTT) to form an orange
formazan dye, whose quantity directly correlates to the number of
living cells. Measurements were carried out on neuronal cell
cultures seeded into 48-well culture plates as described [62,63].
Briefly, control and treated neuronal cultures (2 ꢅ 105 cells/well)
were incubated with the XTT solution at 0.3 mg/ml final concen-
tration for 3 h in a humidified incubator at 37 ꢄC with 5% CO2 and
95% air (v/v) and the soluble orange formazan dye formed was
spectrophotometrically quantified, using a scanning multi-well
spectrophotometer (ELISA plate reader) at 450 nm (reference
650 nm). All XTT assays were performed in triplicate in cells of
different batches. Control cells treated with EMEM alone were
regarded as 100% viability. Controls containing different DMSO
concentrations (0.001e1% DMSO) were performed in all assays.
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