Andrew Patterson

Expedient Synthesis of N-Methyl Tubulysin Analogues with High Cytotoxicity

An optimized and highly efficient synthesis of potent, bioactive N-methyl tubulysin analogues 2 and 4 has been achieved with > 40% overall yields. This synthesis represents a significant improvement over previously reported syntheses of these and related tubulysin analogues. The stereoselective synthesis of the unnatural amino acid tubuvaline is accomplished using tert-butanesulfinamide chemistry. N-Alkylation to form N-methyl tubuvaline is performed without protection of the tubuvaline alcohol by implementing a unique N-methylation strategy via formation and reduction of a 1,3-tetrahydrooxazine heterocycle. Acylation of the hindered N-methyl tubuvaline amine utilizes a novel sequence of O-acylation followed by an O- to N-acyl transfer to form the hindered amide bond between N-methyl tubuvaline and isoleucine. This high-yielding synthesis should enable the production of large quantities of material for biological studies.

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery.

Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism–based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates.

Chemotherapeutic evaluation of a synthetic tubulysin analogue-dendrimer conjugate in c26 tumor bearing mice.

The tubulysins, first isolated by the Hofle/Reichenbach group from myxobacterial cultures,1 are exceptionally potent cell-growth inhibitors that act by inhibiting tubulin polymerization and thereby induce apoptosis. For this reason, the biosynthesis,2 mechanism,3 and anticancer activity4 of the tubulysins have been intensively investigated. Tubulysin D (Figure 1), the most potent of the tubulysins, has activity that exceeds virtually all tubulin modifiers, and in particular is 20- to 10000-fold more cytotoxic than the important, clinically approved anticancer drugs the epothilones, vinblastine, and paclitaxel.5,6 Unfortunately, the extraordinary activity of the most potent tubulysins poses considerable complications for chemotherapeutic applications due to toxicity against healthy cells and tissues. Moreover, the tubulysins are difficult to obtain in large quantities from the myxobacteria from which they are isolated, and total synthesis routes are prohibitively difficult due to the fragile and complex nature of the most potent of the naturally occurring tubulysins, which incorporate the labile N,O-acetal functionality.7 Consequently, both academia and the pharmaceutical industry have focused on the synthesis and evaluation of simpler and more stable tubulysin analogues.8 Similarly, significant efforts have been directed to prodrug strategies for selective tumor delivery of tubulysin and its analogues in order to minimize toxicity.8q