Inhibitory activity on cholinesterases produced by aryl-phthalimide derivatives: green synthesis, in silico and in vitro evaluation

Article abstract of DOI:10.1007/s00044-020-02543-2

Background: Alzheimer’s disease (AD) is characterized by cognitive impairment and loss of immediate memory resulting from neuronal death in different brain areas, mainly those producing acetylcholine. Acetylcholinesterase inhibitors improve cognitive function, delay mental deterioration, and reduce other symptoms. Despite being the cornerstone for treating mild–moderate AD, these compounds are only palliative agents and often have severe adverse effects. Recently, butyrylcholinesterase (BuChE) has been found to be involved in AD. The aim of this study was to synthesize a series of six phthalimides with structural relationship with monoamines and evaluate them in vitro and in silico as AChE and BuChE inhibitors. In addition, a modified version of the Bonting and Featherstone method for determining AChE activity was adapted for the assessment of BuChE activity. Results: Six molecules (dioxoisoindolines A–F) were synthesized in good yields using a green chemistry approach. Dioxoisoindolines E and F were more active for AChE, with a K_i of 232 and 193 μM, respectively. Contrarily, dioxoisoindolines C and D showed up to fivefold greater selectivity for BuChE than AchE, with a K_i of 200 and 100 μM, respectively. The competitive inhibitory activity of the latter two molecules was similar to that of the reference compounds. Molecular docking demonstrated the participation of carbonyl carbons and aromatic rings in the high affinity of dioxoisoindoles for cholinesterases. Conclusion: The modified version of the Bonting and Featherstone method was successfully adapted to quantify BuChE activity. Dioxoisoindolines C and D displayed greater inhibition of BuChE versus AChE, with good inhibition of both enzymes. Thus, they are promising lead compounds for developing new BuChE/AChE inhibitors. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02543-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02543-2
ORIGINAL RESEARCH
Inhibitory activity on cholinesterases produced by aryl-phthalimide
derivatives: green synthesis, in silico and in vitro evaluation
Omar Ruiz-Maciel1 Itzia I. Padilla-Martínez2 Luis A. Sánchez-Labastida1 Marvin A. Soriano-Ursúa3
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Erik Andrade-Jorge1,4 José G. Trujillo-Ferrara1
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Received: 8 November 2019 / Accepted: 4 April 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Background Alzheimer’s disease (AD) is characterized by cognitive impairment and loss of immediate memory resulting
from neuronal death in different brain areas, mainly those producing acetylcholine. Acetylcholinesterase inhibitors improve
cognitive function, delay mental deterioration, and reduce other symptoms. Despite being the cornerstone for treating
mild–moderate AD, these compounds are only palliative agents and often have severe adverse effects. Recently, butyr-
ylcholinesterase (BuChE) has been found to be involved in AD. The aim of this study was to synthesize a series of six
phthalimides with structural relationship with monoamines and evaluate them in vitro and in silico as AChE and BuChE
inhibitors. In addition, a modified version of the Bonting and Featherstone method for determining AChE activity was
adapted for the assessment of BuChE activity.
Results Six molecules (dioxoisoindolines A–F) were synthesized in good yields using a green chemistry approach. Diox-
oisoindolines E and F were more active for AChE, with a Ki of 232 and 193 µM, respectively. Contrarily, dioxoisoindolines
C and D showed up to fivefold greater selectivity for BuChE than AchE, with a Ki of 200 and 100 µM, respectively. The
competitive inhibitory activity of the latter two molecules was similar to that of the reference compounds. Molecular docking
demonstrated the participation of carbonyl carbons and aromatic rings in the high affinity of dioxoisoindoles for
cholinesterases.
Conclusion The modified version of the Bonting and Featherstone method was successfully adapted to quantify BuChE
activity. Dioxoisoindolines C and D displayed greater inhibition of BuChE versus AChE, with good inhibition of both
enzymes. Thus, they are promising lead compounds for developing new BuChE/AChE inhibitors.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02543-2) contains supplementary
material, which is available to authorized users.
* Erik Andrade-Jorge
Politécnico Nacional, Av. Acueducto s/n Barrio la Laguna
Ticomán, 07340 Mexico City, Mexico
andrade136@hotmail.com
3
4
* José G. Trujillo-Ferrara
Laboratorio de Investigación en Fisiología, Sección de Estudios de
Posgrado e Investigación, Escuela Superior de Medicina del
Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n
Casco de Santo Tomás, 11340 Mexico City, Mexico
jtrujillo@ipn.mx
1
Laboratorio de Investigación en Bioquímica, Sección de Estudios
de Posgrado e Investigación, Escuela Superior de Medicina del
Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n
Casco de Santo Tomás, 11340 Mexico City, Mexico
Unidad de Investigación en Biomedicina, Facultad de Estudios
Superiores-Iztacala, Universidad Nacional Autónoma de México,
Av. de los Barrios 1, Los Reyes Iztacala, Tlalnepantla, 54090
Estado de Mexico, Mexico
2
Laboratorio de Química Supramolecular y Nanociencias, Unidad
Profesional Interdisciplinaria de Biotecnología del Instituto

Medicinal Chemistry Research
Graphical Abstract
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Keywords Dioxoisoindolines Acetylcholinesterase Butyrylcholinesterase Inhibitors Alzheimer
Highlights
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Using green chemistry principles, six phthalimides were synthesized in good yields.
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Dioxoisoindolines D and F were potent inhibitors of BuChE and AChE, respectively.
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The improved Bonting and Featherstone method proved useful for evaluating BuChE activity.
Introduction
mild-to-moderate AD, they do not represent a cure for the
disease (Brufani et al. 1997; Huang and Mucke 2012). More-
over, those now on the market have several adverse effects.
Hence, efforts are being made to look for new drugs with
greater potency and less toxicity (Bajda et al. 2013).
In the search for new AChEIs during the last few dec-
ades, many techniques have been developed to measure the
activity of AChE. In 1961, Ellman et al. (1961) published
the ultramicro cholinesterase assay, which was an adapta-
tion of the hydroxamic acid method for the determination of
choline esters (Bonting and Featherstone 1956). Since this
assay is time consuming and expensive, our group made
modifications to more easily assess AChE catalytic activity
(Andrade-Jorge et al. 2018b).
Alzheimer’s disease (AD), a progressive neurodegenerative
disease and the most common form of dementia, is char-
acterized by a decreased cognitive function and several
behavioral abnormalities (Mohammadi-Farani et al. 2013; Liu
et al. 2017). It can also be described as a brain disorder that
leads to a loss of memory and cognitive abilities (Moham-
madi-Farani et al. 2013). There are several hypotheses to
explain the origin of AD, the most studied being the choli-
nergic and the β-amyloid theories (extracellular deposits in the
cerebral parenchyma and intracellular neurofibrillary tangles)
(Lahiri et al. 2002, 2007). An important role of the tau protein
has also been proposed (Mukherjee et al. 2007).
Since the 1970s, the main strategy of many scientists
doing research on AD (Ayazgok et al. 2017) has been the
inhibition of the acetylcholinesterase enzyme (Si et al.
2016; Liu et al. 2017) (AChE, EC 3.1.1.7), which hydro-
lyzes the neurotransmitter acetylcholine in the cholinergic
synapses and therefore nerve transmission ends (Kamkwa-
lala and Newhouse 2016). More recent investigation on AD
has also targeted butyrylcholinesterase (BuChE), another
nonspecific cholinesterase enzyme (Wenk 2003; Liu et al.
2017; Zhang and Zhang 2017) thought to be involved in
neurodegeneration, becoming a therapeutic target for the
treatment of AD (Shen et al. 2017).
Currently, the treatments for AD are all based on acet-
ylcholinesterase inhibitors (AChEIs), especially tacrine, done-
pezil, rivastigmine, galantamine, and memantine (Musiał et al.
2007; Guzior et al. 2015). Several studies indicate that inhi-
biting AChE in patients with AD contributes to improved
cognitive function, delayed progression of mental deterioration,
and a reduction of other characteristic symptoms (Lahiri et al.
2007). Although AChEIs are the most common treatment for
The catalytic site of the AChE enzyme is located inside a
deep throat (Rosenberry 1975; Gilson et al. 2006; Bajda et al.
2013). It is surrounded by many other ligand recognition sites,
including the oxyanionic orifice, an aromatic patch, an anionic
site, and a peripheral anionic site (Dvir et al. 2010). The per-
ipheral anionic site is intimately involved in the formation of
β-amyloid peptide aggregates (Kryger et al. 1999; Colovic et al.
2013). Thus, a molecule capable of interacting at this site may
have the additional benefit of stopping the aggregation of the
β-amyloid peptide (Gupta and Mohan 2011).
According to recent research, isoindoline-1,3-diones are
potent and novel AChEIs. They contain two phthalimide car-
bonyl groups that facilitate hydrogen bonding with the catalytic
active site and the peripheral anionic site simultaneously.
Consequently, these molecules could possibly inhibit both the
formation of the β-amyloid peptide and the hydrolytic activity
of AChE on acetylcholine (Mary et al. 1998; Mohammadi-
Farani et al. 2013). Indeed, an isoindoline-1,3-dione was very
recently crystallized and tested as an AChEI, finding good
inhibitory activity (Ki = 630 μM) (Andrade-Jorge et al. 2018a).

Medicinal Chemistry Research
Albeit if the mechanism(s) of action beyond choinesterases
inhibition is unclear, the structural relationship with mono-
amines is attractive for exerting actions ameliorating AD pro-
gression (Farfán-García et al. 2019).
The aim of the present study was to evaluate the inhi-
bitory activity of a series of six dioxoisoindolines on AChE
and BuChE. The activity of AChE was determined with the
improved Bonting and Featherstone method, as previously
described. The same technique was herein adapted to
measure BuChE activity. The results of the different com-
pounds were compared in order to elucidate the affinity of
dioxoisoindolines for AChE and BuChE.
following a published green chemistry technique, without
modifications (Andrade-Jorge et al. 2017). Briefly, 1.1 mmol
of phthalic anhydride and 1 mmol of the corresponding
amine were placed in a 50 mL round-bottom flask. The
mixture was stirred and heated to melting point
(100–150 °C) for 5–10 min. Then 40 mL of ethyl acetate
were added and the reaction was sonicated until a white
powder formed. The precipitate was filtered, dissolved in
acetone, and precipitated again with water (pH 13). The
reactions were monitored by TLC (0.25 mm thick silica gel,
60 F254 plates, Merck, Darmstadt, Germany) and the spots
detected with UV light. The melting points were determined
on a Stuart® SMP40 automatic melting point apparatus and
are uncorrected. The products were characterized by nuclear
magnetic resonance (NMR) spectra, recorded on a Varian
Materials and methods
1
Mercury 300 spectrometer at 300 MHz for H and 75 MHz
Synthesis and characterization
for 13C NMR, with tetramethylsilane as internal reference. In
addition, spectra were obtained on a Bruker Ascend g750
1
All reagents and solvents were used as received from Sigma-
Aldrich. Dioxoisoindolines were synthesized (Table 1) by
Ultrashield 750 MHz for H and 189 MHz for 13C NMR.
Infrared (IR) spectroscopy was recorded on a Perkin-Elmer
Table 1 Structures and synthetic route for isoindoline-1,3-diones
General Reaction
Reaction
Reaction
t
ime (min)
a
time (min)
Yielld
%
Yield
%
ID
Substituent
nd
temperature
c°)
IDD
Substituennt
and
temper
ature
(
(c°)
DioxoisoindolineA
85
89
89
10/150
10/100
15/250
D
ioxoisoi
n
dol
i
neB
90
85
80
12/20
10/10
12/20
0
DioxoisoindolineC
DioxoisoindolineE
D
i
oxoisoi
n
n
dol
i
neD
0
0
D
ioxoisoinndolineF

Medicinal Chemistry Research
model FT-IR, setting spectral resolution at 4 cm−1. Electro-
spray ionization high-resolution mass spectrometry was
performed on a Bruker micrOTOf-Q-II instrument.
2018b) and the concentration–response curve was con-
structed. The same methodology was adapted to measure the
activity and inhibition of BuChE, following the hydroxamic
acid method (Fig. 1). Dilutions of BuCh were prepared at
distinct concentrations (Scheme 1a), then mixed in an analog
vortex mixer (Thomas Scientific). Twenty microliters of each
solution was placed in a 96-well microplate, according to the
pattern described in Scheme 1b. The microplate was allowed
to incubate for 55 min at 37 °C in a water bath (Felisa) before
stopping the reaction with 40 μL of alkaline hydroxylamine
solution. After adding 100 μL of FeCl3 solution to each well
and premixing for 30 s, the optical density was read at 540 nm
in a microplate reader (Accuris MR9600). With the corre-
sponding values, the standard curve for BuCh was built. With
the same procedure and 20 μL of BuChE in the starting
mixture, the standard curve was constructed for this enzyme.
The process for evaluating the activity of BuChE was repe-
ated, adding 20 μL of one of the test compounds in each assay
before adding BuChE. All assays were performed in triplicate
and the data are expressed as the average of three values.
Solutions
The phosphate buffer solution (0.1 M, pH 8) was prepared by
adding 14.96 g of K2HPO4 and 1.832 g of KH2PO4 to
1000 mL of distilled water, then mixing. The sodium hydro-
xide solution (4.2 M) was obtained by dissolving 84.8 g of
NaOH in distilled water to reach a final volume of 500 mL.
For the hydroxylamine solution (2.4 M), 84.2 g NH2OH•HCl
was dissolved in distilled water to reach a final volume of
500 mL. For the alkaline hydroxylamine solution, equal
volumes of sodium hydroxide and hydroxylamine solution
were mixed immediately before use. The ferric chloride
solution (0.75 M) was afforded by slowly mixing 101.55 g of
FeCl3•6H2O with an HCl solution (61.95%) to a final volume
of 500 mL. Dissolution of 50 mg of ACh iodide in 0.63 mL of
phosphate buffer provided 256 mM of stock acetylcholine
solution, and 30 mg of BuCh iodide in 1 mL of phosphate
buffer furnished 100 mM of stock butyrylcholine solution.
Subsequently, various dilutions were made for the kinetic
experiment. The stock AChE solution (10 U/mL) was pre-
pared with the freeze-dried Electrophorus electricus AChE
powder (EeAChE, Sigma Chemical C3389), and the stock
BuChE solution (0.4 U/mL) with the freeze-dried human
BuChE powder (H-BuChE, Sigma Chemical C-9971)
obtained from the original source. Different concentrations of
the synthesized compounds, neostigmine and galantamine,
were generated by adding DMSO (<1%) or distilled water as
the dissolution medium. All solutions were stored at −20 °C.
Docking
The ligands were optimized with GaussView software
5.0.9, considering the protonation status under physiologi-
cal conditions (pH 7.4). The conformational analysis was
carried out on Gaussian 16 (Frisch et al. 2016) with a
semiempirical method (AM1). The rotational bonds, degree
of torsional freedom, atomic partial loads, and nonpolar H
bonds were assigned with AutoDock 1.5.6 tools (Huey and
Morris 2008). Crystal structures of the enzymes were
downloaded from the Protein Data Bank (PDB: HsBuChE
4P0I, HsAChE 4PQE, EeAChE 1C2B).
Acetylcholinesterase and BuChE activity assay
The coupling conditions were programmed with Auto-
Dock 1.5.6 tools and Raccoon (Cosconati et al. 2010), using
a hybrid Lamarckian genetic algorithm (Morris et al. 1998)
and an initial population of 100 randomly placed indivi-
duals. Kollmann partial charges were assigned for all pro-
teins and Gasteiger charges for the ligands. Subsequently,
After applying 20 μL of neostigmine or galantamine or the
dioxoisoindolines to AChE, its activity and inhibition was
determined with the modified Bonting and Featherstone
method (Bonting and Featherstone 1956; Andrade-Jorge et al.
Fig. 1 Mechanism of reaction of hydroxamic acid with the choline esters in the improved method of Bonting–Featherstone

Medicinal Chemistry Research
Scheme 1 Representation of the procedure involved in the improved
method for BuChE, using a 96-well microplate for BuChE. a Dilutions
performed for the BuCh curve. b The pattern used to construct the
different curves to be used for evaluating the inhibitory activity of
dioxoisoindolines
the grid parameter file was established for the three enzymes
(HsBuChE, HsAChE, and EeAChE) with a grid of 126 ×
126 × 126 Å, coordinates of X = 138.889, Y = 123.537, and
Z = 38.703 for HsBuChE, coordinates of X = −25.301, Y
= 22.446, and Z = 0.649 for HsAChE, coordinates of X =
88.481, Y = 109.042, and Z = 83.026 for EeAChE, and a
mesh separation of 0.375 Å. The calculations were executed
in AutoDock4 in a Linux operating system (Fedora 22).
Finally, the lowest energy state, expressed as Gibbs free
energy (ΔG), was obtained for each compound. The dis-
sociation constant (Kd), −log dissociation constant (pKd),
number of interactions and distance, and type of binding
were determined with the Visual Molecular Dynamics
program (VMD v.1.8.6) (Humphrey et al. 1996) and Dis-
covery studio 4.0 client. The validation of the molecular
docking was performed by reproducing the binding mode of
well-known ligands (acetylcholine, neostigmine, galanta-
mine, rivastigmine).
on GraphPad Prism statistical software in the enzyme
kinetic mode.
Results and discussion
Chemical characterization
Six aryl-phthalimides structurally related to some monoamine
neurotransmitters were synthesized under solventless condi-
tions, this relation is in regard to the presence of the pheny-
lethyl or ethylamine moiety in the structures (Table 1). The
complete chemical characterization of these compounds is
shown below.
2-(2-phenylethyl) isoindolin-1,3-dione (dioxoisoindoline A)
It was obtained as a white solid, 85% yield; mp 130–131 °C
1
(recrystallization solvent ethyl acetate); H NMR (CDCl3,
Statistical analysis
300 MHz) δ (PPM) 7.82 (2H, dd, J = 5.5, 3.0 Hz, H-4,7),
7.70 (2H, dd, J = 5.6, 3.0 Hz, H-5,6), 7.32–7.18 (5H, m, H-
13,14,15,16,17), 3.92 (2H, t, J = 7.4 Hz, H-10), 2.98 (2H, t,
J = 7.4 Hz, H-11); 13C NMR (CDCl3, 75 MHz) δ 168.1 (C-
1,3), 137.9 (C-12), 133.9 (C-5,6), 132.0 (C-8,9), 128.8 (C-
14,16), 128.5 (C-13,17), 126.6 (C-15), 123.2 (C-4,7), 39.2
(C-10), 34.6 (C-11). IR (ATR, cm−1) ύ: 3031 (C–H,
The data from the cholinesterase inhibition assays are
expressed as the mean 95% confidence intervals. Each
treatment was compared with the corresponding control
group. A Michaelis–Menten graph was made for nonlinear
regression analysis, carried out at 95% confidence intervals

Medicinal Chemistry Research
Aromatic), 2935 (C–H, Aliphatic), 1705 (C=O), 1600
(C=C), 1427 (CH2), 1391 (C–N). HRMS (m/z): 274.0752
(M + Na), calculated: 274.0838.
(C=C), 1458 (CH2), 1453 (CH3), 1384 (C–N). HRMS (m/
z): 274.0761 (M + Na), calculated: 274.0838.
2-(2-(4-hydroxyphenyl)ethyl) isoindolin-1,3-dione
(dioxoisoindoline E)
2-(2-(3,4-dimethoxyphenyl)ethyl) isoindolin-1,3-dione
(dioxoisoindoline B)
It was obtained as a white solid, 89% yield; mp 237–239 °C
1
It was obtained as a light yellow solid, 90% yield; mp
171–172 °C (recrystallization solvent ethyl acetate); 1H
NMR (CDCl3, 300 MHz) δ (PPM) 7.81 (2H, dd, J = 5.5,
3.0 Hz, H-4,7), 7.70 (2H, dd, J = 5.4, 3.1 Hz, H-5,6),
6.78–6.73 (3H, m, H-13,16,17), 3.83 (3H, s, H-18), 3.80
(3H, s, H-19), 3.90 (2H, t, J = 7.5 Hz, H-10), 2.93 (2H, t, J
= 7.5 Hz, H-11); 13C NMR (CDCl3, 75 MHz) δ 168.2 (C-
1,3), 148.7 (C-14), 147.6 (C-15), 133.9 (C-12), 132.0
(C-5,6), 130.4 (C-8,9), 123.1 (C-4,7), 120.8 (C-17), 111.8
(C-16), 111.1 (C-13), 55.7 (C-18,19), 39.3 (C-10), 34.0 (C-
11). IR (ATR, cm−1) ύ: 3063 (C–H, Aromatic), 2943 (C–H,
Aliphatic), 2842 (O–CH3, Aliphatic), 1705 (C=O), 1600
(C=C), 1466 (CH2), 1427 (CH3), 1394 (C–N), 1228
(O–CH3). HRMS (m/z): 334.0956 (M + Na), calculated:
334.1049.
(recrystallization solvent ethyl acetate); H NMR (DMSO,
750 MHz) δ 9.18 (1H, s, OH-15), 7.83–7.80 (4H, m, H-
4,5,6,7), 6.96 (2H, d, J = 8.4 Hz, H-13,17), 6.63 (2H, d,
J = 8.4 Hz, H-14,16), 3.73 (2H, t, J = 7.4 Hz, H-10), 2.79
(2H, t, J = 7.4 Hz, H-11); 13C NMR (DMSO, 189 MHz) δ
168.2 (C-1,3), 156.0 (C-15), 134.8 (C-5,6), 131.9 (C-8,9),
130.0 (C-12), 128.6 (C-13,17), 123.6 (C-4,7), 115.6 (C-
14,16), 39.5 (C-10), 33.2 (C-11). IR (ATR, cm−1) ύ:3255
(O–H) 3026 (C–H, aromatic), 2955 (C–H, aliphatic), 1703
(C=O), 1613 (C=C), 1466 (CH2), 1384 (C–N). HRMS (m/
z): 290.0715 (M + Na), calculated: 290.0787.
2-(2-(1H-indol-3-yl)ethyl) isoindolin-1,3-dione
(dioxoisoindoline F)
It was obtained as a light yellow solid, 80% yield; mp
182–184 °C (recrystallization solvent ethyl acetate); 1H
NMR (DMSO, 750 MHz) δ 10.83 (1H, s, NH-14), 7.85
(2H, dd, J = 5.5, 3.0 Hz, H-7,4), 7.81 (2H, dd, J = 5.2,
3.1 Hz, H-5,6), 7.55 (1H, d, J = 7.9 Hz, H-15), 7.32 (1H, d,
J = 8.1 Hz, H-18), 7.17 (1H, s, H-13), 7.06 (1H, t, J =
7.5 Hz, H-16), 6.97 (1H, t, J = 7.4 Hz, H-17), 3.85 (2H, t, J
= 7.5 Hz, H-10), 3.03 (2H, t, J = 7.7 Hz, H-11); 13C NMR
(DMSO, 189 MHz) δ 168.2 (C-1,3), 123.4 (C-4,7), 136.7
(C-20), 134.8 (C-5,6), 132.0 (C-8,9), 127.5 (C-19), 123.4
(C-13), 121.4 (C-16), 118.8 (C-17), 118.4 (C-18), 111.9
(C-12), 111.0 (C-15), 38.7 (C-10), 24.3 (C-11); IR (ATR,
cm−1) ύ: 3374 (N–H) 3031 (C–H, aromatic), 2986 (C-H,
aliphatic), 1703 (C=O), 1611 (C=C), 1466 (CH2), 1384
(C–N). HRMS (m/z): 313.0869 (M + Na), calculated:
313.0947.
(S)-(2-(1-phenyl)ethyl)isoindolin-1,3-dione
(dioxoisoindoline C)
It was obtained as a yellow oil, 89% yield; 1H NMR
(CDCl3, 300 MHz) δ (PPM) 7.80 (2H, dd, J = 5.6, 2.9 Hz,
H-4,7), 7.68 (2H, dd, J = 5.6, 2.9 Hz, H-5,6), 7.52–7.50
(2H, m, H-14,16), 7.49–7.48 (2H, m, H-13,17), 7.35–7.22
(1H, m, H-15), 5.56 (1H, q, J = 7.3 Hz, H-10), 1.93 (3H, d,
J = 7.3 Hz, H-11); 13C NMR (CDCl3,75 MHz) δ 168.1 (C-
1,3), 140.2 (C-12), 133.8 (C-5,6), 131.9 (C-8,9), 128.4 (C-
15), 127.6 (C-14,16) 127.4 (C-13,17), 123.1 (C-4,7), 49.5
(C-10), 17.5 (C-11). IR (ATR, cm−1) ύ: 3063 (C–H, aro-
matic), 2935 (C–H, aliphatic), 1703 (C=O), 1600 (C=C),
1427 (CH2), 1425 (CH3), 1384 (C–N). HRMS (m/z):
274.0774 (M + Na), calculated: 274.0838.
Three new dioxoisoindolines (dioxoisoindoline D,
dioxoisoindoline E, and dioxoisoindoline F) were synthe-
sized with the present green solventless methodology and a
complete chemical characterization was made for each.
(R)-(2-(1-phenyl)ethyl)isoindolin-1,3-dione
(dioxoisoindoline D)
It was obtained as a white solid, 85% yield; mp 65–68 °C
1
(recrystallization solvent ethyl acetate); H NMR (DMSO,
Anticholinesterase activity assay
750 MHz) δ 7.86 (2H, dd, J = 3.3, 1.9 Hz, H-4,7), 7.83 (2H,
dd, J = 3.2, 1.9 Hz, H-5,6), 7.38 (2H, d, J = 7.6 Hz, H-
13,17), 7.33 (2H, t, J = 7.7 Hz, H-14,16), 7.25 (1H, t,
J = 7.3 Hz, H-15), 5.46 (1H, q, J = 7.3 Hz, H-10), 1.83 (3H,
d, J = 7.3 Hz, H-11); 13C NMR (DMSO, 189 MHz) δ 168.1
(C-1,3), 141.0 (C-12), 135.0 (C-5,6), 131.8 (C-8,9), 128.9
(C-15), 127.7 (C-14,16), 127.0 (C-13,17), 123.5 (C-4,7),
49.1 (C-10), 17.9 (C-11). IR (ATR, cm−1) ύ: 3031 (C–H,
aromatic), 2989 (C–H, aliphatic), 1704 (C=O), 1613
Previously, our group reported good inhibitory activity of
dioxoisoindoline A and dioxoisoindoline B on AChE
(Andrade-Jorge et al. 2018a, 2018b), but assays were not
conducted at that time on BuChE. In the current con-
tribution, six dioxoisoindolines derived from arylamines
(dioxoisoindolines A–F) were evaluated for inhibition of
both AChE (Fig. 25s) and BuChE (Fig. 26s). Analysis of
the type of inhibition was carried out with
a

Medicinal Chemistry Research
Lineweaver–Burk plot. Dioxoisoindolines A, C, and D
were found to be uncompetitive for AChE, while diox-
oisoindolines B, E, and F proved to be competitive for the
same enzyme (Table 2). For BuChE, the inhibitory
activity produced by Dioxoisoindolines B–F was com-
petitive and that by dioxoisoindoline A uncompetitive
(Table 3).
Interestingly, dioxoisoindolines E and F showed the best
inhibition of AChE, with a 1.5- and 2.1-fold selectivity for
AChE versus BuChE, respectively. For BuChE, dioxoi-
soindolines C and D gave rise to the best inhibition, with a
3.5- and 5-fold selectivity (BuChE versus AChE), respec-
tively. The latter two molecules are likely more relevant,
because the ratio of these two cholinesterases changes with
the onset of AD. As Savelev et al. observed, the level of
AChE decreases up to 15%, while that of BuChE increases
up to 90%. Therefore, a higher affinity for BuChE than
AChE is a desirable characteristic for an AD medication, as
evidenced by a greater improvement in the cognitive per-
formance of patients (Savelev et al. 2004). The affinity of
the rest of the molecules currently tested was very similar
for both enzymes.
Table 2 Inhibition constant, type
Ligand
Ki (μM)
Interval 95%
Type of inhibition
ΔG (kcal/mol)
of inhibition and Gibbs free
energy obtained for the
interaction of the
dioxoisoindolines and two
references drugs with
Dioxoisoindoline A
Dioxoisoindoline B
Dioxoisoindoline C
Dioxoisoindoline D
Dioxoisoindoline E
Dioxoisoindoline F
Galantamine
504.4
632.3
708.3
531.2
232.6
193.9
436.6–572.2
512–702
Uncompetitive
Competitive
Uncompetitive
Uncompetitive
Competitive
Competitive
Competitive
Competitive
−4.67
−4.53
−4.46
−4.62
−5.15
−5.24
−9.21
−6.56
623.6–729.9
558.4–700.8
202.5–262.8
168.7–219.0
0.270–0.362
20.19–24.93
Electrophorus electricus AChE
0.316
Neostigmine
22.56
Table 3 Inhibition constant, type
of inhibition and Gibbs free
energy obtained for the
interaction of dioxoisoindolines
and two references drugs with
human BuChE
Ligand
Ki (μM)
Interval 95%
Type of inhibition
ΔG (kcal/mol)
Dioxoisoindoline A
Dioxoisoindoline B
Dioxoisoindoline C
DioxoisoindolineD
Dioxoisoindoline E
Dioxoisoindoline F
Galantamine
499.7
596.4
200.5
106.1
501.5
430.2
7.29
413.7–585.7
496.9–695.9
174.4–226.6
76.7–135.7
412–590.1
347.5–512.9
6.50– 8.06
3.28–4.19
Uncompetitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
−4.68
−4.57
−5.24
−5.63
−4.68
−4.77
−7.28
−7.69
Neostigmine
3.74
Table 4 Gibbs free energy (ΔG), the dissociation constant (Kd), and –log10 dissociation constant (pKd) for the interaction of the test and references
molecules with the enzymes
AChE (Electrophorus electricus)
AChE (Homo sapiens)
BuChE (Homo sapiens)
ID
ΔG
(kcal/mol)
Kd (µM)
pKd
ΔG
(kcal/mol)
Kd (µM)
pKd
ΔG
(kcal/mol)
Kd (µM)
pKd
Dioxoisoindoline A
Dioxoisoindoline B
Dioxoisoindoline C
Dioxoisoindoline D
Dioxoisoindoline E
Dioxoisoindoline F
Acetylcholine
–
–
0.283
–
–
6.5
–
−7.19
−5.91
−5.83
−5.65
−6.95
−5.61
−4.56
−5.65
−6.73
−6.74
5.35
46.77
52.95
72.18
8.11
5.3
4.3
4.3
4.1
5.1
4.1
3.3
4.1
4.8
4.9
–
–
4.68
–
−8.93
–
−7.27
−6.12
−6.41
−7.37
−8.22
−4.87
−5.74
−8.7
5.3
4.5
4.7
5.4
6.0
3.6
4.2
6.3
4.6
32.71
20.11
3.93
–
–
–
−8.27
−9.06
−4.52
−5.71
−6.58
−6.32
0.867
0.229
462.23
69.17
17.32
22.18
6.1
6.6
3.3
4.1
4.8
4.6
77.44
454.93
72.7
0.94
268.04
62.14
0.42
Neostigmine
Galantamine
14.2
Rivastigmine
11.49
−6.28
24.88

Medicinal Chemistry Research
Table 5 Amino acid residues and type of interactions involved in the approach of the ligands (phthalamides and reference molecules) to the
enzymes
Ligand
Hydrophobic
π–π interaction
H-bond
π-cation π-anion
BuChE human Dioxoisoindoline A
–
–
–
–
–
–
Dioxoisoindoline B Val288,Trp82,
His438
Trp23, Phe329
Ser198
Glu197
Dioxoisoindoline C
–
Trp82
Gly116
Gly116
Gly116
His438
Ser198
–
–
–
–
–
Glu197
Glu197
Glu197
Glu197
Dioxoisoindoline D Pro84,Tyr114
Dioxoisoindoline E Leu286
Dioxoisoindoline F Gly117
Trp82
Trp82,Trp231,His438
Trp82,Trp231,His438
Trp231
Butyrylcholine
Neostigmine
Galantamine
–
Glu197
(electrostatic)
Glu197
Trp82
Trp82,His438
Tyr128
Tyr128
–
–
Glu197
(electrostatic)
Glu197,
–
Gly116, Thr120
Rivastigmine
AChE Human Dioxoisoindoline A
Dioxoisoindoline B
Trp82
His438
Thr120
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
His447,Trp86,Trp236
–
Glu202
–
Dioxoisoindoline C
–
–
Dioxoisoindoline D
–
–
–
Dioxoisoindoline E Ala204
Trp86.Tyr337,His447
Ser203
Glu202
Glu202
Dioxoisoindoline F
–
Trp86,Trp236, Tyr337,
His447
–
Acetylcholine
His447
Tyr337
Ser203
Tyr337 Glu202
(electrostatic)
Glu202
Neostigmine
Glu202
Trp86,Tyr337
Trp86
Gly122
–
–
–
–
–
–
–
–
–
Galantamine
Tyr337
Glu202
–
Rivastigmine
Ala204,Tyr337
Trp86,Trp236
–
–
Glu202
AChE of Ee
Dioxoisoindoline A
Dioxoisoindoline B
Dioxoisoindoline C
Dioxoisoindoline D
Dioxoisoindoline E
Dioxoisoindoline F
Acetylcholine
–
–
–
–
–
–
–
–
–
Trp86,Tyr337
–
Ser203, Tyr337
–
–
–
–
–
–
Trp86, Tyr337
Trp86,Tyr124
His447
Ser203,Phe338
Asp74
–
Tyr124
Ser203,Trp86
Tyr337 Glu202
(electrostatic)
Tyr337 Glu202
(electrostatic)
Neostigmine
Trp86
Trp86
Ser125
Galantamine
Rivastigmine
Tyr124
Ile451
Trp286,Tyr341
Trp86
Ser203,Arg296
Ser203
–
–
–
–
Another important aspect is the apparent better inhi-
bition of both enzymes generated by the R-configuration
of the molecules, although no significant statistical dif-
ferences were found; another aspect is that dioxoi-
soindolines C and D have different behavior in both
enzyme, this is interesting while for EeAChE the inhibi-
tion is uncompetitive for HuBuChE is competitive, this
could be related to the molecular structure because of the
disposition of the phenylethyl moiety in molecules C and
D is different than molecule A, moreover, we need to take
into account that both compounds possess chiral carbons
that may make interact with the enzymes in a different
way. More research is needed on the inhibitory potential
of the R- and S-configuration. According to the data from
the in vitro assays, all six dioxoisoindolines turned out to
be good inhibitors of AChE and BuChE, having good
affinity and selectivity in some cases. Hence, they can be
considered as dual inhibitors. Finally, the reference
compounds (galantamine and neostigmine) were assayed
to validate the experiments, finding their Ki values and

Medicinal Chemistry Research
type of inhibition to be in agreement with previous
reports.
29s). The interactions of most of the six dioxoisoindolines
with amino acid residues and their binding modes in the
catalytic site were similar to those found for the reference
drugs. Docking simulations were run on reference mole-
cules (acetylcholine, butyrylcholine, neostigmine, rivas-
tigmine, and galantamine) to reproduce the binding mode
and validate the results with the six dioxoisoindolines (Figs.
27s, 28s, and 30s).
On the other hand, the analysis of the clustering of the
docked ligands shown that many orientations are focused on
the catalytic site of the enzymes (AChE and BuChE), dis-
playing an around 68–100% of all conformations in the cata-
lytic site. Moreover, the binding mode of the different ligands
is very similar between them, as is shown in Figs. 2e and 3e.
Molecular docking and theoretical calculations
To provide insights into the in vitro results and to extrapolate
the findings to physiological conditions since in vitro experi-
ments required pH = 8, a molecular docking study was carried
out with the dioxoisoindolines that showed competitive beha-
vior. As is well known, it is not possible to use molecular
docking in cases of uncompetitive inhibition. Apart from fur-
nishing essential information on the binding mode, the mole-
cular docking data on the energy state of the ligand allowed for
an analysis of its correlation to the inhibition of the enzyme.
Accordingly, the values were determined for Gibbs free energy
as well as the dissociation constant of the ligand–enzyme
interactions in physiological conditions (pH = 7.4), compara-
tively with the in vitro results Gibbs free energy in the in silico
results is higher, this may indicate that under physiological
conditions the compounds may have greater affinity and effi-
cacy. Homo sapiens AChE was presently included to extra-
polate the findings to patients (Table 4), interestingly, Gibbs
free energy was higher with Homo sapiens than EeAChE.
Finally, the mean affinity for H-BuChE is very similar to that
obtained for human AChE, so it is expected to have a com-
parable activity with the human enzyme in physiological
conditions.
Conclusions
A series of phthalimides were synthesized in good yields by
using a green chemistry approach. A fast and inexpensive
method for evaluating the inhibition of ligands on AChE
activity was adapted for assessing BuChE activity. The test
molecules displayed good dual inhibition of AChE and
BuChE. Two compounds (dioxoisoindolines C and D) showed
up to fivefold selectivity for BuChE over AChE. Considering
the increased presence of the former enzyme after the onset of
the disease, these two compounds could be advantageous as a
medication for AD. Docking results clearly demonstrate that
the dioxoisoindolines interact preferentially with the catalytic
site of the enzymes, which coincides with the data from the
in vitro assays. In addition, carbonyl carbons play an important
role in the recognition of the ligands by the enzymes. Some
dioxoisoindolines presently tested hold promise as lead com-
pounds for designing new dual cholinesterase inhibitors for AD
The interactions of the test and reference compounds
with the main residues in the catalytic site of AChE and
BuChE were closely observed (Table 5). Many of the
interactions are due to the carbonyl carbon in the structure
of the dioxoisoindolines, as described in other reports
(Mohammadi-Farani et al. 2017). Moreover, hydrophobic
interactions involve the aromatic groups (Figs. 2, 3, and
Fig. 2 Binding mode of two representative compounds with Homo
sapiens BuChE (PDB ID:4P0I), according to the docking simulations.
a, b Lowest energy conformation of dioxoisoindoline C and its cor-
responding amino acid residues. c, d Lowest energy conformation of
dioxoisoindoline D and its amino acid residues. e Binding mode of the
two dioxoisoindolines, observing a great similarity in the arrangement
of the molecules

Medicinal Chemistry Research
Fig. 3 Binding mode of two representative compounds with Homo
sapiens AChE (PDB ID:4PQE), according to the docking simulations.
a, b Lowest energy conformation of dioxoisoindoline A and its cor-
responding amino acid residues. c, d Lowest energy conformation of
dioxoisoindoline E and its amino acid residues. e Binding mode of the
two dioxoisoindolines, observing a great similarity in the arrangement
of the molecules
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