Date of Award

May 2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

JAMES COOK

Committee Members

MAHMUN HOSSAIN, ANDREW PACHECO, ALEXANDER ARNOLD, XIAOHUA PENG, JAMES COOK

Abstract

DESIGN AND SYNTHESIS OF ACHIRAL AND CHIRAL GABA RECEPTOR SUBTYPE SELECTIVE LIGANDS FOR THE TREATMENT OF ANXIETY, DEPRESSION AS WELL AS EPILEPSYby Prithu Mondal The University of Wisconsin-Milwaukee, 2024 Under the Supervision of Professor James M. Cook Gamma-aminobutyric acid type A receptors (GABAAR) are pentameric ligand-gated chloride ion channels located in the membranes, which respond to gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter within the central nervous system (CNS). Benzodiazepines (BZDs) bind to the extracellular interface of the α+γ2-subunits of GABAAR. The binding of ligands to different subunits of GABAA receptors, particularly at α1-6β2/3 γ2 ion channels, can influence a broad spectrum of brain functions. The α1-subtype selective ion channels of GABAARs contribute to sedative, ataxic, amnesic, anticonvulsant, and addictive effects, which should generally be avoided, except for the desirable anti-anxiety and anticonvulsant effects, when designing ligands targeting this BZ allosteric modulatory site. GABAARs containing α2/3 subunits have been associated with anxiolytic, anticonvulsant, and antinociceptive activities. At higher doses, muscle relaxation may be mediated by interactions with α3 subtypes. It is well-established that α5-containing GABAARs play roles in cognition, learning, and memory processes. The dysregulation of GABA activity at α5 subtypes on GABAAR is implicated in the pathophysiology of various CNS disorders, including schizophrenia, major depressive disorder (MDD), bipolar disorder, and specific anxiety disorders like OCD. Additionally, α5 subtypes in the lung play a significant role in exploring potential new treatments for asthma. Drawing from the "privileged imidazodiazepine (IMZD) structures" derived from the unified pharmacophore model of Milwaukee, this research endeavors to design, synthesize, and assess the biological activities of over 120 novel chiral GABAAR α2/α3/α5 or α5 subtype-selective imidazodiazepines (IMDZs) related to SH-053-2'F-R-CH3 and its enantiomer iii SH-053-2'F-S-CH3. The objective is to develop new analogs with enhanced metabolic stability while retaining the desired biological properties with minimal or no side effects for the treatment of CNS disorders and asthma. A subset of compounds from the α5 subtype-selective group demonstrates anxiolytic, antidepressant, and pro-cognitive properties, making them promising candidates for treating major depressive disorders. Approximately 30 grams of a lead compound (GL-II-73) and several analogs were synthesized and assessed. The α5-selective chiral lead amide, GL-II-73, exhibits promising results in rodent models for depression treatment. It demonstrates excellent pharmacokinetic characteristics and displays antidepressant and anxiolytic effects in mice without inducing sedation or motor impairment typically associated with benzodiazepine drugs. Furthermore, it shows improved cognitive effects, which are crucial in treating depression or schizophrenia. Recent studies indicate that administering a single dose of GL-II-73 within 30 minutes can reverse stress-induced or age-related working memory deficits in old mice, restoring their performance levels to approximately 80-90%, nearly equivalent to those of young mice. Typically, old or stressed young mice perform at about 50-60%. Moreover, there was a remarkable reversal of brain cell shrinkage (dendrites and spines) in older mice, typically associated with aging, to a level similar to that of young mice. This exciting development occurred after two months of administering GL-II-73 in the drinking water of the older mice. These findings, presented by collaborator Dr. Etienne Sibille in an interview with the BBC, have resulted in three papers and two patents. There is optimism that the CAMH group led by Sibille can advance this project to human trials within two years, as proposed. Ongoing research by multiple groups continues to explore the potential of these key compounds, including their application in other CNS neurodegenerative diseases. Motivated by the results of GL-II-73( 2′F), a similar analogue was synthesized with the new analogue is different by a single atom (2′Cl). PM-II-26 (2 Cl GL-II-73) showed promising data as an antidepressant and a procognitive compound and further test is going on..Approximately 5g grams of this ligand has been synthesized in good yield to support various biological assays by our collaborators. To further test the 2′Cl series more amides and oxadiazoles were made. One of the oxadiazole PM-II-84E showed promising data as a precognitive compound even at slightly higher doses with no sedation and ataxia. This compound is a very good candidate for further testing. To enhance the iv development of new α2/3 subtype selective agents for treating anxiety disorders, neuropathic pain, epilepsy and asthma we implemented an improved large-scale synthetic method for GL-I-54, MP-III-023, PM-II-26 and PM-II-84E. These ligands, known for their anxiolytic, anticonvulsant, and antinociceptive properties, were synthesized under milder conditions with significantly improved yields. Epilepsy stands as one of the most prevalent neurological conditions, contributing significantly to neurological disability. Approximately 1-2% of the population grapples with this disorder, with about 33% enduring refractory epilepsy. While numerous drugs, including widely prescribed benzodiazepines like Valium and Xanax, are available for neurological disorders such as epilepsy and anxiety, they unfortunately lead to tolerance development within approximately three days, rendering them ineffective. Benzodiazepines bind to the α and γ interface of the gamma-aminobutyric acid type A (GABAA) receptor non-selectively, resulting in adverse effects, including tolerance, addiction, sedation, ataxia, somnolence, and confusion. For the first time, agents like HZ-166 and XHe-II-053, developed in Milwaukee, emerged as α2/α3 receptor subtype-selective ligands with anxiolytic and anticonvulsive activity, devoid of undesired side effects such as sedation and ataxia. However, the ester function in XHe-II-053 and HZ-166 could undergo metabolism into less active corresponding carboxylic acids, resulting in poor exposure, high clearance, and low blood-brain barrier penetration. The replacement of the ester with heterocycles (oxazoles and oxadiazoles) improved metabolic stability and pharmacokinetics, leading to the development of the clinically progressing non-sedating anticonvulsant KRM-II-81. As backup compounds, analogs were designed and synthesized based on the structure of KRM-II-81. Following docking in the human full-length heteromeric α1β3γ2 GABAA receptor subtype CryoEM structure (6HUO), several novel oxazole and oxazoline analogs exhibited potent anticonvulsant activity with enhanced in-vivo and in-vitro stability, free from cytotoxicity, sedation, ataxia, and loss of righting response. Among the many novel KRM-II-81 bioisosteres designed and synthesized, KPP-III-34 (8-bromo substituted imidazodiazepine) demonstrated improved target site (brain) exposure and metabolic stability in plasma and brain, leading to enhanced oral bioavailability ideal for pre-clinical studies. KPP-III-34 exhibited exceptional efficacy in various epilepsy models, including Lamotrigine-resistant seizure models, non-convulsive status epilepticus models, and chronic seizure models, via intraperitoneal (i.p.) or oral v administration in animals (mice or rats). Additionally, KPP-III-34 demonstrated a clean toxicity profile and selectively bound to the rat brain benzodiazepine receptor site without undesired binding to other receptors, according to PDSP (UNC) screens. The facile synthesis of KPP-III-34 was successfully executed on a large scale, requiring no column chromatography or toxic and expensive palladium. KPP-III-34 emerges as a potent and safe backup anticonvulsant agent for the treatment of epilepsy, complementing the clinically progressing KRM-II-81 (ETSP, NINDS). With these improved synthesis methods, we can now supply our collaborators with large quantities of these three α2/3-subtype selective GABAARs PAMs(KRM-II-81,KPP-III-34,DS-II-73 and MP-III-024 efficiently and with high quality. These compounds have demonstrated anxiolytic, anticonvulsant, and antinociceptive effects in numerous animal tests. The availability of gram quantities of these compounds enables further investigation into their ADME toxicity and behavior assays, including those in primates. This facilitates studies in animal models for anxiety, epilepsy, and neuropathic pain, which require substantial amounts of compounds. Additionally, it allows for the execution of long-term safety studies necessary for regulatory approval by the FDA.

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