Date of Award

December 2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Alexander Arnold

Committee Members

Shama Mirza, Nicholas Silvaggi, Christopher Cunningham, Xiaohua Peng

Abstract

Abstract Part I:

Calcitroic acid (CTA) was first isolated and characterized over four decades ago.6 At that time, radiolabeled calcitriol (1,25-dihydroxyvitamin D3) was utilized to facilitate the detection of radioactive CTA generated in vivo. Subsequently, researchers extracted and characterized CTA through derivatization.7 Notably, CTA was predominantly formed in the liver and transported to the gut through the bile duct via enterohepatic circulation, resulting in significant concentrations of this vitamin D metabolite in the intestines.8 However, since it was initially regarded as merely a breakdown product of calcitriol, it remained largely overlooked.Recent groundbreaking experiments have unveiled a new dimension to CTA's biological role. It has been revealed that CTA can bind to the vitamin D receptor (VDR) and initiate the regulation of specific metabolic enzymes.9 VDR, a nuclear hormone receptor found in various tissues, is capable of binding to several endogenous ligands. While calcitriol exhibits the highest affinity for VDR and has undergone extensive investigation, our knowledge regarding the principal end-stage metabolite of vitamin D, calcitroic acid, remains limited. Initially considered biologically inactive due to its weaker binding to VDR compared to calcitriol, recent findings challenge this assumption. Recent research has demonstrated gene regulation in the presence of CTA in epithelial cells, keratinocytes, and prostate cancer cells.8 One proposed role of VDR in the intestine is the regulation of P450 enzymes, critical for detoxification processes, especially concerning the accumulation of harmful endogenous molecules such as lithocholic acid. Our hypothesis states that CTA interacts with VDR to regulate this process, potentially safeguarding against the development of inflammation-related gastrointestinal disorders, including irritable bowel syndrome and colorectal cancer. Here is presented a detailed report of the 12-step synthesis to obtain CTA.

Abstract Part II:

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1β3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator DAW-III-30 with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

Abstract Part III:

The synthesis of 5,5′-bis(trifluoromethyl)-2,2′-bipyridine using 2-bromo-5-(trifluoromethyl) pyridine was achieved at 50 °C using palladium acetate, tetrabutylammonium iodide (TBAI), potassium carbonate, and isopropanol in Cyrene™ (dihydrolevoglucosenone), a bio-renewable “green” solvent formed by a two-step process from cellulose. Improvements were achieved with 50% of γ-valerolactone (GVL) in Cyrene™ resulting in a 95% yield and 99% product purity without the use of column chromatography or recrystallization. At 80 °C, the reaction was completed within 1 h. Full conversion with 1 mol% instead of 15 mol% of palladium acetate was observed within 10 h. We showed that the formed 2,2′-bipyridine product significantly accelerated the reaction probably due to the stabilization of the catalytic species. The addition of TBAI was essential for the rapid homocoupling, however, 20 mol% of TBAI was sufficient to reach full conversion of 2-bromo-5-(trifluoromethyl) pyridine within 6 h at 80 °C. Another improvement was observed with the substitution of isopropanol by 1,4-butanediol achieving full conversion within 6 h. 2-Bromopyridines with electron withdrawing substituents in the 6, 5, 4 ring position reacted under these conditions. 2-Bromopyridines with an electron donating substituent reacted slower. Overall, we demonstrated that the 50% GVL in Cyrene™ blend is a superior “green” and less toxic alternative to dimethylformamide for the reductive homocoupling reaction. Using a quantitative scoring for twelve principles of green chemistry (DOZN™), we found significant improvements that were mediated by higher yield (atom economy), shorter heating time and lower reaction temperature (energy efficiency), safer solvent (hazardous chemical synthesis), and safer chemistry (accident prevention).

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