Development of the substrate activity screening method for the identification of nonpeptidic protease inhibitors, and, Synthesis and biological study of tubulysin D and related analogues

Chapter 1. A new fragment-based method for the development nonpeptidic protease inhibitors, Substrate Activity Screening, is described. Application of the method to cathepsin S resulted in the identification of multiple distinct classes of nonpeptidic substrates. Two of the substrate classes were optimized to substrates with >8000-fold improvements in cleavage efficiency, which were subsequently converted to low molecular weight, novel aldehyde inhibitors with nanomolar affinity.;Chapter 2. Replacement of the metabolically labile aldehyde pharmacophore of inhibitors identified in Chapter 1 with the nitrile pharmacophore provided moderately potent cathepsin S inhibitors. Structure guided optimization yielded compounds that are low molecular weight and potent cathepsin S inhibitors with >1000-fold selectivity over related proteases.;Chapter 3. Aldehyde and nitrile pharmacophores of mechanism-based inhibitors identified in Chapters 1 and 2 are replaced with noncovalent warheads to determine the correlation between substrate activity and inhibitor potency when using noncovalent pharmacophores. The azide pharmacophore was identified as ineffective, and the aminoaryl pharmacophore suspiciously behaved as a time-dependent transition-state analogue inhibitor.;Chapter 4. Addition of lithium acetylides to N-tert-butanesulfinyl ketimines affords a range of alpha,alpha-dibranched propargyl sulfinamides with high diastereoselectivities is described. Conversion to alpha,alpha-dibranched propargylamines enables synthesis of 1,2,3-triazole inhibitors identified in Chapters 1-3.;Chapter 5. The first total synthesis of tubulysin D is reported using N-tert-butanesulfinamide methods for the rapid syntheses of the tubuvaline and tubuphenylalanine fragments. A synthetic route was devised to introduce and carry forward the highly labile N, O-acetal functionality.;Chapter 6. For the first time, analogues of tubulysin D were prepared to establish the essential features for the potent cytotoxicity of tubulysin D. Most surprisingly, replacement of the labile N,O-acetal with the stable N-methyl group results in almost no loss in activity. The results obtained enable the design of potent analogues that are smaller and considerably more stable than tubulysin D.;Chapter 7. A highly efficient synthesis of potent N-methyl tubulysin analogues identified in Chapter 6 using unique strategies for the formation and acylation of N-methyl tubuvaline is described. This high-yielding synthesis should enable the production of large quantities of material for biological studies.