Design, synthesis and evaluation of novel 2-(Aminoalkyl)-isoindoline-1,3- dione derivatives as dual-binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1002/ardp.201100423

A new series of 2-(diethylaminoalkyl)-isoindoline-1,3-dione derivatives intended as dual binding site cholinesterase inhibitors were designed using molecular modeling and evaluated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and the formation of the β-amyloid (Aβ) plaques. For AChE inhibitory activity, the spectrophotometric method of Ellman and the electrophoretically mediated microanalysis assay were used, giving good results. Most of the synthesized compounds had AChE inhibitory activity with IC₅₀ values ranging from IC₅₀ = 0.9 to 19.5 μM and weak Aβ anti-aggregation inhibitory activity. These results support the outcome of docking studies which tested compounds targeting both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. The most promising selective AChE inhibitors are compounds 10 (IC₅₀ = 1.2 μM) and 11 (IC₅₀ = 1.1 μM), with 6-7 methylene chains, which also inhibit Aβ fibril formation. A new series of 2-(diethylaminoalkyl)-isoindoline-1,3- dione derivatives intended as dual-binding site cholinesterase inhibitors were designed using molecular modeling. They were evaluated as inhibitors of acetylcholinesterase, butyrylcholinesterase, and the formation of β-amyloid (Aβ) plaques. The most promising selective AChE inhibitors are compounds 10 (IC₅₀ = 1.2 μM) and 11 (IC₅₀ = 1.1 μM), with 6-7 methylene chains, which also inhibit Aβ fibril formation. Copyright

Full text of DOI:10.1002/ardp.201100423

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Design, Synthesis and Evaluation of Novel 2-(Aminoalkyl)-
isoindoline-1,3-dione Derivatives as Dual-Binding Site
Acetylcholinesterase Inhibitors
Michalina Ignasik1, Marek Bajda1, Natalia Guzior1, Michaela Prinz2, Ulrike Holzgrabe2, and Barbara
Malawska1
1 Chair of Pharmaceutical Chemistry, Department of Physicochemical Drug Analysis, Jagiellonian University
´
Medical College, Krakow, Poland
2 Institut of Pharmacy and Food Chemistry, University of Wu¨rzburg, Wu¨rzburg, Germany
A new series of 2-(diethylaminoalkyl)-isoindoline-1,3-dione derivatives intended as dual binding site
cholinesterase inhibitors were designed using molecular modeling and evaluated as inhibitors of acetyl-
cholinesterase (AChE), butyrylcholinesterase (BuChE), and the formation of the b-amyloid (Ab) plaques.
For AChE inhibitory activity, the spectrophotometric method of Ellman and the electrophoretically
mediated microanalysis assay were used, giving good results. Most of the synthesized compounds had
AChE inhibitory activity with IC50 values ranging from IC50 ¼ 0.9 to 19.5 mM and weak Ab anti-
aggregation inhibitory activity. These results support the outcome of docking studies which tested
compounds targeting both the catalytic active site (CAS) and the peripheral anionic site (PAS) of
AChE. The most promising selective AChE inhibitors are compounds 10 (IC50 ¼ 1.2 mM) and 11
(IC50 ¼ 1.1 mM), with 6–7 methylene chains, which also inhibit Ab fibril formation.
Keywords: 2-(Aminoalkyl)-isoindoline-1,3-dione derivatives / Ab aggregation / Acetylcholinesterase inhibitors /
Electrophoretically mediated microanalysis (EMMA) / Molecular modeling
Received: November 23, 2011; Revised: January 23, 2012; Accepted: February 16, 2012
DOI 10.1002/ardp.201100423
Introduction
as at its peripheral anionic binding site (PAS), the latter of
which is thought to possess the ability to bind to Ab peptides
thus promoting fibrillogenesis. Dual binding site inhibitors
of cholinesterases influence acetylcholine hydrolysis and
PAS-dependent b-amyloid aggregation [6, 7]. Compounds
with these properties can be defined as multi-target ligands.
The multi-target-directed ligand design strategy is an attrac-
tive approach to seeking novel effective drugs for the treat-
ment of disorders with complex pathological mechanisms
such as AD [8–10]. Thus, there is reason to develop novel dual
binding site cholinesterase inhibitors as multipotent anti-
Alzheimer’s agents. In recent years many dual binding site
cholinesterase inhibitors with b-amyloid anti-aggregation
properties have been described and collected into review
articles [11–13]. Structures of selected compounds are pre-
sented in Fig. 1 (see [14] for bis-tacrine).
Alzheimer’s disease (AD) is the most common neurodegener-
ative brain disorder of the 21st century [1]. AD is character-
ized by progressive dementia caused by the deficits in the
cholinergic system in the brain areas related to memory and
learning, brain deposits of amyloid beta (Ab) peptide and
neurofibrillary tangles [2]. Treatment of AD currently focuses
on increasing cholinergic neurotransmission in the brain by
cholinesterase inhibitors: donepezil, rivastigmine, and gal-
antamine [3]. Clinical experience has shown that all these
medications represent forms of symptomatic therapy; how-
ever there is reason to suspect their involvement in certain
disease-modifying effects including b-amyloid formation and
neuroprotection [4, 5]. Acetylcholinesterase (AChE) inhibitors
can interact at the active catalytic site of the enzyme as well
Our research group has been involved in the development
of cholinesterase inhibitors as potential means of treating
AD. The recently described series of carbamate derivatives
of N-benzylpiperidine displayed non-selective BuChE/AChE
inhibitory activities [15]. Results of molecular modeling
Correspondence: Professor Barbara Malawska, Department of
´
Physicochemical Drug Analysis, 30688 Krakow, Medyczna 9, Poland.
E-mail: mfmalaws@cyf-kr.edu.pl
Fax: þ48 12 657 0262
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M. Ignasik et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
O
O
O
donepezil
N
H
N
H
N
7
O
N
O
N
N
nN
bis-tacrine [14]
interactions with
n = 3 - 8
catalityc site (CAS)
Figure 1. Structures of dual binding site cho-
linesterase inhibitors and the designed com-
pounds.
interactions
with PAS
studies showed that these inhibitors can bind to the active
and the peripheral site of the AChE and like donepezil its
N-benzylpiperidine fragment is oriented toward the catalytic
center. In the present study a novel series of isoindoline-1,3-
dione derivatives intended as cholinesterase inhibitors were
designed using molecular modeling. Target compounds
are designed as dual binding site inhibitors with an
alkylamine moiety responsible for the interactions with
the catalytic binding site, connected by an alkyl chain
N-phthalimido fragment. The latter fragment, similarly to
the indan-1-one fragment of donepezil may be able to bind at
the peripheral active site of AChE and therefore prevent
AChE-induced Ab-aggregation (Fig. 1). Herein, we describe
the synthesis, pharmacological evaluation (AChE and
BuChE, inhibition and Ab-anti-aggregation effect) and
molecular modeling on a new series of 2-(diethylamino-
alkyl)-isoindoline-1,3-dione derivatives.
The reactions were carried out in acetonitrile for 20 h under
reflux. Purification by silica gel column chromatography
produced bromoalkyl derivatives of isoindoline-1,3-dione
(1–6) with good yields (51–94%). The bromoalkyl derivatives
of isoindoline-1,3-dione were then used as alkylating agents
in reaction with diethylamine. Reactions were carried out in
acetonitrile in the presence of potassium carbonate for 24 h
under reflux. Following purification by silica gel column
chromatography, the final 2-(3-diethylaminoalkyl)-isoindo-
line-1,3-diones (7–12) were isolated as yellow-colored oils with
satisfactory yields (35–71%) and then converted into hydro-
chloride salts.
Biological activity
The inhibitory potency of the target compounds against
AChE (from electric eels) and BuChE (from horse serum) were
evaluated via the classical method established by Ellman
et al. [16] and by electrophoretically mediated microanalysis
(EMMA, AChE) [17, 18]. For the Ab-anti-aggregating assay,
a modified thioflavine T test was applied.
Results and discussion
Chemistry
The method commonly used for testing anti-cholinesterase
activity is a spectrometric procedure developed by Ellman
et al. [16]. Ellman’s test is based on the reaction of 5,50-dithio-
bis-(2-nitrobenzoic) acid (DTNB, also called Ellman’s reagent)
with the thiocholine (product of hydrolysis of acetylthio-
New isoindoline-1,3-dione derivatives (7–12) were synthesized
via the route outlined in Scheme 1. At the first stage phthal-
imide potassium was alkylated with a,v-dibromoalkane
using tetra-n-butylammonium bromide (TBAB) as a catalyst.
O
O
HN
O
Br
Br
N
n
NK
N
Br
n
N
n
HCl
a) TBAB, MeCN
reflux, ~20 h
b) K2CO3, MeCN
reflux,
~24 h
O
O
_
_
O
(7 12)
(1 6)
c) HCl/isopropanol
solution
_
n = 3 8
Scheme 1. Synthesis of 2-(3-diethylaminoalkyl)-isoindoline-1,3-dione derivatives 7–12.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Dual-Binding Site Acetylcholinesterase Inhibitors
3
Table 1. Inhibitory activity of isoindoline-1,3-dione derivatives 7–12 and reference compounds against AChE and BuChE, determined by
Ellman’s method
Compound
n
AChE
BuChE
% of inhibition
at 100 mM
IC50
[mM]
% of inhibition
at 100 mM
IC50
[mM]
7
8
9
10
11
12
3
4
5
6
7
8
–
19.5
3.4
1.2
1.1
31.5
–
–
–
–
–
3.5
8.9
11.0
26.8
47.0
62.6
0.9
67.5
2.8
0.005
0.9–4.2
Donepezil
Tacrine
Rivastigminea)
0.03
0.02
0.04–0.8
a)
Data from ref. [19].
choline or butyrylthiocholine) which results in generation
of a yellow-colored product, i.e. 2-nitro-5-thiobenzoic acid.
Changes in absorbance determine the activity of the eval-
uated compounds. Experiments were carried out at 100 mM
concentrations of potential inhibitors. The activity measured
is given as a percentage of enzyme inhibition and as IC50
values, which were only determined for compounds with
better than 50% inhibitory activity at 100 mM concentrations
(Table 1). Donepezil and tacrine were used as reference
compounds.
described: this includes EMMA [17, 20] and the immobilized
capillary enzyme reactor method [21]. Capillary electropho-
resis has a number of advantages over other methods, includ-
ing short analysis time and high-efficiency separation;
moreover, it does not require large samples. In our previous
studies, EMMA has been adopted for rapid AChE inhibition
assay [18]. In the presented study the EMMA method was
optimized, enabling us to measure the activity of four of
the most active 2-(3-diethylaminoalkyl)-isoindoline-1,3-dione
derivatives (9–12) and then compare it with reference inhibi-
tors – tacrine and donepezil, whose activity was assessed by
Ellman’s method.
Target compounds 7–12 displayed moderate or good
inhibitory activity against AChE and weak activity against
BuChE. Their anti-AChE activity ranged from IC50 ¼ 0.9 to
19.5 mM with only compound 7 showing inhibitory activity
lower than 50%. Inhibitory potency against BuChE ranged
from 3.49 to 46.99% at 100 mM concentrations, with only
compound 12 showing activity >50% (the corresponding IC50
value being equal to 67.5 mM). These compounds proved less
active than reference substances (donepezil and tacrine),
however they were comparable with rivastigmine in terms
of inhibitory potential. Results indicate that 2-(3-diethyl-
aminoalkyl)-isoindoline-1,3-dione derivatives could be
described as selective AChE inhibitors. Structure–activity
relationship analysis showed that elongation of the alkyl
chain from 3 to 8 methylene groups resulted in an increase
of inhibitory activity against both enzymes. The strongest
activity was observed for compound 12, with an 8-methylene
alkyl chain. It appears that the optimal length of the tether is
between 5 and 8 groups.
In the scope of the EMMA method, solutions of enzymes,
potential inhibitors, and acetylthiocholine (enzyme sub-
strate) were injected into a capillary and mixed electrophor-
etically by applying voltage for a short time. Following 1 min
of incubation, voltage was reapplied to separate the product
from any substrate that did not undergo reaction, as well as
from any potential inhibitor. Detection was performed at UV
230 nm wavelength, with the area bounded by the product
curve peak assumed to represent the enzymatic activity. The
inhibition percentage and IC50 values were then calculated
using nonlinear regression (Table 2). Figure 2 shows the
Table 2. Inhibitory potency (IC50) against AChE obtained by
Ellman’s method and by EMMA
Compound
IC50 [mM]
Ellman’s method
IC50 [mM]
EMMA
Preliminary steps in the development of new drugs typi-
cally include in vitro evaluation of the biological activity of
the candidate compounds – hence efficient and rapid
methods for estimating such activity are needed. Quite
recently, new approaches to testing anti-cholinesterase
activity by means of capillary electrophoresis have been
Donepezil
Tacrine
9
10
11
12
0.03
0.16
3.4
1.2
1.1
0.04
0.6
33.0
9.6
7.23
5.8
0.9
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M. Ignasik et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
tacrine
Thioflavin T fluorescence assay
The thioflavin T (ThT) assay is a well-known test for inhibition
of amyloid b fibrillogenesis as it can be used to visualize
fibril formation. Compounds 7–12 were tested for their
ability to inhibit Ab aggregation in a modified test, using
150
100
50
a
smaller peptide composed of eleven amino acids
(HHQKLVFFAED) [22]. The short peptide containing five
highly conserved amino acids (KLVFF) has been described
as a core peptide responsible for fibril formation [23]. The
test was validated using 4-aminophenol as the reference
compound, whose IC50 value of blocking the Ab fibril
formation was reported to be 83 mM. Donepezil – dual
binding site AChE inhibitor was found to inhibit AChE-
induced Ab1ꢀ42 aggregation by 22% at 100 mM [24] while
in the test against Ab1–40 self-aggregation was not active
(at 100 mM) [25]. In our assays, at 500 mM concentration
six of the tested compounds exhibited anti-Ab aggregation
effects with percentage of inhibition ranging from 19 to 72%.
The most active compounds, 10 and 11, inhibited Ab aggre-
gation by about 34 and 40%, respectively, even at 80 mM
concentration (Table 3). Their activity was higher than
that of the described reference donepezil and promising
for further studies.
0
_
_
_
15
10
5
0
log C
Figure 2. Dose–response plot for tacrine (EMMA method).
inhibition curve for tacrine obtained by plotting the
inhibition percentage versus the logarithm of inhibitory
concentration in the capillary.
In our experiments the IC50 values obtained using the
EMMA method were higher than in Ellman’s test. This dis-
crepancy was probably caused by different concentration of
reagents used in the assay and different reaction conditions
in the capillary. Although the temperature in both methods
was the same (258C), there were dissimilarities in the time of
enzymatic reaction and the volume of mixture. In Ellman’s
method the reaction lasted about 5 min and in EMMA about
1 min. Volume in which reaction took place in EMMA
was much smaller than in colorimetric test (nL vs. mL).
Correlation between both methods is shown in Fig. 3. The
EMMA method is fast and repeatable (RSD area <2%); more-
over, unlike Ellman’s method, it does not require indirect
detection. The migration time for thiocholine was 1.25 min,
with RSD under 2%. In summary, the EMMA method can
be used for rapid screening of new compounds for anti-
cholinesterase inhibitory activity.
Molecular modeling
The presented series of novel isoindoline-1,3-dione deriva-
tives was designed from structural fragments. Some simple
moieties were chosen for docking into the active site of AChE
and BuChE to locate preferable binding areas. Among them,
phthalimide and diethylamine were selected as promising
fragments. It was noted that phthalimide could interact by
p–p stacking and CH–p interactions with aromatic amino
acids at the anionic subsite (Phe330, Phe331, and Trp84)
and/or with residues at the PAS (Trp279, Tyr334) of AChE.
A similar situation occurs with diethylamine, which binds
the same amino acids through cation–p interactions (due to
its protonated form). It can also form H-bonds with Tyr121.
In case of BuChE, both fragments interacted mainly
with Trp82 and Trp430 at the anionic subsite of the
catalytic center. Docking results showed that there were
Table 3. Inhibition of Ab aggregation by isoindoline-1,3-dione
derivatives 7–12
Compound
Inhibition (%)
at 80 mM
Inhibition (%)
at 500 mM
7
8
9
10
11
12
5.7
22.9
5.0
33.8
39.4
7.9
19.8
56.6
54.7
72.3
45.1
30.1
Figure 3. Relation between IC50 values in both methods.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Dual-Binding Site Acetylcholinesterase Inhibitors
5
two interaction sites in AChE but only one in BuChE for such
fragments. Thus, we decided to connect both moieties with
an oligomethylene spacer, thereby obtaining a new series of
isoindoline-1,3-dione derivatives. The structures of these com-
pounds were then docked into the active gorge of both
cholinesterases and it was noted that they interacted with
is involved in p–p stacking with Trp279 and in CH–p inter-
actions with Tyr70. Both carbonyl groups produce H-bonds:
one with Tyr121 and the other one with water. Results of
biological assays confirm that compound 12 is the most
active selective AChE inhibitor in this group.
AChE but not with BuChE. This confirmed our expectations: Conclusion
the new compounds could not bind BuChE because the PAS
in this enzyme is extremely reduced, while interactions with
only one site (anionic subsite in catalytic active site, CAS) are
insufficient to produce activity. The presented compounds
are therefore selective AChE inhibitors. Linker length
analysis showed that the optimal number of methylene
groups is 7–8, ensuring the best arrangement of diethyl-
amine and phthalimide in the AChE active gorge. The
binding potency of novel inhibitors was assessed by the
ChemScore function. The most active compound (12) reached
a score of 40.54, in comparison with 49.48 for the reference
inhibitor – donepezil. This suggests that the presented com-
pounds are less active than reference substances. The binding
mode of compound 12 is presented in Fig. 4. The inhibitor
assumes an extended conformation, lining up with the gorge.
Its protonated amine group produces cation–p interactions
with Trp84 while also integrating itself into the H-bond
network (amine group – water molecule – Tyr121). The tether
is located in the middle of the active gorge, near aromatic
amino acids Phe330, Phe331, and Tyr334, where it
initiates hydrophobic interactions. The phthalimide moiety
A series of new 2-(diethylaminoalkyl)-isoindoline-1,3-dione
derivatives were designed using molecular modeling. Our
goal was to produce dual binding site cholinesterase inhibi-
tors. The synthesized compounds were evaluated in terms of
their inhibitory potency against AChE and BuChE, as well as
against the formation of Ab plaque. For AChE inhibitory
activity, the spectrophotometric methods of Ellman’s and
EMMA assay were used. Both methods produced comparable
results, which lends support for EMMA as a convenient tool
for rapid screening of novel cholinesterase inhibitors. All
synthesized compounds turned out to be moderately potent,
selective inhibitors of AChE, with IC50 values ranging from
0.9 to 19.5, and weak inhibitors of Ab plaque formation.
These results agree with the conclusions of docking studies
aimed at compounds which target both the CAS and PAS of
AChE. The most promising selective AChE inhibitors are
compounds 10 (IC50 ¼ 1.2 mM) and 11 (IC50 ¼ 1.1 mM) with
6–7 methylene spacers, which also inhibit Ab fibril for-
mation. The presented study shows that these compounds
are of interest as prospective multipotent agents.
Figure 4. Binding mode of compound 12 at
the active site of AChE.
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M. Ignasik et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
of K2CO3 (18 mmol, 2.49 g) was stirred in 100 mL acetonitrile
under reflux for 24 h. Subsequently, the solvent was evaporated
in vacuum, producing a residue which was further dissolved in
40 mL of sodium bicarbonate and extracted with ethyl acetate
(3 ꢁ 30 mL). The organic layer was dried with Na2SO4. The sol-
vent was then evaporated and the residue purified by column
chromatography (n-hexane/ethyl acetate/TEA, 5:5:1) to afford the
pure product. The final product was obtained in the form of
hydrochloride salts.
Experimental
Chemistry
General procedure for the synthesis of compounds (1–6)
A mixture of potassium phthalimide (15 mmol, 2.78 g) with 2.5-
fold excess of suitable a,v-dibromoalkane (37.5 mmol) and cata-
lytic amount of TBAB (0.56 mmol, 0.18 g) was stirred in 150 mL
of acetonitrile under reflux for 20 h. Subsequently, the reaction
mixture was filtered and the filtrate concentrated. The resulting
yellow oil was purified by column chromatography (n-hexane/
ethyl acetate; 1:1).
2-(3-Diethylaminopropyl)-isoindoline-1,3-dione (7)
Yellow oil; yield 70%. Rf (n-hexane/ethyl acetate/TEA; 5:5:1)
0.84; 1HNMR (CDCl3) d ppm: 7.82–7.62 (m, 4H, phthalimide);
3.71–3.64 (m, 2H, –CH2–C2H4–N-(C2H5)2); 2.50–2.39 (m, 2H,
–C2H4–CH2–N–(CH2–CH3)2), 1.88–1.70 (m, 2H, –CH2–CH2–CH2
–N–(C2H5)2), 0.9 (t, 6H, –N–(CH2–CH3)2. As the HCl salt:
mp ¼ 1588C; anal. calc. for C15H20N2O2 HCl: C-60.7%; N-9.44%;
H-7.13%, found C-60.64%; N-9.41%; H-7.50%.
2-(3-Bromopropyl)-isoindoline-1,3-dione (1)
White solid; yield: 51%; Rf (n-hexane/ethyl acetate; 1:1) 0.74;
mp ¼ 778C (mp lit. ¼ 74–768C [26]); 1HNMR (CDCl3) d ppm:
7.95–7.67 (m, 4H, phthalimide); 3.9–3.79 (t, 2H, J ¼ 6.85 Hz,
–CH2–C2H2Br–); 3.45–3.38 (t, 2H, J ¼ 6.75 Hz, –C2H4–CH2–Br);
2.31–2.21 (p, 2H, J ¼ 6.78 Hz, –CH2–CH2–CH2Br).
2-(4-Diethylaminobutyl)-isoindoline-1,3-dione (8)
2-(4-Bromobutyl)-isoindoline-1,3-dione (2)
Yellow oil; yield 70%. Rf (n-hexane/ethyl acetate/TEA; 5:5:1) 0.56;
1HNMR (CDCl3) d ppm: 7.91–7.60 (m, 4H, phthalimide); 3.71–3.65
(t, 2H, –CH2–C3H12–); 2.54–2.38 (qd, 6H, –CH2–N–(CH2–CH3)2);
1.73–1.60 (m, 2H, –C2H4–CH2–CH2–); 1.53–1.40 (m, 2H,
–CH2–CH2–C2H4–); 1.05–0.91 (m, 6H, (–CH2–CH3)2). As the HCl salt:
mp ¼ 1708C; anal. calc. for C16H22N2O2 HCl: C-61.83%; N-9.01%;
H-7.46%, found C-61%.92; N-9.04%; H-7.76%.
White solid; yield: 79%; Rf (n-hexane/ethyl acetate; 1:1) 0.85;
mp ¼ 748C (mp lit. ¼ 72.5–748C [27]); 1HNMR (CDCl3) d ppm:
7.96–7.63 (m, 4H, phthalimide); 3.79–3.68 (dd, 2H, J ¼ 6.85,
–CH2–C3H8Br); 3.45–3.38 (t, 2H, J ¼ 6.75, –C3H8–CH2–Br); 2.31–
2.21 (p, 4H, J ¼ 6.78, –CH2–C2H4–CH2Br).
2-(5-Bromopentyl)-isoindoline-1,3-dione (3)
2-(5-Diethylamiopentyl)-isoindoline-1,3-dione (9)
White solid; yield: 76%; Rf (n-hexane/ethyl acetate; 1:1) 0.82;
mp ¼ 61.58C; 1HNMR (CDCl3) d ppm: 7.89–7.65 (m, 4H, phthal-
imide); 3.68 (t, 2H, –CH2–C4H8Br); 3.38 (t, 2H, –C4H8–CH2–Br);
1.94–1.83 (m, 2H, –C3H6–CH2–CH2Br); 1.75–1.64 (m, 2H,
–C2H4–CH2–C2H4Br); 1.54–1.40 (m, 2H, –C2H4–CH2-C2H4Br).
Yellow oil; yield 64%. Rf (n-hexane/ethyl acetate/TEA; 5:5:1) 0.54;
1HNMR (CDCl3) d ppm: 7.88–7.61 (m, 4H, phthalimide); 3.71–3.65
(t, 2H, –CH2–C4H14–); 2.52–2.41 (q, 4H, J ¼ 7.13 Hz, –N–(CH2–CH3)2);
2.39–2.33 (m, 2H, –CH2–N–); 1.73–1.60 (m, 2H, –C3H6–CH2–CH2–);
1.51–1.40 (m, 2H, –CH2–CH2–C3H6–); 1.36–1.26 (m, 2H,
–C2H4–CH2–C2H4–); 1.00–0.93 (t, 6H, J ¼ 7.16 Hz, (–CH2–CH3)2).
As the HCl salt: mp ¼ 1658C; anal. calc. for C17H24N2O2 HCl:
C-62.86%; N-8.62%; H-7.76%, found C-62.73%; N-8.61%; H-8.10%.
2-(6-Bromohexyl)-isoindoline-1,3-dione (4)
White solid; yield: 53%; Rf (n-hexane/ethyl acetate; 1:1) 0.59;
mp ¼ 608C; 1HNMR (CDCl3) d ppm: 7.89–7.62 (m, 4H, phthal-
imide); 3.67 (t, 2H, N–CH2–C6H12Br); 3.40 (t, 2H, C6H12–CH2–Br);
1.91–1.79 (m, 2H, –CH2–CH2–C4H8Br); 1.73–1.62 (m, 2H,
C4H8–CH2–CH2Br); 1.50–1.42 (m, 4H, –C2H4–C2H4–C2H4Br).
2-(3-Diethylaminohexyl)-isoindoline-1,3-dione (10)
Yellow oil; yield 35%. Rf (n-hexane/ethyl acetate/TEA; 5:5:1) 0.54;
1HNMR (CDCl3) d ppm: 7.87–7.61 (m, 4H, phthalimide); 3.81–3.50
(m, 2H, –CH2–C5H10–); 2.53–2.42 (m, 4H, –(CH2–CH3)2); 2.40–2.31
(m, 2H, –C5H10–CH2–N); 1.73–1.57 (m, 2H, –CH2–CH2–C4H8–); 1.48–
1.19 (m, 2H, –C2H4–C3H6–CH2–); 1.00–0.94 (m, 6H, (–CH2–CH3)2). As
the HCl salt: mp ¼ 1728C anal. calc. for C18H26N2O2 HCl: C-63.8%;
N-8.27%; H-8.03%, found C-63.58%; N-8.15%; H-8.25%.
2-(7-Bromoheptyl)-isoindoline-1,3-dione (5)
White solid; yield: 66%; Rf (n-hexane/ethyl acetate; 1:1) 0.85;
mp ¼ 58.58C; 1HNMR (CDCl3) d ppm: 7.89–7.63 (m, 4H, phthal-
imide); 3.70–3.61 (m, 2H, –CH2–C6H12Br); 3.45–3.28 (dt, 2H,
–C6H12–CH2–Br); 1.90–1.76 (m, 2H, –C5H10–CH2–CH2Br); 1.70–1.63
(m, 2H, –CH2–CH2–C5H10Br); 1.40–1.30 (m, 6H, –C2H4–C3H6–C2H4Br).
2-(3-Diethylaminoheptyl)-isoindoline-1,3-dione (11)
Yellow oil; yield 56%. Rf (n-hexane/ethyl acetate/TEA; 5:5:1) 0.75;
1HNMR (CDCl3) d ppm: 7.85–7.64 (m, 4H, phthalimide); 3.73–3.56
(m, 2H, –CH2–C6H12–); 2.52–2.42 (m, 4H, –(CH2–CH3)2); 2.38–2.31
(m, 2H, –C6H12–CH2–N); 1.69–1.59 (m, 2H, –CH2–CH2–C3H6–); 1.45–
1.17 (m, 6H, –C2H4–C3H6–C2H4–); 1.02–0.93 (m, 6H, (–CH2–CH3)2).
As the HCl salt: mp ¼ 132.58C; anal. calc. for C19H28N2O2 HCl:
C-64.83%; N-8.13%; H-8.48%, found C-64.67%; N-7.94%; H-8.28%.
2-(8-Bromooctyl)-isoindoline-1,3-dione (6)
White solid; yield: 94%; Rf (n-hexane/ethyl acetate; 1:1) 0.80;
mp ¼ 55.58C (mp lit. ¼ 54–558C [28]); 1HNMR (CDCl3) d ppm:
7.89–7.67 (m, 4H, phthalimide); 3.72–3.66 (m, 2H, –CH2–C7H14Br);
3.43–3.36 (t, 2H, –CH2–Br); 1.91–1.80 (m, 2H, C6H12–CH2–CH2Br);
1.75–1.64 (m, 2H, –CH2–CH2–C6H12Br); 1.54–1.30 (m, 8H,
–C2H4–C4H8–C2H4Br).
2-(3-Diethylaminooctyl)-isoindoline-1,3-dione (12)
General procedure for synthesis of compounds (7–12)
2-(Bromoalkyl)-isoindoline-1,3-dione (6 mmol) with fourfold
excess of diethylamine (24 mmol, 1.76 g) in the presence
Yellow oil; yield 71%. Rf (n-hexane/ethyl acetate/TEA; 5:5:1) 0.67;
1HNMR (CDCl3) d ppm: 7.82–7.53 (m, 4H, phthalimide); 3.61–3.53
(m, 2H, –CH2–C7H14–); 2.47–2.34 (m, 4H, –(CH2–CH3)2); 2.31–2.27
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Dual-Binding Site Acetylcholinesterase Inhibitors
7
(m, 2H, –C7H14–CH2–N); 1.67–1.46 (m, 2H, –CH2–CH2–C6H12–);
1.33–1.17 (m, 10H, –C2H4–C5H10–CH2–); 0.93–0.89 (m, 6H,
(–CH2–CH3)2). As the HCl salt: mp ¼ 1178C; anal. calc. for
C20H30N2O2 HCl: C-65.47%; N-7.63%; H-8.52%, found C-65.84%;
N-7.83%; H-8.72%.
Thioflavin T assay fibril formation
Inhibitors were diluted in buffer (25 mM NaH2PO4, 120 mM
NaCl, 3 mM ThT, 0.02% NaN3, pH of 7.4) to reach concentrations
between 16 and 500 mM. A 2 mM stock solution of the
peptide HHQKLVFFAED in DMSO was added to reach a final
peptide concentration of 100 mM. Incubations were performed
on 96-well fluorescence microtiter plates (Nunc GmbH,
Wiesbaden, Germany). Fluorescence was measured every
10 min over a period of 12 h (excitation wavelength 450 nm,
emission wavelength 482 nm) on a Cary Eclipse fluorescence
spectrophotometer (Varian, Darmstadt, Germany).
Biology
Ellman’s method AChE/BuChE inhibition activity
Reagents and chemicals: DNTB, acetylthiocholine iodide (ATCh),
butyrylthiocholine iodide (BTCh), AChE from Electrophorus electri-
cus (425.96 U/mg solid), and BuChE from horse serum (2.5 units/
1 mL) – were purchased from Sigma–Aldrich. All assays were
performed using the Perkin Elmer Lambda 12 device. Detection
was performed at 412 nm. The reaction took place in 100 mM
phosphate buffer, pH 8.0, containing 0.25 units of AChE or
BuChE, 0.3 mmol 5,50-dithio-bis-(2-nitrobenzoic) acid (DTNB)
and 0.45 mmol acetylthiocholine or butyrylthiocholine as sub-
strates. The tested compounds were incubated with the enzyme
for 5 min at 258C prior to starting the reaction by adding the
substrate. Enzymatic activity was determined by measuring
absorbance over a period of 5 min. Two kinds of assays were
conducted: the peak activity of the enzyme was determined by
using a blank sample, followed by reference measurements (for
tacrine and donepezil) and measurements for the synthesized
compounds. Data from concentration–inhibition experiments
was integrated through nonlinear regression analysis using
the GraphPad Prism program (GraphPad Prism Software Inc.
Molecular modeling
Three-dimensional representations of ligands were created using
Corina online tool (Molecular Networks) and saved as pdb files.
Using Sybyl 8.0 (Tripos) Gasteiger-Marsili charges were assigned
following checks of atom types and protonation of the com-
pounds. Finally, ligand structures were saved in the mol2 format.
Dockings were performed to AChE from 1EVE and to BuChE from
the 1P0I crystal complex using Gold 4.1 (CCDC) program. In the
preparatory phase all histidine residues were protonated at Ne,
hydrogen atoms added, ligand molecules removed, and binding
sites defined as all amino acid residues within 10 A from done-
˚
pezil (for AChE) and within 20 A from the glycerol molecule
present in the active center of BuChE. The presence of some water
molecules was also taken into account. A standard set of genetic
algorithms with population size 100, number of operations
˚
˚
2005), producing estimates of IC50
.
100 000 and clustering tolerance of 1 A were applied. As a result,
10 ligand conformations were obtained and sorted according to
ChemScore function values. Results were visualized by PyMOL.
EMMA AChE inhibition activity
Reagents and chemicals: AChE from Electrophorus electricus
(425.96 U/mg solid), ATCh, boric acid, and sodium phosphate
monobasic – were purchased from Sigma–Aldrich. A solution of
AChE (0.4 mg/mL) was prepared with 30 mM borate-phosphate
buffer at pH 8.0. A solution of ATCh (10 mM) and solutions of
inhibitors were prepared with 30 mM borate-phosphate buffer
at pH 8.0. All solutions were filtered through 0.45 mm Millex
filters. All separations were performed on a Beckman CE system
(P/ACE MDQ) equipped with a diode-array detector (DAD) and
controlled by 32 Karat software version 8.0. An uncoated fused-
silica capillary with a total length of 30 cm (20.2 cm to detection
window) and internal diameter 50 mm (external diameter
375 mm) was purchased from Beckman. The capillary was
thermostatted at 258C and samples at 108C. Detection was per-
formed at UV 230 nm.
Procedures: A new capillary was pretreated with 0.1 M NaOH
solution for 10 min, followed by water for 10 min and running
buffer for 10 min. The same rinsing sequence was performed
every day prior to starting separations. Between runs, the capil-
lary was rinsed with 0.1 M hydrochloric acid, 0.1 M sodium
hydroxide, water and borate-phosphate buffer (50 psi, 2 min
each). For every rinsing and separation cycle 30 mM borate-
phosphate buffer pH 8.0 was used. Two kinds of assays were
conducted: the peak activity of the enzyme was determined by
using a blank sample, followed by measurements for inhibitors
at different concentrations. The procedure was repeated three
times for each sample. The plug–plug method was used with the
following injection sequence: enzyme solution 0.5 psi, 10 s,
inhibitor or buffer (in blank) 0.5 psi, 15 s, substrate 0.5 psi,
10 s, 1 kV for 15 s, incubation for 1 min, separation at 12 kV
for 4 min.
We wish to thank the Polish Ministry of Science and Higher Education
(grant no. N N405 16339) for financial support.
The authors have declared no conflict of interest.
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