Development of non-sedating antischistosomal benzodiazepines.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970's. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) are not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index, and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

New Imidazodiazepine Analogue, 5-(8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole, Provides a Simplified Synthetic Scheme, High Oral Plasma and Brain Exposures, and Produces Antiseizure Efficacy in Mice, and Antiepileptogenic Activity in Neural Networks in Brain Slices from a Patient with Mesial Temporal Lobe Epilepsy.

KRM-II-81 (1) is an imidazodiazepine GABAA receptor (GABAAR) potentiator with broad antiseizure efficacy and a low sedative burden. A brominated analogue, DS-II-73 (5), was synthesized and pharmacologically characterized as a potential backup compound as KRM-II-81 moves forward into development. The synthesis from 2-amino-5-bromophenyl)(pyridin-2yl)methanone (6) was processed in five steps with an overall yield of 38% and without the need for a palladium catalyst. GABAAR binding occurred with a Ki of 150 nM, and only 3 of 41 screened binding sites produced inhibition ≥50% at 10 µM, and the potency to induce cytotoxicity was ≥240 mM. DS-II-73 was selective for α2/3/5- over that of α1-containing GABAARs. Oral exposure of plasma and brain of rats was more than sufficient to functionally impact GABAARs. Tonic convulsions in mice and lethality induced by pentylenetetrazol were suppressed by DS-II-73 after oral administration and latencies to clonic and tonic seizures were prolonged. Cortical slice preparations from a patient with pharmacoresistant epilepsy (mesial temporal lobe) showed decreases in the frequency of local field potentials by DS-II-73. As with KRM-II-81, the motor-impairing effects of DS-II-73 were low compared to diazepam. Molecular docking studies of DS-II-73 with the α1ß3γ2L-configured GABAAR showed low interaction with α1His102 that is suggested as a potential molecular mechanism for its low sedative side effects. These findings support the viability of DS-II-73 as a backup molecule for its ethynyl analogue, KRM-II-81, with the human tissue data providing translational credibility.

Comparative anticonvulsant activity of the GABAkine KRM-II-81 and a deuterated analog.

A series of imidazodiazepines has been developed that possess reduced sedative liabilities but retain efficacy in anticonvulsant screening models. The latest of these compounds, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole known as KRM-II-81) is currently awaiting advancement into the clinic. A deuterated structural analog (D5-KRM-II-81) was made as a potential backup compound and studied here in comparison to KRM-II-81. In the present study, both compounds significantly prevented seizures in mice induced by 6 Hz (44 mA) electrical stimulation without significantly altering motoric function on a rotarod after intraperitoneal administration. Both compounds also significantly prevented clonic seizures, tonic seizures, and lethality induced by pentylenetetrazol in mice when given orally. D5-KRM-II-81 had a slightly longer duration of action against clonic and tonic seizures than KRM-II-81. Oral administration of 100 mg/kg of either KRM-II-81 or D5-KRM-II-81 was significantly less disruptive of sensorimotor function in mice than diazepam (5 mg/kg, p.o.). The present report documents that D5-KRM-II-81 represents another in this series of imidazodiazepines with anticonvulsant activity at doses that do not impair sensorimotor function.

Rationalizing the binding and α subtype selectivity of synthesized imidazodiazepines and benzodiazepines at GABAA receptors by using molecular docking studies.

The pharmacological actions exerted by benzodiazepines are dependent on the discrete α protein subunits of the γ-aminobutyric acid type A receptor (GABAA R). Recent developments via a cryo-EM structure of the α1ß3γ2L GABAA R ion channel provide crucial insights into ligand efficacy and binding affinity at this subtype. We investigated the molecular interactions of diazepam and alprazolam bound GABAA R structures (6HUP and 6HUO) to determine key binding interaction domains. A halogen bond between the chlorine atoms of diazepam and alprazolam with the group on the backbone of the α1 histidine amino acid 102 is important to the positive allosteric modulatory actions of diazepam and alprazolam in the α1ß3γ2L GABAA R ion channel. In order to gain insight into α subtype selectivity we designed and synthesized close structural analogs of diazepam and alprazolam. These compounds were then docked into the recently publish cryo-EM structures of GABAA Rs (6HUP and 6HUO). This modeling along with radio-ligand binding data resulted in the conclusion that the non-classical bioisosteric replacement of the chlorine atom at C7 with an ethinyl group (compound 5) resulted in an 11-fold gain in α5 binding selectivity over the α1 subtype. Moreover, the potency of compound 5 resulted in a ligand with less sedation than diazepam, while still maintaining the same anxiolytic potency. These modeling data extend our understanding of the structural requirements for α-subtype-selective compounds that can be utilized to achieve improved medical treatments. It is clear that the ethinyl group in place of a halogen atom decreases the affinity and efficacy of benzodiazepines and imidazodiazepines at α1 subtypes, which results in less sedation and ataxia.

Behavioral Deficits Induced by Somatostatin-Positive GABA Neuron Silencing Are Rescued by Alpha 5 GABA-A Receptor Potentiation.

INTRODUCTION: Deficits in somatostatin-positive gamma-aminobutyric acid interneurons (SST+ GABA cells) are commonly reported in human studies of mood and anxiety disorder patients. A causal link between SST+ cell dysfunction and symptom-related behaviors has been proposed based on rodent studies showing that chronic stress, a major risk factor for mood and anxiety disorders, induces a low SST+ GABA cellular phenotype across corticolimbic brain regions; that lowering Sst, SST+ cell, or GABA functions induces depressive-/anxiety-like behaviors (a rodent behavioral construct collectively defined as "behavioral emotionality"); and that disinhibiting SST+ cells has antidepressant-like effects. Recent studies found that compounds preferentially potentiating receptors mediating SST+ cell functions, α5-GABAA receptor positive allosteric modulators (α5-PAMs), achieved antidepressant-like effects. Together, the evidence suggests that SST+ cells regulate mood and cognitive functions that are disrupted in mood disorders and that rescuing SST+ cell function via α5-PAM may represent a targeted therapeutic strategy. METHODS: We developed a mouse model allowing chemogenetic manipulation of brain-wide SST+ cells and employed behavioral characterization 30 minutes after repeated acute silencing to identify contributions to symptom-related behaviors. We then assessed whether an α5-PAM, GL-II-73, could rescue behavioral deficits. RESULTS: Brain-wide SST+ cell silencing induced features of stress-related illnesses, including elevated neuronal activity and plasma corticosterone levels, increased anxiety- and anhedonia-like behaviors, and impaired short-term memory. GL-II-73 led to antidepressant- and anxiolytic-like improvements among behavioral deficits induced by brain-wide SST+ cell silencing. CONCLUSION: Our data validate SST+ cells as regulators of mood and cognitive functions and demonstrate that bypassing low SST+ cell function via α5-PAM represents a targeted therapeutic strategy.

Reversal of Age-Related Neuronal Atrophy by α5-GABAA Receptor Positive Allosteric Modulation.

Aging is associated with reduced brain volume, altered neural activity, and neuronal atrophy in cortical-like structures, comprising the frontal cortex and hippocampus, together contributing to cognitive impairments. Therapeutic efforts aimed at reversing these deficits have focused on excitatory or neurotrophic mechanisms, although recent findings show that reduced dendritic inhibition mediated by α5-subunit containing GABA-A receptors (α5-GABAA-Rs) occurs during aging and contributes to cognitive impairment. Here, we aimed to confirm the beneficial effect on working memory of augmenting α5-GABAA-R activity in old mice and tested its potential at reversing age-related neuronal atrophy. We show that GL-II-73, a novel ligand with positive allosteric modulatory activity at α5-GABAA-R (α5-PAM), increases dendritic branching complexity and spine numbers of cortical neurons in vitro. Using old mice, we confirm that α5-PAM reverses age-related working memory deficits and show that chronic treatment (3 months) significantly reverses age-related dendritic shrinkage and spine loss in frontal cortex and hippocampus. A subsequent 1-week treatment cessation (separate cohort) resulted in loss of efficacy on working memory but maintained morphological neurotrophic effects. Together, the results demonstrate the beneficial effect on working memory and neurotrophic efficacy of augmenting α5-GABAA-R function in old mice, suggesting symptomatic and disease-modifying potential in age-related brain disorders.

An efficient debromination technique using PMHS with a number of ligands containing different functional groups.

Herein is described the strategy to debrominate different aryl bromides selectively, using polymethylhydrosiloxane (PMHS) which tolerates a variety of functional groups. Key elements of this approach include the use of catalytic Pd(OAc)2 and the correct equivalents of polymethylhydrosiloxane (PMHS), in conjunction with aqueous KF. The present reaction process provides a strategic tool for the synthesis of a number of medicinally important molecules.

Design, synthesis and characterization of novel gamma­aminobutyric acid type A receptor ligands.

Antinociceptive ligand HZ-166 is a GABAA α2/α3 receptor subtype-selective potentiator. It has been shown to exhibit anxiolytic-like effects in rodent and rhesus monkeys, as well as reduced sedative/ataxic liabilities. In order to improve the metabolic stability of HZ-166, the ethyl ester moiety was bioisosterically replaced with 2,4-disubstituted oxazoles and oxazolines. The new analogs of HZ-166 were synthesized, characterized, and evalutated for their biological activity and docked in the human full-length heteromeric α1ß3γ2L GABAA receptor subtype CyroEM structure (6HUO). Importantly no sedation nor ataxia was observed on the rotorod for LKG-I-70 (6) or KPP-III-51 (6c) at 100 and 120 mg/kg, respectively. These was also no loss of righting response for either ligand.