Molecular recognition by acetylcholinesterase at the peripheral anionic site: Structure-activity relationships for inhibitions by aryl carbamates

Article abstract of DOI:10.1016/S0968-0896(99)00213-8

Substituted phenyl-N-butyl carbamates (1-9Figure 2Chemical structures of carbamates 1-9 and edrophonium.) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irrev

Full text of DOI:10.1016/S0968-0896(99)00213-8

Bioorganic & Medicinal Chemistry 7 (1999) 2683±2689
Molecular Recognition by Acetylcholinesterase at the Peripheral
Anionic Site: Structure±Activity Relationships for Inhibitions by
Aryl Carbamates
Gialih Lin,* Cheng-Yue Lai and Wei-Cheng Liao
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
Received 1 April 1999; accepted 14 June 1999
AbstractÐSubstituted phenyl-N-butyl carbamates (1±9) are potent irreversible inhibitors of Electrophorus electricus acet-
ylcholinesterase. Carbamates 1±9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the
stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dis-
sociation constant of the enzyme±inhibitor adduct (Ki), the inactivation constant of the enzyme±inhibitor adduct (k2), and the
bimolecular inhibition constant (ki) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1±9 and the
Hammett substituent constant (s), are observed, and the reaction constants (rs) are 1.36, 0.35 and 1.01, respectively. Therefore,
the above reaction may form a positive charged enzyme±inhibitor intermediate at the peripheral anionic site of the enzyme and may
follow the irreversible inactivation by a conformational change of the enzyme. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
part of ACh and of various active site ligands binds
through a preferential interaction of quaternary nitro-
gens with the p electrons of aromatic groups; (d) an
active site-selective aromatic binding site (AACS) that is
contiguous with or near ES and AS and that is important
in binding aryl substrates and active site ligands;2 and (e)
an acyl binding site (ABS), Phe288 and Phe299, that
binds the acetyl group of ACh.4 Besides ®ve active sites,
AChE also has a peripheral anionic site (PAS), Trp279,
Tyr70, Tyr121, Asp72, Glu199 and Phe290,5±7 that may
bind to 9-aminoacridine, 9-amino-1,2,3,4-tetrahydro-
acridine (tacrine) and is >20 A from the active site.2
Acetylcholinesterase (AChE, EC 3.1.1.7) plays a major
role in the termination of impulse transmission at cho-
linergic synpases, where AChE catalyzes the hydrolysis
of the neurotransmitter acetylcholine (ACh).1,2 The
three-dimensional (3-D) X-ray structure of AChE from
Torpedo californica electric organ has been reported.3
The X-ray structure of a transition state analogue com-
plex of AChE has also been reported recently.4 The
active site of AChE also consists of at least ®ve major
binding sites (Fig. 1): (a) an oxyanion hole (OH), Gly118,
Gly119 and Ala201,3 that stabilizes the tetrahedral inter-
mediate; (b) an esteratic site (ES), that is comprised of the
catalytic triad Ser200-His440-Glu327;3 (c) an anionic
substrate (AS) binding site, Trp84, Glu199 and Phe330,3,4
that contains a small number of negative charges but
many aromatic residues, where the quaternary ammonium
In Alzheimer's disease, a neurological disorder, cholin-
ergic de®ciency in the brain has been reported.8,9
Therefore, synthesis and study of inhibitors of AChE
may help to develop useful drugs to treat such disease.
AChE has long been an attractive target for the rational
design of mechanism-based inhibitors such as organo-
phosphus compounds,10,11 ¯uoroketones,12,13 carba-
mates,14±17 quaternary ammonium salts, propidium,
edrophonium, and decamethonium,2,9,18 and amino
compounds, 9-aminoacridine, tacrine and Huperzine-
A.2,6,7,18,19
Key words: Acetylcholinesterase inhibition; peripheral site; carbamates.
Abbreviations: AACS, active site-selective aromatic cation binding site;
ABS, acyl binding site; ACh, acetylcholine; AChE, acetylcholinesterase;
AS, anionic substrate binding site; ATCh, acetylthiocholine; DTNB;
5,50-dithio-bis-2-nitrobenzoate; ES, esteratic site; Ki, dissociation con-
stant of the enzyme-inhibitor adduct; Ki, bimolecular inhibition constant;
k2, inactivation constant of the enzyme-inhibitor adduct; OH, oxyanion
hole; PAS, peripheral anionic site; s, Hammett substituent constant;
QSAR, quantitive structure±activity relationship; r, reaction constants.
* Corresponding author.
The active site binding inhibitors of AChE are well
known.2 9-Aminoacridine,5 tacrine and its derivatives,6
and propidium7 have been characterized as PAS-
directed inhibitors. We have also reported that both
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00213-8

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G. Lin et al. / Bioorg. Med. Chem. 7 (1999) 2683±2689
enantiomers of naphthyl-N-n-butyl carbamates are
PAS-directed inhibitors of AChE.17 In this paper, we
further use substituted phenyl-N-n-butyl carbamates (1±
9, Fig. 2) to probe the structure±reactivity relationships
at the PAS of AChE.
of inhibitors.25,31 Lin and Lai32,33 and Feaster et al.34
have reported the linear free energy relationships for the
inhibition of CEase by aryl carbamates.32±34
Results
The e€ects of structure on reactivity can be divided into
three major types:20±23 inductive, resonance and steric
e€ects. In most cases, two or all three of these are
operating, and it is usually not easy to tell how much of
the rate enhancement or decrease is caused by each of
the three e€ects. Quantitative treatment of the e€ects of
structure on reactivity was ®rst reported by Hammett
and known as the Hammett equation (eq (1)).
Carbamates 1±9 (Fig. 2) are prepared from the con-
densation of the corresponding phenol with 1.2 mol
equivalents of n-butyl isocyanate in the presence of a
catalytic amount of pyridine in dichloromethane at
25ꢀC for 48 h (76±85% yield).
In the presence of substrate, the mechanism of irreversible
inhibition of AChE has been proposed in Scheme 1.10,17
Because the inhibition of AChE follows ®rst-order
kinetics over the observed time period, the rate of
hydrolysis of E0I must be signi®cantly slower than the
rate of formation of E0I. Therefore, values of Ki and kc
can be calculated from eq (2):10,17,32±34,36±39
log k ˆ log k0 ‡ ꢀꢁ
ꢀ1†
In eq (1), the log k0 value and the parameters r and s
are the reaction constant (the intensity factor of the
inductive e€ect) and the Hammett substituent constant,
respectively. The s values are the scales for the mea-
surement of both inductive and resonance e€ects of
substituents. The sign for an electron-donating group
such as p-OCH3 is negative due to the through-reso-
nance e€ect of the methoxyl group at the para-position,
but the sign for an electron withdrawing group such as
m-OCH3 is positive due to the pure inductive e€ect of
the methoxyl group at the meta-position. QSARs in
serine protease catalysis24±29 and acetylcholinesterase30
have been reported. For enzyme inhibition, less atten-
tion has been paid to the correlation with the structure
k2‰IŠ
kapp
ˆ
ꢀ2†
‰SŠ
KIꢀ1 ‡
† ‡ ‰IŠ
Km
In eq (2), kapp values are the ®rst-order rate constants
which can be obtained according to Hosie's method.
Bimolecular rate constant, ki ˆ k2=Ki, is related to
overall inhibitory potency.
Carbamates 1±9 are characterized as irreversible inhibi-
tors of AChE due to the facts that the inhibition is time-
dependent and follows ®rst-order kinetics and that the
enzyme displays saturation kinetics with increasing con-
centration of inhibitor;40 however, carbamates 1±9 do
not meet the third criterion for active site-directed irre-
versible inhibitors as proposed by Abeles and May-
cock.40 In other words, the enzyme is not protected from
inhibition by carbamates 1±9 in the presence of a revers-
ible inhibitor, edrophonium. Therefore, carbamates 1±9
are characterized as PAS-directed inhibitors, like 9-
aminoacridine,6 tacrine,7 and binaphthyl carbamates.17
Table 1 summarizes the inhibition data Ki; k2, and ki for
the AChE-catalyzed hydrolysis of ATCh in the presence
of DTNB and carbamates 1±9. The ki values for elec-
tron-withdrawing groups such as NO2, CF3, Cl, and m-
OCH3 are less than that for the electron-donating one
(p-OCH3). Therefore, an electron-donating substituent
facilitates the inhibition, but an electron-withdrawing
Figure 1. The active site and the PAS of AChE. Numbers refer to
residue positions in Torpedo californica AChE.3
Scheme 1. Kinetic scheme for irreversible inhibition of AChE in the
presence of substrate.
Figure 2. Chemical structures of carbamates 1±9 and edrophonium.

G. Lin et al. / Bioorg. Med. Chem. 7 (1999) 2683±2689
2685
Table 1. The Hammett substituent constants and kinetic data for the
inhibition of AChE by carbamates 1±9
AChE is not protected from inhibition by carbamates 1±9
in the presence of a reversible inhibitor, edrophonium.
It is conceivable that carbamates 1±9 react irreversibly
at PAS, not at the active site.
4
1
1
1
Compound
X=
s
Ki (mM) k2 (10
s
)
ki (M
s )
2
1
3
4
5
7
6
8
9
p-OMe
H
m-OMe
p-Cl
m-Cl
m-CF3
p-CF3
m-NO2
p-NO2
0.27 1.0‹0.1 2.00‹0.04
0
0.12 3.7‹0.4 3.00‹0.06
0.23 4.0‹0.4 3.30‹0.04
0.37 8.1‹0.6 3.60‹0.08
0.43 8.6‹0.7 3.80‹0.07
200‹20
97‹7
81‹9
83‹8
44‹3
44‹4
29‹2
20‹2
17‹1
3.0‹0.2 2.90‹0.05
Tables 1 and 2 summarize both inhibition and correla-
tion data for the inhibition of AChE by carbamates 1±9.
All inhibitory potencies depend upon the s values of
substituents alone; therefore, the inhibitory potency is
predominated by both inductive and resonance e€ects
of substituents. The inhibitory potency for carbamate 1
toward AChE is the greatest due to the fact that the
inhibition is facilitated by the only electron-donating
substituent, p-OCH3. By switching the OCH3 group
from para to meta position, there is a signi®cant di€er-
ence in inhibition due to the fact that the OCH3 group
donates electrons via the through-resonance20±23 at para
position but withdraws electrons via the inductive e€ect
at meta position. By switching the NO2 group from para
to meta position, there is not much di€erence in inhibi-
tion due to the fact that the NO2 group withdraws elec-
trons at both para and meta positions. Since the
correlations (Table 2) applies for the s values for both
meta and para positions, the binding at the PAS of
AChE by carbamates 1±9 is nonspeci®c for the position
of the substituents. Therefore, the direction for the
binding between carbamates 1±9 and the PAS of AChE
may be perpendicular to the benzene ring of the inhi-
bitor molecules. Thus, substituents at either meta or
para position do not have much steric di€erence for this
binding.
0.54 14‹1
0.71 23‹2
0.78 29‹2
4.00‹0.09
4.70‹0.08
5.0‹0.1
substituent detracts the inhibition. From the Ki values,
the binding between the electron-donating substituted
carbamate 1 is bound more tightly to AChE than the
electron-withdrawing substituted carbamates 3±9. The
k2 values are insensitive to the substituents.
After quantitative structure±activity correlations of the
logarithms of the inhibition data (Table 1) by Hammett
equation (eq (1)), the results are shown in Figure 3 and
summarized in Table 2. The logarithms of the Ki; k2, and
ki values are correlated with the s values alone, and the r
values for these inhibition reactions are 1.36, 0.35 and
1.01, respectively. In other words, the inhibitory
potency depends upon the s values alone.
Discussion
Carbamates 1±9 are characterized as irreversible inhibi-
tors of AChE as proposed by Abeles and Maycock.40
The mechanism for the inhibition of AChE by carbamates
1±8 is proposed in Figure 4 according to the negative r
Figure 3. Plot of
log Ki; log k2, and log ki for the inhibition of AChE by carbamates 1±9 against s.

2686
G. Lin et al. / Bioorg. Med. Chem. 7 (1999) 2683±2689
value for
log Ki-s-correlation, the positive r values
PAS of AChE acts as the Lewis base to accept electrons
from the inhibitor molecules. Moreover, the EI adduct
is more positive than both enzyme and inhibitors due to
a negative r value ( 1.36). The second step of this
mechanism is less sensitive to the s values of sub-
stituents than the ®rst step. Therefore, the second step
of this mechanism is probably the conformational
change of the enzyme, which results in a small r value
(0.35). The second step of this mechanism (Fig. 4),
therefore, is proposed to be the conformational change
at Phe330 (the residue of AS near the entrance) of
AChE, which results in the partial blockade for the
entrance of substrate.18,42 The rotation of Phe330 also
results in the donation of electrons from Phe330 to PAS.
Therefore, the character of the Lewis base, PAS, is wea-
kened by this conformational change, and electrons par-
tially feed back to the inhibitor molecules, which results
in a slightly positive r value for this step. The overall
inhibition results in the donation of electrons from the
for
log k2-s-correlation (Table 2), the PAS-directed
inhibitors, and X-ray structural data for the PAS.3,5,18,41
The ®rst step of this mechanism (Fig. 4) is to form the
non-covalent EI adducts through the donation of elec-
trons from the inhibitor molecules to the aromatic rings of
PAS (Trp279, Tyr70, Tyr121 and Phe290). Therefore, the
Table 2. Structure±activity correlation results for the inhibition of
AChE by carbamates 1±9a
logKi
logk2
logki
r
1.36‹0.06
5.61‹0.03
0.99
0.35‹0.02
3.57‹0.01
0.99
1.01‹0.05
2.04‹0.02
0.99
Constantb
Rc
a
Hammett equation (eq (1)) is used in correlations.
b
Calculated
(X=H) from eq (1).
log Ki; log K2, and log Ki values for carbamate 2
c
The correlation coecient.
Figure 4. The proposed mechanism for the inhibition of AChE by carbamates 1±9 at the PAS of the enzyme. Numbers refer to residue positions in
Torpedo californica AChe.3

G. Lin et al. / Bioorg. Med. Chem. 7 (1999) 2683±2689
2687
inhibitor molecules to the PAS of the enzyme due to the
negative r value ( 1.01) for the logarithms of ki.
4-Methoxyphenyl-N-n-butyl carbamate (2). 1H NMR
(CDCl3, 300 MHz) d 0.95 (t, J=7 Hz, 3H, o-CH3), 1.38
(sextet, J=7.2 Hz, 2H, g-CH2), 1.54 (quintet, J=7.2 Hz,
2H, b-CH2), 3.22 and 3.28 (ABq, J=6.6 Hz, 2H, a-CH2),
3.78 (s, 3H, OCH3), 5.04 (s, 1H, NH), 6.84±7.06 (m, 4H,
phenyl-H); 13C NMR (CDCl3, 75.4 MHz) d 13.51 (o-C),
16.79 (g-C), 31.67 (b-C), 40.75 (a-C), 55.37 (OCH3),
114.14 (phenyl C-3,5), 122.37 (phenyl C-2,6), 144.59
(phenyl C-4), 155.06 (phenyl C-1), 156.74 (C=O). High
resolution mass spectrum: exact mass: 223.1208. Found:
223.1201. Anal. calcd for C12H17O3N: C, 64.54; H, 7.68;
N, 6.28. Found: C, 64.47; H, 7.76; N, 6.19.
Conclusion
Carbamates 1±9 are the PAS-directed irreversible inhibi-
tors of Electrophorus electricus AChE. The structure±
activity relationships for the inhibition of AChE by car-
bamates 1±9 allow us to solve, partially, the inhibition
mechanism at the PAS of the enzyme. Linear correlations
between the logarithms of Ki; k2, and ki values and the s
values of m- and p-substituents of carbamates 1±9 are
observed, and the r values are 1.36, 0.35 and 1.01,
respectively. The inhibition reaction may form a positive
charged enzyme±inhibitor intermediate at the peripheral
anionic site of the enzyme and may follow the irreversible
inactivation by a conformational change of the enzyme.
Overall, the structure±activity relationships for this study
allow us to solve, partially, the inhibition mechanism at
the PAS of the enzyme.
3-Methoxyphenyl-N-n-butyl carbamate (3). 1H NMR
(CDCl3, 300 MHz) d 0.95 (t, J=7.5 Hz, 3H, o-CH3),
1.39 (sextet, J=7.5 Hz, 2H, g-CH2), 1.55 (quintet,
J=7.5 Hz, 2H, b-CH2), 3.24 and 3.30 (ABq, J=7.2 Hz,
2H, a-CH2), 3.79 (s, 3H, OCH3), 5.01 (s, 1H, NH),
6.69±7.27 (m, 4H, phenyl-H); 13C NMR (CDCl3, 75.4
MHz) d 13.66 (o-C), 19.83 (g-C), 31.82 (b-C), 40.91 (a-
C), 55.53 (OCH3), 17.55, 111.22, 113.85 and 129.65
(phenyl C-2,4,5,6), 152.09 (phenyl C-3), 154.52 (phenyl
C-1), 160.40 (phenyl C-1), 156.74 (C=O); High resolu-
tion mass spectrum: exact mass: 223.1208. Found:
223.1196. Anal. calcd for C12H17O3N: C, 64.54; H, 7.68;
N, 6.28. Found: C, 64.44; H, 7.77; N, 6.18.
Experimental
Materials. All chemicals were of the highest grade avail-
able. 5,50-Dithio-bis-2-nitrobenzoate (DTNB) and acetyl-
thiocholine (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 chromatography plates (60 F254) were
obtained from Merck; other chemicals and biochemicals
were of the highest quality available commercially.
4-Chlorophenyl-N-n-butyl carbamate (4). 1H NMR
(CDCl3, 300 MHz) d 0.96 (t, J=7.5 Hz, 3H, o-CH3), 1.40
(sextet, J=6.9 Hz, 2H, g-CH2), 1.56 (quintet, J=7.2 Hz,
2H, b-CH2), 3.24 and 3.30 (ABq, J=6.9 Hz, 2H, a-CH2),
5.00 (s, 1H, NH), 7.05±7.32 (m, 4H, phenyl-H); 13C NMR
(CDCl3, 75.4 MHz) d 13.62 (o-C), 19.80 (g-C), 31.77 (b-
C), 40.93 (a-C), 122.95 (phenyl C-3,5), 129.27 (phenyl C-
2,6), 130.48 (phenyl C-4), 149.63 (phenyl C-1), 154.25
(C=O). High resolution mass spectrum: exact mass:
227.7013. Found: 227.7026. Anal. calcd for
C11H14O2NCl: C, 58.13; H, 6.21; N, 6.17. Found: C,
58.03; H, 6.33; N, 6.07.
Instrumental methods. 1H and 13C NMR spectra were
recorded at 300 and 75.4 MHz (Varian-XL 300 spectro-
meter), respectively. The 1H chemical shift was referred to
internal Me4Si. All steady state kinetic data were obtained
from an UV±visible spectrophotometer (HP 8452 or
Beckman DU-650) with a cell holder circulated with a
water bath.
3-Chlorophenyl-N-n-butyl carbamate (5). 1H NMR
(CDCl3, 300 MHz) d 0.96 (t, J=7.5 Hz, 3H, o-CH3), 1.40
(sextet, J=6.9 Hz, 2H, g-CH2), 1.55 (quintet, J=7.2 Hz,
2H, b-CH2), 3.24 and 3.30 (ABq, J=6.9 Hz, 2H, a-CH2),
5.00 (s, 1H, NH), 7.02±7.30 (m, 4H, phenyl-H); 13C NMR
(CDCl3, 75.4 MHz) d 13.64 (o-C), 19.82 (g-C), 31.78 (b-
C), 40.97 (a-C), 119.97, 122.26, 125.48 and 129.98 (phenyl
C-2,4,5,6), 134.49 (phenyl C-3), 151.64 (phenyl C-1),
154.05 (C=O). High resolution mass spectrum: exact
mass: 227.7013. Found: 227.7026. Anal. calcd for
C11H14O2NCl: C, 58.13; H, 6.21; N, 6.17. Found: C,
58.05; H, 6.30; N, 6.08.
Synthesis of carbamates 1±9. Carbamates 1±9 (Fig. 2)
were prepared from the condensation of the correspond-
ing phenol with 1.2 mol equivalents of n-butyl isocyanate
in the presence of a catalytic amount of pyridine in
dichloromethane at 25ꢀC for 48 h (76±85% yield). All
compounds were puri®ed by liquid chromatography on
1
silica gel (hexane±ethyl acetate) and characterized by H
(300 MHz) NMR, 13C (75.4 MHz) NMR, mass spectrum,
and elemental analysis.
1
Phenyl-N-n-butyl carbamate (1). H NMR (CDCl3, 300
MHz) d 0.95 (t, J=7 Hz, 3H, o-CH3), 1.42 (sextet, J=7
Hz, 2H, g-CH2), 1.56 (quintet, J=7 Hz, 2H, b-CH2), 3.22
and 3.27 (ABq, J=7 Hz, 2H, a-CH2), 5.02 (s, 1H, NH),
7.11±7.37 (m, 5H, phenyl-H); 13C NMR (CDCl3, 75.4
MHz) d 13.60 (o-C), 19.76 (g-C), 31.76 (b-C), 40.81 (a-C),
121.57, 125.13 and 129.20 (phenyl C-2,6), 151.01 (phenyl
C-1), 154.66 (C=O). High resolution mass spectrum: exact
mass: 193.1103. Found: 193.1107. Anal. calcd for
C11H15O2N: C, 68.35; H, 7.83; N, 7.25. Found: C, 68.27; H,
7.96, N, 7.15.
4-Tri¯uorophenyl-N-n-butyl carbamate (6). 1H NMR
(CDCl3, 300 MHz) d 0.96 (t, J=7.5 Hz, 3H, o-CH3), 1.40
(sextet, J=7.5 Hz, 2H, g-CH2), 1.55 (quintet, J=7.0 Hz,
2H, b-CH2), 3.26 and 3.32 (ABq, J=6.6 Hz, 2H, a-CH2),
5.04 (s, 1H, NH), 7.24±7.63 (m, 4H, phenyl-H); 13C NMR
(CDCl3, 75.4 MHz) d 13.61 (o-C), 19.80 (g-C), 31.73 (b-
C), 40.96 (a-C), 121.87 (phenyl C-3,5), 124.24 (q,
1JCF=300 Hz, CF3), 126.60 (phenyl C-2,6), 126.63 (q,
2JCF=20 Hz, phenyl C-4), 153.67 (phenyl C-1), 153.82
(C=O). High resolution mass spectrum: exact mass:

2688
G. Lin et al. / Bioorg. Med. Chem. 7 (1999) 2683±2689
261.0976. Found: 261.0988. Anal. calcd for
C12H14O2NF3: C, 55.15; H, 5.40; N, 5.36. Found: C,
55.09; H, 5.48; N, 5.25.
spectrophotometer. First-order rate constants (kapp
values) for inhibition of AChE were calculated as
described by Hosie et al.36 Values of Ki and k2 can be
obtained by ®tting the data of kapp and [I] to eq (1) by
nonlinear least squares regression analyses. Duplicate or
triplicate sets of data were collected for each inhibitor
concentration.
3-Tri¯uorophenyl-N-n-butyl carbamate (7). 1H NMR
(CDCl3, 300 MHz) d 0.96 (t, J=7.5 Hz, 3H, o-CH3), 1.41
(sextet, J=6.9 Hz, 2H, g-CH2), 1.58 (quintet, J=7.2 Hz,
2H, b-CH2), 3.26 and 3.32 (ABq, J=6.6 Hz, 2H, a-CH2),
5.06 (s, 1H, NH), 7.31±7.50 (m, 4H, phenyl-H); 13C NMR
(CDCl3, 75.4 MHz) d 13.56 (o-C), 19.77 (g-C), 31.69 (b-
C), 40.94 (a-C), 118.78, 121.89, 125.17 and 129.77 (phenyl
Return of activity and protection by edrophonium. For
the return of activity study, AChE was incubated with
each carbamate 1±9 (1 mM) in the absence and presence of
edrophonium chloride (2 mM), a known competitive inhi-
bitor of the enzyme,2 before the inhibition reaction. All
the other procedures followed those of Hosie et al.17,36
1
C-2,4,5,6), 123.55 (q, JCF=230 Hz, CF3), 131.24 (q,
2JCF=20 Hz, phenyl C-3), 151.21 (phenyl C-1), 154.02
(C=O). High resolution mass spectrum: exact mass:
261.0976. Found: 261.0985. Anal. calcd for C12H14
O2NF3: C, 55.15; H, 5.40; N, 5.36. Found: C, 55.06; H,
5.49; N, 5.24.
Acknowledgement
3-Nitrophenyl-N-n-butyl carbamate (8). 1H NMR
(CDCl3, 300 MHz) d 0.97 (t, J=7.2 Hz, 3H, o-CH3),
1.41 (sextet, J=6.9 Hz, 2H, g-CH2), 1.59 (quintet,
J=7.2 Hz, 2H, b-CH2), 3.26 and 3.32 (ABq, J=6.6 Hz,
2H, a-CH2), 5.13 (s, 1H, NH), 7.48±8.09 (m, 4H, phen-
yl-H); 13C NMR (CDCl3, 75.4 MHz) d 13.62 (o-C),
19.80 (g-C), 31.70 (b-C), 41.07 (a-C), 117.24, 120.14,
128.08 and 129.81 (phenyl C-2,4,5,6), 148.72 (phenyl C-
1), 151.46 (phenyl C-3), 153.61 (C=O). High resolution
mass spectrum: exact mass: 238.0953. Found: 238.0942.
Anal. calcd for C11H14N2O4: C, 55.44; H, 5.93; N,
11.76. Found: C, 55.33; H, 6.02; N, 11.65.
This work was supported by the National Science
Council of Taiwan.
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