Antineoplastic Agents. 590. The X-ray Crystal Structure of Dolastatin 16 and Syntheses of the Dolamethylleuine and Dolaphenvaline Units

摘要

Three advances necessary to bringing dolastatin 16 (>1) into full-scale preclinical development as an anticancer drug have been accomplished. The X-ray crystal structure of dolastatin 16 has been solved, which allowed stereoselective syntheses of its two new amino acid units, dolamethylleuine (Dml) and dolaphenvaline (Dpv), to be completed as summarized in reaction and , respectively. The X-ray crystal structures of synthetic Z-Dml and TFA-Dpv have also been completed.Very early in the discovery of the biologically remarkable and structurally unique peptides from the sea hare Dolabella auricularia, which we designated dolastatins, it became clear that certain members (e.g. 10–15) exhibited a variety of important properties that include anticancer and antifungal activities. Indeed, dolastatin 10 and three structural modifications are currently in human cancer phase II and phase III clinical trials. Two derivatives of dolastatin 15 are also in cancer clinical trials (phase I–II).When we extended our field collections of D. auricularia from the Indian Ocean to the Western Pacific (Papua New Guinea and the Philippines), we were able to expand the dolastatin series to 16–19. Dolastatin 16 (>1) especially proved to be an exceptionally potent inhibitor of cancer cell growth and a candidate for further development. However, the latter important initiative has been delayed by the need for unequivocal configurational assignments and a practical total synthesis of dolastatin 16. We are pleased to report herein the X-ray crystal structure of dolastatin 16 (>1) and syntheses of the new amino acid units dolamethylleuine (>2) and dolaphenvaline (>3). id="largeobj_idm140174230860288" class="largeobj-link align_right" style="display: none">Other options for obtaining certain dolastatin members appeared likely some 35 years ago when we considered^{href="#R2" rid="R2" class=" bibr popnode">2d} that Dolabella species derived nutrition by consuming marine microalgae and that such exogenous sources might be providing the dolastatins or intermediates. This expectation has been amply realized over the past decade by the isolation of dolastatins 10–16^{href="#R4" rid="R4" class=" bibr popnode">4a,href="#R5" rid="R5" class=" bibr popnode">5} or close analogues from the cyanobacterium Lyngbya majuscula and other such microalgae. Thus, fermentation methods using marine cyanobacteria may eventually be competitive with total syntheses for scale-up production of new anticancer drugs in the family. At present, the yields from these initial experiments remain very low, and for the foreseeable future the provision of dolastatin 16 for cancer clinical trial development will require a practical total synthesis for scale-up production. However, the microalgae investigations continue to be very productive and promising for the future.The three most obvious challenges to finding a useful synthesis of dolastatin 16, namely, an X-ray crystal structure to confirm the configuration and convenient stereoselective syntheses of the new amino acid units >2 and >3, have been met as follows. Dolastatin 16 was originally isolated (in 3.1 × 10⁻⁷% yield) as an amorphous powder, and a long period of attempts at crystallization were unsuccessful. Eventually, we found that very slow (over three years) crystal formation from acetonitrile and water provided X-ray quality crystals. Structurally, dolastatin 16 is a cyclodepsipeptide containing two new amino acids, dolamethylleuine (Dml, >2), a β-amino acid, and dolaphenvaline (Dpv, >3). As reported previously,^{href="#R4" rid="R4" class=" bibr popnode">4a} the structure of >1 without assignment of the configuration of the novel amino acids was achieved by high-field NMR and tandem MS/MS mass spectroscopic interpretations. X-ray crystallographic analysis of >1 has now confirmed its cyclodepsipeptide structure and permitted the configurational assignments of the novel amino acids as 2R,3R for >2 and 2S,3R for >3 (href="/pmc/articles/PMC3111978/figure/F1/" target="figure" class="fig-table-link figpopup" rid-figpopup="F1" rid-ob="ob-F1" co-legend-rid="lgnd_F1">Figure 1). class="fig iconblock whole_rhythm clearfix" id="F1" co-legend-rid="lgnd_F1">href="/pmc/articles/PMC3111978/figure/F1/" target="figure" rid-figpopup="F1" rid-ob="ob-F1"> data-largeobj="" data-largeobj-link-rid="largeobj_idm140174208401104" class="figure">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3111978_nihms294130f1.jpg" target="tileshopwindow"> class="ts_bar small" title="Click on image to zoom"> id="largeobj_idm140174208401104" class="largeobj-link align_right" style="display: none">target="object" href="/pmc/articles/PMC3111978/figure/F1/?report=objectonly">Open in a separate window class="icnblk_cntnt" id="lgnd_F1">>class="figpopup" href="/pmc/articles/PMC3111978/figure/F1/" target="figure" rid-figpopup="F1" rid-ob="ob-F1">Figure 1 class="caption">X-ray structure of dolastatin 16 (>1). The atoms of this cyclic depsipeptide and solvent (one acetonitrile and three water) molecules are displayed as 30% probability thermal ellipsoids