DOI: 10.1002/cmdc.200800396 Structure–Activity Relationships and Binding Mode in the Human Acetylcholinesterase Active Site of Pseudo- Irreversible Inhibitors Related to Xanthostigmine Stefano Rizzo, [a] Andrea Cavalli,* [a, b] Luisa Ceccarini, [a] Manuela Bartolini, [a] Federica Belluti, [a] Alessandra Bisi, [a] Vincenza Andrisano, [a] Maurizio Recanatini, [a] and Angela Rampa* [a] Introduction Alzheimer’s disease (AD), first described by Alois Alzheimer in 1907, [1] has become the most common cause of dementia in the elderly, affecting ~ 3 % of the population between the age of 65 to 74, and nearly 50% of those 85 years and older. [2] The course of the devastating disease is marked by a progressive loss of brain functions such as memory and language skills, and life expectancy after diagnosis is ~ 8–10 years. AD pathology is characterized by the production of extracel- lular senile plaques, mostly composed of aggregated b-amy- loid peptide (Ab), [3] in different areas of the brain, and intracel- lular neurofibrillary tangles, containing hyperphosphorylated Tau protein. [4] During the past decade, treatment of patients with AD has been largely based on a strategy of enhancing acetylcholine (ACh)-mediated transmission. Symptomatic cognitive, function- al and behavioral benefits [5, 6] have been observed in multiple clinical trials with agents known to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), both of which cata- lyze the breakdown of ACh. There is no doubt that significant dysfunction of the basal forebrain ACh system occurs in ad- vanced AD, and therefore the development of cholinesterase (ChE) inhibitors was based on the well-accepted cholinergic hypothesis. [7] As a result, various ChE inhibitors, such as tacrine, donepezil, rivastigmine and galanthamine, have been launched onto the market. Our research involves the development of AChE inhibitors as potential drugs for AD. Previously, we designed and studied a class of inhibitors (N-methyl-N-(3-carbamoyloxyphenyl)methyl- ACHTUNGTRENNUNGamino) characterized by the presence of a heteroaryl moiety linked to the tertiary amino nitrogen through an alkoxy chain. Xanthostigmine (1), the lead compound, showed the highest activity (IC 50 = 0.3 nm). [8, 9] A three-dimensional model of the quaternary complex between AChE and compound 1 showed that the carbamate moiety is located in the active site of the enzyme for interaction with the catalytic triad, while the xan- thone moiety points toward the opening of the gorge, caged within a framework of aromatic residues, but not within reach of the Trp279 residue (Torpedo californica AChE numbering), located at the entrance of the gorge. This aromatic residue, to- gether with others, is considered to constitute an essential part of the AChE peripheral anionic site (PAS), [10–12] and recent studies have shown that inhibitors binding to this site are able to block the Ab aggregation induced by AChE. [13–15] In previous papers, we introduced modifications in the linker chain, the carbamoyl substituent and the aryl moiety of com- pound 1 in order to investigate the structure–activity relation- Structure–activity relationship studies on acetylcholinesterase (AChE) inhibitors were extended to newly synthesized com- pounds derived from the lead compound xantostigmine (1). The xanthone ring of compound 1 was replaced with several different scaffolds based on the benzopyran skeleton, linked to the tertiary amino nitrogen through an heptyloxy chain. These modifications resulted in 19 new compounds, most of them showing activity in the nanomolar–subnanomolar range. Dock- ing and molecular dynamics simulations were carried out to both define a new computational protocol for the simulation of pseudo-irreversibile AChE covalent inhibitors, and to acquire a better understanding of the structure–activity relationships of the present series of compounds. The results of this compu- tational work prompted us to to evaluate the ability of com- pounds 5 and 13 to inhibit acetylACHTUNGTRENNUNGcholinesterase-induced Ab aggregation. [a] S. Rizzo, A. Cavalli, L. Ceccarini, M. Bartolini, F. Belluti, A. Bisi, V. Andrisano, M. Recanatini, A. Rampa Department of Pharmaceutical Sciences, University of Bologna Via Belmeloro 6, 40126 Bologna (Italy) Fax: (+ 39) 051-2099734 E-mail : angela.rampa@unibo.it [b] A. Cavalli Unit of Drug Discovery and Development, Italian Institute of Technology Via Morego 30, 16163 Genova (Italy) Fax: (+ 39) 051-2099734 E-mail : andrea.cavalli@unibo.it Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.200800396. 670  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2009, 4, 670 – 679 MED