New multipotent tetracyclic tacrines with neuroprotective activity

Article abstract of DOI:10.1016/j.bmc.2006.09.025

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5-13) are described. Compounds 7, 8 and 11 sh

Full text of DOI:10.1016/j.bmc.2006.09.025

Bioorganic & Medicinal Chemistry 14 (2006) 8176–8185
New multipotent tetracyclic tacrines with neuroprotective activity
a,
a,b,
a,b
´ ´ ´
Cristobal de los Rıos, Antonio G. Garcıa,
b,c
*
*
´
´
´
Jose Marco-Contelles, Rafael Leon,
b
b,
*
Manuela G. Lopez and Mercedes Villarroya
aLaboratorio de Radicales Libres (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
bInstituto Teo´filo Hernando, Departamento de Farmacologı´a y Terape´utica, Facultad de Medicina, Universidad Auto´noma de Madrid,
C/Arzobispo Morcillo 4, 28029 Madrid, Spain
cServicio de Farmacologı´a Clinica, Hospital Universitario de la Princesa, c/Diego de Leo´n 62, 28006 Madrid, Spain
Received 8 May 2006; revised 1 September 2006; accepted 13 September 2006
Available online 26 September 2006
Abstract—The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/
BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5–13) are described. Com-
pounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca2+ overload or free radical
induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but
proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement exper-
iments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential
agents that can prevent the aggregation of b-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient
neuroprotective agent against 70 mM K+ and 60 lM H2O2. Based on these results, some of these molecules can be considered as
lead candidates for the further development of anti-Alzheimer drugs.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
the most widely studied and effective clinical approach to
treat AD.5 Cholinesterase inhibitors (AChEI), such as
donepezil, rivastigmine and galantamine, have been ap-
proved by the FDA and EMEA for treating the symp-
toms of AD.6 On the other hand, the multifactorial
pathogenesis of AD suggests that drug treatments with
two or more mechanisms of action, acting in a comple-
mentary manner, could be more efficacious to patients
suffering the disease. In fact, galantamine, a drug cur-
rently used to treat AD, is a mild inhibitor of AChE that
sensitizes the nicotinic receptor to ACh7 and prevents cell
death induced by amyloid b (Ab) or thapsigargin.8,9
Alzheimer’s disease (AD)1 is the most frequent of the
primary degenerative dementias. This disease is thought
to be rising dramatically during the next years due to in-
creased longevity of the population. Hallmarks of AD
are different histo-pathological alterations like marked
atrophy of the cerebral cortex with loss of cortical and
subcortical neurons, formation of senile plaques by
accumulation of the amyloid b (Ab) peptide and
neurofibrillary tangles composed by pairs of helical fila-
ments of hyperphosphorylated s protein and neuritic
degeneration.
Tacrine, a potent and reversible AChE inhibitor,10 was
the first drug approved in the USA for the palliative
treatment of AD; however, it exhibited side effects like
hepatotoxicity.11 Current research is focused on devel-
oping new AChE inhibitors with improved activity
and reduced adverse side effects. The strategy leading
to multipotent anti-Alzheimer drugs has given excellent
results and has paved the way for future and more suc-
cessful developments.12
The neuropsychiatric symptoms associated with AD are
related to the reduction in the number of functional neu-
ronal nicotinic receptors (nAChR)2,3 which correlates
with the severity of the disease at the time of death.4 Dur-
ing the past decade, cholinesterase inhibition has become
Keywords: Tetracyclic tacrines; AChE; BuChE; Neuroprotection;
Voltage dependent calcium channels (VDCC); Alzheimer‘s disease;
Multipotent drugs.
*
In this context, and based on the fact that a sustained
elevation of the cytosolic concentration of Ca2+ ions,
Corresponding authors. Tel.: +34 91 5622900; fax: +34 91 5644853
(J.M.C.); e-mail: iqoc21@iqog.csic.es
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.025

J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
8177
X
OMe
NH2
NH2
NH2
N
4
EtO2C
Me
EtO2C
Me
4
N
N
N
O
Tacrine (3)
1 X= F (IC50= 710 nM)
4 X= H (IC50= 820 nM)
2 (IC50= 868 nM)
Figure 1.
leading to Ca2+ overload, seems to play a crucial role in
neuronal degeneration and death,13 we embarked on a
long-term research project aimed at the synthesis of a
series of multipotent compounds designed to target
AChE and neuronal Ca2+ modulation.14 As a result,
we have synthesized and evaluated a number of hybrid
compounds, such as 1 and 2 (Fig. 1), which combine
the tetrahydroaminoquinoline moiety present in tacrine
(3) (Fig. 1) with a pyridine or a 4H-pyran bearing a sub-
stitution pattern similar to that found in the isosteric
1,4-dihydropyridines, well-known calcium channel
blockers.14 Compounds 1 and 2 were less potent as
AChEIs than tacrine (IC50 = 180 nM, using TcA-
ChE),14b,14c but they blocked voltage-dependent Ca2+
channels (38% and 50% inhibition, respectively).14f Fur-
thermore, compounds 1 and 2 also blocked Ca2+ uptake
induced by nicotinic stimulation (63% and 90% inhibi-
tion, respectively) in bovine chromaffin cells, more effi-
ciently than tacrine (17% inhibition of Ca2+ channels
and 55% inhibition of the Ca2+ uptake induced by nico-
tinic stimulation).14f
hydrogen peroxide or Ab in bovine chromaffin or
human neuroblastoma cells;14f these properties make
of compound 4 an interesting candidate with potential
therapeutic effect in Alzheimer’s disease. The neuropro-
tective effect of compound 4 is related to a mild and sus-
tained increase in the cytosolic levels of Ca2+ which, in
turn, has been shown to induce the expression of pro-
teins implicated in cell survival such as those of the anti-
apoptotic Bcl-2 family.
Based on these previous results, and continuing with our
current project directed to the development of new anti-
Alzheimer multipotent drugs, in this paper we describe
the synthesis and pharmacological study of the new
tetracyclic tacrine derivatives 5–13 (Fig. 2). Being struc-
turally similar to compounds 1, 2 or 4, the synthesis and
biological evaluation of compounds 5–13 is the first step
on our projected structure activity relationship (SAR)
analysis, directed to define the key structural and func-
tional elements that might improve the pharmacological
profile of these molecules. The selection of compounds
5–13 was mainly based on the presumed ability of these
molecules to bind the PAS of AChE, as these substrates
retain the basic core present on molecules 1 or 2. Com-
paring with the lead compounds, we hypothesized that
the incorporation of a fourth cyclohexane ring, fused
at the 4H-pyran heterocyclic moiety, should afford more
rigid molecules, giving possibly stronger p–p interac-
tions between the pyridine nucleus present in compounds
5–13 with Trp 279 in the PAS of AChE. In order to eval-
uate the effect of the aromatic substituents we have
located electron-withdrawing (F, CF3, and NO2) and
electron-donating (Me, OMe) groups at different avail-
able positions (C-2, C-3, and C-4) in the aromatic ring.
Molecular modelling studies, carried out by Prof. Luque
and associates, suggested that binding of these AChE
inhibitors to the catalytic centre of the enzyme might
be hampered by unfavourable steric interactions, mainly
due to the aromatic ring moiety installed at C-4; conse-
quently, it was proposed that these tacrine analogues
might interact with Trp279 binding at the PAS of the en-
zyme.14e We have confirmed this hypothesis in our lab-
oratory as shown by the propidium displacement
experiments. Therefore, these drugs have the ability to
bind to both the active and peripheral sites of AChE,
and could prevent the aggregation of Ab8,9 besides
increasing cholinergic neurotransmission.
While Ca2+ overload plays a crucial role in causing cell
death, a mild and sustained elevation of the cytosolic
concentration of Ca2+ as a result of moderate chronic
depolarization, has been shown to increase the survival
of different neuronal types.15–17 Our group has devel-
oped compound 4 (Fig. 1), as a novel compound that
combines both an AChE inhibitory activity and a mod-
erate Ca2+ promotor action, which leads to protection
against a variety of toxic stimuli such as Ca2+ overload
induced by veratridine, endoplasmic reticulum stress
elicited by thapsigargin and free radical generation by
X
O
NH2
N
Me
O
Me
5-13 X= H, F, CF3, Me, NO2, OMe
Figure 2.

8178
J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
Finally, the ready availability of 5,5-dimethyl-1,3.cyclo-
hexadione (‘dimedone’), the starting material for the
presumed synthetic sequence, was an added bonus of
interest in order to develop a simple and good yielding
approach to the desired target molecules.
5–13 in good yields (53–96%) (see Section 4). These prod-
ucts gave satisfactory analytical and spectroscopic data
in good agreement with their structures. Specific NMR
experiments (1H, 13C, DEPT, 1H–1H COSY, HMQC
and HMBC) allowed us to assign the corresponding sig-
nals to the respective protons and carbons.
2. Results and discussion
2.1. Chemistry
With these compounds in hand, we tested them in sever-
al pharmacological assays.
2.2. Pharmacology
According to the standard methodology for the synthesis
of tacrines,14 the new tetracyclic derivatives could also be
easily prepared by the AlCl3 promoted Friedla¨nder reac-
tion18 between the corresponding, known and polyfunc-
tionalized 4H-chromenes19 14–22 and cyclohexanone
(23) (Scheme 1). The 4H-chromenes 14–22 are well-de-
scribed intermediates,20 which were obtained uneventful-
ly from commercially and readily available aldehydes 24,
25 and 27–32, respectively (Scheme 1). Only 4H-chro-
mene 16, with a trifluoromethylphenyl group attached
to carbon C-4, has not been reported before, but we were
able to synthesize it in a ‘one-pot’ reaction protocol start-
ing from aldehyde 26, after a piperidine catalyzed Knoe-
venagel reaction with malonitrile (33), giving the
corresponding, not isolated, arylidenemalononitrile
intermediate (A), followed by reaction with dimedone
(34)20a (Scheme 1) (see Section 4). As expected, interme-
diates 14–22, on treatment with cyclohexanone (23), in
the presence of aluminium trichloride, in 1,2-dicholore-
thane as solvent, at reflux, afforded the target molecules
AChE inhibition by the currently used drugs in the
treatment of AD improves the symptoms,21 but does
not delay the neuronal loss that is characteristic of this
illness. As mentioned in Section 1, treatment strategies
with drugs holding more than one mechanism of action,
acting in a complementary manner, might be more ade-
quate on patients suffering the disease. One of these pos-
sible mechanisms might be neuroprotection. Recently,
several preclinical studies have shown that clinical useful
AChEI, such as galantamine22–24 and donepezil,25,26
display neuroprotective properties.
Therefore, we decided to assess the potential neuropro-
tective effect of the new compounds on SH-SY5Y cells
exposed during 24 h to a medium with a depolarizing
concentration of KCl (70 mM) which induces a Ca2+
overload and the consequent cell death. Drugs, at the
concentration of 0.3 lM, were administered 24 h before
the incubation of cells with high K+ (70 mM; hypertonic)
O
X
X
Me
O
Me
CN
Piperidine, MeOH
rt, 10-20 min
+
CN
CN
34
CN
33
O
H
24-32
A
(X= H, F, CF3,
Me, NO2, OMe)
X
X
O
O
(23)
O
NH2
N
CN
NH2
4
ClCH2CH2Cl, AlCl3
reflux, 10-24 h
Me
O
Me
O
Me
Me
14 X= H
15 X= p-F
5 X= H
6 X= p-F
16 X= o-CF3
17 X= m-NO2
18 X= p-NO2
19 X= p-Me
20 X= o-OMe
21 X= m-OMe
22 X= p-OMe
7 X= -CF
o
3
8 X= m-NO2
9 X= -NO
p
2
10 X= p-Me
11 X= o-OMe
12 X= m-OMe
13 X= p-OMe
Scheme 1.

J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
8179
and maintained during the entire experiment. Thereafter,
release of lactic dehydrogenase (LDH) was measured as
a parameter of cell death, since this enzyme is released to
the extracellular medium as a consequence of cell death.
Basal release of LDH by the cells was subtracted from
the values obtained after incubation with 70 mM K+ in
the presence or absence of the compounds.
SY5Y neuroblastoma cells exposed to 60 lM H2O2 for
24 h.
Compounds 5–13 and their synthetic precursors 2-
amino-4-aryl-3-cyano-5,6,7,8-tetrahydro-7,7-dimethyl-
5-oxo-4H-chromenes (14–22) were evaluated. Results
are shown in Table 2. Fourteen of the total compounds
studied manifested a statistically significant neuropro-
tective effect against H2O2 toxicity, the most potent of
them were compounds 12, 19 and 6 with a 61.3, 55.9
and 54.2% protection, respectively. In general, for the
protection observed against free radicals generated from
H2O2, we have observed that the trend was reversed;
compounds 5–13 were more efficient than their synthetic
precursors 14–22, all of them showing a good neuropro-
tective profile.
Compounds 5–13 and their synthetic precursors 2-ami-
no-4-aryl-3-cyano-5,6,7,8-tetrahydro-7,7-dimethyl-5-
oxo-4H-chromenes (14–22) were tested. The results are
shown in Table 1. All compounds showed a statistically
significant neuroprotective effect at the assayed concen-
tration. Note that tacrine (3), also investigated for com-
parative purposes, showed the poorest (13%) protection.
Compounds 5–13 exhibited protection values in the
range 35–44.8%, the most potent being compounds 5
and 12. It is worth mentioning that precursors 14–22 evi-
denced average higher values than compounds 5–13; in
the range 32.5–61.6%, the most potent was compound
17, bearing a 3-nitrophenylgroup at C-4. Since products
14–22 are inactive towards AChE, we conclude that the
anticholinesterase activity may not display a clear rela-
tionship with the neuroprotective action.
In summary, from these assays we conclude that our
new molecules (5–13) are quite promising from the neu-
roprotection point of view.
Since a significant cytoprotection was achieved with all
compounds against Ca2+ overload induced by 70 mM
K+ (Table 1), next we decided to study if these com-
pounds were blocking voltage-dependent Ca2+ channels
(VDCC) and could therefore explain why they were pre-
venting excess Ca2+ entry into the cells.
Oxidative damage also appears to play an important
role in the progressive neuronal death characteristic in
AD.27 Thus, we considered convenient to study the pos-
sible neuroprotective effect of our compounds on SH-
Neuroblastoma cells previously loaded with the fluores-
cence dye Fluo-4 were incubated in the presence of the
Table 1. Cell viability expressed as reduction in the increase of LDH
released in the presence of 70 mM K+a
Table 2. Cell viability expressed as reduction in the increase of LDH
a
released in the presence of 60 lM H2O2
Compound (0.3 lM)
X
LDH release
(% vehicle)
% protection
Compound (0.3 lM)
X
LDH release
(% vehicle)
% protection
Vehicle
Tacrine (3)
Galantamine
100
—
13.4 7.2
34.80
Vehicle
Tacrine (3)
Galantamine
100
—
0.8 4.1
14.90
90.4 4.1
75.8 2.1
63.2 4.3
65.5 1.9
76.3 2.8
57 1.8
98.0 1.80
89.9 2.3
79.6 2.6
75.5 2.2
87.5 3.7
66.5 2.6
64.3 1,5
56.2 1.3
82.8 3.1
83.9 3.6
77.7 2.6
69.0 8.2
57,87 1.57
85.6 4.1
67.7 1.5
65.5 1.4
87.4 2.9
65.6 2.6
52.2 1.9
83.8 2.5
14
15
16
17
18
19
20
21
22
5
H
4-F
50.3 2.2
48.9 2.1
32.5 3.3
14
15
16
17
18
19
20
21
22
5
H
4-F
28.2 3.8
34.9 2.9
26.2 2.8
49 3.9
53.4 3.9
55.9 1.1
27.5 3.5
27.3 3.5
2-CF3
3-NO2
4-NO2
4-Me
2-OMe
3-OMe
4-OMe
H
2-CF3
3-NO2
4-NO2
4-Me
2-OMe
3-OMe
4-OMe
H
61.6
3
67.4 1.2
62.4 1.4
65.7 2.6
65.1 1.8
64 4.2
47.3 3.2
55.6 4.7
47.3 2.5
49.5 2.6
49.9 5.5
44.8 4,5
42.3 6.6
40.0 3.3
36.4 5.7
39.4 5.8
38.8 1.7
38.9 3.9
43.1 4.3
31.7
3
63.7 2,34
65.2 3.5
69.1 2.8
70.5 3.12
68.5 2.9
70.3 1.5
70 2.4
39.8 2.1
54.2 2.0
19.4 3.2
41.4 1.8
43.8 1.9
15.7 2.9
44.8 2.3
61.3 2.14
20.9 2.9
6
4-F
6
4-F
7
2-CF3
3-NO2
4-NO2
4-Me
2-OMe
3-OMe
4-OMe
7
2-CF3
3-NO2
4-NO2
4-Me
2-OMe
3-OMe
4-OMe
8
8
9
9
10
11
12
13
10
11
12
13
65,7 1.8
75.6 1.7
35
2
a Data are expressed as means SEM of at least three different cultures
in quadruplicate. LDH released was calculated for each individual
experiment considering 100% the extracellular LDH released in the
presence of vehicle with respect to the total. To calculate % protec-
tion, LDH release was normalized as follows: in each individual
triplicate experiment, LDH release obtained in non-treated cells
(basal) was subtracted from the LDH released upon 70 mM K+
treatment and normalized to 100% and that value was subtracted
from 100.
a Data are expressed as means SEM of at least three different cultures
in quadruplicate. LDH released was calculated for each individual
experiment considering 100% the extracellular LDH released in the
presence of vehicle with respect to the total. To calculate % protec-
tion, LDH release was normalized as follows: in each individual
triplicate experiment, LDH release obtained in non-treated cells
(basal) was subtracted from the LDH released upon H2O2 treatment
and normalized to 100% and that value was subtracted from 100.

8180
J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
Table 3. Effect of the different compounds on the [Ca2+
]
increase
c
with an electron-withdrawing group (NO2) at C-3 is
the second most active AChE inhibitor in this series of
compounds. Despite the electronic properties of the aro-
matic substituent, the order of the inhibitory activity
increases on going from substituents at position C-4 to
C-2, the most active being those with these groups at
C-2 position. Also, a good direct correlation was found
between the electron-donating power of the substituent
and the activity of the compound (compare compound
10 with a Me group at C-4, and compound 13 with a
OMe group at C-4). The same trend is observed for
the electron-withdrawing groups (compare compound
6 with 7, bearing a fluorine at C-4, and trifluoromethyl
at C-2, respectively).
elicited by 70 mM K+ in SH-SY5Y cells (% inhibition with respect to a
control without any drug)a
Compound (0.3 lM)
X
% inhibition
14
15
16
17
18
19
20
21
22
5
H
4-F
24.6 4.2
18.5 5.6
32.7 3.0
31.5 4.8
21.3 4.8
28.7 7.0
27.3 3.8
33.2 5.6
31.2 4.0
35.0 3,5
36,5 4.0
43.1 4.8
30.0 4.0
32.0 3.0
25.5 4.0
31.9 4.5
43,5 4.0
32.5 5.0
2-CF3
3-NO2
4-NO2
4-Me
2-OMe
3-OMe
4-OMe
H
6
4-F
7
2-CF3
3-NO2
4-NO2
4-Me
2-OMe
3-OMe
4-OMe
The compounds 5–13 were also assayed to determine its
activity on butyrylcholinesterase, following Ellman’s
method.29 Only two of them, compounds 7 and 11,
inhibited butyrylcholinesterase with IC50s of 2.2 and
8.7 lM, respectively. For the rest of the compounds
the maximal concentration assayed, 100 lM, did not
produce an inhibition sufficient to calculate an IC50.
8
9
10
11
12
13
All compounds were tested at the concentration of 0.3 lM.
a Data are expressed as means SEM of at least three different cultures
in quadruplicate.
Comparing with the parent compound 2 (Fig. 1), the
corresponding new tetracyclic analogue 13 is 50 fold less
active, suggesting that, at least for a 4-OMe substituent
at the aromatic ring, the newly incorporated saturated
cyclohexane ring is deleterious for the AChE inhibitory
activity. We have also investigated the AChE/BuChE
inhibitory activity of precursors 2-amino-4-aryl-3-cya-
no-5,6,7,8-tetrahydro-7,7-dimethyl-5-oxo-4H-chromenes
14–22, but no significant activity was observed. In sum-
mary, compounds 5–13 are AChE inhibitors revealing
modest potency but, with the exception of compounds
7 and 11, high selectivity towards AChE, as they were,
in general, devoid of any inhibitory activity regarding
BuChE.
compounds for 10 min and then stimulated with a con-
centrated solution of KCl so that the final concentration
in the medium was 70 mM K+. Changes in fluorescence
as a consequence of the [Ca2+]c increase elicited by high
K+ concentrations were measured in a fluorescence plate
reader FluoStar Optima (BMG).
Compounds 5–13 and their synthetic precursors 2-ami-
no-4-aryl-3-cyano-5,6,7,8-tetrahydro-7,7-dimethyl-5-
oxo-4H-chromenes (14–22) were tested. The results are
shown in Table 3. All compounds induced a statistically
significant inhibition of the [Ca2+]c increase elicited by
70 mM K+ in neuroblastoma cells, with values ranging
from 18% to 43%, the most potent being compounds
12 (43.5%) and 7 (43.1%), showing similar values to
the one observed for compound 2 (50%) (Fig. 1). These
results clearly point out that our selection of compounds
was correct, and that the new molecules still block Ca2+
channels efficiently. These results suggest that our com-
pounds could be blocking VDCC and thus preventing
Ca2+ overload in neuroblastoma cells.
As stated in Section 1, one of the hallmarks of AD lies in
the formation of senile plaques by accumulation of the
Ab peptide. Different studies have proven that AChE
binds to Ab inducing the formation of fibrils30 and the
PAS of the enzyme is involved in the adhesion func-
tion.31 Ligands, like propidium iodide, that bind to the
PAS are known to block Ab aggregation.31a Thus, we
were interested in investigating whether some of our
compounds (5–13) might be able to bind to the PAS, a
fact that could make them good candidates for the
development of new anti Ab drugs.
Finally, and in view of these results, compounds 5–13
have been evaluated as possible AChEI according to
standard methodology28 (see Section 4).
A propidium competition assay was accomplished for
those compounds which inhibited AChE with an
IC50 < 100 lM at the concentration of 0.3 lM. Propidi-
um, a selective ligand for the PAS of AChE, exhibits an
increase in the fluorescence upon binding to that site.32
A decrease in propidium fluorescence in the presence
of the compounds could be interpreted as a displace-
ment of propidium from the PAS.
In Table 4 we present the IC50 values corresponding to
those compounds. As shown and as expected, all the
compounds inhibit AChE, but comparing with tacrine
(3) (IC50 0.14 lM), the newly synthesized compounds
manifest lower activity, the most active being compound
11 (IC50 7.0 lM) (around 50-fold less active) which has
an electron-donating group (OMe) at C-2. From the
SAR results, in general, compounds (10–13) with elec-
tron-donating groups (Me, OMe) are more active than
those with electron-withdrawing functionalities (F,
CF3 and NO2) (6–9). Note, however, that compound 8
As shown in Table 4, at the concentration of 0.3 lM,
compounds 5–13 showed consistent and significant abil-
ity to displace propidium from the PAS of AChE. These
results clearly confirm our initial hypothesis regarding

J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
8181
Table 4. Inhibition of AChE (IC50 in lM), and displacement of
propidium iodide from the peripheral anionic site, measured as the
inhibition of the fluorescence of propidium bound to AChE, by the
new compounds 5–13
their moderate activity as AChE inhibitors would cause
a more selective interaction with the peripheral site of
the enzyme. In fact, our new compounds displace propi-
dium from the PAS with values around 40% for com-
pounds 6 or 10. This could be an indication of a
possible antiagregant effect of Ab.
Compound
X
AChE inhibition Propidium displacement
(% inhibition at 0.3 lMa)
IC50 (lM)
Tacrine (3)
—
0.14
48.6
69.7
3.9 1.8
17.4 2.6
40 4.1
5
6
H
4-F
A good candidate for further pharmacological studies
could be compound 8 that has the following pharmaco-
logical profile: (1) IC50 to block AChE 11.9 lM; (2) dis-
placement of propidium from the AChE peripheral site,
24% at 0.3 lM; (3) neuroprotection against Ca2+ over-
load, 36% at 0.3 lM; (4) neuroprotection against H2O2,
41% at 0.3 lM; and (5) blockade of Ca2+ entry through
Ca2+ channels, 30% at 0.3 lM. Full concentration-re-
sponse curves against those parameters can allow the pre-
diction of EC50 values of around 1–10 lM, not far away
from the AChE IC50 value. Hence, a nice possibility is
that compound 8 (or some related compounds of those
reported here) could have a polyfunctional pharmacolog-
ical profile that causes: (i) an improvement of cognitive
symptoms by inhibiting AChE and improving cholinergic
neurotransmission; (ii) a neuroprotective effect against
Ca2+ overloading and free radical generation, thus delay-
ing apoptosis and neuronal death and (iii) a prevention of
Ab deposition and the formation of senile plaques, by act-
ing on the AChE peripheric site. These are qualities to be
expected to benefit Alzheimer disease patients by improv-
ing cognition and delaying the progression of the disease.
7
2-CF3 17.8
3-NO2 11.9
4-NO2 94.1
38.4 3.9
23.9 3.4
22.3 3.8
35.4 3.6
32.1 3.9
28.2 2.7
39 4.7
8
9
10
11
12
13
4-Me
51.6
2-OMe 7.0
3-OMe 21.5
4-OMe 40.5
a Data are expressed as means SEM of at least three different
experiments in quadruplicate.
the site where these inhibitors are interacting with
AChE. First of all, the most active compound was the
fluorinated derivative 6, with a 40% inhibition. Both
compounds 6 (69.7 lM) and 10 (51.6 lM) showed high-
er IC50 for AChE than compound 7 (Table 1). Thus,
compounds 6 and 10 are almost 10- and 7-fold, respec-
tively, less active than inhibitor 11. Although the ability
to displace propidium did not follow the increasing or-
der of AChE inhibitory activity,33 two of the most po-
tent AChE inhibitors (11 and 7) showed high
inhibition values, around 32.1% and 38.4%, respectively.
In summary, these results reveal that compounds 5–13
are weaker and more selective AChE inhibitors than
the parent compounds 1, 2 or 4 (Fig. 1) but, interesting-
ly, they still bind significantly the enzyme at the PAS,
confirming that the proposed structural modification
of compounds 1, 2 or 4 leading to the new substrates
was a correct choice.
4. Experimental
4.1. General methods
Reactions were monitored by TLC using precoated sili-
ca gel aluminium plates containing a fluorescent indica-
tor (Merck, 5539). Detection was done by UV (254 nm)
followed by charring with sulfuric–acetic acid spray, 1%
aqueous potassium permanganate solution or 0.5%
phosphomolybdic acid in 95% EtOH. Anhydrous
Na2SO4 was used to dry organic solutions during
work-ups and the removal of solvents was carried out
under vacuum with a rotary evaporator. Flash chroma-
tography was performed using silica gel 60 (230–
400 mesh, Merck). Melting points were determined on
a Kofler block and are uncorrected. IR spectra were ob-
tained on a Perkin-Elmer Spectrum One spectropho-
tometer. 1H NMR spectra were recorded with a
Varian VXR-200S spectrometer, using tetramethylsilane
as internal standard and 13C NMR spectra were record-
ed with a Bruker WP-200-SY. All the assignments for
protons and carbons were in agreement with 2D COSY,
gHSQC, gHMBC and 1D NOESY spectra. Values with
3. Conclusions
To summarize, we have reported the synthesis and biolog-
ical evaluation including the neuroprotective properties,
voltage-dependent calcium channel blockade, AChE/
BuChE inhibitory activities and propidium binding to
the AChE PAS of new tetracyclic tacrines (5–13). Regard-
ing AChE inhibition, none of the assayed compounds
manifested higher activity than tacrine (IC50 0.14 mM),
showing just moderate activity, the most potent com-
pound (7) being 50-fold less active than tacrine. The
majority of these compounds are extremely selective for
AChE as they were devoid of any inhibitory activity
regarding BuChE. From SAR, we have observed that
compounds 5–13, bearing electron-donating substituents
in the aromatic ring, are more active than those substitut-
ed with electron-withdrawing groups; the activity increas-
es from carbon 4 to carbon 3 and to carbon 2 in the
aromatic nucleus. In addition, the stronger this electronic
property was, the more active the compounds were.
( ) can be interchanged. Elemental analyses were con-
*
ducted on a Carlo Erba EA 1108 apparatus.
4.2. General method for the synthesis of 2-amino-4-
aryl-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-
chromenes
In our opinion, the main interest of these new com-
pounds lies in their neuroprotective capacity against
Ca2+ overload or free radical generation. In addition,
To a solution of the corresponding aldehyde (1 equiv) in
dry methanol (1 mL/mmol), under argon, malonodinit-

8182
J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
rile (1 equiv) and a catalytic amount of piperidine were
added. The mixture was stirred at rt for 10–20 min.
Then, 5,5-dimethyl-1,3-cyclohexanedione (1 equiv) and
a catalytic amount of piperidine were added. The mix-
ture was stirred at rt for 30 min–14 h, and the precipitat-
ed solid was isolated by filtration, washed with cold
methanol, dried and recrystallized from mixtures of
dichloromethane/hexane.
product 5 (300 mg, 90%) was obtained: mp 301ꢁC; IR
(KBr) m 3430, 3355, 3246, 3028, 2934, 2862, 1640, 1453,
1371, 1229 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
7.29 (m, 2 H, 2· H20), 7.17 (m, 2H, 2· H30), 7.07 (m,
1H, H40), 5.63 (s, 2H, NH2), 4.95 (s, 1H, H12), 2.55-2.49
(m, 4H, 2H10, 2H7), 2.29 (m, 1H, H4A), 2.26 (d,
J = 16.1 Hz, 1H, H2A), 2.16 (m, 1H, H4B), 2.05 (d,
J = 16.1 Hz, 1H, H2B), 1.65 (br s, 4H, 2H9, 2H8), 1.02
[s, 3H, CH3(C3)], 0.87 [s, 3H, CH3(C3)]; 13C NMR
(DMSO-d6, 75 MHz) d 195.6 (C1), 164.7 (C4a), 153.8
(C5a), 152.1 (C11*), 151.3 (C6a*), 144.2 (C10), 128.0 (2·
C20), 127.8 (2· C30), 126.1 (C40), 113.6 (C10a), 113.1
(C12a), 99.1 (C11a), 50.0 (C2), 44.3 (C4), 32.8 (C12),
31.9 (C3), 31.6 (C7), 28.7 [CH3(C3)], 26.4 [CH3(C3)],
22.9 (C10), 22.2 (C9), 21.9 (C8); MS (APCI+) m/z:
[M+1]+ 375.3; [M+Na]+ 397.3; [2M+Na]+ 771.3. Anal.
Calcd for C24H26N2O2: C, 76.98; H, 7.00; N, 7.48. Found:
C, 76.34; H, 7.00; N, 7.23.
4.3. 2-Amino-3-cyano-4-[2-(trifluoromethyl)phenyl]-
5,6,7,8-tetrahydro-7,7-dimethyl-5-oxo-4H-chromene (16)
Following Section 4.1 for the synthesis of 4H-chromenes,
3-trifluoromethylbenzaldehyde (1 g, 5.74 mmol) was
reacted with malonodinitrile (416 mg, 6.31 mmol), 5,5-
dimethyl-1,3-cyclohexanedione (824 mg, 6.31 mmol),
piperidine (15 drops) and methanol (30 mL); after
45 min, compound 16 (2.05 g, 99%) was obtained: mp
218–220 ꢁC; IR (KBr) m 3481, 3328, 3202, 2964, 2199,
1
1665, 1595, 1366, 1251, 1156 cmꢀ1; H NMR (DMSO-
4.6. 11-Amino-12-(4-fluorophenyl)-2,3,4,7,8,9,10,12-octa-
hydro-3,3-dimethyl-1H-chromeno[2,3-b]quinolin-1-one (6)
d6, 300 MHz) d 7.61 (t, J4 ꢀ3 = 7.9 Hz, 1H, H40), 7.57
0
0
0
0
0
0
0
(d, J6 ꢀ5 = 7.7 Hz, 1H, H6 ), 7.37 (t, J5 ꢀ6 = 7.7 Hz,
1H, H50), 7.27 (d, J3 ꢀ4 = 7.9 Hz, 1H, H30), 7.04 (br s,
2H, NH2), 4.55 (s, 1H, H4), 2.50 (br s, 2H, H8), 2.14
[AB, J = 15.9 Hz, 2H, H6], 1.03 [s, 3H, CH3(C7)], 0.95
[s, 3H, CH3(C7)]; 13C NMR (DMSO-d6, 75 MHz) d
195.6 (C5), 163.3 (C2), 158.4 (C8a), 144.57/144.55/
Following Section 4.4, from compound 15 (250 mg,
0.80 mmol), AlCl3 (159.6 mg, 1.2 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (117.6 mg, 1.2 mmol), after
6.5 h, product 6 (308 mg, 91%) was obtained: mp
313 ꢁC; IR (KBr) m 3418, 3355, 3246, 2939, 2869, 1639,
0
0
144.54/144.52 (JC1 ꢀF = 1.2 Hz, C10), 133.1 (C50), 129.9/
1601, 1567, 1371, 1226, 1205, 1168 cmꢀ1 1H NMR
;
0
126.3/122.6/119.0 (JCꢀF = 275.3 Hz, CF3), 129.7 (C60),
(DMSO-d6, 300 MHz) d 7.30 (t, J = 8.7 Hz, 2H, 2·
H20], 7.00 (t, J = 8.7 Hz, 2H, 2· H30), 5.66 (s, 2H,
NH2), 4.98 (s, 1H, H12), 2.55–2.50 (m, 4H, 2H10, 2H7),
2.29 (m, 1H, H4A), 2.26 (d, J = 16.1 Hz, 1H, H2A),
2.16 (m, 1H, H4B), 2.05 (d, J = 16.1 Hz, 1H, H2B), 1.65
(br s, 4H, 2H9, 2H8), 1.02 [s, 3H, CH3(C3)], 0.86 [s, 3H,
CH3(C3)]; 13C NMR (DMSO-d6, 75 MHz) d 195.6 (C1),
127.1 (C40), 127.1/126.7/ 126.3/125.9 (JC2 ꢀF = 29.7 Hz,
0
C20), 125.7/125.6 / 125.5 / 125.4 (JC3 ꢀF = 5.8 Hz, C30),
0
119.2 (CꢁN), 112.8 (C4a), 58.4 (C3), 50.3 (C6), 40.1
(C8), 32.1 (C7), 31.6 (C4), 28.6 [CH3(C7)], 27.2
[CH3(C7)]; MS (APIES+) m/z: [M + 1]+ 363.3 [M +
Na]+ 385.2; [2M+Na]+ 747.5. Anal. Calcd for
C19H17N2O2F3: C, 62.98; H, 4.73; N, 7.73. Found: C,
62.82; H, 4.76; N, 7.48.
164.7 (C4a), 162.1/159.8 (JC4 ꢀF = 172.5 Hz, C40), 153.8
0
(C5a), 152.2 (C11*), 151.3 (C6a*), 140.3 (C10), 129.8/
129.7 (JC2 ,C6 ꢀF = 8.2 Hz, 2· C20), 114.6/114.3
0
0
(JC3 ,5 ꢀF = 21.0 Hz, 2· C30), 113.4 (C12a), 113.2
(C10a), 98.8 (C11a), 50.4 (C2), 40.7 (C4), 32.0 (C12),
31.9 (C3), 31.8 (C7), 28.6 [CH3(C3)], 26.4 [CH3(C3)],
22.9 (C10), 22.2 (C9), 22.0 (C8); MS (APCI+) m/z:
[M+1]+ 393.2; [2M+1]+ 807.5. Anal. Calcd for
C24H25N2O2: C, 73.45; H, 6.42; N, 7.14. Found: C,
73.37; H, 6.40; N, 7.18.
0
0
¨
4.4. General method for the Friedlander reaction
Aluminium chloride (1.2–1.7 equiv) was suspended in
dry 1,2-dichloroethane (10 mL) at rt under argon. The
corresponding 4H-benzopyran (1 equiv) and cyclohexa-
none (1.2–1.7 equiv) were added. The reaction mixture
was heated under reflux (10–24 h). When the reaction
was over (TLC analysis), a mixture of THF/H2O (1:1)
was added at rt. An aqueous solution of sodium hydrox-
ide (10%) was added dropwise to the mixture until the
aqueous solution was basic. After stirring for 30 min,
the mixture was extracted three times with dichloro-
methane. The organic layer was washed with brine,
dried over anhydrous sodium sulfate, filtered and the
solvent was evaporated. The resultant solid was purified
by silica gel flash chromatography using methanol/di-
chloromethane mixtures as eluent to give pure
compounds.
4.7. 11-Amino-12-(2-trifluoromethylphenyl)-
2,3,4,7,8,9,10,12-octahydro-3,3-dimethyl-1H-
chromeno[2,3-b]quinolin-1-one (7)
Following Section 4.4, from compound16 (250 mg,
0.71 mmol), AlCl3 (141.64 mg, 1.06 mmol), ClCH2-
CH2Cl (5 mL), cyclohexanone (104.4 mg, 1.06 mmol)],
after 6 h, product 7 (287 mg, 91%) was obtained: mp
171–172ꢁC; IR (KBr) m 3500, 3429, 3246, 2939, 2869,
1
1649, 1447, 1368, 1305, 1232 cmꢀ1; H NMR (DMSO-
d6, 300 MHz) d 7.19 (m, 2H, H30, H60), 6.39 (m, 2H,
H40, H50), 5.58 (s, 1H, H12), 5.18 (s, 2H, NH2), 2.70-
2.52 (m, 4H, 2H4, 2H7), 2.38 (m, 1H, H4A), 2.30 (d,
J = 16.1 Hz, 1H, H2A), 2.27 (m, 1H, H4B), 2.07 (d,
J = 16.1 Hz, 1H, H2B), 1.71 (br s, 4H, 2H9, 2H8), 1.04
[s, 3H, CH3(C3)], 0.91 [s, 3H, CH3(C3)]; 13C NMR
(DMSO-d6, 75 MHz) d 195.3 (C1), 164.4 (C4a), 153.5
4.5. 11-Amino–2,3,4,7,8,9,10,12-octahydro-3,3-dimethyl-
12-phenyl-1H-chromeno[2,3-b]quinolin-1-one (5)
Following Section 4.4, from compound 14 (250 mg,
0.85 mmol), AlCl3 (170.67 mg, 1.28 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (125.1 mg, 1.28 mmol), after 5 h,

J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
8183
(C5a), 152.9 (C6a), 151.3 (C11), 142.23/142.20/142.18/
142.16 (JC1 ꢀF = 1.7 Hz, C10), 133.6 (C60), 133.0/129.5/
4.10. 11-Amino–2,3,4,7,8,9,10,12-octahydro-3,3-dimeth-
yl-12-(4-methylphenyl)-1H-chromeno[2,3-b]quinolin-1-
one (10)
0
126.0/123.5 (JCꢀF = 265.1 Hz, CF3), 131.72/ 131.70/
131.69/131.67 (JC4 ꢀF = 1.3 Hz, C40), 128.2 (C50), 127.2/
0
126.8/126.4/126.0 (JC2 ꢀF = 29.6 Hz, C20), 126.88/
Following Section 4.4, from compound19 (250 mg,
0.84 mmol), AlCl3 (211.15 mg, 1.26 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (196.15 mg, 1.26 mmol)], after
8 h, product 10 (246 mg, 91%) was obtained: mp 310ꢁC;
IR (KBr) m 3417, 3244, 2936, 2869, 1638, 1568, 1454,
0
126.80/126.74/126.67 (JC3 ꢀF = 5.0 Hz, C30), 113.8
0
(C12a), 113.0 (C10a), 99.1 (C11a), 49.9 (C2), 40.4 (C4),
31.8 (C12), 31.2 (C3), 30.8 (C7), 28.5 [CH3(C3)], 26.4
[CH3(C3)], 22.8 (C10), 22.1 (C9), 21.9 (C8); MS
(APIES+) m/z: [M + 1]+ 443.1 [M + Na]+ 466.1;
[2M+Na]+ 907.2. Anal. Calcd for C25H25N2O2F3: C,
67.86; H, 5.69; N, 6.33. Found: C, 67.77; H, 5.36; N, 6.21.
1372, 1228 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
7.16 (d, J = 7.9 Hz, 2H, H20), 6.72 (d, J = 7.9 Hz, 1H,
H30), 5.57 (s, 2H, NH2), 4.88 (s, 1H, H6), 2.60 (br s, 4H,
H4, H8), 2.17 [s, 3H, CH3(C40)], 2.42–2.30 (m, 4H,
2H10, 2H7), 2.07 (m, 1H, H4A), 1.92 (m, 1H, H4B),
2.07 (d, J = 16.1 Hz, 1H, H2A), 1.83 (d, J = 16.1 Hz,
1H, H2B), 1.44 (br s, 4H, 2H9, 2H8), 1.02 [s, 3H,
CH3(C3)], 0.87 [s, 3H, CH3(C3)]; 13C NMR (DMSO-d6,
75 MHz) d 195.9 (C1), 164.9 (C4a), 154.1 (C5a), 152.3
(C6a), 151.7 (C11), 141.6 (C10), 135.5 (C40), 128.8 (2 C,
C20), 128.2 (2 C, C30), 114.1 (C12a), 113.5 (C10a), 99.6
(C11a), 50,5 (C2), 40,6 (C4), 32,8 (C12), 32,3 (C3), 29,1
(C7), 28,7 [CH3(C3)], 26,8 [CH3(C3)], 23.3 (C10), 22.6
(C9), 22.4 (C8), 20.9 [CH3(C40)]; MS (APCI+) m/z:
[M+1]+ 389.2; [M+Na]+ 411.2; [2M+1]+ 799.5. Anal.
Calcd for C25H28N2O2: C, 77.29; H, 7.26; N, 7.21. Found:
C, 77.02; H, 7.25; N, 6.69.
4.8. 11-Amino-2,3,4,7,8,9,10,12-octahydro-3,3-dimethyl-
12-(3-nitrophenyl)-1H-chromeno[2,3-b]quinolin-1-one (8)
Following Section 4.4, from compound 17 (250 mg,
0.74 mmol), AlCl3 (146.3 mg, 1.1 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (108.7 mg, 1.11 mmol), after
7 h, product 8 (167.2 mg, 53%) was obtained: mp 293–
295ꢁC; IR (KBr) m 3428, 3246, 2934, 2869, 1637, 1528,
1371, 1205 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
8.35 (s, 1 H, H20), 7.96 (d, J = 7.9 Hz, 1H, H40), 7.64
(d, J = 7.9 Hz,1H, H60), 7.49 (t, J = 7.9 Hz,1H, H50),
5.82 (s, 2H, NH2), 5.19 (s, 1H, H12), 2.71–2.48 (m,
4H, 2H10, 2H7), 2.30 (m, 1H, H4A), 2.15 (m, 1H,
H4B), 2.32 (d, J = 16.0 Hz, 1H, H2A), 2.05 (d,
J = 16.1 Hz, 1H, H2B), 1.66 (br s, 4H, 2H9, 2H8),
1.03 [s, 3H, CH3(C3), 0.85 [s, 3H, CH3(C3)]; 13C
NMR (DMSO-d6, 75 MHz) d 195.6 (C1), 165.3 (C4a),
153.6 (C5a), 152.6 (C10), 151.5 (C11), 147.1 (C6a),
146.4 (C30), 134.4 (C60), 129.5 (C50), 122.9 (C20), 121.3
(C40), 113.3 (C10a), 112.7 (C12a), 97.9 (C11a), 49.9
(C2), 40.4 (C4), 32.5 (C12), 31.9 (2 C, C3, C7), 28.6
[CH3(C3)], 26.2 [CH3(C3)], 22.9 (C10), 22.1 (C9)*, 21.9
(C8)*; MS (APCI+) m/z: [M+1]+ 420.3; [M+Na]+
442.2; [2M+Na]+ 861.5. Anal. Calcd for C24H25N3O4:
C, 68.72; H, 6.01; N, 10.02. Found: C, 68.96; H, 6.22;
N, 9.94.
4.11. 11-Amino–2,3,4,7,8,9,10,12-octahydro-12-(2-meth-
oxyphenyl)-3,3-dimethyl-1H-chromeno[2,3-b]quinolin-1-
one (11)
Following Section 4.4, from compound 20 (250 mg,
0.77 mmol), [AlCl3 (151.70 mg, 1.14 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (112.50 mg, 1.14 mmol)], after 5 h,
product 11 (291 mg, 94%) was obtained: mp 145ꢁC; IR
(KBr) m 3442, 3381, 2934, 2869, 1650, 1490, 1453, 1372,
1241, 1173 cmꢀ1; 1H NMR (DMSO-d6, 300 MHz) d 7.09
(d, J = 8,3 Hz, 1H, H30), 7.07 (t, J = 7,2 Hz, 1H, H60),
6.97 (d, J = 8.3 Hz, 1H, H40), 6.65 (t, J = 7.2 Hz, 1H,
H50), 5.54 (s, 2H, NH2), 5.03 (s, 1H, H12), 3.86 (s, 3H,
CH3O), 2.42–2.30 (m, 4 H, 2H10, 2H7), 2.07 (m, 1H,
H4A), 1.92 (m, 1H, H4B), 2.07 (d, J = 16.1 Hz, 1H,
H2A), 1,83 (d, J = 16,1 Hz, 1H, H2B), 1,44 (br s, 4H, 2
H9, 2H8), 1,04 [s, 3H, CH3(C3)], 0.93 [s, 3H, CH3(C3)];
13C NMR (DMSO-d6, 75 MHz) d 195.7 (C1), 165.8
(C4a), 155.8 (C20), 154.0 (C5a), 152.2 (C6a), 151.7 (C11),
132.5 (C10), 129.8 (C60), 128.1 (C30), 121.5 (C50), 113.2
(C10a), 112.9 (C12a), 112.2 (C40), 99.4 (C11a), 56.6
(CH3O), 50.4 (C2), 40.7 (C4), 32.2 (C12), 31.6 (C3), 29.2
(C7), 27.5 [CH3(C3)], 26.7 [CH3(C3)], 23.2 (C10), 22.6
(C9), 22.3 (C8); MS (APCI+) m/z: [M+1]+ 405.1;
[M+Na]+ 427.1; [2M+1]+ 831.2. Anal. Calcd for
C25H28N2O3: C, 74.23; H, 6.98; N, 6.93. Found: C,
74.53; H, 7.10; N, 7.02.
4.9. 11-Amino-2,3,4,7,8,9,10,12-octahydro-3,3-dimethyl-
12-(4-nitrophenyl)-1H-chromeno[2,3-b]quinolin-1-one (9)
Following Section 4.4, from compound 18 (250 mg,
0.74 mmol), AlCl3 (146.03 mg, 0.11 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (108.78 mg, 1.11 mmol), after 5 h,
product 9 (215 mg, 69%) was obtained: mp 323ꢁC; IR
(KBr) m 3390, 3227, 2936, 2869, 1639, 1517, 1371, 1345,
1
1227 cmꢀ1; H NMR (DMSO-d6, 300 MHz) d 7.87 (d,
J = 8.6 Hz, 2H, H30, H50), 7,35 (d, J = 8.6 Hz, 2H, H20,
H60), 5.53 (s, 2H, NH2), 4.95 (s, 1H, H12), 2.42–2.30
(m, 4H, 2H10, 2H7), 2.07 (m, 1H, H4A), 1.92 (m, 1H,
H4B), 2.07 (d, J = 16.1 Hz, 1H, H2A), 1.83 (d,
J = 16.1 Hz, 1H, H2B), 1.44 (br s, 4H, 2H9, 2H8), 0.81
[s, 3H, CH3(C3)], 0.63 [s, 3H, CH3(C3)]; 13C NMR
(DMSO-d6, 75 MHz) d 195.5 (C1), 165.3 (C4a), 153.7
(C5a)*, 152.6 (C10)*, 151.8 (C11)**, 151.5 (C6a)**, 145.8
(C40), 129.3 (2 C, C20, C60), 123.0 (2 C, C30, C50), 113.4
(C10a), 112.5 (C12a), 97.8 (C11a), 49.9 (C2), 40.3 (C4),
32.8 (C12), 31.9 (C3), 31.8 (C7), 28.6 [CH3(C3)], 26.4
[CH3(C3)], 22.9 (C10), 22.1 (C9), 21.9 (C8); MS (APCI+)
m/z: [M+1]+ 420.3. Anal. Calcd for C24H25N3O4: C, 68.72;
H, 6.01; N, 10.02. Found:C, 68.58; H, 6.12; N, 10.10.
4.12. 11-Amino-12-(3-methoxyphenyl)-3,3-dimethyl-
2,3,4,7,8,9,10,12-octahydro-1H-chromeno[2,3-b]quinolin-
1-one (12)
Following Section 4.4, from compound 21 (200 mg,
0.64 mmol), AlCl3 (127.28 mg, 0.96 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (93.81 mg, 0.96 mmol), after 8 h,
product 12 (246 mg, 96%) was obtained: mp 245ꢁC; IR

8184
J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
(KBr) m 3420, 3347, 3243, 2938, 2833, 1636, 1453, 1372,
4.14.2. Measurement of lactic dehydrogenase (LDH) activ-
ity. Extracellular and intracellular LDH activity was spec-
trophotometrically measured using a Cytotoxicity Cell
Death kit (Roche-Boehringer. Mannheim, Germany)
according to the manufacturer’s indications. Total LDH
activity wasdefined asthe sum of intracellularand extracel-
lular LDH activity; released LDH was defined as the per-
centage of extracellular compared to total LDH activity.
1
1229, 1171 cmꢀ1; H NMR (DMSO-d6,300 MHz) d 7.09
(t, J = 7.8 Hz, 1H, H50), 6.97 (s, 1H, H20), 6.77 (d,
J = 7.8 Hz, 1H, H40), 6.65 (d, J = 7,8 Hz, 1H, H60), 5.65
(s, 2H, NH2), 4.93 (s, 1H, H12), 3.66 (s, 3H, CH3O),
2.42–2.30 (m, 4H, 2H10, 2H7), 2.07 (m, 1H, H4A), 1.92
(m, 1H, H4B), 2.07 (d, J = 16.1 Hz, 1H, H2A), 1.83 (d,
J = 16.1 Hz, 1H, H2B), 1.44 (br s, 4H, 2H9, 2H8), 1.03
[s, 3H, CH3(C3)], 0.89 [s, 3H, CH3(C3)]; 13C NMR
(DMSO, 75 MHz) d 195.5 (C1), 164.8 (C4a), 158.7
(C30), 153.8 (C5a), 152.1 (C11*), 151.3 (C6a*), 145.7
(C10), 128.9 (C50), 120.0 (C40), 114.0 (C20), 113.4 (C10a),
113.1 (C12a), 110.8 (C60), 99.0 (C11a), 54.9 (CH3O),
50.0 (C2), 40.3 (C4), 32.8 (C12), 31.9 (C3), 31.8 (C7),
28.7 [CH3(C3)], 26.3 [CH3(C3)], 22.9 (C10), 22.2 (C9),
22.0 (C8); MS (APCI+) m/z: [M+1]+ 405.2; [2M+1]+
831.7. Anal. Calcd for C25H28N2O3: C, 74.23; H, 6.98;
N, 6.93. Found: C, 74.31; H, 6.56; N, 6.93.
4.14.3. Measurement of cytosolic Ca2+ concentrations.
For these experiments, SH-SY5Y neuroblastoma cells
were grown at confluence in 96-well black dishes. Cells
were loaded with 4 lM fluo 4/AM for 1 h at 37ꢁC in
DMEM. Then cells were washed twice with Krebs-Hepes
solution and kept at room temperature for 30 min before
the beginning of the experiment. Fluorescence was mea-
sured in a fluorescence microplate reader (FLUOstar
Optima, BMG, Germany). Wavelengths of excitation
and emission were 485 and 520 nm, respectively.
4.13. 11-Amino-2,3,4,7,8,9,10,12-octahydro-12-(4-meth-
oxyphenyl)-3,3-dimethyl-1H-chromeno[2,3-b]quinolin-1-
one (13)
4.14.4. Measurement of AChE activity. To assess the
inhibitory activity of the compounds towards AChE, we
followed the spectrophotometric method of Rappaport28
using purified AChE from electric eel (Torpedo californica)
and acetylcholine chloride (29.5 mM) as a substrate. The
reaction took place in a final volume of 2.5 mL of an aque-
ous solution containing 0.78 U AChE and 1.9 mM m-ni-
trophenol to produce a yellow colour which is lost as a
function of enzyme activity. Inhibition curves were made
by incubating with the different compounds for 30 min;
a sample without any compound was always present to
determine the 100% of enzyme activity. After the 30 min
incubation, the loss of yellow colour by m-nitrophenol
was evaluated by measuring absorbance at 405 nm in a
spectrophometric plate reader (iEMS Reader MF, Lab-
systems). The concentration of compound that produces
50% AChE activity inhibition (IC50) was calculated by
transforming the values of absorbance to Rappaport enzy-
matic activity units extrapolating from a calibration curve
previously obtained. Data are means SEM of at least
three different experiments in triplicate.
Following Section 4.4, from compound22 (250 mg,
0.77 mmol), AlCl3 (151.62 mg, 1.14 mmol), ClCH2CH2Cl
(5 mL), cyclohexanone (112.50 mg, 1.14 mmol), after 7 h,
product 13 (289 mg, 92%) was obtained: mp 291ꢁC; IR
(KBr) m 3414, 3241, 2937, 2360, 1635, 1509,1455, 1372,
1295, 1228 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
7.19 (d, J = 8.6 Hz, 2H, H20), 6.72 (d, J = 8.6 Hz, 1H,
H30), 5.58 (s, 2 H, NH2), 4.88 (s, 1H, H6), 3.64 (s, 3H,
CH3O), 2.42–2.30 (m, 4H, 2H10, 2H7), 2.07 (m, 1H,
H4A), 1.92 (m, 1H, H4B), 2.07 (d, J = 16.1 Hz, 1H,
H2A), 1.83 (d, J = 16.1 Hz, 1H, H2B), 1.44 (br s), 4H,
2H9, 2H8), 1.02 [s, 3H, CH3(C3)], 0.88 [s, 3H,
CH3(C3)]; 13C NMR (DMSO-d6, 75 MHz) d 195.9 (C1),
164.7 (C4a), 157.9 (C40), 154.1 (C5a), 152.3 (C6a), 151.6
(C11), 136.6 (C10), 129.3 (2 C, C20), 114.1 (C12a), 113.6
(2 C, C30), 113.5 (C10a), 99.7 (C11a), 55.2 (CH3O), 50.4
(C2), 40.7 (C4), 32.3 (C12), 32.2 (C3), 29.4 (C7), 29.0
[CH3(C3)], 26.8 [CH3(C3)], 23.3 (C10), 22.6 (C9), 22.4
(C8); MS (APCI+) m/z: [M+1]+ 405.1; [M+Na]+ 427.0;
[2M+1]+ 831.2. Anal. Calcd for C25H28N2O3: C, 74.23;
H, 6.98; N, 6.93. Found: C, 74.11; H, 6.98; N, 7.37.
4.14.5. Measurement of BuChE activity. The inhibitory
activity of the compounds towards BuChE was deter-
mined following the method of Ellman29 using BuChE
from human serum and butiryl thiocholine cloride
(5 mM) as a substrate. The reaction took place in a final
volume of 1 mL of a phosphate buffer solution at pH 7.2
containing 0.035 U BuChE and 0.25 mM 5,50-dithiobis-
2-nitrobenzoic acid (DTNB) which produces the yellow
anion 5-thio-2-nitrobenzoic acid. Inhibition curves were
made by incubating with the different compounds for
15 min; a sample without any compound was always
present to determine the 100% of enzymatic activity.
After the 15 min incubation period, the production of
colour, as an indication of enzymatic activity, was evalu-
ated by measuring absorbance at 412 nm in a spectropho-
tometric plate reader (iEMS Reader MF, Labsystems).
4.14. Pharmacological studies
4.14.1. Culture of SH-SY5Y cells. SH-SY5Y cells, at
passages between 3 and 16 after de-freezing, were main-
tained in a Dulbecco’s modified Eagle’s medium
(DMEM) containing 15 non-essential amino-acids
(NEAAs) and supplemented with 10% foetal calf serum
(FCS), 1 mM glutamine, 50 U/mL penicillin and 50 lg/
ml streptomycin (reagents from Gibco, Madrid, Spain).
Cultures were seeded into flasks containing supplement-
ed medium and maintained at 37ꢁC in 5% CO2/humidi-
fied air. Stock cultures were passaged 1:4 twice weekly.
For assays, SH-SY5Y cells were sub-cultured in 24-well
plates at a seeding density of 2 · 105 cells per well, or in
96-well plates at a seeding density of 8 · 104 cells per
well. For the cytotoxicity experiments cells were treated
with drugs before confluence, in DMEM free of serum.
4.14.6. Measurement of propidium iodide displacement
from the peripheral anionic site (PAS) of AChE. A solu-
tion of AChE from bovine erythrocyte at the concentra-
tion of 5 lM in 0.1 mM Tris buffer, pH 8, was used.

J. Marco-Contelles et al. / Bioorg. Med. Chem. 14 (2006) 8176–8185
8185
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(e) Marco, J. L.; de los Rıos, C.; Garcıa, A. G.; Villarroya,
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A.; Orozco, M.; Luque, F. J. Bioorg. Med. Chem. 2004, 12,
´
Aliquots of the compounds to get the final concentra-
tion of 0.3 lM were added, and the solutions were kept
at room temperature for at least 6 h. After that time, the
samples were incubated for 15 min with propidium at a
final concentration of 20 lM and the fluorescence was
measured in a fluorescence microplate reader (FLUO-
star Optima, BMG, Germany). Wavelengths of excita-
tion and emission were 485 and 620 nm, respectively.
´
´
2199; Orozco, C (f) de los Rıos, C.; Arias, E.; Leon, R.;
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The present work has been partially supported by Fun-
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