enantiomeric inhibitors of cholesterol esterase and acetylcholinesterase

Enantiomeric inhibitors of cholesterol esterase and acetylcholinesterase

Gialih Lin *, Yi-Chin Tsai, Hsiao-Chien Liu, Wei-Cheng Liao, Chun-Hui ChangDepartment of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan

Received 9 June 1998; accepted 13 August 1998

Abstract

Enantiomers of N-methyl-N,K-methylbenzylbutyramide (1), 1-butyl-3-methyl-3P-K-methylbenzylurea (2), 1,2,3,4-tetrahy-dro-1-naphthyl-N-butylcarbamate (3), 1,1P-bi-2-naphthyl-2,2P-di-N-butylcarbamate (4), 1,1P-bi-2-naphthyl-2-ol-2P-N-butyl-carbamate (5), and 1,1P-bi-2-naphthyl-2-butyrate-2P-N-butylcarbamate (6) are inhibitors of porcine pancreatic cholesterolesterase-catalyzed hydrolysis of 4-nitrophenyl butyrate and of electric eel acetylcholinesterase-catalyzed hydrolysis ofacetylthiocholine in the presence of 5,5P-dithiobis-2-nitrobenzoate. For competitive inhibitors, values of the inhibitionconstant (Ki) and the enantiomeric ratio (Ecomp:) are investigated. For active site-directed irreversible inhibitors, values of theinhibition constant (Ki), the carbamylation constant (k2), the bimolecular rate constant (ki), and the enantiomeric ratio (E)are investigated. Toward both enzymes, compounds 1 are poor competitive inhibitors (Ki = 102^104 WM) but have goodenantioselectivities (Ecomp: = 10^50, the preference for R). R-2 and S-2 are competitive inhibitors of acetylcholinesterase withKi = 26 and 80 WM, respectively (the preference for R) but are active site-directed irreversible inhibitors of cholesterol esterasewith ki = 4 and 16 M31 sec31, respectively (the preference for S). For those competitive inhibitions, both leaving grouphydrophilic and hydrophobic binding sites of cholesterol esterase or both anionic substrate binding site and peripheralanionic binding site of acetylcholinesterase bind to N,N-methyl-K-methylbenzyl disubstituted amide parts of these inhibitorsand the enzyme does not catalyze the hydrolysis of these inhibitors. The opposite stereopreference (S) for the inhibition ofcholesterol esterase by compounds 2 may be due to the fact that N,N-methyl-K-methylbenzyl disubstituted amide parts ofthese inhibitors bind to the alkyl chain binding site of the enzyme. Compounds 3^6 are active site-directed irreversibleinhibitors of cholesterol esterase (ki = 1^13 000 M31 s31) and peripheral anionic binding site-directed irreversible inhibitors ofacetylcholinesterase (ki = 1.7^1300 M31 s31). Compounds 3 have low enantioselectivities (E = 1.3^1.4) for both enzymes. Thestereopreference for atropisomers 4 and 6 is S-form toward both enzymes (E = 2^30) and is identical to that of cholesterolesterase-catalyzed hydrolysis of 1,1P-bi-2-naphthyl-2,2P-diacylate. This stereopreference (S) may be due to the fact that thebutyryl group or one of two butylcarbamate groups of S-atropisomers binds more effectively to the leaving grouphydrophobic binding site of cholesterol esterase or the peripheral anionic binding site of acetylcholinesterase than that of R-atropisomers. The opposite stereopreference (R) for atropisomers 5 toward both enzymes may be due to a favorableinteraction between the hydroxyl group of the inhibitors and the leaving group hydrophilic binding site of cholesterol esteraseor the peripheral anionic binding site of acetylcholinesterase. ß 1998 Elsevier Science B.V. All rights reserved.

Keywords: Enantiomeric inhibitor; Inhibition of cholesterol esterase and acetylcholinesterase

0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 1 8 4 - 8

Abbreviations: AACS, active site-selective aromatic cation binding site; ACh, acetylcholine; AChE, acetylcholinesterase; ACS, alkylchain binding site; AS, anionic substrate binding site; ATCh, acetylthiocholine; CEase, cholesterol esterase; CRL, Candida rugosa lipase;DTNB, 5,5P-dithiobis-2-nitrobenzoate; ES, esteratic site; LHIS, leaving group hydrophilic binding site; LHOS, leaving group hydro-phobic binding site; OH, oxyanion hole; PAS, peripheral anionic binding site or sites; PNPB, p-nitrophenylbutyrate; TFA, tri£uoro-acetophenone

* Corresponding author. Fax: +886 (4) 2862547.

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Biochimica et Biophysica Acta 1388 (1998) 161^174

1. Introduction

Recently there has been increased interest in pan-creatic cholesterol esterase (CEase, EC 3.1.1.13) dueto correlation between enzymatic activity in vivo andabsorption of dietary cholesterol [1,2]. Since the as-sociation between serum cholesterol levels and ath-erosclerosis has been well documented [3], an investi-gation into the mechanism of action of this enzymemay lead to the design the mechanism-based inhib-itors which could be of future therapeutic use. Thethree-dimensional structure of Candida rugosa lipase(CRL), a homologue of CEase, has been reportedrecently [4]. Therefore, the active site of CEase mayconsist of at least ¢ve major binding sites (Fig. 1): (a)an alkyl chain binding site (ACS) that binds to theacyl chain of the substrate, (b) an oxyanion hole(OH) that stabilizes the tetrahedral intermediate, (c)an esteratic site (ES), comprised of the active siteserine which would attack the re face of the estercarbonyl, (d) a leaving group hydrophobic bindingsite (LHOS) that binds to the hydrophobic part ofthe leaving group and is in a crevice above the cata-lytic site, and (e) a leaving group hydrophilic bindingsite (LHIS) that binds to the hydrophilic part of theleaving group and is located at the opposite directionof ACS.

Acetylcholinesterase (AChE, EC 3.1.1.7) plays avital role in the central and peripheral nervous sys-tems, where it catalyzes the hydrolysis of the neuro-transmitter acetylcholine (ACh) [5,6]. The three-di-mensional structure of AChE from Torpedocalifornica electric organ has been reported recently[7]. The active site of AChE also consists of at least¢ve major binding sites (Fig. 2) [6,7] : (a) an oxyanionhole (OH) that stabilizes the tetrahedral intermedi-ate; (b) an esteratic site (ES), comprised of the activesite serine which would attack the re face of the estercarbonyl; (c) an anionic substrate binding site (AS)that contains a small number of negative charge butmany aromatic residues, where the quaternary am-monium pole of ACh and of various active site lig-ands binds through a preferential interaction ofquaternary nitrogens with the Z electrons of aromaticgroups; (d) an active site-selective aromatic bindingsite (AACS) that is contiguous with or near the es-teratic and anionic loci and that is important in bind-ing aryl substrates and active site ligands; and (e) a

peripheral anionic binding site or sites (PAS) that maybind to the hydrophobic part of the leaving groupand is s 20 Aî from the active site. In Alzheimer'sdisease, a neurological disorder, cholinergic de¢-ciency in the brain has been reported [8,9]. Therefore,synthesis and study of inhibitors of acetylcholinester-ase may aid to development of therapeutically usefulcompounds to treat such neurological disorders.

Both enzymes are characterized as serine hydro-lases and have the Asp-His-Ser or Glu-His-Ser cata-lytic triad [4^7,10]. The reversibly competitive inhib-itors phenyl haloalkylketones [11,12] and phenyl-n-butylborinic acid [13] are potent transition state ana-logues of CEase and AChE. In the presence of sub-strate, the mechanism of this inactivation has beenproposed (Scheme 1). Inhibition constant (Ki) can becalculated from Eq. 1 [11].

V Ii � V0

i

K iKm � �S�

Km� L�I�

K iKm � �S�

Km� �I�

�1�

V Ii is the initial velocity in the presence of inhibitor;

Ki, L, and V0i are the enzyme-inhibitor dissociation

constant, the residual fractional activity at saturating[I], and the initial velocity in the absence of inhibitor,respectively and are the adjustable parameters of thenon-linear least squares ¢t. For linear competitiveinhibition, L values equal to zero.The enantioselec-tivity for competitive inhibitor (Ecomp:) is de¢ned asthe following:

Ecomp: � 1=KAi

1=KBi

� KBi

KAi

�2�

where A and B are enantiomeric inhibitors that com-pete for the same site on the enzyme.

On the other hand, 4-nitrophenyl-N-alkyl carba-mates are potent active site-directed irreversible in-hibitors of CEase [10]. The mechanism for inhibitionin the presence of substrate is shown in Scheme 2

Scheme 1. Kinetic scheme for competitive inhibition of a serinehydrolase in the presence of substrate.

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G. Lin et al. / Biochimica et Biophysica Acta 1388 (1998) 161^174162

[10,14]. Because the inhibition of CEase follows ¢rst-order kinetics over the observed time period, the rateof hydrolysis of EIP must be signi¢cantly slower thanthe rate of formation of EIP(k2Ek3) [15]. Therefore,values of Ki and k2 can be calculated from Eq. 3[10,14]:

kapp � k2�I�K i�1� �S�Km

� � �I��3�

In Eq. 3, kapp values are the ¢rst-order rate constantswhich can be obtained according to Hosie's method[10]. Bimolecular rate constant, ki = k2/Ki, is relatedto overall inhibitory potency. According to the de¢-nition of the enantiomeric ratio (E) [16], E is gov-erned by

E � VAmax=KA

i

VBmax=KB

i

� kA2 =KA

i

kB2 =KB

i

� kAi

kBi

�4�

where A and B are the fast and slow reacting enan-tiomeric inhibitors that compete for the same site onthe enzyme.

As part of an investigation of stereoisomeric inhib-itors as serine hydrolase inhibitors, enantiomeric in-hibitors of N-methyl-N,K-methylbenzylbutyramide(R-1 and S-1), 1-butyl-3-methyl-3P-K-methylbenzylur-ea (R-2 and S-2), 1,2,3,4-tetrahydro-1-naphthyl-N-butylcarbamate (R-3 and S-3), 1,1P-bi-2-naphthyl-2,2P-di-N-butylcarbamate (R-4 and S-4), 1,1P-bi-2-naphthyl-2-ol-2P-N-butylcarbamate (R-5 and S-5),and 1,1P-bi-2-naphthyl-2-butyrate-2P-N-butylcarba-mate (R-6 and S-6) (Fig. 3) are prepared and eval-uated for their e¡ects on CEase and AChE. Theprinciple of stereoelectronic control [17] predictsthat N,N-disubstituted amides are not substrates(no hydrolysis) but competitive inhibitors of K-chy-motrypsin [18,19]. Like K-chymotrypsin, compounds1 may be competitive inhibitors of CEase and AChE.

Compounds 2 are competitive inhibitors if the leav-ing group binding site of the enzyme binds to N,N-disubstituted amide part or active site-directed irre-versible inhibitors if that site binds to the butylamidepart. The latter case may or may not have the samestereopreference as the former case toward the en-zyme. The enantiomeric ratios show that CEase isspeci¢c for the S atropisomer in both steps of thehydrolysis of 1,1P-bi-2-naphthyl-2,2P-O-diacylatewith the enantiospeci¢city of the ¢rst hydrolysis(Es 400) being considerably larger than that of thesecond (E = 4.9) [20]. According to this, atropisomers4^6 are prepared and studied the phenomena on theinhibition of CEase and AChE. The enantiomericratios show that CEase is speci¢c for the R enan-tiomer of the hydrolysis of 1,2,3,4-tetrahydro-1-naphthyl-O-butyrate [21,22]. Thus, compoundsrac-3, R-3, and S-3 are prepared and evaluated fortheir e¡ects on CEase and AChE.

2. Materials and methods

2.1. Materials

CEase from porcine pancreas, AChE from electriceel, p-nitrophenyl butyrate (PNPB), 5,5P-dithiobis-2-nitrobenzoate (DTNB), edrophonium chloride,and acetylthiocholine (ATCh) were obtained fromSigma; other chemicals are obtained from Aldrich;silica gel used in liquid chromatography (LicorpreSilica 60, 200^400 mesh) and thin-layer chromatog-raphy plates (60 F254) were obtained from Merck;other chemicals and biochemicals were of the highestquality available commercially.

2.2. Instrumental methods

1H- and 13C-NMR spectra were recorded at 300and 75.4 MHz (Varian-XR 300 spectrometer), re-spectively. The 1H and 13C chemical shifts were re-ferred to internal Me4Si. UV-visible spectra were re-corded on an UV-visible spectrometer (HP 8452)with a cell holder circulated with a water bath.

2.3. Synthesis of compounds 1^6

R- and S-N-methyl-N,K-methylbenzylbutyramide

Scheme 2. Kinetic scheme for active site-directed irreversible in-hibition of a serine hydrolase in the presence of substrate.

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G. Lin et al. / Biochimica et Biophysica Acta 1388 (1998) 161^174 163

(R-1 and S-1) or rac-, R-, and S-1,1P-bi-2-naphthyl-2-butyrate-2P-N-butylcarbamate (rac-6, R-6, and S-6)were prepared from the condensation of R- and S-N-methyl-N,K-methylbenzylamine or rac-, R-, and S-1,1P-bi-2-naphthyl-2-ol-2P-N-butylcarbamate (rac-5,R-5, and S-5) with 1.2 mole equivalents of n-butyricchloride in the presence of catalytic amount of pyr-idine in dichloromethane at 25³C for 48 h (80^91%yield). R- and S-1-butyl-3-methyl-3P-K-methylbenzy-lurea (R-2 and S-2), R-, S-, and rac-1,2,3,4-tetrahy-dro-1-naphthyl-N-butylcarbamate (R-3, S-3, and rac-3), and R-, and S-, and rac-1,1P-bi-2-naphthyl-2-ol-2P-N-butylcarbamate (R-5, S-5, and rac-5) were pre-pared from the condensation of the correspondingamine or alcohol with 1.2 mole equivalents of n-bu-tylisocyanate in the presence of catalytic amount ofpyridine in dichloromethane at 25³C for 48 h (75^92% yield). R-, S-, and rac-1,1P-bi-2-naphthyl-2,2P-di-N-butylcarbamate (R-4, S-4, and rac-4) were pre-pared from the condensation of the correspond-ing 1,1P-bi-2-naphthol with 5 mole equivalents ofn-butylisocyanate in the presence of catalytic amountof pyridine in dichloromethane at 25³C for 48 h (86^92% yield). All compounds were puri¢ed by liquidchromatography on silica gel (hexane-ethyl acetate)and characterized by 1H- (300 MHz) and 13C-NMR(75.4 MHz), mass, and IR spectra and elementalanalysis.

N-Methyl-N,K-methylbenzylbutyramide (1)-1H-NMR (CDCl3, 300 MHz) N/ppm 0.93 (t, J = 7.2 Hz,3H, g-CH3), 1.26^1.55 (m, 4H, L-CH2 and Q-CH2),1.50 (d, J = 6.5 Hz, 3H, CH3-C(H)Ph), 3.28 (m, 2H,K-CH2), 2.59 (s, 3H, N-CH3), 4.35 (br, 1H, NH),5.66 (q, J = 7.1 Hz, 1H, CH3-C(H)Ph), 7.22^7.36(m, 5H, phenyl-H) ; 13C-NMR (CDCl3, 75.4 MHz)N/ppm 13.73 (g-C), 16.46 and 28.51 (N-CH3 andCH3-C(H)Ph), 20.00 (Q-C), 32.38 (L-C), 40.66 (K-C),51.77 (CH3-C(H)Ph), 126.98 and 128.38 (phenyl C-2-C-6), 141.81 (phenyl C-1), 158.41 (C = O); mass spec-trum (high resolution): calcd. for C14H22N2O234.3406. Found 234.3397. Anal. calcd. forC14H22N2O: C, 71.74; H, 9.47; N, 11.96. FoundC, 71.65; H, 9.35, N, 11.97.

1-Butyl-3-methyl-3P-K-methylbenzylurea (2)-1H-NMR (CDCl3, 300 MHz) N/ppm 1.00 (t, J = 7.2 Hz,3H, g-CH3), 1.47 (d, J = 6.9 Hz, 3H, CH3-C(H)Ph),1.62^1.77 (m, 2H, L-CH2), 2.33 (t, J = 8 Hz, 2H, K-CH2), 2.65 (s, 3H, N-CH3), 6.10 (q, J = 6.6 Hz, 1H,

CH3-C(H)Ph), 7.22^7.40 (m, 5H, phenyl-H); 13C-NMR (CDCl3, 75.4 MHz) N/ppm 13.92 (g-C),15.48 and 29.41 (N-CH3 and CH3-C(H)Ph), 18.52(L-C), 35.86 (K-C), 49.93 (CH3-C(H)Ph), 126.43,127.13, 127.26, and 128.40 (phenyl C-2-C-6), 140.87(phenyl C-1), 173.08 (C = O); mass spectrum (highresolution): calcd. for C13H19NO 205.1467. Found205.1463. Anal. calcd. for C13H19NO: C, 76.04; H,9.33; N, 6.83. Found C, 76.12; H, 9.23, N, 6.95.

1,2,3,4-Tetrahydro-1-naphthyl-N-butylcarbamate(3)-1H-NMR (CDCl3, 300 MHz) N/ppm 0.93 (t,J = 7.2 Hz, 3H, g-CH3), 1.26^1.55 (m, 4H, L-CH2

and Q-CH2), 1.74^2.08 and 2.68^2.89 (m, 6H, cyclicCH2), 3.21 (m, 2H, K-CH2), 4.66 (br, 1H, NH), 5.87(m, 1H, -CH2C(H)Ph-), 7.10^7.35 (m, 4H, phenyl-H) ; 13C-NMR (CDCl3, 75.4 MHz) N/ppm 13.61 (g-C), 18.71, 28.93, and 29.28 (cyclic CH2), 19.80 (Q-C),31.95 (L-C), 40.65 (K-C), 70.03 (-CH2C(H)Ph-),126.01, 127.89, 128.97, 129.45, 135.11, and 137.77(phenyl-C), 156.44 (C = O); mass spectrum (high res-olution): calcd. for C15H21NO2 247.1572. Found247.1562. Anal. calcd. for C15H21NO2 : C, 72.83;H,8.56; N, 5.67. Found C, 72.98; H, 8.49, N, 5.72.

1,1P-Bi-2-naphthyl-2,2P-di-N-butylcarbamate (4)-IR (KBr) 3452 (NH) and 1719 (C = O) cm31 ; 1H-NMR (CDCl3, 300 MHz) N/ppm 0.94 (t, J = 7 Hz,6H, g-CH3), 1.41 (sextet, J = 7 Hz, 4H, Q-CH2), 1.58(m, 4H, L-CH2), 3.26 and 3.32 (ABq, J = 7 Hz, 4H,K-CH2), 5.02 (t, J = 4 Hz, 2H, NH), 7.17^8.00 (m,12H, naphthyl-H) ; 13C-NMR (CDCl3, 75.4 MHz)N/ppm 13.67 (g-C), 19.83 (Q-C), 31.87 (L-C), 41.06(K-C), 115.45, 118.57, 122.31, 124.43, 125.87,127.90, 128.16, and 128.68 (binaphthyl C-3-C-10),134.15 (binaphthyl C-1), 148.93 (binaphthyl C-2), 156.71 (C = O); mass spectrum (EI): m/z (%)484 (3.67), 286 (100). Anal. calcd. for C30H32O4N2 :C, 74.34; H, 6.66; N, 5.78. Found C, 74.26; H, 6.68,N, 5.80.

1,1P-Bi-2-naphthyl-2-ol-2P-N-butylcarbamate (5)-1H-NMR (CDCl3, 300 MHz) N/ppm 0.827 (t,J = 7.5 Hz, 3H, g-CH3), 1.08 (sextet, J = 7 Hz, 2H,Q-CH2), 1.28 (m, 2H, L-CH2), 3.07 (m, 2H, K-CH2),4.71 (t, J = 5 Hz, 1H, NH), 5.94 (br, 1H, OH), 7.00^8.05 (m, 12H, naphthyl-H) ; 13C-NMR (CDCl3, 75.4MHz) N/ppm 13.54 (g-C), 19.43 (Q-C), 31.44 (L-C),40.76 (K-C), 114.97, 119.14, 122.10, 123.44, 124.08,124.57, 125.73, 126.04, 126.55, 127.98, 128.17,129.10, 130.10, 130.53, and 132.10 (binaphthyl C-3-

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C-10), 133.60 and 133.74 (binaphthyl C-1), 148.08and 152.31 (binaphthyl C-2), 155.63 (C = O); massspectrum (high resolution): calcd. for C25H23O3N385.1678. Found 385.1685. Anal. calcd. forC25H23O3N: C, 77.89; H, 6.02; N, 3.64. Found C,77.78; H, 5.97, N, 3.54.

1,1P-Bi-2-naphthyl-2-butyrate-2P-N-butylcarbamate(6)-1H-NMR (CDCl3, 300 MHz) N/ppm 0.66 (t,J = 7.4 Hz, 3H, g-CH3 of butyryl), 0.75 (t, J = 7.1Hz, 3H, g-CH3 of N-butylcarbamate), 1.05 (m, 4H,Q-CH2 of N-butylcarbamate and L-CH2 of butyryl),1.30 (m, 2H, L-CH2 of N-butylcarbamate), 2.90 (t,7.4 Hz, 2H, K-CH2 of butyryl), 3.07 (m, 2H, K-CH2

of N-butylcarbamate), 4.76 (m, 1H, NH), 7.14^7.99(m, 12H, naphthyl-H) ; 13C-NMR (CDCl3, 75.4MHz) N/ppm 13.16 and 13.53 (g-C), 17.91 and19.37 (Q-C of N-butylcarbamate and L-C of butyryl),31.38 (L-C of N-butylcarbamate), 35.87 (K-C of bu-tyryl), 40.44 (K-C of N-butylcarbamate), 121.96,122.32, 123.27, 123.66, 125.33, 125.66, 126.05,126.54, 126.70, 127.84, 127.99, 129.28, 129.41,131.34, and 131.52 (binaphthyl C-3-C-10), 133.28and 133.47 (binaphthyl C-1), 146.80 and 147.29 (bi-naphthyl C-2), 154.13 (C = O of N-butylcarbamate),172.62 (C = O of butyryl); mass spectrum (high res-olution): calcd. for C29H29O4N 455.2096. Found455.2093. Anal. calcd. for C29H29O4N: C, 76.45;H, 6.42; N, 3.08. Found C, 76.32; H, 6.52, N,3.18.

2.4. Enzyme kinetics and data reduction

All kinetic data were obtained by using a UV-visi-ble spectrometer (HP 8452) that was interfaced to apersonal computer. Kaleida Graph was used for all

least squares curve ¢ttings. The CEase inhibition wasassayed by the Hosie method [10] and the AChEinhibition was assayed by the Ellman method [23].The temperature was maintained at 25.0 þ 0.1³C by arefrigerated circulating water bath. All active site-di-rected irreversible inhibition reactions were per-formed in sodium phosphate bu¡er (pH 7.01) con-taining NaCl (0.1 M), acetonitrile (2% v/v), TritonX-100 (0.5% w/w), and substrate (for CEase, 50 WMof PNPB; for AChE, 50 WM of ATCh with 50 WM ofDTNB (chromogenic reagent)) and varying concen-tration of inhibitors. For the competitive inhibition,all procedures were the same as those of the activesite-directed irreversible inhibition except varyingconcentration of substrate. Requisite volumes ofstock solution of substrate and inhibitors in acetoni-trile were injected into reaction bu¡ers via GilsonPipetmen. Porcine pancreatic CEase and electric eelAChE were dissolved in sodium phosphate bu¡er(0.1 M, pH 7.01). Reactions were initiated by inject-ing enzyme and monitored at 410 nm (for CEase) or405 nm (for AChE) on the UV-visible spectrometer.First-order rate constants (kapp values) for inhibitionof CEase were determined as described by Hosie etal. [10]. Values of Ki and k2 were obtained by ¢ttingthe data of kapp and [I] to Eq. 1 by non-linear leastsquares. Duplicate or triplicate sets of data were col-lected for each inhibitor concentration. For thereturn of activity study, CEase or AChE was incu-bated with carbamate 3, 4, 5, or 6 (1 WM) inthe absence and presence of TFA (2 WM) or edro-phonium chloride (2 WM), a known competitive in-hibitor of the enzyme [6,11] as described by Hosie etal. [10].

Fig. 2. The interaction between ACh and active sites of AChE.

Fig. 1. The interaction between cholesterol ester and active sitesof CEase.

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3. Results

3.1. The synthesis of compounds 1^6

Compounds 1^6 were prepared according to gen-eral procedures (75^92% yield). Compounds 1^6were puri¢ed (up to 99.9%) and characterized byIR, 1H- and 13C-NMR, and mass spectra and ele-mental analysis.

3.2. The inhibition of CEase

Table 1 summarizes the inhibition data for CEase-catalyzed hydrolysis of PNPB in the presence ofcompounds 1^6. Compounds R-1 and S-1 were re-versibly competitive inhibitors of CEase becausetheir L values of Eq. 1 were zero (Fig. 4) and theirLineweaver-Burk plots gave lines that intersect at acommon point on the 1/v axis (Fig. 5). Compounds

Fig. 3. Structures of enantiomeric inhibitors.

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2^6 were characteristic of active site-directed irrever-sible inhibition (Fig. 6) since they met some of thecriteria proposed by Abeles and Maycock [24]. First,the inhibition was time-dependent and followed ¢rst-order kinetics; second, with increasing concentrationof inhibitor the enzyme displayed saturation kinetics;third, the enzyme was protected from inhibition bycompounds 2^6 in the presence of a reversible inhib-itor TFA.

Comparing Ki values, we found that R-1 hadbound more tightly to the enzyme by a factor of 10than S-1, therefore the value of Ecomp: was 10. Thestereochemical preference (RsS) for inhibitors 1and 3 toward CEase was the same as that ofCEase-catalyzed hydrolysis of the substrate 1,2,3,4-tetrahydro-1-naphthylbutyrate [20^22] and that ofCRL-(1R,2S,5R)-menthyl hexylphosphonate com-plex [4] ; however, the stereochemical preference forinhibitors 2 toward CEase was opposite (SsR).Comparing Ki values, we found that S-4 had boundmore tightly to the enzyme by a factor of 15 than R-4. The k2 value increased 1.7-fold from R-4 to S-4.

Therefore, a 25-fold increase in ki was observed fromR-4 to S-4 (E = 30 þ 20). The inhibition power (ki)increased 2-fold from R-6 to S-6 but decreased2-fold from R-5 to S-5. The stereochemical prefer-ence (SsR) for carbamates 4 and 6 was the same asthat of CEase-catalyzed hydrolysis of the substrate1,1P-bi-2-naphthyl-2,2P-dibutyrate [20^22]; however,the stereochemical preference for carbamates 5 wasopposite (RsS).

3.3. The inhibition of AChE

Table 2 summarized the inhibition data for AChE-catalyzed hydrolysis of ATCh in the presence ofDTNB and compounds 1^6. Compounds 1 and 2were reversibly competitive inhibitors of AChE be-cause their L values were very close to zero and theirLineweaver-Burk plots gave lines that intersect at acommon point on the 1/v axis. Carbamates 3^6 wereirreversible inhibitors since they met the ¢rst twocriteria of Abeles and Maycock [24]. First, the inhib-ition was time-dependent and followed ¢rst-order ki-

Table 1Kinetic data for the CEase-catalyzed hydrolysis of PNPB in the presence of compounds 1^6a

Inhibitors Ki (WM)b k2 (1033 s31) ki (102 M31 s31) E or Ecomp:

R-1 102 þ 6 ^ ^ 10 þ 5S-1 1000 þ 500 ^ ^ ^R-2 200 þ 100 0.8 þ 0.4 0.04 þ 0.03 ^S-2 50 þ 1 0.80 þ 0.05 0.16 þ 0.03 4 þ 2rac-3 300 þ 200 0.3 þ 0.1 0.010 þ 0.008 ^R-3 250 þ 40 0.26 þ 0.02 0.010 þ 0.002 1.3 þ 0.4S-3 400 þ 100 0.30 þ 0.05 0.008 þ 0.002 ^rac-4 29 þ 7 1.2 þ 0.1 0.4 þ 0.1 ^R-4 30 þ 1 0.95 þ 0.02 0.32 þ 0.01 ^S-4 2 þ 1 1.6 þ 0.9 8 þ 6 30 þ 20rac-5 0.8 þ 0.8 7 þ 5 100 þ 100 ^R-5 0.8 þ 0.1 10 þ 1 130 þ 20 3 þ 1S-5 1.3 þ 0.6 6 þ 1 50 þ 20 ^rac-6 10 þ 5 0.6 þ 0.3 0.6 þ 0.4 ^R-6 20 þ 4 0.8 þ 0.4 0.4 þ 0.1 ^S-6 9 þ 3 0.6 þ 0.2 0.8 þ 0.4 2 þ 1aKinetic runs were done at 25.0 þ 0.1³C and pH 7.01 in 0.1 M sodium phosphate bu¡er that contained 0.1 M NaCl. The CEase-cata-lyzed hydrolysis of PNPB (50 WM) was followed continuously at 410 nm in the presence and absence of inhibitor on an UV-visiblespectrometer (HP 8452) that was interfaced to a personal computer. Kaleida Graph (version 2.0) was used for all least squares curve¢ttings. For competitive inhibition, Eq. 1 and Eq. 2 were used to calculate Ki and Ecomp:. For active site-directed irreversible inhibi-tion, Eq. 3 and Eq. 4 were used to calculate Ki, ki and E. Bovine pancreatic CEase was purchased from Sigma. All the other proce-dures were the same as described by Quinn [10,11].bFor competitive inhibition, these values were in excellent agreement with those determined from a replot of the slope of each Line-weaver-Burk plot versus the corresponding inhibitor concentration. For active site-directed irreversible inhibition, these values were inexcellent agreement with those determined in the absence of substrate (zero time method) [14].

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netics; second, with increasing concentration of in-hibitor the enzyme displayed saturation kinetics.

However, the enzyme was not protected from inhib-ition by carbamates 3^6 in the presence of a rever-

Fig. 5. Lineweaver-Burk plot for CEase-catalyzed hydrolysis of PNPB in the presence of R-1. Reactions were performed under condi-tions described in Section 2.

Fig. 4. Initial rate vs. inhibitor concentration plot of the CEase-catalyzed hydrolysis of PNPB in the presence of compound R-1. Ki

(0.10 þ 0.04 mM) and L= 0 were calculated from this plot by non-linear least squares ¢tting to Eq. 1.

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sible inhibitor edrophonium. Edrophonium, an AScompetitive inhibitor of AChE [6], further inhibitedthe enzyme-carbamates 3^6 complexes. Therefore, itwas conceivable that carbamates 3^6 react irreversi-bly at the PAS, and not the active site.

Comparing Ki values, we found that R-1 and R-2had bound more tightly to the enzyme by a factor of50 and 3 than S-1 and S-2, respectively. The stereo-chemical preference (RsS) for inhibitors 1 and 2toward this enzyme was the same as that of CEase-

Table 2Kinetic data for the AChE-catalyzed hydrolysis of ATCh in the presence of DTNB and compounds 1^6a

Inhibitors Ki (WM)b k2 (1034 s31) ki (M31 s31) E or Ecomp:

R-1 200 þ 100 ^ ^ 50 þ 35S-1 (1.0 þ 0.5)U104 ^ ^ ^R-2 26 þ 5 ^ ^ 3.0 þ 0.9S-2 80 þ 20 ^ ^ ^rac-3 460 þ 250 5.5 þ 0.1 1.9 þ 0.1 ^R-3 700 þ 70 7.6 þ 0.1 2.37 þ 0.08 1.4 þ 0.2S-3 500 þ 110 5.6 þ 0.2 1.7 þ 0.2 ^rac-4 20 þ 10 4.0 þ 0.3 20 þ 10 ^R-4 40 þ 20 1.0 þ 0.5 2 þ 1 ^S-4 10 þ 5 4.00 þ 0.03 40 þ 20 20 þ 10rac-5 1.0 þ 0.3 2.4 þ 0.2 240 þ 70 ^R-5 0.6 þ 0.1 2.4 þ 0.1 420 þ 80 2.2 þ 0.4S-5 1.30 þ 0.01 2.50 þ 0.01 192 þ 2 ^rac-6 0.9 þ 0.1 6.1 þ 0.2 680 þ 80 ^R-6 2.6 þ 0.9 4.6 þ 0.5 180 þ 80 ^S-6 0.2 þ 0.1 2.6 þ 0.3 1300 þ 700 7 þ 5aThe AChE-catalyzed hydrolysis of ATCh chloride (50 WM) in the presence of DTNB (50 WM) was followed continuously at 405 nmon a UV-visible spectrometer. All the other procedures were the same as in Table 1.bSee footnote b in Table 1.

Fig. 6. Michaelis-Menton plot for inhibition of the CEase-catalyzed hydrolysis of PNPB by compound R-5. First-order rate constants(kapp values) were calculated as described by Quinn et al. [10]. 50 WM of substrate PNPB was used. Non-linear least squares ¢tting toEq. 3 of the text gave Ki = 0.8 þ 0.1 WM and k2 = 0.010 þ 0.001 s31.

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catalyzed hydrolysis of the substrate 1,2,3,4-tetrahy-dro-1-naphthylbutyrate [20^22] and that of CRL-(1R,2S,5R)-menthyl hexylphosphonate complex [4].Comparing ki values, we found that S-4 and S-6

had inhibited AChE more potently by a factor of20 and 7 than R-4 and R-6, respectively. The stereo-chemical preferences for atropisomers 4^6 towardAChE were exactly the same as those toward CEase.

Fig. 7. Interactions between compounds 1^3 and CEase. (A) Interaction of R-1 with CEase: R-1 is not a substrate but the preferredenantiomer. The stereopreference for LHOS and LHIS is R-enantiomer. (B) Interaction of S-1 with CEase: S-1 is not a substrate andis not the preferred enantiomer, either. (C) Interaction of R-2 with CEase: R-2 is an active site-directed irreversible inhibitor but notthe preferred enantiomer. (D) Interaction of S-2 with CEase: S-2 is an active site-directed irreversible inhibitor and the preferredenantiomer. The stereochemical preference for ACS is S-enantiomer. (E) Interaction of R-3 with CEase: R-3 is an active site-directedirreversible inhibitor and the preferred enantiomer. (F) Interaction of S-3 with CEase: S-3 is an active site-directed irreversible inhibi-tor but not the preferred enantiomer.

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Fig. 8. Interactions between active site-directed irreversible inhibitors 4^6 and CEase. (A) Interaction of R-4 with CEase: one of thenaphthyl group binds to the catalytic sites (ACS, ES, and OH). R-4 is not the preferred enantiomer because of the repulsion betweenLHIS and the other N-butyl carbamate group. (B) Interaction of S-4 with CEase: S-4 is the preferred enantiomer. (C) Interaction ofR-5 with CEase: R-5 is the preferred enantiomer because of the formation of a hydrogen bond between the hydroxyl group of the in-hibitor and the basic residue of LHIS. (D) Interaction of S-5 with CEase: S-5 is not the preferred enantiomer because of the repul-sion between the hydroxyl group of the inhibitor and LHOS. (E) Interaction of R-6 with CEase: R-6 is not the preferred enantiomerbecause of the repulsion. (F) Interaction of S-6 with CEase: S-6 is the preferred enantiomer.

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Fig. 9. Interactions between active site-directed irreversible inhibitors 4 and 6 and AChE. (A) Interaction of R-4 with AChE: one ofthe N-butylcarbamate groups binds to PAS. R-4 is not the preferred enantiomer because of the repulsion between PAS and the otherN-butylcarbamate group. (B) Interaction of S-4 with AChE: S-4 is the preferred enantiomer because one of the N-butylcarbamategroups binds to PAS and the other one ¢ts into the space between PAS and the active site. (C) Interaction of R-5 with AChE: R-5 isthe preferred enantiomer because of the formation of a hydrogen bond between the hydroxyl group of the inhibitor and basic residuesof PAS. (D) Interaction of S-5 with AChE: S-5 is not the preferred enantiomer. (E) Interaction of R-6 with AChE: R-6 is not thepreferred enantiomer because of the repulsion. (F) Interaction of S-6 with AChE: S-6 is the preferred enantiomer.

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4. Discussion

4.1. The inhibition of CEase

Application of the principle of stereoelectroniccontrol [17] predicts that N,N-disubstituted amidesare not substrates (no hydrolysis) but competitiveinhibitors of K-chymotrypsin [18,19]. It is becausethe amide nitrogen carried an extra alkyl substituentand molecular models show that this alkyl substitu-ent allows the formation of an enzyme-inhibitorcomplex but does not permit the formation of a tet-rahedral intermediate in the required conformationbecause the extra N-alkyl substituent comes too closeto the imidazole ring of His-57. Similarly, N,N-di-substituent amides 1 are competitive inhibitors ofCEase (Figs. 4 and 5). According to the structureof CRL [4], we have proposed CEase contains atleast ¢ve major binding sites (Fig. 1). For com-pounds 1 and 3, it is obviously that both LHOSand LHIS control the stereopreference which isRsS as literature cited for most serine hydrolase[18^20] (Fig. 7A,B,E,F).

Because compounds 2 are active site-directed irre-versible inhibitors and the N-methyl-N,K-methylben-zylamine group cannot be a leaving group, the butyl-amine group of compounds 2 instead binds to theleaving group binding sites LHOS and LHIS (Fig.7C,D) while the N-methyl-N,K-methylbenzylaminegroup binds to ACS. As a result, the stereopreferenceon ACS for compounds 2 is SsR (E = 4 þ 3). Thus,LHOS/LHIS and ACS have opposite stereochemicalrequirement. Therefore, compounds 2 are success-fully used in the determination of stereochemicalpreference at ACS.

It has been suggested that CEase is speci¢c for theS enantiomer in both steps of the hydrolysis of 1,1P-bi-2-naphthyl-2,2P-O-diacylate with the enantiospeci-¢city of the ¢rst hydrolysis (Es 400) being consider-ably larger than that of the second (E = 3.5^5.2) andS-1,1P-bi-2-naphthyl-2,2P-di-O-acylate has a similarthree-dimensional structure to that of cholesterol es-ter [20]. The stereopreference for atropisomers 4 and6 is also S enantiomers; however, that of atropisom-ers 5 is R enantiomer (Fig. 8). In S-4 and S-6, one ofthe N-butylcarbamate group binds to the catalyticsites (ACS, ES, and OH) while the binaphthyl and

the other N-butylcarbamate or the butyrate groupsbind to LHOS (Fig. 8B,F). On the other hand, therepulsion between one of the N-butylcarbamategroup of R-4 or the butyrate group of R-6 andLHIS prohibits bindings of these carbamates to theenzyme (Fig. 8A,E). Therefore, this makes R-4 andR-6 worse. But, atropisomers 5 do not have this typeof interaction but have another. In the event, R-5 isthe most potent active site-directed irreversible inhib-itor we examined. This may be due to the fact thatthe acidity of the hydroxyl group of R-5, on the samesite of LHIS, is relatively high and facilitates theinhibitor to interact with the basic residue (likelyHis) of LHIS by the formation of a hydrogen bondbetween them (Fig. 8D). In S-5, the hydroxyl groupis in LHOS and this makes the binding worse. Di¡er-ences in ki values between R- and S-atropisomers ofcarbamates 5 and 6 are small but between that ofcarbamates 4 are large. A possible reason for thisis the preferred atropisomer carbamate S-4 canbind to the enzyme at either butylcarbamate groups,which doubles the e¡ective concentration of the in-hibitor and makes the inhibitor more accessible tothe enzyme.

4.2. The inhibition of AChE

The inhibition of AChE by compounds 1^6 issummarized in Table 2. Like CEase, N,N-disubstitu-ent amides 1 are competitive inhibitors of AChE andhave the stereopreference for R enantiomer. It is sur-prising that compounds 2 (Fig. 8) are also compet-itive inhibitors of AChE and also have the stereo-preference for R enantiomer. For the AChEcatalysis, the butyrate group of compounds 1 orthe N-butylaminoamide group (n-BuNHCO) of com-pounds 2 binds to AACS (mimics ACS of CEase),and N-methyl-N,K-methylbenzyl groups of com-pounds 1 and 2 bind to both PAS (mimics LHOSof CEase) and AS (mimics LHIS of CEase) wherethe stereospeci¢city takes place (RsS). If com-pounds 2 were active site-directed irreversible inhib-itors, the N-methyl-N,K-methylbenzylaminoamidegroup (PhCH(Me)NMeC(O)) would bind to AACS.In fact, this does not happen because the size ofAACS is relatively small (for small acyl grouponly) at the beginning. Overall, compounds 1 and 2

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are competitive inhibitors for AChE because of theN,N-disubstituted amide character and have the ster-eopreference for R enantiomer.

It is conceivable that carbamates 3 are PAS-di-rected irreversible inhibitors of AChE since the en-zyme is not protected by edrophonium. The enzymeshows low stereoselectivity (E = 1.4 þ 0.2) for bothenantiomers of carbamates 3 since the size of PASis large enough to adapt both enantiomers.

Carbamates 4^6 may also be PAS-directed irrever-sible inhibitors of AChE since the enzyme is notprotected by edrophonium. The enzyme showssame stereopreferences as those of CEase. In S-4and S-6, one of N-butylcarbamate groups binds toPAS while the other N-butylcarbamate or the buty-rate group occupies the space between PAS and theactive site (Fig. 9). On the other hand, one of N-butylcarbamate groups of R-4 and that of R-6 bindsto PAS while the other N-butylcarbamate or the bu-tyrate group contacts the wall of PAS and thereforeprohibits the binding (Fig. 9). Therefore, this makesR-4 and R-6 worse. On the other hand, R-5 does nothave an extra N-butylcarbamate or butyrate group toprohibits the binding at PAS. Further, the acidichydroxyl group of R-5, on the same site of PAS,facilitates the inhibitor to bind to the basic residueof PAS through a hydrogen bond (Fig. 9). In S-5, thehydroxyl group is not in PAS and this makes thebinding worse.

It has been reported that the enzyme exhibits ster-eoselectivity toward acetyl L-methylcholine [26] butdoes not toward sec-butyl acetate [27]. Therefore, itcan be concluded that the stereoselectivity for AChEneeds a strong cation-anion interaction between thesubstrate and AS. All compounds we prepared donot have any charge and therefore do not showgood stereoselectivities.

Acknowledgements

We would like to thank the National ScienceCouncil of Taiwan for ¢nancial support (NSC 85-2113-M005-005).

References

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Biochem. Biophys. Res. Commun. 134 (1986) 386^392.[14] G.J. Hart, R.D. O'Brien, Biochemistry 12 (1973) 2940^2945.[15] W.N. Aldridge, E. Reiner, in: A. Neuberger, E.L. Tatun

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enantiomeric inhibitors of cholesterol esterase and acetylcholinesterase

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