Pretubulysin: a new option for the treatment of metastatic cancer.

Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-xL, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF(Fbw7) E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer.

Anti-angiogenic effects of the tubulysin precursor pretubulysin and of simplified pretubulysin derivatives.

BACKGROUND AND PURPOSE: The use of tubulin-binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti-angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA). EXPERIMENTAL APPROACH: The compounds were tested in cellular angiogenesis assays (proliferation, cytotoxicity, cell cycle, migration, chemotaxis, tube formation) and in vitro (tubulin polymerization). The efficacy of Prt was also tested in vivo in a murine subcutaneous tumour model induced with HUH7 cells; tumour size and vascularization were measured. KEY RESULTS: The anti-angiogenic potency of all the compounds tested ran parallel to their inhibition of tubulin polymerization in vitro. Prt showed nearly the same efficacy as TubA (EC(50) in low nanomolar range in all cellular assays). Some modifications in the Prt molecule caused only a moderate drop in potency, while others resulted in a dramatic loss of action, providing initial insight into structure-activity relations. In vivo, Prt completely prevented tumour growth and reduced vascular density to 30%. CONCLUSIONS AND IMPLICATIONS: Prt, a chemically accessible precursor of some tubulysins is a highly attractive anti-angiogenic compound both in vitro and in vivo. Even more simplified derivatives of this compound still retain high anti-angiogenic efficacy.

Preparation and reactions of stannylated amino acids.

Hydrostannations of propargylic glycine esters with the new hydrostannation catalyst [Mo(CO)3(CNtBu)3] (MoBI3) gave rise to alpha-stannylated allylic esters in good yield and with high regioselectivity. The chelate Claisen rearrangements of these esters allow the syntheses of gamma,delta-unsaturated amino acids with a vinylstannane moiety in the side chain. The amino acids obtained can be further modified by cross-coupling with various types of electrophiles.

Synthesis ofγ,δ-unsaturated amino acids via ester enolate Claisen rearrangement of chelated allylic esters.

Deprotonation ofN-protected amino acid allylic esters with LDA at -78°C and subsequent addition of a metal salt presumably results in the formation of a chelated metal enolate which undergoes Claisen rearrangement upon warming up to room temperature, giving rise to unsaturated amino acid. Many different metal salts can be used for chelation, but in general the best results are obtained with zinc chloride. Due to the fixed enolate geometry, as a result of chelate formation, and a strong preference for thechair like transition state, the rearrangement proceeds with a high degree of diastereoselectivity. This methodology can be applied to acyclic as well as to cyclic substrates, and even to peptides, and allows for the synthesis of amino acids containing quaternary carbon centers.