Ortho Effects for Inhibition Mechanisms of Butyrylcholinesterase by o

Phenyl carbamates are used to treat Alzheimer's disease. These compounds inhibit acetylcholinesterase and butyrylcholinesterase. The goal of this work...
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Chem. Res. Toxicol. 2005, 18, 1124-1131

Ortho Effects for Inhibition Mechanisms of Butyrylcholinesterase by o-Substituted Phenyl N-Butyl Carbamates and Comparison with Acetylcholinesterase, Cholesterol Esterase, and Lipase Gialih Lin,* Yu-Ru Lee, Yu-Chen Liu, and Yon-Gi Wu Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan Received January 20, 2005

Phenyl carbamates are used to treat Alzheimer’s disease. These compounds inhibit acetylcholinesterase and butyrylcholinesterase. The goal of this work was to determine the chemical characteristics of ortho substituents that make some carbamates better inhibitors of butyrylcholinesterase than of acetylcholinesterase, cholesterol esterase, and lipase. The inhibition constants, Ki, Ki′, kc, and ki were measured for nine different carbamates. The values were plotted according to Hammett, Taft-Kutter-Hansch, and Swan-Lupton to obtain constants that correlated the chemical nature of the substituents with inhibition potency. It was found that the negative charges of tetrahedral intermediates were more stabilized by ortho electron-withdrawing substituents of the inhibitors in butyrylcholinesterase than in acetylcholinesterase. This result confirmed formation of 3-pronged hydrogen bonds for the oxyanion hole of butyrylcholinesterase and 2-pronged hydrogen bonds for the oxyanion hole of acetylcholinesterase. Furthermore, it was found that ortho electron-donating substituents of the inhibitors accelerated inhibition of butyrylcholinesterase by ortho polar effects. Conformations of enzyme-inhibitor tetrahedral intermediates for butyrylcholinesterase were different from those for acetylcholinesterase and cholesterol esterase; ortho substituents in the tetrahedral intermediates were located far from the negatively charged carbonyl oxygens in butyrylcholinesterase, but close to the negatively charged carbonyl oxygens in acetylcholinesterase and cholesterol esterase. In conclusion, electron-donating substituents in the ortho position were better inhibitors of butyrylcholinesterase than acetylcholinesterase, while electron-withdrawing substituents were better inhibitors of acetylcholinesterase.

Introduction Butyrylcholinesterase (BChE1, EC 3.1.1.8) is a serine hydrolase related to acetylcholinesterase (AChE, EC 3.1.1.7). Unlike AChE, which plays a vital role in the central and peripheral nervous systems, the physiological function of BChE remains unclear (1, 2). Despite having no identified endogenous substrate, BChE plays a key role in detoxification, by degrading esters such as succinylcholine and cocaine (3). The X-ray structures of BChE and BChE-inhibitor complex have been recently reported (4, 5). Similar to AChE (6-9), the active site of BChE (Figure 1) contains (a) an esteratic site (ES) comprised of the catalytic triad Ser198-His438-Glu325, which is located at the bottom of the gorge (4, 5), (b) an oxyanion * Corresponding author. Fax: 886-4-2286-2547. E-mail: [email protected]. 1 Abbreviation: ABS, acyl group binding site; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; AS, anionic binding site; ATCh, acetylthiocholine; δ, intensity factor for ortho steric constant; DTNB, 5,5′-dithio-bis-2-nitrobenzoate; BCh, butyrylcholine; BChE, butyrylcholinesterase; BTCh, butyrylthiocholine; CEase, cholesterol esterase; CRL, Candida rugosa lipase; ES, esteratic site or catalytic triad; ESo, Taft-Kutter-Hansch ortho steric constant; F, Swain-Lupton-Hansch ortho polar constant or polar constant through space; f, intensity factor to the ortho polar constant; kc, carbamylation constant; kd, decarbamylation constant; Ki, inhibition constant; Ki′, virtual inhibition constant; ki, bimolecular inhibition constant; OAH, oxyanion hole; PAS, peripheral anionic binding sites; PCL, Pseudomonas cepacia lipase; PSL, Pseudomonas species lipase; QSAR, quantitative-structure activity relationship; F, Hammett reaction constant; σp, Hammett para-substituent constant or polar constant through bonds.

Figure 1. The acyl group binding site (ABS), oxyanion hole (OAH), esteratic site or catalytic triad (ES), anionic binding site (AS), and peripheral anionic binding site (PAS) of BChE.

hole (OAH) composed of Gly116, Gly117, and Ala199, that stabilizes the tetrahedral intermediate, (c) an anionic

10.1021/tx050014o CCC: $30.25 © 2005 American Chemical Society Published on Web 06/24/2005

Effects for BChE, AChE, CEase, and Lipase Inhibitions

Chem. Res. Toxicol., Vol. 18, No. 7, 2005 1125 Scheme 1: Kinetic Scheme for Pseudosubstrate Inhibitions of BChE in the Presence of Substrate

intermediate from the intermediate. In the presence of substrate, the pseudosubstrates aryl carbamates serve as inhibitors (Scheme 1) (5, 27). The carbamylation stage is rapid compared to subsequent decarbamylation (kc . kd)2, thus the two stages are easily resolved kinetically (20-27, 29-37). In the presence of a carbamate inhibitor, time courses for hydrolysis of butyrylthiocholine (BTCh) are biphasic, and kapp values can be calculated from eq 1 (31, 32). In eq 1, A0, kapp, vo, and vss are the absorbance

A ) A0 + (vo - vss)(1 - exp(- kappt ))/kapp + vsst Figure 2. Structures of carbamates 1-9, rivastigmine, physostigmine, edrophonium, and carbaryl.

substrate-binding site (AS) composed of Trp82, where the quaternary ammonium pole of butyrylcholine (BCh) and of various active site ligands binds through a preferential interaction of quaternary nitrogens with the π electrons of aromatic groups, (d) an acyl group-binding site (ABS) that binds the acyl or carbamyl group of substrate or inhibitor, and (e) a peripheral anionic binding site (PAS) composed of Phe278 (10), Tyr332 (11), and Asp70, which is located at the entrance (mouth) of the active site gorge that may bind to the tacrine-based heterobivalent ligands (10) and cage amines (12). In Alzheimer’s disease (AD), a neurological disorder, cholinergic deficiency in the brain has been reported (13, 14). Four drugs for treatment of AD, tacrine (Cognex), donepezil (Aricept), rivastigmine (Exelon) (Figure 2), and galantamine (Reminyl), are dual inhibitors of AChE and BChE (14). The additional demonstration that central BChE rather than AChE inhibition is the best correlation of cognitive improvement in AD clinical studies with the dual cholinesterase inhibitor rivastigmine (Figure 2) further suggests that BChE represents an intriguing target to develop drugs for the treatment of neurodegenerative disease (15-17). The derivatives of physostigmine (Figure 2) are also potential drugs for the treatment of AD (18). Since rivastigmine (19) and physostigmine are carbamates, both inhibition mechanisms of AChE (2026) and BChE (5, 27) by carbamates may play important roles for treatment of AD. Carbaryl (1-naphthyl N-methylcarbamate, Sevin) (Figure 2), carbofuran (Furadan), propoxur (Baygon), and aldicarb (Temik) are carbamate pesticides that have activities against a broad range of insects and low mammalian toxicity (28). These carbamate pesticides are also dual inhibitors of AChE and BChE. Therefore, both inhibition mechanisms of AChE and BChE by carbamates may also play important roles in understanding the mechanism of pesticide toxicology. The mechanism for BChE-catalyzed hydrolysis of substrate is formation of the first tetrahedral intermediate via nucleophilic attack of the active site Ser198 (Figure 1) to substrate, then formation of the acyl enzyme

(1)

at t ) 0, observed first-order inhibition rate constant, initial velocity, and steady-state velocity, respectively. Reactions must be followed for at least six half-lives to obtain reliable estimates of the parameters, especially vss and kapp (33). Once kapp values have been determined at various inhibitor concentrations, the resulting data are fit to eq 2 to obtain Ki and kc values. In other words, Ki and kc values are obtained from each nonlinear leastsquares curve fittings of kapp values against [I] according to eq 2 (22-27, 31-38). Determination of the Ki and kc

kapp ) kc[I]/(Ki(1 + [S]/Km) + [I])

(2)

values by this method is called the continuous assay method and is much more rapid than a traditional stopped-time (or dilution) assay method (31). The bimolecular rate constant, ki ) kc/Ki, is related to overall inhibitory potency. Moreover, aryl carbamates meet the third criterion for the pseudosubstrate inhibitors, as proposed by Abeles and Maycock (39), in that enzyme is protected from the inhibitions by carbamates in the presence of a reversible inhibitor, edrophonium (Figure 2). Therefore, carbamates are characterized as pseudosubstrate inhibitors of BChE (5, 27). Quantitative structure-activity relationships (QSARs) represent an attempt to correlate structural properties of compounds with biological activities or chemical reactivities (40, 41). These chemical descriptors, which include parameters to account for hydrophobicity, electronic, inductive, or polar properties, and steric effects, are determined empirically or by calculations. Little additional development of QSAR has occurred until the work of Louis Hammett, who has correlated electronic properties of substituted benzoic acids with their equilibrium constants and reactivities by the Hammett equation (eq 3).

log k ) h + Fσ

(3)

In eq 3, the h value is the log k0 value for the standard reaction (unsubstituted benzoic acid) and the parameters F and σ are the Hammett reaction constant and the 2 The k value of (9 ( 2) × 10-4 s-1 for BChE inhibition by d carbamates 1-9 is calculated from the progress curves (33).

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Table 1. Ortho Substituent Constantsa and Inhibition Constants of the BChE Inhibitions by ortho-Substituted Phenyl N-Butyl Carbamates (1-9) inhibitor

X

σp

ESo

F

Ki (µM)

kc (10-3 s-1)

ki (103 M-1 s-1)

selectivity for BChE over AChEb

1 2 3 4 5 6 7 8 9

o-OMe o-t-Bu o-CH3 o-Et o-Ph H o-Cl o-CF3 o-NO2

-0.27 -0.2 -0.17 -0.15 -0.01 0 0.23 0.54 0.78

-0.55 -2.78 -1.24 -1.31 -1.01 0 -0.97 -2.40 -2.52c

0.26 -0.07 -0.04 -0.05 0.08 0 0.41 0.38 0.67

17 ( 2 22 ( 4 11 ( 3 4.2 ( 0.6 11 ( 2 9(2 13 ( 2 1.7 ( 0.7 0.7 ( 0.1

7.5 ( 0.7 8.0 ( 0.7 9(1 10 ( 1 10 ( 1 12 ( 1 14.8 ( 0.6 15 ( 1 18 ( 1

0.44 ( 0.07 0.36 ( 0.07 0.8 ( 0.2 2.4 ( 0.4 0.9 ( 0.1 1.3 ( 0.5 1.1 ( 0.1 9(4 26 ( 4

2.5 ( 0.5 4(1 1.1 ( 0.4 0.4 ( 0.1 1.0 ( 0.2 0.8 ( 0.3 0.6 ( 0.2 0.11 ( 0.06 0.04 ( 0.01

a

The σp, ESo, and F values were obtained from the literature (42). b ki (BChE)/ki (AChE). c Maximum value for the coplanar orientation.

substituents constant, respectively. The investigation also reveals that meta- and para-substituted compounds generally correlate well, but ortho-substituted ones do not (40) due to complications from direct steric and polar effects (42). According to Fujita and Nishioka’s suggestion, the total ortho effect is composed of the ordinary polar effect or polar effect through bonds, ortho steric effect, and ortho polar effect or polar effect through space (eq 4) (40, 42). In eq 4, the parameters h, F, σp, ESo, δ, F,

log k ) h + Fσp + δESo + fF

(4)

and f are the intercept or calculated value for carbamate 6, Hammett reaction constant for ordinary polar effect, Hammett para-substituent constant, Taft-Kutter-Hansch ortho steric constant, intensity factor to ortho steric constant, Swain-Lupton-Hansch ortho polar constant, and intensity factor to ortho polar constant, respectively. Once the Ki kc, and ki values have been determined from eq 2, the logarithms of 1/Ki, kc, and ki are treated with least-squares fittings with three parameters, ESo, δ, and F (Table 1) against eq 4 (multiple-parameters linear regression analysis) to determine the h, F, δ, and f values. In other words, the F, δ, and f values are the slopes or sensitivity factors for the pKi-, log kc-, and log ki-ESo-δ-F correlations, respectively, and h is the intercept or calculated values for carbamate 6 (Figure 2) for these correlations. Ortho effects for QSARs of AChE (24), Pseudomonas species lipase (PSL) (37), and cholesterol esterase (CEase) (38) inhibitions by ortho-substituted phenyl N-butyl carbamates (1-9) (Figure 2) have been recently reported. In this paper, we further study ortho effects for QSARs of BChE inhibitions by carbamates 1-9 and compare BChE inhibition to AChE, PSL, and CEase inhibition.

Materials and Methods Materials. Horse serum BChE, DTNB, and BTCh 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. All other chemicals were of the highest purity available commercially. Synthesis of Carbamates. Carbamates 1-9 were prepared from the condensation of the corresponding phenol with n-butyl isocyanate in the presence of a catalytic amount of pyridine in toluene (80-95% yield) as described previously (24, 37, 38). All compounds were purified by liquid chromatography on silica gel and characterized by 1H and 13C NMR spectra and highresolution mass spectra. Instrumental Methods. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Varian-

GEMINI 400 spectrometer. HRMS were recorded at 70 eV on a Joel JMS-SX/SX-102A mass spectrometer. All steady-state kinetic data were obtained from a UV-vis spectrometer (Spectronic Genesys 8, Agilent 8453, or Scinco S-3100) with a cell holder circulated with a water bath. Data Reduction. Origin (version 6.0) was used for linear, nonlinear, and multiple-parameters linear least-squares fittings (regression analyses). Steady-State Enzyme Kinetics. BChE inhibition by carbamates 1-9 was assayed by the Ellman method (43). The temperature was maintained at 25.0 °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 vol %), Triton X-100 (0.5 wt %), substrate (50 µM), and varying concentrations of inhibitors. The concentration ranges for carbamates 1-7 were from 0.1 to 50 µM, and those for carbamates 8 and 9 were from 0.01 to 10 µM. Requisite volumes of stock solution of substrate and inhibitors in acetonitrile were injected into reaction buffer via a pipet. BChE was dissolved in sodium phosphate buffer (0.1 M, pH 7.0). First-order rate constant (kapp) for inhibition was determined as described by Hosie et al. (eq 1) (31-33). The Ki and kc values were obtained by nonlinear least-squares curve fittings of the kapp values versus concentration of inhibitor ([I]) plot against eq 2 (21-27, 31-38). Duplicate sets of data were collected for each inhibitor concentration.

Results The synthesis of carbamates 1-9 (Figure 2) was reported previously (24, 37, 38). Carbamates 1-9 were characterized as pseudosubstrate inhibitors of BChE because the inhibitions were time-dependent, the inhibitions followed first-order kinetics, and the BChE activities were protected from a competitive inhibitor (39), edrophonium (Figure 2). These compounds were also pseudosubstrates of AChE (24), CEase (38), and PSL (37). The σp, ESo, and F values (40, 42) for ortho substituents of carbamates 1-9 are listed in Table 1. For the BChE inhibitions by carbamates 1-9, the Ki, kc, and ki values, which were determined from eqs 1 and 2, are summarized in Table 1. In general, the inhibitors with strong electron-withdrawing substituents such as nitro and trifluoromethyl groups were 10-fold more potent inhibitors than the inhibitors with strong electron-donating substituents such as OMe and t-Bu. The selectivity for BChE over AChE (24) inhibitions by carbamates 1-9 was defined as ki (BChE)/ki (AChE) (Table 1). Carbamates 1-9 with strong electron-donating substituents were selective for BChE over AChE inhibitions. On the contrary, carbamates 1-9 with strong electron-withdrawing substituents were selective for AChE over BChE inhibitions.

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Chem. Res. Toxicol., Vol. 18, No. 7, 2005 1127

Figure 3. The proposed mechanism for BChE inhibition by carbamates 1-9.

Since carbamates 1-9 were protonated at pH 7.0 buffer solution (22, 36), the Ki step (Scheme 1) consisted of the preequilibrium protonation Kb step and Ki′ step (Figure 3) (22-25, 37, 38). Thus, the virtual inhibition constant, Ki′ was calculated from eq 5.

pKi′ ) pKi + pKb

(5)

The logarithms of 1/Ki, kc, and ki for BChE inhibitions by carbamates 1-9 were best correlated with threeparameters, σp, ESo, and F (24, 37, 38, 42) of least-squares fittings (Table 2). Moreover, correlations of the pKi′ values were also calculated from eq 5 and summarized in Table 2. Values of F, δ, and f for the pKi′-, pKi-, log kc-, and log ki-σp-ESo-F correlations for BChE inhibitions were 4, 1.4, 0.47, 1.9; 0.0, 0, 0.04, 0.0; and -0.5, -0.5, -0.10, -0.6, respectively (Table 2). The F value of 4 for the pKi′correlation of the BChE inhibitions by carbamates 1-9 indicated that the enzyme-inhibitor tetrahedral intermediates were more negatively charged than the proto-

Table 2. Correlation Results for BChE Inhibition by ortho-Substituted Phenyl N-Butyl Carbamates (1-9) pKi

pKi′ a

log kc

log ki

Fb hb Rb

1.2 ( 0.3 5.08 ( 0.09 0.859

3.7 ( 0.4 9.08 ( 0.09 0.857

0.34 ( 0.05 -1.98 ( 0.02 0.935

1.5 ( 0.3 3.10 ( 0.09 0.912

Fc δc hc Rc

1.1 ( 0.3 0.0 ( 0.3 5.0 ( 0.2 0.862

3.6 ( 0.3 0.0 ( 0.3 9.0 ( 0.2 0.860

0.38 ( 0.04 0.04 ( 0.02 -1.93 ( 0.03 0.964

1.5 ( 0.3 0.0 ( 0.1 3.1 ( 0.2 0.912

Fd δd fd hd Rd

1.4 ( 0.6 0.0 ( 0.1 -0.5 ( 0.2 5.1 ( 0.2 0.872

4(1 0.0 ( 0.1 -0.5 ( 0.2 9.1 ( 0.4 0.870

0.47 ( 0.08 0.04 ( 0.02 -0.10 ( 0.01 -1.90 ( 0.03 0.975

1.9 ( 0.6 0.0 ( 0.1 -0.6 ( 0.3 3.2 ( 0.2 0.923

a pK ′ ) pK + pK (eq 5) (24, 37, 38). b Correlations of pK , log i i b i kc, and log ki with σp. c Multiple-parameter correlations of pKi, log kc, and log ki with σp and ESo. d Multiple-parameter correlation of pKi, log kc, and log ki with σp, ESo, and F.

nated inhibitors (Figure 3). This result also confirmed three-pronged hydrogen bonds for the oxyanion hole of

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Figure 4. The F values for pKi′-correlations of the AChE, BChE, CEase, and PSL inhibition by ortho-, meta-, and parasubstituted phenyl N-butylcarbamates. Enzyme (o) represents inhibition by ortho-substituted inhibitors. Enzyme (m, p) represents inhibition by meta- and para-substituted inhibitors (21, 23). Table 3. Sensitivity Factors for the pKi′-Correlations of the AChE, BChE, CEase, and PSL Inhibitions by ortho-Substituted Phenyl N-Butyl Carbamates (1-9) F δ f a

AChEa

BChE

CEaseb

PSLc

1.9 ( 0.6 -0.16 ( 0.05 0.7 ( 0.3

4(1 0.0 ( 0.1 -0.5 ( 0.2

3(1 -0.07 ( 0.07 0.5 ( 0.3

2.7 ( 0.3 -0.06 ( 0.07 -1.7 ( 0.4

Taken from ref 24. b Taken from ref 38. c Taken from ref 37.

BChE (3) (discuss later). Small δ values for the pKi′-, pKi-, log kc-, and log ki-correlations of the BChE inhibitions by carbamates 1-9 revealed that ortho steric effects did not play important roles for these inhibition reactions. The f value of -0.5 for the pKi′-correlation of the BChE inhibitions indicated that strong ortho electron-donating substituents of the inhibitors accelerated the inhibition reactions by ortho polar effects or polar effects through space. Therefore, ortho substituents were far away from the negatively charged carbonyl oxygens in the tetrahedral intermediates (Figure 3) for BChE inhibitions (discuss later).

Discussion Proposed Mechanisms for BChE Inhibition by Carbamates 1-9. The BChE inhibition mechanism by carbamates 1-9 proposed in Figure 3 is similar to the mechanism for AChE, CEase, and PSL inhibition (24, 37, 38). Since carbamates 1-9 are protonated at pH 7.0 buffer solution (22, 36), the Ki step consists of the protonation, Kb step, and then the virtual inhibition, Ki′ step (Figure 3) (24, 37, 38). Accordingly, the second step in this mechanism, Ki′ step, is formation of the negatively charged enzyme-inhibitor tetrahedral intermediate from the protonated inhibitors (Figure 3). The third step in this mechanism is formation of the carbamyl enzyme from the above tetrahedral intermediate (Figure 3). Virtual Inhibition, Ki′ Step. 1. The G Value. Positive F values for the pKi′ -correlations (Figure 4) in the AChE (21, 24), SPL (37), CEase (34, 38), and BChE (Table 3) inhibitions by substituted phenyl N-butylcarbamates reveal that the enzyme-inhibitor tetrahedral intermediates (Figure 3) are more negatively charged than the protonated inhibitors. More positive F values indicate that the inhibition reactions are more sensitive to the

substituents of the inhibitors and that the enzymes are relatively more nucleophilic to the inhibitors. Since the two closest sites to the reaction center for a serine hydrolase are the nucleophilic serine and OAH of the enzyme (Figure 1) and the nucleophilic serine is in common for all serine hydrolases, the nucleophilicity of the enzyme therefore may depend on the OAHs of the enzymes. Thus, the F values for various serine hydrolase inhibitions by common inhibitors may represent a scale to measure the nucleophilicity of a serine hydrolase. The F value of 4 for the pKi′-correlation of the BChE inhibitions by carbamates 1-9 (Table 2) indicates that the enzyme-inhibitor tetrahedral intermediates (Figure 3) are more negatively charged than the protonated inhibitors. Comparison of this value with the F value of 1.9 for the pKi′-correlation of the AChE inhibitions by carbamates 1-9 (Table 3 and Figure 4) (24) reveals that the negative charges of the enzyme-inhibitor tetrahedral intermediates (Figure 3) are more stabilized by ortho electron-withdrawing substituents of the inhibitors for BChE inhibitions than for AChE inhibitions. Presumably, a major factor that stabilizes the negative charges of the enzyme-inhibitor tetrahedral intermediates is the number of hydrogen bonds formed between the negatively charged carbonyl oxygen and the peptidic NH groups in the oxyanion hole (OAH). Recent combined ab initio quantum mechanical/molecular mechanical calculations indicate that, in the tetrahedral intermediate, only two hydrogen bonds are formed between the carbonyl oxygen of ACh and the peptidic NH groups of Gly118 and Gly119 (44) without the third hydrogen bond between the carbonyl oxygen of ACh and the peptidic NH of Ala201 as suggested from the X-ray structure (9). Recent molecular dynamic calculations also indicate that, in the first tetrahedral intermediate of the BChE-cocaine complex, three hydrogen bonds are formed between the carbonyl oxygen of cocaine and the peptidic NH groups of Gly116, Gly117, and Ala199 (3). Obviously, the larger F value of 4 for the pKi′-correlation of the BChE inhibitions by carbamates 1-9 (Figure 4) implies that three- instead of two-pronged hydrogen bonds are formed between the negatively charged carbonyl oxygens of the enzymeinhibitor tetrahedral intermediates (Figure 3) and the peptidic NH groups of Gly116, Gly117, and Ala199 in the OAH of BChE. On the other hand, the smaller F value of 1.9 for the pKi′-correlation of the AChE inhibitions by carbamates 1-9 (Figure 4) (24) suggests that twopronged hydrogen bonds are formed between the negatively charged carbonyl oxygens of the enzyme-inhibitor tetrahedral intermediates (Figure 3) and the peptidic NH groups of Gly118 and Gly119 in the OAH of AChE. Therefore, this result confirms formations of threepronged hydrogen bonds for the OAH of BChE and formations of two-pronged hydrogen bonds for the OAH of AChE. Since the F values for the pKi′-correlations in both CEase and PSL inhibitions by carbamates 1-9 (Figure 4) (37, 38) are between 4 and 1.9, we cannot tell at this time whether two- or three-pronged hydrogen bonds are formed between the negatively charged carbonyl oxygens of the enzyme-inhibitor tetrahedral intermediates (Figure 3) and the OAHs of both enzymes. However, the F value of 6 (Figure 4) for the pKi′correlations of the CEase inhibitions by meta- and parasubstituted phenyl N-butylcarbamates (34) may suggest that three-pronged hydrogen bonds are formed between the negatively charged carbonyl oxygens of the enzyme-

Effects for BChE, AChE, CEase, and Lipase Inhibitions

Chem. Res. Toxicol., Vol. 18, No. 7, 2005 1129 Table 4. Sensitivity Factors for the log kc-Correlations of the AChE, BChE, CEase, and PSL Inhibitions by ortho-Substituted Phenyl N-Butyl Carbamates (1-9) F δ f a

AChEa

BChE

CEaseb

PSLc

0.11 ( 0.06 0.03 ( 0.02 -0.3 ( 0.1

0.47 ( 0.08 0.04 ( 0.02 -0.10 ( 0.01

0.5 ( 0.1 0.04 ( 0.05 -0.5 ( 0.2

0.8 ( 0.1 0.00 ( 0.03 0.0 ( 0.2

Taken from ref 24. b Taken from ref 38. c Taken from ref 37.

Figure 5. Two putative conformations for the tetrahedral intermediates of the enzyme-carbamates 1-9 adducts in the virtual inhibition, Ki′ step. In the pseudo-cis conformation (top), the substituent X is at the same site as the negatively charged carbonyl oxygen. In the pseudo-trans conformation (bottom), the substituent X is on the opposite site of the negatively charged carbonyl oxygen.

inhibitor tetrahedral intermediates and the peptidic NH groups in the OAH of CEase. 2. The δ Value. Small δ values for the pKi′-correlations of all four enzyme inhibitions by carbamates 1-9 (Table 3) reveal that ortho steric effects are insensitive to these inhibition reactions. Furthermore, a null δ value for the pKi′-correlation of BChE inhibition carbamates 1-9 indicates that the substituent X of the enzyme-inhibitor tetrahedral intermediate (Figure 3), which adapts a pseudo-trans conformation, is far away from the oxygen atom of Ser198 (Figure 5). On the other hand, negative δ values for the pKi′-correlations of the AChE, CEase, and PSL inhibitions by carbamates 1-9 (Table 3) suggest that the substituent X of the enzyme-inhibitor tetrahedral intermediate (Figure 3), which adapts a pseudo-cis conformation, is close to the nucleophilic serine of the enzyme (Figure 5). Thus, small, negative δ values for the AChE, CEase, and PSL inhibitions by carbamates 1-9 are due to little enhancement of the steric effect (24) from the substituent X of the inhibitors to the nucleophilic serine of the enzymes. 3. The f Value. The f value of -0.5 for the pKi′correlation of the BChE inhibitions by carbamates 1-9 (Table 3) indicates that ortho electron-donating substituents of the inhibitors accelerate the inhibition reactions through ortho polar effects or polar effects through space. Comparing this value with those for AChE and CEase inhibitions (Table 3), we suggest that conformations of the enzyme-inhibitor tetrahedral intermediates (Figure 3) for BChE inhibitions are different to those for both AChE and CEase inhibitions (Figure 5). In the enzymeinhibitor tetrahedral intermediates (Figure 3) for BChE inhibitions, the ortho substituents at the phenyl groups are far away from the negatively charged carbonyl oxygens, and thus, the tetrahedral intermediates adapt pseudo-trans conformations (Figure 5). However, in the enzyme-inhibitor tetrahedral intermediates for both AChE and CEase inhibitions, the ortho substituents at

Figure 6. Two putative conformations for the transition states in the carbamylation, kc step. In the pseudo-cis conformation (top), the substituent X is on the same site of the negatively charged carbonyl oxygen. In the pseudo-trans conformation (bottom), the substituent X is on the opposite site of the negatively charged carbonyl oxygen.

the phenyl groups are relatively close to the negatively charged carbonyl oxygens, and thus, the tetrahedral intermediates adapt pseudo-cis conformation (Figure 5). This result is in agreement with that for the δ value (discussed above). As regards PSL, the large, negative f value of -1.7 also implies that the enzyme-inhibitor tetrahedral intermediates adapt pseudo-cis conformations and further suggests that the 1,3-proton transfer may occur during the reaction (37). Carbamylation, kc Step. 1. The Role of n-Butyryl. The carbamylation rates are about 10 times slower in AChE (24) when compared to those in BChE (Table 1). We know that butyrylcholine is virtually not hydrolyzed by AChE because of steric hindrance. So, faster carbamylation in BChE is likely to be a consequence of ‘easier’ accommodation of acyl part of the ligand. 2. The G Value. Positive F values for all log kccorrelations (Table 4) indicate that the transition state of the kc step is slightly more negatively charged than the enzyme-inhibitor tetrahedral intermediate (Figure 3). Since the product of the kc step is neutral orthosubstituted phenol, the transition state of this step (Figure 6) is similar to the enzyme-inhibitor tetrahedral intermediate (Figure 3). This is because negative charges on the carbonyl oxygen redistribute to the phenol oxygen and make them closer to ortho substituents of the inhibitors (24). Therefore, the F values for the log kccorrelations are indeed a measure for this charge redistribution. Notably, that the F values for the log kccorrelations of both BChE and CEase inhibitions are close

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while electron-withdrawing substituents are better inhibitors of AChE.

Acknowledgment. We thank the National Science Council of Taiwan for financial support.

References

Figure 7. Plot of ki (BChE)/ki (AChE) values for the inhibitions by carbamates 1-9 against Hammett substituent constant (σP) of the substituent X. Data are from Table 1.

to each other implies a similar mechanism for both inhibition reactions in their kc steps. For the kc steps of BChE, AChE, and CEase inhibitions, the leaving groups are presumably the ortho-substituted phenoxide ions due to positive F values (Table 3). On the other hand, the products for the kc steps of PSL inhibitions are likely to be the ortho-substituted phenols due to the 1,3-protontransfer mentioned above (37). 3. The δ Value. All ortho steric effects in the kc steps are negligible (Table 4). Thus, the leaving group binding site of the enzyme is large enough to adapt any bulky ortho-substituted phenol. In other words, increasing the C-O bond length (Figure 6) toward the transition state for the kc step weakens ortho steric effects. 4. The f Value. Small negative f values for the log kccorrelations of the BChE, AChE, and CEase inhibitions by carbamates 1-9 (Table 4) indicate that electrondonating substituents facilitate the inhibition reactions through space better than the electron-withdrawing ones. For kc steps, therefore, the transition states for BChE inhibitions retain their pseudo-trans conformations, while those for both AChE and CEase inhibitions change from pseudo-cis to pseudo-trans (Figure 5). Overall Inhibition Reaction. The selectivity for BChE over AChE (24) inhibitions by carbamates 1-9 is defined as ki (BChE)/ki (AChE) (Table 1). In general, carbamates 1-9 with electron-donating substituents are selective for BChE over AChE inhibitions such as tacrine, but carbamates 1-9 with electron-withdrawing substituents are selective for AChE over BChE inhibitions such as rivastigmine (Figure 7). Therefore, selection for BChE or AChE inhibition partially depends on electronic characters of the substituents. Among carbamates 1-9, o-nitrophenyl N-butylcarbamate (9) is the most potent inhibitor of both BChE and CEase and is also a potent inhibitor of both AChE and PSL. The inhibitory potency for carbamate 9 is BChE (ki ) 26 000 M-1 s-1) > CEase (ki ) 1500 M-1 s-1) > AChE (ki ) 1000 M-1 s-1) > PSL (ki ) 110 M-1 s-1) (Table 1 and refs 24, 37, 38). Therefore, carbamate 9 is a good candidate as a standard electrophile in measuring the nucleophilicity of a serine hydrolase or protease. In conclusion, electron-donating substituents in the ortho position are better inhibitors of BChE than AChE,

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