Novel tacrine-melatonin hybrids as dual-acting drugs for alzheimer disease, with improved acetylcholinesterase inhibitory and antioxidant properties

Article abstract of DOI:10.1021/jm050746d

Tacrine and melatonin are well-known drugs with activities as an acetylcholinesterase (AChE) inhibitor and free radical scavenger, respectively. In this work, we report new hybrids of both drugs that display higher in vitro properties than the sum of their parts. As selective inhibitors of human AChE, their IC₅₀ values range from sub-nanomolar to picomolar. They exhibit a higher oxygen radical absorbance capacity than does melatonin and are predicted to be able to cross the blood-brain barrier to reach their targets in the central nervous system.

Full text of DOI:10.1021/jm050746d

J. Med. Chem. 2006, 49, 459-462
459
factor for the initiation and progression of AD.7 Recent research
demonstrated that oxidative damage is an event that precedes
the appearance of other pathological hallmarks of the disease,
namely, senile plaques and neurofibrillary tangles.8 Thus, drugs
that specifically scavenge oxygen radicals may have a particular
therapeutic efficacy,9 and several antioxidants have been tested
in clinical trials.10
Novel Tacrine-Melatonin Hybrids as
Dual-Acting Drugs for Alzheimer Disease, with
Improved Acetylcholinesterase Inhibitory and
Antioxidant Properties
Mar´ıa Isabel Rodr´ıguez-Franco,*,†
Mar´ıa Isabel Ferna´ndez-Bachiller,†,# Concepcio´n Pe´rez,†
Blanca Herna´ndez-Ledesma,‡ and Begon˜a Bartolome´‡
Tacrine (1), the first approved drug for AD, is a potent
nonselective inhibitor of both AChE and butyrylcholinesterase
(BuChE). Although this lack of selectivity and its hepatotoxicity
have reduced its therapeutic use,11 the search for tacrine
analogues is still of interest in AD.12 Melatonin (2) is a pineal
neurohormone whose levels decrease during aging, especially
in AD patients. It has been reported to possess strong antioxidant
actions and is able to directly scavenge a variety of reactive
oxygen species.13 Moreover, recent studies have shown that
melatonin has protective effects against Aâ-induced apoptosis
in microglial cells and improves learning and memory in rats.14
Our research in the AD field15 is currently focused on dual-
acting drugs, capable of combining AChE inhibition and
antioxidant properties in a single small molecule (3-11). In
this work, we planned to use a high-quality moiety for each
biological activity, tacrine and melatonin. According to the
catalytic site of AChE, which is located at the bottom of a deep
gorge,16 we considered binding these fragments using carbon
chains that could be accommodated into the enzyme cavity
(Chart 1).
Instituto de Qu´ımica Me´dica (CSIC) and Instituto de
Fermentaciones Industriales (CSIC), Juan de la CierVa 3,
28006 Madrid, Spain
ReceiVed July 30, 2005
Abstract: Tacrine and melatonin are well-known drugs with activities
as an acetylcholinesterase (AChE) inhibitor and free radical scavenger,
respectively. In this work, we report new hybrids of both drugs that
display higher in vitro properties than the sum of their parts. As selective
inhibitors of human AChE, their IC50 values range from sub-nanomolar
to picomolar. They exhibit a higher oxygen radical absorbance capacity
than does melatonin and are predicted to be able to cross the blood-
brain barrier to reach their targets in the central nervous system.
Alzheimer’s disease (AD), the most common dementia in
elderly people, is a complex neurodegenerative disorder of the
central nervous system. AD patients present a progressive loss
of cholinergic synapses in brain regions associated with higher
mental functions, mainly located in hippocampus and neocortex.
It causes brain atrophy and a progressive failure of intellectual
abilities. The histopathological hallmarks of the disease are
senile plaques and neurofibrillary tangles. Plaques are massive
extracellular deposits of aggregated amyloid-â peptide, while
tangles are intracellular residues of abnormally phosphorylated
protein-tau.1
In the last two decades, several rational pharmacological
strategies have emerged, including cholinergic and noncholin-
ergic interventions.2 Among the cholinergic hypothesis, the first
approved drugs for the management of the disease were
cholinesterase inhibitors tacrine, donepezil, rivastigmine, and
galanthamine that increase neurotransmission at cholinergic
synapses in the brain and thereby improve cognition.3
Despite the many published clinical trials of approved
acetylcholinesterase inhibitors (AChE-I) for AD treatment, the
practical effectiveness of these drugs remains controversial.
Recent AD trials concluded that AChE-I therapies are not cost-
effective.4 However, other recent clinical studies showed that
patients treated with cholinesterase inhibitors did not show the
widespread cortical atrophic changes associated with AD,
providing empirical evidence of neuroprotection by cholinest-
erase inhibitors.5 These effects might be related to their primary
mode of action or their interaction with other neuronal targets,
such as N-methyl-D-asparate (NMDA), nicotinic, or muscarinic
receptors.5c,6 For these reasons, the interest in cholinesterase
inhibitors has increased in the last few years.
Scheme 1 depicts the general procedure for the synthesis of
tacrine-melatonin derivatives. Substituted 9-chloro-1,2,3,4-
tetrahydroacridines 12-15 were synthesized as previously
described.17 Their treatment with the appropriate R,ω-amino acid
in n-pentanol and subsequent hydrolysis of the pentyl ester
derivatives with sodium hydroxide gave the intermediate acids
16-19a,b. Reaction of these acids with different amines
(tryptamine, serotonin, or 5-methoxytryptamine), using (ben-
zotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluo-
rophosphate as a coupling reagent, afforded the desired tacrine-
1
indol derivatives 3-11a,b, which were characterized by H
NMR, 13C NMR, mass spectra, and elemental analyses.
The new tacrine-melatonin derivatives were evaluated as
inhibitors of AChE and BuChE, following the method of
Ellman.18 Initially, compounds were tested with enzymes of
animal source, namely, AChE from bovine erythrocytes and
BuChE from horse serum, which show a high degree of
homology with the corresponding human enzymes.19 Tacrine
and melatonin were also evaluated for comparative purposes
(Table 1).
All the new compounds are potent inhibitors of nonhuman
cholinesterases at the low nanomolar level, better than tacrine.
As expected, melatonin did not inhibit either enzyme. In general,
compounds with a 6-methylene linker between the amine and
the amide groups showed better inhibition of AChE than
molecules with a 5-methylene chain. Compounds derived from
6-chlorotacrine and 6,8-dichlorotacrine combined high potency
and selectivity toward AChE.
Furthermore, several studies have shown that the antioxidant
defense system in elderly people loses its capacity to neutralize
oxidative species, and then oxidative stress can act as a risk
A selection of tacrine-melatonin hybrids was further evalu-
ated using human enzymes. Tacrine was also tested, and data
are presented in Table 2.
* Corresponding author. Phone: 34-91-5622900. Fax: 34-91-5644853.
E-mail: IsabelRguez@iqm.csic.es.
All tested hybrids inhibited the human AChE (h-AChE) more
efficiently than the bovine enzyme, showing IC50 values ranging
† Instituto de Qu´ımica Me´dica (CSIC).
‡ Instituto de Fermentaciones Industriales (CSIC).
# This paper comprises a part of M.I.F.-B.’s Ph.D. Thesis.
from the sub-nanomolar to the picomolar order (8 × 10-10
-
10.1021/jm050746d CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/17/2005

460 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Letters
Table 2. Inhibition of Human AChE and BuChE (nM) and Oxygen
Chart 1. Structures of New Tacrine-Melatonin Hybrids 3-11
and Their Parent Drugs
Radical Absorbance Capacity (ORAC, Trolox Equivalents) by Selected
Tacrine-Melatonin Hybrids 3-11, Tacrine (1), and Melatonin (2)a
IC50 ( SD (nM)b
h-AChE
Trolox
equivd
h-AChE
h-BuChE
selectivityc
3b
4b
5a
6b
7b
10b
11a
1
0.5 ( 0.02
0.1 ( 0.005
0.87 ( 0.04
0.008 ( 0.0004
0.65 ( 0.03
0.04 ( 0.002
0.45 ( 0.02
350 ( 10
6.8 ( 0.3
35 ( 2
14
350
26
975
5
625
2
0.1
3.3 ( 0.1
2.1 ( 0.03
4.0 ( 0.1
2.5 ( 0.1
2.7 ( 0.1
1.7 ( 0.01
3.2 ( 0.2
<0.01
23 ( 2
7.8 ( 0.4
3.0 ( 0.2
25 ( 1
1.0 ( 0.1
40 ( 2
n.d.
2
n.d.
2.3 ( 0.1
a See Figure 1 and Table 1 for structure definitions. b Results are the
mean (n ) 3) ( SD. c Selectivity for h-AChE ) IC50 (h-BuChE)/IC50 (h-
AChE). d Data are expressed as µmol of trolox equivalent/µmol of tested
compound and are the mean (n ) 3) ( SD.
Scheme 1. Synthesis of Tacrine-Melatonin Derivativesa
structure is not well tolerated by the active center. Finally,
disubstituted 6,8-dichlorotacrine derivative 6b was the most
potent and selective h-AChE inhibitor of the series, pointing
out that the two chlorine atoms could work synergistically in
the active center. In relation to structural modifications in the
indole fragment, a methoxy or a hydroxy group in the 5-position
gave rise to compounds with inferior values for both potency
and selectivity toward AChE.
To the best of our knowledge, compound 6b is one of the
most potent inhibitors of human AChE described, although
recently femtomolar inhibitors of eel AChE were obtained by
click chemistry.20 This compound, derived from 6,8-dichloro-
tacrine and unsubstituted indole with a 6-methylene linker,
exhibited an IC50 (h-AChE) ) 0.008 nM and was about 1000-
fold more active toward h-AChE than toward h-BuChE.
Moreover, this inhibitor is 40 000 times more effective than
tacrine and showed the required selectivity.
a Reagents and conditions: (i) H2N(CH2)nCO2H (1 equiv), n-pentanol,
reflux, 18-24 h; (ii) NaOH 2 N, reflux, 6 h; (iii) H2N(CH2)2-indole (1.3
equiv), BOP (1.3 equiv), (CH3CH2)3N (2.6 equiv), CH2Cl2, r.t., 8-16 h.
Table 1. Inhibition of AChE and BuChE by Tacrine-Melatonin
Hybrids 3-11, Tacrine (1), and Melatonin (2)a
Since AChE is mainly located in the central nervous system
and BuChE is more abundant in the peripheral system, the
potency and selectivity toward AChE found in tacrine-
melatonin hybrids are of paramount importance. These new
molecules should be able to activate mostly the central cholin-
ergic transmission improving mental abilities and lack the side
effects related to nonselective cholinesterase inhibitors.21
The antioxidant activity of new tacrine-melatonin hybrids
3-11a,b was determined by the oxygen radical absorbance
capacity assay using fluorescein (ORAC-FL). Following the
method described by Ou et al.,22 recently optimized by Da´valos
et al.,23 peroxyl radicals were thermally generated from 2,2′-
azobis-(amidinopropane) dihydrochloride and reacted with
fluorescein to form nonfluorescent products at 520 nm. The
antioxidant capacity of new compounds was determined by their
competition with fluorescein in the radical capture, using a
fluorescence microplate reader. Vitamin E analogue trolox was
used as a standard, and the results were expressed as trolox
equivalents (µmol of trolox equivalents/µmol of tested com-
pound) (Table 2). Tacrine and melatonin were also checked for
comparison. Tacrine showed negligible radical capture, whereas
melatonin had an ORAC-FL value 2.3-fold higher than that of
trolox. This activity fully agrees with the value previously
described for melatonin (2.0 trolox equiv),24 pointing out the
reliability of our experiments.
IC50 ( SD (nM)b
R
n
R′
AChEc
BuChEd
3a
3b
4a
4b
5a
6a
6b
7a
7b
8a
9a
10b
11a
1
H
H
5
6
5
6
5
5
6
5
6
5
5
6
5
H
H
H
H
H
H
H
4.0 ( 0.2
1.0 ( 0.1
2.0 ( 0.1
0.2 ( 0.01
65 ( 3
12 ( 1
0.95 ( 0.05
5.2 ( 0.3
8.1 ( 0.3
15 ( 1
6-Cl
6-Cl
8-Cl
6,8-diCl
6,8-diCl
H
3.5 ( 0.2
2.0 ( 0.1
9.0 ( 0.3
2.3 ( 0.1
12 ( 1
8.2 ( 0.5
85 ( 4
2.0 ( 0.1
2.5 ( 0.1
55 ( 2
22 ( 1
100
3.5 ( 0.2
10 ( 0.4
>100
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
6-Cl
8-Cl
6,8-diCl
H
25 ( 1
5.0 ( 0.3
35 ( 2
40 ( 2
>100
2
a See Figure 1 for structures. b Results are presented as the mean (n )
3) ( SD. c AChE from bovine erythrocytes. d BuChE from horse serum.
8 × 10-12 M). They inhibited human BuChE (h-BuChE) with
IC50 ) 1 - 250 nM, exhibiting an interesting selectivity toward
h-AChE.
Comparing 3-6, which do not bear any substituent in the
indole ring, it is possible to establish the best modifications in
the tacrine fragment for potency and for selectivity toward
h-AChE. The presence of a chlorine atom in position 6 improved
both factors, compound 4b being a low sub-nanomolar h-AChE
inhibitor with a good selectivity to this enzyme. When the
chlorine atom was attached to position 8 of the tacrine ring (5a)
inhibition of AChE dropped, revealing that this change in the
Compounds were tested in 0.1-1 µM concentrations, showing
potent peroxyl radical absorbance capacities ranging from 1.7-
to 4-fold the value of trolox. In general, best results were
obtained with derivatives bearing an unsubstituted indole or a
5-hydroxyindole, whereas compounds derived from 5-meth-

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 461
Table 3. Permeability (Pe × 10-6 cm s-1) in the PAMPA-BBB Assay
toward human BuChE, displaying an interesting selectivity. In
the oxygen radical absorbance capacity assay, this compound
is 2.5-fold higher than trolox, a vitamin E analogue. In addition,
these tacrine-melatonin hybrids are also predicted to be able
to enter the central nervous system. Although there were
differences in potencies for AChE inhibition and antioxidant
properties, these new compounds can be considered interesting
structures in the search for new agents of potential application
in AD. Work is now in progress to outline the scope of these
new tacrine-melatonin hybrids.
of 20 Commercial Drugs, Used in the Experiment Validation
compd
bibla
expb
compd
bibla
expb
testosterone
verapamil
imipramine
desipramine
astemizole
progesterone
promazine
chlorpromazine
clonidine
17.0
16.0
13.0
12.0
11.0
9.3
8.8
6.5
5.3
5.1
13.0
13.0
9.1
11.0
11.0
8.6
7.3
6.1
6.8
7.2
piroxicam
hydrocortisone
aldosterone
lomefloxacin
enoxacin
atenolol
ofloxacin
isoxicam
theophylline
cimetidine
2.5
1.9
1.2
1.1
0.9
0.8
0.8
0.3
0.1
0.0
2.3
3.2
2.7
1.3
1.7
2.0
1.8
1.6
1.5
1.2
corticosterone
Acknowledgment. This manuscript is dedicated to Prof.
Carmen Ochoa de Oca´riz on the occasion of her retirement.
The authors gratefully acknowledge the financial support of
CICYT (SAF 2003/2262), Comunidad de Madrid (8.5/53.1/
2003), and NeuroPharma S. A., and the predoctoral fellowship
to M.I.F.-B. from CSIC (I3P Program). We also thank Dr. S.
Conde for helpful suggestions and Mr. V. Ga´lvez for technical
assistance. The Sociedad Espan˜ola de Qu´ımica Terape´utica
awarded the “Laboratorios Almirall Prize for Young Researches
- 2005” to a part of this work.
a Taken from ref 25. b Data are the mean of three independent experi-
ments and standard errors are within 5% of the mean.
Table 4. Permeability (Pe × 10-6 cm s-1) in the PAMPA-BBB Assay
for Tacrine-Melatonin Hybrids and Their Predictive Penetration in the
CNS
a
comp
Pe (× 10-6 cm s-1
)
prediction
3a
3b
4a
5a
6a
7a
7b
8a
9a
11a
11.0 ( 0.5
9.2 ( 0.4
7.7 ( 0.3
5.2 ( 0.1
8.4 ( 0.2
7.7 ( 0.1
8.6 ( 0.3
8.0 ( 0.2
8.9 ( 0.1
2.0 ( 0.1
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS-
Supporting Information Available: Experimental details for
new compounds (synthesis and in vitro biological tests). This
material is available free of charge via the Internet at http://
pubs.acs.org.
a Data are the mean (n ) 3) ( SD.
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