Methods for the preparation, isolation and purification of epothilone B, and x-ray crystal structures of epothilone B

FIELD OF INVENTION

The present invention relates to improved methods for the production, isolation and purification of epothilone B. These methods include, for example, a fermentation process for the production of epothilone B, isolation via adsorption onto aresin, and subsequent purification.

BACKGROUND OF INVENTION

Epothilones are a relatively new class of macrolide compounds that were originally obtained by fermentation of myxobacteria (Sorangium cellulosum). These compounds were initially investigated as plant protective agents due to their anti-fungalproperties. Epothilones then became of interest due to their cytotoxic activity on animal cells, and were subsequently characterized as tubulin polymerization agents. It is now known that epothilones exert microtubule-stabilizing effects similar topaclitaxel (TAXOL.RTM.) and cytotoxic activity against rapidly proliferating cells such as tumor cells or other hyperproliferative cellular disease. The use of epothilones as chemotherapeutic agents is described in Bollag et al., Cancer Research 55,2325, 1995.

Epothilones A and B (epo A or epo B, respectively) have the structures,

##STR00001## Epothilone A R=H Epothilone B R=Me

       One scheme for obtaining epothilones was revealed by Hofle et al. in WO 93/10121. Hofle cultured a strain of Sorangium cellulosum in a medium containing carbon sources, nitrogen sources and mineral salts. An adsorber resin was added during theculturing of the strain. The epothilones were eluted with solvent from the adsorbent resin. The various epothilones were separated by reverse-phase chromatography and crystallized. However, Hofle et al. conceded that this method produced only a lowquantity of epothilone B, and also that the ratio of epothilone B to epothilone A in the fermentation was low. This low ratio of epothilone B relative to epothilone A makes recovery of pure epothilone B difficult. Thus, there is a need in the art forimproved methods of fermentation to produce epothilone B in preference to epothilone A, and improved methods of isolation and purification of epothilone B.

SUMMARY OF INVENTION

    The present invention is directed to an improved fermentation process for the production of epothilone B.

      Further included in the present invention are new strains of Sorangium cellulosum obtained by mutagenesis for the production of epothilones.

        Also included in the present invention are methods to improve the ratio of epothilone B to A produced by the new strain of Sorangium cellulosum by providing an additive to the fermentation. In one preferred embodiment, the additive ispropionate, propionic acid with proper pH adjustment, or another propionate precursor.

        Also included in the present invention is an improved extraction process for isolation of epothilone B from the fermentation medium using a resin. Further included are methods for washing epothilone-rich resin to reduce impurity levels andimprove downstream processing.

        Also included in the present invention is an improved process for the purification of epothilone B. In one embodiment, purification is achieved using crystallization. In another embodiment, purification is achieved by chromatographic methodswhich include normal-phase chromatography or reverse-phase chromatography. In yet another embodiment, purification is achieved by a combination of crystallization and purification of samples by chromatography, including normal and reverse-phasechromatography. In a further embodiment, the resin extract is processed by crystallization only.

        Epothilone B ("epo B") is useful as an intermediate in the preparation of derivative 1 ("D1"), (as described in U.S. Pat. No. 6,262,094, herein incorporated by reference), where the 2-methyl on the thiazole ring is substituted with an amine:

##STR00002##

        Epothilone B is also useful in the preparation of derivative 2 ("D2") (such conversion of the lactone of epothilone B to the lactam of derivative 2 is described by Borzilleri et al., J. Amer. Chem. Soc. 122, 8890, 2000, and in WO 99/02514,herein incorporated by reference):

##STR00003##

        Furthermore, epothilone B ("epo B") is useful for the preparation of derivative 3 (epothilone D, "D3") (as described in U.S. Pat. No. 6,320,045, herein incorporated by reference):

##STR00004##

        Further included in the invention are crystal forms of epothilone B produced using the methods and materials described herein.

        It is to be understood that both the foregoing general description and the following detailed description are exemplary, but not restrictive, of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

      The advantages, nature and various features of the invention may appear more fully upon consideration of the accompanying drawings. In the drawings:

        FIG. 1 shows the molecular structure in the monoclinic unit cell of form epoB-EAβ, with two molecules of epothilone B and two molecules of ethyl acetate in the guest channel of the monoclinic unit cell.

        FIG. 2 shows the molecular structure in the monoclinic unit cell of form epoB-ANβ, with two molecules of epothilone B and two molecules of acetonitrile in the guest channel of the monoclinic unit cell.

        FIG. 3 shows the molecular structure in the monoclinic unit cell of form epoB-Ipβ, with two molecules of epothilone B and two molecules of isopropanol in the guest channel of the monoclinic unit cell.

        FIG. 4 shows the molecular structure in the monoclinic unit cell of form epoB-Toβ, with two molecules of epothilone B and two molecules of toluene in the guest channel of the monoclinic unit cell.

        FIG. 5 shows observed (top) and simulated (bottom) PXRD patterns for the ethyl acetate solvate (crystal form epoB-EAβ) of epothilone B. In FIG. 5, the simulated pattern was calculated from the refined atomic parameters in the monocliniccrystal structure at -33° C., and the observed pattern was measured at +23° C.

        FIG. 6 shows observed (top) and simulated (bottom) PXRD patterns for the toluene solvate (crystal form epoB-TOβ) of epothilone B. In FIG. 6, the simulated pattern was calculated from the refined atomic parameters in the monoclinic crystalstructure at -33° C., and the observed pattern was measured at +23° C.

        FIG. 7 shows observed (top) and simulated (bottom) PXRD patterns for the acetonitrile solvate (crystal form epoB-ANβ) of epothilone B. In FIG. 7, the simulated pattern was calculated from the refined atomic parameters in the monocliniccrystal structure at -40° C., and the observed pattern was measured at +23° C.

        FIG. 8 shows observed (top) and simulated (bottom) PXRD patterns for the isopropyl alcohol solvate (crystal form epoB-IPβ) of epothilone B. In FIG. 8, the simulated pattern was calculated from the refined atomic parameters in the monocliniccrystal structure at -3° C., and the observed pattern was measured at +23° C.

        FIG. 9 shows an observed PXRD pattern for a toluene-containing primary grade solvate of epothilone B produced following the method described in Example 7, Step A.

        FIG. 10 shows the thermal analysis (DSC and TGA) for the toluene-containing primary grade solvate of FIG. 9.

        FIG. 11 shows an observed PXRD pattern for a toluene-containing recrystallized solvate of epothilone B, produced following the method described in Example 7, Step B.

        FIG. 12 shows the thermal analysis (DSC and TGA) for the toluene-containing recrystallized solvate of FIG. 11.

        FIG. 13 shows an observed PXRD pattern for the ethyl acetate containing solvate of epothilone B, produced following the method described in Example 7, Step C.

        FIG. 14 shows the thermal analysis (DSC and TGA) for the ethyl acetate containing solvate of FIG. 13.

        FIG. 15 shows an observed (top) PXRD pattern for the toluene-containing solvate prepared following the method described in Example 7C, together with a simulated (bottom) PXRD pattern for the toluene solvate of epothilone B at room temperature.

        FIG. 16 shows the thermal analysis (DSC and TGA) for the toluene-containing solvate of FIG. 15.

        It is to be understood that these drawings are for purposes of illustrating the concepts of the invention and are not limiting in nature. In each of FIGS. 1 through 4, all methyl and methylene hydrogen atoms of the epothilone have been omittedfor clarity. In FIGS. 1-4, intermolecular hydrogen bonds are shown at the bottom right and top left portions of the diagrams as dashed rods, and H-bond distances (Angstroms) designate the intermolecular oxygen--oxygen distances.