Design, synthesis, and biological evaluation of 2-(benzylamino-2-hydroxyalkyl)isoindoline-1,3-diones derivatives as potential disease-modifying multifunctional anti-Alzheimer agents

Article abstract of DOI:10.3390/molecules23020347

The complex nature of Alzheimer's disease calls for multidirectional treatment. Consequently, the search for multi-target-directed ligands may lead to potential drug candidates. The aim of the present study is to seek multifunctional compounds with expected activity against disease-modifying and symptomatic targets. A series of 15 drug-like various substituted derivatives of 2-(benzylamino-2-hydroxyalkyl)isoindoline-1,3-diones was designed by modification of cholinesterase inhibitors toward β-secretase inhibition. All target compounds have been synthesized and tested against eel acetylcholinesterase (eeAChE), equine serum butyrylcholinesterase (eqBuChE), human β-secretase (hBACE-1), and β-amyloid (Aβ-aggregation). The most promising compound, 12 (2-(5-(benzylamino)-4-hydroxypentyl)isoindoline-1,3-dione), displayed inhibitory potency against eeAChE (IC₅₀ = 3.33 μM), hBACE-1 (43.7% at 50 μM), and Aβ-aggregation (24.9% at 10 μM). Molecular modeling studies have revealed possible interaction of compound 12 with the active sites of both enzymes-acetylcholinesterase and β-secretase. In conclusion: modifications of acetylcholinesterase inhibitors led to the discovery of a multipotent anti-Alzheimer's agent, with moderate and balanced potency, capable of inhibiting acetylcholinesterase, a symptomatic target, and disease-modifying targets: β-secretase and Aβ-aggregation.

Full text of DOI:10.3390/molecules23020347

molecules
Article
Design, Synthesis, and Biological Evaluation of
-(Benzylamino-2-Hydroxyalkyl)Isoindoline-1,3-
2
Diones Derivatives as Potential Disease-Modifying
Multifunctional Anti-Alzheimer Agents
Dawid Panek 1 , Anna Wi e˛ ckowska
,†
1,† ID
, Anna Pasieka , Justyna Gody n´ , Jakub Jo n´ czyk ,
1
1
1
1
2
2
1,
Marek Bajda , Damijan Knez , Stanislav Gobec and Barbara Malawska *
1
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30-688 Kraków, Poland; dawid.panek@doctoral.uj.edu.pl (D.P.);
anna.wieckowska@uj.edu.pl (A.W.); pasieka.ann@gmail.com (A.P.); justyna.godyn@doctoral.uj.edu.pl (J.G.);
jakub.jonczyk@doctoral.uj.edu.pl (J.J.); marek.bajda@uj.edu.pl (M.B.)
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Ašker cˇ eva 7,
1
000 Ljubljana, Slovenia; damijan.knez@ffa.uni-lj.si (D.K.); stanislav.gobec@ffa.uni-lj.si (S.G.)
*
†
Correspondence: mfmalaws@cyf-kr.edu.pl; Tel.: +48-12-620-5464
These authors contributed equally to this work.
Received: 17 January 2018; Accepted: 3 February 2018; Published: 7 February 2018
Abstract: The complex nature of Alzheimer’s disease calls for multidirectional treatment.
Consequently, the search for multi-target-directed ligands may lead to potential drug candidates.
The aim of the present study is to seek multifunctional compounds with expected activity
against disease-modifying and symptomatic targets. A series of 15 drug-like various substituted
derivatives of 2-(benzylamino-2-hydroxyalkyl)isoindoline-1,3-diones was designed by modification
of cholinesterase inhibitors toward
synthesized and tested against eel acetylcholinesterase (eeAChE), equine serum butyrylcholinesterase
eqBuChE), human -secretase (hBACE-1), and -amyloid (A -aggregation). The most promising
compound, 12 (2-(5-(benzylamino)-4-hydroxypentyl)isoindoline-1,3-dione), displayed inhibitory
potency against eeAChE (IC50 = 3.33 M), hBACE-1 (43.7% at 50 M), and A -aggregation (24.9%
at 10 M). Molecular modeling studies have revealed possible interaction of compound 12 with the
active sites of both enzymes—acetylcholinesterase and -secretase. In conclusion: modifications of
β-secretase inhibition. All target compounds have been
(
β
β
β
µ
µ
β
µ
β
acetylcholinesterase inhibitors led to the discovery of a multipotent anti-Alzheimer’s agent, with
moderate and balanced potency, capable of inhibiting acetylcholinesterase, a symptomatic target, and
disease-modifying targets: β-secretase and Aβ-aggregation.
Keywords: isoindoline-1,3-dione derivatives; cholinesterase inhibitors; BACE-1 inhibitors;
Aβ-aggregation; molecular modeling; multiple anti-Alzheimer’s ligands
1
. Introduction
Alzheimer’s disease (AD) is one of the neurodegenerative diseases and the most commonly
diagnosed cause of dementia in the elderly. It affects an estimated 36 million people worldwide,
and this figure is expected to increase to 100 million by the end of 2050 [ ]. The lack of an effective
1
disease-modifying treatment for AD, caused, among others, by the disease’s poorly understood
and complex etiopathogenesis, is a major challenge facing modern medicine. Deposits of insoluble
proteins:
AD. A
BACE-1) and
β
-amyloid (A
is the product of enzymatic cleavage of amyloid precursor protein (APP) by
-secretase. Various forms of A , primarily A 1 and A 1–40, have the ability to
β
) and hyperphosporylated tau are regarded as the primary cause of
β
β-secretase
(
γ
β
β
β
–42
Molecules 2018, 23, 347; doi:10.3390/molecules23020347
www.mdpi.com/journal/molecules

Molecules 2018, 23, 347
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aggregate and create extracellular neurotoxic senile plaques [
2
]. In parallel, inside neurons, excessive
phosphorylation of the tau protein leads to the formation of neurofibrillary tangles [
may begin up to 25 years before initial symptoms occur [ ]. As complementary elements of the
etiopathogenesis of AD, they represent obvious biological targets in search for anti-AD treatments.
Progressive neurodegeneration in the course of AD affects mainly cholinergic neurotransmission [ ].
3]. Both processes
4
5–7
Current pharmacotherapy of AD is therefore based on the restoration of cholinergic activity with
cholinesterase inhibitors: donepezil, rivastigmine, and galantamine. These drugs do not prevent,
reverse, or even slow down the neurodegenerative processes, but offer only symptomatic treatment
based on improving cognition and memory [8]. Therefore, the overriding objective for researchers
is to find new, effective disease-modifying agents for the treatment of AD. Complex diseases,
such as AD, require multidirectional treatment. An interesting form of polypharmacology involves
multi-target-directed-ligands (MTDLs), which are compounds that act on two or more biological targets
and mechanisms [
the context of multifactorial diseases such as AD [10
anti-AD molecules has been designed and synthesized by combining chemical fragments responsible
for interaction with desirable biological targets [14 18]. In this paper, we present the design, synthesis,
9
]. This approach has developed intensively over the last few years, particularly in
–
13]. A large number of potential multifunctional
–
biological evaluation, and molecular modeling studies of new phthalimide derivatives linked by a
hydroxyalkyl chain with a benzylamine moiety, as novel, potential, multifunctional anti-AD agents.
Biological profiling of these new compounds includes the assessment of acetylcholinesterase from
Electrophorus electricus (eeAChE), butyrylcholinesterase from equine serum (eqBuChE), BACE-1, and A
β
aggregation inhibition.
2
. Results and Discussion
2
.1. Design
Our main area of interest is the development of new, multifunctional compounds which target
the causes of AD. Previously, we have reported a series of dual binding site donepezil-based inhibitors
of AChE with anti-A aggregating properties. Compound III (Figure 1) was found to be a potent
and selective human AChE inhibitor (IC50 = 0.268 M) with anti-A aggregation activity (66% at
M) and a neuroprotective effect against A toxicity at 1 M and 3 M [19]. Molecular modeling
β
µ
β
1
0
µ
β
µ
µ
studies revealed that the phthalimide fragment of compound III is responsible for interactions with
the peripheral anionic site (PAS), while the benzylamine moiety interacts with the catalytic anionic
site of AChE. The goal of the presented study was modification of lead III to introduce additional
inhibitory activity toward BACE-1. A 2-(benzylamino)ethan-1-ol fragment is frequently incorporated
in potent BACE-1 inhibitors; among others, NVP-BXD552 (I) [20] and GRL-8234 (II) [21] (Figure 1).
Therefore, we introduced a hydroxyl group in the alkyl chain of compound III and assumed that the
resulting hydroxyethylamine fragment will mediate interaction with the catalytic dyad of BACE-1
(composed of two aspartic residues: Asp32 and Asp228), which is the key to the effective inhibition of
BACE-1. Moreover, we calculated the most important physicochemical parameters for the designed
compounds and confirmed that they meet the criteria set by Lipinski’s rule of five [22] and possess
drug-like properties.

Molecules 2018, 23, 347
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Figure 1. Design strategy for multitarget anti-Alzheimer’s agents. CAS: catalytic anionic site, PAS:
peripheral anionic site.
2
.2. Chemistry
We synthesized 15 derivatives with various substituents in the benzylamine fragment and different
linker lengths based on results of molecular modeling. The general procedure for the synthesis
of the final compounds ( 15) is presented in Scheme 1. Alkylation of potassium phthalimide by
-bromobut-1-ene derivatives and subsequent oxidation with meta-chloroperoxybenzoic acid (mCBPA)
1
–
ω
yielded epoxides (20–23). These epoxides were then used as alkylating agents in a reaction with the
appropriate benzylamines, leading to final compounds (1–15) in their racemic form.
◦
1–15. Reagents and conditions: (i) alkenyl halide, DMF, 80 C, 16 h;
Scheme 1. Synthesis of compounds
ii) meta-chloroperoxybenzoic acid, DCM, room temperature (R.T.) 16 h; (iii) benzylamine derivatives,
(
◦
n-propanol, pyridine, 97 C.
2
.3. Biological Evaluation
Target compounds
against acetyl- and butyrylcholinesterase,
1
–
15 were subjected to biological screening to test their inhibitory activity
-secretase, and aggregation of A . We evaluated the
β
β
potency of the compounds against cholinesterases in Ellman’s assay [23] using AChE from Electrophorus
electricus (eeAChE) and BuChE from equine serum (eqBuChE). We used tacrine and donepezil as
reference compounds. Following initial screening at 10
µM, we determined IC50 values for compounds
which exhibited potency greater than 50%. Eleven out of the fifteen compounds display percentages

Molecules 2018, 23, 347
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of inhibition of eeAChE between <10% and 33% at a concentration of 10
µM (Table 1). Similarly,
fourteen out of the fifteen compounds display % inhibition of eqBuChE between <10% and 36%
at a concentration of 10 M. Four compounds exhibit moderate eeAChE activity (low micromolar
µ
IC ) and one compound exhibits moderate eqBuChE inhibitory (low micromolar IC ). The most
50
50
potent AChE inhibitors, with IC50 ranging from 1.95 to 11.07
µ
M, were compounds 12
–
15 with longer
3- and 4-carbon atom linkers. This observation is similar to our previous findings [19] concerning
phthalimide-benzylamine derivatives as cholinesterase inhibitors (compound III). The optimal linker
length, ensuring interaction with both catalytic anionic site CAS and PAS, was five carbon atoms
(compound III), corresponding to n = 3 in compounds discussed in this work. Compounds with
shorter linkers (n = 1 and n = 2) are weak inhibitors of eeAChE (11.5–32.7% inhibition at 10 M),
µ
or are altogether inactive (<10% inhibition). We have not observed any structure-activity relationship
regarding substituents at the benzylamine ring; however, we only synthesized differently substituted
derivatives with the shortest linkers. Regarding inhibition of eqBuChE, we found compound
for which we determined the IC50 value (7.86 M). Unlike the case of AChE inhibition, the most
potent inhibitor of BuChE was those compounds with shorter carbon linkers (n = 1). Comparing the
unsubstituted benzylamine derivative with the 2-methoxy substituted one ( versus ) we conclude
5,
µ
1
5
that this substitution is important for BuChE inhibitory activity. The results obtained show that
introduction of the hydroxyl group to the alkyl chain led to a reduction in inhibitory activity with
respect to both cholinesterase compared to the model compound III.
To determine the inhibitory activity of the reported compounds against human recombinant
BACE-1 (hBACE-1), we used a biochemical spectrofluorometric assay (fluorescence resonance energy
transfer, FRET-based) [24,25]. The assay is based on the cleavage of an amyloid precursor protein
APP analogue with the Swedish mutation, leading to an increase in fluorescence. As a reference, we
included inhibitor IV and established its IC50 value at 46 nM, which is in accordance with published
data [26]. We tested each compound at 50
which ranged from 15.7% for 15 to 45.0% for 6 (Table 1).
We determined A antiaggregating properties for the presented compounds (
thioflavin-T assay [27]. The assay was performed at 10 M inhibitor concentration. Donepezil was
used as the reference, since the presented compounds had been structurally developed from this drug.
We also included resveratrol as a much stronger inhibitor of A aggregation (Table 1). We found
aggregation: , and 12, with inhibition
M) (Figure 2). These results are inferior to
µM concentrations and calculated their inhibitory activity,
β
1–15) in a
µ
β
that three of the tested compounds significantly inhibit A
percentages of 21.1%, 19.9%, and 24.9%, respectively (at 10
resveratrol but superior to donepezil.
β
µ
2, 5
%
Aβ-aggr.
100
8
0
0
0
0
0
6
4
2
control
resveratrol
donepezil
2
5
12
Aβ-aggregation
Compound
Figure 2. Effect of the synthesized compounds on A
standard deviation (SD) for at least two independent experiments.
β
aggregation. Values are expressed as means
±

Molecules 2018, 23, 347
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Table 1. Inhibitory activity on eeAChE, eqBuChE, hBACE-1, and Aβ for the presented compounds.
eeAChE a
eqBuChE b
hBACE-1 c.
Aβ-Aggr. d
% Inh. f
Cmp.
R
n
e
f
e
f
% Inh. f
IC50 (µM) /% Inh.
IC50 (µM) /% Inh.
1
2
3
4
5
6
7
8
9
H
2-F
2-CF3
3-Cl
2-OMe
4-Me
4-OMe
2,4-diCl
H
2-OMe
4-OMe
H
1
1
1
1
1
1
1
1
2
2
2
3
3
4
4
11.5% ± 5.0%
28.1% ± 2.0%
11.8% ± 5.0%
<10%
14.1% ± 3.3%
19.5% ± 2.0%
<10%
24.1% ± 4.7%
7.86 ± 0.29
11.6% ± 3.3%
19.1% ± 1.5%
21.7% ± 1.0%
<10%
37.3% ± 7.5%
39.6% ± 6.5%
41.8% ± 4.5%
34.1% ± 7.7%
39.2% ± 9.2%
45.0% ± 2.0%
35.1% ± 10.0%
37.2% ± 1.3%
<10%
24.4% ± 5.9%
34.8% ± 10.6%
43.7% ± 6.9%
37.1% ± 3.9%
42.1% ± 10.7%
15.7% ± 1.3%
<10%
21.1% ± 6.6%
<10%
<10%
<10%
<10%
<10%
<10%
19.9% ± 1.5%
<10%
<10%
<10%
<10%
<10%
<10%
10
11
12
13
14
15
32.7% ± 4.7%
17.8% ± 2.0%
<10%
<10%
<10%
3.32 ± 0.15
11.07 ± 0.49
2.13 ± 0.07
1.95 ± 0.06
14.1% ± 5.8%
26.2% ± 9.7%
36.0% ± 7.2%
36.0% ± 1.1%
24.9% ± 6.6%
2-OMe
H
2-OMe
<10%
<10%
<10%
Reference compounds
Donepezil
Tacrine
Inhibitor IV
0.011 ± 0.0002
1.83 ± 0.04
n.d. g
n.d.
13.8% ± 6.8
g
g
0.023 ± 0.0004
0.015 ± 0.0001
n.d.
h
g
g
i
g
n.d.
n.d.
0.046 ± 0.001
n.d.
g
g
g
Resveratrol
n.d.
n.d.
n.d.
78.5% ± 5.2
a
b
c
IC50 inhibitory concentration of AChE from electric eel or inhibition percentage at 10
µM inhibitor concentration.
IC50 inhibitory concentration of BuChE from horse serum or inhibition percentage at 10
µ
M inhibitor concentration.
-aggregation at
standard error of the mean (SEM) for at least three
inhibition percentage of hBACE-1 at 50 µ β
M inhibitor concentration. d inhibition percentage of A
e
1
0
µ
M inhibitor concentration. Values are expressed as means
±
f
experiments. Values are expressed as means
±
standard deviation (SD) for at least two independent experiments.
g
Not determined. h Calbiochem, Merck; Nottingham, UK. IC50 value.
i
2
.4. Molecular Modeling Studies
The presented active compounds were docked into the Torpedo californica AChE crystal structure,
as well as into human BuChE and human BACE-1. Docking was performed in order to determine
the causes of potency variations, by finding differences in the potential bonding mode. We used
previously developed methods to dock ligands and assess the binding modes [28
AChE, the length of the linker had a significant influence on ligand arrangement in the enzymatic active
gorge and on docking score value (from 34.01 for inactive compound to 44.47 for active compound
. The presence of a hydroxyl group within the linker made it very difficult for the compounds to
,29]. In the case of
3
1
4
adjust to the AChE active site. Short linkers (n = 1 and n = 2) were halted within the PAS by hydrogen
bonds generated by OH with Tyr334 and Asp72, restricting interactions between benzylamine and
CAS or between phthalimide and PAS. As the linker grows in length, the effect of the hydroxyl group
is compensated for by the flexibility of the compound. The binding mode of the most active inhibitor
1
5 is shown in Figure 3.
Despite hydrogen bonding of the hydroxyl group with Tyr334 and Asp72 at the proximal part of
the active gorge, this compound adopts a conformation which resembles potent donepezil-like AChE
inhibitors. The first key element, is the benzylamine position, providing CH- interaction with Trp84
and cation- interactions with Phe330. Hydrogen bonds between the ligand and the conserved water
π
π
molecule (1159) appear to be significant. The most active compound, with the longest carbon linker,
also provides the best phthalimide-PAS fit. This was the only compound which formed both hydrogen
bonds, with Tyr121 and conserved water molecule (1254), while maintaining optimal π-π interaction
with Trp279 and CH-π interaction with Tyr-70.

Molecules 2018, 23, 347
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Figure 3. Left panel: illustrative location of compound 15 (green sticks) in the active site of AChE. Active
site elements are color-coded: yellow: catalytic triad; magenta: anionic site; orange: acyl pocket; cyan:
oxyanion hole; green: PAS. Right panel: detailed visualization of compound 15 (green) interactions with
amino acids (yellow) belonging to the active site of AChE, including the conserved waters (red balls).
The predicted BuChE binding mode for active compound (5) was very consistent despite
differences observed in biological studies. Interactions with three tryptophan residues—Trp82, Trp231,
and Trp430—appeared to be crucial from the point of view of the molecular modeling results. Similarly
to BuChE substrates, the tested compound exhibited cation-
amine basic center and Trp82 [30]. Phthalimide, in a manner analogous to the BuChE-decomposed
ester, occupied a position close to CAS. The active compound ( ) provides a good illustration of the
π interactions between the protonated
5
presented binding mode (Figure 4). The carbonyl oxygen atom of phthalimide is involved in the
hydrogen bonding network of Ser198 and His438. Depending on the analyzed enantiomer, the short
linker may facilitate binding of the hydroxyl group with the conserved HOH799 water molecule,
and through it, with Thr120 (S-enantiomer shown in Figure 4). The R-enantiomer hydroxyl group
interacts with Glu197. Cation-π interactions of the protonated amine are stabilized by hydrophobic
and aromatic interactions of the aromatic benzyl ring with the hydrophobic pocket formed by Tyr332,
Trp430, and Tyr440. Interactions of the methoxy group with Tyr332 are clearly preferred.
Figure 4. Left panel: illustrative location of compound
site elements are color-coded: yellow: catalytic triad; magenta: anionic site; orange: acyl pocket; cyan:
oxyanion hole; green: PAS. Right panel: detailed visualization of compound (green) interactions with
amino acids (yellow) belonging to the active site of AChE, including the conserved water (red balls).
5 (green sticks) in the active site of BuChE. Active
5

Molecules 2018, 23, 347
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Despite the addition of a hydroxyethylamine moiety associated with the benzyl substituent, which
is a characteristic pharmacophore in BACE-1 inhibitors, the tested compounds did not exhibit strong
enzymatic inhibition potency. Molecular modeling studies indicate that the described pharmacophore
binds at the preferred place (catalytic dyad consisting of Asp32 and Asp221); however, there is a
very broad variation in the predicted conformation of both aromatic systems. The good fit of the
phthalimide fragment within the S3 hydrophobic pocket makes it difficult to direct the benzylic
0
substituent towards the S2 pockets. This situation is illustrated in Figure 5. Compound
6 showed
the highest-rated conformation in the BACE-1 active site. In other conformations obtained during
docking experiments, the benzylamine and hydroxyethyl moieties assumed the same position as the
corresponding reference compound (NVP-BXD552); however, under these conditions phthalimide
provides only weak interaction beyond the S3 sub-pocket.
Figure 5. Left panel: illustrative location of compound
6
(green sticks) in the active site of BACE-1.
0
0
0
Active site elements are color-coded: S3 : violet; S2 : orange; S1 : magenta; S1: yellow; S2: blue; S3:
green; S4: red. Right panel: detailed visualization of compound 6 (green sticks) interactions with amino
acids (yellow) belonging to the active site of BACE-1.
3
3
3
. Materials and Methods
.1. Chemistry
.1.1. General Methods
1H NMR and 13C NMR spectra were recorded on a Varian Mercury 300 (Varian, San Diego, CA,
1
USA) at 300 MHz. The chemical shifts for H NMR are referenced to tetramethylsilane TMS via
1
residual solvent signals ( H, CDCl at 7.26 ppm, DMSO-d at 2.50 ppm). Mass spectra (MS) were
3
6
®
®
recorded on a UPLC-MS/MS system consisting of a Waters ACQUITY UPLC (Waters Corporation,
Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode
ESI-tandem quadrupole). Analytical thin layer chromatography (TLC) was done using aluminum
sheets precoated with silica gel 60 F254. Column chromatography was performed on Merck silica
gel 60 (63–200
µm) (Merck, Darmstadt, Germany). For the TLC and column chromatography,
the following solvents were used: dichloromethane (DCM), methanol (MeOH), petroleum ether,
chloroform, ethyl acetate, and 25% ammonia water solution. The purity of the final compounds
was determined using an analytical RPLC-MS on a Waters Acquity TQD using an Aquity UPLC
BEH C18 column (1.7
.1% HCOOH was used as the mobile phase at a flow rate of 0.3 mL/min. All of the compounds
showed purity above 95%. All of the reagents were purchased from commercial suppliers and were
µm, 2.1
×
100 mm) at 214 nm and 254 nm. CH CN/H O gradient with
3 2
0

Molecules 2018, 23, 347
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used without further purification. The following compounds: 2-allylisoindoline-1,3-dione (16) [31],
2-(but-3-en-1-yl)isoindoline-1,3-dione (17) [32], 2-(pent-4-en-1-yl)isoindoline-1,3-dione (18) [33],
2-(hex-5-en-1-yl)isoindoline-1,3-dione (19) [34], 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione (20) [35],
2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-dione (21) [35], 2-(3-(oxiran-2-yl)propyl)isoindoline-1,3-dione
(22) [35], 2-(4-(oxiran-2-yl)butyl)isoindoline-1,3-dione (23) [36] have been reported previously.
3
.1.2. General Procedure for the Synthesis of Compounds: 1–15 (Procedure A)
-( -(oxiranyl)alkyl)isoindoline-1,3-dione (21) (1.0 equiv.), corresponding benzylamine (1.0
2
ω
equiv.), and a catalytic amount of pyridine in n-propanol were refluxed for 16 h. Then, the solvent was
evaporated and the resulting residue was purified by flash column chromatography using a mixture
of MeOH and DCM (gradient from 1% to 10% of MeOH).
2
-(3-(Benzylamino)-2-hydroxypropyl)isoindoline-1,3-dione (
phenylmethanamine (0.065 mL, 0.591 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione (20
0.120 g, 0.591 mmoL) and a catalytic amount of pyridine in 4 mL n-propanol was performed. Yield:
1). Following the procedure A, reaction of
)
(
3
3
5
6 mg (19.6%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.33. MW 310.35. Formula C H N O . MS m/z
f
18 18 2 3
+
1
11.09 (M + H ). H NMR (300 MHz, CDCl )
δ
7.80–7.89 (m, 2H), 7.66–7.76 (m, 2H), 7.18–7.38 (m,
H), 3.98 (tdd, J = 6.92, 5.26, 3.98 Hz, 1H), 3.69–3.87 (m, 4H), 2.79 (dd, J = 12.31, 3.85 Hz, 1H), 2.65
168.67, 139.62, 134.05, 131.97,
3
(
dd, J = 12.31, 7.18 Hz, 1H), 2.33 (br.s., 2H). 13C NMR (75 MHz, CDCl3)
δ
1
28.47, 128.14, 127.16, 123.38, 68.05, 53.75, 51.84, 41.91.
-(3-((2-Fluorobenzyl)amino)-2-hydroxypropyl)isoindoline-1,3-dione (
of (2-fluorophenyl)methanamine (0.068 mL, 0.591 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione
20) (0.120 g, 0.591 mmoL) and a catalytic amount of pyridine in 4 mL n-propanol was performed. Yield:
2
2). Following the procedure A, reaction
(
5
0 mg (26.0%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.21. MW 328.34. Formula C H FN O . MS m/z
f
18 17 2 3
+
1
3
29.10 (M + H ). H NMR (300 MHz, CDCl )
δ
7.79–7.88 (m, 2H), 7.66–7.75 (m, 2H), 7.35 (td, J = 7.50,
.67 Hz, 1H), 7.19–7.29 (m, 1H), 7.06–7.14 (m, 1H), 7.02 (ddd, J = 10.00, 8.46, 1.03 Hz, 1H), 3.98–4.10
m, 1H), 3.90 (s, 2H), 3.81 (dd, J = 14.11, 6.92 Hz, 1H), 3.73 (dd, J = 13.85, 4.87 Hz, 1H), 3.49 (br.s., 2H),
.81 (dd, J = 12.31, 3.85 Hz, 1H), 2.66 (dd, J = 12.05, 7.44 Hz, 1H). 13C NMR (75 MHz, CDCl3)
168.03,
3
1
(
2
δ
1
1
62.21, 133.44, 131.35, 129.99 (d, JC=F = 1.9 Hz), 128.60 (d, JC=F = 8.29 Hz), 125.04 (d, JC=F = 14.93 Hz),
23.61 (d, JC=F = 3.32 Hz), 122.78, 114.78 (d, JC=F = 22.12 Hz), 67.11, 50.97, 46.23, 41.23.
2-(2-Hydroxy-3-((2-(trifluoromethyl)benzyl)amino)propyl)isoindoline-1,3-dione (3). Following the procedure A,
reaction of (2-(trifluoromethyl)phenyl)methanamine (0.270 mL, 1.969 mmoL) with 2-(oxiran-2-ylmethyl)
isoindoline-1,3-dione (20) (0.400 g, 1.969 mmoL) and a catalytic amount of pyridine in 5 mL n-propanol
was performed. Yield: 150 mg (20.1%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.16. MW 378.35.
f
+
1
Formula C H F N O . MS m/z 379.08 (M + H ). H NMR (300 MHz, CDCl )
δ
7.81–7.90 (m, 2H),
.69–7.77 (m, 2H), 7.62 (t, J = 8.50 Hz, 2H), 7.52 (t, J = 7.40 Hz, 1 H), 7.35 (t, J = 7.20 Hz, 1H), 3.94–4.05
m, 3H), 3.85 (dd, J = 14.11, 6.67 Hz, 1H), 3.77 (dd, J = 13.85, 5.13 Hz, 1H), 2.83 (dd, J = 12.05, 4.10 Hz,
19
17
3
2
3
3
7
(
1
=
1
3
H), 2.68 (dd, J = 12.05, 7.18 Hz, 1H), 2.23 (br.s., 2H) C NMR (75 MHz, CDCl3)
1.11 Hz), 134.08, 132.11, 130.31, 128.50, 127.08, 126.28, 125.94 (JC=F = 6.08 Hz), 123.40, 122.64, 68.25,
2.14, 49.84 (d, JC=F = 2.21 Hz), 41.89.
δ 168.70, 138.43 (d, JC=F
5
2
-(3-((3-Chlorobenzyl)amino)-2-hydroxypropyl)isoindoline-1,3-dione (
of (3-chlorophenyl)methanamine (0.090 mL, 0.738 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione
20) (0.150 g, 0.738 mmoL) and a catalytic amount of pyridine in 4.5 mL n-propanol was performed. Yield:
4). Following the procedure A, reaction
(
7
3
5 mg (29.5%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.22. MW 344.80. Formula C H ClN O . MS m/z
f
18 17
2
3
+
1
45.62 (M + H ). H NMR (300 MHz, CDCl )
δ
7.81–7.89 (m, 2H), 7.69–7.77 (m, 2H), 7.28–7.33 (m,
3
1
(
H), 7.14–7.28 (m, 3H), 3.93–4.05 (m, 1H), 3.71–3.89 (m, 4H), 2.77 (dd, J = 12.31, 3.85 Hz, 1H), 2.64
dd, J = 12.31, 6.92 Hz, 1H), 2.31 (br.s., 2H). 13C NMR (75 MHz, CDCl3)
δ
168.72, 141.99, 134.28, 134.10,
1
31.92, 129.69, 128.16, 127.24, 126.17, 123.41, 68.30, 53.26, 51.88, 41.91.

Molecules 2018, 23, 347
9 of 15
2-(2-Hydroxy-3-((2-methoxybenzyl)amino)propyl)isoindoline-1,3-dione (5). Following the procedure A, reaction
of (2-methoxyphenyl)methanamine (0.096 mL, 0.738 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-
dione (20) (0.150 g, 0.738 mmoL) and a catalytic amount of pyridine in 4.5 mL n-propanol was performed.
Yield: 66 mg (26.3%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.29. MW 340.38. Formula C H N O .
f
19 20
2
4
+
1
MS m/z 341.12 (M + H ). H NMR (300 MHz, CDCl )
(
(
δ
7.83–7.99 (m, 2H), 7.57–7.71 (m, 2H), 7.15–7.23
m, 1H), 7.09 (dd, J = 7.57, 1.41 Hz, 1H), 6.80–6.89 (m, 2H), 3.80–4.11 (m, 7H), 3.63–3.72 (m, 1H), 3.57
dd, J = 11.03, 4.62 Hz, 1H), 2.62–3.01 (m, 2H). 13C NMR (75 MHz, CDCl3)
167.52, 156.76, 132.98,
32.80, 131.94, 129.72, 128.32, 128.19, 125.44, 123.79, 120.54, 110.58, 70.78, 64.58, 55.61, 52.12, 36.79.
3
δ
1
2
-(2-Hydroxy-3-((4-methylbenzyl)amino)propyl)isoindoline-1,3-dione ( ). Following the procedure A, reaction
6
of (4-(methyl)phenyl)methanamine (0.063 mL, 121.18 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-
dione (20) (0.100 g, 0.492 mmoL) and a catalytic amount of pyridine in 3.5 mL n-propanol was performed.
Yield: 47 mg (29.5%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.22. MW 324.38. Formula C H N O .
f
19 20
2
3
+
1
MS m/z 325.11 (M + H ). H NMR (300 MHz, CDCl )
δ
7.78–7.89 (m, 2H), 7.68–7.72 (m, 2H), 7.06–7.21
3
(
m, 4H), 3.93–4.03 (m, 1H), 3.63–3.86 (m, 4H), 3.00 (br.s., 2H), 2.76 (dd, J = 12.31, 3.85 Hz, 1H), 2.62 (dd,
J = 12.31, 7.44 Hz, 1H), 2.30 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ
168.64, 136.75, 136.48, 134.01, 131.97,
1
29.15, 128.14, 123.35, 67.93, 53.41, 51.79, 41.92, 21.07.
2-(2-Hydroxy-3-((4-methoxybenzyl)amino)propyl)isoindoline-1,3-dione (7). Following the procedure A, reaction
of (4-methoxyphenyl)methanamine (0.096 mL, 0.738 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-
dione (20) (0.150 g, 0.738 mmol) and a catalytic amount of pyridine in 4.5 mL n-propanol was performed.
Yield: 92 mg (36.6%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.27. MW 340.38. Formula C H N O .
f
19 20
2
4
+
1
MS m/z 341.13 (M + H ). H NMR (300 MHz, CDCl )
δ
7.79–7.87 (m, 2H), 7.66–7.75 (m, 2H), 7.21–7.27
3
(
2
m, 2H), 6.80–6.88 (m, 2H), 4.01 (ddd, J = 7.05, 3.46, 1.54 Hz, 1H), 3.66–3.85 (m, 7H), 3.09 (br.s., 2H),
13
.79 (dd, J = 12.57, 3.85 Hz, 1H), 2.64 (dd, J = 12.31, 7.44 Hz, 1H) C NMR (75 MHz, CDCl3)
δ 168.63,
1
58.85, 134.02, 131.96, 131.05, 129.54, 123.35, 113.89, 67.75, 55.24, 52.95, 51.58, 41.88.
2
-(3-((2,4-Dichlorobenzyl)amino)-2-hydroxypropyl)isoindoline-1,3-dione (
8). Following the procedure A, reaction
of (2,4-dichlorophenyl)methanamine (0.083 mL, 0.615 mmoL) with 2-(oxiran-2-ylmethyl)isoindoline-1,3-
dione (20) (0.125 g, 0.615 mmol) and a catalytic amount of pyridine in 4.0 mL n-propanol was performed.
Yield: 72 mg (31.4%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.25. MW 379.24. Formula C H Cl N O .
f
18 16
2
2
3
+
1
MS m/z 379.01, 380.00 (M + H ). H NMR (300 MHz, CDCl )
δ
7.79–7.88 (m, 2H), 7.68–7.76 (m, 2H),
.35 (d, J = 2.05 Hz, 1H), 7.30 (d, J = 8.21 Hz, 1H), 7.19 (dd, J = 8.46, 2.31 Hz, 1H), 4.00 (tdd, J = 6.89,
.89, 5.19, 4.10 Hz, 1H), 3.84–3.88 (m, 2H), 3.75–3.82 (m, 2H), 2.76 (dd, J = 12.57, 3.85 Hz, 1H), 2.63
168.69, 135.86, 134.40, 134.09,
3
7
6
(
dd, J = 12.31, 6.92 Hz, 1H) 2.52 (br.s., 2H) 13C NMR (75 MHz, CDCl3)
δ
1
33.43, 131.91, 130.80, 129.33, 127.10, 123.40, 68.24, 51.83, 50.57, 41.91.
-(4-(Benzylamino)-3-hydroxybutyl)isoindoline-1,3-dione ( ). Following the procedure A, reaction of
phenylmethanamine (0.071 mL, 0.650 mmoL) with 2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-dione (21
2
9
)
(0.140 g, 0.650 mmoL) and a catalytic amount of pyridine in 4 mL n-propanol was performed. Yield:
5
5.2 mg (26.2%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.18. MW 324.38. Formula C H N O .
f
19 20 2 3
+
1
MS m/z 325.11(M + H ). H NMR (300 MHz, CDCl )
δ 7.79–7.87 (m, 2H), 7.68–7.76 (m, 2H), 7.19–7.36
3
13
(
(
3
m, 5H), 3.83–3.90 (m, 2H), 3.81 (s, 2H), 3.62–3.74 (m, 1H), 2.54–2.82 (m, 4H), 1.66–1.83 (m, 2H). C NMR
75 MHz, DMSO-d6) 168.35, 141.02, 134.73, 132.17, 128.52, 128.37, 127.00, 123.36, 67.89, 55.33, 53.36,
5.49, 33.95.
δ
2-(3-Hydroxy-4-((2-methoxybenzyl)amino)butyl)isoindoline-1,3-dione (10). Following the procedure A, reaction
of (2-methoxyphenyl)methanamine (0.072 mL, 0.550 mmoL) with 2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-
dione (21) (0.120 g, 0.550 mmoL) and a catalytic amount of pyridine in 4 mL n-propanol was performed.
Yield: 54 mg (27.7%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.21. MW 354.41. Formula C H N O .
f
20 22
2
4
+
1
MS m/z 355.15 (M + H ). H NMR 7.77–7.87 (m, 2H), 7.65–7.75 (m, 2H), 7.27–7.38 (m, 2H), 6.85–6.99
(
(
m, 2H), 4.16 (d, J = 13.48 Hz, 1H), 3.99 (d, J = 13.48 Hz, 1H), 3.86–3.92 (m, 3H), 3.69–3.86 (m, 4H), 2.90
dd, J = 12.31, 2.93 Hz, 1H), 2.66 (dd, J = 12.31, 9.96 Hz, 1H), 1.50–1.86 (m, 3H). C NMR (75 MHz,
13

Molecules 2018, 23, 347
10 of 15
CDCl3)
δ 168.36, 157.46, 134.73, 132.17, 129.35, 128.35, 128.23, 123.36, 120.48, 110.85, 67.78, 55.63, 49.04,
4
7.97, 35.46, 33.95.
2-(3-Hydroxy-4-((4-methoxybenzyl)amino)butyl)isoindoline-1,3-dione (11). Following the procedure A, reaction
of (4-methoxyphenyl)methanamine (0.096 mL, 0.738 mmoL) with 2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-
dione (21) (130 mg, 0.600 mmoL) and a catalytic amount of pyridine in 3 mL n-propanol was performed.
Yield: 55 mg (25.9%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.24. MW 354.41. Formula C H N O .
f
δ
20 22
2
4
+
1
MS m/z 355.08 (M + H ). H NMR (300 MHz, CDCl )
7.77–7.87 (m, 2H), 7.66–7.74 (m, 2H), 7.24
3
(
2
d, J = 8.46 Hz, 2H), 6.84 (d, J = 8.46 Hz, 2H), 3.84 (t, J = 6.67 Hz, 2H), 3.66–3.79 (m, 6H), 3.35 (br.s.,
H), 2.70 (dd, J = 11.80, 3.08 Hz, 1H), 2.58 (dd, J = 12.05, 8.72 Hz, 1H), 1.64–1.83 (m, 2H). C NMR
13
(
75 MHz, CDCl3)
δ
168.67, 158.93, 133.98, 132.05, 130.49, 129.67, 123.27, 113.90, 66.85, 55.23, 53.94, 52.76,
3
4.63, 33.78.
2-(5-(Benzylamino)-4-hydroxypentyl)isoindoline-1,3-dione (12). Following the procedure A, reaction of
phenylmethanamine (0.071 mL, 0.650 mmoL) with 2-(3-(oxiran-2-yl)propyl)isoindoline-1,3-dione (22
0.150 g, 0.650 mmoL) and a catalytic amount of pyridine in 4 mL n-propanol was performed. Yield: 52
mg (23.7%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.21. MW 338.41. Formula C H N O . MS m/z
)
(
f
20 22
2
3
+
1
3
3
1
39.40 (M + H ). H NMR (300 MHz, CDCl )
δ
7.78–7.86 (m, 2H), 7.66–7.74 (m, 2H), 7.27–7.41 (m, 5H),
.84–3.97 (m, 2H), 3.74–3.84 (m, 1H), 3.66–3.73 (m, 2H), 3.30 (br.s., 1H), 2.77 (dd, J = 12.31, 2.93 Hz,
H), 2.55 (dd, J = 12.02, 9.67 Hz, 1H), 1.79–1.94 (m, 1H), 1.63–1.79 (m, 1H), 1.38–1.52 (m, 2H), 1.35 (d,
J = 11.14 Hz, 1H). 13C NMR (75 MHz, CDCl3)
168.43, 137.05, 133.89, 132.08, 128.69, 127.82, 127.51,
3
δ
1
23.18, 68.15, 53.86, 52.92, 37.76, 31.93, 24.81.
2-(4-Hydroxy-5-((2-methoxybenzyl)amino)pentyl)isoindoline-1,3-dione (13). Following the procedure A, reaction
of (2-methoxyphenyl)methanamine (0.085 mL, 0.650 mmol) with 2-(3-(oxiran-2-yl)propyl)isoindoline-1,3-
dione (22) (0.150 g, 0.650 mmol) and a catalytic amount of pyridine in 4 mL n-propanol was performed.
Yield: 52 mg (21.8%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.18. MW 368.43. Formula C H N O .
f
21 24
2
3
+
1
MS m/z 369.17 (M + H ). H NMR (300 MHz, CDCl )
δ
7.76–7.87 (m, 2H), 7.65–7.73 (m, 2H), 7.29–7.39
3
(
m, 2H), 6.86–6.98 (m, 2H), 4.22 (d, J = 12.89 Hz, 1H), 4.01 (d, J = 12.89 Hz, 1H), 3.91 (s, 3H), 3.62–3.72 (m,
2
H), 2.89 (dd, J = 12.31, 1.76 Hz, 1H), 2.62 (dd, J = 12.02, 10.26 Hz, 1H), 1.83 (dt, J = 9.23, 6.52 Hz, 1H),
13
1.58–1.75 (m, 1H), 1.31–1.44 (m, 3H), 1.23–1.30 (m, 2H). C NMR (75 MHz, CDCl3)
δ 168.39, 157.81,
1
33.88, 132.05, 131.65, 131.10, 123.17, 120.88, 119.03, 110.64, 66.10, 55.59, 52.75, 47.77, 37.63, 31.73, 24.73.
2
-(6-(Benzylamino)-5-hydroxyhexyl)isoindoline-1,3-dione (14). Following the procedure A, reaction of
phenylmethanamine (0.062 mL, 0.570 mmoL) with 2-(4-(oxiran-2-yl)butyl)isoindoline-1,3-dione (23
0.140 g, 0.570 mmoL) and a catalytic amount of pyridine in 4 mL n-propanol was performed. Yield: 69
mg (34.1%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.19. MW 352.43. Formula C H N O . MS m/z
)
(
f
21 24
2
3
+
1
3
53.22 (M + H ). H NMR (300 MHz, CDCl )
δ
7.77–7.86 (m, 2H), 7.65–7.73 (m, 2H), 7.27–7.44 (m, 5H),
3
3
.88–4.01 (m, 2H), 3.70–3.84 (m, 2H), 3.66 (t, J = 7.03 Hz, 2H), 2.79 (dd, J = 12.31, 2.93 Hz, 1H), 2.58
13
(
dd, J = 12.02, 9.67 Hz, 1H), 1.57–1.77 (m, 2H), 1.39–1.56 (m, 3H), 1.22–1.39 (m, 2H) C NMR (75 MHz,
CDCl3) 168.42, 135.48, 133.86, 132.11, 129.09, 128.81, 128.23, 123.17, 68.03, 53.44, 52.54, 37.70, 34.31,
8.47, 22.68.
δ
2
2-(5-Hydroxy-6-((2-methoxybenzyl)amino)hexyl)isoindoline-1,3-dione (15). Following the procedure A, reaction
of (2-methoxyphenyl)methanamine (0.057 mL, 0.440 mmoL) with 2-(4-(oxiran-2-yl)butyl)isoindoline-1,3-
dione (23) (0.109 g, 0.440 mmoL) and a catalytic amount of pyridine in 3 mL n-propanol was performed.
Yield: 49.8 mg (29.4%), TLC (DCM/MeOH, 9.5/0.5, v/v) R = 0.16. MW 382.46. Formula C H N O .
f
22 26
2
3
+
1
MS m/z 383.20 (M + H ). H NMR (300 MHz, CDCl )
δ
7.76–7.85 (m, 2H), 7.65–7.73 (m, 2H), 7.29–7.39
3
(
1
m, 2H), 6.87–6.99 (m, 2H), 3.96–4.32 (m, 3H), 3.90 (s, 3H), 3.56–3.68 (m, 2H), 2.88 (dd, J = 12.31, 2.34 Hz,
H), 2.62 (dd, J = 12.31, 9.96 Hz, 1H), 1.56–1.76 (m, 2H), 1.22–1.56 (m, 6H). C NMR (75 MHz, CDCl3)
13
δ
168.36, 157.84, 133.85, 132.08, 131.74, 131.12, 123.15, 120.88, 118.88, 110.61, 66.30, 55.56, 52.52, 47.38,
3
7.69, 34.10, 28.41, 22.58.

Molecules 2018, 23, 347
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3
.2. Biological Activity
3
.2.1. In Vitro Inhibition of AChE and BuChE
Inhibitory activities of the synthesized compounds against the cholinesterases were measured
using the spectrophotometric method described by Ellman et al. [23], as modified for 96-well
microplates. AChE from Electrophorus electricus (eeAChE) and BuChE from equine serum
0
(
eqBuChE) were used. 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATC),
butyrylthiocholine iodide (BTC), and both cholinesterases were purchased from Sigma–Aldrich.
The enzymes were prepared by dissolving 500 U of each in water to give stock solutions of 5 U/mL.
eeAChE and eqBuChE were further diluted before use to a final concentration of 0.384 U/mL. In the
first step of Ellman’s method, 25
in 0.1 M phosphate buffer (200
eeAChE or eqBuChE) at a temperature of 25 C. Then, after an incubation period of 5 min, 20 t
µ
L of the test compound (or water; i.e., blank samples) was incubated
L, pH = 8.0) with DTNB (20 L; 0.0025 M) and the enzyme (20 L;
L of
µ
µ
µ
◦
µ
ATC (0.00375 M) or BTC (0.00375 M) solutions (depending on the enzyme used) were added. After a
further 5 min, the changes in absorbance were measured at 412 nm using a microplate reader (EnSpire
Multimode; PerkinElmer, Hong Kong, China). All compounds were tested at a screening concentration
of 10
respective enzyme activities with and without the test sample, respectively. For compounds with better
than 50% inhibitory activity at 10 M, the IC50 values were determined. To determine the IC50 value,
µM. Percent of inhibition was calculated from 100
−
(S/B)
×
100, where S and B were the
µ
six different concentrations of each compound were used to obtain enzyme activities between 5%
and 95%. All reactions were performed in triplicate. The IC50 values were calculated using nonlinear
regression (GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA, 5.00)) by plotting the residual
enzyme activities against the applied inhibitor concentration.
3
.2.2. In Vitro BACE-1 Inhibitory Activity
All the compounds were tested as hBACE-1 inhibitors using a PanVera’s BACE-1 fluorescence
resonance energy transfer (FRET) [24,25] Assay Kit, which includes purified baclovirus-expressed
BACE-1 and specific peptide substrate (Rh-EVNLDAEFK-quencher), based on the “Swedish” mutant
of APP. The assay was purchased from Life Technologies Polska Sp. z o.o. The analyses were carried
out according to the supplied protocol with small modifications using 384-well black microplates and
a microplate reader (EnSpire Multimode; PerkinElmer).The wavelength was optimized for 553 nm
excitation and 576 nm emission. Stock solutions of all test compounds were prepared in DMSO and
further diluted with assay buffer (50 mM sodium acetate; pH 4.5). In the first step, 10
substrate was mixed with 10 L of test compound (or assay buffer; i.e., blank sample), then 10
of enzyme (1 U/mL) was added to start the reaction. After 60 min of incubation at 25 C, 10
stop solution (2.5 M sodium acetate) was applied to stop the reaction. The fluorescence signal was
read at 576 nm. Percent of inhibition was calculated from [1 (S60 S0)/(C60 C0)] 100, where
µL of BACE-1
µ
µL
L of
◦
µ
−
−
−
×
S0 and S60 are the fluorescence intensities of the test sample (enzyme, substrate, test compound)
at the beginning of the reaction and after 60 min, respectively, while C0 and C60 are the analogical
fluorescence intensities of the blank sample (enzyme, substrate, buffer). All of the compounds were
tested at a screening concentration of 50
µM. Each compound was analyzed in triplicate. BACE-1
Inhibitor IV (Calbiochem, Merck, Nottingham, UK) was used as the reference compound.
3
.2.3. Inhibition of Aβ-Aggregation
In order to investigate the inhibition of
β-amyloid peptide aggregation by the compounds,
a thioflavin T-based fluorometric assay was performed [27]. Recombinant human HFIP-pretreated
peptide (Lot 2691412 and 2718332, Merck Millipore, Darmstadt, Germany) was dissolved in
A
β
1–42
DMSO to give a 75
solution (150 mM HEPES, pH 7.4, 150 mM NaCl) to 7.5
compounds in black-walled 96-well plate and diluted with ThT solution (final concentration of 10
µ
M stock solution. The stock solution was further diluted in HEPES buffered
µ
M. A solution was then added to the test
β
1–42
µM).

Molecules 2018, 23, 347
12 of 15
The final mixture contained 1.5
µM Aβ1–42, 10
µM of test compound, and 3% DMSO. ThT fluorescence
was measured every 300 s (excitation wavelength of 440 nm, emission wavelength of 490 nm), with the
medium continuously shaking between measurements using a 96-well microplate reader (Synergy
H4, BioTek Instruments, Inc., Weinusky, VT, USA). The ThT emission of the A began to rise after
™
β
1
–42
approximately 4 h, reached a plateau after 20 h, and remained almost unchanged for an additional
8 h of incubation. The fluorescence intensities at the plateau in the absence and presence of the
test compounds were averaged, and the average fluorescence of the corresponding wells at t = 0 h
was subtracted. The A aggregation inhibitory potency is expressed as the percentage inhibition
2
β
1
–42
(
% inh = (1
−
Fi / F0)
×
100%), where Fi is the increase in fluorescence of A 1
β
treated with the
–42
test compound and F0 is the increase in fluorescence of A
β
1
alone. The percentage of inhibition is
–42
expressed as mean
different compared to control experiments (A
±
standard deviation of at least two independent experiments; p < 0.05, statistically
+ DMSO); one-way analysis of variance (ANOVA),
β
1
–42
followed by post hoc Bonferroni t-test (SigmaPlot v 12.0).
3
.3. Molecular Modeling
All compounds prepared by the LigPrep program were docked with GOLD 5.3 (CCDC) to AChE
(
PDB code 1EVE), BuChE (PDB code 1P0I) complex, and to BACE-1 (PDB code 4D8C) [37]. Each protein
was prepared using Hermes 1.7 (CCDC) [38]. All histidine residues were protonated at N and the
ε
hydrogen atoms were added. The binding sites were defined as: all amino acid residues within a
radius of 10 Å from the donepezil (E20) present in the AChE, all amino acid residues within a radius
of 20 Å from the glycerol molecule (GOL) present in the active center of BuChE, and all amino acid
residues within a radius of 10 Å from the ligand (BXD) in the BACE-1 complex. Standard settings
of the genetic algorithm were applied in each docking procedure. We received 10 conformations for
each docked ligand sorted by the ChemScore (for AChE and BuChE) and the GoldScore (for BACE-1)
function value. Results of docking were visualized with PyMOL 0.99rc6 (DeLano Scientific LLC) [39].
4
. Conclusions
Modification of the lead structure is a simple way to improve affinity for a selected (desired)
biological target. In the case of multi-target-directed ligands, this strategy is more complicated because
it requires optimization towards more than one target. In this study, new potential MTDLs have
been designed and synthesized based on previously obtained AChE inhibitors. The structure of
lead III (2-(5-(4-flurobenzylamino)pentyl)isoindoline-1,3-dione) was modified with expected activity
against disease-modifying and symptomatic targets. In order to introduce additional inhibitory
activity against BACE-1, we enriched the base compound by adding a hydroxyl group at position
2
of the alkyl chain, which is a common moiety occurring in BACE-1 inhibitors. Results indicate
that the presented modification leads to an overall decrease in anticholinesterase activity; however,
an inhibitory effect against BACE-1 appears. In terms of cholinesterase inhibition, the most interesting
compounds are
However, considering the multifunctional profile, the most valuable compound seems to be 12
2-(5-(benzylamino)-4-hydroxypentyl)isoindoline-1,3-dione) with a balanced activity profile involving
inhibition of eeAChE (IC50 = 3.33 M), hBACE-1(43.7% at 50 M), and A -aggregation (24.9% at
M). Most existing multifunctional, anti-Alzheimer’s agents are cholinesterase inhibitors with some
additional properties, such as inhibition of A aggregation, antioxidative action, a neuroprotective
5 (selective inhibitor of eqBuChE) and 15 (dual action against both cholinesterases).
(
µ
µ
β
10 µ
µ
effect, or metal chelating activity. Relatively few inhibitors of both cholinesterase and BACE-1 have
been identified thus far. Therefore, the presented results represent a promising start towards the
identification and synthesis of new active multifunctional agents.
Acknowledgments: This work was supported by the National Science Center of Poland (grants
UMO-2016/21/B/NZ7/01744 and UMO-2016/21/N/NZ7/03288), the European Cooperation in Science and
Technology COST Action CA15135, and the Slovenian Research Agency (research program P1-0208 and research
project L1-8157).

Molecules 2018, 23, 347
13 of 15
Author Contributions: D.P. designed the research, synthesized most of the compounds, and drafted the
manuscript. A.W. designed the research, supervised part of the synthesis, and wrote a part of the manuscript
and corrected it. A.P. participated in the chemical synthesis. J.G. performed the inhibition potency on the
cholinesterases, performed the BACE-1 inhibitory test, and wrote a part of manuscript. J.J. designed the novel
compounds, performed molecular modelling studies, and wrote a part of manuscript. M.B. supervised the
molecular modelling studies and corrected the manuscript. D.K. performed the A
β
test and corrected the
manuscript. S.G. supervised the A
β tests and corrected the manuscript. B.M. supervised and coordinated all
studies and corrected the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
2
3
4
5
6
7
8
9
1
1
1
.
.
.
.
.
.
.
.
.
Rizzi, L.; Rosset, I.; Roriz-Cruz, M. Global Epidemiology of Dementia: Alzheimer’s and Vascular Types.
Biomed. Res. Int. 2014, 2014, 1–8. [CrossRef] [PubMed]
Selkoe, D.J. Amyloid protein and Alzheimer’s disease. Sci. Am. 1991, 265, 68–71, 74–76, 78. [CrossRef]
[
PubMed]
Guo, T.; Noble, W.; Hanger, D.P. Roles of tau protein in health and disease. Acta Neuropathol. 2017, 133,
65–704. [CrossRef] [PubMed]
Selkoe, D.J.; Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol. Med. 2016, 8,
95–608. [CrossRef] [PubMed]
6
5
Pinto, T.; Lanctôt, K.L.; Herrmann, N. Revisiting the cholinergic hypothesis of behavioral and psychological
symptoms in dementia of the Alzheimer’s type. Ageing Res. Rev. 2011, 10, 404–412. [CrossRef] [PubMed]
Bowen, D.M.; Smith, C.B.; White, P.; Davison, A.N. Neurotransmitter-related enzymes and indices of hypoxia
in senile dementia and other abiotrophies. Brain 1976, 99, 459–496. [CrossRef] [PubMed]
Davies, P.; Maloney, A.J.F. Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet 1976
08, 1403. [CrossRef]
Casey, D.A.; Antimisiaris, D.; O’Brien, J. Drugs for Alzheimer’s disease: Are they effective? Pharm. Ther.
010, 35, 208–211.
Morphy, R.; Rankovic, Z. Designed Multiple Ligands. An Emerging Drug Discovery Paradigm. J. Med. Chem.
005, 48, 6523–6543. [CrossRef] [PubMed]
,
3
2
2
0. Rosini, M.; Simoni, E.; Caporaso, R.; Minarini, A. Multitarget strategies in Alzheimer’s disease: Benefits and
challenges on the road to therapeutics. Future Med. Chem. 2016, 8, 697–711. [CrossRef] [PubMed]
1. Prati, F.; Cavalli, A.; Bolognesi, M. Navigating the Chemical Space of Multitarget-Directed Ligands: From
Hybrids to Fragments in Alzheimer’s Disease. Molecules 2016, 21, 466. [CrossRef] [PubMed]
2. Spilovska, K.; Korabecny, J.; Nepovimova, E.; Dolezal, R.; Mezeiova, E.; Soukup, O.; Kuca, K. Multitarget
Tacrine Hybrids with Neuroprotective Properties to Confront Alzheimer’s Disease. Curr. Top. Med. Chem.
2
017, 17, 1006–1026. [CrossRef] [PubMed]
1
1
3. Mohamed, T.; Shakeri, A.; Rao, P.P.N. Amyloid cascade in Alzheimer’s disease: Recent advances in medicinal
chemistry. Eur. J. Med. Chem. 2016, 113, 258–272. [CrossRef] [PubMed]
4. Ismaili, L.; Refouvelet, B.; Benchekroun, M.; Brogi, S.; Brindisi, M.; Gemma, S.; Campiani, G.; Filipic, S.;
Agbaba, D.; Esteban, G.; et al. Multitarget compounds bearing tacrine- and donepezil-like structural and
functional motifs for the potential treatment of Alzheimer’s disease. Prog. Neurobiol. 2017, 151, 4–34.
[CrossRef] [PubMed]
1
1
5. Mohamed, T.; Rao, P.P.N. 2,4-Disubstituted quinazolines as amyloid-β aggregation inhibitors with dual
cholinesterase inhibition and antioxidant properties: Development and structure-activity relationship (SAR)
studies. Eur. J. Med. Chem. 2017, 126, 823–843. [CrossRef] [PubMed]
6. Sola, I.; Aso, E.; Frattini, D.; López-González, I.; Espargaró, A.; Sabaté, R.; Di Pietro, O.; Luque, F.J.; Clos, M.V.;
Ferrer, I.; et al. Novel levetiracetam derivatives that are effective against the Alzheimer-like phenotype in
mice: Synthesis, in vitro, ex vivo, and in vivo efficacy studies. J. Med. Chem. 2015, 58, 6018–6032. [CrossRef]
[PubMed]
1
7. Darras, F.H.; Pockes, S.; Huang, G.; Wehle, S.; Strasser, A.; Wittmann, H.-J.; Nimczick, M.; Sotriffer, C.A.;
Decker, M. Synthesis, Biological Evaluation, and Computational Studies of Tri- and Tetracyclic
Nitrogen-Bridgehead Compounds as Potent Dual-Acting AChE Inhibitors and hH3 Receptor Antagonists.
ACS Chem. Neurosci. 2014, 5, 225–242. [CrossRef] [PubMed]

Molecules 2018, 23, 347
14 of 15
1
1
8. Bautista-Aguilera, Ó.M.; Hagenow, S.; Palomino-Antolin, A.; Farré-Alins, V.; Ismaili, L.; Joffrin, P.-L.;
Jimeno, M.L.; Soukup, O.; Jano cˇ ková, J.; Kalinowsky, L.; et al. Multitarget-Directed Ligands Combining
Cholinesterase and Monoamine Oxidase Inhibition with Histamine H R Antagonism for Neurodegenerative
3
Diseases. Angew. Chem. Int. Ed. 2017, 56, 12765–12769. [CrossRef] [PubMed]
9. Guzior, N.; Bajda, M.; Skrok, M.; Kurpiewska, K.; Lewi n´ ski, K.; Brus, B.; Pišlar, A.; Kos, J.; Gobec, S.;
Malawska, B. Development of multifunctional, heterodimeric isoindoline-1,3-dione derivatives as
cholinesterase and
β-amyloid aggregation inhibitors with neuroprotective properties. Eur. J. Med. Chem.
2
015, 92, 738–749. [CrossRef] [PubMed]
2
2
2
0. Rueeger, H.; Rondeau, J.-M.; McCarthy, C.; Möbitz, H.; Tintelnot-Blomley, M.; Neumann, U.; Desrayaud, S.
Structure based design, synthesis and SAR of cyclic hydroxyethylamine (HEA) BACE-1 inhibitors. Bioorg.
Med. Chem. Lett. 2011, 21, 1942–1947. [CrossRef] [PubMed]
1. Ghosh, A.K.; Kumaragurubaran, N.; Hong, L.; Kulkarni, S.; Xu, X.; Miller, H.B.; Reddy, D.S.; Weerasena, V.;
Turner, R.; Chang, W.; et al. Potent memapsin 2 (beta-secretase) inhibitors: Design, synthesis, protein-ligand
X-ray structure, and in vivo evaluation. Bioorg. Med. Chem. Lett. 2008, 18, 1031–1036. [CrossRef] [PubMed]
2. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 1997
,
2
3, 3–25. [CrossRef]
2
3. Ellman, G.L.; Courtney, K.D.; Andres, V.; Feather-Stone, R.M. A new and rapid colorimetric determination of
acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef]
2
4. Kennedy, M.E.; Wang, W.; Song, L.; Lee, J.; Zhang, L.; Wong, G.; Wang, L.; Parker, E. Measuring human
β-secretase (BACE1) activity using homogeneous time-resolved fluorescence. Anal. Biochem. 2003, 319, 49–55.
[CrossRef]
2
2
5. Mancini, F.; De Simone, A.; Andrisano, V. Beta-secretase as a target for Alzheimer’s disease drug discovery:
An overview of in vitro methods for characterization of inhibitors. Anal. Bioanal. Chem. 2011, 400, 1979–1996.
[CrossRef] [PubMed]
6. Stachel, S.J.; Coburn, C.A.; Steele, T.G.; Jones, K.G.; Loutzenhiser, E.F.; Gregro, A.R.; Rajapakse, H.A.;
Lai, M.-T.; Crouthamel, M.-C.; Xu, M.; et al. Structure-based design of potent and selective cell-permeable
inhibitors of human beta-secretase (BACE-1). J. Med. Chem. 2004, 47, 6447–6450. [CrossRef] [PubMed]
7. LeVine, H. Thioflavine T interaction with synthetic Alzheimer’s disease beta-amyloid peptides: Detection of
amyloid aggregation in solution. Protein Sci. 1993, 2, 404–410. [CrossRef] [PubMed]
2
2
8. Bajda, M.; Jo n´ czyk, J.; Malawska, B.; Filipek, S. Application of computational methods for the design of
BACE-1 inhibitors: Validation of in silico modelling. Int. J. Mol. Sci. 2014, 15, 5128–5139. [CrossRef]
[PubMed]
2
9. Bajda, M.; Wi e˛ ckowska, A.; Hebda, M.; Guzior, N.; Sotriffer, C.A.; Malawska, B. Structure-based search for
new inhibitors of cholinesterases. Int. J. Mol. Sci. 2013, 14, 5608–5632. [CrossRef] [PubMed]
3
0. Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J.C.; Nachon, F. Crystal Structure of Human
Butyrylcholinesterase and of Its Complexes with Substrate and Products. J. Biol. Chem. 2003, 278,
4
1141–41147. [CrossRef] [PubMed]
3
3
1. Mosley, C.A.; Myers, S.J.; Murray, E.E.; Santangelo, R.; Tahirovic, Y.A.; Kurtkaya, N.; Mullasseril, P.; Yuan, H.;
Lyuboslavsky, P.; Le, P.; et al. Synthesis, structural activity-relationships, and biological evaluation of
novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-D-aspartate receptors.
Bioorg. Med. Chem. 2009, 17, 6463–6480. [CrossRef] [PubMed]
2. Kuznetsov, N.Y.; Tikhov, R.M.; Godovikov, I.A.; Khrustalev, V.N.; Bubnov, Y.N. New enolate-carbodiimide
rearrangement in the concise synthesis of 6-amino-2,3-dihydro-4-pyridinones from homoallylamines.
Org. Biomol. Chem. 2016, 14, 4283–4298. [CrossRef] [PubMed]
3
3
3
3. Nickels, M.; Xie, J.; Cobb, J.; Gore, J.C.; Pham, W. Functionalization of iron oxide nanoparticles with a
versatile epoxy amine linker. J. Mater. Chem. 2010, 20, 4776–4780. [CrossRef] [PubMed]
4. Fraunhoffer, K.J.; Bachovchin, D.A.; White, M.C. Hydrocarbon Oxidation vs C C Bond-Forming Approaches
−
for Efficient Syntheses of Oxygenated Molecules. Org. Lett. 2005, 7, 223–226. [CrossRef] [PubMed]
5. Kanoh, S.; Naka, M.; Nishimura, T.; Motoi, M. Isomerization of cyclic ethers having a carbonyl functional
group: New entries into different heterocyclic compounds. Tetrahedron 2002, 58, 7094–7096. [CrossRef]

Molecules 2018, 23, 347
15 of 15
3
6. Dey, S.; Powell, D.R.; Hu, C.; Berkowitz, D.B. “Cassette” In Situ Enzymatic Screening Identifies
Complementary Chiral Scaffolds for Hydrolytic Kinetic Resolution Across a Range of Epoxides. Angew. Chem.
Int. Ed. 2007, 46, 7010–7014. [CrossRef] [PubMed]
3
3
7. CombiGlide, version 4.1; Schrödinger LLC: New York, NY, USA, 2016.
8. Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and validation of a genetic algorithm
for flexible docking. J. Mol. Biol. 1997, 267, 727–748. [CrossRef] [PubMed]
3
9. PyMOL, version 0.99rc6; DeLano Scientific LLC: Palo Alto, CA, USA, 2006.
Sample Availability: Samples of the selected compounds (1–15) are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(