From micrograms to grams: scale-up synthesis of eribulin mesylate.

The synthesis of eribulin mesylate from microgram to multi-gram scale is described in this Highlight. Key coupling reactions include formation of the C30a to C1 carbon-carbon bond and macrocyclic ring closure through an intramolecular Nozaki-Hiyama-Kishi reaction.

Druggable chemical space and enumerative combinatorics.

BACKGROUND: There is a growing body of literature describing the properties of marketed drugs, the concept of drug-likeness and the vastness of chemical space. In that context, enumerative combinatorics with simple atomic components may be useful in the conception and design of structurally novel compounds for expanding and enhancing high-throughput screening (HTS) libraries. RESULTS: A random combination of mono- and diatomic carbon, hydrogen, nitrogen, and oxygen containing components in the absence of molecular weight constraints but with the ability to form rings affords virtual compounds that fall in bulk physicochemical space typically associated with drugs, but whose ring assemblies fall in new or under-represented areas of chemical shape space. When compared against compounds in the ChEMBL_14, MDDR, Drug Bank and Dictionary of Natural Products, the percentage of virtual compounds with a Tanimoto index of 1.0 (ECFP_4) was found to be as high as 0.21. Depending on therapeutic target, this value may be in range of what might be expected from an experimental HTS campaign in terms of a true hit rate. CONCLUSION: Virtual compounds derived through enumerative combinatorics of simple atomic components have drug-like properties with ring assemblies that fall in new or under-represented areas of shape space. Structures derived in this manner could provide the starting point or inspiration for the design of structurally novel scaffolds in an unbiased fashion.

Simulating the drug discovery pipeline: a Monte Carlo approach.

BACKGROUND: The early drug discovery phase in pharmaceutical research and development marks the beginning of a long, complex and costly process of bringing a new molecular entity to market. As such, it plays a critical role in helping to maintain a robust downstream clinical development pipeline. Despite its importance, however, to our knowledge there are no published in silico models to simulate the progression of discrete virtual projects through a discovery milestone system. RESULTS: Multiple variables were tested and their impact on productivity metrics examined. Simulations predict that there is an optimum number of scientists for a given drug discovery portfolio, beyond which output in the form of preclinical candidates per year will remain flat. The model further predicts that the frequency of compounds to successfully pass the candidate selection milestone as a function of time will be irregular, with projects entering preclinical development in clusters marked by periods of low apparent productivity. CONCLUSIONS: The model may be useful as a tool to facilitate analysis of historical growth and achievement over time, help gauge current working group progress against future performance expectations, and provide the basis for dialogue regarding working group best practices and resource deployment strategies.

Atom-based enumeration: new eribulin analogues with low susceptibility to P-glycoprotein-mediated drug efflux.

A series of eribulin analogues was evolved in silico through iterative atom-based enumeration employing a genetic algorithm-derived survival function to minimize predicted PgP-mediated drug efflux. Representatives of the virtual series were subsequently synthesized in the laboratory and tested in vitro for PgP-susceptibility. These new computer-inspired derivatives were found to exhibit high cell growth inhibitory activity and to be among the least sensitive to P-glycoprotein-mediated drug efflux in the eribulin series, thereby validating this approach to in silico molecular design.

Broad spectrum preclinical antitumor activity of eribulin (Halaven(R)): optimal effectiveness under intermittent dosing conditions.

BACKGROUND: Eribulin is a pharmaceutically and structurally optimized analog of the marine sponge natural product halichondrin B. Its salt form, eribulin mesylate (Halaven®) is clinically used in the United States, the European Union, and Japan for the treatment of heavily pretreated patients with metastatic breast cancer, who previously received an anthracycline and a taxane. Early preclinical studies of this new inhibitor of microtubule dynamics showed high antitumor potency towards several human cancer types in vitro and in vivo. Here we extend those early studies by examining the effects of eribulin against a wider spectrum of human tumor xenografts in vivo, and by directly comparing the in vivo effectiveness of different dosing administration schedules. MATERIALS AND METHODS: In single-schedule studies, in vivo activity of eribulin against HT-1080 fibrosarcoma, U251 glioblastoma, SR-475 head and neck cancer, SK-LMS-1 leiomyosarcoma, NCI-H322M and NCI-H522 non-small cell lung cancer (NSCLC), PANC-1 pancreatic cancer, and NCI-H82 small cell lung cancer (SCLC) xenografts was examined at dose levels of 0.19-4.0 mg/kg using q2d×3(×3), q4d×3, q4d×4, and q7d×2 schedules. Administration schedule dependence was evaluated by directly comparing q1d×5, q2d×3(×3), q4d×3, and q7d×3 schedules in the MDA-MB-435 breast cancer xenograft model, using conditions of equivalent total dosing over the course of the experiment. RESULTS: In single-schedule studies, maximum tolerated dose (MTD) values (or maximal 'at or below MTD' values) ranged from 0.8-1.7 mg/kg. In vivo antitumor responses at these dosing levels included tumor growth inhibition, stasis, and regression; several studies showing regression also yielded long-term tumor-free survivors. Effectiveness of eribulin showed model-to-model variability that appeared to be unrelated to dose level or administration schedule, suggesting that characterization of models with differing eribulin sensitivities may reveal potential biomarker strategies. Results of the dose schedule comparison study in the MDA-MB-435 model suggested the following order of effectiveness and tolerability: q2d×3(×3)>q4d×3≈q7d×3>> q1d×5. Moderately intermittent dosing thus shows optimal preclinical effectiveness, in good agreement with the approved intermittent clinical schedule for eribulin (days 1 and 8 of a 21-day cycle). CONCLUSION: The current results show that eribulin has broad spectrum preclinical antitumor activity against a wide variety of human cancer types, and indicate that maximum effectiveness and optimal tolerability are obtained using moderately intermittent dosing schedules.

Natural product-like virtual libraries: recursive atom-based enumeration.

A new molecular enumerator is described that allows chemically and architecturally diverse sets of natural product-like and drug-like structures to be generated from a core structure as simple as a single carbon atom or as complex as a polycyclic ring system. Integrated with a rudimentary machine-learning algorithm, the enumerator has the ability to assemble biased virtual libraries enriched in compounds predicted to meet target criteria. The ability to dynamically generate relatively small focused libraries in a recursive manner could reduce the computational time and infrastructure necessary to construct and manage extremely large static libraries. Depending on enumeration conditions, natural product-like structures can be produced with a wide range of heterocyclic and alicyclic ring assemblies. Because natural products represent a proven source of validated structures for identifying and designing new drug candidates, mimicking the structural and topological diversity found in nature with a dynamic set of virtual natural product-like compounds may facilitate the creation of new ideas for novel, biologically relevant lead structures in areas of uncharted chemical space.

Novel second generation analogs of eribulin. Part I: Compounds containing a lipophilic C32 side chain overcome P-glycoprotein susceptibility.

Eribulin mesylate (Halaven™), a totally synthetic analog of the marine polyether macrolide halichondrin B, has recently been approved in the United States as a treatment for breast cancer. It is also currently under regulatory review in Japan and the European Union. Our continuing medicinal chemistry efforts on this scaffold have focused on oral bioavailability, brain penetration and efficacy against multidrug resistant (MDR) tumors by lowering the susceptibility of these compounds to P-glycoprotein (P-gp)-mediated drug efflux. Replacement of the 1,2-amino alcohol C32 side chain of eribulin with fragments neutral at physiologic pH led to the identification of analogs with significantly lower P-gp susceptibility. The analogs maintained low- to sub-nM potency in vitro against both sensitive and MDR cell lines. Within this series, increasing lipophilicity generally led to decreased P-gp susceptibility. In addition to potency in cell culture, these compounds showed in vivo activity in mouse xenograft models.

Novel second generation analogs of eribulin. Part III: Blood-brain barrier permeability and in vivo activity in a brain tumor model.

Novel second generation analogs of eribulin mesylate, a tubulin agent recently approved for the treatment of breast cancer, are reported. Our recent efforts have focused on expanding the target indications for this class of compounds to other tumor types. Herein, we describe the design, synthesis and evaluation of eribulin analogs active against brain tumor cell lines in vitro and corresponding brain tumor models in mice. Attenuation of basicity of the amino group(s) in the C32 side-chain region led to compounds with lower susceptibility to P-gp mediated drug efflux, allowing these compounds to permeate through the blood-brain barrier. In preclinical in vivo studies, these compounds showed significantly higher levels in the brain and cerebrospinal fluid as compared to eribulin. In addition, analogs within this series showed antitumor activity in an orthotopic murine model of human glioblastoma.

Novel second generation analogs of eribulin. Part II: Orally available and active against resistant tumors in vivo.

Eribulin mesylate is a newly approved treatment for locally advanced and metastatic breast cancer. We targeted oral bioavailability and efficacy against multidrug resistant (MDR) tumors for further work. The design, synthesis and evaluation of novel amine-containing analogs of eribulin mesylate are described in this part. Attenuation of basicity of the amino group(s) in the C32 side-chain region led to compounds with low susceptibility to PgP-mediated drug efflux. These compounds were active against MDR tumor cell lines in vitro and in xenograft models in vivo, in addition to being orally bioavailable.

Eribulin induces irreversible mitotic blockade: implications of cell-based pharmacodynamics for in vivo efficacy under intermittent dosing conditions.

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 [3H]eribulin accumulates to lower intracellular levels than [3H]ER-076349, yet is retained longer and at higher levels. Similar findings occurred with irreversible vincristine and reversible vinblastine, pointing to persistent cellular retention as a component of irreversibility. 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.

Predicting total clearance in humans from chemical structure.

A conceptually simple, fully in silico model to predict total clearance of new compounds in humans is described. Based on the premise that similar molecules will exhibit similar pharmacokinetic properties, we used a k-nearest-neighbors (kNN) technique to predict total clearance by comparison with known reference agents. Molecular similarity was defined using readily calculated one- and two-dimensional molecular descriptors, and the reference set was obtained by combining the Obach and Berellini sets of human pharmacokinetic data. Neutral molecules and drugs whose biological activity is associated with a metal center were removed from the combined set. The remaining 462 compounds were partitioned into a training and external test set of 370 and 92 compounds, respectively. For acids, bases, zwitterions, and quaternary ammonium/pyridinium ions, average prediction accuracy was within two-fold of observed for the external test set (n = 92). Using a collection of 20 drugs from the literature with > or =3 preclinical animal species allometric scaling data, accuracy of the in silico kNN model was not as good as the rule of exponents, but better than simple allometry (SA), and approached that of combination multiexponential allometry (ME) as defined by the number of predictions with < or =50% error. For a collection of 18 drugs with two species (rat-dog) data, the kNN model outperformed both SA and combination ME using the same performance standard. Since the model is fully in silico and, therefore, capable of generating total clearance predictions in the absence of any experimental data, it can be used to help guide early drug discovery research efforts, such as virtual compound library screening, and analogue prioritization prior to chemical synthesis and biological evaluation. Model validation was accomplished using the external test set, three- and five-fold cross-validation and two different y-randomization techniques (y-shuffling and random number pseudodescriptors).

Structurally simplified macrolactone analogues of halichondrin B.

A structurally simplified macrolactone analogue of halichondrin B was identified that retains the potent cell growth inhibitory activity of the natural product in vitro.

Macrocyclic ketone analogues of halichondrin B.

Structurally simplified macrocyclic ketone analogues of halichondrin B were prepared by total synthesis and found to retain the potent cell growth inhibitory activity in vitro, stability in mouse serum, and in vivo efficacy of the natural product.

Induction of morphological and biochemical apoptosis following prolonged mitotic blockage by halichondrin B macrocyclic ketone analog E7389.

E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G2-M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.