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

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  • JOURNAL OF PHYSICAL ORGANIC CHEMISTRYJ. Phys. Org. Chem. 2004; 17: 707714Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.740

    Ortho effects and cross interaction correlations for themechanisms of cholesterol esterase inhibition by arylcarbamates

    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 (111) were synthesized to evaluate the inhibitionmechanisms of porcine pancreatic cholesterol esterase. All carbamate inhibitors act as the active site-directed pseudosubstrate inhibitors of the enzymes. The logarithms of dissociation constant (Ki), carbamylation constant (k2) andbimolecular inhibition constant (ki) multiply linearly correlate with the Hammett substituent constant (), the TaftKutterHansch ortho steric constant (ES), and the SwanLuptonHansch ortho polar constant (F). For the logKi,log k2 and log ki correlations, the reaction constant for ordinary polar effect (), the intensity factor to ortho stericconstant () 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** correlations are 3, 2, and 1, respectively. The Ki step may be composed of the following two steps: (1)protonation of carbamates 111 and (2) the pseudo-trans to pseudo-cis conformation change of protonated carbamates111 due to a large XR value of 3 and formation of the enzyme-protonated carbamates 111 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 betweenthe carbamate and phenyl groups in all transition states of inhibition reactions are relatively short owing to large jXRjvalues. The Ki step shows little ortho steric enhancement effect; moreover, the k2 step shows little ortho stericinhibition effect. Copyright# 2004 John Wiley & Sons, Ltd.

    KEYWORDS: ortho effects; cross interaction correlations; cholesterol esterase; carbamate inhibitors


    Pancreatic cholesterol esterase (CEase, EC, alsoknown as bile salt-stimulated lipase, pancreatic carboxylester lipase, pancreatic lysophospholipase, non-specificlipase, functions in the hydrolysis of dietary cholesterolesters and other dietary esters.1 In addition to cholesterolesters, triacylglycerols, phospholipids and vitamin estersare also substrates of CEase.2,3 Two x-ray structures ofCEase have been reported recently.4,5 Although different

    bile salt-activation mechanisms have been proposed, theactive site of CEase is similar to that of lipases.68

    Recently, there has been increased interest in CEaseand lipases owing to the correlation between enzymaticactivity in vivo and absorption of dietary cholesterol9,10

    and the use of orlistat (Xenical). Orlistat, whose originalmechanism of action consists of the selective inhibitionof gastrointestinal lipases, has been commercialized forthe treatment of obesity.11,12 It has also been demon-strated that CEase is involved directly in lipoproteinmetabolism, in that the enzyme catalyzes the conversionof large low-density lipoprotein to smaller, denser, morecholesterol ester-rich lipoproteins, and that the enzymemay regulate serum cholesterol levels.13 Both CEase andlipase share the same catalytic mechanism as serineproteases14 in that they have a SerHisAsp (Glu) cata-lytic triad that is involved in nucleophilic and generalacidbase catalysis and a neighboring oxyanion hole(OAH), the hydrogen bonding peptide NH functions ofGly and Ala, that stabilizes the incipient carbonyl CO

    of the ester function during turnover. Both CEase andlipase may well be expected to be inhibited by the sameclasses of mechanism-based inhibitors. In the presence

    Copyright# 2004 John Wiley & Sons, Ltd. J. Phys. Org. Chem. 2004; 17: 707714

    *Correspondence to: G. Lin, Department of Chemistry, NationalChung-Hsing University, Taichung 402, Taiwan.E-mail: gilin@dragon.nchu.edu.twContract/grant sponsor: National Science Council of Taiwan.

    Abbreviations: ACS, alkyl chain binding site; CEase, cholesterolesterase; , intensity factor to ortho steric constant or NMR chemicalshift in ppm; ES, esteratic site; Es, TaftKutterHansch ortho stericconstant; F, SwanLuptonHansch ortho polar constant; f, intensityfactor to ortho polar constant; k2, carbamylation constant; k3, decarba-mylation constant; Ki, inhibition or dissociation constant; K

    0i , virtual

    inhibition or dissociation constant of the enzyme-protonated carbamatetetrahedral intermediate; OAH, oxyanion hole; PNPB, p-nitrophenylbutyrate; QSAR, quantitative structureactivity relationship; , reac-tion constant for ordinary polar effect; SACS; second alkyl chainbinding site; TFA, trifluoroacetophenone.

  • of substrate, the CEase inhibition by pseudo substrateinhibitors such as carbamates have been proposed(Scheme 1).1522 Since the inhibition follows first-orderkinetics over the observed time period for the steady-statekinetics, the rate of hydrolysis of EI0 must be significantlyslower than the rate of formation of EI0 (k2 k3).23Therefore, values of Ki and k2 can be calculated fromthe equation.15

    kapp k2I=Ki1 S=Km I 1

    where kapp is the first-order rate constant and can beobtained according to Hosies method.15 The bimolecularrate constant, Ki k2/Ki, is related to overall inhibitorypotency.

    According to the x-ray structures of CEase and li-pases,4,5,7,8 CEase consists of at least five major bindingsites:20,21 (a) an alkyl chain binding site (ACS) that bindsto the substrate alkyl chain, (b) an oxyanion hole (OAH)that stabilizes the tetrahedral intermediate, (c) an esteraticsite (ES), comprised of the active site serine whichattacks the ester carbonyl, (d) a leaving group hydro-phobic binding site, the peripheral site and/or the secondalkyl chain or group binding site (SACS) that binds to thecholesterol part of cholesterol ester or the second fattyacid chain of triacylglyceols, which is relatively largerthan ACS, and (e) a leaving group hydrophilic bindingsite that binds to the hydrophilic part of the leaving groupand is located at the opposite direction of ACS.

    Quantitative structureactivity relationships (QSARs)represent an attempt to correlate structural properties ofcompounds with activities or reactivities.2426 These che-mical descriptors, which include parameters to account forhydrophobicity, electronic, inductive or polar propertiesand steric effects, are determined empirically or by calcu-lations. Many biological activities and chemical reactiv-ities are correlated with the Hammett equation:2426

    log k h 2

    where h, and are log k0, the reaction constant (orintensity factor for inductive effect) and the Hammettsubstituents constant, respectively. Also, meta- and para-substituted compounds generally correlate well butortho-substituted compounds do not.25 Ortho problems,

    due to complications from direct steric and polar effects,are not generally applicable.27 According to Fujita andNishiokas suggestion, the total ortho effect is composedof the ordinary polar effect, the ortho steric effect and theortho polar effect:25,27

    log k h ES fF 3

    where h, , , ES, , f and F are log k0, the reactionconstant for ordinary polar effect, the Hammett substi-tuent constant, the TaftKutterHansch ortho steric con-stant, the intensity factor to ortho steric constant, theintensity factor to ortho polar constant and the SwainLuptonHansch ortho polar constant, respectively.

    Cross interaction correlations for the CEase inhibitionsby meta- and para-substituted phenyl-N-butylcarbamates(13) and p-nitrophenyl-N-substituted carbamates (12)with Eqn (4)28,29 reveal that the carbamate OC(O)NR geometries in the transition states of all inhibitionreactions retain in the pseudo-trans conformations:

    log k h XR 4

    where h, , , *, *, XR and are log k0, the reactionconstant 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 substituentconstant of R, the cross interaction constant and theweighing factor for the HammettTaft cross interactionterm ( 1 for the Hammett substituent X; 2.54 forthe Taft substituent R), respectively.28 Moreover, thedistance between X and R substituents in the transitionstate of the reaction is inversely proportional to jXRj.29

    Aryl carbamates, such as meta- and para-substitutedphenyl-N-substituted carbamates (12 and 13) (Fig. 1),are characterized as the pseudo substrate inhibitors ofCEase and show the Hammett16,18,21 and TaftIngoldtypes17,20,21 of correlations. In this work, ortho-substi-tuted phenyl-N-butyl carbamates (111) (Fig. 1) weresynthesized to explore the ortho effects for the pre-steady-state CEase inhibition and the cross interactioncorrelations with p-nitrophenyl-N-substituted carbamates(12) (Fig. 1).

    Scheme 1. Kinetic scheme for pseudo substrate inhibitionsof CEase in the presence of substrate

    Figure 1. Structures of carbamates 1--12

    708 G. LIN ET AL.

    Copyright# 2004 John Wiley & Sons, Ltd. J. Phys. Org. Chem. 2004; 17: 707714


    Carbamates 111 (Fig. 1) are characterized as the pseudosubstrate inhibitors of CEase because the inhibitors aretime dependent, the inhibitions follow first-order kineticsand the enzyme activities recover with a competitiveinhibitor, trifluoroacetophenone (TFA).1522 The orthosubstituent constants and inhibition constants for theCEase-catalyzed hydrolysis of p-nitrophenyl butyrate(PNPB) by carbamates 111 are summarized in Table 1.

    Correlations of NH, C----O, logKi, log k2 and log kiwith the Hammett equation [Eqn (2)] are poor (Table 2).These correlations are improved after addition of theTaftKutterHansch steric constant, ES, to the correlationparameters (Table 2). Multiple correlations with three

    parameters, , ES and F [(Eqn (3)],25,27 are much impro-

    ved (Table 2). The bimolecular rate constant, ki k2/Ki, isrelated to overall inhibitory potency.1522 The pKa valuesof carbamates 112 are correlated with the Hammettequation [Eqn (2)] (Table 2).21 The logKi

    0 values arewell correlated with Eqn (3) (Table 2), where the virtualinhibition constant, Ki

    0 KbKi, is the dissociation con-stant of the enzyme-protonated carbamate tetrahedralintermediate.

    The results for the cross interaction correlationbetween the ortho-substituted carbamates 111 and 4-nitrophenyl-N-substituted carbamates (12) (Fig. 1) aresummarized in Table 3. The jXRj values for the crossinteraction between ortho-substituted carbamates 111and carbamates 12 are larger than those between

    Table 1. Ortho substituent constants and inhibition constants for CEase-catalyzed hydrolysis of PNPB in the presence ofcarbamates 1--11

    Inhibitor Substituent a EaS Fb k2 (10

    4 s1)c Ki (mM)c ki (10

    3M1 s1)d

    1 o-t-Bu 0.20 2.78 0.07 15.8 0.4 3.2 0.3 0.5 0.12 o-Cl 0.23 0.97 0.41 16.5 0.8 2.2 0.4 0.8 0.23 o-OMe 0.27 0.55 0.26 7.0 0.05 7 1 0.10 0.024 o-NO2 0.78 2.52 0.67 17.6 0.7 0.8 0.3 2.2 0.45 o-CH3 0.17 1.24 0.04 20.3 0.8 7 1 0.30 0.026 o-C2H5 0.15 1.31 0.05 13.8 0.3 7 1 0.20 0037 o-Ph 0.01 1.01 0.08 11.7 0.8 5 1 0.23 0.038 o-CF3 0.54 2.40 0.38 9.5 0.1 0.7 0.1 1.4 0.29 p-NO2 0.78 0 0 38 2 2.6 0.3 1.5 0.1

    10 o,p-Di-t-Bu 0.4 2.78 0.07 7.5 0.7 18 3 0.04 0.0211 H 0 0 0 16.8 0.9 5 1 0.32 0.02a Hydrogen is taken as standard. These values are calculated from Refs. 2426.b Taken from Ref. 27.c Obtained from fitting the kapp values to Eqn (1).d ki k2/Ki.

    Table 2. QSAR results for NMR chemical shifts of carbamates 1--11 and the inhibition constants for the inhibitions of CEase bycarbamates 1--11

    h f R

    aNH 5.12 0.03 0.27 0.09 0.718bNH 5.13 0.07 0.3 0.1 0.01 0.04 0.719cNH 5.07 0.07 0.0 0.2 0.00 0.03 0.4 0.3 0.813aC----O 154.09 0.07 1.3 0.2 0.913bC----O 154.1 0.2 1.3 0.2 0.01 0.09 0.913cC----O 154.3 0.1 0.5 0.3 0.01 0.06 1.3 0.4 0.969LogKdb 4.00 0.06 2.5 0.1 0.989LogK 0ei 9.2 0.2 3 1 0.07 0.07 0.5 0.3 0.906LogKai 5.35 0.07 0.8 0.2 0.847LogKbi 5.2 0.1 0.9 0.2 0.10 0.07 0.884LogKci 5.2 0.1 0.7 0.2 0.07 0.07 0.5 0.3 0.912Log ka2 2.87 0.06 0.3 0.1 0.559Log kb2 2.78 0.09 0.3 0.1 0.07 0.05 0.654Log kc2 2.76 0.08 0.5 0.1 0.04 0.05 0.5 0.2 0.811Log kai 2.47 0.07 1.1 0.2 0.904Log kbi 2.4 0.1 1.1 0.2 0.03 0.07 0.906Log kci 2.4 0.1 1.1 0.3 0.03 0.08 0.0 0.1 0.906a Correlation with [Eqn (2)] for carbamates 111.b Correlation with and ES for carbamates 111.c Correlation with , ES and F [Eqn (3)] for carbamates 111.d Correlation with , ES and F [Eqn (3)] for carbamates 111 and meta- and para-substituted phenyl-N-butylcarbamates.e 1/KiKb (1/Ki0).


    Copyright# 2004 John Wiley & Sons, Ltd. J. Phys. Org. Chem. 2004; 17: 707714

  • para- and meta-substituted carbamates and carbamates12.28 In other words, the distances between X and R inthe transition states for the CEase inhibitions by orthosubstituted carbamates are much shorter than those inhi-bitions by para- and meta-substituted carbamates. There-fore, this result confirms that the carbamate inhibitorconformations during all inhibition reactions retain thepseudo-cis conformations.28


    Like meta- and para-substituted phenyl-N-butylcarba-mates1618,21 and 1,10-bi-2-naphthyl- 2,20-di-N- butylcar-bamate,19 carbamates 111 are characterized as thepseudo substrate inhibitors of CEase (Scheme 1 andTable 1) and show the same kinetic behavior. Therefore,the leaving group binding site of the enzyme is largeenough to adapt to extremely bulky ortho substituentssuch as 1-naphthyl, tert-butyl, and phenyl.

    The f value of 0.4 for the NHESF correlationsuggests that the NH bonds of carbamates 111 polar-ize to generate a partial negative charge on nitrogen underthe NMR conditions; moreover, the f value of 1.3 for theC----OESF correlation suggests that the CO bondsof carbamates 111 polarize to generate a partial positivecharge on carbon under the NMR conditions.

    A three-step CEase inhibition mechanism by carba-mates 111 is proposed (Fig. 2). The first step (Kb) is thepre-equilibrium protonation of carbamates 111. Thesecond step (Ki

    0) is formation of the enzyme-protonatedcarbamate tetrahedral intermediate. The third step (k2) isformation of the carbamyl enzyme intermediate andrelease of the substituted phenol product.

    Kb step

    For the Kb step, carbamates 111 like carbamates 13and 12,21,28 are bases (Table 2) and are protonated inaqueous solution outside the enzyme active site (pH 7).From the cross interaction correlation results,28 the

    OC(O)NR geometries of carbamates 111 andprotonated carbamates 111 are in the pseudo-transconformations (Fig. 2). The excellent logKb correla-tion (Table 2) indicates that the Kb step is insensitive toortho steric...


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