Design, synthesis and preliminary biological evaluation of rivastigmine-INDY hybrids as multitarget ligands against Alzheimer's disease by targeting butyrylcholinesterase and DYRK1A/CLK1 kinases.

Based on a multitarget approach implementing rivastigmine-INDY hybrids 1, we identified a set of pseudo-irreversible carbamate-type inhibitors of eqBuChE that, after carbamate transfer at the active site serine residue, released the corresponding INDY analogues 2 endowed with hDYRK1A/hCLK1 kinases inhibitory properties. A SAR study and molecular docking investigation of both series of compounds 1 and 2 revealed that appropriate structural modifications at the carbamate moiety and at the N-appendage of the benzothiazole core led to potent and selective eqBuChE inhibitors with IC50 up to 27 nM and potent hDYRK1A and hCLK1 inhibitors with IC50 up to 106 nM and 17 nM respectively. Pleasingly, identification of the matched pair of compounds 1b/2b with a good balance between inhibition of eqBuChE and hDYRK1A/hCLK1 kinases (IC50 = 68 nM and IC50 = 529/54 nM, respectively) further validated our multitarget approach based on a sequential mechanism of action. In addition, target compound 1b exhibited a suitable ADMET profile, including good brain permeability and high stability in PBS, encouraging further biological investigation as a drug candidate.

Straightforward Access to a New Class of Dual DYRK1A/CLK1 Inhibitors Possessing a Simple Dihydroquinoline Core.

The DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) family of protein kinases is involved in the pathogenesis of several neurodegenerative diseases. Among them, the DYRK1A protein kinase is thought to be implicated in Alzheimer's disease (AD) and Down syndrome, and as such, has emerged as an appealing therapeutic target. DYRKs are a subset of the CMGC (CDK, MAPKK, GSK3 and CLK) group of kinases. Within this group of kinases, the CDC2-like kinases (CLKs), such as CLK1, are closely related to DYRKs and have also sparked great interest as potential therapeutic targets for AD. Based on inhibitors previously described in the literature (namely TG003 and INDY), we report in this work a new class of dihydroquinolines exhibiting inhibitory activities in the nanomolar range on hDYRK1A and hCLK1. Moreover, there is overwhelming evidence that oxidative stress plays an important role in AD. Pleasingly, the most potent dual kinase inhibitor 1p exhibited antioxidant and radical scavenging properties. Finally, drug-likeness and molecular docking studies of this new class of DYRK1A/CLK1 inhibitors are also discussed in this article.

Chemical Delivery System of MIBG to the Central Nervous System: Synthesis, 11C-Radiosynthesis, and in Vivo Evaluation.

The norepinephrine transporter (NET) plays an important role in neurotransmission and is involved in a multitude of psychiatric and neurodegenerative diseases. [123I/131I]meta-iodobenzylguanidine (MIBG) is a widely used radiotracer in the diagnosis and follow-up of peripheral neuroendocrine tumors overexpressing the norepinephrine transporter. MIBG does not cross the blood-brain barrier (BBB), and we have demonstrated the "proof-of-concept" that 1,4-dihydroquinoline/quinolinium salt as chemical delivery system (CDS) is a promising tool to deliver MIBG to the brain. To improve BBB passage, various substituents on the 1,4-dihydroquinoline moiety and a linker between CDS and MIBG were added. A series of CDS-MIBG 1a-d was synthesized, labeled with carbon-11, and evaluated in vivo into rats. The in vivo results demonstrated that, although adding substituents on CDS in 1a-c is of no benefit for brain delivery of MIBG, the presence of a linker in CDS-MIBG 1d greatly improved both brain penetration and the release rate of MIBG in the central nervous system.

Access to Highly Enantioenriched Donepezil-like 1,4-Dihydropyridines as Promising Anti-Alzheimer Prodrug Candidates via Enantioselective Tsuji Allylation and Organocatalytic Aza-Ene-Type Domino Reactions.

This work aims at exploiting both the enantioselective Tsuji allylation of allyl carbonate 6 and an organocatalytic aza-ene-type domino reaction between enal 3a and ß-enaminone 4a to develop a straightforward access to all of the four possible stereoisomers of a donepezil-like 1,4-dihydropyridine 1a (er up to 99.5:0.5; overall yield up 64%), an anti-Alzheimer's prodrug candidate. This strategy was extended to the preparation of other enantioenriched 1,4-dihydropyridines 1b-i (eight examples), highlighting its potential in the development of these chiral AChE inhibitors.

Rational design of carbamate-based dual binding site and central AChE inhibitors by a "biooxidisable" prodrug approach: Synthesis, in vitro evaluation and docking studies.

Herein, we report a new class of dual binding site AChE inhibitor 4 designed to exert a central cholinergic activation thanks to a redox-activation step of a prodrug precursor 3. Starting from potent pseudo-irreversible quinolinium salts AChE inhibitors 2 previously reported, a new set of diversely substituted quinolinium salts 2a-p was prepared and assayed for their inhibitory activity against AChE. Structure-activity relationship (SAR) analysis of 2a-p coupled with molecular docking studies allowed us to determine which position of the quinolinium scaffold may be considered to anchor the phtalimide fragment presumed to interact with the peripheral anionic site (PAS). In addition, molecular docking provided insight on the linker length required to connect both quinolinium and phatlimide moieties without disrupting the crucial role of quinolinium salt moiety within the catalytic active site (CAS); namely placing the carbamate in the correct position to trigger carbamylation of the active-site serine hydroxyl. Based on this rational design, the putative dual binding site inhibitor 4 and its prodrug 3 were synthesized and subsequently evaluated in vitro against AChE. Pleasingly, whereas compound 4 showed to be a highly potent inhibitor of AChE (IC50 = 6 nM) and binds to AChE-PAS to the same extent as donepezil, its prodrug 3 revealed to be inactive (IC50 > 10 µM). These preliminary results constitute one of the few examples of carbamate-based dual binding site AChE inhibitors.

Novel donepezil-like N-benzylpyridinium salt derivatives as AChE inhibitors and their corresponding dihydropyridine "bio-oxidizable" prodrugs: Synthesis, biological evaluation and structure-activity relationship.

As an extension of our previous work on donepezil-based "bio-oxidizable" prodrug approach, two new classes of N-benzylpyridinium donepezil analogues in tetralone B2 and acetophenone B3 series and a new set of indanone derivatives B1 were investigated along with the corresponding dihydropyridine prodrugs A1-3. A total of fifty one N-benzylpyridinium quaternary donepezil analogues B1-3 and twenty two prodrugs A1-3 were synthesized and evaluated for their inhibitory activities against hAChE and eqBuChE. While most prodrugs A1-3 were demonstrated to be inactive against AChE (IC50 > 10 µM), a large number of the corresponding N-benzylpyridinium salt B1-3 exhibited appealing three-to-one-digit nanomolar hAChE inhibitory activities and even reaching subnanomolar activity (IC50 = 0.36 nM). In addition, in silico docking studies were conducted for several compounds to explain the more relevant in vitro results. Lastly, the influence of the two stereogenic centers in prodrugs A was also evaluated, highlighting not only marked differences in residual AChE inhibitory activity of the four separated isomers of prodrug 23h (IC50 ranging from 173 nM to 10 µM) but also significant variations of the oxidation rate between two separated diastereoisomers of prodrug 24a. This work provides useful information in the search of a preclinical candidate to conduct further development of this attractive "bio-oxidizable" prodrug strategy.

Delivering FLT to the Central Nervous System by Means of a Promising Targeting System: Synthesis, [11C]Radiosynthesis, and in Vivo Evaluation.

The development of delivery systems to transport some specific radiotracers across the blood-brain barrier (BBB) needs to be investigated for brain imaging. [18F]FLT (3'-deoxy-3'-18F-fluoro-l-thymidine), an analogue substrate of the nucleoside thymidine, has been developed as a proliferation tracer for oncological PET studies. Unfortunately, low-grade brain tumors are poorly visualized due to the low uptake of [18F]FLT in brain tissue, preventing its use in PET imaging to detect brain tumors at an early stage. Based on our previous work, a redox chemical delivery system (CDS) related to Bodor's strategy was developed to enable the penetration of FLT into the brain. To this end, FLT was covalently linked to a series of lipophilic carriers based on a 1,4-dihydroquinoline structure. To determine the best carrier, various sets of [11C]CDS-FLT were prepared and injected into rats. Pleasingly, in vivo results let us suggest that this CDS is a promising approach to overcome the BBB to target low-grade brain tumors for PET imaging.

Donepezil-Based Central Acetylcholinesterase Inhibitors by Means of a "Bio-Oxidizable" Prodrug Strategy: Design, Synthesis, and in Vitro Biological Evaluation.

With the aim of reducing side effects of acetylcholinesterase inhibitors (AChEIs) during symptomatic treatment of Alzheimer's disease, we report herein a new class of donepezil-based "bio-oxidizable" prodrugs 1 designed to be converted into dual binding site AChEIs 2. While most of indanone-derived N-benzylpyridinium salts 2 revealed to be highly potent dual binding site hAChEIs (IC50 up to 3 nM), outperforming the standard drug donepezil (IC50 = 11 nM), most of the corresponding 1,4-dihydropyridines 1 were found to be inactive. Promisingly, whereas the selected prodrug 1r showed good permeability in the PAMPA-BBB model and high in vitro antioxidant activity, its conversion to AChEI 2r could be easily achieved under mild conditions when incubated in various oxidizing media. Lastly, both compounds 1r and 2r did not show genotoxicity in vitro and displayed high LD50 values in mice, making this prodrug 1r/drug 2r couple a good candidate for further in vivo biological experiments.

Palladium-Catalyzed Carbonylation of (Hetero)Aryl, Alkenyl and Allyl Halides by Means of N-Hydroxysuccinimidyl Formate as CO Surrogate.

An efficient Pd-catalyzed carbonylation protocol is described for the coupling of a large panel of aryl, heteroaryl, benzyl, vinyl and allyl halides 2 with the unusual N-hydroxysuccinimidyl (NHS) formate 1 as a CO surrogate to afford the corresponding valuable NHS esters 3. High conversion to the coupling products was achieved with up to 98% yield by means of Pd(OAc)2/Xantphos catalyst system.

Dihydroquinoline Carbamate Derivatives as "Bio-oxidizable" Prodrugs for Brain Delivery of Acetylcholinesterase Inhibitors: [¹¹C] Radiosynthesis and Biological Evaluation.

With the aim of improving the efficiency of marketed acetylcholinesterase (AChE) inhibitors in the symptomatic treatment of Alzheimer's disease, plagued by adverse effects arising from peripheral cholinergic activation, this work reports a biological evaluation of new central AChE inhibitors based on an original "bio-oxidizable" prodrug strategy. After peripheral injection of the prodrug 1a [IC50 > 1 mM (hAChE)] in mice, monitoring markers of central and peripheral cholinergic activation provided in vivo proof-of-concept for brain delivery of the drug 2a [IC50 = 20 nM (hAChE)] through central redox activation of 1a. Interestingly, peripheral cholinergic activation has been shown to be limited in time, likely due to the presence of a permanent positive charge in 2a promoting rapid elimination of the AChE inhibitor from the circulation of mice. To support these assumptions, the radiosynthesis with carbon-11 of prodrug 1a was developed for additional ex vivo studies in rats. Whole-body biodistribution of radioactivity revealed high accumulation in excretory organs along with moderate but rapid brain uptake. Radio-HPLC analyses of brain samples confirm rapid CNS penetration of [(11)C]1a, while identification of [(11)C]2a and [(11)C]3a both accounts for central redox activation of 1a and pseudoirreversible inhibition of AChE, respectively. Finally, Caco-2 permeability assays predicted metabolite 3a as a substrate for efflux transporters (P-gp inter alia), suggesting that metabolite 3a might possibly be actively transported out of the brain. Overall, a large body of evidence from in vivo and ex vivo studies on small animals has been collected to validate this "bio-oxidizable" prodrug approach, emerging as a very promising strategy in the rational design of selective central AChE inhibitors.

New developments in redox chemical delivery systems by means of 1,4-dihydroquinoline-based targetor: application to galantamine delivery to the brain.

The therapeutic efficiency of palliative treatments of AD, mostly based on acetylcholinesterase (AChE) inhibitors, is marred by serious adverse effects due to peripheral activity of these AChE inhibitors. In the literature, a redox-based chemical delivery system (CDS) has been developed to enhance drugs distribution to the brain while reducing peripheral side effects. Herein, we disclose two new synthetic strategies for the preparation of 1,4-dihydroquinoline/quinolinium salt redox-based systems particularly well designed for brain delivery of drugs sensitive to alkylation reactions. These strategies have been applied in the present case to the AChE inhibitor galantamine with the aim of alleviating adverse effects observed with cholinergic AD treatment. The first strategy is based on an intramolecular alkylation reaction as key step, whilst the second strategy relies on a useful coupling between galantamine and quinolinium salt key intermediate. In the course of this work, polymer-supported reagents and a solid-phase synthesis approach revealed to be highly helpful to develop this redox-based galantamine CDS.

(3-Chloro-phen-yl){2-eth-oxy-5-[(Z)-hydroxy(phen-yl)methyl-idene]cyclo-penta-1,3-dien-1-yl}methanone.

The title compound, C(21)H(17)ClO(3), which crystallizes as one of two possible oxo/hy-droxy-fulvene prototropic tautomers, possesses a strong intra-molecular O-H⋯O hydrogen bond that closes a seven-membered ring. The dihedral angles between the central five-membered ring and two pendant rings are 55.05 (9) and 44.51 (10)°. The crystal packing is characterized by weak inter-molecular C-H⋯O inter-actions between an H atom of the oxymethyl-ene unit and the carbonyl group of an adjacent mol-ecule, resulting in formation of chains of mol-ecules along the a axis.