Lin G, Tsai Y C, Liu H C, Liao W C, Chang C H
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan.
Biochim Biophys Acta. 1998 Oct 14;1388(1):161-74. doi: 10.1016/s0167-4838(98)00184-8.
Enantiomers of N-methyl-N,alpha-methylbenzylbutyramide (1), 1-butyl-3-methyl-3'-alpha-methylbenzylurea (2), 1,2,3, 4-tetrahydro-1-naphthyl-N-butylcarbamate (3), 1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (4), 1, 1'-bi-2-naphthyl-2-ol-2'-N-butylcarbamate (5), and 1, 1'-bi-2-naphthyl-2-butyrate-2'-N-butylcarbamate (6) are inhibitors of porcine pancreatic cholesterol esterase-catalyzed hydrolysis of 4-nitrophenyl butyrate and of electric eel acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine in the presence of 5,5'-dithiobis-2-nitrobenzoate. For competitive inhibitors, values of the inhibition constant (Ki) and the enantiomeric ratio (Ecomp.) are investigated. For active site-directed irreversible inhibitors, values of the inhibition 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 microM) but have good enantioselectivities (Ecomp.=10-50, the preference for R). R-2 and S-2 are competitive inhibitors of acetylcholinesterase with Ki=26 and 80 microM, respectively (the preference for R) but are active site-directed irreversible inhibitors of cholesterol esterase with ki=4 and 16 M-1 sec-1, respectively (the preference for S). For those competitive inhibitions, both leaving group hydrophilic and hydrophobic binding sites of cholesterol esterase or both anionic substrate binding site and peripheral anionic binding site of acetylcholinesterase bind to N,N-methyl-alpha-methylbenzyl disubstituted amide parts of these inhibitors and the enzyme does not catalyze the hydrolysis of these inhibitors. The opposite stereopreference (S) for the inhibition of cholesterol esterase by compounds 2 may be due to the fact that N, N-methyl-alpha-methylbenzyl disubstituted amide parts of these inhibitors bind to the alkyl chain binding site of the enzyme. Compounds 3-6 are active site-directed irreversible inhibitors of cholesterol esterase (ki=1-13000 M-1 s-1) and peripheral anionic binding site-directed irreversible inhibitors of acetylcholinesterase (ki=1.7-1300 M-1 s-1). Compounds 3 have low enantioselectivities (E=1.3-1.4) for both enzymes. The stereopreference for atropisomers 4 and 6 is S-form toward both enzymes (E=2-30) and is identical to that of cholesterol esterase-catalyzed hydrolysis of 1,1'-bi-2-naphthyl-2,2'-diacylate. This stereopreference (S) may be due to the fact that the butyryl group or one of two butylcarbamate groups of S-atropisomers binds more effectively to the leaving group hydrophobic 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 favorable interaction between the hydroxyl group of the inhibitors and the leaving group hydrophilic binding site of cholesterol esterase or the peripheral anionic binding site of acetylcholinesterase.
N-甲基-N,α-甲基苄基丁酰胺（1）、1-丁基-3-甲基-3'-α-甲基苄基脲（2）、1,2,3,4-四氢-1-萘基-N-丁基氨基甲酸酯（3）、1,1'-联-2-萘基-2,2'-二-N-丁基氨基甲酸酯（4）、1,1'-联-2-萘基-2-醇-2'-N-丁基氨基甲酸酯（5）和1,1'-联-2-萘基-2-丁酸酯-2'-N-丁基氨基甲酸酯（6）的对映体在5,5'-二硫代双-2-硝基苯甲酸存在下，是猪胰胆固醇酯酶催化4-硝基苯基丁酸水解以及电鳗乙酰胆碱酯酶催化乙酰硫代胆碱水解的抑制剂。对于竞争性抑制剂，研究了抑制常数（Ki）和对映体比例（Ecomp.）的值。对于活性部位定向不可逆抑制剂，研究了抑制常数（Ki）、氨甲酰化常数（k2）、双分子速率常数（ki）和对映体比例（E）的值。对于这两种酶，化合物1是较差的竞争性抑制剂（Ki = 102 - 104 μM），但具有良好的对映选择性（Ecomp. = 10 - 50，对R的偏好）。R-2和S-2是乙酰胆碱酯酶的竞争性抑制剂，Ki分别为26和80 μM（对R的偏好），但它们是胆固醇酯酶的活性部位定向不可逆抑制剂，ki分别为4和16 M-1 s-1（对S的偏好）。对于那些竞争性抑制作用，胆固醇酯酶的离去基团亲水性和疏水性结合位点或乙酰胆碱酯酶的阴离子底物结合位点和外周阴离子结合位点都与这些抑制剂的N,N-甲基-α-甲基苄基二取代酰胺部分结合，并且酶不催化这些抑制剂的水解。化合物2对胆固醇酯酶抑制的相反立体偏好（S）可能是由于这些抑制剂的N,N-甲基-α-甲基苄基二取代酰胺部分与酶的烷基链结合位点结合。化合物3 - 6是胆固醇酯酶的活性部位定向不可逆抑制剂（ki = 1 - 13000 M-1 s-1）和乙酰胆碱酯酶的外周阴离子结合位点定向不可逆抑制剂（ki = 1.7 - 1300 M-1 s-1）。化合物3对这两种酶的对映选择性都较低（E = 1.3 - 1.4）。对映体4和6对这两种酶的立体偏好都是S型（E = 2 - 30），并且与胆固醇酯酶催化1,1'-联-2-萘基-2,2'-二酰化物水解的情况相同。这种立体偏好（S）可能是由于S-对映体的丁酰基或两个丁基氨基甲酸酯基团之一比R-对映体更有效地与胆固醇酯酶的离去基团疏水性结合位点或乙酰胆碱酯酶的外周阴离子结合位点结合。对映体5对这两种酶的相反立体偏好（R）可能是由于抑制剂的羟基与胆固醇酯酶的离去基团亲水性结合位点或乙酰胆碱酯酶的外周阴离子结合位点之间的有利相互作用。
