Propidium-based polyamine ligands as potent inhibitors of acetylcholinesterase and acetylcholinesterase-induced amyloid-β aggregation

Article abstract of DOI:10.1021/jm049156q

Heterodimers 4 and 5 were effective inhibitors of acetylcholinesterase (AChE) activity and AChE-induced amyloid-β (Aβ) aggregation. The peculiar biological profile of 4 can be relevant in studying the molecular basis underlying the nonclassical action of

Full text of DOI:10.1021/jm049156q

24
J. Med. Chem. 2005, 48, 24-27
Propidium-Based Polyamine Ligands as
Potent Inhibitors of Acetylcholinesterase
and Acetylcholinesterase-Induced
Amyloid-â Aggregation
Maria Laura Bolognesi,* Vincenza Andrisano,
Manuela Bartolini, Rita Banzi, and
Carlo Melchiorre*
Alma Mater Studiorum, University of Bologna,
Department of Pharmaceutical Sciences, Via Belmeloro, 6,
40126, Bologna, Italy
Figure 1. Chemical structures of AChEIs referred to in this
study.
Received October 21, 2004
participate in nucleation of amyloid fibrils and in other
noncatalytic functions.
Abstract: Heterodimers 4 and 5 were effective inhibitors of
acetylcholinesterase (AChE) activity and AChE-induced amyl-
oid-â (Aâ) aggregation. The peculiar biological profile of 4 can
be relevant in studying the molecular basis underlying the
nonclassical action of AChE and in addressing the question
whether AChE inhibitors can affect the neurotoxic cascade
leading to Alzheimer’s disease. Compound 4 emerged as the
most potent heterodimer so far available to inhibit AChE-
induced Aâ aggregation.
Considering the nonclassical AChE functions, their
relationships to AD hallmarks, and the putative role of
PAS in all these functions, dual-site inhibitors may
acquire paramount importance for AD treatment.10
Moreover, bivalency is a successful approach in medici-
nal chemistry for improving drug potency and selectiv-
ity.11 Following this rationale, different bivalent ligands
were obtained by linking through a spacer two moieties
able to contact both acylation and peripheral sites of
AChE. The pharmacophoric units resemble the features
of well-known inhibitors, such as 1,12-14 galantamine,15
linked by alkylene tethers, whose prominent role was
the reduction of the entropy loss, although in some other
instances the spacers provide additional sites of inter-
action.16-18
Alzheimer’s disease (AD) is characterized by selective
neuronal loss in cholinergic population and by massive
deposits of aggregated proteins to form, as peculiar
lesions, the intracellular neurofibrillary tangles and the
extracellular senile plaques. In the last two decades,
basic research has tried to bridge the gap between these
pathological hallmarks and rational pharmacological
treatments, through the formulation of the so-called
“cholinergic”,1 “tau”,2 and “amyloid”3 hypotheses. Not-
withstanding, these hypotheses are not lone and not
mutually exclusive, and the cholinergic pharmaco-
therapy has been the subject of criticism; until 2002,
the only marketed drugs for AD were the acetylcho-
linesterase inhibitors (AChEIs) tacrine (1), donepezil,
rivastigmine, and galantamine.
Recently, following mounting evidence that acetyl-
cholinesterase (AChE) may be involved in functions
distinct from the catalytic activity and defined as
“nonclassical”, a renewed interest in the search of
AChEIs has occurred.4 For example, AChE colocalizes
with amyloid-â peptide (Aâ) in neuritic plaques where
an abnormal expression has been detected.5 There is
also evidence that AChE can enhance the rate of
formation of Aâ fibrils, forming with them stable
complexes.6 It has been speculated that the site respon-
sible for this aggregation-promoting action is localized
close to the entrance of the gorge, perhaps overlapping
the peripheral anionic site (PAS). In fact, inhibition of
the active site did not influence this action, whereas
occupancy of the PAS by the specific inhibitor propidium
(2) blocked it.7 Crystallographic8 and computational9
studies of the AChE/2 complex identified the amino acid
residues that form contacts with allosteric inhibitors,
helping to elucidate the nature of interactions that
The aim of our study was to design a new series of
dual binding site inhibitors based on a polyamine
backbone, exploiting the universal template approach,
a design strategy proposed by our group19 and success-
fully applied in the development of the AChEI caproct-
amine (3).19 We planned to synthesize new bivalent
ligands in which the connecting unit does not act only
as spacer, but plays a role in the target recognition
process, being able to establish its own interactions with
the enzyme. This idea was supported by the consider-
ation that the AChE gorge is lined with several con-
served aromatic residues,20 capable, in principle, to form
cation-π interaction with the basic polyamine counter-
part.16 As proof of principle, we chose a triamine
backbone to link the structural motif of 2, as a scaffold
for contacting the peripheral site, to the tetrahydroami-
noacridine system of 1, or to the N-methyl-2-methoxy-
benzylamino group of 3 for fishing the active site,
affording novel heterobivalent polyamine ligands 4 and
5. In particular, the choice of the tether for heterodimers
4 and 5 was based on the observation that tacrine
dimers bear in their structure, like 4, secondary amino
functions,21 whereas the polyamine backbone of caproct-
amine contains, like that of 5, tertiary amino groups.19
However, a detailed account for the effect on the
biological activity of different tethers, linking the two
terminal aromatic moieties of heterodimers related to
4 and 5, will be reported in due course.
* To whom correspondence should be addressed. Phone: +39-051-
2099700. Fax: +39-051-2099734. E-mail: M.L.B., marialaura.bolognesi@
unibo.it or C.M., carlo.melchiorre@unibo.it.
Heterodimers 4 and 5 were prepared by alkylation of
7 and 12, respectively, with bromide 8,22 followed by
10.1021/jm049156q CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/15/2004

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 25
Scheme 1a
a Reagents: (a) 1-pentanol, KI, reflux; (b) MeOH, reflux, then
MeOH, 12 N HCl, reflux.
Figure 2. Steady-state inhibition by 4 of AChE hydrolysis of
acetylthiocholine (ATCh) (a). Reciprocal plots of initial velocity
and substrate concentration (20-550 µM) are reported. Lines
were derived from a weighted least-squares analysis of data
points. Estimation of Ki value for 4, from slope replot vs
inhibitor concentration (b).
Scheme 2a
Table 1. In Vitro Biological Data of Compounds 1-5
Ki a (nM)
AChE
IC50 (nM)
AChE
IC50 (nM)
BChE
IC50 (µM)
Aâ aggregation
no.
1
2
3
4
5
151 ( 16b
424 ( 21
45.8 ( 3.0
nac
7140 ( 620 32300 ( 2200 13200 ( 400
12.6 ( 2.0
104 ( 16b
1.49 ( 0.52
894 ( 81
170 ( 2.0
1.55 ( 0.16
952 ( 34
11600 ( 300
63.7 ( 2.8
2190 ( 60
nac
13.8 ( 1.8
17.7 ( 1.6
a Estimates of the competitive inhibition constants (Ki) were
obtained from replots of the slopes of the graphs of Lineweaver-
Burk vs inhibitor concentration. b Data from ref 20. c na, not
active; no significant inhibition up to 100 µM concentration.
actions) and butyrylcholinesterase (BChE) was assessed
in comparison with reference compounds 1-3. The
inhibitory potency against AChE and isolated serum
BChE was evaluated using the method of Ellman.24 The
graphical analysis of steady-state AChE inhibition data
for 4 is shown in Figure 2, whereas estimates of
competitive inhibition constants Ki and IC50 values are
reported in Table 1. The ability of 4 and 5 to inhibit the
proaggregating action of AChE toward Aâ was assessed
through a thioflavin T-based fluorometric assay reported
earlier.7,25
As postulated and as seen in other series, hetero-
dimerization resulted in a remarkable increase in AChE
potency; compound 4 was nearly 20 000-fold more potent
than 2 and 300-fold more potent than 1, consistent with
the simultaneous binding to both active and peripheral
sites. The same trend was not shown by compound 5,
which displayed a 34-fold increase in potency compared
to 2, whereas it was 5-fold less potent than 3. In
agreement with the IC50, the Ki values showed that 4
a Reagents: (a) EtOH, molecular sieves 3 Å, NaBH4, room
temperature; (b) HCHO, CH3COOH, NaBH3CN, EtOH, room
temperature; (c) CF3COOH, room temperature; (d) MeOH, reflux,
then MeOH, 12 N HCl, reflux.
acidic hydrolysis of protecting groups. Intermediate 7
was obtained from 623 and N1-(3-aminopropyl)propane-
1,3-diamine with negligible amount of polyalkylation
products, due to the low reactivity of 6 (Scheme 1),
whereas 12 was synthesized from 9, which, upon reduc-
tive amination with 2-methoxybenzaldehyde to yield 10,
was sequentially methylated (11) and deprotected at the
terminal amino group (Scheme 2).
The biological profile of 4 and 5 against human
recombinant AChE (catalytic and Aâ proaggregating

26 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Letters
Supporting Information Available: Full experimental
procedures and characterization data for reported compounds.
This material is available free of charge via Internet at http://
pubs.acs.org.
was the most potent among the investigated compounds.
In this respect, we should note that in using the same
triamine spacer, the distance between the two anchoring
points, the phenantridinium nitrogen atom and, in the
two cases, the endocyclic nitrogen atom of 1 and the
benzylamine of 3, is not the same. Probably, in the case
of 4 the tether length is approaching or is close to the
optimum distance, and, consequently, the ligand can
easily achieve the dual binding, whereas the optimum
tether length for a propidium/N-methyl-2-methoxyben-
zylamine heterodimer may not have been realized in 5.
Alternatively, the substantial difference in the inhibi-
tory AChE activity of 4 and 5 might simply reflect that
the tetrahydroacrydine moiety of 4 is a more potent
catalytic site ligand than the N-methyl-2-methoxyben-
zylamine function of 5. Work to define the proper
polyamine spacer between the two sites is in progress.
An analysis of the Lineweaver-Burk reciprocal plots
of 4 and 5 revealed that there are both an increasing
slope and an increasing intercept with higher inhibitor
concentrations. The inhibitory behavior of 4 and 5, as
illustrated in Figure 2 for 4, is strictly similar to that
displayed by some reported bis-tetrahydroaminoacridine
inhibitors of AChE.24 Therefore, from the kinetic profile,
we derived that compound 4 and 5 cause a mixed type
of inhibition.
Furthermore, the most striking result of the present
investigation was that both our inhibitors markedly
prevented the proaggregating effect of AChE toward Aâ
in the fluorometric assay. In particular, 4 and 5 were
found to inhibit AChE-induced Aâ aggregation with IC50
values comparable to those shown by 2, a specific PAS
ligand and the most effective in this action so far
available. On the contrary, 1 and 3 did not show any
inhibitory activity (Table 1), which parallels their
limited ability to interact with the peripheral site of the
enzyme. Moreover, a 100 µM concentration of 4 and 5
inhibits AChE-induced Aâ aggregation by 95% and 79%,
respectively, a percentage which is severalfold higher
than that of all the other inhibitors ever tested25 and
even significantly higher than that of AP2238, an
inhibitor purposely designed to bind both sites of the
enzyme (35% inhibition).26
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The relevant biological profile displayed by 4 and 5
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In conclusion, we have discovered a new dual-binding
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Acknowledgment. This work was supported by
grants from the University of Bologna (Funds for
selected research topics) and MIUR.

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JM049156Q