Isoindoline-1,3-dione derivatives targeting cholinesterases: Design, synthesis and biological evaluation of potential anti-Alzheimer's agents

Article abstract of DOI:10.1016/j.bmc.2015.01.045

Alzheimer's disease is a fatal neurodegenerative disorder with a complex etiology. Because the available therapy brings limited benefits, the effective treatment for Alzheimer's disease remains the unmet challenge. Our aim was to develop a new series of donepezil-based compounds endowed with inhibitory properties against cholinesterases and β-amyloid aggregation. We designed the target compounds as dual binding site acetylcholinesterase inhibitors with N-benzylamine moiety interacting with the catalytic site of the enzyme and an isoindoline-1,3-dione fragment interacting with the peripheral anionic site of the enzyme. The results of pharmacological evaluation lead us to identify a compound 3b as the most potent and selective human acetylcholinesterase inhibitor (hAChE IC₅₀ = 0.361 μM). Kinetic studies revealed that 3b inhibited acetylcholinesterase in non-competitive mode. The result of the parallel artificial membrane permeability assay for the blood-brain barrier indicated that the compound 3b would be able to cross the blood-brain barrier and reach its biological targets in the central nervous system. The selected compound 3b represents a potential lead structure for further development of anti-Alzheimer's agents.

Full text of DOI:10.1016/j.bmc.2015.01.045

Accepted Manuscript
Isoindoline-1,3-dione derivatives targeting cholinesterases: Design, synthesis
and biological evaluation of potential anti-Alzheimer’s agents
Natalia Guzior, Marek Bajda, Jurand Rakoczy, Boris Brus, Stanislav Gobec,
Barbara Malawska
PII:
S0968-0896(15)00069-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.01.045
BMC 12048
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
25 November 2014
22 January 2015
25 January 2015
Please cite this article as: 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, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.01.045
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Isoindoline-1,3-dione derivatives targeting cholinesterases: Design, synthesis and
biological evaluation of potential anti-Alzheimer’s agents
Natalia Guziora, Marek Bajdaa, Jurand Rakoczya, Boris Brusb, Stanislav Gobecb, Barbara
Malawskaa*
aDepartment of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University
b
Medical College, Kraków, Poland, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Ljubljana, Ljubljana, Slovenia
*Corresponding Author Information
Phone: 48-12-620-54-64. E-mail: mfmalaws@cyf-kr.edu.pl.
Abstract
Alzheimer’s disease is a fatal neurodegenerative disorder with a complex etiology.
Because the available therapy brings limited benefits, the effective treatment for Alzheimer’s
disease remains the unmet challenge. Our aim was to develop a new series of donepezil-based
compounds endowed with inhibitory properties against cholinesterases and β-amyloid
aggregation. We designed the target compounds as dual binding site acetylcholinesterase
inhibitors with N-benzylamine moiety interacting with the catalytic site of the enzyme and an
isoindoline-1,3-dione fragment interacting with the peripheral anionic site of the enzyme. The
results of pharmacological evaluation lead us to identify a compound 3b as the most potent
and selective human acetylcholinesterase inhibitor (hAChE IC50 = 0.361 µM). Kinetic studies
revealed that 3b inhibited acetylcholinesterase in non-competitive mode. The result of the
parallel artificial membrane permeability assay for the blood-brain barrier indicated that the
compound 3b would be able to cross the blood-brain barrier and reach its biological targets in
the central nervous system. The selected compound 3b represents a potential lead structure for
further development of anti-Alzheimer’s agents.
1

Key words:
cholinesterase inhibitors; β-amyloid aggregation; Alzheimer’s disease, multi-target-directed
ligands, PAMPA-BBB assay
1. Introduction
Alzheimer’s disease (AD) is a fatal neurodegenerative disorder with complex etiology that
remains the largest unmet challenge in neurology.1 Worldwide, it is estimated that 36 million
people suffer from dementia and this number is still growing.2
AD is the most common form of dementia that is characterized by the loss of memory and
cognitive functions.3 The major pathological changes associated with the disease include the
extensive loss of neurons (particularly cholinergic) and the presence of altered protein deposits:
senile plaques and neurofibrillary tangles.4 These observations led to two major hypotheses of
the disease. Cholinergic hypothesis was proposed in 1976 and it relates memory impairments
in AD with the decreased number of cholinergic neurons that is accompanied by a progressive
decline in the acetylcholine (ACh) level.5,6 The amyloid hypothesis relates the cause of the
disease with β-amyloid (Aβ) cascade. Aβ is cleaved from the amyloid precursor protein (APP)
with two enzymes: β- and γ-secretase7,8 and Aβ species (especially Aβ1-42) are highly
fibrillogenic, able to form oligomers and senile plaques.
The available AD drug therapy is based mainly on cholinergic hypothesis and includes
cholinesterase inhibitors: donepezil, galantamine and rivastigmine. These drugs mostly inhibit
acetylcholinesterase (AChE), the key enzyme in the cholinergic system involved in the
hydrolysis of ACh, and less so butyrylcholinesterase (BuChE), the auxiliary enzyme in the
cholinergic system.9 In addition to its hydrolytic function, AChE performs also other
functions, such as regulation of neurite growth10,11, maintenance of synapses12 and mediation
of cell adhesion.13 Some studies revealed that AChE co-localizes with senile plaques14,
AChE/Aβ complexes display higher neurotoxicity compared with Aβ fibrils15 and AChE
accelerates the assembly of Aβ into amyloid fibrils.16 Crystallographic studies revealed that
AChE has two binding sites: the active site at the bottom of a deep narrow 20 Å gorge – with
the AChE catalytic triad and the anionic subsite called the catalytic anionic site (CAS) – and
the peripheral anionic site (PAS) near the entrance of the gorge.17 PAS is believed to promote
2

the Aβ aggregation process.18 Finding an additional role of PAS resulted in the development
of a novel class of compounds – bivalent AChE inhibitors – that interact with CAS and PAS
at the same time.19 Recently, many cholinesterase inhibitors targeting Aβ aggregation have
been developed.20-24 According to the multi-target-directed ligands (MTDLs) strategy,25
modulation of multiple biological targets with one compound can be beneficial for treatment
of complex diseases.26 Numerous reviews indicate that searching for ligands with a specific
multi-target profile (also called multiple ligands) seems to be the current trend in the
development of anti-AD therapy.27-31
AD requires a novel therapy because the available treatment is insufficient and no new
drugs have been approved since 2004.32 Currently, 94 drug-candidates are in clinical trials and
only 14 in phase III. This relatively small number, followed by high failure rate of AD clinical
trials suggest there is an urgent need to increase the number of compounds that enter the AD
drug-development process.33
Herein, we present the design, synthesis and biological evaluation of a novel series of
isoindoline-1,3-dione derivatives as potential cholinesterase and Aβ inhibitors. All the
compounds were tested against AChE, BuChE and self-induced Aβ1-42 aggregation. We also
tested their blood-brain barrier (BBB) permeability using the parallel artificial membrane
permeability assay (PAMPA).
2. Results and discussion
2.1. Design
Recently, we have developed a new series of AChE inhibitors that were designed as dual
binding site cholinesterase inhibitors able to bind to both the CAS and PAS.34,35 Those
donepezil-based compounds consisted of the isoindoline-1,3-dione moiety, which was
reported to interact with the PAS36 and the benzylamine fragment, which was reported to
interact with the CAS (compound 1, Figure 1).37 In this series, we identified potent and
selective human AChE (hAChE) inhibitors endowed with additional biological properties
such as Aβ aggregation inhibition and neuroprotective effect against Aβ toxicity.
To further explore the biological properties of isoindoline-1,3-dione derivatives, we
designed a new series of donepezil-based compounds with N-methylbenzylamine fragment
(series A, Figure 1). We assumed that the introduction of a methyl group in benzylamine will
3

provide hydrophobic interactions of this group with an aromatic side chain of Phe330 in
AChE. We also introduced a benzene ring into the alkyl chain spacer of compound 1 (series
B, Figure 1). We expected that the presence of the aromatic ring in an alkyl chain would
provide additional π- π stacking and CH- π interaction with Phe331 and Tyr334 in AChE.
Finally, we introduced different substituents in the benzylamine fragment. They were selected
based on our previous knowledge35 that a fluorine atom at position 2 or a chlorine atom at
position 3 in the phenyl ring can improve the inhibitory potency towards AChE. We assumed
that these modifications would allow us to identify new AChE inhibitors with a
multifunctional profile.
Figure 1. Structure of the isoindolino-1,3-dione derivative 1 and general structures of the
designed compounds.
Molecular modeling
In order to examine possible interactions of the designed compounds with the enzyme
we performed molecular modeling studies (Figure 2). According to the docking studies, all
the designed compounds were arranged along the gorge of AChE to create optimal
interactions with both the CAS and PAS at the same time. The phthalimide fragment was
engaged in - stacking with Trp279 and CH- interactions with Tyr70 in the PAS. Both
carbonyl groups of phthalimide formed H-bonds – with water molecule and Tyr121,
respectively. The benzylamine fragment was responsible for - stacking with Trp84 in the
CAS. The protonated amine group formed cation- interactions with Phe330 and a hydrogen
bond network with Tyr121 via water molecule. It was noted that changing secondary amine to
tertiary (with N-methyl substituent) decreased the basicity of the compounds and weakened
4

cation- interaction, but on the other hand provided hydrophobic interactions of N-methyl
group with the aromatic side chain of Phe330. The alkyl linker formed hydrophobic
interactions with aromatic residues such as Phe290, Phe331 and Tyr334 in the middle of the
active gorge. When we introduced a phenyl group into the linker (series B, Figure 1), we
expected that it would provide extra interactions in the middle of the active site gorge. Further
modifications included the introduction of different halogen atoms in the benzylamine moiety.
A fluorine atom at ortho position created a hydrogen bond with a hydroxyl group of Ser200
while a chlorine atom at meta position made interactions with a carboxyl group of Glu199 and
backbone of Gly441.
5

Figure 2. The binding mode of compound 4b in the active site of acetylcholinesterase
obtained by docking. General view (top; orange represents amino acids involved in binding)
and detailed representation (bottom).
2.2. Chemistry
The synthesis of the designed isoindoline-1,3-dione derivatives was accomplished as
shown in Scheme 1. Compounds 2a-e were the key intermediates, prepared as previously
described.38 Phthalimide potassium was alkylated with the appropriate α,ω-dibromoalkane or
α,α′-dibromo-p-xylene in the presence of a phase transfer catalyst, tetra-n-butylammonium
bromide (TBAB). The reactions were carried out in acetonitrile for 15 h under reflux. In the
next step, compounds 2a-e were used as alkylating agents in reactions with N-
methylbenzylamine or its 2-fluoro or 3-chloro derivatives. Reactions were carried out in
acetonitrile in the presence of potassium carbonate for 16 h, under reflux. Following
purification
by
silica
gel
column
chromatography,
the
final
2-(ω-(N-
methylbenzylamino)alkyl)isoindoline-1,3-dione derivatives 3a-e – 5a-e were isolated with
satisfactory yields (72 – 99%) and converted into hydrochloride salts. Compound 2f was used
as an alkylating agent in reactions with an appropriate N-benzylamine according to the
method reported previously.34 Reactions were carried out in acetonitrile in the presence of
potassium carbonate for 48 h at room temperature. After purification by silica gel column
6

chromatography,
the
final
2-(4-((benzylamino)methyl)benzyl)isoindoline-1,3-dione
derivatives 6 – 8 were isolated with satisfactory yields (39 – 48%) and converted into
hydrochloride salts.
Scheme 1. Synthesis of the target isoindoline-1,3-dione derivatives. Reagents and conditions:
(a) α,ω-dibromoalkane or α,α′-dibromo-p-xylene, TBAB, MeCN, reflux, 15 h; (b) N-
methylbenzylamine or substituted N-methylbenzylamine , K2CO3, MeCN, reflux, 16 h; (c)
HCl in 2-propanol (d) N-benzylamine or substituted N-benzylamine, K2CO3, MeCN, rt, 48 h;
(e) HCl in 2-propanol.
2.3.
Biological activity evaluation
We determined the inhibitory potency of the compounds against cholinesterases using the
method of Ellman.39 We tested the compounds against AChE from Electrophorus electricus
7

(EeAChE) and BuChE from equine serum (EqBuChE). In the next step, we tested the active
compounds against human recombinant AChE (hAChE). Compounds with inhibitory potency
lower than 50% at a screening concentration (10 µM) were considered as inactive. The IC50
values are given in Table 1. Compound 1, donepezil and tacrine were included as a positive
control.
In the series A (compounds 3a-e, 4a-e and 5a-e with different lengths of linkers) we
identified potent EeAChE inhibitors with IC50 values in the low micromolar to nanomolar
range. In each subseries we observed that the most active were the compounds with five or six
carbon atom linkers. Compound 4b (a five carbon linker, 2-fluoro derivative) was found to be
the most potent and selective EeAChE inhibitor with an IC50 value of 67 nM. The activity
significantly decreased with shortening of the tether (to a four carbon linker) and slightly
decreased with the elongation of the tether (to seven and eight carbon linkers). Regarding the
influence of substituents in benzylamine on EeAChE inhibitory potency, we confirmed that
the introduction of a fluorine atom at position 2 was beneficial for inhibitory potency
(compound 4b) due to an extra hydrogen bond in the CAS. In the case of m-chloro derivatives
the halogen atom could be engaged in a halogen bond interaction, however it did not increase
the potency.
For hAChE we observed similar structure-activity relationship (SAR) trends with slightly
higher IC50 values. The most active were compounds with five carbon atom linkers and
among them compound 3b with an unsubstituted benzylamine fragment was the most potent
(IC50 = 361 nM). In the case of a six carbon atom alkyl chain, we observed a significant
decrease in potency compared with EeAChE activity. This is due to the difference in the
structure of both enzymes. In the case of hAChE, the six carbon linker prevents phthalimide
and benzylamine fragments from optimal interactions with Trp286 and Trp86, at the same
time. We observed that the introduction of fluorine or chlorine atoms in the phenyl ring
decreased the potency against hAChE.
In the case of EqBuChE, we observed that the potency increased according to the length
of the linker and the most potent EqBuChE inhibitors were compounds 3e and 5e with
octamethylene linkers (IC50 = 0.543 µM and IC50 = 0.909 µM, respectively). The longer
linker provided higher flexibility of a molecule and enabled the compounds occurr in bent
conformation. Benzylamine and phthalimide fragments better fit to Trp82 from CAS and
Leu286, Val288 and Trp231, forming lipophilic pocket, respectively. Moreover, the
octamethylene tether created more hydrophobic interactions.
8

Regarding the introduction of a phenyl group in the alkyl linker in the series B, we
observed that this modification deteriorated the potency against EeAChE but improved the
potency against BuChE. In the case of AChE, the length of the alkyl linker between a phenyl
group and a phthalimide moiety was probably too short to provide a good fit of phthalimide to
PAS. On the contrary in the case of BuChE π- π stacking and CH- π interactions of the phenyl
group in the middle of the active gorge were strong enough to inhibit the enzyme regardless
the length of the linker. Through the replacement of methylene groups by aromatic fragments
we can reach a more balanced profile of inhibition towards both cholinesterases. The
introduction of a fluorine atom was beneficial for inhibition of both AChE and BuChE, while
a chlorine atom was beneficial only for BuChE.
Targeting the self-induced Aβ aggregation represents an emerging approach for
discovering drug candidates for AD. Aβ1-42 has a high tendency to form fibrils and aggregates.
Also, its oligomers are neurotoxic and cause membrane disruption in neuronal cells.40 Within
the previous series of compounds35 we identified potent inhibitors of self-induced Aβ
aggregation (62 to 66.0% at 10 µM), however we did not observed the relevant structure-
activity relationships among the tested compounds. This may suggest that the mechanisms of
Aβ aggregation inhibition are nonspecific. In the presented series of compounds we expected
to identify more Aβ aggregation inhibitors. We tested all the compounds in the thioflavin T
based assay to investigate their ability to inhibit self-induced Aβ aggregation.41 We screened
them at relatively low concentration (10 µM), to obtain the results that are comparable with
the Ellman’s assay. The tested compounds turned out to be very poor inhibitors of self-
induced Aβ aggregation (percentages of inhibition lower than 5% at 10 µM).
Summarizing the results of biological evaluation of the presented series of compounds, we
can conclude that we identified potent and selective AChE inhibitors among N-
methylbenzylamine derivatives. We noticed that changing a secondary amine (benzylamine)
to tertiary amine (N-methylbenzylamine) allowed us to develop potent AChE inhibitors. They
displayed similar potency to a compound 1 and donepezil. We also observed that
incorporation of a benzene ring in an alkyl linker improved the potency against BuChE in
comparison with the compound 1.
9

Table 1. Inhibitory potency of isoindoline-1,3-dione derivatives against EeAChE, hAChE and
EqBuChE.
EeAChE
EqBuChE
IC50 [µM]a
ndc
ndc
ndc
hAChE
SIb
Compd
IC50 [µM]a
2.326±0.090
0.080±0.001
0.089±0.002
0.172±0.009
0.139±0.006
1.522±0.018
0.067±0.001
0.075±0.001
0.525±0.048
0.661±0.012
1.430±0.051
0.110±0.003
0.095±0.001
0.500±0.016
1.363±0.050
ndc
IC50 [µM]a
ndc
-
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6
-
0.361±0.010
1.831±0.048
0.997±0.018
0.860±0.022
ndc
-
1.011±0.025
0.543±0.011
ndc
5.9
3.9
-
ndc
-
0.525±0.010
10.460±0.097 139.5 1.992±0.037
7.157±0.062
4.678±0.047
ndc
13.6 0.625±0.012
7.1
-
0.644±0.010
ndc
5.826±0.057
2.435±0.080
1.499±0.052
0.909±0.017
2.080±0.028
2.899±0.041
1.319±0.079
ndc
53.0 0.537±0.012
25.6 1.189±0.075
3.0
0.7
-
1.303±0.082
1.044±0.028
ndc
ndc
ndc
2.582±0.078
6.311±0.320
0.078±0.001
1.1
0.3
-
7
8
1d
0.202±0.004
0.006±0.001
Donepezil 0.010±0.001
Tacrine 0.024±0.001
1.830±0.176
0.002±0.001
183
<0.1 0.131±0.002
a
IC50 values are expressed as mean ± standard error of the mean (SEM) of at least three
experiments.
b SI – selectivity index; IC50 EqBuChE/IC50 EeAChE
c IC50 value not determined; % inhibition at 10 µM lower than 50%.
d Data from Ref.35
10

2.3. Kinetic studies
We performed kinetic studies to investigate the mechanism of EeAChE inhibition for
compound 3b. Substrate-velocity curves in the absence and presence of the tested compound
were recorded. Analysis of Lineweaver-Burk double reciprocal plots showed that with
increasing concentrations of the tested inhibitor, x-intercepts were the same (unaffected Km)
but slopes were different (decreased Vmax) (Figure. 3). This means that compound 3b is a non-
competitive AChE inhibitor, the same as donepezil42. The non-competitive type of inhibition
may
indicate
preferential
binding
to
the
PAS
rather
than
the
Compound 3b
20
15
10
5
200 nM
150 nM
100 nM
0 nM
-10
10
20
[ATCh]-1 mM-1
-5
CAS.
Figure 3. Lineweaver–Burk plots illustrating non-competitive type of EeAChE inhibition by
compound 3b. ATCh = acetylthiocholine; V = initial velocity rate. Lines were derived from a
weighted least-square analysis of data points.
2.4. Blood brain barrier permeability assay
The permeability through BBB of the anti-AD drug candidates should be assessed as early
as possible in the drug discovery process. We used a parallel artificial membrane permeability
assay for BBB (PAMPA-BBB) to determine the brain permeability of the synthesized
compounds.43 We used 7 commercial drugs as the reference compounds and we established
the following ranges of permeability: logPe > -5.4 for compounds with high BBB
permeability; logPe < -7.3 for compounds with low permeability and -7.3 > logPe > -5.4 for
compounds with uncertain BBB permeability. We chose compounds 3b, 4b and 5b for this
11

assay. The results of the PAMPA-BBB assay (Table 2) indicates that all the tested
compounds would be able to cross the BBB and reach their biological targets in the CNS.
Table 2. Permeability (logPe) in the PAMPA-BBB assay for commercial drugs and the
selected compounds with prediction of their penetration to the CNS.
Compd
LogPea
-7.3
Prediction
CNS-
Compd
3b
LogPeb
-3.7
Prediction
CNS+
Theophylline
Verapamil.HCl
Lidocaine
-3.5
CNS+
CNS+
CNS+
CNS+
CNS±
CNS+
-3.6
CNS+
4b
-4.3
-3.4
CNS+
5b
Quinidine.HCl
Progesterone
Corticosterone
Propranolol.HCl
-3.5
-5.1
-5.4
-3.5
a Results are the mean of three replicates (n = 3).
b Results are the mean of two replicates (n = 2).
3. Conclusions
In this paper we have presented a new series of isoindoline-1,3-dione cholinesterase
inhibitors. We designed the target compounds based on the results of our previous studies and
with the assistance of molecular modeling. The donepezil-based compounds consist of an
isoindoline-1,3-dione fragment connected to N-methylbenzylamine by alkyl linkers of
different lengths or to benzylamine by the alkyl linker containing a phenyl group. Halogen
atoms were introduced in a phenyl group in benzylamine at positions validated in our
previous studies.35 We tested all the compounds against cholinesterases and self-induced Aβ1-
aggregation. The results allowed us to identify selective and potent hAChE inhibitors
42
among N-methylbenzylamine derivatives. The most active was compound 3b, which
comprises of N-methylbenzylamine and a five carbon atom linker. We also identified
compounds with balanced inhibitory activity towards AChE and BuChE: compounds 3e and
5e. Kinetic studies revealed that the developed compounds are non-competitive AChE
inhibitors. According to the results of self-induced Aβ aggregation assay, these compounds
are poor aggregation inhibitors at 10 µM. Finally, the preliminary blood brain barrier
permeability assay results showed that the compounds would be able to reach the CNS.
12

Taking into account all the results presented in this paper, we developed the novel
cholinesterase inhibitors with the ability to reach their targets in the CNS. They serve as the
lead compounds for further development as potential therapeutics for AD.
4. Experimental part
4.1.Molecular modeling
Three-dimensional structures of ligands were built with the Corina online tool.44
Subsequently, by using Sybyl 8.045, Gasteiger-Marsili charges were assigned following
checks of atom types and protonation of compounds. Finally, ligand structures were saved in
the mol2 format. Docking was performed with AChE from 1EVE and BuChE from the 1P0I
crystal structure46 using the Gold 4.1 program.47 The targets were prepared as follows: all
histidine residues were protonated at Ne, hydrogen atoms added, ligand molecules removed,
and binding sites defined as all amino acid residues within 10 Å from donepezil (for AChE)
and within 20 Å from the glycerol molecule present in the active center of BuChE. The
presence of some conserved water molecules was also taken into account. A standard set of
genetic algorithms with population size 100, number of operations 100 000 and 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 by PyMOL.
4.2. Chemistry
4.2.1. General methods
1H NMR spectra were recorded on Varian Mercury 300 at 300 MHz. The chemical shifts for
1H NMR are referenced to TMS via 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 was done using aluminum sheets precoated with silica gel 60
F254. Column chromatography was performed on Merck silica gel 60 (63 – 200 μm). Flash
chromatography was performed on IsoleraTM Spektra (Biotage). 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 x 100 mm) at 214 nm and 254 nm. CH3CN/H2O
13

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%.
The following compounds: 2-(4-bromobutyl)isoindoline-1,3-dione (2a)38, 2-(5-
bromopentyl)isoindoline-1,3-dione (2b)38, 2-(6-bromohexyl)isoindoline-1,3-dione (2c)38, 2-
(7-bromoheptyl)isoindoline-1,3-dione (2d)38, 2-(8-bromooctyl)isoindoline-1,3-dione (2e)38
and 2-(4-(bromomethyl)benzyl)isoindoline-1,3-dione (2f)48 had been previously reported.
4.2.2. General procedure for the preparation of hydrochloride salts
The hydrochloride salts were prepared by dissolving the compounds in a minimum
quantity of dichloromethane. Then the solution was treated with 5 M solution of HCl in 2-
propanol, evaporated under reduced pressure, washed with pentane and dried.
4.2.3. General procedure for the synthesis of compounds (3a-e – 5a-e)
2-(ω-Bromoalkyl)-isoindoline-1,3-dione (1 eq) with N-methylbenzylamine (1 eq) or its
substituted analog in the presence of K2CO3 (3 eq) was stirred in acetonitrile under reflux for
16 h. Once the reaction was finished, the solvent was evaporated under vacuum, producing a
residue that was further dissolved in 50 mL of 5 M NaOH and extracted with ethyl acetate (3
x 50 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The crude product was purified by silica gel column
chromatography (dichloromethane to dichloromethane/methanol 98:2), yielding a yellow oil.
The final product was obtained in the form of hydrochloride salt. The following compounds
were obtained.
4.2.4. 2-(4-(Benzyl(methyl)amino)butyl)isoindoline-1,3-dione (3a)
Following the General procedure, reaction of 2-(4-bromobutyl)isoindoline-1,3-dione
(2a) (564 mg, 2 mmol) with N-methylbenzylamine (260 μL, 2 mmol) and K2CO3 (829 mg, 6
mmol) in acetonitrile (30 mL), after 16 h, column chromatography and preparation the salt
gave product 3a (708 mg, 98%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.29; mp 107 °C;
1H NMR (300 MHz, DMSO-d6) δ ppm: 1.52 – 1.66 (m, 2H), 1.72 – 1.80 (m, 2H), 2.58 (d, 2H,
J = 4.85 Hz), 2.90 – 3.16 (m, 2H), 3.56 (t, 2H, J = 6.86 Hz), 4.19 (dd, 1H, J1 = 6.04, J2=13],
4.33 (dd,1H, J1 = 4 .57, J2 = 12.96),7.40 – 7.42 (m, 3H), 7.60 – 7.63 (m, 2H), 7.81 – 7.89 (m,
4H), 11.06 (s br, 1H); MS: m/z 323.18 [M+H+].
14

4.2.5. 2-(5-(Benzyl(methyl)amino)pentyl)isoindoline-1,3-dione (3b)
Following the General procedure, reaction of 2-(5-bromopentyl)isoindoline-1,3-dione
(2b) (590 mg, 2 mmol) with N-methylbenzylamine (260 μL, 2 mmol) and K2CO3 (829 mg, 6
mmol) in acetonitrile (30 mL), after 16 h, column chromatography and preparation the salt
gave product 3b (719 mg, 97%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.26; mp 102 °C;
1H NMR (300 MHz, DMSO-d6) δ ppm: 1.23 – 1.27 (m, 2H), 1.55 – 1.60 (m, 2H), 1.72 – 1.80
(m, 2H), 2.57 (d, 3H, J = 4.87), 2.84 – 3.02 (m, 2H), 3.54 (t, 2H, J = 6.91), 4.13 – 4.19 (dd,
1H, J1 = 5.99, J2 = 12.99), 4.28 – 4.34 (dd, 1H, J1 = 4.62, J2 = 12.99), 7.40 – 7.42 (m, 3H),
7.58 – 7.61 (m, 2H), 7.79 – 7.86 (m, 4H), 10.95 (s br 1H); MS: m/z 337.20 [M+H+].
4.2.6. 2-(6-(Benzyl(methyl)amino)hexyl)isoindoline-1,3-dione (3c)
Following the General procedure, reaction of 2-(6-bromohexyl)isoindoline-1,3-dione
(2c) (620 mg, 2 mmol) with N-methylbenzylamine (260 μL, 2 mmol) and K2CO3 (829 mg, 6
mmol) in acetonitrile (30 mL), after 16 h, column chromatography and preparation the salt
gave product 3c (771 mg, 99%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.20; mp 130 °C;
1H NMR (300 MHz, DMSO-d6) δ ppm: 1.20 – 1.33 (m, 4H), 1.55 – 1.59 (m, 2H),1.72 – 1.75
(m, 2H), 2.58 (d, 3H, J = 4.85), 2.81 – 3.07 (m, 2H), 3.54 (t, 2H J = 6.97), 4.16 – 4.23 (dd,
1H, J1= 5.87, J2 = 12.98), 4.29 – 4.35 (dd, 1H, J1 = 4.74, J2=12.96), 7.41 – 7.43 (m, 3H),
7.61 – 7.64 (m, 2H), 7.80 – 7.87 (m, 4H), 11.24 ( s br, 1H); MS: m/z 351.23 [M+H+].
4.2.7. 2-(7-(Benzyl(methyl)amino)heptyl)isoindoline-1,3-dione (3d)
Following the General procedure, reaction of 2-(7-bromoheptyl)isoindoline-1,3-dione
(2d) (648 mg, 2 mmol) with N-methylbenzylamine (260 μL, 2 mmol) and K2CO3 (829 mg, 6
mmol) in acetonitrile (30 mL), after 16 h, column chromatography and preparation the salt
gave product 3d (723 mg, 90%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.21; mp 125 °C;
1H NMR (300 MHz, DMSO-d6) δ ppm: 1.20 – 1.27 (m, 6H), 1.52 – 1.56 (m, 2H), 1.67 – 1.72
(m, 2H), 2.57 (d, 3H, J = 4.87), 2.81 – 4.07 (m, 2H), 3.52 (t, 2H, J = 7.09), 4.14 – 4.21 (dd,
1H, J1 = 5.95, J2 = 12.99), 4.28 – 4.34 (dd, 1H, J1 = 4.74, J2 =12.98), 7.39 – 7.42 (m, 3H),
7.59 – 7.62 (m, 2H), 7.79 – 7.85 (m, 4H), 11.01 (s br, 1H); MS: m/z 365.26 [M+H+].
4.2.8. 2-(8-(Benzyl(methyl)amino)octyl)isoindoline-1,3-dione (3e)
Following the General procedure, reaction of 2-(8-bromooctyl)isoindoline-1,3-dione
(2e) (676 mg, 2 mmol) with N-methylbenzylamine (260 μL, 2 mmol) and K2CO3 (829 mg, 6
15

mmol) in acetonitrile (30 mL), after 16 h, column chromatography and preparation the salt
gave product 3e (657 mg, 75%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.19; mp 137 °C;
1H NMR (300 MHz, DMSO-d6) δ ppm: 1.19 – 1.22 (m, 8H), 1.52 – 1.57 (m, 2H), 1.66 – 1.69
(m, 2H), 2.57 (d, 3H, J = 4.86), 2.81 – 3.00 (m, 2H), 3.53 (t, 2H, J =7.07), 4.14 – 4.20 (dd,
1H, J1 = 5.97, J2 = 12.97), 4.28 – 4.34 (dd, 1H, J1 = 4.72, J2 = 12.99), 7.40 – 7.43 (m, 3H),
7.57 – 7.61 (m, 2H), 7.81 – 7.86 (m, 4H), 10.85 (s br, 1H); MS: m/z 379.28 [M+H+].
4.2.9. 2-(4-((2-Fluorobenzyl)(methyl)amino)butyl)isoindoline-1,3-dione (4a)
Following the General procedure, reaction of 2-(4-bromobutyl)isoindoline-1,3-dione
(2a) (282 mg, 1 mmol) with 2-fluoro-N-methylbenzylamine (133 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 4a (278 mg, 74%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.43; mp
1
137 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.59 – 1.67 (m, 2H), 1.72 – 1.82 (m, 2H),
2.64 (d, 3H, J = 4.31), 2.97 – 3.18 (m, 2H), 3.58 (t, 2H, J = 6.78), 4.22 – 4.28 (dd, 1H, J1 =
5.60, J2 = 13.32), 4.35 – 4.41 (dd, 1H, J1 = 3.43, J2 = 13.38), 7.27 – 7.35 (m, 2H), 7.48 –
7.51 (m, 1H), 7.53 – 7.56 (m, 1H), 7.73 – 7.90 (m, 4H), 10.82 (s br, 1H); MS: m/z 341.26
[M+H+].
4.2.10. 2-(5-((2-Fluorobenzyl)(methyl)amino)pentyl)isoindoline-1,3-dione (4b)
Following the General procedure, reaction of 2-(5-bromopentyl)isoindoline-1,3-dione
(2b) (295 mg, 1 mmol) with 2-fluoro-N-methylbenzylamine (133 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 4b (331 mg, 85%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.31; mp
1
138 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.21 – 1.34 (m, 2H), 1.56 – 1.65 (m, 2H),
1.75 – 1.80 (m, 2H), 2.63(s 3H), 2.98 – 3.05 (m, 2H), 3.57 (t, 2H, J = 6.89), 4.22 – 4.27 (dd,
1H, J1 = 3.89, J2 = 13.04), 4.37 (d, 1H, J = 13.11), 7.27 – 7.34 (m, 2H), 7.48 – 7.58 (m, 1H),
7.76 – 7.83 (m, 1H), 7.84 – 7.88 (m, 4H), 11.00 (s br, 1H); MS: m/z 355.29 [M+H+].
4.2.11. 2-(6-((2-Fluorobenzyl)(methyl)amino)hexyl)isoindoline-1,3-dione (4c)
Following the General procedure, reaction of 2-(6-bromohexyl)isoindoline-1,3-dione
(2c) (310 mg, 1 mmol) with 2-fluoro-N-methylbenzylamine (133 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 4c (336 mg, 83%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.33; mp
1
135 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.23 – 1.33 (m, 4H), 1.55 – 1.59 (m, 2H),
16

1.71 – 1.75 (m, 2H), 2.62 (d, 3H, J=2.61), 2.96 – 3.04 (m, 2H), 3.54 (t,2H, J = 6.98), 4.20 –
4.26 (dd, 1H, J1 = 4.59, J2 = 12.95), 4.36 (d, 1H, J = 12.83), 7.25 – 7.32 (m, 2H), 7.47 – 7.50
(m, 1H), 7.52 – 7.54 (m, 1H), 7.69 – 7.85 (m, 4H), 10.80 (s br, 1H); MS: m/z 369.31 [M+H+].
4.2.12. 2-(7-((2-Fluorobenzyl)(methyl)amino)heptyl)isoindoline-1,3-dione (4d)
Following the General procedure, reaction of 2-(7-bromoheptyl)isoindoline-1,3-dione
(2d) (324 mg, 1 mmol) with 2-fluoro-N-methylbenzylamine (133 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 4d (302 mg, 72%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.32; mp
1
142 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.21 – 1.26 (m, 6H), 1.53 – 1.58 (m, 2H),
1.70 – 1.75 (m, 2H), 2.59 (d, 3H, J = 2.74), 2.89 – 2.99 (m, 2H), 3.51 (t, 2H, J = 7.09), 4.21-
4.24 (dd, 1H, J1 = 4.95, J2 = 12.98), 4.36 (d, 1H, J = 12.77), 7.25 – 7.33 (m, 2H), 7.51 – 7.58
(m, 1H), 7.73 – 7.79 (m, 1H), 7.82 – 7.87 (m, 4H), 10.95 (s br, 1H); MS: m/z 385.33 [M+H+].
4.2.13. 2-(8-((2-Fluorobenzyl)(methyl)amino)octyl)isoindoline-1,3-dione (4e)
Following the General procedure, reaction of 2-(8-bromooctyl)isoindoline-1,3-dione
(2e) (338 mg, 1 mmol) with 2-fluoro-N-methylbenzylamine (133 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 4e (402 mg, 93%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.34; mp
1
136 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.20 – 1.27 (m, 8H), 1.53 – 1.58 (m, 2H),
1.70 – 1.75 (m, 2H), 2.62 (d, 3H, J = 2.90), 2.94 – 3.01 (m, 2H), 3.53 (t, 2H, J = 7.06), 4.21 –
4.27 (dd, 1H, J1 = 4.97, J2 = 13.08), 4.36 (d, 1H, J = 12.40), 7.25 – 7.33 (m, 2H), 7.47 – 7.54
(m, 1H), 7.74 – 7.79 (m, 1H), 7.81 – 7.86 (m, 4H), 10.89 (s br, 1H); MS: m/z 397.36 [M+H+].
4.2.14. 2-(4-((3-Chlorobenzyl)(methyl)amino)butyl)isoindoline-1,3-dione (5a)
Following the General procedure, reaction of 2-(4-bromobutyl)isoindoline-1,3-dione
(2a) (282 mg, 1 mmol) with 3-chloro-N-methylbenzylamine (146 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 5a (357 mg, 91%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.48; mp
1
172 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.56 – 1.64 (m, 2H), 1.69 – 1.80 (m, 2H),
2.57 (d, 3H, J = 3.89), 2.91 – 3.09 (m, 2H), 3.56 (t, 2H, J = 6.75), 4.15 – 4.21 (dd, 1H, J1 =
17

5.87, J2=12.84), 4.32 – 4.37 (dd, 1H, J1 = 3.65, J2 = 13.03), 7.41 – 7.49 (m, 2H), 7.55 – 7.58
(m, 1H), 7.74 (s , 1H), 7.81 – 7.85 (m, 4H), 10.93 (s br, 1H); MS: m/z 357.28 [M+H+].
4.2.15. 2-(5-((3-Chlorobenzyl)(methyl)amino)pentyl)isoindoline-1,3-dione (5b)
Following the General procedure, reaction of 2-(5-bromopentyl)isoindoline-1,3-dione
(2b) (295 mg, 1 mmol) with 3-chloro-N-methylbenzylamine (146 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 5b (377 mg, 93%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.62; mp
1
177 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.24 – 1.31 (m, 2H), 1.56 – 1.63 (m, 2H),
1.65 – 1.81 (m, 2H), 2.60 (d, 3H, J = 3.71), 2.90 – 3.05 (m, 2H), 3.57 (t, 2H, J = 6.89), 4.16 –
4.26 (dd, 1H, J1 = 5.70, J2 = 12.84), 4.33 – 4.39 (dd, 1H, J1 = 3.31, J2 = 13.05), 7.46 – 7.49
(m, 2H), 7.59 (d, 1H, J = 6.84), 7.76 (s, 1H), 7.81 – 7.88 (m, 4H), 10.92 (s br, 1H); MS: m/z
371.24 [M+H+].
4.2.16. 2-(6-((3-Chlorobenzyl)(methyl)amino)hexyl)isoindoline-1,3-dione (5c)
Following the General procedure, reaction of 2-(6-bromohexyl)isoindoline-1,3-dione
(2c) (310 mg, 1 mmol) with 3-chloro-N-methylbenzylamine (146 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 5c (399 mg, 95%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.50; mp
1
150 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.25 – 1.29 (m, 4H), 1.56 – 1.61 (m, 2H),
1.69 – 1.75 (m, 2H), 2.58 (d, 3H, J = 4.19), 2.88 – 3.04 (m, 2H), 3.54 (t, 2H, J = 6.99), 4.17 –
4.26 (dd, 1H, J1 = 5.88, J2 = 12.97), 4.33 – 4.39 (dd, 1H, J1=3.97, J2 = 13.00), 7.46 – 7.52
(m, 2H), 7.58 – 7.60 (m, 1H), 7.76 (s, 1H), 7.81 – 7.88 (m, 4H), 10.93 (s br, 1H); MS: m/z
385.33 [M+H+].
4.2.17. 2-(7-((3-Chlorobenzyl)(methyl)amino)heptyl)isoindoline-1,3-dione (5d)
Following the General procedure, reaction of 2-(7-bromoheptyl)isoindoline-1,3-dione
(2d) (324 mg, 1 mmol) with 3-chloro-N-methylbenzylamine (146 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 5d (412 mg, 95%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.58; mp
1
135 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.20 – 1.28 (m, 8 H), 1.55 – 1.62 (m, 2H),
18

1.67 – 1.72 (m, 2H), 2.61 (d, 3H, J = 2.59), 2.90 – 3.06 (m, 2H), 3.56 (t, 2H, J = 7.07), 4.17 –
4.23 (dd, 1H, J1 = 5.37, J2 = 12.74), 4.33 – 4.39 (dd, 1H, J1 = 2.35, J2 = 13.25), 7.45 – 7.59
(m, 3H), 7.75 (s, 1H), 7.83 – 7.88 (m, 4H), 10.74 (s br, 1H); MS: m/z 399.29 [M+H+].
4.2.18. 2-(8-((3-Chlorobenzyl)(methyl)amino)octyl)isoindoline-1,3-dione (5e)
Following the General procedure, reaction of 2-(8-bromooctyl)isoindoline-1,3-dione
(2e) (338 mg, 1 mmol) with 3-chloro-N-methylbenzylamine (146 μL, 1 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (15 mL), after 16 h, column chromatography and preparation
the salt gave product 5e (403 mg, 90%) as a white solid: Rf (DCM/MeOH, 95:5) = 0.51; mp
1
164 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 1.20 – 1.33 (m, 6H), 1.54 – 1.60 (m, 2H),
1.66 – 1.73 (m, 2H), 2.58 (d, 3H, J = 4.68), 2.87 – 3.07 (m, 2H), 3.54 (t, 2H, J = 7.06), 4.15 –
4.21 (dd, 1H, J1 = 6.12, J2 = 12.98), 4.31 – 4.37 (dd, 1H, J1 = 4.39, J2 = 13.01), 7.43 – 7.51
(m, 2H), 7.55 – 7.57 (m, 1H), 7.73 (s, 1H), 7.79 – 7.86 (m, 4H), 10.75 (s br, 1H) MS: m/z
413.32 [M+H+].
4.2.19. General procedure for the synthesis of compounds (6 – 8)
2-(4-(Bromomethyl)benzyl)isoindoline-1,3-dione (1 eq) with threefold excess of
benzylamine (3 eq) in the presence of K2CO3 (3 eq) was stirred in acetonitrile at room
temperature for 48 h. Once the reaction was finished, the solvent was evaporated under
vacuum, producing a residue that was further dissolved in 20 mL of NaHCO3 and extracted
with dichloromethane (4 x 25 mL). The combined organic extracts were dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by
silica gel column chromatography (dichloromethane to dichloromethane/acetone 95:5),
yielding a yellow oil. The final product was obtained in the form of hydrochloride salt. The
following compounds were obtained.
4.2.20. 2-(4-((Benzylamino)methyl)benzyl)isoindoline-1,3-dione (6)
Following the General procedure, reaction of 2-(4-(bromomethyl)benzyl)isoindoline-
1,3-dione (2f) (330 mg, 1 mmol) with benzylamine (327 μL, 3 mmol) and K2CO3 (415 mg, 3
mmol) in acetonitrile (20 mL), after 48 h, column chromatography and preparation the salt
gave product 6 (157 mg, 39%) as a white solid: Rf (DCM/acetone, 95:5) = 0.26; mp 270 °C;
19

1H NMR (300 MHz, DMSO-d6) δ ppm: 3.94 – 4.23 (m, 4H), 4.79 (s, 2H), 7.32 – 7.44 (m,
5H), 7.49 – 7.58 (m, 4H), 7.79 – 8.04 (m, 4H), 9.72 (s br, 2H); MS: m/z 357.28 [M+H+].
4.2.21. 2-(4-(((2-Fluorobenzyl)amino)methyl)benzyl)isoindoline-1,3-dione (7)
Following the General procedure, reaction of 2-(4-(bromomethyl)benzyl)isoindoline-
1,3-dione (2f) (330 mg, 1 mmol) with 2-fluorobenzylamine (343 μL, 3 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (20 mL), after 48 h, column chromatography and preparation
the salt gave product 7 (197 mg, 48%) as a white solid: Rf (DCM/acetone, 95:5) = 0.22; mp
269 °C; 1H NMR (300 MHz, DMSO-d6) δ ppm: 4.10 – 4.24 (m, 4H), 4.79 (s, 2H), 7.17 – 7.74
(m, 8H), 7.98 – 7.81 (m, 4H), 9.70(s br, 2H); MS: m/z 375.36 [M+H+].
4.2.22. 2-(4-(((3-Chlorobenzyl)amino)methyl)benzyl)isoindoline-1,3-dione (8)
Following the General procedure, reaction of 2-(4-(bromomethyl)benzyl)isoindoline-
1,3-dione (2f) (330 mg, 1 mmol) with 2-fluorobenzylamine (365 μL, 3 mmol) and K2CO3
(415 mg, 3 mmol) in acetonitrile (20 mL), after 48 h, column chromatography and preparation
the salt gave product 7 (192 mg, 45%) as a white solid: Rf (DCM/acetone, 95:5) = 0.23; mp
1
283 °C; H NMR (300 MHz, DMSO-d6) δ ppm: 3.95 – 4.33 (m, 4H), 4.79 (s, 2H), 7.31 –
7.55 (m, 7H), 7.67 (s, 1H), 7.81 – 7.96 (m, 4H), 9.74 ( s br, 2H); MS: m/z 391.25 [M+H+]
4.3.
Biological Activity
4.3.1. In vitro inhibition of AChE and BuChE
To assess the inhibitory activity of the target compounds towards cholinesterases, we
performed Ellman’s assay using AChE from Electrophorus electricus (Sigma–Aldrich),
human recombinant AChE (Sigma – Aldrich) and BuChE from horse serum (Sigma–Aldrich).
500 U of AChE or BuChE were 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 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)/butryltiocholine (BTC) iodide
solution were prepared in demineralized water. All assays were performed in 0.1 M phosphate
20

buffer pH 8.0. The tested compounds or water in a blank sample (25 µl) were incubated with
the enzyme (20 µl) for 5 min at 25 °C (hAChE – 37 °C) in 765 µl buffer prior to starting the
reaction. Then, 20 µL of DTNB and 20 µl of ATC/BTC were added. After 5 minutes of the
reaction, changes in absorbance were measured at 412 nM in EnSpire Multimode Microplate
Reader (PerkinElmer). The percentage of inhibition of enzymes was calculated by comparison
with a blank sample (100% activity). Calculation of IC50 values was performed with
GraphPad Prism 5. Each concentration was measured in triplicate. Data is expressed as mean
± SEM.
4.3.2. Inhibition of Aβ1-42 aggregation
To investigate the inhibition of β amyloid peptide aggregation by the tested compounds,we
performed thioflavin T-based fluorometric assay. Recombinant human HFIP-pretreated Aβ1-42
peptide (Merck Millipore, Darmstadt, Germany) was solubilized in DMSO to give a 100 μM
stock solution. Prior to incubation, peptide stock solution was diluted in 150 mM HEPES
buffer (pH 7.4) containing 150 mM NaCl to give a final concentration of 10 μM. A volume of
10 μL Aβ was mixed with 10 μL of the tested compound at the final concentration 10 μM.
The mixture was immediately added to the corresponding wells in black-walled 96-well plate
and filled with the HEPES buffer to the final volume of 100 μL (2 μM final Aβ
concentration). Each sample was prepared in quadruplicate and the content of the DMSO was
always 2%. The plate was sealed and placed on a Synergy™ H4 plate reader at room
temperature. After 24 h of incubation and continuous shaking, 50 μL of the thioflavin-T
solution (15 μM stock solution in HEPES, 5 μM final concentration) was added to each well
using the built-in injector of Synergy™ H4 to quantify the amyloid fibril formation. After
incubating for 5 min, fluorescence was measured at an excitation wavelength of 446 nm and
emission wavelength of 490 nm on a Synergy™ H4 plate reader. Fluorescence intensities at
the plateau were averaged, and the average fluorescence of the buffer and DMSO only, or of
the buffer and the tested compound, was subtracted. The equation, Fi/F0 x 100%, where Fi is
the fluorescence of Aβ treated with the tested compound, and F0 is the fluorescence of Aβ
alone, was used to quantify the inhibition of Aβ1-42 self-induced aggregation.
4.3.3. Kinetic characterization of EeAChE inhibition
To estimate the type of inhibition of EeAChE we performed the same experimental
protocol as reported at section 4.3.1. The different concentrations of the substrate ATC (0.05
21

– 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 0.3, 0.2, 0.1
and 0.05 mM concentrations of substrate. Compound 3b was tested at three different
concentrations. 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.
4.3.4. PAMPA-BBB assay
The brain penetration of compounds 3b, 4b, 5b was assessed using the parallel
artificial membrane permeability assay for blood brain barrier. 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 µl/1 ml). Then, the acceptor 96-well microplate was filled with solution of the tested
compounds in buffer (200 µl/well). The filter membrane in acceptor 96-well microplate was
impregnated with BBB-1 lipid solution (5 µl/well) and the acceptor plate was filled with
Brain Sink Buffer (200 µl/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 (logPe) of the compounds was
calculated using the pION software. Assay validation was made by comparing experimental
permeability of the commercial drugs with the reference value. Taking into account the limits
established by Di et al. for BBB permeation, we established the following ranges of
permeability: compounds with high permeability (CNS+), log Pe > -5.4; compounds with low
permeability (CNS-), logPe < -7.3; compounds with uncertain BBB permeability (CNS±), -7.3
> logPe > -5.4.
Acknowledgments
Financial support from the Polish Ministry for Science and High Education (grant No
N N405 163339), the European Regional Development Fund (POIG.02.01.00-12-023/08) and
22

the Slovene Research Agency is gratefully acknowledged. Ellman’s assays were performed at
the Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Faculty of Pharmacy, Kraków, Poland. We also thank Dr Bálint Sinkó for his help
with establishing and managing PAMPA-BBB assay.
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