Novel Selective Butyrylcholinesterase Inhibitors Incorporating Antioxidant Functionalities as Potential Bimodal Therapeutics for Alzheimer's Disease.

Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 µM when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 µM. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 ± 0.020 Trolox Equivalent.

Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode.

Butyrylcholinesterase (BChE) is a promising target for the treatment of later stage cognitive decline in Alzheimer's disease. A set of pseudo-irreversible BChE inhibitors with high selectivity over hAChE was synthesized based on carbamates attached to tetrahydroquinazoline scaffolds with the 2-thiophenyl compound 2p as the most potent inhibitor of eqBChE (KC = 14.3 nM) and also of hBChE (KC = 19.7 nM). The inhibitors transfer the carbamate moiety onto the active site under release of the phenolic tetrahydroquinazoline scaffolds that themselves act as neuroprotectants. By combination of kinetic data with molecular docking studies, a plausible binding model was probed describing how the tetrahydroquinazoline scaffold guides the carbamate into a close position to the active site. The model explains the influence of the carrier scaffold onto the affinity of an inhibitor just before carbamate transfer. This strategy can be used to utilize the binding mode of other carbamate-based inhibitors.

Cyclic acyl guanidines bearing carbamate moieties allow potent and dirigible cholinesterase inhibition of either acetyl- or butyrylcholinesterase.

A series of cyclic acyl guanidine with carbamate moieties have been synthesized and evaluated in vitro for their AChE and BChE inhibitory activities. Structure-activity relationships identified compound 23 as a nanomolar and selective BChE inhibitor, while compound 32 exhibited nanomolar and selective AChE inhibition, selectivity depending on both the structure of the carbamate substituent as well as the position of guanidines-N substitution. The velocity of enzyme carbamoylation was analyzed and showed similar behavior to physostigmine. Phenolic compounds formed after carbamate transfer to the active site of cholinesterases showed additional neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation.

Identification of a neuroprotective and selective butyrylcholinesterase inhibitor derived from the natural alkaloid evodiamine.

Two sets of carbamates based on the natural alkaloid evodiamine were designed, synthesized and evaluated as potential butyrylcholinesterase inhibitors. Although a set of carbamates of 3-hydroxyevodiamine (10a-f) is inactive both at AChE and BChE, carbamates of 5-deoxo-3-hydroxyevodiamine (11a-f) exhibit much better potency with selectivity toward BChE. The heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c) shows the best potency with an IC50 value of 77 nM and very good selectivity over AChE. ORAC and cell-based assays indicate 11c owns pronounced antioxidant properties with 1.75 Trolox equivalents and strong neuroprotection even from 1 µM onwards. These combined activities might enable compound 11c to be a potential candidate for treatment of Alzheimer's disease.

Flavonoids, flavonoid metabolites, and phenolic acids inhibit oxidative stress in the neuronal cell line HT-22 monitored by ECIS and MTT assay: a comparative study.

A real-time and label-free in vitro assay based on electric cell-substrate impedance sensing (ECIS) was established, validated, and compared to an end-point MTT assay within an experimental trial addressing the cytoprotective effects of 19 different flavonoids, flavonoid metabolites, and phenolic acids and their methyl esters on the HT-22 neuronal cell line, after induction of oxidative stress with tert-butyl hydroperoxide. Among the flavonoids under study, only those with a catechol unit and an additional 4-keto group provided cytoprotection. The presence of a 2,3-double bond was not a structural prerequisite for a neuroprotective effect. In the case of the phenolics, catechol substitution was the only structural requirement for activity. The flavonoids and other phenolics with a ferulic acid substitution or a single hydroxy group showed no activity. Electrochemical characterization of all compounds via square-wave voltammetry provided a rather specific correlation between cytoprotective activity and redox potential for the active flavonoids, but not for the active phenolics with a low molecular weight. Moreover this study was used to compare label-free ECIS recordings with results of the established MTT assay. Whereas the former provides time-resolved and thus entirely unbiased information on changes of cell morphology that are unequivocally associated with cell death, the latter requires predefined exposure times and a strict causality between metabolic activity and cell death. However, MTT assays are based on standard lab equipment and provide a more economic way to higher throughput.

Tacrine-silibinin codrug shows neuro- and hepatoprotective effects in vitro and pro-cognitive and hepatoprotective effects in vivo.

A codrug of the anti-Alzheimer drug tacrine and the natural product silibinin was synthesized. The codrug's biological and pharmacological properties were compared to an equimolar mixture of the components. The compound showed potent acetyl- and butyrylcholinesterase inhibition. In a cellular hepatotoxicity model, analyzing the influence on viability and mitochondria of hepatic stellate cells (HSC), the toxicity of the codrug was markedly reduced in comparison to that of tacrine. Using a neuronal cell line (HT-22), a neuroprotective effect against glutamate-induced toxicity could be observed that was absent for the 1:1 mixture of components. In subsequent in vivo experiments in rats, in contrast to the effects seen after tacrine treatment, after administration of the codrug no hepatotoxicity and no induction of the cytochrome P450 system were noticed. In a scopolamine-induced cognitive impairment model using Wistar rats, the codrug was as potent as tacrine in reversing memory dysfunction. The tacrine-silibinin codrug shows high AChE and BChE inhibition, neuroprotective effects, lacks tacrine's hepatotoxicity in vitro and in vivo, and shows the same pro-cognitive effects in vivo as tacrine, being superior to the physical mixture of tacrine and silibinin in all these regards.

Neuroprotective Tri- and Tetracyclic BChE Inhibitors Releasing Reversible Inhibitors upon Carbamate Transfer.

Tri- and tetracyclic nitrogen-bridgehead compounds were designed and synthesized to yield micromolar cholinesterase (ChE) inhibitors. Structure-activity relationships identified potent compounds with butyrylcholinesterase selectivity. These compounds were selected as starting points for the design and synthesis of carbamate-based (pseudo)irreversible inhibitors. Compounds with superior inhibitory activity and selectivity were obtained and kinetically characterized also with regard to the velocity of enzyme carbamoylation. Structural elements were identified and introduced that additionally showed neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation. We have identified potent and selective pseudoirreversible butyrylcholinesterase inhibitors that release reversible inhibitors with neuroprotective properties after carbamate transfer to the active site of cholinesterases.