Nontoxic and neuroprotective β-naphthotacrines for alzheimer's disease

Article abstract of DOI:10.1021/tx400138s

The synthesis, toxicity, neuroprotection, and human acetylcholinesterase (hAChE)/ human butyrylcholinesterase (hBuChE) inhibition properties of β-naphthotacrines1-14 as new drugs for Alzheimer's disease (AD) potential treatment, are reported. β-Naphthotac

Full text of DOI:10.1021/tx400138s

Article
pubs.acs.org/crt
Nontoxic and Neuroprotective β‑Naphthotacrines for Alzheimer’s
Disease
Mario Esquivias-Perez,† Emna Maalej,‡ Alejandro Romero,*,∥ Fakher Chabchoub,‡ Abdelouahid Samadi,⊥
́
Jose Marco-Contelles,*,⊥,§ and María Jesus Oset-Gasque*,†,§
́
́
†Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense de Madrid (UCM),
28040-Madrid, Spain
‡Laboratoire de Chimie Appliquee
3018-Sfax, Tunisia
∥Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain
⊥Laboratorio de Química Med
ica (IQOG, CSIC), C/Juan de la Cierva 3, 28006-Madrid, Spain
§Instituto Universitario de Investigacion
en Neuroquímica (IUIN), Universidad Complutense de Madrid (UCM), 28040-Madrid,
́ ́ ́ ̀ ́ ́
: Heterocycles, Corps Gras et Polymeres Faculte des Sciences de Sfax, Universite de Sfax,
́
́
Spain.
S
* Supporting Information
ABSTRACT: The synthesis, toxicity, neuroprotection, and human
acetylcholinesterase (hAChE)/ human butyrylcholinesterase
(hBuChE) inhibition properties of β-naphthotacrines 1−14 as new
drugs for Alzheimer’s disease (AD) potential treatment, are reported.
β-Naphthotacrines 1−14 showed lower toxicity than tacrine; more-
over, at the highest concentration assayed (300 μM) compounds 7,
10 and 11 displayed 2.25−2.01-fold higher cell viability than tacrine
in HepG2 cells. A neuroprotective effect was observed for
compounds 10 and 11 in a neuronal cortical culture exposed to a
combination of oligomycin A/rotenone. An efficient and selective
inhibition of hAChE, was only observed for the β-naphthotacrines
bearing electron-donating substituents at the aromatic ring, β-naphthotacrine 10 being the most potent (hAChE: IC50 = 0.083
0.024 μM). Kinetic inhibition analysis clearly demonstrated that β-naphthotacrine 10 behaves as a mixed-type inhibitor (Ki2= 0.72
0.06 μM) at high substrate concentrations (0.5−10 μM), while at low concentrations (0.01−0.1 μM) it behaves as a hAChE
competitive inhibitor (Ki1= 0.007 0.001 μM). These findings identified β-naphthotacrine 10 as a potent and selective hAChE
inhibitor in a nanomolar range, with toxicity lower than that of tacrine both in human hepatocytes and rat cortical neurons, with a
potent neuroprotective activity and, consequently, an attractive multipotent active molecule of potential application in AD
treatment.
level in the cholinergic synapses.6 Unfortunately, instead of
INTRODUCTION
■
curing or preventing the neurodegeneration, AChEI only enable
a palliative treatment,7 and their clinical effectiveness is still under
debate.8 Taking into account AD multifactorial nature, the
traditional approach of single-target molecules generally offers
only limited and transient benefits. Multitarget-directed ligand
strategy (MTDL)9 has been recently applied to AChEIs10
research, which means that the focus has been on pharmaco-
phores with other properties besides cholinesterase inhibition.
In this complex scenario, tacrine (Table 1), the most potent
AChEI clinically effective,11 was approved for clinical use by the
United States Federal Drug Agency (FDA) in 1993. However,
tacrine exhibited hepatotoxicity via elevation of serum alanine
aminotransferase levels and thus showed limited clinical
application. In consequence, tacrine was withdrawn from the
Alzheimer’s disease (AD) is the most prominent form of
dementia in the world affecting about 6% of the population aged
over 65 and whose incidence increases with age.1 Despite huge
efforts and numerous successes in the investigation of AD
pathophysiology, the disease is still incurable.2 AD is clinically
characterized by memory impairment and progressive deficit in
different cognitive domains related to a pronounced cholinergic
system degradation and to an alteration in other neuro-
transmitter systems.3 The cholinergic hypothesis of AD4 asserts
that declination of acetylcholine (ACh) levels leads to cognitive
and memory deficits; therefore, sustaining or recovering
cholinergic function is supposed to be clinically beneficial.5
ACh can be degraded by two types of cholinesterases,
acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE). Indeed, nowadays AD therapy is bolstered mainly
by AChE inhibitors (AChEI), such as tacrine, rivastigmine,
donepezil and galanthamine, which are able to increase the ACh
Received: April 8, 2013
Published: May 15, 2013
© 2013 American Chemical Society
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a
Table 1. In Vitro Toxicity of Tacrine, and the β-Naphthotacrines 1−14 in HepG2 Cells
viability (%) HepG2 cells
e
e
e
e
e
e
cmpd
1 μM
3 μM
10 μM
30 μM
100 μM
300 μM
d
c
b
b
b
b
b
b
b
b
b
b
b
c
1
100.2 0.46ns
96.4 0.75ns
100.2 0.83ns
87.6 2.03
84.5 1.03
75.4 3.84
86.2 1.02
71.5 4.98
67.5 1.81
71.2 0.23
67.7 4.97
52.9 2.58
70.1 0.18
64.9 4.92
65.8 0.77
79.1 0.20
65.8 0.09
b
c
c
2
93.9 2.12ns
99.6 0.26ns
83.7 0.99
3
93.2 1.65ns
b
b
b
b
b
b
d
4
5
80.8 1.26
76.3 0.61
82.6 0.27
80.1 0.07
85.3 0.87
71.8 0.52
90.1 0.33
82.7 0.92
b
b
97.2 1.06ns
99.9 0.50ns
100.1 1.13ns
98.2 0.98ns
99.5 1.37ns
99.2 0.41ns
99.1 0.39ns
100.3 1.42ns
100 1.20ns
100.1 0.79ns
93.4 4.69ns
93.7 0.40ns
84.9 0.87
82.3 1.4 1
c
b
b
6
74.4 0.26
73.0 0.41
74.3 1.58
c
7
97.6 0.92ns
94.3 1.28ns
97.1 1.09ns
93.6 0.47ns
97.3 0.11ns
98.4 0.65ns
99.7 1.08ns
96.2 1.20ns
96.8 0.91ns
84.5 1.20
85.1 1.46
d
b
8
97.0 0.93ns
92.2 0.58
74.9 0.43
c
b
b
b
9
86 0.44
80.9 1.67
74.9 2.78
36 2.60
d
c
b
b
c
10
11
12
13
14
tacrine
91.5 2.14
90.1 0.30
81.8 0.1 0
84.1 0.37
98.0 1.36ns
97.8 0.56ns
98.3 1.68ns
96.7 1.11ns
97.5 0.32ns
93.9 0.62ns
96.1 0.74ns
95.1 0.57ns
90.9 0.68
c
b
90.9 0.37
83.1 0.76
61.5 1.13
77.5 4.61
b
b
b
79.8 2.04
e
e
96.8 0.61ns
90 2.95ns
97.2 1.77ns
92.2 0.38ns
d
b
b
88.7 3.42ns
81.6 4.88
64.3 4.54
40 2.20
a
Cell viability was measured as MTT reduction, and data were normalized as % control. Data are expressed as the means SEM of triplicate of at
least three different cultures. All compounds were assayed at increasing concentrations (1−300 μM). Comparisons between drugs and control group
b
c
d
e
were performed by one-way ANOVA followed by the Newman−Keuls posthoc test. P ≤ 0.001. P ≤ 0.01. P ≤ 0.05. ns = not significant, with
respect to control group.
pharmaceutical market shortly after its approval.12 For this
reason tacrine is not considered as a gold standard for AD drug
discovery. In fact, although new AChEIs continue to be under
investigation, recent efforts are focused on the development of
target molecules in AD’s underlying pathogenic mechanisms,
such as the amyloid cascade hypothesis,13 oxidative stress, and
free radical generation.14 These new approaches and the fact that
today most of the funding agencies declare limited interest in
cholinesterase inhibitor programs make AChEI drug discovery
less relevant from a medicinal chemistry perspective.
In spite of this and because of the high potency of tacrine, this
structure has been widely and successfully used in medicinal
chemistry for application in hybrid or multitarget com-
pounds.15,16 In order to combine tacrine’s AChE inhibition
with other pharmacological properties, tacrine’s structure was
covalently connected to other pharmacophores,17 such as CB1
receptor antagonists and an M1 agonist.18,19 Very well-known
tacrine derivatives include the following: (a) dimer bis(7)-
tacrine,20 which exhibited a 1000-fold higher AChE inhibition
potency, a better pharmacological profile consisting in the
inhibition of the AChE-induced Aβ aggregation through the
interaction with its peripheral binding site (PAS),21 and in
neuroprotective effects related to the interaction with β-secretase
enzyme and NMDA and GABAA receptors;22 (b) cystamine−
tacrine dimer, endowed with a lower toxicity in comparison to
that of bis(7)tacrine, able to inhibit AChE/BuChE, self- and
AChE-induced Aβ aggregation in the same range of the reference
compound, and exerting a neuroprotective action on SH-SY5Y
cell line against H2O2-induced oxidative injury;23 (c) tacrine−
ferulic acid hybrids as potent cholinesterase inhibitor (ChEIs)
which can block the PAS;24 (d) tacrine−organic nitrates,25,26
and (e) tacrine−silibinin codrug which showed high AChE and
BuChE inhibition, neuroprotective effects, no hepatotoxicity in
vitro and in vivo, but with the same in vivo pro-cognitive effects as
tacrine, being superior to the physical mixture of tacrine and
silibinin in all these regards.27 Particularly interesting among all
studied tacrines, 7-MEOTA (9-amino-7-methoxy-1,2,3,4-tetra-
hydroacridine), a Czech cholinergic drug first synthesized by
Patocka et al.,28 is a potent, centrally active ChEI free of the
serious side effects related to tacrine.12
On the basis of these precedents, having worked previously on
this topic15,16 and looking for nontoxic, neuroprotective tacrines
as a MTDL strategy for AD, here we report the biological
evaluation of several racemic β-naphthotacrines (I) (Figure 1),
Figure 1. Structures of tacrine and of β-naphthotacrines (I).29
such as 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]-
chromeno[2,3-b]quinolin-13-amines 1−14 (Table 1).29 On the
basis of this work, we identified β-naphthotacrine 10 {14-(3,4-
dimethoxyphenyl)-10,11,12,14-tetrahydro-9H-benzo[5,6]-
chromeno[2,3-b]quinolin-13-amine} (Table 1) as a potent and
selective human AChEI (hAChEI), neuroprotector in a
nanomolar range and nontoxic in human HepG2 cells and,
consequently, a promising molecule in AD treatment.
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Figure 2. IC50 (μM) calculations for hAChE of tacrine (A) and β-naphthotacrine 10 (B). IC50 values were calculated from hAChE inhibition data, at 10
nM to 100 μM compound concentrations. Fittings were performed by a nonlinear regression analysis by using the function f1 = min + (max − min)/(1 +
̂
(x/IC50)(Hillslope). Values represent the means SEM of three different experiments performed in triplicate.
naphthotacrines 7 and 10 were also less toxic than the
nonsubstituted β-naphthotacrine 1 or β-naphthotacrines 2−6,
substituted with only one Me, OH, or OMe group. In this case
the position of the substituents in the aromatic ring at C-14 is also
important as shown by comparing compound 9 (toxic) with 10
(less toxic), where one of the two methoxy groups moves from
C2′ to C3′. All these effects are also in good agreement with the
low toxicity found in 7-MEOTA.28 The case of β-naphthotacrine
12 bearing a nitro substituent, an electron-withdrawing group at
C4′ in the aromatic ring, is an exception to this general trend, as it
shows a viability value of 83% at 300 μM concentration (see
Table 1). Finally, it is also clear that the substitution of a fused
benzene ring by a benzochromane motif in tacrine, coupled to a
correct selection of the substituents at defined and synthetically
available aromatic positions, produces new tacrines devoid of the
toxic effects present in the reference compound.
Cholinesterase Inhibition. On the basis of these promising
results that enhanced interest in them and in potential
therapeutic application, β-naphthotacrines 1−14 were evaluated
as inhibitors of hAChE and hBuChE, according to Ellman’s
protocol.33 The IC50 values for hAChE were measured at
concentrations between 0.01 and 100 μM. An example of IC50
calculation for β-naphthotacrine 10, compared with that for
tacrine, is represented in Figure 2. The remaining IC50 values for
the tested compounds and tacrine are shown in Table 2.
β-naphthotacrines 1−14 were selective hAChE inhibitors since
they produced poor or no inhibition of hBuChE activity. In fact,
only compounds 5, 6 and 9 had an IC50 < 100 μM, which is very
high as compared with tacrine. With the exception of β-
naphthotacrines 12−14, bearing electron-withdrawing substitu-
ents at the aromatic ring at C-14, the electron-donating
substituted β-naphthotacrines 1−11 were potent hAChE
inhibitors, from 0.048 to 4.48 μM. The most potent, and
insignificantly different from tacrine, were β-naphthotacrine 4
(IC50 = 0.048 0.016 μM), bearing a hydroxyl group at C4′, and
β-naphthotacrine 10 (IC50 = 0.083 0.024 μM), bearing two
methoxy groups at C3′ and C4′.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of known racemic 14-aryl-
10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]-
quinolin-13-amines 2−8, and 10−12 (Table 1) has been carried
out as previously reported.29 New β-naphthotacrines 1, 9, 13, and
14 have been synthesized by Friedlander-type reactions of 3-
̈
amino-1-aryl-1H-benzo[f ]chromene-2-carbonitriles 19−22,
prepared from the corresponding 2-arylidenemalononitriles
15−18, with selected cycloalkanones (see Supporting Informa-
tion). We have obtained these compounds by incorporating
different substituents in the aromatic ring at C14, taking into
account their electronic properties, the number of groups, and
the position at the aromatic nucleus. All the new compounds
showed analytical and spectroscopic data in good agreement with
their structures.
Pharmacology. Hepatotoxicity of β-Naphthotacrines. We
first investigated, as the most critical and crucial point,
hepatotoxicity of β-naphthotacrines 1−14 on HepG2 cells in a
concentration range (1, 3, 10, 30, 100, and 300 μM) as previously
described by Denizot and Lang30 (see Supporting Information).
HepG2 cells are considered to be a reasonable model to study in
vitro xenobiotic metabolism and liver toxicity. In particular,
HepG2 cells are able to activate and detoxify xenobiotics, and
they are employed to study drug mechanisms of action.31
Cytotoxicity of compounds 1−14 was determined using the
MTT assay. These compounds were well tolerated after a 24 h
incubation period in human HepG2 cells compared with tacrine,
as shown in Table 1. The hepatotoxicity of tacrine is associated
with increased levels of intracellular reactive oxygen species
(ROS) and decreased levels of antioxidant defenses.32 As shown
in Table 1, tacrine decreases the number of cells even at the
lowest concentration (30 μM), being more hepatotoxic gradually
at rising doses. It is worth mentioning that β-naphthotacrines 11,
7, and 10, in this order, at the highest concentration used (100
and 300 μM) showed a reduced hepatotoxicity compared with
tacrine. Thus, these compounds exhibited a wide therapeutic
safety range.
Comparing C′4-substituted β-naphthotacrines 2−4 IC50, we
notice that inhibition potency increases gradually from methyl to
hydroxyl up to methoxy electron-donating substituents. With the
same methoxy substituent at different positions, we notice that
AChE inhibition potency was higher for β-naphthotacrine 6
(methoxy at C2′) than for β-naphthotacrine 5 (methoxy at C3′),
which showed higher inhibition potency than β-naphthotacrine 4
(methoxy at C4′). Once again the substituent position in the
From a structure−activity relationship (SAR) perspective, it
seems that electron-donating substituted β-naphthotacrines are
less toxic, in general, than electron-withdrawing substituted ones.
An additive effect also operates, as β-naphthotacrine 11,
substituted with three methoxy groups, is less toxic than β-
naphthotacrines 7 or 10, substituted with two electron-donating
groups, such as OH-OMe or di-OMe, respectively. In addition, β-
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Table 2. IC50 (μM) Values for the Inhibition of hAChE and
a
hBuChE by Tacrine and Racemic β-Naphthotacrines 1−14
b
c
cmpd
R
n
hAChE (μM)
hBuChE (μM)
1
H
1
0.24 0.07ns
>100
>100
>100
>100
∼60
f
2
4′-Me
4′-OH
4′-OMe
3′-OMe
2′-OMe
4′-OH,3′-OMe
4′-OH,3′-OMe
2′,4′-di-OMe
3′,4′-di-OMe
3′,4′,5′-tri-OMe
4′-NO2
1
1
1
1
1
0
1
1
1
1
1
1
1
4.48 1.56
d
3
0.98 0.08
4
0.048 0.016ns
0.47 0.23ns
5
e
6
1.46 0.21
∼50
f
7
4.42 1.28
>100
>100
∼30
>100
>100
>100
>100
>100
0.04 0.002
e
8
1.54 0.22
f
9
2.94 0.11
10
11
12
13
14
tacrine
0.083 0.024ns
f
3.14 1.62
f
11.87 0.53
f
2′,6′-di-Cl
3′,4′-di-Cl
59.41 2.18
f
33.54 1.23
Figure 3. Kinetics of inhibition of hAChE hydrolysis of acetylthiocho-
line (ATCh) by β-naphthotacrine 10. (A) Lineweaver−Burk reciprocal
plots of initial velocity and substrate concentrations (0.01−10 μM) are
presented. Lines were derived from a weighted least-squares analysis of
data. (B) Ki calculation for β-naphthotacrine 10 (P < 0.001 Anova, n = 6).
0.19 0.05
a
Values are expressed as mean standard error of the mean of at least
b
three different experiments in triplicate. nd = not determined. n = 0,
cyclopentil; n = 1, cyclohexyl. Statistical comparisons between IC50’s
for drugs and that for tacrine were performed by one-way ANOVA
followed by the Holm−Sidak posthoc test. ns = not significant. P <
0.05. P < 0.01. P < 0.001.
c
d
Table 3. Kinetics of the hAChE Inhibition for β-
Naphthotacrine 10 (Km’s and Vmax’s)
e
f
[I10] μM
Vmax(ΔDO/min)
P <
Km (mM)
P <
aromatic ring at C14 is critical for AChE inhibition. Thus, not
surprisingly, compound 10, a dimethoxy derivative with these
groups at C3′/C4′ positions, is much more potent than 9, a
dimethoxy derivative with these groups at C2′/C4′ positions.
The effect of the cycloalkane size is also clearly favoring, as in the
case of tacrine, the six-membered vs the five-membered ring
when comparing the IC50 for β-naphthotacrines 8 and 7. To gain
further insight into this family of compounds’ mechanism of
action on hAChE, a kinetic study was carried out for nontoxic
and potent hAChEI β-naphthotacrine 10. Graphical analysis of
the reciprocal Lineweaver−Burk plots (Figure 3A) showed both
increased slopes (decreased Vmax) and intercepts (higher Km) at
increasing concentrations of the inhibitor. This pattern indicates
a mixed-type inhibition. Replots of the slope vs concentration of
compound 10 gave an estimate of the inhibition constant, Ki =
0.35 0.04 μM (Figure 3B).
When we analyzed statistically the Km and Vmax modifications
at different concentrations of β-naphthotacrine 10, we observed
that at low concentrations (from 0.01 to 0.1 μM) Vmax does not
significantly change, but Km does (Table 3), whereas at high
concentrations, both Vmax and Km change in a statistically
significant way (Table 3). These results mean that β-
naphthotacrine 10 behaves as a competitive inhibitor at low
concentrations and as a mixed-type inhibitor at higher
concentrations.
a
control
0.047 0.003
0.052 0.003
0.053 0.002
0.054 0.004
0.042 0.002
0.029 0.001
0.016 0.001
−
ns
0.145 0.014
0.205 0.003
0.254 0.006
0.305 0.016
0.445 0.03
0.456 0.008
0.537 0.014
−
d
d
d
d
d
d
b
0.01
0.05
0.1
0.5
1
b
ns
b
ns
c
d
d
10
a
Statistical comparisons were carried out against the control (one way
ANOVA; n = 6). ns = not significant. P < 0.05. P < 0.001.
b
c
d
Thus, at low concentrations, β-naphthotacrine 10 acts as a
competitive inhibitor, binding the enzyme at positions closely
located to the catalytic active site (CAS), while at high
concentrations, 10 acts as a mixed-type inhibitor, suggesting
that β-naphthotacrine 10 is possibly binding simultaneously to the
CAS and to the PAS of hAChE.33
Neuroprotection Studies. Finally, we investigated the neuro-
protection profile of β-naphthotacrines 2−8, and 10−12 on
primary cortical neurons treated with oligomycin-A (10 μM) and
rotenone (30 μM) (Olig/Rot), two mitochondrial respiratory
chain inhibitors, which block complex V and I, respectively, as
previously described34 by using the XTT (2,3-bis(2-methoxi-4-
nitro-5-sulfophenyl)-2H-tetrazolyl-5-carboxyanilide) assay to
determine neuronal viability (see Supporting Information).
β-Naphthotacrines 2−8 and 10−12 were added 15 min before
Olig/Rot, at concentrations between 0.1 μM and 100 μM, using
tacrine as a reference compound. In Table 4, the highest
neuroprotective effects and the EC50 values are shown. These
values clearly demonstrate that after 24 h, the decrease in cellular
viability [63.15 2.75% (mean SEM; n = 15, p < 0.001 vs
In Figure 4 we show the Lineweaver−Burk kinetic analysis of
β-naphthotacrine 10 hAChE inhibition. This analysis gives two
inhibition constants (Ki): Ki = 0.007 0.001 μM and Ki = 0.72
1
2
0.06 μM, at low and high concentrations, respectively. These
results suggest that, depending on the inhibitor concentration, β-
naphthotacrine 10 is able to bind to different sites of hAChE.
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Figure 4. Lineweaver−Burk representation of the kinetics of the hAChE inhibition for β-naphthotacrine 10, at low concentrations (0.01−0.1 μM) (A)
and at high concentrations (0.5−10 μM) (B).
Table 4. Neuroprotective Effects of β-Naphthotacrines 2−8,
10−12, and Tacrine on the Decrease in Cellular Viability
Induced by Mitochondrial Respiratory Chain Blockers Olig/
compounds 10 and 11, showed a very good non-neurotoxic
profile.
a
Rot in Primary Cultures of Cortical Neurons
CONCLUSIONS
■
Although the AD therapeutics is focused on immunization
procedures against Aβ deposition in senile plaques, antiphos-
phorylating agents to halt neurofibrillary tangle formation and γ-
and β-secretase inhibitors, the results reported with tacrine-
related compounds should not be neglected.8 Thus, this may be
the moment to revise the old-fashioned therapeutic strategies. In
this arena, and not surprisingly, some researchers have clearly
banked on tacrine. The present work clearly shows that tacrine
analogues such as the β-naphthotacrines reported here and,
particularly, those bearing electro-donor groups at aromatic
positions deserve attention in the search for new drugs for AD.
In this work we have reported the synthesis and pharmaco-
logical analysis (including toxicity, neuroprotection, and hAChE
and hBuChE inhibition properties) of racemic β-naphthotacrines
1−14, bearing the 14-aryl-10,11,12,14-tetrahydro-9H-benzo-
[5,6]chromeno[2,3-b]quinolin-13-amine heterocyclic ring skel-
eton (I), as new drugs for the potential treatment of AD. β-
Naphthotacrines 1−14 have been prepared in high yield by a
cmpd
structure (R)
maximal activity (%)
EC50 (μM)
tacrine
−
64.34 17.60
50.02 8.16
65.50 6.21
55.66 1.41
2.29 1.50
23.73 5.49
5.30 0.88
24.79 7.01
15.89 3.45
4.95 2.50
11.41 3.40
10.81 2.70
11.40 2.30
10.09 1.23
−
2
3
4′-Me, n = 1
4′-OH, n = 1
4′-OMe, n = 1
3′-OMe, n = 1
4
b
5
77.24 4.82
c
6
2′-OMe, n = 1
94.58 4.82
b
7
4′−OH-3′-OMe, n = 0
4-OH,3-OMe, n = 1
3′,4′-di-OMe, n = 1
3′,4′,5′-tri-OMe, n = 1
4′-NO2, n = 1
73.33 4.82
b
8
77.28 4.82
c
10
11
12
134.52 21.35
c
107.60 15.36
no neuroprotector
a
Data are expressed as mean SEM of at least three experiments,
Friedlander-type reaction of 3-amino-1-phenyl-1H-benzo[f ]-
̈
each one performed in triplicate, performed in at least three different
cortical neuron cultures. n = 0 cyclopentil, n = 1 cyclohexyl. Statistical
comparisons with tacrine were performed by one-way ANOVA
chromene-2-carbonitriles, 19−22, with selected cyclohexanone
or cyclopentanone. Hepatotoxicity analyses on compounds 1−
14 showed that the β-naphthotacrines are nontoxic derivatives,
and at the highest concentration assayed (300 μM) the HepG2
cell viabilities in the presence of compounds 11, 7, and 10, in this
order, were 2.25−2.01-fold higher compared with that in the
presence of tacrine. Efficient and selective inhibition of hAChE,
in a micromolar range, was only observed for the β-
naphthotacrines bearing electron-donating substituents at the
aromatic ring. One of the most potent was β-naphthotacrine 10
(hAChE: IC50 = 0.083 0.024 μM). Kinetic inhibition analysis
clearly demonstrated that β-naphthotacrine 10 behaves as a
b
c
followed by the Holm−Sidak post-hoc test. P < 0.05. P < 0.001.
100% control, one way ANOVA test)] induced by Olig/Rot was
totally or partially reverted by β-naphthotacrines 2−8, 10−12,
and tacrine, in a concentration-dependent manner, these effects
being statistically significant. As shown in Table 4, at 50 μM, only
compounds 10 and 11 were able to reach 100% cellular viability
after 24 h.
Due to the fact that the neuroprotective effects of some of
these β-naphthotacrines decrease at ≥25−50 μM, we evaluated
the basal neurotoxicity. In contrast to the toxic effects shown by
tacrine (>25−60% at 25 μM; Figure 5), β-naphthotacrines 1−11
and 13 and 14 proved to be non-neurotoxic between 1 and 50
μM; in addition, β-naphthotacrine 10 was neuroprotective
between 10 and 50 μM, but this effect decreased mostly at the
highest (100 μM) concentration analyzed (Figure 5).
From the SAR analysis only β-naphthotacrine 12, bearing the
nitro electron-withdrawing group at C4′, showed a significant
neurotoxic effect of 30−50% at concentrations between 5 and
100 μM, whereas the other β-naphthotacrines bearing electron-
donor substituents in the aromatic ring at C-14, and particularly,
mixed-type (Ki = 0.72 0.06 μM) at high concentrations, while
2
at low concentrations it is a hAChE competitive inhibitor (Ki =
1
0.007 0.001 μM). On the other hand, strong neuroprotection
effect was observed for compounds 10 and 11 in the Olig/Rot
assay. From this work we have identified β-naphthotacrine 10 as a
potent and selective hAChE inhibitor in a nanomolar range,
nontoxic in human HepG2 cells with a strong neuroprotective
activity and no neurotoxic effects. Consequently, β-naphthota-
crine 10 is an attractive, multipotent molecule, anti-AD candidate.
Studies are now in progress to resolve this racemic mixture and
separately analyze each enantiomer in the same pharmacological
tests.
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dx.doi.org/10.1021/tx400138s | Chem. Res. Toxicol. 2013, 26, 986−992

Chemical Research in Toxicology
Article
Figure 5. Neurotoxic effect of β-naphthotacrines 1−14 and tacrine on the cellular viability of cortical neurons in primary cultures represented by the % of
cellular viability in the presence or absence (control; C) of the indicated β-naphthotacrines and tacrine concentrations. The values are the mean SEM of
at least three independent experiments, each one carried out in triplicate, in different cell cultures. The statistical analysis shows the neurotoxic or
neuroprotective effects against controls at * = P < 0.05, ** = P < 0.01, *** = P < 0.001 (one way ANOVA).
MICINN (SAF2010-20337) and UCM-Santander (GR35/10-
B) for financial support.
ASSOCIATED CONTENT
* Supporting Information
Preparation of compounds 1, 9, 13, and 14, and the
pharmacological protocols. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Eva Ramos for her technical assistance and help
improving the manuscript.
■
AUTHOR INFORMATION
Corresponding Author
*Fax: +34-91-3941779, E-mail: mjoset@ucm.es (M.J.O.-G.);
Fax: +34-91-5644853, E-mail: iqoc21@iqog.csic.es (J.M.-C.);
Fax: +34-91-3943840, E-mail: manarome@ucm.es (A.R.).
■
ABBREVIATIONS
■
hAChE, human acetylcholinesterase; hBuChE, human butyr-
ylcholinesterase; ACh, acetylcholine; AD, Alzheimer disease;
MTDL, multitarget-directed ligands strategy; AChEI, acetylcho-
linesterase inhibitor; PAS, peripheral anionic binding site; CAS,
catalytic anionic site; MTT, 3-[4,5 dimethylthiazol-2-yl]-2,5-
diphenyl-tetrazolium bromide; XTT, (2,3-bis(2-methoxi-4-nitro-
Funding
J.M.-C. thanks MICINN (SAF2009-07271; SAF2012-33304) for
support. J.M.C. (Spain) and F.C. (Tunisia) thank AECID
(Ministerio de Asuntos Exteriores (Spain) for a Grant “Accion
Integrada (A1/035457/11)” with Tunisia. M.J.O.-G. thanks
́
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Chemical Research in Toxicology
Article
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5-sulfophenyl)-2H-tetrazolyl-5-carboxyanilide; ROS, reactive
oxygen species; Olig/Rot, oligomycin-A/rotenone
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