Date of Award

May 2022

Degree Type


Degree Name

Doctor of Philosophy



First Advisor

James JC Cook

Committee Members

Alexander AA Arnold, Alan AS Schwabacher, Arsenio AP Pacheco, Nicholas NS Silvaggi


ach acetylcholine, AHR airway hyperresponsiveness, AP-4 4-Aminopyridine, ASM airway smooth muscle, AUC area under curve, BBB blood-brain-barrier


Part I: Design, synthesis, and biological evaluation of non-sedating anti-schistosomal agents

Schistosomiasis is a parasitic flatworm infection with only one drug therapy currently on the market, praziquantel. An old lead identified by Roche, meclonazepam (US Patent 4031078A) displayed antiparasitic efficacy but was not pursued due to dose-limiting sedation in human clinical trials (S. Afr. Med J. 1979 Apr 14;55(16):617-8, PMID: 380021). However, given the superior spectrum of activity meclonazepam displays relative to praziquantel (which is unable to cure immature parasites within the host) and advances in the understanding of benzodiazepine SAR at human GABAARs, the study of this benzodiazepine was resumed. Approximately 50 novel compounds were designed, synthesized, and tested for anti-schistosomal activity and 150 more from our BZR library. This resulted in many compounds that were active on the schistosome mobility assay. Of the various analogs ,which were synthesized MYM-III-10 (69), which contains a hydroxy group at the benzodiazepine C-3 position, was identified as the most active hit. MYM-III-10 (69) displayed anti-parasitic effects (causing parasite contractile paralysis in vitro and clearing the worm burden in vivo) comparable to meclonazepam, but was significantly less sedating in a mouse rotarod assay. It was also found that modification of the C3 position by replacing the ‘methyl’ in meclonazepam with a halogen resulted in the potent MYM-V-56 (84) that retains anti-parasitic effects (causing parasite contractile paralysis in vitro and clearing the worm burden in vivo) comparable to meclonazepam ,but was much less sedating in mice. This is a novel modification ,which eliminates the main side effects of this drug class, sedation and ataxia, which has so far prevented the clinical development of meclonazepam. When the enantiomers of MYM-V-56 (69) were compared, the S-MYM-V-56 was found to potently induce contractile paralysis on the worm muscle in vitro and clear the worms in vivo. A large-scale robust synthetic method was developed for both of these compounds (racemic) without employing any chromatographic purification in any step. Purification of compounds for each step simply required recrystallization by using different solvents such as ethyl acetate etc. Both MYM-III-10 and S-MYM-V-56 presented excellent preclinical data in different assays and further experiments will be performed to move these drugs toward the clinic for the treatment of this parasitic disease and other flatworm diseases (humans,cattle, horses,dogs, etc). Part II: Design, synthesis, and biological evaluation of chiral α5 subtype BZR/GABAAR modulators for the treatment of anxiety, major depressive disorder with agents that are procognitive and active against models of schizophrenia as well

Gamma (γ)-amino butyric acid, a naturally occurring amino acid, is a proteinogenic amino acid that acts as the principal inhibitory neurotransmitter in the CNS, as well as the cerebrum of the mammalian brain. The benzodiazepines (BZDs) bind at the extracellular interface of the α+γ2-subunits of GABAAR. The binding of ligands at different subunits affects a wide variety of brain functions. The α1-subtypes are associated with the anxiolytic sedative, ataxic, amnesic, anticonvulsant and addictive effects, while the α2/3-subtypes are responsible for anxiolytic, anticonvulsant, and antinociceptive activities. The α5-containing GABAARs in the CNS are involved in cognition, as well as learning and memory processes; α5-subtypes in lungs are related with asthma. Their dysfunction in the CNS is also involved in other CNS disorders.The need for improved medications for the treatment of anxiety and depression is essential. Anxiety is comorbid with depression. Currently there are no drugs on the market for depression or schizophrenia that treat the cognition problems. This is an unmet medical need. Based on the "privileged imidazodiazepine (IMZD) structure”, more than 150 novel α5 preferring ligands have been synthesized in Milwaukee for evaluation of pro-cognitive, anxiolytic, as well as anti-depressant activity. The lead, GL-II-73 (developed by Guanguan Li and later Daniel Knutson) was found to be anxiolytic, anti-depressant, as well as procognitive. A key finding. A large scale, industrial friendly synthetic strategy was developed for the lead, GL-II-73, without employing any chromatographic purification except for one-step and it was a flash column. Around 50 grams of the lead, GL-II-73 were synthesized and evaluated for additional biological activity. The ligand,GL-II-73, produced symptomatic, disease modifying, and neurotrophic effects in a mouse model of chronic stress. This ligand reversed the stress-induced or normal aging-related working memory deficits in old mice, as well as young mice. Further studies are ongoing by DAMONA PHARMACEUTICALS to move this ligand toward the clinic for depression and potentially Alzheimer's disease. In addition, more than 30 novel α5 subtype selective ligands were synthesized based on the structure of GL-II-73 and evaluated for pro-cognitive, anxiolytic as well as anti-depressant activity. Of the synthesized derivatives, the amide analogs MYM-III-41 (97) and MYM-III-29(106) showed pro-cognitive effects on primary and secondary screening in the Y-maze assay. These ligands will be tested in the forced-swim test and elevated plus maze to determine their anti-depressive and anxiolytic behavioral profile. Also, the 1,2,4-oxadiazoles MYM-V-28 (129), MYM-III-43 (134), and MYM-IV-47 (128) showed strong pro-cognitive profiles in the Y-maze assay. These ligands may well be key backups for ligand GL-II-73, but require further testing such as the forced-swim test, UCMS, elevated plus maze, metabolism, etc.

Part III: Design, synthesis and biological evaluation of α2/3-GABA(A)R subtype-selective imidazodiazepines to treat epilepsy, anxiety and neuropathic pain

The novel imidazodiazepine, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]-imidazole[1,5-α[1,4]diaze-pin-3-yl) oxazole ( KRM-II-81) is a positive allosteric modulator of GABAA receptors (a GABAkine) undergoing preparation for clinical development. Since salts can be utilized to create opportunities for increased solubility, enhanced absorption and distribution, as well as for efficient methods of bulk synthesis, a hydrochloride salt of KRM-II-81 termed MYM-V-26 (193) was prepared. This salt is 14 times more water soluble than the free base. The oral administration of MYM-V-26 (193) was similar to that of the free base in the brain to plasma ratio in rats. High levels of both compounds were exposed in plasma and brain of rats with about 1 µM concentrations in plasma and 0.5 µM in brain at Cmax. Oral dosing in mice significantly elevated the latency to both clonic and tonic convulsions and decreased pentylenetetrazole-induced lethality. For clonus, the anticonvulsant effects lasted for at least 8 h. The newly synthesized MYM-V-26 (193) exhibited improved physicochemical properties such as high water solubility and a lower partition coefficient value as compared to those of the free base, KRM-II-81. Kinetic solubility experiments also indicated that the HCl salt allows KRM-II-81 to remain in the solution state over a longer period of time. One key to this study is the increased water solubility of the salt permits i.v. dosing in primates instead of just i.p. or i.m. dosing, which will permit better safety and efficacy studies.

Part IV: Design, synthesis, analytical characterization, and biological evaluation of chiral α5 subtype GABAAR modulators for the treatment of asthma

The novel α5-selective ligand MIDD0301, ((R)-8-bromo-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4], diazepine-3-carboxylic acid), GL-II-93, is under development for the treatment of asthma by PANTHERICS. This lead compound, MIDD0301 exhibited relaxation ex vivo on human, guinea pig, and mouse airway smooth muscle (ASM), which express discrete GABAARs.A scalable and reproducible synthetic route for the lead asthma ligand MIDD0301 has been developed on multi-gram scale for large scale toxicity testing and for further biological evaluation. A number of impurities present in less than 2% are formed in each step during the large-scale synthesis. These impurities have been identified, synthesized and characterized by NMR, HPLC and LCMS. The carboxylic acid ligand MIDD0301 forms different metabolites in Phase I and Phase II metabolism studies. One of the phase II metabolites, MIDD0301-glucoronide (170) was synthesized to quantify this metabolite in vivo. In addition, more than 10 carboxylic acid analogs of MIDD0301 were synthesized and evaluated on an ex vivo ASM relaxation assay. Excitedly, MYM-III-100 (167) was the most potent compound in the ASM relaxation assay reported, to date . This ligand relaxed the ex vivo smooth muscle force in guinea pig tracheal rings more potently than the lead, MIDD0301. The 2’Cl version of MIDD0301, MYM-FR-II-88 (178) produced similar effects to MIDD0301 in the ASM relaxation assay. In addition, two other analogs MYM-III-20 (185) and MYM-V-71 (186) showed better ASM relaxation profiles than MIDD0301 in the ex vivo ASM relaxation assay. These newly synthesized ligands require further testing to establish the complete profile and to determine if they are better than MIDD0301 for the treatment of asthma.

Available for download on Friday, June 07, 2024