Chronic stress is a risk factor for Major Depressive Disorder (MDD), and in rodents, it recapitulates human behavioral, cellular and molecular changes. In MDD and after chronic stress, neuronal dysfunctions and deficits in GABAergic signaling are observed and responsible for symptom severity. GABA signals predominantly through GABAA receptors (GABAA-R) composed of various subunit types that relate to downstream outcomes. Activity at α2-GABAA-Rs contributes to anxiolytic properties, α5-GABAA-Rs to cognitive functions, and α1-GABAA-Rs to sedation. Therefore, a therapy aiming at increasing α2- and α5-GABAA-Rs activity, but devoid of α1-GABAA-R activity, has potential to address several symptomologies of depression while avoiding side-effects. This study investigated the activity profiles and behavioral efficacy of two enantiomers of each other (GL-II-73 and GL-I-54), separately and as a racemic mixture (GL-RM), and potential disease-modifying effects on neuronal morphology. Results confirm GL-I-54 and GL-II-73 exert positive allosteric modulation at the α2-, α3-, α5-GABAA-Rs and α5-containing GABAA-Rs, respectively, and separately reduces immobility in the forced swim test and improves stress-induced spatial working memory deficits. Using unpredictable chronic mild stress (UCMS), we show that acute and chronic administration of GL-RM provide pro-cognitive effects, with mild efficacy on mood symptoms, although at lower doses avoiding sedation. Morphology studies showed reversal of spine density loss caused by UCMS after chronic GL-RM treatment at apical and basal dendrites of the PFC and CA1. Together, these results support using a racemic mixture with combined α2-, α3-, α5-GABAA-R profile to reverse chronic stress-induced mood symptoms, cognitive deficits, and with anti-stress neurotrophic effects.

GABA mediates inhibitory actions through various GABA_A receptor subtypes, including 19 subunits in human GABAAR. Dysregulation of GABAergic neurotransmission is associated with several psychiatric disorders, including depression, anxiety, and schizophrenia. Selective targeting of α2/3 GABAARs can treat mood and anxiety, while α5 GABAA-Rs can treat anxiety, depression, and cognitive performance. GL-II-73 and MP-III-022, α5-positive allosteric modulators have shown promising results in animal models of chronic stress, aging, and cognitive disorders, including MDD, schizophrenia, autism, and Alzheimer’s disease. Described in this article is how small changes in the structure of imidazodiazepine substituents can greatly impact the subtype selectivity of benzodiazepine GABAAR. To investigate alternate and potentially more effective therapeutic compounds, modifications were made to the structure of imidazodiazepine 1 to synthesize different amide analogs. The novel ligands were screened at the NIMH PDSP against a panel of 47 receptors, ion channels, including hERG, and transporters to identify on- and off-target interactions. Any ligands with significant inhibition in primary binding were subjected to secondary binding assays to determine their K_i values. The newly synthesized imidazodiazepines were found to have variable affinities for the benzodiazepine site and negligible or no binding to any off-target profile receptors that could cause other physiological problems.

Analgesic and anti-inflammatory properties mediated by the κ opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, µ opioid receptor (MOR), and δ opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC₅₀ of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with ³H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABA_AR) of the α_1-3β_2-3γ_1-2 subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABA_ARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors.