Novel Multipotent Tacrine-Dihydropyridine Hybrids with Improved Acetylcholinesterase Inhibitory and Neuroprotective Activities as Potential Drugs for the Treatment of Alzheimer’s Disease Jose ´ Marco-Contelles,* ,² Rafael Leo ´n, ²,‡ Cristo ´bal de los Rı ´os, ²,‡ Antonio Guglietta, § Jose ´ Terencio, § Manuela G. Lo ´pez, ‡ Antonio G. Garcı ´a, ‡,# and Mercedes Villarroya* ,‡ Laboratorio de Radicales Libres (IQOG, CSIC), C/Juan de la CierVa 3, 28006-Madrid, Spain, Instituto Teo ´ filo Hernando, Departamento de Farmacologı ´a y Terape ´ utica, Facultad de Medicina, UniVersidad Auto ´ noma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain, Ferrer Internacional S.A., Juan de Sada, 28-32, 08028 Barcelona, Spain, and SerVicio de Farmacologı ´a Clı ´nica, Hospital UniVersitario de la Princesa, C/Diego de Leo ´ n 62, 28006 Madrid, Spain ReceiVed September 1, 2006 Abstract: In this work we describe the synthesis and biological evaluation of the tacrine-1,4-dihydropyridine (DHP) hybrids (3-11). These multipotent molecules are the result of the juxtaposition of an acetylcholinesterase inhibitor (AChEI) such as tacrine (1) and a 1,4- DHP such as nimodipine (2). Compounds 3-11 are very selective and potent AChEIs and show an excellent neuprotective profile and a moderate Ca 2+ channel blockade effect. Consequently, these molecules are new potential drugs for the treatment of Alzheimer’s disease. Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by progressive memory loss, decline in language skills, and other cognitive impairments. Although the etiology of AD is not well-known, there are diverse factors such as amyloid- (A) deposits, τ-protein aggregation, oxidative stress, and low levels of acetylcholine (ACh) that are thought to play significant roles in the disease. 1 The cholinergic theory of AD suggests that the selective loss of cholinergic neurons in AD results in a deficit of ACh in specific regions of the brain that mediate learning and memory functions. 2 The primary approach for treating AD has therefore focused on increasing the levels of acetylcholine in the brain by using acetylcholinest- erase inhibitors (AChEI) such as tacrine, donepezil, galantamine, and rivastigmine. 3 On the other hand, it is well-known that Ca 2+ overload is the main factor initiating the processes leading to cell death. Several lines of evidence show that calcium dysfunc- tion, involved in the pathogeny of AD, 4 augments A formation 5a and τ hyperphosphorylation. 5b Ca 2+ entry through L channels causes calcium overload and mitochondrial disruption, which lead to the activation of the apoptotic cascade and cell death. 6 Hence, blocking the entrance of Ca 2+ through this specific subtype of Ca 2+ channel could be a good strategy to prevent cell death. The multitarget approach in drug design 7 for the treatment of AD includes novel tacrine-melatonin hybrids, 8a dual inhibi- tors of AChE and monoamine oxidase (MAO), 8b dual AChEI and serotonin transporters, 8c and potent cholinesterase inhibitors with antioxidant and neuroprotective properties. 8d Since 1,4-dihydropyridines (DHPs) selectively block L-type voltage-dependent Ca 2+ channels (VDCC), we considered the synthesis and pharmacological study of new multipotent hybrid molecules, based on an AChEI and a DHP such as tacrine (1) and nimodipine (2) (Chart 1), to be novel and of great interest. 9 Besides inhibition of AChE and blockade of VDCC, which could prevent Ca 2+ overload and subsequent cell death, we were also interested in compounds targeted to prevent oxidative stress. Recent research has demonstrated that oxidative damage is an event that precedes the appearance of other pathological hallmarks of AD. 10 Thus, drugs that scavenge oxygen radicals may have a particular therapeutic efficacy. 11,12 In this Letter we report our preliminary results on the synthesis and biological evaluation, including AChE/BuChE inhibition, propidium iodide displacement, Ca 2+ blockade, and neuroprotective activity, of novel tacrine-DHP hybrids (“tacripyrines”) (3-11) (Chart 1). From this study, we conclude that tacripyrines (3-11) are very selective and potent AChEIs, show excellent neuroprotective profiles and moderate Ca 2+ channel blockade effects, and consequently, can be considered as new potential drugs for further development, targeted to the treatment of Alzheimer’s disease. The synthesis of tacripyrines (3-11) was easily achieved, in excellent yields, by the Friedla ¨nder reaction 13 between the unknown ethyl esters of 6-amino-4-aryl-5-cyano-2-methyl-1,4- dihydropyridine-3-carboxylic acids (12-20) 14 and cyclohex- anone under standard conditions 15 (Scheme 1). Compounds 3-11 are racemic hexahydrobenzo[b][1,8]naphthyridines sub- stituted at C-4 by an aromatic ring incorporating different types of substituents. These molecules have been conveniently characterized by their analytical and spectroscopic data (see Supporting Information). The new tacripyrines were evaluated as inhibitors of AChE from electric eel (Electrophorus electricus) and of AChE from human serum, following the method of Rappaport, 16 and as inhibitors of BuChE from human serum, following the method of Ellman. 17 To allow comparisons of the results, tacrine (1) was used as the reference compound (Table 1). As shown, all tacripyrines are more potent inhibitors of AChE at the nanomolar * To whom correspondence should be addressed. For J.M.-C.: phone, 34-91-5622900; fax, 34-91-5644853; e-mail, iqoc21@iqog.csic.es. For M.V.: phone, 34-91 4975386; fax, 34-91 4975380; e-mail, mercedes.villarroya@uam.es. ² Laboratorio de Radicales Libres (IQOG, CSIC). ‡ Universidad Auto ´noma de Madrid. § Ferrer Internacional S.A. # Hospital Universitario de la Princesa. Chart 1. Selection of Multipotent Hybrid Molecules (3-11), Based on the Juxtaposition of an AChEI and a 1,4-DHP, Such as Tacrine (1) and Nimodipine (2) 7607 J. Med. Chem. 2006, 49, 7607-7610 10.1021/jm061047j CCC: $33.50 © 2006 American Chemical Society Published on Web 12/02/2006