Structure-reactivity Relationships as Probes to Acetylcholinesterase Inhibition Mechanisms by Aryl Carbamates. II. Hammett-Taft Cross-interaction Correlations

Article abstract of DOI:10.1002/jccs.200400065

Substituted phenyl-N-butyl carbamates (1) and p-nitrophenyl-N-substituted carbamates (2) are characterized as "pseudo-pseudo-substrate" inhibitors of acetylcholinesterase. Since the inhibitors protonate in pH 7.0 buffer solution, the virtual inhibition co

Full text of DOI:10.1002/jccs.200400065

Journal of the Chinese Chemical Society, 2004, 51, 423-429
423
Structure-reactivity Relationships as Probes to Acetylcholinesterase
Inhibition Mechanisms by Aryl Carbamates. II. Hammett-Taft
Cross-interaction Correlations#
Gialih Lin (
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan, R.O.C.
)
Substituted phenyl-N-butyl carbamates (1) and p-nitrophenyl-N-substituted carbamates (2) are character-
ized as “pseudo-pseudo-substrate” inhibitors of acetylcholinesterase. Since the inhibitors protonate in pH 7.0
buffer solution, the virtual inhibition constants (Ki’s) of the protonated inhibitors can be calculated from the
equation, -logKi’ = -logKi – pKa + 14. The -logKi’ and logkc values for acetylcholinesterase inhibitions by car-
bamates 1 correlate with the Hammett equation (log(k/k0) = rs); moreover, those by carbamates 2 correlate
with the Taft equation (log(k/k0) = r* s*). With modified Hammett-Taft cross-interaction variations, multiple
linear regressions of the -logKi’ and logkc values of carbamates 1 and 2 give good correlations, and the
cross-interaction constants (rXR) are 0.5 and 0.0, respectively. The rXR value of 0.5 indicates that the
carbamate O-C(O)-N-R geometries for the transition states that lead to enzyme-carbamate tetrahedral inter-
mediates are all pseudo-trans conformations. Therefore, the carbamate moiety of the inhibitors stretches
along the active site gorge of the enzyme but does not bind in the acyl binding site pocket of the enzyme. Over-
all, the carbamate O-C(O)-N-R geometries for carbamates 1 and 2, protonated carbamates 1 and 2, and the tet-
rahedral intermediate are all retained in pseudo-trans conformations. The rXR value of 0.0 suggests that the
transition states that lead to the carbamyl enzymes are breaking C-O bonds and are excluding the leaving
groups, substituted phenols.
Keywords: Acetylcholinesterase inhibition; LFER; QSAR; Cross-interaction; Carbamates; Enzyme
mechanism.
INTRODUCTION
Acetylcholinesterase (AChEa, EC 3.1.1.7) plays a vital
(AS) composed of Trp84, Glu199, and Phe330, that contains
a small number of negative charges but many aromatic resi-
dues, where the quaternary ammonium pole of Ach and of
various active site ligands binds through a preferential inter-
action of quaternary nitrogens with the p electrons of aro-
matic groups, and (d) an acyl binding site (ABS) composed of
Phe288 and Phe290, that binds the acetyl group of Ach (Fig.
1). Besides the active site, AChE also has a peripheral anionic
binding site (PAS) composed of Trp279, Tyr70, Tyr121, and
Asp72, which is located at the entrance (mouth) of the active
site gorge that may bind to tacrine (Fig. 1).8-10
role in the central and peripheral nervous systems, where it
catalyzes the hydrolysis of neurotransmitter acetylcholine
(ACh).2,3 Many X-ray structures of AChEs and AChE-inhibi-
tor complexes have been reported recently.4-7 The active site
of AChE contains (a) an esteratic site (ES) comprised of the
catalytic triad Ser200-His440-Glu327, which is located at the
bottom of a 20 Å gorge, (b) an oxyanion hole (OAH) com-
posed of Gly118, Gly119, and Ala201, that stabilizes the tet-
rahedral intermediate, (c) an anionic substrate binding site
In Alzheimer’s disease, a neurological disorder, chol-
# Part I. Steady-State Kinetics1
* Corresponding author. Fax: +886-4-2286-2547; e-mail: gilin@dragon.nchu.edu.tw
a
Abbreviation: a, weighting factor for cross-interaction; ABS, acyl binding site; ACh, acetylcholine; AChE, acetyl-
cholinesterase; AS, anionic substrate binding site; ATCh, acetylthiocholine; d, intensity factor for Taft equation; DTNB,
5,5¢-dithio-bis-2-nitrobenzoate; ES, esteratic site; kc, carbamylation constant; kd, decarbamylation constant; Ki, inhibition or
dissociation constant; ki, bimolecular inhibition constant; LFER, linear free energy relationship; OAH, oxyanion hole; PAS, pe-
ripheral anionic binding sites; QSAR, quantitative structure activity relationship; rX, Hammett reaction constant; r*, Taft reac-
tion constant; rXR, Hammett-Taft cross-interaction constant; sX, Hammett substituent constant; s*, Taft substituent constant.

424 J. Chin. Chem. Soc., Vol. 51, No. 2, 2004
Lin
inergic deficiency in the brain has been reported.2,11-13 Be-
cause of the pivotal role that AChE plays in the nervous sys-
tem, the enzyme has long been an attractive target for the ra-
tional design of mechanism-based inhibitors as drugs for
treatment of Alzheimer’s disease. Rivastigmine, trade name
of Exelon, is a carbamate inhibitor of AChE already in use for
the treatment of Alzheimer’s disease.14
Scheme I Kinetic scheme for pseudo-substrate inhi-
bitions of AChE in the presence of sub-
stratea
The mechanism for AChE-catalyzed hydrolysis of sub-
strate is formation of the enzyme-substrate tetrahedral inter-
mediate via nucleophilic attack of Ser200 to the enzyme then
formation of the acyl enzyme intermediate (Scheme I). In the
presence of substrate, the pseudo-substrate inhibition mecha-
nisms of AChE by carbamates 1 and 2 (Fig. 2) have been pro-
posed (Scheme I).1,15 Since the inhibition follows first-order
a
E, AChE enzyme; S, substrate; I, carbamate inhibitor; EI,
the enzyme-carbamate tetrahedral intermediate; EI’, the
carbamyl enzyme intermediate; Ki, inhibition constant; kc,
carbamylation constant; P, the first product; kd, decarbamyl-
ation constant; Q, the second product.
kinetics over the observed time period for the steady-state ki-
netics, the rate of hydrolysis of EI¢ must be significantly
slower than the rate of formation of EI¢ (kc >> kd).16-19 There-
fore, values of Ki and kc can be calculated from Eq (1).17-19
kapp = kc [I] / (Ki (1 + [S] / Km) + [I])
(1)
In Eq (1), the kapp values are the first-order rate constants that
can be calculated according to Hosie's method.17-19 The bimo-
lecular rate constant, ki = k2/Ki, is related to overall inhibitory
potency. Therefore, carbamates 1 and 2 are called pseudo-
substrate or active site-directed irreversible inhibitors of
AChE. However, carbamates 1 and 2 do not meet the third
criterion for the pseudo-substrate inhibitors as proposed by
Abeles and Maycock,20 in that the enzyme does not protect
from inhibition by carbamates 1 and 2 in the presence of a re-
versible inhibitor, edrophonium.5,6 Edrophonium is excluded
from accessing the enzyme active site by the carbamyl moi-
ety of the carbamyl enzyme, which stretches along the active
site gorge and/or due to the conformational change for
His440 and Phe330 of the carbamyl enzyme.7,14 Therefore,
like Rivastigmine,14 carbamates 1 and 2 should be character-
ized as “pseudo-pseudo-substrate” inhibitors of AChE.1
Quantitative structure-activity relationships (QSARs)
represent an attempt to correlate structural properties of com-
pounds with biological activities or chemical reactivities.21-23
These chemical descriptors, which include parameters to ac-
count for hydrophobicity, electronic, inductive, or polar
properties, and steric effects, are determined empirically or
by calculations. AChE inhibitions by carbamates 1 show
Hammett-type correlation (Eq (2)),15 and AChE inhibitions
Fig. 1. The active site and peripheral anionic binding
site (PAS) of AChE. Numbers refer to amino
acid residue positions in Torpedo californica
AChE.4 The active site of AChE contains of (a)
an esteratic site (ES), (b) an oxyanion hole
(OAH), (c) an anionic substrate binding site
(AS), and (d) an acyl binding site (ABS). PAS is
located at the entrance (mouth) of the active site
gorge.8-10
Fig. 2. Chemical structures of carbamates 1 and 2.

Hammett-Taft Cross-interaction for AChE Inhibition
J. Chin. Chem. Soc., Vol. 51, No. 2, 2004 425
by carbamates 2 show Taft-Ingold-type correlation (Eq (3)).1
between X and R substituents in the reaction transition state
is inversely proportional to |rXR|.28
logk = h + rs
(2)
(3)
The Hammett-Taft cross-interaction correlations for
cholesterol esterase inhibitions by carbamates 1 and 2 with
Eq (4) have revealed that the geometry for the carbamate
C(O)-N fragment in the enzyme reaction transition state is all
retained in pseudo-trans conformations.27 In this paper, we
further report the Hammett-Taft cross-interaction correla-
tions for the AChE inhibitions by carbamates 1 and 2.
logk = h + r*s* + dES
In Eqs. (2) and (3), the parameters s, s*, r, r*, d, Es,
and h are Hammett substituent constant, Taft substituent con-
stant, the reaction constant (the intensity factor of inductive
effect) for Hammett substituents, the reaction constant for
Taft substituents, the intensity factor of steric effect, and Taft
substituent steric constant, the calculated logk value (logk0)
for phenyl-N-butylcarbamate (X=H, Eq (2)) or 4-nitrophen-
yl-N-methylcarbamate (R=CH3, Eq (3)), respectively. Cho-
lesterol esterase inhibitions by carbamates 1 show Hammett-
type correlation,19,24,25 and cholesterol esterase inhibitions by
carbamates 2 show Taft-type correlation.19,26
RESULTS
Carbamates 1 and 2 are characterized as pseudo-
pseudo-substrate inhibitors of AChE because all inhibitors
are time-dependent and follow first-order kinetics but the en-
zyme activities are not recovered by a competitive inhibitor,
edrophonium.1 Hammett and Taft substituent constants, Ki,
and kc for the AChE inhibitions by carbamates 115 and 21 are
summarized (Table 1).
Cholesterol esterase inhibitions by carbamates 1 and 2
show Hammett-Taft cross interaction correlation (Eq. (4)).27
logk = h + rs + r*s* + rXR sa s*
(4)
Multiple linear correlations of the -logKi and logkc val-
ues against the Hammett-Taft cross-interaction correlation
equation (Eq (4)) are poor. However, multiple linear correla-
tions of the logkc values against the modified Hammett-Taft
cross-correlation equation (Eq (5)) are much improved
(Table 2).
Multiple correlations of -logKi, logkc and logki against
Hammett-Taft cross-correlation Eq (4) are poor. In Eq (4), the
h, rXR, and a values are the calculated logk value for phenyl-
N-methylcarbamate, Hammett-Taft cross-interaction con-
stant, and the weighing factor for Hammett-Taft cross-inter-
action term (a =1 for Hammett substituent X and a = 2.54 for
Taft substituent R), respectively. The intramolecular distance
logk = h + r s + r*a s* + rXR sa s*
(5)
Table 1. Hammett and Taft Substituent Constants, Ki, and kc for AChE Inhibitions by Carbamates 1
and 2a
X
R
s
s*
Ki (mM)
kc (10-4s-1)
p-OMe
H
m-OMe
p-Cl
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
t-Bu
n-Hex
n-Oct
C2H4Cl
CH2Ph
Allyl
-0.27
0
-0.13
-0.13
-0.13
-0.13
-0.13
-0.13
-0.13
-0.13
-0.13
-0.3
1.0 ± 0.1
3.0 ± 0.2
3.7 ± 0.4
4.0 ± 0.4
8.1 ± 0.6
8.6 ± 0.7
14 ± 1
23 ± 2
29 ± 2
29 ± 2
37 ± 4
2.00 ± 0.04
2.96 ± 0.05
3.00 ± 0.06
3.30 ± 0.04
3.60 ± 0.08
3.80 ± 0.07
4.00 ± 0.09
4.70 ± 0.08
5.0 ± 0.1
0.12
0.23
0.37
0.43
0.54
0.71
0.78
0.78
0.78
0.78
0.78
0.78
0.78
m-Cl
m-CF3
p-CF3
m-NO2
p-NO2
p-NO2
p-NO2
p-NO2
p-NO2
p-NO2
p-NO2
5.0 ± 0.1
-0.4
4.70 ± 0.06
4.60 ± 0.05
6.10 ± 0.08
5.60 ± 0.04
5.30 ± 0.04
-0.33
0.39
0.22
0.1
49 ± 5
120 ± 10
70 ± 6
48 ± 5
a Published previously.1,15

426 J. Chin. Chem. Soc., Vol. 51, No. 2, 2004
Lin
therefore, Ki’ is the virtual inhibition constant. The third step
(kc) is formation of the carbamyl enzyme from the tetrahedral
intermediate.
Table 2. Hammett-Taft Cross-interaction Correlation Results for
AChE Inhibitions by Carbamates 1 and 2
a
d
pKa
-logKi’b,c
logkc
For the Kb step, the O-C(O)-N-R moieties of the inhibi-
tors and the protonated inhibitors are all retained in pseudo-
trans conformations due to small |rXR| values.27
r
-2.5 ± 0.1
-1.5 ± 0.7
0.3 ± 0.3
9.8 ± 0.1
0.9983
1.3 ± 0.1
0.5 ± 0.3
0.5 ± 0.3
9.5 ± 0.1
0.9948
0.35 ± 0.03
0.05 ± 0.08
0.0 ± 0.1
-3.56 ± 0.03
0.9892
r*
rXR
he
Rf
For the Ki’ step, the small |rXR| value of 0.5 (Table 2) in-
dicates that the carbamate O-C(O)-N-R moieties of the tran-
sition states that lead to the tetrahedral intermediates are also
all retained in pseudo-trans conformations (Fig. 4),27,28 in
contrast to the cholesterol esterase inhibitions by carbamates
1 and 2.27 Therefore, the inhibitor R groups do not bind to
ABS of AChE but stretch along the active gorge (Fig. 5), and
this result confirms the X-ray structure of the enzyme
carbamylated by a long chain carbamate inhibitor, 8-(cis-
2,6-dimethylmorpholino)octylcarbamoyleseroline.7 The
binding for the N+H2R moieties of carbamates 1 and 2 to the
active site gorge of the enzyme is probably because the con-
cave ABS of the enzyme is too small to accommodate a bulky
carbamate substituent (Fig. 5).6
a Taken from references.19,27
b -log Ki’ = -logKi – pKa + 14.
c Correlation of the -logKi’ values against Eq (5) (a = 1 for
carbamates 1; a = 2.54 for carbamates 2).
d Correlation of logkc values against Eq (5) (a = 1 for carbamates
1; a = 2.54 for carbamates 2).
e Calculated pKa, -logKi’, and logkc values for phenyl-N-
methylcarbamate.
f Correlation coefficient.
On the other hand, multiple linear correlations of the -logKi
values against Eq (5) are still poor. Since the Ki step is com-
posed of protonation of carbamates 1 and 2 and the Ki’ step
(Fig. 3), the virtual inhibition constant, Ki’, can be calculated
according to Eq (6).
For the kc step, the |rXR| value of 0 (Table 2) indicates
that the distance between X and R substituents of the transi-
tion states that lead to the carbamyl enzyme is very long and
suggests that the transition states are breaking C-O bonds and
excluding the leaving groups, substituted phenols. The leav-
ing group, substituted phenol, may bind to AS and to the
conformational changed H440 (Fig. 3).14
-log Ki’ = –logKi – pKa + 14
(6)
Finally, multiple linear correlations of the –logKi’ values
against Eq (5) are excellent (Table 2).
DISCUSSION
EXPERIMENTAL
The r value of 1.2 for the –logKi’-s-as*-sas*-corre-
lation (Eq (5) & Table 2) agrees with the value for the
-logKi-s-correlation (-1.32)15 minus the value for pKa-s-cor-
relation (-2.5).19 The r* value of 0.5 for the -logKi’-s-as*-
sas* correlation (Eq (5) & Table 2) is close to the value for
-logKi-s*-correlation (-1.2)1 minus the value for pKa-s*-cor-
relation (-1.4).19 Therefore, multiple linear correlations
against the modified Hammett-Taft cross-interaction equa-
tion (Eq (5)) are valid. Furthermore, the positive r and r*
values for the -logKi’-s-as*-sas*-correlation suggest that
the Ki’ step is formation of the negative charge enzyme-
carbamate tetrahedral intermediate (Fig. 3).1
Materials
Electrophorus electricus AChE, DTNB, and ATCh
were obtained from Sigma; other chemicals were obtained
from Aldrich; silica gel used in liquid chromatography
(Licorpre Silica 60, 200-400 mesh) and thin-layer chroma-
tography plates (60 F254) were obtained from Merck. All
other chemicals were of the highest purity available commer-
cially. Synthesis of carbamates 1 and 2 has been reported pre-
viously.19
Instrumental Methods
All steady state kinetic data were obtained from a
UV-VIS spectrophotometer (HP 8452 or Beckman DU-650)
with a cell holder circulated with a water bath.
A three-step mechanism for the AChE inhibition by
carbamates 1 and 2 is proposed (Fig. 3).1 The first step (Kb) is
protonation of the inhibitors. The second step (Ki’) is forma-
tion of the enzyme-carbamate tetrahedral intermediate from
nucleophilic attack of S200 to the protonated inhibitors;
Data Reduction
Origin (version 6.0) was used for linear, nonlinear, and

Hammett-Taft Cross-interaction for AChE Inhibition
J. Chin. Chem. Soc., Vol. 51, No. 2, 2004 427
Fig. 3. The proposed AChE inhibition mechanism by carbamates 1 and 2. The O-C(O)-N-R geometries of carbamates 1 and
2 are pseudo-trans conformations. The first step (Kb) is protonation of the inhibitors.1 The O-C(O)-N-R geometries of
protonated carbamates 1 and 2 are retained in pseudo-trans conformations. The second step (Ki’) is formation of the
enzyme-carbamate tetrahedral intermediate from nucleophilic attack of S200 to the protonated inhibitors; therefore,
Ki’ is the virtual inhibition constant. The O-C(O)-N-R geometries of the tetrahedral intermediates are also retained in
pseudo-trans conformations. Therefore, the R moieties of the tetrahedral intermediates do not bind to ABS but to the
active site gorge of the enzyme. The third step (kc) is formation of the carbamyl enzyme intermediate from the tetra-
hedral intermediate. The leaving group, substituted phenol, may bind to AS and to the conformational changed
H440.14

428 J. Chin. Chem. Soc., Vol. 51, No. 2, 2004
Lin
multiple linear least-squares regression analyses.27
Steady-State Enzyme Kinetics
The AChE inhibition by carbamates 115 and 21 was as-
sayed by Ellman’s method.29 The temperature was main-
tained at 25.0 ± 0.1 °C by a refrigerated circulating water
bath. All inhibition reactions were performed in sodium
phosphate buffer (1 mL, 0.1 M, pH 7.0) containing NaCl (0.1
M), acetonitrile (2% by volume), triton X-100 (0.5% by
weight), substrate (0.2 mM), and varying concentrations of
inhibitors. Requisite volumes of stock solution of substrate
and inhibitors in acetonitrile were injected into reaction
buffer via a pipet. AChE was dissolved in sodium phosphate
buffer (0.1 M, pH 7.0). First-order rate constant (kapp) for in-
hibition was determined as described by Hosie et al.18 The
Ki and kc values were obtained by fitting the kapp values and
[I] to Eq (1) by nonlinear least-squares regression analy-
ses.1,15,17-19,24-27,30-33 Duplicate sets of data were collected for
each inhibitor concentration.
Fig. 4. The proposed transition states that lead to the
AChE-carbamate tetrahedral intermediates.
The O-C(O)-N-R geometries of the transition
states are retained in pseudo-trans conforma-
tions. The R moieties of the transitional states
do not bind to ABS but to the active site gorge
of the enzyme.
ACKNOWLEDGEMENTS
We thank the National Science Council of Taiwan for
financial support.
Received June 9, 2003.
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