Ortho Effects in Quantitative Structure Activity Relationships for Lipase Inhibition by Aryl Carbamates

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  • Ortho Effects in Quantitative Structure Activity Relationships forLipase Inhibition by Aryl CarbamatesGialih Lin*, Yue-Chen Liu, Yon-Gi Wu, and Yu-Ru Lee

    Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan

    Full Paper

    Ortho-substituted phenyl-N-butyl carbamates (1 11) aresynthesized and evaluated for their inhibition effects onPseudomonas species lipase. Carbamates 1 11 are char-acterized as pseudo-substrate inhibitors of the enzyme.The logarithms of dissociation constant (Ki), carbamyla-tion constant (k2), and bimolecular inhibition constant (ki)multiply linearly correlate with Hammett substituentconstant (), Taft-Kutter-Hansch ortho steric constant(ES), and Swan-Lupton field constant (F). For logKi-,logk2-, and logki-correlations, values of , , f, XR are 0.2, 0.06, 1.7, 0.8; 0.0, 0.0, 1.0, 0.07; and 1.8, 7, 0.6, 5;respectively. The enzyme inhibition mechanism is com-posed of four steps: 1) the first step which is protonation ofcarbamates 1 11, 2) the second step (Ki1) which involvesin the proton 1,3-shift of protonated carbamates 1 11 thenthe pseudo-trans to cis conformational change, 3) the third

    step (Ki2) which is formation of a negative chargedenzyme-inhibitor tetrahedral intermediate, and 4) thefourth step (k2) which is the carbamylation step. Theformer three steps are likely composed of the Ki step.There is little ortho steric enhancement effect in the Kistep. From cross-interaction correlations, distance betweencarbamate and phenyl substituents in transition state forthe Ki step is relatively short due to a large XR value of 7.The k2 step is insensitive to ortho steric effect. The k2 stepinvolves in departure of leaving group, substituted phenolin which is protonated from the proton 1,3-shift but notfrom the active site histidine of the enzyme. From cross-interaction correlations, the distance between carbamateand phenyl substituents in transition state for the k2 step isrelatively long due to a small XR value of 0.6.

    1 Introduction

    The commercial potential of organic syntheses catalyzed bylipases (EC 3.1.1.3) underscores the need for a comprehen-sive understanding of lipase structure and function andprovided the impetus for many recent investigations [1, 2].Lipases are lipolytic enzymes, which hydrolyze ester bondsof trilglycerides and many esters [3]. Recently, there has

    been increased interest in lipases due to the use of orlistat(Xenical). Orlistat, whose original mechanism of actionconsists of the selective inhibition of gastrointestinal lipases,has been commercialized for the treatment of obesity [9].Many X-ray structures of lipases such as Pseudomonas

    cepacia lipase (PCL) andCandida rugosa lipase (CRL) havebeen reported [4 8]. Although different activation mecha-nisms are proposed, the active sites of most lipases arestrongly resembled to one another. Most lipases have thesame catalytic mechanism as serine proteases in that theyhave a Ser-His-Asp (orGlu) catalytic triadwhich is involvedin nucleophilic and general acid-base catalyses and aneighboring oxyanion hole (OAH), the hydrogen bondingpeptide NH functions of Gly and Ala, which stabilizes theincipient carbonyl CO of the ester function duringturnover [3]. The conservation of this catalytic triad suggeststhat most lipases share a common mechanism for substratehydrolysis, that is, formation of discrete acyl enzyme speciesvia the serine hydroxyl group. In the presence of substrate,the kinetic scheme for PSL inhibition by carbamates 1 11 isproposed (Scheme 1) [10, 11]. Since this inhibition followsfirst-order kinetics over observed time period for steady-state kinetics, rate of hydrolysis of carbamyl enzyme EI

    852 QSAR Comb. Sci. 22 (2003) DOI: 10.1002/qsar.200330827 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

    * To receive all correspondence

    Key words: QSAR, ortho effects, cross-interactions, lipase inhibi-tion, carbamate inhibitors

    Abbreviations: ACS, alkyl chain binding site; CRL, Candidarugosa lipase; ES, esteratic site; k2, inactivation constant ofenzyme-inhibitor adduct; Ki, dissociation constant of enzyme-inhibitor adduct; ki, bimolecular inhibition constant; OAH, oxy-anion hole; PCL, Pseudomonas cepacia lipase; PNPB, p-nitro-phenyl butyrate; PSL, Pseudomonas species lipase; QSAR,quantitative structure-activity relationship; SACS; second alkylchain binding site; TACS, third alkyl chain binding site; TFA,trifluoroacetophenone.

    G. Lin et al.

  • must be significantly slower than rate of formation of EI(k2k3) [12]. Therefore, values of Ki and k2 can becalculated from Eq. 1 [13].

    kapp k2[I]/(Ki(1 [S]/Km) [I]) (1)

    In Eq. 1 the kapp values are the first-order rate constantswhich are obtainedbyHosiesmethod [13]. Thebimolecularrate constant, ki k2/Ki, is related to overall inhibitorypotency.According to PCL [7] and CRL [8] X-ray structures, the

    active site of PSLmay be consist of at least sixmajor bindingsites (Figure 1) [10, 11]: (a) an alkyl chain binding site (ACS)which binds to the alkyl chain of the substrate, (b) anoxyanion hole (OAH) which stabilizes the tetrahedralintermediates, (c) an esteratic site (ES), comprised of theactive site serine which attacks the ester carbonyl group ofsubstate, (d) a leaving group hydrophobic binding site, theperipheral site, and/or the second alkyl chain or groupbinding site (SACS) which binds to the cholesterol part ofcholesterol ester or the second fatty acid chain of triglycer-ides, which is relatively larger than ACS, (e) a leaving grouphydrophilic binding site which binds to the hydrophilic partof the leaving group and is located at the opposite directionof ACS, and (f) the third alkyl chain binding site (TACS)which binds to the third fatty acid chain of triglyceride, islocated at the opposite direction of ACS, has exposures tothe solvent, and has room to adopt many different con-formations.Quantitative structure-activity relationships (QSARs)

    represent an attempt to correlate structural properties ofcompounds with biological activities and chemical reactiv-ities [14 16]. These chemical descriptors, which includeparameters to account for hydrophobicity, electronic, in-ductive, or polar properties, and steric effects, are deter-mined empirically or by calculations. Many drug activitiesand chemical reactivities are correlated with Hammettequation (Eq. 2) [14 16].

    log k h (2)

    In Eq. 2 the h value is the logk0 value and the parameters and are reaction constant (intensity factor of inductiveeffect) and Hammett substituents constant, respectively.Investigation also reveals that meta and para substitutedcompounds generally correlate well but ortho substitutedcompounds give poor correlation [15].Ortho problems dueto complications from direct steric and polar effects, is notgenerally applicable [17]. According to Fujita andNishiokassuggestion, ortho effect is composed of ordinary polar effect,ortho steric effect, and ortho polar effect (Eq. 3) [15, 17].

    logk h ES fF (3)

    In Eq. 3 the parameters h, , , ES, , f and F are logko,reaction constant for ordinary polar effect, Hammett

    substituent constant, Taft-Kutter-Hansch ortho steric con-stant, intensity factor to ortho steric constant, intensityfactor to ortho polar constant, and Swain-Lupton-Hanschortho polar constant, respectively.Cross-interaction correlations for cholesterol esterase

    inhibitions by substituted phenyl-N- substituted carbamateswith Eq. 4 [18] has revealed that the C(O)N fragmentgeometry of the inhibitors in the transition state is allretained in pseudo-trans conformation.

    logk h * *XR * (4)

    In Eq. 4 [19], the h, , , *, *, XR, and values are logko,reaction constant for substituted phenyl-N-butyl carba-mates,Hammett substituent (X) constant, reaction constantfor p-nitrophenyl-N-substituted carbamates, Taft substitu-ent (R) constant, the cross-interaction constant between Xand R, and the weighing factor for Hammett-Taft cross-interaction ( 1 for the Hammett substituent X; 2.54for the Taft substituent R), respectively [18]. Moreover, theintramolecular distance between X and R in the transitionstate of reaction is inversely proportional to XR [19].Aryl carbamates, such as meta- and para-substituted

    phenyl-N-substituted carbamates, are characterized aspseudo substrate inhibitors of PSL and their inhibitionconstants show QSAR with Taft-Ingold correlation [11]. Inthis paper, ortho-substituted phenyl-N-butyl carbamates(1 8, and 10) (Figure 1) are synthesized to explore steady-state lipase inhibition mechanisms by ortho effects andcross-interaction correlations with p-nitrophenyl-N-substi-tuted carbamates.

    2 Materials and Methods

    2.1 Materials

    PSL and p-nitrophenyl butrate (PNPB) were obtained fromSigma; other chemicals were obtained from Aldrich; silicagel used in liquid chromatography (Licorpre Silica 60, 200 400 mesh) and thin-layer chromatography plates (60 F254)were obtained fromMerck. All other chemicals were of thehighest purity available commercially.

    Instrumental Methods

    1Hand 13CNMRspectrawere recorded at 400 and 100 MHz,respectively, on a Varian-GEMINI 400 spectrometer. Allsteady state kinetic data were obtained from an UV-VISspectrophotometer (HP 8452 or Beckman DU-650) with acell holder circulated with a water bath.

    2.2 Data Reduction

    Origin (version 6.0) was used for linear, nonlinear, andmultiple linear least squares regression analyses.

    QSAR Comb. Sci. 22 (2003) 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 853

    Ortho Effects in Quantitative Structure Activity Relationships for Lipase Inhibition by Aryl Carbamates

  • 2.3 Steady-State Enzyme Kinetics

    The PSL inhibition was assayed by Hosies method [13].Temperature was maintained at 25.0 0.1 C by a refriger-ated circulating water bath. All inhibition reactions werepreformed in sodium phosph