The overall objective of this project are to design, synthesize, and determine relevant physicochemical properties for, new analogs of the benzotriazine-di-N-oxides (tirapazamine; TPZ) and related quinoxaline- di-N- oxides (QNOs), seeking compounds in this class with superior anti- tumor efficacy than the clinical drug tirapazamine itself. An initial set of analogs with widely varying lipophilic, electronic and steric properties will be designed and prepared (by carefully varying the ring substituents). In addition to substituent parameters, we will also measure a number of global physicochemical properties, including lipophilicity, one-electron reduction potential and the lifetime of the critical radical species (the latter two by pulse radiolysis). The activities of these analogs for the nuclear matrix enzyme complex (that results in drug activation) and the mitochondrial enzyme complex (considered to give rise to the major toxic side effects of tirapazamine) will be determined by Project 2. The results of these assays will be used to determine (by techniques such as multiple linear regression, principal component analysis, etc) structure-activity relationships (SAR) that will be used in subsequent syntheses to develop compounds with optimal patterns of enzyme substrate properties to minimize toxicity and maximize activity. The analogs will also be screened in Project 2 for hypoxic cell selectivity. These additional biological assays in Projects 2 and 3. The results from these assays will decide, through a detailed decision tree, whether compounds progress to further testing, and will also provide direct feedback for new drug design. A second major hypothesis to be tested is that DNA-targeting of TPZ analogs (by the attachment of DNA-affinic intercalating or minor groove binding carriers) will provide compounds with superior in vivo activity because of targeting the reactive unit to the activating nuclear enzyme complex ( and away from the mitochondrial enzyme complex). Project 3 will use selected of these compounds to determine the features required to ensure efficient delivery to hypoxic tumor cells in vivo. A third hypothesis to be tested is that N-oxide pro-drug forms of the DNA- targeting units will provide analogs with good distribution properties. We will also evaluate known methods of preparing BTO compounds, and will adapt these to the preparation of analogs of the above types. In both cases, we will explore the use of parallel array synthesis from common precursors from suitable.
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