Reversible and irreversible inhibitory activity of succinic and maleic acid derivatives on acetylcholinesterase

Article abstract of DOI:10.1016/S0928-0987(03)00023-X

Aryl succinic and maleic acid derivatives are potent inhibitors of bovine acetylcholinesterase in vitro. Succinic acid aminophenol derivatives 1b-e and 2b-d act as reversible inhibitors of acetylcholinesterase, while maleic acid aminophenol derivatives 3b-d and 4c-e act as choline subsite-directed irreversible inhibitors, detected by dialysis in the presence of edrophonium. Linear relationships between the logarithm of the velocity of hydrolysis of acetylcholine plotted against the time of incubation at several different inhibitor concentrations were determined. The K_i for reversible competitive inhibitors was determined. For irreversible inhibitors the K_i for the dissociation constant of the enzyme-inhibitor complex at the beginning of the recognition process was also determined as well as the inactivation constant of the enzyme-inhibitor adduct formation k₊₂ and the bimolecular inhibition constant k_i for the inhibition of acetylcholinesterase by aminophenol derivatives 3b-d and 4c-e. The conclusions of this study can be summarized as follows for both families: (a) the aromatic moiety played a critical role in the recognition of the active site; (b) in case of the reversible inhibitor, when the ester function took the place of the hydroxyl fragment, there was an important increase in the affinity; and (c) the distance between phenolic hydroxyl and nitrogen was critical because the inhibition is ortho?meta

Full text of DOI:10.1016/S0928-0987(03)00023-X

European Journal of Pharmaceutical Sciences 18 (2003) 313–322
www.elsevier.com/locate/ejps
Reversible and irreversible inhibitory activity of succinic and maleic acid
derivatives on acetylcholinesterase
a
a
b
b
J. Trujillo-Ferraraa , , Ivan Vazquez , Judith Espinosa , Rosa Santillan , Norberto Farfan ,
*
´
´
´
c
¨
Herbert Hopfl
´
a
´
´
Seccion de Graduados y Departamento de Bioquımica, Escuela Superior de Medicina, Instituto Politecnico Nacional,
´
´
´
Plan de San Luis y Dıaz Miron, Mexico, D.F. 11340, Mexico
b
´
´
´
Departamento de Quımica del Centro de Investigacion y Estudios Avanzados, Instituto Politecnico Nacional, Apartado Postal 14-740,
´
Mexico, D.F. 07000, Mexico
c
´
´
Universidad Autonoma del Estado de Morelos, Centro de Investigaciones Quımicas, Av. Universidad 1001, CP 62210 Cuernavaca, Morelos, Mexico
Received 11 September 2002; received in revised form 3 February 2003; accepted 3 February 2003
Abstract
Aryl succinic and maleic acid derivatives are potent inhibitors of bovine acetylcholinesterase in vitro. Succinic acid aminophenol
derivatives 1b–e and 2b–d act as reversible inhibitors of acetylcholinesterase, while maleic acid aminophenol derivatives 3b–d and 4c–e
act as choline subsite-directed irreversible inhibitors, detected by dialysis in the presence of edrophonium. Linear relationships between
the logarithm of the velocity of hydrolysis of acetylcholine plotted against the time of incubation at several different inhibitor
concentrations were determined. The Ki for reversible competitive inhibitors was determined. For irreversible inhibitors the Ki for the
dissociation constant of the enzyme–inhibitor complex at the beginning of the recognition process was also determined as well as the
inactivation constant of the enzyme–inhibitor adduct formation k12 and the bimolecular inhibition constant ki for the inhibition of
acetylcholinesterase by aminophenol derivatives 3b–d and 4c–e. The conclusions of this study can be summarized as follows for both
families: (a) the aromatic moiety played a critical role in the recognition of the active site; (b) in case of the reversible inhibitor, when the
ester function took the place of the hydroxyl fragment, there was an important increase in the affinity; and (c) the distance between
phenolic hydroxyl and nitrogen was critical because the inhibition is ortho<meta,para.
2003 Published by Elsevier Science B.V.
Keywords: Acetylcholine; Acetylcholinesterase; Succinic and maleic acids; Reversible and irreversible inhibitors
1. Introduction
might lead to a better understanding of the pathology of
this disease and permit the emergence of effective thera-
peutic modalities. In general, compounds containing a
quaternary ammonium group do not penetrate the cell
membrane; hence, anti-AChE agents in this category are
absorbed poorly from the gastrointestinal tract or through
the skin and are excluded from the central nervous system
by the blood–brain barrier (Berman and Leonard, 1991;
Taylor, 2001). In contrast, the more lipid soluble agents
are well absorbed after oral administration and have
ubiquitous effects at both peripheral and central choliner-
gic sites.
Molecular cloning revealed that a single gene encodes
vertebrates. However, multiple genes encode in other
biological systems; this diversity arises from alternative
processing of the mRNA. The different forms differ only
in their carboxyl-termini; the portion of the gene that
encodes the catalytic core of the enzyme is invariant
Acetylcholine (ACh) is an important neurotransmitter
across nerve–nerve and neuromuscular synapses in both
the central and the peripheral nervous system. Acetyl-
cholinesterase (AChE, EC 3.1.1.7) ends the ACh action by
its hydrolysis of it (Quinn, 1987).
Today it is well known that senile dementia of the
Alzheimer’s type is a neurodegenerative disease consistent
with low concentrations of cholinergic markers such as
ACh and cholinetransferase (Taylor, 1998, 2001; Trybulski
et al., 1992). Therefore, the development of potent and
specific AChE inhibitors with low toxicity that cause
increasing ACh levels in the affected regions of the brain
*
Corresponding author. Fax: 152-555-729-6000x62744.
E-mail address: jtrujillo@ipn.mx (J. Trujillo-Ferrara).
0928-0987/03/$ – see front matter
doi:10.1016/S0928-0987(03)00023-X
2003 Published by Elsevier Science B.V.

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J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
(Schumacher et al., 1986; Grisaru et al., 1999; Taylor,
2001).
peripheral anionic binding site (PAS), consisting of
Trp286, Tyr72, Tyr124 and Asp74. Tyrosines 337 and 449
are further removed from the active center but likely
contribute to the stabilization of certain ligands.
In 1991 the molecular structure of AChE from Torpedo
californica (TcAChE) was examined by X-ray crystal-
lography (Sussman et al., 1991), and in 1995 the three
dimensional structure mouse AChE (mAChE) was ex-
amined in a complex with fasciculin 2 (Fas 2) (Bourne et
al., 1995) and only slight differences were observed in the
conformation of Fas2-associated mAChE compared with
uncomplexed TcAChE. The active site of mAChE consists
of: (a) a steratic site (ES) that is comprised of the catalytic
triad Ser203, His447 and Glu334; (b) an acyl binding site
(ABS) located on Phe295 and Phe297, that binds the acetyl
group of ACh and that is important for binding interactions
with aryl substrates and active site ligands (Lin et al.,
1999); (c) a choline subsite with Trp86, Glu202 and
Tyr337 that contains a small number of negative charges,
but many aromatic residues, and where the quaternary
ammonium pole of ACh and various active site ligands
bind through a preferential interaction of quaternary nitro-
gens with the p electrons of the aromatic groups; (d) a
A step forward into the treatment of Alzheimer’s disease
has involved attempts to augment the cholinergic function
of the brain. A somewhat more successful strategy has
been the use of inhibitors of AChE. Nowadays new
inhibitors can be developed by molecular design strategies
in order to take advantage of several important features of
the active-site gorge of AChE so that a drug can be
produced with both a high affinity and a high degree of
selectivity for the enzyme (Recatini et al., 1997; Kryger et
al., 1999).
In this paper we report the in vitro inhibition of AChE
by a variety of carefully designed amide and imide
derivatives of succinic and maleic acids (Fig. 1) that have
been previously synthesized in our laboratory (Trujillo-
Ferrara et al., 1999). Four of them are new compounds
(1d, 2d, 3d and 4e; Fig. 2) and a complete characterization
1
of H and 13C NMR spectroscopy is reported.
Before the design of the inhibitors we considered several
factors that would be important for an optimum activity
(Baker and Alumaula, 1963). Firstly, there should be a
structural relationship between ACh, choline (Ch) and the
inhibitors. Secondly, most of the ligands were prepared
from aromatic starting materials in order to enhance the
selectivity of molecular recognition on the surface of the
active site gorge, which contains 40% of aromatic residues
(Sussman et al., 1991). The structural similarities of
compound 2c with ACh, Ch and edrophonium have been
marked by bold lines.
Although it has been demonstrated by several studies
that molecules with a positive charged ammonium group
like edrophonium and ACh are more selectively bound to
the enzyme than analogous uncharged molecules (Harel et
al., 1993), some of the compounds that we prepared are
neutral molecules (2 and 4) in order to enhance the
lipophilicity of the inhibitors. Nevertheless, the imide
group in compounds 2 and 4 is polarizable and the electron
density of the free electron pair of the nitrogen atom is
delocalized through the carbonyl groups and aromatic ring.
Thus, more than two mesomeric structures with partial
positive charges on the nitrogen atom can be formulated, a
fact that may be important for the recognition between the
inhibitor and the Ch subsite of AChE.
Fig. 1. Structures of the derivatives of succinic (1–2) and maleic acids
(3–4) that have been examined as reversible and irreversible inhibitors of
AChE in this work.

J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
315
Fig. 2. Structures of the new compounds 1e, 2d, 3d and 4e.
There is another important feature during the design of
molecules 1–4, it was considered that succinic amides/
imides (1–2) and maleic amides/imides (3–4) may react
in different ways with bionucleophiles such as thiols and
imidazoles. Both succinic amides and imides are suscep-
tible to 1,2-addition reactions at the carbonyl functions
(Groutas et al., 1995); however, maleic amides and imides
are susceptible to 1,2-addition, and at the same time to
1,4-addition reactions at the a,b-unsaturated carbonyl
function, especially with thiols and imidazole derivatives
(Walker, 1994). Therefore, it could be expected that the
unsaturated compounds 3a–d and 4a–g react as irrevers-
ible inhibitors, while compounds 1a–e and 2a–d without
the C=C double bond could react as reversible inhibitors.
To our knowledge, the following study is the first report
dealing with the AChE inhibitory activity of these types of
compounds.
were recorded on a Joel GSX-270 spectrometer. Approxi-
mately 10% sample solutions in DMSO-d6 were prepared
in 5-mm tubes with samples referred to tetramethylsilane.
Chemical shifts are stated in ppm and the coupling
constants in Hz; they are positive, when the signal is
shifted to higher frequencies than the standard. Infrared
spectra were measured with a MIDAC M2000 FT-IR
spectrophotometer from KBr pellets. Melting points were
determined in open ended capillary tubes in an Electrother-
mal 9300 digital apparatus and are uncorrected.
2.1.1. Synthesis of 4-(49-acetoxy-phenylamino)-4-oxo
propanoic acid 1e (Fig. 2)
A solution of 4-(49-hydroxy-phenylamino)-4-oxo pro-
panoic acid 1c (3 g, 14.4 mmol) in the presence of
anhydrous tetrahydrofuran (60 ml), pyridine (50 ml) and
an excess of acetic anhydride (60 ml) was heated and
stirred at 60 8C for 5 h, followed by evaporation of the
solvent under vacuum. The residue was washed three times
with 0.001 M HCl, yielding 1e (2.7 g, 90%). Melting point
of 1e 260–262 8C; 1H NMR (DMSO-d6, 270 MHz) d
(ppm), 2.50 (1H, t, J56, H-2), 2.52 (1H, t, J56, H-3),
7.00 (2H, d, J59, H-29,69), 6.76 (2H, d, J59, H-39,59);
13C NMR (67.5 MHz) d (ppm), 170.00 (C-1), 128.40
(C-29,69), 115.40 (C-39,59); IR (KBr) n (cm21) 3264 (OH,
carboxylic acid), 1686 (C=0), 1204 (C–N), 1189 (C–H).
2. Materials and methods
2.1. Synthesis
The synthesis of amides and imides 1–4 was achieved
by slight modifications of a previously reported method
(Sauers, 1969; Walker, 1994; Trujillo-Ferrara et al., 1999),
which briefly consists of the following.
Reaction of maleic or succinic anhydride with the
appropriate arylamide in the presence of anhydrous tetra-
hydrofuran at room temperature generated the corre-
sponding amides (1a–c) or vinyl amides (3a–c) in nearly
quantitative yields (94–98%). The starting materials were
subsequently transformed to the corresponding imides (2a–
c and 4a–c) by heating them in acetic anhydride with an
equimolecular amount of sodium acetate.
2.1.2. Synthesis of 1H-(49-hydroxy-phenyl)-2,5-
pyrrolidine dione 2d (Fig. 2)
A solution of 4-(49-hydroxy-phenylamino)-4-oxo pro-
panoic acid 1c (3 g, 14.4 mmol) in the presence of
anhydrous tetrahydrofuran (60 ml) and equimolecular
amount of sodium acetate (1.14 g) and acetic anhydride
(1.5 ml) was heated and stirred at 60 8C for 8 h, followed
by evaporation of the solvent under vacuum. The residue
was washed three times with 0.001 M HCl, yielding 2d
(2.8 g, 95%). Melting point of 2d .300 8C; 1H NMR
(DMSO-d6, 270 MHz) d (ppm), 2.73 (4H, s, H-3), 7.01
(2H, d, J58.6, H-29,69), 6.82 (2H, d, J58.6, H-39,59);
All synthesized and assayed compounds were character-
ized by 1H and 13C nuclear magnetic resonance (NMR)
1
and infrared spectra (IR). H and 13C NMR spectra were
compared to our recently reported data on these molecules
1
(Trujillo-Ferrara et al., 1999). H and 13C NMR spectra

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J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
13C NMR (67.5 MHz) d (ppm), 177.76 (C-1), 28.87 (C-2,
C-5), 157.65 (C-49), 128.83 (C-29,69), 135.84 (C-39,59). IR
(KBr) n (cm21), 3268 (OH), 1720 (C=O), 1686 (C=O).
sary; corrections were made for Lorentz and polarization
effects. Direct methods (SHELXS-86) were used for
structure solution and the CRYSTALS (version 9, 1994)
software package for refinement and data output. Non
hydrogen atoms were refined anisotropically. Hydrogen
atoms were determined by differences in the Fourier maps,
and one overall isotropic thermal parameter as well as their
coordinates were refined. I . 3s(I) (R 5 o(iFou 2 uFci)/
ouFou, Rw 5 [ow(uFou 2 uFcu)2 /owF2o]1 / 2. In all cases only
independent reflections on the basis of Friedel’s law have
been collected and a reflection–parameter ratio .5 has
been considered sufficient for the type of structural studies
performed here. The asymmetric unit of compounds 1c, 2a
and 2b contains two independent molecules.
2.1.3. Synthesis of 4-(49-acetyloxyphenylamino)-4-
oxobut-Z-2-enoic acid 3d (Fig. 2)
A solution of 4-(49-hydroxy-phenylamino)-4-oxobut-Z-
2-enoic acid 3c (3 g, 14.5 mmol) in the presence of
anhydrous tetrahydrofuran (60 ml), pyridine (50 ml) and
an excess of acetic anhydride (60 ml) was heated and
stirred at 60 8C for 8 h, followed by evaporation of the
solvent under vacuum. The residue was washed three times
with 0.001 M HCl, yielding 3d (2.7 g, 90%). Melting point
1
of 3d 186–188 8C. H NMR (DMSO-d6, 270 MHz) d
The most important crystallographic data have been
summarized in Table 1. Lists with atomic parameters, bond
lengths, bond angles and thermal parameters have been
deposited at the Cambridge Crystallographic Data Center.
(ppm), 6.48 (1H, s, J512, H-2), 6.60 (1H, s, J512, H-3),
6.80 (2H, d, J59, H-29,69), 7.43 (2H, d, J59, H-39,59);
13C NMR (67.5 MHz) d (ppm), 167.00 (C-1), 131.00
(C-2), 130.00 (C-3), 160.30 (C-4), 121.20 (C-29,69),
113.00 (C-39,59). IR (KBr) n (cm21), 3334 (OH, car-
boxylic acid), 1728 (C=0), 1233 (C–N), 1193 (C–H).
2.3. Enzyme kinetics
2.1.4. Synthesis of 1H-pyrrole-1-(49-hydroxyphenyl)-2,5-
dione 4e (Fig. 2)
For all in vitro kinetic experiments true bovine erythro-
cyte AChE (EC 3.1.1.7), type XII from Sigma, with a
specific activity of hydrolysis of 1.0 mmol/min per mg of
protein in 0.10 M phosphate buffer at pH 8.0 and T537 8C
were used. Acetylcholine iodide from Sigma was used as a
substrate at various concentrations both below and above
but near Km in a phosphate buffer at pH 8. The inhibitors
were dissolved in ethanol (3% v/v) and immediately
diluted to the desired working concentration with the same
phosphate buffer used for the preparation of the AChE
suspension.
A solution of 4-(49-hydroxy-phenylamino)-4-oxobut-Z-
2-enoic acid 3c (3 g, 14.4 mmol) in the presence of
anhydrous tetrahydrofuran (60 ml) and equimolecular
amount of sodium acetate (1.14 g) and acetic anhydride
(1.5 ml) was heated and stirred at 60 8C for 8 h, followed
by evaporation of the solvent under vacuum. The residue
was washed three times with 0.001 M HCl, yielding 4e
1
(2.5 g, 83.3%). Melting point 4e 185–187 8C. H NMR
(DMSO-d6, 270 MHz) d (ppm), 7.10 (1H, s, H-3), 6.82
(1H, d, J58.8, H-3959), 7.07 (1H, d, J58.8, H-29,69); 13C
NMR (67.5 MHz) d (ppm) 171.19 (C-3, C-4), 135.39
(C-2), 123.30 (C-19), 157.88 (C-49), 129.28 (C-29,69),
116.25 (C-39,59). IR (KBr) n (cm21), 3485 (OH), 1703
(C=O).
In order to confirm the stability of phenyl acetates, the
reaction of the compounds 2c and 4c in the buffer and
enzyme solution was tested at pH 8 and 37 8C for 24 h and
the reaction was followed by thin layer chromatography;
the starting material and the hydrolysis products were not
observed. The hydrolysis was undertaken in ETOH/H2O
pH 1 at 50 8C and only in these conditions was the acetate
group lost. The hydrolysis products were characterized by
1H and 13C NMR spectra.
The enzymatic inhibition measurements of the different
inhibitors were carried out in the presence and absence of a
fixed concentration of inhibitors and at different substrate
concentrations.
For the dialysis kinetic studies, a solution of enzyme
(0.04 ml, 0.5 M) and fixed inhibitor concentration (1.2 ml)
was prepared and dialyzed against 100 ml buffer at 37 8C
for 2 h using regenerated cellulose dialysis membranes
SPECTRAPOR . Aliquots of 0.08 ml of the enzyme–
inhibitor mixture were taken in time intervals of 0, 20, 40,
60, 90, 120 min and added to 0.12 ml of substrate (2.853
1024 M), and after an hour of incubation the enzyme
activity was measured. A control solution prepared with
enzyme (0.04 ml) and buffer (1.2 ml) was treated similar-
ly.
2.2. X-ray crystallography
For the time–concentration-dependence kinetic, test and
control solutions were prepared as before, but without
dialysis, and the enzyme activity was measured in the same
time intervals.
At the end of incubation, the concentration of the
remaining ACh was detected by a modified version of the
colorimetric method of Bonting and Featherstone (1956).
Therefore, 0.08-ml aliquots were transferred under rapid
X-ray diffraction studies of single crystals were realized
on an Enraf-Nonius CAD4 diffractometer (lMo Ka
5
˚
0.71069 A, monochromator: graphite, T5293 K, v–2u
scan). Cell parameters were determined by least squares
refinement on diffractometer angles for 24 automatically
centered reflections. Absorption correction was not neces-

J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
317
Table 1
Crystallographic data for compounds 1d, 2a–c and 3a
Crystal data
1da
2a
2ba
2c
3a
Formula
C10H10ClNO4
0.530.630.6
243.64
C12H11NO4
0.330.530.5
233.22
C12H11NO4
0.430.530.6
233.22
C12H11NO4
0.230.230.4
233.22
C10H9NO4
0.330.330.3
207.19
Crystal size (mm)
Mw (g mol21
)
Space group
P-1
Pcab
P21 /a
P21
P212121
Cell parameters
˚
a (A)
14.302(1)
5.344(1)
14.322(1)
90.23 (1)
101.22(1)
89.79(1)
1073.77(1)
4
9.686(1)
12.318(4)
19.072(9)
90
90
90
2275.5(9)
8
0.097
1.36
12.199(1)
12.017(1)
16.291(1)
90
110.69(1)
90
2234.2(3)
8
0.099
7.827(2)
6.250(2)
12.068(4)
90
106.34(3)
90
566.5(3)
2
0.097
6.793(1)
10.682(1)
12.853(1)
90
90
90
932.6(1)
4
0.108
1.48
˚
b (A)
˚
c (A)
a (8)
b (8)
g (8)
3
˚
V (A )
Z
m (mm21
)
0.350
1.51
rcalcd (g cm23
)
1.39
1.37
Data collection
No. collected refl.
No. ind. refl.
No. observed refl.
3436
3294
2500
2834
2477
1576
5957
4841
3531
1558
1490
971
1211
1127
955
Refinement
R
Rw
W
0.045
0.045
1/s2
351
0.037
0.032
1/s2
189
0.038
0.032
1/s2
375
0.031
0.024
1/s2
189
0.039
0.044
1/s2
165
No. of variables
GOOF
2.73
2.63
1.85
1.99
1.97
a Two independent molecules in the asymmetric unit.
stirring to a 3.0-ml cuvette of 1.0 cm pathlength with 2.0
ml of ferric chloride (0.086 mmol) and hydroxyl amine
(0.47 mmol). As a colorimetric blank, a cuvette with
exactly the same components including the inhibitor but no
ACh was prepared. Absorption measurements were real-
ized at l5540 nm, both with a control and a test sample.
The extinction of the test sample was subtracted from the
value obtained for the blank. Data are quoted as mean
value of three experiments, which varied by less than 8%
in relation to each other.
In the case of the irreversible inhibitors, competitive
experiments with edrophonium (3-hydroxyphenyl-
ethyldimethylammonium iodide), a well-known reversible
inhibitor, were also carried out. The competitive inhibitor
was added to the enzyme–substrate mixture just before the
inhibitor under study.
triplet from the H-2. The magnitude of coupling constant is
found to be identical between H-2 and H-3 (6 Hz). In case
of 2d its formation was confirmed by the disappearance of
the characteristic triplets of 1c and generation of the A4
system. For 3d the assignment of H-2 and H-3 in the
vinylic moiety was achieved by COLOC experiments
which exhibit an AB system with a J512 Hz and the
signals of vinylic carbons were assigned unambiguously by
1H-13C HETCOR. 4e is the cyclical product derived from
3c in which the AB vinylic system is transformed to an A2
system and that absorbs at 7.12 ppm.
Due to the difference in reactivity between ligands 1–2
and the a,b-unsaturated ligands 3–4, two different kinetic
models had to be considered for the inhibition. Compounds
1a–e and 2a–d behaved as competitive reversible in-
hibitors and their inhibitory kinetics were evaluated by the
Lineweaver and Burk (1934) method; different substrate
concentrations above and below the Michaelis constant Km
of AChE for ACh were assayed. The inhibitor concen-
tration was always kept close to one which corresponds to
50% inhibition of the enzyme activity (IC50). Straight lines
were obtained in the case of compounds 1b–c and 2b–c
(Fig. 3), whereas for compounds 1a and 2a (ortho deriva-
tives) the inhibition was not observed even at concen-
trations as high as 10 mM. For the active compounds, the
existence of a covalent bond between inhibitor and enzyme
could be excluded, because the enzyme activity could be
recovered by dialysis within 2 h. In the case of these
reversible inhibitors, Vmax did not change at high substrate
3. Results and discussion
1
All compounds were characterized by H and 13C NMR
spectroscopy in order to verify their purity, and the data
were compared with the reported values (Trujillo-Ferrara
et al., 1999; Hargreaves et al., 1970). In the case of the
new compounds 1e, 2d, 3d and 4e all the aromatic moieties
showed an AA9BB9 system and J58.7 Hz. For the non-
aromatic moiety of the new compounds the results were as
follows: 1e, H-2 and H-3 protons appear as triplets, the
downfield triplet arising from the H-3 and the highfield

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J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
concentration, indicating that inhibition is competitive. The
inhibitory effects of the two families of compounds on
AChE were compared among themselves and then with
several known reversible inhibitors, as shown in Table 2.
The inhibitory activity of the meta-substituted amino-
phenol derivatives 1b and 2b was lower than that of the
para-substituted derivatives. The most potent inhibitor
among this class of compounds was succinimide 2c with
Ki 57.831028 mol/l. It is therefore more active than
edrophonium, physostigmine (Gregor et al., 1992) and,
however, it approximately presents eight times less activity
than tacrine, Ki 5131028 mol/l, and 27 times less activity
than E2020, Ki 5331029 mol/l (Kawakami et al., 1996).
In contrast, aminophenol derivatives with vinylic moiety
3b–d and 4b–e behaved as irreversible inhibitors since
they met the three criteria of Abeles and Maycock (1976)
for irreversible inhibition. The most definitive way of
testing irreversible inhibition requires the complete charac-
terization of the enzyme–inhibitor adduct. Such inves-
tigation is time-consuming and frequently very difficult to
do because of the limited amounts of enzyme which are
generally available. However, relatively simple kinetic
experiments can give reasonable assurance that our pro-
posed inhibitors were acting in a covalent form when the
following conditions were met: (a) time dependence pro-
vides good but not definitive evidence that covalent
modification had taken place and the demonstration that
the loss of enzyme activity at a constant inhibitor con-
centration was of the first order provided evidence that
inactivation occurred before the inhibitor was released
from the enzyme; (b) the rate of inhibition should have
been proportional to the inhibitor concentration at low
concentrations of the same; (c) the rate of inactivation at a
given inhibitor concentration should have diminished as
Fig. 3. Lineweaver–Burk plot of in vitro acetylcholinesterase inhibition
by 1H-pyrrolidine-1-(39-acetyloxyphenyl)-2,5-dione (2c). Each point
represents the mean value of three determinations, which varied by less
than 6% in relation to it.
Table 2
Kinetic data for reversible and irreversible inhibition on acetylcholinesterase by aminophenol derivatives of succinic and maleic acid
Compound
Type of inhibition
Ki (M)
k12 (1022 s21
)
ki (104 M21 s21
)
1b
1c
1e
2b
2c
2d
3b
3c
3d
4b
4c
4d
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Irreversible
Reversible
Reversible
Reversible
Reversible
46.060.3
1.360.5
6.660.3
40.060.3
0.00560.4
28.560.4
2.361.2
1.261.3
0.860.5
Unstable
0.5460.2
2.761.5
1.861.0
18.5
0.01360.02
6.060.4
3.060.8
Unstable
1.060.3
3.062.3
2.060.8
0.005660.0001
5.060.3
3.861.0
Unstable
1.8561.0
1.160.1
4e
1.160.8
Edrofonioa
Neostigminaa
Tacrinea
E2020
14.4
0.01
0.003
a Kinetic data on bovine erythrocyte AChE, type XII from Sigma.

J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
319
the substrate or competitive reversible inhibitor concen-
tration increased. These two kinetic phenomena, saturation
kinetic and substrate or competitive inhibitor protection
against inhibition, are necessary consequences of the
involvement of the enzyme’s active site in the inhibition
process for the aforementioned. The inhibitory kinetics of
the compounds 3b–d and 4c–e were evaluated by the
method established by Kitz and Wilson (1962) and they all
met these conditions and therefore it may be suggested that
they are active site-directed irreversible inhibitors, because
after exposure to edrophonium (a reversible inhibitor
which binds at the Ch subsite) and then dialysis, the
enzyme recovered its activity.
were less potent than para-substituted ones. The most
potent inhibitor was maleimide 4c with values of k12 5
1.031022 s21 and Ki 55.431027 mol/l. Therefore 4c is
more active than neostigmine (Jaen and Moos, 1992),
diisopropylfluorophosphate and paraoxon, but less active
than tacrine, 131028 M, and E2020, 331029 M (Boyd et
al., 2000). Compounds 4d and 4e had a significantly lower
activity than 4c, so the presence of an acetyl group in the
aromatic para position increased recognition; compounds
3e and 4f–g that have neither aromatic rings nor acetyl
groups were clearly inactive. The importance of the
aromatic ring in active compounds is shown by the results
of 3e and 4f–g which have alkyl substituents instead of an
aromatic ring and which did not inhibit the enzyme even at
a concentration of 10 mM.
The inhibition of AChE by maleic acid aminophenol
derivatives was lower with edrophonium than without it, as
would be expected in the event that the active site is
involved in the inhibition. With this observation it is
possible to conclude that all active irreversible molecules
found in this study block the active site of AChE,
particularly the choline subsite. Also it can be proposed
that the first step for both types of inhibitors is the
formation of a reversible enzyme–inhibitor complex with
the a,b-unsaturated derivatives 3b–d and 4c–e; this re-
versible enzyme–inhibitor complex by addition reactions
with nucleophilic substituents of the enzyme, or a p–p
recognition of high affinity is possible in an irreversible
interaction.
For this reason, the following reaction sequence between
the enzyme and the inhibitor was considered for these
types of compounds
k
k
12
11
E 1 IáEIáEI9
k
k
22
21
whereby EI is a reversible enzyme–inhibitor complex and
EI9 an irreversible one. The existence of an irreversible
complex was proved by exhaustive dialysis of the en-
zyme–substrate complex after inhibition and later mea-
surement of the enzyme activity, the result of which was
that it could not be recovered. Therefore k12 must be very
small. The kinetics of the enzyme inhibition were evalu-
ated by Eqs. (1) and (2) (Kitz and Wilson, 1962)
k
12t
with [ET] 5 [E] 1 [EI9]
and [E] 5 [E] 1 [EI]
[E]
[ET]
]
]
In 5
5 2
1
(1)
(2)
1 1 K1[I]21
The X-ray structure of AChE has established the exist-
ence of Trp84. In addition, the active site contains imida-
zole groups, which are known to undergo nucleophilic
addition reactions with a,b-unsaturated carbonyl deriva-
tives. Firstly, there are imidazol residues in the catalytic
triad (His440) and at the gorge rim (His287) (Sussman et
al., 1991). Additionally, there are two Tyr residues, one at
the bottom and the other one half way up the gorge of the
active site. So the reaction between imidazol and a,b-
unsaturated derivatives is possible (Fig. 5).
K1
k12
1
1
1
[I]
]
]
5
5
with kapp 5
.
1 1 K1[I]21
kapp k12 k12
The kinetic data obtained were plotted according to Eq.
(1). From Fig. 4 (plot A) the concentration and time
dependence of the irreversible inhibition of compound 4c
can be seen. The kinetic of the inhibition of AChE is of
first order as evidenced by a plot of the inverse of kapp
versus the inverse of the inhibitor concentration [I]. Eq. (2)
is the equation of a straight-line graph. As in the case of
the ortho derivatives, 1a and 2a, 3a and 4a did not cause
any inhibition at 10 mM, even after 1 day of contact with
the enzyme. The same behavior was observed with com-
pounds 3e and 4f–g.
In Fig. 4 (plot B) the linear plot of compound 4c
according to Eq. (2) is shown and a reasonable correlation
was obtained. The regression line did not pass through the
origin, but intercepted the positive y-axis, a fact which
indicates that initially reversible enzyme–inhibitory com-
plexes were formed (Kitz and Wilson, 1962). The rate
constants for the inhibitory activity k12 and the equilib-
rium constants for the enzyme inhibition Ki of the a,b-
unsaturated compounds 3b–d and 4c–e are listed in Table
2. As in the case of the above studied reversible inhibitors,
meta-substituted maleamide and maleimide derivatives
In order to support this hypothesis we reacted histidine
with compound 4c in H2O/EtOH (1:1) and clearly a
1
1,2-addition product was observed and characterized by H
and 13C NMR spectroscopy, showing that the vinylic
protons that absorbed at 7.2 ppm disappeared and in their
place appeared an AB system at 6.3 and 6.5 ppm with
J512 Hz; also the imide carbonyl carbon disappeared and
the amide carbonyl at 164 ppm appeared which are typical
values of chemical shifts for amide carbonyls.
In order to obtain more information about the type of
interaction between the inhibitors and the active site of
AChE, the molecular structures of compounds 2a–c (Fig.
6) were determined by X-ray crystallography. The struc-
tures of these compounds are outlined together with the
structure of the trans,trans conformer of ACh, which has
been found to be the most adequate conformation of the
complex with AChE (Canepa et al., 1966).

320
J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
Fig. 4. Time- and concentration-dependent inhibition of acetylcholinesterase by 1H-pyrrole-1-(49-acetyloxyphenyl)-2,5-dione (4c). Plot A shows how the
concentration of the enzyme–inhibitor complex [EI] decreases with enhanced incubation time. Plot B shows that the inhibition kinetics is of first order. The
line corresponds to the best fit by linear regression. All points are the mean values of three determinations.
The length and width of molecules 1d and 3a are listed
in Table 3 in comparison to the corresponding data of
trans,trans-ACh and compounds 2a–c.
An analysis of the molecular structures of compounds
2a–c shows that both the acetyl group and the succinimide
function are twisted out of the molecular plane of the

J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
321
at first sight it may be surprising that they are inactive. A
possible explanation might be related to the presence of a
polar hydroxyl group at the lateral site of the molecules
that reduces its lipophilicity. This reduced lipophilicity at
the lateral site of the molecule may inhibit the passage of
the compound through the highly hydrophobic gorge of the
enzyme to the active site.
Fig. 5. Possible mechanisms for the formation of a covalent bond
between enzyme and inhibitor molecules 3 and 4. Nu-H is acetyl-
cholinesterase with one of its nucleophilic imidazole groups.
Another explanation for the inactivity of the ortho-
substituted derivatives of compounds 1a and 3a could be
the observation that these molecules have a close structural
relationship to the gauche conformation of ACh. It has
been demonstrated that the gauche conformation of ACh is
probably inactive at the catalytic site of AChE, although it
is the most stable conformation of ACh (Canepa et al.,
1966). The inactivity results from a sterical inhibition in
the formation of the tetrahedral intermediate generated
during the acylation in the hydrolysis of ACh by AChE
(Harel et al., 1996; Silman et al., 1992).
In contrast, the structures of the para-substituted deriva-
tives of 2c and 4c are more related to the anti-conforma-
tion of ACh, the conformer with the most adequate
structure for comparison in the catalytic triad prior to
hydrolysis.
Fig. 6. Molecular structure of 2c by X-ray crystallography.
phenyl ring. Although compound 2a is the compound with
the highest structural similarity to ACh, this molecule has
been found to be inactive as an inhibitor. One reason may
be its large and compact molecular structure with a length
˚
˚
of 7.9 A and a width of 7.3 A that may inhibit diffusion
through the gorge to the active site. The gorge has a width
˚
of 4.5 A at its narrowest point. Although compound 2b is
as broad as 2a, diffusion through the gorge may be easier
for this molecule because the acetyl and succinimide
groups are more separated (at the ortho position, O–N 2.78
˚
˚
A; and at the meta position, O–N 4.84 A). The para-
˚
substituted derivative 2c with a width of only 4.5 A and a
distance O–N of 5.78 A should pass through the gorge
˚
more easily and this may be the reason why this molecule
has been found to be an |103 more potent reversible
inhibitor than compound 2b. In the same context as the
aforementioned, with compounds 4, the para-substituted
aminophenol derivative of maleamic acid 4c is also thinner
than 4b, a fact that might explain the higher activity of the
para-substituted derivative. The ortho-substituted amino-
phenol derivatives 1a and 3a are also small molecules and
A further result of the present study is the observation
that the molecular dimensions, as well as the presence of
an aromatic ring system and the polarity of functional
groups at the hydrophobic part of the molecules used as
inhibitors must be important factors for their inhibitory
activities and that the non-aromatic imide molecule is not
active as an inhibitor at all.
Table 3
The conclusions of this study can be summarized as
follows for both families: (a) the aromatic moiety plays a
critical role in the recognition of the active site; (b) in case
of the reversible inhibitor, when the ester function took the
place of the hydroxyl fragment, there was an important
increase in the affinity; (c) the distance between phenolic
hydroxyl and nitrogen was critical because the inhibition is
ortho<meta,para. These conclusions are in agreement
with a recent comparative QSAR analysis of AChE
inhibitors currently being studied for the treatment Al-
zheimer’s disease (Recatini et al., 1997).
Comparison of some important structural data between trans,trans-ACh,
1a, 2a–c and 3a. Only atom to atom distances have been considered. The
structural data of compound 1a have been estimated on the basis of the
molecular structure of 1d
Compound
Length of the
molecule (A)
Width of the
molecule (A)
O12/O13 . . . O17
distance (A)
˚
˚
˚
trans,trans-ACh
8.7
9.0
10.4
7.9
9.4
10.9
3.9
5.3
5.5
7.3
7.3
4.5
–
–
–
1a
3a
2a
2b
2c
3.50/6.34
4.22/7.07
5.95/7.45
Finally, further studies are important in order to modify

322
J. Trujillo-Ferrara et al. / European Journal of Pharmaceutical Sciences 18 (2003) 313–322
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Acknowledgements
The authors wish to thank Arturo Bustamante Quezada
for technical assistance and CONACYT and SEGEPI for
financial support.
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