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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 Ki

step. 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(Xenical¾). 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 C�O� 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 ¹ 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.

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must be significantly slower than rate of formation of EI�(k2��k3) [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 obtainedbyHosie×smethod [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 andNishioka×ssuggestion, 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.

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

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2.3 Steady-State Enzyme Kinetics

The PSL inhibition was assayed by Hosie×s method [13].Temperature was maintained at 25.0� 0.1 �C by a refriger-ated circulating water bath. All inhibition reactions werepreformed in sodium phosphate buffer (1 mL, 0.1 M,pH 7.0) containing NaCl (0.1 M), acetonitrile (2% byvolume), triton X-100 (0.5% by weight), substrate(0.2 mM), and varying concentration of inhibitors. Requi-site volumes of stock solution of substrate and inhibitors inacetonitrile were injected into reaction buffer via a pipet.PSL was dissolved in sodium phosphate buffer (0.1 M,pH 7.0). First-order rate constants (kapp×s) for inhibitionwere determined as described by Hosie et al. [13]. Ki×s andk2×s were obtained by fitting kapp×s and [I] to Eq (1) bynonlinear least squares regression analyses [10,11,13,18,20 ±25]. Duplicate sets of data were collected for each inhibitorconcentration.

2.4 Synthesis of Carbamates

Carbamates 1 ± 11 were prepared from condensation ofsubstituted phenol with n-butyl isocyanate in the presenceof a catalytic amount of pyridine in toluene at roomtemperature for 48 h (60 ± 90% yield) [11, 20, 21, 25]. Allcompounds were purified by liquid chromatography onsilica gel and characterized by 1H and 13C NMR spectra andhigh resolution mass spectra (HRMS).

o-t-Butylphenyl-N-butylcarbamate (1)1H NMR (CDCl3, 400 MHz) �/ppm 0.96 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.32 (s, 9H, C(CH3)3), 1.38 (sextet, J� 7 Hz,2H, CH2CH2CH3), 1.58 (quintet, J� 7 Hz, 2H,CH2CH2CH3), 3.31 (q, J� 7 Hz, 2H, NHCH2), 5.02 (br s,1H,NH), 7.04 ± 7.37 (m, 4H, aromaticH); 13CNMR(CDCl3,100 MHz) �/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.

o-Chlorophenyl-N-butylcarbamate (2)1H NMR (CDCl3, 400 MHz) �/ppm 0.92 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.37 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.52 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.23 (q, J� 7 Hz,2H, NHCH2), 5.37 (br s, 1H, NH), 7.12 ± 7.41 (m, 4H,aromatic H); 13C NMR (CDCl3, 100 MHz) �/ppm 13.72(CH2CH2CH3), 19.84 (CH2CH2CH3), 31.77 (CH2CH2CH3),41.04 (NHCH2), 123.92, 126.23, 127.32, 129.87 (phenylCH),127.08 (phenyl C-2), 146.92 (phenyl C-1), 153.35 (C�O).HRMS calculated for C11H14NO2Cl: 227.0713, found:227.0721.

o-Methoxyphenyl-N-butylcarbamate (3)1H NMR (CDCl3, 400 MHz) �/ppm 0.94 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.39 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.57 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.27 (q, J� 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) �/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 C-1), 154.17 (C�O). HRMS calculated forC12H17NO3: 223.1208, found: 223.1211.

o-Nitrophenyl-N-butylcarbamate (4)1H NMR (CDCl3, 400 MHz) �/ppm 0.94 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.32 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.51 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.28 (t, J� 7 Hz,2H, NHCH2), 5.28 (br s, 1H, NH), 7.22 ± 8.05 (m, 4H,aromatic H); 13C NMR (CDCl3, 100 MHz) �/ppm 13.70(CH2CH2CH3), 19.80 (CH2CH2CH3), 32.03 (CH2CH2CH3),41.20 (NHCH2), 125.40, 125.70, 125.90, 134.20 (phenylCH),142.10 (phenyl C-2), 144.20 (phenyl C-1), 153.10 (C�O).HRMS calculated for C11H14N2O4: 238.0954, found:238.0959.

o-Methylphenyl-N-butylcarbamate (5)1H NMR (CDCl3, 400 MHz) �/ppm 0.94 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.37 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.54 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 2.21 (s, 3H, o-CH3), 3.25 (q, J� 7 Hz, 2H, NHCH2), 5.09 (br s, 1H, NH),7.05 ± 7.25 (m, 4H, aromatic H); 13C NMR (CDCl3,100 MHz) �/ppm 13.81 (CH2CH2CH3), 16.16 (o-CH3),19.96 (CH2CH2CH3), 32.00 (CH2CH2CH3), 41.00(NHCH2), 122.02, 125.34, 126.58, 130.77 (phenyl CH),130.42 (phenyl C-2), 149.29 (phenyl C-1), 154.24 (C�O).HRMS calculated for C12H17NO2: 207.1260, found:207.1252.

o-Ethylphenyl-N-butylcarbamate (6)1H NMR (CDCl3, 400 MHz) �/ppm 0.94 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.19 (t, 3H, J� 7 Hz, o-CH2CH3), 1.37(sextet, J� 7 Hz, 2H, CH2CH2CH3), 1.54 (quintet, J�7 Hz, 2H, CH2CH2CH3), 2.59 (q, J� 7 Hz, 2H, o-CH2CH3), 3.24 (q, J� 7 Hz, 2H, NHCH2), 5.10 (br s, 1H,NH), 7.05 ± 7.25 (m, 4H, aromatic H); 13C NMR (CDCl3,100 MHz) �/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, 129.02(phenyl CH), 136.07 (phenyl C-2), 148.81 (phenyl C-1),154.49 (C�O). HRMS calculated for C13H19NO2: 221.1416,found: 221.1407.

o-Biphenyl-N-butylcarbamate (7)1H NMR (CDCl3, 400 MHz) �/ppm 0.96 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.32 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.43 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.19 (q, J� 7 Hz,2H, NHCH2), 5.03 (br s, 1H, NH), 7.27 ± 7.51 (m, 9H,aromatic H); 13C NMR (CDCl3, 100 MHz) �/ppm 13.74(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-1×), 137.62(phenyl C-2), 147.71 (phenyl C-1), 154.31 (C�O). HRMScalculated for C17H19NO2: 269.1416, found: 269.1419.

o-Trifluoromethylphenyl-N-butylcarbamate (8)1H NMR (CDCl3, 400 MHz) �/ppm 0.93 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.38 (sextet, J� 7 Hz, 2H, CH2CH2CH3),

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G. Lin et al.

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1.54 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.26 (q, J� 7 Hz,2H, NHCH2), 5.21 (br s, 1H, NH), 7.26 ± 7.64 (m, 4H,aromatic H); 13C NMR (CDCl3, 100 MHz) �/ppm 13.75(CH2CH2CH3), 19.86 (CH2CH2CH3), 31.84 (CH2CH2CH3),41.11 (NHCH2), 122.10 (q, 1JCF� 180 Hz, CF3), 123.05 (q,2JCF� 20 Hz, phenyl C-2), 125.07, 126.48, 132.65 (phenylCH), d, 148.38 (phenyl C-1), 153.42 (C�O). HRMS calcu-lated for C12H14NO2F3: 261.0977, found: 261.0969.

p-Nitrophenyl-N-butylcarbamate (9) [11, 20, 21, 25]1H NMR (CDCl3, 400 MHz) �/ppm 0.97 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.39 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.59 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.30 (q, J� 7 Hz,2H, NHCH2), 5.12 (br s, 1H, NH), 7.27 ± 8.26 (m, 4H,aromatic H); 13C NMR (CDCl3, 100 MHz) �/ppm 13.78(CH2CH2CH3), 19.96 (CH2CH2CH3), 31.81 (CH2CH2CH3),41.15 (NHCH2), 121.75, 124.91 (phenylCH), 144.45 (phenylC-4), 152.86 (phenyl C-1), 155.78 (C�O). HRMS calculatedfor C11H14N2O4: 238.0954, found: 238.0959.

2,4-di-t-Butylphenyl-N-butylcarbamate (10)1H NMR (CDCl3, 400 MHz) �/ppm 0.95 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.32 (s, 9H, o-C(CH3) 3), 1.38 (s, 9H, p-C(CH3) 3), 1.38 (sextet, J� 7 Hz, 2H, CH2CH2CH3), 1.55(quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.29 (q, J� 7 Hz, 2H,NHCH2), 5.01 (br s, 1H, NH), 6.95 ± 7.36 (m, 3H, aromaticH); 13C NMR (CDCl3, 100 MHz) �/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), 146.92 (phenyl C-4),147.31 (phenyl C-1), 154.61 (C�O). HRMS calculated forC19H31NO2: 305.2355, found: 305.2358.

Phenyl-N-butylcarbamate (11) [20, 25]1H NMR (CDCl3, 400 MHz) �/ppm 0.96 (t, J� 7 Hz, 3H,

CH2CH2CH3), 1.35 (sextet, J� 7 Hz, 2H, CH2CH2CH3),1.56 (quintet, J� 7 Hz, 2H, CH2CH2CH3), 3.27 (q, J� 7 Hz,2H, NHCH2), 4.99 (br s, 1H, NH), 7.12 ± 7.37 (m, 4H,aromatic H); 13C NMR (CDCl3, 100 MHz) �/ppm13.81(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), 150.89(phenyl C-1), 154.37 (C�O). HRMS calculated forC11H15N2O4: 193.1103, found: 193.1104.

3 Results

o-Substituted phenyl-N-butyl carbamates (1 ± 8), p-nitro-phenyl-N-butyl carbamates (9) [11, 20, 21, 25], o, p-di-t-butylphenyl-N-butyl carbamates (10), and phenyl-N-butylcarbamates (11) [20, 25] (Fig. 1) are synthesized fromcondensation of substituted phenol with n-butyl isocyanatein the presence of pyridine at room temperature (60 ± 90%yield).Carbamates 1 ± 11 are characterized as pseudo substrate

inhibitors of PSL (Scheme 1) because carbamates 1 ± 11 aretime-dependent and follow first-order kinetics, and the

enzyme activity recovers by a competitive inhibitor, tri-fluoroacetophenone (TFA) [20 ± 25].Substituent constants and inhibition data for the inhib-

ition of PSL-catalyzed hydrolysis of PNPB by carbamates1 ± 11 are summarized (Table 1). The bimolecular rateconstant, ki � k2/Ki, is related to overall inhibitory potency[20 ± 25]. p-Nitrophenyl-N-butyl carbamate (9) [11] is themost potent PSL inhibitor with the ki value of 900 M�1s�1.All ortho substituted phenyl-N-butyl carbamates (1 ± 8) arenot as potent PSL inhibitors as carbamate 9. For orthosubstituted carbamates 1 ± 8, electron donating substitutedcarbamates (1, 5, 6) are more potent PSL inhibitors thanelectron withdrawing carbamates (2 ± 4, 7, 8).Multiple correlations of � logKi, logk2 and logki with �,

ES, and F ((Eq. (3)) are summarized (Table 2). Cross-interaction correlations between ortho-substituted carba-mates 1 ± 11 and p-nitrophenyl-N-substituted carbamates[11] are summarized (Table 3).


Figure 1. Structures of inhibitors 1–11.

Scheme 1. Kinetic scheme for pseudo-substrate inhibitions ofPSL in the presence of substrate.


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4 Discussion

p-Nitrophenyl-N-butyl carbamate (9) [11] is themost potentPSL inhibitor with the ki value of 900 M�1s�1 (Table 1). Allortho substituted phenyl-N-butyl carbamates (1 ± 8), o, p-di-t-butylphenyl-N-butyl carbamate (10), and phenyl-N-butylcarbamate (11) are not as potent PSL inhibitors ascarbamate 9. It is possible that bent-shaped ortho carba-mates 1 ± 8 are more difficult to enter narrow active sitetunnel of the enzyme [4 ± 8] than linear para carbamate 9.Therefore, potent lipase inhibitors should exclude orthosubstituents. For ortho substituted carbamates 1 ± 8, carba-mates 1, 5, and 6 with electron donating substituents aremore potent PSL inhibitors than carbamates 2 ± 4, 7, and 8with electron withdrawing substituents. Thus, potent lipaseinhibitors should include electron donating substituents.A 4-step PSL inhibition mechanism by carbamates 1 ± 11

is proposed (Figure 2). The first step is protonation of

carbamates 1 ± 11 because carbamates 1 ± 11 are basic inaqueous solution [25]. The second step (Ki1) is formation ofnon-covalent enzyme-pseudo-cis-inhibitor complex (Fig-ure 2), which involves in the proton 1,3-shift of protonatedcarbamates 1 ± 11 from carbamate quaternary amine tocarbamate ether oxygen (Figure 3), followed by a pseudo-trans to pseudo-cis conformational change (discussionbelow). The third step (Ki2) involves in formation ofcovalent enzyme-inhibitor tetrahedral intermediate bynucleophilic attack of the active site Ser87ofPSL (Figure 2).The fourth step is carbamylation k2 step and forms thecovalent carbamyl enzyme intermediate (Figure 2).The � value of 0.2 for the � logKi-�-ES-F correlation

(Table 2) confirms that the Ki step involves in nucleophilicattack of the active site Ser 87 of the enzyme to carbamates1 ± 11 and that enzyme-inhibitor tetrahedral intermediate ismore negative charge than non-covalent enzyme- inhibitorcomplex (Figure 2). There is little ortho steric enhancementin theKi step due to a small negative � value (� 0.06) for the� logKi-�-ES-F correlation (Table 2).The f value of � 1.7 for the � logKi-�-ES-F correlation

(Table 2) suggests that Ki may be divided into three steps:protonation, Ki1, and Ki2 (Figure 2). The f value fordeprotonation of protonated carbamates 1 ± 11 is about 0.6[17]; therefore, the f value for protonation of carbamates 1 ±11 is about � 0.6. Deprotonations of protonated anilines orphenols give an f value around 1.8 [17]; therefore, proto-nations of anilines or phenols should give an f value around� 1.8. Thus, the f value for the 1,3-proton shift (Figure 3) isabout � 1.2 (� 0.6 ± 1.8). The f value for theKi1 step (� 1.2)


Table 1. Substituent constants and inhibition data for PSL-catalyzed hydrolysis of PNPB in the presence of carbamates 1 ± 11

Inhibitor Substituent � ESa Fa kc (10�4s�1) Ki (�M) ki (103 M�1s�1)

1 o-t-Bu � 0.20 � 2.78 � 0.07 6.0� 0.3 2.0� 0.4 0.30� 0.052 o-Cl 0.23 � 0.97 0.41 15.1� 0.4 17� 2 0.09� 0.013 o-OMe � 0.27 � 0.55 0.26 3.50� 0.05 18� 1 0.019� 0.0024 o-NO2 0.78 � 2.52 0.67 22.5� 0.8 14� 2 0.16� 0.015 o-CH3 � 0.17 � 1.24 � 0.04 4.30� 0.03 2.0� 0.2 0.22� 0.026 o-C2H5 � 0.15 � 1.31 � 0.05 4.3� 0.3 2.0� 0.1 0.215� 0.027 o-Ph � 0.01 � 1.01 0.08 5.6� 0.6 4.0� 0.4 0.14� 0.028 o-CF3 0.54 � 2.40 0.38 16� 1 26� 4 0.06� 0.019 p-NO2 0.78 0 0 32� 2 3.0� 0.4 0.11� 0.02

10 o,p-di-t-Bu � 0.4b � 2.78 � 0.07 3.2� 0.2 3.0� 0.4 0.11� 0.0211 o-H 0 0 0 5.6� 0.5 3� 1 0.15� 0.05

a Obtain from reference [17]b Sum of �(o-t-Bu) and �(p-t-Bu)

Table 2. QSAR results for PSL inhibitions by carbamates 1 ± 11

h � � f Rb

logk2 � 3.19� 0.06 0.8� 0.1 0.00� 0.03 0.0� 0.2 0.969� logKi 5.4� 0.1 0.2� 0.2 � 0.06� 0.07 � 1.7� 0.4 0.890logki 2.2� 0.1 1.0� 0.2 � 0.07� 0.08 � 1.8� 0.4 0.879

a Correlations of � logKi, logks, and logki with �, ES and F (Eq (3)) for all data of carbamates 1 ± 11.b Correlations coefficient.

Table 3. Cross-interaction correlation results for PSL inhibitionsby o-substituted phenyl-N-butylcarbamates and p-nitrophenyl-N-substituted carbamatesa

Parameters � logKi logk2 logki

�* � 15� 4 � 1� 1 � 11� 3� 0.6� 0.6 1.0� 0.2 0.9� 0.5�XR 7� 2 0.6� 0.7 5� 2h 4.7� 0.5 � 3.3� 0.4 2.0� 0.2R 0.912 0.959 0.812

a Correlations with Eq (4). [19]

G. Lin et al.

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is about the f value for proton 1,3-shift because the f valuefor conformational change from pseudo-trans to pseudo-cisshould be negligible. Formation of protonated enzyme-inhibitor tetrahedral intermediate (Ki2 step, Figure 2) issimilar to that of hydrolysis of ethyl phenylacetate [17];therefore, the f value for Ki2 step is about � 0.2. The f valueof � 1.7 for the � logKi-�-ES-F correlation (Table 2) is closeto calculated f value of � 1.6 [� 0.6 (for protonation ofcarbamates 1 ± 11) � 1.2 (for Ki1)� 0.2 (for Ki2)]. Overall,

these f values confirm that Ki is composed of protonation,Ki1 ,and Ki2 (Figure 2).From previous cross-interaction correlation results, pro-

tonated carbamates 1 ± 11 are in pseudo-trans conformation[18]. A large �XR value of 7 from the ±logKi-Hammett-Taftcross-interaction correlation of carbamates 1 ± 11 and p-nitrophenyl-N-substituted carbamates [11] (Table 3) indi-cates that the distance between X and R in transition statefor the Ki step is relatively short [18]. Therefore, a pseudo-trans to pseudo-cis conformational change involves in theKi1 step (Figure 2). Moreover, that the X-ray structure ofphosphonate inhibitor-PCL tetrahedral intermediate [7] isbent confirms this conformational change.Thek2 step is formation of carbamyl enzyme intermediate

and releases substituted phenol product which is notprotonated from the active site His 286 of the enzyme(Figure 2). The � value of 0.8 for the logk2-�-ES-F correla-tion (Table 2) indicates that substituted phenol product ofthis step is less positive charge than protonated enzyme-inhibitor tetrahedral intermediate (Figure 2). Moreover,that both ortho steric and polar effects in this step arenegligible (�� f� 0.0) (Table 3) indicates that leaving groupbinding site of the enzyme is large enough to adapt anybulkyortho substituted phenol and that no proton transfer isobserved in this step. Since the leaving group hydroxylproton is not from the active site His 286 of ES, the leavinggroup does not need to constrain to the same side of ES.Therefore, the leaving groupmay rotate to far away pseudo-trans conformation to depart from the enzyme active site(Figure 2).The �XR value of 0.6 for the k2 step (Table 3) suggests that

the distance between X and R in the transition state of thisstep is relatively long and confirms that the k2 step involvesin C�O bond breaking and product releasing (Figure 2).In conclusion, ortho effects inQSAR for steady-state PSL

inhibition by ortho substituted phenyl-N-butyl carbamatesare more understood than before, and the PSL inhibitionmechanism based on these results is proposed (Figure 2).

Acknowledgements

We thank the National Science Council of Taiwan forfinancial support.

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Figure 2. The proposed mechanism for the inhibition of PSL bythe carbamate inhibitors.

Figure 3. The proton 1,3-shift from the carbamate nitrogen toether oxygen of the inhibitors.


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Received on March 26, 2003; Accepted on September 12, 2003


G. Lin et al.

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