Arylazobenzimidazoles: versatile visible-light photoswitches with tuneable Z-isomer stability.

Benzimidazole heterocycles are of great importance in medicinal chemistry due to their applicability to a wide range of pharmacological targets, therefore representing a prototypical "privileged structure". In photopharmacology, azoheteroarene photoswitches have emerged as valuable tools for a variety of applications due to the high tuneability of their photophysical properties. Benzimidazole-based photoswitches could therefore enable the optically-controlled investigation of many pharmacological targets and find application in materials science. Here we report a combined experimental and computational investigation of such arylazobenzimidazoles, which allowed us to identify derivatives with near-quantitative bidirectional photoswitching using visible light and highly tuneable Z-isomer stability. We further demonstrate that arylazobenzimidazoles bearing a free benzimidazole N-H group not only exhibit efficient bidirectional photoswitching, but also excellent thermal Z-isomer stability, contrary to previously reported fast-relaxing Z-isomers of N-H azoheteroarenes. Finally, we describe derivatives which can be reversibly isomerized with cyan and red light, thereby enabling significantly "red-shifted" photocontrol over prior azoheteroarenes. The understanding gained in this study should enable future photopharmacological efforts by employing photoswitches based on the privileged benzimidazole structure.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides.

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily.

Bridging the Binding Sites 2.0: Photoswitchable Dualsteric Ligands for the Cannabinoid 2 Receptor.

The cannabinoid receptor 2 (CB2R) has high, unexploited therapeutic potential in several central nervous system disorders due to its involvement in neuroinflammatory processes and pathologies like neurodegeneration. Dualsteric/bitopic ligands are currently developed to achieve receptor subtype selectivity and biased signaling. To obtain a molecular tool compound with photoswitchable potential dualsteric properties, we applied two different approaches to link a positive allosteric modulator with an orthosteric agonist via a photochromic unit. We characterized the photophysical properties of all compounds and determined efficacy in internalization, calcium mobilization, and BRET studies. We report the first potentially dualsteric photoswitchable ligand for studying molecular mechanisms of CB2R-associated pathologies. Compound 17-para is a submicromolar "cis-on" agonist with >10-fold higher potency compared to its trans photoisomer and allows high spatiotemporal control of CB2R activation.

Visible-Light Photoswitchable Benzimidazole Azo-Arenes as ß-Arrestin2-Biased Selective Cannabinoid 2 Receptor Agonists.

The cannabinoid 2 receptor (CB2 R) has high therapeutic potential for multiple pathogenic processes, such as neuroinflammation. Pathway-selective ligands are needed to overcome the lack of clinical success and to elucidate correlations between pathways and their respective therapeutic effects. Herein, we report the design and synthesis of a photoswitchable scaffold based on the privileged structure of benzimidazole and its application as a functionally selective CB2 R "efficacy-switch". Benzimidazole azo-arenes offer huge potential for the broad extension of photopharmacology to a wide range of optically addressable biological targets. We used this scaffold to develop compound 10 d, a "trans-on" agonist, which serves as a molecular probe to study the ß-arrestin2 (ßarr2) pathway at CB2 R. ßΑrr2 bias was observed in CB2 R internalization and ßarr2 recruitment, while no activation occurred when looking at Gα16 or mini-Gαi . Overall, compound 10 d is the first light-dependent functionally selective agonist to investigate the complex mechanisms of CB2 R-ßarr2 dependent endocytosis.

Dual-Acting Small Molecules: Subtype-Selective Cannabinoid Receptor 2 Agonist/Butyrylcholinesterase Inhibitor Hybrids Show Neuroprotection in an Alzheimer's Disease Mouse Model.

We present the synthesis and characterization of merged human butyrylcholinesterase (hBChE) inhibitor/cannabinoid receptor 2 (hCB2R) ligands for the treatment of neurodegeneration. In total, 15 benzimidazole carbamates were synthesized and tested for their inhibition of human cholinesterases, also with regard to their pseudoirreversible binding mode and affinity toward both cannabinoid receptors in radioligand binding studies. After evaluation in a calcium mobilization assay as well as a ß-arrestin 2 (ßarr2) recruitment assay, two compounds with balanced activities on both targets were tested for their immunomodulatory effect on microglia activation and regarding their pharmacokinetic properties and blood-brain barrier penetration. Compound 15d, containing a dimethyl carbamate motif, was further evaluated in vivo, showing prevention of Aß25-35-induced learning impairments in a pharmacological mouse model of Alzheimer's disease for both short- and long-term memory responses. Additional combination studies proved a synergic effect of BChE inhibition and CB2R activation in vivo.

Novel benzimidazole-based pseudo-irreversible butyrylcholinesterase inhibitors with neuroprotective activity in an Alzheimer's disease mouse model.

As levels of acetylcholinesterase (AChE) decrease while levels of butyrylcholinesterase (BChE) increase in later stages of Alzheimer's disease (AD), BChE stands out as a promising target for treatment of AD. Therefore, several benzimidazole-carbamates were designed based on docking studies to inhibit BChE selectively over AChE, while retaining a reasonable solubility. Synthesized molecules exhibit IC50 values from 2.4 µM down to 3.7 nM with an overall highly hBChE-selective profile of the designed compound class. After evaluation of potential neurotoxicity, the most promising compound was further investigated in vivo. Compound 11d attenuates Aß25-35-induced learning impairments in both spontaneous alternation and passive avoidance responses at a very low dosage of 0.03 mg kg-1, proving selective BChE inhibition to lead to effective neuroprotectivity in AD.

Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CB1R) "Cis-On" Agonist.

Activation of the human cannabinoid receptor type 1 (hCB1R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for hCB1R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB1R (Ki (cis-form) = 0.18 µM), with a marked difference in affinity with respect to its inactive "trans-off" form (CB1R Ki trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT ßarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of hCB1R in disorders associated with the endocannabinoid system.