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The design, synthesis, and biological evaluation of novel protein kinase C ligands based on the bryostatin and diacylglycerol scaffolds [electronic resource]

The protein kinase C isozyme family has been implicated in a number of diseases representing the majority of the most significant challenges to global human health, including cancer, neurodegenerative diseases (Alzheimer's disease, depression, schizophrenia, etc.), cardiovascular disease, HIV/AIDS, diabetes, and chronic pain. As such, potent and selective modulation of this enzyme family using small molecule ligands designed for a particular function has tremendous therapeutic potential. A number of agents have been identified as ligands for the C1 domain of protein kinase C. Many of these compounds, including the endogenous ligand diacylglycerol and the complex bryostatin family of natural products, act by inducing an initial activation event, resulting in the translocation of protein kinase C to the plasma membrane where it can participate in the phosphorylation of downstream serine and threonine residues. The bryostatins are complex macrolides isolated from the marine organism Bugula neritina. Although a number of agents from this family of natural products have been shown to be biologically active, bryostatin 1 in particular has garnered tremendous therapeutic interest owing to its remarkable potency and activity profile. Specifically, bryostatin 1 has been shown to stimulate apoptosis, bolster the immune system, reverse multidrug resistance, synergize with other anticancer agents, enhance memory and learning in rodent models through the induction of synapse formation (synaptogenesis), reverse the effects of stroke in animal models, and induce latent HIV in vitro. As a result of this activity profile, bryostatin 1 is currently in phase I and II clinical trials for cancer treatment and is being advanced to the clinic for the treatment of Alzheimer's disease. However, despite its remarkable clinical potency (often ~ 1 mg is required for a 8 week treatment cycle in humans), the extremely low supply of bryostatin 1 prohibits its continued human clinical use and investigation for the treatment of additional therapeutic indications. In an effort to address the issues associated with the supply and unoptimized nature of a number of complex natural product protein kinase C ligands, the Wender group developed a pharmacophore model for C1 domain binding in the mid 1980s. This resulted in the design and synthesis of highly simplified, functional protein kinase C ligands based on the diacylglycerol scaffold. Additionally, structurally simplified, synthetically accessible bryostatin analogs were designed and shown to have comparable or even superior potency relative to bryostatin 1 for protein kinase C binding and in vitro anticancer activity. Described herein is the design, synthesis, and biological evaluation of a series of macrocyclic diacylglycerol analogs in an effort to improve protein kinase C affinity by reducing the entropic penalty of the binding event relative to the endogenous linear diacylglycerols. The binding affinity was found to be highly dependent on macrocycle size, with the most potent analog being up to two orders of magnitude more potent than the linear diacylglycerols (consistent with previous reports). Moreover, these analogs were prepared in a step-economical fashion (3-4 steps from commercial materials). Additionally, the design and synthesis of members of the first series of B-ring tetrahydropyran bryostatin analogs produced in the Wender group is reported. The use of a novel, high yielding Prins macrocyclization allowed for the retention of the synthetic convergency that has become a hallmark of the B-ring dioxane analogs produced previously in the Wender group. Several compounds from this B- ring tetrahydropyran class were found to be among the most potent analogs produced to date (with respect to protein kinase C affinity and in vitro anticancer activity). Despite the high potency and synthetic accessibility of the previously reported bryostatin analogs, these compounds lacked the ability to activate protein kinase C isozymes (as measured by their ability to induce the translocation of the enzyme from the cytosol to the plasma membrane) with a high degree of selectivity but were not equally non-selective either. The ability to tune isozyme selectivity has tremendous therapeutic potential and, in an effort to address this challenge, a series of A-ring functionalized, B-ring tetrahydropyran analogs was designed and synthesized using the Prins macrocyclization methodology. It was found that the C8 geminal dimethyl group on the A-ring in combination with C7 hydroxyl functionality imparts selectivity for the conventional protein kinase C [Beta]I over the novel protein kinase C [lowercase Delta}. Alternatively, C8 geminal dimethyl functionality in combination with C7 acetate functionality results in a high degree of non-selectivity for these isoforms. C13 functionalization was found to increase the potency of this already highly active analog class. Finally, several of the B-ring tetrahydropyran bryostatin analogs were shown to synergize with taxol in a human leukemia cell line. Additionally, a lead B-ring dioxane bryostatin analog was shown to be capable of inhibiting tumor growth in vivo in a transgenic mouse lymphoma model. Protein kinase C was implicated in this activity by monitoring the phosphorylation of downstream proteins as well as by performing inhibitor studies. This lead analog was also shown to induce apoptosis in a number of human B- and T-lymphocytes. The apoptotic induction observed in the murine cell line used for this pilot in vivo study was found to be independent of direct cell cycle effects. Significantly, this represents the first academic report of bryostatin analog efficacy and safety in vivo.