Acetylcholinesterase inhibition by products generated in situ from the transformation of N-arylisomaleimides

Article abstract of DOI:10.1007/s00044-017-2122-4

When N-arylisomaleimides were transformed under enzymatic reaction conditions, the transformation reaction proved to be influenced by electronic effects. This was demonstrated qualitatively by ¹H NMR spectroscopy and quantitatively by monitoring the kinetic of isomerization of N-phenylisomaleimide to N-phenylmaleimide. Subsequently, the first pseudo-order and activation energy (E_a) of the process were determined. The compounds showed in situ influence on AChE inhibition. The derivatives with electron-withdrawing groups exhibited a better effect than those having electron-donating groups. The in silico experiments show that the ligands evaluated established interactions with the CAS site. This suggests that these compounds could be useful for generating better reversible and competitive inhibitors of AChE.

Full text of DOI:10.1007/s00044-017-2122-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2122-4
ORIGINAL RESEARCH
Acetylcholinesterase inhibition by products generated in situ from
the transformation of N-arylisomaleimides
Juan A. Guevara1 José G. Trujillo2 Delia Quintana3 Hugo A. Jiménez4
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Mónica G. Arellano5 José R. Bahena2 Feliciano Tamay2 Fabiola J. Ciprés6
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Received: 12 July 2017 / Accepted: 23 November 2017
© Springer Science+Business Media, LLC, part of Springer Nature 2017
Abstract When N-arylisomaleimides were transformed
under enzymatic reaction conditions, the transformation
reaction proved to be influenced by electronic effects. This
was demonstrated qualitatively by H NMR spectroscopy
and quantitatively by monitoring the kinetic of isomeriza-
Subsequently, the first pseudo-order and activation energy
(Ea) of the process were determined. The compounds
showed in situ influence on AChE inhibition. The deriva-
tives with electron-withdrawing groups exhibited a better
effect than those having electron-donating groups. The in
silico experiments show that the ligands evaluated estab-
lished interactions with the CAS site. This suggests that
these compounds could be useful for generating better
reversible and competitive inhibitors of AChE.
1
tion of N-phenylisomaleimide to N-phenylmaleimide.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2122-4) contains supplementary
material, which is available to authorized users.
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Keywords Alzheimer’s disease Acetylcholinesterase
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inhibitors Isomerization kinetics N-arylimides N-
* Juan A. Guevara
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arylisomaleimides Catalysis
jguevaras@ipn.mx
* José G. Trujillo
jtrujillo@ipn.mx
1
Academy of Pharmacology, Escuela Superior de Medicina,
Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón,
11340 México, D.F., Mexico
Introduction
2
Department of Biochemistry and Sección de Estudios de Posgrado
The acetylcholinesterase (AChE) enzyme catalyzes the
acetylcholine (ACh) hydrolysis reaction in acetate and
choline that terminates cholinergic neurotransmission.
This is the most efficient enzyme known (EC: 1.6 × 108
M−1 s−1) (Fersht 1999; Taylor 1991) and its inhibition
produces an important change in cholinoceptor stimulation.
The inhibition of AChE is a strategy that has been employed
to treat several diseases, such as smooth muscle atony
(Zeinali et al. 2009; Durai 2009; Breine et al. 1958), glau-
coma (Kolko 2015), myasthenia gravis (Mehndiratta et al.
2014), neuromuscular blockers-induced paralysis (Fortier
et al. 2015; Schreiber 2014), and Alzheimer’s disease (AD)
(Desmidt et al. 2016; Graipaspong et al. 2016; Cummings
et al. 2016).
e Investigación, Escuela Superior de Medicina, Instituto
Politécnico Nacional, Plan de San Luis y Díaz Mirón, 11340
México, D.F., Mexico
3
Laboratory of Organic Chemistry, CICATA Unidad Legaria,
Instituto Politécnico Nacional, Legaria No. 694, 11500 México,
D.F., Mexico
4
Department of Organic Chemistry, ENCB, Instituto Politécnico
Nacional, Prolongación Manuel Carpio y Plan de Ayala, 11350
México, D.F., Mexico
5
Laboratory of Chronic-Degenerative Diseases and Sección de
Estudios de Posgrado e Investigación, Escuela Superior de
Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz
Mirón, 11340 México, D.F., Mexico
6
Academy of Physiology and Sección de Estudios de Posgrado e
Investigación, Escuela Superior de Medicina, Instituto Politécnico
Nacional, Plan de San Luis y Díaz Mirón, 11340 México, D.F.,
Mexico
AD is a neurodegenerative disease that involves a dete-
rioration of cognitive abilities and an irreversible and

Med Chem Res
gradual loss of memory. These symptoms are accompanied
by atrophy of the cerebral cortex as well as a loss of cortical
and subcortical neurons, including cholinergic neurons
(Standaert and Roberson 2011). The symptoms of dementia
associated with AD are closely related to those of a choli-
nergic deficit, which implies the accumulation of the neu-
rotoxic αβ-amyloid peptide (Sadigh-Eteghad et al. 2015;
Inestrosa et al. 2005; Pakaski and Kasa 2003). AChE
inhibitors have been used to delay the advance of the dis-
ease and improve cognitive functions (Davies and Maloney
1976; Perry 1986).
The AChE inhibitors currently approved for treating AD
are donepezil, rivastigmine, galantamine, and tacrine. These
drugs effectively increase the half-life of ACh in the
synaptic space (Standaert and Roberson 2011; Davies and
Maloney 1976; Perry 1986; Kelly 1999). However, it is
important to seek new drugs that are more selective for the
enzyme and less toxic to the patient. The enzyme reportedly
has different sites of recognition for ligands, including the
catalyist active site (CAS), aromatic patch (AP), anionic site
(AS), acyl site, oxyanionic hole, and peripheral anionic site
(PAS). The latter site is involved in the rate of αβ-amyloid
aggregation (Gupta and Mohan 2014). Thus, it is important
to consider the physicochemical properties of each sites to
increase the potency and selectivity of ligands on the
enzyme (Kiametis et al. 2017; Shakil et al. 2012).
Accordingly, our research team has designed several
compounds showing competitive inhibition of AChE,
including N-arylmaleamic acids (Trujillo-Ferrara et al.
2003a, b; Correa-Basurto et al. 2007), N-arylmaleimides
(Trujillo-Ferrara et al. 2003a, b), and N-arylisomaleimides
(Correa-Basurto et al. 2006). Of these compounds, N-ary-
lisomaleimides are the ones that exhibit important activity,
although the isomaleimides are less stable molecules than
maleimides and maleamic acids derivatives (Kirino et al.
1985) because of being favored thermodynamically (Ivanov
and Constantinescu 2005; Constantinescu and Ivanov
2005).
sodium and benzophenone under nitrogen atmosphere.
Aniline, ethyl acetate, acetone, hexane, and DCM were
purified by fractional distillation. Analytical thin-layer
chromatography was carried out on Merck-DC-F254 alu-
minum plates, using short-wave UV light for visualization.
The melting points were determined in a Melt-Temp II
apparatus and Electrothermal 9300. The IR spectra were
obtained in KBr disks, in a Midac Corporation M Series
spectrophotometer. The IT absorption frequencies are
reported in cm−1. The UV–Vis spectra, chemical kinetics,
and enzymatic kinetics were evaluated on a UV–Vis
Beackman Coulter DU 650 spectrophotometer, with phos-
phate buffer and CH3OH or CH2Cl2 as solvents. The 1H and
13C NMR spectra were recorded in JEOL FX-270Q, Varian
VNMRS-300, and VNMRS-500 spectrometers, at 270
(67.89 MHz for 13C), 300 (75 MHz for 13C), and 500 MHz
(125.787 MHz for 13C), respectively. The samples were
dissolved in CDCl3 or DMSO-d6, using TMS as internal
reference.
General method for the preparation of the N-arylmaleamic
acids 3a–c
Each of the anilines 1a–1c (~5.0 g) and an approximately
1.1 equivalent of maleic anhydride 2 were dissolved sepa-
rately in 100 mL round-bottom flasks fitted with rubber
septa, adding about 20 mL of anhydrous THF to each
(Cotter et al. 1961; Oishi et al. 1989; Barba et al. 1999; Fruk
and Graham 2003). The flask with aniline, containing a
magnetic stirrer, was introduced into an ice bath at 0 °C.
The solution of maleic anhydride was slowly added through
a cannula and the mixture was stirred for 16 h at RT. The
product was filtered and washed twice with 20 mL of n-
hexane and then characterized by spectroscopy. No further
purification was carried out.
This study evaluates the stability of the para-mono-
substituted N-phenylisomaleimides under enzymatic reac-
tion conditions, as well as the inhibitory activity of the
products of their transformation, observing the influence of
the electronic effects for different substituents on AChE.
(2Z)-3-(N-phenylcabamoyl)-2-propenoic acid (3a) From
the general method, with 5.0 mL of 1a (0.0548 mol) and
6.226 g of 2 (0.06028 mol), the reaction produced a yel-
lowish powder in 98.21% yield (10.29 g, 0.05382 mol); m.
p.: 204–205 °C; Rf = 0.67 (MeOH/AcOEt 1:1); IR (KBr):
νmax = 3274, 2680, 2613, 2240, 2071, 1963, 1701, 1632,
1586, 1544, 1537, 1488 (cm−1); 1H NMR (500 MHz,
3
Material and methods
Chemistry
DMSO-d6): δ = 10.44 (1H, s, NH), 7.65 (2H, d, J = 7.5
3
Hz, H-2′, H-6′), 7.32 (2H, t, J = 8.0 Hz, H-3′, H-5′), 7.08
3
3
(1H, t, J = 7.0 Hz, H-4′), 6.47 (1H, d, J = 12.0 Hz, H-2),
6.29 (1H, d, 3J = 12.0 Hz, H-3) ppm; 13C NMR (125 MHz;
DMSO-d6): δ = 167.63 (CO, C-1), 164.07 (CO, C-4),
138.95 (CH, C-2), 132.43 (CH, C-3), 131.13 (C, C-1′),
129.52 (CH, C-2′, C-6′), 124.801 (CH, C-4′), 120.370 (CH,
C-3′, C-5′) ppm.
All reagents and solvents, except aniline, AcOEt, dichlor-
omethane (DCM), acetone, hexane, and THF, were pur-
chased and used without additional purification. THF was
distilled from a deep-blue solution of the solvent containing

Med Chem Res
(2Z)-3-[N-(4-methoxyphenyl)-cabamoyl]-2-propenoic acid
(3b) From the general method, with 5.0 g of 1b (0.0406
mol) and 4.378 g of 2 (0.04465 mol), the reaction produced
a yellow powder in 98.79% yield (8.87 g, 0.04010 mol); m.
p.: 189–190 °C; IR (KBr): νmax = 3258, 3071, 2921, 2500,
1701, 1624, 1556, 1503, 1399, 1488, 1240 (cm−1); 1H
NMR (270 MHz, DMSO-d6): δ = 10.45 (1H, s, NH), 7.55
(2H, d, 3J = 8.91 Hz, H-3′, H-5′), 6.89 (2H, d, 3J = 8.91 Hz,
(KBr): νmax = 3294, 3090, 3031, 2544, 2613, 1920, 1863,
1804, 1707, 1637, 1561, 1508, 1454, 1334 (cm−1); 1H
NMR (270 MHz, DMSO-d6): δ = 10.85 (1H, s, NH), 8.19
(2H, d, 3J = 9.15 Hz, H-3′, H-5′), 7.84 (2H, d, 3J = 9.15 Hz,
3
H-2′, H-6′), 6.51 (1H, d, J = 12.00 Hz, H-2), 6.34 (1H, d,
3J = 11.88 Hz, H-3) ppm; 13C NMR (67.89 MHz; DMSO-
d6): δ = 167.41 (CO, C-1), 164.64 (CO, C-2), 145.42 (C, C-
4′), 142.98 (CH, C-2), 132.26 (CH, C-3), 130.77 (C, C-1′),
125.48 (CH, C-3′, C-5′), 119.60 (CH, C-2′, C-6′) ppm.
3
H-2′, H-6′), 6.48 (1H, d, J = 12.12 Hz, H-2), 6.30 (1H, d,
3J = 12.12 Hz, H-3), 3.70 (3H, s, O-CH3) ppm; 13C NMR
(67.89 MHz; DMSO-d6): δ = 167.13 (CO, C-1), 163.54
(CO, C-4), 156.51 (C, C-4′), 132.26 (CH, C-2), 131.82
(CH, C-3), 131.66 (C, C-1′), 121.91 (CH, C-3′, C-5′),
114.51 (CH, C-2′, C-6′), 55.68 (CH3, O-CH3) ppm.
General method for the preparation of the N-
arylisomaleimides 4a–c
Each of the N-arylmaleamic acids 3a–3c (~3.0 g) and an
approximately 1.1 equivalent of dicyclohexylcabordiimide
(DCC) were dissolved separately in 50 mL round-bottom
flasks fitted with rubber septa, adding about 20 mL of
anhydrous DCM to each (Scheme 1). The flask with N-
arylmaleamic acid, containing a magnetic stirrer, was
(2Z)-3-[N-(4-nitrophenyl)-cabamoyl]-2-propenoic
acid
(3c) From the general method, with 5.0 g of 1c (0.0362
mol) and 3.905 g of 2 (0.03982 mol), the reaction produced
a light yellow powder in 95.52% yield (8.16 g, 0.03457
mol); m.p.: 194–195 °C; Rf = 0.67 (MeOH/AcOEt 1:1); IR
Scheme 1 Synthesis pathway of
N-phenylmaleamic acids (3a–c)
and N-phenylisomaleimides
(4a–c)
R
R
O
O
+
O
NH
O
THF / 16 h / RT
OH
NH2
O
2
3a-3c
1a-1c
R: a = -H
R: b = -OCH3
R: c = -NO2
DCC / CH2Cl2 /
18 h / RT
R
N
O
O
4a-4c

Med Chem Res
introduced into an ice bath at 0 °C. The solution of DCC
was slowly added through a cannula for 30 min and the
mixture was stirred for 18 h at RT. The subproduct dicy-
clohexylurea was eliminated by filtration and washed with
DCM. Subsequently, the dissolution was evaporated under
vacuum in the rotavapor, and then the product was dis-
solved in DCM and precipitated by using cold n-hexane.
The solid was filtered and washed twice with 20 mL of n-
hexane before being characterized by spectroscopy. No
further purification was carried out.
General method for the preparation of the N-
arylmaleimides 5a
The N-arylmaleamic acid 3a (~1.0 g) was deposited in a 20
mL ACE pressure tube in 10 mL of toluene (Guevara-Sal-
azar et al. 2011). The tube was closed, introduced into a
sand bath at 160 °C, and kept under constant stirring for 96
h. Afterward, the product was precipitated by using cold n-
hexane, filtered, washed twice with 20 mL of n-hexane at
RT, purified, and then characterized by spectroscopy.
5-(N-phenyl)-imino-5H-furan-2-one (4a) From the general
method, with 3.0 g of 3a (0.0157 mol) and 3.553 g of DCC
(0.01727 mol), the reaction produced a light yellow powder
in 97.69% yield (2.656 g, 0.01533 mol); m.p.: 60–62 °C; Rf
= 0.65 (n-Hexane/AcOEt 1:1); UV–Vis (MeOH): λmax (ε)
= 334.6 1.53 nm (4141.37 M−1 cm−1); IR (KBr): νmax =
3085, 1964, 1899, 1784, 1669, 1567, 1482, 1446 (cm−1);
1H NMR (300 MHz, CDCl3): δ = 7.395 (5H, m, H-3, H-2′,
N-phenyl-pyrrole-2,5-dione (5a) From the general
method, with 1.0 g of 3a (0.00523 mol), the reaction pro-
duced a light yellow powder in 18.32% yield (0.1658 g,
0.00096 mol); m.p.: 84–86 °C; Rf = 0.65 (n-Hexane/AcOEt
1:1); UV–Vis (MeOH): λmax (ε) = 305.0 + 0.22 nm
(4141.37 M−1 cm−1); IR (KBr): νmax = 3097, 2987, 1775,
1714, 15978, 1502, 1381, 1189, 894 (cm−1); 1H NMR (270
MHz, CDCl3): δ = 7.54–7.31 (5H, m, H-2′, H-3′, H-4′, H-
5′, H-6′), 6.80 (2H, s, H-2, H-3) ppm; 13C NMR (67.89
MHz; CDCl3): δ = 167.26 (CO, C-1, C-4), 134.200 (CH, C-
3′, C-5′), 131.27 (C, C-1′), 129.95 (CH, C-2, C-3), 128.25
(CH, C-4′), 126.03 (CH, C-2′, C-6′) ppm.
3
H-3′, H-5′, H-6′), 7.21–7.28 (1H, m, H-4′), 6.68 (1H, d, J
= 5.4 Hz, H-2) ppm; 13C NMR (75 MHz; CDCl3): δ =
167.46 (CO, C-1), 150.37 (C = N, C-4), 143.70 (C, C-1′),
143.47 (CH, C-3), 129.35 (CH, C-2′, C-6′), 128.21 (CH, C-
2), 127.63 (CH, C-4′), 125.36 (CH, C-3′, C-5′) ppm.
5-[N-(4-methoxyphenyl)-imino]-5H-furan-2-one
(4b)
From the general method, with 3.0 g of 3b (0.0135 mol) and
3.069 g of DCC (0.01485 mol), the reaction produced a
yellow powder in 99.59% yield (2.7318 g, 0.01344 mol), m.
p.: 72–73 °C; Rf = 0.54 (n-Hexane/AcOEt 1:1); IR (KBr):
νmax = 3061, 2928, 1788, 1715, 1591, 1563, 1502, 1454,
1262 (cm−1); 1H NMR (270 MHz, CDCl3): δ = 7.47 (2H, d,
Enzymatic kinetic assay
The inhibition of AChE induced by the compounds was
herein evaluated in vitro with the modified Bonting and
Featherstone colorimetric method (Bonting and Feath-
erstone 1956; Ellman et al. 1961). AChE (EC 3.1.1.7 of
Electrophorus electricus Sigma-Aldrich) and acetylcholine
iodide (ACh+I−, Merck) were used as received. The opti-
mum concentration of AChE (0.1 U/mL) was diluted in 0.1
M of phosphate buffer (pH = 8.0). One unit of this enzyme
hydrolyzes 1.0 mmol of ACh per min at 37 °C (pH = 8.0).
ACh iodide was used as a substrate at several concentra-
tions (0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, 4.8, and 6.4 mM) that
are slightly above and below of the Km value of AChE. The
stock solutions were prepared at 0.1 M of the ligands, and
then several concentrations were prepared (from 1 × 10−7
to 1 × 10−3 M). Neostigmine (neostigmine bromide, Sigma-
Aldrich) was tested as the positive control at several con-
centrations (from 1 × 10−9 to 1 × 10−5 M), in phosphate
buffer (0.1 M, pH = 8.0).
3
3J = 8.91 Hz, H-3′, H-5′), 7.29 (1H, d, J = 5.44 Hz, H-3),
3
3
6.84 (2H, d, J = 8.66 Hz, H-2′, H-6′), 6.54 (1H, d, J =
5.44 Hz, H-2), 3.75 (3H, s, O-CH3) ppm; 13C NMR (67.89
MHz; CDCl3): δ = 167.70 (CO, C-1), 159.37 (C, C-4′),
148.52 (C = N, C-4), 143.52 (CH, C-3), 136.54 (C, C-1′),
128.42 (CH, C-3′, C-5′), 126.65 (CH, C-2), 114.32 (CH, C-
2′, C-6′), 55.50 (O-CH3) ppm.
5-[N-(4-nitrophenyl)-imino]-5H-furan-2-one
(4c) From
the general method, with 3.0 g of 3c (0.0127 mol) and
2.882 g of DCC (0.01397 mol), the reaction produced a
yellow powder in 28.86% yield (0.80 g, 0.00367 mol); m.p.:
107–108 °C; Rf = 0.47 (n-Hexane/AcOEt 1:1); IR (KBr):
ν
max = 3109, 3054, 1801, 1686, 1598, 1522, 1392, 1346
(cm−1); 1H NMR (270 MHz, CDCl3): δ = 8.24 (2H, d, 3J =
The enzymatic inhibition measurements were carried out
with and without ligands at the differents substrate con-
centrations mentioned. At the end of each incubation (25
min at 37 °C), the reaction was stopped by adding 40 μL of
alkaline hydroxylamine (0.048 mmol). Subsequently, 0.08
mL aliquots were mixed with 1.5 mL of 0.05 M of FeCl3
solution dissolved in 0.5 N HCl, and the solution was
3
8.91 Hz, H-3′, H-5′), 7.43 (1H, d, J = 5.69 Hz, H-3), 7.36
(2H, d, 3J = 8.91 Hz, H-2′, H-6′), 6.78 (1H, d, 3J = 5.69 Hz,
H-2) ppm; 13C NMR (67.89 MHz; CDCl3): δ = 165.94
(CO, C-1), 152.44 (C=N, C-4), 145.01 (C, C-4′), 142.47
(CH, C-3), 134.54 (C, C-1′), 129.68 (CH, C-2), 124.25
(CH, C-3′, C-5′), 123.99 (CH, C-2′, C-6′) ppm.

Med Chem Res
centrifuged for 3 min at 3000 rpm at 4 °C. Finally, the
absorption measurements were made at λmax = 500 nm.
The Km and Vmax values were determined with the
Lineweaver–Burk method because the data had
Michaelis–Menten behavior. The Ki values for all com-
pounds and neostigmine were obtained by Schild regression
analysis.
regression analysis). Statistical significance was considered
at p < 0.05. Each experiment was repeated 3–5 times.
Molecular docking studies
Protein structure and sequence alignment The protein
crystal structure of Electrophorus electricus AChE (PDB I.
D 1C2O) (Bourne et al. 1999) was obtained from the RCSB
Protein Data Bank (www.rcsb.org) (Berman et al. 2000).
The crystal obtained was visualized and optimized using
Discovery Studio Visualizer v 2016 (Dassault 2016). The
sequence for this protein was aligned with human AChE
(Uniprot entry P22303) (Apweiler et al. 2004) in order to
analyze the conservation rate among orthosteric and allos-
teric sites.
1
Qualitative analysis by H NMR spectroscopy of the
transformation from N-phenylisomaleimides to N-
phenylmaleimides
A 1-mL solution of each compound, 4a–4c, was prepared in
0.1 M acetone. To each solution was added 1.4 mL of
phosphate buffer at 0.1 M (pH = 8.0). After the mixture was
gently stirred for 2 min, the two phases (liquid and solid,
formed by 4b and 4c) were separated. Compound 4a
1
Ligand preparation and docking set up The synthesized
compounds as well as ACh and neostigmine were drawn
using Gaussview 5.0.9 (Frisch et al. 2009) software to
obtain a minimal energy conformation. A geometry opti-
mization calculation was then performed using Gaussian 09
(Frisch et al. 2009) through semi-empirical molecular
orbital calculations (PM3). The AutoDock tools 1.5.4 pro-
gram (Morris et al. 2009) was used to prepare the docking
simulations. The receptor was transformed into a PDBQT
format file containing Kollman’s partial charges. For the
ligands files, merged nonpolar hydrogen atoms, rotatable
bonds, torsions, and Gasteiger atomic partial charges were
assigned. The grid box size was set with the AutoGrid
program in the orthosteric site at 58, 50, and 60 (for the x, y,
and z-axis). The spacing between grid points was 0.375 Å.
The center of the grid was set to the coordinates 10.598,
67.943, and 63.006 (x, y, and z). AutoDock was run with the
Linux Fedora operating system and all the AutoDock
docking runs were performed 100 times. The Lamarckian
genetic algorithm (Morris et al. 1998) was used for the
conformational search with an initial population of 100
randomly placed individuals, and the maximum number of
energy evaluations was 1 × 107. The conformations
obtained with a RMSQ lower than 2 Å were clustered
together. After the simulations were completed, interaction
modes were investigated using AutoDock Tools and Dis-
covery Studio 4.5 software.
formed a single phase (liquid). The H NMR spectra were
acquired for all phases. The solids were filtered and dried
under vacuum, followed by dissolution in deuterated
chloroform (CDCl3). Meanwhile, the liquid fraction was
dried under vacuum and dissolved in deuterated dimethyl-
sulfoxide (DMSO-d6).
The kinetics of isomerization was monitored with 1H
NMR spectroscopy at 37 °C and at several different time
points: 0.17, 72.00, 144.00, and 168.00 h. This experiment
was carried out by using 40 mg of 4a in 1.0 mL of CDCl3
and 100 μL of phosphate buffer at 0.1 M (pH = 8.0).
Isomerization kinetics of 4a
Reaction order determination (n) In a ball flask were
added 49.0 mL of phosphate buffer solution at 0.1 M (pH =
8.0). The flask was placed in a water bath at 22 °C to
equilibrate. Subsequently, 1.0 mL of stock solution of 4a in
methanol was added to get the final concentrations of 0.1,
0.2, and 0.4 mM. Samples were then taken every 30 s for
10 min and the absorbance of 4a was measured at λmax
=
334.6 nm. The reaction-order evaluation was made by the
differential method.
Activation energy determination (Ea) The isomerization
reaction was carried out under the aforementioned condi-
tions at a constant concentration of 4a (C = 0.4 mM) and
variable temperatures 22, 30, 35, and 40 °C. Samples were
taken every minute for 15 min. The absorbance was mea-
sured for 5a at λmax = 305.0 nm. The Ea was estimated with
the Arrhenius equation.
Results and discussion
Synthesis
Statistical analysis All results of enzymatic kinetics and
isomerization kinetics were reported as the mean standard
error (SEM) and evaluated with the Student’s t-test (lineal
N-arylmaleamic acids 3a–3c and N-arylisomaleimides
4a–4b were synthesized by previously reported methods

Med Chem Res
(Cotter et al. 1961; Oishi et al. 1989; Barba et al. 1999; Fruk
and Graham 2003; Scheme 1), affording yields greater than
95.0 and 97.0%, respectively. N-arylmaleamic acids were
obtained by treating para-substituted anilines 1a–1c with
maleic anhydride 2 in THF, resulting in good yields. N-
arylisomaleimides were obtained by treating 3a–3c with
DCC in DCM, also giving good yields. Finally, N-phe-
nylmaleimide 5a was synthesized by treating 1a with 2 in a
pressure tube at 160 °C in toluene (Guevara-Salazar et al.
2011) with an 18.3% yield (Scheme 2). At this stage, all the
Km value of AChE was 0.6832 0.0017 mM, while the Ki
values for 4a, 4b, and 4c were 193.34 28.56, 65.85
0.26, and 0.0888 0.1114 μM, respectively. As can be
appreciated, compound 4c, which has an –NO2 group,
showed greater inhibition of the enzyme than 4a and 4b.
These results are moderate in comparison to neostigmine,
which has 1250 times more affinity than 4c (Table 1).
On the other hand, a correlation exists between the pKi
values of N-arylisomaleimides obtained with Hammett’s σH,
1
derivatives were fully characterized by H and 13C NMR
spectroscopy and IR spectrophotometry analysis.
Enzymatic kinetic assay
Judging by enzymatic kinetics, compounds 4a–4c exert
competitive inhibition (see the pattern in the
Lineweaver–Burk plots, Figs. 1–3). The slope increased as
did the concentration of each N-arylisomaleimide deriva-
tive, while the intercept did not substantially change. The
Fig. 2 Lineweaver–Burk plot for compound 4b clearly displaying
competitive inhibition. Statistical simple regression analysis: ♦[I] =
0.00 μM (n = 15), b = 2.1900 0.2697 (*p < 0.001), m = 1.4963
0.0833 (*p < 0.001) and r = 0.9818 (*p < 0.001); ■[I] = 10.0 μM (n
= 3), b = 2.7376 0.3889 (*p < 0.001), m = 1.6564 0.0417 (*p <
0.001) and r = 0.9671 (*p < 0.001); ▲[I] = 100.0 μM (n = 3), b =
1.8813 0.2201 (*p < 0.001), m = 2.3825 0.6632 (*p < 0.001) and
r = 0.9896 (*p < 0.001); ●[I] = 1000.0 μM (n = 3), b = −0.0122
0.0413 (p > 0.05), m = 3.6497 0.2056 (*p < 0.001) and r = 0.9952
(*p < 0.001). *p < 0.05 represents a statistically significant difference
at 95.0% confidence assessed with the Student’s t-test
Toluene
96 h / 160 °C
O
NH
N
O
O
OH
O
5a
3a
Scheme 2 Synthesis pathway of N-phenylmaleimide (5a)
Fig. 3 Lineweaver–Burk plot for compound 4c clearly evidencing
competitive inhibition. Statistical simple regression analysis: ♦[I] =
0.00 μM (n = 15), b = 2.1900 0.2697 (*p < 0.001), m = 1.4963
0.0833 (*p < 0.001) and r = 0.9818 (*p < 0.001); ■[I] = 0.1 μM (n =
5), b = 2.2506 0.0856 (*p < 0.001), m = 3.0404 0.6002 (*p <
0.001) and r = 0.9612 (*p < 0.001); ▲[I] = 1.0 μM (n = 5), b =
1.8556 0.3031 (*p < 0.001), m = 3.4832 0.0812 (*p < 0.001) and
r = 0.9667 (*p < 0.001); ●[I] = 1000.0 μM (n = 5), b = −1.1231
0.0645 (*p < 0.001), m = 5.8733 0.2253 (*p < 0.001) and r =
0.9805 (*p < 0.001). *p < 0.05 represents a statistically significant
difference at 95.0% confidence assessed with the Student’s t-test
Fig. 1 Lineweaver–Burk plot for compound 4a clearly depicting
competitive inhibition. Statistical simple regression analysis: ♦[I] =
0.00 μM (n = 15), b = 2.1900 0.2697 (*p < 0.001), m = 1.4963
0.0833 (*p < 0.001) and r = 0.9818 (*p < 0.001); ■[I] = 1.0 μM (n =
5), b = 1.9805 0.3002 (*p < 0.001), m = 1.7435 0.2457 (*p <
0.001) and r = 0.9735 (*p < 0.001); ▲[I] = 1000.0 μM (n = 5), b =
1.2740 0.1172 (*p < 0.001), m = 2.1534 0.2506 (*p < 0.001) and
r = 0.9768 (*p < 0.001). *p < 0.05 represents a statistically significant
difference at 95.0% confidence assessed with the Student’s t-test

Med Chem Res
including the N-(4-acetyl-phenylisomaleimide) derivative
(Correa-Basurto et al. 2006). This suggests that electronic
effects participate in the recognition of the active site by the
enzyme. Furthermore, it is the electron-withdrawing groups
that improved affinity (Fig. 4).
N-phenylmaleimide (5a) (Table 2), while 4b and 4c formed
two phases, a solid and a liquid. Whereas the solid phase
contained a mixture of N-arylisomaleimide and N-arylma-
leimide, the liquid phase had only N-arylmaleamic acid salts
(Table 2).
Because there was complete transformation with 4a, it
was chosen for monitoring isomerization kinetics. Thus, a
qualitative analysis was conducted by applying H NMR
1
Qualitative analysis H NMR spectroscopy of the
1
transformation from N-phenylisomaleimides to N-
spectroscopy at 37 °C and at distinct reaction times to verify
the change from the AA′BB′ system of 4a to the singlet
signal of N-phenylmaleimide at 6.48 ppm (Table 3; Fig. 5).
There was a correlation between the time and the inte-
gration signal at 6.48 ppm. That is, the integration signal
increased as time went along. This observation is very
important because no evidence exists of N-phenylmaleamic
acid formation.
phenylmaleimides
N-arylisomaleimides 4a–4c reacted in phosphate buffer
at 0.1 M (pH = 8.0). Compound 4a was isomerized into
Table 1 Parameters of enzymatic kinetics for N-arylisomaleimides
acting on AChE of Electrophorus electricus
Compounds
σH
pKi SEM (M)
Vmax (μmol/U min)
SEM
Isomerization kinetics of 4a
ACh
–
pKm = 3.165 0.002 0.456 0.114
4b (–OCH3) −0.27 4.181 0.002
0.448 0.044
0.645 0.151
–
Reaction order determination (n)
4a (–H)
Acetyla
4c (–NO2)
Neostigmine
a
0.00
0.31
0.78
–
3.713 0.064
5.443 0.238
7.051 0.054
10.151 0.320
The isomerization reaction was performed while monitoring
the decrease in absorbance of 4a, finding a negative expo-
nential relationship (Fig. 6) that was similar to a first-order
plot, according to the integral graph method. Subsequently,
the reaction order was determined for each tested con-
centration through a differential method, showing a trend to
first-order that was supported by statistical analysis (Fig. 7).
The mean of the reaction order for the three curves was n =
1.26 0.26.
0.491 0.181
0.619 0.277
Correa-Basurto et al. (2006)
Table 3 Qualitative analysis of the transformation from N-
phenylisomaleimide to N-phenylmaleimide was carried out by
monitoring the singlet signal at 6.48 ppm in CDCl3 with 100 μL of
phosphate buffer (C = 0.1 M, pH = 8.0 at 37 °C)
t (h)
Integration
Fig. 4 Quantitative structure–activity relationship between inhibition
constants (pKi) of N-arylisomaleimides, illustrated with Hammett’s σH:
(n = 3), b = 4.46 0.075 (*p < 0.001), m = 3.08 0.0691 (*p <
0.001) and r = 0.9303 (*p < 0.001). *p < 0.05 represents a statistically
significant difference at 95.0% confidence assessed with the Student’s
t-test
0.17
0.075
0.316
0.397
0.653
72.00
144.00
168.00
Table 2 Changes in the chemical shifts of the AA′BB′ system of N-arylisomaleimides to the AA′BB′ system of N-arylmaleamics acids and the
singlet signal of N-arylmaleimides
Compounds
4a δ (3JH-H in Hz) ppm
4b δ (3JH-H in Hz) ppm
4c δ (3JH-H in Hz) ppm
Isomaleimide AA′BB′ system 7.39 (overlapping signals) and 6.678 (5.4) 7.29 (5.44) and 6.54 (5.44)
7.43 (5.69) and 6.78 (5.69)
Maleamic acid AA′BB′ system
–
6.3557 (12.12) and 5.9615 (12.34) 6.24 (13.36) and 5.79 (13.34)
6.84 7.23
Maleimide singlet system
6.84

Med Chem Res
Fig. 5 Changes in the chemical shifts of the AA′BB′ system of N-phenylisomaleimide to a singlet signal of N-arylmaleimide at several points in
time. a 0.17 h; b 72.00 h; c 144.00 h; d 168.00 h
Fig. 8 Activation energy determination by the Arrhenius equation for
Fig. 6 Isomerization kinetics of N-phenylisomaleimide to N-phe-
nylmaleimide. The N-phenylisomaleimide concentration decrease: ♦C
= 0.1 mM (n = 3); ■C = 0.2 mM (n = 3); ▲C = 0.4 mM (n = 3). *p <
0.05 represents a statistically significant difference at 95.0% con-
fidence assessed with the Student’s t-test
the isomerization kinetics of N-phenylisomaleimide to N-phenylma-
leimide. (n = 3) b = 15.59 1.01 (*p < 0.001), m = −5321.4 335.3
(*p < 0.001) and r = −0.9871 (*p < 0.001). *p < 0.05 represents a
statistically significant difference at 95.0% confidence assessed with
the Student’s t-test
Activation energy determination (Ea)
The activation energy, calculated with the Arrhenius equa-
tion (Fig. 8), was +10.57 1.33 kcal/mol. This energy is
sufficient for producing the isomerization from 4a to 5a, a
process that is temperature-dependent. That is, the reaction
is favored when the temperature increases.
Molecular docking studies
Validation of the model was achieved by the binding of
ACh and neostigme as standard ligands, observing a bind-
ing mode similar to that previously (Bourne et al. 1999).
The repetitions on this methodology estimate an RMSD for
the ACh in 100 observed conformers in the range between
0.1 and 0.9 Å. Considering the stabilty experiments for the
N-arylisomaleimides, docking studies were performed on
the AChE of Electrophorus electricus with compounds 3a–
d, 4a–d, and 5a–d, which established hydrogen bond,
Fig. 7 Reaction order determination for the differential method plot
for isomerization kinetics of N-phenylisomaleimide to N-phenylma-
leimide. ♦C = 0.1 mM (n = 3), b = −0.5861 0.1766 (*p < 0.001),
m = 0.7502 0.0938 (*p < 0.001) and r = 0.9116 (*p < 0.001); ■C =
0.2 mM (n = 3), b = −0.1440 0.1695 (p > 0.05), m = 1.4224
0.1323 (*p < 0.001) and r = 0.9426 (*p < 0.001); ▲C = 0.4 (n = 3),
b = −0.1987 0.1000 (*p < 0.001), m = 1.6305 0.1153 (*p <
0.001) and r = 0.9656 (*p < 0.001). *p < 0.05 represents a statistically
significant difference at 95.0% confidence assessed with the Student’s
t-test

Med Chem Res
Table 4 Intermolecular interactions of compounds 3a–d, 4a–d, and 5a–d on different pockets of AChE of Electrophorus electricus
Compounds
Active site
Oxyanionic site
Gly143:H---Oa
Aromatic patch
Acyl pocket
Anionic site
PAS
3a
Ser225:H---Oa
His494:H---Oa
His494:N---Ob
Ser225:H---Oa
His494:H---Oa
His494:N---Ob
Ser225:H---Oa
His494:H---Oa
His494:N---Ob
Ser225:H---Oa
His494:H---Oa
His494:N---Ob
Ser225:H---Oa
Phe356:π---πc
Ser225:H---Oa
Phe356:π–πc
Ser225:H---Oa
Phe356:π---πc
Ser225:H---Oa
His494:H---Oa
His494:N---Ob
His494:π---πc
Ser225:H---Oa
Phe356:π---πc
His494:H---Oa
Ser225:H---Oa
Phe356:π---πc
His494:H---Oa
Ser147:H---Oa
Tyr355:π---πc
Trp108:π---πc
Tyr146:H---Oa
3b
3c
3d
Gly143:H---Oa
Gly143:H---Oa
Gly143:H---Oa
Gly143:C---πc
Tyr355:π---πc
Tyr355:π---πc
Tyr355:π---πc
Trp108:π---πc
Tyr146:H---Oa
Tyr146:H---Oa
Tyr146:H---Oa
Trp108:H---Oa
Trp108:π---πc
Trp108:H---Oa
Trp108:H---Oa
Trp108:π---πc
Glu224:O---πb
4a
4b
4c
4d
Tyr355:H---Oa
Tyr355:H---Oa
Tyr355:π---πc
Phe313:H---Oa
Phe313:H---Oa
Tyr146:H---Oa
Gly143:H---Oa
Gly143:H---Oa
5a
5b
5c
5d
Gly143:HN---Oa
Gly143:HN---Oa
Tyr355:π---πc
Tyr355:π---πc
Tyr355:π---πc
Tyr355:π---πc
Trp108:π---πc
Trp108:π---πc
Tyr146:H---Oa
Tyr146:H---Oa
Ser147:H---Oa,d
Gly143:H---Oa
Gly144:H---Oa
Ala226:H---Oa
Gly143:H---Oa
Ser225:H---Oa
His494:H---Oa
His494:N---Ob
His494:π---πc
Ser225:H---Oa
His494:H---Oa
His494:π---Na
Acetylcholine
Neostigmine
Gly143:HN---Oa
Gly143:H---Oa
Trp108:π---Nb
Trp108:C---Cc
Trp108:C---Cc
Glu224:O---Nc
Trp108:π---Cc
Trp108:π---Nb
Trp108:π---Nb
His494:H---Oa
His494:N---Ob
Tyr355:π---Cc
Tyr355:π---Nb
a
Hydrogen bond
b
Electrostatic interaction
Hydrophobic interaction
Near PAS
c
d

Med Chem Res
R
R
R
+
N
O
NH
Phosphate buffer
0.1 M, pH = 8.0
N
O
O
O
O
O
OH
4a-4c
5b-5c
3b-3c
R: a = -H
R: b = -OCH3
R: c = -NO2
Phosphate buffer
0.1 M, pH = 8.0
R
N
O
O
5a-5c
Scheme 3 Transformation of N-arylisomaleimides to N-arylmaleamic acids and N-arylmaleimide derivatives under enzymatic reaction
conditions
electrostatic and hydrophobic interactions at some relevant
sites of AChE (Table 4).
However, it is important to mention that the N-aryliso-
maleimides are transformed under enzymatic reaction con-
ditions, which is demonstrated qualitatively by applying 1H
NMR spectroscopy (Table 2). Hence, 4b and 4c are sus-
ceptible to incomplete transformation generating a mixture
of N-arylmaleimides and N-arylmaleamic acids (Scheme 3).
Meanwhile, 4a is totally transformed to N-phenylmaleimide
(5a) (Scheme 3), which can be observed in the qualitative
The N-arylmaleamic acids, N-arylsuccinamic acids,
and derivatives of N-arylmaleimides, N-arylsuccnimides
(Trujillo-Ferrara et al. 2003a; Correa-Basurto et al. 2006,
2007; Guevara-Salazar et al. 2007) and N-arylisomalei-
mides (Trujillo-Ferrara et al. 2003a; Correa-Basurto et al.
2006, 2007) have produced inhibition on the AChE
enzyme. In the present study, compounds 4a–4c could bind
to the active site and exert competitive inhibition, evidenced
by the fact that the Vmax value is constant for N-aryliso-
maleimide derivatives (Table 1).
The relationship that exists between the pKi values of N-
arylisomaleimides, found with Hammett’s σH, suggests that
the electron-withdrawing groups improved the affinity and
recognition of the active site by AChE (Fig. 4). Likewise,
the ρ = 3.08 value confirms that electron-withdrawing
groups favor enzyme recognition of the active site and the
sensitivity of the system for electronic effects. This suggests
that the chemical environment in the AChE active site has
low electronic density.
1
kinetics evaluated by H NMR spectroscopy (Fig. 5). That
is, as time passed the double signal of proton in the α-
position with respect to the carbonyl-carbon decreased,
while the singlet signal of protons of the vinyl group
increased.
In general, the transformation of N-arylisomaleimides
occured under enzymatic reaction conditions, implying
that the phosphate ion was involved in the isomerization
reaction. In addition, the mixture of products resulting
from 4b and 4c indicate that these compounds are sensi-
tive to electronic effects. Contrarily, 4a is less sensitive to
the same affects, evidenced by the fact that only 5a was
obtained. Consequently, the proposed reaction mechanism

Med Chem Res
Scheme 4 Transformation pathways to obtain N-arylmaleamic acids (a) and N-arylmaleimides (b) from N-arylisomaleimides in phosphate buffer
0.1 M (pH = 8.0)
(Scheme 4) is similar to that of the isomerization reactions
taking place during the transformation of N-arylisoma-
leimides to N-arylmaleimides, mediated by a leaving
group like acetate (Sauers 1969) or aziridine (Joseph-
Nathan et al. 1974). According to this proposed
mechanism, the nucleophilic attack of the phosphate ion
on the α,β-unsaturated carbonyl-carbon is determinant for
the generation of N-arylmaleamic acids (pathway A) as
well as the isomerization to N-arylmaleimides (pathway
B). The electroactive (withdrawal and donor) groups of
4b–4c influence negative nitrogen intermediary nucleo-
philicity. This intermediary reacts unspecifically whether
with a water molecule to generate the maleamic acids
(pathway A) or through a nucleophilic attack on the α,β-
unsaturated carbonyl-carbon to generate intramolecular
cyclization and N-arylmaleimide formation. In both cases,
the phosphate ion acts as a catalyst and a leaving group.
Particularly, the electron-donating group of compound
4b increases the nucleophilicity of negative nitrogen, gen-
erating the mixture of 3b, 5b, and 4b in a proportion
less selective, according of 1H NMR spectrum, which
shows that 3c and 5c are in a proportion of 0.2:1, respec-
tively. Thus, the –NO2 –para position group stabilizes the
negative charge of the nitrogen, decreasing its nucleophili-
city and favoring the intramolecular reaction velocity.
The kinetics assay of the complete transformation from
4a to 5a indicates that the reaction is rapid and occurs
through a first-order process (according to first pseudo-order
experiments). In addition, the reaction velocity is tempera-
ture-dependent, taking half the time at 40 vs. 22 °C (3.01 vs.
8.12 min, respectively). Likewise, the isomerization reac-
tion from isomaleimide to maleimide for 4a is thermo-
dynamically stable and its activation energy (Ea) of +10.6
kcal/mol is congruent with an isomerization process.
Based on the stability of the N-arylisomaleimides sug-
1
gested by H NMR spectroscopy, it is possible that there is
a mixture of the transformation products of isomaleimides
in enzymatic reaction medium. Thus, the docking experi-
ments were carried out to explore the binding mode on
AChE for each of the possible ligands in the medium. The
results confirm that the N-arylmaleamic acids, N-aryliso-
maleimides, and N-arylmaleimides interact mainly in the
CAS site, which correlates with the experimental results
showing that inhibition is competitive regardless of the type
1
0.005:1:1, respectively, according to the H NMR integral
values. This means that the reaction is slower and less
selective than which occurs when using 4a. With the
electron-withdrawing group of 4c, the reaction is faster and

Med Chem Res
ligand. The ΔG and Kd values obtained through in silico
experiments suggest a higher affinity for all derivatives in
comparison with ACh; this tendency is in general similar to
that observed with the experimental data (Table 5). The 3a–
d derivatives recognize the CAS site, establishing an
ion–dipole interaction between the –NH group of the imi-
dazole of His494 and the carboxylate anion of the N-
arylmaleamic acids, and a hydrogen bond (H---O) with
Ser225. Other hydrogen bonds were identified with in the
oxyanionic hole among the Gly143 and the carboxylate.
Apparently, the orientation of the carboxylate toward CAS
and the oxyanionic hole is in function of a greater electro-
negativity or molecular dipole moment. Interesringly, the
Tyr146 belongings to PAS binds through a hydrogen bond
with the oxygen atom of the carbon-cabonyl of the amide
group, which is attractive for the dual inhibition of enzyme
(Fig. 9). The aromatic moiety is oriented to AP in a face-to-
face mode. Nevertheless, the derivatives 3c–d undergo a
partial reorientation of the electron-withdrawing group
toward the acyl pocket.
Compounds 4a–d interact with CAS and the oxyanionic
hole, although the binding orientation is not uniform. For
example, the orientation of the 4a molecule to CAS depends
on the greatest electronegative moiety, which is located in
the lactone group, while the aromatic ring is stabilized
through interaction with Glu224. Meanwhile, the 4b–c
molecules cannot adopt this orientation because the sub-
stituent in the –para position of the phenyl presents steric
impediment with a portion of the CAS site. Therefore, since
the phenyl cannot be oriented by the double bond of the
imine, the substituent is forced to interact with Ser225,
while the lactone group fits into the AS. The 4d compound
cannot orient itself in the same way as the 4b–c derivatives
because the –acetyl subtituent is bulkier and consequently
Table 5 Affinity parameters estimated through in silico experiments
Compounds
ΔG (kcal/mol)
Kd (μM)
pKd (μM)
Acetylcholine
−5.05
−5.74
−6.15
−5.29
−7.06
−6.430
−7.04
−7.22
−7.67
−5.95
−6.37
−5.94
−7.29
−6.68
198.92
61.94
30.91
131.93
6.65
−3.70
−4.20
−4.51
−3.88
−5.17
−4.71
−5.16
−5.29
−5.62
−4.35
−4.66
−4.35
−5.34
−4.89
3a
3b
3c
3d
4a
19.23
6.9
4b
4c
5.09
4d
2.38
5a
43.84
21.49
44.51
4.54
5b
5c
5d
Neostigmine
12.68
Fig. 9 Binding mode of 3a–d
on the CAS of AChE of E.
electricus. All molecules are
oriented in a similar
conformation and exhibit similar
interactions. 3a yellow, 3b red,
3c blue, 3d green (color figure
online)

Med Chem Res
Fig. 10 Binding mode of 4a–d
on the CAS of AChE of E.
electricus. The binding modes
for all the compounds are
heterogeneous, but all the
molecules are oriented toward
CAS. 4a yellow, 4b red, 4c blue,
4d green (color figure online)
Fig. 11 Binding mode of 5a–d
on the CAS of AChE of E.
electricus. The binding modes
for all the compounds are
heterogeneous, with compound
5c interacting mainly on
oxyanionic hole. 5a yellow, 5b
red, 5c blue, 5d green (color
figure online)
presents steric impediment, resulting in the adoption of
another orientation, which is reinforced by His494 and
Tyr355 (Fig. 10).
Compounds 5a–d orient themselves to CAS according to
the greatest electronegative or molecular dipole moment. In
particularl, compounds 5a–b bind by the imide moiety,

Med Chem Res
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