ortho effects for inhibition mechanisms of butyrylcholinesterase by o -substituted phenyl n -butyl...

Ortho Effects for Inhibition Mechanisms ofButyrylcholinesterase by o-Substituted Phenyl N-ButylCarbamates and Comparison with Acetylcholinesterase,

Cholesterol Esterase, and LipaseGialih 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 inhibitacetylcholinesterase and butyrylcholinesterase. The goal of this work was to determine thechemical characteristics of ortho substituents that make some carbamates better inhibitors ofbutyrylcholinesterase than of acetylcholinesterase, cholesterol esterase, and lipase. Theinhibition constants, Ki, Ki′, kc, and ki were measured for nine different carbamates. The valueswere plotted according to Hammett, Taft-Kutter-Hansch, and Swan-Lupton to obtainconstants that correlated the chemical nature of the substituents with inhibition potency. Itwas found that the negative charges of tetrahedral intermediates were more stabilized by orthoelectron-withdrawing substituents of the inhibitors in butyrylcholinesterase than in acetyl-cholinesterase. This result confirmed formation of 3-pronged hydrogen bonds for the oxyanionhole of butyrylcholinesterase and 2-pronged hydrogen bonds for the oxyanion hole ofacetylcholinesterase. Furthermore, it was found that ortho electron-donating substituents ofthe inhibitors accelerated inhibition of butyrylcholinesterase by ortho polar effects. Conforma-tions of enzyme-inhibitor tetrahedral intermediates for butyrylcholinesterase were differentfrom those for acetylcholinesterase and cholesterol esterase; ortho substituents in thetetrahedral intermediates were located far from the negatively charged carbonyl oxygens inbutyrylcholinesterase, but close to the negatively charged carbonyl oxygens in acetylcholines-terase and cholesterol esterase. In conclusion, electron-donating substituents in the orthoposition were better inhibitors of butyrylcholinesterase than acetylcholinesterase, whileelectron-withdrawing substituents were better inhibitors of acetylcholinesterase.

Introduction

Butyrylcholinesterase (BChE1, EC 3.1.1.8) is a serinehydrolase related to acetylcholinesterase (AChE, EC3.1.1.7). Unlike AChE, which plays a vital role in thecentral and peripheral nervous systems, the physiologicalfunction of BChE remains unclear (1, 2). Despite havingno identified endogenous substrate, BChE plays a keyrole in detoxification, by degrading esters such as succi-nylcholine and cocaine (3). The X-ray structures of BChEand 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) comprisedof the catalytic triad Ser198-His438-Glu325, which islocated at the bottom of the gorge (4, 5), (b) an oxyanion

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

* 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, anionicbinding site; ATCh, acetylthiocholine; δ, intensity factor for ortho stericconstant; DTNB, 5,5′-dithio-bis-2-nitrobenzoate; BCh, butyrylcholine;BChE, butyrylcholinesterase; BTCh, butyrylthiocholine; CEase, cho-lesterol esterase; CRL, Candida rugosa lipase; ES, esteratic site orcatalytic triad; ES

o, Taft-Kutter-Hansch ortho steric constant; F,Swain-Lupton-Hansch ortho polar constant or polar constant throughspace; f, intensity factor to the ortho polar constant; kc, carbamylationconstant; kd, decarbamylation constant; Ki, inhibition constant; Ki′,virtual inhibition constant; ki, bimolecular inhibition constant; OAH,oxyanion hole; PAS, peripheral anionic binding sites; PCL, Pseudomo-nas cepacia lipase; PSL, Pseudomonas species lipase; QSAR, quantita-tive-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.

1124 Chem. Res. Toxicol. 2005, 18, 1124-1131

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

substrate-binding site (AS) composed of Trp82, where thequaternary ammonium pole of butyrylcholine (BCh) andof various active site ligands binds through a preferentialinteraction of quaternary nitrogens with the π electronsof aromatic groups, (d) an acyl group-binding site (ABS)that binds the acyl or carbamyl group of substrate orinhibitor, and (e) a peripheral anionic binding site (PAS)composed of Phe278 (10), Tyr332 (11), and Asp70, whichis located at the entrance (mouth) of the active site gorgethat 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), andgalantamine (Reminyl), are dual inhibitors of AChE andBChE (14). The additional demonstration that centralBChE rather than AChE inhibition is the best correlationof cognitive improvement in AD clinical studies with thedual cholinesterase inhibitor rivastigmine (Figure 2)further suggests that BChE represents an intriguingtarget to develop drugs for the treatment of neurodegen-erative disease (15-17). The derivatives of physostigmine(Figure 2) are also potential drugs for the treatment ofAD (18). Since rivastigmine (19) and physostigmine arecarbamates, both inhibition mechanisms of AChE (20-26) and BChE (5, 27) by carbamates may play importantroles for treatment of AD.

Carbaryl (1-naphthyl N-methylcarbamate, Sevin) (Fig-ure 2), carbofuran (Furadan), propoxur (Baygon), andaldicarb (Temik) are carbamate pesticides that haveactivities against a broad range of insects and lowmammalian toxicity (28). These carbamate pesticides arealso dual inhibitors of AChE and BChE. Therefore, bothinhibition mechanisms of AChE and BChE by carbam-ates may also play important roles in understanding themechanism of pesticide toxicology.

The mechanism for BChE-catalyzed hydrolysis ofsubstrate is formation of the first tetrahedral intermedi-ate via nucleophilic attack of the active site Ser198(Figure 1) to substrate, then formation of the acyl enzyme

intermediate from the intermediate. In the presence ofsubstrate, the pseudosubstrates aryl carbamates serveas inhibitors (Scheme 1) (5, 27). The carbamylation stageis 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

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 toobtain reliable estimates of the parameters, especiallyvss and kapp (33). Once kapp values have been determinedat various inhibitor concentrations, the resulting data arefit to eq 2 to obtain Ki and kc values. In other words, Ki

and kc values are obtained from each nonlinear least-squares curve fittings of kapp values against [I] accordingto eq 2 (22-27, 31-38). Determination of the Ki and kc

values by this method is called the continuous assaymethod and is much more rapid than a traditionalstopped-time (or dilution) assay method (31). The bimo-lecular rate constant, ki ) kc/Ki, is related to overallinhibitory potency. Moreover, aryl carbamates meet thethird criterion for the pseudosubstrate inhibitors, asproposed by Abeles and Maycock (39), in that enzyme isprotected from the inhibitions by carbamates in thepresence of a reversible inhibitor, edrophonium (Figure2). Therefore, carbamates are characterized as pseu-dosubstrate inhibitors of BChE (5, 27).

Quantitative structure-activity relationships (QSARs)represent an attempt to correlate structural propertiesof compounds with biological activities or chemical reac-tivities (40, 41). These chemical descriptors, which in-clude parameters to account for hydrophobicity, elec-tronic, inductive, or polar properties, and steric effects,are determined empirically or by calculations. Littleadditional development of QSAR has occurred until thework of Louis Hammett, who has correlated electronicproperties of substituted benzoic acids with their equi-librium constants and reactivities by the Hammettequation (eq 3).

In eq 3, the h value is the log k0 value for the standardreaction (unsubstituted benzoic acid) and the parametersF and σ are the Hammett reaction constant and the

2 The kd value of (9 ( 2) × 10-4 s-1 for BChE inhibition bycarbamates 1-9 is calculated from the progress curves (33).

Figure 2. Structures of carbamates 1-9, rivastigmine, phys-ostigmine, edrophonium, and carbaryl.

Scheme 1: Kinetic Scheme for PseudosubstrateInhibitions of BChE in the Presence of Substrate

A ) A0 + (vo - vss)(1 - exp(- kappt ))/kapp + vsst (1)

kapp ) kc[I]/(Ki(1 + [S]/Km) + [I]) (2)

log k ) h + Fσ (3)

Effects for BChE, AChE, CEase, and Lipase Inhibitions Chem. Res. Toxicol., Vol. 18, No. 7, 2005 1125

substituents constant, respectively. The investigation alsoreveals that meta- and para-substituted compoundsgenerally correlate well, but ortho-substituted ones do not(40) due to complications from direct steric and polareffects (42). According to Fujita and Nishioka’s sugges-tion, the total ortho effect is composed of the ordinarypolar effect or polar effect through bonds, ortho stericeffect, and ortho polar effect or polar effect through space(eq 4) (40, 42). In eq 4, the parameters h, F, σp, ES

o, δ, F,

and f are the intercept or calculated value for carbamate6, Hammett reaction constant for ordinary polar effect,Hammett para-substituent constant, Taft-Kutter-Han-sch ortho steric constant, intensity factor to ortho stericconstant, Swain-Lupton-Hansch ortho polar constant,and intensity factor to ortho polar constant, respectively.Once the Ki kc, and ki values have been determined fromeq 2, the logarithms of 1/Ki, kc, and ki are treated withleast-squares fittings with three parameters, ES

o, δ, andF (Table 1) against eq 4 (multiple-parameters linearregression analysis) to determine the h, F, δ, and f values.In other words, the F, δ, and f values are the slopes orsensitivity factors for the pKi-, log kc-, and log ki-ES

o-δ-Fcorrelations, respectively, and h is the intercept orcalculated values for carbamate 6 (Figure 2) for thesecorrelations.

Ortho effects for QSARs of AChE (24), Pseudomonasspecies lipase (PSL) (37), and cholesterol esterase (CEase)(38) inhibitions by ortho-substituted phenyl N-butylcarbamates (1-9) (Figure 2) have been recently reported.In this paper, we further study ortho effects for QSARsof BChE inhibitions by carbamates 1-9 and compareBChE inhibition to AChE, PSL, and CEase inhibition.

Materials and Methods

Materials. Horse serum BChE, DTNB, and BTCh wereobtained from Sigma; other chemicals were obtained fromAldrich. Silica gel used in liquid chromatography (Licorpre Silica60, 200-400 mesh) and thin-layer chromatography plates (60F254) were obtained from Merck. All other chemicals were of thehighest purity available commercially.

Synthesis of Carbamates. Carbamates 1-9 were preparedfrom the condensation of the corresponding phenol with n-butylisocyanate in the presence of a catalytic amount of pyridine intoluene (80-95% yield) as described previously (24, 37, 38). Allcompounds were purified by liquid chromatography on silica geland characterized by 1H and 13C NMR spectra and high-resolution mass spectra.

Instrumental Methods. 1H and 13C NMR spectra wererecorded at 400 and 100 MHz, respectively, on a Varian-

GEMINI 400 spectrometer. HRMS were recorded at 70 eV on aJoel JMS-SX/SX-102A mass spectrometer. All steady-statekinetic data were obtained from a UV-vis spectrometer (Spec-tronic Genesys 8, Agilent 8453, or Scinco S-3100) with a cellholder 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 car-bamates 1-9 was assayed by the Ellman method (43). Thetemperature was maintained at 25.0 °C by a refrigeratedcirculating water bath. All inhibition reactions were performedin sodium phosphate buffer (1 mL, 0.1 M, pH 7.0) containingNaCl (0.1 M), acetonitrile (2 vol %), Triton X-100 (0.5 wt %),substrate (50 µM), and varying concentrations of inhibitors. Theconcentration ranges for carbamates 1-7 were from 0.1 to 50µM, and those for carbamates 8 and 9 were from 0.01 to 10 µM.Requisite volumes of stock solution of substrate and inhibitorsin 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 determinedas described by Hosie et al. (eq 1) (31-33). The Ki and kc valueswere obtained by nonlinear least-squares curve fittings of thekapp values versus concentration of inhibitor ([I]) plot againsteq 2 (21-27, 31-38). Duplicate sets of data were collected foreach inhibitor concentration.

Results

The synthesis of carbamates 1-9 (Figure 2) wasreported previously (24, 37, 38). Carbamates 1-9 werecharacterized as pseudosubstrate inhibitors of BChEbecause the inhibitions were time-dependent, the inhibi-tions followed first-order kinetics, and the BChE activi-ties were protected from a competitive inhibitor (39),edrophonium (Figure 2). These compounds were alsopseudosubstrates of AChE (24), CEase (38), and PSL (37).The σp, ES

o, and F values (40, 42) for ortho substituentsof carbamates 1-9 are listed in Table 1. For the BChEinhibitions by carbamates 1-9, the Ki, kc, and ki values,which were determined from eqs 1 and 2, are sum-marized in Table 1. In general, the inhibitors with strongelectron-withdrawing substituents such as nitro andtrifluoromethyl groups were 10-fold more potent inhibi-tors than the inhibitors with strong electron-donatingsubstituents such as OMe and t-Bu. The selectivity forBChE over AChE (24) inhibitions by carbamates 1-9 wasdefined as ki (BChE)/ki (AChE) (Table 1). Carbamates1-9 with strong electron-donating substituents wereselective for BChE over AChE inhibitions. On the con-trary, carbamates 1-9 with strong electron-withdrawingsubstituents were selective for AChE over BChE inhibi-tions.

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

inhibitor X σp ESo F

Ki(µM)

kc(10-3 s-1)

ki(103 M-1 s-1)

selectivity forBChE over AChEb

1 o-OMe -0.27 -0.55 0.26 17 ( 2 7.5 ( 0.7 0.44 ( 0.07 2.5 ( 0.52 o-t-Bu -0.2 -2.78 -0.07 22 ( 4 8.0 ( 0.7 0.36 ( 0.07 4 ( 13 o-CH3 -0.17 -1.24 -0.04 11 ( 3 9 ( 1 0.8 ( 0.2 1.1 ( 0.44 o-Et -0.15 -1.31 -0.05 4.2 ( 0.6 10 ( 1 2.4 ( 0.4 0.4 ( 0.15 o-Ph -0.01 -1.01 0.08 11 ( 2 10 ( 1 0.9 ( 0.1 1.0 ( 0.26 H 0 0 0 9 ( 2 12 ( 1 1.3 ( 0.5 0.8 ( 0.37 o-Cl 0.23 -0.97 0.41 13 ( 2 14.8 ( 0.6 1.1 ( 0.1 0.6 ( 0.28 o-CF3 0.54 -2.40 0.38 1.7 ( 0.7 15 ( 1 9 ( 4 0.11 ( 0.069 o-NO2 0.78 -2.52c 0.67 0.7 ( 0.1 18 ( 1 26 ( 4 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.

log k ) h + Fσp + δESo + fF (4)

1126 Chem. Res. Toxicol., Vol. 18, No. 7, 2005 Lin et al.

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

The logarithms of 1/Ki, kc, and ki for BChE inhibitionsby carbamates 1-9 were best correlated with three-parameters, σp, ES

o, and F (24, 37, 38, 42) of least-squaresfittings (Table 2). Moreover, correlations of the pKi′ valueswere also calculated from eq 5 and summarized in Table2. Values of F, δ, and f for the pKi′-, pKi-, log kc-, and logki-σp-ES

o-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 1-9indicated that the enzyme-inhibitor tetrahedral inter-mediates were more negatively charged than the proto-

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

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

pKi′ ) pKi + pKb (5)

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

pKi pKi′ a log kc log ki

Fb 1.2 ( 0.3 3.7 ( 0.4 0.34 ( 0.05 1.5 ( 0.3hb 5.08 ( 0.09 9.08 ( 0.09 -1.98 ( 0.02 3.10 ( 0.09Rb 0.859 0.857 0.935 0.912

Fc 1.1 ( 0.3 3.6 ( 0.3 0.38 ( 0.04 1.5 ( 0.3δ c 0.0 ( 0.3 0.0 ( 0.3 0.04 ( 0.02 0.0 ( 0.1hc 5.0 ( 0.2 9.0 ( 0.2 -1.93 ( 0.03 3.1 ( 0.2Rc 0.862 0.860 0.964 0.912

Fd 1.4 ( 0.6 4 ( 1 0.47 ( 0.08 1.9 ( 0.6δd 0.0 ( 0.1 0.0 ( 0.1 0.04 ( 0.02 0.0 ( 0.1fd -0.5 ( 0.2 -0.5 ( 0.2 -0.10 ( 0.01 -0.6 ( 0.3hd 5.1 ( 0.2 9.1 ( 0.4 -1.90 ( 0.03 3.2 ( 0.2Rd 0.872 0.870 0.975 0.923

a pKi′ ) pKi + pKb (eq 5) (24, 37, 38). b Correlations of pKi, logkc, and log ki with σp. c Multiple-parameter correlations of pKi, logkc, and log ki with σp and ES

o. d Multiple-parameter correlation ofpKi, log kc, and log ki with σp, ES

o, and F.


BChE (3) (discuss later). Small δ values for the pKi′-, pKi-,log kc-, and log ki-correlations of the BChE inhibitionsby carbamates 1-9 revealed that ortho steric effects didnot play important roles for these inhibition reactions.The f value of -0.5 for the pKi′-correlation of the BChEinhibitions indicated that strong ortho electron-donatingsubstituents of the inhibitors accelerated the inhibitionreactions by ortho polar effects or polar effects throughspace. Therefore, ortho substituents were far away fromthe negatively charged carbonyl oxygens in the tetrahe-dral intermediates (Figure 3) for BChE inhibitions(discuss later).

Discussion

Proposed Mechanisms for BChE Inhibition byCarbamates 1-9. The BChE inhibition mechanism bycarbamates 1-9 proposed in Figure 3 is similar to themechanism for AChE, CEase, and PSL inhibition (24, 37,38). Since carbamates 1-9 are protonated at pH 7.0buffer solution (22, 36), the Ki step consists of theprotonation, Kb step, and then the virtual inhibition, Ki′step (Figure 3) (24, 37, 38). Accordingly, the second stepin this mechanism, Ki′ step, is formation of the negativelycharged enzyme-inhibitor tetrahedral intermediate fromthe protonated inhibitors (Figure 3). The third step inthis mechanism is formation of the carbamyl enzymefrom the above tetrahedral intermediate (Figure 3).

Virtual Inhibition, Ki′ Step. 1. The G Value. PositiveF 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-butylcarbamatesreveal that the enzyme-inhibitor tetrahedral intermedi-ates (Figure 3) are more negatively charged than theprotonated inhibitors. More positive F values indicatethat the inhibition reactions are more sensitive to the

substituents of the inhibitors and that the enzymes arerelatively more nucleophilic to the inhibitors. Since thetwo closest sites to the reaction center for a serinehydrolase are the nucleophilic serine and OAH of theenzyme (Figure 1) and the nucleophilic serine is incommon for all serine hydrolases, the nucleophilicity ofthe enzyme therefore may depend on the OAHs of theenzymes. Thus, the F values for various serine hydrolaseinhibitions by common inhibitors may represent a scaleto measure the nucleophilicity of a serine hydrolase. TheF value of 4 for the pKi′-correlation of the BChE inhibi-tions by carbamates 1-9 (Table 2) indicates that theenzyme-inhibitor tetrahedral intermediates (Figure 3)are more negatively charged than the protonated inhibi-tors. Comparison of this value with the F value of 1.9 forthe pKi′-correlation of the AChE inhibitions by carbam-ates 1-9 (Table 3 and Figure 4) (24) reveals that thenegative charges of the enzyme-inhibitor tetrahedralintermediates (Figure 3) are more stabilized by orthoelectron-withdrawing substituents of the inhibitors forBChE inhibitions than for AChE inhibitions. Presumably,a major factor that stabilizes the negative charges of theenzyme-inhibitor tetrahedral intermediates is the num-ber of hydrogen bonds formed between the negativelycharged carbonyl oxygen and the peptidic NH groups inthe oxyanion hole (OAH). Recent combined ab initioquantum mechanical/molecular mechanical calculationsindicate that, in the tetrahedral intermediate, only twohydrogen bonds are formed between the carbonyl oxygenof ACh and the peptidic NH groups of Gly118 and Gly119(44) without the third hydrogen bond between the car-bonyl oxygen of ACh and the peptidic NH of Ala201 assuggested from the X-ray structure (9). Recent moleculardynamic calculations also indicate that, in the firsttetrahedral intermediate of the BChE-cocaine complex,three hydrogen bonds are formed between the carbonyloxygen of cocaine and the peptidic NH groups of Gly116,Gly117, and Ala199 (3). Obviously, the larger F value of4 for the pKi′-correlation of the BChE inhibitions bycarbamates 1-9 (Figure 4) implies that three- insteadof two-pronged hydrogen bonds are formed between thenegatively charged carbonyl oxygens of the enzyme-inhibitor tetrahedral intermediates (Figure 3) and thepeptidic NH groups of Gly116, Gly117, and Ala199 in theOAH of BChE. On the other hand, the smaller F value of1.9 for the pKi′-correlation of the AChE inhibitions bycarbamates 1-9 (Figure 4) (24) suggests that two-pronged hydrogen bonds are formed between the nega-tively charged carbonyl oxygens of the enzyme-inhibitortetrahedral intermediates (Figure 3) and the peptidic NHgroups of Gly118 and Gly119 in the OAH of AChE.Therefore, this result confirms formations of three-pronged hydrogen bonds for the OAH of BChE andformations of two-pronged hydrogen bonds for the OAHof AChE. Since the F values for the pKi′-correlations inboth CEase and PSL inhibitions by carbamates 1-9(Figure 4) (37, 38) are between 4 and 1.9, we cannot tellat this time whether two- or three-pronged hydrogenbonds are formed between the negatively charged car-bonyl oxygens of the enzyme-inhibitor tetrahedral in-termediates (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 para-substituted phenyl N-butylcarbamates (34) may suggestthat three-pronged hydrogen bonds are formed betweenthe negatively charged carbonyl oxygens of the enzyme-

Figure 4. The F values for pKi′-correlations of the AChE,BChE, CEase, and PSL inhibition by ortho-, meta-, and para-substituted phenyl N-butylcarbamates. Enzyme (o) representsinhibition by ortho-substituted inhibitors. Enzyme (m, p) rep-resents inhibition by meta- and para-substituted inhibitors (21,23).

Table 3. Sensitivity Factors for the pKi′-Correlations ofthe AChE, BChE, CEase, and PSL Inhibitions by

ortho-Substituted Phenyl N-Butyl Carbamates (1-9)

AChEa BChE CEaseb PSLc

F 1.9 ( 0.6 4 ( 1 3 ( 1 2.7 ( 0.3δ -0.16 ( 0.05 0.0 ( 0.1 -0.07 ( 0.07 -0.06 ( 0.07f 0.7 ( 0.3 -0.5 ( 0.2 0.5 ( 0.3 -1.7 ( 0.4

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


inhibitor tetrahedral intermediates and the peptidic NHgroups in the OAH of CEase.

2. The δ Value. Small δ values for the pKi′-correlationsof all four enzyme inhibitions by carbamates 1-9 (Table3) reveal that ortho steric effects are insensitive to theseinhibition reactions. Furthermore, a null δ value for thepKi′-correlation of BChE inhibition carbamates 1-9indicates that the substituent X of the enzyme-inhibitortetrahedral intermediate (Figure 3), which adapts apseudo-trans conformation, is far away from the oxygenatom of Ser198 (Figure 5). On the other hand, negativeδ values for the pKi′-correlations of the AChE, CEase,and PSL inhibitions by carbamates 1-9 (Table 3) suggestthat the substituent X of the enzyme-inhibitor tetrahe-dral intermediate (Figure 3), which adapts a pseudo-cisconformation, is close to the nucleophilic serine of theenzyme (Figure 5). Thus, small, negative δ values for theAChE, CEase, and PSL inhibitions by carbamates 1-9are due to little enhancement of the steric effect (24) fromthe substituent X of the inhibitors to the nucleophilicserine 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 substit-uents of the inhibitors accelerate the inhibition reactionsthrough ortho polar effects or polar effects through space.Comparing this value with those for AChE and CEaseinhibitions (Table 3), we suggest that conformations ofthe enzyme-inhibitor tetrahedral intermediates (Figure3) for BChE inhibitions are different to those for bothAChE and CEase inhibitions (Figure 5). In the enzyme-inhibitor tetrahedral intermediates (Figure 3) for BChEinhibitions, the ortho substituents at the phenyl groupsare far away from the negatively charged carbonyloxygens, and thus, the tetrahedral intermediates adaptpseudo-trans conformations (Figure 5). However, in theenzyme-inhibitor tetrahedral intermediates for bothAChE and CEase inhibitions, the ortho substituents at

the phenyl groups are relatively close to the negativelycharged carbonyl oxygens, and thus, the tetrahedralintermediates 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 fvalue of -1.7 also implies that the enzyme-inhibitortetrahedral intermediates adapt pseudo-cis conforma-tions and further suggests that the 1,3-proton transfermay occur during the reaction (37).

Carbamylation, kc Step. 1. The Role of n-Butyryl.The carbamylation rates are about 10 times slower inAChE (24) when compared to those in BChE (Table 1).We know that butyrylcholine is virtually not hydrolyzedby AChE because of steric hindrance. So, faster carbamyl-ation 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 kc-correlations (Table 4) indicate that the transition stateof the kc step is slightly more negatively charged thanthe enzyme-inhibitor tetrahedral intermediate (Figure3). Since the product of the kc step is neutral ortho-substituted phenol, the transition state of this step(Figure 6) is similar to the enzyme-inhibitor tetrahedralintermediate (Figure 3). This is because negative chargeson the carbonyl oxygen redistribute to the phenol oxygenand make them closer to ortho substituents of theinhibitors (24). Therefore, the F values for the log kc-correlations are indeed a measure for this charge redis-tribution. Notably, that the F values for the log kc-correlations of both BChE and CEase inhibitions are close

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

Table 4. Sensitivity Factors for the log kc-Correlations ofthe AChE, BChE, CEase, and PSL Inhibitions by

ortho-Substituted Phenyl N-Butyl Carbamates (1-9)

AChEa BChE CEaseb PSLc

F 0.11 ( 0.06 0.47 ( 0.08 0.5 ( 0.1 0.8 ( 0.1δ 0.03 ( 0.02 0.04 ( 0.02 0.04 ( 0.05 0.00 ( 0.03f -0.3 ( 0.1 -0.10 ( 0.01 -0.5 ( 0.2 0.0 ( 0.2

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

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


to each other implies a similar mechanism for bothinhibition reactions in their kc steps. For the kc steps ofBChE, AChE, and CEase inhibitions, the leaving groupsare presumably the ortho-substituted phenoxide ions dueto positive F values (Table 3). On the other hand, theproducts for the kc steps of PSL inhibitions are likely tobe the ortho-substituted phenols due to the 1,3-proton-transfer mentioned above (37).

3. The δ Value. All ortho steric effects in the kc stepsare negligible (Table 4). Thus, the leaving group bindingsite of the enzyme is large enough to adapt any bulkyortho-substituted phenol. In other words, increasing theC-O bond length (Figure 6) toward the transition statefor the kc step weakens ortho steric effects.

4. The f Value. Small negative f values for the log kc-correlations of the BChE, AChE, and CEase inhibitionsby carbamates 1-9 (Table 4) indicate that electron-donating substituents facilitate the inhibition reactionsthrough space better than the electron-withdrawing ones.For kc steps, therefore, the transition states for BChEinhibitions retain their pseudo-trans conformations, whilethose for both AChE and CEase inhibitions change frompseudo-cis to pseudo-trans (Figure 5).

Overall Inhibition Reaction. The selectivity forBChE over AChE (24) inhibitions by carbamates 1-9 isdefined as ki (BChE)/ki (AChE) (Table 1). In general,carbamates 1-9 with electron-donating substituents areselective for BChE over AChE inhibitions such as tacrine,but carbamates 1-9 with electron-withdrawing substit-uents are selective for AChE over BChE inhibitions suchas rivastigmine (Figure 7). Therefore, selection for BChEor AChE inhibition partially depends on electronic char-acters of the substituents. Among carbamates 1-9,o-nitrophenyl N-butylcarbamate (9) is the most potentinhibitor of both BChE and CEase and is also a potentinhibitor of both AChE and PSL. The inhibitory potencyfor 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 electro-phile in measuring the nucleophilicity of a serine hydro-lase or protease.

In conclusion, electron-donating substituents in theortho position are better inhibitors of BChE than AChE,

while electron-withdrawing substituents are better in-hibitors of AChE.

Acknowledgment. We thank the National ScienceCouncil of Taiwan for financial support.

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