Background of the Invention Of great importance to man is the control of pathological cellular proliferation.

While certain methods and chemical compositions have been developed which aid in inhibiting, remitting, or controlling cellular proliferation, new methods and compositions are needed.

In searching for new natural product leads for potential biomedical applications, it has been found that some organisms are sources for chemical structures of great diversity and having useful biological activity. For example, the diterpene commonly known as taxol, isolated from several species of yew trees, is a mitotic spindle poison that stabilizes microtubules and inhibits their depolymerization to free tubulin (Fuchs, D. A. , R. K.

Johnson (1978) Cancer Treat. Rep. 62: 1219-1222; Schiff, P. B. , J. Fant, S. B. Horwitz (1979) Nature (London) 22: 665-667). Taxol is also known to have antitumor activity and has undergone a number of clinical trials which have shown it to be effective in the treatment of a wide range of cancers (Rowinski, E. K., R. C. Donehower (1995) N. Engl. J Med. 332: 1004-1014). See also, e. g., U. S. Patent Nos. 5,157, 049 ; 4,960, 790 ; and 4,206, 221.

Marine sponges have also proven to be an important source of biologically active compounds. A number of publications disclose organic compounds derived from marine sponges including Scheuer, P. J. (ed.) Marine Natural Products, Chemical and Biological Perspectives, Academic Press, New York, 1978-1983, Vol. I-V ; Uemura, D. , K.

Takahashi, T. Yamamoto, C. Katayama, J. Tanaka, Y. Okumura, Y. Hirata (1985) J Am.

Chem. Soc. 107: 4796-4798; Minale, L. et al. (1976) Fortschr. Chez. org. Naturst.

33: 1-72; Faulkner, D. J. (1998) Natural Products Reports 15: 113-158; Gunasekera, S. P., M. Gunasekera, R. E. Longley and G. K. Schulte (1990) J : Org Chem., 55: 4912-4915.

A prime target for the discovery and design of novel therapeutic agents against cancer is the mitotic apparatus of the cell and more specifically, microtubule assembly and its function (Wilson, L. (1975) "Microtubules as drug receptors: pharmacological properties of microtubule protein"Ann. N. Y. Acad. Sci. 253: 213-231). Ancillary functions of microtubules, including intracellular transport, signal transduction and the maintenance of cellular shape and motility are important factors which contribute to the overall growth of tumor cells and resulting metastases (Dustin, P. (1980) Sci. Am.

243: 66-76). Taxol is a microtubule interactive agent whose mechanism of action includes the premature polymerization of tubulin, resulting in hyperstable microtubule formation, blockage of cellular proliferation in the G2/M phase of the cell cycle, mitotic spindle disorganization and cell death. Additional compounds, which are chemically unrelated to taxol, are rapidly coming onto the scene which share a similar mechanism of action with taxol and are the subject of intense research into their potential as novel antitumor agents. These include the epothilones A and B, macrolides isolated from a myxobacterium, So°ahgiura cellulosum (Bollag, D. M. , P. A. McQueney, J. Zhu et al.

(1995) Cancer Res. 55: 2325-2333); eleutherobin, obtained from a marine soft coral (Lindel, T. , P. R. Jensen, W. Fenical et al. (1997) J. Am. Chem. Soc. 119: 8744-8745); laulimalide, isolated from a marine sponge (Mooberry, S. L. , G. Tien, A. H. Hernandez et al. (1999) Cancer Res. 59: 653-660); and discodermolide isolated from a marine sponge (Gunasekera, S. P. , M. Gunasekera, R. E. Longley (1990) J ; Org Chem. 55: 4912-4915 and Ter Haar E. , R. J. Kowalski, E. Hamel, et. al. (1996) Biochemist7y 3: 243-250). All of these compounds induce microtubule hyperstabilizing activity and are cytotoxic in vitro to tumor cells in the nanomolar range.

The success of chemotherapy for the treatment of various cancers can be substantially negated though cellular mechanisms which have evolved to enable neoplastic cells to subvert the cytotoxic effects of the drug. Some cells have developed mechanisms, which confer resistance to a number of structurally unrelated drugs. This multi-drug resistance (or MDR) phenomenon may arise through a number of different mechanisms. One of these involves the ability of a cell to reduce intracellular concentrations of a given drug through efflux from cytoplasm through and out the cell membrane by a series of unique ATP-dependent transporter proteins called-P-glycoproteins (Pgp) (Casazza, A. M. and C. R. Fairchild (1996) Cancer Treat Res. 87: 149-171). The surface membrane, 170 kDa Pgp, is encoded by the mdr-l gene and appears to require substrate binding before transport begins. A wide range of compounds. including a number of structurally-unrelated chemotherapeutic agents (adriamycin, vinblastine, colchicine, etoposide and taxol), are capable of being transported by Pgp and render the cell resistant to the cytotoxic effects of these compounds. While many normal cell types possess Pgp, in general, tumor cell lines, which possess high levels of mRNA specific for Pgp, also exhibit overexpression of membrane Pgp and demonstrate resistance to various drugs. This intrinsic resistance can be increased multifold by incubation of cells with stepwise increasing doses of a particular drug over a period of several months. This can be further facilitated by the addition of the MDR reversal agent, verapamil (Casazza, A. M. and C. R. Fairchild (1996) supra) in combination with the particular drug. Drug resistant cell lines produced in this fashion exhibit resistance to drug cytotoxicity from 20 to 500 fold, compared to parental cell lines.

An additional target for cancer drug discovery is a high molecular weight membrane protein associated with multi-drug resistance properties of certain tumor cells known as the multidrug resistance-associated protein (MRP). MRP is a 190 kD membrane-bound glycoprotein (Bellamy, W. T. (1996), Amnu. Rev. Pharmacol.

Toxicol., 36: 161-183) which belongs to the same family of proteins as the p-glycoprotein pump P-gp (Broxterman, H. J. , Giaccone, G. , and Lankelma, J. (1995), Cuvent Opinion in Oncology, 7: 532-540) but shares less than 15% homology of amino acids with P-gp (Komorov, P. G. , Shtil, A. A. , Holian, O., Tee, L. , Buckingham, L., Mechetner, E. B. , Roninson, I. B. , and Coon, J. S. (1998), Oncology Research, 10: 185-192). MRP has been found to occur naturally in a number of normal tissues, including liver, adrenal, testis, and peripheral blood mononuclear cells (Krishan, A. , Fitz, C. M. , and Andritsch, 1. (1997), Cytomet7y 29: 279-285). MRP has also been identified in tissues of the lung, kidney, colon, thyroid, urinary bladder, stomach, spleen (Sugawara 1. (1998) The Cancer Journal 8 (2) ) and skeletal muscle (Kruh, G. D. , Gaughan, K. T., Godwin, A. , and Chan, A. (1995) Journal of the Natioñãl~Cañcer Institute 87 (16) :- 1256-1258). High levels of MRP have been implicated in multidrug resistance (MDR) in cancers of the lung and pancreas (Miller, D. W. , Fontain, M., Kolar, C. , and Lawson, T.

(1996) Cancer Letters 107: 301-306), and in neuroblastomas, leukemias and cancer of the thyroid (Kruh, G. D., Gaughan, K. T. , Godwin, A. , and Chan, A. (1995) Journal of the National Cancer Institute 87 (16): 1256-1258), as well as bladder, ovarian and breast cancers (Barrand, M. , Bagrij, T. , and Neo, S. (1997) General Pharmacology 28 (5): 639-645). MRP-mediated MDR involves some of the same classes of compounds as those which are mediated by P-gp, including vinca alkaloid, epipodophyllotoxins, anthracyclins and actinomycin D (Barrand, M., Bagrij, T. , and Neo, S. (1997) General Pharmacology 28 (5): 639-645). However, the substrate specificity has been demonstrated to differ from that of P-gp (Komorov, P. G. , Shtil, A. A. , Holian, O., Tee, L. , Buckingham, L. , Mechetner, E. B. , Roninson, I. B. , and Coon, J. S. (1998) Oncology Research 10: 185-192). Drugs which would inhibit or which are not substrates for the MDR pump would, therefore, be useful as chemotherapeutic agents.

Some cancer cell lines, which have been induced to develop resistance to one type of microtubule interactive agent such as taxol, have been found to be sensitive to other types of microtubule agents. For example, the chemically unrelated compounds epothilones (A and B), which are isolated from a myxobacterium, Sorangium cellulosen and are composed of 16 membered macrolides (Bollag, D. M. , P. A. McQueney, J. Zhu et al. (1995) Cancer Res. 55: 2325-2333) enhance microtubule stability, block cells in the G2/M phase of the cell cycle and prevent microtubule depolymerization in cancer cells, similar to taxol. The epothilones also have a much greater cytotoxicity against p-glycoprotein expressing, multidrug resistant cells compared to non-multi-drug resistant cell lines.

Laulimalide and isolaulimalide, are two compounds which share taxol's microtubule-stabilizing activity (Mooberry, S. L. , G. Tien, A. H. Hernandez et al. (1999) Cancer Res. 59: 653-660), but are not chemically related to taxol. Laulimalide is a potent inhibitor of cellular proliferation with IC50 values in the low nanomolar range, whereas isolaulimalide is much less potent with ICso values in the low micromolar range. Both compounds inhibit cellular replication at the G2/M phase of the cell cycle. Laulimalide and isolaulimaIide inhibit~the~proliferation~of SKVLB-1 cells, a Pgp overexpressing multidrug-resistant cell line, again, suggesting that they are poor substrates for transport by Pgp.

Discodermolide, a compound derived from the marine sponge, Discodermia dissoluta (Gunasekera, S. P. , M. Gunasekera, R. E. Longley (1990) J. Org. Chem.

55: 4912-4915), is a potent inhibitor of cellular proliferation and has a similar mechanism of action to taxol. Discodermolide blocks cells in the G2/M phase of the cell cycle (Longley, R. E. , S. P. Gunasekera, D. Faherty et al. (1993) Immunosuppression by discodermolide. In : A. C. Allison ed. Annals of the New York Academy of Sciences Conference Proceedings, "Immunosuppressive and Anti-inflammatory Drugs"Vol. 696, April 12-15) and induces the hyperstabilization of microtubules in cells, leading to cell death (ter Harr, E., Kowalski et al. (1996) Biochemistry 35: 243-250). Discodermolide also inhibits the proliferation of Pgp overexpressing, multidrug-resistant cell lines (Kowalski, R. J. et al. (1997) Mol. Pharmacol. 52: 613-622).

The prevention and control of inflammation is also of great importance for the treatment of humans and animals. Much research has been devoted to development of compounds having anti-inflammatory properties. Certain methods and chemical compositions have been developed which aid in inhibiting or controlling inflammation, but additional anti-inflammatory methods and compositions are needed.

Immunomodulation is a developing segment of immunopharmacology.

Immunomodulator compounds and compositions, as the name implies, are useful for modulating or regulating immunological functions in animals. Immunomodulators may be immunostimulants for building up immunities to, or initiate healing from, certain diseases and disorders. Conversely, immunomodulators may be immunoinhibitors or immunosuppressors for preventing undesirable immune reactions of the body to foreign materials, or to prevent or ameliorate autoimmune reactions or diseases.

Immunomodulators have been found to be useful for treating systemic autoimmune diseases, such as lupus erythematosus and diabetes, as well as immunodeficiency diseases. Further, immunomodulators may be useful for immunotherapy of cancer or to prevent rejections of foreign organs or other tissues in transplants, e. g., kidney, heart, or bone marrow.

Various immunomodulator compounds have been discovered, including FK506, muramylic acid dipeptide derivatives, levamisole, niridazole, oxysuran, flagyl, and others from the groups of interferons, interleukins, leukotrienes, corticosteroids, and cyclosporins. Many of these compounds have been found, however, to have undesirable side effects and/or high toxicity. New immunomodulator compounds are therefore needed to provide a wider range of immunomodulator function.

Dictyostatin 1 is a macrolide of polyketide origin which was first reported by Pettit et al. [l] from a sponge of the genus Spongia collected in the Republic of the Maldives. U. S. Patent No. 5,430, 053pu (incorporated herein in its entirety by reference) describes the isolation and structure of dictyostatin 1 as well as its ability to inhibit the growth of various cancer cell lines in vitro. These reports do not disclose any utility for the compound against multi-drug resistant tumors in animals or humans or the ability of dictyostatin 1 to induce microtubule hyperstabilizing activity.

More recently, dictyostatin was isolated from a Caribbean sponge (Corallistidae <BR> <BR> <BR> sp. ) E2] and demonstrated to inhibit human cancer cell proliferation at nanomolar concentrations, retaining activity against multidrug-resistant cell lines and displaying a taxol-like mechanism of action, by binding to tubulin and promoting microtubule assembly. Dictyostatin now joins an elite group of microtubule-stabilising polyketides of marine origin that includes laulimalides, peloruside AE61 and discodermolide «, as natural product leads for the development of improved anti-cancer drugs (Myles, D. C. Myles (2001) Anne. Rep. Med. Chenu., 37: 125; and Altmann, K. -H. (2001) Cu77 ». Opin. Chem.

Biol. 5: 424). Use of dictyostatin to inhibit cellular proliferation, including in the case of multi-drug resistant cancer, has been described in U. S. Patent No. 6,576, 658.

Although dictyostatin has been previously reported, the correct stereochemistry of this product has not previously been determined. The planar structure of this unsaturated 22-membered macrolactone, featuring 11 stereogenic centers, an endocyclic (2Z, 4E)- dienoate and a pendant (Z) -diene moiety at C21, was deduced by the Pettit group primarily on the basis of 2D-NMR data. Structural similarities with discodermolide have been noted[4].<BR> <P> Unfortunately, evaluation of its antitumor, immunomodulator and other properties has been precluded so far by its very low natural abundance. It has also contributed to the difficulty in determining its relative and absolute configuration. Consequently, there is a need for an efficient process for the total synthesis of dictyostatin and for the relative and absolute configuration of this rare polyketide metabolite to be firmly established. A synthetic approach is disclosed in WO-A-2004/022552. Unfortunately, this was based on a wrong assignment of the relative and absolute configuration of dictyostatin and, furthermore, does not provide a high degree of flexibility that would enable the simple synthesis of a range of dictyostatin derivatives and analogues. Indeed, a particularly desirable aspect of any method for the total synthesis of dictyostatins is for it to be a modular synthetic approach that is flexible, highly convergent and stereocontrolled, which then offers the potential to provide useful quantities of dictyostatin, as well as a range of structural derivatives and analogues to initiate SAR studies.

Objects of the Invention It is one object of the present invention to provide a sterochemical characterisation of dictyostatin compounds.

It is a further object of the present invention to provide a method for the synthesis of dictyostatin as well as derivatives, analogues and salts thereof.

It is a further object of the present invention to provide novel dictyostatin derivatives and analogues thereof and uses therefor, including the control of cellular proliferation, cytotoxicity against human tumor cells resistant to chemotherapeutic agents, immunomodulation, and the control of inflammation (said uses arise from the role of dictyostatin compounds as tubulin polymerizers and microtubule stabilizers).

It is a further object of the present invention to provide substantially pure dictyostatin.

It is yet a further object of the present invention to provide intermediate compounds particularly useful in the synthesis of dictyostatin as well as derivatives, analogues and salts thereof.----- Summary of the Present Invention"" These and other objects are satisfied by the methods, compounds and intermediates of the present invention.

Thus, in a first aspect of the present invention there is provided dictyostatin 1 or a salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4. In particular, there is provided dictyostatin 1 having the following formula (If) or a salt, derivative or analogue thereof : (If) In a second aspect of the present invention there is provided a method for the synthesis of dictyostatin or an analogue or derivative thereof having the following formula (I) : wherein: each of Ra, Rb, RC, Rd, Re and Rf is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a hydroxy group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Ra and Rb together and/or Rc and Rd together and/or Re and Together represent a single bond, a group of formula-CH2-or a group of formula-0- ; each of Rg, Rh, R'and Ri is the same or different and is selected from the group consisting of a hydrogen atom, a hydroxy protecting group, an alkyl group, an aralkyl group, an aryl group and an acyl group ; Rk is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group ; RP and Rum are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, an alkoxy group, a haloalkoxy group and an aryloxy group, or RP and Rm together represent a single bond or a group of formula ; R"is selected from the group consisting of a hydrogen atom, an alkyl group (said alkyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkenyl group (said alkenyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkynyl group, a dienyl group and an aryl group ; R° is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group ; Rq is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rq and Rh together represent a single bond ; Rr and Rs are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, a hydroxy group, an alkenyl group, an alkynyl group, a hydroxyalkyl group, a haloalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rr and R'together represent a single bond or a group of formula ; Rt and Ru are the same or different and each is selected from the group consisting of a hydrogen atom, a hydroxy group, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rt and Ru together represent a single bond or a group of formula-CH2-or-O-, or le and Ru together with the 2 carbon atoms to which they are connected form an aryl group; each of RV, RW, RX, RY and RZ is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom, an aryl group or an alkyl group), an alkoxy group and a haloalkoxy group; and X represents a group of formula-0-, a group of formula-CH2-or a group of formula -N R'-, wherein R'is as defined above; or a salt thereof, said method comprising coupling units of the following formulae (II), (III), (IV) and (V) in any appropriate order, making any necessary changes to the functional groups of the intermediate obtained after each coupling step before performing the next coupling step, subjecting the resulting compound obtained by the coupling of said units of formulae (II), (III), (IV) and (V) to macrocyclisation and, if necessary, subjecting one or more of the functional groups of the resulting macrocyclised compound to one or more reactions to convert said group or groups to a different desired group or groups to give said compound of formula (I) : wherein: R1 is a hydrogen atom and R2 is selected from the group consisting of-O-Pl (wherein Pl represents a hydroxy protecting group), a leaving group and a group of formula -CH2-L (wherein L represents a leaving group), or R1 and R2 together represent a group of formula =0, =NR' (wherein R'is as defined above), =N-N (R') 2 (wherein each R'is the same or different and is as defined above), or a group of formula =CHM wherein M is selected from the group consisting of a halogen atom, a triflate, Li, Cu, Si (R') 3 (wherein each R'is the same or different and is as defined above), Sn (R') 3 (wherein each R'is the same or different and is as defined above), B (OR') 2 (wherein each R'is the same or different and is as defined above), MgR' (wherein R'is as defined above), Zn, Na and K, and R16 is selected from the group consisting of a hydrogen atom, an alkyl group, a chiral auxiliary group and a group of formula-CH2-P (R19) 3 wherein each R'9 is the same or different and is selected from the group consisting of=0, an aryl group, an alkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or RI, R and R16 together with the carbon atom to which they are attached represent an ethynyl group; represents a group selected from a hydroxy protecting group, an alkyl group, an aralkyl group and an aryl group; represents a group of forriiula-O-P2 (wherein P2 represents a hydroxy protecting group) or a group of formula P (R19) 3 wherein each R19 is the same or different and is as defined above, and RUZ represent a hydrogen atom, or R4 and R together represent a group of formula =O or a group of formula =CHM wherein M is as defined above, or a group of formula =CH-C (=O) (CH3), and Rls represents a hydrogen atom, or R4, Rs and R15 together with the carbon atom to which they are attached represent an ethynyl group; R6 is selected from the group consisting of a hydrogen atom, an alkyl group, a leaving group and a group of formula-CH2-P (R19) 3 wherein each R19 is the same or different and is as defined above, and R7 and R8 each represent a hydrogen atom, or R7 and R8 together represent a group of formula =O, =NR' (wherein R'is as defined above) or a dithiane group of formula -S- (CH2) nl-S- wherein n'= 3; R9 represents a group selected from a hydroxy protecting group, an alkyl group, an aralkyl group and an aryl group; R10 is a hydrogen atom, Rllis a group of formula-O-P3, wherein P3 represents a hydroxy protecting group and R12 is a formyl group, or R10 is selected from the group consisting of a hydrogen atom, a leaving group and a group of formula-CH2-P (R19) 3 wherein each Rl9 is the same or different and is as defined above, and Rll and R12 together represent a group of formula =O ; n2 is 0 or 1 (if it is 0, the place of the group in brackets is taken by a hydrogen atom); R13 is selected from the group consisting of a hydroxy protecting group, an alkyl group, an aralkyl group and an aryl group, and R13 is a hydrogen atom, or R13 and R13' together represent a single bond; R14 is a hydrogen atom or a carboxy protecting group; R17 and Rl8 are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rl7 and R'8 together represent a single bond or a group of formula-CH2-or-0-, or R17 and R18 together with the 2 carbon atoms to which they are connected form an aryl group ; Y is selected from the group consisting of a halogen atom, a group of formula Sn (R) s (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (R20) 3 (wherein each R20 is the same or different and is an alkyl group), and a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group; Z is selected from the group consisting of a group of formula Sn (R20)3 (wherein each R2o is the same or different and is an alkyl group), a halogen atom and a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group ; and Rk, Rn, R°, Rr, Rs, Rv, Rw, Rx, Ry and Rz are as defined above.

This method can, for example, be used for the total synthesis of dictyostatin of formula (If) or a salt thereof : (If) In a further aspect of the present invention, there is provided a compound having the following formula (Io) or a salt thereof : wherein: each of Ra, Rb, RC, Rd, Re and Rf is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group ; an alkoxyalkyl group ;-an aryl group, an-aralkyl group, a halogen atom, a hydroxy group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Ra and Rb together and/or R° and Rd together and/or Re and Together represent a single bond, a group of formula-CH2-or a group of formula-0- ; each of Rg, Rh, R'and Ri is the same or different and is selected from the group consisting of a hydrogen atom, a hydroxy protecting group, an alkyl group, an aralkyl group, an aryl group and an acyl group; Rk is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group; RP and R ! n are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, an alkoxy group, a haloalkoxy group and an aryloxy group, or RP and Rm together represent a single bond or a group of formula ; R° is selected from the group consisting of a hydrogen atom, an alkyl group (said alkyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkenyl group (said alkenyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkynyl group, a dienyl group and an aryl group ; R° is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group; Rq is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, R and Rs are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a hydroxy group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rr and Rs together represent a single bond or a group of formula ; Rt and Ru are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rt and Ru together represent a single bond or a group of formula-CH2-or-O-, or Rt and Ru together with the 2 carbon atoms to which they are connected form an aryl group; each of RV, RW, RX, RY and RZ is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom or an alkyl group), an alkoxy group and a haloalkoxy group; and X represents a group of formula-0-, a'group of formula-CH2-or a group of formula- NR', wherein R'is as defined above; or a salt thereof.

Further dictyostatins and derivatives, analogues and salts thereof are also provided, as is explained in greater detail below.

Intermediates particularly suitable for use in the methods of the present invention_ are also provided, such as the intermediates of formulae (In, (II), (IV) and (V) defined in the method of the present invention as defined above.

The method of the present invention also enables the production for the first time of dictyostatin of formula (If) above in high purity and confirms both the relative and absolute stereochemistry of dictyostatin, and this also forms another aspect of the present invention.

The present invention also provides a pharmaceutical composition comprising an effective amount of a pharmacologically active compound together with a carrier or diluent therefor, wherein said pharmacologically active compound is dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4.

The present invention also provides a pharmaceutical composition comprising an effective amount of a pharmacologically active compound together with a carrier or diluent therefor, wherein said pharmacologically active compound is a compound of formula (Io) or a pharmacologically acceptable salt thereof as defined above.

The present invention also provides a pharmaceutical composition comprising an effective amount of a pharmacologically active compound together with a carrier or diluent therefor, wherein said pharmacologically active compound is substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined above.

The present invention also provides a method for the prophylaxis or treatment of cancer, said method comprising administering an effective amount of a cytotoxic compound to a patient suffering from said cancer, wherein said cytotoxic compound is dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4.

The present invention also provides a method for the prophylaxis or treatment of cancer, said method comprising administering an effective amount of a cytotoxic compound to a patient suffering from said cancer, wherein said cytotoxic compound is a compound of formula (Io) or a pharmacologically acceptable salt as defined above.

The present invention also provides a method for the prophylaxis or treatment of cancer, said method comprising administering an effective amount of a cytotoxic compound to a patient suffering from said cancer, wherein said cytotoxic compound is substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined above.

The present invention also provides a method for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules, said method comprising administering to a patient in need thereof a pharmacologically effective amount of a compound of formula (Io) or a pharmacologically acceptable salt as defined above.

The present invention also provides a method for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules, said method comprising administering to a patient in need thereof a pharmacologically effective amount of dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4.

The present invention also provides a method for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules, said method comprising administering to a patient in need thereof a pharmacologically effective amount of substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined above.

The present invention also provides use of dictyostatin 1 or a pharmacologically acceptable'salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4 in the manufacture of a medicament for the prophylaxis or treatment of cancer.

The present invention also provides use of a compound of formula (Io) or a pharmacologically acceptable salt as defined above in the manufacture of a medicament for the prophylaxis or treatment of cancer.

The present invention also provides use of substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined above in the manufacture of a medicament for the prophylaxis or treatment of cancer.

The present invention also provides use of dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4 in the manufacture of a medicament for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules.

The present invention also provides use of a compound of formula (Io) or a pharmacologically acceptable salt as defined above in the manufacture of a medicament for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules.

The present invention also provides use of substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined above in the manufacture of a medicament for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules.

Detailed Description of the Invention The first aspect of the present invention provides dictyostatin 1 or a salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4. This first aspect of the invention provides the first full stereochemical characterization of dictyostatin compounds. Methods of use for these compounds are also provided, including the control of cellular proliferation, cytotoxicity against human tumor cells resistant to chemotherapeutic agents, immunomodulation, and the control of inflammation (said uses arise from the role of dictyostatin compounds as tubulin polymerizers and microtubule stabilizers).

The relative stereochemistry of the 22-membered marine macrolide dictyostatin, a taxol-like antimitotic agent, was determined based on a combination of extensive high field NMR studies, including J-based contiguration analysis, and molecular modeling.

The dictyostatin class of compounds can be isolated from Dictyoceratid sponges of the genus Spongia as well as from a lithistid sponge of the family Corallistidae. See U. S. Patent No. 6, 576, 658, which is incorporated herein, in its entirety, by reference.

Specifically exemplified herein in the first aspect of the present invention is dictyostatin 1 or a salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4. In particular, there is provided dictyostatin 1 having formula (If) or a salt, derivative or analogue thereof.

There is also provided the use of dictyostatin 1 or a salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and in particular dictyostatin 1 having formula (If) or a salt, derivative or analogue thereof, for immunomodulation, control of inflammation, stabilization of microtubules, induction of polymerization of tubulin, inhibiting cellular proliferation, and/or inhibiting cellular proliferation of multi-drug resistant tumor cells.

As used in this application, the terms"analogues"and"derivatives"refer to compounds which are substantially the same as another compound but which may have been modified by, for example, adding side groups, oxidation or reduction of the parent structure. Salts are also within the scope of the present invention. Analogues or derivatives of the exemplified compounds can be readily prepared using commonly known standard reactions. These standard reactions include, but are not limited to, hydrogenation, alkylation, acetylation, and acidification reactions.

The subject invention particularly contemplates-analogues of dictyostatin 1 having the stereochemical structure shown in Figures 1 through 4, and in particular dictyostatin 1 having formula (If), that retain the stereochemistry reported herein. Thus, one aspect of the subject invention provides analogues of dictyostatin 1 having the stereochemical structure shown in Figures 1 through 4, and in particular dictyostatin 1 having formula (If), that retain the stereochemistry in any, or all, points in the molecule but differ from natural dictyostation by one or more additions or deletions of side groups.

A further aspect of the subject invention is the use of the stereochemistry reported herein, in conjunction with similar information with respect to taxol and/or discodermolide to provide analogues that have the desired anti-proliferative activity.

In further preferred embodiments of the invention, salts within the scope of the invention are made by adding mineral acids, e. g., HC1, H2S04, or strong organic acids, e. g., formic, oxalic, in appropriate amounts to form the acid addition salt of the parent compound or its derivative. Further suitable salts are detailed below in relation to the compounds of formula (I).

As stated above, the full stereochemical assignment for the antimitotic macrolide dictyostatin is proposed as (If) (2Z, 4E, 6R, 7S, 9S, 12S, 13R, 14S, 16S, 19R, 20S, 21S, 22S, 23Z), based on the results of extensive high field NMR studies, including J-based configuration analysis, and molecular modelling. This assignment is consistent with a common biogenesis for discodermolide and suggests that both these sponge-derived polyketides interact in a similar fashion (Shin, N. Choy, R. Balachandran, C. Madiraju, B. W. Day and D. P. Curran (2002) Org Lett. 4: 4443) with the taxol binding site on tubulin.

Unfortunately, evaluation of the antitumor, immunomodulator and other properties of dictyostatin has been precluded so far by its very low natural abundance, and it has also contributed to the difficulty in determining its relative and absolute configuration.

Consequently, there is a need for an efficient process for the total synthesis of dictyostatin and for confirmation of the relative and absolute configuration as discussed above to be confirmed.

Thus, in a second aspect of the present invention there is provided a method for the synthesis of dictyostatin or an analogue or derivative thereof having the following formula (I) : wherein: each of Ra, Rb, RC, Rd, Re and Rf is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a hydroxy group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Ra and Rb together and/or Rc and Rd together and/or Re and Together represent a single bond, a group of formula-CH2-or a group of formula-0- ; each of Rg, Rh, Ri and Rj is the same or different and is selected from the group consisting of a hydrogen atom, a hydroxy protecting group, an alkyl group, an aralkyl group, an aryl group and an acyl group ; Rk is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group; RP and Rm are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, an alkoxy group, a haloalkoxy group and an aryloxy group, or RP and Rm together represent a single bond or a group of formula-CH2-or ; Rn is selected from the group consisting of a hydrogen atom, an alkyl group (said alkyl group being optionally substituted with at least one substituent selected from the group- consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkenyl group (said alkenyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkynyl group, a dienyl group and an aryl group; R° is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group; Rq is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rq and Rh together represent a single bond; Rr and Rs are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, a hydroxy group, an alkenyl group, an alkynyl group, a hydroxyalkyl group, a haloalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rr and Rs together represent a single bond or a group of formula-CH2-or-O-; Rt and Ru are the same or different and each is selected from the group consisting of a hydrogen atom, a hydroxy group, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rt and Ru together represent a single bond or a group of formula-CH2-or-O-, or Rt and Ru together with the 2 carbon atoms to which they are connected form an aryl group; each of RV, R9W, RX, RY and RZ is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom, an aryl group or an alkyl group), an alkoxy group and a haloalkoxy group; and--- X represents a group of formula-0-, a group of formula WH2-or a group of formula -NR'-, wherein R'is as defined above ; or a salt thereof, said method comprising coupling units of the following formulae (II), (III), (IV) and (V) in any appropriate order, making any necessary changes to the functional groups of the intermediate obtained after each coupling step before performing the next coupling step, subjecting the resulting compound obtained by the coupling of said units of formulae (II), (III), (IV) and (V) to macrocyclisation and, if necessary, subjecting one or more of the functional groups of the resulting macrocyclised compound to one or more reactions to convert said group or groups to a different desired group or groups to give said compound of formula (I): wherein: Rl is a hydrogen atom and R2 is selected from the group consisting of 4 pl (wherein Pl represents a hydroxy protecting group), a leaving group and a group of formula - CH2-L (wherein L represents a leaving group), or R1 and R2 together represent a group of formula =O, =NR' (wherein R'is as defined above), =N-N (R') 2 (wherein each R'is the same or different and is as defined above), or a group of formula =CHM wherein M is selected from the group consisting of a halogen atom, a triflate, Li, Cu, Si (R') 3 (wherein each R'is the same or different and is as defined above), Sn (R') 3 (wherein each R'is the same or different and is as defined above), B (OR') 2 (wherein each R'is the same or different and is as defined above), MgR' (wherein R'is as defined above), Zn, Na and K, and RI6 is selected from the group consisting of a hydrogen atom, an alkyl group, a chiral auxiliary group and a group of formula-CH2-P (RI9) 3 wherein each Rl9 is the same or different and is selected from the group consisting of=0, an aryl group, an alkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or R', R2 and R16 together with the carbon atom to which they are attached represent an ethynyl group ; R3 represents a group selected from a hydroxy protecting group, an alkyl group, an aralkyl group and an aryl group ; R4 represents a group of formula-O-P2 (wherein p2 represents a hydroxy protecting group) or a group of formula P (Rt9) 3 wherein each Rl9 is the same or different and is as defined above, and RS represent a hydrogen atom, or R4 and Rs together represent a group of formula =O or a group of formula =CHM wherein M is as defined above, or a group of formula =CH-C (=O) (CH3), and Rl5 represents a hydrogen atom, or R4, R and Rls together with the carbon atom to which they are attached represent an ethynyl group ; R6 is selected from the group consisting of a hydrogen atom, an alkyl group, a leaving group and a group of formula-CH2-P (RI9) 3 wherein each Rl9 is the same or different and is as defined above, and R7 and R each represent a hydrogen atom, or R7 and R8 together represent a group of formula =O, =NR' (wherein R'is as defined above) or a dithiane group of formula -S-(CH2)n1-S- wherein n1 = 3; R9 represents a group selected from a hydroxy protecting group, an alkyl group, an aralkyl group and an aryl group ; Rl° is a hydrogen atom, R11 is a group of formula-0-P3, wherein P3 represents a hydroxy protecting group and R12 is a formyl group, or Rl° is selected from the group consisting of a hydrogen atom, a leaving group and a group of formula-CH2-P (R19) 3 wherein each R19 is the same or different and is as defined above, and Ri 1 and R12 together represent a group of formula =0 ; n2 is 0 or 1 (if it is 0, the place of the group in brackets is taken by a hydrogen atom) ; R13 is selected from the group consisting of a hydroxy protecting group, an alkyl group, an aralkyl group and an aryl group, and R13' is a hydrogen atom, or R13 and R13 together represent a single bond; R14 is a hydrogen atom or a carboxy protecting group ; R17 and R18 are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rl7 and Rl8 together represent a single bond or a group of formula-CH2-or-0-, or R17 and R18 together with the 2 carbon atoms to which they are connected form an aryl group; Y is selected from the group consisting of a halogen atom, a group of formula Sn (R2°) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (R20) 3 (wherein each R20 is the same or different and is an alkyl group), and a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group ; Z is selected from the group consisting of a group of formula Sn (R20) 3 (wherein each R20 is the same or different and is an alkyl group), a halogen atom and a group of formula B (OR21) 2 wherein each Ral is the same or different and is an alkyl group or an aryl group; and Rk, Rn, R°, Rr, Rs, Rv, RW, R", Ry and RZ are as defined above.

This method can, for example, be used for the total synthesis of dictyostatin of formula (If) or a salt thereof : The synthesis of said dictyostain of formula (If) and comparison of the NMR data obtained therefor with that of naturally obtained dictyostatin 1 have enabled the inventors to confirm that naturally obtained dictyostatin 1 does indeed have the stereochemistry shown in formula (If). Furthermore, it has enabled the inventors to show that the absolute configuration of dictyostatin 1 is (-) -dictyostatin of formula (If), and (-)- dictyostatin-of formula (I) or-a salt, analogue or derivative thereof also form a part of the present invention.

The alkyl groups in the definitions of substituents Ra, Rb, RO, Rd, Re, Rf, Rg, Rh, Ri, Rj, Rk, Rp, Rm, Rn, Ro, Rq, Rr, Rs, Rt, Ru, Rv, Rw, Rx, Ry, Rz, R', R", R3, R6, R9, R13, R16, R17, R18, R19, R20 and R21 above are straight or branched alkyl groups having from 1 to 6 carbon atoms. Examples of said lower alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2- methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2, 3-dimethylbutyl and 2-ethylbutyl groups. Alkyl groups having from 1 to 4 carbon atoms are preferred, methyl, ethyl, propyl, isopropyl and butyl groups are more preferred, and methyl, ethyl and propyl groups are most preferred. For RV, RW, RX, RY and Rz, methyl groups are particularly preferred.

The alkenyl groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rk, RP, Rm, Rn, R°, Rq, Rr, Rs, Rt, Ru, R", RW, R", Ry, RZ, Rl and Rl8 above are straight or branched alkenyl groups having from 2 to 6 carbon atoms. Examples of said alkenyl groups include vinyl, 2-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 2-butenyl, 1- methyl-2-butenyl, 2-methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2- pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4- pentenyl, 2-methyl-4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl groups.

Alkenyl groups having from 2 to 4 carbon atoms are preferred, and alkenyl groups having 2 or 3 carbon atoms are most preferred.

The alkynyl groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rk, Rp, Rm, Rn, Ro, Rq, Rr, Rs, Rt, Ru, R, RW, RX, Ry, RZ, Rl7 and Rl8 above are straight or branched alkynyl groups having from 2 to 6 carbon atoms. Examples of said alkynyl groups include ethynyl, 2-propynyl, 1-methyl-2-propynyl, 2-butynyl, 1-methyl-2-butynyl, 1-ethyl-2-- butynyl, 3-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-ethyl-3-butynyl, 2- pentynyl, 1-methyl-2-pentynyl, 3-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 4- pentynyl, 1-methyl-4-pentynyl, 2-methyl-4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl groups. Alkynyl groups having from 2 to 4 carbon atoms are preferred, and alkynyl groups having 2 or 3 carbon atoms are most preferred.

The dienyl group in the definition of is a straight or branched dienyl group having from 4 to 10 carbon atoms. Preferred are groups having from 4 to 6 carbon atoms and 1,3-butadienyl groups are particularly preferred.

The halogen atoms in the definitions of Ra, Rb, RC, Rd, Re, Rf, RP, Rm, Rn, RV, RW, RX, RY, RZ, Y and Z above include fluorine, chlorine, bromine and iodine atoms.

The haloalkyl groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rk, Rp, Rm, Ro, Rq, Rr, Rs, Rt, Ru, Rv, Rw, Rx, Ry, Rz, R17 and Rl8 above are alkyl groups as defined and exemplified above which are substituted with at least one halogen atom as exemplified above. It is preferably a straight or branched halogenoalkyl group having from 1 to 4 carbon atoms, examples of which include a trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl, 2,2, 2-trichloroethyl, 2, 2, 2-trifluoroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl and 2,2-dibromoethyl groups.

The alkoxy groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rp, Rm, Rn, Rq, Rr, Rs, Rt, RU, RV, RW, Rx, RY, Rz, R17, R18and R19 above are alkyl groups as defined and exemplified above which are bonded to an oxygen atom. The alkoxy groups are preferably straight or branched alkoxy groups having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy and butoxy groups.

The haloalkoxy groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rp, Rm, Rq, Rr, Rs, Rt, Ru, RV, RW, RX, Ry, RZ, R 17, and Rl9 above are alkoxy groups as defined above that are substituted with at least one halogen atom as exemplified above. The haloalkoxy groups preferably have from 1 to 4 carbon atoms, such as difluoromethoxy, trifluoromethoxy and 2, 2, 2-trifluoroethoxy groups.

The alkoxyalkyl groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rk, Rp, Rm, Ro, Rq, Rr, Rs, Rt, Ru, Rv, Rw, Rx, Ry, Rz, R17 and Rl8 above are alkyl groups as defined and exemplified above that are substituted with at least one alkoxy group as defined and exemplified above. Methoxymethyl, ethoxyethyl groups, groups, 1-methoxyethyl groups and 1-ethoxyethyl groups are particularly preferred.

The aryl groups in the definitions of Ra, Rb, RC, Rd, Re, Rf, Rg, Rh, Ri, Ri, Rk, RP, R, Rn, Ro, Rq, Rr, Rs, Rt, Ru, Rv, Rw, Rx, Ry, Rz, R3, R9, R13, R17, R18, R19 and R21 above are aryl groups that may optionally be substituted with at least one substituent selected from the group consisting ot an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom or an alkyl group), an alkoxy group and a haloalkoxy group, said aryl groups being aromatic hydrocarbon groups having from 6 to 14 carbon atoms in one or more rings, preferably from 6 to 10 carbon atoms, and examples include phenyl, naphthyl, phenanthryl and anthracenyl groups. Of these, we prefer phenyl and naphthyl groups, most preferably phenyl groups.

The aryl groups defined and exemplified above may be fused with a cycloalkyl group having from 3 to 10 carbon atoms. Examples of such a fused ring group include 5- indanyl groups.

Where the aryl groups are substituted, we prefer those that are substituted with from 1 to 4 substituents selected from the group above. Examples of such substituted aryl groups include 4-fluorophenyl, 3-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 3,4- difluorophenyl, 3,4-dichlorophenyl, 3,4, 5-trifluorophenyl, 3-chloro-4-fluorophenyl, 3- difluoromethoxyphenyl, 3-trifluoromethoxyphenyl and 3-trifluoromethylphenyl.

The aralkyl groups in the definitions of Ra, Rb, RC, Rd, Re, Rf, Rg, Rh, Ri, Ri, Rk, Rp, Rm, Ro, Rq, Rr, Rs, Rt, Ru, Rv, Rw, Rx, Ry, Rz, R3, R9, R13, R17 and R18 above are alkyl groups as defined above that are substituted by at least one aryl group as defined and exemplified above. Examples of such an aralkyl group include benzyl, indenylmethyl, phenanthrylmethyl, anthrylmethyl, a-naphthylmethyl, (3-naphthylmethyl, diphenylmethyl, n-iphenylmethyla-naphthyldiphenylmethyl, 9-antbrylmethyl, piperonyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2- phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1- phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2- naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3- phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3- naphthylpentyl, 4-naphthylpentyl, 5-naphthylpenthyl, 1-phenylhexyl, 2-phenylhexyl, 3- phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylhexyl, 2- naphthylhexyl, 3-naphthylhexyl, 4-naphthylhexyl, 5-naphthylhexyl and 6-naphthylhexyl.

Of these, benzyl, phenanthrylmethyl, anthrylmethyl, a-naphthylmethyl, (3--_ naphthylmethyl, diphenylmethyl, triphenylmethyl, 9-anthrylmethyl, piperonyl, 1- phenethyl, 2-phenethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl and 4-phenylbutyl are preferred.

The aryloxy groups in the definitions of Ra, Rb, Rc, Rd, Re, Rf, Rp, Rm, Rq, Rr, Rs, Rt, RU, Rl7, Rl8 and Rl9 above are aryl groups as defined and exemplified above that are bonded to an oxygen atom. Examples include phenoxy, naphthyloxy, phenanthryloxy and anthracenyloxy groups.

The acyl groups in the definitions of Rg, Rh, Rl, Ri and Rn are selected from aliphatic acyl groups, aromatic acyl groups and alkoxycarbonyl groups as defined and exemplified below: (i) aliphatic acyl groups, examples of which include alkylcarbonyl groups having from 1 to 25 carbon atoms, examples of which include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methyl- pentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl and heneicosanoyl groups, halogenated alkylcarbonyl groups having from 1 to 25 carbons in which the alkyl moiety thereof is substituted by at least one halogen atom, examples of which include chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl groups, alkoxyalkylcarbonyl groups which comprise an alkylcarbonyl group having from 1 to 25 carbon atoms in which the alkyl moiety thereof is substituted with at least one alkoxy group as defined above, examples of said alkoxyalkylcarbonyl groups including methoxyacetyl groups, and--- unsaturated alkylcarbonyl groups having from 1 to 25 carbon atoms, examples of which include acryloyl, propioloyl, methacryloyl, crotonoyl, isocrotonoyl and (E)-2- methyl-2-butenoyl groups; of these, alkylcarbonyl groups having from 1 to 6 carbon atoms are preferred and acetyl groups are particularly preferred ; (ii) aromatic acyl groups, examples of which include arylcarbonyl groups which comprise a carbonyl group which is substituted with an aryl group as defined above, examples of which include benzoyl, a-naphthoyl and P-naphthoyl groups, halogenated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one halogen atom, examples of which include 2-bromobenzoyl, 4-chlorobenzoyl and 2,4, 6-trifluorobenzoyl groups, alkylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one alkyl group as defined above, examples of which include 2,4, 6-trimethylbenzoyl and 4-toluoyl groups, alkoxylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one alkoxy group as defined above, examples of which include 4-anisoyl groups, nitrated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one nitro group, examples of which include 4- nitrobenzoyl and 2-nitrobenzoyl groups, alkoxycarbonylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with a carbonyl group which is itself substituted with an alkoxy group as defined above, examples of which include 2- (methoxycarbonyl) benzoyl groups, and- arylated arylcarbonyl groups which comprise an arylcarboriyl group as defined---- above which is substituted with at least one aryl group as defined above, examples of which include 4-phenylbenzoyl groups; (iii) alkoxycarbonyl groups, examples of which include alkoxycarbonyl groups which comprise a carbonyl group substituted with an- alkoxy group as defined above, examples of which include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, s-butoxy-carbonyl, t-butoxycarbonyl and isobutoxycarbonyl groups, and alkoxycarbonyl groups as defined above which are substituted with at least one substituent selected from the group consisting of halogen atoms and trialkylsilyl groups (wherein said alkyl groups are as defined above), examples of which include 2,2, 2- trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl groups.

The hydroxy protecting groups in the definitions of Rg, Rh, R', R, P1, p2, P3, R3, R9 and Rl3 above are not particularly limited provided that they can protect a hydroxy group in a reaction. Such a protecting group is a group which can be removed by a chemical reaction such as hydrogenolysis, hydrolysis, electrolysis and photolysis and may be, for example, an aliphatic acyl group, examples of which include an alkylcarbonyl group such as a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a pivaloyl group, a valeryl group or an isovaleryl group, an alkoxyalkylcarbonyl group such as a methoxyacetyl group, or an unsaturated alkylcarbonyl group such as an (E)-2-methyl-2-butenoyl group ; an aromatic acyl group, examples of which include an arylcarbonyl group such as a benzoyl group, an a- naphthoyl group or a p-naphthoyl group, a halogenated arylcarbonyl group such as a 2- bromobenzoyl group or a 4-chlorobenzoyl group, an alkylated arylcarbonyl group such as a 2,4, 6-trimethylbenzoyl group or a 4-toluoyl group, an alkoxy arylcarbonyl group such as an 4-anisoyl group, a nitrated arylcarbonyl group such as a 4-nitrobenzoyl group or a 2-nitrobenzoyl group, an alkoxycarbonyl arylcarbonyl group such as a 2- (methoxycarbonyl) benzoyl group or an arylated arylcarbonyl group such as a 4- phenylbenzoyl group ; an alkyl group as defined above such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group or a t-butyl group; an unsaturated alkyl group such as a vinyl group, an allyl group or a 2-butenyl group; an alkoxyalkyl group as defined above such as a methoxymethyl group and an ethoxyethyl group; a tetrahydropyranyl group; a tetrahydrofuranyl group; an alkoxyalkoxyalkyl group such as a methoxyethoxymethyl group; a benzyl group optionally substituted with an alkyl group as defined above, an alkoxy group as defined above or a halogen atom such as a benzyl group, a 4-methylbenzyl group, a 4-methoxybenzyl group or a 4-chlorobenzyl group; and a tri-substituted silyl group substituted with an alkyl group as defined above or a phenyl group such as a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group or a triphenylsilyl group. Protection of a hydroxy group with the above described protecting groups or removal of these protecting groups can be accomplished according to standard methods (described in, for example, T. H. Green et al., Protective Groups in Organic Synthesis, JOHN WILEY & SONS, INC.).

The carboxy protecting group in the definition of R14 above are not particularly limited provided that they can protect a carboxy group in a reaction. Such a protecting group is a group which can be removed by a chemical reaction such as hydrogenolysis, hydrolysis, electrolysis and photolysis and may be, for example, an alkyl group as defined and exemplified above; an alkenyl group as defined and exemplified above; an alkynyl group as defined and exemplified above; a haloalkyl group as defined and exemplified above; a hydroxyalkyl group such as a 2-hydroxyethyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, 3,4-dihydroxybutyl or 4-hydroxybutyl group; an"aliphatic acyl"-"alkyl group"such as an acetylmethyl group; an aralkyl group as defined and exemplified above; and a tri-substituted silyl group substituted with an alkyl group as defined above or a phenyl group such as a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group or a triphenylsilyl group.

The leaving groups in the definitions of L and Rl° are not particularly limited provided that they can be substituted in a reaction by a nucleophilic reagent. Suitable leaving groups include, for example, a hydroxy group; a halogen atom as described above; an alkylsulfonyloxy group such as a methanesulfonyloxy group or an ethanesulfonyloxy group; a halogenated alkylsulfonyloxy group such as a trifluoromethanesulfonyloxy group; an aromatic sulfonyloxy group, which is for example, an arylsulfonyloxy group such as a benzenesulfonyloxy group, an alkylated arylsulfonyloxy group such as a p-toluenesulfonyloxy group, or a halogenated arylsulfonyloxy group such as a p-chlorobenzenesulfonyloxy group. Preferably, the leaving group L in the definitions of L and Rl° is a halogen atom, a trifluoromethanesulfonyloxy group (a triflate group) or a methanesulfonyloxy group (a mesylate group).

Where the compounds of formula (I) of the present invention have an acidic or basic group, they can form a salt and these are also encompassed within the scope of the present invention. Where the compound of formula (I) is to be used as an anti-cancer agent, any such salt should be a pharmacologically acceptable salt.

Preferred examples of the salt formed with an acidic group include alkali metal salts such as a sodium salt, potassium salt or lithium salt, alkaline earth metal salts such as a calcium salt or magnesium salt, metal salts such as an aluminum salt or iron salt; amine salts, e. g. , inorganic salts such as an ammonium salt and organic salts such as a t- octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'- dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N- benzylphenethylamine salt, piperazine salt, tetramethylammonium salt or tris (hydroxymethyl) aminomethane salt; and amino acid salts such as a glycine salt, lysine salt, arginine salt, ornithine salt, glutamate or aspartate.

Preferred examples of the salt formed with a basic group include hydro-halides such as a hydrofluoride, hydrochloride, hydrobromide or hydroiodide, inorganic acid salts such as a nitrate, perchlorate, sulfate or phosphate; lower alkanesulfonates such as a methanesulfonate, trifluoromethanesulfonate or ethanesulfonate, arylsulfonates such as a benzenesulfonate or p-toluenesulfonate, organic acid salts such as an acetate, malate, fumarate, succinate, citrate, ascorbate, tartrate, oxalate or maleate; and amino acid salts such as a glycine salt, lysine salt, arginine salt, ornithine salt, glutamate or aspartate.

If the compounds of the present invention are allowed to stand in the atmosphere for some time, they sometimes absorb water to form a hydrate. Such a hydrate is also embraced in the present invention.

The compounds of the present invention such as those of formulae (I), (II), (III), (IV) and (V) contain a number of asymmetric centres, and can therefore form optical isomers (including diastereoisomers). For the broadest definitions of the compounds of the present invention, such as in formulae (I), (II), (III), (IV) and (V), each of said isomers and mixtures of said isomers are depicted by a single formula. Accordingly, the present invention covers both the individual isomers and mixtures thereof in any proportion, including racemic mixtures.--- (1) The method for the synthesis of dictyostatin or a derivative thereof having the formula (I) or a salt thereof as defined above is particularly effective for the synthesis of the desired dictyostatins and analogues and derivatives thereof. Preferred examples of the method of the present invention include the following: (2) A method according to (1), wherein said compound of formula (I) is a compound of formula (Ia) or a salt thereof : (Ia) (3) A method according to (1), wherein said compound of formula (I) is a compound of formula (Ib) or a salt thereof : (Ib) (4) A method according to (1), wherein said compound of formula (I) is a compound of formula (Ic) or a salt thereof : (5) A method according to (1), wherein said compound of formula (I) is a compound of formula (Id) or a salt thereof : (Id) (6) A method according to any one of (1) to (5), wherein Rk is a methyl group.

(7) A method according to any one of (1) to (6), wherein each of RP and Rm represents a hydrogen atom.

(8) A method according to (1), for the synthesis of dictyostatin having the following formula (Ie) or a salt thereof : (9) A method according to (1), for the synthesis of dictyostatin having the following formula (If) or a salt thereof : (It (10) A particularly preferred method according to (1), comprises the following steps: (a) coupling of a compound of formula (IIa) and a compound of formula (IIIa) wherein each of said groups of formula R19a are the same or different and represents an alkoxy group or a haloalkoxy group, to give a compound of formula (VI) : (b) converting the double bond of the enone of said compound of formula (VI) to a single bond and reducing the carbonyl group thereof to a protected hydroxyl group of formula _op4 to give a group of formula (VII) : (c) deprotecting the group-OP2 to give a hydroxyl group and then oxidising the resulting hydroxyl group to give a compound of formula (VIII) : (d) coupling the compound of formula (VIII) with a compound of formula (IVa): wherein Y1 is a halogen atom, a group of formula Sn(R20)@ 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (R2°) 3 (wherein each Ro is the same or different and is an alkyl group), or a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group, to give a compound of formula (IX): (e) coupling said compound of formula (IX) with a compound of formula (Va): to give a compound of formula (X): (X) (f) subjecting said compound of formula (X) to a macrolactonisation reaction to give a compound of formula (XI): (XI) (g) reducing the ketone group at the C-9 position to a hydroxyl group to give a compound of formula (Ig) or a salt thereof : (h) and, if desired, removing any protecting groups R3, R13 and P4 from said compound of formula (Ig) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, R', Ri and Rh respectively to give a compound of formula (Ih) or a salt thereof : () h) In method (10) above, P4 is a hydroxy-protecting group and is not particularly limited provided that it can protect a hydroxy group in a reaction. Such a protecting group is a group which can be removed by a chemical reaction such as hydrogenolysis, hydrolysis, electrolysis and photolysis. Examples of such a protecting group are as given for protecting groups pl, P2 and P3 above. All other groups are as defined and exemplified previously. Preferred methods according to method (10) include: (11) A method according to (10), wherein said coupling step (a) is performed under Horner-Wadsworth-Emmons olefination conditions.

(12) A method according to (10) or (11), wherein said coupling step (d) is performed under Still-Gennari Horner-Wadsworth-Emmons olefination conditions, the group of formula P (R19) 3 in said compound of formula (IVa) being a group of formula P (O) (ORt9a) 2 wherein each of said groups of formula OR' is the same or different and represent an alkoxy group, an aryloxy group or a haloalkoxy group (preferably a 2,2, 2- trifluoroethoxy group).

(13) A method according to any one of (10) to (12), wherein said coupling step (e) is performed under Liebeskind or Stille coupling conditions, the substituent RI4 being a trialkylsilyl group (preferably a triisopropylsilyl group) and each substituent Ro being an n-butyl group in said compound of formula (Va).

(14) A method according to any one of (10) to (13), wherein said macrolactonisation step (f) is performed under Yamaguchi conditions.

(15) A method according to any one of (10) to (14), wherein the reduction of the C-9 carbonyl group is conducted under Luche conditions using sodium borohydride and caesium trichloride.

(16) A method according to any one of (10) to (15) for the synthesis of a compound of formula (Ih) or a salt thereof, wherein each of R°, Rr and R ! is the same or different and is a hydrogen atom or an alkyl group (preferably each is a hydrogen atom).

(17) A method according to any one of (10) to (16) for the synthesis of a compound of formula (Ih) or a salt thereof, wherein each of Rk, RV, RW, RX, RY and RZ is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group and an aralkyl group.

(18) A method according to (17) for the synthesis of a compound of formula (Ih) or a salt thereof, wherein each of Rk, RV, RW, RX, RY and RZ is the same or different and is an alkyl group.

(19) A method according to (18) for the synthesis of a compound of formula (Ih) or a- salt thereof, wherein each of Rk, RV, RW, RX, RY and Ra is a methyl group.

(20) A method according to any one of (10) to (19) for the synthesis of a compound of formula (Ih) or a salt thereof, wherein each of Rg, Rh, R'and Ri is the same or different and is selected the group-consisting of a hydrogen-atom, an~alkyl group, an aralkyl group, an aryl group, an alkylcarbonyl group, a haloalkylcarbonyl group, an arylcarbonyl group and a trialkylsilyl group.

(21) A method according to (20) for the synthesis of a compound of formula (Ih) or a salt thereof, wherein each of Rg, Rh, R'and Ri is a hydrogen atom.

(22) A method according to any one of (10) to (21) for the synthesis of a compound of formula (Ih) or a salt thereof, wherein each of Rl7 and Rl8 is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an aralkyl group or an aryl group, or Rl7 and Rl8 together with the 2 carbon atoms to which they are connected form an aryl group.

(23) A method according to (22) for the synthesis of a compound of formula (Ih), wherein each of R17 and R'8 is a hydrogen atom.

(24) A method according to (10) for the synthesis of a compound of formula (Ii) or a salt thereof : (25) A method according to (10) for the synthesis of a compound of formula (Ij) or a salt thereof : (Ij) (26) A method according to (10) for the synthesis of a compound of formula (If) or a salt thereof : (H) The approach adopted in the method of (10) to (26) above is outlined retrosynthetically in Schemes 1 to 5 below, using the compound of formula (If) as an example. The late stage reduction of the enone 2 is controlled by the macrocyclic conformation adopted by the 22-membered ring.

Scheme 1 Retrosynthetic analysis for dictyostatin (If). OH OH d 0 z // OH OH n : Dictyostatin TBS 0 TBS,, '.,' Yamaguchi macrolactonization Slõ+SS 10 9/ ; / Still-Gennari < HWE olefination 2, Liebeskind-Stille coupling 9 TIPSO 0 (F3CH2co) 2, p, + j TIPSO¢O (FsCHCOP-YY'+ f O O OTBS Bu3Sn 3'C. ¢-Cosubunit 5 HWE olefination \ TBS 26 19 TBSO,, nsrren ne H 11 oh 0 4 C1 t-C26 subunif O Myers (MeO) 2P"-o alkylation 16 0 10 0 TBSO,,, + +"ff" , +.B OPMB PMBO 6'C11-c17 subunit 7 C18-C26 subunit PMBO OH OH 8 The (lOZ)-olefin in 2'is produced via a coupling reaction, preferably a Still- Gennari-type olefination between the ß-Keto phosphonate 3'and aldehyde 4' ; in conjunction with further coupling reaction, preferably a Stille cross-coupling using vinyl stannane 5 to deliver the (2Z, 4E) -dienoate. In principle, either of these two reactions could be employed to close the macrocycle, as an alternative to a more conventional Yamaguchi-macrolactonization,---offering considerable flexibility-in _ the-synthesis endgame. In turn, the Cji-C26 subunit 4'is accessible via a coupling reaction, preferably a Horner-Wadsworth-Emmons (HWE) coupling, between aldehyde 6'and phosphonate 7, containing the terminal (Z) -diene. As these latter segments share a common stereochemical triad, it was anticipated that both 6 and 7 could be prepared from common intermediate 8, available in multigram quantities using efficient aldol methodology reported previously. 181 Scheme 2 Synthesis of C11-C26 subunit 4'.

In the above, TBS is a tert-butyldimethylsilyl group and PMB is a p- methoxybenzyl group Synthesis of the Cll-C26 subunit 4', as shown in Scheme 2, commences with conversion of 1,3-diol 8 into bis-TBS ether 9 and liberation of the primary hydroxyl group under mildly acidic conditions. The resulting alcohol 10 is readily converted into <BR> <BR> <BR> iodide 11 via a modified Mitsunobu protocol (PPh3, Is, Imid. ). E93 Subsequent alkylation of the lithium enolate of Myers' propionamide 12[10] effects a three carbon homologation, delivering 13 with excellent diastereoselectivity and yield (19 : 1 dr, 88%). Reductive removal of the pseudoephedrine auxiliary (LiNH2BH3) affords a primary alcohol which is, in turn, oxidized with Dess-Martin periodinane to provide aldehyde 6'. The synthesis of HWE coupling-partner-7 is achieved by addition of (MeO) 2P (O) CH2Li to aldehyde 14, prepared from 1,3-diol 8 as described previously, [9,12] delivering, after careful work-up, an epimeric mixture of alcohols, which is converted into ß-keto phosphonate 7 (Dess-Martin periodinane, 83% over 2 steps). Following a HWE coupling between aldehyde 6'and phosphonate 7, employing Ba (OH) 2 in wet THF, enone 15 is isolated in 92% yield. With the entire Cll-C26 backbone assembled, stereocontrolled reduction of the Cl9 ketone directed by the proximal hydroxyl-bearing stereocenter using metal chelation is required. To this end, conjugate reduction of enone 15 using Stryker's reagent {[Ph3PCuH]6}[14] and oxidative removal of both PMB groups with DDQ (2,3- dichloro-5, 6-dicyano-1, 4-benzoquinone) provides the intermediate (3-hydroxy ketone, which undergoes 1,3-syn selective reduction when treated with a cold (-30 °C) ethereal solution of Zn (BH4) 2 (151 generating triol 16 (>20: 1 dr). [161 Finally, the synthesis of subunit 4'is completed by a 3-step sequence involving selective TBS protection of the Cl, and Ci9 hydroxyl groups over that at 21 (TBSOTf), cleavage (TBAF, AcOH) of the primary TBS ether in 17 and oxidation of resulting alcohol 18 (TEMPO/PhI (OAc) 2). [l71 Scheme 3 Synthesis of C4-C10 phosphonate 3'.

As the primary objective in this example of synthetic method (1) is realizing an efficient and convergent synthesis of dictyostatin (If), it is crucial to install the (1OZ)- olefin employing advanced coupling partners. While a number of protocols exist to effect such coupling, most require strongly basic conditions. There are obviously concerns regarding the epimerizable center at C12 in aldehyde 4', which led to the investigation of the mild Still-Gennari modification of the HWE olefination. 1181 The requisite CF3CH20-substituted phosphonate 3 was prepared, as detailed in Scheme 3, starting from alcohol 19, obtained in 95% ee and 20: 1 dr through a Brown asymmetric crotylation of aldehyde 20. 1"1 Silyl ether formation (TBSOTf), ozonolysis and Takai methylenation [201 of the intermediate aldehyde provides (E) -vinyl iodide 21 in 71% yield.

After selective cleavage of the primary silyl ether, oxidation of the resulting alcohol affords acid 22 which is transformed into its acid chloride using the Ghosez reagent (Me2C=C (Cl) NMe2). 12'1 Next, (F3CCH20) 2P (O) CH2Li, [223 generated at-100 °C, is added to the acid chloride affording phosphonate 3 containing the required functionality for the pivotal fragment assembly (Scheme 4).

Scheme 4 Synthesis of seco-acid 26. 3'4' a 77%, 2 steps } Z : E=5 : 1 Z : E = 5 : 1 TBS 0 TBSO,, OH I O OTBS 23 TIPS02C d H02C d Bu3Sn 90% Bu3Sn Bu3Sn s 5 24 TBS u TBSO,,, HO HO COR II l O OTBS c 25 : R = TIPS 83%, 2 steps 26 : R = H When a mixture of aldehyde 4'and phosphonate 3'is subjected to an excess of K2C03 in the presence of 18-crown-6 in toluene, E181 the HWE coupling proceeds smoothly producing (Z) -enone 23 efficiently, with good levels of selectivity (Z: E = 5 : 1, 77%). Notably, this constitutes one of the first examples of a (Z) -selective intermolecular Still-Gennari olefination employing such an elaborate 3-keto phosphonate. Construction of the dictyostatin carbon backbone is completed through a copper-mediated, Liebeskind- type Stille coupling, [23] using copper (1) thiophenecarboxylate (CuTC) in 1-methyl-2- pyrrolidinone (NMP), between vinyl iodide 23 and (Z)-vinyl stannane 5, readily accessible from known acid 24. E241 Finally, conversion of TIPS ester 25 into acid 26 is achieved (KF, THF/MeOH) in 83% overall yield.

Scheme 5 Completion of the synthesis of dictyostatin (If). 26 77% | a TBS O,/ TBSO. TBS0", 0 O O g O OTBS 2' 70% I b i. R o e RO,,, ß 0s<0 O O // OH OR CL27 : R=TBS 87% (M : Dictyostatin, R = H 87 A) With 26 in hand, completion of the synthesis is achieved via a macrolactonizaton reaction, as shown in Scheme 5 above. Seco-acid 26 is subjected to a standard Yamaguchi macrolactonization protocol (2,4, 6-trichlorobenzoyl chloride, NEt3, DMAP, PhMe, 60 °C, 2 h) to furnish the 22-membered macrocycle 2 cleanly in 77% yield. [25] The reduction of the Cg-ketone is achieved by exploiting macrocyclic stereocontrol. E8) Luche reduction of enone 2 (NaBH4, CeCl3, EtOH)[26] provides the expected alcohol 27 (70%). Finally, global deprotection is achieved with 3N HCI in methanol to give dictyostatin (If) in 87%-yield. The spectroscopic data of the synthetic material are in complete agreement with those of an authentic sample obtained from the Corallistidae sponge source (700 MHz 1H and 125 MHz 13C NMR, MS), while the specific rotation of [a] D =-32.7 (c = 0.22, McOH) agrees with that measured in our laboratory for the authentic sample, [273 thus allowing confident assignment of the absolute configuration of dictyostatin as shown in (If).

This expedient total synthesis of (-)-dictyostatin unequivocally establishes the relative and absolute configuration as shown in (If).

Method (10) is highly flexible and allows functional group modifications at various points in the synthesis, allowing access to further dictyostatin derivatives and analogues. Examples include the following: (27) A method according to any one of (10) to (26), wherein one or more of the double bonds in said compound of formula (If), (Ig), (Ih), (Ii) or (Ij) is reduced to give a single bond or bonds. Examples include those set out in Scheme 6 below: Scheme 6 OH OH OH HO JJ hydroxy-directed HOJ, % ,,/u hydrogenation 0 0 0 0 1 y v V. Y. v v OH OH-OH OH dictyostatin (Ifl 10, 11-dihydro dictyostatin conjugate reduction conjugate reduction if OH OH HO,,, hydroxy-directed HO,,, hydrogenation 0 0 0 0 _ OH OH OH OH 2, 3, 4, 5-tetrahydro dictyostatin 2, 3, 4, 5, 10, 11-hexahydro dictyostatin hydrogenation OH HO,,, 0 0 I OH OH (28) A method according to any one of (10) to (27), wherein one or more of the hydroxyl groups in said compound of formula (If), (Ig), (Ih), (li) or (Ij) is oxidised to give one or more carbonyl groups. Examples include those illustrated in Scheme 7 below: Scheme 7 OFi, s R7 R8,/ 16v 19 HO., 4 .,, 2 , R5R6 12 O O chemoselective i and/ non-selective OH OH oxidation dictyostatin (It R1 = H ; R2 = OH or Ri = R2 = 0 R4=H ; R3=OHorR4=R3=O R6=H ; R5=OHorR6=R5=O R7=H ; R8=OHorR7=Rs=0 OH,/OH,/ 16 19 v Mn02 v v "i 4 i, 21 is, 8yIC "i, 'i, , i 12 oxidation) (. O O '1 _ Y vv OH OH dictyostatin (if (29) A method according to any one of (10) to (28), wherein one or more of the double bonds in said compound of formula (If), (Ig), (Ih), (li) or (Ij) is subjected to epoxidation or cyclopropanation to give a compound of formula (I) wherein one or more of Ra and Rub together and/or R and Rd together and/or Re and Rftogether represents a group of formula-CH2-or a group of formula-0-. Examples include those illustrated in Scheme 8 below: Scheme 8 Oh-, *- OH OH 21, hou If/I'll, epoxidation 124 0 0 1 cyclopropanation _ OH OH OH OH dictyostatin (If) X = O, CH2 (30) A method according to any one of (10) to (29), wherein one or more hydroxyl groups in said compound of formula (If), (Ig), (Ih), (li) or (Ij) or in intermediates in said method are subjected to etherification or esterification to give a compound of formula (I) having one or more etherified or esterified groups. Examples include those illustrated in Scheme 9 below: Scheme 9 OP,/OP,/ conjugate PO. <% reduction % O O O O _ _ I 9-., 5 . ___ _ _ . 0 OP 0 OP o op o op advanced intermediate from | NaBH4, CeCI3 total synthesis of dictyostatin (2 in Scheme 5) OH OP OH o'op lo, O O O O _ //// OH OH OH OP A'-dihydro dictyostatin) etherification or esterification OH OP HOJ k POJ % 0 0 HCI, MEOH 0 0 w erv OR OH OR OP R = alkyl, benzyl, acyl R = alkyl, benzyl, acyl Cg-ethers and esters of dictyostatin Scheme 9 (Continued) H O H O 'O'alkylation---- R = silyl protecting group or alkyl group OP OP a similar sequence of reactions to that reported in the total synthesis of dictyostatin Y H H (RO) z. P/ ici O O OR OP R = alcohol protecting group R = silyl protecting group or coupling partner C alkyl group coupling partner B a similar sequence of reactions to that reported in the total synthesis alkylation of dictyostatin OP OH OP OR (RO) 2s p vI O O OR R = silyl protecting group or alkyl group coupling partner A Scheme 9 (continued) Using coup ! ring partners A B and C and either protecting C19 alcohol after reduction or alkylating C19 alcohol after reduction and/or alkylating C9 alcohol or not alkylating C9 alcohol after reduction-a total of 24 ethers of dictyostatin may be prepared selectively alkyl, benzyl, acyl etc (31) A method according to (10), wherein the intermediate of formula (XI): (Xi) is subjected to a conjugate addition reaction with a compound of formula RalMl wherein Ra1 is selected from the group consisting of an alkyl group, an aryl group and an aralkyl group as defined and exemplified above and Ml is selected from copper and magnesium to give an intermediate of formula (all) : (XII) the C-9 ketone of said intermediate of formula (XII) then being reduced to give a compound of formula (Ik) or a salt thereof : (Ik) and, if desired, removing any protecting groups R3, R13 and P4 from said compound of formula (Ik) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, R', Ri and Rh respectively to give further compounds of formula (I) or salts thereof. Examples include those illustrated in Scheme 10 below: Scheme 10 OP,/OP,/ 19 ou conjugate p0... 11, I'll, addition 12 0 0 Ra 0 Ra O O -..... O OP O OP advanced intermediate from total synthesis of dictyostatin (2 from Scheme 5) 1. NaBH4 2. HCI, MeOH Oh HO," al Ra1 O O OH OH 11-substituted 10, 11-dihydro dictyostatin (32) A method according to (10), wherein the intermediate of formula (XI) : is subjected to a 1,2 nucleophilic reaction with a compound of formula RqlMl, wherein Rql is selected from the group consisting of an alkyl group, an aryl group and an aralkyl group as defined and exemplified above and Ml is selected from lithium, sodium, magnesium and potassium, to give a compound of formula (I1) or a salt thereof : (ici) and, if desired, removing any protecting groups R3, R13 and P4 from said compound of formula (Il) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, Ri, Ri and Rh respectively to give further compounds of formula (I) or salts thereof. Examples include those illustrated in Scheme 11 below: Scheme 11 OP h-õto PW, n, 21, 12 p O I 9 _ 5/ 12 0 0 0 OP advanced intermediate from total synthesis of dictyostatin (2 from Scheme 5) 1, 2-addition of nucleophiles Rq1M1 in presence of CeCI3 OP PO., P 0 Rqe OH OP HCI, MeOH if OH HO,,, 0 0 Rq1H Ru1 _ OH OH 9-substituted dictyostatin (33) A method according to (10), wherein in step (b), instead of reducing the double bond of the enone of the intermediate of formula (VI) to a single bond it is subjected to a conjugate addition reaction with a compound of formula Rp1M2, wherein Rp1 is selected from the group consisting of an alkyl group, an aryl group and an aralkyl group as defined and exemplified above and M is selected from copper and magnesium, before reducing the carbonyl group to a protected hydroxy group, to give an intermediate of formula (VIIa) : said intermediate of formula (VIIa) then being subjected to the remaining steps (c) to (h) of (10) to give a compound of formula (Im) or a salt thereof : (Im) Examples include those illustrated in Scheme 12 below: Scheme 12 /conjugate addition I 1 PO 0 OH RM' PO R 0 6H OU similar sequence of reactions to that used in the total synthesis of dictyostatin OH H HO",/"", R _"" 8 _ 0se0 O + OH OH Further examples of the method (1) include the following: (34) A method according to (1) above comprising the following steps: (a) subjecting an intermediate of formula (VIII) as defined in (10) above to a Stork- Wittig reaction to give the following intermediate of formula (XIII) : wherein Hal is a halogen atom (preferably an iodine atom) ; (b) coupling the compound of formula (XIII) with a compound of formula (IVg) : wherein y2 is a halogen atom, a group of formula Sn (R20) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si(R20) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group, a group of formula CHCHC02R14 or a group of formula CCC02R14 and R°, RV, R13 and Rl4 are as defined in (1) above, to give a compound of formula (XIV) : (c) where y2 is a halogen atom, coupling said compound of formula (XIV) with a compound of formula (Va) as defined in (10) : to give a compound of formula (XV): (XV) (d) subjecting said compound of formula (XV) or said compound of formula (XIV) wherein y2 is a group of formula CHCHCO2R14 or a group of formula CCCO2RI4 to a macrolactonisation reaction to give a compound of formula (Ig) : (e) and, if desired, removing any protecting groups R3, R13 and P4 from said compound of formula (Ig) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, Ri, Ri and Rh respectively to give further compounds of formula (Ih) or a salt thereof : (Ih) Examples of method (34) include those illustrated in Scheme 13 below: Scheme 13 // i i v y y v gtork-Witti-' OP OH 9 P'i's,, OP OH PO., .,, H O intermediate in dictyostatin synthesis (4 in Scheme 1) 1. Nozaki-Kishi coupling (with or without chiral catalyst) or lithium iodine exchange followed by transmetallation with Mg, Ce, K, Na, Cu 2-H y 0 OP O OP Y = halogen, CHCHCO2R, CCCO2R, etc OH i OH//pp OH HO,,, f PO,, J 5p 5H O O /Y similar sequence OH OP OH OH of reactions to that used in the total synthesis of dictyostatin (35) A method according to (1) above comprising the following steps: (a) subjecting an intermediate of formula (VIII) as defined in (10) above to a reaction to convert the aldehyde group thereof to an alkynyl group to give the following intermediate of formula (XVI) : (b) converting said compound of formula (XVI) to the metallated alkynyl derivative thereof and reacting this with a compound of formula (IVb) : wherein Ll is a leaving group (preferably a halogen atom, an alkoxy group, a triflate, a mesylate or a tosylate), Y2 is a halogen atom, a group of formula Sn (R2°) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (fez) 3 (wherein each Wo is the same or different and is an alkyl group), a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group, a group of formula CHCHC02R14 or a group of formula CCCO2RI4 and R°, RV, R13 and R14 are as defined in (1) above, to give a compound of formula (XVII) : (XVII) (c) hydrogenating the triple bond group to a double bond to give a compound of formula (XVIII) : (XVIII) (d) where Y2 is a halogen atom, coupling said compound of formula (XVIII) with a compound of formula (Va) as defined in (10) above: (Va) to give a compound of formula M as defined in (10) above ; (e) subjecting said compound of formula (X) or said compound of formula (XVIII) wherein Y2 is a group of formula CHCHCo2R94 or a group of formula CCC02R14 to a macrolactonisation reaction to give a compound of formula (XI) as defined in (10) above ; (f) subjecting said compound of formula (XI) to step (g) as defined in (10) above to give a compound of formula (Ig) or a salt thereof as defined in (10) above and, if desired, removing any protecting groups R3, Rl3 and P4 from said compound of formula (Ig) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, R', Ri and Rh respectively to give a compound of formula (Ih) or a salt thereof as defined in (10) above.

Examples of method (35) include those illustrated in Scheme 14 below: Scheme 14 /Corey-Fuchs or other means po, OP OH POJ OP OH------- OP OH of transforming H alehyde to alkyne 0 intermediate in dictyostatin synthesis 1. RM (R = H, alkyl, aryl, M = Na, Li, (4 in Scheme 1 above) K, Mg) also transmetallation may provide M = Ce, Cu, etc. 2-L1 Y2 2 O OP M f L1 = halogen or alkoxy y2 = halogen, CHCHCo2R14, CCCo2R14 // OH Po", 0""ÕP ÕH Po", 0""ÕP ÕH . hydrogenation il ty2 over Lindlar wy2 O OP O OP 0 OP 0 OP similar sequence of reactions to that used in the total synthesis of dictyostatin OH,/ HO,,, O O / OH OH (36) A method according to (1) above, comprising the following steps: (a) reacting a compound of formula (IX) as defined in (10) above: with a compound of formula (Va) as defined in (10) above: (Va) under Yamaguchi esterification conditions to give a compound of formula (XIX): (XIX) (b) subjecting said compound of formula (XIX) to a coupling reaction (preferably a Stille coupling or a coupling using copper I thiophenecarboxylate) to give a compound of formula (XI) as defined in (10) above; and (c) subjecting said compound of formula (XI) to step (g) of (10) above to give a compound of formula (Ig) or a salt thereof and, if desired, subjecting said compound of formula (Ig) to step (h) of (10) above to give a compound of formula (Ih) or a salt thereof. Examples of method (36) include those illustrated in Scheme 15 below: Scheme 15 i o i Sn tohu OP OH SnR2o>3 POs si, > 20 _ Yamaguchi/9 O Sn (R) 3 esterification t1 +1 O OP O OP Stille intermediate in dictyostatin synthesis coupling (23 in Scheme 4) or CuTC coupling OP ou Viz HO,,, 0 0 0 0 milarsequence of reactions to that used in the total 0 OP synthesis of OH OH dictyostatin (37) A method according to (1) above comprising the following steps: (a) reacting a compound of formula (VII) as defined in (10) above with a compound of formula (Vb) under Yamaguchi esterification conditions: (Vb) wherein Rl7 and Rl8 are as defined in (1) above and zl is a halogen atom or a group of formula Sn (R2°) 3 wherein R2° is as defined in (1) above, to give a compound of formula (XX): (b) selectively removing the protecting group p2 and oxidising the resulting hydroxy group to give a compound of formula (XXI) : (XXI) (c) coupling said compound of formula (XXI) with a compound of formula (IVb) : wherein Y2 represents a halogen atom or a group of formula Sn (leo) 3 wherein R20 is as defined in (1) above, to give a compound of formula (XXII) : (XXII) (d) subjecting said compound of formula (XXII) to a coupling reaction (preferably a Stille coupling or a coupling using copper I thiophenecarboxylate) to give a compound of formula (XI) as defined in (10) above; and (e) subjecting said compound of formula (XI) to step (g) of (10) above to give a compound of formula (Ig) or a salt thereof and, if desired, subjecting said compound of formula (Ig) to step (g) of (10) above to give a compound of formula (Ih) or a salt thereof.

Examples of method (37) include those illustrated in Scheme 16 below: Scheme 16 0 OH v y y v I = _ Zl 0 zi Yamaguchi OP esterification op OP intermediate in dictyostatin synthesis 1. selective (17 in Scheme 2) deprotection 2. oxidation 000, // vvvv _ p0.,.,, _.-_ _ PO,,,, op 01, Op on _ H 0 z o z, 0 Y2 (RO) 21 O OP p O OP Stille coupling or CuTC coupling OH ou oh POI,, HO,,, similar sequence of reactions to that 0 O used in the total synthesis of O OP dictyostatin E) H OH OH OH (38) A method according to (1) above, comprising the following steps: (a) deprotecting the OP2 group of a compound of formula (VII) as defined in (10) above, and converting the resulting hydroxyl group to give a compound of formula (XXIII) wherein said group Q is a group of formula P (R19b)3+I-, wherein each Rl9b is the same or different and is an aryl group as defined and exemplified above, or to a group of formula P (O) (OR19c)2 wherein each Rl9c is the same or different and is an alkyl group, a haloalkyl group or an aryl group as defined and exemplified above: (b) coupling said compound of formula (XXIII) with a compound of formula (IVc) under Wittig conditions: wherein p5 is a hydrogen atom or a hydroxy protecting group as defined and exemplified above, Hal is a halogen atom and R°, R and R13 are as defined in (1) above, to give a compound of formula (XXIV) : (c) subjecting said compound of formula (XXIV) to steps (e) and (f) as defined in (10) above to give a compound of formula (Ig) or a salt thereof and, if desired, subjecting said compound of formula (Ig) to step (h) as defined in (10) above to give a compound of formula (Ih).

Examples of method (38) include those illustrated in Scheme 17 below: Scheme 17 i i OH - = ='Ph3P, 12, imid pp OH POs, i, OP OI-I PO OH intermediate in dictyostatin synthesis 19b 19c (18 in Scheme 2) t P (R) 3 or (R O) 3P _ 4 coupling fragment 1 Po,, J 6p 6H Q= P (R'9b) 3+ 1-or Q PO (OR19C) 2 1. alcohol protection 2. selective deprotection o OR 0 BR2 OR OH 3. oxidation OR OP H Brown H allylation OR Takai olefination I OR P 1. deprotect 1 2. oxidation 'OU or coupling fragment 2 Scheme 17 (continued) vvvv wittig coupling'' 20 PO H PO,,, Q= OP H ON PO,,, OP OH Ph Q= PRsb ? 3+ _ Q or PO (OR9) 2 OH OR similar sequence of reactions to that used in the total synthesis of dictyostatin OH HO",/"", AJ"", HO, WS v v OH OH (39) A method according to (1) above, comprising the following steps: (a) reacting compounds of formulae (IIb) and (IIIb) : wherein Ll is a leaving group (preferably a halogen atom, an alkoxy group, a triflate, a mesylate or a tosylate) and all other groups are as defined in (1) above to give a compound of formula (XXV) : (b) reducing the ketone group of said compound of formula (XXV) to a hydroxy group and protecting said group to give a compound of formula (VII) as defined in (10) above; (c) subjecting said compound of formula (VII) to steps (c), (d), (e), (f), (g) and optionally (h) as defined in (10) above to give a compound of formula (Ig) or (Ih) or salts thereof.

Examples of method (39) include those illustrated in Scheme 18 below: Scheme 18 oxidation Diastereoselective PMBO OH HWE-PMBO, reduction OH protection OTBS OH OTBS :) I Reduction PMBO-----"C02Et FGI PNIBO L _ 1 _ OTBS OTBS L = Br, I, OTf, OMs, OTs PMBO L2 OTBS O,/ L2= Br, I, OTf, OMs, OTs Alkylation TBSO.,,, ,",, PMB Methyl addition OPMB H Oxidation intermediate in Dictyostatin synthesis O OPMB (15 in Scheme 2) intermediate in Dictyostatin synthesis (14 in Scheme 2) dictyostatin (40) A method according to (1) above comprising the following steps: (a) reacting compounds of formulae (IIc) and (IIIc) : wherein L 2 is a leaving group (preferably a halogen atom, a triflate, a mesylate or a tosylate) and all other groups are as defined in (1) above in a dithiane alkylation and then removing the dithiane group to give a compound of formula (XXV) as defined in (39) above; (b) reducing the ketone group of said compound of formula (XXV) to a hydroxy group and protecting said group to give a compound of formula (VII) as defined in (10) above; (c) subjecting said compound of formula (VII) to steps (c), (d), (e), (f), (g) and optionally (h) as defined in (10) above to give a compound of formula (Ig) or (Ih) or salts thereof.

Examples of method (40) include those illustrated in Scheme 19 below: Scheme 19 Oxidation Hydroboration PMBO OH Asymmetric allylation PMBO Protection PMB/ ! j Y Deoxygenation OTBS O OH OTBS 0 OH Early intermediate in TBS dictyostatin Synthesis (10 in Scheme 2) Deprotection PBS OP FGI PMBO L2 L O O TBS TBS L2= I, Br, OTf, OMS, OTs Dithiane formation H °''Dithiane alkylation Dithiane removal 0 OPMB ? ? OPMB ' "''' intermediate in Dictyostatin synthesis n = 0, 1 (14 in Scheme 2) 0 TBSO,,,, Dictyostatin OPMB OPMB intermediate in Dictyostatin synthesis (15 in Scheme 2) (41) A method according to (1) above, comprising the following steps: (a) reacting compounds of formulae (IId) and (IIId) : wherein M2 is a selected from the group consisting of a halogen atom, Si (R') 3 (wherein R' is as defined in (1) above), Sn (R') 3 (wherein R'is as defined is as defined in (1) above) and B (OR') 2 (wherein R'is as defined is as defined in (1) above), Q2 represents a hydrogen atom, a halogen atom or an alkoxy group as defined and exemplified above, and all other substituents are as defined in (1) above, to give a compound of formula (VI) as defined in (10) above ; (c) subjecting said compound of formula (VI) to steps (b), (c), (d), (e), (f), (g) and optionally (h) as defined in (10) above to give a compound of formula (Ig) or (Ih) or salts thereof.

Examples of method (41) include those illustrated in Scheme 20 below: Scheme 20 Oxidation PMBO OH Asymmetric allylation PMBO PMBOv J OTBS O OH Early intermediate in TBS dictyostatin Synthesis (10 in Scheme 2) 1. Deoxygenation 1. Deoxygenation 2. Metathesis with/M2 M2 = I, Br, SiR'3, SnR3, B (OR) 3 1. Oxidation H", FGI Q 2 PMBO m2 O PMB O PMB intermediate in Dictyostatin synthesis Q2 = CI, Br, OR 0 TBSO,,, Dictyostatin OPMB OPMB Advance intermediate in Dictyostatin synthesis (15 in Scheme 2) (42) A method according to (1) above, comprising the following steps: (a) coupling compounds of formulae (IVd) and (Vc) : to give a compound of formula (XXVI): (b) removing the protecting group R13 from said compound of formula (XXVI) and oxidising the resulting hydroxyl group to give a formyl group, optionally hydrogenating the triple bond of said compound before doing so, to give a compound of formula (XXVIIa) or (XXVIIb) : (c) reacting said compound of formula (XXVIIa) or (XXVIIb) with a compound of formula (IIe) : wherein Alk is an alkyl group as defined and exemplified above and all other groups are as defined in (1) above, to give a compound of formula (XXVIIIa) or (XXVIIIb) : (d) for said compound of formula (XXVIIIa), hydrogenating the triple bond thereof to give a compound of formula (XXVIIIb) ; (e) reducing the COzAlk group of said compound of formula (XXVIIIb) to an aldehyde group and the C-9 carbonyl group to a hydroxy group to give a compound of formula (XXIX): reacting said compound of formula (XXIX) with a compound of formula (IIIa) as defined in (10) above, to give a compound of formula (XXX) : (XXX) (g) selectively reducing the double bond of the enone of said compound of formula (XXX) to a single bond, converting the carbonyl group of said enone to a protected hydroxyl group and oxidizing the C-9 hydroxy group to a carbonyl group to give a compound of formula (X) as defined in (10) above ; (h) subjecting said compound of formula (X) to the reactions of steps (f) and (g) of (10) above to give a compound of formula (Ig) or a salt thereof and, if desired, further subjecting said compound of formula (Ig) to the reactions of step (h) of (10) above to give a compound of formula (Ih).

Examples of method (42) include those illustrated in Scheme 21 below: Scheme 21 Me Reduction Coupling with R Reduction _ PMB'/ R P CO R PMBO\ ^/N. Me methylenation Z 14 PMBO Hydrogenation FGI FGI Fui -j FGI FGI C02Et C02Et TBSO \ +CO2Et H/ \ w O \ R I/R O OH COZEt Ester Reduction Similar sequence of transformations to that reported in the oxidation total synthesis (10) of Dictyostatin oxidation above olefination Dictyostatin r-COzEt TBSO PMB COZEt OTBS O OH R Ester Reduction Similar sequence of transformations to that reported in the total synthesis (10) of Dictyostatin above Dictyostatin (43) A method according to (1) above, comprising the following steps: (a) converting the aldehyde group in a compound of formula (VIII) as defined in (10) above to an ethynyl group to give a compound of formula (XXXI) : (b) converting said compound of formula (XXXI) to the metallated alkynyl derivative thereof and reacting this with a compound of formula (IVe) : wherein y2 is a halogen atom, a group of formula CHCHC02R14 or a group of formula CCCO2R14 and R°, RV, Rl3 and R14 are as defined in (1) above, and Hal is a halogen atom, to give a compound of formula (XXXII): (c) hydrogenating the triple bond group to a double bond to give a compound of formula (XXXIII) : (XXXIII) (d) where y2 is a halogen atom, coupling said compound ofibrmula (XXXIII) with a compound of formula (Va) as defined in (10) above: (Va) to give a compound of formula (X) as defined in (10) above; (e) subjecting said compound of formula (X) or said compound of formula (XXXIII) wherein Y2 is a group of formula CHCHC02R14 or a group of formula CCC02R14 to a macrolactonisation reaction to give a compound of formula (XI) as defined in (10) above; (f) subjecting said compound of formula (XI) to the reaction of step (g) of (10) above to give a compound of formula (Ig) or a salt thereof and, if desired, further subjecting said compound of formula (Ig) to the reactions of step (h) of (10) above to give a compound of formula (Ih).

Examples of method (43) include those illustrated in Scheme 22 below: Scheme 22 TBS I TBS TBAF.,, ,, TBSO,,,, H OH OH TBSO,,, FGI TBSO,,, Advance intermediate Alkyne addition to in the Dictyostatin synthesis (4 in Scheme 2) 0 OTBS Hal = Cl, Br Y'-COR TBS TBS TBS TBSO,,, Selective,, TBSO,,, hydrogenation OH 6H 6H Y2 MY O O BS O OTBS 1 Dictyostatin (44) A method according to (1) above, comprising the following steps: (a) reacting a compound of formula (IIf) : wherein Rk, Rr, R, Rw, Rx, R3 and P1 are as defined in (1) above, with a compound of formula (IVa) as defined in (10) above, to give a compound of formula (XXXIV) : (XXXIV) (b) reducing the C-7 carbonyl of said compound of formula (XXXIV) and protecting the resulting hydroxy group to give a compound of formula (XXXV) : (XXXV) wherein R13 is a hydroxy protecting group ; (c) converting the group opt in said compound of formula (XXXV) to an aldehyde group to give a compound of formula (XXVI) : (XXXVI) (d) reacting said compound of formula (XXXVI) with a compound of formula (IIIa) as defined in (10) above to give a compound of formula (XXXVII): (XXXVII) (e) converting the double bond of the enone of said compound of formula (XXXVII) to a single bond and reducing the carbonyl group thereof to a protected hydroxyl group of formula-OP4, wherein P4 is as defined and exemplified above, to give a group of formula (XXXVIII) : (XXXVfH) (f) coupling said compound of formula (XXXVIII) with a compound of formula (Va) as defined in (10) to give a compound of formula (XV) as defined in (34) above; (g) subjecting said compound of formula (XV) to a macrolactonisation reaction to give a compound of formula (Ig) or a salt thereof as defined in (10) above; (h) and, if desired, removing any protecting groups R3, R13 and P4 from said compound of formula (Ig) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, Ri, Ri and Ruz respectively to give a compound of formula (Ih) or a salt thereof as defined in (10) above.

Examples of method (44) include those illustrated in Scheme 23 below: Scheme 23 1. LiAIH4 OP 2. protection of PO,,, 0 primary a) coho ! po ; DDQ O OPMB OPMB X = chiral auxillary intermediate in total synthesis of dictyostatin (11 in. Scheme 2) Still-Gennari coupling with .. C-C phosphonate PO., ., oxidation pp,, .,, 3'C'10 Phosphonate H H OH 0 OH OP I °P looP 1. deprotect C17 PO,,, PO/,, alcohol Iî ~ 2. oxidation Y Y/ 1 v I 1 v I O OP OH OP Horner-Wadsworth- Emmons coupling -C26 phosphonate /I /I OH OP OH OP Scheme 23 (continued) / zip POJ'0 OP reduction PO., 0 6p 0 0 p I OH OP OH OP 1. C21-deprotection/~< protection of 2. 1, 3-syn reduction P°, Xt"'ÕH ÕHdl C19 alcohol 1 I OH OH OH OP pOS", ÕP ÕHgß C02R .. OR C2R14 + n (R2) 3 r = f) r = cop OH OP OH OP 0 OP macrolactonisation PO,,, common intermediate with current route to 0 O dictyostatin OH OP In addition to the method (1) and variations thereon above, the present also provides further methods for the synthesis of disctyostatin analogues as defined below: (45) A method for the synthesis of a dictyostatin analogue of formula (XXXIX) or a salt thereof : (XXXIX) wherein R22 is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom or an alkyl group), an alkoxy group and a haloalkoxy group as defined and exemplified above and the other groups are as defined in (1) above, said method comprising the following steps: (a) reducing the double bond of the enone of a compound of formula (XXXVII) as defined in (44) above to a single bond to give a compound of formula (XL) : (b) subjecting said compound of formula (XL) to a vinyl halide coupling reaction to give a compound of formula (XLI) : (XLI) (c) converting the carbonyl group of said compound of formula (XLI) to a group of formula-OR'and said groups R3 and R13 to groups of formulae Rg and R'respectively to give a dictyostatin analogue of formula (XXXIX) or a salt thereof.

Examples of method (45) include those illustrated in Scheme 24 below: Scheme 24 0 OP R PO,,, 0 6P R zu Suzuki, Sonagashira, Heck, Stille, Negishi or Buchwald-Hartwig OH OP coupling of vinyl iodide T OH OP similar sequence of reactions to that used OH R" 0 in syntheses of dictyostatin above HO,,, OH R22 OH OH (46) A method for the synthesis of a dictyostatin analogue of formula (XLII) or a salt thereof : (XLII) wherein R23 is selected from the group consisting of a hydrogen atom, a hydroxy protecting group, an alkyl group, an aralkyl group, an aryl group and an acyl group as defined and exemplified above and the other groups are as defined in (1) above, said method comprising the following steps: (a) deprotecting the C-19 alcohol in the compound of formula (X) as defined in (10) above to give a compound of formula (XLIII): (XLIII) (b) subjecting said compound of formula (XLIII) to a macrolactonisation reaction to give a compound of formula (XLIV) : (XLIV) (c) reducing the ketone group at the C-9 position to a hydroxyl group to give a compound of formula (XLV) or a salt thereof : (xuv) (d) and, where needed, removing any protecting groups R3, R13 and R9 from said compound of formula (XLIV) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, R', R23 and Rh respectively to give a compound of formula (XLIII) or a salt thereof.

Examples of method (46) include those illustrated in Scheme 25 below: Scheme 25 // // remove POw, J""ÕP ÕH protecting group HO",/", OH OH r from C19aicohpl l fro COZH // cl 2H O OP O OH If'P 0 OH advanced intermediate in total synthesis of dictyostatin (26 in Scheme 4) macrolactonisation t f e HO Ho HO,,, // O OH NaBH4 CeCI3 if fuzz HO., H 0,,, OH OH (47) A method for the synthesis of a dictyostatin analogue of formula (XLV) or a salt thereof : wherein Rg, Ruz R', Ri Ruz Ru Ru Rs, Rv, Rw, Rx, Ry and RZ are as defined in (1) above, said method comprising the following steps: (a) performing a palladium catalysed cabonylation reaction on a compound of formula (IX) as defined in (10) above to give a compound of formula (XLVI) : (XLVI) wherein R3, R9, R13, P4, Rk, Rn, Rr, Rs, Rv, Rw, Rx, Ry and RZ are as defined in (10) above ; (b) subjecting said compound of formula (XLVI) to a macrolactonisation reaction to give a compound of formula (XLVII) : (XLVII) (c) reducing the ketone group at the C-7 position to a hydroxyl group to give a compound of formula (XLVIII) or a salt thereof : (XLVIII) (h) and, where needed, removing any protecting groups R3, R13 and P4 from said compound of formula (XLVIII) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-7 position to other functional groups of formulae Rg, R', Ri and Rh respectively to give a compound of formula (XLV) or a salt thereof.

Examples of method (47) include those illustrated in Scheme 26 below: Scheme 26 w-w- / PO,, ÕP ~ ÕH ~ ~ ~ Pd catalysed PO", J""ÕP ÕH ) carbonylation - _ "0'OH O OP O OP O advanced intermediate in total synthesis of dictyostatin (23 in Scheme 4) 1. macrolactonisation 2. NaBH4, CeCI3 3. HCI, MeOH '' HO., ° /O OH OH O (48) A method for the synthesis of a dictyostatin analogue of formula (In) or a salt thereof: (In) wherein Rg, Rh, R', Rj, Rk, Rn, Rr, Rs, RV, Rw, RX, Ry and RZ are as defined in (1) above, said method comprising the following steps: (a) performing a Sonagashira coupling reaction on a compound of formula (IX) as defined in (10) to give a compound of formula (IL): (IL) wherein R3, R9 R", P4, Ruz Ruz Ruz Rus, kv, Ru, RXS Ry and RZ are as defined in (10) above; (b) subjecting said compound of formula (IL) to a macrolactonisation reaction to give a compound of formula (L): (L) (c) reducing the ketone group at the C-9 position to a hydroxyl group to give a compound of formula (LI) or a salt thereof : (d) and, where needed, removing any protecting groups R3, R13 and P4 from said compound of formula (LI) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, Ri, Ri and Rh respectively to give a compound of formula (In) or a salt thereof.

Examples of method (48) include those illustrated in Scheme 27 below: Scheme 27 / v v v v PO", J""ÕP ÕH < r Sonagashira J. coupting PO OP OH i 0 ou advanced intermediate in total 0 OP OH synthesis of dictyostatin (23 in Scheme 4) 1. macrolactonisation 2. NaBH4, CeCI3 3. HCI, MEOH I OH H oOHgH 0 0 // OH OH (49) A method for the synthesis of a dictyostatin analogue of formula (LII) or a salt thereof : (Lll) wherein Rg, Rh, Ri, Rj, Rk, Rn, Rr, Rs, R, Rw, Rx, Ry and RZ are as defined in (1) above and n3 is an integer of from 1 to 4, said method comprising the following steps: (a) performing a palladium catalysed coupling reaction (preferably Suzuki or Negishi) on a compound of formula (IX) as defined in (10) above with a compound of formula M3-(CH2)n3-CH2OP6, wherein M3 is selected from the group consisting of B (OR') 2 (wherein R'is as defined is as defined in (1) above) and ZnR' (wherein R'is as defined is as defined in (1) above), P6 is a hydroxy protecting group and n3 is as defined above, followed by deprotection of the CH2OP6 group and oxidation of the resulting primary hydroxy group to a carboxy group to give a compound of formula (LIII): (LIII) wherein R3 R9, R", P4, Rk, R", Rr, Rs, RV, Rw, Rx, Ry and RZ are as defined in (10) above ; (b) subjecting said compound of formula (LIII) to a macrolactonisation reaction to give a compound of formula (LIV) : (Llp (c) reducing the ketone group at the C-9 position to a hydroxyl group to give a compound of formula (LV) : (LV) (d) and, where needed, removing any protecting groups R3, R13 and P4 from said compound of formula (LV) to give hydroxyl groups and/or converting one or more of said groups and the hydroxyl group at the C-9 position to other functional groups of formulae Rg, R', Ri and 10 respectively to give a compound of formula (LII) or a salt thereof Examples of method (49) include those illustrated in Scheme 28 below: Scheme 28 i i POQR", °P ÕH pO",/d"ÕP ÕH 91 ¢H0 OH H O OP O P v M3 O advanced intermediate in total synthesis of dictyostatin (23 in Scheme 4) 1. macrolactonisation 2. NaBH4, CeCI3 3. HCI, MeOH OH HO,,, an3 OH OH OH OH The flexible methods of the present invention allow the synthesis of many novel dictyostatins and derivatives, analogues and salts thereof, which also form a part of the present invention.

(50) A compound having the following formula (Io) or a salt thereof : wherein: each of Ra, kb, Rc, R, W and Rf is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a hydroxy group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Ra and Rb together and/or R° and Rd together and/or Re and Rftogether represent a single bond, a group of formula-CH2-or a group of formula-0- ; each of Rg, Rh, R'and Ri is the same or different and is selected from the group consisting of a hydrogen atom, a hydroxy protecting group, an alkyl group, an aralkyl group, an aryl group and an acyl group; Rk is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group; RP and Rm are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, an alkoxy group, a haloalkoxy group and an aryloxy group, or RP and Rm together represent a single bond or a group of formula-CH2-or-0- ; Rn is selected from the group consisting of a hydrogen atom, an alkyl group (said alkyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkenyl group (said alkenyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom, an aryl group, an acyl group and an alkoxy group), an alkynyl group, a dienyl group and an aryl group; R° is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group and an aralkyl group; Rq is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, Rr and Rs are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a hydroxy group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rr and Rs together represent a single bond or a group of formula ; Rt and Ru are the same or different and each is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, an alkoxy group, a haloalkoxy group and an aryloxy group, or Rt and Ru together represent a single bond or a group of formula-CH2-or-O-, or Rt and Ru together with the 2 carbon atoms to which they are connected form an aryl group; each of RV, RW, RX, RY and RZ is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a hydroxyalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom or an alkyl group), an alkoxy group and a haloalkoxy group; and X represents a group of formula-0-, a group of formula-CH2-or a group of formula- NR', wherein R'is as defined above. (51) A compound according to (50), wherein said compound of formula (Io) is a compound of formula (Ip) or a salt thereof : (Ip) (52) A compound according to (50), wherein said compound of formula (Io) is a compound of formula (Iq) or a salt thereof : (Iq) (53) A compound according to (50), wherein said compound of formula (Io) is a compound of formula (Ir) or a salt thereof : (54) A compound according to any one of (50) to (53) or a salt thereof, wherein each of R°, Rr and Rs is the same or different and is a hydrogen atom or an alkyl group.

(55) A compound according to any one of (50) to (53) or a salt thereof, wherein each of R°, Rr and Rs is a hydrogen atom.

(56) A compound according to any one of (50) to (55) or a salt thereof, wherein each of Rk, RV, RW, RX, RY and RZ is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group and an aralkyl group.

(57) A compound according to (56) or a salt thereof, wherein each of Rk, Rv, Rw, Rx, Ry and Rz is the same or different and is an alkyl group.

(58) A compound according to (56) or a salt thereof, wherein each of Rk, RV, RW, RX, Ry and Ra is a methyl group.

(59) A compound according to any one of (50) to (58) or a salt thereof, wherein each of Rg, Rh, R'and R'is the same or different and is selected the group consisting of a hydrogen atom, an alkyl group, an aralkyl group, an aryl group, an alkylcarbonyl group, a haloalkylcarbonyl group, an arylcarbonyl group and a trialkylsilyl group. (60) A compound according to (59) or a salt thereof, wherein each of Rg, Rh, R'and Ri is a hydrogen atom.

(61) A compound according to any one of (50) to (60) or a salt thereof, wherein each of Rt and Ru is the same or different and is selected from the group consisting of a hydrogen atom, an alkyl group, an aralkyl group or an aryl group.

(62) A compound according to (61) or a salt thereof, wherein each of Rt and Ru is a hydrogen atom.

(63) A compound according to any one of (50) to (62) or a salt thereof, wherein Rn is selected from the group consisting of a hydrogen atom, an alkyl group (said alkyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom and an aryl group), an alkenyl group (said alkenyl group being optionally substituted with at least one substituent selected from the group consisting of a halogen atom and an aryl group), a dienyl group and an aryl group.

(64) A compound according to (63) or a salt thereof, wherein Rn is a dienyl group.

(65) A compound according to (63) or a salt thereof, wherein R"is a 1,3-butadienyl group.

(66) A compound according to (50) or a salt thereof having the following formula (Is): (67) A compound according to (50) or a salt thereof having the following formula (It): (It) (68) A dictyostatin analogue of formula (XXXIX) or a salt thereof : wherein R22 is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, an alkoxyalkyl group, an aryl group, an aralkyl group, a halogen atom, a cyano group, a group of formula-NR'R" (wherein R'and R"are the same or different and each is a hydrogen atom or an alkyl group), an alkoxy group and a haloalkoxy group as defined and exemplified above and R9, Rg, Rh, R', Ri, Rk, Rn, Rr, Rs, RV, RW, RX, Ry and RZ are as defined in (1) above.

(69) A dictyostatin analogue of formula (XLII) or a salt thereof : (XLII) wherein R23 is selected from the group consisting of a hydrogen atom, a hydroxy protecting group, an alkyl group, an aralkyl group, an aryl group and an acyl group as defined and exemplified above and R17, R18, Rg, Rh, Ri, Rk, Rn, Rr, Rs, Rv, Rw, Rx, Ry and RZ are as defined in (1) above.

(70) A dictyostatin analogue of formula (XLV) or a salt thereof : (XLV) wherein Rg, Rh, R', Ri Rk, Rn, Rr, Rs, RV, Rw, Rx, Ry and RZ are as defined in (1) above.

(71) A dictyostatin analogue of formula (In) or a salt thereof : wherein Rg, Rh, R', Ri Ruz Ruz Rr, Rs, RV, RW, Rx, Ry and RZ are as defined in (1) above.

(72) A dictyostatin analogue of formula (LII) or a salt thereof : (Lll) wherein Rg, Ruz R', Ri Rk, R", Rr, Rs, Rv,Rw,Rx,Ry and RZ are as defined in (1) above and n3 is an integer of from 1 to 4.- In compounds (50) to (72) above, substituents Ra, Rb, RO, Rd, Re, Rf,Rg,Rh,Ri,Rj, Rk, R, Rm,Rn,Ro,Rq,Rr,Rs,Rt,Ru,Rv,Rw,Rx,Ry,Rz,R17,R18,R22,R23 and X are as defined and exemplified in the definitions and examples of the methods of the invention above.

The present invention also provides a number of intermediate compounds that are particularly suitable for the synthesis of dictyostatins and derivatives, analogues and derivatives thereof, said intermediates being as follows: (73) A compound of formula (II) : wherein R1,R2,R3,R4,R5,R15,R16,Rk,Rr,Rs,Rw and Rx are as defined in (1) above.

(74) A compound according to (73) of formula (IIg) : (75) A compound according to (73) of formula (IIa): wherein p2 is a hydroxy protecting group and R3, Rk, Rr, Rs, Ruz and Rare as defined in (1) above.

(76) A compound according to (75) of formula (IIa'): (77) A compound according to (73) of formula (IIb) : wherein L1 is a leaving group (preferably a halogen atom, an alkoxy group, a triflate group, a mesylate group or a tosylate group), P2 is a hydroxy protecting group and R3, Rk, Rr, Rs, Rw and Rx are as defined in (1) above.

(78) A compound according to (77) of formula (IIb') : (79) A compound according to (73) of formula (IIc) : wherein L2 is a leaving group (preferably a halogen atom, a triflate group, a mesylate group or a tosylate group), P2 is a hydroxy protecting group and R3, Rk, Rr, Rs, Rw and R" are as defined in (1) above.

(80) A compound according to (79) of formula (IIc') : (81) A compound according to (73) of formula (IId) : wherein Ma is a selected from the group consisting of a halogen atom, Si (R') 3 (wherein R' is as defined in (1) above), Sn (R') 3 (wherein R'is as defined is as defined in (1) above) and B (OR') 3 (wherein R'is as defined is as defined in (1) above), p2 is a hydroxy protecting group and R3, Rk, Rr, R, Rw and Rx are as defined in (1) above.

(82) A compound according to (81) of formula (IId') : (83) A compound according to (73) of formula (IIe) : wherein Alk is an alkyl group as defined and exemplified above and R3, Rk, Rr, Rs, Rw and R are as defined in (1) above.

(84) A compound according to (83) of formula (IIe') : (85) A compound according to (73) of formula (III) : wherein pl, R3, Rk, Rr, Rs, Rw and Rx are as defined in (1) above.

(86) A compound according to (85) of formula (IIf) : (nf) (87) A compound of formula (III): (III) wherein R6,R7,R8,R9,Rn,Ry and Rz are as defined in (1) above.

(88) A compound according to (87) of formula (IIIe): (89) A compound according to (87) of formula (IIIa) : (IIIa) wherein R9, Rn, RI and Rz are as defined in (1) above and each of said groups of formula R'9a is the same or different and represents an alkoxy group or a haloalkoxy group as defined and exemplified above.

(90) A compound according to (89) of formula (IIIa') : (IIIa') (91) A compound according to (87) of formula (IIIb) : wherein R9, Rn, RY and RZ are as defined in (1) above.

(92) A compound according to (91) of formula (IIIb') : (93) A compound according to (87) of formula (IIIc): wherein R9, Rn, Ry and RZ are as defined in (1) above.

(94) A compound according to (93) above of formula (IIIc') : (95) A compound according to (87) of formula (IIId) : wherein R, Rn, Ry and RZ are as defined in (1) above and Q represents a hydrogen atom, a halogen atom or an alkoxy group as defined and exemplified above.

(96) A compound according to (95) of formula (IIId') : (97) A compound of formula (IV) : wherein R'°, R", R12,R13,Ro,Rv, Y and n2 are as defined in (1) above.

(98) A compound according to (97) of formula (IVf) : (99) A compound according to (97) of formula (IVa): wherein R13, Roi9, R° and Rv are as defined in (1) above and yl is a halogen atom, a group of formula Sn (le) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (R20) 3 (wherein each R20 is the same or different and is an alkyl group), or a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group.

(100) A compound according to (99) of formula (IVa'): (IVa') (101) A compound according to (97) of formula (IVb): wherein Ll is a leaving group (preferably a halogen atom, an alkoxy group, a triflate, a mesylate or a tosylate), Y2 is a halogen atom, a group of formula Sn (R2°) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (fez) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group, a group of formula CHCHCO2RI4 or a group of formula CCCO2R14 and R°, RV, R13 and R14 are as defined in (1) above, (102) A compound according to (101) above of formula (IVb') : (103) A compound according to (97) of formula (IVc) : (IVc) wherein R13, R° and Rv are as defined in (1) above, Hal is a halogen atom and p5 is a hydrogen atom or a hydroxy protecting group as defined and exemplified above.

(104) A compound according to (103) of formula (IVc') : (IVc') (105) A compound according to (97) of formula (IVd) : (IVd) wherein R13 and R are as defined in (1) above and Hal is a halogen atom.

(106) A compound according to (105) of formula (IVd') : (107) A compound according to (97) of formula (IVe): (IVe) wherein R13, Ro and Rv are as defined in (1) above, Hal is a halogen atom and y2 is a halogen atom, a group of formula Sn (R20) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (leo) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group, a group of formula CHCHCO2R14 or a group of formula CCC02R, wherein R14 is as defined in (1) above.

(108) A compound according to (107) of formula (IVe'): (IVe') (109) A compound according to (97) of formula (IVg): wherein Y2 is a halogen atom, a group of formula Sn (R2°) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula Si (R2°) 3 (wherein each R20 is the same or different and is an alkyl group), a group of formula B (OR21) 2 wherein each R21 is the same or different and is an alkyl group or an aryl group, a group of formula CHCHC02R14 or a group of formula CCC02R14 and R°, RV, R13 and R14 are as defined in (1) above (110) A compound according to (109) of formula (IVg'): (111) A compound of formula (V) : (V) wherein Rl4, Rl7, Rl8 and Z are as defined in (1) above.

(112) A compound according to (111) of formula (Va): (Va) wherein R14,R17,R18 and R20 are as defined in (1) above.

(113) A compound according to (111) of formula (Vb): wherein Rl7 and R's are as defined in (1) above and zl is a halogen atom or a group of formula Sn (leo) 3 wherein R20 is as defined in (1) above.

(114) A compound according to (111) of formula (Vc): (Vc) wherein R14 is as defined in (1) above.

In the intermediates in (73) to (114) above, the substituents therein are as defined and exemplified in the definitions and examples of the methods of the invention above.

The methods of the present invention allows for the first time the synthesis of dictyostatin in substantially pure form, and this also forms a part of the present invention.

(115) Dictyostatin of formula (If) or a salt thereof in substantially pure form: (116) (-)-Dictyostatin of formula (If) or a salt thereof in substantially pure form: (117) Dictyostatin according to (115) or (116) having a specific rotation of [a] D =-32. 7 (c = 0.22, MeOH).

(118) Dictyostatin according to (115) or (116) having a purity of at least 99 %.

(119) Dictyostatin according to (118) having a purity greater of at least 99. 3 %.

(120) Dictyostatin according to (118) having a purity greater of at least 99. 5 %.

(121) Dictyostatin according to (115) or (116) having high performance liquid chromatography traces as shown in Figures 10 to 12.

(122) Dictyostatin according to (115) or (116) having a'H mnr spectrum as shown in Figure 13.

(123) Dictyostatin according to (115) or (116) having a 13C nmr spectrum as shown in Figure 14.

The present invention provides dictyostatin compounds, and uses thereof. These compounds specifically include dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, especially dictyostatin having the formula (If) or a salt, derivative or analogue thereof, a compound of formula (Io) or a pharmacologically acceptable salt as defined above and substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined above. These new uses include the control of cellular proliferation, cytotoxicity against human tumor cells resistant to chemotherapeutic agents, immunomodulation, and the control of inflammation. These uses arise from the role of dictyostatin compounds as tubulin polymerizers and microtubule stabilizers.

When tubulin is treated with dictyostatin, a rapid onset of polymerization occurs in the absence of cells. This effect is not reversed upon temperature change indicating a long term stabilization of the microtubules. Also, PANC-1 human pancreatic adenoma cells treated with dictyostatin 1 do not undergo mitosis and show pronounced rearrangement of the microtubules in the cells.

The compounds of the present invention have utility in the treatment of various human cancers that may have developed resistance to certain chemotherapeutic agents.

Thus, the compounds of the subject invention are useful in the treatment of multi-drug resistant cancers.

In view of the mode of action of the dictyostatin compounds, these compounds can be used in the treatment of a number of conditions in which aberrant cellular proliferation occurs. These conditions include, for example, autoimmune disorders and inflammatory diseases. In addition to use in the treatment of these disorders as well as other conditions involving pathological cellular proliferation, the compounds of the current invention can also be used as biochemical tools to study the process of tubulin polymerization/depolymerization and drug resistance.

In one embodiment, the present invention pertains to the immunosuppressive use of the dictyostatins compounds of the present invention. The dictyostatin compounds of the present invention can be used to reduce, suppress, inhibit, or prevent unwanted immune responses. Thus, the dictyostatin compounds of the present invention are useful for treatments of humans or animals requiring immunosuppression. Examples of conditions for which immunosuppression is desired include, but are not limited to, treatment or prevention of autoimmune diseases such as diabetes, lupus, and rheumatoid arthritis. Immunosuppression is also frequently needed in conjunction with organ transplants. Immunosuppressive agents can also be utilized when a human or animal has been, or may be, exposed to superantigens or other factors known to cause overstimulation of the immune system. The dictyostatin compounds of the present invention are also useful as standards to assess the activity of other putative immunosuppressive agents.

The dictyostatin compounds of the present invention are useful for various non-therapeutic and therapeutic purposes. It is apparent from the testing that the dictyostatin compounds of the present invention are'effective for inhibiting cell growth." Because of the antiproliferative properties of the compounds, they are useful to prevent unwanted cell growth in a wide variety of settings including in vitro uses. They are also useful as standards and for teaching demonstrations. They can also be used as ultraviolet screeners in the plastics industry since they effectively absorb UV rays. As disclosed herein, they are also useful prophylactically and therapeutically for treating cancer cells in animals and humans.

Therapeutic application of the dictyostatin compounds of the present invention and compositions containing them can be accomplished by any suitable therapeutic method and technique presently or prospectively known to those skilled in the art.

Further, the dictyostatin compounds of the present invention have use as starting materials or intermediates for the preparation of other useful compounds and compositions.

As mentioned earlier, the dictyostatins and derivatives, analogues and salts thereof of the present invention can be used in the prophylaxis or treatment of cancer and the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules. They are potentially of particular use against multi- drug resistant cancers where resistance is caused by either pGp or MRP resistance mechanisms. Furthermore, they can also be used in the inhibition of cellular proliferation, e. g. in the prophylaxis or treatment of autoimmune diseases or inflammatory diseases such as diabetes, lupus, rheumatoid arthritis or organ transplant rejection. Thus, the present invention also provides: (124) A pharmaceutical composition comprising an effective amount of a pharmacologically active compound together with a carrier or diluent therefor, wherein said pharmacologically active compound is a compound as defined in any one of (50) to (72) above.

(125) A pharmaceutical composition comprising an effective amount of a pharmacologically active compound together with a carrier or diluent therefor, wherein said pharmacologically active compound is substantially pure dictyostatin of formula (If) or a pharmacologically acceptable salt thereof as defined in any one of (115) to (123) above.

(126) A pharmaceutical composition comprising an effective amount of a pharmacologically active compound together with a carrier or diluent therefor, wherein said pharmacologically active compound is dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof.

(127) A method for the prophylaxis or treatment of cancer, said method comprising administering an effective amount of a cytotoxic compound to a patient suffering from said cancer, wherein said cytotoxic compound is a compound as defined in any one of (50) to (72) above.

(128) A method according to (127), wherein said cancer is selected from the group consisting of leukemia, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, uterine cancer, brain cancer, renal cancer, prostate cancer, breast cancer and melanoma.

(129) A method for the prophylaxis or treatment of cancer, said method comprising administering an effective amount of a cytotoxic compound to a patient suffering from said cancer, wherein said cytotoxic compound is a compound as defined in any one of (115) to (123) above.

(130) A method according to (129), wherein said cancer is selected from the group consisting of leukemia, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, uterine cancer, brain cancer, renal cancer, prostate cancer, breast cancer and melanoma.

(131) A method for the prophylaxis or treatment of cancer, said method comprising administering an effective amount of a cytotoxic compound to a patient suffering from said cancer, wherein said cytotoxic compound is dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof.

(132) A method according to (131), wherein said cancer is selected from the group consisting of leukemia, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, uterine cancer, brain cancer, renal cancer, prostate cancer, breast cancer and melanoma.

(133) A method for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules, said method comprising administering to a patient in need thereof a pharmacologically effective amount of a compound as defined in any one of (50) to (72) above.

(134) A method for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules, said method comprising administering to a patient in need thereof a pharmacologically effective amount of a compound as defined in any one of (115) to (123) above.

(135) A method for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules, said method comprising administering to a patient in need thereof a pharmacologically effective amount of dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof.

(136) A method for inhibiting cellular proliferation in an animal, that may be human, said method comprising administering to said animal a pharmacologically effective amount of a compound as defined in any one of (50) to (72) above.

(137) A method according to (136), wherein said method is for the prophylaxis or treatment of an autoimmune disease or an inflammatory disease.

(138) A method for inhibiting cellular proliferation in an animal, that may be human, said method comprising administering to said animal a pharmacologically effective amount of a compound as defined in any one of (115) to (123) above.

(139) A method according to (138), wherein said method is for the prophylaxis or treatment of an autoimmune disease or an inflammatory disease.

(140) A method for inhibiting cellular proliferation in an animal, that may be human, said method comprising administering to said animal a pharmacologically effective amount of dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof.

(141) A method according to (140), wherein said method is for the prophylaxis or treatment of an autoimmune disease or an inflammatory disease.

(142) Use of a compound as defined in any one of (50) to (72) above in the manufacture of a medicament for the prophylaxis or treatment of cancer.

(143) Use according to (142), wherein said cancer is selected from the group consisting of leukemia, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, uterine cancer, brain cancer, renal cancer, prostate cancer, breast cancer and melanoma.

(144) Use of a compound as defined in any one of (115) to (123) above in the manufacture of a medicament for the prophylaxis or treatment of cancer.

(145) Use according to (144), wherein said cancer is selected from the group consisting of leukemia, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, uterine cancer, brain cancer, renal cancer, prostate cancer, breast cancer and melanoma.

(146) Use of dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof in the manufacture of a medicament for the prophylaxis or treatment of cancer.

(147) Use according to (146), wherein said cancer is selected from the group consisting of leukemia, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, uterine cancer, brain cancer, renal cancer, prostate cancer, breast cancer and melanoma.

(148) Use of a compound as defined in any one of (50) to (72) above in the manufacture of a medicament for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules.

(149) Use of a compound as defined in any one of (115) to (123) above in the manufacture of a medicament for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules.

(150) Use of dictyostatin 1 or a pharmacologically acceptable, salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof in the manufacture of a medicament for the prophylaxis or treatment of diseases that can be prevented or treated by compounds capable of stabilising microtubules.

(151) Use of a compound as defined in any one of (50) to (72) above in the manufacture of a medicament for use in a method of inhibiting cellular proliferation in an animal, that may be human.

(152) Use according to (151), wherein said method is for the prophylaxis or treatment of an autoimmune disease or an inflammatory disease.

(153) Use of a compound as defined in any one of (115) to (123) above in the manufacture of a medicament for use in a method of inhibiting cellular proliferation in an animal, that may be human.

(154) Use according to (153), wherein said method is for the prophylaxis or treatment of an autoimmune disease or an inflammatory disease.

(155) Use of dictyostatin 1 or a pharmacologically acceptable salt, derivative or analogue thereof having the stereochemical structure shown in Figures 1 through 4, and preferably dictyostatin having the formula (If) or a salt, derivative or analogue thereof in the manufacture of a medicament for use in a method of inhibiting cellular proliferation in an animal, that may be human.

(156) Use according to (155), wherein said method is for the prophylaxis or treatment of an autoimmune disease or an inflammatory disease.

When the dictyostatins and derivatives, analogues and salts thereof of the present invention are used as a medicament for the treatment or prevention of cancer and diseases that can be prevented or treated by compounds capable of stabilising microtubules, as described above, the dictyostatins and derivatives, analogues and salts thereof of the present invention can be administered alone, and can also be administered orally in a pharmaceutical formulation such as a tablet, capsule, granule, powder or syrup or parenterally in a pharmaceutical formulation such as an injection, suppository, stick preparation or external preparation, by combination with a pharmaceutically acceptable excipient, diluent and the like. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention.

These pharmaceutical formulations can be prepared by well known methods using additives such as a excipients, lubricants, binders, disintegrants, emulsifiers, stabilizers, corrigents, diluents and the like.

The excipient may be, for example, an organic excipient or an inorganic excipient. Organic excipients include, for example, a sugar derivative such as lactose, sucrose, glucose, mannitol or sorbitol; a starch derivative such as corn starch, potato starch, a-starch, or dextrin ; a cellulose derivative such as crystalline cellulose; acacia; dextran ; or pullula. Inorganic excipients may be, for example, a silicate derivative such as light silicic acid anhydride, synthesized aluminum silicate, calcium silicate or magnesium metasilicate aluminate ; a phosphate such as calcium hydrogenphosphate; a carbonate such as calcium carbonate; or a sulfate such as calcium sulfate.

The lubricant may be, for example, stearic acid; a metal stearate such as calcium stearate or magnesium stearate ; talc; colloidal silica; a wax such as beeswax or spermaceti ; boric acid; adipic acid; a sulfate such as sodium sulfate; a glycol; fumaric acid; sodium benzoate; DL-leucine ; a lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; a silicic acid derivative such as silicic acid anhydride or silicic acid hydrate; or a starch derivative as described above.

The binder may be, for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, poly (ethylene glycol) or the derivatives described in the above excipient.

The disintegrant may be, for example, a cellulose derivative such as a lower- substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose or sodium internally cross-linked carboxymethyl cellulose; a chemically modified starch-cellulose derivative such as carboxymethyl starch or sodium carboxymethyl starch; or cross-linked polyvinylpyrrolidone.

The emulsifier may be, for example, a colloidal clay such as bentonite or veegum; a metal hydroxide such as magnesium hydroxide or aluminum hydroxide; an anionic surfactant such as sodium lauryl sulfate or calcium stearate; a cationic surfactant such as benzalkonium chloride; or a non-ionic surfactant such as a polyoxyethylenealkylether, a polyoxyethylene sorbitan ester of fatty acid or a sucrose ester of a fatty acid.

The stabilizer may be, for example, a parahydroxybenzoic acid ester such as methylparaben or propylparaben; an alcohol such as chlorobutanol, benzyl alcohol or phenethyl alcohol; benzalkonium chloride; a phenol derivative such as phenol or cresol; thimerosal; dehydroacetic acid; or sorbic acid.

The corrigent may be, for example, a conventional sweetening, souring, flavoring agent or the like.

The dosage level of the dictyostatins and derivatives, analogues and salts thereof of the present invention varies depending on the disease being treated, the age of the patient, weight, health, kind of concurrent treatment, if any, frequency of treatment, and therapeutic ratio.

To provide for the administration of such dosages for the desired therapeutic treatment, new pharmaceutical compositions of the present invention will advantageously comprise between about 0. 1% and 45%, and especially, 1 and 15%, by weight of the total of one or more of the new compounds based on the weight of the total composition including carrier or diluent. Illustratively, dosage levels of the administered active ingredients can be: intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about 200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation, 0. 01 to about 20 mg/kg; and aerosol, 0. 01 to about 20 mg/kg of animal (body) weight.

The work of the present inventors shows that the dictyostatins and derivatives, analogues and salts thereof of the present invention are potent inducers of tubulin polymerization and stabilizers of the microtubule network. This activity is useful in the treatment of diseases caused by proliferation of cells including autoimmune and inflammatory processes. Moreover, their work indicates that the dictyostatin class of metabolites are useful in the treatment of multi-drug resistant tumors where resistance is caused by either the pGp or MRP resistance mechanisms.

Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art by consideration of the following non-limiting examples. The compound numbering in the Examples is as shown in Schemes 1 to 5 above. Reference is made to the following Figures 1 to 15: Figure 1 shows the planar structure of dictyostatin.

Figure 2 shows aspects of the stereochemistry of dictyostatin.

Figure 3 shows aspects of the stereochemistry of dictyostatin.

Figure 4 shows aspects of the stereochemistry of dictyostatin.

Figure 5 shows the chemical structure of dictyostatin having formula (If) and discodermolide.

Figure 6 shows IH NMR spectrum of naturally obtained dictyostatin having formula (If) in CD30D (700 MHz).

Figure 7 shows NOESY spectrum of naturally obtained dictyostatin having formula (If) in CD30D (700 MHz).

Figure 8 shows HSQC-HECADE spectrum of naturally obtained dictyostatin having formula (If) in CD30D (700 MHz).

Figure 9 shows perspective drawings of the lowest energy conformation of dictyostatin having formula (If) generated by Macromodel V 7.2.

. Figure 10 is an HPLC trace observed at 225 nm for dictyostatin having formula (If) prepared according to the method of the present invention.

Figure 11 is an HPLC trace observed at 256 nm for dictyostatin having formula (If) prepared according to the method of the present invention.

Figure 12 is an HPLC trace observed at 263 nm for dictyostatin having formula (If) prepared according to the method of the present invention.

Figure 13 is a IH nmr speckum (CD30D, 500 MHz) for dictyostatin having formula (If) prepared according to the method of the present invention.

Figure 14 is a 13C nmr spectrum (CD30D, 125 MHz) for dictyostatin having formula (If) prepared according to the method of the present invention.

Figure 15 shows immunofluorescence images of stained A549 cells treated with control, natural and synthetic dictyostatin 1 and paclitaxel.

References [1] Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Boyd, M. R.; Schmidt, J. M. J Chem. Soc., Chem. Commun. 1994,1111.

[2] Isbrucker, R. A.; Cummins, J. ; Pomponi, S : A. ; Longley ; R ; E.; Wright, A. E.

Biochem. Pharm. 2003, 66, 75.

[3] Pettit, G. R.; Cichacz, Z. A., U. S. Patent No 5,430, 053, 1995, contains a partial stereochemical assignment for dictyostatin which differs significantly from our findings.

[4] The structural similarities between discodermolide and dictyostatin and the scope for preparing hybrid structures have also been highlighted by Curran and Day, see: Shin, Y.; Choy, N.; Balachandran, R.; Madiraju, C.; Day, B. W. ; Curran, D. P. Org. Lett. 2002,4, 4443.

[5] Mooberry, S. L.; Tien, G.; Hernandez, A. H.; Plubrukarn, A.; Davidson, B. S. Cancer Res. 1999, 59, 653.

[6] Hood, K. A.; West, L. M.; Rouwe, B.; Northcote, P. T.; Berridge, M. V.; Wakefield, S. J.; Miller, J. H. Cancer Res. 2002, 62, 3356.

[7] a) ter Haar, E. ; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Longley, R. E.; Gunasekera, S.

P.; Rosenkranz, H. S. ; Day, B. W. Biochemistry 1996, 35, 243. b) Review: Paterson, I.; Florence, G. J. Eur. J. Org Che7n. 2003, 12, 2193.

[8] Paterson, I. ; Delgado, 0. ; Florence, G. J.; Lyothier, I.; Scott, J. P.; Sereinig, N. Org.

Lett. 2003, 5, 35.

[9] Garegg, P. J.; Samuelson, B. J Chem. Soc., Perkin Trans. I 1980, 2866.

[10] Myers, A. G. ; Yang, B. H.; Chen, H.; McKinstry, L. ; Kopecky, D. J.; Gleason, J. L.

J. Am. Chem. Soc. 1997, 119, 6496. [11] The 1, 3-syn stereochemistry of amide 13 was confirmed by nOe enhancements on lactone i obtained via a 2-step sequence (1. 2N HCl, EtOH ; 2. TEMPO, PhI (OAc) 2) from alcohol ii (see Scheme 29 below).

Scheme 29 1. 2N HCI, EtOH 2. TEMPO, Phl (OAC) 2 PMBO-- OTBS ii i nOeX OTBS [12] a) Paterson, I. ; Florence, G. J.; Gerlach, K.; Scott, J. P. Angew. Chem. vint. Ed. 2000, 39, 377. b) Paterson, I.; Florence, G. J.; Gerlach, K.; Scott, J. P.; Sereinig, N. J. Am.

Chem. Soc. 2001, 123, 9535.

[13] Paterson, I. ; Yeung, K. -S. ; Smaill, J. B. Synlett 1993,774.

[14] Mahoney, W. S.; Brestensky, D. M.; Stryker, J. M. J. Am. Chem. Soc. 1988, 110, 291.

[15] Oishi, T.; Nakata, T. Acc. Chem. Res. 1984, 17, 338.

[16] The relative stereochemistry of 16 was confirmed unequivocally by NMR experiments performed on the corresponding acetonide.

[17] De Mico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G. J. Org. Chem.

1997, 62, 6974.

[18] Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405.

[19] a) Brown, H. C.; Bhat, K. S.; Randad, R. S. J. Org. Chem. 1989, 54, 1570. b) Paterson, I; Ashton, K.; Britton, R.; Knust, H. Org. Lett. 2003, 5, 1963.

[20] Takai, K. ; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408.

[21] Devos, A.; Remion, J.; Frisque-Hesbain, A. M.; Colens, A.; Ghosez, L. J. Chem.

Soc., Chem. Commun. 1979,1180.

[22] Patois, C.; Savignac, P.; About-Jadet, E.; Collignon, N. Synth. Commun. 1991, 21, 2391.

[23] Allred, G. D.; Liebeskind, L. S. J Am. Chem. Soc. 1996, 118, 2748.

[24] Crichter, D. J.; Pattenden, G. Tetrahedron Lett. 1996, 37, 9107.

[25] Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn.

1979,52, 1989.

[26] Luche, J. L. J. Am. Chem. Soc. 1978, 100, 2226.

[27] In comparison, the specific rotation for dictyostatin-1 reported by Pettit et al. (ref 1) was-20 (c 0. 12, MeOH).

Example 1 Preparation of Keto-Phosphonate 7 l (a) Preparation of hydroxyphosphonate 28 from aldehyde 14 A solution of dimethyl methyl phosphonate (412 1L, 3.80 mmol) in 25 mL THF cooled to-78 °C was treated with a 1.6M solution of n-BuLi in hexanes (2.25 mL, 3.61 mmol). After 30 min, a solution of freshly prepared aldehyde 14 (287 mg, 1.0 mmol) (prepared from 1,3-diol 8 as described in references 9 and 12) in THF (10 mL), precooled to-78 °C, was added via cannula. After 20 min, the reaction was quenched with 60 mL brine. The aqueous layer was extracted with EtOAc (3x). The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (Si02, EtOAc) to afford recovered aldehyde (42 mg, 14%) and a 1: 1 mixture of hydroxyphosphonates 28 (345 mg, 84%) as a yellow oil. Rf 0.28 (EtOAc) ; 1H NMR (500 MHz, CDC13) 5 7.29-7. 23 (4H, m), 6.60-6. 84 (4H, m), 6.75-6. 63 (2H, m), 6.10-6. 03 (2H, m), 5.58-5. 49 (2H, m), 5.27-5. 20 (2H, m), 5.17-5. 11 (2H, m), 4.70-4. 60 (2H, m), 4.50-4. 53 (2H, m), 4.20- 4.12 (1H, m), 3. 98-3. 89 (1H, m), 3.80 (6H, s), 3.78-3. 74 (12H, m), 3.66 (1H, dd, J = 8.5, 2.5 Hz), 3.51-3. 45 (3H, m), 3.08 (1H, ddq, J = 8.4, 8.3, 6.9 Hz), 2.97 (1H, ddq, J = 10.0, 7.1, 6.9 Hz), 2.13-1. 97 (2H, m), 1.90-1. 80 (2H, m), 1.07 (3H, d, J= 6.9 Hz), 1.02 (3H, d, J = 7.0 Hz), 0.99 (3H, d, J = 6.9 Hz), 0.93 (3H, d, J = 6.9 Hz); 13C NMR (125 MHz, CDC13) 8 159.1, 159.0, 135. 8, 134.8, 132.6, 132.3, 140.0, 130.5, 129.4 (2C), 129.3, 129.1, 85.6, 82.2, 74.2, 73.8, 68.6 (2C), 68.2 (2C), 55.2 (2C), 52.5-52. 2 (m, 4C), 41.4 (d, J = 15.0 Hz), 41.2 (d, J = 14.0 Hz), 35.7, 35.4, 31. 3, 30.8, 30.2, 29. 8, 18.3, 18.0, 10.0, 7.2 ; IR (Thin Film) 3357,2958, 1613,1514, 1459,1247 ; HRMS (+ESI) calcd for C2lH3406P [M+H] : 413.2088. Found: 413.2085.

Example 1 (b) Conversion of hydroxyphosphonate 28 to ketophosphonate 7 A solution of hydroxyphosphonates 28 prepared in Example 1 (a) above (345 mg, 0.84 mmol) in 16 mL CH2C12 was treated with Dess-Martin periodinane (424 mg, 1.0 mmol). After 30 min, the slurry was partitioned between sodium thiosulphate (50 mL), NaHCO3 (50 mL) and Et20 (40 mL). The organic layer was washed with brine (20 mL), dried (MgS04) and the volatiles were removed in vacuo. The residue was purified by flash chromatography (SiO2, EtOAc) to afford ketophoshonate 7 (345 mg, 100 %) as a yellow oil. Rf 0.42 (EtOAc); [a] D20-2. 8 (c 1.7, CHC13) ; 1H NMR (400 MHz, CDCl3) 8 7.25 (2H, d, J = 8.6 Hz), 6.86 (2H, d, J = 8.6 Hz), 6.50 (1H, ddd, J= 16.8, 11.6, 10.6 Hz), 6.02 (1H, dd, J = 11.6, 10.8 Hz), 5.51 (1H, dd, J = 10.8, 10.7 Hz), 5.21 (1H, dd, J = 16.8, 2.0 Hz), 5.12 (1H, d, J= 10.2 Hz), 4. 58-4. 49 (2H, m), 3.80 (3H, s), 3.76 (3H, d, J = 11. 3 Hz), 3.74 (3H, d, J = 11. 3 Hz), 3.59 (1H, dd, J = 6.3, 4.0 Hz), 3.30 (1H, dd, J= 22.1, 14.6 Hz), 3.02-2. 92 (2H, m), 2.86-2. 76 (1H, m), 1.20 (3H, d, J= 7.0 Hz), 1.06 (3H, d, J= 7.0 Hz) ; 13C NMR (125 MHz, CDC13) 5 204.6 (d, J= 3.8 Hz), 159.2, 133.5, 132.1, 130.4, 129.9, 129.4, 118.1, 113.7, 83.5, 74.2, 55.2, 52.9, 50.6, 41.3, 40.3, 35.8, 18.9, 12.7 ; IR (Thin Film) 2959,1711, 1574, 1248; HRMS (+ESI) calcd for C21H3206P [M+H] : 411. 1931. Found: 411.1932.

Example 2 Preparation of bis-silyl ether 9 from 1, 3-diol 8 A solution of diol 8 (4.267 g, 15.91 mmol) in 150 mL CHUCK cooled to-78 °C was successively treated with 2,6-lutidine (4.3 mL, 36.59 mmol) and TBSOTf (8.0 mL, 35.01 mmol). After warming to RT over 2 h, the volatiles were removed in vacuo and the residue was purified by flash chromatography (SiO2, hexanes/EtOAc, 9: 1) to provide bis-TBS-ether 9 (7.88 g, 99%) as a colorless oil. Rf 0.73 (hexanes/EtOAc, 9: 1); [a] D20 =-4. 5 (c 0.5, CH2C12) ; 1H NMR (CDCl3, 500 MHz) 6 7.27 (d, J= 8.5 Hz, 2H), 6.89 (d, J= 8.5 Hz, 2H), 4.44 (d, J= 12.6 Hz, 2H), 3.82 (s, 3H), 3.75 (dd, J = 2. 2,6. 6 Hz, 1H), 3. 55 (dd, J= 4.7, 9.1 Hz, 1H), 3.48 (dd, J= 7.5, 9.7 Hz, 1H), 3. 39 (dd, J= 6.9, 9. 8 Hz, 1H), 3.24 (app t, J = 8. 1 Hz, 1H), 1.93-2. 01 (m, 1H), 1. 78 (d-hex, J= 1.9, 6.9 Hz, 1H), 0. 98 (d, J= 6.9 Hz, 3H), 0.91 (d, J= 4.7 Hz, 9H), 0. 85 (d, J= 6.9 Hz, 3H), 0.05 (app t, J = 5.0 Hz, 6H) ; 13C NMR (CDCl3, 125 MHz) 6 159.0, 131.0, 129.0, 113.7, 73.1, 73.0, 72.7, 66.1, 55.3, 38.7, 38.5, 26.1, 25.9, 25.7, 18.4, 18.1, 14.7, 10.8,-4. 1, -5.3 ; IR (CH2Cl2) v 2989,1393, 1249,1066 cm~l ; HRMS (+ESI) calcd for C27Hs204Si2 [M+H]: 497.3477. Found: 497.3468.

Example 3 Preparation of alcohol 10 from bis-silyl ether 9 A solution of bis-TBS ether 9 (9.76 g, 19.67 mmol) prepared in Example 2 above in 100 mL THF/H20 (20: 1) was treated withp-toluenesulfonic acid (1.12 g, 5.9 mmol). After 18 h at RT, 50 mL of saturated NaHCO3 was added to the reaction mixture. The aqueous layer was extracted with EtOAc (2x). The combined organics were dried (MgSO4), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (Si02, hexanes/EtOAc, 7: 3) to provide alcohol 10 (6.99 g, 93%) as a colorless oil. Rf 0.13 (hexanes/EtOAc, 9: 1); [a] D20 =-0. 3 (c 0.5, CHzClx) ; 1H NMR (CDCl3, 400 MHz) 6 7.25 (d, J= 7.7 Hz, 2H), 6.87 (d, J= 7.7 Hz, 2H), 4.42 (d, J= 11.8 Hz, 2H), 3.80 (s, 3H), 3.74 (dd, J= 2.8, 5.6 Hz, 1H), 3.53 (dd, J= 5.6, 8.9 Hz, 1H), 3.45-3. 50 (m, 2H), 3.27 (dd, J= 7.1, 9.0 Hz, 1H), 2.04 (app heptet, J= 5.1 Hz, 1H), 1.97 (br t, J= 5.2 Hz, 1H), 1.83-1. 91 (m, 1H), 0.96 (d, J= 7.0 Hz, 3H), 0.88 (s, 9H), 0.86 (d, J= 7.0 Hz, 3H), 0.05 (d, J= 8.5 Hz, 6H) ; 13C NMR (CDC13, 100 MHz) # 159.1, 130.6, 129.2, 113.7, 74.8, 72.7, 66.2, 55.2, 39.0, 37.6, 26.0, 18.3, 15.0, 11.9,-4. 2; IR (CH2Cl2) v 3426,2929, 1513,1247, 1035 cm- ; HRMS (+ESI) calcd for C21H3sO4Si [M+H]: 383.2612. Found: 383.2605.

Example 4 Preparation of iodide 11 from alcohol 10 A solution of alcohol 10 (1.21 g, 3.18 mmol) prepared in Example 3 above, triphenylphosphine (833.9 mg, 3.18 mmol) and imidazole (216.5 mg, 3.18 mmol) in 30 mL toluene cooled to 0 °C was treated with a solution of iodine (806.9 mg, 3.18 mmol) in 10 mL toluene. After 45 min, the reaction mixture was poured over Et2O/H2O. The aqueous layer was extracted with Et2O. The combined organics were dried (MgSO4), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (SiO2, Pet. Ether/CH2Cl2, 7: 3) to provide iodide 11 (1.35 g, 86%) as a colorless oil. Rf 0.72 (hexanes/EtOAc, 3: 2); [a] D20 = +15.1 (c 1.04, CH2Cl2) ; 1H NMR (CDCl3, 400 MHz) 8 7.25 (d, J= 7.1 Hz, 2H), 6.88 (d, J = 8. 7 Hz, 2H), 4.41 (d, J= 11.3 Hz, 2H), 3.81 (s, 3H), 3.68 (dd, J= 2.8, 5.9 Hz, 1H), 3. 50 (dd, J= 4.7, 9.2 Hz, 1H), 3.19-3. 26 (m, 2H), 3.11 (dd, J= 7.3, 9.4 Hz, 1H), 1. 86- 1. 99 (m, 2H), 0.99 (d, J= 6. 8 Hz, 3H), 0.94 (d, J= 6. 8 Hz, 3H), 0.88 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H) ; 13C NMR (CDCl3, 125 MHz) 8 159.1, 130.7, 129.2, 113.8, 76.2, 72.7, 72.3, 55.3, 39.6, 38.1, 26.1, 18.8, 15.3, 14.5,-4. 1; IR (CH2Cl2) v 2989,1394, 1066 cm-1, HRMS (+ESI) calcd for C2lH37IO3Si [M+NH4]: 510.1895. Found : 510.1902.

Example 5 Preparation of aldehyde 6' 5 (a) Preparation of alcohol 29 from iodide 11 A solution of diisopropylamine (2 mL, 14.177 mmol) in 20 mL THF cooled to-78 °C and LiCl (1.43 g, 33.754 mmol) was treated with a 1.6M solution of n-BuLi in hexanes (8.6 mL, 13.839 mmol). The mixture was allowed to stir at 0 °C for 20 min, was cooled to-78 °C and treated with a solution of (+)-pseudoephedrine propionamide 12 (1.49 g, 6.750 mmol) in 10 mL THF. The reaction mixture was stirred at-78 °C for 1 h, at 0 °C for 15 min and rt for 5 min. The enolate was cooled to 0 °C and treated with a solution of iodide 11 (831.2 mg, 1. 688 mmol) in 10 mL THF.

After 18 h at RT, the reaction was partitioned between half saturated NH4Cl (20 mL) and EtOAc (2x). The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (Si02, hexanes/EtOAc, 3: 2) to afford amide 13 (872.4 mg, 88%) as a mixture of rotamers. A suspension of BH3-NH3 (184.0 mg, 5.961 mmol) in 20 mL THF at 0 °C was treated with freshly prepared LDA (3.9 mmol) and allowed to stir at rt. Thre resulting LAB solution was cooled to 0 °C and treated with a solution of amide 13 (872.4 mg, 1.490 mmol). After 18 h at rt, the reaction was quenched at 0 °C with 15 mL 3N HCI. After 10 min at 0 °C, the aqueous layer was extracted with Et20. The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (SiO2, hexanes/EtOAc, 4: 1) to give the target alcohol 29. Rf 0.50 (hexanes/EtOAc, 3: 2); [a] D20 = -14. 3 (c 0.45, CH2Cl2) ;'H NMR (CDC13, 500 MHz) 8 7.27 (d, J= 8.5 Hz, 2H), 6. 89 (d, J= 8.5 Hz, 2H), 4.42 (d, J= 11.9 Hz, 2H), 3.82 (s, 3H), 3.55 (dd, J= 5.3, 9.1 Hz, 1H), 3.49-3. 53 (m, 1H), 3.46 (dd, J= 2.8, 5.6 Hz, 1H), 3. 36-3. 41 (m, 1H), 3.24 (dd, J= 6.9, 9.1 Hz, 1H), 2.00 (app heptet, J= 5.9 Hz, 1H), 1.71-1. 79 (m, 1H), 1.65-1. 71 (m, 1H), 1.47 (br t, J= 6.0 Hz, 1H), 1.39-1. 45 (m, 1H), 0.96 (d, J= 4.7 Hz, 3H), 0.88 (d, J= 4.4 Hz, 3H), 0.05 (s, 3H), 0.04 (s, 3H); 13C NMR (CDCl3, 125 MHz) 8 159.1, 130.8, 129.2, 113.7, 77.4, 72.7, 67.3, 55. 3, 38.2, 37.9, 33.5, 33.1, 26.2, 18.5, 17.8, 15.4,-3. 6, -4.0 ; IR (CH2Cl2) v 3397,2929, 1513,1247, 1037 cm~l ; HRMS (+ESI) calcd for C24H4404Si [M+Na] : 447.2907. Found: 447.2935.

5 (b) Preparation of aldehyde 6'from alcohol 29 A solution of alcohol 29 (223.2 mg, 0.526 mmol) in 10 mL CH2C12 cooled to 0 °C was treated with TEMPO (16.4 mg, 0.105 mmol) and BAIB (254.1 mg, 0.789 mmol).

After 18 h at rt, the volatiles were removed in vacuo and the residue was purified by flash chromatography (Si02, hexanes/EtOAc, 9: 1) to afford aldehyde 6' (177.5 mg, 80%) as a colorless oil. Rf 0.30 (hexanes/EtOAc, 9: 1); [a] D20 = -18.2 (c 0.44, CH2Cl2) ; 1H NMR (CDCl3, 400 MHz) 8 9.53 (d, J= 2.4 Hz, 1H), 7.24 (d, J= 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.40 (d, J= 11. 5 Hz, 2H), 3.80 (s, 3H), 3. 50 (d, J= 5. 6 Hz, 1H), 3.49 (dd, J = 5. 6,12. 7 Hz, 1H), 3.25 (dd, J= 7.5, 9.0 Hz, 1H), 2.40 (app hexet, J = 6.6 Hz, 1H), 1.94 (app heptet, J= 6.1 Hz, 1H), 1.79 (ddd, J= 5.4, 8.0, 13.4 Hz, 1H), 1.62-1. 73 (m, 1H), 1.19 (ddd, J= 5.7, 8.9, 14.4 Hz, 1H), 1.07 (d, J= 7.0 Hz, 3H), 0.94 (d, J= 7.0 Hz, 3H), 0.89 (s, 9H), 0.87 (d, J= 7.0 Hz, 3H), 0.03 (s, 6H) ; 13C NMR (CDC13, 100 MHz) 8 205.1, 159.1, 130.8, 129.1, 113.7, 72.7, 55.2, 44.2, 38.0, 35. 9,33. 7,26. 1,18. 4,15. 1,14. 5,14. 3, -3.7,-4. 1; IR (CH2C12) v 2930, 1726,1513, 1247,1037 cm7', HRMS (+ESI) calcd for C24H4204Si [M+Na]: 445.2750. Found: 445.2750.

Example 6 Preparation of enone 15 from aldehyde 6' A suspension of dry Ba (OH) 2-8H20 (753.9 mg, 2. 389 mmol) in 20 mL anhydrous THF was treated with a solution of phosphonate 7 produced in Example 1 above (980.2 mg, 2.389 mmol) in 10 mL THF. After 1 h, a solution of aldehyde 6'produced in Example 5 above (841.0 mg, 1.991 mmol) in 10 mL THF/HsO (40: 1) was added to the phosphonate. After 18 h, 50 mL CH2Cl2 and MgS04 was added and the mixture was filtered through celite. The volatiles were removed in vacuo and the residue was purified by flash chromatography (SiO2, hexanes/EtOAc, 4: 1) to afford enone 15 (1.361 g, 97%) as a colorless oil. Rf 0.30 (hexanes/EtOAc, 9: 1); [a] D20 = +16.1 (c 0. 21, CH2Cl2) ; 1H NMR (CDC13, 400 MHz) 8 7.27 (d, J= 8.7 Hz, 2H), 7.24 (d, J= 8.7 Hz, 2H), 6.87 (d, J= 8.7 Hz, 4H), 6.68 (dd, J= 8. 3, 15.8 Hz, 1H), 6.39 (app dt, J = 10. 6,16. 7 Hz, 1H), 6.03 (d, J = 15. 8 Hz, 1H), 6.00 (app t, J= 11.0 Hz, 1H), 5.52 (app t, J= 10.6 Hz, 1H), 5.14 (d, J= 16.7 Hz, 1H), 5.00 (d, J= 10.3 Hz, 1H), 4.54 (d, J= 10.6 Hz, 2H), 4.39 (d, J= 11.7 Hz, 2H), 3.78 (s, 6H), 3. 68 (dd, J= 3.1, 8.2 Hz, 1H), 3.48 (dd, J = 4. 7,9. 0 Hz, 1H), 3.43 (dd, J = 2. 3,6. 3 Hz, 1H), 3.23 (app t, J= 8.5 Hz, 1H), 2.92 (app pentet, J= 7.8 Hz, 1H), 2.72-2. 81 (m, 1H), 2.33 (app heptet, J= 7.0 Hz, 1H), 1.85-1. 96 (m, 1H), 1.54-1. 63 (m, 1H), 1.40 (ddd, J= 4.7, 8.9, 13.4 Hz, 1H), 1.20-1. 28 (m, 1H), 1.18 (d, J= 6.8 Hz, 3H), 1.08 (d, J= 6.8 Hz, 3H), 1.00 (d, J= 6.8 Hz, 3H), 0.92 (d, J= 6. 8 Hz, 3H), 0.88 (s, 9H), 0.82 (d, J= 6.8 Hz, 3H), 0.01 (s, 3H), 0.00 (s, 3H); 13C NMR (CDCl3, 100 MHz) 6 203.1, 159.0, 152. 8, 134.0, 132. 4, 130.8, 129.6, 129.3, 129.1, 127.9, 117.4, 113.7, 84.2, 75.3, 72.7, 55.2, 48.5, 41.1, 37.9, 36.4, 34.3, 33.8, 26.1, 20.2, 28.9, 28.4, 15.3, 14.4, 14.1,-3. 6, -4.1 ; IR (CH2Cl2) v 2958, 1690, 1664,1613, 1513,1247, 1038 cm~l, HRMS (+ESI) calcd for C43H6606Si [M+H] : 707.4701. Found: 707.4696.

Example 7 Preparation of ketone 30 from enone 15 A solution of enone 15 prepared in Example 6 above (469.2 mg, 0.664 mmol) in 25 mL deoxygenated toluene was cannulated directly into a flask containing [Ph3PCuH] 6 (390. 7 mg, 0.199 mmol). After 18 h under argon, the volatiles were removed in vacuo and the residue was purified by flask chromatography (SiO2, hexanes/EtOAc, 9: 1) to afford ketone 30 (438.9 mg, 93%) as a colorless oil. Rf 0.38 (hexanes/EtOAc, 9: 1); [α]D20 = +1.2 (c 0.60, CH2C12) ; 1H NMR (CDC13, 500 MHz) 8 7.28 (d, J= 8.5 Hz, 2H), 7.27 (d, J= 8.5 Hz, 2H), 6.89 (d, J= 8.5 Hz, 4H), 6.46 (app dt, J= 10.7, 17.0 Hz, 1H), 6.05 (app t, J= 11. 0 Hz, 1H), 5. 55 (app t, J = 10. 7 Hz, 1H), 5.20 (d, J= 16.1 Hz, 1H), 5.09 (d, J= 10.4 Hz, 1H), 4. 58 (d, J= 10.4 Hz, 1H), 4.52 (d, J= 10.4 Hz, 1H), 4.43 (d, J= 11.37 Hz, 2H), 3. 82 (s, 6H), 3.66 (dd, J= 3.4, 8.2 Hz, 1H), 3.54 (dd, J= 4.4, 9.1 Hz, 1H), 3.46 (dd, J= 2.5, 6.3 Hz, 1H), 3.26 (app t, J= 8.5 Hz, 1H), 2.79 (ddd, J = 3. 1,6. 9,10. 1 Hz, 1H), 2.75 (app pentet, J= 7.6 Hz, 1H), 2. 37-2. 44 (m, 1H), 1.92-1. 99 (m, 1H), 1.70-1. 76 (m, 1H), 1.62-1. 69 (m, 1H), 1. 37-1. 46 (m, 1H), 1.23- 1.30 (m, 1H), 1.19 (d, J= 7.2 Hz, 3H), 1.11 (d, J= 6.9 Hz, 3H), 1.00-1. 08 (m, 1H), 0.97 (d, J= 6.9 Hz, 3H), 0.90 (s, 9H), 0.85 (d, J= 6.9 Hz, 3H), 0.82 (d, J= 6.6 Hz, 3H), 0.05 (s, 3H), 0.04 (s, 3H) ; 13C NMR (CDCl3, 125 MHz) 8 214.3, 159.1, 134.0, 132.1, 130.9, 130.8, 129.6, 129.3, 129.1, 117.8, 113.7, 83.8, 75.3, 73.0, 72.7, 55.3, 50.4, 42.6, 40.3, 38.2, 36.2, 33. 2, 29.7, 26.2, 20.0, 18. 8, 18.5, 15.2, 14.7, 14.0,-3. 6, - 4.0 ; IR (CH2Cl2) v 2929,1709, 1613, 1513,1247, 1037 cm7l ; HRMS (+ESI) calcd for C43H6806Si [M+Na] : 731.4677. Found: 731.4665.

Example 8 Preparation of diol 31 from ketone 30 Ketone precursor to 15. A biphasic mixture of ketone 30 prepared in Example 7 above (438. 9 mg, 0.619 mmol) in 20 mL CH2Cl2 and 6 mL pH 7 buffer cooled to 0 °C was treated with DDQ (351.6 mg, 1.549 mmol). After 4 h at RT, the aqueous layer was extracted with CH2Cl2. The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (SiO2, Pet. Ether/Et2O, 1: 1) to provide diol 31 (226.5 mg, 78 %) as a colorless oil. Rf 0.44 (hexanes/EtOAc, 3: 2); [a] D = _ 5. 2 (c 0.13, CH2Cl2) ; 1H NMR (CDC13, 500 MHz) 8 6.56 (app dt, J= 10.7, 17.0 Hz, 1H), 6.12 (app t, J= 11.0 Hz, 1H), 5.41 (app t, J= 10.7 Hz, 1H), 5.23 (d, J= 16.0 Hz, 1H), 5.13 (d, J= 10.1 Hz, 1H), 3.74 (dd, J= 4.7, 6.6 Hz, 1H), 3. 58-3. 61 (m, 2H), 3. 48 (dd, J= 3.7, 5.3 Hz, 1H), 2.75 (app dp, J = 7.2, 9.8 Hz, 1H), 2.67 (app d heptet, J= 2.2, 7.2 Hz, 1H), 2.48 (app t, J= 7.9 Hz, 2H), 1.86 (app heptet, J= 6.3 Hz, 1H), 1.66-1. 79 (m, 2H), 1.41-1. 51 (m, 1H), 1.31-1. 138 (m, 1H), 1.18-1. 25 (m, 1H), 1.17 (d, J= 7.2 Hz, 3H), 1.04-1. 11 (m, 1H), 1.00 (d, J= 6.6 Hz, 3H), 0.95 (d, J= 6.9 Hz, 3H), 0.92 (s, 9H), 0.89 (d, J= 6.6 Hz, 3H), 0.87 (d, J= 6.6 Hz, 3H), 0.11 (s, 3H), 0.09 (s, 3H) ; 13C NMR (CDCl3, 125 MHz) 8 214.9, 133.9, 132.0, 130.6, 118.3, 80.7, 75.1, 66.1, 48.5, 41.3, 39.1, 38.2, 35.5, 35.1, 29.8, 29.4, 26.1, 20.3, 18.3, 17.6, 16.2, 15.4, 10.6,-3. 8, -4.0 ; IR (CH2Cl2) v 3405, 2968, 1709,1613, 1513,1247, 1066 cm-1 ; HRMS (+ESI) calcd for C27H5204Si [M+H] : 469.3708. Found: 469.3710.

Example 9 Preparation of triol 16 from diol 31 A solution of hydroxy-ketone 31 prepared in Example 8 above (79.2 mg, 0.169 mmol) in 2 mL Et20 cooled to-30 °C was treated with a 0.5 M solution of Zn (BH4) 2 (1.4 mL, 0.676 mmol). After 30 min, the reaction was quenched with 2 mL saturated NH4Cl and 2 mL 1N HCl. After 20 min at rt, the aqueous layer was extracted with Et20 (3x). The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo.-The residue was purified by flash chromatography (SiO2, hexanes/EtOAc, 3: 2) to provide triol 16 (70.2 mg, 88%) as a colorless oil. Rf 0.30 (hexanes/EtOAc, 3: 2); [a] D20 =-3. 4 (c 0.27, CH2Cl2) ; 1H NMR (CDC13, 500 MHz) 5 6.65 (app dt, J= 10.4, 17 Hz, 1H), 6.21 (app t, J= 11.0 Hz, 1H), 5.27 (dd, J= 5.0, 9.7 Hz, 1H), 5.19 (d, J = 10. 7 Hz, 1H), 3.78 (app t, J = 5.6 Hz, 1H), 3.61 (d, J= 4.7 Hz, 2H), 3.45-3. 51 (m, 2H), 3.39 (br s, 1H), 2.83 (app hex, J= 6.9 Hz, 1H), 2.54 (br s, 1H), 2.33 (br s, 1H), 1.87 (app heptet, J= 5.3 Hz, 1H), 1.68-1. 80 (m, 2H), 1.45-1. 59 (m, 3H), 1.33-1. 41 (m, 1H), 1.04-1. 12 (m, 1H), 0.94-0. 98 (m, 12H), 0.93 (s, 9H), 0.89-0. 92 (m, 6H), 0.12 (s, 3H), 0.10 (s, 3H) ; 13C NMR (CDC13, 125 MHz) 8 134.3, 131.9, 118.9, 113.7, 81. 0, 80. 6,77. 2,66. 1,41. 4, 38. 1,37. 1,36. 4,35. 3,32. 5,32. 3, 30.5, 26.1, 20.6, 18.3, 16.7, 16.2, 15.5, 4.2,-3. 8,-4. 0; IR (CH2Cl2) v 3425,2958, 1711,1610, 1513,1247, 1037 cm-1; HRMS (+ESI) calcd for C27H5404Si [M+H]: 471.3864. Found: 471.3861.

Example 10 Preparation of tris-silyl ether 17 from triol 16 A solution of triol 16 prepared in Example 9 above (84.1 mg, 0.179 mmol) in 3 mL CHUCK cooled to 0 °C was treated with 2,6-lutidine (73 pL, 0.626 mmol) and TBSOTf (135 µL, 0.590 mmol. After 1 h at RT, the volatiles were removed in vacuo and the residue was purified by flash chromatography (SiO2, hexanes/EtOAc, 9: 1) to provide TBS-ether 17 (114.3 mg, 91%) as a colorless oil. Rf 0.52 (hexanes/EtOAc, 9: 1); [α]D20 = -11. 4 (c 0.73, CH2Cl2) ; 1H NMR (CDC13, 400 MHz) b 6.63 (app dt, J= 10.4, 17.1 Hz, 1H), 6.09 (app t, J= 10.8 Hz, 1H), 5.41 (app t, J= 10.4 Hz, 1H), 5.20 (d, J= 16.7 Hz, 1H), 5.12 (d, J= 9.7 Hz, 1H), 3.73-3. 78 (m, 1H), 3.65 (dd, J= 5.0, 9.7 Hz, 1H), 3.44-3. 52 (m, 2H), 3.40 (dd, J= 7.1, 9.7 Hz, 1H), 2.74-2. 85 (m, 1H), 2.26 (d, J= 1.9 Hz, 1H), 1.59-1. 82 (m, 5H), 1. 37-1. 47 (m, 2H), 1. 20-1. 32 (m, 2H), 1.01-1. 08 (m, 1H), 0.96 (d, J= 6.8 Hz, 3H), 0. 84-0. 93 (m, 36H), 0.82 (d, J= 6.8 Hz, 3H), 0.01-0. 09 (m, 18H) ; 13C NMR (CDC13, 100 MHz) 8 135.3, 132.3, 130.0, 117.7, 77.6, 76.5, 65.7, 43.0, 40.6, 37.6, 36.1, 32.7, 32.0, 31.7, 30.3, 26.2, 25.9, 20.1, 18.5, 18.3, 18.1, 17.7, 14.4, 14. 3, 6.9,-3. 7, -3.9,-4. 4, -5.3 ; IR (CH2Cl2) v 2929,1472, 1387, 1253,1085 ; HRMS (+ESI) calcd for C39Hs204Si3 [M+Na]: 721.5413. Found: 721.5424.

Example 11 Preparation of diol 18 from tris-silyl ether 17 A solution of TBS-ether 17 prepared in Example 10 above (95 mg, 0.136 mmol) in 2 mL THF was treated with 800 uL of a 0.9 M solution of TBAF/AcOH (1: 1) in THF.

After 18 h, another 500 IlL of the TBAF solution was added to the reaction mixture.

After 18 h at RT, the crude product was directly purified by flash chromatography (SiO2, hexanes/EtOAc, 10: 1) to afford diol 18 (80.0 mg, 100%) as a crystalline solid.

Rf (hexanes/EtOAc, 9: 1), 1H NMR (CDC13, 500 MHz) 8 6.65 (app dt, J= 10.4, 17.0 Hz, 1H), 6.12 (app t, J = 11.0 Hz, 1H), 5.43 (app t, J = 10. 1 Hz, 1H), 5.23 (d, J= 17.0 Hz, 1H), 5.14 (d, J= 10.1 Hz, 1H), 3.74-3. 79 (m, 1H), 3.61 (app t, J= 5.4 Hz, 1H), 3.49 (dd, J = 3.4, 5.6 Hz, 1H), 2.82 (app hex, J= 6.9 Hz, 1H), 2.43 (t, J= 5.6 Hz, 1H), 2.28 (br s, 1H), 1.86 (app heptet, J= 6.0 Hz, 1H) 1.69-1. 77 (m, 52H), 1.60-1. 68 (m, 1H), 1.39-1. 48 (m, 2H), 1. 27-1. 37 (m, 2H), 1.04-1. 11 (m, 1H), 0.98 (d, J= 6.9 Hz, 3H), 0.97 (d, J= 7.2 Hz, 3H), 0.94 (s, 9H), 0.93 (d, J= 6.8 Hz, 3H), 0.91 (s, 9H), 0.88-0. 90 (m, 6H), 0.08-0. 13 (m, 12H).

Example 12 Preparation of aldehyde 4'from alcohol 18 A stirred solution of alcohol 18 prepared in Example 11 above (10 mg, 17. 24 J. mol) in CH2Cl2 (1.5 ml) at RT was treated with BAIB (7.77 mg, 24.14 pLmol) and TEMPO (0.27 mg, 1.72 pLmol). After 18 h, the volatiles were removed in vacuo and the crude product was purified by flash column chromatography (Si02, PE/EtOAc 10 : 1) to yield aldehyde 4' (9.8 mg, 97 %) as colourless oil. Rf 0.66 (PE/EtOAc, 4: 1) ; [a] =-28. 3 (c = 1.43 in CHCl3); 1H NMR (CDCl3, 500 MHz) 8 9.76 (d, J= 2.8 Hz, 1H) 6.64 (app dtd, J= 16.9, 10.6, 0.9 Hz, 1H), 6.10 (app td, J= 11.0, 0.7 Hz, 1H), 5.41 (app t, J= 10.6 Hz, 1H), 5.23 (dd, J= 16.8, 2.0 Hz, 1H), 5.13 (d, J= 10.1 Hz, 1H), 3.75 (dd, J= 7.9, 4.6 Hz, 1H) 3.74 (dd, J= 7.9, 4.6 Hz, 1H), 3.47 (app dt, J= 5.0, 2.5 Hz, 1H), 2.84-2. 76 (m, 1H), 2.57-2. 52 (m, 1H), 2.23 (d, J= 2.3 Hz, 1H), 13C NMR (CDCl3, 125 MHz) 8 205.1, 135.3, 132.3, 130.0, 117. 8, 78.0, 77.4, 76.4, 50.1, 41.2, 37.8, 36.1, 35.0, 31.9, 30.5, 26.0, 25.9, 20.4, 18.3, 18.1, 17.7, 15.1, 12.3, 7.0,-3. 83,-3. 78, -4.2,- 4. 4 ; IR v 2952,2929, 2857,1725 cm-1; HRMS calculated for C33H6604Si2 [M+Na] : 605.4397. Found: 605.4395.

Example 13 Preparation of bis-silyl ether 32 from alcohol 19 TBSOTf (3.69 ml, 16.07 mmol) was added to a cooled (-78 °C) solution of alcohol 19 (2.8 g, 11.48 mmol) (prepared as described in reference 19) and 2,6-lutidine (4.00 ml, 34.43 mmol) in CH2C12 (100 ml). After 30 min at-78 °C, the reaction was quenched with saturated NH4Cl solution (100 mL). The aqueous was extracted with CH2C12 (3 x 100 ml). The combined organic layers were dried (MgS04) and the volatiles were removed in vacuo. The residue was purified by flash column chromatography (SiO2, PE/EtOAc, 30 : 1) to yield bis-TBS-ether 32 (3.66 mg, 89%) as a colourless oil. Rf 0.74 (PE/EtOAc, 9: 1); [α]D20 =-9.8 (c 1.11, CHCl3) ; 1H NMR (CDC13, 500 MHz) 8 5.81- 5.74 (m, 1H), 5.06-5. 01 (m, 1H), 4.99-4. 97 (m, 1H), 3.76 (ddd, J= 6.9, 5.4, 3.8 Hz, 1H), 3.69-3. 59 (m, 2H), 2.31 (qndt, J= 6.9, 3.8, 1.3 Hz, 1H), 1.60-1. 56 (m, 2H), 1.01 (d, J= 6.9 Hz, 3H), 0.894 (s, 9H), 0.889 (s, 9H), 0.052 (s, 6H), 0.036 (s, 6H) ; 13C NMR (CDCl3, 125 MHz) 8 140.9, 114.4, 72.3, 60.2, 43.3, 36.3, 25.94, 25.91, 18.27, 18.13, 14.7,-4. 48, -4.51,-5. 29.

Example 14 Preparation of aldehyde 33 from bis-silyl ether 32 Ozone was bubbled through a cooled (-78 °C) solution of bis-silyl ether 32 prepared in Example 13 above (2.47 g, 6.89 mmol) in CH2Cl2 (150 ml) until the reaction mixture turned dark blue. The ozone stream was replaced by a stream of oxygen until the solution was clear. Triphenylphosphine (1.98 g, 7.57 mmol) was added at-78 °C and the reaction mixture was allowed to warm to RT. After stirring for 4 h, silica gel was added to the reaction and the solvent was removed in vacuo. The on silica absorbed crude product was purified by flash column chromatography (SiO2, PE/EtOAc 30 : 1) to yield aldehyde 33 as a slight yellow oil which was contaminated with a little triphenylphosphine. Rf 0.47 (PE/EtOAc, 9: 1) ;'H NMR (CDC13, 500 MHz) 5 9.74 (d, J= 1.9 Hz, 1H), 4.19-4. 13 (m, 1H), 3.72-3. 67 (m, 2H), 2.59-2. 53 (m, 1H), 1.81-1. 63 (m, 2H), 1.12 (d, J= 6.9 Hz, 3H), 0.89 (s, 9H), 0. 88 (s, 9H), 0. 082 (s, 3H), 0.068 (s, 3H). 0.045 (s, 3H), 0.041 (s, 3H).

Example 15 Preparation of vinyl iodide 21 from aldehyde 33 A stirred suspension of chromium (II) chloride (5.92 g, 48.20 mmol) in dioxane/THF (40 ml, 1: 1) at 0 °C was treated with a solution of aldehyde 33 prepared in Example 14 above (2. 48 g, 6.89 mmol) in dioxane/THF (15 ml, 1 : 1). After 5 min at 0 °C, iodoform (9.49 g, 24.10 mmol) was added to the reaction mixture. After 18 h at 0 °C, the reaction was quenched with water (50 ml) and EtOAc (50 ml). The aqueous layer was extracted with EtOAc (4 x 50 ml). The combined organics were dried (MgSO4) and the volatiles were removed in vacuo. The crude product was purified by flash column chromatography (SiO2, PE/EtOAc 1: 0~20 : 1) to yield 21 as a colourless oil (2.67 g, 78 % over 2 steps). Rf 0.70 (PE/Et2O, 10: 1); [a] = +6. 4 (c 1.05, CHC13) ; 'H NMR (CDC13,500 MHz) 8 6.48 (dd, J= 14.5, 8.2 Hz, 1H), 6.00 (d, J= 15.1 Hz, 1H), 3.75-3. 71 (m, 1H), 3.67-3. 59 (m, 2H), 2.38-2. 31 (m, 1H), 1.62-1. 57 (m, 2H), 1.02 (d, J= 6.9 Hz, 3H), 0.89 (s, 18H), 0.05 (s, 3H), 0.047 (s, 3H), 0.041 (s, 6H); 13C NMR (CDC13, 125 MHz) 8 148.7, 75.0, 71.9, 59.7, 46.0, 37.0, 25.93, 25.89, 18. 3, 18.1, 15.1,-4. 46, -4.50,-5. 30, -5.32 ; IR v 2955,2929, 2857,1603 cm-1; HRMS calcd for Cl9H4lIO2Si2 [M+H] : 485. 1763. Found: 485. 1766.

Example 16 Preparation of alcohol 34 from vinyl iodide 21 To a stirred solution of vinyl iodide 21 prepared in Example 15 above (2.62 g, 5.41 mmol) in THF (60 ml) at RT was added a premixed solution of TBAF (1M in THF, 10.8 ml) and acetic acid (1.08 ml). After 18 h, the reaction was quenched with saturated NaHCO3 solution (60 ml). The aqueous layer was extracted with EtOAc (3 x 60 ml). The combined organic layers were dried (MgS04) and the volatiles were removed in vacuo. The crude product was purified by flash column chromatography (SiO2, PE/EtOAc 9: 1) to yield alcohol 34 (1.66 g, 83 %) as a colourless oil. Rf 0.17 (PE/EtOAc, 9: 1); [α]D20 = +8.0 (c 1.18, CHCl3) ; 1H NMR (CDC13, 500 MHz) 6 6.46 (dd, J= 14.5, 8.2 Hz, 1H), 6.04 (dd, J= 14.5, 1.0 Hz, 1H), 3.77 (app q, J= 5.4 Hz, 1H), 3.76-3. 68 (m, 1H), 2.44-2. 37 (m, 1H), 1.69 (app q, J= 6.3 Hz, 1H), 1.02 (d, J= 6.9 Hz, 3H), 0.90 (s, 18H), 0.09 (s, 3H), 0.08 (s, 3H) ;"C NMR (CDC13, 125 MHz) 8 <BR> <BR> <BR> <BR> <BR> <BR> 148.5, 75.5, 73.4, 59.9, 45.9, 35.4, 25.8, 18.0, 14.5,-4. 4, -4. 5 ; IR v3339,2954, 2 929, 2857,1603 cm~l, HRMS calcd for C13H27IO2Si [M+NH4] : 388.1163. Found: 388.1165.

Example 17 Preparation of aldehyde 35 from alcohol 34 A solution of alcohol 34 prepared in Example 16 above (1.65, 4.46 mmol) and pyridine (1. 08 ml ; 13. 37mmbl) inCH2Cl2 (20 ml) at RT was treated with Dess Martin periodinane (2.82 g, 6.68 mmol). After 2 h, the reaction was quenched with saturated NaHCO3 (10 ml) and Na2S203 solution (10 ml, 20 %, aq. ). The aqueous layer was extracted with CH2C12 (3 x 20 ml). The combined organic layers were dried (MgSO4) and the volatiles were removed ive vacuo. The crude product was purified by flash column chromatography (SiO2, PE/EtOAc 20: 1) to yield aldehyde 35 (1.49 g, 91 %) as a colourless oil. Rf 0.42 (PE/EtOAc, 9: 1); [α]D20 = +15.8 (c 1.02, CHCl3); 1H NMR (CDC13, 500 MHz) 8 9.79-9. 77 (m, 1H) 6.46 (dd, J= 14.6, 8.4 Hz, 1H), 6.07 (dd, J= 14.5, 1.3 Hz, 1H), 4.14-4. 10 (m, 1H), 2. 55 (dd, J= 6.3, 2.5 Hz, 1H), 2. 50 (dd, J= 5.4, 1.9 Hz, 1H), 2.41-2. 34 (m, 1H), 1.04 (d, J= 6.9 Hz, 3H), 0.88 (s, 9H), 0.08 (s, 3H), 0.05 (s, 3H) ; 13C NMR (CDCl3, 125 MHz) 8 201.3, 147.4, 70.4, 48.4, 46.6, 25.8, 18.0, 15.0,-4. 6 ; IR v 2955,2929, 2857,1725, 1602 cm-1; HRMS calcd for C13H25IO2Si [M+NH4] : 386. 1011. Found: 386. 1007.

Example 18 Preparation of acid 22 from aldehyde 35 A stirred solution of aldehyde 35 prepared in Example 17 above (1.47g, 3.99 mmol) in tert-butanol (15 ml) and 2-methyl-2-butene (1.5 ml) was treated with a mixture of NaC102 (1.44 g, 15.96 mmol) and Na2H2P04 (2.49 g, 15.96 mmol) in water (6 ml).

The reaction mixture was stirred for 2 h at RT, thinned by the addition of brine (15 ml) and EtOAc (15 ml) and extracted with CHUCK (3 x 20 ml). The combined organic layers were dried (MgS04) and the volatiles were removed in vacuo. to yield acid 22 (1.40 g, 92 %) as a colourless oil. Rf 0.31 (PE/EtOAc, 9 : 1), [a] D20-+10. 0 (c 1.22, CHCl3) ; 1H NMR (CDC13, 500 MHz) 8 6.47 (dd, J= 14. 5,8. 2 Hz, 1H), 6.10 (dd, J= 14.5, 1.3 Hz, lH), 4.08-4. 03 (m, 1H), 2.46 (d, J= 6.0 Hz, 2H), 2.44-2. 37 (m, 1H), 1.04 (d, J= 6.9 Hz, 3H), 0.89 (s, 9H), 0.09 (s, 3H), 0.06 (s, 3H) ; 13C NMR (CDCl3, 125 MHz) 8 176.6, 147.3, 76.5, 71.7, 46.2, 39.4, 25. 8, 18.0, 15.0,-4. 67, -4.70 ; IR v 2955,2929, 2857,1709, 1603 cm~l ; HRMS calcd for Cl3Hz5I03Si [M+H]: 385.0690.

Found: 385. 0693.

Example 19 Preparation of ß-ketophosphonate 3'from acid 22 A stirred solution of acid 22 prepared in Example 18 above (0.8 g, 2.08 mmol) in CH2C12 (6 ml) at RT was treated with 1-chloro-N, N-trimethylpropenylamine (551 ul, 4.16 mmol). After 1 h, the volatiles were removed in vacuo under inert gas and the yellow residue was dried for 2 h under high vacum. A stirred solution of bis (2,2, 2- trifluoroethyl) methylphosphonate in THF (10 ml) at-100 °C was treated with a 1M solution of LiHMDS (6.25 mL, 6.25 mmol) in THF. After 10 min, a solution of the previously prepared mixed anhydride in THF (2 ml) was added via cannula to the reaction. After 2 h, the reaction was quenched with saturated NH4C1 (20 ml). The aqueous layer was extracted with Et2O (3 x 20 ml). The combined organic layers were washed with brine (6 ml), dried (MgSO4) and the volatiles were removed in vacuo.

The crude product was purified by flash column chromatography (Si02, PE/EtOAc 9: 1 # 5: 1) to yield ketone 3'as a colourless oil (711.0 mg, 55 %). Rf 0.60 (PE/EtOAc, 3: 2); [α]D20 = 8.3 (c = 1. 07 in CHCl3); 1H NMR (CDCl3, 500 MHz) 8 6.44 (dd, J= 14.5, 8.2 Hz, 1H), 6.07 (dd, J= 14.6, 0.9 Hz, 1H), 4.49-4. 40 (m, 4H) 4.10 (ddd, 1H, J = 6. 2,5. 3,3. 9 Hz), 3.29 (dd, J = 21. 6,15. 4 Hz, 1H), 3.25 (dd, J= 22.0, 15.5 Hz, 1H), 2.69 (dd, J= 16.7, 6.5 Hz, 2H), 2.60 (dd, J= 16. 9, 5.2 Hz, 2H), 2.34 (app. qnd, 1H, J= 7.1, 3.9 Hz), 1.03 (d, J= 6.9 Hz, 3H), 0.87 (s, 9H), 0.08 (s, 3H), 0.02 (s, 3H), 13C NMR (CDC13, 125 MHz) 6 199.6 (d, J= 7.0 Hz), 147.3, 122.4 (qdd, J= 279.4, 8.8, 3.2 Hz), 76.4, 62.4 (qdd, J= 53.7, 15.6, 5.4 Hz), 48.8 (d, J = 4. 8 Hz), 46.1, 43.2, 42.1, 18.0, 14.8, 14.0.-4. 70,4. 77; IR v 2954,2932, 2856,1721 cm' ; HRMS calcd for C18H3oIPOSSi [M+Na]: 649.0447. Found: 649.0440.

Example 20 Preparation of vinyl iodide 23 from P-ketophosphonate 3'and aldehyde 4' 23 A suspension of 18-crown-6 (169 mg, 0.64 mmol) and freshly ground, dry K2CO3 (74 mg, 0.53 mmol) in toluene (1 ml) was stirred for 2 h until the mixture turned clear.

The reaction was cooled to-30 °C and treated with a solution of phosphonate 3' prepared in Example 19 above (50 mg, 79.75 µmol) and aldehyde 4'prepared in Example 12 above (31 mg, 53.17 u. mol) in toluene (0.5 ml). After 2 h at RT the reaction was quenched with saturated NH4Cl (3 ml). The aqueous layer was extracted with CH2C12 (3 x 4 ml). The combined organic layers were dried (MgS04) and concentrated in vacuo. The crude product was purified by-flash column chromatography (SiO2, PE/EtOAc 30: 1) to yield vinyl iodide 23 as a colourless oil (31 mg, 47 %, Z : E 4: 1). Rf 0.46 (PE/EtOAc, 9: 1) ; [a] D20 = 21. 5 (c 1.52, CHC13) ; 1H NMR (CDC13, 500 MHz) 8 6.63 (dtd, J= 16. 8, 10.6, 0. 8 Hz, 1H), 6.49 (dd, J= 14.5, 8.6 Hz, 1H), 6. 31 (dd, J= 11.6, 9.8 Hz, 1H), 6.09 (t, J= 11.0 Hz, 1H), 6.03 (d, J = 11.7 Hz, 1H), 6.00 (dd, J= 14.5, 1.0 Hz, 1H), 5.41 (t, J= 10.2 Hz, 1H), 5.21 (dd, J= 16.9, 1.9 Hz, 1H), 5.12 (d, J = 10.1 Hz, 1H), 4.14 (td, J = 6.0, 3.5 Hz, 1H), 3.76-3. 73 (m, 1H), 3.72-3. 65 (m, 1H), 3.49-3. 45 (m, 2H), 2. 84-2. 76 (m, 1H), 2.55 (s, 1H) 2.54 (d, J= 0.6 Hz, 1H), 2.36-2. 29 (m, 1H), 2.32 (d J= 2.1 Hz, 1H), 1.73-1. 67 (m, 1H), 1.65-1. 59 (m, 2H), 1.41-1. 33 (m, 2H), 1.30-1. 21 (m, 2H), 1.03 (d, J= 6.9 Hz, 3H), 1.00 (d, J= 6.9 Hz, 3H), 0.95 (d, J= 6.8 Hz, 3H), 0.91 (s, 9H), 0. 91-0. 90 9 (m, 1H), 0.90 (d, J= 6. 5 Hz, 3H), 0.89 (s, 9H), 0. 88-0. 86 (m, 1H), 0.87 (s, 9H), 0.83 (d, J= 6. 2 Hz, 3H), 0. 82 (d, J= 6. 8 Hz, 3H), 0.083 (s, 3H), 0.079 (s, 3H), 0.076 (s, 3H), 0.069 (s, 3H), 0.054 (s, 3H), 0.013 (s, 3H); 13C NMR (CDCl3, 125 MHz) S 199. 3, 152.4, 147.9, 135.4, 132.3, 129.9, 125.3, 117.7, 79.7, 77.7, 75.7, 71.1, 49.4, 46.3, 41.1, 37.6, 36.4, 36.1, 35.8, 32.1, 31.3, 30.6, 26.2, 25.94, 25.88, 20.5, 19.0, 18.4, 18.1, 18.0, 17.7, 15.9, 15.7, 6. 8,-3. 65, -3.72,-3. 91, -4.36,-4. 40,4. 80; HRMS calculated for C47H9lIO5Si3 [M+Na]: 969.5117. Found: 969.5101. IR v 2955,2928, 2856,1689, 1611 cmi 1 ; HRMS calcd for C47H91IO5Si3 [M+Na] : 969.5117. Found: 969.5101.

Example 21 Preparation of seco acid 26 from vinyl iodide 23 A solution of vinyl iodide 23 prepared in Example 20 above (30.0 mg, 0.031 mmol) and stannane 5 (65.5 mg, 0.127 mmol) in 750 µL of deoxygenated NMP was treated with copper I thiophenecarboxylate (61.0 mg, 0.316 mmol). After 18 h, the reaction was quenched with saturated NH4Cl, the aqueous layer was extracted with CH2C12 (2x). The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. A solution of crude TIPS ester in 5 mL THF/MeOH (4: 1) was treated with KF (37 mg, 0.637 mmol). After 30 min, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with CHzClz (3x). The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (SiO2, hexanes/Et20, 1: 1) to provide seco-acid 26 (23.5 mg, 83%) as a colorless oil. [a] D20 = +8.7 (c 6.7, CHCl3) ; 1H NMR (500 MHz, CDC13) 8 7.31 (dd, 1H, J= 11.2, 15.4 Hz), 6.65 (dd, 1H, J= 11.2, 11.2 Hz), 6.63 (ddd, 1H, J= 10.5, 10.5, 17.0 Hz), 6.29 (dd, 1H, J= 9.9, 11.5 Hz), 6.09 (m, 1), 6.09 (dd, 1H, J= 10.4, 10.5 Hz), 6.03 (d, 1H, J= 11.5 Hz), 5.61 (d, 1H, J= 11.2 Hz), 5.42 (dd, 1H, J= 10.4, 10.4 Hz), 5.21 (dd, 1H, J= 1. 6,17. 0 Hz), 5.12 (d, 1H, J= 10.5 Hz), 4.25 (ddd, 1H, J= 3.4, 5.6, 6.2 Hz), 3.74 (m, 1H), 3.69 (m, 1H), 3. 48 (m, 1H), 3.46 (m, 1H), 2.80 (m, 1H), 2. 55 (dd, 1H, J= 6.2, 16.4 Hz), 2.50 (dd, 1H, J = 5.6, 16.4 Hz), 1.69 (m, 1H), 1.58-1. 68 (m, 2H), 1.20-1. 42 (m, 6H), 1.09 (d, 3H, J= 6. 8 Hz), 1.00 (d, 3H, J = 7.0 Hz), 0.95 (d, 3H, J= 6. 9 Hz), 0.91 (s, 9H), 0.90 (d, 3H, J = 7.1 Hz), 0.89 (s, 9H), 0.87 (s, 9H), 0.86 (m, 1H), 0.82 (d, 3H, J= 6.9 Hz), 0.82 (d, 3H, J= 6. 8 Hz), 0.09 (s, 3H), 0.08 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H), 0.05 (s, 3H), 0.01 (s, 3H); 13C NMR (125 MHz, CDCl3) 8 199.3, 170.6, 152.3, 147.6, 147.3, 135.4, 132.3, 130.0, 127.4, 125.4, 117.7, 115.1, 79.7, 77.6, 76.7, 71.5, 49.5, 43.2, 41.1, 37.6, 36.4, 36.1, 35.8, 32.1, 31. 3, 30.5, 26.2, 25.9, 25.9, 20.5, 19.0, 18.4, 18.1, 18.1, 17.7, 16.0, 15.7, 6.8,-3. 7, -3.7,-3. 9, -4.3,-4. 4, -4.8 ; IR (neat) v 2957,2857, 1691,1601, 1462,1253, 1082 cm' ; HRMS (+ESI) calcd. for C50Hg407Si3 [M+Na]: 913.6205.

Found: 913.6221.

Example 22 Preparation of macrocycle 2'from seco acid 26 A solution of seco acid 26 prepared in Example 21 above (13.0 mg, 0.015 mmol) and NEt3 (15 IL 0.105 mmol) was successively treated with 2,4, 6- trichlorobenzoylchloride (12 aL, 0.075 mmol) and a solution of DMAP (4.6 mg, 0.037 mmol) in 4 mL toluene. After 30 min at 50 °C, DMAP (4.6 mg, 0.037 mmol) was added to the reaction mixture. After 30 min, the reaction was quenched with saturated NaHC03. The aqueous layer was extracted with EtOAc (3x). The combined organics were dried (NaS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (SiO2, Pet ether/Et20, 30 : 1) to provide macrolactone 2 (10.0 mg, 77%) as a colorless oil and recovered seco-acid 26 (1.0 mg, 8%). [a] D =-11. 2 (c 1.7, CHCl3), lH NMR (500 MHz, CDC13) 5 7.14 (dd, 1H, J= 11.0, 15.6 Hz), 6. 58 (ddd, 1H, J= 10.7, 10.7, 16.5 Hz), 6.54 (dd, 1H, J= 11.3, 11.3 Hz), 6. 36 (dd, 1H, J = 10.3, 11.8 Hz), 6.09 (d, 1H, J= 11.9 Hz), 6.04 (dd, 1H, J = 12. 3, 12.3 Hz), 6.01 (dd, 1H, J= 8.1, 15.2 Hz), 5. 58 (d, 1H, J= 11.6 Hz), 5.45 (dd, 1H, J= 10.4, 10.4 Hz), 5.22 (dd, 1H, J= 4.1, 7.9 Hz), 5.21 (d, 1H, J= 16.8 Hz), 5.15 (d, 1H, J 10.4 Hz), 4.11 (m, 1H), 3.67 (m, 1H), 3.63 (m, 1H), 3. 33 (dd, 1H, J = 1.7, 5.4 Hz), 3.07 (m, 1H), 2.59 (dd, 1H, J= 6.3, 13.9 Hz), 2.53 (dd, 1H, J= 5.6, 13.9 Hz), 2.40 (m, 1H), 1.73 (m, 1H), 1.39-1. 53 (m, 3H), 1.05-1. 28 (m, 3H), 1.11 (d, 3H, J= 6.9 Hz), 1.04 (d, 3H, J= 7.0 Hz), 1.00 (d, 3H, J= 6.89 Hz), 0.94 (s, 9H), 0.92 (s, 9H), 0.89 (s, 9H), 0.83 (d, 3H, J= 7.0 Hz), 0.78 (d, 3H, J= 7.1 Hz), 0.78 (d, 3H, J= 6.8 Hz), 0.68 (m, 1H) ; 13C NMR (125 MHz, CDC13) 8 199.1, 166.5, 150.9, 144.6, 142.9, 133.2, 131.7, 129.8, 128.1, 125.4, 118.2, 117.9, 80.6, 77.6, 76.7, 72.7, 51.8, 44.1, 42.3, 39.2, 37.5, 35.8, 33.8, 31.2, 30.2, 29.7, 26.2, 26. 1 ; 25. 8,20. 5,18. 7,18. 5,18. 1, 18.1, 18.0, 15.7, 14.1, 9.2,-3. 5, -3.6,-4. 2, -4.5,-4. 7, -4.9 ; IR (neat) v 2956,2857, 1707,1472, 1255,1026 cm- ; HRMS (+ESI) calcd for CSoH9206Si3 [M+Na]: 895.6099. Found: 895.6071.

Example 23 Preparation of tris-TBS-dictyostatin 27 from macrocycle 2' 21 27 A premixed solution of macrocycle 2'prepared in Example 22 above (2.6 mg, 2.97 µmol) and CeCls-VHzO (22.2 mg, 59.5 umol) in 1 mL EtOH at-78 °C was treated with NaBH4 (2.25 mg, 59. 5 amol) and then warmed to-30 °C. After 30 min, the reaction was quenched with pH 7 buffer. The aqueous layer was extracted with CH2C12 (3x). The combined organics were dried (MgS04), filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography (Si02, Pet.

Ether/EtOAc, 15: 1) to provide tris-TBS-dictyostatin 27 (1.9 mg, 73 %) as a colorless oil. [a] D20 = + 17. 1 (c 0. 31, CHC13) ; 1H NMR (500 MHz, CDC13) 8 7.07 (dd, 1H, J= 11.2, 12.0 Hz), 6.57 (ddd, 1H, J = 10.5, 10.5, 16.4 Hz), 6.52 (dd, 1H, J = 10.9, 10.9 Hz), 5.98-6. 08 (m, 2H), 5.64 (dd, 1H, J= 9.6, 9.6 Hz), 5.58 (d, 1H, J= 11.4 Hz), 5.37 (m, 2H), 5.20 (d, 1H, J= 16.4 Hz), 5.10 (m, 2H), 4. 58 (m, 1H), 4. 08 (m, 1H), 3. 55 (m, 1H), 3.24 (m, 1H), 3.02 (m, 1H), 2.66 (m, 1H), 2.57 (m, 1H), 1.83 (m, 1H), 1.20-1. 60 (m, 7H), 1.09 (m, 1H), 1.07 (d, 3H, J = 7.0 Hz), 1.02 (d, 3H, J = 6.6 Hz), 0.97 (d, 3H, J = 6. 9 Hz), 0.96 (d, 3H, J = 6.8 Hz), 0.93 (s, 9H), 0.93 (s, 9H), 0.90 (s, 9H), 0.79 (d, 3H, J = 6.9 Hz), 0. 78 (d, 3H, J = 7. 1 Hz), 1.00 (m, 1H), 0.62 (m, 1H), 0.14 (s, 3H), 0.12 (s, 3H), 0.07 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H) ; 13C NMR (125 MHz, CDCl3) 8 166. 3,143. 4,142. 2,132. 4,131. 8,130. 9,129. 7,128. 8,127. 7, 118. 1, 118.0, 80. 3,76. 7-77.2 (3C), 70.0, 53.4, 42.7, 41.5, 39.3, 35.5, 34.9, 34.6, 31. 8, 30.3, 28. 9, 26. 2,25. 9,25. 8,20. 3,19. 8, 18.3, 18.1, 18.0, 15.9, 14.0, 12.1, 10.1,-3. 5, -3.6,- 3.9,-4. 2, -4.4,-4. 8; IR (neat) v 2955, 2858, 1715,1462, 1379,1255, 1074 cm-1; HRMS (+ESI) calcd. for CsoH9406si3 [M+Na]: 897.6256. Found: 897. 6205.

Example 24 Preparation of Dictyostatin (if) To a stirred solution of the tris-TBS-ether 27 obtained in Example 23 above (3 mg, 3. 43 µmol, 1 eq) in MeOH (0.5 ml) at 0 gC was added acidified MeOH (3: 1 MeOH: conc. HCl). After stirring for 5 h at r. t. the reaction mixture was thinned with water (3 ml) and EtOAc (3 ml). The phases were separated and the aqueous phase was extracted with EtOAc (3 x 3 ml). The combined organic layers were dried (MgS04) and concentrated in vacuo. The crude product was purified by flash column chromatography (PE/EtOAc 2: 1 # 1 ;1 #2 : 1) to yield dictyostatin of formula (If) as a white solid (1.4 mg, 2.63 mmol, 76 %).

Rf 0.43 (EtOAc); [a] D20 =-32. 7 (c = 0.22 in CHC13) ; 1H NMR (CDCl3, 500 MHz) 8 7.21 (dd, J = 15.5, 11.4 Hz, 1H), 6.70 (ddd, J = 15.2, 10.4, 10.4 Hz, 1H), 6.65 (dd, J = 11.4, 11.4 Hz, 1H), 6. 18 (dd, J= 15.5, 6.7 Hz, 1H), 6.06 (dd, J = 11. 1,11. 1 Hz, 1H), 5. 55 (d, J= 11.4 Hz, 1H), 5. 55 (dd, J= 10.0, 10.0 Hz), 5.41 (dd, J= 10.8, 8.9 Hz, 1H), 5. 33 (dd, J= 11. 1, 10.6 Hz, 1H), 5.24 (dd, J= 15.2, 1.7 Hz, 1H), 5.14 (dd, J = 10. 4, 1. 7 Hz, 1H), 5.13 (dd, J = 6. 9,5. 1 Hz, 1H), 4.65 (dddd, J= 10.1, 9.5, 2.9, 0. 8 Hz, 1H), 4.05 (ddd, J= 10. 6,4. 0,2. 7 Hz, 1H), 3.34 (m, 1H), 3.16 (ddq, J= 10.6, 6.9, 6.8 Hz, 1H), 3.10 (dd, J= 8.1, 2. 9 Hz, 1H), 2. 76 (m, 1H), 2.60 (m, 1H), 1. 88 (m, 1H), 1.83 (m, 1H), 1.59 (m, 1H), 1.57 (m, 1H), 1.53 (m, 1H), 1.49 (ddd, J= 14.0, 10.6, 2.9 Hz, 1H), 1.42 (ddd, J= 14. 0,10. 1,2. 7 Hz, 1H), 1.24 (ddd, J= 13. 8,10. 3,3. 8 Hz, 1H), 1.15 (d, J= 6.9 Hz, 3H), 1.13 (d, J= 7.0 Hz, 3H), 1.10 (m, 1H), 1. 07 (d, J = 6.9 Hz, 3H), 1.01 (d, J= 6. 8 Hz, 3H), 0.95 (d, J= 6.5 Hz, 3H), 0.93 (d, J= 6.6 Hz, 3H), 0.89 (m, 1H), 0.69 (m, 1H) ; 13C NMR (CDCl3, 125 MHz) 8 168.0, 146.3, 144.8, 134. 9, 134. 5, 133.4, 131. 3,131. 1, 128. 5,118. 5,118. 0,80. 3, 73.7, 70.3, 65.4, 44.0, 35.8, 35. 7, 35.3, 31.2, 21. 8, 19.3, 18.0, 16.0, 10.3 ; HRMS calculated for C32H52O6 [M+Na] : 555.3662. Found: 555. 3663.

High performance liquid chromatography (HPLC) was performed on the dictyostatin prepared above and observed at 225 nm, 256 nm and 263 nm, using an analytical 305 pump from Gilson, a 305 UNIS detector and a 806 manometric module using a CHIRACEL OD (4.6 x 250 mm) column. A filtered solvent mixture of hexane with 20 % isopropanol was used at a flow rate of 1.0 ml per min. The HPLC traces shown in Figures 10 to 12 indicates a purity of higher than 99% for the synthetic dictyostatin.

The lH and 13C nmr spectra are shown in Figures 13 and 14 and also indicate a purity of higher than 99%.

Example 25 Stereochemical assignment of the structure of dictyostatin The planar structure 1 (Figure 1) of dictyostatin, featuring a 22-membered macrolactone ring with five alkenes (2Z, 4E, 1 OZ, 23Z) and a characteristic sequence of methyl and hydroxyl-bearing stereocentres, was determined from spectroscopic data (H and 13C NMR, COSY and HMQC) obtained for a Corallistidae derived sample (1 mg).

Optimum lu signal dispersion was realised in CD30D at the highest available field strength (700 and 800 MHz). The 3JH,H coupling constants were extracted from a combination of 2D J-resolved spectra and homonuclear decoupling experiments, while measurement of heteronuclear coupling constants (23JC, H) relied on analysis of HSQC-HECADE spectra (Nanz, D. and W. Kozminski, J. Magn (2000) Reson.

142: 294 and Marquez, B. , W. H. Gerwick and R. T. Williamson (2001) Magn. Peson.

Chem. 39: 499). A series of 1D and 2D NOESY experiments proved invaluable in defining relationships between the three isolated stereoclusters indicated in 1.

Notably, the coupling constants and NOESY correlations suggested the C-2 to C-16 region is relatively rigid, while at least two rapidly interconverting conformations must be considered for the C-17 to C-21 region.

As depicted in Figure 2, J-based configuration analysis indicated a 6, 7-anti- 7, 9-anti relationship within the C-5 to C-10 segment 2. The small coupling between H-6 and H-7 suggested a gauche relationship, while a large heteronuclear coupling from H-6 to C-7 and a relatively large coupling from H-7 to Me-6 supported an anti relationship between the adjacent methyl and hydroxyl substituents, confirmed by a number of strong NOESY correlations. The diastereotopic methylene protons at C-8 could be related to H-7 and H-9 through large dipolar couplings, H-8a to H-7 and H- 8b to H-9, and small couplings, H-8a to H-9 and H-8b to H-7. A large coupling, H-8a to C-7, and a small coupling, H-8a to C-9, established the relationship between H-8a and the two carbinol stereocentres. Couplings of similar magnitude were observed from H-8b to C-9 and H-8b to C-7, securing the 7, 9-anti diol relationship.

The homo-and heteronuclear coupling constants observed for the C-11 to C- 16 subunit of dictyostatin 3 are listed in Figure 3. Assignment of an anti relationship between the adjacent methyl and hydroxyl substituents at C-12 and C-13 was determined by the small couplings observed from H-12 to C-13, H-12 to H-13 and H- 13 to Me-12. A large coupling between H-13 and H-14 suggested an antiperiplanar relationship between these protons, supported by small couplings from H-13 to C-15 and H-13 to Me-14. A series of NOESY correlations (H-12 and H-15a, H-13 and H- 15b, and H-13 and Me-14) confirmed the 13, 14-syn relationship. Connectivity between the 1,3-related methyl-bearing stereocentres at C-14 and C-16 relied on a confident assignment of the diastereotopic methylene protons at C-15. Large dipolar couplings, H-15ato H-16 and H-15b to H-14, and small couplings, H-15ato H-14 and H-15b to H-16, together with small heteronuclear couplings between both methylene protons and C-13 and Me-16 supported the relative orientation depicted (14, 16-syn).

Analysis of the NOESY spectra revealed a series of correlations, H-14 to H-11, H-11 to H-10 and H-10 to H-8b, indicating these protons are oriented on the same face of the macrolide ring. Additional correlations from H-15a to H-12, H-15b to H-13 and H-12 to H-9, in combination with the relative stereochemistry determined by J-based configuration analysis established the connectivity between the isolated C-6 to C-9 and C-12 to C-16 stereoclusters.

As depicted in Figure 4, both the homo-and heteronuclear couplings observed for the C-17 to C-21 segment of dictyostatin suggested the contribution of two or more rapidly interconverting conformations. In particular, medium couplings from H- 19 to H-18a, H-19 to H-20, H-19 to Me-20 and H-20 to H-21 supported a degree of conformational flexibility. Establishing a relationship between the substituents at C- 21 and C-22 relied on both J-based configuration analysis and relevant NOESY correlations. A relatively large homonuclear coupling between H-21 and H-22 and small couplings, H-21 to C-23 and H-21 to Me-22, supported an antiperiplanar relationship. Two key NOESY correlations, between H-20 and Me-22 and between H-23 and H-4, supported the relative assignment as depicted. Analysis of models representing potential C-19 and C-20 stereoisomers suggested this was most consistent with an all-syn relationship for the substituents at C-19, C-20 and C-21.

The strong NOESY correlations from H-17b to H-20 and H-18a to H-21, along with the observed couplings in this region, were rationalised by the conformational reorganisation required to accommodate both the C-1/C-2 s-trans and s-cis rotamers 4A and 4B indicated in Fig. 4. While the determination of the relationship between this isolated stereocluster and the C-1 to C-16 segment was not possible through J- based configuration analysis, molecular modelling of the two possible stereochemical permutations favoured the assignment of structure (If) (Figure 5).

Using Macromodel (Version 7.2) Mohamadi, F. , N. G. J. Richards, W. C.

Guida, R. Kiskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson and W. C.

Still (1990) J Comput. Chem. 11: 440), a 10,000 step Monte Carlo search was performed with the MM2* force field and the generalised Born/surface area (CB/SA) water solvent model (Still, W. C. , A. Tempczyk, R. C. Hawley and T. Hendrickson (1990) J Am. Chem. Soc. 112: 6127). For structure (If), a series of discrete families of low energy conformations were found, with only two conformers within 2.00 kcal/mol of the global minimum. The lowest energy conformation 4A, in which the lactone adopts a C-1/C-2 s-trans arrangement, accounted for the observed NOESY correlations from H-17b to H-20, H-19 to H-22, and H-22 to H-25 (4B (accounting for the strong NOESY correlation from H-18a to H-21) was found 3.9 kcal/mol above the lowest energy conformation 4A). The energy difference seems too high for the ready exchange observed between the two conformations by NMR. However, for complex, polar molecules, the energetic profile obtained depends on the force field employed: N. Nevins, D. Cicero and J. P. Snyder (1990) J. Org. Chem. 64: 3979) Furthermore, examination of the calculated dihedral anglest and correlation to a corresponding series of 3JH, H coupling constants, resulted in an acceptable match with the experimental NMR data. Finally, the remarkable homology between the relative stereochemistry, as assigned by these studies, and that of the structurally related polyketide discodermolide (6) (ter Haar, E. , R. J. Kowalski, E. Hamel, C. M. Lin, R.

E. Longley, S. P. Gunasekera, H. S. Rosenkranz and B. W. Day (1996) Biochemistry 35: 243) suggests the assignment of the full absolute configuration for dictyostatin, as shown in (If), based on a common biogenesis.

A comparison of the 1H and 13C nmr spectra for this naturally obtained dictyostatin 1 and that of the dictyostatin prepared in Examples 1 to 24 above confirm that the relative and absolute configuration of dictyostatin 1 is indeed as predicted above, i. e. (-)-dictyostatin 1 has the formula (If).

Example 26 Effect of natural dictyostatin 1 and synthetic dictyostatin 1 on cell cycle progression of A549 human lung adenocarcinoma cells in comparison to paclitaxel A549 human lung adenocarcinoma cells were used as cell cycle targets to compare the effects on perturbation of the cell cycle of synthetic dictyostatin 1 to that of natural dictyostatin 1 and the known mitotic spindle inhibitor paclitaxel. Cell cycle analyses were performed as follows: A549 human lung adenocarcinoma cells were incubated in tissue culture media (TCM = Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 60 mg/ml 1-glutamine, 18 mM HEPES, 0.05 mg/ml gentamicin and 10% fetal bovine serum) at 37°C in 5% C02 in air in the presence or absence of varying concentrations of natural dictyostatin 1, synthetic dictyostatin 1 or paclitaxel for 24 hours.

Cells were harvested, fixed in ethanol and stained with 0.02 mg/ml of propidium iodide (P. I. ) together with 0.1 mg/ml of RNAse A. This procedure permeabilizes cells and allows entry of P. I. to stain DNA (propidium iodide also stains double stranded RNA, so RNAse is included in the preparation to exclude this possibility). Stained preparations were analyzed on a Coulter EPICS ELITE with 488 nm excitation. Fluorescence measurements and resulting DNA histograms were collected from at least 3,000 P. I. stained cells at an emission wavelength of 690 nm.

Raw histogram data was further analyzed using a cell cycle analysis program (Multicycle, Phoenix Flow Systems).

The results of these experiments are shown in Table 1 below. Non-treated control A549 cells exhibited a typical pattern of cell cycling, with a large percentage (51%) of the cell population comprising the Gl population (first peak) with lesser percentages comprising both the S (12%) and G2/M (23 %) phases of the cell cycle.

A549 cells treated with 100 nM paclitaxel exhibited decreased percentages of cells comprising the Gl population (8%) and corresponding increased percentages in both S (8%) and G2/M (77%) phases of the cell cycle indicating paclitaxel's ability to induce G2/M block. A549 cells treated with 100 nM natural dictyostatin 1 exhibited decreased percentages of cells comprising the Gl population (19%) and corresponding increased percentages in both S (12%) and G2/M (60%) phases of the cell cycle indicating natural dictyostatin's ability to induce G2/M block. A549 cells treated with 100 nM synthetic dictyostatin 1 exhibited decreased percentages of cells comprising the Gl population (16%) and corresponding increased percentages in both S (12%) and G2/M (62%) phases of the cell cycle indicating synthetic dictyostatin's ability to induce G2/M block. The effect on the cell cycle of natural and synthetic dictyostatin 1 is indistinguishable in this analysis.

Table 1. Effect on Cell Cycle Progression of A549 Human Lung Adenocarcinoma Cells of natural and synthetic dictyostatin-1 in Comparison to Paclitaxel Treatment G1 S G2/M Non-treated 51% 12% 23% 100nM Paclitaxel 8% 8% 77% 1000nM Natural 10% 9% 76% Dictyostatin 1 100nM Natural 19% 12% 60% Dictyostatin 1 lOnM Natural Dictyostatin 1 46% 15% 23% 1000nM Synthetic 12% 9% 75% Dictyostatin 1 100nM Synthetic 16% 12% 62% Dicytostatin 1 10nM Synthetic 46% 15% 24% Dicytostatin 1 Example 27 Immunofluoreseent detection of effects on the microtubule matrix in tumor cells Natural and synthetic-dictyostatin 1 were evaluated as to their effects on the microtubule network of cells using anti-alpha-tubulin monoclonal antibodies in order to compare their effects with those of the mitotic spindle inhibitor paclitaxel. Cells treated with the anti-cancer drug paclitaxel routinely exhibit abnormal formation of multiple centriolar-radiating microtubules with extensive clusters of associated microtubular"bundles", unlike the fine"mesh"of individual microtubules which make up the cytoskeletal network.

A549 human lung adenocarcinoma cells were maintained in tissue culture media [TCM = Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 60 mg/ml 1-glutamine, 18 mM HEPES, 0.05 mg/ml gentamicin and 10% fetal bovine serum] and cultured in plastic culture flasks at 37°C in humidified air containing 5% C02. Stock cultures of A549 cells were subcultured 1: 10 every 3 to 4 days. On day 1,1. 25 x 105 A549 cells were- sub-cultured in TCM overnight at 37°C in 5% C02 on 22 mm2 coverslips in 6-well microtiter plates. On day 2, TCM was removed and replaced with various concentrations of natural dictyostatin 1, synthetic dictyostatin 1 or paclitaxel, in TCM or TCM without drug (control) and incubated overnight at 37°C in 5% CO2. On day 3, TCM was removed and cells attached to coverslips were fixed with a 3.7% formaldehyde solution in Dulbecco's PBS for 10 minutes at room temperature. Cells were permeabilized with a 2% Triton X-100 solution, 2 ml per well, for 5 minutes at room temperature and washed twice in Dulbecco's PBS prior to staining.

To each well containing cells attached to coverslips a 2 ml volume of mouse monoclonal anti-alpha-tubulin (Cat # T-5168, Sigma Chemical Co. ) diluted 1: 1000 in Dulbecco's phosphate buffered saline (D-PBS) was added and the cells incubated at room temperature for 45 minutes. A 2 rnl volume of goat-anti-mouse-IgG-FITC conjugate (Cat # T-5262, Sigma Chemical Co. ) diluted 1: 1000 in D-PBS was added and the cells incubated at room temperature for 45 minutes. Coverslips were rinsed three times with sterile distilled water, air-dried and mounted on slides and observed under the microscope using epifluorescence illumination for the presence of abnormal aster and microtubule formation.

The results of these experiments are shown in Figures 15A, 15B, 15C and 15D. Figure 15A shows a non-treated control A549 cell preparation with characteristic staining of individual microtubules with fluorescent anti-alpha-tubulin indicated by a fine network"mesh"of microtubular material. Nuclei are uniform and rounded as indicated by red staining with propidium iodide. Figure 15B shows corresponding A549 cells treated with 100 nM Paclitaxel and exhibits a characteristic formation of microtubular"bundles"but also a substantial amount of"non-bundled" microtubular material still remained in the cytoplasm. Figure 15C shows corresponding A549 cells treated with 100 nM natural dictyostatin 1 and exhibiting extensive microtubule bundling resulting in almost complete depletion of non- bundled microtubular material in the cytoplasm. Figure 15D shows corresponding A549 cells treated with 100 nM synthetic dictyostatin 1 and also exhibits extensive microtubule bundling resulting in almost complete depletion of non-bundled microtubular material in the cytoplasm. The results observed for natural and synthetic dictyostatin 1 are indistinguishable in this assay.- Example 28 Cytotoxic effects of dictyostatin 1 on tumor cell lines Natural dictyostatin 1 and synthetic dictyostatin 1 were analyzed as to their effects on the proliferation of a panel of tumor cell lines including both cell lines which are sensitive and resistant to the anticancer drug paclitaxel. The cell lines tested include: A549 human lung adenocarcinoma, NCI-ADR-RES (Formerly MCF-7/ADR) human breast cancer, PANC-1 human pancreatic cancer, AsPC-1 pancreatic adenocarcinoma, MIA PaCa2 human pancreatic carcinoma, DLD-1 human colorectal carcinoma, and P388 murine leukemia cell lines. P388 cells were obtained from Dr.

R. Camalier, National Cancer Institute, Bethesda, MD, and A549, PANC-1, AsPC-1, MIA PaCa2, DLD-1 and NCI-ADR-RES cells were obtained from American Type Culture Collection, Rockville, MD. The A549, NCI-ADR-RES, PANC-1, AsPC-1, DLD-1 and P388 cell lines are maintained in Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 100 U/mL penicillin 100 u, g/ml streptomycin, 60 llg/ml L-glutamine, 18 mM HEPES, 0.05 mg/mL gentamycin and 10% fetal bovine serum (for the PANC-1, AsPC-1 and DLD-1 cell lines the media is also supplemented with 100 llg/ml sodium pyruvate and 2.5 mg/ml glucose). MIA PaCa2 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 4 mM L-glutamine, 4500 mg/L glucose and 1500 mg/L sodium bicarbonate and additionally supplemented with 100 U/mL penicillin 100 pg/ml streptomycin, 0.05 mg/mL gentamycin, 2.5% horse serum, and 10% fetal bovine serum. Cell lines-are cultured in plastic tissue culture flasks-and kept in an incubator at 37°C in humidified air containing 5% C02. To assess the antiproliferative effects of agents against the various cell lines, 200 pil cultures (96-well tissue culture plates, Nunc, Denmark) are first established at 3 x 104 cells/ml for adherent lines (A549, NCI ADR-RES, PANC-1, AsPC-1, MIA-PaCa2, and DLD-1) and 1 x 105 for non- adherent lines (P388) in tissue culture medium and incubated for 24 hr at 37°C in 10% C02 in air in order to allow cells to attach. A volume of 100 ul of medium is removed from each test well and 100 gel of medium containing serial, two-fold dilutions of the test agent is added to each well containing tumor cells. Medium without drug is also added to wells containing tumor cells which serve as no drug controls. Positive drug controls are included to monitor drug sensitivity of each of the cell lines. These include varying dilutions of 5-fluorouracil, doxorubicin and Paclitaxel. After 72-h exposures (Adherent cell lines) or 48-hr exposure (Non- adherent cell lines), tumor cells are enumerated using 3- [4, 5-Dimethylthiazol-2-yl] - 2,5-diphenyltetrazolium bromide (MTT) (M. C. Alley, et al. , Cancer Res. 48: 589, 1988) as follows : A volume of 75 ul of warm growth media containing 5 mg/ml MTT is added to each well, cultures returned to the incubator, and left undisturbed for 3 hours. To spectrophotometrically quantitate formation of reduced formazan, plates are centrifuged (900 x g, 5 minutes), culture fluids removed by aspiration, and 200 ul of acidified isopropanol (2 ml concentrated HCl/liter isopropanol) added per well. The absorbance of the resulting solutions is measured at 570 nm with a plate reader (Spectra II (Tecan Laboratories). The absorbance of tests wells is divided by the absorbance of drug-free wells, and the concentration of agent that results in 50% of the absorbance of untreated cultures (ICso) is determined by linear regression of logit-transformed data (D. J. Finney, Statistical Method in Biological Assay, third ed., pp. 316-348, Charles Griffin Co. , London, 1978). A linear relationship between tumor cell number and formazan production has been routinely observed over the range of cell densities observed in these experiments. The two standard drug controls (indicated above) are included in each assay to monitor the drug sensitivity of each of the cell lines and IC50 values are determined for each drug-cell combination. The results in Table 2 below show that the cytotoxic activity of synthetic dictyostatin 1 is essentially the same as that of natural dictyostatin 1. The data also indicates that both natural and syntheticdictyostatin1-show significant-activity against the-paclitaxel- resistant NCI/ADR-Res tumor cell line.

Table 2. ICso Values for growth inhibition in nM P388 A549 NCI-ADR-PANC-1 DLD-1 AsPC- Res PaCa2 1 Natural 7. 5 1. 6 20. 7 3. 8 1. 5 1. 2 9. 8 Dictyostatin 1 Synthetic 4.1 1.1 14.8 0.6 1.3 1.4 14. 2 Dictyostatin 1 Paclitaxel 34.0 4.7 2892.5 41 15.2 28.7 22. 9