M. Victòria Clos

Pyrano(3,2-c)quinoline-6-Chlorotacrine Hybrids as a Novel Family of Acetylcholinesterase- and β-Amyloid-Directed Anti-Alzheimer Compounds

Two isomeric series of dual binding site acetylcholinesterase (AChE) inhibitors have been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase, AChE-induced and self-induced beta-amyloid (Abeta) aggregation, and beta-secretase (BACE-1) and to cross blood-brain barrier. The new hybrids consist of a unit of 6-chlorotacrine and a multicomponent reaction-derived pyrano[3,2-c]quinoline scaffold as the active-site and peripheral-site interacting moieties, respectively, connected through an oligomethylene linker containing an amido group at variable position. Indeed, molecular modeling and kinetic studies have confirmed the dual site binding of these compounds. The new hybrids, and particularly 27, retain the potent and selective human AChE inhibitory activity of the parent 6-chlorotacrine while exhibiting a significant in vitro inhibitory activity toward the AChE-induced and self-induced Abeta aggregation and toward BACE-1, as well as ability to enter the central nervous system, which makes them promising anti-Alzheimer lead compounds.

Synthesis and multitarget biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents.

We have synthesized a family of rhein-huprine hybrids to hit several key targets for Alzheimers disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase, butyrylcholinesterase, and BACE-1, dual Aβ42 and tau antiaggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and (-)-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction, preventing the loss of synaptic proteins and/or have a positive effect on the induction of long-term potentiation. In vivo studies in APP-PS1 transgenic mice treated ip for 4 weeks with (+)- and (-)-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and (-)-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Huprine–Tacrine Heterodimers as Anti-Amyloidogenic Compounds of Potential Interest against Alzheimer’s and Prion Diseases

A family of huprine-tacrine heterodimers has been developed to simultaneously block the active and peripheral sites of acetylcholinesterase (AChE). Their dual site binding for AChE, supported by kinetic and molecular modeling studies, results in a highly potent inhibition of the catalytic activity of human AChE and, more importantly, in the in vitro neutralization of the pathological chaperoning effect of AChE toward the aggregation of both the β-amyloid peptide (Aβ) and a prion peptide with a key role in the aggregation of the prion protein. Huprine-tacrine heterodimers take on added value in that they display a potent in vitro inhibitory activity toward human butyrylcholinesterase, self-induced Aβ aggregation, and β-secretase. Finally, they are able to cross the blood-brain barrier, as predicted in an artificial membrane model assay and demonstrated in ex vivo experiments with OF1 mice, reaching their multiple biological targets in the central nervous system. Overall, these compounds are promising lead compounds for the treatment of Alzheimers and prion diseases.

Tacrine-based dual binding site acetylcholinesterase inhibitors as potential disease-modifying anti-Alzheimer drug candidates.

Two novel families of dual binding site acetylcholinesterase (AChE) inhibitors have been developed, consisting of a tacrine or 6-chlorotacrine unit as the active site interacting moiety, either the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone fragment of donepezil (or the indane derivative thereof) or a 5-phenylpyrano[3,2-c]quinoline system, reminiscent to the tryciclic core of propidium, as the peripheral site interacting unit, and a linker of suitable length as to allow the simultaneous binding at both sites. These hybrid compounds are all potent and selective inhibitors of human AChE, and more interestingly, are able to interfere in vitro both formation and aggregation of the beta-amyloid peptide, the latter effects endowing these compounds with the potential to modify Alzheimers disease progression.

Novel huprine derivatives with inhibitory activity toward β-amyloid aggregation and formation as disease-modifying anti-Alzheimer drug candidates.

A new family of dual binding site acetylcholinesterase (AChE) inhibitors has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), AChE‐induced and self‐induced β‐amyloid (Aβ) aggregation and β‐secretase (BACE‐1), and to cross the blood–brain barrier. The new heterodimers consist of a unit of racemic or enantiopure huprine Y or X and a donepezil‐related 5,6‐dimethoxy‐2‐[(4‐piperidinyl)methyl]indane moiety as the active site and peripheral site to mid‐gorge‐interacting moieties, respectively, connected through a short oligomethylene linker. Molecular dynamics simulations and kinetics studies support the dual site binding to AChE. The new heterodimers are potent inhibitors of human AChE and moderately potent inhibitors of human BChE, AChE‐induced and self‐induced Aβ aggregation, and BACE‐1, and are predicted to be able to enter the central nervous system (CNS), thus constituting promising multitarget anti‐Alzheimer drug candidates with the potential to modify the natural course of this disease.

Effect of Huprine X on β-Amyloid, Synaptophysin and α7 Neuronal Nicotinic Acetylcholine Receptors in the Brain of 3xTg-AD and APPswe Transgenic Mice

Background: Several studies implicate acetylcholinesterase (AChE) in the pathogenesis of Alzheimer’s disease (AD), raising the question of whether inhibitors of AChE also might act in a disease-modifying manner. Huprine X (HX), a reversible AChE inhibitor hybrid of tacrine and huperzine A, has shown to affect the amyloidogenic process in vitro. In this study, the aim was to investigate whether HX could affect the AD-related neuropathology in vivoin two mouse models. Methods:Tg2576 (K670M/N671L) (APPswe) and 3xTg-AD (K670M/N671L, PS1M146V, tauP301L) mice were treated with HX (0.12 µmol/kg, i.p., 21 days) or saline at 6–7 months. Human β-amyloid (Aβ) was measured by ELISA, synaptophysin by Western blot and α7 neuronal nicotinic acetylcholine receptors (nAChRs) were analyzed by [125I]α-bungarotoxin autoradiography. Results: Treatment with HX reduced insoluble Aβ1–40 (about 40%) in the hippocampus of 3xTg-AD mice, while showing no effect in APPswe mice. Additionally, HX markedly increased cortical synaptophysin levels (about 140%) and decreased (about 30%) the levels of α7 nAChRs in the caudate nucleus of 3xTg-AD mice, while increasing (about 10%) hippocampal α7 nAChRs in APPswe mice. Conclusion: The two mouse models react differently to HX treatment, possibly due to their differences in brain neuropathology. The modulation of Aβ and synaptophysin by HX in 3xTg-AD mice might be due to its suggested interaction with the peripheral anionic site on AChE, and/or via cholinergic mechanisms involving activation of cholinergic receptors. Our results provide further evidence that drugs targeting AChE affect some of the fundamental processes that contribute to neurodegeneration, but whether HX might act in a disease-modifying manner in AD patients remains to be proven.

Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting β-amyloid, tau, and cholinesterase pathologies.

Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5b-d have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5a-d has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC50 values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5a-d, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities.

Novel Levetiracetam Derivatives That Are Effective against the Alzheimer-like Phenotype in Mice: Synthesis, in Vitro, ex Vivo, and in Vivo Efficacy Studies.

We have synthesized a series of heptamethylene-linked levetiracetam-huprine and levetiracetam-(6-chloro)tacrine hybrids to hit amyloid, tau, and cholinergic pathologies as well as β-amyloid (Aβ)-induced epileptiform activity, some of the mechanisms that eventually lead to cognitive deficits in Alzheimers disease patients. These hybrids are potent inhibitors of human acetylcholinesterase and butyrylcholinesterase in vitro and moderately potent Aβ42 and tau antiaggregating agents in a simple E. coli model of amyloid aggregation. Ex vivo determination of the brain acetylcholinesterase inhibitory activity of these compounds after intraperitoneal injection to C57BL6J mice has demonstrated their ability to enter the brain. The levetiracetam-huprine hybrid 10 significantly reduced the incidence of epileptic seizures, cortical amyloid burden, and neuroinflammation in APP/PS1 mice after a 4-week treatment with a 5 mg/kg dose. Moreover, the hybrid 10 rescued transgenic mice from cognitive deficits, thereby emerging as an interesting disease-modifying anti-Alzheimer drug candidate.

Multicomponent reaction-based synthesis and biological evaluation of tricyclic heterofused quinolines with multi-trypanosomatid activity

Human African trypanosomiasis (HAT), Chagas disease and leishmaniasis, which are caused by the trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania species, are among the most deadly neglected tropical diseases. The development of drugs that are active against several trypanosomatids is appealing from a clinical and economic viewpoint, and seems feasible, as these parasites share metabolic pathways and hence might be treatable by common drugs. From benzonapthyridine 1, an inhibitor of acetylcholinesterase (AChE) for which we have found a remarkable trypanocidal activity, we have designed and synthesized novel benzo[h][1,6]naphthyridines, pyrrolo[3,2-c]quinolines, azepino[3,2-c]quinolines, and pyrano[3,2-c]quinolines through 2–4-step sequences featuring an initial multicomponent Povarov reaction as the key step. To assess the therapeutic potential of the novel compounds, we have evaluated their in vitro activity against T. brucei, T. cruzi, and Leishmania infantum, as well as their brain permeability, which is of specific interest for the treatment of late-stage HAT. To assess their potential toxicity, we have determined their cytotoxicity against rat myoblast L6 cells and their AChE inhibitory activity. Several tricyclic heterofused quinoline derivatives were found to display an interesting multi-trypanosomatid profile, with one-digit micromolar potencies against two of these parasites and two-digit micromolar potency against the other. Pyranoquinoline 39, which displays IC50 values of 1.5 μM, 6.1 μM and 29.2 μM against T. brucei, L. infantum and T. cruzi, respectively, brain permeability, better drug-like properties (lower lipophilicity and molecular weight and higher CNS MPO desirability score) than hit 1, and the lowest AChE inhibitory activity of the series (IC50 > 30 μM), emerges as an interesting multi-trypanosomatid lead, amenable to further optimization particularly in terms of its selectivity index over mammalian cells.

Shogaol-huprine hybrids: dual antioxidant and anticholinesterase agents with β-amyloid and tau anti-aggregating properties.

Multitarget compounds are increasingly being pursued for the effective treatment of complex diseases. Herein, we describe the design and synthesis of a novel class of shogaol-huprine hybrids, purported to hit several key targets involved in Alzheimers disease. The hybrids have been tested in vitro for their inhibitory activity against human acetylcholinesterase and butyrylcholinesterase and antioxidant activity (ABTS.+, DPPH and Folin-Ciocalteu assays), and in intact Escherichia coli cells for their Aβ42 and tau anti-aggregating activity. Also, their brain penetration has been assessed (PAMPA-BBB assay). Even though the hybrids are not as potent AChE inhibitors or antioxidant agents as the parent huprine Y and [4]-shogaol, respectively, they still exhibit very potent anticholinesterase and antioxidant activities and are much more potent Aβ42 and tau anti-aggregating agents than the parent compounds. Overall, the shogaol-huprine hybrids emerge as interesting brain permeable multitarget anti-Alzheimer leads.