Natural products continue to influence society at large as many drugs are of natural product origin. Consequently, the development and study of new chemical reactions that lead to the efficient preparation of natural products is a central goal in organic chemistry today. To this end, we have investigated the reactivity and stereoselectivity of cyclic 2-(N-acylamino)-1,3-dienes toward a total synthesis of stenine. Chapter I describes the isolation and characterization of stenine, in addition to comparing five published total syntheses of stenine. Chapter II discusses synthetic and theoretical investigations of cyclic 2-(Nacylamino)- 1,3-dienes. We have shown that vinylazepenes react with Nphenylmaleimide to afford exo cycloadducts exclusively. Previous studies of vinylpiperidenes revealed a preference for endo selectivity. We confirmed the contrasting stereoselectivity by X-ray analysis of compounds i and ii. The stereoselectivity of vinylazepenes, vinylcycloalkenes, and vinyl piperidenes in Diels-Alder reactions varies depending on the detailed structure of the diene. In collaboration with Dr. Daniel Singleton and Jennifer Hirschi, DFT calculations were utilized to study model reactions of these dienes to provide insight into controlling the stereochemistry of this class of Diels-Alder reactions. Chapter III describes the synthesis of compounds such as iv and v that were utilized in intra and intermolecular Diels-Alder approaches toward the synthesis of stenine. We demonstrated that intermediate viii could be constructed from vinyl stannane vii in ten steps via an intermolecular Diels-Alder reaction of v and dimethyl fumarate (vi). Finally, we propose a synthetic plan for the completion of stenine from advanced intermediate viii.

Cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species and a decreased expression of enzymatic antioxidants. Prodrugs inducing additional oxidative stress can selectively induce apoptosis in cancer cells already having a high level of intrinsic oxidative stress. This study focused on the rational design and in vitro evaluation of a novel prodrug ester, (4- [(nitrooxy)methyl]phenyl-N-acetyl-L-alaninate or NPAA) activated by acylamino-acidreleasing enzyme (AARE, EC 3.4.19.1) to yield a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. NPAA shares structural features with both nitric oxide donating aspirin (NO-ASA), a wellcharacterized QM releasing anticancer prodrug, and N-acetyl-L-alanine-4-nitroanailide (AANA), a known specific substrate for AARE. AARE is a serine peptidase that is overexpressed in some tumors and cancer cell lines. The overall approach was to first predict the 3-dimensional structure of both rat (rAARE) and human AARE (hAARE) and then use the resulting low-resolution models to determine if NPAA was a plausible prodrug by estimating its affinity to hAARE and rAARE in comparison to AANA. The AARE models were constructed using a bioinformatic-based protein structure prediction webserver (I-TASSER) followed by energy minimization and refinement. The resulting models were subjected to a variety of structural quality assessments. The optimal models of hAARE and rAARE were found to have similar three-dimensional structures with a ß- propeller domain and an a/ß-hydrolase domain containing an exopeptidase catalytic site with active site residue distances typically found in serine peptidases. Protein-ligand docking studies showed that both AANA and NPAA could bind to the exopeptidase catalytic site of the hAARE and rAARE models with reasonable affinities and in a region with a highly druggable pocket. In order to validate the in silico results, NPAA was synthesized, purified, physically characterized and evaluated for its in vitro ability to deplete GSH in the presence of rAARE. As anticipated, NPAA was found to deplete GSH and this effect was completely blocked by diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, including rAARE. These studies support further efforts to optimize the design of QM releasing anticancer prodrugs targeted to AARE. Moreover, the molecular models presented here could be useful for the rational design of AARE inhibitors, which could also be exploited as potential anticancer agents.