Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates as conformationally constrained inhibitors of acetylcholinesterase

Article abstract of DOI:10.1002/jbt.20202

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are synthesized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively. All carbamates 1-15 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. For a series of geometric isomers, the inhibitory potencies are as follows: benzene-1,4-di-N-substituted carbamate (para compound) > benzene-1,3-di-N-substituted carbamate (meta compound) > benzene-1,2-di-N-substituted carbamate (ortho compound). Therefore, benzene-1,4-di-N-substituted carbamates (para compounds), with the angle of 180° between two C(benzene)-O bonds, mimic the preferable anti C-O/C-N conformers of acetylcholine for the choline ethylene backbone in the acetylcholinesterase catalysis.

Full text of DOI:10.1002/jbt.20202

J BIOCHEM MOLECULAR TOXICOLOGY
Volume 21, Number 6, 2007
Benzene-1,2-, 1,3-, and 1,4-di-N-Substituted
Carbamates as Conformationally Constrained
Inhibitors of Acetylcholinesterase
Ming-Cheng Lin,1 Mei-Ting Hwang,2 Han-Ging Chang,2 Chung-Sheng Lin,1
and Gialih Lin2
1Institute of Medicine, Department of Cardiology, Chung Shan Medical University Hospital, Taichung 402, Taiwan
2Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan; E-mail: gilin@dragon.nchu.edu.tw
Received 23 May 2007; accepted 20 June 2007
ABSTRACT: Benzene-1,2-, 1,3-, and 1,4-di-N-
substituted carbamates (1–15) are synthesized as
the conformationally constrained inhibitors of
acetylcholinesterase and mimic gauche, eclipsed, and
anti-conformations of acetylcholine, respectively. All
carbamates 1–15 are characterized as the pseudo sub-
strate inhibitors of acetylcholinesterase. For a series
of geometric isomers, the inhibitory potencies are as
follows: benzene-1,4-di-N-substituted carbamate (para
compound) > benzene-1,3-di-N-substituted carbamate
and 3 in humans, respectively. Both subtypes unselec-
tive cholinesterase and AChE-selective inhibitors have
been used in Alzheimer’s disease to amplify the ac-
tion of acetylcholine at remaining cholinergic synapses
within the Alzheimer’s disease brain. The X-ray crystal
structures of AChE have revealed that AChE contains
a catalytic triad similar to that present in other serine
hydrolases. They have also revealed that this triad is lo-
cated near the bottom of a deep and narrow gorge about
˚
20 A in depth [2]. The X-ray crystal structures of BChE
(meta
compound) > benzene-1,2-di-N-substituted
carbamate (ortho compound). Therefore, benzene-
1,4-di-N-substituted carbamates (para compounds),
with the angle of 180◦ between two C(benzene) O
bonds, mimic the preferable anti C O/C N con-
formers of acetylcholine for the choline ethylene
have been recently reported [4,5,7]. AChE and BChE
have a common catalytic triad, Ser–His–Glu. The active
sites of both enzymes are located at the bottom of a cav-
ity and act as nucleophiles while attacking the carbonyl
groups of substrates or pseudo substrate inhibitors.
Carbamate inhibitors, such as Alzheimer’s disease
drug Rivastigmine (Exelon) and aryl carbamates, are
characterized as the pseudo substrate inhibitors of
AChE, BChE, cholesterol esterase, and lipase [3,8–15].
In the presence of substrate, the kinetic scheme for
pseudo substrate inhibitions of serine hydrolases by
carbamate inhibitors is proposed (Figure 1) [8]. Since
this inhibition follows first-order kinetics over the ob-
served time period for steady-state kinetics, the hydrol-
ysis rate of carbamyl enzyme EI’ must be significantly
slower than the EI’ formation rate (k2 ꢀ k3). Therefore,
values of Ki and k2 can be calculated from Eq. (1) [8].
In Eq. (1), the kapp values are first-order rate constants
which are obtained by Hosie’s method. The bimolec-
ular rate constant, ki = k2/Ki , is related to overall in-
hibitory potency:
backbone in the acetylcholinesterase catalysis. ꢀ 2007
C
Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:348–
353, 2007; Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10:1002/jbt.20202
KEYWORDS: Acetylcholinesterase; Carbamate; Confor-
mation; Inhibitor
INTRODUCTION
Two forms of cholinesterase coexist ubiquitously
throughout the body, acetylcholinesterase (AChE, EC
3.1.1.7) [1–3] and butyrylcholinesterase (BChE, EC
3.1.1.8) [4–7], and although highly homologous, >65%,
they are products of different genes on chromosomes 7
kapp = k2[I]/(Ki (1 + [S]/Km) + [I])
(1)
Correspondence to: Gialih Lin.
Contract Grant Sponsor: Department of Pharmaceutical Sci-
ences, St. John’s University, Jamaica, New York.
2007 Wiley Periodicals, Inc.
Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carba-
mates (1–15) are synthesized as the conformationally
constrained analogs [16,17] of acetylcholine since
c
ꢀ
348

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CONFORMATIONALLY CONSTRAINED INHIBITORS OF AChE
349
MATERIALS AND METHODS
Materials
All chemicals were of the highest grade available.
Silica gel used in liquid chromatography and thin-layer
chromatography plates were obtained from Merk. Elec-
trophorus electricus AChE, acetylthiocholine, and 5,5ꢁ-
dithio-bis-2- nitrobenzoate were obtained from Sigma.
FIGURE 1. Kinetic scheme for the pseudo substrate inhibition of
carbamates in the presence of substrate. E, enzyme; S, substrate; ES,
acylenzyme intermediate; I, carbamate, EI, enzyme-inhibitor tetra-
hedral intermediate; EIꢁ, carbamyl enzyme intermediate; P, the first
product; Q, the second product.
Chemistry
these compounds mimic gauche, eclipsed, and anti-
conformations of acetylcholine at the choline-ethylene
backbone), respectively (Figure 2). In other words,
with the angles of 60◦, 120◦, and 180◦ between two
C(benzene) O bonds, benzene-1,2-di-N-substituted
carbamates (1–5), benzene-1,3-di-N-substituted carba-
mates (6–10), and benzene-1,4-di-N-substituted car-
bamates (11–15) mimic gauche, eclipsed, and anti-
C O/C N conformers of acetylcholine in the acetyl-
cholinesterase catalysis, respectively.
Benzene-1,2-di-N-substituted carbamates (1–5),
benzene-1,3-di-N-substituted carbamates (6–10), and
benzene-1,4-di-N-substituted carbamates (11–15)
(Figure 2) were synthesized from the condensation of
catechol, resorcinol, and hydroquinone, respectively,
with three equivalents of the corresponding isocyanate
in triethylamine at 25◦C for 24 h (65–90% yield). The
products were purified by liquid chromatography
(silica gel, hexane-ethyl acetate) and characterized by
1H and 13C NMR spectra.
FIGURE 2. Possible conformations of acetylcholine and chemical structures of inhibitors 1–15.
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LIN ET AL.
Volume 21, Number 6, 2007
1
C-6 of benzene ring), 129.0 (C-2 of benzene ring), 151.4
(C-1 and C-3 of benzene ring), and 154.2 (carbamate
C O).
Benzene-1,2-di-N-n-butylcarbamate (1): H NMR
(CDCl3, 400 MHz) δ/ppm 0.93 (t, J = 7 Hz, 6H, ω-
CH3), 1.35 (sextet, J = 7 Hz, 4H, γ-CH2), 1.52 (quintet,
J = 7 Hz, 4H, β-CH2), 3.24 (q, J = 7 Hz, 4H, α-CH2),
5.09 (s, 2H, NH), and 7.15–7.24 (m, 4H, benzene-H).
13C NMR (CDCl3, 100 MHz, assignment from DEPT
experiments) δ/ppm 13.5 (ω-CH3), 19.6 (γ-CH2), 31.6
(β-CH2), 40.8 (α-CH2), 123.4 (C-4 and C-5 of benzene
ring), 125.8 (C-3 and C-6 of benzene ring), 143.0 (C-1
and C-2 of benzene ring), and 153.9 (carbamate C O).
1
Benzene-1,3-di-N-n-hexylcarbamate (7): H NMR
(CDCl3, 400 MHz) δ/ppm 0.88 (t, J = 7 Hz, 6H, ω-CH3),
1.30–1.35 (m, 12H, γ- to ω-1-CH2), 1.52–1.57 (m, 4H, β-
CH2), 3.23 (q, J = 7 Hz, 4H, α-CH2), 4.98 (s, 2H, NH),
and 6.90–7.35 (m, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
13.9 (ω-CH3), 22.4, 26.3, 29.6 (γ-, δ-, and ε-CH2), 31.3 (β-
CH2), 41.2 (α-CH2), 115.3 (C-5 of benzene ring), 118.1
(C-4 and C-6 of benzene ring), 129.1 (C-2 of benzene
ring), 151.4 (C-1 and C-3 of benzene ring), and 154.2
(carbamate C O).
1
Benzene-1,2-di-N-n-hexylcarbamate (2): H NMR
(CDCl3, 400 MHz) δ/ppm 0.88 (t, J = 7 Hz, 6H, ω-CH3),
1.26–1.35 (m, 12H, γ- to ω-1-CH2), 1.51–1.55 (m, 4H, β-
CH2), 3.23 (q, J = 7 Hz, 4H, α-CH2), 5.08 (s, 2H, NH),
and 7.15–7.24 (m, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
13.9 (ω-CH3), 22.4, 26.3, 29.6 (γ-, δ-, and ε-CH2), 31.3
(β-CH2), 41.2 (α-CH2), 123.4 (C-4 and C-5 of benzene
ring), 125.9 (C-3 and C-6 of benzene ring), 143.0 (C-1
and C-2 of benzene ring), and 153.9 (carbamate C O).
Benzene-1,2-di-N-n-octylcarbamate (3): 1H NMR
(CDCl3, 400 MHz) δ/ppm 0.89 (t, J = 7 Hz, 6H, ω-CH3),
1.26–1.30 (m, 20H, γ- to ω-1-CH2), 1.51–1.57 (m, 4H, β-
CH2), 3.23 (q, J = 7 Hz, 4H, α-CH2), 5.06 (s, 2H, NH),
and 7.15–7.24 (m, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
14.0 (ω-CH3), 22.5, 26.2, 26.3, 29.1, 29.7 (γ- to ω-1-CH2),
31.7 (β-CH2), 41.2 (α-CH2), 123.4 (C-4 and C-5 of ben-
zene ring), 126.0 (C-3 and C-6 of benzene ring), 143.0
(C-1 and C-2 of benzene ring), and 153.9 (carbamate
C O).
Benzene-1,3-di-N-n-octylcarbamate (8): 1H NMR
(CDCl3, 400 MHz) δ/ppm 0.88 (t, J = 7 Hz, 6H, ω-CH3),
1.26–1.33 (m, 20H, γ- to ω-1-CH2), 1.52–1.57 (m, 4H, β-
CH2), 3.23 (q, J = 7 Hz, 4H, α-CH2), 4.97 (s, 2H, NH),
and 6.90–7.35 (m, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
14.0 (ω-CH3), 22.6, 26.7, 29.1, 29.2, 29.7 (γ- to ω-1-CH2),
31.7 (β-CH2), 41.2 (α-CH2), 115.3 (C-5 of benzene ring),
118.2 (C-4 and C-6 of benzene ring), 129.2 (C-2 of ben-
zene ring), 151.5 (C-1 and C-3 of benzene ring), and
154.2 (carbamate C O).
Benzene-1,3-di-N-t-butylcarbamate (9): 1H NMR
(CDCl3, 400 MHz) δ/ppm 1.36 (s, 18H, CH3), 5.09 (s,
2H, NH), and 6.90–7.35 (m, 4H, benzene-H). 13C NMR
(CDCl3, 100 MHz, assignment from DEPT experiments)
δ/ppm 28.7 (CH3), 50.8 (C(CH3)3), 115.3 (C-5 of ben-
zene ring), 118.3 (C-4 and C-6 of benzene ring), 129.2
(C-2 of benzene ring), 151.3 (C-1 and C-3 of benzene
ring), and 152.3 (carbamate C O).
Benzene-1,2-di-N-t-butylcarbamate (4): 1H NMR
(CDCl3, 400 MHz) δ/ppm 1.36 (s, 18H, CH3), 5.09 (s,
2H, NH), and 7.15–7.24 (m, 4H, benzene-H). 13C NMR
(CDCl3, 100 MHz, assignment from DEPT experiments)
δ/ppm 28.7 (CH3), 50.8 (C(CH3)3), 123.5 (C-4 and C-5
of benzene ring), 125.9 (C-3 and C-6 of benzene ring),
142.9 (C-1 and C-2 of benzene ring), and 151.8 (car-
bamate C O). Benzene-1,2-di-N-benzylcarbamate (5):
1H NMR (CDCl3, 400 MHz) δ/ppm 4.33 (d, J = 6 Hz,
4H, CH2Ph), 5.09 (s, 2H, NH), and 7.15–7.24 (m, 4H,
1,2-benzene-H), 7.27–7.30 (m, 10H, phenyl-H of benzyl
group). 13C NMR (CDCl3, 100 MHz, assignment from
DEPT experiments) δ/ppm 45.3 (benzyl CH2), 123.5,
126.2, 127.4, 127.7, 128.6, 138.1, 142.9 (aromatic Cꢁs),
and 153.9 (carbamate C O).
1
Benzene-1,3-di-N-benzylcarbamate (10): H NMR
(CDCl3, 400 MHz) δ/ppm 4.43 (d, J = 2 Hz, 4H,
CH2Ph), 5.09 (s, 2H, NH), 6.90–7.35 (m, 4H, benzene-
H), and 7.27–7.37 (m, 10H, phenyl-H of benzyl group).
13C NMR (CDCl3, 100 MHz, assignment from DEPT
experiments) δ/ppm 45.3 (benzyl CH2), 115.3, 118.4,
127.7, 127.8, 128.8, 129.4, 137.9, 151.4 (aromatic Cꢁs),
and 154.2 (carbamate C O).
Benzene-1,4-di-N-n-butylcarbamate (11): 1H NMR
(CDCl3, 400 MHz) δ/ppm 0.93 (t, J = 7 Hz, 6H,
ω-CH3), 1.37 (sextet, J = 7 Hz, 4H, γ-CH2), 1.53 (quin-
tet, J = 7 Hz, 4H, β-CH2), 3.24 (q, J = 7 Hz, 4H, α-
CH2), 4.98 (s, 2H, NH), and 7.07 (s, 4H, benzene-H).
13C NMR (CDCl3, 100 MHz, assignment from DEPT
experiments) δ/ppm 13.7 (ω-CH3), 19.9 (γ-CH2), 31.8
(β-CH2), 40.9 (α-CH2), 122.2 (C-2, C-3, C-5, and C-6 of
benzene ring), 148.0 (C-1 and C-4 of benzene ring), and
154.5 (carbamate C O).
1
Benzene-1,3-di-N-n-butylcarbamate (6): H NMR
(CDCl3, 400 MHz) δ/ppm 0.93 (t, J = 7 Hz, 6H, ω-CH3),
1.37 (sextet, J = 7 Hz, 4H, γ-CH2), 1.52 (quintet, J =
7 Hz, 4H, β-CH2), 3.24 (q, J = 7 Hz, 4H, α-CH2), 4.99 (s,
2H, NH), and 6.90–7.40 (m, 4H, benzene-H). 13C NMR
(CDCl3, 100 MHz, assignment from DEPT experiments)
δ/ppm 13.5 (ω-CH3), 19.7 (γ-CH2), 31.6 (β-CH2), 40.7
(α-CH2), 115.3 (C-5 of benzene ring), 118.0 (C-4 and
Benzene-1,4-di-N-n-hexylcarbamate (12): 1H NMR
(CDCl3, 400 MHz) δ/ppm 0.88 (t, J = 7 Hz, 6H, ω-
CH3), 1.30–1.35 (m, 12H, γ- to ω-1-CH2), 1.52–1.55 (m,
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CONFORMATIONALLY CONSTRAINED INHIBITORS OF AChE
351
TABLE 1. The Inhibition Constants of AChE by Inhibitors
1–15
4H, β-CH2), 3.24 (q,J = 7 Hz, 4H, α-CH2), 4.97 (s, 2H,
NH), and 7.07 (s, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
14.0 (ω-CH3), 22.5, 26.4, 29.7 (γ-, δ-, and ε-CH2), 31.4
(β-CH2), 41.3 (α-CH2), 122.2 (C-2, C-3, C-5 and C-6 of
benzene ring), 148.0 (C-1 and C-4 of benzene ring), and
154.5 (carbamate C O).
a
a
Inhibitors
Ki (nM)a
k2 (10−3 s−1
)
ki (105 M−1 s−1
)
1
2
3
4
5
6
7
8
23
13
44
29
70 20
2
7
6
5
8
6
9.6 0.4
6.1 0.1
5.3 0.1
7.8 0.2
6.1 0.1
6.9 0.3
7.5 0.3
6.5 0.1
5.47 0.03
5.91 0.08
8.2 0.2
7.2 0.2
6.8 0.1
4
5
1
2
1.2 0.2
2.7 0.6
0.8 0.3
30 10
11
1.7 0.2
6.0 0.7
1
Benzene-1,4-di-N-n-octylcarbamate (13): H NMR
1
2
5
1
(CDCl3, 400 MHz) δ/ppm 0.87 (t, J = 7 Hz, 6H, ω-
CH3), 1.26–1.30 (m, 20H, γ- to ω-1-CH2), 1.52–1.55 (m,
4H, β-CH2), 3.24 (q, J = 7 Hz, 4H, α-CH2), 4.97 (s, 2H,
NH), and 7.07 (s, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
14.0 (ω-CH3), 22.6, 26.7, 29.1, 29.2, 29.8 (γ- to ω-1-CH2),
31.7 (β-CH2), 41.3 (α-CH2), 122.2 (C-2, C-3, C-5 and C-6
of benzene ring), 148.0 (C-1 and C-4 of benzene ring),
and 154.5 (carbamate C O).
3
39
9
9
10
11
12
13
14
15
930 80
1.7 0.3
5
16
3.6 0.6
0.06 0.01
48
15
4
16
3
7
4
1
3
1
1
4
5.90 0.08
2.06 0.01
7
3
a Obtained from the nonlinear least-squares curve fittings of kp vs [I] plot
against Eq. (1) [8].
1
Benzene-1,4-di-N-t-butylcarbamate (14): H NMR
(CDCl3, 400 MHz) δ/ppm 1.36 (s, 18H, CH3), 5.09 (s, 2H,
NH), and 7.06 (s, 4H, benzene-H). 13C NMR (CDCl3,
100 MHz, assignment from DEPT experiments) δ/ppm
29.0 (CH3), 51.1 (C(CH3)3), 122.6 (C-2, C-3, C-5 and C-6
of benzene ring), 148.1 (C-1 and C-4 of benzene ring),
and 152.9 (carbamate C O).
lowed continuously at 410 nm on a UV–visible spec-
trometer at 25◦C, pH 7.1. The Ki and k2 values were
obtained from the nonlinear least-squares of curve fit-
tings of the kapp values vs inhibition concentration ([I])
plot against Eq. (1) (Figure 3 and Table 1).
1
Benzene-1,4-di-N-benzylcarbamate (15): H NMR
(CDCl3, 400 MHz) δ/ppm 4.34 (d, J = 6 Hz, 4H,
CH2Ph), 5.09 (s, 2H, NH), and 7.09 (s, 4H, 1,2-benzene-
H), 7.28–7.35 (m, 10H, phenyl-H of benzyl group). 13
C
RESULTS
NMR (CDCl3, 100 MHz, assignment from DEPT ex-
periments) δ/ppm 45.3 (benzyl CH2), 122.1 (C-2, C-3,
C-5 and C-6 of benzene ring), 127.4, 127.9, 128.7, 140.0
(phenyl C’s of benzyl group), 149.0 (C-1 and C-4 of
benzene ring), and 154.9 (carbamate C O).
In order to mimic different conformations of
acetylcholine at the choline ethylene backbone
(Figure 2), benzene-1,2-di-N-substituted carbamates
(1–5), benzene-1,3-di-N-substituted carbamates (6–10),
and benzene-1,4-di-N-substituted carbamates (11–15)
(Figure 2) were synthesized from condensation of cat-
echol, resorcinol, hydroquinone, respectively, with the
corresponding isocyanate in the presence of excess tri-
ethylamine.
Inhibitors 1–15 were all characterized as the pseudo
substrate inhibitors (Figure 1) of AChE (Figure 3 and
Table 1). For a series of geometric isomers, the
inhibitory potencies are as follows: benzene-1,4-
Instrumental Methods
1H and 13C NMR spectra were recorded at 400 and
100 MHz, respectively, on a Varian-Gemini 400 spec-
trometer. All steady state kinetic data were obtained
from a UV–visible spectrometer (Agilent 8453) with a
cell holder circulated with a water bath.
di-N-substituted carbamate (para compound)
>
benzene-1,3-di-N-substituted carbamate (meta com-
pound) > benzene-1,2-di-N-substituted carbamate
(ortho compound).
Data Reduction
Origin (version 6.0) was used for the linear and
nonlinear least-squares curve fittings.
DISCUSSION
AChE Inhibition
Inhibitory Potencies for Varied
Di-Substituted Geometries
of Carbamates 1–15
The inhibition reactions of AChE were determined
by the Ellman assay [18] as described before [12].
The AChE-catalyzed hydrolysis of acetylthiocholine
(0.1 mM) in the presence of 5,5ꢁ-dithio-bis-2-
nitrobenzoate (0.1 mM) and inhibitors 1–15 were fol-
Benzene-1,2-di-N-substituted carbamates (1–5),
benzene-1,3-di-N-substituted carbamates (6–10), and
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352
LIN ET AL.
Volume 21, Number 6, 2007
FIGURE 4. Comparisons of the ki values of the pseudo substrate
inhibitions of AChE by inhibitors 1–15.
benzene-1,4-di-N-substituted carbamates (11–15)
(Figure 2) are characterized as the pseudo sub-
strate inhibitors (Figure 1) of AChE (Figure 3 and
Table 1). For a series of geometric isomers, for
example, benzene-1,2-di-N-n-butylcarbamate (1),
benzene-1,3-di-N-n-butylcarbamate (6), and benzene-
1,4-di-N-n-butylcarbamate (11), para compound 11 is
a more potent AChE inhibitor than meta compound
6 and meta compound 6 is more potent than ortho
compound 1 (Figure 4 and Table 1). Therefore, para
carbamates 11–15, with the angle of 180◦ between
two C(benzene) O bonds, mimic the preferable anti
C O/C N conformers of acetylcholine in the acetyl-
cholinesterase catalysis (Figure 2). In other words, the
ethylene backbone conformations of acetylcholine for
acetoxyl and trimethyl amine groups (Figure 2) may
fully extend in space.
Inhibitory Potencies for Varied Substituents
of Carbamates 1–15
When different carbamate substituents in 1–15 are
compared, compounds with less bulky substituents
such as n-butyl- and n-hexyl-carbamates are more po-
tent than those with bulky substituents such as n-octyl-,
t-butyl-, and benzyl-carbamates (Figure 4 and Table 1).
The possible reason for this is that the acetyl binding
site of AChE is relatively small when compared to other
serine hydrolases and therefore is suitable for small car-
bamate substituents [2].
FIGURE 3. Nonlinear least-squares curve fittings of kapp vs inhibitor
concentration ([I]) plots against Eq. (1) for the pseudo substrate in-
hibitions [8] of AChE by benzene-1,2-di-N-n-butylcarbamate (1) (A),
benzene-1,3-di-N-n-butylcarbamate (6) (B), and benzene-1,4-di-N-n-
butylcarbamate (11) (C). For A, the parameters of the fit were k2
=
0.0096 0.00004 s−1 and Ki = 23 6 nM (R2 = 0.98337). For B, the
parameters of the fit were k2 = 0.0069 0.00003 s−1 and Ki = 2.4
1.1 nM (R2 = 0.95018). For C, the parameters of the fit were k2
0.0082 0.00002 s−1 and Ki = 1.7 0.3 nM (R2 = 0.99085).
=
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CONFORMATIONALLY CONSTRAINED INHIBITORS OF AChE
353
cholinesterase and of its complexes with substrate and
products. J Biol Chem 2003;278:41141–41147.
8. Hosie L, Sutton LD, Quinn DM. p-Nitrophenyl and
cholesteryl-N-alkyl carbamates as inhibitors of choles-
terol esterase. J Biol Chem 1987;262:260–264.
The k2 Values
In contrast to Ki and ki values, the k2 values for
inhibitors 1–15 are about the same (Table 1). Therefore,
both sizes and geometries of the leaving groups for the
AChE-carbamate complexes (benzene-1-N-substituted
carbamate-2-, 3-, and 4-ols) (Figure 1) do not discrimi-
nate the k2 values.
9. Lin G, Lai C-Y. Hammett analysis of the inhibition of
pancreatic cholesterol esterase by substituted phenyl-N-
butylcarbamate. Tetrahedron Lett 1995;36:6117–6120.
10. Lin G, Lai C-Y. Linear free energy relationships
of the inhibition of pancreatic cholesterol esterase
by 4-nitrophenyl-N-alkylcarbamate. Tetrahedron Lett
1996;37:193–196.
11. Lin G, Chen G-H, Ho H-C. Conformationally re-
stricted carbamate inhibitors of horse serum butyryl-
cholinesterase. Bioorg Med Chem Lett 1998;8:2747–2750.
12. Lin G, Lai C-Y, Liao W-C. Molecular recognition
by acetylcholinesterase at the peripheral anionic site:
Structure-activity relationships for inhibitions by aryl
carbamates. Bioorg Med Chem 1999;7:2683–2689.
13. Lin G, Shieh C-T, Ho H-C, Chouhwang J-Y, Lin W-Y,
Lu C-P. Structure-reactivity relationships for the in-
hibition mechanism at the second alkyl-chain-binding
site of cholesterol esterase and lipase. Biochemistry
1999;38:9971–9981.
14. Lin G, Lai C-Y, Liao W-C, Kuo B-H, Lu C-P. Structure-
reactivity relationships as probes for the inhibition mech-
anism of cholesterol esterase by aryl carbamates: I.
Steady-state kinetics. J Chin Chem Soc 2000;47:489–500.
15. Lin G, Chouhwang J-Y. Quantitative structure-activity
relationships for the inhibition of Pseudomonas species
lipase by 4-nitrophenyl-N-substituted carbamates.
J Biochem Mol Biol Biophys 2001;5:301–308.
16. Lin G, Lin Y-F, Hwang M-T, Lin Y-Z. Benzene-di-N-
octylcarbamates as conformationally constrained phos-
pholipase A2 inhibitors. Bioorg Med Chem 2004;14:751–
755.
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