3-(4-{[benzyl(methyl)amino]methyl}-phenyl)-6,7-dimethoxy-2H-2-chromenone (AP2238) inhibits both acetylcholinesterase and acetylcholinesterase-induced β-amyloid aggregation: A dual function lead for Alzheimer's disease therapy

Article abstract of DOI:10.1021/jm0340602

In recent years, the investigation of acetylcholinesterase (AChE) inhibitors has gained further interest, because the involvement of the peripheral site of the enzyme in the β-amyloid (Aβ) aggregation process has been disclosed. We present here, for the f

Full text of DOI:10.1021/jm0340602

J . Med. Chem. 2003, 46, 2279-2282
2279
catalytic and peripheral AChE binding sites (i.e., dual
inhibitors) can prevent the proaggregating activity of
AChE toward Aâ.5,7 Therefore, peripheral and dual
binding AChE inhibitors might represent a new thera-
peutic option, as these compounds should be able, at
least in principle, both to contrast the cognitive defi-
ciency and to avoid Aâ aggregation.
3-(4-{[Ben zyl(m eth yl)a m in o]m eth yl}-
p h en yl)-6,7-d im eth oxy-2H-2-ch r om en on e
(AP 2238) In h ibits Both
Acetylch olin ester a se a n d
Acetylch olin ester a se-In d u ced â-Am yloid
Aggr ega tion : A Du a l F u n ction Lea d for
Alzh eim er ’s Disea se Th er a p y§
AChE inhibitors targeted at the peripheral or both
sites have already been reported,8 but their effects on
the Aâ proaggregating activity has never been experi-
mentally determined. In this paper, we report design,
synthesis, and in vitro biological properties of a com-
pound that represents a novel class of AChE inhibitors
endowed with the ability to partially block the action
of AChE on Aâ. To this aim, we first designed the new
inhibitors following a computational approach based on
docking simulations carried out on the structure of
human AChE (HuAChE). Then, we selected and syn-
thesized 3-(4-{[benzyl(methyl)amino]methyl}phenyl)-
6,7-dimethoxy-2H-2-chromenone (AP2238, 4, Scheme 1)
through a fast and convenient method. Finally, we
tested on the isolated enzyme the ability of 4 to inhibit
both the catalytic and the Aâ proaggregating actions of
AChE. The results of the in vitro assays compared to
the corresponding data of the leading AD drug, done-
pezil, reveal that 4 can be considered as the protoype
of a new class of compounds endowed with an AChE
inhibitory profile favorable for a therapeutic application
in the treatment of AD.
Lorna Piazzi, Angela Rampa, Alessandra Bisi,
Silvia Gobbi, Federica Belluti, Andrea Cavalli,
Manuela Bartolini, Vincenza Andrisano,
Piero Valenti,* and Maurizio Recanatini
Department of Pharmaceutical Sciences, University of
Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
Received March 10, 2003
Abstr a ct: In recent years, the investigation of acetylcho-
linesterase (AChE) inhibitors has gained further interest,
because the involvement of the peripheral site of the enzyme
in the â-amyloid (Aâ) aggregation process has been disclosed.
We present here, for the first time, a direct evidence of the Aâ
antiaggregating action of an AChE inhibitor (AP2238) pur-
posely designed to bind at both the catalytic and the peripheral
sites of the human enzyme.
In tr od u ction . Alzheimer’s disease (AD) is a progres-
sive, chronic, neurodegenerative disorder, characterized
by loss of cognitive ability, severe behavioral abnormali-
ties, and ultimately death. Treatments of AD are most
likely to succeed if they are based on an understanding
of the complex biology of the disease and its effects on
cognition. At this regard, Aâ is considered to be critical
for inducing the pathology, as its accumulation may
result in a cascade of biochemical events leading to
neuronal dysfunction.1 Therapeutic approaches may be
directed to decreasing Aâ production or aggregation, or
increasing Aâ removal. Alternatively, more distal path-
ways may be targeted, by either modulating down-
stream events possibly due to the presence of Aâ fibrils,
such as free radical toxicity, inflammation, cell mem-
brane damage, calcium dishomeostasis, and excitotox-
icity, or blocking the cellular response to injury through
inhibition of neuronal apoptosis.2 However, AChE in-
hibition is currently the most widely studied and
developed approach for treating the symptoms of AD,
and tacrine, donepezil, galantamine, and rivastigmine
are the AChE inhibitors in clinical use.3
In h ibitor Design . To obtain an inhibitor able to bind
at both the catalytic and the peripheral sites of AChE,
we designed a molecule composed by two moieties
optimal for the binding at each site and linked by an
appropriate spacer. The two selected moieties were a
benzylamino group and a coumarin (2H-2-chromenone)
heterocycle. The first one is present in many potent
AChE inhibitors,9 and its binding capability at the
catalytic center of AChE is demonstrated by the X-ray
crystallographic studies of the AChE/donepezil and
AChE/galantamine complexes.10,11 The coumarin ring
is an heterocyclic moiety we and others demonstrated
to be compatible with a high anti-AChE potency,12,13 and
its choice was also favored by the optimal synthetic
accessibility. As regards the spacer, we reasoned that
a linking chain bearing a phenyl ring might have the
chance to favorably interact with some of the numerous
aromatic residues lining the wall of the AChE gorge.
Recently, it has been pointed out that AChE is also
responsible for several noncatalytic actions.4 Among
these, the proaggregating activity toward Aâ is intrigu-
ing, as some evidences suggest that the enzyme may
play a key role in the development of the AD plaques
by accelerating Aâ deposition.5 Likely, AChE interacts
with Aâ and promotes amyloid fibril formation through
a pool of amino acids located in proximity of the
peripheral anionic binding site (PAS) of the enzyme.6
Moreover, it has been shown that molecules able to
interact either exclusively with PAS or with both
Different combinations of these structural fragments
were modeled, and the docking of the resulting mol-
ecules into the HuAChE gorge was studied by means
of the DOCK 4.1 software.14 Atomic partial charges of
the compounds, whose geometries were optimized at
PM3 semiempirical level,15 were calculated by carrying
out single point ab initio calculations at HF/6-31G(d)
level using Gaussian98 package (Gaussian98, Gaussian,
Inc., Pittsburgh PA, 1994) and then by applying the
restrained electrostatic potential (RESP) procedure.16
The all-atom Amber force field17 was used, and the
energy estimation was carried out without using the
grid approach, to properly represent the π-cation inter-
action. Actually, this enabled us to account also for the
* Corresponding author: Phone: +39 051 2099701.Fax: +39 051
2099734. E-mail: pvalenti@alma.unibo.it.
§ Dedicated to the memory of Professor Paolo Da Re.
10.1021/jm0340602 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/13/2003

2280 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Sch em e 1. Synthesis of Compound 4 (AP2238)a
Letters
a
Reagents: (i) BCl3, CH2Cl2, -78 °C; (ii) p-tolylacetic acid sodium salt, Ac2O, 180 °C; (iii) N-bromosuccinimide, (PhCOO)2, CCl4, reflux;
(iv) N-benzyl-N-methylamine, toluene, reflux. Analytical data: mp 230 °C (MeOH), 1H NMR (CDCl3) δ: 2.41 (s, 3H), 3.68 (s, 2H), 3.69
(s, 2H), 3.93 (s, 3H), 3.95 (s, 3H), 6.84 (d, 2H), 7.19-7.48 (m, 7H), 7.63 (d, 2H), 7.77 (s, 1H). ESMS m/z 416 (M + 1).
multipole/ion contributions, which are fundamental
when modeling π-cation interactions by means of clas-
sical force field-based calculations.18
HuAChE inhibition data for 4 in comparison to done-
pezil is shown in Figure 2, whereas estimates of Ki and
IC50 values are reported in Table 1 along with the
corresponding values for donepezil. From the kinetic
analysis, we concluded that both compounds caused a
mixed type of inhibition, implying binding at both the
catalytic and the peripheral sites of AChE. This was
already known for donepezil,23 while for 4 it was an
indirect confirmation of the inhibitor/enzyme interaction
hypothesis involving both AChE sites described above.
The best results of the docking simulations (Figure
1a) were obtained with compound 4. In the lowest
energy conformation of the ligand/enzyme complex, the
following interactions could be detected between 4 and
some HuAChE residues: (i) the benzyl group of 4
interacts by means of a π-π stacking with the indole
ring of Trp86; (ii) the spacer phenyl ring can establish
both a π-π interaction with the phenol ring of Tyr341
and an OH-π interaction19 with the hydroxyl group of
Tyr124; (iii) the protonated ammonium group of 4 is
involved in a π-cation interaction with the phenol ring
of Tyr337; (iv) the carbonyl group of the coumarin
moiety and eventually the endocyclic oxygen can estab-
lish H-bond interactions with the backbone amide group
of Phe295; (v) the aromatic moiety of the coumarin ring
interacts with Trp286, by means of a π-π stacking.
Indeed, 4 seemed to achieve the desired property. It
contacted the catalytic site of AChE and apparently it
reached the PAS of the enzyme (Trp286 and surround-
ing residues) better than donepezil (Figure 1b).
The IC50 values for inhibition of both cholinesterases
were determined for 4, allowing an estimation of the
eventual selectivity of the compound. From the data
shown in Table 1, a factor of 3 log units appears to favor
AChE with respect to BuChE inhibition.
The ability of 4 to inhibit the proaggregating action
of AChE toward Aâ was assessed through a thioflavin
T-based fluorometric assay early reported by Inestrosa5
and recently validated through circular dichroism.7 The
results are reported in Table 1. Although 4 shows a
lower degree of antiaggregating potency with respect
to the exclusive PAS ligand propidium (82% inhibition7),
it is still able to significantly prevent the AChE-induced
Aâ aggregation. This result is striking, and again it
confirms the design hypothesis pointing to AChE dual-
site inhibitors. Remarkably, we previously reported a
similar (but lower) effect for donepezil,7 that was shown
by X-ray crystallographic analysis to contact both the
catalytic and the peripheral AChE binding sites.10
Ch em istr y. The synthesis of the new inhibitor was
obtained in a rather straightforward way, owing to the
good accessibility of the coumarin nucleus. The target
compound was synthesized by standard procedures
1
(Scheme 1) and was characterized by H NMR, mass
spectra, and elemental analysis. Partial demethylation
of 2,4,5-trimethoxybenzaldehyde with BCl3 gave 2-hy-
droxy-4,5-dimethoxybenzaldehyde (1),20 whose conden-
sation with the sodium salt of p-tolylacetic acid in the
presence of acetic anhydride afforded 6,7-dimethoxy-3-
(4-methylphenyl)-2H-2-chromenone (2). This tolyl in-
termediate was treated with N-bromosuccinimide and
a catalytic amount of benzoyl peroxide to give 3-[4-(bro-
momethyl)phenyl]-6,7-dimethoxy-2H-2-chromenone (3).
The bromo derivative 3 was condensed with N-benzyl-
N-methylamine to afford the final compound.
Biology. The biological profile of both 4 and donepezil
against HuAChE (catalytic and Aâ proaggregating
actions) and butyrylcholinesterase BuChE was deter-
mined.
The inhibitory potency of 4 against recombinant
human AChE and isolated serum BuChE was evaluated
by studying the hydrolysis of acetylthiocholine (ATCh)
following the method of Ellman21 as reported in detail
elsewhere.22 The graphical analysis of steady-state
Discu ssion . Despite their far from optimal pharma-
cological profile, AChE inhibitors are at the moment the
only drugs available for the clinical treatment of AD.
Therefore, besides the exploration of alternative ap-
proaches targeting early events in the neurotoxic cas-
cade, it still seems worthwhile to try to improve this
class of drugs, for which a vast amount of pharmaco-
logical evidence and expertise exists. A suitable strategy
to achieve this goal might be that of simultaneously
targeting another system involved in the pathogenesis
of the disease in addition to the cholinergic one. At this
regard, two ways are open: either to associate to an
AChE inhibitor a drug able to control the clinical
symptoms of AD through a different mechanism, or to
combine into a single compound the ability of acting at
both the AChE and another target.24 Recently appeared
examples of the latter approach13,25,26 consisted of mul-
tipotent compounds able, at least in principle, to provide

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2281
F igu r e 2. Steady-state inhibition by (a) 4 and (b) donepezil
of AChE hydrolysis of ATCh. Reciprocal plots of initial velocity
and substrate concentrations (20-550 µM) are reported. Lines
were derived from a weighted least-squares analysis of data
points.
Ta ble 1. In Vitro Biological Data of AP2238 (4) and Donepezil
% inhibition
Kia (nM)
AChE
IC50 (nM)
AChE
IC50 (nM)
BuChE
of Aâ
compound
AP2238
aggregationb
21.7 ( 1.4 44.5 ( 6.5 48900 ( 3700
35 ( 1
donepezil 20.5 ( 3.3 23.1 ( 4.8c 7420 ( 390
22 ( 3c
a
Estimates of the competitive inhibition constants (Ki) were
obtained from replots of the slopes of the graphs of Figure 2 vs
b
inhibitor concentration. Inhibition of AChE-induced Aâ aggrega-
tion produced by the tested compound at 100 µM concentration.
c Data obtained from ref 7.
ing site.6 Following this line, articles started appearing
in the literature reporting the design and synthesis of
putative dual-binding AChE inhibitors.8
F igu r e 1. (a) Docking model of 4 (carbon atoms are green)
within the HuAChE gorge. The H-bond between the carbonyl
of the coumarin moiety of 4 and the hydrogen (cyan) of the
Phe295 backbone NH is shown as a yellow dotted line. The
hydrogen (cyan) of the hydroxyl group of Tyr124, which can
establish OH-π interaction with a phenyl ring of 4, is also
shown. (b) The X-ray structure of donepezil (carbon atoms are
magenta) within the Torpedo californica AChE gorge (PDB
code: 1eve) is reported for comparison: 4 seems to contact the
peripheral Trp better than donepezil.
In this letter, we report for the first time a direct
evidence of the Aâ antiaggregating action of an AChE
inhibitor purposely designed to bind at both the catalytic
and the peripheral sites of the human enzyme. The
ability of the compound 4 to contrast the Aâ aggregation
is higher than that of other AChE inhibitors tested,7
with the exception of propidium, which is a bis-
quaternary ammonium PAS ligand in no way useful for
the treatment of AD.
In addition, 4 shows a potency against HuAChE
which is comparable to that of donepezil, and a quite
high selectivity for AChE with respect to BuChE. This
remarkable selectivity, which largely overcomes that
shown by donepezil, suggests some considerations.
Actually, a peculiar structural difference between the
a synergistic effect toward different molecular and
biochemical targets of AD.
As one of the central events in the AD pathogenesis
is the Aâ fibrillization, an intervention against this
phenomenon seems crucial. Interestingly, research ef-
forts pioneered by Inestrosa5 and recently reappraised
by us7 have pointed out a direct link between AChE and
Aâ aggregation: AChE was shown to exert an Aâ
proaggregating action mediated by its peripheral bind-

2282 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Letters
(9) Sugimoto, H.; Yamanishi, Y.; Iimura, Y.; Kawakami, Y. Done-
pezil hydrochloride (E2020) and other acetylcholinesterase
inhibitors. Curr. Med. Chem. 2000, 7, 303-339.
two cholinesterases is the lack of the PAS (in particular
of Trp286) in BuChE.27 This seems to prevent the
interaction of BuChE with Aâ, such that no role on Aâ
aggregation can be played by this enzyme.5 For instance,
tacrine, a nonselective mixed-type AChE inhibitor, binds
more tightly to BuChE and is almost inactive in inhibit-
ing the AChE-promoted Aâ aggregation.7 This inhibitor
mostly interacts with AChE catalytic site,28 as it likely
does with BuChE. In contrast, propidium, a well-known
AChE inhibitor binding exclusively at the PAS, is almost
inactive against BuChE and strongly inhibits the AChE-
induced Aâ aggregation.7 The results presented in Table
1 confirm the phenomenological correlation between
AChE selectivity and inhibition of the AChE-induced
proaggregating action. It might be concluded that
inhibitors of AChE able to strongly interact with the
PAS show high AChE/BuChE selectivity and eventually
an inhibitory action against AChE-induced Aâ aggrega-
tion. We might therefore advance that AChE/BuChE
selectivity is key information when searching new
potential inhibitors of AChE-induced Aâ aggregation.
In conclusion, here we present evidence of the in vitro
Aâ antiaggregating action of an AChE inhibitor that
was designed in such a way as to be able to contact
simultaneously both the catalytic and the peripheral
binding sites of the enzyme. This action combines with
an elevated AChE inhibitory potency to provide a
compound endowed with a potentially useful biological
profile for the treatment of AD. By the way, we also
showed how the integration of focused computational,
synthetic and biochemical efforts led to the identification
and in vitro characterization of a new lead compound
bearing the desired biological properties.
(10) Kryger, G.; Silman, I.; Sussman, J . L. Three-dimensional
structure of a complex of E2020 with acetylcholinesterase from
Torpedo californica. J . Physiol. Paris 1998, 92, 191-194.
(11) Bartolucci, C.; Perola, E.; Pilger, C.; Fels, G.; Lamba, D. Three-
dimensional structure of a complex of galanthamine (Nivalin)
with acetylcholinesterase from Torpedo californica: implications
for the design of new anti-Alzheimer drugs. Proteins 2001, 42,
182-191.
(12) Rampa, A.; Bisi, A.; Valenti, P.; Recanatini, M.; Cavalli, A.;
Andrisano, V.; Cavrini, V.; Fin, L.; Buriani, A.; Giusti, P.
Acetylcholinesterase inhibitors: synthesis and structure-activ-
ity relationships of omega-[N-methyl-N-(3-alkylcarbamoyloxy-
phenyl)methyl]aminoalkoxyheteroaryl derivatives. J . Med. Chem.
1998, 41, 3976-3986.
(13) Bruhlmann, C.; Ooms, F.; Carrupt, P. A.; Testa, B.; Catto, M.;
Leonetti, F.; Altomare, C.; Carotti, A. Coumarins derivatives as
dual inhibitors of acetylcholinesterase and monoamine oxidase.
J . Med. Chem. 2001, 44, 3195-3198.
(14) Kuntz, I. D.; Blaney, J . M.; Oatley, S. J .; Langridge, R.; Ferrin,
T. E. A geometric approach to macromolecule-ligand interactions.
J . Mol. Biol. 1982, 161, 269-288.
(15) Stewart, J . P. P. Optimization of parameters for semiempirical
methods I. Methodol. J . Comput. Chem. 1989, 10, 209-220.
(16) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A well-
behaved electrostatic potential based method using charge
restraints for determining atom-centered charges: the RESP
model. J . Phys. Chem. 1993, 97, 10269-10280.
(17) Wendy, D. C.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.
M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J . W.;
Kollman, P. A. A second generation force field for the simulation
of proteins, nucleic acids, and organic molecules. J . Am. Chem.
Soc. 1995, 117, 5179-5197.
(18) Dougherty, D. A. Cation-pi interactions in chemistry and biol-
ogy: a new view of benzene, Phe, Tyr, and Trp. Science 1996,
271, 163-168.
(19) Sulpizi, M.; Carloni, P. Cation-pi versus OH-pi interactions in
proteins: A density functional study. J . Phys. Chem. B 2000,
104, 10087-10091.
(20) Sullivan, B.; Djura, P.; McIntyre, D. E.; Faulkner, D. J .
Antimicrobial constituents of the Sponge Siphonodictyon Cor-
raliphagum. Tetrahedron 1981, 37, 979-982.
(21) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M.
A new and rapid colorimetric determination of acetylcholin-
esterase activity. Biochem. Pharmacol. 1961, 7, 88-95.
(22) Rampa, A.; Bisi, A.; Belluti, F.; Gobbi, S.; Valenti, P.; Andrisano,
V.; Cavrini, V.; Cavalli, A.; Recanatini, M. Acetylcholinesterase
inhibitors for potential use in Alzheimer’s disease: molecular
modeling, synthesis and kinetic evaluation of 11H-indeno-[1,2-
b]-quinolin-10-ylamine derivatives. Bioorg. Med. Chem. 2000,
8, 497-506.
(23) Snape, M. F.; Misra, A.; Murray, T. K.; De Souza, R. J .; Williams,
J . L.; Cross, A. J .; Green, A. R. A comparative study in rats of
the in vitro and in vivo pharmacology of the acetylcholinesterase
inhibitors tacrine, donepezil and NXX-066. Neuropharmacology
1999, 38, 181-193.
(24) Recanatini, M.; Cavalli, A. Acetylcholinesterase inhibitors in the
context of therapeutic strategies to combat Alzheimer’s disease.
Expert Opin. Ther. Pat. 2002, 12, 1853-1865.
(25) Melchiorre, C.; Andrisano, V.; Bolognesi, M. L.; Budriesi, R.;
Cavalli, A.; Cavrini, V.; Rosini, M.; Tumiatti, V.; Recanatini, M.
Acetylcholinesterase noncovalent inhibitors based on a polyamine
backbone for potential use against Alzheimer’s disease. J . Med.
Chem. 1998, 41, 4186-4189.
(26) Ildiko, M. B.; Gyorgy, T.; Sandor, M.; Tivadar, T.; Gyorgy, S.;
Eva, M. Preparation of N-disubstituted carbamoyloxy flavones
as therapeutic agents. PCT Int. Appl. WO 0224677, A1 20020328,
2002.
(27) Massoulie, J .; Sussman, J .; Bon, S.; Silman, I. Structure and
functions of acetylcholinesterase and butyrylcholinesterase.
Progr. Brain Res. 1993, 98, 139-146.
(28) Sussman, J . L.; Harel, M.; Silman, I. Three-dimensional struc-
ture of acetylcholinesterase and of its complexes with anticho-
linesterase drugs. Chem. Biol. Interact. 1993, 87, 187-197.
Ack n ow led gm en t . This investigation was sup-
ported by University of Bologna (funds for selected
research topics).
Refer en ces
(1) Hardy, J .; Selkoe, D. J . The amyloid hypothesis of Alzheimer’s
disease: progress and problems on the road to therapeutics.
Science 2002, 297, 353-356.
(2) Dominguez, D. I.; De Strooper, B. Novel therapeutic strategies
provide the real test for the amyloid hypothesis of Alzheimer’s
disease. Trends Pharmacol. Sci. 2002, 23, 324-330.
(3) Moghul, S.; Wilkinson, D. Use of acetylcholinesterase inhibitors
in Alzheimer’s disease. Expert Rev. Neurotherapeutics 2001, 1,
61-69.
(4) Soreq, H.; Seidman, S. Acetylcholinesterasesnew roles for an
old actor. Nat. Rev. Neurosci. 2001, 2, 294-302.
(5) Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente,
M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J . Acetyl-
cholinesterase accelerates assembly of amyloid-beta-peptides
into Alzheimer’s fibrils: possible role of the peripheral site of
the enzyme. Neuron 1996, 16, 881-891.
(6) De Ferrari, G. V.; Canales, M. A.; Shin, I.; Weiner, L. M.; Silman,
I.; Inestrosa, N. C. A structural motif of acetylcholinesterase that
promotes amyloid beta-peptide fibril formation. Biochemistry
2001, 40, 10447-10457.
(7) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Beta-
amyloid aggregation induced by human acetylcholinesterase:
inhibition studies. Biochem. Pharmacol. 2003, 65, 407-416.
(8) Castro, A.; Martinez, A. Peripheral and dual binding site
acetylcholinesterase inhibitors: implications in treatment of
Alzheimer’s disease. Mini Rev. Med. Chem. 2001, 1, 267-272.
J M0340602