MXPA06009792A - Synthesis of epothilones, intermediates thereto, analogues and uses thereof - Google Patents

Abstract

The present invention provides compounds of formula (I):as described generally and in classes and subclasses herein. The present invention additionally provides pharmaceutical compositions comprising compounds of formula (I) and provides methods of treating cancer comprising administering a compound of formula (I).

Description

SYNTHESIS OF EPOTILONES, INTERMEDIARIES OF THESE, ANALOGUES AND USES OF THEM BACKGROUND OF THE INVENTION Epothilones and B (2a and 2b, Reaction Scheme 1) are naturally occurring cytotoxic macrolides that were isolated from a cellulose degrading mycobacterium, Sorangium cell? Osum (Höfle et al., Angew. Chem. Int. Ed. Engl 1996, 35, 1567 and J. Antibiot, 1996, 49, 560, each of which is incorporated herein by reference). Despite their vastly different structures, epothilones B share the same mechanism of action as paclitaxel (Taxol®) which involves inhibition of growth of tumorigenic cells by tubulin polymerization and stabilization of microtubule mounts (Bollag et al., Cancer Res. 1395, 55, 2325, incorporated for reference). Despite its unchallenged clinical value as a front-line chemotherapeutic agent, Taxol® is far from an ideal drug. Its solubility in marginal water necessitates the resource for the formulation of vehicles such as Cre oforos that propose their own risks and management emissions (Essayan et al., J. Allergy Clin.Immunol., 1996, 91, 42; incorporated herein by reference). ). In addition, Taxol® is vulnerable to deactivation through multiple drug resistance Ref. 175425 (RFM) (Giannakakou et al., J. Biol. Chem. 1997., 272, 17118, incorporated herein by reference). However, it has also been shown that epothilones A and B retain remarkable potency against tumorigenic RFM cells (Kowalski et al., Mol. Biol. Cell 1995, 6, 2137, incorporated herein by reference). Additionally, the increased water solubility compared to paclitaxel may be useful for the formulability of the epothilones. While the naturally occurring compound, epothilone B (2b, EpoB, in Reaction Scheme 1), is a potent member of the natural product epothilone family, it unfortunately possesses, at least in mice xenograft, a therapeutic index worryingly narrow (Su et al., Angew., Chem. Jnt. Ed. Engl., 1997, 36, 1093; Harris et al., J. Org. Chem., 1999, 64, 8434, each of which is incorporated for reference).

R = Ph, Paclitaxel (Taxol) 2a Rj. = H, R2 = CH3, Epothilone A (EpoA) Ib R = t-Bu, Docetaxel (Taxotere) 2b Ri = CH3, R2 = CH3, Epothilone B (EpoB) 2c Rx = H, R2 = CH2OH, Epothilone E (EpoE) ) 2d Rx = CH3, R2 = CH2OH, Epothilone F (EpoF) Reaction Scheme 1: Taxoids and Epothilones Given the limited therapeutic index of EpoB, other epothilone analogues, in particular the 12,13-deoxyiepothones, were investigated for ability to provide an improved therapeutic profile (see, U.S. Patent No.: 6,242,469, 6,284,781, 6,300,355, 6,369,234, 6,204,388, 6,316,630, each of which is incorporated herein by reference). In vivo experiments conducted on several mouse models demonstrated that 12, 13-deoxyhepothilone B (3b, dEpoB in Reaction Scheme 2) has therapeutic potential against several sensitive and resistant human tumors in mouse xenografts (Chou et al. Nati, Acad. Sci. USA 1998, 95, 9642 and 15798, incorporated herein by reference). Recently, the therapeutic superiority of these deoxiepotilones over other anticancer agents has been conclusively demonstrated by complete comparative studies (Chou et al., Proc. Nati, Acad. Sci., S.A. 2001, 98, 8113, incorporated herein by reference). Due to its impressive in vivo profile, dEpoB has progressed through toxicology evaluations in dogs, and now in human trials it is like an anticancer drug.

Reaction Scheme 2. Various Desoxiepotilone Analogs In view of the promising therapeutic use of the 12,13-deoxieotilones, it may be desirable to investigate additional analogs as well as additional synthetic methodologies for the synthesis of existing epothilones, deoxyepotilones, and analogues thereof, as well as new analogs of the same. In particular, given the interest of the therapeutic utility of this class of compounds, it may also be desirable to develop methodologies capable of providing significant amounts of some epothilones or deoxy-hypothones previously described, or those described herein, for clinical trials and for large-scale preparation. scale.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a synthesis of 12-trifluoromethyl-9, 10-dehydro-deoxyepotilone D.

Figures 2A-2B show various methods of preparing 12-trifluoromethyl-9, 10-dehydrodeoxy-potilone A from Epo A. Figure 3 shows the preparation of 9,10-dehydro-epoD from Epo 490 by isomerization from the double bond C10-C11 to the position C9-C10. Figures 4A-4B depict various epothilone analogues with modifications at the 9,10 position. Figure 5 shows the synthesis of various 9,10-dehydro-epothilone analogues using a ring closure metathesis reaction in the 9,10 position. Figures 6A-6C show the results of an experiment in which an extra-large MX-1 xenograft was implanted in nude mice. Tumor tissue MX-1 (50 mg) was implanted s.c. on day 22. On day 22 (D22), when the tumor size reached 960 ± 132 mg (approximately 3.4% of body weight), 25 mg / kg of Fludelone, i.v. of 6 hr, Q3Dx5 the D22, D25, D28, D31 and D34 as indicated by the arrows. The second treatment cycle, after 9 days of rest, gave D43, D46, D49 and D52. Changes in tumor size in the control treated with vehicle (•) and group treated with Fludelone (G) (n = 5 each). The observation was continued D3D until D180 when the experiment was terminated (128 days after cessation of treatment on D52). Figure 6A shows the therapeutic effect as demonstrated by a reduction in tumor size. Figure 6B shows photographs of nude mice (one mouse each selected from the control group and the treated group) taken on D25, D31, D37, D43 and D52. No relapse was seen in D180 when the experiment was finished. Figure 6C shows changes in body weight. Figures 7A-7B show the results of an experiment in which nude mice were implanted with human T-cell lymphoblastic leukemia (CCRF-CEM / Taxol) which is 44-fold resistant to Taxol. Tumor tissue from CCRF-CEN / Taxol (44-fold resistant in vi tro), 50mg / mouse was implanted s.c. in nude mice on Day 0. The infusion treatment i.v. of 6 hr initiated the D8 with 15 mg / kg of Fludelone (D) (n = 3) and 30 mg / kg (?) (n = 4), or started the D16, Q2Dx3 and then the dose was increased to 40 mg / kg, Q2Dx3 (D22-26) and then 60 mg / kg, Q2Dx3 (D28-40). Figure 10A shows the therapeutic effect as demonstrated by a reduction in tumor size. Figure 10B shows changes in body weight. Figures 11A-11B show the treatment of nude mice bearing human lung carcinoma (A549 / Taxol) which is 44 times resistant to Taxol using 26-trifluoro-9, 10-dehydro-dEpoB and Taxol (6-hour iv infusion, Q3Dxll). Figure HA shows the therapeutic effect as demonstrated by a reduction in tumor size. Figure 11B shows changes in body weight. Figures 12A-12B show the treatment of nude mice bearing human lung carcinoma A549 / Taxol which is 44 times resistant to Taxol using 9,10-dehydro-dEpoB, (iv infusion for 6 hours, Q4Dx5, x3). Figure 12A shows the therapeutic effect as demonstrated by a reduction in tumor size. Figure 12B shows changes in body weight before and after treatment. Figures 13A-13B show a comparison of 26-trifluoro-9, 10-dehydro-dEpoB, dEpoB, and Taxol in the treatment of nude mice carrying MX-1 xenografts of human mammary carcinoma using an oral formulation (PO, Q2Dx7, x5). Figure 13A shows the therapeutic effect as demonstrated by a reduction in tumor size. Figure 13B shows changes in body weight. Figure 14 is a table summarizing various therapeutic and pharmacokinetic parameters of epothilone derivatives. Figures 15A-15B show the treatment of nude mice carrying human lung carcinoma xenograft (A549) using 26-trifluoro-9, 10-dehydro-dEpoB and dEpoB (iv infusion of 6 hr, (Q2Dx6) x2, x2) . Figure 15A shows the therapeutic effect as demonstrated by the reduction of tumor volume. Figure 15B shows changes in body weight during and after treatment.

Figure 16 shows the stabilization of microtubule formation to 10 μM drug. The microtubules formed in the presence of 10 μM taxol were defined as 100%. Bovine brain tubulin was a product of Sigma. The tubulin assembly test was performed in accordance with the manufacturer's specifications. Tubulin (1 mg in 100 μl) was incubated with 790 μl of buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl 2, 0.1 mM EDTA, and 2.5 M glycerol, pH 6.5) and with 10 μl of drug (final concentration 10 μM). For the assembly, the incubation was carried out at 35 ° C for 40 minutes, and for the disassembly of the same samples, the incubation was carried out at 4 ° C for 40 minutes. Absorbance at 350 nm was measured for microtubule stabilization. The solvent blank (DMSO) was subtracted from the absorbance. Figure 17 shows the potency of epothilones against tumorigenic cell growth in vitro and the relative therapeutic index. Figure 18 is cell cycle analysis as determined by staining DNA with propidium iodide. The cell cycle was stopped in the G2M phase starting from 6 hours and absolutely blocked at 24 hours by OPM-2 myeloma cells after treatment with dEpoB (upper panel) and Fludelone (lower panel). The two drugs induced the same configuration of cell cycle arrest. Figure 19 shows staining with Annexin V on the myeloma cell line RPMI8226. In premature apoptotic cells, membrane phospholipids phosphatidylserine (PS) are translocated from the inner leaf to the outer side of the plasma membrane, thereby exposing the PS to the external cellular environment. Annexin V is a Ca2 + -dependent phospholipid binding protein 35-36 kD that has a high affinity for PS, and binds to cells with exposed PS. "In conjunction with a vital dye such as 7-amino-actinomycin (7-) AAD) staining removes cells, this assay allows the identification of premature apoptotic cells.The data show that most cells either enter apoptosis or die after 24 hours of treatment with dEpoB or Fludelone. 20B show micrographs of myeloma and lymphoma cell lines treated with Fludelone (125 nM) for 24 hours.The ring-like structures characteristic of cells arrested in G2 / M are seen Figure 21 shows the cell count of myeloma cells RPMI8226 treated with Fludelona and dEpoB (125 nM) .At different time points (1, 2, 4, 8, 24 hours), the drug was washed, and the cells continued to be incubated for up to 48 hours.Note that with so little com or 1 hour of exposure to Fludelone the tumorigenic cell numbers progressively decreased with most of the cells undergoing apoptosis at 24 hours while with dEpoB the cells continued to expand. Figure 22 shows staining with a-tubulin of myeloma cells RPMI8226. Fludelone seems to stabilize the microtubules and greatly increases the polymer mass of microtubules at an early stage of treatment (at 12 hours with myeloma cells RPMI8226). After 24 hours of drug treatment, the microtubule mass was decreased and interrupted while the cells underwent apoptosis. Figure 23 is the cell cycle analysis determined by staining DNA with propidium iodide. The solid lines describe the values for Gl and G2, the gray shaded area represents the cells in S phase, the dotted line delineates the complete curve that produces the number of doublets especially evident with Taxol (top left image). The upper row demonstrates the G2M phase arrest after 24 hours of incubation with Fludelone (10 nM), dEpoB (100 nM), and Taxol (100 nM) of the ovarian cancer cell line IGROV. The lower row demonstrates the increase in cell cycle arrest of HT-29 with increased concentrations of Fludelone. Incubation with 100 nM of Fludelone resulted in massive apoptosis after 24 hours which prevents cell cycle analysis in various cell lines including IGROV and HT-29. Figure 24 shows staining with Annexin V of the ovarian cancer cell line 0vcar3 after 24 hours of incubation with 100 nM of Fludelone, Taxol, or dEpoB. The percentages given in the lower left quadrant are the percentage of Annexin vV negative cells Pl ", which resemble the cells in the early apoptotic stage Figures 25A-25F are cytospin of colon cancer cells HT-29 after 24 hours of staining treatment with HEMA 3 (200x magnification) Fig. 25A Control cells treated with solvent (DMSO) Fig. 25B-25D shows the increased concentrations of Fludelone 1, 10, and 100 nM. -29 after the application of 100 nM of dEpoB and 25F after 100 nM of taxol.All three drugs produce the same phenotype after 24 hours evident in a ring-like structure of the nucleus resulting in apoptosis. experimental design of a system to evaluate the action of Fludelone and dEpoB in disseminated and metastatic human tumor xenograft models Figure 27 shows IC50 assays (the concentration for 50% inhibition cellular) and cell proliferation. Cell proliferation was determined using 3 '- [1- (phenylamino-carbonyl) -3,4-tetrazolium] -bis (4-methoxy-6-nitro) benzenesulfonic acid sodium hydrate (XTT), which measured the conversion of a tetrazolium compound into formazan by a mitochondrial dehydrogenase enzyme in living cells. The amount of formaza is proportional to the number of living cells present in the test mixture. Each data point was the average of four independent determinations. The IC50 of Fludelone is approximately 7.6 ~ 36.67 nM and dEpoB 36.67-61.34 n for myeloma cell lines (RPMI8226, CAG). The IC50 of Fludelone is approximately 60 ~ 80 nM for lymphoma lines (SKIDLBCL and RL). Figure 28 shows the IC50 assays and cell proliferation in normal stromal cells. The same method was used as described in Figure 104. Human bone marrow stromal cell lines, HS-27A and HS-5 immortalized by E6 / E7 genes, have normal bone marrow stromal function, which supports the auto- stem cell renewal, and proliferation. The IC50 of Fludelone and dEpOB is approximately 90-100 nM for these stromal lines with a population doubling time comparable to tumor lines. Figure 29 shows the titration of the concentrations of Fludelone and dEpoB in cell cycle arrest. CAG myeloma cell lines were used. At the concentration of 31.25 nM, both drugs can absolutely block the cell cycle in the G2M phase (data below 31.25 nM are not shown).

Figure 30 shows the titration of the concentration of Fludelone and dEpoB in cell cycle arrest. Myeloma cell lines were used RPMI8226. At the concentration of 31.25 nM, both drugs can absolutely block the cell cycle in G2M phase (Data below 31.25 nM is not shown). Figure 31 shows staining with Annexin V in CAG myeloma cell lines. In premature apoptotic cells, membrane phospholipids phosphatidylserine (PS) were translocated from the internal leaflet to the outer membrane of the plasma, thereby exposing the PS to the external cellular environment. Annexin V is a protein that binds Ca2 + -dependent phospholipids of 35-36kD that has a high affinity for PS, and binds to cells with exposed PS. In conjunction with a vital dye such as 7-amino-actinomycin (7-AAD) staining eliminates cells, this assay allows the identification of premature apoptotic cells. The data show that most cells either enter apoptosis or die after 24 hours of treatment with Fludelone or dEpoB. Note that the X axis is spotted with Annexin and the Y axis is spotted with 7-ADA. Figure 32 is a DNA fragmentation assay. A major biochemical marker of nucleosome excision of chromanthin apoptosis. Nucleosome cleavage originates by endonuclease-mediated digestion of DNA binding sites exposed between nucleosomes in antinean. Since base pairs 180-200 of DNA around a histone nucleus are conformably protected from digestion, this endonuclease-mediated nucleosome cleavage is observable as a DNA marker on agarose gels. The data shows that the Typical DNA markers were detected - after treatment of myeloma cells RPMI8226 and CAG with Fludelone or dEpoB for 24 hours. The data imply that cellular apoptosis is induced by epothilones through the trajectory of caspase. Figure 33 shows the alternation of body weight and the effect after treatment with epothilone (20 mg / kg) of mice with disseminated CAG xenograft myeloma. The data show that control mice die in approximately 30 days and significant body weight loss was observed after 20 days of treatment. There is no significant difference in lifespan between mice treated with dEpoB and controls; however, the mice treated with Fludelone showed a significant extension of survival days than either the control and dEpoB groups. The number of mice used in each group was four, and the mice were irradiated with 300 Rad prior to the injection of CAG cells. Figure 34 shows the treatment of mice with CAG xenograft myeloma disseminated at week 4. 10x106 CAG myeloma cells modified with Luc-eGFP-TK fusion genes were injected intravenously into the tail vein of the mouse, and treatment initiated when implantation of appropriate myeloma cells was detected by bioluminescence imaging on day 7. The figure shows the 'bioluminescence images in mice treated either with control, or dEpoB, or Fludelone, respectively. Figure 35 shows quantification of average tumor photon emissions in xenoin and CAG mice (N = 4). The figure shows that mice treated with Fludelone had significantly lower tumor photon emissions than mice treated with either vehicle or dEpoB alone. The emission of photons in tumor correlates positively with tumor burden. Figure 36 shows alternations of body weight and the effect after treatment with epothilone (20 mg / kg) of mice with disseminated CAG xenograft myeloma. The figure shows a similar characteristic as figure 33 in the first 30 days; however, the only surviving group of mice treated with Fludelona received 5 additional doses of Velcade booster (6.25 ug / mouse, I.V. ). Figure 37 shows the treatment of mice with CAG xenograft myeloma disseminated on day 18. The figure shows the significant difference of images between the groups, which reflects their tumor load in the group. Figure 38 shows the quantification of photon emissions in average tumor in the treatment of mice with CAG xenograft myeloma disseminated on day 18. The figure shows the significant difference of images between the groups, which reflects their tumor load in the group . Figure 39 shows the treatment of mice with CAG xenograft myeloma disseminated at day 40. The figure shows the difference in images, which reflects the tumor burden in three groups of mice. Figure 40 shows the quantification of photon emissions in average tumor in the treatment of mice with CAG xenograft myeloma disseminated on day 40. Figure 40 shows the difference in images, which reflects the tumor burden in the three groups of mice. Figure 41 shows mice with CAG xenograft myeloma treated with Fludelone in combination with Velcade. Mice treated with Fludelone from day 0 to 28 (20 mg / kg, Q2d) and Velcade from day 35 to 45 (6.25 ug / mouse, I.V., 3 /). The treatment started on day 10 and the images were taken after 12 dosages of Fludelone and 5 dosages of Velcade on day 59. Compared to the images on day 40, two of three mice had significantly decreased tumor burden in both femurs as an invertebrate column. Figure 42 is a Kaplan-Meier survival curve of seven xenoin NOD / SCID mice harvested with CAG myeloma cells. The control mice died within 40 days after transplantation of CAG cells, and mice treated with dEpoB died within 50 days. Except for a mouse that died in 70 days, all mice treated with Fludelone survived a 1 beyond 80 days. Figure 43 shows the trajectories of cellular apoptosis. Figure 44 shows the epothilone-induced time-dependent processing of caspase 3 in CAG myeloma cells, showing an increased 17 kd cleavage form of caspase 3 with drug treatment time. Figure 45 shows the stain in unohistochemical of CAG myeloma cells using cleaved caspase-3 antibody, showing cytoplasmic and perinuclear localization in apoptotic cells (low and high amplifications are shown). Figure 46 shows the immunohistochemical staining of CAG myeloma cells using cleaved caspase-3 antibody, showing cytoplasmic and perinuclear localization in apoptotic cells (low and high amplifications are shown).

Figure 47 indicates that the activity of caspase 8 increased after treatment with epothilone, and that this increase can be inhibited by specific inhibitors of caspase 8. Figure 48 indicates that the activity of caspase 8 increased after treatment with epothilone, and that this increase can be inhibited by specific inhibitors of caspase 8. Figure 49 indicates that the activity of caspase 9 was increased after treatment with epothilone. Figure 50 shows staining with Annexin V of CB CD34 + cells incubated with dEpoB or Fludelone in the absence or presence of KL for 24 hours. Then the drugs were washed and staining with Annexin V was performed. The black area represents vehicle control and the open area represents the group treated with drug. There is no obvious increased apoptosis of non-cyclized human CD34 + cells with a short period of exposure to epothilones. Figure 51 shows the influence of epothilones on human CD34 + cells not cyclized in colony formation. There is no significant difference of progenitor cells evaluated by colony formation in 2 weeks between controls and drug treatment. Figure 52 shows the influence of epothilones on human CD34 + cells not cyclized in colony formation. There is no significant difference of progenitor cells evaluated by colony formation in 2 weeks between controls and drug treatment. Figure 53 shows the therapeutic effects against the CCRF-CEM / Paclitaxel xenograft of drug-resistant human T-cell lymphoblastic leukemia by Fludelone and Paclitaxel (loss of body weight). There was no death due to toxicity for all treatments despite the marked decreases in body weight.

DEFINITIONS Certain compounds of the present invention, and definitions of specific functional groups are also described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 Ed., Internal cover, and the specific functional groups are generally defined as described in I presented. In addition, the general principles of organic chemistry, as well as the specific functional portions and reactivity, are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein. reference. In addition, it will be appreciated by one of ordinary skill in the art that synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group", as used herein, it is understood that a particular functional portion, eg, O, γ, or N, is temporarily blocked so that a reaction can be selectively carried out at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group selectively reacts in good yield to produce a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable with good performance by preferably non-toxic reagents, readily available / not attacking the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid additional reaction sites. As detailed herein, oxygen, sulfur, nitrogen and carbon protecting groups can be used. Exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be easily identified using the above criteria and used in the method of the present invention. Additionally, a variety of protecting groups is described in "Protective Groups in Organic Synthesis" Third Ed. Greene, T.W. and Wuts, P.G., Eds., John Wiley & amp;; Sons, New York: 1999, the complete contents of which are incorporated herein for reference. It will be appreciated that the compounds, as described herein, can be substituted with any number of substituents or functional portions. In general, the term "substituted" if preceded by the term "optionally" or not, and the substituents contained in the formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specific substituent. When more than one position in any given structure can be substituted with more than one substituent selected from a specific group, the substituent can be either the same or different in each position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any of the permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Furthermore, this invention is not intended to be limited in any way by the permissible substituents of organic compounds. The combinations of substituents and variables provided by this invention are preferably those which result in the formation of stable compounds useful in the treatment, for example of proliferative disorders, including, but not limited to, cancer. The term "stable", as used herein, preferably refers to compounds which possess sufficient stability to allow manufacture and which maintain the integrity of the compound for a period of time sufficient to be detected and preferably for a period of time. sufficient time to be useful for the purposes detailed herein. The term "aliphatic", as used herein, includes both saturated and unsaturated, straight chain (ie, unbranched), branched, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups . As will be appreciated by one of ordinary skill in the art, "aliphatic" is proposed herein to include, but not be limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl portions. Accordingly, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl", and the like. In addition, as used herein, the terms "alkyl", "alkenyl", "alkynyl", and the like comprise both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched and unbranched) having 1-6 carbon atoms. In certain embodiments, the alkenyl, alkenyl and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkenyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups therefore include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, ~ CH2-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH2-cyclobutyl. , n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, -CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH2-cyclohexyl and the like, which again, may carry one or more substituents . Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-1-yl; and similar. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. The term "alkoxy" or "thioalkyl" as used herein refers to an algeryl group, as previously defined, attached to the parent molecular moiety through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-4 aliphatic carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like. The term "alkylamino" refers to a group having the structure -NHR1 wherein R1 is alkyl, as defined herein. In certain embodiments, the alkyl group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-4 aliphatic carbon atoms. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino and the like. Some examples of substituent of the aliphatic (and other) portions of compounds described above of the invention include, but are not limited to, aliphatic, heteroaliphatic; aril; heteroaryl; Arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -N02; -CN; -CF3; -CH2F3; -CHC12; -CH20H; -CH2CH20; -CH2NH2; -CH2S02CH3; -C (0) Rx; -C02 (Rx);Hen -C0N (Rx) 2; -0C (0) Rx; -OC02Rx; -0C0N (Rx) 2; -N (RX) 2; -S (0) 2Rx; -NRx (CO) Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein. In general, the terms "aryl" and "heteroaryl," as used herein, refer to mono- or polycyclic, heterocyclic, polycyclic, and unsaturated polyheterocyclic portions preferably having 3-14 carbon atoms, each of which which can be substituted or not replaced. The substituents include, but are not limited to, any of the aforementioned substituents, i.e., substituents cited for aliphatic portions, or for other portions as described herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, pl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl," as used herein, refers to a cyclic aromatic radical tending from five to ten ring atoms of which one ring atom is selected from S, 0, and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, 0, and N; and the remaining ring atoms are carbon, the radical is attached to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiopl, furanyl, quinolinyl, isoquinolinyl, and the like. It will be appreciated that the aryl and heteroaryl groups (including bicyclic aryl groups) may be unsubstituted or substituted, wherein the substitution includes the replacement of one, two, three or more of the hydrogen atoms in these independently with any one or more of the following portions including, but not limited to: aliphatic; heteroaliphatic; aril; heteroaryl; Arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -N02; -CN; -CF3; -CH2CF3; - CHC12; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2S02CH3; -C (0) Rx; -C02 (Rx); -CON (Rx) 2; -OC (O) Rx; -0C02Rx; -OCON (Rx) 2; -N (Rx) Z! S (O) 2RX; -NRx (CO) Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein. The term "cycloalkyl", as used herein, specifically refers to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl ,. cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic or heterocyclic portions, may optionally be substituted with substituents including, but not limited to, aliphatic; heteroaliphatic; aril; heteroaryl; Arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -N02; -CN; -CF3; -CH2CF3; -CHC12; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2S02CH3; -C (0) Rx; -C02 (Rx); -CON (RJ 2; -OC (O) Rx; -OC02Rx; -OCON (Rx) 2; -N (RJ2; -S (0) 2Rx; -NRx (CO) Rx where each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched , cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted The additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described in The term "heteroaliphatic", as used herein, refers to aliphatic portions containing one or more oxygen atoms, sulfur, nitrogen, phosphorus or silicon, for example, instead of atoms The heteroaliphatic portions may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, the heteroaliphatic portions are replaced by independent replacement of one or more hydrogen atoms in these with one or more portions including, but not limited to, aliphatic; heteroaliphatic; aril; heteroaryl; Arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; T; -OH; -N02; -CN; -CF3; -CH2CF3; -CHC12; -CH20H; -CH2CH2OH; -CH2NH2; -CH2S02CH3; -C (0) Rx; -C02 (Rx); -CON (Rx) 2; -OC (0) Rx; -OC02Rx; -OCON (RJ2; -N (RX) 2; -S (0) 2R ?; -NRx (CO) Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl , or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted The additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.The terms "halo" and "halogen" as used in present refer to an atom selected from fluoro, chloro, bromo and iodo The term "haloalkyl" denotes an alkyl group, as defined above, having one, two or three atoms of halogen attached to it and exemplified by such groups as chloromethyl, bromomethyl, trifluoromethyl, and the like. The term "cycloalkyl" or "heterocycle", as used herein, refers to a 5-, 6- or 7-membered, non-aromatic polycyclic or ring group, including, but not limited to, a bi- or tri- group. cyclic comprising rings of six fused members having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatom may be optionally quaternized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a "heterocycloalkyl or substituted heterocycle" group is used and as used herein, refers to a heterocycloalkyl group or heterocycle, as defined above, substituted by the independent replacement of one, two or three of the atoms of hydrogen in this with, but not limited to, aliphatic; heteroaliphatic; aril; heteroaryl; Arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -N02; -CN; -CF3; -CH2CF3; -CHC12; -CH2OH; -CH2CH20H; -CH2NH2; -CH2S02CH3; -C (0) Rx; -C02 (Rx); -C0N (Rx) 2; -0C (0) Rx; -OC02Rx; -OCON (Rx) 2; -N (RJ2; -S (0) 2Rx; -NRx (CO) Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted The additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples which are described herein. "Labeling": As used herein, the term "labeling" is intended to mean a compound having at least one element, isotope or chemical compound bound to enable the detection of the compound. falls into three classes: a) isotopic labels, which can be radioactive or heavy isotopes, including, but not limited to, "" "H, 3H, 32P, 35S, 67Ga, 99mTc (Tc-99m), lxlIn, 123I, 12I, 169Yb, and 186Re; b) immune labels, which may be antibodies or antigens, which can bind to enzymes (such as horseradish peroxidase) that produce detectable agents; and c) colored, luminescent, phosphorescent, or fluorescent dyes. It will be appreciated that labels can be incorporated into the compound in any position that does not interfere with the biological activity or characteristic of the compound being detected. In certain embodiments of the invention, the photoaffinity labeling is used for the direct elucidation of intermolecular interactions in biological systems (eg, to probe the epothilone binding site in a tubulin dimer). A variety of photophores can be employed, more dependent on the photoconversion of diazo compound, azides, or diazirines to nitrenes or carbenes (See, Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam), complete contents of which are incorporated by this means for reference. In certain embodiments of the invention, the photoaffinity labels employed are o-, m- and p-azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to, 4-azido-2,3,5,6-acid. -tetrafluorobenzoic acid "Polymer": The term "polymer", as used herein, refers to a composition comprising open, closed, linear, branched or crosslinked chains of repeating units (monomers) which may be the same or different . It will be appreciated that in certain embodiments the term polymer refers to biopolymers, which, when used herein, are proposed to refer to polymeric materials found in nature or based on those materials found in nature, including, but not limited to, limited to nucleic acids, peptides and mimetics thereof. In certain other embodiments, the term polymer refers to synthetic polymers, such as biodegradable polymers or other polymeric materials. It will be appreciated that solid polymeric supports are also included by the polymers of the present invention. The compounds of the invention can be attached to polymeric supports and consequently certain synthetic modifications can be conducted in the solid phase. As used herein, the term "solid support" is understood to include, but is not limited to, pellets, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, porous glass beads, silica gels, polystyrene beads optionally crosslinked with divinylbenzene, co-poly grafted beads, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with NN '- bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer. One of ordinary skill in the art will understand that the choice of the particular solid support will be limited by the compatibility of the support with the reaction chemistry that is used. An exemplary solid support is an amino resin Tentagel, a compound of 1) a polystyrene bead crosslinked with divinylbenzene and 2) PEG (polyethylene glycol). The Tentagel is a particularly useful solid support because it provides versatile support for use in pearl or out-of-pearl trials, and also undergoes excellent expansion in solvents ranging from toluene to water.

DETAILED DESCRIPTION OF THE INVENTION In recognition of the need to develop new and effective cancer therapies, the present invention provides novel synthetic methodologies enabling access to macrocycles having a broad range of biological and pharmacological activity, as well as novel compounds with such activity, new therapeutic compositions, and methods of using these compounds and compositions. In certain embodiments, the compounds of the invention are useful in the treatment of cancer. Certain compounds of the invention exhibit inhibitory effects on cancer and cytotoxic cancer cell lines, exhibit an ability to polymerize tubulin and stabilize microtubule assemblies, and / or lead to shrinkage or disappearance of tumors in xenograft models of cancer cell. In certain embodiments, the compounds may have reduced or minimal side effects including toxicity to vital organs, nausea, vomiting, diarrhea, alopecia, weight loss, weight gain, liver toxicity, skin disorders, etc. The compounds may also be easier to formulate because of the increased water solubility, decreased toxicity, increased therapeutic range, increased efficacy, etc.

General Description of the Compounds of the Invention The compounds of the invention include compounds of the general formula (0) as further defined below: wherein Ro is an aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, or substituted or unsubstituted heteroarylalkynyl moiety; in certain embodiments, R a is an arylalkyl, arylalkenyl, heteroarylalkyl, or heteroarylalkenyl moiety; in other embodiments, Ro is a heteroarylalkenyl portion; in certain embodiments, R0 is a heteroarylalkyl portion; in other embodiments, Ro is an aryl or heteroaryl portion of 5-7 members; in still other embodiments, R0 is an aryl or bicyclic heteroaryl portion of 8-12 members; in still other embodiments, Ro is a bicyclic moiety wherein a phenyl ring is fused to a heteroaryl or aryl moiety; in other embodiments, Ro is a bicyclic moiety wherein a phenyl ring is fused to a thiazole, oxazole, or imidazole moiety; in still other embodiments, Ro is a substituted or unsubstituted phenyl portion; R3 and R are each independently hydrogen; or aliphatic portion, heteroaliphatic, aryl, heteroaryl, arylalkyl, or substituted or unsubstituted, linear or branched, cyclic or acyclic heteroarylalkyl, optionally substituted by one or more of hydroxy, protected hydroxy, alkoxy, carboxy, carboxaldehyde, cyclic acetal or linear alkyl or branched, fluoro, amino, protected amino, amino substituted with one or two alkyl or aryl portions, N-hydroxyimino, or N-alkoxyimino; in certain embodiments, R3 and R4 are each independently hydrogen, fluoro, or lower alkyl; in other embodiments, R3 and R4 are each independently hydrogen or methyl; in still other embodiments, R3 is methyl, and R4 is hydrogen; R5 and R6 are each independently hydrogen or a protecting group; in certain embodiments, R5 and R6 are both hydrogen; X is 0, S, C (R7) 2, or NR7, where each occurrence of R? is independently hydrogen or lower alkyl; in certain modalities, X is 0; in other embodiments, X is NH; Y is 0, S, NH, C (R7) 2, CH2, N (R7), or NH, wherein each occurrence of R7 is independently hydrogen or lower alkenyl; in certain modalities, Y is 0; in other modalities, Y is NH; in still other modalities, Y is CH2; each R3 is independently hydrogen; halogen, hydroxy, alkoxy, amino, dialkylamino, alkylamino, fluoro, cyano, or aliphatic portion, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, or heteroarylalkyl, heteroarylalkenyl, substituted or unsubstituted, straight or branched heteroarylalkynyl, cyclic or acyclic optionally substituted by one or more hydroxy, protected hydroxy, alkoxy, carboxy, carboxaldehyde, cyclic acetal or linear or branched alkyl, fluoro, amino, protected amino, amino substituted with one or two servings of alkyl or aryl, N-hydroximino, or N-alkoxyimino; in certain embodiments, Rs is hydrogen; in other embodiments, Re is hydroxy; in still other modalities, Rs is fluoro; in still other embodiments, Ra is lower alkyl such as methyl; in other embodiments R8 is -CF3, -CF2H, or -CFH2; in other embodiments, Rs is perfluorinated or fluorinated alkyl group; in still other embodiments, Re is halogenated or perhalogenated alkyl group; R9 and Rio are each independently hydrogen; or aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, or substituted or unsubstituted, linear or branched, cyclic or acyclic heteroaryl, optionally substituted by one or more of hydroxy, protected hydroxy, alkoxy, carboxy , carboxaldehyde, cyclic acetal or linear or branched alkyl, fluoro, amino, protected amino, amino substituted with one or two alkyl or aryl portions, N-hydroxyimino, or N-alkoxyimino; in certain modalities, one of Rg and Rio is methyl; in other embodiments, both Rg and R10 are methyl; in still other modalities, one of Rg and Rio is methyl, and the other is hydrogen; in other modalities, both R9 and Rio are hydrogen; AB represents CRA = CRB-, C (RJ2-C (RB) 2-, or -C = C-; CD represents -CRC = CRD-, ~ C (Rc) 2-C (RD) 2-, or -C = C-, where each occurrence of RA is independently hydrogen, halogen, -0RA, -SRA-; ~ N (RA.) 2; -C (0) ORA; -C (0) RA.; -CONHRA ,; -0 (C = 0) RA; -0 (C = 0) ORA> -NRA (C = 0) RA;; N3; N2RA; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen, halogen, -0RA, -SRA <-N (RA.) 2; -C (0) ORA; -C ( 0) RA; -CONHRA> -0 (C = 0) RA;; 0 (C = 0) ORA> -NRA. (C = 0) RA;; N3; N2RA; aliphatic portion, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted, RB is, independently for each case, hydrogen, halogen, -0RB> -SRB «; -N (RB«) 2; -C ( 0) 0RB; -C (0) RB; -C0NHRB-; -0 (C = 0) RB-; -0 (C = 0) ORB; -NRB. (? = 0) RB-; N3; N2RB-; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RB-; -SRB-; -N (RB.) 2; ~ C (0) 0RB >; -C (0) RB >; -CONHRB-; -0 (C = 0) RB .; -0 (C = 0) ORB; NRB > (C = 0) RB-; N3; NRB 'aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; in certain modalities, RB is < , hydrogen, °, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each unsubstituted or optionally substituted with one or more occurrences of halogen, -OH, -ORB > , NH2, or N (RB ') 2, or any combination thereof, wherein each occurrence of RB- is independently hydrogen, alkyl, aryl, or a protecting group, in other embodiments, RB is hydrogen, methyl, or ethyl , in still other modalities, RB is methyl, in other modalities, ~ CY3, -CHY / -CH2Y, where Y is F, Br, Cl, I, 0RB., NHRB-, N (RB.) 2, or SRB.; in still other embodiments, RB is -CF3, -CH2F, or CHF2; in other embodiments, RB is perfluorinated or fluorinated alkyl group; in still other embodiments, RB is halogenated or perhalogenated alkyl group; Rc is, independently for each occurrence, hydrogen; halogen; -ORc; -SRc; -N (RC ') 2; -C (0) 0Rc; -C (0) Rc >; -CONHRc-; -0 (C = 0) Rc «; ~ 0 (C = 0) 0Rc-; -NRc (C = 0) Rc >; N3; N2Rc «; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RC; -SRC-; -N (RC.) 2; -C (0) 0Rc >; -C (0) Rc >; -CONHRc-; -0 (C = 0) Rc >; -0 (C =?) ORc >; -NRCAC = 0) RC; N3; N2RC >; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; in certain embodiments, R c is halogen, alkyl, hydroxy, or amino; in other modalities, Rc is fluoro; in still other modalities, Rc is hydroxy; RD is, independently for each occurrence, hydrogen; halogen; -OR <; -MR. D-; -N (RD.) 2; -C (0) ORD >; -C (0) RD >; -CONHRD; -0 (C = 0) RD «; ~ 0 (C = 0) ORD; -NRD. (C = 0) RD >; N3; N2RD; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RD «; -SRD >; -N (RD.) 2; -C (0) ORD >; -C (0) RD «; -CONHRD-; - 0 (C = 0) RCD '; -0 (C = 0) ORD-; -NRD. (C = 0) RD >; N3; N2RD; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; or wherein some two of RA, RB, Rc or RD taken together can form a cyclic portion and can be linked through an oxygen, sulfur, carbon or nitrogen atom, or some two adjacent groups RA, RB, Rc, or RD, taken together, can form an aliphatic, heteroaliphatic, aryl or substituted or unsubstituted heteroaryl ring of 3-6 members; in certain embodiments, RA and RB taken together form a 3-membered ring linked through an oxygen, sulfur, carbon, or nitrogen atom; in other embodiments Rc and RD taken together form a 3-membered ring linked through one atom - oxygen, sulfur, carbon or nitrogen; where each occurrence of RA ?, RB > , e and RD- is independently hydrogen; a protective group; an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalguyl, arylalkenyl, arylalkynyl, or heteroarylalkyl, heteroarylalkenyl, straight or branched heteroarylalkyl, substituted or unsubstituted, cyclic or acyclic moiety; and pharmaceutically acceptable derivatives thereof.

The compounds of the invention include compounds of the general formula (I) as further defined below: wherein Ri is hydrogen or lower alkyl; in certain modalities, Ri is methyl; in certain embodiments, Ri is -CF3, -CF2H, or CH2F; in other embodiments, Ri is perfluorinated or fluorinated alkyl group; in still other embodiments, Ri is halogenated or perhalogenated alkyl group; R 2 is an unsubstituted or substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety; in certain embodiments, R2 is substituted or unsubstituted oxazole; in other embodiments, R2 is substituted or unsubstituted thiazole; and A, B, C, D, R3, R4, R5, Re and X are as defined above. In certain embodiments, the compounds of the invention include compounds of the general formula (II) with the stereochemistry defined as shown: where A, B, C, D, Ri, R2, R3, R4, R5, Re and X are as defined above. In certain embodiments, the compounds of the invention include compounds of the general formula (III) as shown: p) wherein Z is an oxygen atom, a sulfur atom, -NRZ-, or -C (RZ) 2-; and A, B, Ri, R2, R3, R4, Rs, Re and X are as defined above. In certain preferred embodiments, 2 is oxygen. In other modalities, Z is -NH-. In still other modalities, Z is -CH2-. In other embodiments, Rz is hydrogen, alkenyl, halogen or acyl. In certain modalities, Rz is fluoro. In certain embodiments, the compounds of the invention include compounds of the general formula (IV) with the stereochemistry defined as shown: wherein A, B, R1 R2, R3, R4, R5, Re, X, and Z are as defined above. In certain modalities, RB is methyl. In other modalities, RB is -CF3. In certain embodiments, the compounds of the invention include compounds of the general formula (V) or (VI) as shown: wherein Z is an oxygen atom, a sulfur atom, -NRZ-, or -C (RZ) 2-; and A, B, Ri, R2, R3, R, R5, Re and X are as defined - above. In certain preferred embodiments, Z is oxygen. In other modalities, Z is -NH-. In still other embodiments, Z is -CH2-, In other embodiments, Rz is hydrogen, alkyl, halogen or acyl. In certain modalities, Rz is fluoro. In certain embodiments, the compounds of the invention include compounds of the general formula (VII) as shown: (vp) where A, B, Rc, RD, Ri, R2, 3, R4, Rs, Re and X are as , defined previously. In certain preferred embodiments, each Rc is independently hydrogen, halogen, or lower alkyl. In other embodiments, each RD is independently hydrogen, halogen or lower alkyl. In certain modalities, X is O. In other modalities, X is NH. In other modalities, X is CH2. In some embodiments, R2 is substituted or unsubstituted thiazole. In certain embodiments, R 2 is 2-methyl-thiazo-4-yl. In other embodiments, R2 is 2-hydroxymethyl-thiazo-4-yl. In still other embodiments, R2 is 2-aminomethyl-thiazo-4-yl. In other embodiments, R2 is 2-thiolmethyl-thiazo-4-yl. In certain embodiments, R2 is substituted or unsubstituted oxazole. In certain embodiments, R2 is 2-methyl-oxazo-4-yl. In other embodiments, R 2 is 2-hydroxymethyl-oxazo-4-yl. In still other embodiments, R2 is 2-aminomethyl-oxazo-4-yl. In other embodiments, R2 is 2-thiolmethyl-oxazo-4-yl. In certain embodiments, RB is hydrogen, methyl, ethyl, -CF3, -CH2F, -CF2H. In certain modalities, RB is methyl. In still other modalities, RB is -CF3. In certain embodiments, B is hydrogen. In other embodiments, B is ethyl. Certain preferred compounds include, for example: The compounds of this invention include those specifically described above and described herein, and are illustrated in part by the various classes, subgenres, and species described elsewhere herein. Without wishing to be bound by any particular theory, certain compounds of the present invention have been modified in C9 to CIO to restrict the conformation of the molecule in much the same way as a carbon-carbon double bond at the C9-C10 position could. The electronic effects, steric effects, hydrogen bonding, dipolar effects, or a combination thereof can be used to create this conformational restriction. For example, cyclic ring systems such as oxiranes, cyclopropyl, and aziridine can be used to restrict the conformation of the molecule. In other embodiments, rings of 4, 5, or 6 members are used to perform the same effect. The effect can also be performed by a conjugated p-orbital system such as that found in an ester, thioester, or amide. The effect can also be realized by an extended delocalized pi system such as those found in an aromatic ring system. In other embodiments, the steric effects of substituents around the C9 and CIO positions are used to restrict the conformation of the molecule in the same way that the molecule is restricted by a carbon-carbon double bond at C9-C10. It will be appreciated by one of ordinary skill in the art that asymmetric centers may exist in the compounds of the present invention. Accordingly, the compounds of the invention and pharmaceutical compositions thereof can be in the form of an individual enantiomer, diastereomer or geometric isomer, or they can be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the invention are enantiopure compounds. In certain other embodiments, mixtures of stereoisomers or diastereomers are provided. It will be appreciated that some of the above classes and subclasses of compounds may exist in various isomeric forms. The invention includes the compounds as individual isomers substantially free of other isomers and alternatively, as mixtures of various isomers, for example, racemic mixtures of stereoisomers. Additionally, the invention includes both (Z) and (E) double bond isomers unless specifically designated otherwise. Accordingly, the compounds of the invention generally represented in structures herein include those structures in which the double bonds are (Z) or (E). In certain preferred embodiments, the double bond at the C12-C13 position is in the cis or Z configuration. In some embodiments, the double bond at the C9-C10 position is in the trans or E configuration. In still other embodiments, the link double at position C12-C13 is in the cis or Z configuration, and the double bond at position C9-C10 is in the trans or E configuration. The invention also includes tautomers of specific compounds as described above. Additionally, the present invention provides pharmaceutically acceptable derivatives of the compounds of the invention, and methods of treating a subject using these compounds, pharmaceutical compositions thereof, or any of these in combination with one or more additional therapeutic agents. The phrase "pharmaceutically acceptable derivative", as used herein, denotes any salt, ester, or salt of such an ester, or such compound, or any other adduct or pharmaceutically acceptable derivative which, when administered to a patient, is capable of providing (directly or indirectly) a compound as described in another form herein, or a metabolite or residue thereof. Pharmaceutically acceptable derivatives therefore include among other prodrugs. A "pro-drug" is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional portion that is susceptible to in vivo removal producing the parent molecule as the pharmacologically active species. An example of a prodrug is an ester that is cleaved in vivo to produce a compound of interest. The pro-drugs of a variety of compounds, and materials and methods for deriving the compounds of origin to create the pro-drugs, are known and can be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below. The compounds of this invention which are of particular interest include those which: • exhibit growth inhibitory or cytotoxic effect in cancer cell lines maintained in animal studies or in vitro using a scientifically acceptable cancer cell xenograft model; • exhibit a capacity to polymerize tubulin and stabilize microtubule mounts; • exhibit minimum levels of toxicity to vital organs; • lead to the disappearance of tumor in scientifically acceptable cancer cell xenograft models; • lead to tumor shrinkage in scientifically acceptable cancer cell xenograft models; • lead to tumor disappearance in scientifically acceptable cancer cell xenograft models and delay / or no tumor occurrence after stopping treatment; • exhibit momentary and reversible body weight decreases and show therapeutic effects in scientifically acceptable cancer cell xenograft models; exhibit improved water solubility on epothilones A, B, C or D, or paclitaxel, or additionally or alternatively exhibit sufficient solubility to be formulated in an aqueous medium using reduced proportion of cremophor; and / or • exhibit a therapeutic profile (eg, optimal safety and curative effect) that is superior to that of epothilone B, epothilone D, or paclitaxel. A variety of epothilone analogs as described above has been prepared, characterized and tested as exemplified herein. It has been found that 9,10-dehydro-epothilone analogues are useful in the treatment of cancer, and in particular the compounds have been prepared and found to possess one or more of the desired characteristics listed above.

Synthetic Methodology The syntheses of certain epothilones, deoxypyotilones and analogs thereof have been previously described (see, U.S. Patents 6,242,469, 6,284,781, 6,300,355, 6,204,388, 6,316,630 and 6,369,234; U.S. Patent Applications 09 / 797,027, 09 / 796,959, and 10 / 236,135; and PCT Publication Nos. WO 99/01124, WO 99/43653, and WO 01/64650, the entire contents of which are hereby incorporated by reference). In recognition of the need for additional or improved synthetic methodologies to efficiently generate epothilones, deoxyepotilones, and analogs thereof in large amounts, the present invention provides a modular, and efficient route for the synthesis of epothilones, deoxyepotilones, and analogs thereof. Although the synthesis of certain exemplary compounds is described in the Exemplification herein, it will be appreciated that this methodology is generally applicable to the generation of analogs and conjugates as discussed above for each of the classes and subclasses described herein. In particular, the 9,10-dehydroepothilone compounds of the present invention can be prepared in a variety of ways using synthetic methodologies useful in the synthesis of epothilones. In certain embodiments, the compounds are prepared using a convergent synthetic route. For example, epothilone can be synthesized by preparing two or three intermediates which are put together to produce the desired compound. In one embodiment, one of the intermediates is an acyl portion containing 1-9 carbon, and another intermediate contains 10-15 carbons and may also contain the thiazole side chain. These two fairly equal portions of the epothilone can be put together by first using an esterification reaction between C-1 and an oxygen of C-15. The macrocycle can then be closed using a carbon-carbon coupling reaction such as a Suzuki coupling reaction or ring closure metathesis. In one embodiment, the final ring closure step is performed using a ring closure metathesis reaction to form the double bond 9,10- and close the macrocycle. The ring closure metathesis reaction is performed using an organometallic catalyst such as the Grubbs catalyst as shown in the subsequent reaction scheme. In certain embodiments, the double bond 9,10- is reduced or oxidized, or the double bond 9,10 can be further functionalized to prepare additional epothilone derivatives. In certain embodiments, the double bond 9,10- is converted to a cycloproyl moiety by the treatment of the double bond 9,10-with a carbene or carbenoid reagent such as CH 2 N 2. In certain embodiments, the 9,10-dehydroepothilone compound is prepared by double isomerization from position 10,11 (e.g., Epo490) to the 9,10 position. This isomerization can be catalyzed by the presence of a transition metal such as palladium. In other embodiments, the final ring closure step is performed using a ring closure metathesis reaction to form the double bond 12,13 and close the macrocycle. In certain embodiments, the double bond 12,13 is reduced or oxidized. In other embodiments, a macroaldolization reaction or macrolactonization is used to form the macrocycle. Certain exemplary syntheses of the compounds of the invention are provided in the figures and in the examples. As would be appreciated by one of ordinary skill in the art, a variety of analogs and derivatives can be prepared using the synthetic procedures described herein. For example, one could perform many of the synthetic steps with different protecting groups or different substituents on the 16-membered ring.

Pharmaceutical Compositions This invention also provides a pharmaceutical preparation comprising at least one of the compounds as described and herein, or a pharmaceutically acceptable derivative thereof, the compounds are capable of inhibiting the growth or elimination of cancer cells, and, in certain modalities of special interest, they are capable of inhibiting the growth or eliminating cancer cells resistant to multiple drugs. In certain embodiments, the pharmaceutical preparation also comprises solubilizing or emulsifying agent such as Cremofor (polyoxyl 35 castor oil) or Solutol (polyethylene glycol 1260 hydroxystearate). As discussed above, the present invention provides novel compounds having anti-tumor or antiproliferative activity, and therefore the compounds of the invention are useful for the treatment of cancer. Accordingly, in another aspect of the present invention, pharmaceutical compositions are provided, wherein these compositions comprise some of the compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally additionally comprise one or more additional therapeutic agents. In certain other embodiments, the additional therapeutic agent is an anticancer agent, as discussed in more detail herein. It will also be appreciated that certain compounds of the present invention may exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof. In accordance with the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, salts, esters, salts of such esters, or any other pharmaceutically acceptable adduct or derivative which upon administration to a patient in need is capable of providing , directly or indirectly, a compound as described herein in another form, or a metabolite or residue thereof, eg, a prodrug. As used herein, the term "pharmaceutically acceptable salt" refers to those salts which, within the scope of correct medical judgment, are suitable for use in contact with the tissues of human and lower animals without undue toxicity, irritation. , allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66 1-19 (1977), incorporated herein by reference. The salts can be prepared in situ during the isolation and final purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of non-toxic, pharmaceutically acceptable acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid , maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include salts of adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorrate, camphorsulfonate, citrate, cyclopentapropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate , heptanoate, hexanoate, iodhydrate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, piccrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. Additionally, as used herein, the term "pharmaceutically acceptable ester" refers to esters which are hydrolyzed in vivo and include those that readily decompose in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl portion advantageously has no more than 6 carbon atoms. Examples of particular esters include formates, acetates, propionates, butyrates, acrylates and ethylsuccinates. In addition, the term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of correct medical judgment, suitable for use in contact with human tissues. and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their proposed use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds that are rapidly transformed in vivo to produce the parent compound of the above formula, for example by hydrolysis in blood. A full discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both are incorporated herein by reference. As described above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, suspension or dispersion aids, agents active surface, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like, as is suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa, 1975) describes various carriers used in the formulation of pharmaceutical compositions and techniques known for the preparation thereof. Except to where any conventional carrier medium is incompatible with the anti-carcinogenic compounds of the invention, such as by producing some undesirable biological effect or otherwise interacting in a noxious manner with some other components of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose.; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; jelly; talcum powder; Cremofor; Solutol; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and perfuming agents, preservatives and antioxidants they may also be present in the composition, according to the judgment of the formulator.

USES OF COMPOUNDS AND PHARMACEUTICAL COMPOSITIONS The invention further provides a method for inhibiting tumor growth and / or tumor metastasis. In certain embodiments of special interest, the invention provides a method of treating cancers by inhibiting tumor growth and / or tumor metastasis for tumors of cancer cells resistant to multiple drugs. The method involves administering a therapeutically effective amount of the compound or a pharmaceutically acceptable derivative thereof to a subject (including, but not limited to a human or anima) in need thereof. In certain embodiments, specifically for treating cancers comprising multiple drug resistant cancer cells, the therapeutically effective amount is an amount sufficient to eliminate or inhibit the growth of cancer cell lines resistant to multiple drugs. In certain embodiments, the compounds of the invention are useful for the treatment of solid tumors. The compounds and pharmaceutical compositions of the present invention can be used in the treatment or prevention of any disease or conditions including proliferative diseases (e.g., cancer), autoimmune diseases (e.g., rheumatoid arthritis), and infections (e.g., bacterial) , fungal, etc.). The compounds and pharmaceutical compositions can be administered to animals, preferably mammals (eg, domesticated animals, cats, dogs, mice, rats), and more preferably humans. Any method of administration can be used to deliver the compound of pharmaceutical compositions to the animal. In certain embodiments, the compound or pharmaceutical composition is administered parenterally. In still another aspect, according to the methods of treatment of the present invention, the tumorigenic cells are killed, or their growth is inhibited by contacting the tumorigenic cells with a composition or compound of the invention, as described herein. Accordingly, in yet another aspect of the invention, there is provided a method for the treatment of cancer comprising administering a therapeutically effective amount of a compound of the invention, or a pharmaceutical composition comprising a compound of the invention to a subject in need. of the same, in such quantities and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention a "therapeutically effective amount" of the compound or pharmaceutical composition of the invention is that amount effective to eliminate or inhibit the growth of tumorigenic cells. The compounds and compositions, according to the method of the present invention, can be administered using any amount and any effective route of administration to eliminate or inhibit the growth of tumorigenic cells. Therefore, the expression "amount effective to eliminate or inhibit the growth of tumorigenic cells", as used herein, refers to a sufficient amount of agent to eliminate or inhibit the growth of tumorigenic cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular anticancer agent, its mode of administration, and the like. The anticancer compounds of the invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" as used herein refers to a physically discrete unit of anticancer agent appropriate for the patient to be treated. It will be understood, however, that the total daily use of the compounds and compositions of the present invention will be decided by the attending physician within the scope of the correct medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend on a variety of factors including the disorder to be treated and the severity of the disorder; the activity of the specific compound used; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincident with the specific compound used; and similar factors well known in the medical arts. In addition, after formulation with a suitable pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments) , or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection to be treated. In certain embodiments of the invention, the compounds of the invention as described herein are formulated by conjugation with water-soluble chelators, or water-soluble polymers such as polyethylene glycol such as poly (l-glutamic acid), or poly (acid). -aspartic), as described in U.S. Patent 5,977,163, the entire contents of which are hereby incorporated by reference. In certain embodiments, the compounds of the invention can be administered orally or parenterally at sufficient dosage levels to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg kg, and more preferably from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times per day, to obtain the desired therapeutic effect. The desired dosage can be supplied as supplied every two days, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, or ten administrations). Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavors, and perfuming agents. In certain embodiments for parenteral administration, the compounds of the invention are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof. In certain embodiments, the compound is mixed with an alcohol, such as ethanol, and Cremofor (polyethoxylated castor oil). Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable wetting and dispersing agents and suspending agents. The sterile injectable preparation can also be a solutionsterile injectable suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable solvents and vehicles that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any soft soft fixed oil may be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria retention filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile prior injectable medium. to use. To prolong the effect of a drug, it is often desirable to delay the absorption of the subcutaneous or intramuscular injection drug. This can be done by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of drug absorption then depends on its rate of dissolution which, in turn, may depend on the crystal size and crystal form. Alternatively, the delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oily vehicle. Depot injectable forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orothers) and poly (anhydrides). Depot injectable formulations are also prepared by trapping the drug in liposomes or microemulsions which are compatible with body tissues. Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are both solid at room temperature but liquid. at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Solid dosage forms for oral administration include capsules, tablets, pills, powder, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and salicylic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato starch or tapioca, alginic acid, certain silicates, and sodium carbonate, e) agents that retard the solution such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as bentonite clay and kaolin, and i) lubricants such as talc, calcium stearate, magnesium stearate, polye solid tilene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type can also be used as fillers in soft or hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredients only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of inlay compositions which may be used include waxes and polymeric substances. Solid compositions of a similar type can also be used as fillers in hard and soft fill gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The active compounds may also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, coatings that control release and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, for example, tabletting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Optionally they may contain opacifying agents and may also be of a composition that they release the active ingredients only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of inlay compositions which may be used include waxes and polymeric substances. Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers or patches. The active component is mixed under sterile conditions with a pharmaceutically acceptable carrier and any of the necessary buffers or preservatives as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the appropriate medium. Absorption enhancers can also be used to increase the flow of the compound through the skin. The speed can be controlled either by providing a membrane that controls the speed or by dispersing the compound in a gel or polymer matrix. As discussed above, the compounds of the present invention are useful as anticancer agents, and therefore may be useful in the treatment of cancer, by killing the tumorigenic cell or inhibiting the growth of tumorigenic cells. In general, the anticancer agents of the invention are useful in the treatment of cancers and other proliferative disorders, including, but not limited to, breast cancer, brain cancer, skin cancer, cervical cancer, colon and rectal cancer, leukemia, lung cancer, melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, and gastric cancer, to name a few. In certain modalities, the anticancer agents of the invention are active against leukemia cells and melanoma cells, and therefore are useful for the treatment of leukemias (for example, myeloid, lymphocytic, promyelocytic, myelocytic and lymphoblastic leukemias, if they are acute or chronic forms) and malignant melanomas. In still other embodiments, the anticancer agents of the invention are active against solid tumors and also eliminate and / or inhibit the growth of multiple drug resistant cells (RFM cells). In certain embodiments, the anticancer agents of the invention are active against cancers which are resistant to other known anti-neoplastic agents or which have been found not to respond clinically to other known anti-neoplastic agents. In other embodiments, the anticancer agents of the invention are active against cancers which are resistant to other anti-neoplastic microtubule stabilizing agents (e.g., paclitaxel). It will also be appreciated that the compounds and pharmaceutical compositions of the present invention may be employed in combination therapies, ie, the compounds and pharmaceutical compositions may be administered concurrently with, prior to, or subsequent to, one or more other medical or therapeutic procedures. desired. The combination of particular therapies (therapeutics or procedures) for use in a combination regimen will take into account the compatibility of the desired therapeutic and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (eg, a compound of the invention may be administered concurrently with another anticancer agent), or may achieve different effects (eg, control of any of the effects). adverse). For example, other therapies or anticancer agents that can be used in combination with the anticancer agents of the invention of the present invention include surgery, radiotherapy (except for a few examples,? -radiation, neutron beam radiotherapy, electron beam radiotherapy , proton therapy, brachytherapy, and systemic radioactive isotopes, to name a few), endocrine therapy, biological response modifiers (interferons, interleukins, and tumor necrosis factor (TNF) by a few), hyperthermia and cryotherapy, agents to attenuate any of the adverse effects (e.g., antiemetics), and other approved chemotherapeutic drugs, including, but not limited to, alkylation drugs (mecloartamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (methotrexate), purine antagonists and pyrimidine antagonists (6-mercaptopurine, 5-fluorouracil, citarabil, gemcitab ina), needle poisons (vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel), podophyllotoxins (etoposide, irinotecan, topotecan), antibiotics (doxorubicin, bleomycin, mitomycin), nitrosoureas (carmustine, lomustine), inorganic ions (cisplatin, carboplatin) , enzymes (asparaginase), and hormones (tamoxifen, leuprolide, flutamide, and megestrol), to name a few. For a more comprehensive discussion of up-to-date cancer therapies, see http://www.nci.nih.gov/, a list of oncology drugs approved by the FDA at http://www.fda.gov/cder/cancer /druglistframe.htm, and the Merck Manual, Seventeenth Ed. 1999, the contents of which are hereby incorporated by reference. In another aspect, the present invention also provides a kit or pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for the use as a combination therapy. Optionally associated with such containers may be a notice in the form pre-written by a government agency that regulates the manufacture, use or sale of pharmaceutical products, the notice reflects the approval by the manufacturing, use or sale agency for human administration.

EQUIVALENTS The representative examples which follow are proposed to help illustrate the invention, and are not proposed, nor should they be constructed, to limit the scope of the invention. Indeed, several modifications of the invention and many additional embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the complete contents of this document, including the examples which follow and references to the patent and scientific literature cited herein. It should be further appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art. The following examples contain important additional exemplification and guidance information which can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

EXAMPLES Example 1: Oral and Parenteral Administration of 26-trifluoro-9,10-trans-dehydro-epothilone D in the Treatment of Human Tumors implanted in Naked Mice In this example, it was demonstrated that the 16-member microtubule stabilizing agent macrolide structurally designed, 26-trifluoro-9, 10-trans-dehydro-epothilone D, shrinks tumors, makes tumors disappear, and achieves no long-term relapse when given for 6 hr. - infusion i.v. or orally. Mice bearing large size tumor (Figure 6) or taxol-resistant tumor xenografts (Figure 7) are shown to be curable with 26-trifluoro-9,10-trans-dehydro-epothilone D as a single agent monotherapy. The curative therapeutic spectrum includes leukemia as well as carcinomas of .mama, colon, and lung (figures 6, 9 and 13). All the in vivo chemotherapeutic experiments reported here were performed using human tumor xenografts in nude immunodeficient mice. This animal model is commonly used in the evaluation of anti-tumorigenic compounds prior to clinical trials in patients with cancer. The MX-1 and HCT-116 experiments lasted 5.5 and 6.5 months, respectively (Figures 6 and 9). There was no tumor relapse in both experiments with 3.8 and 5.2 tumor-free months, respectively, after cessation of treatment. For the HCT-116 experiment (Figure 9), both paclitaxel and 26-trifluoro-9, 10-trans-dehydro-epothilone D at 20 mg / kg were used and both achieved tumor disappearance. However, the group treated with paclitaxel relapsed at 1.1 months after stopping treatment, while the animals treated with 26-trifluoro-9, 10-trans-dehydro-epothilone D were tumor free for more than 5.2 months. Assuming a 4-day tumor doubling time (based on vehicle-treated control), treatment with HCT-116 tumor paclitaxel resulted in 99.7% tumor suppression or 2.56 log cell elimination, while elimination of cells by 26-trifluoro-9, 10-trans-dehydro-epothilone D in the MX-1 and HCT-116 experiments could be > 8.5-log and > 11.6 log, respectively, when the experiments were finished. Based on the healthy active conditions of the mice treated with body weight returned to pre-treatment levels, it is expected that the "cure" can be assured in all 2 years of life of the mice if prolonged treatment or additional cycles of treatment were reinforced. This result should be achieved despite the fact that "cure" may be more difficult to achieve in immunodeficient mice than in immunocompetent mice. For our recognition, 'these are the longest therapeutic xenograft studies performed on nude mice in the biodical literature, and the longest complete remission that has been reported either with parenteral or oral administration for a single antitumor agent. It is pertinent to establish that it is relatively easy to find a compound that suppresses tumor growth. But it is relatively rare to find a compound that achieves tumor shrinkage. The discovery with our compound, 26-trifluoro-9, 10-trans ~ dehydro-epothilone D, which achieves disappearance of complete tumor in all mice without relapse after 5.2 months for our recognition has never been previously reported, indicating the great potential of 26-trifluoro- 9, 10-trans-dehydro-epothilone D for therapeutic development.

The additional significant benefit of oral therapy is that the use of Cremofor formulation that can cause severe allergic reactions can be avoided. It is well known that the use of Cremophor in the formulation in Taxol, deoxy-EpoB, and 15-aza-Epo 13 induces annoying allergic reactions which make it necessary to pretreat with antihistamine and / or steroid. The oral effectiveness of 26-trifluoro-9, 10-trans-dehydro-epothilone D is consistent with its observable metabolic stability in mouse plasma and in the microsomal fraction S9 of human liver in vitro. This metabolic stability is attributable to the trifluorination at the C-26 position of the epothilone molecule (FIG. 14). The introduction of the double bond at C9-C10 also increases the metabolic stability (Figure 14). The proximity of the optimal dose of 26-trifluoro-9, 10-trans-dehydro-epothilone D for infusion i.v. (20-30 mg / kg, Q2D) and for oral administration (20 mg / kg, QD or 30 mg / kg Q2D) suggests that the compound is well absorbed and has excellent bioavailability in vivo. It is worth noting that many of our in vivo therapeutic studies on Fludelone (figures 13, 7, 9, 10, 11 and 16) against xenografts were performed in parallel with Taxol, which is one of the most important therapeutic agents against cancer currently in use in clinics. The profound discoveries with fludelone, in comparison with Taxol, indicate the promising potential of this compound for the treatment of cancer.

Materials and Chemical Methods: All epothilones were synthesized as indicated herein. Paclitaxel (Taxol®) and vinblastine sulfate (VBL) were purchased from Sigma. All these compounds were dissolved in dimethyl sulfoxide for the in vitro tests, (except VBL in saline). For in vivo studies, all epothilones and paclitaxel were dissolved in Cremofor / ethanol vehicle (1: 1) and then diluted with saline for i.v. infusion. for 6 hrs via the tail vein using a custom-designed mini-catheter and a programmable pump (Chou et al. (1998) Proc. Nati, Acad Sci USA 95, 15798-15802; Chou et al. (2001) Proc. Nati Acad. Sci. USA 98, 8113-8118; each of which is incorporated herein by reference). Oral administration of the drugs was prepared by dissolving the compound in ethanol and was suspended with equal volume of Tween-80 and the suspension was diluted with 5 volumes of saline prior to administration to nude mice. Forced feeding was performed using a 1 ml syringe and a # 22 ball tip animal feeding needle (Popper &Sons, Inc. New Hyde Park, NY).

Tumor and Cell Lines: The human lymphoblastic leukemia cells CCRF-CEM and its vinblastine-resistant subline (CCRF-CEM / VBL? 00, resistance 720 times) were obtained from Dr. William Beck of the University of Illinois, Chicago, and CCRF -CEM / Taxol (resistance 44 times) exposing the CCRF-CEM cells to increased sub-lethal concentration (IC50-IC90) of paclitaxel for six months. The degree of resistance is shown in Figure 14. Human mammary carcinoma (MX-1), human lung carcinoma cells (A549), and human colon carcinoma (HCT-116) were obtained from American Type Culture Collection (ATCC , Rockville, MD). Animals: Athymic nude mice carrying the nu / nu genes were obtained from NCI, Frederick, MD and used for all human tumor xenografts. Male nude mice 6 weeks or older weighing 20-22 g or more were used. The drugs were administered via the tail vein for 6 hours by i.v. using a homemade mini infusion catheter and containment tube. A Harvard PHD2000 syringe pump programmable with multivies was used for i.v infusion. A typical 6-hour infusion volume for each drug in Cremofor / ethanol (1: 1) was 100 μl in 2.0 ml of saline. For oral administration, both fludelone and taxol were dissolved in ethanol and diluted 5 times with Tween-80. The taxol solution should be used within 5 min to avoid precipitation. Tumor volume was assessed by measuring length x width x height (or width) using a calibrator. For nude mice that carry tumor during the course of the experiment, body weight refers to the total weight minus the weight of the tumor. All animal studies were conducted in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Animáis and the protocol approved by the Memorial Sloan-Kettering Cancer Center's Institutional Animal Care and Use Committee. Cytotoxicity Assays: In the preparation for in vitro cytotoxicity assays, the cells were cultured at an initial density of 2-5 x 10 4 cells per milliliter. They were kept in a humidified atmosphere with 5% CO2 - at 37 ° C in RPMI 1640 medium (GIBCO / BRL) containing penicillin (100 units / ml), - streptomycin (100 μg / ml, GIBCO / BRL), and % FBS inactivated with heat. For solid tumor cells growing in a monolayer (such as HCT-116 and A549), the cytotoxicity of the drug was determined in 96-well microtiter plates using the sulforhodamine B method (Skehan et al (1990) J. Nati. Cancer Inst. 82, 1107-1112; incorporated herein by reference). For cells grown in suspension (such as CCRF-CEM and its sub-lines), cytotoxicity was measured, in duplicate, using the micro-culture method in 2,3-bis- (2-methoxy-4-nitro-) hydroxide. 5-sulfophenyl) -5-carboxanilide) -2H-tetrazodium (XTT) (Scudiero et al. (1988) Cancer, J. 48, 4827-4833, incorporated herein by reference) into 96-well microtiter plates. For both methods, the absorbance of each cavity was measured with a microplate reader (Power Wave XS, Bio-Tek, Winooski, VT). The data of dose-effect relationship of 6 to 7 concentrations of each drug, in duplicate, were analyzed with the medium-effect graph using a computer program (Chou et al. (1997) CalcuSyn for Windows (Biosoft, Cambridge, United Kingdom), each of which is incorporated herein by reference). Stability of epothilones in the S9 fraction of human and mouse liver: The stability study was performed with a fully automated HPLC system which consisted of a sample preparation system Prospekt-2 (Spark Holland, The Netherlands) and a system of CLAR Agilent 1100. Briefly, Prospekt 2 collected a C8 extraction cartridge and washed it with acetonitrile and water. The Agilent autosampler, adjusted to 37 ° C, collected 20 μl of the sample, loaded it on the cartridge, washed it with water, then the Prospekt-2 divided the mobile phase current through the extraction cartridge on the analytical column, Reliance Stable Bond C8 4 x 80 mm with protected column (MacMod, Chadds Ford, PA) and the eluent was monitored at 250 nm. The mobile phase consists of 53 or 65% acetonitrile / 0.1% formic acid at 0.4 ml / min, so that the reaction time of the compound of interest is about 6 minutes. The sample preparation involves the addition of equal volumes of plasma to PBS for a total volume of 400 μl, was filtered, and the addition of 0.5-2 μl of the substrate (20 mM) to achieve approximately 30-50 AU at 250 nm in the analysis of CLAR. For the S9 fraction of pooled human liver microsomes (Xeno Tech, Lenex, KS), 20 μl (400 μg) or fraction S9 was mixed with 280 μl of PBS then proceed as above. The sampling period was controlled by the autosampler and the peak area data were collected to compare the disappearance rate of the source compound.

Results Structure-Activity Relations in vitro against Human Leukemic Cells, Leukemic Cells Resistant to Taxol and Vinblastine and Solid Tumor Cells. The potency in terms of ICso (in μM) for twelve representative epothilones against the growth of human leukemic CCRF-CEM cells and their sub-lines resistant to vinblastine CCRF-CEM / VBL, and resistant to paclitaxel, CCRF-CEM / Taxol is they are listed in the table in figure 17. IC50 values are also listed for two human solid tumor cell lines: lung carcinoma A549 and colon carcinoma HCT-116. It is shown that deH-EpoB replaces EpoB as the most potent epothilone known. It is also shown that i) The modification of 9,10-dehydro- in dEpoB, dEpoF, or F3-dEpoF always results in marked increase in power, while the trifluorination in position C-26 reduces power slightly, however , the metabolic stability is greatly increased by the trifluorination (figure 14); and ii) Most of the epothilones are not cross-resistant with vinblastine, a typical substrate for multidrug resistance P-glycoprotein (RFM) without cross-resistance with Taxol. Taxol is a good substrate for the RFM phenotype but taxol resistance can also be generated by mutation in the tubulin gene. The exceptions are 15-aza-EpoB, 15-aza-deH-dEpoB which show considerable cross-resistance to both vinblastine and paclitaxel. The dEpoF and its derivative showed some cross resistance with vinblastine but not paclitaxel. The leukemic cells CCRF-CEN and solid tumor cells A549 and HCT-116 are almost equally susceptible to epothilones. Iv) F3-deH-dEpoB, deH-dEpoB, and deH-EpoB, which are our new major compounds for further pharmacological evaluations, are not appreciably cross-resistant to either vinblastine or paclitaxel. In early studies (Schiff et al. (1979) Nature (London) 277, 665-667; Meng et al. (1997) J. Am. Chem. Soc. 119, 2733-2734, each of which is incorporated in the present for reference) it was concluded that the epothilone molecules can be divided into three zones. Therefore, in the acyl sector C -1 ~ 8, structural changes are not tolerated in terms of in vitro cytotoxicity and microtubule stabilization capacity. This is in contrast to the O-alkyl sector C ~ 9 ~ 15 and the pendant aryl sectors C-15 where considerable modification of the structures is tolerated (Haar et al (1996) Biochemistry 35, 243-250; Kowalski et al. (1997) J. Biol. Chem. 272, 2354-2541, each of which is incorporated herein by reference). The epothilones with 12, 13-epoxyl portion, such as EpoB, and deH-EpoB, although they are the most potent agents in the epothilone series but possess rather poor therapeutic index in their maximum tolerated doses. This hypothesis is illustrated with the therapeutic data given in the table in figure 14.

Physical-chemical, Metabolic, Pharmacological and Therapeutic Results of Epothilone Derivatives. The different inter-related properties of a series of epothilones facilitates the understanding of the factors that contribute to the final therapeutic results for the major compounds. The table in figure 14 summarizes the profiles of nine epothilone derivatives. The in vitro cytotoxic structure-activity relationship provides initial assessment based on potency. For example, the new class of 9, 10-dehydro modification greatly increases the potency in vitro and in vivo. In these pre-selected compounds, it is more difficult to correlate the structure-microtubule stabilizing potency because the potencies are all quite high (ie, similar to the potency of paclitaxel). The solubility in water and lipophilicity play a varied role in the therapeutic effect and can be important for the formulation design. The deH-dEpoF, F3-deH-dEpoF, and deH-dEpoB greatly increase the solubility in water relative to other epothilones. The discovery that F3 ~ deH-dEpoB is orally effective reduces the interest of insolubility in water in the drug formulation and the use of Cremofor that causes allergies can be avoided. In vitro, deH-EpoB, deH-dEpoF, EpoB, and deH-dEpoB, in this order have sub-nanomolar IC50 while F3-deH-dEpoF, dEpoF, F3-deH-dEpoB, dEpoB, dEpoB and F3- dEpoB, in this order, have IC 50 intervals of 1.3 to 9.3 nM (Figure 17). DeH-EpoB and EpoB are the most potent epothilones known in vitro and in vivo but do not produce the best therapeutic index. Apparently, the epoxy portion in C-12 ~ 13 of EpoB and deH-EpoB contributes greatly to host toxicity as evidenced by the lower value in the percent fall in maximum body weight without a death (Figure 14). In contrast, F3-deH-dEpoB and dEpoB tolerate the highest values of fallen body weight and show excellent therapeutic results such as complete tumor remission in all animals. In general, the in vitro potency and the optimal therapeutic dose revealed good correlations with i.v. 6 hours of treatment. Of special interest is that the F3-deH-dEpoB (Fludelone) has the widest curative therapeutic dose range (10-30 mg / kg) (Chou et al. (2003) Angew Chem. Int. Ed. Engl. 42, 4762-4767; incorporated herein by reference), excellent metabolic stability and the best total therapeutic results among the listed epothilones (Figure 14). In addition, F3-deH-dEpoB provides curative therapeutic effect via oral route of administration. It is concluded that the most potent epothilones do not necessarily perform better as antitumor agents and that F3-deH-dEpoB is the main candidate for therapeutic development.

MX-1 Extra Long Tumor Xenograft Therapy. As shown in Figure 6A, the treatment of MX-1 xenografts as large as 3.4% body weight with F3-deH-dEpoB, 25 mg / kg, i.v. of 6 hr Q3Dx5 that started on D22 after tumor implantation, led to marked tumor shrinkage (> 97.4%). During the remaining period of 9 days without treatment (D34-D43), the tumor size continued to shrink (> 99.3%) and the tumor disappeared in 2 out of 5 studied mice, while the body weight of the treated group recovered to the level of pre-treatment during the same remaining period (Figure 6B). The continuation of treatment in D43, Q3Dx4, led to the disappearance of tumor in the remaining 3 mice in D50, D50, and D51. After D52 (ie, date of last dose), animals were observed every three days until DI65 when the experiment was determined. There was no tumor reaction in all 5 animals in the DI65 (ie, for 3.7 months after cessation of treatment). Photographs of nude mice from this experiment were taken on D25, D31, D37, D43, and D52 of a mouse each of the control and treated groups are shown in Figure 6B.

Xenograft Cancer Healing Therapy MX-1 by Fludelone via Oral Administration. As shown in Figure 13A, the F3-deH-dEpoB given orally at 30 mg / kg every other day for 7 times led to MX-1 tumor shrinkage and tumor disappearance in 2 out of 4 mice. Two additional doses (after the dose jump) led to the disappearance of tumor in 4 out of 4 mice. In contrast, oral treatment of Taxol at the same dose and the same program suppressed tumor growth MX-1 but did not lead to tumor shrinkage. Two days after the last dose of Taxol (D36), it induced tumor suppression of 66.9%. Treatment with F3 ~ deH-dEpoB induced moderate yet persistent decreases in body weight with maximum fall of 15% of body weight (Figure 13B). Treatment with Taxol induced few changes in body weight, suggesting that oral administration of Taxol was not an adequate treatment, apparently due to the metabolic inactivation of the drug or poor bioavailability. In contrast to the oral administration route, our previous report (Chou et al. (2001) Proc. Nati. Acad. Sci. USA 98, 8113-8118, incorporated herein by reference), Taxol treatment of tumor MX -1 to 20 mg / kg via iv infusion 6 hr could lead to tumor shrinkage and tumor disappearance.

Healing Therapeutic Effect of Fludelone against CCRF-CEM / Taxol Xenograft Resistant to Taxol. In our recent report (Chou et al. (2003) Angew Chem. Int. Ed. Engl 42, 4762-4767, incorporated herein by reference), it is shown that F3-deH-dEpoB at 20 mg / kg, Q2Dx7, iv infusion of 6 hr against human lung carcinoma resistant to Taxol A549 / Taxol (44-fold resistance to paclitaxel in vitro) provided complete suppression of tumor growth but failed to achieve tumor disappearance. Another paclitaxel-resistant xenograft, CCRF-CEM / Taxol (44-fold resistance to paclitaxel in vitro) is now used. As shown in figure 7, fludelone at 30 mg / kg Q2Dx7, infusion i.v. of 6 hr led to the disappearance of tumor in 2 out of 4 mice. Additional Q2Dx5 (after one dose jump) led to the disappearance of tumor in 3 out of 4 mice, with the final tumor suppression of 99.8% (Figure 7A). At the reduced dose of 15 mg / kg, the disappearance of tumor occurred in only 1 of 4 mice on the 5th day after two cycles of treatment. The final tumor suppression in D34 was 98.8%. The parallel experiment with taxol at 20 mg / kg provided suppression of tumor growth but with little or no tumor shrinkage. The final tumor suppression in D34 was 75.6%. Both F3-deH-dEpoB (at 15 mg / kg and 30 mg / kg) and Taxol (20 mg / kg) persistently reduced body weight during the first cycle of 7 treatment via i.v. infusion. of 6 hr every two days. The jump of one treatment in D22 led to immediate gain of body weight in all mice. The second treatment cycle of Q2Dx5 again persistently reduced body weight but without lethality to all mice tested (Figure 7B).

Healing Therapy against HCT-116 Xenografts of Human Colon Carcinoma by Fludelone. As shown in Figure 9A, F3-deH-dEpoB at 20 mg / kg and 30 mg / kg or Taxol at 20 mg / kg, Q2Dx4, infusion i.v. of 6 hr for 3 cycles, all led to tumor disappearance in 4 out of 4 mice. The treatment initiated D9 after tumor implantation. There was a jump of a dose in the D17 between the 1st and 2nd cycles and there was a jump of two doses in D27 and D29 between the 2nd and 3rd cycles. The D9-D37 was the 3-cycle therapy period and D37-D200 was the consecutive period. The experiment lasted 200 days which represent more than a quarter of the average life span of the mice. For F3-deH-dEpoB at 30 mg / kg, the tumor disappeared on D21, 23, 33, and 41 and at 20 mg / kg, the tumor disappeared on D31, 35, 41, and 45. For both doses of F3-deH -dEpoB, there was no tumor relapse in 4 of 4 mice in the D200 when the experiment was finished. For Taxol at 20 mg / kg, the tumor disappeared in D33, 33, 41, and 45 which were similar to the observation with F3-deH-dEpoB at 20 mg / kg. However, in the group treated with Taxol, tumor relapse occurred in D71, 75, 81 and 81. The treatment schedule that includes the remaining period is dictated by decreases in body weight and physical conditions of the mice. For treatment with F3-deH-dEpoB at a dose of 30 mg / kg, the maximum body weight loss was 30% but no lethality which occurred at the end of the 2nd treatment cycle (ie, D29) (Figure 9B). The decreases in body weight and recovery of Taxol 20 mg / kg and F3-deH-dEpoB 20 mg / kg were similar in both configuration and magnitude.

Example 2: Determination of the Mechanism of Action of Fludelone (F3-deH-dEpoB) and How it Differ from that of the EpoD; Therapeutic Implications of these Differences Drug sensitivity in Tumor Cell Lines of Non-Hodgkins Lymphoma (NHL) and Human Multiple Myeloma (MM) MM accounts for 1% of all cancers and 10% of hematologic malignancies, with 15,500 new classes diagnosed and XLS, 000 deaths in 2002. Treatment of MM with conventional chemotherapy is not curative, with a median survival of approximately 3 years (Barlogie et al., "Treatment of multiple myeloma" Blood 2004; 103: 20-32; incorporated herein by reference). Although high-dose chemotherapy with hematopoietic stem cell support increases the rate of complete remission and event-free survival, almost every patient relapsed, sending the crucial need for forced feeding therapy options. Paclitaxel has been used to treat multiple myeloma and non-Hodgkin's lymphoma (Miller et al, "Paclitaxel as the initial treatment of multiple myeloma: an Eastern Cooperative Oncology Group Study (E1A93)" Am. J.

Clin. Oncol. 1998; 21: 553-556; Jazirehi et al. "Resveratrol modifies the expression of apoptotic regulatory proteins and sensitizes non-Hodgkin's lymphoma and multiple myeloma cell Unes to paclitaxel-induced apoptosis" Mol. Cancer Ther. 2004; 3: 71-84; each of which is incorporated herein by reference). However, the application was limited due to its high toxicity and resistance to multiple drugs. Fludelone and dEpoB have been evaluated against a panel of human MM and NHL lines that have been used in a number of recent studies in NOD / SCID xenograft models for evaluation of new therapies, including 10-propargyl-10-deazaaminopterin (PDX) (Wang et al. "Activity of a novel anti-folate (PDX, 10-propargyl-10-deazaaminopterin) against human lymphoma is superior to methotrexate and correlates with tumot RFC-1 gene expression" Leukemia & lymphoma 2003, 44: 1027-1035), anti-telomerase (Wang et al. "Telomerase inhibition with an oligonucleotide telomerase antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma" Blood 2004; 103: 258-66; incorporated herein by reference) and anti-VEGFR Mab (Wang et al. "Targeting autocrine and paracrine VEGF receptor pathways regresses human lymphoma xenographs in vivo" Blood, in press, incorporated herein by reference). Both Fludelone and dEpoB can significantly inhibit myeloma cell proliferation as lymphoma. The myeloma cell lines are very sensitive to Fludelone and dEpoB with extremely low IC50, while two lines of NHL were inhibited at doses of Fludelone that were 5-10 times higher than what was effective in MM (Table 2-1).

Table 2-1. Inhibition of cell growth and IC50 of Fludelone and dEpoB against a panel of human nodal tumors and normal human cell populations as determined by the tetrazonium XTT assay.

Normal bone marrow stromal cells (lines HS-27A and HS-5) showed relative resistance to these compounds, indicating that Fludelone and dEpoB have a safe therapeutic window in MM. (Table 2-1). Fludelone is ~ 5 times more potent than dEpoB in MM cell lines whereas both groups had a toxicity comparable to normal bone marrow stromal cells. The fetal human lung fibroblasts (MRC-5) were sensitive to dEpoB and Flu but a clear therapeutic window was evident with Flu even with these normal cells very sensitive. Fludelone and dEpoB cause the myeloma and lymphoma cells to stop in the G2M phase (Figure 18) and induce tumor cell apoptosis (Figures 19 and 20). The duration of in vitro drug exposure necessary to cause apoptosis in myeloma cells was evaluated. Exposure to pulses for 1, 2, 4, 8 and 24 hours was followed by a collapse of the drug and incubation continued for up to 40 hours. Exposure of the cells to either Fludelone or dEpoB for 24 hrs resulted in all cells being removed for 48 hours (Figure 21). With dEpoB, incubation for 4-8 hours delayed cell expansion while the same duration of exposure to Fludelone decreased the number of cells from entry by ~ 50%. A one-hour exposure to dEpoB reduces but does not prevent tumor cell expansion for 48 hours, whereas with Flu, tumor numbers decreased by 50% (Figure 21). Fludelone and dEpoB shared paclitaxel's ability to improve microtubule bundle formation in tumor cells without appreciably changing the mass of microtubules in the cell shortly after exposure (Figure 22). At a later time (~ 24 hrs) the microtubules broke and cellular apoptosis occurred. In a comparison of Fludelone and dEpoB in primary CD138 MM cells obtained from patent bone marrow it was shown that Fludelone but not dEpoB induces tumor apoptosis within 24 hrs.

Drug Sensitivity in Human Solid Tumor Cell Lines The IC50 of Fludelone and dEpoB were determined in a panel of human solid tumor lines (colon, breast and ovaries) (Table 2-1). In each case the IC50 of fludelone was lower than that of the dEpoB. The ovarian cancer lines were particularly sensitive to Fludelone and 4/5 of the ovarian lines had an average IC50 of 1.6 nM compared to an average IC50 of 16.5 nM with dEpoB. Both groups originated tumor cells to stop in the G2M phase (figure 23) and rapidly induced apoptosis (figures 24 and 25).

Global gene expression of the myeloma cell line RPMI-8226 at 6 and 18 hrs after treatment with dEpoB or Fludelone at xl O of their respective IC50s Global gene expression (Affymetric chip - AU133 2.0), comparison of gene expression differential in the human multiple myeloma cell line RPMI-8226 was undertaken 6 or 18 hrs after treatment with dEpoB or Fludelone (at dose xlO IC50) • In the comparison of RPMI-08226 treated with Fludelone with untreated control cells, at 6 hours, 5 genes were upregulated (Table 2-2) and at 18 hrs, 48 genes were upregulated (Table 2-3). JUN overregulated both times (+3.25, +2.64). At 6 hours, 3 genes were low-regulated (Table 2-2) and at 18 hrs, 16 genes were low-regulated (Table 2-3). The HNRPD (Heterogeneous nuclear D-similar ribonucleoprotein) was low-regulated both times (-2.46, -3.25). In the comparison of RPMI-08226 treated with dEpoB with untreated control cells at 6 hrs, 21 genes were upregulated (Table 2-2) and at 18 hrs, 26 genes were upregulated (Table 2-3). JUN (+5.66, +3.73) and Tubulina a3 (+2.64, +2.30) were overregulated both times. At 6 hrs, three genes were low-regulated (Table 2-2), and at 18 hrs, sixteen genes were low-regulated (Table 2-3). The HNRPD was downregulated twice (-4.92, -2.00). The gene expression altered by Fludelone against dEpoB was then compared (Table 2-2 and 2-3).

Table 2-2: Global gene expression (Affymetrix-AU133 2.0 chip), comparison of differential gene expression in RPMI-8226 (human multiple myeloma cell line). Control comparison with cells treated with Fludelone or dEpoB (at dose 10 IC50) for 6 hrs. Fludelone 6 hr Desoxiepotilone change B 6 hrs Double change Double +3.25 IF127 (Protein inducible by +18.38 SKIL (SKI-Equal-) +2. 83 interferon-) BASP1. (signaling +2.83 JON +5.66 bound to membrane G1P3 * (inducible protein +5.28 abundant brain) interferon-) APP (Precursor protein +2.14 IFIT 1 * (Protein 1 +4. 92 ß amyloid) transmembrane induced by SHMGS1 (3-0H-3- +200 interferon) methylglutaryl-Co-epzima APP (β2.83 precursor protein synthase) amyloid) CC 5 (chemokine R & NTES) +2.64 TOBA3 (Tubulin c3) +2. 64 INSIG1 (Gene induced by +2.46 insulin) TRI 22 (contains 22 portions +2.30 tripartite) HIA-DPA1 * +2.30 (Histocompatibility class II DPal) IFITl * (Protein induced by +2.30 interferon with tetratricopeptide repeats) HMGCS1 (3- 0H-3-methylglutaryl +2.30 Coenzyme A) MARCKS (Substrate PKC +2 .14 rich in meristolated alanine) ABCG1 (activity of +2.14 transporter ABC) MX1 * (p78 inducible by +2.00 interferon resistance to myxovirus) OAS1 * (2 ', 5'-oligoadenylate +2.00 siptetase) P SCR1 (phospholipid +2.00 escramblase) STS (steroid sulphatase) +2. 00 DÜSP4 (phosphatase of +2.00 dual specificity) AP1S1 (complex-l of protein +2.00 related to adapter) Underlined: genes over- or under-regulated in cells treated with both Fludelone as dEpoB at 6 hrs. * Interferon-inducible genes Table 2-3: Global gene expression (Affymetrix-AU133 2.0 chip), comparison of differential gene expression in RPMI-8226 (human multiple myeloma cell line). Control comparison with cells treated with Fludelone or dEpoB (at dose xlO IC50) for 18 hrs. Fludelona 18 hr Desoxiepotilona B change 18 hrs Double change Double PRKDC (repair of +21.11 REPSl (associated Eps domain +9 .85 double strand break) with RABP1) Fibrillin 2 +12.3 PKD1 (kidney disease +5 .6 REPS1 (domain Eps +9.85 polycystic 1) associated with RALP1) JON +3 .73 PKD1 (disease of +7.46 SEMA3D (Ig domain sema) +3 .73 polycystic kidney 1) PRKCBP1 (protein link +3 .03 TRIO (guanil NEF domain +4.29 kinase C) triple function) PCNX ( homology of Pecanex) +3 03 F J20241 (protein that +3.73 GTF2H2 (factor of +2 83 activates NFkB) transcription general IIH) CCL3 (chemokine MIP-la) +3.48 KIAA1025 protein +2 83 PCNX (homology of Pecanex) +3.48 TRIO (guanil NEF domain of +2 83 KIA &1025 protein +3.25 triple function) TRA2A (transformer 2a, +3.03 CCL3 (chemokine MIP-la) +2 64 splicing of mRNA) VEZATIN (protein +2.46 GTF2H2 (transmembrane +3.03 factor) general transcription IIH) TR & 2A (transformer 2a, +2.46 SLC13A1 (transporter +3.03 splicing of mRNA) of sodium ion) APRIN (inhibition) proliferin ion +2. 46 PHC3 (polyhameotic equal) +2. 83 induced by androgen) Fludelone 18 hr Desoxiepotilone B change 18 hrs Double change Double C ar £ 29 (reading frame 6 06 Cyclin E2 -3.03 open of crcmonone 1) MARS (methionine-tRNA -2.83 FLJ20130 (protein 5 66 synthetase) hypothetical) P0O4F1 (factor -2.46 Fll (coagulation factor XI) 4 0 transcription of Pou domain) G1P3 (inducible protein 3 73 NRTN (Neurturin) -2.14 for interferon) PABPN1 (binding protein -2.00 HNRPD (Ribunocleoprotein -3.25 Poly (A), nuclear) D-equal nuclear HNRPD -2.00 heterogeneous) (Ribunocleoprotein D-equal) Cyclin E2 -2.64 heterogeneous nuclear) FLJ20045 (protein -2.46 hypothetical) CEB1 (cyclin link -2.30 E, ubiquitin ligase) FBX05 (F box only -2.30 protein 5) NRIN (Neurturin) -2.3 DUSP6 (phosphatase of -2.14 dual specificity) P0O4F1 (factor of -2.14 transcription of Pou domain) MARS (methionine-tRNA -2.00 synthetase) HDAC9 ( histone -2.00 deacetylase 9) CENPC1 (-2.00 centromere Cl protein) Underlined: over- or under-regulated genes in cells treated with both Fludelone as dEpoB at 18 hrs. * Interferon-inducible genes At 6 hrs, five genes were upregulated by Fludelone, 21 by dEpoB, and three genes were upregulated by both while three genes were downregulated by Fludelone, four by dEpoB, and no genes by both. At 18 hrs., 48 genes were upregulated by Fludelona, 26 by dEpoB and 18 genes were upregulated by both while 16 genes were low-regulated by Fludelona, 7 by dEpoB and 6 genes were low-regulated by both. The proposal is to test the hypothesis that the increased antitumorigenic efficacy of Fludelone on dEpoB and epothilones was previously due to some additional tumor target mechanism in addition to microtubule stabilization. Therefore it was determined that the genes were differentially on or low-regulated by Flu when compared to MM cells treated with dEpoB (Table 2-4).

Table 2-4: Global gene expression (Affymetrix-AU133 2.0 chip), comparison of differential gene expression in RPMI-8226 (human multiple myeloma cell line) treated with dEpoB or Fludelone (at dose xlO IC50) by 6 or 18 hrs. The genes show where those changed selectively by Fludelona.

Underlined: low -regulated genes in cells treated with both Fludelone and dEpoB both at 6 and 18 hrs. * Interferon-inducible genes At 6 hrs, two genes were upregulated, and at 18 hrs, one gene was upregulated. At 6 hrs, ten genes were downregulated, and at 18 hrs, eleven genes were downregulated and 4 genes were downregulated twice (IFI27, -7.46, -5.28), GIP3 (-6.06, -5.28), IFTM1 (-4.00, -3.25 ), MX-1 (-2.00, -2.30). These are all the genes inducible by interferon. The gene expression profiles of ovaries 1A9 and ovarian xenografts 1A9PTX22 have been reported to show decreased expression of IFN response genes (G1P3, IFI27, IFITM1, IFII6, ISG15) 24 hrs after treatment with paclitaxel (Bani et al. "Gene expression correlating with response to paclitaxel in ovarian carcinoma xenografts" Mol. Cancer Thera, 2004; 3: 111-121; incorporated herein by reference). Three of these genes (G1P3, IFI27, IFITM1) were overexpressed strongly at 6 hrs after treatment with dEpoB of RPMI-8226 cells but not with Fludelona, and these were also the genes whose expression most strongly distinguished the profiles of the Fludelona and dEpoB. Overexpression of IFN-responsive genes (eg, IFR9) has recently been associated with paclitaxel resistance in tumor cell lines and the findings support the possible involvement of IFN signaling mediators other than IFN itself, in response to paclitaxel ( Luker et al. "Overexpression of - IRF9 confers resistance to antimicrotubule agents in breast cancer cells" Cancer Res. 2001; 61: 6540-7; incorporated herein by reference). This new independent paper. of INF of IRF9 in the development of resistance to antimicrotubule agents was reported in approximately one-half of breast and uterine cancers (Luker et al., "Overexpression of IRF9 confers resistance to antimicrotubule agents in breast cancer cells" Cancer Res. 2001; 61: 6540-7; incorporated herein by reference). Microarray analysis of tumor lines that were resistant to eEpoA or Taxol showed that most of the genes which were highly expressed in EpoA-resistant tumors but not in Taxol-resistant tumors encode the interferon-inducible genes (Atadja et al. "Gene expression profiling of epothilone A-restitant cells" Novartis Found Symp. 2002; 243: 119-32; incorporated herein by reference). Notably, it was found that 8/21 (38%) of all genes upregulated after treatment with dEpoB of RPMI-8226 were inducible by IFN and none of these were changed by treatment with Flu (Table 2-2 to 2-4). It was shown that the following IFN-inducible genes are up-regulated in 8226 MM cells after 6 hrs or 18 hrs of treatment with dEpoB and no change with treatment with Fludelone. In order of degree of upregulation following the dEpoB were 1). IFI 27 inducible by interferon (IFN) or i (increase of +18.83). This belongs to a family of small interferon-a inducible genes of unknown function that are upregulated in inflammatory skin disease, epithelial cancers and wound regeneration (Suomela et al. "Inferieron alpha-inducible protein 27 (IFI27) is upregulated in Psoriatic skin and certain epithelial cancers "J Invest, Dermatol, 2004; 122: 717-721; incorporated herein by reference). 2). IFNa-inducible protein (clone IFI-6-16) GIP3 (Bani et al. "Gene expression correlating with response to paclitaxel in ovarian carcinoma xenografts" Mol. Cancer, Thera. 2004; 3: 111-121; incorporated herein by reference). reference) (increase of +5.28). 3) . Transmembrane protein-1 induced by INF, IFTM1 (Bani et al) (increase of +4.92). 4) . MX1 inducible by IFN of myxovirus resistance (increase from +2 to 6 hrs and +2.3 to 18 hrs). MX1 genes confer selective innate resistance to influenza virus (Staeheli, Ad. Viral Res. 38: 147, 1990; incorporated herein by reference). They can also serve as basic cellular functions possibly as a GTP binding protein (Arnheiter H and Meier E. "Mx proteins: antiviral proteins by chance or necessity?" New Biol. 1990; 90 851; incorporated herein by reference). 5) . PLSCR1 gene of phospholipid escramblase. The transcriptional control of the genes is completely regulated by a unique IFN-stimulated response element located in the first exon (Zhou et al. "Transcriptional control of the human plasma membrane phospholipid scramplase 1 gene is mediated by interferon-alpha" Blood 2000; 95: 2593-2599, incorporated herein by reference). The PLSCR1 was involved in the regeneration of plasma membrane phospholipids and mobilization of phosphatidylserine to the cell surface. It may contribute to the rapid trans-bilayer movement of plasma membrane phospholipids that was observed in injured or apoptotic cells that are exposed to elevated intracellular Ca ++. Studies show that it can also translocate to the nucleus and can act as a transcription factor (Ben-Efrai et al. "Phospholipid Scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interactions with DNA" Biochem 2004; 43: 35181 incorporated herein by reference). 6). Interferon-induced protein with tetrapeptide repeats, IFITI (+2.3 increase). It has been associated with systemic lupus erythematosus and protein-protein studies show that it can activate Rho proteins by interaction with guanine Rho / Rac nucleotide exchange factors (Ye et al. "Protein interaction for an interferon-inducible syste-3526mic lupus associated gene, IFIT1"Rheumatology 2003; 42: 1155-63; incorporated herein by reference). 7). 2'-5f-oligoadenylate synthetase-2, 0AS2 (increase of +2). This family (OASI, QAS2, 0AS3, OAS4) of the interferon-induced genes are involved in stress responses and are important for the anti-viral activity of interferon (Chebath, Nature 330: 587, 1987; Meurs et al., J. Viral 66: 5804, 1992, each of which is incorporated herein by reference). OAS2 catalyzes the synthesis of adenosine oligoes (2-5A). It then activates RNase L, a latent endoribonuclease in most mammalian cells, which in turn can inactivate viral (picornovirus, for example engovirus) and cellular RNA (Anderson et al., Eur. J. Biochem., 2004; 271: 628-36; incorporated herein by reference). 8). Histocompatibility class II DPal, HLA-DPAl (increase of +2). MHC-Class II are known mediators of the biological functions of IFN (Tissot et al., "Molecular cloning of a new Interferon-induced factor that represses human ipittunodeficiency virus Type 1 long terminal repeat expression" J. Biol. Chem. 1995; 270 : 14891-14898; incorporated herein by reference).

Brief Summary The CAG myeloma cell line exhibits many features of clinical multiple myeloma with both phenotype (CD38 + CD138 + CD45-) and grafting in vivo. A mouse model with NOD-SCID xenograft myeloma was established by intravenous injection of 10 ~ 15 million CAG cells modified with HSV-TK-eGFP-Luciferase fusion gene. Therefore, complete animal imaging by luciferase bioluminescence can be applied to evaluate tumor burden and tumor efficacy in real time, reporting non-invasively in living mice. The mouse with myeloma exhibits bone marrow infiltration and pathological osteolytic bone lesion after 7 to 20 days of tumor injection. After implantation of tumor cells, the mice were randomized and divided into treatment groups with dEpoB and Fludelone, vehicle control. The dose of both dEpoB and Fludelone was 20 mg / kg, and the drugs were administered by the intraperitoneal route. The average number of doses was 10 for dEpoB and 12 for Fludelone during the first 30 days of treatment. In addition, five doses of Velcade (6.25 ug / mouse, i.v.) were administered in 3 of 7 mice initially treated with Fludelone between 35 to 45 days. The results show that myeloma mice treated with Fludelone had significantly increased tumor load evaluated by bioluminescence imaging against dEpoB treated mice and controls. All the control mice died within 30 days after the start of treatment, however, the mice treated with Fludelone had at least double the survival time than the controls. There was no difference in survival between the mice treated with dEpoB and control. In combination with the proteasome inhibitor, Velcade, tumors located in the spine and femurs can be greatly reduced, indicating that this combination can attack myeloma cells even under microenvironmental protection of bone marrow. Previous data have shown that myeloma cells treated with epothilones exhibited typical apoptosis characteristics. It is not clear if dandruff is involved in this process. First, the activation of caspase 3, an effector caspase in a caspase pathway, in myeloma cells, was investigated by Western staining. Either with dEpoB or Fludelone treatment, the myeloma cells showed increased 17 kd caspase 3 cleavage, which correlates with their activity. Immunohistochemical staining of CAG myeloma cells using antibody specific for cleaved caspase-3 was used to show the characteristics of cytoplasmic and perinuclear localization in apoptotic cells. These data confirm that caspase was inactivated in cells treated with epothilone. Second, the activity of caspase 8 and 9, initiator caspase in the caspase path, was intensified by fluorometric method. It was again found that both activities of caspase 8 and 9 are increased in myeloma cells. Note that the increased caspase-9 activity was more prominent than caspase-8. The signals that trigger the activation of caspase 8 or 9 are actively under investigation. The cytotoxicity of drug to hematopoietic stem cells is always a main interest. Human CD34 + stem cells were isolated from umbilical cord blood and incubated with either dEpoB or Fludelone alone for 24 hours. After washing the drugs, apoptosis assays and 2 weeks of parental assays were performed to evaluate the influence of epothilone on stem cells. The data show that there is no significant toxicity of dEpoB or Fludelone in uncyclic stem cells. Example 3: (E) -9,10-dehydroepothilones: A New Class of Microtubule Stabilizing Antitumorigenic Agents with Highly Promising Characteristics in Murine Xenoinjero Models The synthesis of 26-F3- [16] dEpOB (2) containing the ring of 16 members can usually be done via a highly convergent strategy, related to that used in the synthesis - of 27-F3- [17] ddEpoB (19). This strategy contemplates the formation of an E-9, 10-olefin via a ring closure metathesis reaction shown below (for an earlier example of macrocyclization in a complex setting via an RCM see: Sinha, S. C; Sun, J, Angew. Chem. Int. Ed. 2002, 41, 1381; incorporated herein by reference). It is anticipated that the chemoselective reduction of E-9, 10-olefin of 28 and 29 could provide dEpoB (1) and the desired 26-F3-dEpoB (2). The RCM precursor could be prepared by joining the two fragments (21 or 24) and 123 through an esterification reaction. The coupling configuration 123 could be constructed by suppressing the methylene spacer group (found in the above routes, compound 18) between the secondary methyl group at C8. As noted earlier, in vitro level discoveries with the 17-member epothilones containing the skipped diene array (see 18), emphasize the need for a corresponding investigation of the biological consequences of such diene in the lactone setting of 16 family members. Given the presence of cis-12,13-olefin in a context of dEpoB, such a skipped diene could necessarily contain a double bond 9,10. Retrosynthesis of the 9,10-dehydroepotilones and 26-F3-dEpoB The alkylation of oxazolidinone 7 (Lee et al., J. Am. Chem. Soc. 2001, 123, 5249; incorporated herein by reference) with the easily synthesized methyl allyl and trifluoro iodides (8 and 124, respectively) allowed the stereocenter C15 to be adjusted in the appropriate absolute configuration (Evans, DA; Morrissey, MM; Dorow, RLJ Am. Chem. Soc. 1985, 107, 4346; Paterson, I .; Bower, S.; McLeod, MD Tetrahedron Lett, 1995, 36, 175, each of which is incorporated herein by reference) with high diastereomeric access, (see products 9 or 125). The latter were converted to their corresponding Weinreb amides (Nahm et al Tetrahedron Lett, 1981, 22, 3815, Levin et al., Synth, Commun, 1982, 12, 989, each of which is incorporated herein by reference). ) and thence to 126 and 42 en route to 24 and 21 by nucleophilic methylation (MeMgBr) and appropriate Hoerner Wittig olefininations (Lythgoe et al., Tetrahedron Lett., 1975, 40, 3863; Lythgoe, Chem. Soc. Rev. 1981, 449; Toh et al., J. Org. Chem. 1983, 48, 1414; Baggiolini et al., J. Org. Chem. 1986, -51, 3098, each of which is incorporated herein by reference). While the use of such oxazolidinones as chiral auxiliaries has been promoted by Evans and associates (Evans et al., J. Am. Chem. Soc. 1985, 107, 4346; Paterson et al., Tetrahedron Lett., 1995, 36, 175, each of which is incorporated herein by reference), its application to the Synthesis of optically defined glycollates by alkylation (rather than by hydroxylation) has not been developed.

Synthesis of Fragments 21 and 24 R = CF3 (126) R = CF3 (24) R = Me (42) R = Me (21) The synthesis of the polypropionate fragment 25 was enabled by two critical aldol reactions, which establish the relative configuration of the stereocenters C3, C6 and C7. The first aldol reaction involves the reaction of Z-enolate of ethyl ketone 30 with Roche 31 aldehyde (Cohen et al., Org. Chem. 1976, 41, 3505; Nagaoka et al., Tetrahedron 1981, 37, 3873; Roush et al. J. Am. Chem. Soc. 1990, 112, 6348, each of which is incorporated herein by reference) to provide the desired 32 with high diastereoselectivity. The recourse to 31, allowed by this synthesis, is a significant advantage over the use of first aldehydes which require resolution for the achievement of enantiomerically pure starting materials. The protection of C7-alcohol followed by hydrolysis of the acetal provides the desired aldehyde 34 and adjusts the stage for the second aldol reaction. Reaction of 34 with Ti-enolate of DAG (diacetone glucose) from Duthaler (Duthaler et al., Angew, Chem. Int. Ed. En., 1989, 28, 495; incorporated herein by reference) provides the desired tert-butyl hydroxy ester with very high (> 95%) diastereoselectivity. The latter is then converted to the desired acid 25 in direct steps.

Acid Synthesis 25 The allylic alcohols 24, 21 and the C1-C9 acid fragment 23 were linked through an EDCI esterification protocol, thus providing the RCM precursors 26 and 27, respectively. Ring closure metathesis reactions 26 and 27 are made using an RCM catalyst (Scholl et al Tetrahedron Lett, 1999, 40, 2247, Grubbs et al., Acc. Chem. Res., 1995, 28, 446; Trnka et al. al. Acc. Chem. Res. 2001, 34, 18, Alkene Metathesis in Organic Chemstry Ed.: Fürstner, A., Springer, Berlin, 1998, Fürstner A. Angew, Chem. Int. Ed. Engl. 2000, 39, 3012; Schrock, Top, Organomet, Chem. 1998, 1, 1; each of which is incorporated herein by reference) into toluene. These reactions indeed provide trans isomers 39a and 40a. However, the main products were 39b and 40b which have emerged from the obvious alternate RCM trajectory. These unwanted RCM isomers predominate over the 39a and 40a desired by the ratios of almost 3: 1. Finally, deprotection of the silyl ethers of 39a and 40a with HF-pyridine leads to the desired E-9, 10-dehydro-epothilones 28 (White et al., J. Am. Chem. Soc. 2001, 123, 5407; White et al. J. Am. Chem. Soc. (Addition / Correction) 2003, 125, 3190; each of which is incorporated herein by reference. With the compound 28 of structure rigorously tested at disposal, it was surprisingly found that its spectral properties were not congruent with those previously reported for a compound that is presumed to be the same entity. The current structure of the compound previously assigned as 28 has now been re-evaluated. In hindsight, it is clear that 28 has not been previously prepared and, in fact, the entire family of (E) -9, 10-dehydroepothilones reported here is a new genre) and 29. As planned, the latter were converted to dEpoB ( 1) and 26-trifluoro-dEpoB (2) via diimide reduction of E-9, 10-olefin (Corey et al., Tetrahedron Lett, 1961, 347; Pasto et al, Org React, 1991, 40, 91; each of which is incorporated herein by reference). By corresponding methodology, 9,10-dehydro-dEpoF (57) was synthesized, see below. The selective di-imide reductions of E-9,10-olefins validate the structures of the various synthetic intermediates described above, thus promoting the re-evaluation of previous assignments in the literature.

Synthesis of 9,10-Dehydroepothilones HF-Pyridine 39a. { RisMe, R2 = H, 35%), 30b (^ Me, 62%) THF (1: 3) I 40a. { R1 =: CF3. R2-H, 22%) > 4Db (R1 = CF3, eQ%) 0 ° C rt rt T iñ ^ ß? R2 »OTBS, 2?% > , 56 (R1 = * Se, 57%) The examination of synthetic analogues (28, 29, and 57), in cell culture settings (see Complementary Edition), revealed that they exert stronger inhibitory effects in several MRD and sensitive tumor cell lines that are exhibited by the clinical candidate. current, dEpoB (1). The impressive cell growth inhibition exhibited by the epothilones 28, 29 and 57 through a range of several drug resistant tumors, promotes the determination of the blood plasma stability of these new congeners (E) -9,10. It is recalled that (E) -10, 11-dehydro-dEpoB (Epo490) exhibits stability in very poor plasma. Indeed, it was this instability in plasma which blocked the further development of Epo490 (6). In contrast, on exposure of 28, 29, and 57 to murine plasma, a very slow drug degradation was observed when compared to dEpoB (I) (by a factor of almost 7). This stability constitutes a substantial advance, from a perspective of drug availability, in relation to dEpoB (1), not to mention the epo 490 (6). Based on preliminary cell culture and pharmacokinetic data of (E) -9, 10-dehydro derivatives 28 and 29, it may be appropriate to promote them for in vivo investigations. Such studies, of course, are rather more drug-intensive than in vitro measurements (Rivkin et al., J. Am. Chem. Soc. 2003, 125, 2899; Chou et al., Angew. Chem., Int. Ed. 2003, 42, 4761-4767, Yoshimura et al., Angew, Chem. Int. Ed. 2003, 42, 2518-2521, each of which is incorporated herein by reference, for recent examples of peotilone analogues. potent alternatives, see: Altmann et al., Bioorg, Med Chem. Lett 2000, 10, 2765, Nicolaou et al., Chem. Biol., 2000, 7, 593, each of which is incorporated herein by reference. This requirement, and indeed the possible eventual need to prepare multigram quantities of 9, 10-dehydro derivatives for further development, promotes a significant re-evaluation of the total synthesis route.Of course, the single most serious problem is that the RCM reaction in 26, 27 and 54 produces 39b, 40b, and 56 only as minor products. Ncipal involves a reaction of RCM which is strictly limited to the O-alkyl sector of 26, 27 and 54, leading mainly to the unwanted 39b and 40b. Consequently, it was decided to try to delay the introduction of thiazole (by olefination) subsequent to the RCM (see below). With eventual processability in multigrade scales as target, the synthesis of the alkyl and acyl fragments entering the RCM reaction were restructured. Compound 86 was easily synthesized as shown. Oxazolidinone 7 was used to rent with high diatereomeric excess. After deprotection of the OTES group, and nucleophilic methylation, compound 90 was available. This cc-hydroxyketone could serve as the acyl acceptor group in the formation of the central ester to prepare all the critical RCM. An obvious interest is the possible vulnerability of such hydroxyketone as an acyl acceptor to racemization or partial deviation to regioisomeric oc-ketones.

Fragment Processable Synthesis 90 7 A serious problem in the most anticipated synthesis of acid fragment 25 was the very expensive and technically demanding Duthaler chemistry (Duthaler et al, Angew.Chem.Inter.

Ed. Engl. 1989, 28, 495; incorporated herein by reference) to generate the desired S-stereochemistry in the C3. To overcome this problem, the aldol reaction was carried out without any auxiliary chiral to provide a 1: 1 mixture of the corresponding β-hydroxy ketone. The asymmetric reduction controlled by reagent of the derived keto function (see compound 69), using Noyori conditions (Noyori et al., J. Am. Chem. Soc. 1987, 109, 5856, incorporated herein by reference) generating the desired S-stereochemistry in C3 in high diastereomeric excess. The now available β-hydroxy ester 70 was converted to acid 25 in several steps followed by more anticipated protocols (Chou et al., Proc. Nati, Acad.Sci.U.S.A. 2001, 98, 8113, incorporated herein by reference).

Acceptable Process Synthesis 25 70 Notably, esterification of the resulting hydroxyketones 43 and 44 with the C1-C9 acid fragment provides the corresponding RCM cyclization precursors 45 and 46 without evident racemization at C15, or lower integrity of the initial α-ketol bond. The ring closure metathesis reaction of 45 and 46 was performed using an RCM catalyst (Scholl et al., Tetrahedron Lett, 1999, 40, 2247; Grubbs et al., Acc. Chem. Res., 1995, 28, 446; Trnka. et al., Acc. Chem. Res., 2001, 34, 18, Alkene Metathesis in Organic Chemistry Ed.: Fürstner, A., Springer, Berlin, 1998, Fürstner, A. Angew, Chem. Int. Ed. Engl. 2000, 39, 3012; Schrock, Top. Organomet. Chem. 1998, 1, 1; each of which are incorporated herein by reference) into toluene. This reaction, now not complicated by alternating metathesis trajectories, exclusively provides the trans isomers 47 and 48 in high yields. The installation of the thiazole portion via a Wittig reaction continues with high selectivity and E / Z yield, to provide 28 and 29 followed by deprotection of the two syl-lyl esters (Hindupur et al., Tetrahedron Lett 2000, 2, 7341 incorporated herein by reference.The Avery protocol is intended to be applied for the installation of the thiazole providing the desired product in low yield and with poor E / Z selectivity). This route meets the standards of agreement towards large-scale synthesis. It is considered that if the incorporation of olefin C9-C10 in epothilone B (51, EpoB) could alter its biological profile in the same direction as was the case with its 12,13 deoxy counterparts. Towards this end, the epoxidation of 28 with 2, 2'-dimetidioxirane (DMDO) is studied. The reaction in effect continues with high chemoselectivity in the most substituted C12-C13 olefin. An 87% yield of a 1: 2.6 ratio of (E) -9, 10-dehydroepothilone was obtained B (49) and its diastereomer bearing the α-12, 13-oxirane (structure not shown). In vitro studies with 49 (those configurations at C12 and C13 were established by their reduction to produce Epo B) reveal that it is approximately 2-4 times more potent than the EpoB of origin (51) in several cell lines. Although compound 49 proves to be the most potent epothilone found in the program, its narrower therapeutic index in xenografts, as well as its difficult accessibility. { vide supra) serves to further discourage its pre-clinical development. Interestingly, the non-natural a-oxirane was virtually devoid of activity.

Processible Synthesis of (E) -9,10-Dehydroepoylones 43, R- Me, 83% 4S, R = Me, 81% 47, R = Me, 78% 44, R = CF3, 78% 4ß, R = CF3.86% 48, R = CF3, 71% 39a, R = Me, 76% 2ß, R - e, S7% 49, R = Me, 24% 4Qa, R = CF3, 70% 23, R = CF3, g8% - isomer a, 63% (main) Other advantage of the restructured synthesis (second generation), described above is that a variety of heterocycles can be installed via ketone intermediates 39a and 40a. This point is well highlighted by the synthesis of 9, 10-dehydro-dEpoF. The Wittig reaction of the ketone with the appropriate phosphonium-ylides produced the desired 9, 10-dehydro-dEpoF compounds 57 and 59 in high yield and with high E / Z selectivity. In addition, they were able to efficiently convert 21-hydroxy 59 to derivatives of type 96 and 97, which contain amino functionality to C21 in several stages as shown below.

Diversification of C-21 from (E) -9,10-Dehydroepothilones Completely synthetic epothilone analogs have been evaluated against a variety of cell types to determine their antitumorgenic potential. The most salient features of the findings are as follows: One can expect a loss of almost an order of magnitude in replacement of the C12-C13 ß-epoxide with a double bond E-12.13 (compare EpoB and dEpoB in the cell line CCRF-CEM sensitive). Another expectation is that the inclusion of a double bond E-9,10, in addition to Z12, 13-olefin leads to a significant increase in cytoxicity through various cell lines. Still another instructive tendency is seen in the comparison of 12-trifluoro-E-9, 10-dehydro-dEpoB (29) (Fludelone), with the corresponding compound E-9, 10-dehydro 28. The inclusion of the three fluoro atoms in C26 attenuates the cito-toxicity by up to a factor of 4, in relation to (1). This attenuation effecting the function of 12-trifluoromethyl is also seen in the compounds lacking the 9,10-unsaturation (compare dEpoB (1) and 12-trifluoro-dEpoB (2)). Given these data, and given the accessibility of these 9, 10-dehydro compounds (including 12-trifluoro congeners). Through chemical synthesis, one is in a position to initiate in vivo experiments on the most promising compounds. We describe here some particularly impressive and promising results with compound 29 (Fludelone), which has emerged as a more exciting possibility for promotion to clinical evaluation. Experiments in vivo were performed using human tumor xenografts in nude immunodeficient mice. For all its imperfections, such models in oncology were widely used in the evaluation (Fiebig, HH, Berger, DP, Preclinical Phase II triais, In: Boven, E. and Winograd, B., Editors, The Nude Mouse in Oncology Research, CRC Press, Boca Ratón (1995), 318; incorporated herein by reference) of the potential of anti-tumorigenic main compounds en route to clinical development. Obviously, the treatment of MX-1 xenografts with dosages of 30 mg / kg of Fludelone resulted in the disappearance of a complete tumor and the absence of any relapse during two months after stopping the treatment (See Figure 6A). More importantly, these therapeutic successes can be achieved either by i.v. infusion. of 6 hr or by oral administration (see Figures 6B and 10A). On the other hand, the treatment of MX-1 xenografts by oral administration of taxol does not affect the tumor (see FIGS. 6B and 10A). Pass without saying that if it is transferable to the human clinical setting, the achievement of oral activity may be of significant advantage. Tumor xenografts resistant to taxol (Figure 7A) as well as human colon carcinoma (HCT-116, Figure 9A) can also be cured with Fludelone by i.v. Experiments using xenografts of human mammary carcinoma (MX-1) and human colon carcinoma (HCT 116) in nude mice lasted 6.0 and 6.6 months, respectively. There was no tumor relapse in any experiment during 4..3 and 5.3 months, respectively, after cessation of treatment. For the HCT-116 experiment, taxol and Fludelone were compared at 20 mg / kg and both achieved tumor disappearance. The group treated with taxol relapsed 1.1 months after the treatment was discontinued, while the animals treated with fludelone were tumor free for 5.3 months. These results have involved a particularly long and complete therapeutic study using xenografts and report remarkably long periods of complete remission with parenteral or oral administration of a single antitumor agent.

Experimental: Methods of General Pharmacology: Cell Lines and Tumor: "The cells of human lymphoblastic leukemia CCRF-CEM and its sub-line resistant to vinblastine (CCRF-CEM / VBLioo, resistance of 720 times) were obtained from Dr. William Beck of the University of Illinois, Chicago, and CCRF-CEM / Taxol (44-fold in vitro resistance) were produced by exposing the CCRF-CEM cells to sub-lethal, increased concentration (IC50-IC90) of paclitaxel during the six months. Human mammary carcinoma (MX-1), human lung carcinoma cells (A549), and human colon carcinoma (HCT-116) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Animals: Athymic nude mice carrying the nu / nu genes were obtained from NCI, Frederick, MD and used for all human tumor xenografts. Except in another manner indicated for mice, female nudes, male nude mice of 6 weeks or older weighing 20-22 g or more were used. The drugs were administered via the tail vein for 6 hours by i.v. using a homemade infusion mini-catheter and restrictor. A Harvard PHD2000 syringe pump programmable with multivies was used for i.v infusion. A typical 6-hour infusion volume for each drug in Cremofor / ethanol (1: 1) was 100 μl in 2.0 ml of saline. For oral administration, both fludelone and taxol were dissolved in ethanol and diluted 5 times with T een-80. The taxol solution should be used within 5 min to avoid precipitation. Tumor volume was assessed using a calibrator to measure length x width x height (or width). For nude mice carrying a tumor the body weight during the course of the experiment refers to the total weight minus the tumor weight. All animal studies were conducted in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Animáis and the protocol approved by the Memorial Sloan-Kettering Cancer Center's Institutional Animal Care and Use Committee. Cytotoxicity Assays: In the preparation for in vitro cytotoxicity assays, the cells were cultured at an initial density of 2-5 x 10 4 cells per milliliter. They were kept in a humidified atmosphere with 5% C02 at 37 ° C in RPMI 1640 medium (GIBCO / BRL) containing penicillin (100 units / ml), streptomycin (100 μg / ml, GIBCO / BRL), and 5% FBS inactivated with heat. For solid tumor cells growing in a monolayer (such as HCT-116 and A549), the cytotoxicity of the drug was determined in 96-well microtiter plates using the sulforhodamine B method (Skehan et al., J. Nati. Cancer Inst. 1990, 82, 1107; incorporated herein by reference). For cells grown in suspension (such as CCRF-CEM and their sub-lines), cytotoxicity was measured, in duplicate, using the micro-culture method in 2,3-bis- (2-methoxy-4-nitro-) hydroxide. 5-sulfophenyl) -5-carboxanilide) -2H-tetrazodium (XTT) (Scudiero et al., Cancer, Res. 1988, 48, 4827, incorporated herein by reference) into 96-well microtiter plates. For both methods, the absorbance of each cavity was measured with a microplate reader (Power Wave XS, Bio-Tek, Winooski, VT). The dose-effect ratio data of 6 to 7 concentrations of each drug, in duplicate, were analyzed with the medium-effect graph using a computer program (Chóu, T.-C. &Talalay, PT Adv. Enzyme Reg. 1984, 22, 27, Chou, T.-C. &Hayball, M. CalcuSyn for Windows (Biosoft, Cambridge, United Kingdom), each of which is incorporated herein by reference). General Chemical Methods: Reagents obtained from commercial suppliers were used without further purification unless otherwise stated. The following solvents were obtained from a dry solvent system (passed through a pre-packed alumina column) and used without additional drying: tetrahydrofuran, methylene chloride, diethyl ether, benzene, and toluene. Triethylamine, N, N-diisopropylethylamine, diisopropylamine, pyridine, and 2,6-lutidine were distilled from calcium hydride. All reactions sensitive to air and water were carried out on flame-dried glassware under a positive pressure of argon gas or prepurified nitrogen gas. The NMR spectra ("" "H and 13C) were recorded in Bruker AMX-400 MHz or Bruker Avance DRX-500 MHz as indicated individually, referenced to CDC13 (7.27 ppm for 1H and 77.1 ppm for 13C). (IR) were obtained on a Perkin-Elmer FT-IR model 1600 spectrometer, optical rotations were obtained on a JASCO DIP-370 digital polarimeter, and low-resolution mass spectra (electro-rot) were recorded on PE SCIEX API. 100. Analytical thin-layer chromatography was performed on plates 60 F254 of E. Merck silica gel The compounds which were not active to UV were visualized by immersing the plates in ethanolic para-anisaldehyde solution, ethanolic phosphomolybdic acid, or molybdate of cerium ammonium or / and heating Preparative thin layer chromatography was performed using the indicated solvent on TLC plates (LK6F Silica gel 60A) Whatman®. Silica gel chromatography was performed using the indicated in silica gel (grade 1740, type 60A, 170-400 mesh) Davisil®. Chemical shifts are reported in d values in relation to chloroform (d 7.24 for protons and d 77.0 for carbon NMR).

To a solution of ethyl 4,4,4-trifluoroacetoacetate (24.0 ml, 0.164 mol) in THF-water (3: 1 = V: V, 320 ml) at room temperature was added allyl bromide (20.0 ml, 1.4 equiv. .) followed by indium (powder, -100 mesh, 25 g, 1.3 equiv.) and the resulting mixture was stirred at 48 ° C for 15 h. The reaction mixture was cooled to room temperature, quenched with 2N aqueous HCl (400 mL) and extracted with CH2C12 (400 mL, 2 x 200 mL). The combined organic products were dried (MgSO4), filtered, and concentrated in vacuo. Flash chromatography (nes-nes-ether 10: 1 → 8: 1 → 6: 1 → 4: 1) produced the alcohol as light oil (31.64 g, 85% yield): IR (film) 3426 (br m) ), 2986 (), 1713 (s), 1377 (m), 1345 (), 1301 (m), 1232 (m), 1173 (s), 1095 (m), 1023 (m), 927 () aa " 1; aH NMR (400 MHz, CDC13) d 5.82 (m, 1H), 5.15 (m, 3H), 4.17 (m, 2H), 2.59 (m, ÍH), 2.58 (d, J = 3.4 Hz, 2H) , 2.29 (dd, J = 14.2, 8.6 Hz, ÍH), 1.24 (t, J = 7.2 Hz, 3H), 13C NMR (100 MHz, CDC13) d 172.08, 130.89, 125.65 (q, J = 280 Hz), 120.27, 73.79 (q, J = 28 Hz), 61.55, 38.97, 35.65, 13.82, high resolution mass spectrum m / z 227.0895 [(M + H) +, calculated for CgH OsFs: 227.0895] Alcohol is volatile After the column chromatography, the alcohol was not completely concentrated, this performance was determined from the total weight and the ratio between product and solvents obtained by NMR integration.

A mixture of alcohol (16.71 g, 0.07386 mol) and pyridine (15.0 ml, 2.5 equiv.) Was cooled to -10 ° C and treated with thionyl chloride (11.3 ml, 2.1 equiv.) Slowly for 11 minutes (yellow precipitate). ). The resulting mixture was heated to 55 ° C (heating to 75 ° C for 17 h producing complex mixture of chlorinated products) and stirred for 12 h. The reaction mixture was cooled to -5 ° C, quenched with water (200 ml) and extracted with CH2C12 (2 x 200 ml, 2 x 150 ml). The combined organic products were washed with saturated NaHCO 3 (2 x 200 ml), and brine (200 ml), dried (Mg ?0), and concentrated in vacuo. Instant chromatography (pentane: ether 15: 1) yielded the ester (11.90 g, 77% yield) as yellow oil: IR (film) 2986 (w), 1731 (s), 1308 (s), 1265 (w), 1227 (m), 1197 (s), 1133 (s), 1025 (m), 920 (w), 896 (w) cm-1; XH NMR (400MHz, CDC13) d 6.36 (s, 1H), 5.79 (ddt, J = 16.9, 10.2, 6.6 Hz, ÍH), 5.15 (dd, J = 17.1, 1.5 Hz, 1H), 5.08 (dd, J = 10.0, 1.4 Hz, 1 H), 4.22 (q, J = 7.1 Hz, 2 H), 3.44 (d, J = 6.5 Hz, 2 H), 1.29 (t, J = 7.1 Hz, 3 H); 13 C NMR (100 MHz, CDCl 3) d 164.22, 143.37 (q, J = 29 Hz), 132.71, 123.21 (q, J = 274 Hz), 122.60 (q, J = 6 Hz), 117.32, 60.85, 30.54, 13.85; high resolution mass spectrum m / z 209.0788 [(M + H) +; calculated for C9H? 202F3: 209.0789]. The ester is volatile. After column chromatography, the ester was not completely concentrated.

To a cooled (~75 ° C) solution of the ester (7.12 g, 0.3442 mol) in CH2C12 (120 ml) was added a solution of DIBAL-H (75 ml, 2.2 equiv.) In CH2C12 ( 1. 0 M) for 35 minutes and the resulting mixture was warmed to room temperature for 3 h. The reaction mixture was cooled to 0 ° C, quenched with saturated NH 4 Cl (12 mL) and stirred at room temperature for 20 minutes. The reaction mixture was diluted with ether (200 ml), dried (MgSO 4), and concentrated in vacuo. Flash chromatography (pentane: ether 3: 1 → 1: 1) yielded alcohol (5.86 g, 99%) as light oil: IR (film) 3331 (br s), 2929 (), 1642 (m), 1445 (m), 1417 (w), 1348 (s), 1316 (s), 1217 (s), 1175 (s), 1119 (s), 1045 (), 985 (s), 921 (m), 831 (w) cm-1; ? E NMR (400 MHz, CDC13) d 6.33 (td, J = 6.1, 1.6 Hz, 1 H), 5.75 (ddt, J = 17.2, 10.0, 6.2 Hz, 1 H), 5.07 (m, 2 H), 4.29 (ddd, J = 6.3, 4.3, 2.1 Hz, 2 H), 2.95 (d, J = 6.2 Hz, 2 H); 13 C NMR (100 MHz, CDCl 3) d 134.45 (q, J = 6 Hz), 133.38, 127.97 (q, J = 29 Hz), 123.76 (q, J = 271 Hz), 116.25, 57.87, 29.79. Alcohol 56 is volatile. After column chromatography, 56 was not completely concentrated.

A solution of cooled alcohol (0 ° C) (5.97 g, 0. 0358 mol) in CH2C12 (50 ml) was treated with PPh3 (11.17 g, 1.2 equiv.), Imidazole (3.55 g, 1.5 equiv.) And I2 (9.10 g, 1.1 equiv.) (The addition of I2 was the last) and the resulting mixture (cloudy yellow) was stirred at 0 ° C for 10 minutes.

The reaction mixture was quenched with saturated Na 2 S 2 3 3-saturated NaHCO 3 (1: 1 = V: V, 200 ml) and extracted with pentane (3 × 200 ml). The combined organic product was washed with saturated Na2S203-saturated NaHCO3 (1: 1 = V: V, 200 mL), and brine (100 mL). ml), dried (MgSO4), and concentrated in vacuo. Flash chromatography (pentane) yielded the iodide (6.69 g, 68%) as a pale red oil (stored in a freezer at 78 ° C): (IR film) 3083 (w), 2982 (w), 1636 (w), 1558 (w), 1456 (w), 13'67 (w), 1317 (s), 1216 (), 1181 (s), 1151 0 (s), 1120 (s), 989 (m), 921 (m) , 896 () cpA; ? E NMR (400MHz, - 'CDC13) d 6.45 (td, J = 8.9, 1.5 Hz, 1 H), 5.79 (ddt, J = 16.8, . 3, 6.2 Hz, 1 H), 5.12 (, 2 H), 3.85 (ddd, J = 8.9, 2.9, 1. 4 Hz, 2 H), 3.00 (dt, J = 6.1, 1.4 Hz, 2 H); 13C NMR (100 MHz, CDCI3) d 132.42, 131.64 (q, J = 6 Hz), 129.63 (q, J = 29 5 Hz), 123.64 (q, J = 272 Hz), 117.00, 29.32, -4.27; low resolution mass spectrum m / z 298.7 [(M + NaA; calculated for C7H8F3INa: 299.0]. Allyl iodide is volatile. After column chromatography, allyl iodide was not completely concentrated. , - a-Hydroxyoxazolidinone. To a cooled solution (-78 ° C) of 4-benzyl-3-hydroxyacetyl-oxazolidin-2-one protected with TES (16.28 g, 1.92 equiv.) In THF (160 ml) was added a solution of LHMDS (42.0 ml). , 1.73 equiv.) In THF (1.0 M) in drops for 15 minutes and the resulting mixture was stirred at -78 ° C for 35 minutes. The reaction mixture was treated with a solution of the allyl iodide (6.69 g, 24.2 mmol) in THF (10 ml) for 15 minutes and the resulting mixture was allowed to warm to room temperature slowly overnight. The reaction mixture was quenched with saturated NaHCO3 (200 mL) and extracted with EtOAc (3 x 200 mL). The combined organics were washed with saturated NHC1 (150 mL), brine (150 mL), dried (MgSO4), and concentrated in vacuo. Flash chromatography (hexanes-EtOAc 6: 1 → 3: 1) yielded a mixture of alkylation product (13.6 g) which were used for the next reaction without further purification (diastereomers were not separable at this stage). A solution of the alkylation products in HOAc-water-THF (3: 1: 1 = V: V: V, 200 ml) was stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo to remove HOAc, quenched with saturated NaHCO3 (400 mL), and extracted with EtOAc (3 x 200 mL). The combined organic products were dried (MgSO4), and concentrated in vacuo. Flash chromatography (hexanes: EtOAc 3: 1 → 2: 1) yielded the OI-hydroxyoxazolidinone (7.55 g, 81% yield by two steps) as light oil: [] ^ (25 ° C) -48.2 ° (c 1.08 , CHC13); IR (film) 3486 (br s), 3030 (), 2983 (s), 2925 (m), 1790 (s), 1682 (s), 1481 (), 1393 (m), 1360 (), 1217 () , 1171 (), 1113 (m), 992 (), 919 (), 847 (w) crrfA 4? NMR (400 MHz, CDCl 3) d 7.32 (m, 3 H), 7.17 (m, 2 H), 6.33 (td, J = 7.2, 1.5 Hz, 1, H), 5.77 (ddt, J = 16.6, 10.1, 6.2 Hz, 1 H), 5.08 (, 3 H), 4.74 (ddt, J = 4.8, 3.7, 4.4 Hz, 1 H), 4.33 (dd, J = 8.6, 8.6 Hz, 1 H), 4.26 (dd, J = 9.2, 3.4 Hz, 1 H), 3.42 (br d, J = 6.4 Hz, 1 H), 3.24 (dd, J = 13.5, 3.4 Hz, 1 H), 2.99 (, 2 H), 2.79 (dd) , J = 13.5, 9.4 Hz, 1 H), 2.70 (, 1 H), 2.50 (m, 1 H); 13 C NMR (125 MHz, CDCl 3) d 173.93, 153.05, 134.43, 133.64, 129.98 (q, J = 6 Hz), 129.82 (q, J = 28 Hz), 129.29, 120.01, 127.58, 124.00 (q, J = 272 Hz), 116.34, 69.60, 67.31, 54.95, 37.78, 32.29, 29.84; high resolution mass spectrum m / z 384.1421 [(M + H) A calculated for Ci9H2? N04F3: 384.1423].

-Hydroxyamide A suspension of (MeO) NHMe «HC1 (10.1 g, 5.25 equiv.) In THF (100 ml) at 0 ° C was treated with an AlMe3 solution (50 ml)., 5.1 equiv.) In toluene (2.0 M) by dripping and the resulting clear solution was stirred at room temperature for 34 minutes, then added slowly to a cooled (0 ° C) solution of the α-hydroxyoxazolidinone (7.55 g, 19.7 mmol) in THF (70 ml) (turbid? light, pale yellow). The resulting mixture was warmed to room temperature and stirred for 12 h. The reaction mixture was cooled to 0 ° C, quenched by the slow addition of IN aqueous tartaric acid (100 ml), stirred at room temperature for 25 minutes, and extracted with EtOAc (3 x 200 ml). The combined organic products were dried (MgSO4), and concentrated in vacuo. Flash chromatography (hexanes: EtOAc 2: 1 → 1: 1) yielded a-hydroxyamide (5.12 g, 97% yield) as light oil: IR (film) 3432 (br s), 3084 (w), 2980 ( m), 2943 (), 1652 (s), 1464 (m), 1373 (m), 1318 (m), 1214 (), 1171 (m), 1112 (m), 991 (m), 919 (m), 818 (w) cm-1; XH NMR (400 MHz, CDC13) d 6.32 (td, J = 7. 3.1, 1.5 Hz, 1 H), 5.74 (ddt, J = 16.9, 10.3, 6.1 Hz, 1 H), 5.05 (m, 2 H), 4.43 (dd, J = 7.6, 3 .5 Hz, 1 H), 3 .70 (s, 3 H), 3 .35 (br s, 1 H), 3 .24 (s, 3 H), 2.94 (d, J = 6.1 Hz, 2 H), 2.59 (m, 1 H), 2.36 (m, 1 H); 13 C NMR (100 MHz, CDC13) d 173.43, 133.68, 130 59 (q, J = 6 Hz), 129.25 (q, J = 28 Hz), 124.05 (q, J = 271 Hz), 116.17, 67.57, 61.44, 32.56, 32.38, 29.75; high resolution mass spectrum m / z 268.1161 [(M + H) +; calculated for C H ^ NOsF3: 268.1161]. a-Hydroxyketone. To a cooled (0 ° C) solution of oxyhydroxyamide (4.87 g, 18.2 mmol) in THF (150 mL) was added a solution of MeMgBr (75 mL, 12 equiv.) In ether. (3.0 M). After 5 minutes, the reaction mixture was quenched with saturated NHC1 (250 mL), and extracted with EtOAc (5 x 200 mL). The combined organic products were dried (MgSO4), and concentrated in vacuo. Instant chromatography (hexanes: EtOAc 4: 1 → 2: 1 → 1: 2) produced the α-hydroxyketone (2.16 g, 53% yield, 73% yield based on the recovered starting material) as light oil and the a-hydroxyamide starting material (1.30 g, 27% yield): [a] ^ '(23 ° C) + 58.5 ° (c 1.30, CHC13); GO (film) 3460 (br s), 3085 (), 2984 (m), 2926 (m), 1716 (s), 1679 (), 1641 (m), 1417 (m), 1361 (m), 1319 (s), 1247 (m), 1216 (s), 1172 (s), 1113 (s), 1020 (s) m), 994 (), 968 (w), 919 (m) cm "1; 4" NMR (500 MHz, CDC13) d 6.21 (t, J = 7.0 Hz, 1 H), 5.75 (ddt, J = 16.7, 10.4, 6.2 Hz, 1 H), 5.07 (m, 2 H), 4.26 (dt, J = 7.1, 4.5 Hz, 1 H), 3.51 (d, J = 4.7 Hz, 1 H), 2. 96 (d, J = 6.1 Hz, 2 H), 2.66 (m, 1 H), 2.42 (m, 1 H), 2.19 (s, 3 H); 13C NMR (100 MHz, CDC13) d 208.53, 133.43, 129. 80 (q, J = 28 Hz), 129.76 (q, J = 6 Hz), 123.85 (q, J = 271 Hz), 116. 32, 75.36, 31.22, 29, 81, 25.11; high resolution mass spectrum m / z 223. 0945 [(M + H) A- calculated for C? 0H? NO2F3: 223. 0946]. This reaction was not completed despite the excess amount of MeMgBr. 2,2- Trichloroethyl ester of l- (2-benzyloxy-1-methylethyl) -5,5-diisopropoxy-2,4,4-trimethyl-3-oxopentyl ester of carbonic acid. To a solution of 7-benzyloxy-5-hydroxy-1, l-diisopropoxy-2,2,4,6-tetramethyl-heptan-3-one (1.0 g, 2.4 mmol) and pyridine (0.8 mL, 7.3 mmol) in CH2C12 (10.0 ml) at 0 ° C was added 2, 2, 2-trichloroethyl chloroformate (668.0 μl, 4.9 mmol) and the mixture was then allowed to warm to rt. After 1 h, the reaction mixture was quenched with brine and then extracted with CH2C12. The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient hexane to hexane / EtOAc 93: 7) to produce the desired product (1285 g, 92%) as a clear oil: 1 H NMR (400 MHz, CDC13) d 1.03-1.09 (m, 12H), 1.15 (d, J = 1.8 Hz, 3H), 1.17 (d, J = 1.9 Hz, 3H), 1.19-1.21 (m, 6H) ', 1.97-2.11 (m, ÍH), 3.2 (dd, J = 6.2 and 9.0 Hz, 1H), 3.54 ( dd, J = 4.8 and 9.1 Hz, 1H), 3.57-3.60 (m, 1H), 3.82 (qd, J = 3.6 and 5.9 Hz, 2H), 4.47 (s, 2H), 4.57 (s, 1H), 4.72 (d, J = 11.9 Hz, ÍH), 4.81 (d, J = 11.9 Hz, 1H), 5.08 (t, J = 6.0 Hz, 1H), 7.29-7.35 (m, 5H); 13C NMR (100 MHz, CDC13) d 11.9, 15.0, 18.8, 21.4, 21.7, 22.3, 23.2, 23.4, 35.7, 42.5, 53.4, 53.9, 69.4, 70.9, 71. 4, 73.3, 81. 3, 94. 7, 103.4, 127.5, 127. 6, 128.2, 138.2, 154. 0, 215. 6; IR (film, NaCl, cm "1) 2966, 1760, 1698, 1247; LRMS (ESI) calculated for C27H4? 07Cl3Na [M + Na +] 605.2, found 605. 2; [oc] 23D = -20. (c = 1.0., CHCl3. 2,2,2-Trisloroethyl ester of 1- (2-benzyloxy-1-methylethyl) -2,4,4-trimethyl-3,5-dioxopentyl ester of carbonic acid. To the solution of the starting material (1.28 g, 2.25 mmol) in 4: 1 THF / H20 (25 mL) was added p-TsOH (111.0 mg, 0.6 mmol). After heating at 70 ° C for 5 h, the reaction mixture was poured into an aqueous solution of saturated NaHCO 2, cooled (0 ° C) (12 ml) and then extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient hexane to hexane / EtOAc 84:16) to yield the product (793.2 mg, 76%) as a clear oil: XH NMR (400 MHz, CDC13) d 0.90 (d, J = 5.8 Hz, 3H), 1.0 (d, J = 6.9 Hz, 3H), 1.24 (s, 6H), 1.97-2.04 (m, ÍH), 3.24 (dd, J = 4.8 and 9.2 Hz, 1H), 3.34 ( , 1H), 3.42 (dd, J = 5.8 and 9.2 Hz, 1H), 4.35 (d, J = 11.9 Hz, ÍH), 4.39 (d, J = 11.9 Hz, ÍH), 4.64 (d, J = 11.9 Hz , ÍH), 4.69 (d, J = 11.9 Hz, ÍH), 4.96 (t, J = 6.0 Hz, ÍH), 7.19-7.28 (, 5H), 9.49 (s, 1H); 13 C NMR (100 MHz, CDC13) - 12.0, 14.8, 19.5, 19.6, 35.4, 43.3, 60.9, 71.1, 73.3, 80.37, 94.5, 127.7, 127.8, 128.3, 137.9, 154.1, 201.0, 210.1; IR (film, NaCl, cm "1) 2973, 2880, 1758, 1701, 1453, 1380, 1248; LRMS (ESI) calculated for C2? H2706Cl3Na [M + Na +] 503.0, found 503.0; [a] 23D - -18.5 (c = 0.8, CHC13).

Tert-butyl ester of 9-bensyloxy-4,4,8-tetramethyl-3,5-dioxo-7- (2,2,2-trichloroethoxycarbonyloxy) nonanoic acid. To a solution of LDA (1.17 mmol, 0.3 M Et2) at -78 ° C was added t-butyl accetate (1.0 mmol, 135.0 μl). After 30 minutes, a solution of starting material (464.0 mg, 1 mmol) in Et20 (2 ml) was added slowly for 15 minutes. After stirring for 1 h, the reaction was quenched with an ac solution of saturated NH 4 Cl and then extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient hexane to hexane / EtOAc 86:14) to yield the product (1: 1 of epimeric mixture, 461.4 mg, 80%) as a clear oil: XH NMR (400 MHz, CDC13 ): d 0.87 (d, J = 5.3 Hz, 3H), 0.89 (d, J = 5. 5 Hz, 3H), 1.02-1.10 (, 18H), 1.38 (s, 18H), 1.97-2.2 (m, 2H), 2.27-2.31 (, 2H), 3.22-3.27 (m, 3H), 3.39-3.48 (m, 5H), 4.03-4.06 (m, ÍH), 4.11-4.14 (m, 1H), 4.38-4.45 (, .4H), 4.58-4.73 (m, 4H), 4. 97 (t, J = 5.8 Hz, ÍH), 5.02 (t, J = 5.8 Hz, 1H), 7.18-7.27 (m, 10H); 13C NMR (100 MHz, CDC13) d 11.9, 12.7, 14.9, 15.2, 18.7, 19.3, 21.4, 21.6, 28.0, 35.6, 37.4, 41.7, 42.0, 51.8, 51. 9, 71.3, 71.3, 72.5, 73.0, 73.3, 73.3, 80.6, 81.2, 81.3, 94. 6, 127.5, 127.7, 127.8, 128.3, 138.0, 138.1, 154.0, 154.1, 172. 3, 172.4, 216.0, 216.3; IR (film, NaCl, cm "1) 3509, 2975, 1759, 1707, 1368, 1248, 1152; LRMS (ESI) calculated for C27H39? 8Cl3 to [M + Na +] 619.1, found 619.2.

To a solution of 0 ° C of starting material (350.0 mg, 0.6 mmol) in CH2C12 (10 ml) was added periodinone from Dess-Martin (398.0 mg, 0.9 mmol). The mixture was stirred at rt by 1 h and then poured into a well stirred mixture of saturated Na 2 S 203 / saturated NaHCO 3 1: 1. The layers separated after 30 minutes. The aqueous layer was extracted three times with Et20- The combined organic extracts were washed with saturated NaHCO3, brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash chromatography (gradient hexane to hexane / EtOAc 91: 9) to yield the product (258.4 mg, 74%) as a clear oil: "'' H NMR (400 MHz, CDC13) d 0.80 (d. , J = 6.9 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H), 1.13 (s, 3H), 1.19 (s, 3H), 1.23 (if 9H), 2.04- 2.12 (, 1H), 3.09 -3.28 (m, 5H), 4.23 (s, 2H), 4.48 (d, J = 11.9 Hz, 1H), 4.55 (d, J = 11.9 Hz, 'ÍH), 4.79 (dd, J = 4.6 and 7.3 Hz , HH), 7.04-7.13 (m, 5H); 13C NMR (100 MHz, CDC13) d 11.7, 14.6, 20.7, 21.5, 27.9, 35.5, 42.2, 43.4, 63.3, 71.3, 73.3, 79.9, 81.5, 90.5, 94.5, 127.6, 127.7, 128. 2, 138.0, 154.0, 166.2, 202.9, 210.0; IR (film, NaCl, c "1) 2977, 1758, 1697, 1368, 1248, 1154; LMRS (ESI) calculated for C27H37? 8Cl3Na [M + Na +] 617.1, found 617.1; [a] 23D = -49.1 (c = 0.9, CHC13). 9-Benzyloxy-3-hydroxy-4,4,6,8-tetramethyl-5-oxo-7- (2,2,2-trichloroethoxycarbonyloxy) -nonanoic acid tert-butyl ester. A pump jacket was loaded with catalyst (R) -RuBINAP (16.8 mg, 10.0 μmol). HCl (555 μl, 0.2N in MeOH) was added and the mixture was then sonicated for 15 seconds. Then a solution of starting material (59.4 mg, 0.1 mmol) in MeOH (555 μl) was added and the mixture transferred to a Parr apparatus. The vessel was purged with H2 for 5 minutes and then pressurized to 1200 psi. After 17 hours, the reaction was returned to atmospheric pressure and poured into an aqueous solution of saturated NaHCO 3. The aqueous layer was extracted three times with EtOAc. The combined organic extracts were dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient hexane to hexane / EtOAc 88:12) to yield the product (47.6 mg, 80%) as a clear oil: XH NMR (400 MHz, CDC13) d 1.06 (d, J = 6.9 Hz, 3H), 1.11 (d, J = 6.8 Hz, 3H), 1.14 (s, 3H), 1.18 (s, 3H), 1.47 (s, 9H), 2.05-2.12 (m, ÍH), 2.35- 2.40 (m, ÍH), 3.31-3.37 (, 2H), 3.51-3.54 (m, 2H), 4.11-4.14 (m, ÍH), 4.46 (s, 2H), 4.72 (d, J = 11.9 Hz, ÍH ), 4.80 (d, J = 11.9 Hz, 'ÍH), 5.05 (dd, J = 5.0 and 6.7 Hz, ÍH), 7.27-7.35 (m, 5H); 13C NMR (100 MHz, CDC13) d 12.0, 15.0, 19.3, 21.7, 28.0, 35.6, 37.5, 41.7, 51.8, 71.3, 73.0, 73.3, 80.6, 81.3, 94.7, 127.7, 127.3, 138.2, 154.1, 172.7 , 216; IR (film, NaCl, cm "1) 3849, 2974, 2879, 1758, 1701, 1454, 1368, 1248, 1152, 926, 734; LRMS (ESI) calculated for [M + Na +] 619.1, found 619.2; ] 23D = -13.0 (c = 0.4, CHC13). 9-Benzyloxy-4,4,6,8-tetramethyl-5-oxo-7- (2,2,2-trichloroethoxycarbonyloxy) -3- (triethylsilanyloxy) -nonanoic acid tert-b ester. To a solution of starting material (37.6 mg, 6.3 μmol) and imidazole (9.4 mg, 13.8 μmol) in DMF (0.4 ml) at 0 ° C was added TESCI (11.6 μl, 69.3 μmol). After 3 h, the mixture was diluted with saturated aqueous NaHCO3. The aqueous layer was extracted three times with hexanes. The combined organic extracts were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient hexane to hexane / EtOAc 93: 7) to produce, in order of elution, the product (22.9 mg, 51%), and the starting material was recovered (12.9 g-, 34 %) as light oils. 1 H NMR (400 MHz, CDC13) d 0.66 (q, J = 7.9 Hz, 6H), 0.96 (t, J = 7.9 Hz, 9H), 1.01 (s, 3H), 1.05 (d, J = 5.2 Hz, 3H -), 1.07 (d, J = 5.3-Hz, 3H), 1.35 (s, 3H). 1.44 (s, 9H), 2. 05 r 2.11 (m, 2H), 2.50 (dd, J = 3.5 and 17.2 Hz, ÍH), 3.35 (dd, J = 5.9 and 9.0 Hz, ÍH), 3.49 (dd, J = 4.0 and 9.0 Hz, ÍH), 3. 53 (dd, J = 3.8 and 6.7 Hz, ÍH), - 4.18 (dd, J = 3.5 and 6.5 Hz, 1H), 4.45 (s, 2H), 4.65 (d, J = 11.9 Hz, lH), 4.79 (d, J = 11.9 Hz, ÍH), 4.97 (dd, J = 3.7 and 8.1 Hz, 1H), 7.29- 7.52 (m, 5H); ^ C NMR (125 MHz, CDC13) d 5.3, 7.3, 10.9, 14.9, 21.3, 22.6, 28.4, 35.9, 41.1, 42.7, 53.7, 71.9, 73.7, 75.7, 80.1, 80. 9, 95.1, 127.9, 128.0, 128.7, 138.6, 154.3, 171.7, 215.7; IR (film, NaCl, cm "1) 2956, 2876, 1732, 1694, 1456, 1366, 1257, 1154, 1098, 988, 835, 774, 741; LRMS (ESI) calculated for C ^ HssOsSiClaNa [M + Na +] 733.2, found 733.3 [a] 23D = -16.1 (c = 0. 1, CHC13).

Tert-b ester of 9-bensyloxy-3- (diethyl-ethylsilanyloxy) -7-hydroxy-4,4,6,8-tetramethyl-5-oxo-nonanoic acid. To a solution of starting material (22.9 mg, 3.2 μmol) in 1: 1 THF / AcOH (1.4 ml) was added Zn (5.0 mg, 7.8 μmol, nanodimetre). The mixture was subjected to sonication for 15 minutes. Additional Zn (5.0 mg, 7.8 μmol, nanodimetre) was added, followed by sonication for an additional 15 minutes. The suspension was filtered through celite pad, washed with EtOAc several times. The filtrates were washed with saturated NaHCO3, brine, dried over MgSO4 and concentrated in vacuo. The crude residue was passed through a short plug of silica gel eluting with 4: 1 hexane / EtOAc to yield 17.1 mg (99% yield) of product as a colorless oil; ZH NMR (400 MHz, CDC13) d (m, 6H), 0.96 (t, J = 7.9 Hz, 9H), 0.97 (d, J = 6.8 Hz, 3H), 1.05 (d, J = 6.8 Hz, 3H) , 1.11 (s, 3H), 1.26 (s, 3H), 1.44 (s, 9H), 1.84-1.90 (m, 1H), 2.21 (dd, J = 6.7 and 17.0 Hz, ÍH), 2.36 (dd, J = 6.7 and 17.0 Hz, ÍH), 3.24-3.29 (, 1H), 3.44-3.52 (m, 2H), 3.67 (dd, J = 3.9 and 8.9 Hz, ÍH), 4.36 (dd, J = 3.5 and 6.5 Hz , ÍH), 4.50 (d, J = 12.0 Hz, 1H), 4.54 (d, J = 12.0 Hz, HH), 7.32-7.36 (, 5H); 13C NMR (100 MHz, CDC13) d 5.0, 6.9, 9.7, 13.9, 20.2, 21.8, 28.0, 36.3, 40.8, 41.5, 53.7, 72.5, 72.9, 73.2, 73.6, 73.6, 80.7, 127.4, 127.5, 128.2, 138.6, 171.0 221.4; IR (film, NaCl, c "1) 3502, 2959, 2875, 1731, 1683, 1456, 1366, 1154, 1098, 996, 739; LRMS (ESI) calculated for C3oH52? 6SiCl3Na [M + Na +] 559.3, found 559.3; [a] 23 D = -41.0 (c = 0.4, CHC13). 9-Benzyloxy-7- (tert-bdimethylsilanyloxy) -3- (diethylmethylsilanyloxy) -4,4 / 6,8-tetramethyl-5-oxo-nonanoic acid tert-b ester. To a solution of the starting material (4.1 mg, 7.6 μmol) and 2,6-lutidine (10.0 μl, 43.5 mmol) in CH2C12 (0.2 ml) at -78 ° C was added TBSOTf (10.0 μl, 85.8 mmol). After 2 h, more 2,6-lutidine was added (10.0 μl, 43.5 mmol) and TBSOTf (10.0 μl, 85.8 mmol). After 6 hours, the mixture was diluted with saturated aqueous NaHCO3. The aqueous layer was extracted three times with EtOAc. The combined organic extracts were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (Hexane to hexane / EtOAc gradient 91: 9) to produce the product (5.4 g, 82%) with a clear oil. The spectroscopy data also agreed with the reported values.

The acid and alcohol were subjected to azeotropy with dry benzene (5 ml x 2) and dried under high vacuum prior to the reaction. To a solution of alcohol (639 mg, 2.63 mmol) in CH2C12 (13 mL) was added EDCI (576 mg, 3.09 mmol) and DMAP (366 mg, 3.09 mmol) at 0 ° C. To a mixture was added a solution of acid (1.11 g, such as 1.88 mmol) in CH2C12 (5 ml + 2 ml of rinse) by dripping for 16 minutes at 0 ° C. After stirring at 0 ° C for 1.5 h, the mixture was stirred at rt for 3.5 h. Following the concentration of the reaction mixture, the residue was purified by flash column chromatography (Si02, hexane / EtOAc = 30: 1 to 20: 1) to yield ester (1.20 g, 1.61 mmol, 80% t-butyl ester) ) as a colorless oil. 1. 30, CHC13); IR (film) v 2955, 2925, 2872, 1732, 1696, 1461, 1378, 1290, 1243, 1173, 1091, 985, 873, 773 cm "1;? E NMR (400 MHz, CDC13) d 0.06 (3H, s), 0.06 (3H, s), 0.58-0.66 (6H,), 0.92 (9H, s), 0.95 (9H, t, J = 8.0 Hz), 1.02 (3H, d, J = 6.5 Hz), 1.03 (3H, d, J = 6.5 Hz), 1.07 (3H, s), 1.21 (3H, s), 1.67 (3H, s), 2.07 (3H, s), 2.05-2.12 (ÍH,), 2.30 (1H, dd, J = 16.9, 7.5 Hz), 2.39 (1H, dt, J = .14.8, 6.7 Hz), 2.49 (1H, dd, J = 17.0, 3.0 Hz), 2.50 (HI, dt, J = 14.8, 6.7 Hz), 2.70 (3H, s), 2.74 -2.30 (2H,), 3.07 (1H, dd, J- 7.0 Hz), 3.83 (HH, dd, J = 7.1, 2.0 Hz), 4.35 (HH, dd, J = 7.4, 2.8 Hz), 4.98-5.07 (4H, m), 5.16 (HH, brt, J = 7.0 Hz), 5.23 (1H, t, J = 6.9 Hz), 5.74 (HH, ddt, J = 16.7, 10.2, 6.5 Hz), 5.91 (HH, ddd, J = 17.8, 10.5, 7.8 Hz), 6.50 (1H, s), 6.95 (HH, s); 13C NMR (100 MHz, CDC13) d '-3.7, -3.3, 5.3 (3C), 7.2 (3C), 14.8, 15.2, 18.7, 18.9, 19.4, 20.3, 23.6, 23.7, 26.4 (3C), 31.7, 36.7 , 40.1, 43.8, 46.4, 53.3, 74.2, 76.5, 79.-6, 115.5, 115.6, 116.5, 120.5, 121.3, 135.8, 136.1, 137.4, 140.2, 152.9, 164.7, 171.5, 218.4; LMRS (ESI) calculated for C4? H? N05SSi2Na [M + Na +] 768.5, found 768.5; HRMS calculated for C41H72NO5SSÍ2 [M + H +] 746.4670, found 746.4680.

A solution of diene (26.9 mg, 36.1 μmol) in toluene (70 ml) was heated to reflux and treated with a solution of Grubbs catalyst (3.1 mg, 3.61 μmol) in toluene (2 ml). The mixture was stirred for 25 minutes, cooled to 0 ° C, filtered through a pad of silica gel, which was rinsed with hexane / EtOAc = 2/1. The combined filtrate was concentrated and purified by flash column chromatography (Si02, hexane / Et20 = 40: 1 to 5: 1) to yield the desired product (9.9 mg, 13.8 μmol, 38%) and cycloheptadiene (14.4 mg, 22.3 μmol, 62%) both as a colorless oil. [a] D25 -41.5 (c 0.715, CHC13); IR (film) v 2955, 2884, 1737, 1690, 1467, 1378, 1249, 1179, 1102, 1014, 979, 879, 826, 773 c "1; XH NMR (400 MHz, CDC13) d 0.08 (3H, s ), 0.12 (3H, s), 0.57 (6H, q, J = 7.8 Hz), 0.89 (9H, t, J = 8.0 Hz), 0.93 (9H, s), 1.04 (3H, s), 1.06 (3H) , d, J = 7.1 Hz), 1.12 (3H, 's), 1.17 (3H, d, J = 7.1 Hz), 1.68 (3H, s), 2.15 (3H, d, J = 0.8 Hz), 2.14- 2.27 (2H, m), 2.45 (ÍH, dd, J = 14.0, 4.8 Hz), 2.50 (1H, dd, J = 14.9, 3.2 Hz), 2.64-2.74 (2H, m), 2.72 (3H, s) , 3.02 (HH, quintet, J = 7.0 Hz), 3.10 (1H, dd, = 14.4, 7.3 Hz), 3.96 (HH, d, J = 8.7 Hz), 4.43 (HH, dd, J = 8.3, 2.9 Hz ), 5.22 (ÍH, dd, J = 9.8, 5.7 Hz), 5.33-5.42 (2H, m), 5.69 (ÍH, dd, J = 15. 8, 8. 2 Hz), 6.57 (ÍH, s), 6.96 (ÍH, s); 13C NMR (100 MHz, CDCI3) d -3.3, -3.2, 5.6 (3C), 7.1 (3C), 15.0, 17.2, 18.8, 19.4, 21.4, 21.7, 23.8, 24.3, 26.5 ( 3C), 33.2, 35.6, 41.3, 41.8, 48.2, 54.0, 74.4, 77.4, 79.3, 116.4, 120.5, 121.0, 129.3, 132.1, 137.8, 138.0, 152.7, 164.8, 170.7, 216.8, LRMS (ESI) calculated for C39H68N05SS i2 [M + H +] 718.4, found 718.3; HRMS calculated for C39H68N05SSi2 [M + H +] 718.4357, found 718.4355. [o]] D26 -38.5 (c 0.400, CHC13), \ IR (film) v 2955, 2878, 1741, 1693, 1472, 1458, 1385, 1295, 1253, 1169, 1098, 988, 871, 837, 775 cm "1; XH NMR (400 MHz, CDC13) d 0.07 (6H, s), 0.61-0.68 (6H, m), 0.93 (9H, s), 0.97 (9H, t, J = 8.0 Hz), 1.03 (3H , d, 'J = 7.0 Hz), 1.04 (3H, d, J = 7.0 Hz), 1.10 (3H, s), 1.21 (3H, s), 1.65 (3H, s), 1.75 (3H, s), 2.06-2.14 (1H, m), 2.31 (ÍH, dd, J = 17.2, 7.2 Hz), 2.34-2.51 (2H, m), 2.49 (1H, dd, J = 17.1, 2.8 Hz), 2.65-2.81 ( 2H, m), 3.07 (1H, quintet, J = 7.0 Hz), 3.84 (IH, dd, J = 7.2, 2.1 Hz), 4.40 (HH, dd, J = 7.2, 2.8 Hz), 4.98-5.09 (2H, m), 5.38-5.42 (1H, m), 5.65 (HH, t, J = 5.9 Hz), 5.93 (1H, ddd, J = 17.9, 10.1, 7. 8 Hz); 13C NMR (100MHz, CDC13) d -3.6, -3.3, 5.4 (3C), 7.3 (3C), 15.3, 18.7, 19.0, 20.0, 22.1, 23.8, 25.8, 26.4 (3C), 31.3, 32.3, 40.0, 43.8 , 46.3, 54.0, 72.5, 73.8, 76.5, 115.6, 119.8, 125.6, 136.5, 140.1, 140.6, 171.9, 218.5; LRMS (ESI) calculated for C35H605Si2Na [M + Na +] 643.4, found 643.3; HRMS calculated for C35H6405SÍ2 to [M + Na +] 643.4190, found 643.4219.

Via Wittig reaction: To a reagent solution of Wittig (19.1 mg, 54.7 μmol) in THF (0.4 ml) was added KHMDS (109 μl of a 0.5 M solution in toluene, 54.7 μmol) at 0 ° C.

The mixture was stirred at 0 ° C for 0.5 h and then cooled to -78 ° C. To the mixture was added dropwise a ketone solution (5.7 mg, 9.12 μmol) in THF (0.3 ml), and the resulting mixture was allowed to warm to -20 ° C for 1.5 h. The reaction was quenched with saturated aqueous NH4C1 (2 mL) and extracted with EtOAc (7 mL x 3). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography (Si02, hexane / Et20 = 10: 1) to afford 5.6 mg of the inseparable olefin mixture. { E / Z - 9: 1). The mixture was purified by preparative CCD (hexane / Et20 = 4: 1) to provide the desired pure isomer (5.0 mg, 6.96 μmol, 76%) as a colorless oil.

XH NMR (400 MHz, CDC13): d 0.08 (3H, s), 0.12 (3H, s), 0.51 (6H, q, J = 7.9 Hz), 0.86 (9H, t, J = 7.9 Hz), 0.97 ( 9H, s), 1. 01 (3H, s), 1.06 (3H, d, J = 7.1 Hz), 1.12 (3H, s), 1.18 (3H, d, J = 7.1 Hz), 1.69 (3H, s), 1.97 (3H, s ), 2.10-2.18 (1H,), 2.24-2.31 (1H, m), 2.38-2.59 (3H, m), 2.68-2.78 (1H, m), 2.72 (3H, s), 2.98-3.14 (2H, m), 3.97 (ÍH, d, J = 9.0 Hz), 4. 45-4.48 (ÍH, m), 5.29-5.41 (2H,), 5.73 (ÍH, dd, J = 15.6, 8. 3 Hz), 6.30 (1H, s), 6.73 (ÍH, d, J = 8.7 Hz), 6.56 (1H, s); LRMS (ESI) calculated for C39H68N05SSÍ2 [M + H +] 718.4, found 718.1.

To a solution of silyl ether (298.8 mg, 0.416 mmol) in THF (6.5 ml) in a plastic tube was added HF "pyridine (3.2 ml) at 0 ° C, and the mixture was stirred at rt for 3 h. The reaction was quenched with dropwise addition of TMSOMe (30 mL) at 0 ° C and the mixture was stirred at rt for 3 h. After concentration and drying under high vacuum, the residue was purified by flash column chromatography (SiO2, hexane / EtOAc = 1: 1) to yield alcohol (196.6 mg, 0.402 mmol, 97%) as a colorless solid. [ex] 25 -96.6 (c 0.235, CHC13); IR (film) v 3502, 2970, 2927, 1733, 1685, 1506, 1456, 1375, 1251, 1152, 1040, 977 cm "1; XH NMR (400 MHz, CDCI3) d 1.06 (3H, s), 1.11 (3H, d, J = 7.0 Hz) , 1.22 (3H, d, J = 6.8 Hz), 1.28 (3H, s), 1.72 (3H, s), 2.10 (3H, s), 2.31-2.40 (2H, m), 2.43 (ÍH, dd, J = 16.0, 3.7 Hz), 2.49 (ÍH, dd, J = 16.0, 9.2 Hz), 2.55-2.68 (2H, m), 2.71 (3H, s), 2.98 (1H, dd, J = 14.4, 6.4 Hz), 3.16 (ÍH, quintet, J = 6.2 Hz), 3.76 (1H, dd, J = 5.9, 3.2 Hz), 4.30 (ÍH, dd, J = 9. 2, 3.7 Hz), 5.18 (1 H, brt, J 7.3 Hz), 5.32 (1 H, dd, J = 8. 4, 2.5 Hz), 5.63 (1H, dd, J = 15.7, 6.4 Hz), 5.60 (HH, ddd, J = 15.7, 6.9, 5.1 Hz), 6.60 (1H, s), 6.98 (HH, s); 13C NMR (100 MHz, CDCI3) d 15.1, 16.0, 17.7, 19.2, 19.5, 22.5, 23.6, 32. 0, 35.0, 39.6, 40.3, 44.8, 53.3, 71.8, 75.6, 78.3, 116.1, 119. 6, 120.5, 129.9, 131.3, 137.5, 138.2, 152.2, 165.0, 170.7, 218.8; LRMS (ESI) calculated for C27H40 O5S [M +? ] 490.3, found 490.2; HRMS calculated for C27H40NO5S [M + tf] 490.2627, found 490.2602.

To a solution of olefin (1.2 mg, 2.5 μmol) and TrisNHNH2 (29.3 mg, 98 μmol) in C1 CH2CH2C1 (0.7 ml) at 50 ° C was added Et3N (13.7 μl, 98 μmol). The reaction was monitored by HPTLC (hexane / EtOAc / CH2Cl2 = 1/1/2). After stirring for 7 h, the mixture was cooled to rt, diluted with EtOAc and filtered through a pad of silica gel, which was rinsed with EtOAc. After concentration, the residue was purified by preparative CCD (hexane / EtOAc / CH2Cl2 = 1- / 1/2) to afford the reduced product (1.1 mg, 2.2 μmol, 91%) as a white solid. The spectral data of this compound were identical to those reported for dEpoB.

The acid and alcohol were subjected to azeotropy with dry benzene (5 ml x 2) and dried under high vacuum prior to the reaction. To a solution of alcohol (10: 1 mixture of isomers, 240 mg, 0.756 mmol) in CH2C12 (5 mL) were added EDCl (192.7 mg, 1.01 mmol) and DMAP (122.8 mg, 1.01 mmol) at 0 ° C. To the mixture was added a solution of acid (314.6 mg, 0.628 iraaol) - in CH2C1 (2 ml + 1 ml of rinse) by dripping for 15 minutes at 0 ° C. After stirring at 0 ° C for 2 h, the mixture was stirred at rt for 2 h. After concentration, the residue was carefully purified by flash column chromatography (Si02, hexane / EtOAc = 20: 1 to 15: 1) to yield ester (340.1 mg, 0.425 mmol, 68% based on acid) as a colorless oil . [a] D -27.5 (c 0.28, CHC13); IR (film) v 2956, 2878, 1740, 1692, 1472, 1378, 1317, 1253, 1174, 1118, 988, 915, 872, 837, 775 cm "1; XH NMR (400 MHz, 'CDC13) d 0.06 (6H, .s), 0.57-0.65 (6H , m), 0.92 (9H, s), 0.94 (9H, t, J = 7.9 Hz), 1.02 (3H, d, J = 6.9 Hz), 1.03 (3H, d, J = 6.8 Hz), 1.07 (3H , s), 1.22"(3H, s), 2.07-2.10 (ÍH, m), 2.09 (3H, s), 2.31 (1H, dd, J = 16.9, 7.3 Hz), 2.51 (1H, dd, J = 16.8, 3.0 Hz), 2.49-2.65 (2H,), 2.71 (3H, s), 2.96-2.99 (2H, m), 3.06 (ÍH, quintet, J = 7.1 Hz), 3.83 (1H, dd, J = 7.3, 2.1 Hz), 4.35 (1H, dd, J = 7.2, 3.0 Hz), 4.98-5.12 (4H, m), 5.30 (ÍH, t, J = 6.7 Hz), 5.76 (1H, ddt, J = 16.7 , 10.2, 6.2 Hz), 5.92 (1H, ddd, J = 17.8, 9.9, 7.8 Hz] 6.19 (H, t, J = 7.0 Hz), 6.51 (H, s), 6.97 (H, s) 13, NMR (100 MHz, CDCl 3) d -3.7, -3.4, 5.2 (3C), 7.1 (3C), 14.7, . 2, 18.6, 18.9, 19.3, 19.9, 23.8, 26.3 (3C), 30.1, 31.2, 40. 0, 43.7, 46.3, 53.3, 73.9, 76.5, 77.9, 115.5, 116.5, 117.0, 121.5, 124.1 [q, XJ (C, F) = 273.4 Hz], 129.6 [q, 2J (C, F) = 28.5 Hz ], 130.5 [q, 3J (C, F) = 6.1 Hz], 133.6, 136.3, 140.1, 152.4, 164.8, 171.3, 218.3; XRMS (ESI) calculated for C4? H68F3N? 5SSi2 a [M + Na +] 822.4, found 822.4; HRMS calculated for C4? H69F3? 5SS2 [M + H +] 800.4387, found 800. 4374 A solution of diene (57.6 mg, 72.0 μmol) in toluene (142 ml) was heated to reflux and treated with a solution of Grubbs catalyst (6.1 mg, 7.20 μmol) in toluene (2 ml). The mixture was stirred for 28 minutes, cooled to 0 ° C, and filtered through a pad of silica gel, which was rinsed with hexane / EtOAc = 2/1 (300 ml).

The combined filtrate was concentrated and purified by flash column chromatography (Si02, hexane / Et20 = 40: 1 to 15: 2) to yield the desired product (12.0 mg, 15.5 μmol, 22%) and cycloheptadiene (29.2 mg, 43.3 μmol , 60%) both as a colorless oil.

[OI] D25 -17.1 (c 0.14, CHC13); IR (film) v 2955, 2884, 1743, 1690, 1472, 1320, 1173, 1114, 1038, 1008, 873, "832, 773 cm" 1; XE NMR (400 MHz, CDCl 3) d 0.09 (3H, s), 0.12 (3H, s), 0.55 (6H, q, J = 7.7 Hz), 0.88 (9H, t, J = 8.0 Hz), 0.96 (9H , s), 1.01 (3H, s), 1.06 (3H, d, J = 7.1 Hz), 1.12 (3H, s), 1.20 (3H, d, J = 7.1 Hz), 2.07-2.17 (ÍH, m) , 2.19 (3H, s), 2.38 (ÍH, dd, J = 14.3, 3.5 Hz), 2.39-2.49 (1H,), 2.50 (ÍH, dd, J = 14.3, 7.3 Hz), 2.73 (3H, s) , 2.77-2.91 (2H, m), 2.96-3.09 (2H,), 3.98 (1H, dd, J = 8.9 Hz), 4.54 (IH, dd, J = 7.3, 3.4 Hz), 5.28-5.38 (IH, ), 5.63 (HH, dd, J = 9.6, 2.3 Hz), 5.77 (1H, dd, J = 15.9, 8.5 Hz "), 6.21-6.28 (1H, m), 6.60 (HH, s), 6.99 (HH) , s); 13C NMR (100MHz, CDCI3) d -3.4, -3.3, 5.5 (3C), 7.0 (3C), 14.6, 17.1, 18.7, 19.4, 19.9, 21.3, 24.8, 26.4 (3C), 29.6, 32.8, 42.0, 42.1, 48.2, 54.1, 73.4, 76.9, 77.8, 117.0, 121.6, 124.3 [q XJ (C, F) = 273.5 Hz], 127.2, '130.6 [q, 2J (C, F) = 28.2 Hz ], 130.8 [q, 3J (C, F) = 6.1 Hz], 133.2, 136.5, 152.3, 165.0, 170.1, 217.1; LRMS (ESI) calculated for C39H55F3 05SS2 [M + H +] 772.4, found 772.4; HRMS calculates for C39H65F3NO5SS2 [M + H "] 772.4074, found 772.4102. [a] D26 -35.6 (c 0.365, CHCl3); IR (film) v 2956, 2878, 1737, 1693, 1472, 1458, 1305, 1279, 1252, 1173, 1116, 988, 871, 775 cm-1; XH NMR (400 MHz, CDC13) d 0.08 (6H, s), 0.64 (6H, q, J = 7.8 Hz), 0.93 (9H, s), 0.96 (9H, t, J = 7.8 Hz), 1.04 (6H , d, J = 7.0 Hz), 1.10 (3H, s), 1.22 (3H, s), 1.70 (3H, s), 2.05-2.14 (1H, m), 2.32 (ÍH, dd, J = 17.0, 7.1 HzA 2.50 (1H, dd, J = 17.0, 3.0 Hz), 2.51-2.63 (2H, m), 2.87 (1H, dd, J = 18.4, 6.7 Hz), 2.90-3.02 (1H, m), 3.07 (1H , quintet, J = 7.1 Hz), 3.85 (ÍH, dd, J = 7.2, 2.0 Hz), 4.39 (ÍH, dd, J = 7.1, 2.9 Hz), 4.98-5.08 (2H, m), 5-.51 (1H, dd, J = 8.1, 3.8 Hz), 5.67 (IH, t, J = 5.9 Hz), 5.93 (1H, ddd, J = 17.8, 10.5, 7.8 Hz), 6.29 (IH, t, J = 5.5 Hz); 13C NMR (100 MHz, CDC13) d -3.7, -3.4, 5.3 (3C), 7.1 (3C), 15.3, 18.7, 18.9, 19.6, 21.3, 23.9, 24.1, 26.3 (3C), 30.3, 40.0, 43.7, 46.3, 53.4, 70.9, 73.7, 76.5, 115.5, 123.4, 123.8 [q, XJ (C, F) = 272.2 Hz], 129.1 [q, 3J (C, F) = 6.1 Hz], 131.5 [q, 2J ( C, F) 28.8 Hz], 138. 1, 140.0, 171.7, 218.5; LRMS (ESI) calculated for C 35 H 6? F 305 Si 2 Na [M + Na +] 697.4, found 697.4; HRMS calculated for C35HS? F305 Si2 Na [M + Na +] 697.3907, found 697.3892.

To a solution of silyl ether (1.78 g, 2.31 mmol) in THF (25 ml) in a plastic tube was slowly added HF "pyridine (12.5 ml) at 0 ° C, and the mixture was stirred at rt for 4 h. The reaction was quenched with dropwise addition of TMSOMe (80 ml) for 10 minutes at 0 ° C. The mixture was stirred vigorously at rt for 2.5 h After concentration and drying under high vacuum for 2 h, the residue was purified by flash column chromatography (Si? 2 ~ 50 g, hexane / EtOAc = 1: 1) to yield diol (1.20 g, 2.21 mmol, 96%) as a colorless solid. [α] D25 -54.6 (c 0.28, CHC13); IR (film) v 3478, 2974, 2929, 1736, 1689, 1449, 1381, 1318, 1247, 1169, 1113, 1039, 983, 867, 736 cm "1; XH NMR (400 MHz, CDC13) d 1.05 (3H, s), 1.12 (3H, d, J = 7.0 Hz), 1.23 (3H, d, J = 6.8 Hz), 1.37 (3H, s), 2. 04 (1H, brd, J = 3.8 Hz, -OH), 2.12 (3H, s), 2.25-2.33 (1H, m), 2.38 (1H, dd, J = 15.3, 3.0 Hz), 2.48 (1H, dd , J = 15.4, 9.8 Hz), 2.54-2.61 (1H, m), 2.66-2.76 (1H, m), 2.71 (3H, s), 2. 96 (1H, dd, J = 16.5, 4.5 Hz), 3.02 (ÍH, dd, J = 16.3, 6.5 Hz), 3.11 (1H, quintet, J = 6.7 Hz), 3.19 (1H, brs, = OH), 3. 74 (ÍH, brs), 4.35 (ÍH, brd, J = 9.5 Hz), 5.42 (ÍH, dd, J = 6. 2, 4.1 Hz), 5.60 (1H, ddd, J = 15.8, 5.6, 4.5 Hz), 5. 66 (ÍH, dd, J = 15.8, 5.8 Hz), 6.24 (ÍH, t, J = 7.2 Hz), 6.64 (1H, s), 7.00 (1H, s); 13C NMR (100 MHz, CDC13) d 15.1, 16.1, 17.7, 18.5, 19.3, 22.5, 28.8, 31.1, 39.6, 39.7, 45.0, 53.7, 71.4, 75.3, 76.8, 116.7, 120.2, 124.3 [q, XJ (C , F) = 273.4 Hz], 127.9, 130.2 [q, 3J (C, F) = 6.0 Hz], 130.6 [q, 2J (C, F) = 28.4 Hz], 132.5, 136.7, 152.0, 165.4, 170.2, 218.4; LRMS (ESI) calculated for C 27 H 37 F 3 N 0 S [M + H +] 544.2, found 544.2; HRMS calculated for C27H37F3NO5S [M + H +] 544.2345, found 544.2346.

To a solution of diol (1.22 mg, 2.24 μmol) and TrisNHNH2 (26.7 mg, 89.6 μmol) in ClCH2CH2Cl (1 ml) at 50 ° C was added Et3N (12.5 μl, 89.6 μmol). The reaction was monitored by HPTLC (hexane / EtOAc / CH2Cl2 = 1/1/2). After stirring for 6.5 h, TrisNHNH (26.7 g, 89.6 μmol) and EtN (12.5 μl, 89.6 μmol) were further added to the mixture. After stirring for 14 h, the mixture was cooled to rt, diluted with EtOAc and filtered through a pad of silica gel, which was rinsed with EtOAc. After concentration, the residue was purified by preparative CCD (hexane / EtOAc / CHCl2 = 1/1/2) to yield the reduced product (1.16 mg, 2.13 μmol, 94%) as a white solid, [a] D24 -75.1 (c 0.35, CHC13); IR (film) v 3483, 2968, 1337, 1685, 1466, 1381, 1322, 1247, 1168, 1113, 1010, 833, 736 c "1; 4 * NMR (400 MHz, CDC13) d 1.03 (3H, d, J = 7.0 Hz), 1.08 (3H, s), 1.19 (3H, d, J = 6.8 Hz), 1.25-1.35 (2H, m), 1.37 (3H, s), 1. 42-1.55 (2H,), 1.65-1.82 (2H,), 2.10 (3H, d, J = 0.8 Hz), 2.21-2.47 (2H, m), 2.27 (ÍH, dd, J = 14.2, 2.6 Hz), 2.48 (1H, dd, J = 14.3, 10.8 Hz), 2.70 (3H, s), 2.70-2.28 (1H, m), 3. 02 (1H, d, J = 2.0 Hz, -OH), 3.19 (HH, qd, J = 6.9, 2.2 Hz), 3.65 (1H, d, J = 6.2 Hz, -OH), 3.69-3.72 (HH, m), 4.34 (1H, ddd, J = 10.8, 6.2, 2.6 Hz), 5.28 (IH, dd, J = 10.2, 2.2 Hz), 6.12 (IH, dd, J = 10.2, 5.2 Hz), 6.61 (1H , s), 6.98 (1H, s); 13C NMR (100 MHz, CDCI3) d 13.0, 15.9, 16.0, 17.7, 19.1, 23.0, 25.6, 26.2, 31.3, 32.3, 37.4, 39.8, 41.6, 53.9, 72.3, 73.6, 77.7, 116.2, 119.9, 124.3 [q , XJ (C, F) = 274.4 Hz], 129.8 [q, 3J (C, F) = 6.1 Hz], 132.6 [q, 2J (C, F) = 27.8 Hz], 138.3, 151.7, 165.4, 170.2, 220.7; LRMS (ESI) calculated for C 27 H 39 F 3 NO 5 S [M + H +] 546.3, found 546.2; HRMS calculated for C27H39F3N05S [M + H +] 546.2501, found 546.2496.

The acid and alcohol was subjected to azeotropy with dry benzene (3 ml x 2) and dried under high vacuum prior to the reaction. To a solution of alcohol (68.0 mg, 0.173 mmol) in CH2C12 (1.3 mL) was added EDCI (37.8 mg, 0.197 mmol) and DMAP (24.1 mg, 0.197 mmol) at 0 ° C. To the mixture was added a solution of acid (72.6 mg, as 0.123 mmol) in CH2C12 (0.7 ml) by dropping for 5 minutes at 0 ° C. After stirring at 0 ° C for 1 h, the mixture was stirred at rt for 2.5 h. After concentration, the residue was purified by flash column chromatography (Si02, hexane / EtOAc = 30: 1) to yield ester (99.5 mg, 0.114 mmol, 92% t-butyl ester) as a colorless oil. [a] D25 -23.4 (c 0.56, CHCl3); IR (film) v 2955, 2931, 2880, 1735, 1696, 1506, 1472, 1386, 1362, 1294, 1254, .1174, 1104, 988, 878, 776, 742 cm "1; XH NMR (400 MHz, CDC13 ) d 0.06 (3H, s), 0.06 (3H, s), 0.14 (6H, s), 0.63 (6H, q, J = 8.0 Hz), 0.92 (9H, s), 0.94 (9H, t, J = 8.0 Hz), 0.97 (9H, s), 1.02 (3H, d, J = 6.6 Hz), 1.05 (3H, d, J = 6.5 Hz), 1.07 (3H, s), 1.21 (3H, s), 1.67 (3H, -s), 2.06 (3H, d, J = 0.8 Hz), 2.05-2.14 (ÍH, m), 2.30 (1H, dd, J = 16.9, 7.5 Hz), 2.33-2.53 (2H, m) , 2.50 (1H, dd, J = 16.9, 2.7 Hz), 2.76-2.80 (2H,), 3.07 (1H, quintet, J = 7.0 Hz), 3.83 (ÍH, dd, J = 7.0, 2.2 Hz), 4 35 (ÍH, dd, J = 7.4, 2.8 Hz), 4.97 (2H, s), 4.97-5.07 (4H, m), 5.16 (1H, t, J = 7.2 Hz), 5.24 (ÍH, t, J = 6.9 Hz), 5.74 (ÍH, ddt, J = 16.6, 10.0, 6.5 Hz), 5.91 (1H, ddd, J = 17.6, 9.9, 7.7 Hz), 6.50 (1H, s), 7.06 (ÍH, s); 13C NMR (100 MHz, CDC13) d -5.2 (2C), -3.7, -3.3, 5.3 (3C), 7.2 (3C), 14.7, 15.2, 18.5, 18.7, 18.9, . 3, 23.6, 23.7, 26.0 (3C), 26.4 (3C), 31.7, 36.7, 40.1, 43. 8, 46.4, 53.3, 63.4, 74.2, 76.5, 79.6, 115.5, 115.6, 116.6, 120.5, 121.3, 135.8, 136.1, 137.4, 140.1, 153.0, 171.5, 172.2, 218.4; LRMS (ESI) calculated for. C47H85N06SSi3 [M + H +] 876.6, found 876.5; HRMS calculated for C47H86N06SS3 [M + H +] 876.5484, found 876.5482.

A solution of diene (69.7 mg, 79.5 μmol) in toluene (158 ml) was heated to reflux and treated with a solution of Grubbs catalyst (6.7 mg, 7.95 μmol) in toluene (2 ml). The mixture was stirred for 11 minutes, cooled to 0 ° C, filtered through a pad of silica gel, which was rinsed with hexane / EtOAc = 3/1 (280 ml).

The combined filtrate was concentrated and purified by flash column chromatography (Si02, hexane / Et2? = 20: 1 to 15: 1) to yield the desired product (18.4 mg, 21.7 μmol, 27%) and cycloheptadiene (28.3 mg, 45.5 μmol, 57%) both as a colorless oil. [a] D24 -40.4 (c 0.26, CHC13); IR (film) v 2955, 2930, 2879, 1740, 1694, 1472, 1387, 1362, 1253, 1200, 1107, 1007, 838, 776, 742 cm "1; XH NMR (400 MHz, CDC13) d 0.08 (3H , s), 0.12 (3H, s), 0.15 (6H, s), 0. 57 (6H, q, J = 7.9 Hz), 0.88 (9H, t, J = 8.0 Hz), 0.95 (9H, s), 0.97 (9H, s), 1.04 (3H, s), 1.06 (3H, d, J = 7.1 Hz), 1.12 (3H, s), 1.17 (3H, d, J = 7.0 Hz), 1. 69 (3H, s), 2.06-2.30 (2H, m), 2.14 (3H, s), 2.45 (1H, dd, J = 15.6, 3.6 Hz), 2.50 (1H, dd, J = 14.9, 3.1 Hz) , 2.63-2.75 (2H, m), 2.97-3.06 (1H, m), 3.10 (ÍH, dd, J = 14.6, 7.7 Hz), 3. 97 (ÍH, d, J = 8.5 Hz), 4.44 (ÍH, dd, J = 8.4, 2.9 Hz), 4. 97 (2H, s), 5.22 (HH, dd, J = 8.7, 5.2 Hz), 5.33-5.44 (2H, m), 5.70 (1H, dd, J = 15.6, 8.1 Hz), 6.57 (HH, s) 7.07 (1H, s); 13 C NMR (125 MHz, CDC13) d -5.2, -3. 3, -3 .2, 5 6, 7 2, 7.3, 15.0, 17.2, 18.5, 18.8, 21.4, 23.9, 24.4, 26.0, 26.5, 33.3, 35.6, - 41.4, 41.8, 48.2, 54.0, 63.5, 74.4, 78.1, 79.3, 116. 6, 120.6, 121.0, 129.3, 132.1, 137.8, 137.9, 153.0, 170. 7, 172.3, 216.8; LRMS (ESI) calculated for C45H82N06SSi3 [M + H +] 848.5, found 848.5; HRMS (ESI) calculated for C45H82N05SS3 [M + H +] 848.5171, found 848.5161.

To a solution of silyl ether (61.8 mg, 72.8 μmol) in THF (2 ml) in a plastic tube was added HF'pyridine (1 ml) at 0 ° C, and the mixture was stirred at rt for 3.2 h. The reaction was quenched with dropwise addition of TMSOMe (15 mL) at 0 ° C. The mixture was stirred at rt for 2 h. After concentration and drying under high vacuum, the residue was purified by flash column chromatography (SiO2, hexane / EtOAc = 1: 3) to give triol (32.4 mg, 64.1 μmol, 88%) as a white solid. [a] D25 -108.4 (c 0.285, CHC13); IR (film) v 3422, 2968, 2919, 2729, 1689, 1449, 1377, 1252, 1152, 1064, 978 cm "1; XH NMR. {400 MHz, CDC13) d 1.05 (3H, s), 1.12 ( 3H, d, J = 6.9 Hz), 1.22 (3H, d, J = 6.8 Hz), 1.32 (3H, s), 1.72 (3H, s), 2.08 (3H, s), 2.31-2.40 (3H, m), 2.43 (1H, dd, J = 15.5, 3.5 Hz), 2.49 (1H, dd, J = 15.5, 9.5 Hz), 2.55-2.67 (2H, m ), 2.95 (ÍH, dd, J = 14.6, 6.3 Hz), 3.13 (ÍH, quintet, J = 6.6 Hz), 3.34 (1H, brs, -OH), 3.75 (1H, dd, J = 6.6, 2.4 Hz), 4.06 (HH, brs, -OH), 4.33 (HH, dd, J = 9.4, 3.0 Hz), 4.92 ( 2H, s), 5.18 (HH, t, J = 6.9 Hz), 5.33 (1H, rdd, J = 8.0, 2.5 Hz), 5.52 (1H, dd, J = 15.8, 6.4 Hz), 5.59 (HH, ddd , J = 15.8, 6.6, 5.0 Hz), 6.63 (ÍH, s), 7.13 (ÍH, s); 13C NMR (100 MHz, CDC13) d 15: 3, 16.3, 17.8, 19.2, 22.8, 23.7, 31.9, 35.1, 39.7, 40.2, 45.0, 53.4, 61.8, 71.7, 75.8, 78.1, 116.7, 119.0, 120.5, 130.0 , 131.2, 137.6, 138.9, 152.5, 170.0, 170.7, 218.7; LRMS (ESI) calculated for C27H39N06SNa [M + Na +] 528.2, found 528.0; HRMS calculated for C27H4oN06S [M + H +] 506.2576, found 506.2552.

The crude acid (4.65 g, as 7.27 mmol) and alcohol (2.18 g, 9.84 mmol) was subjected to azeotropy with dry benzene and then dried under high vacuum for 20 minutes before the reaction. To a solution of alcohol (2.18 g, 9.84 mmol) in CH2C12 (65 mL) was added EDC1 (2.09 g, 10.9 mmol) and DMAP (1.33 g, 10.9 mmol) at 0 ° C. To the mixture was added a solution of crude acid (4.65 g, as 7.27 mmol) in CH 2 Cl 2 (20 ml + 5 ml of rinse) by dripping for 20 minutes at 0 ° C.

After stirring at 0 ° C for 40 minutes, the mixture was stirred at rt for 4 h. After concentration, the residue was purified by flash column chromatography (SiO2 ~ 160 g, hexane / EtOAc = 20: 1) to yield ester (4.85 g, 6.87 mmol, 94% t-butyl ester) as a colorless oil. . [a] D25 -22.7 (c 0.26, CHCl3); IR (film) v 2958, 2936, 2800, 1748, 1732, 1693, 1473, 1416, 1360, 1317, 1296, 1254, 1174, 1119, 989, 916, 872, 838, 776 cm "1; 2H NMR (400 MHz, CDC13) d 0.08 (3H, s), 0.08 (3H, s), 0.60 (6H, q, J = 7.8 Hz), 0.93 (9H, s), 0.94 (9H, t, J = 8.0 Hz), 1.04 (3H, d, J = 7.0 Hz), 1.04 (3H, d, J = 7.0 Hz), 1.11 (3H, s), 1.23 (3H, s), 2.05-2.14 (1H, m), 2.17 (3H , s), 2.40 (1H, dd, J = 16.9, 7.0 Hz), 2.59 (ÍH, dd, J = 17.0, 3.6 Hz), 2.56-2.64 (2H,), 2.90-3.01 (2H,), 3.06 ( 1H, quintet, J = 7.0 Hz), 3.85 (1H, dd, J = 7.3, 2.0 Hz), 4.38 (1H, d, J = 7.0, 3.4 Hz), 4.97-5.14 (5H, m), 5.75 (H) , ddt, J = 16.0, 9. 9, 6.2 Hz), 5.92 (1H, ddd, J = 17.8, 10.5, 7.8 Hz), 6.21 (HI, td, J = 7.2, 1.5 Hz); 13C NMR (100 MHz , CDC13) d -3.7, -3.4, 5.2 (3C), 7.1 (3C), 15.4, 18.7, 18.9, 19.5, 23.9, 26.3 (3C), 26.6, 28.5, 30.0, 39.8, 43.7, 46.3, 53.3, 73.6 , 76.5, 77.1, 115.6, 117.8, 124.0 [q, XJ (C, F) = 273.5 Hz], 129.2 [q, 3J (C, F) = 6.1 Hz], 130.6 [q, 2J (C, F) = 28.7 Hz], 133.4, 140.0, 171.8, 204.6, 218.4; LRMS (ESI) calculated for C3sHS3F306SÍ2Na [M + Na +] 727.4, found 727.3; HRMS calculated for C36H64F306SÍ2 [M + H +] 705.4194, found 705.4193.

A solution of diene (510.0 mg, 0.723 mmol) in toluene (500 ml) was heated to reflux and treated with a solution of Grubbs catalyst (92.1 mg, 0.109 mmol) in toluene (10 ml). The mixture was stirred for 17 minutes under reflux and immediately cooled to 0 ° C and maintained at 0 ° C before filtration through a pad of silica gel. A second batch of diene (510.0 mg, 0.723 mmol) was processed identically and simultaneously. The combined reaction mixture was filtered through a pad of silica gel (100 g), which was rinsed with hexane / EtOAc = 3/1 (1.4 L). The combined filtrate was concentrated and purified by flash column chromatography (Si0 -65 g, hexane / Et20 = 10: 1 to 5: 1) to yield macrolide (742.4 mg, 1.10 mmol, 76%) as a colorless amorphous oil. [a] D25 -7.5 (c 0.12, CHC13); IR (film) v 2956, 2979, 1748, 1732, 1695, 1472, 1415, 1384, 1252, 1170, 1119, 1018, 986, 876, 835 c "1; XH NMR (400 MHz, CDC13) d 0.08 (3H , s), 0.10 . { 3E-, s), 0.60 (6H, q, J = 7.8 Hz), 0.93 (9H, s), 0.94 (9H, t, J = 7.8 Hz), 1.03 (3H, d, J = 7.1 Hz), 1.08 (3H, s), 1.13 (3H, d, J = 7.0 Hz), 1.17 (3H, s), 2.26 (3H, s) , 2.25-2.34 (ÍH, m), 2. 64 (HH, dd, J = 15.5, 5.0 Hz), 2.68-2.75 (2H,), 2.76 (HH, dd, J = 15.6, 6.4 Hz), 2.85 (HH, dd, J = 15.6, 5.7 Hz), 2.97 (1H, dq, J = 8.3, 6.9 Hz), 3.04 (1H, dd, J = 15.6, 6.3 Hz), 3. 92 (ÍH, dd, J = 8.3, 1.2 Hz), 4.36 (ÍH, t, J = 5.3 Hz), 5.30- 5.39 (2H, m), 5.58 (1H, dd, J = 15.5, 8.0 Hz), 6.13 (ÍH, brt, J = 7.2 Hz); 13C NMR (100 MHz, CDC13) d -3.6, -3.6, 5.4 (3C), 7.0 (3C), 17.5, 18.5, 19.0, 21.6, 23.5, 26.3 (3C), 26.5, 28. 6, 29.1, 41.0, 42.3, 47.3, 54.1, 74.2, 76.8, 77.7, 124.0 [q, XJ (C, F) = 273.7 Hz], 126.0, 128.7 [q, 3J (C, F) = 5.9 Hz], 132. 2 [q, 2J (C, F) = 28.1 Hz], 133.8, 170.5, 204.1, 216.1; LRMS (ESI) calculated t for C34H59F306SÍ2 a [M + Na +] 699.4, found 699.4; HRMS calculated for C34H60F3O6SÍ2 [M + H +] 677.33881, found for 677. 3892, via the Wittig reaction: the ketone was subjected to azeotropy with benzene (5 ml x 2) and then dried under high vacuum for 0.5 h. To a solution of Wittig's salt (907 mg, 2.59 mmol) in THF (19 ml) was added t-BuOK (2.4 ml of a 1.0 M solution in THF, 2.43 mmol) by dripping for 5 minutes at 0 ° C. The mixture was stirred at 0 ° C for 0.5 h and then cooled to -78 ° C. To the mixture was added dropwise a solution of ketone (1.10 g, 1.62 mmol) in THF (13 ml) for 10 minutes, and the The resulting mixture was allowed to warm to -20 ° C during h. The reaction was quenched with rated aqueous NH4C1 (15 ml) and extracted with EtOAc (50 ml x 3). The combined organic layers were washed with brine (20 ml), dried over Na 2 SO 4 and concentrated. The residue was purified by flash column chromatography (Si02, hexane / Et20 = 20: 1 to 10: 1) to yield the desired 16 (E) -isomer (940 mg, 1.22 mmol, 75%) and 16 (Z) undesired isomer (140.9 mg, 0.182 mmol, 11%) both as a colorless amorphous oil. [a] D25 62.7 (c 0.33, CHC13); IR (film) v 2955, 2878, 1743, 1692, 1472, 1379, 1320, 1253, 1169, 1114, 1007, 956, 877, 835, 775 c "1; XH NMR (400 MHz, CDC13) d 0.09 (3H , s), 0.13 (3H, s), 0.49 (6H, q, J = 7.8 Hz), 0.85 (9H, t, J = 7.8 Hz), 0.97 (9H, s), "0.99 (3H, s), 1.06 (3H, d, J = 7.1 Hz), 1.11 (3H, s), 1.20 (3H, d, J = 7.1 Hz), 2.00 (3H, s), 2.03-2.13 (1H, m), 2.35 (1H , dd, J = 14.3, 3.0 Hz), 2.46 (HH, dd, J = 14.3, 7.8 Hz), 2.41-2.50 (HH, m), 2.73 (3H, s), 2.71-2.90 (2H,) 2.98- 3.12 (2H, m), 3.99 (1H, d, J = 9.2 Hz), 4.56 (1H, dd, J = 7.7, 2.8 Hz), 5.33 (ΔI, ddd, J = 15.6, 8.9, 4.1 Hz), 5.82 (1H, dd, J = 15.6, 8. 4 Hz), 6.29 (1H, s), 6.33-6.40 (1H, m), 6.94 (1H, m), 7.09 (HH, brd, J = 8.4 Hz); 13C NMR (100 MHz, CDC13) d -3.2, -3.2, 5.5 (3C), 7.0 (3C), 17.2, 18.7, 19.3, 19.6, 20.0, 22.3, 24.9, 26.4 (3C), 29.7, 32.9, 41.9, 42.0, 48.6, 54.0, 72.2, 73.3, 77.0, 116.7, 120.7, 124.5 [q,? J (C, F) = 273.3 Hz], 127.9, 129.7 [q, 2J (C, F) = 28.0 Hz], 131.9 [q, 3J (C, F) = 6.1 Hz], 132.9, 136.6, 152.1, 165.4, 170.2, 217.4; LRMS (ESI) calculated for C39H65F3N05SS2 [M + H +] 772.4, found 772.4; HRMS calculated for C39HS5F3N05SSi2 [M + H +] 772.4074, found 772.4044.

A solution of deH-dEpoB (12.2 mg, 24.9 μmol) in CH2C12 (1.25 ml) was cooled to -78 ° C and treated with a cooled solution of DMDO (-78 ° C, 0.06 M in acetone, 914 μl, 54.8 μmol). The mixture was allowed to warm to -50 ° C and was stirred at -50 ° C for 2.7 h. The excess DMDO was quenched at -50 ° C by the addition of dimethyl sulfide (117 μl) and the mixture was stirred at this temperature for 0.5 h. The solvent was removed in vacuo. Purification by thin layer chromatography. preparative (hexane / EtOAc = 1/2) produced ß-epoxide (3.0 mg, 5.93 μmol, 24%) and -epoxide (7.9 mg, 15.6 μmol, 63%) both as a colorless solid. [α] D 25 -78.5 (c 0.33, CHCL3); IR (film) v 3454, 2974, 2928, 1734, 1689, 1450, 1379, 1250, 1152, 1061, 978, 735 cm-1; XH NMR (400 MHz, CDC13) d 1.03 (3H, s), 1.11 (3H, d, J = 7.0 Hz), 1.14 (3H, d, J = 6.9 Hz), 1.34 (3H, s), 1.36 (3H , s), 2.00 (1H, ddd, J = 15.1, 7.3, 4.0 Hz), 2.14 (1H, dt, J = 15.1, 5.2 Hz), 2.14 (3H, s), 2.21 (1H, dd, J = 14.6 , 8.0 Hz), 2.33 (1H, dd, J = 14.7, 4.8 Hz), 2.47 (HH, dd, J = 13.8, 3.3 Hz), 2.59 (HH, dd, J = 13.8, 9.4 Hz), 2.73 (3H , s), 2.77 (ÍH, brs, OH), 2.93 (1H, dd, J = 7.3, 4.8 Hz), 3.34 (ÍH, qd, J = 6.9, 3.8 Hz), 3.75-3.82 '(1H, m) , 4.12-4.24 (2H, m, which includes OH), 5.54 (1H, ddd, J = 15.7, 7.4, 5.0 Hz), 5.54-5.60 (ÍH,), "5.64 (ÍH, dd, J = 15.7, 5.6 Hz), 6.94 (HH, s), 7.01 (HH, s); XE NMR (500 MHz, CD2C12) d 0.91 (3H, s), 1.01 (3H, d, J = '6.9 Hz), 1.03 (3H, d, J = 6.9 Hz), 1.22 (3H, s), 1.27 (3H, s), 1.96-2.02 (HH, m), 2.04 (3H, d, J = 0.7 Hz), 2.16-2.23 (2H, m ), 2.33 (ÍH, dd, J = 14.2, 3.1 Hz), 2.30-2.35 (1H, m), 2.44 (1H, dd, J = 14.4, 10.3 Hz), 2.69 (3H, s), 2.77 (ÍH, t, J = 5.9 Hz), 3.24 (HH, qd, J = 6.9, 4.5 Hz), 3.63 (HH, t, J = 4. 1 Hz), 4.18-4.26 (HH, m), 5.37 (HH, t, J = 4.5 Hz), 5.48 (ÍH, dtd, J = 15.7, 6.7, 0.5 Hz), 5.58 (1H, dd, J = 15.7, 6.2 Hz), 6.58 (H, s), 7.00 (H, s); 13C NMR (100 MHz, CDC13) d 14.4, 16.3 (2C), 19.3, 19.7, 21.6, 22.6, 31.8, 35.9, 38.7, 39. 6, 44.1, 52.8, 60.8, 61.8, 74.0, 75.7, 75.9, 116.5, 119.6, 124. 3, 135.8, 136.2, 152.1, 165.2, 170.8, 221.5; LRMS cale. for C27H40N06S [M + H +] 506.3, found 506.3; HRMS (ESI) cale. for C27H4oN06S [M + H +] 506.2576, found 506.2566. [a] D25 -53.9 (c 0.700, CHC13); IR (film) v 3460, 2976, 2928, 1735, 1688, 1506, 1451, 1378, 1252, 1186, 1151, 1087, 1042, 976, 879, 735 cm "1; XH NMR (400 MHz, CDC13) d 1.00 - (3H, s), 1.04 (3H, d, J = 6.9 Hz), 1.12 (3H, d, J = 7.0 Hz), 1.35 (3H, s), 1.35 (3H, s), 1.87 (ÍH, dt , J = 15.0, 9.2 Hz), 2.03 (1H, dd, J = 13.9, 9.2 Hz), 2.13 (3H, s), 1.13-2.19 (HH, m), 2.36 (HH, dd, J = 13.9, ' 3.4 Hz), 2.39 (HH, dd, J = 12.2, 2.1 Hz), 1.42-2.51 (1H, m), 2.49 (1H, dd, J = 12.4, 10.9 Hz), 2.69 (HH, d, J = 2.7 Hz), 2.72 (3H, s), 3.06 (HH, dd, J = 9.1, 3.1 Hz), 3.54 (HH, qd, J = 7.0, 1.8 Hz), 3.76-3.80 (1H,), 4.07-4.14 ( HH, m), 4.31 (HH, d, J = 4.1 Hz), 5.52 (1H, dd, J = 15.5, 8.7 Hz), 5.60 (1H, ddd, J = 15.1, 9.4, 3.4 Hz), 5.71 (HH) , d, J = 8.4 Hz), 6.63 (1H, s), 6.99 (HH, s); 13C NMR (10OMHz, CDC13) d 13.7, 15.3, 15.7, 18.5, 19.4, 21.2, 22.4, 32.5, 35.5, 39.1 , 43.4, 43.8, 51.9, 61.3, 64.8, 73.5, 75.9, 76.4, 116.7, 120.1, 124.3, 137.5, 137.7, 152.3, 165.2, 171.0, 222.3, LRMS (ESI) cale for C27H33 05S to [M + N a +] 528.2, found 128.2; HRMS cale, for C27H40NO6S [M + H +] 506.2576, found 506.2583.

To a solution of epoxide (0.7 mg, 1.38 μmol) and TrisNHNH2 (20.6 mg, 69 μmol) in C1 CH2CH2C1 (0.4 ml) at 50 ° C was added Et3N (9.6 μl, 69 μmol). The reaction was monitored by CCDHP (hexane / EtOAc = 1/2). After stirring for 6 hours, the mixture was cooled to room temperature, diluted with EtOAc and filtered through a pad of silica gel, which was rinsed with EtOAc. After concentration, the residue was purified by preparative CCD (hexane / EtOAc = 1/2) to give the reduced product (0.5 mg, 0.985 μmol, 71%) as a white solid. The spectrum data of this compound are identical to those reported by EpoB.

To a solution of epoxide (14.0 mg, 27.7 μmol) and TrsiNHNH2 (165 mg, 0.554 mmol) in C1CH2CHC1 (3.3 ml) at 50 ° C was added Et3N (77.0 μl, 0.554 mmol). The reaction was monitored by CCDHP (hexane / EtOAc = 1/2). After stirring for 6 hours, the mixture was cooled to room temperature, diluted with EtOAc and filtered through a pad of silica, which was rinsed with EtOAc. After concentration, the residue was purified by preparative CCD (hexane / EtOAc = 1/2) to give the reduced product (12.3 mg, 24.2 μmol, 87%) as a colorless solid. [a] D24-13.8 (c 0.61, CHC13); IR (film) v 3475, 2971, 2875, 1735, 1689, 1456, 1382, 1253, 1181, 1151, 1056, 980, 884, 735 cm-1; XH NMR (400 MHz, CDC13) d 0.95 (3 H, d, J = 7.1 Hz), 1.04 (3 H, s), 1.11 (3 H, d, J = 1.0 Hz), 1.28 (3 H, s), 1. 37 (3H, s), 1.25-1.44 (2H, m), 1.45-1.59 (2H,), 1.71-1.82 (3H,), 1.86 (HH, dt, J = 15.3, 9.5 Hz), 2.10 (HH, dd, J = . 3, 3.6 Hz), 2.13 (3H, s), 2.40 (1H, dd, J = 12.5, 2.5 Hz), 2. 49 (HH, dd, J = 12.5, 11.0 Hz), 2.74 (3H, s), 2.80 (HH, brs, OH), 3.07 (HH, dd, J = 10.3, 3.3 Hz), 3.34 (1H, qd, J = 7.0, 0.5 Hz), 3.89 (HH, brs, OH), 4.03-4.09 (HH, m), 4.12-4.17 (1H, m), 5.69 (IH, d, "7 = 9.1 Hz), 6.63 ( 1H, s), 7.00 (1H, s); 13C NMR (100 MHz, CDC13) d 12.9, 15.4, 16.3, 18.8, 19.3, 21.6, 22.0, 23.0, 31.5, 32.1, 33.6, 38.6, 38.9, 42.6, 51.7, 62.6, 65.5, 71.2, 74.5, 76.3, 116.6 , 119.9, 138.0, 152.2, 165.2, 170.6, 222.7; LRMS (ESI) cale. for C27H42N06SNa [M + Na +] 530.3, found 530.2; HRMS cale, for C27H42N06S [M + H +] 508.2733, found 508.2754.

. Acetone was azeotroped with benzene (5 ml x 2) and then dried under high vacuum for 0.5 hour. To a Wittig salt solution (1.19 g, 2.27 mmol) in THF (18 ml), t-BuO (2.2 to 1.0 M of a solution in THF, 2.20 mmol) was added dropwise for 5 minutes at 0 ° C. The resulting mixture was stirred at 0 ° C for 20 minutes and then cooled to -78 ° C. To the mixture, a solution of ketone (1.06 g, 1.51 mmol) in THF was added dropwise (10 ml + 2 ml was rinsed) for 10 minutes, and the resulting mixture was allowed to warm to -20 ° C for 2 hours. The reaction was quenched with saturated aqueous NH4C1 (15 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (20 ml), dried over Na 2 SO and concentrated. The residue was purified by flash column chromatography (SÍO2 ~ 65 g, hexane / Et20 = 30: 1 to 20: 1) to give the desired 16 (E) -isomer (1.01 g, 1.11 mmol, 74%) and the (Z) - unwanted isomer (154. 5 mg, 0.182 mmol, 11%) both as a colorless amorphous oil. [a] D24 -19.0 (c 0.10, CHCl3); IR (film) v 2954, 2930, 2880, 1744, 1692, 1472, 1381, 1321, 1252, 1171, 1114, 1038, 1006, 837, 776 c -1; X H NMR (400 MHz, CDCl 3) d 0.09 (3 H, s), 0.12 (3 H, s), 0.15 (6 H, s), 0.55 (6 H, q, J = 7.8 Hz), 0.87 (9 H, t, J = 8.0 Hz), 0.96 (9H, s), 0.97 (9H, s), 1.01 (3H, s), 1.06 (3H, d, "7 = 7.1 Hz), 1.12 (3H, s), 1.20 [3H, d , J = 7.1 Hz), 2.07-2.16 (ÍH, m), 2.18 (3H, d, J = 1.0 Hz), 2.38 (1H, dd, J = 14.4, 3.3 Hz), 2.34-2.46 (1H, m) , 2.49 (ÍH, dd, J = 14.4, 7.4 Hz), 2.78-2.90 (2H, m), 2.97-3.09 (2H, m), 3.98 (HH, d, J = 8.9 Hz), 4.54 (HH, dd, J = 7.3, 3.3 Hz), 4.97 (2H, s), 5.33 (HH, ddd, J = 15.8, 8.6.4.9 Hz), 5.63 ( 1H, dd, J = 9. 6, 2.4 Hz), 5.78 (HH, dd, J = 15.8, 12 Hz), 6.22-6.27 (HH, m), 6.60 (HH, s), 7. 09 (ÍH, s); 13C NMR (100 MHz, CDC13) d -5.3 (2C), -3.4, -3.3, 5.5 (3C), 7.0 (3C), 14.6, 17.1, 18.4, 18.7, 19.8, 21.3, 24.8, 25.9 (3C), 26.4 (3C), 29.6, 32.9, 42.0, 42.1 / 48.2, 54.1, 63.4, 73.4, 76.9, 77.8, 117.2, 121.7, 124.3 [q, 1J (C, F) = 273.6 Hz], 127.2, 130.7 [q, J (C, F) = 27.5 Hz], 130.8 [q, 3J (C, F) = 6.2 Hz], 133.2, 136.4, 152.6, 170.1, 172.4, 217.1; LRMS (ESI) cale, for C4sH7gF3N06SSi3Na [M + Na +] 924.5-, found 924.5; HRMS cale, for C45H79F3N? 6SSi3 [M + H +] 902.4888, found 902.4887. [a] D26 65.7 (c 1.76, CHC13); IR (film) v 2955, 2931, 2879, 1743, 1692, 1472, 1380, 1321, 1253, 1170, 1113, 1007, 836, 776 cm "1; XE NMR (400 MHz, CDC13) d 0.07 (3H, s), 0.13 (3H, s ), 0.16 (6H, s), 0.48 (6H, q, J = 7.8 Hz), 0.84 (9H, t, J = 7.9 Hz), 0.97 (18H, s), 0.98 (3H, s), 1.06 (3H) , d, J = 7.1 Hz), 1.11 (3H, s), 1.20 (3H, d, J = 7.2 Hz), 2.00 (3H, s), 2.03-2.11 (1H,), 2.33 (1H, dd, J = 14.1.2.8 Hz), 2.43 (HH, dd, J = 14.0, 7.8 Hz), 2.40-2.48 (HH, m), 2.76-2.89 (2H, m), 2.97-3.10 (2H, m), 3.99 (HH, d, J = 9.3 Hz ), 4.57 (1H, dd, J = 7.8.2.6 Hz), 4.95 (HH, d, J = 14.6 Hz), 5.00 (1H, d, J = 14.6 Hz), 5.33 (1H, ddd, J = 15.6, 9.1, 3.8 Hz), 5.82 ( HH, dd, J = 15.6, 8.3 Hz), 6.30 (HH, s), 6.32-6.38 (HH, m), 7.04 (HH, s), 7.11 (HH, dd, J = 11.0.2.3 Hz); 13C NMR (100 MHz, CDC13) d -5.3 (2C), -3.2, -3.2, 5.5 (3C), 7.0 (3C), 17.2, 18.4, 18.8, 19.3, 19.8, 22.4, 25.1, 25.9 (3C), 26.5 (3C), 29.7, 33.0, 41.9, 42.1, 48.6, 54.0, 63.5, 72.1, 73.3, 76.9, 117.0, 120.8, 124.5 [q, XJ (C, F) = 273.5 Hz], 127.9, 129.7 [q, 2J (C, F) - 27.6 Hz], 131.9 [q, 3J (C, F) = 6.1 Hz], 132.9, 136.4, 152.4, 170.1, 172.9, 217.5; . LRMS (ESI) cale, for C45H78F3N06SNa [M + Na +] 924.5, found 924.5.

To a solution of silyl ether (1.04 g, 2.25 mmol) in THF (22 ml) in a plastic tube was slowly added HF * pyridine (11 ml) at 0 ° C, and the mixture was stirred at room temperature for 3.3 hours. The reaction was quenched with dropwise addition of TNSOMe (75 ml) for 10 minutes at 0 ° C. The mixture was stirred vigorously at room temperature for 4.2 hours. After concentration and drying under vacuum for 1 hour, the residue was purified by flash column chromatography (Si0 ~ 25 g, hexane / EtOAc = 3: 4 to 1: 2) to give triol (615. 7 mg, 1.00 mmol, 96%) as a colorless powder. [a] D25 -57.7 (c 1.20, CHC13); IR (film) v 3441, 2974, 2932, 1734, 1685, 1507, 1456, 1374, 1318, 1248, 1169, 1112, 1054, 982, 888, 737 cm "1; 1 H NMR (400 MHz, CDC13) d 1.04 (3H, s), 1.12 (3H, d, J = 6.9 Hz), 1.25 (3H, d, J = 6.8 Hz), 1.36 (3H, s), 1.90 (1H, d, J = 6.6 Hz, OH) / 2.08 (3H, s), 2.23-2.32 (1H,), 2.34 (1H, dd, J = 15.7, 2.4 3z), 2.49 (ÍH, dd, J = 15.7, 10.1 Hz), 2.59-2.69 (2H, m), 2.95-3.01 (2'H,), 3.04 (ÍH, quintet, J- 6.8 Hz), 3.72 (1H, td, J = 7.0, 3.0 Hz), 3.78 (ÍH, d, = 5.7 Hz, OH ), 4.38 IH, ddd, J = 10.1, 5.7, 2.4 Hz), 4.90 (2H, d, J = 6.1 Hz), 5.10 (IH, t, J = 6.1 Hz, OH), 1.44 (1H, t, J = 4.7 Hz), 5.60 (HH, dd, J = 15.9, 4.4 Hz), 5.66 (HH, dd, J = 15.9, 5.0 Hz), 6.28 (HH, t, J = 6.7 Hz), 6.73 (HH) , s), 7.16 (ÍH, s); 13C NMR (100 MHz, CDCI3) d 16.0, 6.5, 17.4, 17.5, 22.9, 28.5, 30.3, 39.0, 39.6, 45.6, 54.0, 60.9, 70.6, 75.6, 75.7, 116.8, 19.2, 124.2 [q, JJ (C, F) = 273.6 Hz], 127.9, 129.8 [q, 2J (C, F) = 28.4 Hz], 130.3 [q, 3J (C, F) = 5.9 Hz] , 131.2, 137.0, 152.2, 169.8, 170.0, 218.3; LRMS (ESI) cale, for C27H37F3N06SNa [M + H +] 560.2, found 560.1; HRMS cale, for [M + H +] 560.2294, found 560.2299.

To a solution of silyl ether (42.8 mg, 55.4 μmol) in THF (1 ml) in a plastic tube was slowly added HF-pyridine (0.-5 ml) at 0 ° C, and the mixture was stirred at room temperature for 4.3 hours. The reaction was quenched with dropwise addition of TMSOMe (3.2 ml) for 10 minutes at 0 ° C. The mixture was stirred vigorously at room temperature for 1.5 hours. After concentrating and drying under high vacuum for 1 hour, the residue was purified by flash column chromatography (SiO2, hexane / EtOAc = 1: 1) to give diol (23.6 mg, 43.4 μmol, 78%) as a colorless oil. [a] D25 31.6 (c 1.00, CHC13); IR (film) v 2955, 287.8, 1743, 1692, 1471, 1379, 1320, 253, 1169, 1114, 1007, 877, 835, 741 cm-1; XE NMR (400 MHz, CDCl 3) d 1.04 (3 H, s), 1.11 (3 H, d, J = 6. 9 Hz), 1.20 (3 H, d, J = 6.9 Hz), 1.30 (3 H, s), 1.93 (3H, brs), 2.22 (1H, d, J = 4.3 Hz, OH), 2.25-2.33 (HH, m), 2.38-2.41 (2H, m), 2.51-2.59 (2H,), 2.70 3H, s ), 2.80-2.90 (HH, m), 2.94 (HH, dd, J = 15.6, 4.7 Hz), 3.06 (HH, dd, J = 15.6, 7.4 Hz), 3.19 (HH, quintet, J = 6.6 Hz) , 3.71- 3.76 (HH, m), 4.26-4.32 (1H, m), 5.57 (HH, dd, "7 = 15.8, 7.2, 5.0 Hz), 5.67 (HH, dd, J = 15.8, 6.8 Hz), 6.27 (1H, s), 6.33 (HH, dd, J = 7.6, 6.3 Hz), 6.76 (HH, dd, J = 8.3, 2.9 Hz), 6.94 (1H, s); 13C NMR (100 MHz, CDC13) d 14.5, 17.9, 19.2, 19.5, 19.8, 22.2, 28.7, 32.4, 39.8 (2C), 44.7, 53.3, 71.9, 74.1, 75.1, 117.0, 120.4, 124.4 [q, V ( C, F) = 272.7 Hz], 128.4,130.1 Eq, 2J (C, F) = 28.9 Hz], 131.5 [q, 3J (C, F) = 5.9 Hz], 133.0, 136.9, 152.2, 165.5, 170.7, 218.5; LRMS (ESI) cale, for C27H36F3? 5S to [M + Na +] 566.2, found 566.3.

R = OTso Cl To a solution of alcohol (18.9 mg, 33.8 μmol) and Et 3 N (18.8 μl, 0.135 mmol) in CH 2 Cl (1 ml) was added TsCl (12.9 mg, 67.5 μmol) followed by DMAP (2.1 MG, 16.9 μmol) a 0 ° C. After stirring at room temperature for 1.5 hours, the mixture was filtered through a pad of silica gel (rinse EtOAc). After concentrating, the residue was purified by preparative CCD (hexane / EtOAc = 1/1) to give tosylate (8.5 mg, 11.9 μmol, 35%) and chloride (4.3 mg, 7.44 μmol, 22%) both as colorless powders. .

XE NMR (400 MHz, CDC13) d 1.06 (3H, s), 1.12 (3H, d, J = 7.0 Hz), 1.23 (3H ,. d, J = 6.7 Hz), 1.33 (3H, s), 1.99 ( HH, d, J = 5.5 Hz), 2.10 (3H, s), 2.25-2.34 (HH, m) 2.41 (1H, dd, J = 15.5, 3.3 Hz), 2.47 (3H, s), 2.48 (1H, dd, J = 15.7, 9.4 Hz), 2.51-2.63 (HH, m), 2.63 (1H, d, J = 6.1 Hz, OH), 2.64-2.75 (HH,), 2.91-3.05 (2H, m), 3.10 (ÍH, quintet, J = 6.8 Hz), 3.70-3.75 (1H, m), 4.30 (ÍH, ddd, J = 9.3, 6.1, 3.2 Hz), 5.32 (2H, s), 5.41 (1H, dd, J = 5.8, 4.5 Hz), - 5.57 (HH, ddd, J = 15.8, 6.4, 4.6 Hz), 5.65 (1H, dd, J = 15.8, 6.0 Hz), 6.21 (HH, t, J = 7.1 Hz) , 6.59 (1H, s), 7.18 (1H, s), 7.37 (2H, d, J = 8.1 Hz), 7.84 (2H, d, J = 8.3 Hz); LRMS (ESI) cale, for C34H42F3N08S2Na [M + Na +] 736.2, found 736.3.

IR (film) v 3494, 2975, 2935, 1734, 1689, 1319, 1248, 1170, 1113, 1040, 979, 738 c "1; XE NMR (400 MHz, CDC13) d 1.06 (3H, s), 1.12 ( 3H, d, J = 6.9 Hz), 1.23 (3H, d, J = 6.7 Hz), 1.34 (3H, s), 2.00 (ÍH, d, J = 5.6 Hz, OH), 2.15 (3H, s), 2.25-2.35 (1H, m), 2.41 (HH, dd, J = 15.5, 3.2 Hz), 2.49 (HH, dd, J = 15.5, 9.4 Hz), 2.53-2.62 (1H,), 2.69 (1H, d , J = 6.1 Hz, OH), 2.66-2.76 (1H,), 2.92-3.05 (2H, m), 3.11 (1H, quintet, J = 6.4 Hz), 3.70-3.76 (1H, m), 4.32 (ÍH , ddd, J = 9.2, 5.9, 3.1 Hz), 4.85 (2H, s), 5.43 (HH, dd, < J = 6.0, 4.4 Hz), 5.59 (HH, ddd, J = 15.9, 6.4, 4.5 Hz ), 5.66 (HH, dd, J = 15.9, 6.1 Hz), 6.23 (HH, t, J = 6.8 Hz), 6.63 (HH, s), 7.20 (HH, s); LRMS (ESI) cale, for C27H35ClF3N05SNa [M + Na +] 600.2, found 600.2.

To a solution of triol (50.4 mg, 90.1 μmol) in THF (1 ml) was added (PhO) 2PON3 (27.2 μl, 0.126 mmol) at 0 ° C.

After 5 minutes, DBU (16.2 μl, 0.108 mmol) was added, and the mixture was stirred at 0 ° C for 2 hours, then at room temperature for 20.5 hours. The mixture was diluted with EtOAc and quenched with water (2 ml), extracted with EtOAc (three times), and the combined organic layers were dried over a2SO4.

After concentration, the residue was purified by flash column chromatography (Si02, hexane / EtOAc = 3: 2) to give azide (45.6 mg, 78.0 μmol, 87% as a colorless powder. [a] D24 -60.3 (c 0.345, CHC13); IR (film) v 3492, 2975, 2931, 2105, 1732, 1688, 1319, 1248, 1169, 1113, 982, 733 c -1; XE NMR (400 MHz, CDC13) d 1.05 (3 H, s), 1.12 (3 H, d, J = 7.0 Hz), 1.23 (3 H, d, J = 6.8 Hz), 1.33 (3 H, s), 2.01 (H) , d, J = * 5.5 Hz, OH), 2.17 (3H, s), 2.25-2.35 (ÍH, m), 2.41 (lH, dd, J = 15.5, 3.2 Hz), 2.49 (1H, dd, J = 15.5, 9.5 Hz), 2.54-2.60 (ÍH, m), 2.66 (1H, d, J = 6.0 Hz), 2.65-2.76 (1H,), 2.96 (ÍH, dd, J = 16.0, 4.2 Hz), 3.03 (1H, dd, J = 16.1, 6.7 Hz), 3.11 (ÍH, quintet, J = 6.8 Hz), 3.71-3.76 (lH, m), 4.31 (HH, ddd, J = 9.2, 5.9, 3.2 Hz), 4.65 (2H, s), 5.43 (HH, dd, J = 6.0, 4.3 Hz), 5.58 (ÍH, ddd, J = 15.8, 6.4, 4.6 Hz), 5.66 (1H, dd, J = 15.8, 6.1 Hz), 6.23 (1H, t, J = 7.3 Hz), 6.63 (1H, s), 7.18 (1H, s); 13C NMR (100 MHz, CDC13) d 15.4, 15.9, 17. 8, 18.6, 22.8, 28.7, 30.9, 39.5, 39.7, 45.1, 51.3, 53.5, 71. 5, 75.4, 76.8, 118.2, 119.6, 122.7 [q, ^ (C) = 273.6 Hz], 127. 9, 130.0 [q, 3J (C, F) = 6.1 Hz], 130.6 [q, 2J (C, F) = 27.9 Hz], 132.3, 137.2, 153.1, 163.9, 170.0, 218.3; LRMS (ESI) cale, for C27H35F3N4? 5S to [M + Na +] 607.2, found 607.2; HRMS cale, for C27H36F3 405S [M + H +] 585.2359 found 585.2344.

To a solution of tosylate (8.9 mg, 12.5 μmol) in DMF (0.4 ml) was added NaN3 (12.2 mg, 0.188 mmol). After stirring at room temperature for 21 hours, the mixture was quenched with saturated NHC1 (aq) and extracted with EtOAc (three times). After concentration, the residue was purified by preparative CCD (hexane / EtOAc = 1: 1) to give azide (6.9 mg, 11.8 μmol, 94%) as a colorless powder.

To a solution of azide (21.0 mg, 35.9 μmol) in THF (0.6 ml) was added PMe3 (1.0 M in THF, 43.1 μl, 43.1 μmol). After 2 minutes, water (0.1 ml) was added and the mixture was stirred at room temperature for 3 hours. Additional PMe3 (1.0 M in THF, 7.2 μl, 7.2 μmol) was added, and the mixture was stirred at room temperature for 1.5 hours. NHOH was added accusing to 28% (54.5 μl). After stirring at room temperature for 1 hour, the mixture was directly purified by preparative CCD (CH2Cl2 / MeOH = 100: 7.5) to give amine (15.9 mg, 28.5 μmol, 79%) as a colorless powder. [a] D26-64.2 (c 0.815, CHC13); IR (film) v 3504, 3363, 2975, 2931, 1733, 1688, 1450, 1383, 1318, 1248, 1169, 1113, 1054, 984, 736 c "1; XE NMR (400 MHz, CDCl 3) d 1.05 (3H, s), 1.12 (3H , d, J = 7.0 Hz), 1.23 (3H, d, J = 6.8 Hz), 1.34 (3H, s), 2.12 (3H, d, J = 0.7 Hz), 2.24-2.35 (1H,), 2.39 ( HH, dd, J = 15.4, 3.0 Hz), 2.49 (HH, dd, J = 15.4, 9.8 Hz), 2.54-2.63 (HH,), 2.66-2.76 (HH, m), 2.97 (HH, dd, J = 16.2, 4.2 Hz), 3.03 (ÍH, dd, J = 16.3, 6.5 Hz), 3.10 (ÍH, quintet, = 6.8 Hz), 3.74 (1H, dd, J = 6.1, 3.5 Hz), 4.18 (2H, s), 4.34 (HH, dd, J = 9.8.2.9 Hz), 5.43 (1H, dd, J = 6.0.4.3 Hz), 5.55- 5.64 (HH, m), 5.67 (1H, dd, J = 15.9, 5.8 Hz), 6.24 (HH, brt, = 7.3 Hz), 6.66 (1H, s), 7.10 (HH, s); 13C NMR (100 MHz, CDCls) d 15.3, 16.1, 17.7, 18.2, 22.6, 28.7, 30.9, 39.4, 39.7, 43.9, 45.1, 53.8, 71.2, 75.3, 76.6, 116.8, 120.1, 124.2 [q, aJ (C, F) = 273.5 Hz], 127.8, 130.2 [q, 3J (C, F) = 6.1 Hz], 130.4 [q, 2J (C, F) = 28.6 Hz], 132.2, 136.6, 152.3, 170.1, 172.7, 218.4; LRMS (ESI) cale for C27H38F3N2O5S [M + H +] 559.2, find do 559.2; HRMS cale, for C27H38F3 2? 5S [M + H +] 559.2454 found 559.2440.

To a stirred solution of amine (15.9 mg, 28.5 μmol) in CH3CN (0.78 ml) was added HCHO (37% aqueous solution, 31.4 μl, 0.143 mmol) followed by NaBH3CN (1.0 M in THF, 85.5 μl, 85.5 μmol) . The mixture was stirred at room temperature for 20 minutes. AcOH (1 drop) was added, and the mixture was stirred for 40 minutes. The mixture is directly purified by preparative CCD (CH2Cl2 / MeOH = 100: 8) to give triethylamine (15.6 mg, 26.6 μmol, 93%) as a colorless powder. [a] D24-49.9 (c 0.74, CHC13); IR (film) v 3424, 2974, 1729, 1689, 1468, 1318, 1247, 1169, 1112, 754 cm "1; XE NMR (400 MHz, CDC13) d 1.05 (3H, s), 1.12 (3H, d, J = 6.9 Hz), 1.23 (3H, d, J = 6.8 Hz), 1.33 (3H, s), 2.17 (3H, s), 2.24-2.35 (HH, m), 2.43 (HH, dd, J = 15.7 , 3.6 Hz), 2.49 (1H, dd, J = 15.6, 9.1 Hz), 2.55-2.64 (2H,, which includes OH), 2.68-2.77 (ÍH, m), 2.80 (3H, s), 2.81 (3H , s), 2.92-3.06 (2H, m), 3.10 (ÍH, quintet, J = 6.8 Hz), 3.69-3.76 (1H, m), 4.25-4.34 (1H, m), 4.33 (2H, s), 5.42 (HH, t, J = 5.5 Hz), 5.57 (HH, dt, J = 15.8, 6.3 Hz), 5.66 (HH, dd, J = 15.7, 6.4 Hz), 6.22 (HH, brt, J = 7.2 Hz), 6.64 (1H, s), 7.30 (HH, s); 13C NMR (100 MHz, CDCI3) d 15.3, 15.8, 17.8, 18.8, 22.3, 28.8, 30.9, 39.6, 39.6, 45.2, 49.7, 49.7, 53.4, 61.5, 71.7, 75.4, 77.4, 119.2, 120.2, 124.2 [q, JJ (C, F ) = 273.5 Hz], 127.8, 129.9 [q, 3J (C, F) = 6.2 Hz], 130.7 [q, 2J (C, F) = 28.4 Hz], 132.4, 137.6, 154.2, 157.2, 170.0, 218.3; LRMS (ESI) cale, for C ^^ FsN-OsS [M + H +] 580.2, found 580.2. Melting points; both samples did not crystallize, but were purified by Si02. mp 90-94 ° C mp 78-81 ° C It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (56)

1. CLAIMS Having described the invention as above, the contents of the following claims are claimed as property: 1. Compound of the formula: characterized in that Ri is hydrogen or lower alkyl; R 2 is an unsubstituted or substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety; R5 and R6 are each independently hydrogen or a protecting group; X is O, S, C (R7) 2, 'or NR7, wherein each occurrence of R7 is independently hydrogen or lower alkyl; A-B represents CRA = CRB-, C (RA) 2-C (RB) 2-, or -C = C-; C-D represents -CRC = CRD-, -C (Rc) 2-C (RD) z ~, or -C = C-; wherein each occurrence of RA is independently hydrogen; halogen; -ORA; -MRS.; -N (RA.) 2; -C (0) ORA.; -C (0) RA.; -CONHRA; -0 (C = 0) RA «; ~ 0 (C = 0) ORA; -NRA (C = 0) RA-; N3; N2RA.; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -ORA-; -SRA >; -N (RA.) 2, * -C (0) ORA; -C (0) RA; -C0NHRA; - 0 (C = 0) RA >; -O (C = 0) 0RA '; -NRA (C = 0) RA >; N3; N2RA-; cyclic acetal; or aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; RB is, independently for each case, hydrogen; halogen; -0RB >; -SRB >; -N (RB.) 2; -C (0) 0RB <; -C (0) RB >; -CONHRB; - 0 (C = 0) RB .; -O (C = 0) 0RB; -NRBAC = 0) RB.; N3; N2RB; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -ORB-; -SRB >; -N (RB >) 2; - C (0) 0RB .; -C (0) RB >; -CONHRB; -0 (C = 0) RB .; -0 (C = 0) ORB; NRB > (C = 0) RB ',' N3; N2RB >; cyclic acetal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; Rc is, independently for each occurrence, hydrogen; halogen; -ORc; -SRC >; -N (RC.); -C (0) 0Rc >; -C (0) Rc >; -CONHRc; -0 (C = 0) Rc; -0 (C =?) ORc; -NRC (C = 0) Rc-; N3; N2Rc-; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RC-; -SRC <; -N (Rc-) 2; -C (0) ORc .; -C (0) Rc-; -CONHRc >; -0 (C = 0) Rc; -0 (C = 0) 0Rc-; -NRcAC = 0) Rc-; N3; N2Rc; cyclic acetal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; RD is, independently for each occurrence, hydrogen; halogen; -0RD.; -MR. D-; -N (RD.) 2; -C (0) ORD-; -C (0) RD .; -CONHRD ,; -0 (C = 0) R .; -0 (C = 0) 0RDA ANRD. (C = 0) RD.; N3; N2RD,; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -ORD.; -MR. D.; -N (RD-) 2; -C (0) 0RD .; -C (0) RD .; -CONHRD.; - 0 (C = 0) RD; -0 (C = 0) ORD.; -NRD- (C = 0) RD.; N3; N2RD.; cyclic acetal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; or wherein some two of RA, RB, Rc or RD taken together can form a cyclic portion and can be linked through an oxygen, sulfur, carbon or nitrogen atom, or some two adjacent groups RA, RB, Rc, or RD, taken together, can form an aliphatic, heteroaliphatic, aryl or heteroaryl-substituted or unsubstituted 3-6 membered ring; wherein each occurrence of RA-, RB-, He and RD- is independently hydrogen; a protective group; an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, or heteroarylalkyl, heteroarylalkenyl, linear or branched heteroarylalkyl, substituted or unsubstituted, cyclic or acyclic moiety; and pharmaceutically acceptable derivatives thereof.
2. 2. Compound of the formula: characterized in that X is O, S, C (R7) 2; or NR7, wherein each occurrence of R7 is independently hydrogen or lower alkyl; And it is O or S; R5 and R-6 are each independently hydrogen or a protecting group; Rs is independently hydrogen; halogen, -ORg, -SR9, -N (R9) 2, CZ3, -CHZ2, -CH2Z, where Z is F, Br, Cl, I, ORB-, NHRB., N (RB.) 2, or SRB; - (CV2) nOR9, - (CV2) nN (R9) 2, - (CV2) nSR9, - (C = 0) R9, -0 (C = 0) R9, - (C = 0) OR9, -0 ( C = 0) OR9; -NH (C = 0) R9, NH (C = 0) OR9, - (C = 0) NHR9, or an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, cyclic or acyclic, linear or branched, optionally substituted with one or more occurrence of halogen, -0R9, -SR9, -N (R9) 2, - (CV2) nOR9, - (CV2) nN (R9) 2, - (CV2) nSR9, - (C = 0) R9, ~ 0 (C = 0) R9, - (C = 0) OR9, -0 (C = 0) OR9; NH (C = 0) R9, ANH (C = 0) OR9, - (C = 0) NHR9, or an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, cyclic or acyclic, linear or branched, substituted or unsubstituted, wherein each occurrence of Rg is independently hydrogen; a protective group; an aliphatic, heteroaliphatic, aryl, heteroaryl, cyclic or acyclic, linear or branched, substituted or unsubstituted moiety; or is it a -epothilone, deoxyepotilone or analogues thereof; a polymer; carbohydrate; photoaffinity label; or radiolabel; wherein each occurrence of V is independently hydrogen, halogen, hydroxyl, thio, amino, alkylamino, or protected hydroxyl, thio or amino; each occurrence of t is independently 0, 1 or 2; and each occurrence of n is independently 0-10; A-B represents CRA = CRB-, C (RA) 2-C (RB) 2-, or -C = C-; C-D represents -CRC = CRD-, -C (Rc) 2-C (RD) z ~, or -C = C-; wherein each occurrence of RA is independently hydrogen; halogen; -ORA; -SRA >; -N (RA.) 2; -C (0) ORA .; -C (0) RA >; -CONHRA.; -O (C = 0) RA.; -O (C = 0) ORA-; -NRA (C = 0) RA.; N3; N2RA.; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RA; -MRS.; -N (RA >) 2; -C (0) 0RA .; -C (0) RA; -CONHRA; - 0 (C = 0) RA >; -0 (C = 0) ORA-; -NRA (C = 0) RA >; N3; N2RA-; cyclic acetal; or aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; RB is, independently for each case, hydrogen; halogen; -0RB .; -SRB .; -N (RB ') 2; -C (0) ORB >; -C (0) RB .; -CONHRB; - 0 (C = 0) RB .; -0 (C = 0) 0RB >; -NRB (C = 0) RB >; N3; 2RB '; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -ORB ?; -SRB-; -N (RB ') 2, "-C (0) ORB> -C (0) RB> -CONHRβ; -0 (C = 0) RB; -0 (C = 0) 0RB.; NRB. N3; N2RB '"cyclic acetal: aliphatic, heteroaliphatic, aryl or cyclic or acyclic, linear or branched, substituted or unsubstituted, Rc is, independently for each occurrence, hydrogen; halogen; -ORc; -SRc; -N (Rc02; -C (0) ORc > -C (0) Rc-; -CONHRc; -O (C = 0) Re; -O (C = 0) ORc; -NRC- (C = 0) Re; N3; N2Rc, cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RC-; -SRc »; -N (RC.) 2, * -C (0) ORc; -C (0) Rc-; -CONHRc .; - 0 (C = 0) Rc; -0 (C = 0) ORc; -NRc- (C = 0) Rc; N3; N2Rc; cyclic acetal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; RD is, independently for each occurrence, hydrogen; halogen; -0RD >; -SRD >; -N (RD.) 2; -C (0) ORD ', * -C (0) RD; -C0NHRD .; -0 (C = 0) RD »; -0 (C = 0) ORD; -NRD- (C = 0) RD.; N3; N2RD-; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -ORD .; -SRD >; -N (RD.) 2; -C (0) ORD «; -C (0) RD .; -CONHRD; - 0 (C = 0) RCD ', * -0 (C = 0) ORD-; -NRD. (C = 0) RD >; N3; N2RD «; cyclic ace.tal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; or wherein some two of RA, RB, Rc or RD taken together can form a cyclic portion and can be linked through an oxygen, sulfur, carbon or nitrogen atom, or some two adjacent groups RA, RB, Rc, or RD, taken together, can form a 3-6 membered aliphatic, heteroaliphatic, aryl or substituted or unsubstituted heteroaryl ring; where each occurrence of RA > , RB > , Re and R-D- is independently hydrogen; a protective group; an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, or heteroarylalkyl, heteroarylalkenyl, linear or branched heteroarylalkyl, substituted or unsubstituted, cyclic or acyclic moiety; and pharmaceutically acceptable derivatives thereof. Compound according to claim 2, characterized in that A-B is -CHC = C (RB) -. 4. Compound in accordance with the claim 2, characterized in that A-B is 5. Compound in accordance with the claim 2, characterized in that RB is methyl. 6. Compound according to claim 2, characterized in that RB is -CF3. Compound according to claim 2, "3 or 4, characterized in that Rs is methyl 8. Compound according to claim 2, 3 or 4, characterized in that R3 is -CH2OH 9. Compound in accordance with claim 2, 3 or 4, characterized in that Rs is -CH2NH2 10. Compound in accordance with the claim 2, 3 or 4, characterized in that C-D is where Z is 0, S, NRZ or C (Rz) 2, .where Rz is hydrogen, halogen, lower acyl, or lower alkyl. 11. Compound in accordance with the claim 10, characterized in that Z is NH. 12. Compound according to claim 10, characterized in that Z is CH2. 13. Compound according to claim 10, characterized in that Z is O. 14. Compound according to claim 10, characterized in that Z is S. 15. Compound in accordance with the claim 2, 3 or 4, characterized in that C-D is where Z is O, S, NRZ, or C (RZ) 2, wherein Rz is hydrogen, halogen, lower acyl, or lower alkyl. 16. Compound according to claim 15, characterized in that Z is NH. 17. Compound in accordance with the claim 15, characterized in that Z is CH2. Compound according to claim 2, characterized in that Y is S. 19. Compound according to claim 2, characterized in that Y is O. 20. Compound according to claim 2, 3 or 4, characterized in that CD is -C (Rc) 2 ~ C (RD) z ~ • 21. Compound according to claim 20, characterized in that each occurrence of Rc and RD is selected from the group consisting of hydrogen, lower alkyl, hydroxyl, halogen, or alkoxy lower. 22. The compound according to claim 20, characterized in that each occurrence of Rc and D is selected from the group consisting of hydrogen and methyl. 23. Compound according to claim 20, characterized in that each Rc is "methyl and each hydrogen RD 24. Compound according to claim 20, characterized in that each Rc is hydrogen and each RD is methyl. Claim 20, characterized in that one Rc is hydrogen and the other Rc is methyl 26. The compound according to claim 20, characterized in that one RD is hydrogen and the other RD is methyl 27. The compound according to claim 1 , characterized by is selected from the group consisting of the formulas: 28. Compound according to claim 1, characterized in that it is selected from the group consisting of the formulas: 29. Compound according to claim 1, characterized in that it is selected from the group consisting of the formulas: 30. Compound according to claim 1, characterized in that it is selected from the group consisting of the formulas: 31. Compound according to claim 1, characterized in that it is selected from the group consisting of the formulas: Compound according to claim 1, characterized in that it is selected from the group consisting of 25 25 33. Pharmaceutical composition for the treatment of cancer, characterized in that it comprises a compound according to claim 1 and a pharmaceutically acceptable excipient. 34. Pharmaceutical composition according to claim 33, characterized in that it additionally comprises Cremophor. 35. Pharmaceutical composition according to claim 33, characterized in that it additionally comprises Cremophor and ethanol. 36. Pharmaceutical composition according to claim 33, characterized in that the compound is suspended in 1: 1 Cremophor / EtOH. 37. Pharmaceutical composition according to claim 33, characterized in that it additionally comprises an additional cytotoxic agent. 38. Pharmaceutical composition for the treatment of cancer, characterized in that it comprises: a therapeutically effective amount of a compound according to claim 1, or pharmaceutically acceptable salts thereof.; and a pharmaceutically acceptable carrier or diluent, wherein the therapeutically effective amount of the compound is an amount sufficient to deliver about 0.001 to about 40 mg of compound per kg of body weight of a subject. 39. Pharmaceutical composition for the treatment of cancer, characterized in that it comprises a compound according to claim 1; and a pharmaceutically acceptable excipient; wherein the pharmaceutical composition is suitable for oral administration to a subject. 40. Pharmaceutical composition for the treatment of cancer, characterized in that it comprises a compound according to claim 2; and a pharmaceutically acceptable excipient; wherein the pharmaceutical composition is suitable for oral administration to a subject. 41. Pharmaceutical composition according to claim 40, characterized in that Re is -CH2OH. 42. Pharmaceutical composition according to claim 40, characterized in that RB is -CHF2, -CH2F, or CF3. 43. Pharmaceutical composition according to claim 40, characterized in that C-D is -CH = CH-, wherein the double bond is in the trans configuration. 44. Pharmaceutical composition according to claim 40, characterized in that the compound is of the formula: 45. Pharmaceutical composition according to claim 40, characterized in that the composition is in solid form. 46. Pharmaceutical composition according to claim 40, characterized in that the composition is in liquid form. 47. Method of cancer treatment, characterized in that it comprises: administering a therapeutically effective amount of a compound according to claim 1 to a subject in need thereof. 48. Method according to claim 47, characterized in that the therapeutically effective amount of the compound is an amount sufficient to deliver about 0.001 mg to about 40 mg of compound per kg of body weight. 49. Method according to claim 47, characterized in that the therapeutically effective amount of the compound is an amount sufficient to deliver about 0.1 mg to about 25 mg of compound per kg of body weight. 50. Cancer treatment method, characterized in that it comprises: orally administering a therapeutically effective amount of a compound of the formula: to a subject in the sameness of it. 51. Method for preparing a compound of the formula: wherein Ri is hydrogen or lower alkyl; R 2 is an unsubstituted or substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety; R5 and d are each independently hydrogen or a protecting group; X is O, S, C (R7) 2; or NR7, wherein each occurrence of R7 is independently hydrogen or lower alkyl; and RB is, independently for each case, hydrogen; halogen; -0RB .; -SRB .; -N (RB.) 2; -CY3, -CHY2, -CH2Y, where Y is F, Br, Cl, I, 0RB., NHRB-, N (RB-) 2, or S B-; -C (0) ORB .; -C (0) RB .; -CONHRß .; -0 (C = 0) RB .; -0 (C = 0) ORB; -NRB (C = 0) RB.; N3; N2RB-; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -ORB >; -SRB >; -N (RB.) 2; -C (0) ORB .; -C (0) RB .; -CONHRB; -0 (C = 0) RB-; -0 (C = 0) ORB; -NRB? (C = 0) RB .; N3; 2RB '; cyclic acetal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; or is an epothilone, deoxyepotilone, or analogs thereof; or is a polymer; carbohydrate; photoaffinity label; or radiolabel; where each occurrence of RB > is independently hydrogen; a protective group; an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalguyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, or heteroarylalkynyl, linear or branched, substituted or unsubstituted, cyclic or acyclic moiety; characterized in that it comprises isomerizing a compound of the formula: in the presence of a metal and a reducing people. 52. Method according to claim 51, characterized in that the metal is a transition metal. 53. Method according to claim 51, characterized in that the metal is palladium. 54. Method according to claim 51, characterized in that the reducing agent is selected from the group consisting of LiAlH ?, NaBH4, and NaBH3CN. 55. Method for preparing a compound of the formula: wherein Ri is hydrogen or lower alkyl; R 2 is an unsubstituted or substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety; R5 and R-6 are each independently hydrogen or a protecting group; X is O, S, C (R7) 2 * or NR7, wherein each occurrence of R7 is independently hydrogen or lower alkyl; and RB is, independently for each case, hydrogen; halogen; -ORB >; -SRB .; -N (RB.) 2; -CY3, -CHY2, -CH2Y, where Y is F, Br, Cl, I, 0RB., NHRB., N (RB.) 2, or SRB >; -C (0) ORB >; -C (0) RB .; -CONHRß-; -0 (C = 0) RB .; -0 (C = 0) 0RB; -NRB (C = 0) RB.; N3; N2RB; cyclic acetal; or aliphatic, heteroaliphatic, aryl, or heteroaryl, cyclic or acyclic, linear or branched, optionally substituted with one or more of hydrogen; halogen; -0RB <; -SRB >; -N (RB.) 2; -C (0) ORB .; -C (0) RB .; -CONHRB; -0 (C = 0) RB .; -0 (C = 0) ORB; -NRB > (C = 0) RB >; N3; N2RB >; cyclic acetal; aliphatic, heteroaliphatic, aryl or cyclic or acyclic heteroaryl, linear or branched, substituted or unsubstituted; or is an epothilone, deoxyepotilone, or analogs thereof; or is a polymer; carbohydrate; photoaffinity label; or radiolabel; where each occurrence of RB > is independently hydrogen; a protective group; an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, or heteroarylalkynyl, linear or branched, substituted or unsubstituted, cyclic or acyclic moiety; characterized in that it comprises reacting a compound of the formula: with a carbene or carbenoid reagent. 56. Method according to claim 55, characterized in that the carbene is CH2N2.