Lead optimization of a series of tryptophan-based nanomolar butyrylcholinesterase (BChE) inhibitors led to tertiary amines as highly potent, achiral, sp³-rich analogues with better synthetic accessibility and high selectivity over acetylcholinesterase (one to ten thousandfold). Taking it one step further, the introduction of a carbamate warhead on the well-explored reversible scaffold allowed conversion to pseudoirreversible inhibitors that bound covalently to BChE and prolonged the duration of inhibition (half-life of 14.8 h for compound 45a-carbamoylated enzyme). Additionally, N-hydroxyindole was discovered as a novel leaving group chemotype. The covalent mechanism of action was confirmed by time-dependency experiments, progress curve analysis, and indirectly by co-crystallization with the human recombinant enzyme. Two crystal structures of BChE-inhibitor complexes were solved and coupled with the supporting molecular dynamics simulations increased our understanding of the structure-activity relationship, while also providing the necessary structural information for future optimization of this series. Overall, this research demonstates the high versatility and potential of this series of BChE inhibitors.

The multi-target-directed ligands (MTDLs) strategy is encouraged for the development of novel modulators targeting multiple pathways in the neurodegenerative cascade typical for Alzheimer’s disease (AD). Based on the structure of an in-house irreversible monoamine oxidase B (MAO-B) inhibitor, we aimed to introduce a carbamate moiety on the aromatic ring to impart cholinesterase (ChE) inhibition, and to furnish multifunctional ligands targeting two enzymes that are intricately involved in AD pathobiology. In this study, we synthesized three dual hMAO-B/hBChE inhibitors 13–15, with compound 15 exhibiting balanced, low micromolar inhibition of hMAO-B (IC₅₀ of 4.3 µM) and hBChE (IC₅₀ of 8.5 µM). The docking studies and time-dependent inhibition of hBChE confirmed the initial expectation that the introduced carbamate moiety is responsible for covalent inhibition. Therefore, dual-acting compound 15 represents an excellent starting point for further optimization of balanced MTDLs

A library of 31 butyrylcholinesterase (BChE) and cathepsin B (CatB) inhibitors was screened in vitro for inhibition of deoxyribonuclease I (DNase I). Compounds 22, 8 and 7 are among the most potent synthetic non‐peptide DNase I inhibitors reported to date. Three 8‐hydroxyquinoline analogues inhibited both DNase I and BChE with IC₅₀ values below 35 μM and 50 nM, respectively, while two nitroxoline derivatives inhibited DNase I and Cat B endopeptidase activity with IC₅₀ values below 60 and 20 μM. Selected derivatives were screened for various co‐target binding affinities at dopamine D₂ and D₃, histamine H₃ and H₄ receptors and inhibition of 5‐lipoxygenase. Compound 8 bound to the H₃ receptor and is highlighted as the most promising multifunctional ligand with a favorable pharmacokinetic profile and one of the most potent non‐peptide DNase I inhibitors. The present study demonstrates that 8‐hydroxyquinoline is a structural fragment critical for DNase I inhibition in the presented series of compounds.

Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action.

The currently approved drugs for the treatment of Alzheimer’s disease (AD) fail to address its interconnected pathological processes. Inhibition of butyrylcholinesterase (BChE) and p38α mitogen-activated protein kinase (p38α MAPK) offers an innovative dual approach to mitigate two major drivers of neurodegeneration in AD: cholinergic deficit and neuroinflammation. Using structure-based drug design and a library of known p38α MAPK inhibitors, we developed first-in-class, selective dual BChE/p38α MAPK inhibitors with balanced activity against both targets. The X-ray crystal structures of the two most promising molecules bound to both enzymes were solved. Those ligands effectively reduced the production of proinflammatory markers in vitro and ex vivo in phytohemagglutinin/lipopolysaccharide neuroinflammation models. Remarkably, these compounds also significantly improved cognition in scopolamine- and lipopolysaccharide-induced models of cognitive dysfunction in mice. Because our dual-acting inhibitors target both the symptoms and the underlying neuropathology, they offer an innovative and comprehensive strategy to combat AD.

The untapped potential of carbamoyl fluorides for various chemico/biological applications is hampered by the scarcity of straightforward and benign methods for their synthesis. In this report, we disclose a novel mild three-step procedure that avoids exotic, corrosive, and highly toxic reagents. Briefly, commercially available secondary amines are carbamoylated with 1,1′-carbonyldiimidazole, followed by alkylation to improve nucleofugality, and exchange with inorganic KF. This procedure works on a gram scale without chromatographic purification. It is however limited to basic, sterically unhindered secondary amines without alkylation-prone functional groups.