Efficient access to functional bryostatin analogs [electronic resource] : the design, synthesis, and biological evaluation of novel bryostatin analogs

The marine-derived bryostatin family of natural products represents a structurally complex molecular scaffold that has inspired a large body of synthetic and mechanistic research relevant to a number of human disease indications. Interest in the therapeutic use of bryostatin began in 1968 following a report by Pettit and coworkers that an organic extract from Bugula neritina demonstrated anticancer activity. Since its subsequent isolation and structural characterization in 1982, bryostatin has garnered increasing attention from synthetic chemists, biologists, and clinicians due to both its inherent molecular complexity as well as the wide variety of biological activities this natural product has exhibited. Bryostatin has demonstrated several anticancer activities including the promotion of apoptosis, reversal of multidrug resistance, stimulation of the immune system, and synergism with other oncolytic agents. As a result of this astounding biological activity, bryostatin 1 has been evaluated in over 30 Phase I and II clinical trials for anticancer treatment, primarily in combination with other anticancer therapeutics. Bryostatin has more recently proven capable of enhancing memory and learning in several animal models, attributable in part to its ability to induce the formation of new synaptic contacts in the brain. Bryostatin has also exhibited neuroprotective effects in animal models of cerebral ischemia. This collection of impressive neurological activities suggests a potential therapeutic use of bryostatin in the treatment of stroke, Alzheimer's disease, and other neurodegenerative disorders. Indeed, a Phase II clinical trial to explore the use of bryostatin in the treatment of Alzheimer's disease has been opened. Even more recently, bryostatin has been reported to activate latent HIV reservoirs, providing a potential first-in-class strategy for the eradication of HIV/AIDS. Unfortunately, the low natural abundance of the bryostatins from their natural source or from biological and total synthetic efforts has limited their availability for further preclinical and clinical research and development. To circumvent issues related to the supply problem of bryostatin, while providing opportunities for improved therapeutic function, the Wender research group has focused upon the design and synthesis of structurally-simplified, step-economical bryostatin analogs that exhibit biological activities comparable or superior to the natural product. This strategy, dubbed "function-oriented synthesis" (FOS), relies upon recapitulation of the biologically-active lead structure on a simplified scaffold, retaining elements necessary for function, while simplifying or eliminating those that are functionally unnecessary, thereby rendering the target more readily accessible through chemical synthesis. Application of this approach has resulted in the synthesis of a library of over 100 bryostatin analogs, the most promising of which are currently undergoing preclinical investigation for their use in the treatment of cancer, Alzheimer's disease, and the activation of latent HIV reservoirs. Chapter 1 provides the background and justification for the remainder of the dissertation. A brief overview of the bryostatins, their potent biological activities, and an in-depth discussion of the various biological and total synthetic efforts aimed at their access is provided for context. Also included is a description of the pharmacophore hypothesis first advanced by our group in 1988, and its application in in the design and synthesis of non-natural functional bryostatin analogs. The general synthetic routes utilized to access these simplified bryostatin analogs is included, as well as a brief description of the biological activities and preclinical investigation of these agents. Chapter 2 provides a review on the principal intracellular target of the bryostatins, protein kinase C (PKC). In addition to an overview of its primary structure, a detailed discussion is provided of the published structural studies of the PKC C1 binding domain, how these studies have aided in determining how our designed analogs might interact with PKC, and a brief discussion of the shortcomings of these studies. Chapters 3 and 4 detail the design and synthesis of a family of novel peripherally-functionalized bryostatin analogs. Preliminary observations suggested that the design of highly simplified analogs lacking functionalization around the northern periphery of the bryostatin scaffold might be an oversimplification, and that A-ring functionality in particular could be utilized to modulate PKC selectivity. A library of diversified A-ring functionalized analogs was designed to further probe the role of peripheral functionalities upon PKC affinity and selectivity. While each analog displayed a potent binding affinity for PKC, this collection of analogs covered a range of selectivities in which some members emulated the PKC functional selectivity of the natural product, bryostatin 1, whereas others exhibited complementary functional selectivities. In addition to these preliminary results, Chapter 4 presents the evaluation of a subset of these analogs in several in vitro and ex vivo assays relevant to HIV/AIDS eradication efforts. These simplified bryostatin analogs demonstrated a potent ability to activate latent HIV viral reservoirs without the concomitant toxic production of high levels of proinflammatory cytokines. In addition, these analogs exhibited the ability to downregulate the expression of CD4, CCR5, and CXCR4 cell surface receptors, indicating that they may help inhibit de novo infection of healthy cells. Finally, these analogs efficiently induced viral reactivation in ex vivo samples isolated from HIV-infected patients on viral suppressive therapy, indicating for the first time that simplified analogs of bryostatin function as desired in the ultimate target of HIV eradication efforts. Finally, Chapter 5 provides an introduction to an exciting collaboration that has been established between the Wender and Cegelski research groups directed at the first experimental determination of the bryostatin binding conformation when bound to PKC in a phospholipid membrane environment using rotational-echo double-resonance (REDOR) solid-state NMR. This project brings together several different areas from computational modeling, target design, chemical synthesis, biosynthesis, biological activity assays, and solid-state NMR. Included in this chapter is the design and synthesis of a novel series of isotopically labeled bryostatin analogs, as well as preliminary solid-state NMR results.

Keywords: Chemistry.
Advisor: Wender Paul A. primary advisor. Thesis advisor
Huestis Wray advisor. Thesis advisor
Wandless Thomas advisor. Thesis advisor