Heterocyclic inhibitors of AChE acylation and peripheral sites

Article abstract of DOI:10.1016/j.farmac.2005.03.010

Notwithstanding the criticism to the so called "cholinergic hypothesis", the therapeutic strategies for the treatment of Alzheimer's disease (AD) have been mainly centered on the restoration of cholinergic functionality and, until the last year, the only

Full text of DOI:10.1016/j.farmac.2005.03.010

Il Farmaco 60 (2005) 465–473
http://france.elsevier.com/direct/FARMAC/
Heterocyclic inhibitors of AChE acylation and peripheral sites
Maria Laura Bolognesi *, Vincenza Andrisano, Manuela Bartolini, Andrea Cavalli,
Anna Minarini, Maurizio Recanatini, Michela Rosini, Vincenzo Tumiatti, Carlo Melchiorre
Dipartimento di Scienze Farmaceutiche, Via Belmeloro, 6, 40126 Bologna, Italy
Received 2 September 2004; accepted 30 March 2005
Available online 05 May 2005
Abstract
Notwithstanding the criticism to the so called “ cholinergic hypothesis”, the therapeutic strategies for the treatment of Alzheimer’s disease
(AD) have been mainly centered on the restoration of cholinergic functionality and, until the last year, the only drugs licensed for the man-
agement of AD were the acetycholinesterase (AChE) inhibitors. Target enzyme AChE consists of a narrow gorge with two separate ligand
binding sites: an acylation site at the bottom of the gorge containing the catalytic triad and a peripheral site located at the gorge rim, which
encompasses binding sites for allosteric ligands. The aim of this short review is to update the knowledge on heterocyclic AChE inhibitors able
to interact with the two sites of enzymes, structurally related to the well known inhibitors physostigmine, rivastigmine and propidium. The
therapeutic potential of the dual site inhibithors in inhibiting amyloid-ß aggregatrion and deposition is also briefly summarised.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Alzheimer’s disease; Acetylcholinesterase; Cholinesterase inhibitors; Rivastigmine; Propidium; Amyloid-b
1. Introduction
fibrillogenesis in the etiology of AD [1]. In recent years, sig-
nificant research attention has also been devoted to the role
of free radical formation, oxidative cell damage, and inflam-
mation in the pathogenesis of AD, providing new promising
targets and validated animal models [2]. To date, however,
the therapeutic strategies for the treatment of AD have been
mainly centered on the restoration of cholinergic functional-
ity [3,4] and, until the last year, the only drugs licensed for
the management ofAD were the acetylcholinesterase (AChE)
inhibitors (AChEI) tacrine, [5] donepezil, [6] rivastigmine,
and galantamine [7]. More recently, the role of the overacti-
vation of glutamate receptors in the neuronal death that char-
acterizes AD has been definitely cleared out, and the antago-
nist memantine has been approved in the US in the late 2003
(Chart 1) [8].
Alzheimer’s disease (AD), the most common cause of
senile dementia, is a complex neurological affection that was
described for the first time in 1906 by the Austrian psychia-
trist Alois Alzheimer. Today AD affects 13 million people
worldwide and, with the aging of Western countries popula-
tion, represents a major public health issue. It is clinically
characterized by a relentless irreversible brain degeneration
leading to memory loss, impaired judgment, difficulty in
speech, inability to word and finally death. Besides these
behavioral manifestations, Alzheimer described neuropatho-
logical stigmata in the brain of AD patients that after a cen-
tury, are still considered the definitive diagnosis of the dis-
ease: appearance of amyloid plaques around nerve cells and
neurofibrillary tangles within the cells. Another key charac-
teristic is a dramatic atrophy of certain sections of the brain,
with a specific degeneration of the cholinergic neurons that
appear the most vulnerable cellular population in AD. Even
if the primary cause ofAD is still speculative, several lines of
evidence point toward a central role for the amyloid-b (Ab)
2. AChE acylation site inhibitors
Target enzyme AChE consists of a narrow gorge with two
separate ligand binding sites: an acylation site (A-site) at the
bottom of the gorge that contains the catalytic triad (His-447,
Glu-334, and Ser-203) as well as Trp-86 which binds the tri-
methylammonium head of acetylcholine (ACh), and a peri-
* Corresponding author.
E-mail address: marialaura.bolognesi@unibo.it (M.L. Bolognesi).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.03.010

466
M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
Chart 1
.
Chemical structures of drugs currently approved for the treatment of AD.
pheral site (P-site) located at the gorge rim, which encom-
passes binding sites for allosteric ligands.
The aim of this short review is to update the knowledge on
heterocyclic AChE inhibitors able to interact with the two
sites of the enzyme.
for AD, because of a short half-life, variable bioavailability
and narrow therapeutic index.All these considerations stimu-
lated our interest in designing new physostigmine-related
inhibitors, which showed improved pharmacokinetic proper-
ties over physostigmine, with respect to stability, duration of
action and oral bioavailability. To verify whether the new ring
system may act as a suitable molecular scaffold inAChE rec-
ognition, we synthesized carbamates 4–6 (see Fig. 1). Fur-
thermore, the enantiomers of the most potent derivative 4 were
investigated to verify the importance, if any, of stereochem-
istry on the affinity for AChE. The quaternary derivative 7
was included in this study to verify further the role of a per-
manent positive charge on affinity.
2.1. Physostigmine-related inhibitors
In a previous project, aimed to the development of inhibi-
tors ofAChE based on a polyamine backbone, we discovered
caproctamine (1), a diamine diamide endowed with an inter-
esting affinity profile against AD [9]. In a related study on
constrained polyamines, [10] the terminal 2-methoxybenzyl
groups were included into a hexahydrochromeno[4,3-
b]pyrrole system (2), to verify whether the spatial relation-
ship of the methoxy moiety relative to the amine function
differently affected affinity for cholinergic receptors.
Aryl-functionalized tricyclic pyrrolidines have very few
synthetic precedents in the literature, [13] so this prompted
us to develop an efficient route to target compounds
(Scheme 1).
At that time, the new ring system reminded us the
hexahydropyrrolo[2,3-b]indole system of physostigmine (3),
the prototype of pseudo-irreversible inhibitors ofAChE, which
is able to form a carbamoylated complex with the Ser of the
A- site [11,12]. In spite of the first encouraging results, 3 was
discontinued after the completion of phase III clinical trials
The substituted hexahydrochromeno[4,3-b]pyrrole sys-
tem required for the synthesis of physostigmine-related com-
pounds was synthesized by following a powerful and stereo-
controlled procedure found in the literature for related phenyl-
unsubstituted compounds [14]. It involved a condensation
between an oleifin aldehyde with a secondary amino acid (tri-
Fig. 1. Design strategy for the synthesis of compounds 4–6 by replacing the pyrroline ring of physostigmine (3) with the dihydropyran ring of 2.

M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
467
Scheme 1
.
methylsylil)ester to give an intermediate, which first decar-
boxilates and then undergoes a [3 + 2] cycloaddition to the
internal oleifin (cis fusion only). Thus, starting from
N-methylglycine, chlorotrimethylsilane, and the appropriate
substituted 2-allyloxybenzaldehyde under dehydrating con-
ditions, compounds 11–13 were obtained, which, upon treat-
ment with BBr3, afforded phenols 8–10. The carbamate func-
tionality was introduced by reaction of methyl isocyanate with
8–10, affording 4–6. Quaternary derivative 7 was obtained
by treatment of 5 with methyl iodide.
The stereochemistry of the ring-fusion in compounds
11–13, and as a consequence in the compounds that were
obtained starting from them, was confirmed from the cou-
pling constant of hydrogens at the ring junction. Enanti-
omers (+)-4 and (–)-4 were obtained by HPLC chromato-
graphic resolution of racemic 4.
To determine the potential interest of compounds 4–8 and
enantiomers (+)-4 and (–)-4 for the treatment of AD, their
AChE inhibitory activity was determined by the method of
Ellman et al. [15] in comparison to those of caproctamine
(1), its constrained analog (2), and physostigmine (3). Fur-
thermore, to establish the selectivity of 4–8, their butyrylcho-
linesterase (BChE) inhibitory activity was also calculated by
the same method on BChE from human erythrocytes. An
analysis of the results shown in Table 1 reveals that the posi-
tion of the carbamate moiety in the phenyl ring has a pivotal
role in determining anticholinesterase activity. The
6-substituted derivative 4 is 60–550-fold more potent than 5
and 6, which bear the carbamate group at position 7 and 8,
respectively, at both AChE and BChE. Substituted
hexahydrochromeno[4,3-b]pyrroles 4–6 did not discrimi-
nate, like physostigmine (3), between AChE and BChE. As
Table 1
Inhibition of AChE and BChE activities by physostigmine-related compounds
Number
R
X
IC50 (nM) S.E.M. a
AChE
k3 (min–1
)
BChE
1
2
3
4
caproctamine
170
2
11600 14
903 55
nd b
9740 330
13.4 5.4
nd
physostigmine
6-OCONHCH3
6-OCONHCH3
6-OCONHCH3
7-OCONHCH3
8-OCONHCH3
7-OCONHCH3
6-OH
Me
26.1 1.8
59.4 2.3
53.8 3.9
78.1 11.7
3560 320
10,200 300
2970 300
788,000 26,000
9.0 0.7
NHCH3
NHCH3
NHCH3
NHCH3
NHCH3
N+(CH3)2
NHCH3
30.0 1.7
26.5 1.8
(–)-4
(+)-4
5
6
7
8
29.8 4.7
nd
nd
nd
nd
nd
nd
74.4 7.0
1940 100
16,200 600
1810 70
39,800 2500
a AChE and BChE from human erythrocytes were used. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and
are the mean of two independent measurements, each performed in triplicate.
b nd, not determined.

468
M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
expected, the quaternary derivative 7 was as active as the par-
ent tertiary amino derivative 5. Similarly, the finding that the
phenolic compound 8, lacking the carbamate function, was
more than three orders of magnitude less potent than 4 at
both AChE and BChE parallels the result observed following
removal of the carbamate group from the physostigmine struc-
ture [12].
It is known that inhibition of bothAChE and BChE by 3 is
highly enantioselective, resting almost exclusively on the 3aS
enantiomer [12]. However, enantiomers (–)-4 and (+)-4 did
not follow the same trend as revealed by a comparison of
their pIC50 values atAChE and BChE. It turned out that (–)-4
was 3- and 14-fold more potent than enantiomer (+)-4 atAChE
and BChE, respectively.Although the reason for this discrep-
ancy is not clear yet, the low enantioselectivity observed for
the enantiomers of 4 is not surprising as other physostigmine
derivatives also showed a similar pattern [12].
As previously reported, [16–18] the inhibition of AChE
by 3 involves a reversible complex formation followed by
carbamylation of the enzyme, yielding a covalent adduct. To
compare the mode of action of 4 with that of 3 their equilib-
rium (Kc = k2/k1) and rate (k3) constants were calculated by
performing a traditional stopped assay [17]. The rate con-
stant k3 calculated for 4 were comparable with those found
for 3 (Table 1). This would suggest a similar ‘time depen-
dent’ pattern of inhibition for the two examined inhibitors
[16]. Similarly, Kc value calculated for 3 was in agreement
with the value reported in literature [18].
In conclusion, the hexahydropyrrolo[2,3-b]indole moiety
of physostigmine (3) can be replaced by a hexahydro-
chromeno[4,3-b]pyrrole, as in 4, without affecting the affin-
ity for AChE: clearly, compound 4 could form a lead for the
development of new AChE inhibitors.
Fig. 2. Design strategy for the synthesis of 16–21 by inserting the
dimethylamino-ethyl-phenyl moiety of 14 in different tricyclic systems rela-
ted to 4.
b]pyrrole carbamates as AChE inhibitors, we set about con-
structing a series of conformationally restricted analogs of
14 and 15 by including the dimethylamino-ethyl-phenyl moi-
ety in different tricyclic systems related to 4 (Fig. 2) [22]. In
order to support this basic idea, a superimposition between
the conformation of 14 and the carbon derivative 16, as
obtained from Monte Carlo simulations, was carried out. Low
energy conformations of each molecule were selected and
fitted, as shown in Fig. 3. Clearly, the overlap was satisfac-
tory (RMSD = 0.28 Å), confirming that the tricyclic deriva-
tives might act as rigid analogs of 14. Thus, the isosteric
2.2. Rivastigmine-related inhibitors
Rivastigmine (14; Fig. 2) represents a second-generation
pseudo-irreversibleAChE inhibitor, [16] endowed again with
a carbamate moiety, able to react covalently with the serine
of the A- site. It belongs to a series of miotine (15; Fig. 2)
derivatives, [17] and shows an inhibitory action towardAChE
less marked than physostigmine. However, its superior glo-
bal pharmacological profile, including a good combination
of longer duration of action, good tolerability and lower tox-
icity, led to its approval by FDA in 2000. In addition, 14 is
referred as a ‘brain-region’ selective cholinesterase inhibitor,
[18] since it preferentially inhibits AChE and BChE of the
hippocampus and cortex [19]. Recent evidence suggests that
in the AD brain, BChE activity rises, while AChE activity
remains unchanged or declines. Therefore, both enzymes are
likely involved in regulating ACh levels and, consequently,
may represent legitimate therapeutic targets for the develop-
ment of agents such as 14, which, with the ability to inhibit
BChE in addition to AChE, should lead to improved clinical
outcomes [20].
Fig. 3. Superimposition of a PM3 minimized low energy conformation of 16
onto a PM3 minimized low energy conformation of 14. The pharmacophoric
functions of 16 fit very well those of 14. The rigid analog 16 might indicate
the bioactive conformation of rivastigmine. Figure adapted from [22].
On the basis of these findings and in connection with our
previous studies [21] on the series of benzopyrano[4,3-

M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
469
Scheme 2
.
replacement of the endocyclic oxygen of 4 with a sulfur or a
carbon atom, leading to 17 and 16, respectively, might reveal
information about the physicochemical requirements of the
enzyme binding site, which are still on debate. In addition,
the role of the carbamoyl nitrogen substituent of 4 was inves-
tigated through the synthesis of different aryl and alkyl car-
bamates (18–21).
stant observed for the ring-fusion protons (J = 6.5 Hz) was
consistent with the reported value for cis-isomer 4 [21] and
the cis stereochemistry was further secured from detailed NOE
studies. Methylation of the amine function of 24 and subse-
quent dealkylation of the methoxyl gave phenol 26, which,
upon reaction with the appropriate isocyanate afforded final
compounds 16 and 21.
The previous pathway to 4, namely the [3 + 2] dipolar
cycloaddition of a substituted 2-allyloxibenzaldeyde, was not
deemed practical for the synthesis of the carbon analog 16,
since the preparation of the correspondent 2-butenyl-3-
methoxy-benzaldheyde was not as feasible. Thus, a different
synthetic strategy, starting from the commercially accessible
tetralone 22, was followed (Scheme 2).
Alkylation of the lithium salt of 5-methoxytetralone with
bromoacetonitrile in rigorously anhydrous conditions fur-
nished nitrile 23, which, in turn, was cyclized to 24 through
catalytic hydrogenation over Raney Ni. The coupling con-
On the contrary, the synthesis of 17 could be performed
using the strategy previously described for 4, [21] through
reaction of the key intermediate 2-allylsulfanyl-3-methoxy-
benzaldehyde (29) and sarcosine (trimethylsiliyl)ester
(Scheme 3).
The preparation of 29 was readily accomplished in one
pot by sequential treatment of 28, [23,24] obtained, in turn,
by the Newman–Kwart rearrangement of O-dimethyl-
thiocarbamoylated vanillin (27), with sodium hydroxide and
allyl bromide. After formation of the tricyclic system 30, the
synthesis proceeded as depicted for 4 in Scheme 1 and, in
Scheme 3
.

470
M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
and the parent derivative miotine (15), confirming the ratio-
nal design carried out. Among the restricted analogs, 17
showed the highest inhibitory activity, being 370 and 12-fold
more potent than 14 and 15, respectively.A reasonable expla-
nation for this phenomenon is that the tricyclic system of the
rigid series acts as conformationally restricted mimic of the
3-[1-(dimethylamino)ethy]phenyl fragment of 14, as revealed
by Monte Carlo conformational analyses. In this respect, the
conformation of 16, shown in Fig. 3, might indicate the bio-
active conformation of 14. The restricted compounds have
higher affinity because they do not incur the entropic penalty
experienced when 14 and 15, having a freely rotating skel-
eton, bind.
Scheme 4
.
agreement with the parent system, the cis fusion ring was
assigned through the coupling constant of the benzylic
methine proton.
However, it is well established that several structural ele-
ments concur to determine AChE inhibitory activity of car-
bamate derivatives. For instance, the alkyl substituent on the
carbamoyl nitrogen strongly affects the affinity profile. In our
series of rigid compounds, the most potent inhibitors resulted
methyl derivatives 4, 16–17, all endowed with potency in the
nanomolar range. Increasing the length of the alkyl chain to
ethyl resulted in 40-fold reduction in both the oxygen and
carbon series (4 vs. 20 and 16 vs. 21). A similar behavior was
shown by 18, carrying a bulky R-1-phenylethyl carbamate on
the [1]benzopyrano[4,3-b]pyrrole scaffold. The potency was
restored to a value similar to that of the methyl derivative 4
by lengthening the chain to n-heptyl, affording 19, which dis-
played an IC50 value of 20.3 nM. These results are in good
agreement with the higher potency reported for mono-
substituted carbamate derivatives of 14 carrying a methyl
group instead of an ethyl one [25,26]. The anomalous “ethyl
effect” was also observed by Lieske et al. [27], who found in
a series of indolinylcarbamates, the diethyl derivative about
7400-fold less potent than the dimethylcarbamoyl analog.
Actually, to support these experimental data, the crystal struc-
ture ofAChE/rivastigmine complex has disclosed a steric hin-
drance effect between the ethyl group of 1 and His440 in the
active site, causing a significant movement of this ammi-
noacid away from its normal partner, Glu327, and resulting
in the disruption of the catalytic triad [28].
Finally, carbamoylation of 8 to give the different carbam-
ates 18–20, was accomplished as reported for the preparation
of 4 (Scheme 4).
It should be mentioned that this time the enantiomer sepa-
ration of the different racemic mixtures was not performed,
since we [21] and others [25] have already noticed that the
chirality seems to not strongly affect the inhibitory activity
of this class of compounds.
The inhibitory potency, expressed as IC50 values, of com-
pounds 16–21 of AChE and BChE is reported in Table 2 in
comparison with that of the analog 4, and the two reference
compounds 14 and 15.
The most interesting finding was that the potency towards
AChE is generally increased in the rigidified [1]benzo-
pyrano[4,3-b]pyrrole derivatives compared to the flexible pro-
totype 14. In particular, derivatives 16, 17, and 19 turned out
to be significantly more potent than both rivastigmine (14)
Table 2
Inhibition of AChE and BChE activities by rivastigmine-related compounds
Moreover, the carbamate moiety also affects the kinetic of
AChE carbamoylation, as revealed by the pseudo first order
rate constants kobs, determined for 17 and 19–21, following
the method reported by Feaster and Quinn [29]. From the
data in Table 2, it appears that all of tested compounds showed
a time-dependent pattern of inhibition, which is characteris-
tic of pseudo-irreversible inhibitors and which was similar to
that of 14. The time course of the inhibition was character-
ized by an increase up to a steady state, which was reached
after 20 min in the case of 17, and almost 40 min for 14 and
19–21. This clearly indicates that, once again, the nature of
the substituent of the N-carbamoyl group of tested molecules
plays a role in differentiating the kinetic mechanism of action
and the inhibitory potency as well (IC50 values). As postu-
lated from the crystallographic study on 14, the N-alkyl car-
bamic chain of compounds 19–21 could increase the bulk of
the molecules to an extent where the interaction with the active
Number
X
R
IC50
kobs
(nM) S.E.M.a
AChE
(min–1
)
BChE
14
rivastig-
mine
miotine
O
1535 64
301 14 0.040
15
4
100 33
30 1.7
406 13 nd b
59.4 2.3 nd
24.5 1.6 nd
10.5 0.5 0.365
Me
Me
Me
16
17
18
19
20
21
CH2
S
17.3 1.2
8.11 0.28
O
CH(CH3)Ph 1870 230
(CH2)6CH3 20.3 3.1
209
nd
3
nd
O
0.047
0.049
0.052
O
Et
420
5
nd
CH2
Et
393 33
nd
a Human recombinant AChE and BChE from human serum were used.
IC50 values represent the concentration of inhibitor required to decrease
enzyme activity by 50% and are the mean of two independent measure-
ments, each performed in triplicate.
b nd, not determined.

M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
471
site is disturbed. Thus, the nucleophilic attack of the catalytic
serine might be hampered, because the steric clash is not expe-
rienced by the methyl-carrying derivative 3. In this regard,
compound 17 was the fastest inhibitor of the series, while
ethyl (20 and 21 likewise 14) and heptyl (19) derivatives
showed a much slower enzyme inactivation, as revealed by
their kobs values (Table 2).
phonium, clearly identifying this pro-aggregating activity as
a non-classical AChE function. All these results constitute
the premise for medicinal chemists to the design of periph-
eral and dual binding site inhibitors, which able to simulta-
neously interact with the two sites, might alleviate the cogni-
tive deficit in AD and, what is more important, address the
etiology of the disease. A recent crystal structure of phe-
nylphenantridinium ligands complexed with mouse AChE
unveiled new structural determinants contributing to P-site
ligand interactions, and permitted a detailed topographic
delineation of this site. These structure confirmed propidium
specific binding to the P-site and definitely highlighted the
significant interaction with Trp-286 [36]. Thus, the two Trp
of the A- and P-sites represent the target residues for dual
inhibitors that, spanning the active center and the peripheral
site, might exhibit tighter binding to AChE and a better phar-
macological profile in AD.
Following this rationale, several strategies have been
employed to design high affinity dual inhibitors with dimeric
structure, and all show a marked enhanced potency, com-
pared to the monomer, which interacts with a single site
[37–40].
A very interesting piece of work is represented by the
approach of Sharpless and coworkers who exploited AChE
as a reaction vessel for the selective assembly of building
blocks derived from the two known inhibitors tacrine and pro-
pidium. The triazole-linked bivalent ligand resulting from the
cycloaddition reaction is the most potent noncovalent AChE
inhibitor known to date [41].
Based on these findings and aware thatAChE gorge is lined
with several aromatic amminoacid side chains capable of
forming cation-p interaction with a basic counterpart, we
planned to synthesize bivalent ligands in which the molecu-
lar feature of well known A- and P-site inhibitors were con-
nected through a polyamine chain. In this case, as postulated
by the universal template design strategy, [42] the nature and
the length of the spacer becomes critical, since it plays an
active role in target recognition process. As proof of prin-
ciple, we choose a triamine backbone to link the structural
motif of propidium to the tetrahydroaminoacridine system of
tacrine or to the methoxybenzylamino group of caproc-
tamine, affording novel heterobivalent polyamine ligands
(Fig. 4). The synthesis and the biological activity of these
compounds will be published in due course.
The results at BChE had a potency trend similar to that
observed at AChE: rigid carbamates 4, 16 and 17 were one
order of magnitude more potent than flexible compounds 14
and 15, but, unlike 14, which displayed a preferential BChE
selectivity, showed similar IC50 values for AChE and BChE
inhibition. Recent evidence suggests that, beyond the regula-
tion of synapticACh levels, BChE may also have a role in the
etiology and progression ofAD. Consequently, the new com-
pounds, which have a dual inhibitory action on both AChE
and BChE, might show a better therapeutic profile in AD and
related dementia [20]. The unfavorable effect of the car-
bamic N-alkyl chain on AChE inhibition was less striking
when considering BChE inhibition, as revealed by compar-
ing the IC50 values of the bulky derivative 18 for the two
enzymes. A reasonable explanation might be that BChE is
characterized by a bigger acyl binding pocket than AChE,
[30] thus accounting for the lower clash effect of a wider sub-
stituent towards BChE.
Exchanging the oxygen or the carbon atom with a sulfur
(17 vs. 4 and 16) resulted in a slight increase of both AChE
and BChE inhibition, suggesting a favorable short range inter-
action between the aromatic moiety of Trp86 (AChE) and
Trp82 (BChE) of the active sites and the sulfur atom of the
inhibitor [31].
3. Peripheral and dual binding site AChE inhibitors
Parallel to the challenges towards validity of cholinergic
hypothesis and to the use of AChEI to treat AD, in recent
years much research effort has been devoted to identify new
roles for AChE in neurodegeneration, other than the regula-
tory function in terminating ACh-mediated neurotransmis-
sion. A unique structural feature of this enzyme is the pres-
ence of the peripheral binding site, that was identified in
kinetic studies, and that seems to be fundamental for some of
its so called “non-classical action”.Although our understand-
ing of additional role of AChE is still incomplete, different
experimental evidences point towards a remarkable variety
ofAChE functions. Neuritic plaques fromAlzheimer patients
include catalitically active AChE [32] and AchE–amyloid-b
complexes were shown to be more neurotoxic than amyloid
peptides alone, [33] suggesting that AChE imbalances might
have implications for AD pathogenesis [34]. Furthermore it
has been reported thatAChE promotes amyloid fibrils assem-
bly, by binding to Ab and inducing a conformational transi-
tion to the amyloidogenic conformer [35]. The same authors
found that this activity was blocked by the peripheral site
inhibitor propidium, but not by the active site inhibitor edro-
4. Conclusions
A renewed interest in the search of AChEI has occurred,
following recent evidences which showed thatAChE has sec-
ondary “non cholinergic” functions and that peripheral anionic
site might be somehow related with aggregation and deposi-
tion of Ab peptide.
Accordingly, novel compounds that might increase the
level of ACh and simultaneously interfere with processing of
amyloid appears to be a promising strategy to be explored by
medicinal chemists.

472
M.L. Bolognesi et al. / Il Farmaco 60 (2005) 465–473
Fig. 4
.
Universal template approach to the design of dual binding site AChE inhibitors derived from propidium, tacrine and caproctamine.
[13] S. Hanessian, G. Papeo, M.Angiolini, K. Fettis, M. Beretta,A. Munro,
Moreover, due to the multifaceted pathology ofAD, in the
field of AChEI pharmacotherapy future trends should point
to the development of multipotent drugs, that exploiting inhi-
bition of AChE and hitting different selected targets in the
neurodegeneration cascade, could represent a valuable phar-
macological treatment.
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