Function oriented synthesis of PKC modulators : tigilanol tiglate, bryostatin 1, and analogs; and function oriented synthesis of bio-orthogonal ruthenium catalyst linkers and cyclopropanated charge-altering releasable transporters
- While natural products have served as the inspiration and basis for the development of therapeutically relevant molecular agents, semisynthetic derivatives of natural products or synthetic compounds inspired by natural products outnumber natural products themselves in the clinic by almost tenfold. This highlights the potential for chemical synthesis to introduce structural modifications, which, small or great, have the potential to augment biological activity and function and to offer opportunities to enhance or change selectivity, potency, and tolerability of such compounds for various clinical indications. Moreover, the development of scalable, step-economical, and supply-impacting synthetic routes has in many cases eclipsed the ability of conventional means of isolation to produce target compounds in an economical and practical fashion, and this has indeed been the case in the current development of our group's total synthesis of Bryostatin 1, which has led to a GMP scaleup endeavor to supply clinical trials in the United States and elsewhere. Protein kinase C (PKC), a membrane-associated kinase protein that is endogenously activated by (S)-diacylglycerol, lies at the crossroads of several biochemical pathways related to treatment of a diverse array of clinical indications, including HIV/AIDS eradication, neurodegenerative disease, and various cancers. Through the sans of time, nature has developed and fine-tuned complex natural product scaffolds that effect selective and potent modulatory activity against PKC, and several of these compounds have advanced to preclinical and clinical trials, including prostratin, Bryostatin 1, and, most recently, tigilanol tiglate (EBC-46), among a myriad of other natural products. Unlike previously developed inhibitors of various cellular pathways, compounds that modulate protein kinase C have the unique ability to exert control over PKC activation and formal inhibition, and by extension, PKC-related pathways that reach a much broader scope of clinical indications, as outlined above. Thus, the driving motivation for developing robust syntheses of natural products and their analogs is twofold - to not only provide scalable access to supply-impacting quantities of biologically active and therapeutically relevant complex natural products, but also to develop means of creating new structures with novel and perhaps comparable or superior function that are not accessible through natural means. Specifically, our group has been inspired by the structural complexity and biological potency of the aforementioned ligands that modulate the activity of protein kinase C. In this dissertation, I hope to detail my and our efforts in using synthesis, coupled with computer modeling and chemical biology, to enable access to and expand nature's arsenal of a host of complex bioactive natural products. In Chapter 1 of this dissertation, a brief survey of the clinical relevance of protein kinase C will be presented, along with compounds which modulate its activity. Several of these compounds have demonstrated remarkable clinical and/or preclinical activity in human trials and in animal trials and present highly compelling solutions to a host of global biomedical challenges, including HIV-AIDS, cancer, neurodegenerative disease, and others. With respect to small molecule modulators of PKC, I will present a brief history on the identification of diverse natural products and synthetic compounds that play unique roles in modulating the activity and function of PKC, as well as the current competitive landscape surrounding PKC as a therapeutic target for small molecule discovery. Finally, Chapter 1 will conclude with a brief synopsis of Function-Oriented Synthesis (FOS) as a unifying and driving force in modern organic chemistry. In Chapter 2, I will detail the first laboratory synthesis of EBC-46 (tigilanol tiglate), an epoxytigliane natural product first isolated from F. picrosperma in the remote Atherton tablelands of northeastern Australia, which has since been approved by the FDA and EMA for treatment of mast cell cancers in canines, and is quickly advancing through Phase II clinical trials for the treatment of human cancers. The synthetic route described herein, which was developed with co-workers Zachary Gentry, David Fanelli, Quang Luu-Nguyen, and Owen McAteer, accomplished in twelve steps and 12% overall yield (more recently, further optimized to eleven steps, and 14% overall yield), features a high-efficiency photo-oxidation of the tigliane B-ring, a highly selective rhenium (VII)-catalyzed allylic rearrangement, and a reversal of substrate-controlled diastereoselective epoxidation with installation of a key acetonide protecting group. Moreover, this synthesis presents a practical solution to the supply of this remarkable compound for advancing clinical and preclinical studies. More broadly, we demonstrate the first synthetic installation of a 5-β-hydroxy-6,7-α-epoxy B-ring oxidation pattern characteristic of several tigliane and daphnane diterpenoids with unique biological activities. Finally, initial biological studies on the isoform-selective PKC binding by EBC-46 is presented. With the first laboratory synthesis of EBC-46 accomplished, Chapter 3 will first describe our efforts in the chemical synthesis of novel analogs of EBC-46 to more closely interrogate its structure-activity relationship. Specifically, I will describe the preparation of B-ring analogs developed with Zach Gentry, which systematically probe which B-ring structural features are particularly responsible for the unique biological activities observed in the natural product. Secondly, I will describe the first preparation of heteroatom-isotere analogs at C-13, where what was once an ester responsible for forging a critical trans-annular hydrogen bond with a proximal hydroxyl is exchanged for carbonate or carbamate moieties. Finally, Chapter 3 will describe a parallel effort where the photo-oxidation of the tigliane B-ring first described in Chapter 2 is exploited in the laboratory synthesis of three antiviral tigliane natural products: Trigowiin A, a tigliane diterpenoid with anti-chikungunya virus activity, pursued with co-workers David Fanelli and Quang Luu-Nguyen, and studies towards the synthesis of two related 12-benzoyl anti-HIV tigliane diterpenoids, Dapholosericin A and Wikstorcin A, each accessed in 3-4 steps from phorbol. Additionally, analogs of these are prepared and evaluated for PKC binding activity. Chapter 4 outlines progress on the development of simplified analogs of another class of PKC-modulating small molecules: the bryostatin family of macrolides first isolated from B. neretina, and their synthetic analogs. The first part of Chapter 4 will detail an updated synthetic route to access SUW-133 with a greatly reduced step count, featuring application of an improved protocol for an asymmetric allylation published by other co-workers in the group. Additionally, this section will outline preliminary results from the use of SUW-133 as an adjuvant in activation of CD22 surface markers as an indication of its potential efficacy in enhancing cancer immunotherapy for patients suffering from acute lymphoblastic leukemia with low antigen density; and of Nf-kB, a key transcription factor implicated in the eradication of HIV in being connected to activation of pathways connected to Bryostatin 1's role in activation of latent reservoirs of HIV-infected cells. The second half of Chapter 4 will outline the chemical synthesis of six novel des-A-ring simplified analogs of bryostatin 1, which are synthesized in collaboration with co-worker Alok Ranjan, and elaborating on earlier studies with an aryl bromide in place of the A-ring pyran as a tunable site for late-stage cross coupling modifications. These simplified analogs are synthesized through a diversification node, which is accessed in 22 steps (15 LLS) in 8% overall yield. With this novel series of analogs, which all exhibit potent pan-PKC binding but only selective isoform translocation, we show that PKC binding affinity is decoupled from translocation. Further, in assays led by co-worker Zhijian Li, we demonstrate the activity of these compounds in eliciting surface antigen expression of CD22 and intracellular activation of Nf-kB in connection to their potential application in enhanced cancer immunotherapy and HIV latency reversal, respectively. Chapter 5 highlights progress towards the development of a bio-orthogonal organometallic ruthenium catalyst to be conjugated to a biomacromolecule targeting system such as a monoclonal antibody or cell targeting peptide. Towards this end, I describe the chemical synthesis of two novel ruthenium catalyst-maleimide linkers which can be efficiently conjugated to thiol-containing peptides or proteins, synthesized in five steps from commercially available (and inexpensive) starting materials. Finally, as a preliminary proof-of-concept study, both catalyst-linker constructs are demonstrated to deprotect allyl carbonate-protected prodrugs of PKC modulators and other clinically relevant compounds such as taxol. Remarkably, catalyst activity is found to be greatly perturbed by the presence of a single methylene unit in the linker. Further, this chapter will discuss preliminary progress towards conjugation of these constructs onto a model monoclonal antibody and to a polyarginine cell-penetrating peptide. Finally, in Chapter 6, I describe an effort in the synthesis of metabolically stable cyclopropanated lipids within the context of nucleic acid delivery systems. Inspired by the structural and functional diversity of cyclopropanated fatty acids found in some bacterial lipid membranes putatively responsible for membranes with exceptional tolerance to heat, acid, and oxidative stress, we applied this bio-inspired design and prepared a selection of cis- and
- trans-, and linear and branched cyclopropanated lipid MTC monomers. These were subsequently oligomerized by Harrison Rahn into charge-altering releasable transporters (CARTs) and evaluated by Gillian Sun for efficacy in the delivery and release of mRNA cargoes in Jurkat cells. Remarkably, a cyclopropanated nonenyl CART outperforms our previous lead CART system, which featured a coblock oligomer of cis-unsaturated oleyl and nonenyl lipid side chains. Importantly, this finding suggests that this new lead cyclopropane-CART may present a more metabolically and oxidatively durable system for mRNA delivery than our previous lead systems.
|Type of resource
|electronic resource; remote; computer; online resource
|1 online resource.
|Wender, Paul A
|Wender, Paul A
|Du Bois, Justin
|Degree committee member
|Degree committee member
|Du Bois, Justin
|Stanford University, School of Humanities and Sciences
|Stanford University, Department of Chemistry
|Statement of responsibility
|Submitted to the Department of Chemistry.
|Thesis Ph.D. Stanford University 2023.
- © 2023 by Edward Njoo
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