Eribulin mesylate (E7389), a synthetic analog of the marine natural product halichondrin B, is in Phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening. Using [ 3 H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K d of 46 μM, but also showing a real or apparent very high affinity (K d , 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (K d , 3.5 μM), strongly suggesting the presence of a relatively high affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per 2 microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Pre-incubation of microtubules with 2 or 4 μM vinblastine induced additional lower affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability. Keywords eribulin; halichondrin; tubulin; microtubule; binding Eribulin mesylate is a tubulin/microtubule-targeting chemotherapeutic drug that inhibits the proliferation of multiple cancer cell types (1,2). It is a synthetic analog of the natural compound, halichondrin B (Fig. 1A), initially isolated from the sea sponge Halichondria okadai (3). Eribulin is currently in Phase III clinical trials for the treatment of metastatic breast cancer. Phase I and Phase II clinical trials have demonstrated that eribulin is active in heavily pretreated individuals while maintaining a tolerable therapeutic index, with the most frequent adverse effects being neutropenia and fatigue (4-6). Neuropathy, a common dose-limiting toxicity of other microtubule-targeting drugs like paclitaxel and some vinca alkaloids (7-9), has a low incidence in eribulin-treated patients, and no grade 4 neuropathy occurred (4)(5)(6) Eribulin exerts its anticancer properties through a novel action on tubulin and microtubules (1,(10)(11)(12). In MCF7 cells, eribulin inhibited microtubule dynamic instability at low concentrations and induced depolymerization of the microtubule network at high concentrations (10 × IC 50 for inhibition of cell proliferation) (11). At significantly lower eribulin concentrations, eribulin potently inhibited microtubule dynamics, resulting in prolonged mitotic arrest and subsequent apoptosis [for a review of microtubule structure and dynamic instability, see (13,14)]. Eribulin binds at or near the vinca domain, a region that is located at the interface of two tubulin heterodimers when arranged end-to-end and overlaps the ex...

Eribulin (E7389), a mechanistically unique microtubule inhibitor in phase III clinical trials for cancer, exhibits superior efficacy in vivo relative to the more potent compound ER-076349, a fact not explained by different pharmacokinetic properties. A cell-based pharmacodynamic explanation was suggested by observations that mitotic blockade induced by eribulin, but not ER-076349, is irreversible as measured by a flow cytometric mitotic block reversibility assay employing full dose/response treatment. Cell viability 5 days after drug washout established relationships between mitotic block reversibility and long-term cell survival. Similar results occurred in U937, Jurkat, HL-60, and HeLa cells, ruling out cell type-specific effects. Studies with other tubulin agents suggest that mitotic block reversibility is a quantifiable, compound-specific characteristic of antimitotic agents in general. Bcl-2 phosphorylation patterns parallel eribulin and ER-076349 mitotic block reversibility patterns, suggesting persistent Bcl-2 phosphorylation contributes to long-term cell-viability loss after eribulin's irreversible blockade. Drug uptake and washout/retention studies show that [ Our results suggest that eribulin's in vivo superiority derives from its ability to induce irreversible mitotic blockade, which appears related to persistent drug retention and sustained Bcl-2 phosphorylation. More broadly, our results suggest that compound-specific reversibility characteristics of antimitotic agents contribute to interactions between cell-based pharmacodynamics and in vivo pharmacokinetics that define antitumor efficacy under intermittent dosing conditions. Cancer Res; 71(2); 496-505. Ó2011 AACR.

The absolute stereochemistry of fostriecin (1, CI-920), a potent antitumor antibiotic presently in Phase I clinical trials at NCI, was determined to be 5R,8R,9R,11R. 2D 1 H-1 H NMR NOE experiments conducted on the cyclic phosphate derivative 2 and acetonide 4 revealed a syn-diol stereochemical relationship between C8 and C9 and an anti-diol stereochemical relationship between C9 and C11, respectively. The 5R absolute configuration assignment was confirmed by synthesis of the degradation product 8 previously disclosed. Additional degradation studies of 1 to provide 7 and chiral-phase HPLC comparison with a sample of known chirality established the absolute stereochemistry of C11 to be R. This, along with the relative stereochemical assignments established the full set of absolute stereochemistry assignments for 1.

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The evolution of the synthesis of Halaven ® (E7389, INN eribulin mesylate) from a medicinal chemistry process to the execution of the final process on pilot scale is described. The completion of the synthesis of Halaven ® from C1-C13 ester and C14-C35 sulfone alcohol involves a series of chemo-, regio-, and stereoselective transformations. Furthermore, a high-dilution macrocyclization presented a number of challenges for industrial-scale manufacture (throughput, processing time, and side reactions). This paper describes studies at Eisai leading to an understanding, optimization, and control of the chemistry that realized the reproducible commercial production of Halaven ® .Amongst the synthetic issues to be addressed in order to provide the necessary quantities of Halaven ® (1) 1-3 for full clinical evaluation are: (i) procurement of the fragments 3 (C14-C35) 4 and 4 (C1-C13), 5 (ii) stereo-and chemoselective coupling of these fragments, (iii) carbon-carbon bond formation to generate the macrocycle, and (iv) controlled establishment of the polycyclic ketal and amino alcohol functionalities. This paper describes the current status of synthetic studies at Eisai which address these issues and have enabled commercial supply of 1 in a totally synthetic manner (Scheme 1). The synthesis of 1 from C14-C35 sulfone 3 and C1-C13 ester 4 is depicted in Scheme 2. The synthesis begins with DIBAL reduction of 4 in toluene solution at -78 °C. Aldehyde 5 is isolated in 93% yield following quench with methanol, workup, and chromatographic purification for removal of the overreduction byproduct. The aldehyde is prone to oxidation to the carboxylic acid during isolation and storage, which had previously been managed by careful handling of the neat preparation or solutions under inert atmospheres, usually argon. However, after several sampling cycles, the carboxylic acid impurity had increased and repurification of the aldehyde was required. Addition of 0.5-1 wt% 3,5-di-tert-butyl-4-hydroxytoluene (BHT) to the reaction mixture and to chromatographic fractions prior to concentration inhibits the oxidation of the aldehyde. The aldehyde is then readily stored and delivered as a solution for the coupling with 3.