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 LeeDepartment of Chemistry, National Chung-Hsing University, Taichung 402, TaiwanFull PaperOrtho-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 thirdstep (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 IntroductionThe commercial potential of organic syntheses catalyzed bylipases (EC 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 hasbeen 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 Pseudomonascepacia 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 EI852 QSAR Comb. Sci. 22 (2003) DOI: 10.1002/qsar.200330827 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim* To receive all correspondenceKey words: QSAR, ortho effects, cross-interactions, lipase inhibi-tion, carbamate inhibitorsAbbreviations: 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, theactive 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, Hammettsubstituent 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 esteraseinhibitions 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-substitutedphenyl-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 Methods2.1 MaterialsPSL 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 Methods1Hand 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 ReductionOrigin (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 853Ortho Effects in Quantitative Structure Activity Relationships for Lipase Inhibition by Aryl Carbamates2.3 Steady-State Enzyme KineticsThe 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 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 (kapps) for inhibitionwere determined as described by Hosie et al. [13]. Kis andk2s were obtained by fitting kapps 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 CarbamatesCarbamates 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 (CO). 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 (CO).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 (CO). 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 (CO).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 (CO).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, J7 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 (CO). 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 (CO). 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),854 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim QSAR Comb. Sci. 22 (2003)G. Lin et al.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 (CO). 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 (CO). 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 (CO). 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 (CO). HRMS calculated forC11H15N2O4: 193.1103, found: 193.1104.3 Resultso-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 substrateinhibitors of PSL (Scheme 1) because carbamates 1 11 aretime-dependent and follow first-order kinetics, and theenzyme 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 M1s1.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).QSAR Comb. Sci. 22 (2003) 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 855Figure 1. Structures of inhibitors 111.Scheme 1. Kinetic scheme for pseudo-substrate inhibitions ofPSL in the presence of substrate.Ortho Effects in Quantitative Structure Activity Relationships for Lipase Inhibition by Aryl Carbamates4 Discussionp-Nitrophenyl-N-butyl carbamate (9) [11] is themost potentPSL inhibitor with the ki value of 900 M1s1 (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 11is proposed (Figure 2). The first step is protonation ofcarbamates 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)856 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim QSAR Comb. Sci. 22 (2003)Table 1. Substituent constants and inhibition data for PSL-catalyzed hydrolysis of PNPB in the presence of carbamates 1 11Inhibitor Substituent ESa Fa kc (104s1) Ki (M) ki (103 M1s1)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 0.0210 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 0.05a Obtain from reference [17]b Sum of (o-t-Bu) and (p-t-Bu)Table 2. QSAR results for PSL inhibitions by carbamates 1 11h f Rblogk2 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 0.879a 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 carbamatesaParameters logKi logk2 logki* 15 4 1 1 11 3 0.6 0.6 1.0 0.2 0.9 0.5XR 7 2 0.6 0.7 5 2h 4.7 0.5 3.3 0.4 2.0 0.2R 0.912 0.959 0.812a Correlations with Eq (4). [19]G. Lin et al.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 forKi2 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 intermediateand 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 thatthe distance between X and R in the transition state of thisstep is relatively long and confirms that the k2 step involvesin CO bond breaking and product releasing (Figure 2).In conclusion, ortho effects inQSAR for steady-state PSLinhibition by ortho substituted phenyl-N-butyl carbamatesare more understood than before, and the PSL inhibitionmechanism based on these results is proposed (Figure 2).AcknowledgementsWe thank the National Science Council of Taiwan forfinancial support.References[1] W. Boland, C. Frl, N. Lorenz, Esterolytic and LipolyticEnzymes in Organic Synthesis, Synthesis 1991, 12, 1049 1072.[2] F. Theil, Lipase-Supported Synthesis of Biologically ActiveCompounds, Chem. Rev. 1995, 95, 2203 2227.[3] A. Svendsen, in: Lipases. Their Structure Biochemistry andApplication, P. Woolley, S. B. Petersen (Eds.), CambridgeUniversity Press, Cambridge, 1994, pp. 1 21.[4] H. van Tilbeurgh, M.-P. Egloff, C. Martinez, N. Rugani, R.Verger, C. Cambillau, Interfacial Activation of the Lipase-QSAR Comb. Sci. 22 (2003) 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 857Figure 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.Ortho Effects in Quantitative Structure Activity Relationships for Lipase Inhibition by Aryl CarbamatesProcolipase Complex by Mixed Micelles Revealed by X-rayCrystallography, Nature 1993, 362, 814 820.[5] K. K. Kim, H. K., Song, D. H. Shin, K. Y. Hwang, S. W. Suh,The Crystal Structure of a Triacylglycerol Lipase fromPseudomonas cepacia Reveals a Highly Open Conformationin the Absence of a Bound Inhibitor, Structure 1997, 5, 173 185.[6] J. D. Schrag, Y. Li, M. Cygler, D. Lang, T. Burgdorf, H.-J.Hecht, R. Schmid, D. Schomburg, T. J. Rydel, J. Day, A.McPherson, The Open Conformation of a PseudomonasLipase, Structure 1997, 5, 187 202.[7] D. A. Lang, M. L. M. Mannesse, G. H. De Haas, H. M.Verheij, B. W. Dijkstra, Structure Basis of the Chiral Selec-tivity of Pseudomonas cepacia Lipase, Eur. J. Biochem. 1998,254, 333 340.[8] P. Grochulski, F. Bouthillier, R. J. Kazlauskas, A. N. Serreqi,J. D. Schrag, E. Ziomek, M. Cygler, Analogs of ReactionIntermediates Identify a Unique Substrate Binding Candidarugosa Lipase, Biochemistry 1994, 33, 3494 3500 (1994).[9] M. L. Drent, E. A. Vanderveen, First Clinical Studies withOrlistat: a Short Review, Obes. Res. 1995, 3, S623 S625.[10] G. Lin, C.-T. Shieh, H.-C. Ho, J.-Y. Chouhwang, W.-Y. Lin, C.-P. Lu, Structure- Reactivity Relationships for the InhibitionMechanism at the Second Alkyl-Chain-Binding Site ofCholesterol Esterase and Lipase, Biochemistry 1999, 38,9971 9981.[11] G. Lin, J.-Y. Chouhwang, Quantitative Structure-ActivityRelationship for the Inhibition of Pseudomonas speciesLipase by 4-Nitrophenyl-N-Substituted Carbamates. I. TheSteady-State Kinetics, J. Biochem. Mol. Biol. & Biophys.2001, 5, 301 308.[12] W. N. Aldridge, E. Reiner, in: Enzyme Inhibitors as Sub-strates, A. Neuberger, E. L. Tatun, (Eds.), North-HollandPublishing Co., Amsterdam, 1972, pp. 123 145.[13] L. Hosie, L. D. Sutton, D. M. Quinn, p-Nitrophenyl andCholestery-N-alkyl Carbamates as Inhibitors of CholesterolEsterase, J. Biol. Chem. 1987, 262, 260 264.[14] J. March, Advanced Organic Chemistry, 4th ed., John Wiley &Sons, New York, 1992.[15] N. Isaacs, Physical Organic Chemistry, 2nd ed., Longman,U. K., 1995.[16] T. H. Lowry, K. S. Richardson, Mechanism and Theory inOrganic Chemistry, 3rd ed., Harper & Row, New York, 1992.[17] T. Fujita, T. Nishioka, The Analysis of the Ortho Effect, Prog.Phys. Org. Chem. 1976, 12, 49 89.[18] G. Lin, Hammett-Taft Cross-Interaction Correlations for theInhibition Mechanism of Cholesterol Esterase by SubstitutedPhenyl N-Substituted Carbamates, J. Phys. Org. Chem. 2000,13, 313 321.[19] I. Lee, Cross-interaction Constants and Transition-stateStructure in Solution, Adv. Phys. Org. Chem. 1992, 27, 57 117.[20] G. Lin, C.-Y. Lai, Hammett Analysis of the Inhibition ofPancreatic Cholesterol Esterase by Substituted Phenyl-N-Butylcarbamate, Tetrahedron Lett. 1995, 36, 6117 6120.[21] G. Lin, C.-Y. Lai, Linear Free Energy Relationships of theInhibition of Pancreatic Cholesterol Esterase by 4-Nitro-phenyl-N-Alkylcarbamate, Tetrahedron Lett. 1996, 37, 193 196.[22] S. R. Feaster, K. Lee, N. Baker, D. Y. Hui, D. M. Quinn,Molecular Recognition by Cholesterol Esterase of Active SiteLigands: Structure-Reactivity Effects for Inhibition by ArylCarbamates and Subsequent Carbamylenzyme Turnover,Biolchemistry 1996, 35, 16723 16734.[23] G. Lin, Y.-C. Tsai, H.-C. Liu, W.-C. Liao, C. H. Chang,Enantiomeric Inhibitors of Cholesterol Esterase and Acetyl-cholinesterase, Biochem. Biophys. Acta 1998, 1388, 161 174.[24] G. Lin, C.-T. Shieh, Y.-C. Tsai, C.-I. Hwang, C.-P. Lu, G.-H.Chen, Structure-Reactivity Probes for Active Site Shapes ofCholesterol Esterase by Carbamate Inhibitors, Biochem.Biophys. Acta 1999, 1431, 500 511.[25] G. Lin, C.-Y. Lai, W.-C. Liao, B.-H. Kuo, C.-P. Lu, Structure-Reactivity Relationships As Probes for the Inhibition Mech-anism of Cholesterol Esterase by Aryl Carbamates. I. Steady-State Kinetics, J. Chin. Chem. Soc. 2000, 47, 489 500.Received on March 26, 2003; Accepted on September 12, 2003858 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim QSAR Comb. Sci. 22 (2003)G. Lin et al.


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