Multiple Ligands Targeting Cholinesterases and β-Amyloid: Synthesis, Biological Evaluation of Heterodimeric Compounds with Benzylamine Pharmacophore

Article abstract of DOI:10.1002/ardp.201500117

Alzheimer's disease (AD) is a fatal and complex neurodegenerative disorder for which effective treatment remains the unmet challenge. Using donepezil as a starting point, we aimed to develop novel potential anti-AD agents with a multidirectional biological profile. We designed the target compounds as dual binding site acetylcholinesterase inhibitors, where the N-benzylamine pharmacophore is responsible for interactions with the catalytic anionic site of the enzyme. The heteroaromatic fragment responsible for interactions with the peripheral anionic site was modified and three different heterocycles were introduced: isoindoline, isoindolin-1-one, and saccharine. Based on the results of the pharmacological evaluation, we identified compound 8b with a saccharine moiety as the most potent and selective human acetylcholinesterase inhibitor (IC₅₀=33 nM) and beta amyloid aggregation inhibitor. It acts as a non-competitive acetylcholinesterase inhibitor and is able to cross the blood-brain barrier in vitro. We believe that compound 8b represents an important lead compound for further development as potential anti-AD agent.

Full text of DOI:10.1002/ardp.201500117

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Full Paper
Multiple Ligands Targeting Cholinesterases and b-Amyloid:
Synthesis, Biological Evaluation of Heterodimeric Compounds with
Benzylamine Pharmacophore
Natalia Szałaj1, Marek Bajda1, Katarzyna Dudek1, Boris Brus2, Stanislav Gobec2, and Barbara Malawska1
1
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical
College, Krakow, Poland
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana,
Slovenia
ꢀ
2
Alzheimer’s disease (AD) is a fatal and complex neurodegenerative disorder for which effective treatment
remains the unmet challenge. Using donepezil as a starting point, we aimed to develop novel potential
anti-AD agents with a multidirectional biological profile. We designed the target compounds as dual
binding site acetylcholinesterase inhibitors, where the N-benzylamine pharmacophore is responsible for
interactions with the catalytic anionic site of the enzyme. The heteroaromatic fragment responsible
for interactions with the peripheral anionic site was modified and three different heterocycles were
introduced: isoindoline, isoindolin-1-one, and saccharine. Based on the results of the pharmacological
evaluation, we identified compound 8b with a saccharine moiety as the most potent and selective human
acetylcholinesterase inhibitor (IC50 ¼ 33 nM) and beta amyloid aggregation inhibitor. It acts as a non-
competitive acetylcholinesterase inhibitor and is able to cross the blood–brain barrier in vitro. We believe
that compound 8b represents an important lead compound for further development as potential anti-AD
agent.
Keywords: Alzheimer’s disease / Beta amyloid aggregation inhibitors / Cholinesterase inhibitors /
Multitarget-directed ligand
Received: March 31, 2015; Revised: March 31, 2015; Accepted: April 24, 2015
DOI 10.1002/ardp.201500117
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
aged 65 years and over. In addition to being a leading cause of
death, AD is a leading cause of disability and morbidity [2].
Introduction
Alzheimer’s disease (AD) is a progressive and fatal neurode-
generative disorder which requires an effective therapy. The
World Health Organization estimates that 36 million people
suffer from dementia worldwide and projects that the
number of patients will double by 2030 and more than triple
by 2050 [1]. AD is the fifth-leading cause of death for those
AD is characterized by the loss of central cholinergic neurons,
and disruption of protein folding and aggregation [3].
According to the cholinergic hypothesis proposed in 1976,
memory impairments in AD are associated with a decreased
number of cholinergic neurons [4, 5]. The reduced number of
these neurons results in a lower level of acetylcholine.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
are enzymes involved in the hydrolysis of acetylcholine. Thus,
the accessible treatment for mild to moderate AD includes
cholinesterase inhibitors—donepezil, rivastigmine, and galant-
amine—that enhance cholinergic neurotransmission [6]. For a
long time, AChE was considered as a symptomatic target;
however, numerous studies have shown that AChE is involved
also in non-hydrolytic processes such as neurite growth [7, 8],
Correspondence: Prof. Barbara Malawska, Department of Physi-
cochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian
ꢀ
University Medical College, Medyczna 9, Krakow 30-688, Poland.
E-mail: mfmalaws@cyf-kr.edu.pl
Fax: þ48126205458
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synaptic maintenance [9], and beta amyloid (Ab) aggrega-
them, there are compounds that influence cholinesterases as
a symptomatic target and Ab as a disease-modifying target. In
many cases, these compounds also possess neuroprotective,
antioxidant, or metal chelating properties [29–35].
tion [10]. Crystallographic studies revealed that AChE has two
̊
bindingsites:theactivesiteatthebottomofadeepnarrow20 A
gorge—with the AChE catalytic triad and the anionic subsite
called the catalytic anionic site (CAS)—and the peripheral
anionic site(PAS) near the entranceofthegorge[11]. ThePASis
believed to accelerate the assembly of Ab into amyloid
fibrils [12, 13].
According to hypotheses related to the cause of the disease,
Ab and t protein are two proteins altered in AD [14]. The
amyloid cascade hypothesis has become the conceptual
framework for investigating disease-modifying therapy since
its proposal in 1991 [15–18]. It states that Ab deposition results
from an imbalance between the production and clearance of
Ab. The deposition of Ab in the brain triggers t protein
phosphorylation, neurofibrillary tangles formation, and
neurons death. Thus, disruption of the amyloid cascade can
positively affect the course of the disease and has become one
of the most researched strategies in the development of
disease modifying therapy for AD [19].
We aimed to develop novel potential anti-AD agents with a
multifunctional profile among heterodimeric compounds with
a benzylamine pharmacophore. The purpose of this study was
to explore the structure–activity relationship (SAR) in a new
series of benzylamine derivatives and to select the lead
compound for further development. In this article, we present
thedesign, synthesis, andbiologicalevaluationof a novel series
of compounds. We assessed their inhibitory potency against
AChE, BuChE, and Ab1–42 aggregation. We also tested their
blood–brain barrier (BBB) permeability using the parallel
artificial membrane permeation assay (PAMPA-BBB).
Results and discussion
Design
Unfortunately, all attempts to discover an effective therapy
have failed; only one drug has been approved since 2003
(memantine—NMDA receptor antagonist) [20]. An appraisal
of the 30 years of drug development for AD revealed that
there is still a need to better understand the complex nature
of the disease [21]. The wide range of biological targets make
it unlikely that affecting one alone will lead to satisfactory
therapeutic effects [22]. Therefore, the development of
multitarget-directed ligands (MTDLs, multiple ligands), which
simultaneously hit crucial targets responsible for the causes of
the disease, seems to be an emerging approach in searching
for the treatment of AD. In recent years, many multifunctional
compounds, which affect different targets involved in
pathology of AD, have been developed [23–28]. Among
Recently, we developed a new series of potent AChE inhibitors
composed of an isoindoline-1,3-dione moiety connected by
alkyl linkers to benzylamine [36, 37]. We designed the target
compounds as dual binding site AChE inhibitors capable of
bindingtobothCASandPAS(compound1, Fig. 1).Benzylamine
part of compound 1 was selected as an analog of benzylpiper-
idine in donepezil, where it is responsible for interactions with
the CAS [38]. Isoindoline-1,3-dione was selected as the second
moiety, as it was reported that it is able to interact with the
PAS [39]. Among those hybrid molecules, we identified potent
and selective human AChE (hAChE) inhibitors with additional
biological properties such as Ab aggregation inhibition and
neuroprotective effect against Ab toxicity. Our further studies
confirmed the importance of the benzylamine pharmacophore
Figure 1. Structure of isoindoline-1,3-dione
derivative 1 and general structures of the
designed compounds.
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for the inhibitory potency against AChE [40]. But those studies
did not confirm that substituents in a phenyl ring of benzyl-
amine (chlorine and fluorine atoms) may bring beneficial
effects for their inhibitory potency. Thus, we used compound 1
with the unsubstituted benzylamine pharmacophore as a
model inhibitor in the design of new series of donepezil-based
compounds.
two other water molecules. The benzylamine fragment was
responsible for p–p stacking with Trp84 in the CAS. The
protonated amino group formed cation–p interactions with
Phe330 and a hydrogen bond network with Tyr121 via a water
molecule. The alkyl linker formed hydrophobic interactions
with aromatic residues such as Phe290, Phe331, and Tyr334 in
the middle of the active gorge.
In the presented study, we decided to systematically modify
the heteroaromatic fragment, which is presumably responsi-
ble for interactions with the PAS, in order to investigate the
SAR in a novel series of compounds. We designed a series of
compounds where the benzylamine moiety is connected by
alkyl linkers of different lengths to isoindolin-1-one, isoindo-
line, or 2,3-dihydro-1,2-benzisothiazol-3-one-1,1-dioxide. We
assumed that the reduction of both carbonyl groups in
phthalimide (series A, Fig. 1) would increase the basicity and
provide additional cation–p interactions in the PAS. We also
reduced a single carbonyl group in phthalimide (series B,
Fig. 1) for the purpose of SAR analysis, although isoindolin-1-
one was reported to reduce the potency against AChE in
comparison with phthalimide [41]. We also postulated that
the replacement of a phthalimide fragment in compound 1
with saccharin (series C, Fig. 1) would provide additional
hydrogen bond interactions in the PAS.
According to the docking studies, all the designed
compounds presented similar binding modes with their
benzylamine fragments and alkyl linkers as compound 8b.
The designed compounds only revealed differences in the
binding mode of their heteroaromatic fragments. The
reduction of carbonyl groups in phthalimide provided a
new basic center with potential to form cation–p interactions
with Trp279 in the PAS, but also removed two hydrogen
bonds, previously formed by CO groups with Tyr121 and a
water molecule. It was also noted that an elongation of a
linker in this series of compounds provided favorable
hydrophobic and cation–p interactions of isoindoline with
Trp279. Binding modes of the target compounds showed that
these inhibitors can bind to both CAS and PAS in AChE and
therefore could be described as dual binding site inhibitors.
The dual binding mode is characteristic for donepezil [38] as
well as for previously described isoindoline-1,3-dione deriv-
atives [36, 37, 39].
Molecular modeling
In order to examine the possible interactions of the compounds
presented in this article with AChE, we performed molecular
modeling studies(a dockingposition for thetypicalinhibitor8b
is presented in Fig. 2). Compound 8b was oriented in the gorge
of AChE to create interactions with the CAS and PAS. The
saccharine fragment was engaged in p–p stacking with Trp279
and CH–p interactions with Tyr70 in the PAS. Three oxygen
atomsfromcarbonylandsulfonegroupscreatedfourhydrogen
bonds. The carbonyl group formed a H-bond with a water
molecule while oxygen atoms from sulfone with Tyr121 and
Chemistry
The synthesis of the designed isoindoline and isoindolin-1-one
derivatives was accomplished as shown in Scheme 1. Com-
pounds 2a–e were the key intermediates and were prepared as
previously described [42]. Phthalimide potassium was alkylated
with the appropriate a,v-dibromoalkane in the presence of a
phase transfer catalyst, tetra-n-butylammonium bromide
(TBAB). The reactions were carried out in acetonitrile for 15h
under reflux. In the next step, compounds 2a–e were used as
alkylating agents in reactions with benzylamine according to
thepreviouslyreportedmethod[36].Thereactionswerecarried
out in acetonitrile in the presence of potassium carbonate for
48 h at room temperature. Following purification by silica gel
column chromatography, the final 2-(v-(benzylamino)alkyl)-
isoindoline-1,3-dione derivatives 3a–e were isolated with
satisfactory yields. Then, compounds 3a–e were reduced with
lithium aluminum hydride (LiAlH4) [43]. The reactions were
carried out in THF for 3 h at reflux, followed by silica gel column
chromatography purification, which afforded the expected
compounds4a–ethatwerefinallyconvertedintohydrochloride
salts.
Subsequently, imides 2a–e were selectively reduced to
corresponding isoindolinones using silane derivatives as
reducing agents in the presence of catalytic amounts of
fluoride ions [44, 45]. The reactions of compounds 2a–e with
poly(methylhydrosiloxane) in THF in the presence of tetra-n-
butylammonium fluoride (TBAF) were carried out at room
temperature. After 24 h DCM, trifluoroacetic acid and
triisopropylsilane were added and stirred for 15 min. The
expected lactams 5a–e were isolated after silica gel column
Figure 2. The proposed binding mode of compound 8b in the
active site of acetylcholinesterase obtained by docking.
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Scheme 1. Synthesis of the target isoindoline and isoindolin-1-one derivatives. Reagents and conditions: (a) TBAB, MeCN, reflux,
15 h; (b) benzylamine, K2CO3, MeCN, rt, 48 h; (c) LiAlH4, THF, reflux, 3 h; (d) HCl in 2-propanol; (e) i: PMHS, TBAF, THF, rt, 24 h; ii: i-
PrSiH3, TFA, DCM, rt, 0.25 h.
chromatography purification. In the next step, compounds
5a–e were used as alkylating agents in reactions with
benzylamine. Reactions were carried out in acetonitrile in
the presence of potassium carbonate for 48 h at room
temperature. Following purification by silica gel column
chromatography, the final 2-(v-(benzylamino)alkyl)isoindo-
lin-1-one derivatives 6a–e were isolated with satisfactory
yields and then converted into hydrochloride salts.
2,3-Dihydro-1,2-benzisothiazol-3-one 1,1-dioxide deriva-
tives were prepared according to the pathway described in
Scheme 2. In the first step, alkylation of saccharin sodium salt
with the appropriate a,v-dibromoalkane gave intermediate
compounds 7a–e [46]. The reactions were carried out in DMF
for 24 h under reflux, followed by purification by flash
chromatography. Subsequently, compounds 7a–e were used
as alkylating agents in reactions with benzylamine in DMSO.
N-Alkylation of benzylamine with compounds 7a–e provided
after purification by flash chromatography the target
compounds 8a–e with satisfactory yield [47]. The compounds
8a–e were converted into hydrochloride salts.
are detailed in the experimental section. The biological
evaluation was also performed with the hydrochloride salts.
Biological activity
In vitro evaluation of the inhibition of cholinesterases
To test the potency against cholinesterases, we used Ellman’s
protocol [48]. We assessed the potency against AChE from
Electrophorus electricus (EeAChE), human recombinant AChE
(hAChE), and BuChE from equine serum (EqBuChE). Com-
pound 1, donepezil, and tacrine were included as the
references. The potency measured is given as IC50 values for
the active compounds (Table 1).
We found that the majority of compounds were potent or
moderate inhibitors of EeAChE, with IC50 values ranging from
0.036 to 2.847mM (Table 1). Analyzing the impact of the
modifications in a heteroaromatic fragment, we found that
the introduction of saccharin with an additional carbonyl
group in a place of phthalimide (series C) increased the potency
against EeAChE. Compound 8b (EeAChE IC50 ¼ 36 nM) was two
times more potent than its analog 1 (EeAChE IC50 ¼ 78 nM)
and also displayed selectivity toward AChE. The results
confirmed our previous findings that a five carbon atom
All the final compounds were characterized in the form of
hydrochloride salts and the analytical and spectroscopic data
Scheme 2. Synthesis of the target 2,3-dihy-
dro-1,2-benzisothiazol-3-one-1,1-dioxide
derivatives. (a) DMF, reflux, 24 h; (b) benzyl-
amine, DMSO, 60°C, 3.5 h; (c) HCl in 2-
propanol.
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Table 1. Inhibitory potency against EeAChE, hAChE, and EqBuChE.
EeAChE
EqBuChE
hAChE
Ab1–42 aggreg.
Compounds
n
IC50 (mM)a)
IC50 (mM)a)
IC50 (mM)a)
inh. (%)b)
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
8a
8b
8c
8d
4
5
6
7
8
4
5
6
7
8
4
5
6
7
8
5
–
–
>10c)
6.582 ꢀ 0.262
7.691 ꢀ 0.222
3.401 ꢀ 0.075
2.134 ꢀ 0.030
1.807 ꢀ 0.029
>10c)
>10c)
26.36 ꢀ 19.78
<10d)
>10c)
>10c)
>10c)
2.847 ꢀ 0.079
0.461 ꢀ 0.008
>10c)
>10c)
3.452 ꢀ 0.134
0.920 ꢀ 0.037
>10c)
27.43 ꢀ 14.60
<10d)
<10d)
<10d)
0.460 ꢀ 0.007
0.407 ꢀ 0.005
0.306 ꢀ 0.003
1.119 ꢀ 0.028
0.727 ꢀ 0.025
0.036 ꢀ 0.001
0.288 ꢀ 0.013
0.405 ꢀ 0.024
0.991 ꢀ 0.040
0.078 ꢀ 0.001
0.010 ꢀ 0.001
0.024 ꢀ 0.001
>10c)
0.577 ꢀ 0.017
3.261 ꢀ 0.111
1.261 ꢀ 0.030
2.264 ꢀ 0.069
1.475 ꢀ 0.104
0.033 ꢀ 0.001
0.906 ꢀ 0.039
0.519 ꢀ 0.022
0.890 ꢀ 0.055
0.202 ꢀ 0.004
0.006 ꢀ 0.001
0.131 ꢀ 0.002
<10d)
2.306 ꢀ 0.067
1.914 ꢀ 0.051
0.363 ꢀ 0.010
>10c)
<10d)
25.00 ꢀ 13.34
<10d)
23.15 ꢀ 6.87
22.19 ꢀ 16.68
<10d)
>10c)
2.735 ꢀ 0.070
1.222 ꢀ 0.076
1.776 ꢀ 0.072
>10c)
18.12 ꢀ 13.42
8e
<10d)
1e)
<10d)
Donepezil
Tacrine
1.830 ꢀ 0.176
13.80 ꢀ 6.8
0.002 ꢀ 0.001
–
a) IC50 values are expressed as mean ꢀ standard error of the mean (SEM) of at least three experiments.
b) % Inhibition of self-induced Ab1–42 aggregation at 10 mM; values are expressed as mean ꢀ standard error (SD) of at least four
independent experiments.
c) % Inhibition at 10 mM lower than 50%.
d) % Inhibition at 10 mM lower than 10%.
e) Data from Ref. [36].
linker was the most suitable and elongation or shortening of a
linkercausedadecreaseinpotencyagainstEeAChE(8a:EeAChE
IC50 ¼ 727 nM vs. 8b: EeAChE IC50 ¼ 36nM). In series A and B,
we noticed that the reduction of carbonyl groups in a
phthalimide fragment adversely affected potency against
EeAChE. The compounds with an isoinodolin-1-one fragment
were 5–14 times less potent EeAChE inhibitors than compound
1. Among them, the most potent were compounds 6c and 6d
with six and seven carbon atom linkers. The reduction of a
second carbonyl group resulted in a lack of potency against
EeAChE among compounds with shorter linkers (4a–c). Only
compounds with longer linkers (4d–e) inhibited EeAChE in
the submicromolar range. We expected that reduction of two
carbonyl groups would improve the potency against AChE,
but we observed an opposite effect. Presumably, the inter-
actions between a protonated amine and Tyr279 in the PAS
did not compensate for the lack of two hydrogen bonds
between carbonyl groups and Tyr121 and water in the PAS.
SincemostofthecompoundswerepotentEeAChEinhibitors,
we also tested them against hAChE. We found that the overall
results were related to those obtained for EeAChE and we
observed similar SAR trends. The most potent and selective
hAChE inhibitor was compound 8b with an IC50 value of 33nM.
Only compounds 6c, 6d, and 8c displayed much lower potency
in comparison to potency against EeAChE.
IC50 values in the low micromolar range. The potency against
EqBuChE increased with the length of the linker. The most
potent BuChE inhibitor was compound 6e comprising an eight
carbon atom spacer and isoinodolin-1-one fragment with an
IC50 value of 0.363 mM. It also inhibited AChE, but was
preferential toward BuChE. We identified also selective
inhibitors of EqBuChE among the compounds with an
isoindoline fragment (compounds 4a–c).
Kinetic studies
To investigate the mechanism of EeAChE inhibition, kinetic
studies with the representative compounds from each series
were performed. Substrate–velocity curves in the absence and
the presence of the compounds 4e,6d, and 8b were recorded.
Analysis of Lineweaver–Burk double reciprocal plots showed
that with increasing concentrations of the tested inhibitors,
x-intercepts were the same (Km is unaffected) but slopes were
different (Vmax is decreased) (Fig. 3). This means that the
tested compounds display non-competitive type of inhibition
which is also characteristic for donepezil [49]. Non-competi-
tive type of inhibition may indicate prevailing interactions
with the PAS rather than with the CAS.
Inhibition of self-induced Ab1–42 aggregation
Although senile plaques are one of the hallmarks of AD in
patients’ brains, they are not specific to AD [50]. Senile plaques
are composed of the neurotoxic Ab that is cleaved from amyloid
Regarding the inhibition of EqBuChE, most of the
compounds displayed moderate inhibitory potency with
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Benzylamine Derivatives Targeting Cholinesterases and Ab
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Figure 3. Lineweaver–Burk plots illustrating non-competitive type of EeAChE inhibition by compounds 4c, 6d, and 8b. ATCh,
acetylthiocholine; V, initial velocity rate. Lines were derived from a weighted least-square analysis of data points.
precursor protein (APP). The accumulation and aggregation of
Ab species (mainly Ab1–42) is considered to be responsible for
neurotoxicity in AD. Ab1–42 is highly fibrillogenic and its
oligomers cause membrane disruption in neuronal cells [51].
Therefore, targeting the Ab self-induced aggregation repre-
sentsanemergingapproachindiscoveringdrugcandidateswith
neuroprotective properties. Among the previous benzylamine
derivatives [36], we identified potent inhibitors of self-induced
Ab aggregation (62–66.0% at 10mM). In the presented series of
compounds, we expected to identify more Ab aggregation
inhibitors. We tested all the compounds in thioflavin T-based
assay [52] to investigate their ability to inhibit self-induced Ab
aggregation. Among the tested compounds, six of them
displayed inhibition of Ab1–42 aggregation at 10 mM with the
percentages of inhibition ranging from 18.12 to 27.43%
(Table 1). They were found to be slightly more potent inhibitors
of Ab1–42 aggregation than donepezil but not as potent as our
previousinhibitors.WedidnotobservetherelevantSARsamong
thetested compounds. This may suggest thatthemechanisms of
Ab aggregation inhibition are non-specific.
enzymes, and compounds 8a and 8d inhibited AChE. We found
compound 8bthemostpromising,asitisselectiveAChEinhibitor
with potency similar to donepezil and improved anti-aggrega-
tion properties. This compound is an interesting multifunctional
agentforfurtherdevelopmentasapotentialtherapeuticforAD.
Blood–brain barrier permeation assay
Permeation through the BBB of the CNS drug candidates
should be assessed as early as possible in the drug discovery
process. Therefore, we performed a parallel artificial mem-
brane permeation assay for the BBB (PAMPA-BBB) to
determine the brain permeability of the synthesized benzyl-
amine derivatives (Table 2) [53]. Seven commercial drugs were
used as the reference compounds and the following ranges of
permeability were established: logPe > ꢁ5.4 for compounds
with high BBB permeability; logPe < ꢁ7.3 for compounds with
low permeability, and ꢁ7.3 > logPe > ꢁ5.4 for compounds
with uncertain BBB permeability. We tested the compounds
4e, 6d, 8b, and 1. The results of the PAMPA-BBB assay indicate
that all the tested compounds would be able to cross the BBB
and reach their biological targets in the CNS.
Summarizing the results of biological assays against chol-
inesterases and self-induced Ab aggregation, we found that
compounds 4a, 4c, 6d, 8a, 8b, and 8d displayed moderate
potencytowardtwoorthreetargets.Eachoftheminhibitedself-
induced Ab-aggregation at 10mM. They also inhibited chol-
inesterases in the low micromolar range. Compounds 4a and 4c
selectively inhibited BuChE, compound 6d inhibited both
Conclusions
We have presented here a continuation of our studies in the
group of heterodimeric compounds with a benzylamine
Table 2. Permeability (logPe) in the PAMPA-BBB assay for commercial drugs and the selected compounds with prediction
of their penetration in the CNS.
a)
b)
Compounds
LogPe
Prediction
Compounds
LogPe
Prediction
Theophylline
Verapamil HCl
Lidocaine
Quinidine HCl
Progesterone
Corticosterone
Propranolol HCl
ꢁ7.3
ꢁ3.5
ꢁ4.3
ꢁ3.5
ꢁ5.1
ꢁ5.4
ꢁ3.5
CNSꢁ
CNSþ
CNSþ
CNSþ
CNSþ
CNSꢀ
CNSþ
4e
6d
8b
1
ꢁ4.2
ꢁ3.9
ꢁ3.2
ꢁ3.9
CNSþ
CNSþ
CNSþ
CNSþ
a) Results are the mean of three replicates (n ¼ 3).
b) Results are the mean of two replicates (n ¼ 2).
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moiety.Wedesignednovelseriesofcompoundsasdualbinding
site cholinesterase inhibitors based on the results of our
previousstudiesandwiththeassistanceofmolecularmodeling.
We found that most of the synthesized compounds are potent
AChE inhibitors or dual AChE/BuChE inhibitors with IC50 values
in thelow micromolar to nanomolar range. The introduction of
a saccharine fragment increased the potency against AChE.
Compound 8b (hAChE IC50 ¼ 33 nM) is the most potent and
selective hAChE inhibitor with an IC50 value of 33nM, which is
comparabletodonepezilandseventimesmorepotentthanthe
prototype compound 1 (hAChE IC50 ¼ 202 nM). Kinetic studies
revealedthatthedevelopedcompoundsactasnon-competitive
AChE inhibitors. We also identified selective BuChE inhibitors
among the compounds with an isoindoline fragment. These
compounds (4a–c) inhibit EqBuChE in the low micromolar
range. All the compounds were also assessed for their Ab anti-
aggregation potency in a self-induced aggregation assay. Six of
them(4a,4c,6d,8a,8b,and8d)inhibit Abaggregationat10 mM
concentration. The results of the PAMPA-BBB assay indicate
that the synthesized compounds will be able to cross the BBB.
This study proves that our novel compounds, especially
compound 8b, are viable candidates for further development
as potential anti-AD agents. Moreover, the presented
modifications of the heteroaromatic fragment provided us
with some interesting conclusions about the interactions of
molecules with AChE, which allowed us to expend our AChE
computational model. We found that the introduced saccha-
rine fragment can be a novel lead pharmacophore due to its
positive effect on potency against hAChE.
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Financial support from the Polish Ministry for Science and
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Research Agency (to B.B., and grant No. P1-0208) is gratefully
acknowledged. Ellman’s assays were performed at the
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Krakow, Poland. We also thank Dr. Balint Sinko for his help
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