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Notwithstanding the combination of cholinesterase (ChE) inhibition and calcium channel blockade within a multitarget therapeutic approach is envisaged as potentially beneficial to confront Alzheimer's disease (AD), this strategy has been scarcely investigated. To explore this promising line, a series of 5- amino-4-aryl-3,4,6,7,8,9-hexahydropyrimido [4,5-b]quinoline-2(1H)-thiones (tacripyrimidines) (4a-l) were designed by juxtaposition of tacrine, a ChE inhibitor (ChEI), and 3,4-dihydropyrimidin-2(1H)-thiones, as efficient calcium channel blockers (CCBs). In agreement with their design, all tacripyrimidines, except the unsubstituted parent compound and its p-methoxy derivative, acted as moderate to potent CCBs with activities generally similar or higher than the reference CCB drug nimodipine and were modest-to-good ChEIs. Most interestingly, the 30-methoxy derivative (4e) emerged as the first well balanced ChEI/CCB agent, acting as low micromolar hChEI (3.05 mM and 3.19 mM on hAChE and hBuChE, respectively) and moderate CCB (30.4% at 1 mM) with no significant hepatotoxicity toward HepG2 cells and good predicted oral absorption and blood brain barrier permeability, EB and FP thank Erasmus for support. JMC thanks MINECO (Government of Spain) for grants SAF2012-33304 and CTQ-68380- R. JMC and MLB thank EU (COST Action 15135). MB and MLB gratefully acknowledges the University of Bologna and the Italian Ministry of Education, Universities and Research (MIUR)

The synthesis and pharmacological analyses of a number of pyrazolo[3,4-b]quinoline and benzo[b]pyrazolo[4,3-g][1,8]naphthyridine derivatives are reported. We have synthesized the diversely substituted tacrine analogues 1-6, by Friedländer-type reaction of readily available o-amino-1-methyl-pyrazole-dicarbonitriles with cyclohexanone. The biological evaluation showed that pyrazolotacrines 1-6 are inhibitors of Electrophorus electricus acetylcholinesterase (EeAChE), in the micromolar range, and quite selective in respect to serum horse butyrylcholinesterase (eqBuChE) inhibition; the most interesting inhibitor is N-(5-amino-1-methyl-6,7,8,9-tetrahydro-1H- benzo[b]pyrazolo[4,3-g][1,8]naphthyridin-3-yl)acetamide (5) [IC50 (EeAChE) = 0.069 ± 0.006 μM; IC50 (eqBuChE) = 6.3 ± 0.6 μM]. Kinetic studies showed that compound 5 is a mixed-type inhibitor of EeAChE (Ki = 155 nM). Inhibitor 5 showed a 45% neuroprotection value against rotenone/oligomycin A-induced neuronal death. © 2011 Elsevier Masson SAS. All rights reserved., We thank V. Gómez and L. González-Lafuente for technical support. D. Silva thanks Fundação para a Ciência e Tecnologia do Ministério da Ciência, Tecnologia e Ensino Superior of Portugal for his grant belonging to project PTDC/SAU-NEU/64151/2006. M. Chioua thanks Instituto de Salud Carlos III (MICINN) for a “Sara Borrell” post-doctoral contract. JMC thanks MICINN (SAF2006-08764-C02-01; SAF2009-07271) financial support. MGL thanks MICINN Ref. SAF2009-12150. DS thanks COST Action D34. This work was partly supported by grants RD06/0026/1002 (RENEVAS), ISCIII, MICINN, S/SAL-0275-2006, Comunidad de Madrid, and Fundación CIEN, project 1004040042. JMC thanks Prof. A. García for his continuous interest and support

The synthesis, biological assessment and molecular modeling of new pyridonepezils 1–8, able to inhibit human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE), are described. The new compounds have been designed as hybrids resulting from a conjunctive approach that combines the N-benzylpiperidine moiety, present in donepezil, and the 2-amino-6-chloropyridine heterocyclic ring system, connected by an appropriate polymethylene linker. Compounds 1–8 were prepared by reaction of 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2-amino-6-chloropyridine-3,5-dicarbonitrile (14)] with 2-(1-benzylpiperidin-4-yl)alkylamines (9–12). The biological evaluation of molecules 1–8 showed that compounds 1–6 are potent AChE inhibitors, in the submicromolar, while compounds 7 and 8 are on the nanomolar range, the most potent, 2-amino-6-((3-(1-benzylpiperidin-4-yl)propyl)amino)pyridine-3,5-dicarbonitrile (7), showing a IC50 (hAChE) = 9.4 ± 0.4 nM. Inhibitors 2–8 are permeable as determined in the PAMPA assay. Compared to donepezil, compound 7 is in the same range of inhibitory activity for hAChE, and 703-fold more selective for hAChE than for hBuChE. Molecular modeling investigation on pyridonepezil 7 supports its dual AChE inhibitory profile, binding simultaneously at the catalytic active and at peripheral anionic sites of the enzyme. The theoretical ADME analysis of pyridonepezils 1–8 has been carried out. Overall, compound 7, a potent and selective dual AChEI, can be considered as a candidate with potential impact for further pharmacological development in Alzheimer's therapy.

The synthesis, pharmacological analysis and molecular modeling of the readily available racemic tacrine analogs 21-30, bearing the 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine heterocyclic ring system (II), prepared by Friedlander reaction of 2-amino-4-aryl-4H-benzo[h]chromene-3-carbonitriles (11-20) with cyclohexanone, are described in this paper. Molecules 21-30 are potent and selective inhibitors of hAChE, in the low micromolar range, one of the most potent inhibitors, 4-(8-amino-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-7-yl)-2-methoxyphenol (25), showing a IC(50) (hAChE) = 0.33 ± 0.04 μM. Kinetic studies of compound 25 proved that this compound is a mixed type inhibitor for EeAChE (K(i) = 81 nM). Accordingly, molecular modeling of inhibitor 25 showed that both enantiomers have two major predicted binding modes at the active and at the peripheral anionic sites of AChE. Inhibitor 25 has an excellent antioxidant profile as determined in the ORAC experiment (1.47 ± 0.10 Trolox equiv). Inhibitors 26-28 and 30 are permeable to BBB as determined in the PAMPA assay. Compared to tacrine, selected compounds 26-28 and 30 showed less hepatic toxicity in HepG2 cells. Moreover, cell viability-related studies in cortical neurons in primary cultures show that compounds 26-28 and 30 (0.1-50 μM) have significant neuroprotective effects against mitochondrial chain blockers-induced cell death, and, unlike tacrine, are not neurotoxic at concentrations lower than 50 μM. It is worth highlighting that compound 27 has the best neuroprotective properties out of all assayed compounds and shows no neurotoxicity. To sum up, these tacrine analogs can be considered as attractive multipotent therapeutic molecules on pharmacological receptors playing key roles in the progress of Alzheimer's disease.

The synthesis, pharmacological evaluation and molecular modeling of heterocyclic substituted alkyl and cycloalkyl propargyl amines 1 – 7 of type I , and 9 – 12 of type II , designed as multipotent inhibitors able to simultaneously inhibit monoamine oxidases (MAO-A/B) as well as cholinesterase (AChE/BuChE) enzymes, as potential drugs for the treatment of Alzheimer's disease, are described. Indole derivatives 1 – 7 of type I are well known MAO inhibitors whose capacity to inhibit AChE and BuChE was here investigated for the first time. As a result, compound 7 was identified as a MAO-B inhibitor (IC 50 = 31 ± 2 nM) and a moderately selective eqBuChE inhibitor (IC 50 = 4.7 ± 0.2 μM). Conversely, the new and readily available 5-amino-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydropyrido[2,3- b ][1,6]naphthyridine derivatives 9 – 13 of type II are poor MAO inhibitors, but showed AChE selective inhibition, compound 12 being the most attractive as it acts as a non-competitive inhibitor on Ee AChE (IC 50 = 25 ± 3 nM, K i = 65 nM). The ability of this compound to interact with the AChE peripheral binding site was confirmed by kinetic studies and by molecular modeling investigation. Studies on human ChEs confirmed that 12 is a selective AChE inhibitor with inhibitory potency in the submicromolar range. Moreover, in agreement with its mode of action, 12 was shown to be able to inhibit A β aggregation induced by hAChE by 30.6%.

The synthesis, biological evaluation and molecular modeling of new multipotent inhibitors of type I and type II , able to simultaneously inhibit monoamine oxidases (MAO) as well as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), is described. Compounds of type I were prepared by sequential reaction of 2,6-dichloro-4-phenylpyridine-3,5-dicarbonitrile ( 14 ) [or 2,6-dichloropyridine-3,5-dicarbonitrile ( 15 )] with prop-2-yn-1-amine (or N -methylprop-2-yn-1-amine) and 2-(1-benzyl-piperidin-4-yl)alkylamines 22–25 . Compounds of type II were prepared by Friedlander type reaction of 6-amino-5-formyl-2-(methyl(prop-2-yn-1-yl)amino)nicotinonitriles 32 and 33 with 4-(1-benzylpiperidin-4-yl)butan-2-one ( 31 ). The biological evaluation of molecules 1–11 showed that most of these compounds are potent, in the nanomolar range, and selective AChEI, with moderate and equipotent selectivity for MAO-A and MAO-B inhibition. Kinetic studies of compound 8 proved that this is a Ee AChE mixed type inhibitor (IC 50 = 16 ± 2; Ki = 12 ± 3 nM). Molecular modeling investigation on compound 8 confirmed its dual AChE inhibitory profile, binding simultaneously at the catalytic active site (CAS) and at the peripheric anionic site (PAS). In overall, compound 11 , as a potent and selective dual AChEI, showing a moderate and selective MAO-A inhibitory profile, can be considered as an attractive multipotent drug for further development on two key pharmacological targets playing key roles in the therapy of Alzheimer’s disease.

The synthesis and pharmacological analyses of a number of furo[2,3-b]quinolin-4-amine, and pyrrolo[2,3-b]quinolin-4-amine derivatives are reported. Thus, we synthesized diversely substituted tacrine analogues 1-11 and 12-16 by Friedlander-type reaction of readily available o-amino(furano/pyrrolo)nitriles with suitable and selected cycloalkanones. The biological evaluation of furanotacrines1-11 and pyrrolotacrine13 showed that these are good, in the micromolar range, and highly selective inhibitors of BuChE. In the furanotacrine group, the most interesting inhibitor was 2-(p-tolyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-amine (3) [IC(50) (eqBuChE)=2.9 ± 0.4 μM; IC(50) (hBuChE)=119 ± 15 μM]. Conversely, pyrrolotacrines 12 and 14 proved moderately equipotent for both cholinesterases, being 1,2-diphenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]quinolin-4-amine (12) the most potent for the inhibition of both enzymes [IC(50) (EeAChE)=0.61 ± 0.04 μM; IC(50) (eqBuChE)=0.074 ± 0.009 μM]. Moreover, pyrrolotacrine 12, at concentrations as low as 300 nM can afford significant neuroprotective effects against Aβ-induced toxicity. Docking studies show that compounds 3 and 12 bind in the middle of the AChE active site gorge, but are buried deeper inside BuChE active site gorge, as a consequence of larger BuChE gorge void. All these data suggest that these new tacrine analogues could be used for the potential treatment of Alzheimer's disease.