Ortho effects and cross interaction correlations for the mechanisms of cholesterol esterase inhibition by aryl carbamates

Article abstract of DOI:10.1002/poc.740

Ortho-substituted phenyl-N-butyl carbamates (1-11) were synthesized to evaluate the inhibition mechanisms of porcine pancreatic cholesterol esterase. All carbamate inhibitors act as the active site-directed pseudo substrate inhibitors of the enzymes. The logarithms of dissociation constant (K _i), carbamylation constant (k₂) and bimolecular inhibition constant (k_i) multiply linearly correlate with the Hammett substituent constant (σ), the Taft-Kutter-Hansch ortho steric constant (E_s), and the Swan-Lupton-Hansch ortho polar constant (F). For the -log K_i, log k₂ and log k_i correlations, the reaction constant for ordinary polar effect (ρ), the intensity factor to ortho steric constant (δ) and the intensity factor to ortho polar constant (f) are 0.7, -0.07, and 0.5; 0.5, 0.04 and -0.5; and 1.1, -0.03 and 0.0, respectively. The cross interaction reaction constant (ρ_XR) for the -log k_i-, log k₂- and log k_i-σ- ασ*-ασσ* correlations are 3, -2, and 1, respectively. The K_i step may be composed of the following two steps: (1) protonation of carbamates 1-11 and (2) the pseudo-trans to pseudo-cis conformation change of protonated carbamates 1-11 due to a large ρ_XR value of 3 and formation of the enzyme-protonated carbamates 1-11 tetrahedral intermediate. The k₂ step involves departure of the leaving group, which is protonated by the active site histidine of the enzyme, from the tetrahedral intermediate to solution and formation of the carbamyl enzyme. Moreover, the distances between the carbamate and phenyl groups in all transition states of inhibition reactions are relatively short owing to large |ρ_XR| values. The K_i step shows little ortho steric enhancement effect; moreover, the k₂ step shows little ortho steric inhibition effect. Copyright

Full text of DOI:10.1002/poc.740

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2004; 17: 707–714
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.740
Ortho effects and cross interaction correlations for the
mechanisms of cholesterol esterase inhibition by aryl
carbamates
Gialih Lin,* Yu-Chen Liu, Yon-Gi Wu and Yu-Ru Lee
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
Received 23 February 2003; revised 4 November 2003; accepted 5 December 2003
ABSTRACT: Ortho-substituted phenyl-N-butyl carbamates (1–11) were synthesized to evaluate the inhibition
mechanisms of porcine pancreatic cholesterol esterase. All carbamate inhibitors act as the active site-directed pseudo
substrate inhibitors of the enzymes. The logarithms of dissociation constant (Ki), carbamylation constant (k2) and
bimolecular inhibition constant (ki) multiply linearly correlate with the Hammett substituent constant (ꢀ), the Taft–
Kutter–Hansch ortho steric constant (ES), and the Swan–Lupton–Hansch ortho polar constant (F). For the –log Ki,
log k2 and log ki correlations, the reaction constant for ordinary polar effect (ꢁ), the intensity factor to ortho steric
constant (ꢂ) and the intensity factor to ortho polar constant (f) are 0.7, ꢀ0.07, and 0.5; 0.5, 0.04 and ꢀ0.5; and 1.1,
ꢀ 0.03 and 0.0, respectively. The cross interaction reaction constant (ꢁXR) for the –log ki–, log k2– and log ki–ꢀ–ꢃꢀ*–
ꢃꢀꢀ* correlations are 3, ꢀ2, and 1, respectively. The Ki step may be composed of the following two steps: (1)
protonation of carbamates 1–11 and (2) the pseudo-trans to pseudo-cis conformation change of protonated carbamates
1–11 due to a large ꢁXR value of 3 and formation of the enzyme-protonated carbamates 1–11 tetrahedral intermediate.
The k2 step involves departure of the leaving group, which is protonated by the active site histidine of the enzyme,
from the tetrahedral intermediate to solution and formation of the carbamyl enzyme. Moreover, the distances between
the carbamate and phenyl groups in all transition states of inhibition reactions are relatively short owing to large jꢁXR
j
values. The Ki step shows little ortho steric enhancement effect; moreover, the k2 step shows little ortho steric
inhibition effect. Copyright # 2004 John Wiley & Sons, Ltd.
KEYWORDS: ortho effects; cross interaction correlations; cholesterol esterase; carbamate inhibitors
INTRODUCTION
bile salt-activation mechanisms have been proposed, the
active site of CEase is similar to that of lipases.6–8
Pancreatic cholesterol esterase (CEase, EC 3.1.1.13), also
known as bile salt-stimulated lipase, pancreatic carboxyl
ester lipase, pancreatic lysophospholipase, non-specific
lipase, functions in the hydrolysis of dietary cholesterol
esters and other dietary esters.1 In addition to cholesterol
esters, triacylglycerols, phospholipids and vitamin esters
are also substrates of CEase.2,3 Two x-ray structures of
CEase have been reported recently.4,5 Although different
Recently, there has been increased interest in CEase
and lipases owing to the correlation between enzymatic
activity in vivo and absorption of dietary cholesterol9,10
and the use of orlistat (Xenical). Orlistat, whose original
mechanism of action consists of the selective inhibition
of gastrointestinal lipases, has been commercialized for
the treatment of obesity.11,12 It has also been demon-
strated that CEase is involved directly in lipoprotein
metabolism, in that the enzyme catalyzes the conversion
of large low-density lipoprotein to smaller, denser, more
cholesterol ester-rich lipoproteins, and that the enzyme
may regulate serum cholesterol levels.13 Both CEase and
lipase share the same catalytic mechanism as serine
proteases14 in that they have a Ser–His–Asp (Glu) cata-
lytic triad that is involved in nucleophilic and general
acid–base catalysis and a neighboring oxyanion hole
(OAH), the hydrogen bonding peptide NH functions of
Gly and Ala, that stabilizes the incipient carbonyl C—Oꢀ
of the ester function during turnover. Both CEase and
lipase may well be expected to be inhibited by the same
classes of mechanism-based inhibitors. In the presence
*Correspondence to: G. Lin, Department of Chemistry, National
Chung-Hsing University, Taichung 402, Taiwan.
E-mail: gilin@dragon.nchu.edu.tw
Contract/grant sponsor: National Science Council of Taiwan.
Abbreviations: ACS, alkyl chain binding site; CEase, cholesterol
esterase; ꢂ, intensity factor to ortho steric constant or NMR chemical
shift in ppm; ES, esteratic site; Es, Taft–Kutter–Hansch ortho steric
constant; F, Swan–Lupton–Hansch ortho polar constant; f, intensity
factor to ortho polar constant; k2, carbamylation constant; k3, decarba-
mylation constant; Ki, inhibition or dissociation constant; Ki0, virtual
inhibition or dissociation constant of the enzyme-protonated carbamate
tetrahedral intermediate; OAH, oxyanion hole; PNPB, p-nitrophenyl
butyrate; QSAR, quantitative structure–activity relationship; ꢁ, reac-
tion constant for ordinary polar effect; SACS; second alkyl chain
binding site; TFA, trifluoroacetophenone.
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2004; 17: 707–714

708
G. LIN ET AL.
due to complications from direct steric and polar effects,
are not generally applicable.27 According to Fujita and
Nishioka’s suggestion, the total ortho effect is composed
of the ordinary polar effect, the ortho steric effect and the
ortho polar effect:25,27
log k ¼ h þ ꢁꢀ þ ꢂES þ fF
ð3Þ
where h, ꢁ, ꢀ, ES, ꢂ, f and F are log k0, the reaction
constant for ordinary polar effect, the Hammett substi-
tuent constant, the Taft–Kutter–Hansch ortho steric con-
stant, the intensity factor to ortho steric constant, the
intensity factor to ortho polar constant and the Swain–
Lupton–Hansch ortho polar constant, respectively.
Cross interaction correlations for the CEase inhibitions
by meta- and para-substituted phenyl-N-butylcarbamates
(13) and p-nitrophenyl-N-substituted carbamates (12)
with Eqn (4)28,29 reveal that the carbamate O—C(O)—
N—R geometries in the transition states of all inhibition
reactions retain in the pseudo-trans conformations:
Scheme 1. Kinetic scheme for pseudo substrate inhibitions
of CEase in the presence of substrate
of substrate, the CEase inhibition by pseudo substrate
inhibitors such as carbamates have been proposed
(Scheme 1).15–22 Since the inhibition follows first-order
kinetics over the observed time period for the steady-state
kinetics, the rate of hydrolysis of EI0 must be significantly
slower than the rate of formation of EI0 (k2 ꢁ k3).23
Therefore, values of Ki and k2 can be calculated from
the equation.15
log k ¼ h þ ꢁꢀ þ ꢁꢃꢃꢀꢃ þ ꢁXRꢀꢃꢀꢃ
ð4Þ
kapp ¼ k2½Iꢂ=ðKið1 þ ½Sꢂ=KmÞ þ ½IꢂÞ
ð1Þ
where h, ꢁ, ꢀ, ꢁ*, ꢀ*, ꢁXR and ꢃ are log k0, the reaction
constant for the substituted phenyl part (varied X, Fig. 1)
of the inhibitor, the Hammett substituent constant of X,
the reaction constant for the substituted carbamate part
(varied R, Fig. 1) of the inhibitor, the Taft substituent
constant of R, the cross interaction constant and the
weighing factor for the Hammett–Taft cross interaction
term (ꢃ ¼ 1 for the Hammett substituent X; ꢃ ¼ 2.54 for
the Taft substituent R), respectively.28 Moreover, the
distance between X and R substituents in the transition
state of the reaction is inversely proportional to jꢁXRj.29
Aryl carbamates, such as meta- and para-substituted
phenyl-N-substituted carbamates (12 and 13) (Fig. 1),
are characterized as the pseudo substrate inhibitors of
CEase and show the Hammett16,18,21 and Taft–Ingold
types17,20,21 of correlations. In this work, ortho-substi-
tuted phenyl-N-butyl carbamates (1–11) (Fig. 1) were
synthesized to explore the ortho effects for the pre-
steady-state CEase inhibition and the cross interaction
correlations with p-nitrophenyl-N-substituted carbamates
(12) (Fig. 1).
where kapp is the first-order rate constant and can be
obtained according to Hosie’s method.15 The bimolecular
rate constant, Ki ¼ k2/Ki, is related to overall inhibitory
potency.
According to the x-ray structures of CEase and li-
pases,4,5,7,8 CEase consists of at least five major binding
sites:20,21 (a) an alkyl chain binding site (ACS) that binds
to the substrate alkyl chain, (b) an oxyanion hole (OAH)
that stabilizes the tetrahedral intermediate, (c) an esteratic
site (ES), comprised of the active site serine which
attacks the ester carbonyl, (d) a leaving group hydro-
phobic binding site, the peripheral site and/or the second
alkyl chain or group binding site (SACS) that binds to the
cholesterol part of cholesterol ester or the second fatty
acid chain of triacylglyceols, which is relatively larger
than ACS, and (e) a leaving group hydrophilic binding
site that binds to the hydrophilic part of the leaving group
and is located at the opposite direction of ACS.
Quantitative structure–activity relationships (QSARs)
represent an attempt to correlate structural properties of
compounds with activities or reactivities.24–26 These che-
mical descriptors, which include parameters to account for
hydrophobicity, electronic, inductive or polar properties
and steric effects, are determined empirically or by calcu-
lations. Many biological activities and chemical reactiv-
ities are correlated with the Hammett equation:24–26
log k ¼ h þ ꢁꢀ
ð2Þ
where h, ꢁ and ꢀ are log k0, the reaction constant (or
intensity factor for inductive effect) and the Hammett
substituents constant, respectively. Also, meta- and para-
substituted compounds generally correlate well but
ortho-substituted compounds do not.25 Ortho problems,
Figure 1. Structures of carbamates 1--12
J. Phys. Org. Chem. 2004; 17: 707–714
Copyright # 2004 John Wiley & Sons, Ltd.

ORTHO EFFECTS AND CROSS INTERACTION CORRELATIONS
709
Table 1. Ortho substituent constants and inhibition constants for CEase-catalyzed hydrolysis of PNPB in the presence of
carbamates 1--11
Inhibitor
Substituent
ꢀa
ESa
Fb
k2 (10ꢀ4 sꢀ1 c
)
Ki (mM)c
ki (103 ꢀ1 sꢀ1 d
M )
1
2
3
4
5
6
7
8
9
o-t-Bu
o-Cl
ꢀ0.20
0.23
ꢀ2.78
ꢀ0.97
ꢀ0.55
ꢀ2.52
ꢀ1.24
ꢀ1.31
ꢀ1.01
ꢀ2.40
0
ꢀ0.07
0.41
0.26
0.67
ꢀ0.04
ꢀ0.05
0.08
0.38
0
15.8 ꢄ 0.4
16.5 ꢄ 0.8
7.0 ꢄ 0.05
17.6 ꢄ 0.7
20.3 ꢄ 0.8
13.8 ꢄ 0.3
11.7 ꢄ 0.8
9.5 ꢄ 0.1
38 ꢄ 2
3.2 ꢄ 0.3
2.2 ꢄ 0.4
7 ꢄ 1
0.5 ꢄ 0.1
0.8 ꢄ 0.2
o-OMe
o-NO2
o-CH3
o-C2H5
o-Ph
o-CF3
p-NO2
o,p-Di-t-Bu
H
ꢀ0.27
0.10 ꢄ 0.02
2.2 ꢄ 0.4
0.78
0.8 ꢄ 0.3
7 ꢄ 1
ꢀ0.17
ꢀ0.15
ꢀ0.01
0.54
0.30 ꢄ 0.02
0.20 ꢄ 003
0.23 ꢄ 0.03
1.4 ꢄ 0.2
7 ꢄ 1
5 ꢄ 1
0.7 ꢄ 0.1
2.6 ꢄ 0.3
18 ꢄ 3
5 ꢄ 1
0.78
1.5 ꢄ 0.1
10
11
ꢀ0.4
ꢀ2.78
ꢀ0.07
7.5 ꢄ 0.7
16.8 ꢄ 0.9
0.04 ꢄ 0.02
0.32 ꢄ 0.02
0
0
0
a
Hydrogen is taken as standard. These values are calculated from Refs. 24–26.
Taken from Ref. 27.
Obtained from fitting the kapp values to Eqn (1).
b
c
d ki ¼ k2/Ki.
RESULTS
parameters, ꢀ, ES and F [(Eqn (3)],25,27 are much impro-
ved (Table 2). The bimolecular rate constant, ki ¼ k2/Ki, is
related to overall inhibitory potency.15–22 The pKa values
of carbamates 1–12 are correlated with the Hammett
equation [Eqn (2)] (Table 2).21 The –log Ki0 values are
well correlated with Eqn (3) (Table 2), where the virtual
inhibition constant, Ki0 ¼ KbKi, is the dissociation con-
stant of the enzyme-protonated carbamate tetrahedral
intermediate.
The results for the cross interaction correlation
between the ortho-substituted carbamates 1–11 and 4-
nitrophenyl-N-substituted carbamates (12) (Fig. 1) are
summarized in Table 3. The jꢁXRj values for the cross
interaction between ortho-substituted carbamates 1–11
and carbamates 12 are larger than those between
Carbamates 1–11 (Fig. 1) are characterized as the pseudo
substrate inhibitors of CEase because the inhibitors are
time dependent, the inhibitions follow first-order kinetics
and the enzyme activities recover with a competitive
inhibitor, trifluoroacetophenone (TFA).15–22 The ortho
substituent constants and inhibition constants for the
CEase-catalyzed hydrolysis of p-nitrophenyl butyrate
(PNPB) by carbamates 1–11 are summarized in Table 1.
Correlations of ꢂNH, ꢂ
, –log K , log k and log k
i 2 i
C--O
--
with the Hammett equation [Eqn (2)] are poor (Table 2).
These correlations are improved after addition of the
Taft–Kutter–Hansch steric constant, ES, to the correlation
parameters (Table 2). Multiple correlations with three
Table 2. QSAR results for NMR chemical shifts of carbamates 1--11 and the inhibition constants for the inhibitions of CEase by
carbamates 1--11
h
ꢁ
ꢂ
f
R
ꢂNa H
ꢂNb H
ꢂNc H
5.12 ꢄ 0.03
5.13 ꢄ 0.07
5.07 ꢄ 0.07
154.09 ꢄ 0.07
154.1 ꢄ 0.2
154.3 ꢄ 0.1
4.00 ꢄ 0.06
9.2 ꢄ 0.2
0.27 ꢄ 0.09
0.3 ꢄ 0.1
0.0 ꢄ 0.2
ꢀ1.3 ꢄ 0.2
ꢀ1.3 ꢄ 0.2
ꢀ0.5 ꢄ 0.3
ꢀ2.5 ꢄ 0.1
3 ꢄ 1
—
0.01 ꢄ 0.04
0.00 ꢄ 0.03
—
ꢀ0.01 ꢄ 0.09
0.01 ꢄ 0.06
—
—
0.718
0.719
0.813
0.913
0.913
0.969
0.989
0.906
0.847
0.884
0.912
0.559
0.654
0.811
0.904
0.906
0.906
—
0.4 ꢄ 0.3
—
ꢂCa --O
--
ꢂCb--O
—
ꢀ1.3 ꢄ 0.4
--
ꢂCc --O
--
Log Kbd
ꢀLog Ki0e
ꢀLog Kia
ꢀLog Kib
ꢀLog Kic
Log k2a
—
ꢀ0.07 ꢄ 0.07
0.5 ꢄ 0.3
—
—
5.35 ꢄ 0.07
5.2 ꢄ 0.1
0.8 ꢄ 0.2
0.9 ꢄ 0.2
0.7 ꢄ 0.2
0.3 ꢄ 0.1
0.3 ꢄ 0.1
0.5 ꢄ 0.1
1.1 ꢄ 0.2
1.1 ꢄ 0.2
1.1 ꢄ 0.3
—
ꢀ0.10 ꢄ 0.07
ꢀ0.07 ꢄ 0.07
—
0.07 ꢄ 0.05
0.04 ꢄ 0.05
—
5.2 ꢄ 0.1
0.5 ꢄ 0.3
ꢀ2.87 ꢄ 0.06
ꢀ2.78 ꢄ 0.09
ꢀ2.76 ꢄ 0.08
2.47 ꢄ 0.07
2.4 ꢄ 0.1
—
—
Log k2b
Log k2c
ꢀ0.5 ꢄ 0.2
Log kia
—
—
Log kib
ꢀ0.03 ꢄ 0.07
Log kic
2.4 ꢄ 0.1
ꢀ0.03 ꢄ 0.08
0.0 ꢄ 0.1
a
Correlation with ꢀ [Eqn (2)] for carbamates 1–11.
Correlation with ꢀ and ES for carbamates 1–11.
Correlation with ꢀ, ES and F [Eqn (3)] for carbamates 1–11.
Correlation with ꢀ, ES and F [Eqn (3)] for carbamates 1–11 and meta- and para-substituted phenyl-N-butylcarbamates.
1/Ki ¼ Kb (1/Ki0).
b
c
d
e
Copyright # 2004 John Wiley & Sons, Ltd.
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710
G. LIN ET AL.
Table 3. Cross interaction correlation results for the CEase
inhibitions by ortho-substituted phenyl-N-butylcarbamates
(1--11) and p-nitrophenyl-N-substituted carbamates (12)
Parametera
ꢀLog Ki
Log k2
Log ki
ꢁ*
ꢁ
ꢀ2.4 ꢄ 0.8
1.1 ꢄ 0.3
3 ꢄ 1
ꢀ0.9 ꢄ 0.1
0.0 ꢄ 0.3
ꢀ2 ꢄ 1
ꢀ1.2 ꢄ 0.8
1.1 ꢄ 0.3
1 ꢄ 1
ꢁbXR
h
4.6 ꢄ 0.2
0.827
ꢀ 2.4 ꢄ 0.3
0.728
2.2 ꢄ 0.3
0.872
Rc
a
Correlation of the ꢀlog Ki, log k2 and log ki values for the CEase inhibi-
tions by ortho-substituted phenyl-N-butylcarbamates (1–11) and p-nitro-
phenyl-N-substituted carbamates (12)17,21,28 on Eqn (4).28,29
Cross interaction constant.29
Correlation coefficient.
b
c
para- and meta-substituted carbamates and carbamates
12.28 In other words, the distances between X and R in
the transition states for the CEase inhibitions by ortho
substituted carbamates are much shorter than those inhi-
bitions by para- and meta-substituted carbamates. There-
fore, this result confirms that the carbamate inhibitor
conformations during all inhibition reactions retain the
pseudo-cis conformations.28
DISCUSSION
Figure 2. The proposed CEase inhibition mechanisms by
carbamates 1--11. The Kb step is protonation of carbamates
1--11. The ꢁ value for the logKb--ꢀ correlation is ꢀ2.5.
Therefore, protonated carbamates 1--11 are more positively
charged than carbamates 1--11. The O---C(O)---N---R geo-
metries of carbamates 1--11 and protonated of carbamates
1--11 are all pseudo-trans conformations. The Ki0 step is
formation of the enzyme--protonated carbamate tetrahedral
intermediate. The ꢁXR value for the --log Ki--ꢀ--ꢃꢀ*--ꢃꢀꢀ*
cross interaction correlation is 3. Hence the O---C(O)---N---R
geometry of the tetrahedral intermediate changes to
the pseudo-cis conformation. Values of ꢁ, ꢂ, and f for the
--log Ki0--ꢀ--Es--F correlation are 3, ꢀ0.07 and 0.5, respec-
tively. The k2 step is formation of the carbamyl enzyme
intermediate. The ꢁXR value for the log k2--ꢀ--ꢃꢀ*--ꢃꢀꢀ*
cross interaction correlation is ꢀ2. Hence, the O---C(O)---
N---R geometry for the transition state which leads to the
carbamyl enzyme retains in the pseudo-cis conformation.
Values of ꢁ, ꢂ and f for the k2 step are 0.5, 0.04 and ꢀ0.5,
respectively
Like meta- and para-substituted phenyl-N-butylcarba-
mates16–18,21 and 1,10-bi-2-naphthyl- 2,20-di-N- butylcar-
bamate,19 carbamates 1–11 are characterized as the
pseudo substrate inhibitors of CEase (Scheme 1 and
Table 1) and show the same kinetic behavior. Therefore,
the leaving group binding site of the enzyme is large
enough to adapt to extremely bulky ortho substituents
such as 1-naphthyl, tert-butyl, and phenyl.
The f value of 0.4 for the ꢂNH–ꢀ–ES–F correlation
suggests that the N—H bonds of carbamates 1–11 polar-
ize to generate a partial negative charge on nitrogen under
the NMR conditions; moreover, the f value of –1.3 for the
—
–ꢀ–E –F correlation suggests that the C O bonds
—
S
ꢂ
C--O
--
of carbamates 1–11 polarize to generate a partial positive
charge on carbon under the NMR conditions.
A three-step CEase inhibition mechanism by carba-
mates 1–11 is proposed (Fig. 2). The first step (Kb) is the
pre-equilibrium protonation of carbamates 1–11. The
second step (Ki0) is formation of the enzyme-protonated
carbamate tetrahedral intermediate. The third step (k2) is
formation of the carbamyl enzyme intermediate and
release of the substituted phenol product.
O—C(O)—N—R geometries of carbamates 1–11 and
protonated carbamates 1–11 are in the pseudo-trans
conformations (Fig. 2). The excellent log Kb–ꢀ correla-
tion (Table 2) indicates that the Kb step is insensitive to
ortho steric and polar effects because the ortho substi-
tuents are far away from the carbamate nitrogen reaction
centers.
Kb step
Ki0 step
For the Kb step, carbamates 1–11 like carbamates 13
and 12,21,28 are bases (Table 2) and are protonated in
aqueous solution outside the enzyme active site (pH 7).
From the cross interaction correlation results,28 the
For the Ki0 step, the ꢁ value of 3 for the –log Ki0–ꢀ–ES–F
correlation (Table 2) suggests that the enzyme–proto-
nated carbamate tetrahedral intermediates are more
Copyright # 2004 John Wiley & Sons, Ltd.
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ORTHO EFFECTS AND CROSS INTERACTION CORRELATIONS
711
negative charges than protonated carbamates 1–11
(Fig. 2). There is little ortho steric enhancement effect
in the CEase inhibitions by protonated carbamates 1–11
owing to a negative, small ꢂ value (ꢀ0.07) for this
correlation (Table 2). Therefore, the enzyme active site
prefers to adapt bulky ortho substituents of the inhibitors.
Since the f value for the Kb step is zero, the f value (0.5)
for the Ki0 step should be identical with that for the Ki step
(discussed later). The carbamate O—C(O)—N—R geo-
metry for the Ki0 step changes from the pseudo-trans to
pseudo-cis conformation (Fig. 2, discussed later).
ꢀ0.5 (Table 2), which is not the value for formation of the
substituted phenoxide ion (f ¼ 2.6).27 Therefore, the
pseudo leaving groups, phenoxide ions, are assumed to
be protonated by His-435 of the enzyme soon after their
formation (Fig. 2). Hence the true leaving group for this
reaction is substituted phenol. Therefore, the O—C(O)—
N—R geometry of the tetrahedral intermediate must be
retained in the pseudo-cis conformation in order to obtain
the proton from His345 of the enzyme (Fig. 2). The f
1
value for the H NMR spectra of substituted phenols in
DMSO is about 0.6, which may indicate the ortho polar
effect for a polarization process of the hydroxyl bond
(Fig. 3).27 Accordingly, the f value of ꢀ0.5 suggests that
the retro-polarization process for the hydroxyl bond
(Fig. 3) strongly resembles the formation mechanism
for substituted phenol in the k2 step (Fig. 2). The ꢁXR
value of ꢀ2 for the –log k2–ꢀ–ꢃꢀ*–ꢃꢀꢀ*-correlation
(Table 3) indicates that the distance between X and R
substituents (Fig. 1) in the transition state of the k2 step is
relatively short and that the carbamate O—C(O)—N—R
geometry in the transition state of this step retains in the
pseudo-cis conformation (Fig. 2).28
Ki step
The Ki step consists of the Kb and Ki0 steps. The ꢁ value of
0.7 for the –log Ki–ꢀ–ES–F correlation (Table 2) suggests
that the enzyme–protonated carbamate tetrahedral inter-
mediates are slightly more negative charges than carba-
mates 1–11 (Fig. 2). There is also little ortho steric
enhancement effect in the CEase inhibitions by carba-
mates 1–11 (Table 2). For the alkaline hydrolyses of
benzoyl esters and amides, the f values are very similar,
the average being 1.1.27 The f value of 0.5 for the Ki or Ki0
step is less than 1.1 because the distance of the carbamate
carbonyl reaction center to the phenyl ring is longer than
that of the benzoyl carbonyl reaction center to the phenyl
ring. For phenylacetic acid ester formation and hydro-
lysis, the f value is almost unchanged regardless of the
catalytic condition of reactions, being ꢀ0.2 to ꢀ0.4.27
Therefore, the carbamate ether oxygen probably transi-
mits the proximity effect much easier than the phenyla-
cetic methylene. The ꢁXR value of 3 for the –log Ki–ꢀ–
ꢃꢀ*–ꢃꢀꢀ* cross interaction correlation (Table 3) indi-
cates that the distance between X and R substituents
(Fig. 1) in the transition state of the Ki step is relatively
short and that the carbamate O—C(O)—N—R geometry
in the tetrahedral intermediate changes to the pseudo-cis
conformation (Fig. 2).28 Moreover, this ꢁXR value is
greater than that for the cross interaction between carba-
mates 13 and 12 (Fig. 1) (ꢁXR ¼ 2).28 Apparently, the
distance between X and R substituents for the pseudo-cis
conformation in the ortho-substituted carbamate tetrahe-
dral intermediate is shorter than that in the meta- and
para-substituted intermediates.
ki step
The ki step consists of both Ki (consists of Kb and Ki0) and
k2 steps. The f value of 0.0 for the log Ki–ꢀ–ES–F cor-
relation (Table 2) indicates that the sum of both f values
for the log Ki–ꢀ–ES–F (f ¼ 0.5) and log k2–ꢀ–ES–F
(f ¼ ꢀ0.5) correlations. Moreover, as most data for ki
(Tables 1–3) can also be calculated from those for Ki and
k2, this confirms that the ki step consists of both Ki and k2
steps. However, ꢁ* value of the log ki–ꢀ–ꢃꢀ*–ꢃꢀꢀ*
correlation differs slightly from the sum of that of –
log Ki– and log k2–ꢀ–ꢃꢀ*–ꢃꢀꢀ* correlations probably
because these correlations are poor and ortho effects
are not taken into account in Eqn (4) (Table 3).
k2 step
Figure 3. Polarization and retro-polarization processes of
the substituted phenol hydroxyl bonds. The polarization
process (top) of the substituted phenol hydroxyl bond gen-
erates a partial negative charge at oxygen in the NMR
condition. The f value for the polarization process is 0.6
from NMR spectra. The retro-polarization process (bottom)
of the substituted phenol hydroxyl bond neutralizes the
partial negative charge at oxygen. The f value for the
retro-polarization process should be ꢀ0.6. The retro-polar-
ization process of substituted phenol in aqueous solution
mimics the k2 step (Fig. 2) (f ¼ ꢀ0.5)
For the k2 step, the ꢁ value for the log k2–ꢀ–ES–F
correlation is 0.5 (Table 2). Therefore, the substituted
phenol product for this step is more negatively charged
than the tetrahedral intermediate. The bulky ortho sub-
stituents of the tetrahedral intermediate slightly inhibit
the CEase inhibition reactions owing to a small positive ꢂ
value (0.04) (Table 2). Hence the leaving group binding
site of CEase slightly prefers to adapt less bulky ortho-
substituted phenol. The f value in this correlation is about
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2004; 17: 707–714

712
G. LIN ET AL.
EXPERIMENTAL
o-tert-Butylphenyl-N-butylcarbamate (1). 1H NMR
(CDCl3, 400 MHz), ꢂ (ppm) 0.96 (t, J ¼ 7 Hz, 3H,
CH2CH2CH3), 1.32 [s, 9H, C(CH3)3], 1.38 (sextet,
J ¼ 7 Hz, 2H, CH2CH2CH3), 1.58 (quintet, J ¼ 7 Hz,
2H, CH2CH2CH3), 3.31 (q, J ¼ 7 Hz, 2H, NHCH2),
5.02 (br s, 1H, NH), 7.04–7.37 (m, 4H, aromatic H).
Materials
CEase from porcine pancreas and p-nitrophenyl butyrate
(PNPB) were obtained from Sigma; other chemicals were
obtained from Aldrich. Silica gel used in liquid chroma-
tography (Licorpre silica 60, 200–400 mesh) and thin-
layer chromatographic plates (60 F254) were obtained
from Merck. All other chemicals were of the highest
purity available commercially.
13C NMR (CDCl3, 100 MHz),
ꢂ
(ppm) 13.84
(CH2CH2CH3), 19.98 (CH2CH2CH3), 30.34 [C(CH3)3],
32.09 (CH2CH2CH3), 34.23 [C(CH3)3], 41.06 (NHCH2),
124.05, 125.04, 126.63, 126.76 (phenyl CH), 141.08
—
(phenyl C-1), 149.35 (phenyl C-2), 154.42 (C O).
—
HRMS, calculated for C15H23NO2 249.1729, found
249.1733.
Instrumental methods
o-Chlorophenyl-N-butylcarbamate (2). 1H NMR (CDCl3,
400 MHz), ꢂ (ppm) 0.92 (t, J ¼ 7 Hz, 3H, CH2CH2CH3),
1.37 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3), 1.52 (quintet,
J ¼ 7 Hz, 2H, CH2CH2CH3), 3.23 (q, J ¼ 7 Hz, 2H,
NHCH2), 5.37 (br s, 1H, NH), 7.12–7.41 (m, 4H, aro-
matic H); 13C NMR (CDCl3, 100 MHz), ꢂ (ppm) 13.72
1H and 13C NMR spectra were recorded at 400 and
100 MHz, respectively, on a Varian Gemini 400 spectro-
meter. All steady-state kinetic data were obtained using a
UV–visible spectrophotometer (HP 8452 or Beckman
DU-650) with a cell holder circulated with a water-bath.
(CH2CH2CH3),
(CH2CH2CH3), 41.04 (NHCH2), 123.92, 126.23,
19.84
(CH2CH2CH3),
31.77
Data reduction
127.32, 129.87 (phenyl CH), 127.08 (phenyl C-2),
—
146.92 (phenyl C-1), 153.35 (C O). HRMS, calculated
—
for C11H14NO2Cl 227.0713, found 227.0721.
Origin (version 6.0) was used for linear, non-linear and
multiple linear least-squares regression analyses.
o-Methoxyphenyl-N-butylcarbamate (3). 1H NMR
(CDCl3, 400 MHz), ꢂ (ppm) 0.94 (t, J ¼ 7 Hz, 3H,
CH2CH2CH3), 1.39 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3),
1.57 (quintet, J ¼ 7 Hz, 2H, CH2CH2CH3), 3.27 (q,
J ¼ 7 Hz, 2H, NHCH2), 3.85 (s, 3H, OCH3), 5.05 (br s,
1H, NH), 6.91–7.20 (m, 4H, aromatic H). 13C NMR
(CDCl3, 100 MHz), ꢂ (ppm) 13.82 (CH2CH2CH3),
19.93 (CH2CH2CH3), 31.93 (CH2CH2CH3), 41.07
(NHCH2), 55.88 (OCH3), 112.22, 120.53, 123.12,
126.17 (phenyl CH), 139.83 (phenyl C-2), 151.47 (phenyl
Steady-state enzyme kinetics
The CEase inhibition was assayed by Hosie’s method.15
The temperature was maintained at 25.0 ꢄ 0.1 ꢅC by a
refrigerated circulating water-bath. All inhibition reac-
tions were performed in sodium phosphate buffer (1 ml,
0.1 M, pH 7.0) containing NaCl (0.1 M), CH3CN (2 vol%),
Triton X-100 (0.5 wt%), substrate (0.2 mM) and varying
concentration of inhibitors. Requisite volumes of stock
solutions of substrate and inhibitors in acetonitrile were
injected into the reaction buffer via a pipet. CEase was
dissolved in sodium phosphate buffer (0.1 M, pH 7.0).
First-order rate constants (kapp) for inhibition were
determined as described by Hosie et al.15 The Ki and
k2 values were obtained by fitting kapp and [I] to Eqn (1)
by non-linear least-squares regression analyses.15–22
Duplicate sets of data were collected for each inhibitor
concentration.
—
C-1), 154.17 (C O). HRMS, calculated for C H NO
12 17
—
223.1208, found 223.1211.
3
1
o-Nitrophenyl-N-butylcarbamate (4). H NMR (CDCl3,
400 MHz), ꢂ (ppm) 0.94 (t, J ¼ 7 Hz, 3H, CH2CH2CH3),
1.32 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3), 1.51 (quintet,
J ¼ 7 Hz, 2H, CH2CH2CH3), 3.28 (t, J ¼ 7 Hz, 2H,
NHCH2), 5.28 (br s, 1H, NH), 7.22–8.05 (m, 4H, aro-
matic H). 13C NMR (CDCl3, 100 MHz), ꢂ (ppm) 13.70
(CH2CH2CH3),
(CH2CH2CH3), 41.20 (NHCH2), 125.40, 125.70,
19.80
(CH2CH2CH3),
32.03
125.90, 134.20 (phenyl CH), 142.10 (phenyl C-2),
—
Synthesis of carbamates
144.20 (phenyl C-1), 153.10 (C O). HRMS, calculated
—
for C11H14N2O4 238.0954, found 238.0959.
Carbamates 1–11 were prepared by condensation of the
corresponding phenol with n-butyl isocyanate in the
presence of a catalytic amount of pyridine in toluene.
All compounds were purified by liquid chromatography
1
o-Methylphenyl-N-butylcarbamate (5). H NMR (CDCl3,
400 MHz), ꢂ (ppm) 0.94 (t, J ¼ 7 Hz, 3H, CH2CH2CH3),
1.37 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3), 1.54 (quintet,
J ¼ 7 Hz, 2H, CH2CH2CH3), 2.21 (s, 3H, o-CH3), 3.25 (q,
J ¼ 7 Hz, 2H, NHCH2), 5.09 (br s, 1H, NH), 7.05–7.25 (m,
4H, aromatic H). 13C NMR (CDCl3, 100MHz), ꢂ (ppm)
1
on silica gel and characterized by H and 13C NMR
spectrometry and high-resolution mass spectrometry
(HRMS).
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2004; 17: 707–714

ORTHO EFFECTS AND CROSS INTERACTION CORRELATIONS
713
13.81 (CH2CH2CH3), 16.16 (o-CH3), 19.96 (CH2CH2
CH3), 32.00 (CH2CH2CH3), 41.00 (NHCH2), 122.02,
125.34, 126.58, 130.77 (phenyl CH), 130.42 (phenyl C-
2,4-Di-tert-butylphenyl-N-butylcarbamate (10). 1H NMR
(CDCl3, 400 MHz), ꢂ (ppm) 0.95 (t, J ¼ 7 Hz, 3H,
CH2CH2CH3), 1.32 [s, 9H, o-C(CH3)3], 1.38 [s, 9H, p-
C(CH3) 3], 1.38 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3), 1.55
(quintet, J ¼ 7 Hz, 2H, CH2CH2CH3), 3.29 (q, J ¼ 7 Hz,
2H, NHCH2), 5.01 (br s, 1H, NH), 6.95–7.36 (m, 3H,
aromatic H). 13C NMR (CDCl3, 100 MHz), ꢂ (ppm)
13.84 (CH2CH2CH3), 19.98 (CH2CH2CH3), 30.42
[o-C(CH3)3], 31.59 [p-C(CH3)3], 32.12 (CH2CH2CH3),
34.69 [o-C(CH3) 3], 34.78 [p-C(CH3)3], 41.04 (NHCH2),
123.27, 123.54, 123.71 (phenyl CH), 139.99 (phenyl C-
—
2), 149.29 (phenyl C-1), 154.24 (C O). HRMS, calcu-
—
lated for C12H17NO2 207.1260, found 207.1252.
1
o-Ethylphenyl-N-butylcarbamate (6). H NMR (CDCl3,
400 MHz), ꢂ (ppm) 0.94 (t, J ¼ 7 Hz, 3H, CH2CH2CH3),
1.19 (t, 3H, J ¼ 7 Hz, o-CH2CH3), 1.37 (sextet, J ¼ 7 Hz,
2H, CH2CH2CH3), 1.54 (quintet, J ¼ 7 Hz, 2H,
CH2CH2CH3), 2.59 (q, J ¼ 7 Hz, 2H, o-CH2CH3), 3.24
(q, J ¼ 7 Hz, 2H, NHCH2), 5.10 (br s, 1H, NH), 7.05–7.25
(m, 4H, aromatic H). 13C NMR (CDCl3, 100 MHz), ꢂ
(ppm) 13.79 (CH2CH2CH3), 14.31 (o-CH2CH3),
19.93 (CH2CH2CH3), 23.18 (CH2CH2CH3), 31.98
(o-CH2CH3), 41.00 (NHCH2), 122.28, 125.46, 126.49,
2), 146.92 (phenyl C-4), 147.31 (phenyl C-1), 154.61
—
(C O). HRMS, calculated for C H NO 305.2355,
19 31
—
found 305.2358.
2
Phenyl-N-butylcarbamate (11). 1H NMR (CDCl3,
400 MHz), ꢂ (ppm) 0.96 (t, J ¼ 7 Hz, 3H, CH2CH2CH3),
1.35 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3), 1.56 (quintet,
J ¼ 7 Hz, 2H, CH2CH2CH3), 3.27 (q, J ¼ 7 Hz, 2H,
NHCH2), 4.99 (br s, 1H, NH), 7.12–7.37 (m, 4H,
aromatic H). 13C NMR (CDCl3, 100 MHz), ꢂ (ppm)
13.81(CH2CH2CH3), 19.99 (CH2CH2CH3), 31.96
(CH2CH2CH3), 41.00 (NHCH2), 121.42 (phenyl C-3,
C-5), 125.00 (phenyl C-4), 129.05 (phenyl C-2, C-6),
129.02 (phenyl CH), 136.07 (phenyl C-2), 148.81 (phenyl
—
C-1), 154.49 (C O). HRMS, calculated for C H NO
—
13 19
221.1416, found 221.1407.
2
o-Biphenyl-N-butylcarbamate (7). 1H NMR (CDCl3,
400 MHz), ꢂ (ppm) 0.96 (t, J ¼ 7 Hz, 3H, CH2CH2CH3),
1.32 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3), 1.43 (quintet,
J ¼ 7 Hz, 2H, CH2CH2CH3), 3.19 (q, J ¼ 7 Hz, 2H,
NHCH2), 5.03 (br s, 1H, NH), 7.27–7.51 (m, 9H, aro-
matic H). 13C NMR (CDCl3, 100 MHz), ꢂ (ppm) 13.74
—
150.89 (phenyl C-1), 154.37 (C O). HRMS, calculated
—
for C11H15N2O4 193.1103, found 193.1104.
(CH2CH2CH3),
19.73
(CH2CH2CH3),
31.81
(CH2CH2CH3), 40.78 (NHCH2), 127.93, 128.13,
129.07, 123.10, 125.57, 127.01, 130.48 (phenyl CH),
134.82 (phenyl C-10), 137.62 (phenyl C-2), 147.71 (phe-
Acknowledgment
—
nyl C-1), 154.31 (C O). HRMS, calculated for
—
C17H19NO2 269.1416, found 269.1419.
We thank the National Science Council of Taiwan for
financial support.
o-Trifluoromethylphenyl-N-butylcarbamate (8). 1H NMR
(CDCl3, 400 MHz), ꢂ (ppm) 0.93 (t, J ¼ 7 Hz, 3H,
CH2CH2CH3), 1.38 (sextet, J ¼ 7 Hz, 2H, CH2CH2CH3),
1.54 (quintet, J ¼ 7 Hz, 2H, CH2CH2CH3), 3.26 (q,
J ¼ 7 Hz, 2H, NHCH2), 5.21 (br s, 1H, NH), 7.26–7.64
(m, 4H, aromatic H). 13C NMR (CDCl3, 100 MHz), ꢂ
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—
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—
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2 4
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J. Phys. Org. Chem. 2004; 17: 707–714