Synthesis, molecular modelling and biological evaluation of novel heterodimeric, multiple ligands targeting cholinesterases and amyloid Beta

Article abstract of DOI:10.3390/molecules21040410

Cholinesterases and amyloid beta are one of the major biological targets in the search for a new and efficacious treatment of Alzheimer's disease. The study describes synthesis and pharmacological evaluation of new compounds designed as dual binding site

Full text of DOI:10.3390/molecules21040410

molecules
Article
Synthesis, Molecular Modelling and Biological
Evaluation of Novel Heterodimeric, Multiple Ligands
Targeting Cholinesterases and Amyloid Beta
Michalina Hebda 1, Marek Bajda 1, Anna Wie˛ckowska 1, Natalia Szałaj 1, Anna Pasieka 1,
1
Dawid Panek 1, Justyna Godyn´ , Tomasz Wichur 1, Damijan Knez 2, Stanislav Gobec 2
and Barbara Malawska 1,
*
1
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical
College, Kraków 30-688, Poland; hebdamichalina@gmail.com (M.H.); marek.bajda@uj.edu.pl (M.B.);
anna.wieckowska@uj.edu.pl (A.W.); natalia.szalaj@uj.edu.pl (N.S.); anna.pasieka@uj.edu.pl (A.P.);
dawid.panek@uj.edu.pl (D.P.); justyna.godyn@uj.edu.pl (J.G.); tomasz.wichur@doctoral.uj.edu.pl (T.W.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000,
Slovenia; damijan.knez@ffa.uni-lj.si (D.K.); Stanislav.Gobec@ffa.uni-lj.si (S.G.)
2
*
Correspondence: mfmalaws@cyf-kr.edu.pl; Tel.: +48-12-62-05-464
Academic Editors: Michael Decker and Diego Muñoz-Torrero
Received: 27 February 2016; Accepted: 23 March 2016; Published: 26 March 2016
Abstract: Cholinesterases and amyloid beta are one of the major biological targets in the search
for a new and efficacious treatment of Alzheimer’s disease. The study describes synthesis and
pharmacological evaluation of new compounds designed as dual binding site acetylcholinesterase
inhibitors. Among the synthesized compounds, two deserve special attention—compounds 42 and
13. The former is a saccharin derivative and the most potent and selective acetylcholinesterase
inhibitor (EeAChE IC50 = 70 nM). Isoindoline-1,3-dione derivative 13 displays balanced inhibitory
potency against acetyl- and butyrylcholinesterase (BuChE) (EeAChE IC50 = 0.76
µM, EqBuChE
IC50 = 0.618 µM), and it inhibits amyloid beta aggregation (35.8% at 10 M). Kinetic studies show
µ
that the developed compounds act as mixed or non-competitive acetylcholinesterase inhibitors.
According to molecular modelling studies, they are able to interact with both catalytic and peripheral
active sites of the acetylcholinesterase. Their ability to cross the blood-brain barrier (BBB) was
confirmed in vitro in the parallel artificial membrane permeability BBB assay. These compounds can
be used as a solid starting point for further development of novel multifunctional ligands as potential
anti-Alzheimer’s agents.
Keywords: cholinesterase inhibitors; molecular modelling;
β-amyloid aggregation inhibitors;
Alzheimer’s disease; multi-target-directed ligands (MTDL); PAMPA-BBB assay
1. Introduction
Alzheimer’s disease (AD) is a fatal neurodegenerative disorder and the most frequent cause of
dementia. World Health Organization estimates the global prevalence of the disease at 36 million [
1]
and annual costs at $604 billion [ ]. These figures are expected to rise, yet we have no new drugs
2
to ease the burden of the disease. Currently, only four drugs are used for the treatment of AD, the
last of them being approved a decade ago. Also, an alarmingly low number of drug candidates are
undergoing clinical trials, with only a dozen compounds in Phase III [3,4]. These facts highlight the
necessity of new effective therapeutic agents for AD.
The etiology of AD is not entirely understood due to the heterogeneity of the disease where ageing,
genetic and environmental risk factors play a very important role [ ]. Two pathological changes in AD
5
are senile plaques—deposits of amyloid beta peptide (Aβ) and neurofibrillary tangles (NFTs)—and
Molecules 2016, 21, 410; doi:10.3390/molecules21040410
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Molecules 2016, 21, 410
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aggregates of hyperphosphorylated tau protein [6]. These lesions accumulate in the affected brains and
damage mainly the cholinergic neurons [7,8]. Severe loss of cholinergic neurons in the basal forebrain
reduces the cholinergic activity in the cerebral cortex and hippocampus, and impairs memory and
cognitive functions [9].
Restoration of memory impairments can be achieved by enhancing cholinergic neurotransmission
through cholinesterase inhibition. Cholinesterases are enzymes responsible for acetylcholine (ACh)
hydrolysis and thus termination of signal transmission. The first enzyme, acetylcholinesterase (AChE),
hydrolyzes the majority (80%) of ACh, and the second, butyrylcholinesterase (BuChE), accounts for
the remaining cholinesterase activity [10]. It was found that apart from its hydrolytic function, AChE
is implicated in non-cholinergic functions such as amyloid deposition, cell adhesion and neurite
outgrowth [11,12]. It is assumed that these functions of AChE are connected with its peripheral anionic
site (PAS) [13]. The peripheral anionic site together with a catalytic anionic site (CAS) are the main
binding sites of the enzyme [14].
Currently in AD therapy, there are either selective AChE inhibitors (donepezil, galantamine)
or nonselective cholinesterase inhibitors (rivastigmine). Additionally to primary anti-cholinesterase
activity, these drugs exhibit neuroprotective properties against A
β toxicity, ischemia and glutamate
excitotoxicity [15 16]. Senile plaques and neurofibrillary tangles are not the only culprits that
,
exacerbate cholinergic neurotransmission. The pathogenesis of AD is far more complex, and other
mechanisms—namely inflammation [17], oxidative stress [18], and immune suppression [19]—are
also involved.
Taking into the consideration the wide range of factors involved in the onset and progress of
AD, it is reasonable to apply the multiple ligand approach to discover new efficacious drugs for
AD [20]. Multi-target-directed ligands (MTDL) are compounds which act on several biological targets
simultaneously and possess noticeable advantages over single-target-directed ligands. With these
drugs, the risk of drug-drug interactions, poor patient compliance and pharmacokinetic differences
between the individual drugs can be avoided. Also, MTDL can be more efficacious and less vulnerable
to resistance [21]. In recent years, many multifunctional compounds, which act on AD-relevant targets
have been discovered and reviewed [22
biological properties, such as A -aggregation inhibition [28
serotonergic activity [34 35] or neuroprotective properties [36], still represent the mainstay of research.
These studies represent a continuation of a project focused on the development of the MTDL as
potential anti-AD agents [37 41]. Herein, we describe the design, molecular modelling and synthesis
–27]. Among these, cholinesterase inhibitors with additional
β
–
30], monoamine oxidase inhibition [31 33],
–
,
–
of a new series of heterodimeric compounds. Their biological activity was evaluated on three potential
targets: acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and Aβ1–42 aggregation. Finally,
their blood–brain barrier (BBB) permeability was assessed using the parallel artificial membrane
permeation assay (PAMPA—BBB).
2. Results and Discussion
2.1. Design
Two series of compounds were designed as dual binding site acetylcholinesterase inhibitors based
on our previous research. Compound
and a weak self-induced Aβ12–23 aggregation inhibitor (30.10% at 50
for series A [37]. Diethylamine moiety of compound was replaced with cyclic analogues (pyrrolidine,
morpholine and tetrahydroisoquinoline). Phthalimide on the left-hand side of was replaced with
I, a selective and moderate EeAChE inhibitor (IC50 = 0.90
µM),
µM) were used as a starting point
I
I
tetrahydroisoquinoline and an indole group. Both fragments were connected by alkyl linkers of various
lengths (Figure 1).
Series
B was designed on the basis of a potent and selective saccharin-benzylamine based AChE
inhibitor (compound II, EeAChE IC50 = 36 nM and 22.19% of inhibition of self-induced Aβ1–42

Molecules 2016, 21, 410
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aggregation at 10
µM) [40]. The chemical space was probed by modifications of the benzylamine
fragment (Figure 1).
Figure 1. The general structures of the designed compounds.
2.2. Chemistry
The synthesis of the designed isoindoline-1,3-dione derivatives was accomplished as shown in
Scheme 1. 2-( -Bromoalkyl)isoindoline-1,3-diones were used as alkylating agents in a reaction of
ω
nucleophilic substitution with secondary amines (pyrrolidine, morpholine and tetrahydroisoquinoline).
The reactions were carried out in acetonitrile (MeCN) in the presence of potassium carbonate
for 24 h, under reflux. Following purification by silica gel column chromatography, the final
2-(
hydrochloride salts. Compounds 10
of hydrazine under reflux. After 2 h, the reaction mixture was cooled down to 0 ˝C and 36% HCl
was added and mixed for the next 2 h, under reflux. Amines 16 19 were isolated and alkylated with
ω-(N-amino)alkyl)isoindoline-1,3-dione derivatives
1–15 were isolated and converted into their
–13 were further hydrolyzed in reaction with ethanolic solution
–
bromoethane in the presence of potassium carbonate. The reactions were carried out in acetonitrile
for 48 h, at room temperature. Following purification by silica gel column chromatography, the final
ω
-(3,4-dihydroisoquinolin-2(1H)-yl)-N,N-diethylalkyl-1-amine derivatives 20–23 were isolated and
subsequently converted into hydrochloride salts.
Scheme 1. Synthesis of the isoindoline-1,3-dione derivatives 1–15 and tetrahydroisoquinoline
derivatives 20
–
23. Reagents and conditions: (
a
) pyrrolidine or morpholine, K2CO3, MeCN, reflux,
) tetrahydroisoquinoline, K2CO3, MeCN, reflux, 24 h; ( ) i: NH2NH2,
) bromoethane, K2CO3, MeCN, rt, 48 h; ( ) HCl
20 h; (
b) HCl in 2-propanol; (
c
d
EtOH, reflux, 2 h; ii: 0 ˝C, 36% HCl, reflux, 2 h (
e
f
in 2-propanol.

Molecules 2016, 21, 410
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A subseries of indole derivatives was synthesized according to the reaction protocol depicted
in Scheme 2. In the first step, indole was alkylated with the appropriate -dibromoalkane in the
presence of potassium tert-butoxide ((CH3)3CO´K+). The reactions were carried out in anhydrous
tetrahydrofuran (THF) at 0 ˝C for 3 h. In the next step, compounds 24
29 were used as alkylating
α,ω
–
agents in a reaction with diethylamine. The reactions were carried out in acetonitrile, in the presence
of potassium carbonate, stirring for 24 h under reflux. After purification by silica gel column
chromatography, the final N,N-diethyl-ω-(1H-indol-1-yl)alkyl-1-amine derivatives 30–35 were isolated
and converted into oxalates.
Scheme 2. Synthesis of the indole derivatives 30
–
35. Reagents and conditions: (
a) α,ω-dibromoalkane,
(CH3)33CO´K+, THF, 0 ˝C, 3 h (
b
) diethylamine, K2CO3, MeCN, reflux 24 h; (
c) Oxalic acid in acetone.
Series
pathway described in Scheme 3. In the first step, alkylation of a saccharin sodium salt with the
appropriate -dibromoalkane gave intermediate compounds 36 38. Reactions were carried out in
N,N-dimethylformamide (DMF) for 24 h under reflux, followed by purification with flash column
chromatography. Subsequently, compounds 36 38 were used as alkylating agents in reaction of
nucleophilic substitution with 2-fluorobenzylamine or 3-chlorobenzylamine. The reactions were
B of benzo[d]isothiazol-3(2H)-one 1,1-dioxide derivatives was prepared according to the
α
,ω
–
–
˝
carried out in dimethyl sulfoxide (DMSO) for 3.5 h at 60 C, followed by silica gel flash chromatography
purification, which afforded the expected 2-(ω-(N-benzylamino)alkyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxides 39–44.
Scheme 3. Synthesis of the saccharin derivatives 39–44. Reagents and conditions: (a) α,ω-dibromoalkane,
DMF, reflux, 24 h; (b) 2-fluorobenzylamine or 3-chlorobenzylamine, DMSO, 60 ˝C, 3.5 h.
2.3. Biological Evaluation
2.3.1. Cholinesterase Inhibitory Potency
Ellman’s method [42] was used to determine the activity of the synthesized compounds against
AChE from electric eel (Electrophorus electricus, EeAChE) and BuChE from equine serum (EqBuChE).
The results of the assays are presented in Table 1. Tacrine, donepezil as well as compounds
were included as references.
I and II

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Table 1. Inhibitory potency of the synthesized compounds against EeAChE, EqBuChE and against
Aβ1–42 aggregation.
EeAChE IC50 [µM] a
EqBuChE IC50 [µM] a
b
Compd
R
n
Aβ
Aggreg.
1–42
1
2
3
4
3
4
5
6
0.781 ˘ 0.009
0.643 ˘ 0.006
0.276 ˘ 0.003
0.282 ˘ 0.011
18.117 ˘ 0.402
17.249 ˘ 0.474
4.114 ˘ 0.066
4.503 ˘ 0.064
>50 c
>50 c
-
-
-
>50 c
3.241 ˘ 0.141
15.17 ˘ 3.06
5
6
7
8
3
4
5
6
>50 c
>50 c
>50 c
>50 c
-
-
17.06 ˘ 6.13
-
9
2
3
4
5
6
8
10
5.596 ˘ 0.087
1.266 ˘ 0.011
0.387 ˘ 0.003
0.688 ˘ 0.017
0.760 ˘ 0.017
>50 c
12.818 ˘ 0.107
5.314 ˘ 0.160
5.124 ˘ 0.090
1.169 ˘ 0.027
0.618 ˘ 0.009
1.727 ˘ 0.038
3.662 ˘ 0.085
-
-
-
-
10
11
12
13
14
15
35.80 ˘ 5.39
-
-
>50 c
20
21
22
23
3
4
5
6
>50 c
>50 c
-
-
-
>50 c
>50 c
17.303 ˘ 0.401
4.722 ˘ 0.149
37.129 ˘ 0.878
<10 d
14.002 ˘ 0.343
30
31
32
33
34
35
1
2
3
4
5
6
>50 c
>50 c
>50 c
-
-
-
-
25.995 ˘ 0.813
22.571 ˘ 0.602
28.733 ˘ 0.723
1.472 ˘ 0.593
3.471 ˘ 0.100
>50 c
>50 c
22.185 ˘ 0.424
-
9.557 ˘ 0.289
16.64 ˘ 2.74
39
40
41
3
4
5
0.150 ˘ 0.003
1.145 ˘ 0.015
0.735 ˘ 0.009
>50 c
>50 c
12.27 ˘ 2.63
-
-
4.971 ˘ 0.127
42
43
44
3
4
5
0.070 ˘ 0.001
0.596 ˘ 0.006
0.923 ˘ 0.012
>50 c
16.54 ˘ 3.01
6.535 ˘ 0.163
-
-
2.459 ˘ 0.032
Compd. I e
Compd. II e
Donepezil
Tacrine
0.900 ˘ 0.004
0.036 ˘ 0.001
0.010 ˘ 0.001
0.024 ˘ 0.001
>50 c
30.10 ˘ 5.78 f
22.19 ˘ 16.6
11.48 ˘ 4.51
-
>50 c
1.830 ˘ 0.176
0.002 ˘ 0.001
a
IC50 values are expressed as mean
M compound concentration and 1.5
deviation (SD); c % Inhibition at 50 M lower than 50%; d % Inhibition at 10
defined as not active; e Data from ref. [37] for compound and from [40] for compound II, respectively; f
˘
standard error of the mean (SEM) of at least three experiments; b % of
inhibition at 10
µ
µM Aβ1–42; values are expressed as mean
˘
standard
µ
µM lower than 10%, compound
I
%
of inhibition at 50
deviation (SD).
µ
M compound concentration, data for Aβ12–23, values are expressed as mean
˘
standard
In the series
were identified. When assessing the impact of the amine moiety on EeAChE inhibitory potency,
we found that the replacement of diethylamine (compound ) by pyrrolidine (compounds ) or
tetrahydroisoquinoline (compounds 11 13) increased the potency on EeAChE. Only in the case of
morpholine, we observed a significant decrease in potency (compounds ). The most potent was
compound (EeAChE IC50 = 0.276 M) with the pyrrolidine moiety. Regarding the length of the linker,
A, moderate EeAChE inhibitors with IC50 values ranging from 0.276 µM to 22.185 µM
I
1–4
–
5–8
3
µ
the most potent were the compounds with six to eight carbon atom linkers. The potency decreased
with shortening and further elongation of the tether. Compounds 14 and 15 with the longest linkers
(10 and 12 carbon atoms) were not active. Modifications of the heteroaromatic fragment also led to

Molecules 2016, 21, 410
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reduced potencies of the tested compounds. These compounds (20
–
23,
30
–
35) were less potent in
comparison with parent compound I and inhibited the enzyme in high micromolar range.
On the basis of the Ellman’s assay for series , we found that this series inhibited EeAChE in
B
the low micromolar to nanomolar range. Compounds with a five carbon atom linker: 39 (2-fluoro
derivative) and 42 (3-chloro derivative) were the most potent EeAChE inhibitors with IC50 values
of 150 and 70 nM, respectively. Comparing these compounds with their unsubstituted analogue
(compound II, EeAChE IC50 = 36 nM), it was established that neither the introduction of a fluorine
atom nor the introduction of a chlorine atom led to significant benefits in the terms of inhibitory
potency. Compounds 39 and 42 were also the most potent and most selective EeAChE inhibitors
presented in this paper.
EqBuChE inhibitory studies showed that the half of the tested compounds were weak EqBuChE
inhibitors with IC50 values ranging from 0.618 to 37.129
µM and the other half were selective EeAChE
inhibitors. The most potent were compounds with seven and eight carbon linkers while shortening
or elongation of the linker resulted in the decrease of potency similar to series A. Compound 13
bearing phthalimide and tetrahydroisoquinoline moieties, was the most potent EqBuChE inhibitor
(IC50 = 0.618 µM) and a moderate EeAChE inhibitor (IC50 = 0.760 µM) from this series.
,
2.3.2. Kinetic Studies of AChE Inhibition
For kinetic studies, we have chosen compounds
3 and 4 with the pyrrolidine moiety and
11 with the tetrahydroisoquinoline moiety, as the most potent AChE inhibitors from series A.
Compound 42 was chosen as the most potent AChE inhibitor from series B. Initial velocity dependence
on different substrate concentration in the absence and presence of the tested compounds at six
different concentrations was established using Ellman’s method. Analysis of Lineweaver–Burk double
reciprocal plots showed that compounds 3, 4 and 11 display mixed type of enzyme inhibition (partial
competitive and pure non-competitive) as demonstrated by increased slopes (decreased Vmax) and
decreased intercepts (lower Km) at increasing concentration of the inhibitor (Figure 2a–c). For the
compound 42, we observed increased slopes and preserved intercepts at increasing concentrations of
the inhibitor, which characterize a linear non-competitive type of inhibition (Figure 2d). Both types
of inhibition were further confirmed by Cornish–Bowden plots (S/V vs. [I]) (A. in Supplementary
Materials) [43].
2.3.3. Aβ1–42 Aggregation Inhibitory Potency
Mechanisms of A
β aggregation remain unclear. Besides the self-induced assembly of Aβ,
several other factors affect its aggregation, including metal ions [44], AChE [45], and oxidative
stress [46]. Therefore, we evaluated our compounds using the most versatile and commonly used
Aβ1–42 aggregation Thioflavin T assay [47]. Seven structurally diverse compounds were selected (one
from each subseries) to test their ability to inhibit self-induced Aβ1–42 aggregation. The results of
this assay showed that these derivatives are rather weak inhibitors of A
β
aggregation at 10
µM. Only
compound 13 was found to be a moderate inhibitor with the 35.80%
˘
5.39% inhibition of Aβ1–42
aggregation. Even though it displayed higher potency than donepezil in this assay, compound 13
was a less potent cholinesterase inhibitor than the reference drug, so the multitarget profile of this
compound still needs to be optimized.

Molecules 2016, 21, 410
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(a)
(b)
Compound 3
10
Compound 4
10
0.00 μM
0.25 μM
0.50 μM
1.00 μM
2.50 μM
0.00 μM
0.35 μM
1.00 μM
2.00 μM
3.00 μM
5
5
-20
-10
10
[ATC]-1 mM-1
20
-20
-10
10
[ATC]-1 mM-1
20
-5
-5
(c)
(d)
Compound 11
10
Compound 42
10
0.00 μM
0.10 μM
0.20 μM
0.30 μM
0.50 μM
0.00 μM
0.50 μM
1.00 μM
2.00 μM
3.00 μM
5
5
-20
-10
10
[ATC]-1 mM-1
20
-20
-10
10
[ATC]-1 mM-1
20
-5
-5
Figure 2.
compounds 3 (a); 4 (b); 11
Lineweaver–Burk plots illustrating mixed type of EeAChE inhibition by
) and non-competitive type of EeAChE inhibition by compound 42 ).
(c
(d
ATC = acetylthiocholine; V = initial velocity rate.
2.4. Molecular Modelling Studies
The structure of Torpedo californica AChE (TcAChE) and the previously described fragment-based
approach were used in molecular modelling studies [48]. Our biological assay was performed on
Electrophorus electricus AChE. However, for docking TcAChE of resolution 2.5 Å, obtained from Protein
Data Bank (PDB code: 1EVE), was utilized. It contained donepezil molecule—reference ligand which
was structurally similar to our compounds. Sequence alignment of TcAChE and EeAChE revealed a
very high degree of identity (above 60%). Further analysis showed that there was only one residue
difference in the active site. The Phe330 residue in Torpedo californica was replaced by Tyr in Electric eel
enzyme [49]. This justified application of TcAChE for the docking studies.
Designed compounds were docked into the active site of AChE to find possible interactions with
the enzyme. The binding potency of novel inhibitors was assessed by the ChemScore function and the
poses were inspected visually.
The first subgroup of inhibitors (compounds
and interacted with AChE in a similar way [37]. The binding mode for the most active inhibitor
this subgroup is presented in Figure 3. The protonated amine group formed cation-
1
–
4
) were simple analogues of parent compound
I
3
from
π
interactions with
aromatic amino acids in the anionic subsite (Phe330 and Trp84) and a hydrogen bond network with
the hydroxyl group of Tyr121 via a water molecule. The pyrrolidine, a cyclic analogue of diethylamine,
provided stronger hydrophobic interactions with Trp84 and, therefore, derivatives
inhibitors of AChE in comparison with compound . The phthalimide moiety of these inhibitors was
engaged in interactions with aromatic amino acids in the PAS: stacking with Trp279 and CH–
1–4 were stronger
I
π
–
π
π
interactions with Tyr70. Moreover, both carbonyl groups formed hydrogen bonds: one with Tyr121
and the other with a water molecule. The optimal linker length was equal to 7–8 methylene groups.
Such tether enabled the terminal fragments to interact with Trp84 and Trp279 in an optimal way. The
aliphatic linker also formed hydrophobic interactions with aromatic residues (Phe290, Phe331 and
Tyr334) half-way down the active site gorge.

Molecules 2016, 21, 410
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Figure 3. The binding mode of compound 3 (pink) within the active site of AChE.
The second subset (compounds ) had an enlarged heterocyclic ring—morpholine. Morpholine
5–8
could provide an extra hydrogen bond between the oxygen atom and the hydroxyl group of Ser200
from the catalytic triad of AChE. However, it required a shift of heterocyclic ring towards the serine in
comparison with pyrrolidine. This change led to reduced cation–
resulting in lower potency.
π interactions, especially with Phe330
In the case of the third subgroup (derivatives 9–15), tetrahydroisoquinoline was introduced
instead of diethylamine. The most potent inhibitor 11 with tetrahydroisoquinoline ring docked into
acetylcholinesterase is shown in Figure 4. This modification was beneficial because the combination
of an aromatic ring and aliphatic amine gave
π–π stacking with Trp84 as well as cation–π interaction
with Phe330. The optimal linker was found to be a six-carbon atom aliphatic chain.
Figure 4. The binding mode of compound 11 (green) within the active site of AChE.

Molecules 2016, 21, 410
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Further structural modifications of parent inhibitor
(compounds 20 23) and the fifth (derivatives 30
with heteroaromatic moiety: tetrahydroisoquinoline and indole, respectively. Tetrahydroisoquinoline
provided stacking as well as cation– interaction with Trp279, and indole stronger stacking.
I
gave the next two subsets: the fourth
–
–35). In both cases, the phthalimide was replaced
π–
π
π
π–π
However, the lack of hydrogen bonds which were previously formed with two carbonyl groups of
phthalimide reduced the potency.
The last two subgroups were based on compound II [40]. The binding mode of the most potent
inhibitor 42 is presented in Figure 5. The sixth subset [39–41] contained a fluorine atom while the
seventh one [42 44] had a chlorine atom on the benzylamine fragment. Introduction of the fluorine
–
atom at ortho position might provide a hydrogen bond with Ser200 while a chlorine atom at meta
position a halogen bond with the carboxyl group of Glu199 or backbone of Gly441 upon small shift
and/or rotation of benzyl substituent. However, the halogen substituted derivatives revealed the
same binding mode as parent inhibitor II. The benzyl moiety was
CAS. Orientation of this fragment remained the same as for parent compound II, and no beneficial
interactions were observed with halogen atoms. The saccharin fragment was engaged in stacking
with Trp279 and CH– interactions with Tyr70 in the PAS. The carbonyl group formed an H-bond with
a water molecule while the oxygen atoms of sulfone formed H-bonds with Tyr121 and two other water
molecules. The protonated amino group formed cation– interactions with Phe330 and a hydrogen
π–π stacked with Trp84 in the
π–π
π
π
bond network with Tyr121 via a water molecule. The alkyl linker formed hydrophobic interactions
with aromatic residues such as Phe290, Phe331, and Tyr334 located halfway down the active gorge.
Figure 5. The binding mode of compound 42 (dark salmon) within the active site of AChE.
Summing up, all subseries were able to interact simultaneously with both the catalytic and
peripheral active sites of acetylcholinesterase. However, the quality of the predicted interactions
varies substantially and may thus lead to the diverse range of activity. The dual binding
mode is characteristic for donepezil as well as for previously described isoindoline-1,3-dione and
benzo[d]isothiazol-3(2H)-one 1,1-dioxide derivatives.
2.5. Blood–Brain Barrier Permeability Assay
For anti-Alzheimer drug candidates, the ability to cross the blood–brain barrier and to enter the
central nervous system (CNS) is crucial to achieve their pharmacological target and activity. Therefore,

Molecules 2016, 21, 410
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the blood–brain barrier permeability of the selected compounds
4,
7
,
13
,
39 and
4
2 was assessed using
the parallel artificial membrane permeability assay (PAMPA-BBB) [50]. We used seven commercial
drugs as references that allowed us to establish the following ranges of permeability: log Pe > 4.5
for compounds with high permeability, log Pe ď ´6.3 for compounds with low permeability and
´
´
6.3 < log Pe ď ´4.5 for compounds with uncertain permeability (Table 2). According to the results
summarized in Table 2, all off the tested compounds should be able to cross BBB and reach CNS.
Table 2. Permeability (log Pe) in the PAMPA-BBB assay for commercial drugs and the selected
compounds with prediction of their BBB penetration.
a
Compound
Log Pe
Prediction
Verapamil
Lidocaine
Quinidine
Progesterone
Propranolol
Corticosterone
Theophylline
–3.9
–4.3
–4.0
–3.8
–3.7
–4.5
–6.3
–4.0
–4.2
–4.2
–4.0
–4.3
CNS+
CNS+
CNS+
CNS+
CNS+
CNS˘
CNS–
CNS+
CNS+
CNS+
CNS+
CNS+
4
7
13
39
42
a
Data are means of two replicates (n = 2); CNS+, log Pe
>
´
4.5, high permeability (i.e., can enter the
CNS); CNS–, log Pe ď ´6.3, low permeability (i.e., excluded from the CNS); CNS
˘, ´6.3 < log Pe ď ´4.5,
uncertain permeability
3. Materials and Methods
3.1. Chemistry
3.1.1. General Methods
1H-NMR spectra were recorded on Varian Mercury 300 (Varian, San Diego, CA, USA) at 300 MHz.
The chemical shifts for 1H-NMR are referenced to residual solvent signals (1H, CDCl3 at 7.26 ppm,
DMSO-d6 at 2.50 ppm). Mass spectra (MS) were obtained on an 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 (Merck,
Darmstadt, Germany). Column chromatography was performed on Merck silica gel 60 (63–200 µm)
(Merck). For the TLC and column chromatography following solvent systems were used: S1 (n-hexane,
ethyl acetate, trimethylamine (TEA); 5:5:1, v/v), S2 (petroleum ether, DCM; 8:2, v/v), S3 (DCM, MeOH;
9.5:0.5, v/v/v), S4 DCM, MeOH; 9:1, v/v), S5 (DCM, MeOH; 10:1, v/v), S6 (DCM, petroleum ether,
MeOH, TEA; 5:3.5:1.5:3 drops, v/v/v/v). The purity of the final compounds was determined using
an analytical RPLC-MS on Waters Acquity TQD using an Aquity UPLC BEH C18 column (1.7 µm,
2.1 ˆ 100 mm) at 214 nm and 254 nm. CH3CN/H2O gradient with 0.1% HCOOH was used as
the mobile phase at a flow rate of 0.3 mL/min. All the compounds showed purity above 95%, as
determined by RPLC. Melting points were determined in open capillaries on an Electrothermal 9300
apparatus (Electrothermal, Staffordshire, UK) and are uncorrected. Elemental analyses were performed
on Vario EL III Elemental analyser (Elementar Analysensysteme GmbH, Hanau, Germany). All the
reagents were purchased from commercial suppliers and were used without further purification.
Tetrahydrofuran (THF) and dichloromethane (DCM) were distilled under nitrogen immediately before
use. The drying agent used for THF was sodium benzophenone ketyl, and for DCM, calcium hydride.

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The following compounds: 2-(5-bromopentyl)isoindoline-1,3-dione [37], 2-(6-bromohexyl)
isoindoline-1,3-dione [37], 2-(7-bromoheptyl)isoindoline-1,3-dione [37], 2-(8-bromooctyl)isoindoline-1,3-
dione [37], 2-(10-bromodecyl)isoindoline-1,3-dione [51], 2-(12-bromododecyl)isoindoline-1,3-dione [51],
2-(4-(bromomethyl)benzyl)isoindoline-1,3-dione [52], 1-(3-bromopropyl)-1H-indole [53], 1-(4-
bromobutyl)-1H-indole [53], 1-(5-bromopentyl)-1H-indole [53], 1-(6-bromohexyl)-1H-indole [54],
1-(7-bromoheptyl)-1H-indole [54], 1-(8-bromooctyl)-1H-indole [54], 2-(5-(pyrrolidin-1-yl)pentyl)
isoindoline-1,3-dione
morpholinooctyl)isoindoline-1,3-dione (
1,3-dione ( ) [58], 2-(5-(3,4-dihydroisoquinolin-2(1H)-yl)pentyl)isoindoline-1,3-dione (10) [59], 2-(6-
(3,4-dihydroisoquinolin-2(1H)-yl)hexyl)isoindoline-1,3-dione (11) [59], 2-(12-(3,4-Dihydroisoquinolin-
2(1H)-yl)dodecyl)isoindoline-1,3-dione 15 60], 5-(3,4-dihydroisoquinolin-2(1H)-yl)pentan-1-
(1)
[55], 2-(5-morpholinopentyl)isoindoline-1,3-dione
(5)
[56], 2-(8-
8) [57], 2-(4-(3,4-dihydroisoquinolin-2(1H)-yl)butyl)isoindoline-
9
(
) [
amine (16) [61], 6-(3,4-dihydroisoquinolin-2(1H)-yl)hexan-1-amine (17) [53], 7-(3,4-dihydroisoquinolin-
2(1H)-yl)heptan-1-amine (18) [54], 8-(3,4-dihydroisoquinolin-2(1H)-yl)octan-1-amine (19) [54], 2-(5-
bromopentyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (36) [62], 2-(6-bromohexyl)benzo[d]isothiazol-
3(2H)-one 1,1-dioxide (37) [62], 2-(7-bromoheptyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (38) [59
]
have been reported previously.
3.1.2. General Procedure for the Preparation of Hydrochloride Salts
The hydrochloride salts were prepared by dissolving the compounds in a minimum quantity of
dichloromethane. The solution was then treated with 5 M solution of HCl in 2-propanol, evaporated
under reduced pressure, washed with diethyl ether and dried.
3.1.3. General Procedure for the Synthesis of Compounds (1–8)
Procedure M1. A mixture of the appropriate 2-(bromoalkyl)isoindoline-1,3-dione (1 equiv) with
amine (pyrrolidine or morpholine) (1.1 equiv) in the presence of K2CO3 (3 equiv) was stirred in
acetonitrile under reflux for 20 h. Subsequently, the solvent was evaporated under reduced pressure,
producing a residue which was further dissolved in 20 mL of water and extracted with DCM
(3 ˆ 30 mL). The organic layer was dried with anhydrous Na2SO4. The solvent was then evaporated
and the residue was purified by silica gel column chromatography (S3) yielding a yellow oil. The final
product was transformed into hydrochloride salt. The following compounds were obtained.
2-(5-(Pyrrolidin-1-yl)pentyl)isoindoline-1,3-dione (1) [55]. Procedure M1. Reaction of 2-(5-bromopentyl)
isoindoline-1,3-dione [37] (0.5 g, 1.69 mmol) with pyrrolidine (0.13 g, 1.86 mmol) and K2CO3 (0.7 g,
5.1 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.35 g
1
(73%). TLC (S3) Rf = 0.13. MW 286.17. Formula: C17H22N2O2. MS: m/z 287.28 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.89–7.77 (m, 2H), 7.78–7.65 (m, 2H), 3.69 (t, J = 6.9 Hz, 2H), 3.00 (m, 4H),
2.06–1.88 (m, 4H), 1.81–1.65 (m, 4H), 1.48–1.21 (m, 4H). Hydrochloride salt: M.p. 190 ˝C. Elemental
analyses (%) for C17H22N2O2¨ HCl Calc. C 63.25; N 8.63; H 7.18, found: C 62.73; N 8.54; H 7.27.
2-(6-(Pyrrolidin-1-yl)hexyl)isoindoline-1,3-dione (2). Procedure M1. Reaction of 2-(6-bromohexyl)
isoindoline-1,3-dione [37] (0.65 g, 2.1 mmol) with pyrrolidine (0.16 g, 2.3 mmol) and K2CO3 (0.87 g,
6.28 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.44 g
1
(70%). TLC (S3) Rf = 0.15. MW 300.18. Formula: C18H24N2O2. MS: m/z 301.31 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.87–7.77 (m, 2H), 7.74–7.65 (m, 2H), 3.66 (t, J = 7.1 Hz, 2H), 2.59 (t, J = 6.7 Hz,
4H), 2.49 (t, J = 7.2 Hz, 2H), 1.87–1.76 (m, 4H), 1.73–1.50 (m, 4H), 1.41–1.31 (m, 4H). Hydrochloride
salt: M.p. 151 ˝C. Elemental analyses (%) for C18H24N2O2
C 64.07; N 8.13; H 7.73.
¨
HCl Calc. C 64.18; N 8.32; H 7.48, found:
2-(7-(Pyrrolidin-1-yl)heptyl)isoindoline-1,3-dione (
3). Procedure M1. Reaction of 2-(7-bromoheptyl)
isoindoline-1,3-dione [37] (0.648 g, 2 mmol) with pyrrolidine (0.156 g, 2.2 mmol) and K2CO3 (0.83 g,

Molecules 2016, 21, 410
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6 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.49 g
1
(78%). TLC (S3) Rf = 0.24. MW 314.20. Formula: C19H26N2O2. MS: m/z 315.40 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.88–7.75 (m, 2H), 7.75–7.62 (m, 2H), 3.65 (t, J = 7.2 Hz, 2H), 2.57 (t, J = 7.6 Hz,
4H), 2.46 (t, J = 6.7 Hz, 2H), 1.86–1.73 (m, 4H), 1.71–1.47 (m, 4H), 1.39–1.23 (m, 6H). Hydrochloride
salt: M.p. 115 ˝C. Elemental analyses (%) for C19H26N2O2
C 65.24; N 7.76; H 7.88.
¨
HCl Calc. C 65.04; N 7.98; H 7.76, found:
2-(8-(Pyrrolidin-1-yl)octyl)isoindoline-1,3-dione (4). Procedure M1. Reaction of 2-(8-bromooctyl)
isoindoline-1,3-dione [37] (0.678 g, 2 mmol) with pyrrolidine (0.156 g, 2.2 mmol) and K2CO3 (0.83 g,
6 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.53 g (80%).
1
TLC (S3) Rf = 0.23. MW 328.22. Formula: C20H28N2O2. MS: m/z 329.43 [M + H]+. H-NMR (300 MHz,
CDCl3) δ ppm: δ 7.88–7.75 (m, 2H), 7.75–7.62 (m, 2H), 3.65 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 6.9 Hz, 4H),
2.44 (t, J = 7.8 Hz, 2H), 1.85–1.72 (m, 4H), 1.70 – 1.43 (m, 4H), 1.36 – 1.21 (m, 8H). Hydrochloride salt:
˝
M.p. 110 C. Elemental analyses (%) for C20H28N2O2
N 7.73; H 8.17.
¨
HCl Calc. C 65.83; N 7.68; H 8.01, found: C 65.88;
2-(5-Morpholinopentyl)isoindoline-1,3-dione (
5) [56]. Procedure M1. Reaction of 2-(5-bromopentyl)
isoindoline-1,3-dione [37 (0.51 g, 1.72 mmol) with morpholine (0.17 g, 1.89 mmol) and K2CO3 (0.75 g
]
,
5.15 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.51 g
1
(89%). TLC (S3) Rf = 0.41. MW 302.16 Formula: C17H22N2O3. MS: m/z 303.24 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.89–7.76 (m, 2H), 7.76–7.63 (m, 2H), 3.73–3.61 (m, 6H), 2.40 (t, J = 4.4 Hz,
4H), 2.30 (t, J = 7.5 Hz, 2H), 1.77–1.60 (m, 2H), 1.60–1.44 (m, 2H), 1.44–1.26 (m, 2H). Hydrochloride salt:
˝
M.p. 210 C. Elemental analyses (%) for C17H22N2O3
N 8.11; H 6.97.
¨
HCl Calc. C 60.26; N 8.27; H 6.84 found: C 60.37;
2-(6-Morpholinohexyl)isoindoline-1,3-dione (
6
).
Procedure M1.
Reaction of 2-(6-bromohexyl)
isoindoline-1,3-dione [37] (0.65 g, 2.1 mmol) with morpholine (0.2 g, 2.3 mmol) and K2CO3 (0.87 g,
6.28 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.58 g
1
(88%). TLC (S3) Rf = 0.34. MW 316.18. Formula: C18H24N2O3. MS: m/z 317.26 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.86–7.80 (m, 2H), 7.73–7.66 (m, 2H), 3.72 (t, J = 4.5 Hz, 4H), 3.67 (t, J = 7.3 Hz,
2H), 2.45 (t, J = 4.7 Hz, 4H), 2.34 (t, J = 7.1 Hz, 2H), 1.75–1.59 (m, 2H), 1.58–1.46 (m, 2H), 1.44–1.19
˝
(m, 4H). Hydrochloride salt: M.p. 195 C. Elemental analyses (%) for C18H24N2O3
N 7.94; H 7.14 found: C 60.93; N 7.87; H 7.23.
¨
HCl Calc. C 61.27;
2-(7-Morpholinoheptyl)isoindoline-1,3-dione (7). Procedure M1. Reaction of 2-(7-bromoheptyl)
isoindoline-1,3-dione [37] (0.648 g, 2 mmol) with morpholine (0.19 g, 2.2 mmol) and K2CO3 (0.83 g,
6 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.58 g
1
(88%). TLC (S3) Rf = 0.58. MW 330.19. Formula: C19H26N2O3. MS: m/z 331.42 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.88–7.75 (m, 2H), 7.76–7.62 (m, 2H), 3.70 (t, J = 4.7 Hz, 4H), 3.65 (t, J = 7.1 Hz,
2H), 2.41 (t, J = 4.6 Hz, 4H), 2.30 (t, J = 7.4 Hz, 2H), 1.77–1.56 (m, 2H), 1.53–1.20 (m, 8H). Hydrochloride
˝
salt: M.p. 142.5 C. Elemental analyses (%) for C19H26N2O3
C 62.11; N 8.06; H 7.48.
¨
HCl Calc. C 62.20; N 7.64; H 7.42, found:
2-(8-Morpholinooctyl)isoindoline-1,3-dione (8) [57]. Procedure M1. Reaction of 2-(8-bromooctyl)
isoindoline-1,3-dione [37] (0.676 g, 2 mmol) with morpholine (0.19 g, 2.2 mmol) and K2CO3 (0.83 g,
6 mmol) in acetonitrile (25 mL), after 20 h, column chromatography gave oil product. Yield 0.59 g
1
(85%). TLC (S3) Rf = 0.46. MW 344.21. Formula: C20H28N2O3. MS: m/z 345.38 [M + H]+. H-NMR
(300 MHz, CDCl3) δ ppm: 7.86–7.78 (m, 2H), 7.74–7.63 (m, 2H), 3.75–3.68 (m, 4H), 3.65 (t, J = 7.2 Hz,
2H), 2.48–2.39 (m, 4H), 2.30 (t, J = 7.3 Hz, 2H), 1.71–1.59 (m, 2H), 1.51–1.42 (m, 2H), 1.35–1.24 (m, 8H).
Hydrochloride salt: M.p. 133 ˝C. Elemental analyses (%) for C20H28N2O3
H 7.67, found: C 63.21; N 7.38; H 7.75.
¨
HCl Calc. C 63.06; N 7.35;

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3.1.4. General Procedure for the Synthesis of Compounds (9–15)
Procedure M2. A mixture of the appropriate 2-(bromoalkyl)-isoindoline-1,3-dione (1 equiv)
with 1,2,3,4-tetrahydoisoquinoline (1 equiv) in the presence of K2CO3 (2.5-3 equiv) was stirred in
acetonitrile under reflux for 20 h. Subsequently, the solvent was evaporated under vacuum, producing
a residue which was further dissolved in 40 mL of sodium bicarbonate and extracted with ethyl acetate
(3 ˆ 30 mL). The organic layer was acidified 2 M¨ HCl and extracted with distilled water (3 ˆ 30 mL).
Then, the combined aqueous extracts were alkalized using 4M NaOH, extracted with DCM and dried
¨
with anhydrous Na2SO4. The solvent was then evaporated and the residue was purified by silica gel
column chromatography (S4) yielding a yellow oil. The final product was obtained in the form of
hydrochloride salt. The following compounds were obtained.
2-(7-(3,4-Dihydroisoquinolin-2(1H)-yl)heptyl)isoindoline-1,3-dione (12). Procedure M2. Reaction of
2-(7-bromoheptyl)isoindoline-1,3-dione [37] (2 g, 6.2 mmol) with 1,2,3,4-tetrahydroisoquinoline (0.83 g,
6.2 mmol) and K2CO3 (2.57 g, 18.6 mmol) in acetonitrile (100 mL), after 20 h, column chromatography
gave oil product. Yield 2.15 g (93%). TLC (S4) Rf = 0.62. MW 376.22. Formula: C24H28N2O2. MS:
m/z 377.29 [M + H]+. 1H-NMR (300 MHz, CDCl3)
δ ppm: 7.89–7.77 (m, 2H), 7.76–7.63 (m, 2H),
7.15–6.94 (m, 4H), 3.72–3.56 (m, 4H), 2.89 (t, J = 5.9 Hz, 2H), 2.72 (t, J = 5.9 Hz, 2H), 2.53–2.41 (m, 2H),
˝
1.76–1.50 (m, 4H), 1.43–1.28 (m, 6H). Hydrochloride salt: M.p. 167 C. Elemental analyses (%) for
C24H28N2O2¨ HCl Calc. C 69.8; N 6.78; H 7.08, found: C 70.10; N 6.83; H 7.47.
2-(8-(3,4-Dihydroisoquinolin-2(1H)-yl)octyl)isoindoline-1,3-dione (13). Procedure M2. Reaction of
2-(8-bromooctyl)isoindoline-1,3-dione [37] (2.1 g, 6.2 mmol) with 1,2,3,4-tetrahydroisoquinoline (0.83 g,
6.2 mmol) and K2CO3 (2.57 g, 18.6 mmol) in acetonitrile (100 mL), after 20 h, column chromatography
gave oil product. Yield 2.33 g (96%). TLC (S4) Rf = 0.64. MW 390.23. Formula: C25H30N2O2. MS:
m/z 391.26 [M + H]+. 1H-NMR (300 MHz, CDCl3)
δ ppm: 7.89–7.77 (m, 2H), 7.77–7.66 (m, 2H),
7.16–7.08 (m, 3H), 7.01–6.96 (m, 1H), 3.67 (t, J = 7.4 Hz, 2H), 3.61 (s, 2H), 2.90 (t, J = 5.9 Hz, 2H), 2.71
(t, J = 5.9˝Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H), 1.73–1.52 (m, 4H), 1.38–1.30 (m, 8H). Hydrochloride salt:
HCl Calc. C 70.32; N 6.56; H 7.32, found: C 70.12;
M.p. 200 C. Elemental analyses (%) for C25H30N2O2
N 6.48; H 7.41.
¨
2-(10-(3,4-Dihydroisoquinolin-2(1H)-yl)decyl)isoindoline-1,3-dione (14). Procedure M2. Reaction of
2-(10-bromodecyl)isoindoline-1,3-dione [51] (0.324 g, 1 mmol) with 1,2,3,4-tetrahydroisoquinoline
(0.13 g, 1 mmol) and K2CO3 (0.41 g, 3 mmol) in acetonitrile (15 mL), after 20 h, column chromatography
gave oil product. Yield 0.26 g (62%). TLC (S4) Rf = 0.71. MW 418.26. Formula: C27H34N2O2. MS:
m/z 419.43 [M + H]+. 1H-NMR (300 MHz, CDCl3)
δ ppm: 7.90–7.77 (m, 2H), 7.76–7.63 (m, 2H),
7.16–6.96 (m, 4H), 3.67 (t, J = 7.2 Hz, 2H), 3.61 (s, 2H), 2.90 (t, J = 5.9 Hz, 2H), 2.72 (t, J = 5.9 Hz, 2H), 2.48
(t, J = 7.1 Hz, 2H), 1.73–1.50 (m, 4H), 1.38–1.24 (m, 12H). Hydrochloride salt: M.p. 140 ˝C. Elemental
analyses (%) for C27H34N2O2¨ HCl Calc. C 71.27; N 6.16; H 7.75, found: C 7.35; N 6.18; H 7.83.
2-(12-(3,4-Dihydroisoquinolin-2(1H)-yl)dodecyl)isoindoline-1,3-dione (15) [60]. Procedure M2. Reaction of
2-(12-bromododecyl)isoindoline-1,3-dione [51] (0.31 g, 1 mmol) with 1,2,3,4-tetrahydroisoquinoline
(0.13 g, 1 mmol) and K2CO3 (0.41 g, 3 mmol) in acetonitrile (15 mL), after 20 h, column chromatography
gave oil product. Yield 0.25 g (57%). TLC (S4) Rf = 0.73. MW 446.29. Formula: C29H38N2O2. MS:
m/z 447.35 [M + H]+. 1H-NMR (300 MHz, CDCl3)
δ ppm: 7.90–7.77 (m, 2H), 7.76–7.64 (m, 2H),
7.16–7.06 (m, 3H), 7.01–6.96 (m, 1H), 3.67 (t, J = 7.2 Hz, 2H), 3.62 (s, 2H), 2.90 (t, J = 5.9 Hz, 2H), 2.72
(t, J = 5.9 ˝Hz, 2H), 2.49 (t, J = 7.5 Hz, 2H), 1.73–1.53 (m, 4H), 1.36–1.23 (m, 16H). Hydrochloride salt:
M.p. 128 C. Elemental analyses (%) for C29H38N2O2¨ HCl Calc. C 72.1; N 5.8; H 8.14, found: C 71.87;
N 5.91; H 8.29.

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3.1.5. General Procedure for the Synthesis of Compounds (20–23)
Procedure M3. A mixture of an appropriate aminoalkyl-1,2,3,4-tetrahydroisoquinoline derivative
(1 equiv) with dibromoethane (2 equiv) in the presence of K2CO3 (2 equiv) was stirred in acetonitrile
at room temperature for 48 h. Subsequently, the solvent was evaporated under vacuum, producing
a residue which was further dissolved in 20 mL of water and extracted with DCM (3
ˆ
30 mL). The
organic layer was dried with anhydrous Na2SO4. The solvent was then evaporated and the residue
was purified by silica gel column chromatography (DCM, petroleum ether, MeOH, TEA; 5:3.5:1:3
drops). The final product was obtained in the form of hydrochloride salt. The following compounds
were obtained.
5-(3,4-dihydroisoquinolin-2(1H)-yl)-N,N-diethylpentan-1-amine (20). Procedure M3. Reaction of
5-(3,4-dihydroisoquinolin-2(1H)-yl)pentan-1-amine [61] (16) (0.15 g, 0.69 mmol), bromoethane (0.15 g,
1.38 mmol) and K2CO3 (0.29 g, 2.07 mmol) in acetonitrile (30 mL), after 48 h, column chromatography
gave oil product. Yield 0.16 g (84%). TLC (S11) Rf = 0.54. MW 274.24. Formula: C18H30N2. MS:
1
m/z 275.39 [M + H]+ 276.24. H-NMR (300 MHz, CDCl3)
δ ppm: 7.16–7.04 (m, 3H), 7.01–6.96 (m, 1H),
3.61 (s, 2H), 2.90 (t, J = 6.1 Hz, 2H), 2.80–2.66 (m, 6H), 2.61 (t, J = 8.2 Hz, 2H), 2.50 (t, J = 7.5 Hz, 2H),
˝
1.71–1.54 (m, 4H), 1.45–1.29 (m, 2H), 1.14 (t, J = 7.2 Hz, 6H). Hydrochloride salt: M.p. 245 C. Elemental
analyses (%) for C18H30N2¨ 2HCl Calc. C 62.24; N 8.06; H 9.29, found: C 62.19; N 8.01; H 8.98.
6-(3,4-Dihydroisoquinolin-2(1H)-yl)-N,N-diethylhexan-1-amine (21). Procedure M3. Reaction of
6-(3,4-dihydroisoquinolin-2(1H)-yl)hexan-1-amine (17) [53] (0.6 g, 2.58 mmol), bromoethane (0.57 g,
5.16 mmol) and K2CO3 (1.07 g, 7.74 mmol) in acetonitrile (30 mL), after 48 h, column chromatography
gave oil product. Yield 0.55 g (74%). TLC (S11) Rf = 0.38. MW 288.26. Formula: C19H32N2. MS [M + H]+
1
289.39. H-NMR (300 MHz, CDCl3)
δ
ppm: 7.14–7.03 (m, 3H), 6.98–6.94 (m, 1H), 3.60 (s, 2H), 2.89 (t,
J = 5.9 Hz, 2H), 2.71 (t, J = 6.0 Hz, 2H), 2.61–2.35 (m, 6H), 1.65–1.39 (m, 4H), 1.37–1.26 (m, 6H), 1.02
(m, 6H). Hydrochloride salt: M.p. 215 ˝C. Elemental analyses (%) for C19H32N2
N 7.75; H 9.48, found: C 63.12; N 7.68; H 9.28.
¨
2HCl Calc. C 63.15;
7-(3,4-Dihydroisoquinolin-2(1H)-yl)-N,N-diethylheptan-1-amine (22). Procedure M3. Reaction of
7-(3,4-dihydroisoquinolin-2(1H)-yl)heptan-1-amine (18) [54] (0.16 g, 0.65 mmol), bromoethane (0.14 g,
1.3 mmol) and K2CO3 (0.27 g, 1.95 mmol) in acetonitrile (25 mL), after 48 h, column chromatography
gave oil product. Yield 0.06 g (31%). TLC (S6) Rf = 0.40. MW 302.27. Formula: C20H34N2. MS [M + H]+
1
303.41. H-NMR (300 MHz, CDCl3)
δ ppm: 7.12–7.07 (m, 3H), 7.04–6.98 (m, 1H), 3.61 (s, 2H), 2.89
(t, J = 5.9 Hz, 2H), 2.78–2.67 (m, 4H), 2.60 (t, J = 7.3 Hz, 2H), 2.48 (t, J = ˝7.3 Hz, 2H), 1.65–1.50 (m, 4H),
1.41–1.23 (m, 8H), 1.14 (t, J = 7.1 Hz, 6H). Hydrochloride salt: M.p. 197 C. Elemental analyses (%) for
C20H34N2¨ 2HCl Calc. C 63.99; N 7.46; H 9.67, found: C 63.97; N 7.44; H 9.37.
8-(3,4-Dihydroisoquinolin-2(1H)-yl)-N,N-diethyloctan-1-amine (23). Procedure M3. Reaction of
8-(3,4-dihydroisoquinolin-2(1H)-yl)octan-1-amine (19) [54] (0.6 g, 2.31 mmol), bromoethane (0.503 g,
4.62 mmol) and K2CO3 (0.96 g, 6.93 mmol) in acetonitrile (30 mL), after 48 h, column chromatography
gave oil product. Yield 0.45 g (62%). TLC (S6) Rf = 0.44. MW 316.29. Formula: C21H36N2. MS [M + H]+
1
317.39. H-NMR (300 MHz, CDCl3)
δ ppm: 7.14–7.09 (m, 3H), 7.03–6.97 (m, 1H), 3.61 (s, 2H), 2.89
(t, J = 5.9 Hz, 2H), 2.71 (t, J = 5.7 Hz, 2H), 2.60–2.35 (m, 6H), 1.67–1.52 (m, 2H), 1.50–1.39 (m, 2H),
˝
1.39–1.23 (m, 10H), 1.02 (t, J = 7.2 Hz, 6H). Hydrochloride salt: M.p. 183 C. Elemental analyses (%) for
C21H36N2¨ 2HCl Calc. C 64.77; N 7.19; H 9.83, found: C 64.66; N 7.09; H 9.56.
3.1.6. General Procedure for the Synthesis of Diethylamine Derivatives of N-alkyl-1H-indole (30–35)
Procedure M4. A mixture of the appropriate 1-(bromoalkyl)-1H-indole (1 equiv) with diethyl
amine (4 equiv) in the presence of K2CO3 (1 equiv) was stirred in acetonitrile under reflux for 24 h.
Subsequently, the solvent was evaporated under vacuum, producing a residue which was further

Molecules 2016, 21, 410
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dissolved in 40 mL of sodium bicarbonate and extracted with ethyl acetate (3
ˆ
30 mL). The organic
layer was dried with anhydrous Na2SO4. The solvent was then evaporated and the residue was
purified by silica gel column chromatography (S3) yielding a yellow oil. The final product was
obtained in the form of oxalic salt. The following compounds were obtained.
N,N-diethyl-3-(1H-indol-1-yl)propan-1-amine (30). Procedure M4. Reaction of 1-(3-bromopropyl)-
1H-indole [60] (0.35 g, 1.47 mmol) with diethylamine (0.43 g, 5.8 mmol) and K2CO3 (0.2 g, 1.47 mmol)
in acetonitrile (25 mL), after 24 h, column chromatography gave oil product. Yield 0.21 g (62%). TLC
1
(S1) Rf = 0.60. MW 230.18. Formula: C15H22N2. MS: m/z 231.31 [M + H]+. H-NMR (300 MHz, CDCl3)
δ
ppm: 7.63 (dd, J = 7.8, 0.8 Hz, 1H), 7.37 (dd, J = 8.2, 0.9 Hz, 1H), 7.24–7.16 (m, 1H), 7.15–7.01 (m, 2H),
6.49 (dd, J = 3.2, 0.9 Hz, 1H), 4.18 (t, J = 7.0 Hz, 2H), 2.51 (q, J = 7.2 Hz, 4H), 2.43 (t, J = 7.2, 6.8 Hz, 2H),
2.04–1.90 (m, 2H), 1.00 (t, J = 7.1 Hz, 6H). Oxalic acid salt: M.p. 156 ˝C. Elemental analyses (%) for
C15H22N2¨ (COOH)2 Calc. C 63.73; N 8.74; H 7.55; found: C 63.89; N 8.82; H 7.63.
N,N-diethyl-4-(1H-indol-1-yl)butan-1-amine (31). Procedure M4. Reaction of 1-(4-bromobutyl)-
1H-indole [53] (1.51 g, 6 mmol) with diethylamine (1.76 g, 24 mmol) and K2CO3 (0.83 g, 6 mmol) in
acetonitrile (100 mL), after 24 h, column chromatography gave oil product. Yield 1.1 g (76%). TLC (S1)
1
Rf = 0.56. MW 244.19. Formula: C16H24N2. MS: m/z 245.45 [M + H]+. H-NMR (300 MHz, CDCl3)
δ ppm: 7.65 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.27–7.18 (m, 1H), 7.16–7.05 (m, 2H), 6.50
(d, J = 3.1 Hz, 1H), 4.15 (t, J = 7.1 Hz, 2H), 2.50 (q, J = 7.2 Hz, 4H), 2.42 (t, J = 7.8, 7.2 Hz, 2H), 1.95–1.78
˝
(m, 2H), 1.56–1.38 (m, 2H), 1.00 (t, J = 7.2 Hz, 6H). Oxalic acid salt: M.p. 107 C. Elemental analyses (%)
for C16H24N2¨ (COOH)2 Calc. C 64.65; N 8.38; H 7.84, found: C 64.73; N 8.35; H 7.96.
N,N-Diethyl-5-(1H-indol-1-yl)pentan-1-amine (32). Procedure M4. Reaction of 1-(5-bromopentyl)-
1H-indole [53] (1.33 g, 5 mmol) with diethylamine (1.5 g, 20 mmol) and K2CO3 (0.69 g, 5 mmol)
in acetonitrile (80 mL), after 24 h, column chromatography gave oil product. Yield 0.89 g (69%). TLC
(S1) Rf = 0.61. MW 258.21. Formula: C17H26N2. MS: m/z 259.47 [M + H]+. 1H-NMR (300 MHz,
CDCl3) δ ppm: 7.58–7.43 (m, 2H), 7.37 (d, J = 3.1 Hz, 1H), 7.18–7.07 (m, 1H), 7.06–6.94 (m, 1H), 6.42 (d,
J = 3.1 Hz, 1H), 4.18 (t, J = 6.9 Hz, 2H), 3.02 (q, J = 7.2 Hz, 4H), 2.90 (t, J = 8.7, 8.2 Hz, 2H), 1.87–1.71 (m,
˝
2H), 1.71–1.53 (m, 2H), 1.37–1.19 (m, 2H), 1.09 (t, J = 7.0 Hz, 6H). Oxalic acid salt: M.p. 82 C. Elemental
analyses (%) for C17H26N2¨ (COOH)2 Calc. C 65.49; N 8.04; H 8.10, found: C 65.69; N 7.96; H 8.18.
N,N-Diethyl-6-(1H-indol-1-yl)hexan-1-amine (33). Procedure M4. Reaction of 1-(6-bromohexyl)-
1H-indole [54] (0.8 g, 2.85 mmol) with diethylamine (0.83 g, 11.4 mmol) and K2CO3 (0.39 g, 2.85 mmol)
in acetonitrile (45 mL), after 24 h, column chromatography gave oil product. Yield 0.59 g (76%). TLC
(S1) Rf = 0.68. MW 272.23. Formula: C18H28N2. MS: m/z 273.35 [M + H]+. 1H-NMR (300 MHz,
CDCl3) δ ppm: 7.64 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.29–7.16 (m, 1H), 7.14–7.05 (m, 2H),
6.49 (d, J = 3.2 Hz, 1H), 4.12 (t, J = 7.1 Hz, 2H), 2.51 (q, J = 7.2 Hz, 4H), 2.38 (t, J = 8.0, 7.0 Hz, 2H),
1.51–1.21 (m, 8H), 1.02 (t, J = 7.2 Hz, 6H). Oxalic acid salt: M.p. 80 ˝C. Elemental analyses (%) for
C18H28N2¨ (COOH)2 Calc. C 66.27; N 7.73; H 8.34, found: C 66.37; N 7.64; H 8.36.
N,N-Diethyl-7-(1H-indol-1-yl)heptan-1-amine (34). Procedure M4. Reaction of 1-(7-bromoheptyl)-
1H-indole [54] (1.03 g, 3.5 mmol) with diethylamine (1.02 g, 14 mmol) and K2CO3 (0.48 g, 3.5 mmol)
in acetonitrile (50 mL), after 24 h, column chromatography gave oil product. Yield 0.63 g (63%). TLC
(S1) Rf = 0.63. MW 286.24. Formula: C19H30N2. MS: m/z 287.35 [M + H]+. 1H-NMR (300 MHz,
CDCl3)
δ ppm: 7.69–7.59 (m, 1H), 7.42–7.31 (m, 1H), 7.25–7.17 (m, 1H), 7.14–7.07 (m, 2H), 6.49
(d, J = 3.1 Hz, 1H), 4.11 (t, J = 7.1 Hz, 2H), 2.52 (q, J = 7.2 Hz, 4H), 2.39 (t,˝J = 7.7 Hz, 2H), 1.92–1.75
(m, 2H), 1.43–1.23 (m, 8H), 1.02 (t, J = 7.2 Hz, 6H). Oxalic acid salt: M.p. 84 C. Elemental analyses (%)
for C19H30N2¨ (COOH)2 Calc. C 66.99; N 7.44; H 8.67, found: C 67.07; N 7.60; H 8.61.

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N,N-Diethyl-8-(1H-indol-1-yl)octan-1-amine (35). Procedure M4. Reaction of 1-(8-bromooctyl)-
1H-indole [54] (0.96 g, 3.1 mmol) with diethylamine (0.91 g, 12.4 mmol) and K2CO3 (0.43 g, 3.1 mmol)
in acetonitrile (50 mL), after 24 h, column chromatography gave oil product. Yield 0.69 g (73%). TLC
1
(S1) Rf = 0.65. MW 300.26. Formula: C20H32N2. MS: m/z 301.39 [M + H]+. H-NMR (300 MHz, CDCl3)
δ
ppm: 7.64 (d, J = 7.8 Hz, 1H), 7.40–7.31 (m, 1H), 7.25–7.16 (m, 1H), 7.14–7.06 (m, 2H), 6.49 (dd, J = 3.1,
0.8 Hz, 1H), 4.12 (t, J = 7.3 Hz, 2H), 2.52 (q, J = 7.2 Hz, 4H), 2.42–2.35 (m, 2H), 1.51–1.16 (m, 12H), 1.02
(t, J = 7.2 Hz, 6H). Oxalic acid salt: M.p. 87 ˝C. Elemental analyses (%) for C20H32N2
(COOH)2 Calc.
¨
C 67.66; N 7.17; H 8.78; found: C 67.47; N 7.05; H 8.71.
3.1.7. General Procedure for the Synthesis of Compounds (39–44)
Procedure M5. To a solution of the appropriate 2-(bromoalkyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxide (1 equiv) in DMSO was added˝threefold access of appropriate benzylamine (3 equiv)
and the reaction mixture was heated to 60 C for 3.5 h. Once the reaction was finished, 50 mL of
water was added. The reaction mixture was extracted with dichloromethane (6
ˆ
15 mL) and the
combined organic extracts were washed with water (5 50 mL). Then, the organic phase was dried
ˆ
over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Purification by flash
chromatography (silica, dichloromethane to dichloromethane/methanol 94:6), afforded a yellow oil.
The following compounds were obtained.
2-(5-(2-fluorobenzylamino)pentyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (39). Procedure M5. Reaction
of 2-(5-bromopentyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide [62] (36) (166 mg, 0.5 mmol) with
2-fluorobenzylamine (187 mg; 1.5 mmol) in DMSO (6.5 mL), after 3.5 h then purification by flash
chromatography and column chromatography (CHCl3:MeOH:NH3—10:0.2:0.05) gave product 39
Yield 38 mg (20%). TLC (S3) Rf = 0.23. MW 376.45. Formula: C19H21FN2O3S. MS: m/z 377.08 [M + H]+.
1H-NMR (300 MHz, CDCl3)
ppm: 8.08–8.02 (m, 1H), 7.94–7.78 (m, 3H), 7.33 (td, J = 7.57, 1.80 Hz,
1H), 7.26–7.16 (m, 1H), 7.13–6.96 (m, 2H), 3.83 (s, 2H), 3.81–3.73 (m, 2H), 2.63 (t, J = 7.05 Hz, 2H), 1.87
(dt, J = 15.13, 7.57 Hz, 2H), 1.64–1.52 (m, 3H), 1.52–1.39 (m, 2H). 13C-NMR (75 MHz, CDCl3)
ppm:
.
δ
δ
161.1 (d, JC-F = 245.0 Hz), 158.9, 137.6, 134.6, 134.2, 130.3 (d, JC-F = 4.4 Hz), 128.5 (d, JC-F = 7.7 Hz), 127.3,
127.2 (d, JC-F = 15.0 Hz), 125.0, 124.0 (d, JC-F = 3.3 Hz), 120.8, 115.2 (d, JC-F = 21.5 Hz), 48.9, 47.2, 39.3,
29.6, 29.4, 28.3, 28.7, 24.5.
2-(6-(2-fluorobenzylamino)hexyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (40). Procedure M5. Reaction
of 2-(6-bromohexyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide [62] (37) (173 mg, 0.5 mmol) with
2-fluorobenzylamine (187 mg; 1.5 mmol) in DMSO (6.5 mL), after 3.5 h and purification by flash
chromatography gave product 40. Yield 133 mg (68 %). TLC (S3) Rf = 0.24. MW 390.47. Formula:
1
C20H23FN2O3S. MS: m/z 391.11 [M + H]+. H-NMR (300 MHz, CDCl3)
δ ppm: 8.13–7.99 (m, 1H),
7.98–7.71 (m, 3H), 7.34 (td, J = 7.57, 1.80 Hz, 1H), 7.25–7.18 (m, 1H), 7.14–6.98 (m, 2H), 3.84 (s, 2H), 3.77
(t, J = 7,44 Hz, 2H), 2.62 (t, J = 7.05 Hz, 2H), 1.85 (quin, J = 7.37 Hz, 2H), 1.65 (br. s., 1H), 1.59–1.48
(m, 2H), 1.46–1.31 (m, 4H). 13C-NMR (75 MHz, CDCl3)
δ ppm 161.1 (d, JC-F = 245.0 Hz), 158.9, 137.6,
134.6, 134.2, 130.4 (d, JC-F = 4.4 Hz), 128.6 (d, JC-F = 8.3 Hz), 127.4, 127.1 (d, JC-F = 14.9 Hz), 125.1, 124.0
(d, JC-F = 3.9 Hz), 120.8, 115.2 (d, JC-F = 22.1 Hz), 49.0, 47.2, 39.3, 29.7, 28.3, 26.7, 26.6.
2-(7-(2-fluorobenzylamino)heptyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (41). Procedure M5. Reaction
of 2-(7-bromoheptyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide [62] (38) (180 mg, 0.5 mmol) with
2-fluorobenzylamine (187 mg; 1.5 mmol) in DMSO (6.5 mL), after 3.5 h then purification by flash
chromatography and column chromatography (CH2Cl2:MeOH—9.5:0.5) gave product 41. Yield 90 mg
1
(45%). TLC (S3) Rf = 0.19. MW 404.50. Formula: C21H25FN2O3S. MS: m/z 405.13 [M + H]+. H-NMR
(300 MHz, CDCl3)
(m, 1H), 7.14–6.98 (m, 2H), 3.85 (s, 2H), 3.76 (t, J = 7,44 Hz, 2H), 2.62 (t, J = 7.18 Hz, 2H), 2.04 (br. s., 1H),
1.85 (quin, J = 7.44 Hz, 2H), 1.52 (quin, J = 6.99 Hz, 2H), 1.46–1.30 (m, 6H). 13C-NMR (75 MHz, CDCl3)
δ ppm: 8.08–8.02 (m, 1H), 7.95–7.78 (m, 3H), 7.35 (td, J = 7.44, 1.80 Hz, 1H), 7.26–7.19
δ

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ppm 161.2 (d, JC-F = 245.5 Hz), 158.9, 137.7, 134.6, 134.2, 130.4 (d, JC-F = 5.0 Hz), 128.6 (d,
J
C-F = 8.3 Hz),
127.4, 126.9 (d, JC-F = 15.5 Hz), 125.1, 124.0 (d, JC-F = 3.3 Hz), 120.8, 115.2 (d, JC-F = 21.5 Hz), 49.0, 47.1,
39.4, 29.7, 28.9, 28.3, 27.1, 26.7.
2-(5-(3-chlorobenzylamino)pentyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (42). Procedure M5. Reaction
of 2-(5-bromopentyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide [62] (36) (166 mg, 0.5 mmol) with
3-chlorobenzylamine (212 mg; 1.5 mmol) in DMSO (6.5 mL), after 3.5 h and purification by flash
chromatography gave product 42. Yield 98 mg (50%). TLC (S3) Rf = 0.29. MW 392.90. Formula:
1
C19H21ClN2O3S. MS: m/z 393.10 [M + H]+. H-NMR (300 MHz, CDCl3)
δ ppm 8.10–8.03 (m, 1H),
7.95–7.81 (m, 3H), 7.34–7.30 (m, 1H), 7.26–7.17 (m, 3H), 3.84–3.74 (m, 4H), 2.64 (t, J = 6.92 Hz, 2H), 1.88
(dt, J = 15.07, 7.47 Hz, 2H), 1.65–1.40 (m, 5H). 13C-NMR (75 MHz, CDCl3)
δ
ppm 158.93, 142.45, 137.60,
134.65, 134.27, 134.16, 129.58, 128.14, 127.36, 127.00, 126.17, 125.09, 120.86, 53.33, 48.98, 39.25, 29.38,
28.24, 24.43.
2-(6-(3-chlorobenzylamino)hexyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (43). Procedure M5. Reaction
of 2-(6-bromohexyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide [62] (37) (166 mg, 0.48 mmol) with
3-chlorobenzylamine (212 mg; 1.5 mmol) in DMSO (6.5 mL), after 3.5 h and purification by flash
chromatography gave product 43. Yield 112 mg (58%). TLC (S3) Rf = 0.31. MW 406.93. Formula:
1
C20H23ClN2O3S. MS: m/z 407.06 [M + H]+. H-NMR (300 MHz, CDCl3)
δ ppm 8.10–8.02 (m, 1H),
7.96–7.77 (m, 3H), 7.36–7.30 (m, 1H), 7.26–7.17 (m, 3H), 3.83–3.70 (m, 4H), 2.62 (t, J = 7.05 Hz, 2H),
1.86 (quin, J = 7.37 Hz, 2H), 1.62 (br. s., 1H), 1.59–1.47 (m, 2H), 1.47–1.38 (m, 4H). 13C-NMR (75 MHz,
CDCl3)
δ ppm 158.92, 142.42, 137.67, 134.61, 134.23, 134.18, 129.58, 128.17, 127.41, 127.02, 126.19, 125.08,
120.84, 53.33, 49.12, 39.31, 29.73, 28.30, 26.65, 26.59.
2-(7-(3-chlorobenzylamino)heptyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (44). Procedure M5. Reaction
of 2-(7-bromoheptyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide [62] (38) (180 mg, 0.5 mmol) with
3-chlorobenzylamine (212 mg; 183 µL, 1.5 mmol) in DMSO (6.5 mL), after 3.5 h and purification
by flash chromatography gave product 44. Yield 134 mg (64%). TLC (S3) Rf = 0.18. MW 420.95.
1
Formula: C21H25ClN2O3S. MS: m/z 421.08 [M + H]+. H-NMR (300 MHz, CDCl3)
δ
ppm: 8.09–8.02 (m,
1H), 7.95–7.78 (m, 3H), 7.37–7.30 (m, 1H), 7.26–7.17 (m, 3H), 3.82–3.71 (m, 4H), 2.61 (t, J = 7.05 Hz, 2H),
1.85 (quin, J = 7.37 Hz, 2H), 1.72 (br. s., 1H), 1.58–1.45 (m, 2H), 1.45–1.30 (m, 6H). 13C-NMR (75 MHz,
CDCl3)
δ ppm 158.90, 142.26, 137.66, 134.61, 134.23, 134.18, 129.59, 128.20, 127.41, 127.05, 126.23, 125.06,
120.83, 53.30, 49.18, 39.36, 29.72, 28.84, 28.29, 27.03, 26.64.
3.2. Molecular Modelling
The three-dimensional ligand structures were built with Corina online tool. Subsequently, atom
types and protonation states were checked and Gasteiger-Marsili charges were assigned using Sybyl 8.0
Finally, ligand structures were saved in the mol2 format. Docking to Torpedo californica AChE (PDB
code: 1EVE) was performed using the Gold 4.1. The target was prepared as follows: all histidine
.
residues were protonated at Nε, hydrogen atoms added, ligand molecules removed, and binding
sites defined as all amino acid residues within 10 Å from donepezil. The presence of conserved water
molecules was also taken into account. A standard set of genetic algorithms with a population size
of 100, number of operations being 100,000 and with a clustering tolerance of 1 Å were applied. As a
result, 10 ligand conformations were obtained and sorted according to ChemScore function values.
Results were visualized using PyMOL.
3.3. Biological Evaluation
3.3.1. In vitro Inhibition of AChE and BuChE
To assess the inhibitory activity of the target compounds towards cholinesterases, we followed
Ellman’s assay (as modified for 24-well microplates) using AChE from Electrophorus electricus (EeAChE)

Molecules 2016, 21, 410
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(Sigma–Aldrich) and BuChE from equine serum (EqBuChE, Sigma–Aldrich). 500 U of AChE or BuChE
was dissolved in 1 ml of a gelatine solution (1% in water) and diluted with demineralized water
to give a stock solution of 5 U/mL. The AChE solution was further diluted before use to give a
1
final concentration of 3.125 U/mL. The 0.0125 M 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s
reagent) solution containing 0.15% (w/v) sodium carbonate and the 0.01875 M acetylthiocholine
(ATC) iodide solution were prepared in demineralized water. All assays were performed in 0.1 M
phosphate buffer pH 8.0. The tested compounds or water in a blank sample (25
µ
L) were incubated
L) prior to starting the reaction. Then,
L) were added. After 5 min of the reaction, changes in absorbance were
˝
µL) for 5 min at 25 C in buffer (765 µ
with the enzyme (20
DTNB (20 µL) and ATC (20
µ
measured at 412 nm using a microplate reader (EnSpire Multimode, PerkinElmer). Each condition was
measured in triplicate. The percentages of enzyme inhibition were calculated from the equation
100%—Ai/A0 ˆ 100%, where Ai is the absorbance of a sample with an inhibitor and A0 is the
absorbance of a blank sample (100% activity of the enzyme). The IC50 values were calculated using
GraphPad Prism 5. Data is expressed as mean ˘ SEM.
3.3.2. Kinetic Characterization of EeAChE Inhibition
To estimate the type of inhibition of EeAChE, the same experimental protocol as reported above
(4.3.1) was performed. Different concentrations of the substrate ATC (0.067–0.5 mM) were used to
create Lineweaver-Burk plots by plotting the inverse initial velocity (1/V) as a function of the inverse
of the substrate concentration (1/[S]). The stock solution of ATC (0.5 mM in a well) was prepared in
water and diluted before use to obtained 0.4, 0.3, 0.2, 0.1and 0.067 mM concentrations of substrate. The
double reciprocal plots were assessed by a weighted least square analysis that assumed the variance
of V to be a constant percentage of V for the entire data set. Each experiment was performed in
triplicate. Then, to confirm the mode of inhibition, Cornish-Bowden plots were obtained by plotting
S/V (substrate concentration/velocity ratio) versus the inhibitor concentration [I]. Data analysis was
performed with GraphPad Prism 5.
3.3.3. In vitro Inhibition of Aβ1–42 Aggregation
Thioflavin-T (ThT) fluorometric assay was performed to investigate the effect of the test
compounds on the self-aggregation of Aβ1–42. Recombinant human HFIP-pretreated Aβ1–42 peptide
(Lot number 2387442, Merck Millipore, Darmstadt, Germany) was dissolved in DMSO. Prior to the
incubations, the Aβ1–42 peptide stock solution was diluted in 150 mM HEPES buffer (pH 7.4) containing
150 mM NaCl, to give a concentration of 7.5
compounds (10 L, 100 M stock in HEPES; 10
wells in black-walled 96-well plates, and diluted with ThT solution (70
µ
M. Then Aβ1–42 (20
M final concentration), added to the corresponding
L, 14.3 M stock solution in
µL) was mixed with of the test
µ
µ
µ
µ
µ
HEPES; 10 µM final concentration), to the final volume of 100 µL (1.5 µM final Aβ1–42 concentration).
Each sample was prepared in quadruplicate, and the DMSO was always at 3%. To quantify amyloid
fibril formation, the ThT fluorescence was measured through the bottom of the plate every 180 s at an
excitation wavelength of 440 nm and emission wavelength of 490 nm, with the medium continuously
shaking between measurements using a 96-well microplate reader (Synergy™ H4, BioTek Instruments,
Inc., Winooski, VT, USA). The ThT emission of the Aβ1–42 began to rise after 4 h, reached a plateau after
36 h, and remained almost unchanged for an additional 12 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 Aβ1–42 self-induced aggregation
inhibitory potencies are expressed as the percentage inhibition (%inh = (1
Fi is the increase in fluorescence of Aβ1–42 treated with the test compounds, and F0 is the increase in
fluorescence of Aβ1–42 alone.
´
Fi/F0)
ˆ
100%), where

Molecules 2016, 21, 410
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3.3.4. PAMPA-BBB Assay
The brain penetration of compounds
4,
7
,
13
,
39 and 42 was assessed using the parallel artificial
membrane permeability assay for blood brain barrier (BBB-PAMPA). The BBB-PAMPA Explorer Test
System was purchased from pION Inc. The in vitro permeability through BBB-1 lipid membrane was
determined for 7 commercial drugs and the tested compounds. The compounds were dissolved in
DMSO (10 mM stock solution) and diluted with Prisma HT buffer (5
96-well microplate was filled with solution of the tested compounds in buffer (200
membrane in acceptor 96-well microplate was impregnated with BBB-1 lipid solution (5
µ
L/1 mL). Then, the acceptor
L/well). The filter
L/well) and
µ
µ
the acceptor plate was filled with Brain Sink Buffer (200
µ
L/well). The acceptor˝plate was carefully
placed on the donor plate to form a sandwich that was left undisturbed for 2 h at 37 C. After incubation,
the donor plate was carefully removed. The concentration of compounds in the acceptor, the donor,
and the reference wells were measured using EnSpire Multimode Microplate Reader (PerkinElmer).
Logarithm of the effective permeability (log Pe) of the compounds was calculated using the pION
software. Assay validation was done by comparison of the experimental permeability of the seven
commercial drugs with their reference values established for this assay by pION. We established the
following ranges of permeability: CNS+, log Pe >
´
4.5, high permeability (i.e., can enter the CNS);
CNS–, log Pe ď´6.3, low permeability (i.e., excluded from the CNS); CNS˘, –6.3 < log Pe ď´4.5,
uncertain BBB permeability.
4. Conclusions
Herein, we presented a continuation of our studies focused on the search for multitarget
compounds as potential anti-AD agents. With the aid of molecular modelling, we designed new
dual binding site inhibitors of AChE. The designed compounds were synthesized and evaluated
in vitro. We found that most of the target compounds are moderate or potent AChE inhibitors or
dual AChE/BuChE inhibitors with IC50 values in the low micromolar and submicromolar range.
Structure–activity relationship analysis revealed that among the tested compounds containing different
heteroaromatic moieties, the most potent inhibitors were derivatives with a saccharin fragment with
the most potent compound 42 (EeAChE IC50 = 70 nM). Regarding the amine part of the molecules, we
found that pyrrolidine, benzylamine and its rigid analogue—tetrahydroisoquinoline—are beneficial
for AChE inhibitory potency when compared to morpholine. Compound 13, a tetrahydroisoquinoline
derivative, was found to be a dual inhibitor of AChE and BuChE (EeAChE IC50 = 0.76
IC50 = 0.618 M). This balanced potency seems to be a promising starting point for further studies given
the fact that currently used anti-AD drugs inhibit these two enzymes with inhibitory potencies in the
same order of magnitude (i.e., rivastigmine: EeAChE IC50 = 3.01–3.4 M, EqBuChE IC50 = 0.30–5.5 µM;
galantamine: EeAChE IC50 = 0.665–2.41 M, EqBuChE IC50 = 17.38–19.78 M) [63 65]. Compound 13
was also the most potent inhibitor of self-induced Aβ1–42 aggregation (35.80% at 10 M) comparable
with other multifunctional agents described recently [22] and a more potent inhibitor than donepezil
(11.48% at 10 M). Kinetic studies revealed that the developed derivatives are mixed or non-competitive
µM, EqBuChE
µ
µ
µ
µ
–
µ
µ
AChE inhibitors. Molecular modelling studies indicated that all the compounds were dual binding site
inhibitors able to interact with catalytic and peripheral active sites of AChE. The results of both kinetic
studies and molecular modelling showed that the tested compounds display a similar mechanism of
action to that of donepezil. The results from the PAMPA-BBB assay indicated that the tested compounds
are able to cross the BBB in vitro. In conclusion, our studies have provided a better understanding of
the structure–activity relationships in a group of heterodimeric cholinesterase inhibitors and allowed
the identification of compounds 42 and 13 as interesting agents which can be used in the further
development of potential anti-Alzheimer’s drugs.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/4/410/s1.

Molecules 2016, 21, 410
20 of 24
Acknowledgments: Financial support from: the Polish Ministry for Science and High Education (grant No.
N N405 163339) and the National Science Center of Poland (grant No. 2012/07/B/NZ7/04253) is gratefully
acknowledged. We thank Bálint Sinkó for his help with establishing and managing PAMPA-BBB assay, this study
received financial support from the Jagiellonian University Collegium Medicum (grant No. K/Z S/004657).
Author Contributions: M.H. synthesized most of the compounds and evaluated their inhibitory potency on the
cholinesterases; M.B. designed the novel compounds, performed molecular modelling studies, wrote a part of
manuscript and corrected the manuscript; A.W. supervised part of the synthesis, performed PAMPA-BBB assay
and corrected the manuscript; N.S. performed PAMPA-BBB assay, wrote a part of the manuscript and corrected it;
A.P. did a part of chemical synthesis; D.P. performed a part of the kinetic study; T.W. wrote a part of the manuscript
and corrected it; J.G. performed a part of the inhibition potency on the cholinesterases and the kinetic study; D.K.
performed the Aβ test and corrected the manuscript; S.G. supervised the Aβ test and corrected the manuscript;
B.M. supervised and coordinated all studies, wrote a part of the manuscript and corrected it.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
Ach
acetylcholine
AChE
AD
acetylcholinesterase
Alzheimer’s disease
Aβ
amyloid beta peptide
BBB
blood-brain barrier
BuChE
BuChE
CAS
butyrylcholinesterase
butyrylcholinesterase
catalytic anionic site in acetylcholinesterase
central nervous system
CNS
EeAChE
EqBuChE
MTDL
NFTs
acetylcholinesterase from electric eel
equine serum butyrylcholinesterase
multi-target-directed ligands
neurofibrillary tangles
PAMPA
PAS
TcAChE
parallel artificial membrane permeation assay
peripheral anionic site in acetylcholinesterase
acetylcholinesterase from Torpedo californica
References
1.
2.
3.
4.
Prince, M.; Prina, M.; Guerchet, M. World Alzheimer Report 2013 Journey of Caring: An Analysis of Long-Term
Care for Dementia; Alzheimer’s Disease International: London, UK, 2013; pp. 1–92.
Wimo, A.; Jönsson, L.; Bond, J.; Prince, M.; Winblad, B. The worldwide economic impact of dementia 2010.
Alzheimer’s Dement. 2013, 9. [CrossRef] [PubMed]
Cummings, J.L.; Morstorf, T.; Zhong, K. Alzheimer’s disease drug-development pipeline: Few candidates,
frequent failures. Alzheimers. Res. Ther. 2014, 6. [CrossRef] [PubMed]
Godyn´, J.; Jon´czyk, J.; Panek, D.; Malawska, B. Therapeutic strategies for Alzheimer’s disease in clinical
trials. Pharmacol. Rep. 2015, 68, 127–138. [CrossRef] [PubMed]
5.
6.
7.
Blennow, K.; de Leon, M.J.; Zetterberg, H. Alzheimer’s disease. Lancet 2006, 368, 387–403. [CrossRef]
Querfurth, H.W.; LaFerla, F.M. Alzheimer’s disease. N. Engl. J. Med. 2010, 362, 329–344. [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]
8.
9.
Davies, P.; Maloney, A.J.F. Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet 1976
308. [CrossRef]
Mufson, E.J.; Counts, S.E.; Perez, S.E.; Ginsberg, S.D. Cholinergic system during the progression of
Alzheimer’s disease: Therapeutic implications. Expert Rev. Neurother. 2008, 8, 1703–1718. [CrossRef]
[PubMed]
,
10. Giacobini, E. Cholinesterase inhibitors: New roles and therapeutic alternatives. Pharmacol. Res. 2004, 50,
433–440. [CrossRef] [PubMed]

Molecules 2016, 21, 410
21 of 24
11. De Ferrari, G.V.; Canales, M.A.; Shin, I.; Weiner, L.M.; Silman, I.; Inestrosa, N.C. A Structural Motif of
Acetylcholinesterase That Promotes Amyloid
[CrossRef] [PubMed]
β-Peptide Fibril Formation. Biochemistry 2001, 40, 10447–10457.
12. Johnson, G.; Moore, S.W. Identification of a structural site on acetylcholinesterase that promotes neurite
outgrowth and binds laminin-1 and collagen IV. Biochem. Biophys. Res. Commun. 2004, 319, 448–455.
[CrossRef] [PubMed]
13. Inestrosa, N.C.; Alvarez, A.; Pérez, C.A.; Moreno, R.D.; Vicente, M.; Linker, C.; Casanueva, O.I.; Soto, C.;
Garrido, J. Acetylcholinesterase Accelerates Assembly of Amyloid-β-Peptides into Alzheimer’s Fibrils:
Possible Role of the Peripheral Site of the Enzyme. Neuron 1996, 16, 881–891. [CrossRef]
14. Sussman, J.L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Atomic structure of
acetylcholinesterase from Torpedo californica: A prototypic acetylcholine-binding protein. Science 1991, 253,
872–879. [CrossRef] [PubMed]
15. Akasofu, S.; Kimura, M.; Kosasa, T.; Sawada, K.; Ogura, H. Study of neuroprotection of donepezil, a therapy
for Alzheimer’s disease. Chem. Biol. Interact. 2008, 175, 222–226. [CrossRef] [PubMed]
16. Akaike, A.; Takada-Takatori, Y.; Kume, T.; Izumi, Y. Mechanisms of Neuroprotective Effects of Nicotine and
Acetylcholinesterase Inhibitors: Role of
211–216. [CrossRef] [PubMed]
α4 and α7 Receptors in Neuroprotection. J. Mol. Neurosci. 2009, 40,
17. Verdile, G.; Fuller, S.J.; Martins, R.N. The role of type 2 diabetes in neurodegeneration. Neurobiol. Dis. 2015,
84, 22–38. [CrossRef] [PubMed]
18. Zhao, Y.; Zhao, B. Oxidative stress and the pathogenesis of Alzheimer’s disease. Oxid. Med. Cell. Longev.
2013, 2013. [CrossRef] [PubMed]
19. Kan, M.J.; Lee, J.E.; Wilson, J.G.; Everhart, A.L.; Brown, C.M.; Hoofnagle, A.N.; Jansen, M.; Vitek, M.P.;
Gunn, M.D.; Colton, C.A. Arginine Deprivation and Immune Suppression in a Mouse Model of Alzheimer’s
Disease. J. Neurosci. 2015, 35, 5969–5982. [CrossRef] [PubMed]
20. Morphy, R.; Kay, C.; Rankovic, Z. From magic bullets to designed multiple ligands. Drug Discov. Today 2004
,
9, 641–651. [CrossRef]
21. Zimmermann, G.R.; Lehár, J.; Keith, C.T. Multi-target therapeutics: When the whole is greater than the sum
of the parts. Drug Discov. Today 2007, 12, 34–42. [CrossRef] [PubMed]
22. Guzior, N.; Wie˛ckowska, A.; Panek, D.; Malawska, B. Recent development of multifunctional agents as
potential drug candidates for the treatment of Alzheimer’s disease. Curr. Med. Chem. 2015, 22, 373–404.
[CrossRef] [PubMed]
23. Dias, K.S.T.; Viegas, C., Jr. Multi-Target Directed Drugs: A Modern Approach for Design of New Drugs for
the treatment of Alzheimer’s Disease. Curr. Neuropharmacol. 2014, 12, 239–255. [CrossRef] [PubMed]
24. Agis-Torres, A.; Sölhuber, M.; Fernandez, M.; Sanchez-Montero, J.M. Multi-Target-Directed Ligands and
other Therapeutic Strategies in the Search of a Real Solution for Alzheimer’s Disease. Curr. Neuropharmacol.
2014, 12, 2–36. [CrossRef] [PubMed]
25. Calza, L.; Antonio Baldassarro, V.; Giuliani, A.; Lorenzini, L.; Fernandez, M.; Mangano, C.; Sivilia, S.;
Alessandri, M.; Gusciglio, M.; Torricella, R.; et al. From the Multifactorial Nature of Alzheimer’s Disease
to Multitarget Therapy: The Contribution of the Translational Approach. Curr. Top. Med. Chem. 2013, 13,
1843–1852. [CrossRef] [PubMed]
26. Carreiras, M.; Mendes, E.; Perry, M.; Francisco, A.; Marco-Contelles, J. The Multifactorial Nature of
Alzheimer’s Disease for Developing Potential Therapeutics. Curr. Top. Med. Chem. 2013, 13, 1745–1770.
[CrossRef] [PubMed]
27. León, R.; Garcia, A.G.; Marco-Contelles, J. Recent advances in the multitarget-directed ligands approach for
the treatment of Alzheimer’s disease. Med. Res. Rev. 2013, 33, 139–189. [CrossRef] [PubMed]
28. 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]
29. Knez, D.; Brus, B.; Coquelle, N.; Sosicˇ, I.; Šink, R.; Brazzolotto, X.; Mravljak, J.; Colletier, J.P.; Gobec, S.
Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.
Bioorg. Med. Chem. 2015, 23. [CrossRef] [PubMed]

Molecules 2016, 21, 410
22 of 24
30. Tonelli, M.; Catto, M.; Tasso, B.; Novelli, F.; Canu, C.; Iusco, G.; Pisani, L.; de Stradis, A.; Denora, N.;
Sparatore, A.; et al. Multitarget Therapeutic Leads for Alzheimer’s Disease: Quinolizidinyl Derivatives of Bi-
and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β-Amyloid (Aβ) Aggregation. ChemMedChem
2015, 10, 1040–1053. [CrossRef] [PubMed]
31. Farina, R.; Pisani, L.; Catto, M.; Nicolotti, O.; Gadaleta, D.; Denora, N.; Soto-Otero, R.; Mendez-Alvarez, E.;
Passos, C.S.; Muncipinto, G.; et al. Structure-Based Design and Optimization of Multitarget-Directed 2 H
-Chromen-2-one Derivatives as Potent Inhibitors of Monoamine Oxidase B and Cholinesterases. J. Med. Chem.
2015, 58, 5561–5578. [CrossRef] [PubMed]
32. Xie, S.S.; Wang, X.; Jiang, N.; Yu, W.; Wang, K.D.G.; Lan, J.S.; Li, Z.R.; Kong, L.Y. Multi-target tacrine-coumarin
hybrids: Cholinesterase and monoamine oxidase B inhibition properties against Alzheimer’s disease. Eur. J.
Med. Chem. 2015, 95, 153–165. [CrossRef] [PubMed]
33. Bolea, I.; Juárez-Jiménez, J.; de los Ríos, C.; Chioua, M.; Pouplana, R.; Luque, F.J.; Unzeta, M.;
Marco-Contelles, J.; Samadi, A. Synthesis, Biological Evaluation, and Molecular Modeling of Donepezil
and N-[(5-(Benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine Hybrids as New
Multipotent Cholinesterase/Monoamine Oxidase Inhibitors for the Treatment of Alzheimer’s Disease.
J. Med. Chem. 2011, 54, 8251–8270. [PubMed]
34. Pau, A.; Catto, M.; Pinna, G.; Frau, S.; Murineddu, G.; Asproni, B.; Curzu, M.M.; Pisani, L.; Leonetti, F.;
Loza, M.I.; et al. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and
cholinesterase inhibitors. ChemMedChem 2015, 10, 1054–1070. [CrossRef] [PubMed]
35. Rochais, C.; Lecoutey, C.; Gaven, F.; Giannoni, P.; Hamidouche, K.; Hedou, D.; Dubost, E.; Genest, D.;
Yahiaoui, S.; Freret, T.; et al. Novel Multitarget-Directed Ligands (MTDLs) with Acetylcholinesterase (AChE)
Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities As Potential Agents against
Alzheimer’s Disease: The Design of Donecopride. J. Med. Chem. 2015, 58, 3172–3187. [CrossRef] [PubMed]
36. Xie, S.S.; Lan, J.S.; Wang, X.B.; Jiang, N.; Dong, G.; Li, Z.R.; Wang, K.D.G.; Guo, P.P.; Kong, L.Y. Multifunctional
tacrine-trolox hybrids for the treatment of Alzheimer’s disease with cholinergic, antioxidant, neuroprotective
and hepatoprotective properties. Eur. J. Med. Chem. 2015, 93, 42–50. [CrossRef] [PubMed]
37. Ignasik, M.; Bajda, M.; Guzior, N.; Prinz, M.; Holzgrabe, U.; Malawska, B. Design, synthesis and evaluation
of novel 2-(aminoalkyl)-isoindoline-1,3-dione derivatives as dual-binding site acetylcholinesterase inhibitors.
Arch. Pharm. 2012, 345, 509–516. [CrossRef] [PubMed]
38. Guzior, N.; Bajda, M.; Skrok, M.; Kurpiewska, K.; Lewin´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.
2015, 92, 738–749. [CrossRef] [PubMed]
39. Guzior, N.; Bajda, M.; Rakoczy, J.; Brus, B.; Gobec, S.; Malawska, B. Isoindoline-1,3-dione derivatives
targeting cholinesterases: Design, synthesis and biological evaluation of potential anti-Alzheimer’s agents.
Bioorg. Med. Chem. 2015, 23, 1629–1637. [CrossRef] [PubMed]
40. Szałaj, N.; Bajda, M.; Dudek, K.; Brus, B.; Gobec, S.; Malawska, B. Multiple Ligands Targeting Cholinesterases
and b-Amyloid: Synthesis, Biological Evaluation of Heterodimeric Compounds with Benzylamine
Pharmacophore. Arch. Pharm. Chem. Life Sci. 2015, 348, 556–563. [CrossRef] [PubMed]
41. Wie˛ckowska, A.; Wie˛ckowski, K.; Bajda, M.; Brus, B.; Sałat, K.; Czerwin´ska, P.; Gobec, S.; Filipek, B.;
Malawska, B. Synthesis of new N-benzylpiperidine derivatives as cholinesterase inhibitors with β-amyloid
anti-aggregation properties and beneficial effects on memory in vivo. Bioorg. Med. Chem. 2015, 23, 2445–2457.
[CrossRef] [PubMed]
42. Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric determination of
acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef]
43. Cornish-Bowden, A. A simple graphical method for determining the inhibition constants of mixed,
uncompetitive and non-competitive inhibitors. Biochem. J. 1974, 137, 143–144. [CrossRef] [PubMed]
44. Faller, P.; Hureau, C.; Berthoumieu, O. Role of metal ions in the self-assembly of the Alzheimer’s amyloid-
β
peptide. Inorg. Chem. 2013, 52, 12193–12206. [CrossRef] [PubMed]
45. Inestrosa, N.C.; Dinamarca, M.C.; Alvarez, A. Amyloid-cholinesterase interactions. Implications for
Alzheimer’s disease. FEBS J. 2008, 275, 625–632. [CrossRef] [PubMed]

Molecules 2016, 21, 410
23 of 24
46. Huang, L.; Liu, X.; Cheng, B.; Huang, K. How our bodies fight amyloidosis: Effects of physiological factors
on pathogenic aggregation of amyloidogenic proteins. Arch. Biochem. Biophys. 2015, 568, 46–55. [CrossRef]
[PubMed]
47. LeVine, H. Biotin-avidin interaction-based screening assay for Alzheimer’s beta-peptide oligomer inhibitors.
Anal. Biochem. 2006, 356, 265–272. [CrossRef] [PubMed]
48. Bajda, M.; Wie˛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]
49. Sopkova-de Oliveira Santos, J.; Lesnard, A.; Agondanou, J.-H.; Dupont, N.; Godard, A.-M.; Stiebing, S.;
Rochais, C.; Fabis, F.; Dallemagne, P.; Bureau, R.; et al. Virtual Screening Discovery of New
Acetylcholinesterase Inhibitors Issued from CERMN Chemical Library. J. Chem. Inf. Model. 2010, 50,
422–428. [CrossRef] [PubMed]
50. Di, L.; Kerns, E.H.; Fan, K.; McConnell, O.J.; Carter, G.T. High throughput artificial membrane permeability
assay for blood–brain barrier. Eur. J. Med. Chem. 2003, 38, 223–232. [CrossRef]
51. Apelt, J.; Ligneau, X.; Pertz, H.H.; Arrang, J.M.; Ganellin, C.R.; Schwartz, J.C.; Schunack, W.; Stark, H.
Development of a new class of nonimidazole histamine H3 receptor ligands with combined inhibitory
histamine N-methyltransferase activity. J. Med. Chem. 2002, 45, 1128–1141. [CrossRef] [PubMed]
52. Fisher, M.J.; Gunn, B.; Harms, C.S.; Kline, A.D.; Mullaney, J.T.; Nunes, A.; Scarborough, R.M.; Arfsten, A.E.;
Skelton, M.A.; Um, S.L.; et al. Non-peptide RGD surrogates which mimic a Gly-Asp
β-turn: Potent
antagonists of platelet glycoprotein IIb-IIIa. J. Med. Chem. 1997, 40, 2085–2101. [CrossRef] [PubMed]
53. Dehaen, W.; Hassner, A. Cycloadditions. 45. Annulation of heterocycles via intramolecular nitrile
oxide-heterocycle cycloaddition reaction. J. Org. Chem. 1991, 56, 896–900. [CrossRef]
54. Plocki, S.; Aoun, D.; Ahamada-Himidi, A.; Tavarès-Camarinha, F.; Dong, C.-Z.; Massicot, F.; Huet, J.;
Adolphe-Pierre, S.; Chau, F.; Godfroid, J.-J.; et al. Molecular Modeling, Design, and Synthesis of Less
Lipophilic Derivatives of 3-(4-Tetradecyloxybenzyl)-4H-1,2,4-oxadiazol-5-one (PMS1062) Specific for Group
II Enzyme. Eur. J. Org. Chem. 2005, 2005, 2747–2757. [CrossRef]
55. Choi, J.Y.; Seo, H.N.; Lee, M.J.; Park, S.J.; Park, S.J.; Jeon, J.Y.; Kang, J.H.; Pae, A.N.; Rhim, H.; Lee, J.Y.
Synthesis and biological evaluation of novel T-type calcium channel blockers. Bioorg. Med. Chem. Lett. 2007
17, 471–475. [CrossRef] [PubMed]
,
56. Dickson, J.K. Jr.; Hodge, C.N.; Mendoza, J.S.; Chen, K. 2-Amido-Thiazole-based Compounds Exhibiting
ATP-Utilizing Enzyme Inhibitory Activity, and Compositions, and Uses Thereof. U.S. Patent No 7,410,988,
12 August 2008.
57. Alisi, M.A.; Brufani, M.; Cesta, M.C.; Filocamo, L.; Gostoli, G.; Lappa, S.; Pagella, P.G.; Ferrari, E.; Maiorana, S.;
Marchesini, D. Mediolanium Farmaceutucu S.P.A. Milan, Italy, Aminoalkylcarbamic Esters of Eseroline
Suitable for Use As Chlorinesterase Activity Inhibitors. U.S. Patent No 5,302,593, 12 April 1994.
58. Mokrosz, J.L.; Deren´-Wesołek, A.; Tatarczyn´ska, E.; Duszyn´ska, B.; Bojarski, A.J.; Mokrosz, M.J.;
Chojnacka-Wójcik, E. 8-[4-[2-(1,2,3,4-Tetrahydroisoquinolinyl)]butyl]-8-azaspiro[4.5]decane-7,9-dione: A
New 5-HT1A Receptor Ligand with the Same Activity Profile as Buspirone. J. Med. Chem. 1996, 39, 1125–1129.
[CrossRef] [PubMed]
59. Kowalski, P.; Mitka, K.; Jas´kowska, J.; Duszyn´ska, B.; Bojarski, A.J. New Arylpiperazines with Flexible versus
Partly Constrained Linker as Serotonin 5-HT1A/5-HT7 Receptor Ligands. Arch. Pharm. (Weinheim). 2013
346, 339–348. [CrossRef] [PubMed]
,
60. Tsotinis, A.; Afroudakis, P.A.; Davidson, K.; Prashar, A.; Sugden, D. Design, Synthesis, and Melatoninergic
Activity of New Azido- and Isothiocyanato-Substituted Indoles. J. Med. Chem. 2007, 50, 6436–6440. [CrossRef]
[PubMed]
61. Contreras, J.M.; Rival, Y.M.; Chayer, S.; Bourguignon, J.J.; Wermuth, C.G. Aminopyridazines as
acetylcholinesterase inhibitors. J. Med. Chem. 1999, 42, 730–741. [CrossRef] [PubMed]
62. Commerford, J.D.; Donahue, H.B. Notes -N-(-Bromoalkyl)saccharins and N,N’-Undecamethylenedisaccharin.
J. Org. Chem. 1956, 21, 583–584. [CrossRef]
63. Meng, F.C.; Mao, F.; Shan, W.J.; Qin, F.; Huang, L.; Li, X.S. Design, synthesis, and evaluation of indanone
derivatives as acetylcholinesterase inhibitors and metal-chelating agents. Bioorg. Med. Chem. Lett. 2012, 22,
4462–4466. [CrossRef] [PubMed]

Molecules 2016, 21, 410
24 of 24
64. Tasso, B.; Catto, M.; Nicolotti, O.; Novelli, F.; Tonelli, M.; Giangreco, I.; Pisani, L.; Sparatore, A.; Boido, V.;
Carotti, A.; et al. Quinolizidinyl derivatives of bi- and tricyclic systems as potent inhibitors of acetyl-
and butyrylcholinesterase with potential in Alzheimer’s disease. Eur. J. Med. Chem. 2011, 46, 2170–2184.
[CrossRef] [PubMed]
65. Shaik, J.B.; Palaka, B.K.; Penumala, M.; Kotapati, K.V.; Devineni, S.R.; Eadlapalli, S.; Darla, M.M.;
Ampasala, D.R.; Vadde, R.; Amooru, G.D. Synthesis, pharmacological assessment, molecular modeling and
in silico studies of fused tricyclic coumarin derivatives as a new family of multifunctional anti-Alzheimer
agents. Eur. J. Med. Chem. 2016, 107, 219–232. [CrossRef] [PubMed]
Sample Availability: Samples of the selected compounds (39–44) are available from the authors.
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