Date of Award

May 2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

James M Cook

Committee Members

Alexander E Arnold, Alan W Schwabacher, Arsenio Andrew Pacheco, Graham Moran

Keywords

Asthma, Depression, GABA(A)R, Imidazodiazepine, Pain, Schizophrenia

Abstract

Gamma-aminobutyric acid type A receptors (GABAAR) are transmembrane pentameric ligand-gated chloride ion channels that respond to GABA, the major inhibitory neurotransmitter in the central nervous systems (CNS). The benzodiazepines (BZDs) bind at the extracellular interface of the α+γ2-subunits of GABAAR. The binding of ligands at different subunits of GABAA receptors specifically at α1-6β2/3 γ2 ion channels, can affect a wide variety of brain functions. The α1-subtype selective ion channels of GABAARs are involved in the sedative, ataxic, amnesic, anticonvulsant and addictive effects, which should be avoided, with the exception of the anticonvulsant effects, when designing ligands for this BZ allosteric modulatory site. The α2/3-containing GABAARs have been implicated by many, in the anxiolytic, anticonvulsant, and antinociceptive activities. At higher doses, muscle relaxation may be mediated by interaction at α3 subtypes. It is known that α5-containing GABAARs are involved in cognition, as well as learning and memory processes. The disruption of GABA activity at α5 subtypes on GABAAR plays a role in the pathophysiology of CNS disorders such as schizophrenia, major depressive disorder (MDD), bipolar disorder and certain anxiety disorders such as OCD. The α5 subtypes in the lung play a major role in potential new treatments for asthma.

Based on the "privileged imidazodiazepine (IMZD) structures" derived from the unified pharmacophore model of Milwaukee, the design, synthesis and biological activities of more than 120 novel chiral GABAAR α2/α3/α5 or α5 subtype selective imidazodiazepines (IMDZs) related to SH-053-2'F-R-CH3, and its enantiomer SH-053-2'F-S-CH3, as well as the key achiral ligand Hz-166 are described. The goal of this research is to develop new analogs with improved metabolic stability that also retain the desired biological properties with little or no side effects for the treatment of CNS disorders, as well as asthma.

Several compounds from the α5 subtype selective group are anxiolytic, antidepressant, as well as pro-cognitive, and are targeted toward the treatment of major depressive disorders. About 20 grams of one lead compound (GL-II-73), and more than 80 analogs were designed, synthesized and evaluated. The α5 selective chiral lead amide, GL-II-73, looks very good in rodent models for the treatment of depression. It exhibited an excellent pharmacokinetic profile, as well as anti-depressant and anxiolytic activities in mice. This antidepressant effect has been realized with no side effects such as sedation nor motor impairment that benzodiazepine drugs would normally effect. In addition, the much sought after improved cognitive effects were clear in mice; this is a key in treating depression or schizophrenia. Examination of the data in the most recent study indicated that the administration of a single dose of GL-II-73 within 30 minutes could reverse the stress-induced or normal aging-related working memory deficits in old mice back up to 80-90 %, which is almost at the level of a young mouse. Whereas the old mice or stressed young mice will normally perform at about a level of 50-60 %. Furthermore, the brain cells, which had shrunk in older mice associated with aging, grew back (dendrites and spines) to a level that is similar to the young mice. This is extremely exciting and occurred after two months of the administration of GL-II-73 to the old mice in their drinking water. These results were presented by our collaborator, Dr. Etienne Sibille in an interview with the BBC, but also resulted in three papers and two patents. It is hoped the CAMH group of Sibille can push this project forward to human trials within two years as proposed. Additional research on these key compounds by several groups is ongoing. One can imagine the use of GL-II-73 or analogs in other neurodegenerative diseases of the CNS.

After evaluation of a series of novel IMDZs that were α5 subtype selective for the treatment of asthma, one compound MIDD0301 (originally named GL-II-93) was found to be orally active and also as an inhalant in rodent asthma models. Ligand MIDD0301 was active in the airway smooth muscle (ASM) relaxation assay, in the airway hyper-responsiveness model, in the human and guinea pig tracheal bath assays. Moreover, it does not induce any immunotoxicity in animal models. Taken together this data suggests that this novel ligand can be a potential candidate for the treatment of asthma. About 50 grams of the ligand has been synthesized in good yield to support all the different biological assays from our collaborators. Furthermore, the large scale total synthesis of MIDD0301 was presented to the Alcami Corporation for a proposed kilogram scale synthesis of GMP material for further research on toxicity and safety. This research has resulted in three new publications over the last year. This compound provided some of the impetus to found a new company, Panthergenics, to try to get it through the FDA. Dr. Arnold and Dr. Stafford founded the company via UW-Milwaukee.

In regard to the design of new α2/3 subtype selective agents to treat anxiety disorders, neuropathic pain and epilepsy, an improved large scale synthesis of Hz-166, MP-III-080, and KRM-II-81 was executed. These ligands are anxiolytic, anticonvulsant and antinociceptive agents. They were prepared under milder conditions and with much improved yields. Moreover, an alternative reduction process, which was developed for the synthesis of the key aldehyde intermediate from the ethyl ester, was optimized. This new process overcame the long-standing problem of imine reduction in good yield. These methods can be employed for larger scale reactions. In addition, there is no need for column chromatography for most of the compounds, which provided a much more practical route. With the improved synthesis, large quantities of these three α2/3-subtype selective GABAARs PAMs, which exhibited anxiolytic, anticonvulsant and antinociceptive effects in many animal tests, can be supplied with high-efficiency and quality to our collaborators. The large-scale synthesis and the analog research will permit investigation of ADME toxicity and for other behavior assays including those in primates. Studies in animal models for anxiety and epilepsy, as well as for neuropathic pain require gram quantities of compounds, which can now be provided. This will also permit the execution of long-term safety studies in order to approach the FDA.

Available for download on Thursday, May 20, 2021

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