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Title:Improving the process synthesis, potency, and pharmacokinetics of the anticancer compound PAC-1
Author(s):Roth, Howard Steven
Director of Research:Hergenrother, Paul J.
Doctoral Committee Chair(s):Hergenrother, Paul J.
Doctoral Committee Member(s):Katzenellenbogen, John A.; Mitchell, Douglas A.; Fan, Timothy M.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):first procaspase activating compound (PAC-1)
Abstract:PAC-1 is an ortho-hydroxy-N-acylhydrazone that induces apoptosis by chelation of antiapoptotic labile zinc, relieving zinc-mediated inhibition of procaspase-3. Favorable results from cell culture and in vivo experiments with PAC-1 and its derivative S-PAC-1 have indicated the potential for translation to the clinic. However, several challenges exist in using PAC-1 in these experiments. This report describes the attempts to improve upon the process synthesis, potency, pharmacokinetics, and safety of PAC-1. The synthetic route to access PAC-1 was successful for the generation of batches of approximately 25 grams, but adaptation to multi-kilogram scale, as would be necessary for a human clinical trial, was not feasible using the existing route. In particular, the route included three chromatographic purification steps, as well as the use of anhydrous hydrazine. Optimization studies indicated that the active pharmaceutical ingredient could be synthesized without purification of any intermediates, and hydrazine monohydrate could be used in place of the anhydrous reagent. A batch of 155 grams of PAC-1 was synthesized using the improved route, and the route was scaled up to produce a batch of greater than 10 kilograms by a contract research organization. The lessons learned through this optimization effort were general and should be applicable if an alternative PAC-1 derivative is identified. In an effort to discover PAC-1 derivatives with improved potency, a combinatorial library of 837 compounds was synthesized. Each of 31 hydrazides was condensed with each of 27 aldehydes. Because standard techniques for isolation and purification of products are impractical for libraries of this size, a solid-phase purification strategy was employed that yielded the library members with an average purity of 91%. Compounds were screened in cell culture, and six potent hits were identified. One of these compounds, B-PAC-1, was studied further in primary isolates from leukemia patients, and animal experiments are currently in progress. An additional challenge is the relatively rapid clearance of PAC-1 from circulation. Many sites of metabolism exist, giving metabolites that arise from oxidative N-dealkylation, olefin oxidation, and arene oxidation. Several derivatives were synthesized containing modifications that prevent the formation of these metabolites. The compounds were evaluated in cell culture, liver microsomes, and mice, and four lead compounds were identified. Pharmacokinetic analysis indicated that each of these four compounds displayed extended elimination half-lives compared to PAC-1 and S-PAC-1, making these compounds viable candidates as next-generation PAC-1 derivatives. One of the most significant challenges in working with PAC-1 in vivo is the neuroexcitation observed upon treatment with elevated doses of compound. Preliminary experiments suggested that the neurotoxicity was not related to the ability of the compound to bind zinc. In order to confirm these results and attempt to determine the portion of the molecule responsible for neurotoxicity, a series of compounds was designed and synthesized containing systematic modifications to PAC-1. Evaluation in cell culture and in vitro further defined the essential nature of the ortho-hydroxy-N-acylhydrazone for activity. Evaluation of the compounds in mice confirmed previous experiments that the neurotoxicity is most likely not a result of metal chelation, but no further conclusions could be drawn from these experiments, and further study will be necessary to fully define the observed neurotoxicity.
Issue Date:2015-06-17
Rights Information:Copyright 2015 Howard Roth
Date Available in IDEALS:2015-09-29
Date Deposited:August 201

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