Total Synthesis and Guanase Inhibition Studies of Azepinomycin and Its Analogs

The overall objectives of my dissertation project are to (a) provide a convenient and efficient total synthesis of the natural product azepinomycin, a known transition state analog inhibitor of the enzyme guanase, (b) separate and characterize the two enantiomers of azepinomycin, which have never been done before, (c) synthesize both heterocyclic and nucleoside analogs of azepinomycin, (d) determine the Ki of azepinomycin against an isolated mammalian guanase, which has never been done before, (e) assess the Ki's of the mentioned synthetic analogs, and (f) assess the effect of translocation of the 6-OH group of azepinomycin on the enzyme activity. Guanine deaminase (guanase) is an important enzyme involved in the nucleotide metabolism, and therefore, an important target in anticancer, antiviral, and antibacterial therapy. There have been reports of abnormally high levels of serum guanase activity in patients with liver diseases, and with multiple sclerosis. Increased levels of guanase have also been detected in cancerous kidney and breast tissue cells. These observations suggest that a potent guanase inhibitor is necessary for exploring the biochemical mechanisms of the above metabolic disorders as well to understand the specific physiological role played by guanase, and not to mention its potential therapeutic use in treating these disorders. Guanase catalyzes the hydrolysis of guanine to xanthine through an intermediate which contains a quaternary carbon at the site of hydrolysis, with a geminal hydroxy/amino functionality. While many studies on guanase inhibition have been reported in the literature, a potent guanase inhibitor with a submicromolar or nanomolar Ki has yet to be discovered. The natural product azepinomycin, a known transition state analog inhibitor of guanase, has so far been studied only in tissue culture systems. So, while its IC50 value is known, the Ki of azepinomycin against guanase has never been determined biochemically against an isolated pure enzyme. As a step forward in exploring this potent guanase inhibitor, an efficient and convenient synthetic strategy has been developed to access not only azepinomycin but also its two diastereomeric nucleoside analogs. In addition, some [5:7]-fused ring expanded heterocycles containing the imidazo[4,5-e][1,4]-diazepine-5,8-dione ring system, with appropriate substituents mimicking the transition state of the enzyme-catalyzed reaction, have also been successfully synthesized. The biochemical investigations were done to assess their Ki values of all target compounds against a mammalian guanase