DOI: 10.1002/cmdc.201000086 Bis(7)-tacrine Derivatives as Multitarget-Directed Ligands: Focus on Anticholinesterase and Antiamyloid Activities Maria Laura Bolognesi,* Manuela Bartolini, Francesca Mancini, Gianpaolo Chiriano, Luisa Ceccarini, Michela Rosini, Andrea Milelli, Vincenzo Tumiatti, Vincenza Andrisano, and Carlo Melchiorre [a] Dedicated to Prof. Pelayo Camps on the occasion of his 65 th birthday and in recognition of his research on cholinesterase inhibitors. A vast array of drug discovery research focuses on the devel- opment of innovative drugs targeting Alzheimer’s disease (AD) and related dementias. [1] However, despite several lead candi- dates progressing into AD pre-clinical testing in the last decade, to date none of these agents have been successful in late-stages clinical trials. Beta-secretase (BACE-1) inhibition has been a strategy actively pursued, however, progress in moving inhibitors to the clinic has been slow, partly as a consequence of its aspartic proteinase character, which hampers the devel- opment of potent, selective and brain-permeable com- pounds. [2] Therefore, it was of great interest to know that a nonpeptidic inhibitor CTS-21166 entered the clinic. [3] At pres- ent, however, AD pharmacotherapy mainly relies on acetylcho- linesterase (AChE) inhibitors (AChEIs): donepezil, rivastigmine and galantamine. [4, 5] Although their effectiveness in delaying disease progression is still a matter of debate, recent clinical date provides evidence of their disease-modifying role. [6] With this in mind, we and others envisaged the development of multitarget-directed ligands (MTDLs) as an innovative and ef- fective strategy to face the multifactorial etiopathogenesis of AD. [7–9] This strategy, embodied by single chemical entities able to simultaneously modulate multiple targets involved in the neurodegenerative cascade, has proven particularly fruitful in recent years and has led to the discovery of several promising anti-AD drug candidates. [10, 11] Among them, bis(7)-tacrine (also called bis(7)cognitin; 1), the heptylene-linked analogue of tacrine, is a rationally de- signed dual binding AChEI, [12] which is 1000-times more potent against rat brain AChE than tacrine. The multimodal mecha- nism-of-action of compound 1 against AD has retrospectively been disclosed. [13] This includes anti-NMDA receptors and anti- nitric oxide synthase (NOS) signaling, the regulation of the downstream signal of NMDA receptors, and the reduction of Ab neurotoxicity. [13] Furthermore, compound 1 was shown to inhibit both BACE-1 activity in cultured cells and the isolated recombinant enzyme in a noncompetitive manner, with an IC 50 value of 7.5 mm. [14] In 2007, we argued that by binding to the allosteric peripheral anionic site (PAS) of AChE, compound 1 might prevent AChE-induced Ab aggregation. [15] The report that AChE facilitates amyloid fibril assembly by forming stable complexes with Ab at the PAS region [16, 17] renewed the interest in dual binding AChEIs as potential modulators of amyloid neuropathology. [18, 19, 6, 20] Indeed, we demonstrated that com- pound 1 inhibits AChE-induced Ab aggregation with an IC 50 value of 41.7 mm, only slightly higher than that of propidium (12.6 mm), one of the most potent reference compounds com- monly used in this assay. [15] Altogether, these scientific findings make 1 a valuable drug candidate for the treatment of AD and an interesting lead compound for the design of novel MTDLs. [13] Here, we focus on three activities that we consider important for therapeutic benefit: cholinesterase inhibition, amyloid formation and amy- loid aggregation. With this concept in mind, we decided to modify the structure of 1 by replacing the heptamethylene chain with more rigid aromatic spacers, such as in compounds 2–4 (Figure 1). In principle, this modification might have posi- tive consequences on the profile of the new hybrids: 1) a tight- er binding at AChE through possible additional p–p interac- tions with several aromatic residues present in the enzyme gorge; 2) a planar aromatic surface available for protein–pro- tein interactions ; [21] 3) a higher molecular hindrance to better address the extended substrate binding site requirements of BACE-1. [22] On the basis of this rationale, derivatives 2–4 should possess a better inhibition profile against AChE and, more im- portantly, against amyloid formation and aggregation. Dimers 2–4 were synthesized by exploiting an efficient pro- tocol developed for a similar series of compounds, [15] which relies on a bis-amination reaction of the 9-chlorotetrahydroacri- dine 5 by diamines 6–8 in phenol (Scheme 1). Figure 1. Chemical structures of 1 and its derivatives 2–4. [a] Prof. Dr. M. L. Bolognesi, Prof. Dr. M. Bartolini, Dr. F. Mancini, G. Chiriano, L. Ceccarini, Prof. Dr. M. Rosini, Dr. A. Milelli, Prof. V. Tumiatti, Prof. Dr. V. Andrisano, Prof. Dr. C. Melchiorre Dipartimento di Scienze Farmaceutiche, Università di Bologna Via Belmeloro 6, 40126 Bologna (Italy) Fax: (+ 39) 051-2099734 E-mail : marialaura.bolognesi@unibo.it ChemMedChem 2010, 5, 1215 – 1220  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1215