Tetrahydropyranodiquinolin-8-amines as new, non hepatotoxic, antioxidant, and acetylcholinesterase inhibitors for Alzheimer's disease therapy

Article abstract of DOI:10.1016/j.ejmech.2016.11.050

Herein we report an efficient two step synthesis and biological assessment of 12 racemic tetrahydropyranodiquinolin-8-amines derivatives as antioxidant, cholinesterase inhibitors and non-hepatotoxic agents. Based on the results of the primary screening, we identified 7-(3-methoxyphenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine (2h) as a particularly interesting non-hepatotoxic compound that shows moderate antioxidant activity (1.83 equiv Trolox in the ORAC assay), a non competitive inhibition of hAChE (IC₅₀= 0.75 ± 0.01 μM), and brain permeable as determined by the PAMPA-Blood Brain Barrier assay.

Full text of DOI:10.1016/j.ejmech.2016.11.050

Accepted Manuscript
Tetrahydropyranodiquinolin-8-amines as new, non hepatotoxic, antioxidant, and
acetylcholinesterase inhibitors for Alzheimer's disease therapy
Youssef Dgachi, Olga Sokolov, Vincent Luzet, Justyna Godyń, Dawid Panek,
Alexandre Bonet, Hélène Martin, Isabel Iriepa, Ignacio Moraleda, Cristina García-
Iriepa, Jana Janockova, Lysiane Richert, Ondrej Soukup, Barbara Malawska, Fakher
Chabchoub, José Marco-Contelles, Lhassane ismaili
PII:
S0223-5234(16)30988-6
10.1016/j.ejmech.2016.11.050
EJMECH 9083
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 July 2016
Revised Date: 21 November 2016
Accepted Date: 22 November 2016
Please cite this article as: Y. Dgachi, O. Sokolov, V. Luzet, J. Godyń, D. Panek, A. Bonet, H. Martin, I.
Iriepa, I. Moraleda, C. García-Iriepa, J. Janockova, L. Richert, O. Soukup, B. Malawska, F. Chabchoub,
J. Marco-Contelles, L. ismaili, Tetrahydropyranodiquinolin-8-amines as new, non hepatotoxic,
antioxidant, and acetylcholinesterase inhibitors for Alzheimer's disease therapy, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.050.
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ACCEPTED MANUSCRIPT
OMe
NH2
N
is a permebale, in the low micromolar range
IC = 0.75 ± 0.01 µM),
(
5
0
selective, hAChE non-competitive inhibitor,
O
showing antioxidant, and non-hepatotoxic properties,
fulfilling thus the requirements to be considered a very
promising new tacrine for Alzheimer's disease therapy.
N
Compound 2h

ACCEPTED MANUSCRIPT
Tetrahydropyranodiquinolin-8-amines as New, non Hepatotoxic,
Antioxidant, and Acetylcholinesterase Inhibitors for Alzheimer’s Disease
Therapy
1
2
2
3
3
Youssef Dgachi, Olga Sokolov, Vincent Luzet, Justyna Godyń, Dawid Panek, Alexandre
4
4
5
5
6
Bonet, Hélène Martin, Isabel Iriepa, Ignacio Moraleda, Cristina García-Iriepa, Jana
Janockova, Lysiane Richert, Ondrej Soukup, Barbara Malawska, Fakher Chabchoub, *
José Marco-Contelles, * and Lhassane ismaili *
7
4
7
3
1
8
2
1
Laboratory of Applied Chemistry: Heterocycles, Lipids and Polymers, Faculty of Sciences of Sfax,
University of Sfax. B. P 802. 3000 Sfax-Tunisia
2
Laboratoire de Chimie Organique et Thérapeutique, Neurosciences intégratives et cliniques EA 481,
Univ. Franche-Comté, Univ. Bourgogne Franche-Comté, UFR SMP, 19, rue Ambroise Paré, F-25000
Besançon, France.
3
Department of Physicochemical Drug Analysis, Jagiellonian University Medical College, Medyczna
9
Street, 30-688 Krakow, Poland.
4
Laboratory of Cell Toxicology, EA 4267, Univ. Bourgogne Franche-Comté, 19 rue Ambroise Paré,
F-25030 Besançon Cedex, France.
5
Department of Organic Chemistry and Inorganic Chemistry, School of Biology, Enviromental
Sciences and Chemistry, University Alcala, Ctra. Barcelona, Km. 33.6, 28871, Alcala de Henares,
Spain
6
Department of Analytical Chemistry, Physical Chemistry, and Chemical Engineering, School of
Biology, Enviromental Sciences and Chemistry, University Alcala, Ctra. Barcelona, Km. 33.6, 28871,
Alcala de Henares, Spain
7
Biomedical Research Center, University Hospital Hradec Kralove, Czech Republic
Laboratory of Medicinal Chemistry (IQOG, CSIC) C/ Juan de la Cierva 3, 28006-Madrid, Spain
8
*
*
*
Fakher Chabchoub: fakher.chabchoub@yahoo.fr: Tel.: +216 74676606
José Marco-Contelles,: iqoc21@iqog.csic.es; Tel.: + 34915622900
Lhassane ismaili: lhassane.ismaili@univ-fcomte.fr Tel.: +33381665543
Abstract
Herein we report an efficient two step synthesis and biological assessment of 12 racemic
tetrahydropyranodiquinolin-8-amines derivatives as antioxidant, cholinesterase inhibitors and
non-hepatotoxic agents. Based on the results of the primary screening, we identified 7-(3-
methoxyphenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine (2h) as a
particularly interesting non-hepatotoxic compound that shows moderate antioxidant activity
(
1.83 equiv Trolox in the ORAC assay), a non competitive inhibition of hAChE (IC = 0.75 ±
50
0
.01µM), and brain permeable as determined by the PAMPA-Blood Brain Barrier assay.
Keywords: Alzheimer’s disease, acetylcholinesterase inhibitors, antioxidants, hepatotoxicity,
brain blood barrier, molecular modeling, multifunctional agents
1

ACCEPTED MANUSCRIPT
1
. Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting round 35
million people worldwide [1]. AD is characterized by progressive memory loss, decline in
language skills and other cognitive impairments [2,3]. Although the causes and mechanisms
underlying the progress of AD are still not fully understood [4], there is a general consensus
about the complexity of the disease. Accordingly, a number of multiple factors, such as the
aggregation of beta-amyloid peptide, hyperphosphorylation of tau protein, low levels of
acetylcholine (ACh), oxidative stress [5,6] and accumulations of biometals (Cu, Fe, Zn), are
playing key roles in the development of AD [7,8].
Nowadays, three acetylcholinesterase inhibitors (AChEIs), donepezil, rivastigmine, and
galantamine [9], able to restore ACh levels, and one N-methyl-D-aspartate receptor
antagonist, memantine [10], are the only available drugs to treat AD patients. These drugs
afford modest improvements in memory and cognitive functions, and do not cure or slow the
progress of the neurodegeneration [11]. Consequently, there is an urgent need for new drugs
for AD therapy.
Figure 1 Structures of tacrine, clioquinol, and the new racemic 7-aryl-9,10,11,12-tetrahydro-
7
H-pyrano[2,3-b:5,6-h']diquinolin-8-amine derivatives (I) described in this work.
One of the current therapeutic strategies is based on the development of Multi-Target-
Directed Ligands (MTDLs), to take into account the multifactorial nature of AD, and
consequently, being able to simultaneously interact with the different enzymatic systems or
2

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receptors involved in the pathology [12–17]. In our laboratory, and by using multicomponent
reactions (MCR), we have recently prepared a number of new MTDLs as AChEIs showing
strong antioxidant power [18–20].
II
We have now designed new MTDLs based on clioquinol, as an antioxidant, and Cu -
complexing agent [21], and tacrine, one of the most potent known ChEIs to date, although
discontinued shortly after its approval due to its hepatotoxicity [22]. Thus, the new
tetrahydropyranodiquinolin-8-amine hybrids (I) (Figure 1) result from the juxtaposition of
both reference molecules and are targeted to act as antioxidant, biometal chelator, and ChEIs.
Thus, in this work we have synthesized and evaluated 7-aryl-9,10,11,12-tetrahydro-7H-
pyrano[2,3-b:5,6-h']diquinolin-8-amine derivatives 2a-l (Scheme 1), and identified 7-(3-
methoxyphenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine (2h) as a
new, non-hepatotoxic, antioxidant, AChEI able to cross the brain-blood-barrier (BBB) for the
potential AD therapy.
Scheme 1. Synthesis of racemic tetrahydropyranodiquinolin-8-amines 2a-l.
3

ACCEPTED MANUSCRIPT
2
. Results and discussion
2
.1. Synthesis
The synthesis of racemic 7-aryl-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amines 2a-l has been carried out in two steps as shown in scheme 1. The first step, a MCR
between malononitrile, selected substituted benzaldehydes or pyridincarbaldehydes, and 8-
hydroxyquinoline, in the presence of catalytic amounts of piperidine, in ethanol, at 100 °C,
for 15 min, gave the known 4H-pyrans 1a-l in very good yield [23], whose Friedlander-type
reaction [24] with cyclohexanone, under the usual experimental conditions, afforded the
expected racemic compounds 2a-l (Scheme 1). All new compounds gave analytical and
spectroscopic data in good agreement with their structures (Experimental Part).
Table 1. Inhibition of EeAChE, hACHE, eqBuChE, (IC , µM) and ORAC-FL values for
5
0
compounds 2a-l , tacrine, clioquinol and ferulic acid.
a
a
Compound
R
H
EeAChE
0.06±0.01
0.11±0.01
0.09±0.01
0.14±0.01
hAChE
eqBuChE
BBB
ORAC
c
2
a
1.73 ± 0.02
1.11±0.00
0.63±0.08
-
1.47±0.25
1.49±0.09
1.36±0.05
2.01±0.05
2.49±0.19
1.22±0.07
1.60±0.22
1.83±0.10
1.71±0.10
c
c
2
b
2-F
3-F
2-Br
4-NO
2,6-di-Cl
2-OCH
3-OCH3
4-OCH
-
-
d
c
2
c
2.20 ± 0.03
-
-
c
c
2
d
-
0.21±0.02
-
c
d
c
2
e
2
0.24±0.01
0.10±0.01
0.08±0.01
0.04±0.01
0.04±0.01
0.04±0.01
0.47±0.07
0.19±0.00
0.03±0.01
-
-
-
c
d
c
2f
-
-
-
2
g
3
1.51±0.10
1.30±0.22
1.30±0.19
-
b
2
h
0.75 ± 0.01
1.51 ± 0.02
2.85 ± 0.05
5.41 ± 0,3
c
2
i
3
-
c
4
-CH3
2
j
0.53±0.03
-
1.88±0.35
1.70±0.12
d
d
c
-
-
2
k
-
-
d
c
-
-
-
-
2
l
2.86±0.18
-
2.35±0.26
c
Tacrine
0.13±0.00
0.05±0.01nM
-
0.2±0.1
3.74±0.22
c
c
c
c
-
Ferulic acid
-
-
-
a
Inhibition curves were obtained by nonlinear regression. Ee: electric eel, eq: equine. Each IC value is the mean ±
50
b
SEM of quadruplicate of at least three different experiments. Results are expressed in µM; PAMPA method (Pe ± SEM
(
-6
-1
c
d
*10 cm s ); Not determined ; No inhibition.
4

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2
2
.2. Biological evaluation
.2.1. Evaluation of AChE and BuChE inhibition
The preliminary evaluation of the inhibitory potencies against Electrophorus electricus AChE
EeAChE) and serum horse BuChE (eqBuChE) was carried out following the Ellman’s
(
protocol [25], using tacrine as reference compound for comparative purposes. As shown in
table 1, these compounds exhibited low micromolar inhibitory potencies against EeAChE
with IC values ranging from 0.04 and 0.47 µM, for compounds 2h and 2j, respectively.
5
0
Notably, compounds 2a,c,g-i showed IC not exceed 100 nM. Among them, compound 2c
5
0
(
IC = 0.09 µM) was 3-fold less active than tacrine (IC = 0.03 µM), but totally selective
50 50
against eqBuChE. The most potent EeAChEIs were 2h-j, all with the same IC value equal to
5
0
0
.04 µM, in the same range of tacrine (IC = 0.03 µM). Regarding the eqBuChE inhibition
50
eight products (2a, 2b, 2d, 2g-j, and 2l) showed micromolar inhibition ranging between 0.21
and 2.86 µM, the most potent being 2d (IC = 0.21 µM), whereas compounds 2c, 2e, 2f and
5
0
2
k showed no inhibition at all.
Regarding the structure-activity relationship, and for the inhibition of AChE, several
conclusions can be drawn. First of all, compounds bearing substituted aryl rings attached to
C7 at the pyran core (2a-d, 2f-j) are more potent AChEIs than those bearing a pyridine ring
(2k,l), except for compound 2e, substituted with a nitro group at C4’. Among the substituted
aryl derivatives, it is clear that the unsubstituted (2a) or those bearing electron-donor
substituents (2g-j) are more potent AChEIs than those bearing electron-withdrawing rings
(2b-f). To sum up, and regarding the positions of substituents at the aromatic ring at C7,
compounds 2b-f bearing a substituent at position C3’, and those (2g-j) bearing it at positions
C3’ or C4’, gave the best inhibition. It is also apparent that among compounds 2g-j, the type
of the substituent (Me vs OMe) or the position of the methoxy group in the aromatic ring has
no effect on the AChE inhibition power, showing similar IC values. For the inhibition of
5
0
5

ACCEPTED MANUSCRIPT
BuChE, no evident SAR could have been established. The tested compounds exhibited a
lower inhibitory potency against eqBuChE compared to EeAChE or complete lack of activity.
Therefore, only the most potent inhibitors 2a,c,h-j against EeAChE were tested for their
ability to inhibit human AChE (hAChE). As shown in table 1, these five compounds had an
activity ranking from 2.85 µM for 2j to 0.75 µM for 2h, being again the most potent hAChE
inhibitor, only 5.7 fold less active than tacrine. In summary, the best inhibitor was compound
2
h, bearing methoxygroup at position C3’ of aryl ring attached to C7 at the pyran core, as
strongly inhibited EeAChE, hAChE and eqBuChE in the low micromolar range, showing IC50
values equal to 0.04 ± 0.01 µM, 0.75 ± 0.01 µM, and 1.30 ± 0.22 µM, respectively.
2
.2.2. Kinetic study of the hAChE inhibition by compound 2h
The kinetic mechanism of hAChE inhibition of compound 2h has been investigated by
obtaining the Lineweaver-Burk double reciprocal plots (Figure 2), whose analysis showed
that K appeared unaltered while V decreased at increasing concentrations of the inhibitor.
m
max
This pattern indicates that inhibitor 2h acts as a non-competitive enzyme inhibitor. Replots of
the slope versus concentration of compound 2h give an estimate of the inhibition constant,
which is equal to 0.87 µM.
Figure 2. Lineweaver – Burk plot illustrating non-competitive type of
hAChE inhibition by compound 2h. S = acetylthiocholine; V = initial
velocity rate.
6

ACCEPTED MANUSCRIPT
2
.2.3. ORAC test
Next, we assessed the antioxidant activity of racemic compounds 2a-l using the Oxygen
Radical Absorbance Capacity (ORAC-FL) method [26,27]. Tacrine and ferulic acid [28] were
used as negative and positive reference molecules, respectively. Results are expressed in
relation to radical scavenging properties of Trolox yielding the Trolox equivalents (TE) unit.
As shown in table 1, all compounds presented good radical scavenging properties with ORAC
values ranging from 1.22 to 2.49 TE, but lower than the value found for ferulic acid (3.7 TE).
The three most potent antioxidants were compounds 2d (2.01 TE), 2l (2.35 TE) and 2e (2.49
TE), bearing a 2’-bromophenyl, 2’-bromopyridyl, and a 4’-nitrophenyl group attached to
pyran ring at C7, respectively. This trend clearly means that stronger the electron-
withdrawing effect of the aromatic ring at C7 is, higher the antioxidant capacity observed.
Thus, for compounds of type 2, we hypothesize that the presence of a free (C8)-NH could
2
explain the observed antioxidant effect, due the lability of the N-H bond against any radical
oxygenated species.
2
.2.4. Studies of metal-chelating properties
2
+
2+
The ability of compound 2h to chelate biometals such as Cu , and Fe was next studied by
UV–vis spectrometry. As shown, the maximum absorption wavelength remained unchanged
when CuSO was added and incubated for 60 min and 20 h (Supplementary Material). The
4
same trend was observed for FeSO4 (Supplementary Material), suggesting that
unfortunately little or no complex formation between compound 2h with these cations took
place. In order to explain these unexpected results, a Density Functional Theory (DFT) study
was conducted to investigate the complexation between the ligand and the Cu(II) metal ion,
and understand the intrinsic factors affecting cation complexation.
7

ACCEPTED MANUSCRIPT
Thus, the pyranopyridoquinoline (PPQ) system was used as a simplified model of compound
2
+
2
h. As shown in figure 3, two distinct complexes of Cu with PPQ have been considered. In
the Cu(PPQ)SO , the metal cation is bonded to one ligand, PPQ, in a bidentate chelating
4
mode and to two oxygens atoms of the sulphate anion resulting in a square-planar
environment. On the other hand, modelled Cu(PPQ)SO (MeOH) adduct consists of five
4
coordinated copper (II) complex with the oxygen and the nitrogen of PPQ bonded to the metal
ion and the two oxygen atoms of the sulphate anion. One further oxygen atom of one
methanol molecule is finally coordinated to the copper ion resulting in a square based
pyramidal environment.
Cu(PPQ)SO4
Cu(PPQ)SO (MeOH)
4
2
+
Figure 3. B3LYP-optimized structures for Cu complexes of PPQ.
The total energy of each complex was calculated and compared with that of the sulphate
complex [Cu (II) (MeOH) (SO )] as a reference to quantitatively evaluate the stability of each
x
4
complex. Table 2 shows the formation energy of each complex, calculated using equations I
and II.
8

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Table 2. Thermodynamic functions of the copper (II)
complexes with PPQ.
Complex
∆H(Kcal/mol) ∆G(Kcal/mol)
Cu(PPQ)(SO4)
0.61
-7.85
-2.77
Cu(PPQ)(SO )(MeOH) 5.69
4
[
E (Cu(PPQ)(SO ) + 2 E(MeOH)] – [E(PPQ) + E (Cu (II) (MeOH) (SO )] (I)
4 2 4
[
E (Cu(PPQ)(SO )(MeOH) ) + 2 E(MeOH)] – [E(PPQ) + E (Cu (II) (MeOH) (SO )]
(II)
4
3
4
Scheme 2. Chemical equations used to measure the formation energies of the complexes
Cu(II)(PPQ)(MeOH) (SO )].
[
x
4
The most important point gathered from table 2 is that the PPQ system does not have the
2
+
capability to form stable metal complexes with Cu since it has positive formation energy of
.61 and 5.69 Kcal/mol. Interestingly, a comparison between formation energies values of
Cu(PPQ)(SO ) and Cu(PPQ)(SO )(MeOH) complexes shows that these values depend on the
0
4
4
9

ACCEPTED MANUSCRIPT
number of molecules coordinated to the metal. Therefore, the formation of the complex with
four ligands is clearly favored with respect to the complex with five ligands, suggesting that
an increase of the steric hindrance around the metal decrease the stability of the corresponding
complex.
Both complexations are endothermic, however the Gibbs free energy of the complex
formation was calculated as -7.85 and -2.77 Kcal/mol, which are negative values. The
positive formation energy of the two complexes could be explained by the destabilizing effect
of the pyridine ring fused to the pyran ring. On one hand, the electron-withdrawing ability of
this ring makes the oxygen-pyran a worse electron donor, decreasing the interaction strength
between the cooper and the oxygen atoms. On the other hand, its nitrogen atom is far enough
from the cooper atom to coordinate but close enough to probably generate an electrostatic
repulsion between the lone pair of this nitrogen atom and the electronic density (e.g. electrons
located in d orbitals) of the cooper atom.
To explore the effect of pyridine ring on the complex formation, a phenyl ring has substituted
it leading to the ligand PhePQ (Figure 4). In scheme 3 we show the equations used to compute
the formation energies of the complexes starting from this ligand.
Cu(PhePQ)(SO4)
Cu(PhePQ)(SO )(MeOH)
4
2
+
Figure 4. B3LYP-optimized structures for Cu complexes of PhePQ.
10

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Scheme 3. Chemical equations used to measure the formation energies of the complexes
[
Cu(PhePQ)(MeOH) (SO )].
x 4
∆
H and ∆G of [Cu(II)(PhePQ)(MeOH) (SO )] complexes (Table 3) were computed and
x 4
compared with the corresponding values for [Cu(II)(PPQ)(MeOH) (SO )] complexes (Table
x
4
3
).
Table 3. Thermodynamics functions of the copper (II)
complexes with PhePQ.
Complex
∆H(Kcal/mol) ∆G(Kcal/mol)
Cu(PhePQ)(SO4)
-2.02
-9.99
-4.22
Cu(PhePQ)(SO )(MeOH) 3.52
4
The energy of formation for the less crowded system, Cu(PhePQ)(SO ), is more favorable
4
than for Cu(PhePQ)(SO )(MeOH) by ∼ 5 Kcal/mol with respect to both ∆H and ∆G.
4
Moreover, comparing the energies of formation of complexes Cu(PPQ)(SO ) and
4
11

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Cu(PhePQ)(SO ) it is observed that ∆H becomes negative for the later, indicating that in this
4
case the formation of Cu(PhePQ)(SO ) is energetically favorable, suggesting a clear effect of
4
the pyridine ring.
To explore the effect of the pyridine ring on the geometrical parameters of chelated ring and
the metal-oxygen structure, the geometrical parameters of Cu(PhePQ)(SO ) were compared
4
with the corresponding values for Cu(PPQ)(SO ). The O-cα-pyrane ring bond length in
4
Cu(PhePQ)(SO ) slightly increases while the Cu-O and Cu-N bond lengths slightly decrease
4
compared to the corresponding values in Cu(PPQ)(SO4).
These results indicate the formation of more stable complex between Cu(II) and PhePQ
compared with that between Cu(II) and PPQ (see Table 4).
Table 4. Selected theoretical geometrical parameters
a
for Cu(PPQ)(SO ) and Cu(PhePQ)(SO ) complexes .
4
4
Complex
dC-O
dCu-O
dCu-N
1.994
1.971
Cu(PPQ)(SO4)
1.397
2.321
2.260
Cu(PhePQ)(SO ) 1.410
4
a
d, bond distance in Å.
In addition, comparison between the atomic charges in the Cu(PPQ)(SO ) and
4
Cu(PhePQ)(SO ) complexes show that the O atom in Cu(PhePQ)(SO ) is more negative than
4
4
that in Cu(PPQ)(SO ) (Table 5). This difference has been attributed to the resonance
4
conjugation between the electron-withdrawing pyridine ring and the chelated ring of PPQ.
This resonance conjugation, which causes less electron delocalization in the chelated ring of
Cu(PPQ)(SO ) compared to that of Cu(PhePQ)(SO ), is responsible for the shorter O-cα-
4
4
pyrane ring bond in the former (Table 5).
12

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Charge transfer from the pyrane to the pyridine acceptor, will decrease the oxygen-electron
density that is available to transfer to the metal, therefore the electron-withdrawing ability of
substituents influences ligand affinity.
Table 5. Selected atomic charges for
Cu(PPQ)(SO ) and Cu(PhePQ)(SO )
4
4
complexes.
Complex
O charge
-0.476
Cu(PPQ)(SO4)
Cu(PhePQ)(SO ) -0.517
4
2
.2.5. In vitro cytotoxicity of compounds 2a, 2c and 2h-j in HepG2 cells
For an effective lead drug, the cytotoxicity should be at the lowest possible level. In this
sense, we submitted the five most promising compounds 2a, 2c and 2h-j to an in vitro
toxicologic evaluation (MTT assay) using human hepatocellular carcinoma cell line (HepG2)
[
29] which represents a well accepted probe to evaluate hepatotoxic effect, using different
dose concentrations (1, 10, 30, 100, 300, 1000 µM). As shown in table 6, where, tacrine
showed no decrease in cell viability up to 100 µM, but exerts a marked cytotoxicity above
3
00 µM. The five tested compounds, except
Table 6. In vitro cytotoxicity of tacrine, 2a, 2c and 2h-j in HepG2 cells.
3
0 µM
106.4 ± 6.1
108.6 ± 12.8 102.2 ± 11.2 93.7 ± 11.5
100 µM
300 µM
1000 µM
Tacrine
110.5 ± 5.8 49.3 ± 3.7*** 13.7 ± 3.1***
2
a
91.2 ± 6.4
75.8 ± 5.7*
94.1 ± 3.0
104.7 ± 1.4
96.2 ± 4.8
2
c
92.4 ± 3.4
110.0 ± 3.5
115.4 ± 1.7
115.9 ± 4.9
95.4 ± 11.5
102.2 ± 5.2
110.7 ± 3.8
106.0 ± 1.5
91.6 ± 13.9
94.0 ± 7.4
113.8 ± 4.0
92.5 ± 5.9
2
h
2
i
2
j
Means ± SEM of triplicates from at least three different cultures. *p<0.05,
**p<0.001, as compared to the control cultures (one-way ANOVA).
*
13

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2
c, had no significant cytotoxic effects measured in concentrations up to 1000 µM on HepG2
cells and could be considered as non-hepatotoxic. Although compound 2c displayed a
hepatotoxic effect at 1000 µM it is still much less cytotoxic than tacrine.
2
.2.6 In vitro cytotoxicity of compounds 2h in primary human hepatocytes
Primary human hepatocytes are considered the gold standard for the evaluation and prediction
of hepatotoxicity in humans [30], therefore we evaluated the most active compound 2h on
human hepatocytes. Pools of cryopreserved primary human hepatocytes used in suspension
have been described as useful for the screening of hepatotoxicants [31,32]. Thus a suspension
of human hepatocytes cells from a pool of 9 donors was used with seven concentrations (0.1,
0
.3, 1.0, 3.0, 10.0, 30. 0 and 100.0 µM) with respect of the solubility limits of compounds in
treatement medium. As shown in Table 7, tacrine, displayed cytotoxicity towards the pool of
primary human hepatocytes from 30.0 µM while compound 2h did not exhibit any
cytotoxicity until 100.0 µM.
Table 7. In vitro concentration-response of tacrine and 2h on primary human hepatocyte
viability
Concentration 00 µM
0.1 µM
94 ± 3
89 ± 4
1.0 µM
86 ± 3
84 ± 8
3.0 µM
83 ± 5
83 ± 8
10.0 µM
81 ± 7
30.0 µM
75 ± 14
99 ± 11
100.0 µM
70 ± 3
Tacrine
100 ± 11
100 ± 11
2
h
89 ± 3
89 ± 2
Results expressed in Means ± SD of triplicates from from a pool of 9 donors
.2.7. PAMPA assay
2
Prediction of blood-brain barrier penetration for the most potent hAChEI 2h shows high
probability to cross the BBB via passive diffusion (Table 1). The data obtained have been
correlated to standard drugs, where CNS availability is known and also reported using the
14

ACCEPTED MANUSCRIPT
PAMPA assay [33], showing high resemblance with previously reported penetrations as well
as with a general knowledge about the availability in the CNS of such standard drugs.
3
. Molecular modeling of compound 2h
To help us understand the observed experimental activities, molecular docking studies were
carried out using AutoDock Vina software[34] to obtain the plausible binding mode of the
compounds, within the active site gorge of hAChE. The kinetic data provide evidence that
compound 2h displays a non-competitive inhibition and argue in favour of interactions of 2h
with the PAS of hAChE. Molecular modeling studies have been carried on the most potent
hAChE, compound 2h, in order to validate this assumption.
The C7 carbon of the pyran ring is chiral and two isomers are possible. As the inhibitors were
tested as racemic mixtures in the assay, both enantiomeric forms were built up and used for
docking to check the effect of chirality on potency and selectivity towards hAChE. The most
favorable docking pose for (R)-2h in the binding site of hAChE is illustrated in figure 5.
Figure 5. Binding mode of (R)-2h at the active site of hAChE: Binding affinity = -11.3 Kcal
-1
mol . The compound is rendered as balls and sticks and illustrated in blue. The side chains
conformations of the mobile residues are illustrated in the same light color that the ligand.
Different sub-sites of the active site were coloured: catalytic triad (CT) in green, oxyanion
hole (OH) in pink, anionic sub-site (AS) in orange, except Trp86, acyl binding pocket (ABP)
in yellow, except Phe297 and Phe338, and peripheral anionic sub-site (PAS) in violet. Red
dashed lines are drawn among atoms involved in hydrogen bond interactions.
15

ACCEPTED MANUSCRIPT
The docking results reveal that the R-enantiomer is accommodated rather far from the active
site, at the top of the gorge (PAS), within the hydrophobic pocket formed by several aromatic
residues Tyr72, Tyr124, Trp286, Phe295, Phe297, Tyr337, Phe338 and Tyr341 (Figure 6).
The intermolecular interactions are mostly contributed by aromatic-aromatic ring interactions
since the ligand contains aromatic rings favoring the ring-ring interactions. The phenyl moiety
showed T-shaped interactions with Tyr72 and face to face interaction with Trp286. The
quinoline moiety is also interacting with Trp286 and Trp341, through π−π interactions. This
enantiomer is bound to the AChE in a manner through its amino group projecting toward the
carboxylate group of Asp74 and the amino group of Leu76 forming two hydrogen bonds
between the amino group and Asp74 and Leu76 (Figure 6).
b
a
Figure 6. a) Schematic representation of different interactions of (R)-2h with hAChE.
b) Top view of the accessible surface of the active site gorge.
16

ACCEPTED MANUSCRIPT
Figure 7. Binding mode of (S)-2h at the active site of hAChE: Binding affinity = -10.8
-1
Kcal mol ; The compound is rendered as balls and sticks and illustrated in pink. The side
chains conformations of the mobile residues are illustrated in the same light color that the
ligand. Different sub-sites of the active site were coloured: catalytic triad (CT) in green,
oxyanion hole (OH) in pink, anionic sub-site (AS) in orange, except Trp86, acyl binding
pocket (ABP) in yellow, except Phe297 and Phe338, and peripheral anionic sub-site (PAS)
in violet. Red dashed lines are drawn among atoms involved in hydrogen bond interactions.
b
a
Figure 8. a) Schematic representation of different interactions of (S)-2h with hAChE.
b) Top view of the accessible surface of the active site gorge.
Docking simulations revealed that the best-scored docking conformation for (S)-2h showed a
binding pattern very similar to that of (R)-2h (Figure 7). At the top of the gorge, the phenyl
moiety showed T-shaped interactions with Tyr72 and face-to-face interactions with Trp286.
The pyridine moiety is also interacting with Trp286 through π−π interactions. Cyclohexane
17

ACCEPTED MANUSCRIPT
moiety was well fitted in the hydrophobic pocket composed by Phe295, Phe297 and Phe338.
The amino group of the ligand forms hydrogen bond with Asp74 carboxylate group and with
the hydroxyl group of the Tyr124 (Figure 8).
Figure 9. Overlay of binding mode of
compound (R)-2h (blue) and (S)-2h
(pink) in hAChE.
The best docked poses of both enantiomers were superposed and showed to be similarly
docked in the gorge of hAChE. The detailed picture of the superimposed docking poses of
(R)-2h and (S)-2h is shown in figure 9. They share the position of the central cores of the
ligands (methoxyphenol-pyran moieties) and this implies that quinoline moiety of each
enantiomer occupies a similar position as the tetrahydroquinoline moiety of the other. This
binding pattern to the hAChE has already been observed on 7-aryl-9,10,11,12-tetrahydro-7H-
benzo[7,8]chromeno[2,3-b]quinolin-8-amines [35].
4
. Conclusions
To sum up, we synthesized and evaluated twelve new racemic tetrahydropyranodiquinolin-8-
amines derivatives as promising multifunctional compounds for the potential AD therapy. As
a result, we have identified racemic 7-(3-methoxyphenyl)-9,10,11,12-tetrahydro-7H-
pyrano[2,3-b:5,6-h']diquinolin-8-amine (2h) as suitable compound for further development,
as this compound is non hepatotoxic, shows moderate antioxidant activity (1.83 equiv
Trolox), crosses the BBB in the PAMPA assay, is a good hAChEI (IC = 0.75 ± 0.01 µM) in
5
0
18

ACCEPTED MANUSCRIPT
the low micromolar range, showing non-competitive inhibition mechanism, binding
presumably at the peripheral binding site of AChE, an hypothesis that has been confirmed by
carrying out molecular modeling of both enantiomers.
5
. Materials and Methods
5
.1. Chemistry methods
Melting points were determined on a Kofler apparatus (Wagner Munz), and are uncorrected.
The reactions were monitored with TLC using aluminium sheets with silica gel 60 F254 from
Merck. IR spectra were performed on a Perkin-Elmer PARAGON FT-IR spectrometer. NMR
1
13
H and C were recorded on a Bruker spectrometer using CDCl or DMSO d as solvents.
3
- 6
The chemical shifts are reported in parts per million (ppm), using tetramethylsilane (TMS) as
internal reference. The multiplicities of the signals are indicated as follow: br, broad; s,
singlet; d, doublet; t, triplet; q, quadruplet; and m, multiplet, the coupling constants are
expressed in Hz. Elemental analysis were performed on Thermofinnigan Flash EA 1112. The
microwave assisted reactions were carried out in synthetic microwave (microwave Anton
Paar 300) with a maximum power of 300 W.
5
.1.1. General procedure for the of compounds 1a-l.
A mixture of aromatic aldehyde (0.01 mmol), malononitrile (0.01 mmol), and 8-
hydroxyquinoline (0.01 mmol) in ethanol (15 ml) in the presence of piperidine (0.5 equiv)
was warmed at 100 °C until complete precipitation. (reaction times: 15 min). The solid
obtained was collected by filtration and recrystallised from ethanol, and dried, to give
compounds 1a-l in good yield.
5
.1.1.1. 2-Amino-4-phenyl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1a). Yield: 92 %; mp
-1 1
1
96-198 °C; IR (KBr) νmax 2225 (CN), 3526, 3445 (NH ) cm ; H NMR (300 MHz, DMSO-
2
13
d ) δ 8.94-7.22 (m, 10Harom), 7.12 (br s, 2H, NH ), 4.95 (s, H ); C NMR (75 MHz,
pyran
6
2
19

ACCEPTED MANUSCRIPT
DMSO-d ) δ 160.6 (C), 150.6 (C), 146.1 (CH), 143.3 (C), 137.8 (C), 136.4 (CH),133.5 (CH),
6
1
29.1 (CH), 128.1 (CH), 127.4 (C), 127.3 (C), 126.9 (CH), 123.9 (CH), 122.5 (CH), 122.3
(CH), 121.4 (CH), 120.8 (CN), 56.2 (C), 41.1 (CH).
5
.1.1.2. 2-Amino-4-(2-fluorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1b). Yield:
-1 1
9
3%; mp 251-2 °C; IR (KBr) νmax 2190 (CN), 3409, 3310 (NH ) cm ; H NMR (300 MHz,
2
13
DMSO-d ) δ 8.90-7.12 (m, 9Harom), 5.06 (br s, 2H, NH ), 4.93 (s, H ); C NMR (75
pyran
6
2
MHz, DMSO-d ) δ 161.9 (C), 157.2 (C), 151.6 (C), 144.1 (C), 137.9 (C), 136.8 (CH), 135.2
6
(CH), 129.1 (CH), 128.2 (C), 127.8 (C), 126.1 (CH), 123.8 (CH), 122.1 (CH), 121.7 (CH),
1
21.0 (CH), 120.3 (CH), 120.1 (CN), 58.2 (C), 41.6 (CH).
5
.1.1.3. 2-Amino-4-(3-fluorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1c). Yield:
-1
1
8
4%; mp 249-250 °C; IR (KBr) νmax in cm : 2190 (CN), 3409, 3310 (NH ). H NMR (300
2
1
3
MHz, DMSO-d ) δ 8.96-7.14 (m, 9Harom), 5.06 (br s, 2H, NH ), 4.92 (s, H ); C NMR
pyran
6
2
(
75 MHz, DMSO-d ) δ 160.9 (C), 157.9 (C), 151.8 (C), 143.7 (C), 137.6 (C), 136.2 (CH),
6
1
35.3 (CH), 129.7 (CH), 128.2 (C), 127.8 (C), 126.4 (CH), 123.8 (CH), 122.9 (CH), 121.8
(CH), 121.3 (CH), 121.0 (CH), 120.1 (CN), 58.2 (C), 39.8 (CH).
5
.1.1.4. 2-Amino-4-(2-bromophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1d). Yield:
-1 1
8
9%; mp 230-1 °C; IR (KBr) νmax 2196 (CN), 3395, 3320 (NH ) cm ; H NMR (300 MHz,
2
13
DMSO-d ) δ 9.00-7.09 (m, 9Harom), 5.22 (br s, 2H, NH ), 4.89 (s, H ); C NMR (75
pyran
6
2
MHz, DMSO-d ) δ 159.4 (C), 156.8 (C), 150.5 (C), 143.3 (C), 137.9 (C), 136.3 (CH), 135.2
6
(CH), 131.6 (CH), 129.6 (C), 128.3 (C), 127.0 (CH), 126.9 (CH), 125.2 (CH), 122.1 (CH),
1
5
7
21.6 (CH), 121.1 (CH), 119.4 (CN), 59.8 (C), 41.2 (CH).
.1.1.5. 2-Amino-4-(4-nitrophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1e). Yield:
-1 1
7%; mp 230-1 °C; IR (KBr) νmax 2196 (CN), 3395, 3320 (NH ) cm ; H NMR (300 MHz,
2
13
DMSO-d ) δ 9.00-7.09 (m, 9Harom), 5.22 (br s, 2H, NH2), 4.89 (s, H
); C NMR (75
6
pyran
MHz, DMSO-d ) δ 165.1 (C), 154.3 (C), 152.1 (C), 150.6 (C), 142.5 (C), 138.4 (CH), 136.3
6
20

ACCEPTED MANUSCRIPT
(C), 135.7 (CH), 134.5 (C), 131.4 (C), 129.1 (CH), 128.3 (CH), 127.5 (CH), 126.4 (CH),
1
24.5 (CH), 123.1 (CH), 122.7 (CN), 59.3 (C), 37.8 (CH).
5
.1.1.6. 2-Amino-4-(2,6-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1f).
-1 1
Yield: 87%; mp 244-5 °C; IR (KBr) νmax 2190 (CN), 3390, 3330 (NH ) cm ; H NMR (300
2
1
3
MHz, DMSO-d ) δ 9.06-7.11 (m, 8Harom), 5.26 (br s, 2H, NH ), 4.93 (s, H ); C NMR
pyran
6
2
(
75 MHz, DMSO-d ) δ 161.2 (C), 158.8 (C), 157.6 (C), 151.5 (C), 144.3 (C), 136.4 (C), 136.0
6
(CH), 135.2 (C), 131.2 (CH), 129.5 (C), 128.1 (CH), 127.6 (CH), 125.4 (CH), 122.8 (C),
1
5
8
21.6 (CH), 121.0 (CH), 119.8 (CN), 59.1 (C), 40.7 (CH).
.1.1.7. 2-Amino-4-(2-methoxyphenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1g). Yield:
-1 1
8%; mp 217-9 °C; IR (KBr) νmax 2217 (CN), 3436, 3320 (NH ) cm ; H NMR (300 MHz,
2
DMSO-d ) δ 8.90-7.33 (m, 9Harom), 7.21 (br s, 2H, NH ), 5.28 (s, Hpyran), 3.77 (s, 3H,
6
2
1
3
OCH ); C NMR (75 MHz, DMSO-d ) δ 161.4 (C), 153.2 (C), 147.6 (C), 143.7 (C), 137.8
3
6
(CH), 136.7 (C), 134.1 (CH), 129.8 (CH), 128.1 (CH), 127.6 (CH), 126.9 (CH), 123.7 (CH),
1
23.1 (C), 122.1 (CH), 121.7 (CH), 121.0 (CH), 120.1 (CN), 56.3 (C), 55.5 (OCH ), 40.4
3
(CH).
5
.1.1.8. 2-Amino-4-(3-methoxyphenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1h). Yield:
-1 1
8
7%; mp 219-220 °C; IR (KBr) νmax 2217 (CN), 3430, 3321 (NH ) cm ; H NMR (300 MHz,
2
DMSO-d ) δ 8.93-7.29 (m, 9Harom), 7.16 (br s, 2H, NH ), 5.28 (s, Hpyran), 3.78 (s, 3H,
6
2
1
3
OCH ); C NMR (75 MHz, DMSO-d ) δ 161.9 (C), 155.9 (C), 149.8 (C), 143.7 (C), 137.8
3
6
(C), 136.1 (CH), 134.3 (C), 129.2 (CH), 128.5 (CH), 127.4 (CH), 126.9 (CH), 123.9 (CH),
1
22.9 (CH), 121.6 (CH), 121.0 (CH), 121.3 (CH), 119.8 (CN), 57.3 (C), 49.7 (OCH ), 39.8
3
(CH).
5
.1.1.9. 2-Amino-4-(4-methoxyphenyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1i). Yield:
-1 1
9
0%; mp 217-9 °C; IR (KBr) νmax 2222 (CN), 3430, 3330 (NH ) cm ; H NMR (300 MHz,
2
DMSO-d ) δ 8.95-7.33 (m, 9Harom), 7.20 (br s, 2H, NH ), 4.90 (s, Hpyran), 3.70 (s, 3H,
6
2
21

ACCEPTED MANUSCRIPT
1
3
OCH ); C NMR (75 MHz, DMSO-d ) δ 161.4 (C), 156.8 (C), 150.5 (C), 143.7 (C), 137.8
3
6
(C), 136.4 (CH), 133.7 (C), 129.5 (CH), 128.8 (CH), 128.0 (CH), 126.9 (CH), 123.9 (CH),
1
22.8 (C), 122.6 (CH), 122.4 (CH), 121.3 (CH), 121.1 (CN), 56.3 (C), 53.7 (OCH ), 41.7
3
(CH).
5
.1.1.10. 2-Amino-4-(p-tolyl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1j). Yield: 82%; mp
-1 1
2
19-220 °C; IR (KBr) νmax 2200 (CN), 3420, 3300 (NH ) cm ; H NMR (300 MHz, DMSO-
2
13
d ) δ 9.02-7.38 (m, 9Harom), 7.16 (br s, 2H, NH ), 5.04 (s, H
), 2.41 (s, 3H, CH ); C
3
6
2
pyran
NMR (75 MHz, DMSO-d ) δ 160.6 (C), 150.6 (C), 146.1 (C), 143.3 (C), 137.8 (CH), 136.4
6
(CH), 135.6 (CH), 133.5 (C), 129.1 (C), 128.1 (CH), 127.4 (CH), 127.3 (C), 123.9 (CH),
1
22.5 (CH), 122.3 (CH), 121.4 (CH), 120.8 (CN), 56.2 (C), 41.1 (CH), 28.6 (CH3).
5
.1.1.11. 2-Amino-4-(pyridin-4-yl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1k). Yield:
-1 1
8
1%; mp 196-8 °C; IR (KBr) νmax 2225 (CN), 3526, 3445 (NH ) cm ; H NMR (300 MHz,
2
13
DMSO-d ) δ 8.94-7.22 (m, 9Harom), 7.12 (br s, 2H, NH ), 4.95 (s, H ); C NMR (75
pyran
6
2
MHz, DMSO-d ) δ 160.7 (C), 150.6 (C), 145.8 (C), 143.3 (C), 137.6 (C), 136.4 (CH), 133.1
6
(C), 129.1 (CH), 128.6 (C), 127.2 (CH), 126.5 (CH), 122.8 (CH), 122.4 (CH), 122.3 (CH),
1
21.6 (CH), 120.8 (CN), 57.2 (C), 39.9 (CH).
5
.1.1.12. 2-Amino-4-(2-bromopyridin-3-yl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1l).
-1 1
Yield: 81%; mp 232-3 °C; IR (KBr) νmax 2196 (CN), 3395, 3320 (NH ) cm ; H NMR (300
2
1
3
MHz, DMSO-d ) δ 8.97-7.11 (m, 8Harom), 5.17 (br s, 2H, NH ), 4.77 (s, H ); C NMR
pyran
6
2
(
75 MHz, DMSO-d ) δ 159.4 (C), 156.6 (C), 150.5 (C), 143.3 (C), 137.7 (C), 136.3 (C), 135.1
6
(CH), 131.5 (CH), 129.8 (CH), 128.3 (CH), 127.1 (CH), 126.9 (CH), 122.5 (CH), 121.6 (CH),
1
21.0 (CN), 59.2 (C), 39.6 (CH).
5
.1.2. General procedure for the of compounds 2a-l.
The 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile (1.0 equiv) was dissolved in
distilled 1,4-dioxane (30 mL), and AlCl (1.5 equiv) and cyclohexanone (1.5 equiv) were
3
22

ACCEPTED MANUSCRIPT
added to this solution. The reaction mixture was heated at 110 °C for 2 h. When the reaction
was complete (TLC analysis, CH Cl /EtOH, 10/1, v/v), the reaction mixture was diluted with
2
2
a solution of dichloromethane/water (1/1), and treated with an aqueous solution of sodium
hydroxide (10%) until pH 11-12. After stirring for 30 min, the mixture was extracted with
CH Cl , dried over anhydrous sodium sulfate, filtered and the solvent was evaporated. The
2
2
solid obtained washed with ether and filtered give pure product.
.1.2.1. 7-Phenyl-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine (2a).
5
Following the general method, reaction of compound 1a (0.5 g, 1.6 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.23 g, 2.4 mmol) and AlCl (0.31 g, 2.4 mmol), after 2 h, product
3
-1 1
2
a (0.6 g) was obtained: yield: 95%; mp >260 ºC; IR (KBr) νmax 3333, 1517 cm ; H NMR
(
300 MHz, DMSO-d ) δ 8.94 (d, 1H, J = 8.4 Hz), 8.27 (d, 1H, J= 7.8 Hz), 7.56 (d, 2H, J= 8.4
6
Hz), 7.39-7.11 (m, 6Harom), 5.63 (br s, 2H, NH ), 5.54 (s, 1H), 2.65-2.60 (m, 2H), 2.32-2.28
2
1
3
(
m, 1H), 2.27-2.18 (m, 1H), 1.76-1.73 (m, 4H); C NMR (75 MHz, DMSO-d ) δ 155.7 (C),
6
1
1
53.3 (C), 153.0 (C), 151.9 (CH), 150.6 (C), 150.4 (C), 145.7 (C), 138.8 (CH), 129.0 (2 CH),
28.0 (CH), 127.9 (2 CH), 127.8 (C), 123.7 (C), 122.8 (C), 122.2 (CH), 121.9 (CH), 112.9
(
CH), 112.7 (CH), 98.4 (C), 35.2 (CH), 32.6 (CH ), 23.5 (CH ), 22.8 (CH ), 22.5 (CH ). Anal.
2 2 2 2
Calcd. for C H N O.H O: C, 75.55; H, 5.83; N, 10.57. Found: C, 75.26; H, 5.86; N, 10.57.
2
5
21
3
2
5
.1.2.2.
7-(2-Fluorophenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amine (2b).
Following the general method, reaction of compound 1b (0.5 g, 1.5 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.22 g, 2.2 mmol) and AlCl (0.29 g, 2.2 mmol), after 2 h, product
3
-1 1
2
b (0.56 g) was obtained: yield: 90%; mp >260 ºC; IR (KBr) νmax 3328, 1586 cm ; H NMR
(
300 MHz, DMSO-d ) δ 8.94 (d, 1H, J= 8.1 Hz), 8.29 (d, 1H, J= 7.8 Hz), 7.57-7.10 (m, 7H
6
arom), 5.75 (s, 1H), 5.46 (br s, 2H, NH ), 2.64-2.60 (m, 2H), 2.38-2.34 (m, 1H), 2.28-2.25 (m,
2
1
3
1
H), 1.76-1.72 (m, 4H); C NMR (100 MHz, DMSO-d ) δ 159.1 (C), 155.9 (C), 153.4 (C),
6
23

ACCEPTED MANUSCRIPT
1
1
51.8 (C), 150.4 (CH), 146.3 (C), 138.8 (C), 136.2 (CH), 131.6 (C), 130.6 (CH), 129.4 (CH),
28.3 (C), 127.3 (CH), 125.2 (CH), 122.6 (CH), 122.4 (CH), 121.6 (C), 116.5 (CH), 113.1
(
C), 97.0 (C), 35.2 (CH), 32.7 (CH ), 23.4 (CH ), 22.9 (CH ), 22.6 (CH ). Anal. Calcd. for
2 2 2 2
C H FN O.H O: C, 72.27; H, 5.34; N, 10.11. Found: C, 72.10; H, 5.23; N, 9.96.
25
20
3
2
5
.1.2.3.
7-(3-Fluorophenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amine (2c).
Following the general method, reaction of compound 1c (0.5 g, 1.5 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.22 g, 2.2 mmol) and AlCl (0.29 g, 2.2 mmol), after 2 h, product
3
-1
1
2
c (0.58 g) was obtained: yield: 93%; mp >260 ºC; IR (KBr) νmax 3331, 1592 cm ; H NMR
(
300 MHz, DMSO-d ) δ 8.95 (d, 1H, J= 8.3 Hz), 8.30 (d, 1H, J= 7.8 Hz), 7.57 (d, 2H, J= 8.1
6
Hz), 7.42-6.94 (m, 5Harom), 5.71 (br s, 2H, NH ), 5.59 (s, 1H), 3.56-3.50 (m, 2H), 2.36-2.32
2
13
(
m, 1H), 2.23-2.20 (m, 1H), 1.76-1.73 (m, 4H); C NMR (100 MHz, DMSO-d ) δ 163.8 (C),
6
1
61.4 (C), 155.9 (C), 153.4 (C), 151.9 (C), 150.5 (CH), 148.4 (C), 138.9 (C), 136.2 (CH),
31.1 (CH), 128.3 (C), 127.4 (CH), 124.0 (CH), 123.1 (C), 122.9 (CH), 122.4 (CH), 114.6
1
(
CH), 113.8 (CH), 113.8 (C), 98.1 (C), 32.7 (CH ), 23.4 (CH ), 22.9 (CH ), 22.6 (CH ). Anal.
2 2 2 2
Calcd. for C H FN O.H O: C, 72.27; H, 5.34; N, 10.11. Found: C, 71.94; H, 5.30; N, 9.98.
2
5
20
3
2
Calcd. for C H FN O.H O: C, 72.27; H, 5.34; N, 10.11. Found: C, 71.94; H, 5.30; N, 9.98.
2
5
20
3
2
5
.1.2.4.
7-(2-Bromophenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amine (2d).
Following the general method, reaction of compound 1d (0.5 g, 1.3 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.19 g, 1.95 mmol) and AlCl (0.25 g, 1.95 mmol), after 2 h,
3
-1
product 2d (0.60 g) was obtained: yield: 89%; mp >260 ºC; IR (KBr) νmax 3312, 1591 cm ;
1
H NMR (300 MHz, DMSO-d ) δ 8.95 (d, 1H, J= 8.1 Hz), 8.29 (d, 1H, J= 7.8 Hz), 7.59 (d,
6
2
H, J= 8.4 Hz), 7.62-7.13 (m, 5Harom), 5.83 (s, 1H), 5.16 (br s, 2H, NH ), 2.64-2.60 (m, 2H),
2
13
2
.31-2.28 (m, 1H), 2.28-2.19 (m, 1H), 1.77-1.73 (m, 4H); C NMR (100 MHz, DMSO-d ) δ
6
24

ACCEPTED MANUSCRIPT
1
1
55.5 (C), 153.8 (C), 151.7 (C), 150.5 (CH), 145.6 (C), 144.5 (C), 138.9 (C), 136.2 (CH),
33.3 (CH), 136.7 (CH), 129.7 (CH), 129.6 (CH), 128.4 (C), 122.9 (CH), 122.5 (CH), 122.0
(
C), 121.8 (C), 113.3 (C), 98.9 (C), 32.7 (CH ), 23.4 (CH ), 22.8 (CH ), 22.6 (CH ). Anal.
2 2 2 2
Calcd. for C H BrN O.H O: C, 63.03; H, 4.66; N, 8.82. Found: C, 62.85; H, 4.55; N, 8.85.
2
5
20
3
2
5
.1.2.5. 7-(4-Nitrophenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine
(2e).
Following the general method, reaction of compound 1e (0.5 g, 1.4 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.19 g, 1.96 mmol) and AlCl (0.26 g, 1.96 mmol), after 2 h,
3
-1 1
product 2e (0.55 g) was obtained: yield: 90%; mp >260 ºC; IR (KBr) νmax 3311, 1597 cm ; H
NMR (300 MHz, DMSO-d ) δ 8.95 (d, 1H, J= 8.4 Hz), 8.27 (d, 1H, J= 7.8 Hz), 7.90 (d, 2H
6
J= 8.4 Hz), 7.57-7.12 (m, 5Harom), 5.91 (s, 1H), 5.57 (br s, 2H, NH ), 2.66-2.62 (m, 2H),
2
1
3
2
1
.33-2.29 (m, 1H), 2.28-2.20 (m, 1H), 1.77-1.73 (m, 4H); C NMR (100 MHz, DMSO-d ) δ
6
55.7 (C), 154.0 (C), 152.0 (C), 150.7 (CH), 148.8 (C), 145.6 (C), 138.8 (C), 138.5 (C), 136.3
(CH), 134.6 (2 CH), 131.4 (CH), 129.6 (CH), 128.4 (C), 126.6 (CH), 124.3 (CH), 123.2 (CH),
1
22.7 (CH), 121.4 (C), 113.3 (C), 97.0 (C), 35.6 (CH), 32.7 (CH ), 23.4 (CH ), 22.8 (CH ),
2 2 2
2
1.5 (CH ). Anal. Calcd. for C H N O .H O: C, 67.86; H, 5.01; N, 12.66. Found: C, 68.02;
2
25 20
4
3
2
H, 4.88; N, 12.61.
.1.2.6. 7-(2,6-Dichlorophenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
5
amine (2f).
Following the general method, reaction of compound 1f (0.5 g, 1.3 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.19 g, 1.95 mmol) and AlCl (0.25 g, 1.95 mmol), after 2 h,
3
-1 1
product 2f (0.56 g) was obtained: yield: 92%; mp >260 ºC; IR (KBr) νmax 3332, 1590 cm ; H
NMR (300 MHz, DMSO-d ) δ 8.94 (d, 1H, J= 8.4 Hz), 8.29 (d, 1H, J= 7.8 Hz), 7.69 (d, 2H,
6
J= 8.4 Hz), 7.60-7.08 (m, 4Harom), 5.90 (s, 1H), 5.59 (br s, 2H, NH ), 2.65-2.61 (m, 2H),
2
13
2
.32-2.29 (m, 1H), 2.27-2.22 (m, 1H), 1.76-1.73 (m, 4H); C NMR (100 MHz, DMSO-d ) δ
6
25

ACCEPTED MANUSCRIPT
1
1
2
55.9 (C), 153.6 (C), 151.6 (C), 150.5 (CH), 146.8 (C), 138.4 (C), 137.7 (C), 130.7 (2xCH),
28.6 (2xC), 126.1 (CH), 122.5 (CH), 117.9 (C), 113.2 (C), 95.6 (C), 37.6 (CH), 32.6 (CH2),
3.3 (CH ), 22.8 (CH ), 22.6 (CH ). Anal. Calcd. for C H Cl N O.2H O: C, 61.99; H, 4.79;
2
2
2
25 19
2
3
2
N, 8.68. Found: C, 62.09; H, 4.64; N, 8.46.
.1.2.7. 7-(2-Methoxyphenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amine (2g).
5
Following the general method, reaction of compound 1g (0.5 g, 1.5 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.22 g, 2.25 mmol) and AlCl (0.29 g, 2.25 mmol), after 2 h,
3
-1 1
product 2g (0.57 g) was obtained: yield: 92%; mp >260 ºC; IR (KBr) νmax 3307, 1589 cm ; H
NMR (300 MHz, DMSO-d ) δ 8.94 (d, 1H, J= 8.3 Hz), 8.30 (d, 1H, J= 7.8 Hz), 7.56 (d, 2H,
6
J= 8.1 Hz), 7.27-6.82 (m, 5Harom), 5.73 (s, 1H), 5.36 (br s, 2H, NH ), 3.92 (s, 3H, OCH ),
2
3
3
2
.57-3.50 (m, 2H), 2.65-2.61 (m, 2H), 2.33-2.29 (m, 1H), 2.26-2.22 (m, 1H), 1.76-1.73 (m,
1
3
H); C NMR (75 MHz, DMSO-d ) δ 155.5 (C), 155.8 (C), 153.1 (C), 152.9 (C), 150.4
6
(
(
(
CH), 147.2 (C), 145.7 (C), 138.8 (C), 136.2 (CH), 130.2 (CH), 128.1 (C), 127.4 (CH), 123.8
C), 122.7 (CH), 122.6 (CH), 120.2 (CH), 114.5 (CH), 112.8 (C), 111.6 (CH), 98.4 (C), 53.2
OCH ), 32.5 (CH), 23.3 (CH ), 22.8 (CH ), 22.7 (CH ), 22.4 (CH ). Anal. Calcd. for
3
2
2
2
2
C H N O .2H O: C, 70.09; H, 6.11; N, 9.43. Found: C, 70.13; H, 5.91; N, 9.20.
26
23
3
2
2
5
.1.2.8.
7-(3-Methoxyphenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amine (2h).
Following the general method, reaction of compound 1h (0.5 g, 1.5 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.22 g, 2.25 mmol) and AlCl (0.29 g, 2.25 mmol), after 2 h,
3
-1
product 2h (0.57 g) was obtained: yield: 93%; mp >260 ºC; IR (KBr) νmax 3327, 1596 cm ;
1
H NMR (300 MHz, DMSO-d ) δ 8.94 (d, 1H, J= 8.3 Hz), 8.30 (d, 1H, J= 7.8 Hz), 7.58 (m,
6
2
H), 7.39 (d, 1H, J= 7.8 Hz), 7.16 (d, 2H, J= 8.9 Hz), 6.71 (d, 2H, J= 8.7 Hz), 5.62 (br s, 2H,
NH ), 5.49 (s, 1H), 3.68 (s, 3H, OCH ), 2.64-2.60 (m, 2H), 2.54-2.50 (m, 2H), 2.35-2.31(m,
2
3
26

ACCEPTED MANUSCRIPT
1
3
1
H), 2.29-2.26 (m, 1H), 1.76-1.73 (m, 2H); C NMR (75 MHz, DMSO-d ) δ 159.7 (C), 155.8
6
(
C), 153.1 (C), 151.8 (C), 150.4 (CH), 147.2 (C), 145.7 (C), 138.8 (C), 136.2 (CH), 130.2
CH), 128.1 (C), 127.4 (CH), 123.5 (C), 122.7 (CH), 122.2 (CH), 120.2 (CH), 114.5 (CH),
(
1
12.8 (C), 111.6 (CH), 98.3 (C), 55.4 (OCH ), 32.6 (CH), 23.4 (CH ), 22.8 (CH ), 22.7
3 2 2
(
CH ), 22.6 (CH ). Anal. Calcd. for C H N O .2H O: C, 70.09; H, 6.11; N, 9.43. Found: C,
2 2 26 23 3 2 2
7
5
0.20; H, 6.02; N, 9.39.
.1.2.9. 7-(4-Methoxyphenyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-
amine (2i).
Following the general method, reaction of compound 1i (0.5 g, 1.5 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.22 g, 2.25 mmol) and AlCl (0.29 g, 2.25 mmol), after 2 h,
3
-1 1
product 2i (0.55 g) was obtained: yield: 89%; mp >260 ºC; IR (KBr) νmax 3336, 1592 cm ; H
NMR (300 MHz, DMSO-d ) δ 8.93 (d, 1H, J= 8.1 Hz), 8.28 (d, 1H, J= 7.8 Hz), 7.57 (m, 2H),
6
7
.36 (d, 1H, J= 7.8 Hz), 7.25 (d, 2H, J= 8.9 Hz), 6.80 (d, 2H, J= 8.7 Hz), 5.58 (br s, 2H,
NH ), 5.46 (s, 1H), 3.64 (s, 3H, OCH ), 2.65-2.60 (m, 2H), 2.54-2.51 (m, 2H), 2.44-2.39 (m,
2
3
1
3
1
H), 2.36-2.32 (m, 1H), 1.78-1.75 (m, 2H); C NMR (75 MHz, DMSO-d ) δ 158.3 (C), 155.7
6
(
C), 152.9 (C), 151.8 (C), 150.3 (C), 145.6 (CH), 138.8 (C), 136.2 (CH), 131.8 (C), 128.8 (2
CH), 128.0 (C), 127.6 (2 CH), 123.9 (C), 123.4 (C), 122.6 (CH), 114.3 (C), 112.8 (CH), 98.6
C), 55.4 (OCH ), 32.6 (CH), 26.6 (CH ), 23.5 (CH ), 23.1 (CH ), 22.8 (CH ). Anal. Calcd.
(
3
2
2
2
2
for C H N O .H O: C, 73.05; H, 5.89; N, 9.83. Found: C, 73.19; H, 5.97; N, 9.60.
2
6
23
3
2
2
5
.1.2.10. 7-(p-Tolyl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine (2j).
Following the general method, reaction of compound 1j (0.5 g, 1.6 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.23 g, 2.4 mmol) and AlCl (0.31 g, 2.4 mmol), after 2 h, product
3
-1 1
2
j (0.56 g) was obtained: yield: 90%; mp >260 ºC; IR (KBr) νmax 3300, 1597 cm ; H NMR
(
300 MHz, DMSO-d ) δ 8.93 (d, 1H, J = 8.2 Hz), 8.28 (d, 1H, J= 7.8 Hz), 7.55 (m, 2H), 7.35
6
(d, 1H, J= 7.8 Hz), 7.24 (d, 2H, J= 8.9 Hz), 7.02 (d, 2H, J= 8.7 Hz), 5.57 (br s, 2H, NH2),
27

ACCEPTED MANUSCRIPT
5
.47 (s, 1H), 2.64-2.61 (m, 2H), 2.53-2.50 (m, 2H), 2.43-2.39 (m, 1H), 2.36-2.33 (m, 1H),
1
3
2
.16 (s, 3H, CH ), 1.76-1.72 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 155.7 (C), 153.0
3
6
(
C), 151.8 (C), 150.3 (CH), 145.6 (C), 142.8 (C), 138.8 (C), 136.2 (C), 131.8 (CH), 129.6
CH), 128.0 (2 CH), 127.8 (C), 127.5 (2 CH), 123.7 (C), 122.6 (CH), 122.2 (CH), 112.8 (C),
(
9
8.6 (C), 39.8 (CH), 32.6 (CH ), 23.4 (CH ), 22.8 (CH ), 22.6 (CH ), 20.9 (CH ). Anal.
2 2 2 2 3
Calcd. for C H N O.H O: C, 75.89; H, 6.12; N, 10.21. Found: C, 76.02; H, 5.96; N, 10.07.
2
6
23
3
2
5
.1.2.11. 7-(Pyridin-4-yl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-8-amine
(2k).
Following the general method, reaction of compound 1k (0.5 g, 1.3 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.19 g, 1.95 mmol) and AlCl (0.25 g, 1.95 mmol), after 2 h,
3
-1
product 2k (0.55 g) was obtained: yield: 87%; mp >260 ºC; IR (KBr) νmax 3302, 1591 cm ;
1
H NMR (300 MHz, DMSO-d ) δ 8.93 (d, 1H, J= 8.1 Hz), 8.27 (d, 1H, J= 7.8 Hz), 7.67 (d,
6
2
2
1
1
H, J= 8.4 Hz), 7.62-7.07 (m, 5Harom), 5.92 (s, 1H), 5.59 (s, 2H, NH ), 2.64-2.61 (m, 2H),
2
1
3
.32-2.29 (m, 1H), 2.29-2.24 (m, 1H), 1.76-1.72 (m, 4H); C NMR (75 MHz, DMSO-d ) δ
6
55.8 (C), 153.7 (C), 153.5 (C), 152.0 (C), 150.6 (CH), 150.3 (C), 148.6 (C), 147.2 (CH),
46.1 (C), 138.7 (CH), 136.3 (CH), 128.3 (2 CH), 123.1 (2 CH), 122.5 (C), 122.2 (C), 97.1
(
C), 38.1 (CH), 32.6 (CH ), 23.5 (CH ), 22.8 (CH ), 22.5 (CH ). Anal. Calcd. for
2 2 2 2
C H N O.H O: C, 72.34; H, 5.57; N, 14.06. Found: C, 72.59; H, 5.38; N, 13.88.
24
20
4
2
5
8
.1.2.12. 7-(2-Bromopyridin-3-yl)-9,10,11,12-tetrahydro-7H-pyrano[2,3-b:5,6-h']diquinolin-
-amine (2l).
Following the general method, reaction of compound 1l (0.5 g, 1.3 mmol) in 1,4-dioxane (30
mL) with cyclohexanone (0.19 g, 1.95 mmol) and AlCl (0.25 g, 1.95 mmol), after 2 h,
3
-1 1
product 2l (0.49 g) was obtained: yield: 81%; mp >260 ºC; IR (KBr) νmax 3317, 1597 cm ; H
NMR (300 MHz, DMSO-d ) δ 8.96 (d, 1H, J= 8.1 Hz), 8.31 (d, 1H, J= 7.8 Hz), 7.60 (d, 2H,
6
J= 8.4 Hz), 7.39-6.92 (m, 4Harom), 5.84 (s, 1H), 5.25 (br s, 2H, NH ), 2.39-2.36 (m, 2H),
2
28