Bolognesi Maria Laura, Bartolini Manuela, Cavalli Andrea, Andrisano Vincenza, Rosini Michela, Minarini Anna, Melchiorre Carlo
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
J Med Chem. 2004 Nov 18;47(24):5945-52. doi: 10.1021/jm049782n.
Rivastigmine (1), an acetylcholinesterase (AChE) inhibitor approved in 2000 for the treatment of Alzheimer disease, bears a carbamate moiety in its structure, which is able to react covalently with the active site of the enzyme. Kinetic and structural studies on the interaction of 1 with different cholinesterases have been published, giving deeper, but not definitive, insights on the catalysis mechanism. On the basis of these findings and in connection with our previous studies on a series of benzopyrano[4,3-b]pyrrole carbamates as AChE inhibitors, we designed a series of conformationally restricted analogues of 1 by including the dimethylamino-alpha-methylbenzyl moiety in different tricyclic systems. A superimposition between the conformation of 1 and the carbon derivative 4, as obtained from Monte Carlo simulations, supported the idea that the tricyclic derivatives might act as rigid analogues of 1. The biological profile of 4-9, assessed in vitro against human AChE and BChE, validated our rational design. Compound 5, bearing a sulfur-containing system, showed the highest inhibitory activity, being 192-fold more potent than 1. In the present study, the most potent inhibitors were always methyl derivatives 3-5, endowed with a nanomolar range potency, whereas the ethyl ones were 40 times less potent. A reasonable explanation for this finding might be a steric hindrance effect between the ethyl group of 1 and His440 in the active site, as already suggested by the crystal structure of the complex AChE/1. The unfavorable influence of the carbamic N-alkyl chain on AChE inhibition is less striking when considering BChE inhibition, since BChE is characterized by a bigger acyl binding pocket than AChE. In fact, methyl carbamates 3-5 did not show AChE/BChE selectivity, whereas compounds 6-9 were significantly more potent in inhibiting BChE than AChE activity. At 100 microM, 5 was found to inhibit the AChE-induced aggregation only by 19% likely because it is not able to strongly interact with the peripheral anionic site of AChE, which plays an essential role in the Abeta aggregation mediated by the enzyme but is lacking in BChE structure.
卡巴拉汀（1）是一种2000年被批准用于治疗阿尔茨海默病的乙酰胆碱酯酶（AChE）抑制剂，其结构中含有一个氨基甲酸酯部分，该部分能够与酶的活性位点发生共价反应。关于1与不同胆碱酯酶相互作用的动力学和结构研究已经发表，对催化机制有了更深入但并非决定性的见解。基于这些发现，并结合我们之前对一系列苯并吡喃并[4,3 - b]吡咯氨基甲酸酯作为AChE抑制剂的研究，我们通过在不同的三环体系中引入二甲基氨基-α-甲基苄基部分，设计了一系列1的构象受限类似物。从蒙特卡罗模拟获得的1和碳衍生物4的构象之间的叠加，支持了三环衍生物可能作为1的刚性类似物的观点。在体外针对人AChE和BChE评估的4 - 9的生物学特性，验证了我们的合理设计。带有含硫体系的化合物5显示出最高的抑制活性，其效力比1高192倍。在本研究中，最有效的抑制剂总是甲基衍生物3 - 5，具有纳摩尔级别的效力，而乙基衍生物的效力则低40倍。对此发现的一个合理解释可能是1的乙基与活性位点中的His440之间的空间位阻效应，正如AChE/1复合物的晶体结构所暗示的那样。当考虑对BChE的抑制时，氨基甲酸N - 烷基链对AChE抑制的不利影响不太明显，因为BChE的特征是其酰基结合口袋比AChE大。事实上，甲基氨基甲酸酯3 - 5没有显示出AChE/BChE选择性，而化合物6 - 9在抑制BChE活性方面比AChE活性显著更强。在100微摩尔浓度下，发现5仅能抑制AChE诱导的聚集19%，这可能是因为它无法与AChE的外周阴离子位点强烈相互作用，该位点在由该酶介导的Aβ聚集过程中起重要作用，但在BChE结构中不存在。
