Date of Award
May 2023
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
James M. Cook
Committee Members
Alan Schwabacher, Xiaohua Peng, Alexander E. Arnold, Arsenio A. Pacheco
Abstract
Abstract-Part I
Nature has been an extensive and sustainable pool of biologically active compounds. Many molecules from nature are secondary metabolites, which are, presumably, used in the defense mechanisms of plants or organisms. These biologically active molecules have been a major source of medications including treatment of cancer, malaria, and antiarrhythmic agents for centuries. It is not a surprise that many clinical drugs are either directly inspired or derived from natural products. The natural bioactive alkaloids such as morphine, vincristine and reserpine exhibit biological activity, and contain their particular stereochemistry. Plants or organisms have evolved to produce, ordinarily, only one of the enantiomers. As a result, it is possible to isolate and screen for biological activity only one of the enantiomers, i.e., the natural enantiomer. Unnatural enantiomers might also be as good as the natural enantiomer or even better in activity, as well as in their toxicity profile depending on the metabolic pathway. For example, an unnatural L-nucleoside-based drug, lamivudine, has been saving millions of lives, and it is also listed in WHO’s essential drugs. The Indole scaffold is one of the few ‘privileged structures’ in modern medicinal chemistry because it has a structural similarity with the molecules of life such as tryptamine and serotonin. The Milwaukee group have developed an efficient and practical method for the total synthesis of a number of sarpagine/macroline/ajmaline- related bioactive natural products (indole alkaloids). Moreover, the ambidextrous Pictet-Splenger reaction/Dieckmann-cyclization process allows the synthesis of natural and unnatural enantiomers of sarpagine/macroline/ajmaline-type indole alkaloids either from D or L-tryptophan. However, the total synthesis of unnatural enantiomers of the C-19 methyl substituted sarpagine/macroline/ajmaline indole alkaloids was not previously executed. By employing the Pictet-Spengler reaction/Dieckmann-cyclization strategy on large scale, the total synthesis of C-19 methylated macroline-type indole alkaloid, (+)-talcarpine, was completed for the first time. The opposite absolute stereochemistry for the unnatural enantiomer was successfully controlled in each step of the synthetic pathway. The desired stereochemistry was confirmed by data from X-ray crystallography of key intermediates (tetracyclic ketone core and pentacyclic ketone core), as well as by comparison of the optical rotation values to that reported for the natural enantiomer. In conclusion, both strategies developed in Milwaukee: the unified Pictet-Spengler reaction/Dieckmann cyclization process, and the ambidextrous Pictet-Spengler reaction/Dieckmann cyclization process work for the synthesis of both natural, as well as unnatural C-19 methylated sarpagine/macroline/ajmaline-type indole alkaloids.
Abstract-Part-II
Epilepsy is one of the most commonly occurring neurological conditions and the ultimate cause of neurological disability. It affects 1-2 % of the total the population and among those patients, about 33% suffer from refractory epilepsy. Many drugs including the highly prescribed benzodiazepine class (such as Valium and Xanax) are available on the market for the treatment of neurological disorders such as epilepsy and anxiety. Unfortunately, the patients develop tolerance to the benzodiazepines, and they are useful in status epilepticus for about 3 days and are then no longer effective. It is well understood that the benzodiazepine class of drugs bind to the alpha and gamma interface of the gamma-aminobutyric acid type A (GABAA) receptor without selectivity at the subtypes. This non-selectivity results in adverse effects, and thus these drugs contain a plethora of side effects such as tolerance, addiction, sedation, ataxia, somnolence, and confusion. For the first time, agents such as HZ-166 (6) and XHe-II-053 (5) (developed in Milwaukee) were found to be alpha 2/alpha 3 receptor subtype-selective ligands with anxiolytic and anticonvulsive activity, importantly, devoid of unwanted side effects including sedation and ataxia. However, the ester function in XHe-II-053 (5) and HZ-166 (6) could undergo metabolism into the less active corresponding carboxylic acid. This result 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, and this led to the clinically progressing non-sedating anticonvulsant KRM-II-81 (7). As back-up compounds, analogs were designed and synthesized in this work based on the structure of KRM-II-81 (7). After docking in the human full-length heteromeric alpha1/beta3/gamma2 GABAA receptor subtype CryroEM structure (6HUO) [1], by following the published procedures by Masiulis et al. Nature, 2019, we tested their biological activity. Importantly, several novel oxazole and oxazoline analogs exhibited potent anticonvulsant activity with improved in-vivo and in-vitro stability in the absence of cytotoxicity, sedation, ataxia, and loss of righting response. Among many novel KRM-II-81 (7) bioisosteres designed and synthesized, KPP-III-34 (9) (8-bromo substituted imidazodiazepine) exhibited an improved target site (brain) exposure, as well as metabolic stability in plasma and brain. This improved oral bioavailability as exactly as desired for pre-clinical studies. This KPP-III-34 (9) demonstrated exceptional efficacy (anticonvulsant activity) in many epilepsy models including the pentylenetetrazole (metrazole) induced clonic and tonic seizure models (in mice), two acute seizure models (6 Hz and maximal electroshock seizure model), corneal kindled seizure models (in mice), and it antagonized convulsions in the mesial temporal lobe epilepsy model (in mice). More importantly, KPP-III-34 (9), was found fully potent in the therapy (Lamotrigine)-resistant seizure model (in rats), non-convulsive status epilepticus model (in mice), and chronic seizure model (in rats). KPP-III-34 (9) exhibited very potent anticonvulsant activity either via intraperitoneal (i.p.), or oral administration in animals (mice or rats). This 1, 4-oxazole also showed impressively clean results in a toxicity profile. In addition, in the PDSP (UNC) screens, out of the 46 receptor and ion channel panel, it selectively bound to the rat brain benzodiazepine receptor site (homogenate) with >92 mean % inhibition in the primary screen, and without undesired binding to the other receptors including HERG. The KPP-III-34 (9) did not show sedation, ataxia, and loss of rightening response (up to 120 mg/kg), and it was non-toxic in the HEK-293 cell line (Dr. Arnold group, UWM). There were no tremors observed in this mouse model, whereas in KRM-II-81 (7) in mice, tremors were observed at 100 mg/kg dose. This is indirect evidence that mouse metabolism affected the ethynyl function, but not in rats, and this was the source of tremors. Moreover, no behavioral effects were seen up to the very high oral dose of 500 mg/kg in rats. The facile synthesis of KPP-III-34 (9) was explored and executed successfully on large scale in 28.7% overall yield without employing column chromatography in any synthetic step and required no toxic and expensive palladium. Also, the intermediates and product were either crystallized or precipitated out to facilitate scale up. The synthesis of KPP-III-34 (9) is one step shorter than the synthesis of KRM-II-81 (7). And thus, the large-scale synthesis of KPP-III-34 (9) can be executed easier and quicker than KRM-II-81 (7). Thus, GABAA potentiator and alpha2/alpha3 receptor subtype-selective (presumably), KPP-III-34 (9) is undoubtedly a potent and safe back up anticonvulsant agent of the clinically progressing KRM-II-81 (7) for the treatment of epilepsy (ETSP, NINDS).
Recommended Citation
Pandey, Kamal Prasad, "Part I Enantiospecific Total Synthesis of Unnatural Enantiomers of C-19 Methylated Sarpagine/Macroline/Ajmaline-Type Biologically Active Indole Alkaloids via the Pictet-Spengler Reaction/Dieckmann Cyclization Process. Part II Design, Synthesis, and Characterization of Novel Gamma-Aminobutyric Acid Type A (GABAA) Receptor Ligands for the Treatment of Neurological Disorders Including Epilepsy, Depr" (2023). Theses and Dissertations. 3109.
https://dc.uwm.edu/etd/3109