RU2822750C2 - Macrocyclic analogues and methods of their application and preparation - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: the invention relates to a macrocyclic compound having the formula

where A denotes a C3 saturated hydrocarbon skeleton, and the specified skeleton has 2 substituents independently selected from Q1; each Q1 is independently selected from OR1 and NR2R1; each of R1 and R2 represents H; each of D and D' is independently selected from R3 and R3, where R3 stands for H or C 1 alkyl; n is equal to 0; E is missing; G stands for O; J and J' taken together represent =CH2; Q stands for C1 alkyl; T stands for ethylene; U and U' taken together denote =CH2; X stands for H; each of Y and Y' stands for H; Z and Z' taken together denote =O; or its pharmacologically acceptable salt.

EFFECT: new compound with anticancer or antimitotic (blocking mitosis) activity has been obtained, which may be suitable for pharmaceutical use.

2 cl, 2 tbl

Description

BACKGROUND OF THE INVENTION

This invention relates to pharmaceutically active macrolides. Halichondrin B is a potent anticancer agent originally isolated from the marine sponge Halichondria okadai and subsequently found in Axinella spp., Phakellia carteri spp., and Lissondendryx spp.

The total synthesis of Halichondrin B was published in 1992 (Aicher, TD et al., J. Am. Chem. Soc. 114:3162-3164). Halichondrin B has demonstrated inhibition of tubulin polymerization, microtubule assembly, beta ^s -tubulin cross-linking, GTP and vinblastine binding to tubulin, and tubulin-dependent GTP hydrolysis in vitro and has shown anticancer properties in vivo and in vitro.

The essence of the invention

The present invention provides halichondrin analogues having pharmaceutical activity, such as anticancer or antimitotic (mitosis blocking) activity. These compounds are significantly smaller than halichondrin B. This invention describes a compound having formula (I):

In formula (I), A is a C _1-6 saturated or a C _2-6 unsaturated hydrocarbon skeleton, which skeleton is unsubstituted or has 1-13 substituents, preferably 1-10 substituents, for example at least one substituent selected from cyano , halogen, azido, Q ₁ and oxo. Each Q ₁ is independently selected from OR ₁ , SR ₁ , SO ₂ R ₁ , OSO ₂ R ₁ , NR ₂ R ₁ , NR ₂ (CO) R ₁ , NR ₂ (CO) (CO) R ₁ , NR ₄ (CO )NR ₂ R ₁ , NR ₂ (CO)OR ₁ , (CO)OR ₁ , O(CO)R ₁ , (CO)NR ₂ R ₁ and O(CO)NR ₂ R ₁ . The number of substituents can be, for example, 1-6, 1-8, 2-5 or 1-4. Throughout the description, numerical ranges are intended to include their limiting values.

Each of R ₁ , R ₂ , R ₄ , R ₅ and R ₆ is independently selected from H, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _6-10 aryl , C _6-10 haloaryl (for example, p-fluorophenyl or p-chlorophenyl), C _6-10 hydroxyaryl, C _1-4 alkoxy-C ₆ -aryl (for example, p-methoxyphenyl, 3,4,5-trimethoxyphenyl, p -ethoxyphenyl or 3,5-diethoxyphenyl), C _6-10 aryl-C _1-6 alkyl (for example, benzyl or phenethyl), C _1-6 alkyl-C _6-10 aryl, C _6-10 haloaryl-C _{1- 6} alkyl, C _1-6 alkyl-C _6-10 haloaryl, (C _1-3 alkoxy-C6-aryl)-C _1-3 alkyl, C _2-9 heterocyclic radical, C _2-9 heterocyclic radical-C _{1- 6} alkyl, C _2-9 heteroaryl and C _2-9 heteroaryl-C _1-6 alkyl. There may be more than one R ₁ , for example if A is substituted with two different alkoxy (OR ₁ ) groups such as butoxy and 2-aminoethoxy.

Examples A include 2,3-dihydroxypropyl, 2-hydroxyethyl, 3-hydroxy-4-perfluorobutyl, 2,4,5-trihydroxypentyl, 3-amino-2-hydroxypropyl, 1,2-dihydroxyethyl, 2,3-dihydroxy -4-perfluorobutyl, 3-cyano-2-hydroxypropyl, 2-amino-1-hydroxyethyl, 3-azido-2-hydroxypropyl, 3,3-difluoro-2,4-dihydroxybutyl, 2,4-dihydroxybutyl, 2-hydroxy -2(p-fluorophenyl)ethyl, _-CH2 (CO) (substituted or unsubstituted aryl), _-CH2 (CO)(alkyl or substituted alkyl such as haloalkyl or hydroxyalkyl) and 3,3-difluoro-2 -hydroxypent-4-enyl.

Examples of Q ₁ include -NH(CO) (CO) - (heterocyclic radical or heteroaryl), -OSO ₂ - (aryl or substituted aryl), -O(CO)NH - (aryl or substituted aryl), amino-alkyl , hydroxyalkyl, -NH(CO)(CO) - (aryl or substituted aryl), -NH(CO)(alkyl)(heteroaryl or heterocyclic radical), O (substituted or unsubstituted alkyl) (substituted or unsubstituted aryl) and -NH (CO) (alkyl) (aryl or substituted aryl).

Each of D or D' is independently selected from R ₃ and OR ₃ , where R ₃ is H, C _1-3 alkyl or C _1-3 haloalkyl. Examples of D and D' are methoxy, methyl, ethoxy and ethyl. In some embodiments, one of D and D' is H.

The value n is 1 or preferably. 0, resulting in either a six-membered or five-membered ring. This ring may be substituted or unsubstituted, for example where E is _R5 or _OR5 , or may be a heterocyclic radical or cycloalkyl, for example where G is S, _CH2 , _NR6 or preferably O.

Each of J and J' is independently H, C _1-6 alkoxy or C _1-6 alkyl; or J and J' taken together are =CH ₂ or -O- (straight or branched C _1-5 alkylene or alkylidene) -O-, such as exocyclic methylidene, isopropylidene, methylene or ethylene. Q is C _1-3 alkyl and is preferably methyl. T is ethylene or ethenylene, optionally substituted with (CO)OR ₇ where R ₇ is H or C _1-6 alkyl. Each of U and U' is independently H, C _1-6 alkoxy or C _1-6 alkyl; or U and U' taken together denote =CH ₂ or -O- (straight or branched C _1-5 alkylene or alkylidene) -O-. X is H or C _1-6 alkoxy. Each of Y and Y' is independently H or C _1-6 alkoxy; or Y and Y' taken together denote =O, =CH ₂ or -O- (straight or branched C _1-5 alkylene or alkylidene) -O-. Each of Z and Z' is independently H or C _1-6 alkoxy; or Z and Z' taken together denote =O, =CH ₂ or -O- (straight or branched C _1-5 alkylene or alkylidene) -O-.

This invention provides compounds of sufficient stability to be suitable for pharmaceutical use. This invention also describes pharmaceutically acceptable salts of the described compounds, describes new synthetic intermediates, pharmaceutical compositions containing one or more of the described compounds, methods for preparing the described compounds or intermediates, and methods of using the described compounds or compositions. Methods of use include methods for reversibly or irreversibly inhibiting mitosis in a cell and inhibiting cancer or tumor growth in vitro, in vivo or in a patient. The present invention also describes methods for identifying an antimitotic or anticancer agent, such as an agent having a reversible effect or, preferably, an irreversible effect.

Brief description of the figures

Fig. 1 is a graph showing the percentage of cells that completed mitosis and returned to G ₁ phase as a function of the concentration of compound B1939 in a mitotic block assay. The minimum concentration required for complete mitotic block at 0 hours is 10 nM. The minimum concentration required for complete mitotic block at 10 hours (after washing) is also 10 nM. The reversibility coefficient is therefore equal to 1 for B1939. Superimposed on this graph is a curve showing the percentage of viable cells after 5 days as a function of the concentration of compound B1939. Viability drops to very low levels at the same concentration at which 10-hour mitotic block occurs.

Fig. 2 is a graph showing the percentage of cells that completed mitosis and returned to G ₁ phase as a function of the concentration of compound B2042 in the mitotic block reversibility assay. The minimum concentration required for complete mitotic block at 0 hours is 3 nM. The minimum concentration required for complete mitotic block at 10 hours (after washing) is 100 nM. The reversibility coefficient for B2042 is therefore 33. Superimposed on this graph is a curve showing the percentage of viable cells after 5 days as a function of the concentration of compound B2042. Viability drops to very low levels at the same concentration at which 10-hour mitotic block occurs.

Fig. 3 is a graph showing the average tumor volume in microliters as a function of time (days) in the LOX melanoma xenograft growth inhibition assay. This illustrates the antitumor activity of the compound of formula (I), compound B1939. Paclitaxel and vehicle were used as controls.

Fig. 4 is a graph showing average body weight per mouse as a function of time (days) in the test described in FIG. 3.

Fig. 5 is a graph showing the average tumor volume in microliters as a function of time (days) in the COLO 205 human colon cancer xenograft growth inhibition assay demonstrating the antitumor activity of vinblastine and vincristine.

Detailed Description of the Invention

A. Definitions

B. Halichondrin analogues

C. Synthesis of halichondrin analogues

D. Pharmacological activity

E. Applications

A. Definitions

The following terms, as used herein, are defined in part below.

The hydrocarbon skeleton contains carbon and hydrogen atoms and can be linear, branched or cyclic. Unsaturated hydrocarbons include one, two, three or more C-C double bonds (sp ² ) or C-C triple bonds (sp). Examples of unsaturated hydrocarbon radicals include ethynyl, 2-propynyl, 1-propenyl, 2-butenyl, 1,3-butadienyl, 2-pentenyl, vinyl(ethenyl), allyl and isopropenyl. Examples of bivalent unsaturated hydrocarbon radicals include alkenylenes and alkylidenes such as methylidine, ethylidene, ethylidene, vinylidene and isopropylidene. Typically, the compounds of this invention have hydrocarbon skeletons ("A" in formula (I)) that are substituted, for example, with hydroxy, amino, cyano, azido, hetroaryl, aryl and other radicals described herein. Hydrocarbon skeletons may have two geminal hydrogens substituted with an oxo group, a divalent carbonyl oxygen (=O), or a ring-forming substituent such as -O- (straight or branched alkylene or alkylidene) -O-, to form an acetal or ketal .

C _1-6 alkyl includes linear, branched and cyclic hydrocarbons such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, neopentyl, tert -pentyl, cyclopentyl, hexyl, isohexyl, sec-hexyl, cyclohexyl, 2-methylpentyl, tert-hexyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,3-dimethylbutyl and 2,3-dimethylbut-2-yl . Alkoxy (-OR), alkylthio (-SR) and other alkyl-derived radicals (substituted, unsaturated or bivalent) are analogous to alkyl groups (R). Alkyl groups and alkyl-derived groups, such as the typical alkoxy, haloalkyl, hydroxyalkyl, alkenyl, alkylidene and alkylene groups, can be _C2-6 , _C3-6 or _C2-4 .

Alkyls substituted with halogen, hydroxy, amino, cyano, azido, etc., may have 1, 2, 3, 4, 5 or more substituents, which are independently selected (may or may not be the same) and may or may not be not be at the same carbon atom. For example, haloalkyl groups are alkyl groups with at least one substituent selected from fluorine, chlorine, bromine and iodine. haloalkyls may have two or more halogen substituents, which may or may not be the same halogen and may or may not be on the same carbon atom. Examples include chloromethyl, periodomethyl, 3,3-dichloropropyl, 1,3-difluorobutyl and 1-bromo-2-chloropropyl.

Heterocyclic radicals and heteroaryls include furyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, 2H-pyrrolyl, pyrrolyl, imidazolyl (for example, 1-, 2- or 4-imidazolyl), pyrazolyl, isothiazolyl, isoxazolyl, pyridyl (e.g. 1-, 2- or 3-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl (e.g. 1-, 2- or 3-indolyl), indazolyl, purinyl, 4n-hinolixil, isokhinolil, chinolil, phthalazi-nan, naphthyiridinyl, chinzolinyl, hinasolinyl, zinolinyl, pteridinyl, pyrrolinyl, pyrrolidyl, imidazolidyl, feast _-3 olidyl, pyrazolinyl, piperidil, pipeperasil, isoindolized and morpholinide. Heterocyclic radicals and heteroaryls can be bonded to the rest of the molecule in any position on the ring. Heterocyclic radicals and heteroaryls may be _C2-9 or less, for example _C3-6 , _C2-5 or _C3-7 .

Aryl groups include phenyl, benzyl, naphthyl, tolyl, mesityl, xylyl and cumenyl.

It is understood that "heterocyclic radical", "aryl" and "heteroaryl" include radicals having 1, 2, 3, 4 or more substituents independently selected from lower alkyl, lower alkoxy, amino, halogen, cyano, nitro, azido and hydroxyl. Heterocyclic radicals, heteroaryls and aryls can also be bivalent substituents on the hydrocarbon skeleton "A" in formula (I). B. Halichondrin analogues

According to formula (I) in the Summary of the Invention section, embodiments of the invention include compounds wherein n is 0; wherein each of D and D' is independently selected from R ₃ , C _1-3 alkoxy and C _1-3 haloalkyloxy; in which R ₅ is selected from H, C _2-6 alkyl, C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _6-10 aryl, C _6-10 haloaryl, C _6-10 hydroxyaryl, C _1-3 alkoxy-C ₆ aryl, C _6-10 aryl-C _1-6 alkyl, C _1-6 alkyl-C _6-10 aryl, C ₆ -hohalogenaryl-C _1-6 alkyl, C _{1- 6} alkyl-C _6-10 haloaryl, (C _1-3 alkoxy-C ₆ aryl)-C _1-3 alkyl, C _2-9 heterocyclic radical, C _2-9 (heterocyclic radical) -C _1-6 alkyl, C _2-9 heteroaryl and C _2-9 heteroaryl-C _1-6 alkyl; and their combinations.

Another embodiment includes compounds having one or more of the following characteristics: compounds (a) wherein A is a C _1-6 saturated or a C _2-6 unsaturated hydrocarbon skeleton, which skeleton has at least one substituent selected from cyano, halogen, azido, Q ₁ and oxo; (b) each Q ₁ is independently selected from OR ₁ , SR ₁ , SO ₂ R ₁ , OSO ₂ R ₁ , NR ₂ R ₁ , NH ₂ (CO) R ₁ , NR ₂ (CO) (CO) R ₁ and O (CO,NR ₂ R ₁ ; (c) Z and Z' taken together denote =O or =CH ₂ ; (d) where each Q ₁ is independently selected from OR ₁ , SR ₁ , SO ₂ R ₁ , OSO ₂ R ₁ , NH (CO) R ₁ , NH(CO) (CO) R ₁ and O(CO)NHR ₁ ; (e) each R ₁ is independently selected from C _1-6 alkyl, C _1-6 haloalkyl, C ₆ aryl, C ₆ haloaryl, C _1-3 alkoxy-C ₆ aryl, C ₆ aryl-C _1-3 alkyl, C _1-3 alkyl-C ₆ aryl, C ₆ haloaryl-C _1-3 alkyl, C _1-3 alkyl-C ₆ haloaryl, (C _1-3 alkoxy-C ₆ aryl) -C _1-3 alkyl, C _2-9 heterocyclic radical, C _2-9 heteroaryl and C _2-9 heteroaryl-C _1-6 alkyl (; f) one of D and D' is methyl or methoxy and the other is H (g) n is 0; (h) G is O; (i) J and J' taken together are =CH ₂ ; j) Q is methyl; (k) T is ethylene; (1) U and U' taken together are =CH ₂ ; (m) X is H; (n) each of Y and Y' is H and ( o) Z and Z' taken together represent =O. Examples of combinations are the combination of (h)-(m), the combination of (a) and (b), the combination of (f) and (h), and the combination of (h) and the option where one of D and D' is methyl and the other is H. Two particularly preferred compounds are B1793 and B1939.

Another embodiment includes compounds in which Q ₁ is independently selected from OR ₁ , SR ₁ , SO ₂ R ₁ and OSO ₂ R ₁ ; and each R ₁ is independently selected from C _1-6 alkyl, C _1-6 haloalkyl, C ₆ aryl, C ₆ haloaryl, C _1-3 alkoxy-C ₆ aryl, C ₆ aryl-C _1-3 alkyl, C _{1- 3} alkyl-C ₆ aryl, C ₆ haloaryl-C _1-3 alkyl, C _1-3 alkyl-C ₆ haloaryl, (C _1-3 alkoxy-C ₆ aryl)-C _1-3 alkyl. Other embodiments include compounds in which: one of D and D' is alkyl or alkoxy, wherein n is 1; (f) as above, where n is 1; E is alkoxy, where n is 1; n is 0, where one of D and D' is hydroxy and the other is H; and (f) as described above, where n is 1 and E is methyl.

This invention also relates to compounds in which: (1) A has at least one substituent selected from hydroxyl, amino, azido, halogen and oxo; (2) A is a saturated hydrocarbon skeleton having at least one substituent selected from hydroxyl, amino and azido (for example, B1793, B1939, B2042, B1794 and B1922); (3) A has at least two substituents independently selected from hydroxyl, amino and azido (eg, B2090 and B2136); (4) A has at least two substituents independently selected from hydroxyl and amino (eg, B2042 and B2090); (5) A has at least one hydroxyl substituent and at least one amino substituent (for example, B1939 and B2136); (6) A has at least two hydroxyl substituents (for example, B1793 and B1794)/ (7) A is a C2-4 hydrocarbon skeleton that is substituted (for example, B2004, B2037, B1920, B2039, B2070, B2090 and B2043 ); (8) A is a C3 hydrocarbon skeleton that is substituted (for example, B1793, B1920, B1984, B1988, B1939, B1940, B2014); (9) A has an (S)-hydroxyl at the alpha position relative to the carbon atom linking A to the ring containing G (for example, B1793, B1939 or E1920), or an (R)-hydroxyl (for example, B2102, B2013, B2042); and (10) A is a C _1-6 saturated hydrocarbon skeleton having at least one substituent selected from hydroxyl and cyano (eg, B2013, B2037, B2102, B2086 and B2091). By (S)-hydroxyl it is meant that the configuration of the carbon atom having a hydroxyl group is (S)-configuration. Embodiments of the present invention also include compounds that have at least two substituents on the carbon atoms (1) alpha and gamma, (2) beta and gamma, or, preferably, (3) alpha and beta relative to the carbon atom linking A to the ring containing G. These alpha, beta and gamma carbon atoms can be in the (R) or (S) configuration.

The present invention further provides preferred compounds having formula (1)-A shown below, wherein the substituents are identical to those defined above.

The present invention further relates to the following monosaccharide intermediate having formula (II):

where R is methyl or methoxy and each of P1, P2 and P3 is independently selected from H and primary alcohol protecting groups. Preferably, the diol side chain is below the page plane and OP2 is above the page plane. Primary alcohol protecting groups include esters, ethers, silyl ethers, alkyl ethers and alkoxy-sialkyl ethers.

Examples of esters include formates, acetates, carbonates and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate, crotonate, 4-methoxycrotonate, benzoate, p- phenyl benzoate, 2,4,6-trimethyl benzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl and p- nitrobenzyl.

Examples of silyl ethers include trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl and other trialkylsilyl ethers. Alkyl esters include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, tert-butyl, allyl and allyloxycarbonyl esters or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl and tetrahydropyranyl ethers. Examples of benzyl esters include p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. Preferably, P1 and P2 are each TBS and P3 is MPM (see alcohol 19 below). In one aspect, formula (II) can be modified such that the hydroxyethyl side chain can also have a protected hydroxyl, -CH ₂ CH ₂ O-P4, where P4 is independently selected from the values for P1. A related intermediate is alcohol 17, where the hydroxyethyl side chain is replaced by a hydroxy-methyl side chain. Likewise, the corresponding hydroxypropyl side chain or amino alkyl side chain can be prepared.

P1 and P2 taken together may be a diol protecting group such as cyclic acetals and ketals (methylene, ethylidene, benzylidene, isopropylidene, cyclohexylidene and cyclopentylidene), silylene derivatives such as di-tert-butylsilylene derivatives and 1,1, 3,3-tetraisopropyl-disiloxanilidene, cyclic carbonates and cyclic boronates. The method for attaching and removing such hydroxyl protecting groups and additional protecting groups are well known in the art and are described, for example, in P. Kocienski, Protecting Groups, Thieme, 1994, and in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley & Sons, 1992.

The following section offers representative syntheses of intermediates of formula (II) and halichondrin analogues of formula (I).

C. Synthesis of halichondrin analogues

Below is an overview followed by synthetic diagrams 1-16 and several detailed protocols.

Compounds of general formula 4 can be prepared by the method described in Synthesis Scheme 1. Key fragment F-2, exemplified by vinyl iodide compound X2, can be prepared according to the method of Kischi et al., (Total synthesis of halichondrin B and norhalichondrin B. Aicher , T.D.; Fang, F.G.; Jung, S.H.; Matelich, M.; 3162-4).

The key fragment F-3 can be obtained by reduction with DIBALH of the corresponding methyl ester, XF3, prepared according to the method of Stamos et al., (Scheme 2). [Synthetic studies on halichondrins: a practical synthesis of the C.1-C13 segment. Stamos, D.P.; Kischi, Y. Tetrahedron Lett. 1996, 37, 8643-8646]. Synthesis of the key fragment F-1, exemplified by compound 20, can be performed as described in Scheme 3 or Scheme 4.

Using B1793 as a representative example, coupling of these three key moieties proceeded as described in Scheme 5: Nozaki-Hiyama-Kischi coupling of fragments 20 and X2 followed by intramolecular Williamson ether formation yielded tetrahydropyran B2318. Modification of the protecting group as described in Scheme 5 or alternatively in Scheme 6 gave the primary iodide B2313. The halogen-metal exchange reaction and binding to the key fragment F-3 gave a mixture of diastereomeric alcohols B2308. Additional protecting groups and oxidation followed by an intramolecular Nozaki-Hiyama-Kischi reaction yielded an intermediate that, when oxidized and treated with TBAF, underwent an intramolecular Michael reaction to close the heterocycle. PPT-mediated acetal formation yielded B1793.

Aryl groups can be introduced into the C32 side chain (eg, B2043), as exemplified in Scheme 7. Intermediate B2318 was deprotected and the resulting diol was oxidatively cleaved to the corresponding aldehyde. Treatment with a Grignard reagent (eg p-F-PhMgBr), separation of the resulting diastereomers and silylation gave 204, which was converted to the final product in a manner similar to that described in scheme b.

Ether analogs can be prepared from B1793 by treatment with a suitable alkylating agent (eg, Scheme 8). Likewise, sulfonates, esters, carbamates, etc. can be obtained from B1793 by treatment with an activated carbonyl component. Oxidative cleavage of diols and reduction or selective oxidation of hydroxyl groups could give derivatives such as B2037 and B1934, respectively.

Alternatively, one or more hydroxyl groups can be converted to the corresponding amino groups, followed by coupling to the activated carbonyl moiety (Scheme 9). Displacement of a sulfonyl intermediate (eg B1920) by compounds with a nucleophilic carbon or heteroatom can also be easily accomplished (Scheme 10).

Methyl analogues of C31 can be prepared as described in Scheme 11. Coupling of allyl bromide ester with indium to 2,3-O-(3-isopropylidene)-D-glyceraldehyde gave lactone 103. Michael heteroaddition, lactone reduction, coupling by Wittig and intramolecular Michael addition yielded tetrahydrofuran 107. Pummerer rearrangement, adjustment of protecting groups, and reduction with DIBALH yielded key fragment 1 (e.g., 114), which was converted to the final compound in a manner similar to that described in Scheme 6.

Fluorine atoms can be introduced as described in Schemes 12-14. Using the appropriate tetrahydrofuran intermediate as starting material, fluorinated key fragment 1 was prepared and converted to the final compound in a manner similar to that illustrated in Scheme 6.

Triol derivatives can be similarly prepared from the tetrahydrofuran intermediate. For example, as described in Scheme 15, addition of allyltributylstannane to aldehyde X32 gave homoallyl alcohol 33, which was converted to the final compound in a manner similar to that described in Scheme 6. These triols can be modified further, as exemplified in Scheme b.

Derivatives of 1,3-diols can be prepared from the intermediates described previously. For example, B208 6 can be subjected to oxidative cleavage and reduced to yield 1,3-diol B2091 (Scheme 16).

Synthesis of key fragment F-3:

Key fragment F-3. DIBALH (1M in toluene, 3.86 mL) was added to a solution of XF-3 (1.46 g, 1.93 mmol) in toluene (37 mL) at -78°C. After stirring for 10 minutes, the reaction was quenched by careful addition of MeOH (0.46 ml) and H ₂ O (0.21 ml), warmed to room temperature and stirred for 15 minutes. The white suspension was filtered through Celite with 1:1 CH ₂ Cl ₂ /Et ₂ O. The filtrate was concentrated and purified by column chromatography (10% EtOAc-hexanes) to give key fragment F-3 (1.34 g, 96%) as oils

Synthesis B17 93:

Triol 1. A solution of TBDPSC1 (444 ml, 1.7 mol) in DMF (0.5 L) was added in three portions to a suspension of L-arabinose (250.0 g, 1.66 mol), imidazole (231.4 g, 3.40 mol) and DMF (2.5 l). Each batch took 1.5 hours to add, with 30-minute and 15-hour intervals separating the second and third batches, respectively. The resulting solution was stirred for 3 hours, concentrated and purified by flash chromatography (5%-33% EtOAc/hexanes) to give triol 1 (394 g, 61%).

Triacetate 2. Acetic anhydride (6.06 mol) was added over 1.5 hours to triol 1 (1.01 mol) in pyridine (1.0 L) at 15°C. The solution was stirred for 1 hour, concentrated and purified by flash chromatography (15%-25% EtOAc-hexanes) to give triacetate 2 (518 g, 97%).

Diacetates 3. Allyltrimethylsilane (1.11 mol) and then BF ₃ ⋅OEt ₂ (1.11 mmol) were added over 1.5 hours to triacetate 2 (164 g, 0.32 mol) in toluene (1.5 L) at 0°C. The orange solution was stirred for 1 hour at 0°C and for 2 hours at room temperature. The mixture was slowly poured into saturated aqueous NaHCO ₃ (1.7 L) at 0°C and stirred for 30 minutes. The separated aqueous layer was extracted with EtOAc (3 x 600 ml) and the combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified flash chromatography (5%-10% EtOAc-hexanes) to give diacetate mixture 3 (108 g, 69%).

Diol 4a. Solid K ₂ CO ₃ (72 mmol) was added to diacetam 3 (108 g, 218 mmol) in MeOH (0.5 L) at room temperature. The suspension was stirred for 2.5 hours and then concentrated. The orange precipitate was suspended in saturated aqueous NH ₄ Cl (150 ml), extracted with EtOAc (3 x 150 ml) and the combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (15%-50% EtOAc-hexanes) with yielding alpha isomer 4a (33.86 g, 37%) and beta isomer 4b (58 g, 63%).

Alcohol 5. Imidazole (16.75 g, 246 mmol) and TBSC1 (16.08 g, 107 mmol) were added to a solution of diol 4a (33.86 g, 82 mmol) in CH ₂ Cl ₂ (250 ml) at 0 °C. After 18 hours at 0°C and 5 hours at room temperature, the reaction mixture was diluted with saturated aqueous NaHCO ₃ solution (250 ml), stirred for 30 minutes and the layers were allowed to separate. The aqueous layer was extracted with EtOAc (3×250 ml) and the combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (2%-50% EtOAc-hexanes) to give alcohol 5 (36.0 g, 83%) .

Methyl ether 6. Iodomethane (16.5 ml, 265 mmol) and NaH (60% in mineral oil, 5.28 g, 132 mmol) were added to a solution of alcohol 5 (34.93 g, 66 mmol), THF (320 ml ) and DMF (80 ml) at 0°C. After 19 hours at 0°C, the reaction was quenched with saturated aqueous NH ₄ Cl and saturated aqueous Na ₂ S ₂ O ₃ . The resulting mixture was stirred for 20 minutes and the layers were allowed to separate. The aqueous phase was extracted with EtOAc (3×200 ml) and the combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (3% EtOAc-hexanes) to give methyl ester 6 (34.23 g, 96%).

Diol 7. HCl (37% aqueous solution, 12.75 mL, 153 mmol) was added to a solution of methyl ester 6 (32.93 g, 61 mmol) in MeOH (110 mL) at room temperature. After 17 hours, NaHCO ₃ (17 g) was added to the reaction mixture. The mixture was stirred for 30 minutes, concentrated, suspended in EtOAc and filtered. The filtrate was concentrated and purified by flash chromatography (50% EtOAc-hexanes - EtOAc) to give diol 7 (10.0 g, 87%).

Alcohol 8. A solution of pivaloyl chloride (8.4 ml, 67 mmol) in pyridine (50 ml) was added over 1.5 h to a solution of diol 7 (12.24 g, 65 mmol) in pyridine (100 ml) at 0°C. . After 1 hour at 0°C and 18 hours at room temperature, the mixture was diluted with saturated aqueous NH ₄ Cl and extracted with EtOAc (3×800 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (50% EtOAc-hexanes) to give alcohol 8 (16.9 g, 96%).

Olefin 9. Benzyl bromide (62 ml, 521 mmol) and Bu ₄ NHSO ₄ (10.6 g, 31 mmol) were added to a solution of alcohol 8 (16.9 g, 62 mmol) in CH ₂ Cl ₂ (100 ml) at 0 °C. A solution of NaOH (9.95 g, 248 mmol) in H ₂ O (10 ml) was added to the reaction mixture over 15 minutes. After 30 minutes at 0°C and 18 hours at room temperature, the reaction mixture was diluted with saturated aqueous NH ₄ Cl and extracted with CH ₂ Cl ₂ (3×100 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (hexanes - 30% EtOAc-hexanes) to give olefin 9 (22.1 g, 98%).

Diol 10. OsO ₄ (0.1 M solution in toluene, 7.3 ml, 0.73 mmol) and a solution of olefin 9 (24.9 g, 69 mmol) in t-BuOH (165 ml) were added to the K ₂ solution CO ₃ (31.2 g, 161 mmol), K ₃ Fe(CN) ₆ (74.4 g, 161 mmol), (DHQ) ₂ PYR (1.33 g, 1.50 mmol), H ₂ O ( 500 ml) and tert-BuOH (330 ml) at 0°C. After 3 hours at 0°C, Na ₂ S ₂ O ₃ ⋅5H ₂ O (37.3 g, 150 mmol) was added. The reaction mixture was warmed to room temperature, stirred for 1 hour and extracted with EtOAc (3 x 300 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (5% isopropanol-CH ₂ Cl ₂ ) to give diol 10 (17.98 g, 75%).

Silyl ester 11. Imidazole (21 g, 308 mmol) and TBSC1 (26.5 g, 176 mmol) were added to a solution of diol 10 (17.4 g, 44 mmol) in DMF (90 ml) at room temperature. After 18 hours, the reaction mixture was diluted with saturated aqueous sodium bicarbonate (250 ml), stirred for 1 hour and extracted with CH ₂ Cl ₂ (3×100 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (5% EtOAc-hexanes) to give silyl ether 11 (25.7 g, 94%).

Alcohol 12. A mixture of silyl ether 11 (21.2 g, 33.8 mmol), Pd(OH) ₂ (20%, 4.7 g, 33.8 mmol) and EtOAc (200 ml) was stirred at room temperature at atmosphere H ₂ for 3 hours. The mixture was filtered through celite, concentrated and purified by flash chromatography (10%-20% EtOAc-hexanes) to give alcohol 12 (17.4 g, 96%).

Olefin 13. 4-Methylmorpholine N-oxide (7.66 g, 65 mmol) and TPAP (1.15 g, 3.26 mmol) were added in four portions over 20 minutes to a solution of alcohol 12 (17.4 g, 32.5 mmol) in CH ₂ Cl ₂ (145 ml) at 0°C. After 20 minutes, the reaction mixture was diluted with Et ₂ O (50 ml) and saturated aqueous Na ₂ S ₂ O ₃ (50 ml) and filtered through celite. The organic layer was separated, washed successively with saturated aqueous CuSO ₄ brine (1:1) and brine, dried over Na ₂ SO ₄ , filtered through celite and concentrated to give the desired crude ketone.

Tebbe's reagent was prepared by stirring bis(cyclopentadienyl)titanium (11.36 g, 45.6 mmol) and Me ₃ Al (2.0 M in toluene, 45.6 ml, 91.2 mmol) for 4 days at room temperature. This material was cooled to -25°C and a solution of the crude ketone in THF (150 ml) was added. The reaction mixture was warmed to 0°C, stirred for 30 minutes, quenched by slow addition of 0.1 N NaOH (3.5 ml) and then stirred for an additional 20 minutes at room temperature. The mixture was diluted with Et ₂ O, filtered through celite and concentrated. The residue was dissolved in CH ₂ Cl ₂ , filtered through basic Al ₂ O ₃ , concentrated and purified by flash chromatography (5% EtOAc-hexanes) to give olefin 13 (12.8 g, 74% over two steps).

Alcohol 14. 9-BBN (0.5 M in HHF, 165 mL, 83 mmol) was added to a solution of olefin 13 (12.78 g, 24 mmol) in THF (280 mL) at 0°C. After stirring for 5 hours at room temperature, the reaction mixture was re-cooled to 0°C at which point H ₂ O (200 ml), THF {100 ml) and NaBO ₃ ⋅ 4H ₂ O (75 g) were added. The mixture was warmed at room temperature, stirred for 16 hours and then concentrated. The aqueous residue was extracted with EtOAc (4×300 ml) and the combined organic layers were dried over Na ₂ SO ₄ . Concentration and purification by flash chromatography (20%-35% EtOAc-hexanes) gave alcohol 14 (12.05 g, 91%).

Alcohol 15. DMSO (9 ml, 127 mmol) was added to a solution of oxalyl chloride (5.6 ml, 64 mmol) in CH ₂ Cl ₂ (350 ml) at -78°C. After stirring for 15 minutes, a solution of alcohol 14 (11.7 g, 0.021 mmol) in CH ₂ Cl ₂ (50 ml) was added and stirring was continued for 1 hour, after which Et ₃ N (26.7 ml, 192 mmol) was added ). The reaction mixture was heated to 0°C, stirred for 15 minutes, diluted with saturated aqueous NH ₄ Cl and extracted with CH ₂ Cl ₂ (3×200 ml). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give the desired crude aldehyde.

This material was dissolved in CH ₂ Cl ₂ (200 ml) and treated with Et ₃ N (20 ml) at room temperature. After stirring overnight, the reaction mixture was diluted with saturated aqueous NH ₄ Cl and extracted with CH ₂ Cl ₂ (3×200 ml). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and filtered through a short column of SiO ₂ (20% EtOAc-hexanes) to obtain the crude epimerized product.

This aldehyde was dissolved in a mixture of Et ₃ N-EtOH (1:1, 100 ml), cooled to 0°C and treated with sodium borohydride (1.21 g, 32 mmol). The mixture was stirred for 20 minutes, carefully diluted with saturated aqueous NH ₄ Cl, stirred for 30 minutes at room temperature and extracted with CH ₂ Cl ₂ (3×150 ml). The combined extracts were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (20% EtOAc-hexanes) to give alcohol 15 (9.95 g, 85% for three steps).

MFM ester 16. BF ₃ ⋅OEt ₂ (0.1 M in CH ₂ Cl ₂ , 1.8 ml, 0.18 mmol) was added to a solution of alcohol 15 (9.87 g, 18 mmol), MFM trichloroimidate (4, 9 ml, 27 mmol) and CH ₂ Cl ₂ (175 ml) at 0°C. After 40 minutes, a second portion of BF ₃ ⋅OEt ₂ (0.1 M in CH ₂ Cl ₂ , 0.9 mL, 0.09 mmol) was added to the reaction mixture. After 20 minutes, the reaction was quenched with saturated aqueous NH ₄ Cl, stirred for 1 hour at room temperature and diluted with Et ₂ O (600 ml). The organic layer was separated and the aqueous layer was extracted with Et ₂ O (150 ml). The combined organic layers were washed successively with 0.1 N aqueous NaOH, saturated aqueous NaHCO ₃ , brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (20% EtOAc-hexanes) to obtain MPM ester 16 (10, 20 g, 85%).

Alcohol 17. LAH (1 M in THF, 22.5 ml, 22.5 mmol) was added to a solution of MPM ester 16 (10.05 g, 15 mmol) in Et ₂ O (1.0 L) at 0°C . After 30 minutes, the reaction was carefully quenched with H ₂ O (1.3 ml) and 1 N aqueous NaOH (1.3 ml). After stirring for 1 hour at room temperature, the suspension was filtered through celite, concentrated and purified by flash chromatography (20% EtOAc-hexanes) to give alcohol 17 (8.18 g, 93%).

Olefin 18: DMSO (5.8 g, 82.4 mmol) was added to a solution of oxalyl chloride (3.6 mL, 41.2 mmol) in CH ₂ Cl ₂ (100 mL) at -78°C. After 15 minutes, a solution of alcohol 17 (7.94 g, 13.5 mmol) in CH ₂ Cl ₂ (35 ml) was added to the reaction mixture. After stirring for 1 hour, Et ₃ N (17 ml, 122 mmol) was added, the mixture was heated to 0° C., stirred for 20 minutes, diluted with saturated aqueous NH ₄ Cl and then extracted with CH ₂ Cl ₂ (3 x 100 ml ). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and filtered through a short column of SiO ₂ (20% EtOAc-hexanes) to obtain the desired crude aldehyde.

n-BuLi (1.6 M, 20 ml, 30 mmol) was added dropwise to a solution of CH ₃ PPh ₃ Br (10.1 g, 30 mmol) in TTP (350 ml) and DMSO (100 ml) at 0°C . After 1 hour, a solution of the crude aldehyde in THF (50 ml) was added. The reaction mixture was warmed to room temperature and stirred for 3 hours. Saturated aqueous NH ₄ Cl was added and the mixture was extracted with EtOAc (3×500 ml). The combined extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (7% EtOAc-hexanes) to give olefin 18 (5.57 g, 71% yield over 2 steps).

Alcohol 19. 9-BBN (0.5 M in THF, 65 mL, 33 mmol) was added to a solution of olefin 18 (5.56 g, 9.6 mmol) in THF (85 mL) at 0°C. The mixture was stirred for 5 hours at room temperature and then re-cooled to 0°C. H ₂ O (200 ml), THF (100 ml) and NaBO ₃ ⋅ 4H ₂ O (30 g) were added successively. After stirring overnight at room temperature, the organic volatiles were removed under reduced pressure. The aqueous residue was extracted with EtOAc (3×200 ml) and the combined organic layers were dried over Na ₂ SO ₄ . Concentration and purification by flash chromatography (30% EtOAc-hexanes) gave alcohol 19 (12.05 g, 92%).

Aldehyde 20. DMSO (1.36 ml, 19.2 mmol) was added dropwise over 4 minutes to a solution of oxalyl chloride (1.26 ml, 14.4 mmol) in CH ₂ Cl ₂ (120 ml) at -78°C . After stirring for 10 minutes, a solution of alcohol 19 (5.76 g, 9.61 mmol) in CH ₂ Cl ₂ (20 ml) was added via cannula. The transfer was completed by rinsing with additional CH ₂ Cl ₂ (2×5 ml). After stirring for 20 minutes, the mixture was treated with Et ₃ N (5.36 ml, 38.4 mmol) and stirred for 10 minutes at -78°C, 30 minutes at 0°C and 10 minutes at room temperature. The reaction mixture was poured into saturated aqueous NaHCO ₃ (200 ml) and the separated aqueous layer was extracted with CH ₂ Cl ₂ (3x) and then with EtOAc (100 ml). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10%-20% EtOAc-hexanes) to give aldehyde compound 20 (5.28 g, 92%) as an oil.

B2318. 0.1% NiCl ₂ /CrCl ₂ (w/w, 3.21 g) and 1% NiCl ₂ /CrCl ₂ (w/w, 4.31 g) were added to the solution of aldehyde 20 (3.73 g, 6. 25 mmol), key fragment F-2, represented by vinyl iodide X2 (5.10 g, 9.16 mmol), THF (85 ml) and DMF (21 ml) at room temperature in a glove box. The reaction mixture was stirred for 24 hours, removed from the glove box, cooled to 0°C, diluted with EtOAc (100 ml), quenched with saturated aqueous NH ₄ Cl (200 ml) and stirred for 30 minutes. The separated aqueous phase was extracted with EtOAc (6x) and the combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes) to obtain B2318 (~3 g), contaminated with closely related impurities and a non-cyclized intermediate product (4.61 g). The latter (4.61 g, 4.48 mmol) was dissolved in THF (150 ml), cooled to 0°C and treated with KHMDS (0.5 M in toluene, 14 ml, 7.0 mmol) over a 2-minute period . After stirring at 0°C for 15 minutes, the reaction was quenched with saturated aqueous NH ₄ Cl (150 ml) and warmed to room temperature. The separated aqueous layer was extracted with EtOAc (3x) and the combined organic phases were dried over Na ₂ SO ₄ , concentrated and combined with the partially purified product obtained as described above. Column chromatography (10% EtOAc-hexanes) gave B2318 (3.17 g, 55%) as an unresolved ~3:1 mixture of C27 diastereoisomers.

B2317. DDQ (1.45 g, 6.42 mmol) was added in portions over 30 minutes to a stirred solution of B2318 (3.12 g, 3.35 mmol) in CH ₂ Cl ₂ (50 ml) and phosphate buffer pH 7 (5 ml) at room temperature. The reaction was quenched with saturated aqueous NaHCO ₃ (50 ml), stirred for 5 minutes, diluted with additional saturated aqueous NaHCO ₃ (100 ml), H ₂ O (200 ml) and extracted with Et ₂ O (5x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (15%-30% EtOAc-hexanes) to give recovered B2318 (1.40 g) and a mixture of C27 isomeric products. The recovered B2318 was resubjected to the reaction conditions described above to obtain additional product. The recovered starting material was recycled again and subjected to deprotection conditions. All target material was combined and separated by medium pressure liquid chromatography to give B2317 (1.65 g, 61%).

B2316. TsCl (0.63 g, 3.30 mmol) was added to a solution of B2317 (1.60 g, 1.97 mmol) in CH ₂ Cl ₂ (8 ml) and pyridine (2 ml) at room temperature. After stirring for 29 hours, the reaction was quenched with a saturated aqueous solution of NaHCO ₃ (30 ml) and H ₂ O (10 ml). The separated aqueous layer was extracted with Et ₂ O and the combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (15%-30% EtOAc-hexanes) to give B2316 (2.01 g, 92%) as an oil along with extracted B2317 (92 mg, 5.8%).

B2315. LAH (1 M in THF, 2.61 ml, 2.61 mmol) was added over 1 minute to a solution of B2316 (1.68 g, 1.74 mmol) in Et ₂ O (80 ml) at 0°C. After stirring for 7 minutes, the reaction was quenched by careful addition of MeOH (0.42 g, 10.4 mmol) and H ₂ O (0.19 ml, 10 mmol), warmed to room temperature and stirred for 20 minutes. Filtration through Celite with 1:1 CH ₂ Cl ₂ -Et ₂ O, concentration and purification by column chromatography (30%-40% EtOAc-hexanes) gave B2315 (1.38 g, 90%) as an oil.

B2314. MMTrCl (0.70 g, 2.26 mmol) was added to a solution of B2315 (1.33 g, 1.51 mmol) in CH ₂ Cl ₂ (25 ml) and iso-Pr ₂ NEt (0.79 ml, 4. 53 mmol) at room temperature. The resulting mixture was stirred for 1 hour and then poured into a mixture of saturated aqueous NaHCO ₃ (20 ml), H ₂ O (10 ml) and Et ₂ O (50 ml). The separated aqueous layer was extracted with Et ₂ O (3x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (CH ₂ Cl ₂ then 15%-30% EtOAc-hexac) to give B2314 (1.65 g, 95%) as a solid foam.

B2313. A mixture of B2314 (1.60 g, 1.39 mmol) and NaI (3.10 g, 20.8 mmol) in acetone (50 ml) was refluxed for 13 hours. After cooling to room temperature, the reaction mixture was diluted with EtOAc and concentrated. H ₂ O (5 ml), brine (20 ml) and Na ₂ S ₂ O ₃ (200 mg) were added and the resulting mixture was extracted with Et ₂ O (4x). The combined extracts were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10% EtOAc-hexanes) to give B2313 (1.50 g, 97%) as an oil.

B2308. Tert-BuLi (1.7 m in pentane, 1.00 ml, 1.7 mmol) was added over 1 minute to a solution of E2313 (0.90 g, 0.81 mmol) in Et ₂ O (14 ml) at - 78°C. After stirring for 9 minutes, the mixture was cannulated over 4 minutes into a solution of key fragment F-3 (0.83 g, 1.14 mmol) in Et ₂ O (4 ml) at -78°C. The transfer was completed by rinsing with additional Et ₂ O (2 ml). The resulting mixture was stirred at -78°C for 5 minutes and then at 0°C for 10 minutes, quenched with saturated aqueous NaHCO ₃ solution (30 ml) and warmed to room temperature. The separated aqueous layer was extracted with Et ₂ O (3x) and the combined organic phases were dried over Na ₂ SO ₄ and concentrated. The residue was combined with the residues of two other batches (corresponding to 0.11 g and 0.44 g of B2313) and purified by column chromatography (10%-20% EtOAc-hexanes) to give a mixture of B2307 and B2308 (1.86 g, 83%) in in the form of hard foam. Although these isomers could be separated by preparative TLC (20% EtOAc-hexanes), they were used further as a mixture.

B2305 and B2306. A mixture of B2307/B2308 (1.80 g, 1.05 mmol) was dissolved in EtOH (20 ml), treated with PPTS (10.0 mg, 0.04 mmol), stirred at room temperature for 11 hours and then quenched with NaHCO ₃ (20.0 mg, 0.24 mmol). After stirring for 15 minutes, the mixture was concentrated, azeotroped with toluene (15 ml) and purified by column chromatography (20%-30% EtOAc-hexanes) to give a mixture of B2305 and B2306 (1.22 g, 81%) as a solid foam . Although these isomers could be separated by preparative TLC (30% EtOAc-hexanes), they were used further as a mixture.

B2304. A mixture of B2305/B2306 (1.16 g, 0.68 mmol) and Dess-Martin periodinane (0.61 g, 1.44 mmol) in CH ₂ Cl ₂ (35 ml) was stirred at room temperature for 1 hour. Additional Dess-Martin periodinane (0.54 g, 1.27 mmol) was added to this mixture and stirring was continued for an additional 1 hour. The mixture was diluted with Et ₂ O (100 ml), stirred for 20 minutes and filtered through celite with Et ₂ O. The colorless filtrate was washed with saturated aqueous NaHCO ₃ (100 ml) and the separated aqueous layer was extracted with Et ₂ O (3x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10%-15% EtOAc-hexanes) to give B2304 (0.98 g, 84%) as a solid foam.

Alternatively, B2304 can be prepared as follows, and in fact the synthesis described below has advantages over the synthesis described above.

To a solution of alcohol, 2.4 g, in methylene chloride, 29 ml, was added trifluoromethanesulfonic anhydride, 770 mg. The mixture was stirred for 15 minutes, extracted with saturated aqueous sodium bicarbonate, dried and chromatographed to obtain 2.737 g, 100%.

To a solution of mesylate, 405 mg, in DMF, 0.06 ml, was added diisopropylethylamine, 0.130 ml, followed by the addition of benzenethiol, 0.061 ml. After 4 hours and after 22 hours, additional amine, 0.03 ml, and benzenethiol, 0.015 ml, were added. After 24 hours, this mixture was diluted with 5% ethyl acetate/hexane, 1 ml, and chromatographed to give 409 mg.

To a solution of sulfide, 1.97 g, in acetonitrile, 16 ml, was added N-methylmorpholine oxide (NMO), 38 mg, followed by a solution of 1.02 g of tetrapropylammonium perruthenate (VII) (TPAP), in acetonitrile, 1 ml. After 3.5 hours at room temperature, the mixture was heated to 40°C for 1 hour. The mixture was cooled and an aqueous saturated sodium thiosulfate solution was added and the mixture was partitioned between water and ethyl acetate. Conventional processing yielded 1.567 g of brown butter.

To a solution of pivaloate ether, 1.567 g, in methylene chloride, 11.2 ml, was added DIBAL, 2.5 ml of a 1 M solution in toluene, at -78°C. After 15 minutes, an additional amount of DIBAL, 0.8 ml, was added. After an additional 5 minutes, methanol, 0.46 ml, was slowly added, followed by the addition of water, 0.2 ml. The mixture was filtered through celite and chromatographed to obtain 1.386 g of oil.

To a solution of sulfone, 36 mg, in DME, 1 ml, n-butyllithium, 2.8 eq., was added at -40°C. After 35 minutes, a solution of aldehyde, 42 mg, in DME, 0.5 ml was added. After 40 minutes, saturated aqueous ammonium chloride was added and the mixture was extracted with ethyl acetate. Conventional workup followed by chromatography gave 52 mg of oil.

To a solution of alcohol, 42 mg, in methylene chloride, 2 ml, was added Dess-Martin reagent, 3-6.4 mg. The mixture was stirred for 30 minutes and ether was added. The mixture was filtered through celite, washed with saturated aqueous sodium bicarbonate, saturated sodium thiosulfate, worked up in the usual manner and chromatographed to give 38 mg of an oil.

Preparation of SmI ₂ solution

A solution of 1,2-diiodoethane in 10 ml THF was added to a suspension of Sm, 0.16 g, in THF, 1 ml. The mixture was stirred for 1 hour. An aliquot of this solution, 0.03 ml, was added to a solution of the sulfone in THF at -78°C. After 5 minutes, additional SmI reagent, 0.05 ml, was added. After a few additional minutes, this reagent was added again, 0.25 ml. The cooling bath was removed and saturated aqueous sodium bicarbonate, 3 ml, was added. The mixture was partitioned between ether and water and normal workup gave 9.1 mg, 81%, oil.

B2302 and B2303. In a glove box, NiCl ₂ /CrCl ₂ (1% w/v, 1.09 g, 8.86 mmol) was added to a solution of B2304 (1.01 g, 0.70 mmol) in THF (600 ml) and DMF ( 150 ml) at room temperature. After stirring for 2 days, the reaction mixture was removed from the glove box, cooled to 0°C, quenched with saturated aqueous NH ₄ Cl (300 ml) and stirred at 0°C for 20 minutes. After adding H ₂ O (100 ml), the two layers were separated and the aqueous layer was extracted with EtOAc (5x). The combined organic phases were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (15% EtOAc-hexanes) to give a mixture of B2302 and B2303 (0.84 g, 92%) as a solid foam. Although these isomers could be separated by preparative TLC (20% EtOAc-hexanes), they were used further as a mixture.

B2301. A mixture of B2302/B2303 (0.79 p, 0.60 mmol) and Dess-Martin periodinane (0.26 g, 0.60 mmol) in CH ₂ Cl ₂ (30 ml) was stirred at room temperature for 30 minutes. Additional Dess-Martin periodinane (0.26 g, 0.60 mmol) was added to the mixture and stirring was continued for an additional 1.5 hours. The mixture was then diluted with Et ₂ O (100 ml), stirred for 5 minutes and filtered through celite. The filtrate was washed with saturated aqueous NaHCO ₃ (100 ml) and the separated aqueous layer was extracted with Et ₂ O (3x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10%-15% EtOAc-hexanes) to give B2301 (0.67 g, 85%) as an oil.

B1793. TBAF (1 M in THF containing 0.5 M imidazole-HCl, 4.60 ml, 4.60 mmol) was added over 2 minutes to a solution of B2301 (0.62 g, 0.48 mmol) in THF (29 ml ) at room temperature and the resulting mixture was stirred for 18 hours. After dilution with hexanes (10 ml), the reaction mixture was immediately applied to a SiO ₂ column loaded with 50% EtOAc-hexanes and eluted with 50% EtOAc-hexanes (1 L) and then with 10% MeOH/EtOAc, collecting the mixture of intermediates products. After removal of the solvent, the residue was dissolved in CH ₂ Cl ₂ (15 ml) and treated with PPTS (645 mg). After stirring for 1 hour at room temperature, additional PPTS (414 mg) was added and the resulting white suspension was stirred for 4.5 hours. The reaction mixture was then applied directly to a SiO ₂ column loaded with 70% EtOAc-hexanes and eluted with 70% EtOAc-hexanes (0.5 L), EtOAc (1 L). Elution with a 5%-10% MeOH/EtOAc gradient gave pure B1793 (181 mg), and eluting with 15% MeOH-EtOAc gave additional semi-purified product, which after purification by preparative TLC (10% MeOH-EtOAc) gave additional pure B1793 (42 mg ). B1793 (total 223 mg, 64%) was obtained as a white solid. High-resolution mass spectrometry (HRMS): calc. for C ₄₀ H ₅₈ O ₁₂ + Na 753.3826. Found: 753, 3808. Synthesis of B1794:

B1794. Except for stereochemical differences and differences in protecting groups (Schemes 3 and 5), arabinose was converted to B1794 in a manner similar to that described for B1793 (see Schemes 4 and 5). HRMS: calc. for C ₄₀ H ₅₈ O ₁₂ + Na 753.3826. Found: 753.3856.

Synthesis of characteristic analogues of B1793:

B1920 and B1921. TsCl (9.9 mg, 0.052 mmol) was added to a solution of diol B1793 (7.6 mg, 0.010 mmol) in CH ₂ Cl ₂ (1 ml) and pyridine (0.1 ml) at room temperature. After 48 hours, the reaction was quenched with a 1:4 mixture of saturated aqueous NaHCO ₃ -salt solution and extracted with CH ₂ Cl ₂ (4x), The combined extracts were dried over Na ₂ SO ₄ and concentrated. Purification by preparative TLC (80% EtOAc-hexanes) provided A1920 monotosylate (6.0 mg, 67%) and B1921 ditosylate (1.8 g, 18%).

B2294. MsCl (0.3 M in CH ₂ Cl ₂ , 98 μl, 0.030 mmol) was added dropwise over 40 minutes to a mixture of collidine (7 μl, 0.054 mmol), B1793 (20.8 mg, 0.028 mmol) and CH ₂ Cl ₂ (1 ml) at 0°C. After 76 hours at 4°C, the reaction was quenched with a 1:4 mixture of saturated aqueous NaHCO ₃ -salt solution and extracted with CH ₂ Cl ₂ (4x). The combined extracts were dried over Na ₂ SO ₄ and concentrated. The crude product was dissolved in toluene (3 ml), concentrated and purified by preparative TLC (1.5% MeOH-EtOAc to give mesylate B2294 (21.4 mg, 95%).

B2014 and B2015. A 0.016 M solution of 4-fluorobenzyl bromide in Et ₂ O (800 µl, 13 µmol) and Ag ₂ O (10 mg, 43 µmol) was each added in three portions at 1 hour intervals at room temperature to the B1793 solution (1.7 mg, 2.3 µmol) in Et ₂ O (1.2 ml). The mixture was protected from light, stirred for 7 hours and then filtered through celite. Concentration and purification by preparative TLC (EtOAc) yielded primary ester B2014 (1.1 mg, 56%) and secondary ester B2015 (0.6 mg, 31%). HRMS (FAB): calc. for C ₄₇ H ₆₃ FO ₁₂ + Na 861, 4201. Found: for B2014 861.4178, for B2015 861.4160.

General part: A mixture of B1793 (1 mg, 1.37 µmol), Et ₃ N (10 µl, 72 µmol) and ArNCO (2-4 eq.) in CH ₂ Cl ₂ (0.2 ml) was stirred at room temperature in for 4 hours - overnight until the reaction was completed (according to TLC data). The reaction mixture was diluted with saturated NaHCO ₃ (3 ml), extracted with CH ₂ Cl ₂ (3x) and EtOAc (2x), dried over Na ₂ SO ₄ and purified by preparative TLC (5% MeOH-CH ₂ Cl ₂ ) to obtain the products:

B1984. (1.0 mg, 86%) HRMS (FAB): calc. for C ₄₇ H ₆₃ NO ₁₃ + Na 872.4197. Found: 872.4214.

B1990. (1.1 mg, 92%) HRMS (FAB): calc. for C ₄₇ H ₆₂ ClNO ₁₃ + Na 906.3807. Found: 906.3826.

B1992. (1.0 mg, 83%) HRMS (FAB): calc. for C ₄₈ H ₆₅ NO ₁₄ + Na 902.4303. Found: 902.4269.

B2042. DEAD (0.4 M in ether, 50 μl, 19 μmol) was added to a solution of B1793 (2.0 mg, 2.7 μmol), triphenylphosphine (5 mg, 19 μmol), 4-nitrobenzoic acid (3.2 mg, 19 µmol) and Et ₂ O (500 µl) at room temperature. After 22 hours, the reaction mixture was applied directly to a preparative TLC plate and eluted with 60% EtOAc-hexanes to give the diester intermediate (3.0 mg). This material was placed in MeOH (300 µl) and treated with K ₂ CO ₃ (approximately 1 mg). After stirring at room temperature for 30 minutes, the reaction mixture was diluted with brine and extracted with CH ₂ Cl ₂ (5x). The combined extracts were dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (5% MeOH-EtOAc to give B2042 (1.2 mg, 60% for two steps). HRMS (FAB): calc. for C ₄₀ H ₅₈ O ₁₂ + Na 753.3826 Found: 753.3810.

B1896 and B1897. NaBH ₄ (3 mg, 0.08 mmol) was added to a solution of B1793 (2.30 mg, 3.15 μmol) in a 1:1 mixture of CH ₂ Cl ₂ -MeOH (0.2 ml) at room temperature. Concentration of the reaction mixture and purification by preparative TLC (8% MeOH-EtOAc) gave B1896 (0.80 mg, 35%) and B1897 (2:1 mixture, 0.15 mg, 6.5%). HRMS (FAB) for B1896: calc. for C ₄₀ H ₆₀ O ₁₂ + Na 755.3983. Found: 753.3969.

B1918. A mixture of B1793 (2.0 mg, 2.74 µmol), NaIO ₄ (35 mg, 0.16 mmol), MeOH (0.8 ml) and H ₂ O (0.2 ml) was stirred at room temperature for 40 minutes. The reaction mixture was diluted with H ₂ O (1 ml), extracted with CH ₂ Cl ₂ (6x), dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (5% MeOH-CH ₂ Cl ₂ ) to obtain B1918 (1.9 mg , 100%).

B2037. A 0.034 M solution of NaBH ₄ (0.1 ml, 3.4 µmol) in EtOH was added in portions to a solution of B1918 (1.9 mg, 2.72 µmol) in MeOH (0.8 ml) and CH ₂ Cl ₂ (0. 2 ml) at -78°C to room temperature until the reaction was complete according to TLC. The reaction was quenched with a saturated aqueous solution of NH ₄ Cl (2 ml) at -78°C, warmed to room temperature, extracted with CH ₂ Cl ₂ (6x), dried over Na ₂ SO ₄ and purified by preparative TLC (5% MeOH-CH ₂ Cl ₂ ) to obtain B2037 (1.7 mg, 89%). HRMS (FAB): calc. for C ₃₉ H ₅₆ O ₁₁ + Na 723, 3720. Found: 723.3749.

NaIO ₄ (35 mg, 0.16 mmol) was added to a solution of B1793 (1.7 mg, 0.0023 mmol), MeOH (800 μl) and H ₂ O (200 μl) and after 15 minutes this mixture was diluted with H ₂ 0 and extracted with CH ₂ Cl g (5x). Organic extracts were dried over Na ₂ SO ₄ , concentrated and the aldehyde intermediate was immediately dissolved in DMF (300 µl). 3-bromo-3,3-difluoropropene (3 µl, 0.023 mmol) and indium powder (3 mg, 0.23 mmol) were added and after 24 hours additional 3-bromo-3, 3-difluoropropene (1 µl, 0.008 mmol). After 18 hours, H ₂ O was added and the mixture was extracted with EtOAc (3x). The combined organic extracts were washed successively with _H2O and _brine , dried over _Na2SO4 , concentrated and purified by preparative TLC (80% EtOAc-hexanes) to give B2035 (0.74 mg, 41% for 2 steps) as a mixture of isomers . HRMS (FAB): calc. for C ₄₂ H ₆₅ F ₂ O ₁₁ + Na 799.3845. Found: 799.3883.

NaBH ₄ (2 mg, 0.05 mmol) was added to a solution of B1793 (2.2 mg, 0.003 mmol) in a 1:1 mixture of CH ₂ Cl ₂ -MeOH (200 μl) at room temperature. After 15 minutes, saturated aqueous NH ₄ Cl and H ₂ O were added and the mixture was extracted with CH ₂ Cl ₂ (6x) and EtOAc (2x). The combined organic layers were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10% MeOH-EtOAc) to give the triol intermediate, which was dissolved in MeOH (800 μl) and H ₂ O (200 μl). NaIO ₄ (35 mg, 0.16 mmol) was added and after 20 minutes the mixture was diluted with H ₂ O and extracted with CH ₂ Cl ₂ (6x). Organic extracts were dried over Na ₂ SO ₄ , concentrated and the aldehyde intermediate was immediately dissolved in THF (500 µl). 4-fluorophenylmagnesium bromide (2Mb Et ₂ O, 12 µl, 0.024 mmol) was added and after 20 minutes the reaction was quenched with saturated aqueous NH ₄ Cl. The mixture was extracted with CH ₂ Cl ₂ (6x) and the combined organic extracts were dried over Na ₂ SO ₄ and concentrated. Purification by preparative TLC (EtOAc) gave the desired product as a mixture of 4 isomers (1.32 mg, 55% for 3 steps).

Dess-Martin periodinan (~3 mg, 0.007 mmol) was added to a solution of the above product (0.95 mg, 0.0012 mmol) in CH ₂ Cl ₂ (300 μl) and the mixture was stirred at room temperature for 20 minutes. Additional Dess-Martin periodinane (~3 mg, 0.007 mmol) and CH ₂ Cl ₂ (300 µl) were added and after an additional 40 minutes, Et ₂ O, saturated aq NaHCO ₃ (4 ml) and saturated aq Na ₂ S ₂ O were added ₃ (1 ml). The mixture was extracted with Et ₂ O (3x) and the combined extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give B2008 (0.58 mg, 61%). HRMS (FAB): calc. for C ₄₁ H ₅₇ FO ₁₁ + Na 815, 3783. Found: 815.3758.

B2011. Similarly, B1793 (1.9 mg, 0.003 mmol) was converted to B2011 (0.87 mg, 42% for 4 steps). HRMS (FAB): calc. for C ₄₅ H ₅₈ O ₁₁ + Na 797, 3877. Found: 797, 3877.

IN 2013

A solution of B1920 (1.4 mg, 0.0016 mmol), KCN (1 mg, 0.016 mmol) and DMSO (500 μl) was heated at 60°C for 8 hours. After cooling to room temperature, H ₂ O was added and the mixture was extracted with EtOAc (3x). The combined organic extracts were washed successively with _H2O and _brine , dried over _Na2SO4 , concentrated and purified by preparative TLC (80% EtOAc-hexanes) to give B2013 (0.78 mg, 67%). HRMS (FAB): calc. for C ₄₁ H ₅₇ NO ₁₁ +Na 762, 3829. Found: 762, 3848.

A mixture of B1920 (1.3 mg, 1.47 µmol), NaI (30 mg, excess) and acetone (1 ml) was stirred at 60°C for 3.5 hours. After cooling to room temperature, the reaction mixture was diluted with saturated aqueous NaHCO ₃ (3 ml), extracted with CH ₂ Cl ₂ (5x) and EtOAc, dried over Na ₂ SO ₄ and purified by column chromatography (50% EtOAc-CH ₂ Cl2 - 80% EtOAc-hexanes) to obtain iodide X1920 (1.3 mg, 100%).

A common part. A mixture of X1920 iodide (1.0 eq.), iso-Pr ₂ EtN (11-22 eq.), ArSH (9-46 eq.) and DMF (0.3 ml) was stirred at room temperature until the reaction was completed according to TLC data (usually 24-48 hours). The reaction mixture was diluted with a saturated aqueous solution of NaHCO ₃ (2 ml), extracted with CH ₂ Cl ₂ and EtOAc, dried over Na ₂ SO ₄ and purified by preparative TLC (80% EtOAc-hexanes or 5% MeOH-CH ₂ Cl ₂ ) to obtain sulfide products:

B1998. (1.3 mg gave 1.1 mg, 85%) HRMS (FAB): calc. for C ₄₆ H ₆₁ ClO ₁₁ S+Na 897, 3521. Found: 897.3533.

B2010. (1.1 mg gave 0.7 mg, 59%) HRMS (FAB): calc. for C ₄₇ H ₆₄ O ₁₂ S+Na 875.4016. Found: 875, 4,057.

B2019. (1.1 mg gave 0.7 mg, 61%) MS (FAB): M+Na

A common part. A 0.01 M solution of mCPBA (1.2 eq) in CH ₂ Cl ₂ was added to a solution of sulfide (1.0 eq) in CH ₂ Cl ₂ (0.5 ml) at 0°C for 30 minutes. The reaction mixture was diluted with saturated aqueous NaHCO ₃ (2 ml), extracted with CH ₂ Cl ₂ and EtOAc, dried over Na ₂ SO ₄ and purified by preparative TLC (80% EtOAc-hexanes or EtOAc) to obtain the products:

B2016. (0.9 mg gave 0.7 mg, 74%)

B2030. (1.0 mg gave 0.6 mg, 61%) HRMS (FAB): calc. for C ₄₇ H ₆₄ O ₁₄ S+Na 907, 3914. Found: 907, 3950.

B1934. TBDPSC1 (3.0 μl, 12 μmol) was added to a solution of 31793 (1.3 mg, 1.78 μmol), imidazole (10 mg, 166 μmol) and DMF (0.10 ml) at room temperature. After stirring for 1 hour, the reaction mixture was diluted with saturated aqueous NaHCO ₃ (2 ml), extracted with CH ₂ Cl ₂ (3x) and EtOAc (2x), dried over Na ₂ SO ₄ and purified by preparative TLC (5% MeOH-CH ₂ Cl ₂ to give the intermediate silyl ether (1.3 mg, 77%).

This material was dissolved in CH ₂ Cl ₂ (0.5 ml) and treated with Dess-Martin periodinane (10 mg, 24 µmol) for 1.5 hours at room temperature, diluted with Et ₂ O and filtered through celite. The filtrate was concentrated and purified by preparative TLC (50% EtOAc-hexanes) to give a diketone intermediate (1.0 mg, 77%), which was dissolved in THF (0.5 ml) and treated with 0.02 M TBAF containing 0 .01 M imidazole hydrochloride (THF solution, 75 µl, 1.5 µmol) at room temperature for 15 minutes. The reaction mixture was eluted through a SiO ₂ column (50% EtOAc-hexanes - 5% MeOH-CH ₂ Cl ₂ ) and the target product was further purified by preparative TLC (5% MeOH-CH ₂ Cl g to obtain B1934 (0.75 mg, 100% ). HRMS (FAB): calculated for C ₄₀ H ₅₆ O ₁₂ + Na 751.3669 Found: 751.3690.

B1922. Tetra-n-butylammonium azide (0.2 M in DMF, 0.5 mL, 0.10 mmol) was added to a solution of B2294 mesylate (21.4 mg, 0.026 mmol) in DMF (2 mL) at room temperature. After stirring at 83°C for 3.5 hours, the reaction mixture was cooled to room temperature, diluted with toluene, concentrated and purified by preparative TLC (80% ethyl acetate-hexanes) to give B1922 (18 mg, 92%).

B1939. Me ₃ P (1 M in THF) and H ₂ O (0.8 ml) were successively added to a solution of B1922 azide (24.6 mg, 0.032 mmol) in THF (3.2 ml) at room temperature. The mixture was stirred for 22 hours, diluted with toluene, concentrated and purified by flash chromatography [step gradient, 10% MeOH-EtOAc then MeOH-EtOAc-30% aqueous NH ₄ OH (9:86:5)] to give the target primary amine (23.3 mg), which according to ¹ H-NMR contained ~1% trimethylphosphine oxide. Lyophilization from benzene and high vacuum for 2 days gave B1939 (20.3 mg, 87%).

Synthesis of characteristic analogues of B1939:

В1930, В1940, В1973, В1987, В1988, В1991, В2003, В2004

B1930. Me ₃ P (1 M in THF, 13 μl, 0.013 mmol) was added to a solution of B1922 (1.6 mg, 2.1 μmol), THF (400 μl) and H ₂ O (100 μl) at room temperature. The mixture was stirred for 22 hours, diluted with toluene, concentrated and azeotroped with toluene (2x) to obtain the crude amine, which was used immediately in the next step.

EDC (0.06 M in CH ₂ Cl ₂ , 100 µl, 11 µmol) was added to a solution of crude amine, benzoylformic acid (0.8 mg, 5.3 µmol) and CH ₂ Cl ₂ (200 µl) at room temperature. After 30 minutes, the reaction was quenched with a 1:4 mixture of saturated aqueous NaHCO ₃ -salt solution and extracted with CH ₂ Cl ₂ (5x). The combined extracts were dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (EtOAc) to give B1930 (1.5 mg, 83% for two steps). HRMS (FAB): calc. for C ₄₈ H ₆₃ NO ₁₃ + Na 884.4197. Found: 884.4166.

B1940. Using the procedure described above for B1930, B1922 was reduced, coupled with 3-pyridylacetic acid hydrochloride and purified by preparative TLC [(MeOH-EtOAc-30% aqueous NH ₄ OH (9:86:5)] to give B1940 (0.8 mg, 67% for two stages) HRMS (FAB): calc. for C ₄₇ H ₆₄ N ₂ O ₁₂ +Na 871.4357.

B1973. Using the procedure described above, B1922 (0.9 mg, 1.2 μmol) was reduced, coupled with phenylacetic acid and purified by preparative TLC (5% MeOH-EtOAc) to give B1973 (0.44 mg, 44% for two steps ). HRMS (FAB): calc. for C ₄₈ H ₆₅ NO ₁₂ + Na 870.4404. Found: 870.4447.

B1987. Using the procedure described above, B1922 (0.9 mg, 1.2 μmol) was reduced, coupled with 3-indoleglyoxylic acid and purified by preparative TLC (3% MeOH-EtOAc) to give B1987 (0.8 mg, 75% for two stages). HRMS (FAB): calculated for C ₅₀ H ₆₄ N ₂ O ₁₂ +Na 923, 4306. Found: 923.4338.

B1991. Using the procedure described above, B1922 (1.0 mg, 1.3 μmol) was reduced, coupled with 4-chlorobenzoic acid and purified by preparative TLC (3% MeOH-EtOAc) to give B1991 (0.8 mg, 70% for two stages). HRMS (FAB): calc. for C ₄₇ H ₆₂ NO ₁₂ +Na 890, 3858. Found: 890.3843.

B2003. Using the procedure described above, B1922 (1.0 mg, 1.3 μmol) was reduced, coupled with 3,4,5-trimethoxybenzoylformic acid and purified by preparative TLC (EtOAc) to give B2003 (0.7 mg, 56% for two stages). HRMS (FAB): calc. for C ₅₁ H ₆₉ NO ₁₆ +Na 974, 4514. Found: 974.4525.

B2004. Using the procedure described above, B1922 (1.0 mg, 1.3 μmol) was reduced, coupled with 3,4,5-trimethoxybenzoic acid and purified by preparative TLC (5% Me OH-EtOAc) to give B2004 (0.7 mg, 58% for two stages). HRMS (FAB): calc. for C ₄₉ H ₆₅ NO ₁₃ + Na 946.4565. Found: 946, 4599.

B1988. Dess-Martin periodinan (1 mg, 2.3 µmol) was added to a solution of B1930 (0.80 mg, 0.93 µmol) in CH ₂ Cl ₂ (500 µl) at room temperature. After 1 hour the reaction was diluted with Et ₂ O and filtered through celite. The filtrate was washed successively with a 1:9 mixture of saturated aqueous NaHCO ₃ -Na ₂ S ₂ O ₃ and brine, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (80% EtOAc-hexanes) to obtain B1988 (0.45 mg, 56%). HRMS (FAB): calc. for C ₄₈ H ₆₁ NO ₁₃ + Na 882.4041. Found: 884.4012. Synthesis B2 090:

Compound 103 Indium powder (1.35 g, 11.8 mmol) was added to a solution of 102 (3.38 g, 17.6 mmol) in DMF (20 ml) at room temperature. Neat aldehyde 101 (3.72 g, 28.6 mmol) was then added and the mixture was stirred overnight, allowing the temperature to rise to room temperature. The reaction mixture was re-cooled to 0°C and then carefully quenched with saturated aqueous NH ₄ Cl (100 ml). After stirring for 30 minutes, the resulting mixture was extracted with Et ₂ O (3x), dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10%-20% EtOAc-hexanes) to give pure crystalline 103 (2.20 g, 59 %).

Compound 104. Et ₃ N (72 μl, 0.51 μmol) was added to a solution of 103 (1.09 g, 5.1-3 mmol) and thiophenol (0.63 ml, 7.16 mmol) in CH ₂ Cl ₂ and the resulting mixture was stirred at 0°C for 1 hour. Filtration through SiO ₂ gave a mixture of 104 and 105, which after medium pressure liquid chromatography (15%-20% EtOAc-hexanes) gave 104 (0.53 g, 32%) and 105 (0.92 g, 56%).

Compound 106. DIBALH (1 M in toluene, 3.28 ml, 3.28 mmol) was added to a solution of 104 (0.53 g, 1.64 mmol) in toluene (10 ml) at -78°C and the mixture was stirred at -78°C for 10 minutes. The reaction was quenched by careful addition of MeOH (0.40 ml, 9.84 mmol) and H ₂ O (0.17 ml, 9.8.4 mmol), warmed to room temperature and stirred for 20 minutes. The white suspension was filtered through a mixture of celite and SiO ₂ with a mixture of 1:1 CH ₂ Cl ₂ -Et ₂ O and concentrated to give 106 (0.53 g, 100%) as an oil.

Compound 107 A mixture of 106 (0.53 g, 1.64 mmol) and ethyl (triphenylphosphoranylidene) acetate (1.15 g, 3.29 mmol) in toluene (10 ml) was heated to 80°C for 15 hours. The mixture was cooled to room temperature and DBU (25 μL, 0.16 mmol) was added. The mixture was heated to 80°C for 1.5 hours, cooled to room temperature, concentrated and purified by column chromatography (10%-20% EtOAc-hexanes) to give 107 (0.54 g, 83%) as an oil (ratio isomers α:β 3:1).

Compound 108. A solution of mCPBA (~55%, 450 mg in 4.5 ml CH ₂ Cl ₂ , 1.44 mmol) was added to solution 107 (0.54 g, 1.36 mmol) c. CH ₂ Cl ₂ (10 ml) at -78°C. The reaction mixture was diluted with a saturated aqueous solution of NaHCO ₃ (50 ml), H ₂ O (10 ml) and Et ₂ O (60 ml) and then warmed to room temperature. The separated aqueous layer was extracted with EtOAc (4x) and the combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (50% EtOAc-hexanes) to give 108 (0.51 g, 92%) as an oil.

Compound 109 A mixture of 108 (0.51 g, 1.24 mmol) and NaOAc (1.00 g, 12.4 mmol) in Ac20 (10 ml) was stirred at 140°C for 12 hours, cooled to room temperature and then concentrated. The residue was partitioned between saturated aqueous NaHCO ₃ (20 ml) and Et ₂ O (30 ml) and stirred vigorously at room temperature for 30 minutes. The separated aqueous layer was extracted with Et ₂ O (2x) and the combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (5%-15% EtOAc-hexanes) to give 109 (0.41 g, 73%) as oils

Compound 110 A mixture of 109 (0.41 g, 0.91 mmol) and K ₂ CO ₃ (44.3 mg, 0.32 mmol) in EtOH (5 ml) was heated to 60-70°C for 1 day. After cooling to room temperature, the reaction mixture was concentrated and eluted through a SiO ₂ column (10%-20% EtOAc-hexanes) to obtain a partially purified aldehyde intermediate. This material was dissolved in EtOH (2.5 ml), treated with NaBH ₄ (50 mg, 1.32 mmol) and stirred at room temperature for 30 minutes. The mixture was concentrated and purified by column chromatography (40% EtOAc-hexanes) to give 110 (181 mg, 66%).

Compound 111. BF ₃ ⋅OEt ₂ (0.05 M in CH ₂ Cl ₂ , 175 µl, 8.75 µmol) was added to solution 110 (181 mg, 0.60 mmol) and p-methoxybenzyl-2,2,2 -trichloroacetimidate (0.50 ml, 1.80 mmol) in CH ₂ Cl ₂ (5 ml) at 0°C. The resulting mixture was stirred for 1.5 hours at 0°C and for 2 hours at room temperature until the reaction was complete. The mixture was quenched with saturated aqueous NaHCO ₃ (25 ml) and extracted with Et ₂ O (5x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (CH ₂ Cl ₂ and then 20% EtOAc-hexanes) to give semi-purified 111 (0.37 g, >100%) as an oil.

Compound 112 A mixture of 111 (0.37 g, max = 0.60 mmol) and TsOH⋅H ₂ O (36 mg) in EtOH (5 ml) was stirred first at room temperature overnight and then at 60° C. within 1 hour. Additional TsOH⋅H ₂ O (31 mg) was added at room temperature and the reaction mixture was stirred for 1 hour at room temperature. The mixture was then concentrated, quenched with saturated aqueous NaHCO ₃ and extracted with EtOAc (5x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-50% EtOAc-hexanes and then 5% Me-OH-CH ₂ Cl ₂ ) to give 112 (121 mg, 53%) as an oil , along with 111 extracted (49 mg, 21%).

Compound 113. TBSOTf (250 µl, 1.09 mmol) was added to a solution of 112 (121 mg, 0.32 mmol) and Et ₃ N (17 6 µl, 1.26 mmol) in CH ₂ Cl ₂ at 0°C and the resulting mixture was stirred for 25 minutes. The reaction was quenched with saturated aqueous NaHCO ₃ (15 ml) and the separated aqueous layer was extracted with ether (3x). The combined organic phases were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (5%-10% EtOAc-hexanes) to give 113 (165 mg, 85%) as an oil.

Compound 114. DIBALH (1 M in toluene, 0.54 ml, 0.54 mmol) was added to a solution of 113 (165 mg, 0.27 mmol) in toluene (5 ml) at -78°C and the resulting mixture was stirred at - 78°C for 10 minutes. The reaction was quenched by careful addition of MeOH (65 μl, 0.81 mmol) and H ₂ O (29 μl, 0.81 mmol), warmed to room temperature and stirred for 25 minutes. The white suspension was filtered through Celite with 1:1 CH ₂ Cl ₂ -Et ₂ O. Concentration and purification by column chromatography (10%-20% EtOAc-hexanes) gave 114 (153 mg, 100%) as an oil.

B2090. Similar to the method described in Scheme 6 for the synthesis of B17 94, intermediate 114 was converted to B2090. HRMS (FAB): calc. for C ₃₉ H ₅₆ O ₁₁ +Na 723, 3720. Found: 723.3731.

B2136. Similar to the method described for B1939, B2090 was converted to B2136. HRMS (FAB): calc. for C ₃₉ H ₅₇ NO ₁₀ + Na 722.3880. Found: 722.3907.

Synthesis of B2039/B2043:

Diol 201. TBAF (1 M in THF, 383 μL, 0.383 mmol) was added to a solution of X2318 (350-LS-218) (80.8 mg, 0.0765 mmol) in THF (7 mL) and stirred at room temperature in within 16 hours. After partial concentration, the residue was applied directly to a SiO ₂ column loaded with 30% EtOAc-hexanes. Gradient elution (30% EtOAc-hexanes - EtOAc) gave diol 201 (49.7 mg, 92%).

Aldehyde 202. A mixture of diol 201 (497 mg, 0.0707 mmol), NaIO ₄ (100 mg, 0.47 mmol), MeOH (10 ml) and H ₂ O (2.5 ml) was stirred at room temperature for 30 minutes. H ₂ O was added and the mixture was extracted with CH ₂ Cl ₂ (4x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (30% EtOAc-hexanes) to give aldehyde 202 (41.7 mg, 88%).

Alcohol 203.4-fluorophenylmagnesium bromide (2 M in Et ₂ O, 155 µl, 0.31 mmol) was added to a solution of addehyde 202 (41.7 mg, 0.062 mmol) in THF (6 ml). After 15 minutes at room temperature, the reaction was quenched with saturated aqueous NaHCO ₃ and extracted with CH ₂ Cl ₂ (4x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (4 0% EtOAc-hexanes) to give alcohol 203 (32.4 mg, 68%) as a 1:1 mixture of C34 isomers. The minor undesired C27 isomer was separated at this stage and was also isolated as a 1:1 mixture of C34 isomers (8.4 mg, 18%).

Ether 204. Et ₃ N (18 μl, 0.13 mmol) and TBSOTf (15 μl, 0.063 mmol) were added to a solution of alcohol 203 (32.4 mg, 0.042 mmol) in CH ₂ Cl ₂ (5 ml) at 0° WITH. After 20 minutes, the reaction was quenched by adding saturated aqueous NH ₄ Cl and extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give ester 204 (33.1 mg, 89%).

Alcohol 205 LAH (1 M in THF, 113 µl, 0.113 mmol) was added dropwise to a solution of ether 204 (33.1 mg, 0.0375 mmol) in Et ₂ O (10 ml) at 0°C. After 20 minutes, H ₂ O and 1 M NaOH were added and the mixture was stirred at room temperature for 10 minutes. Filtration through Celite, concentration and purification by column chromatography (4 0% EtOAc-hexanes) gave alcohol 205 (28.4 mg, 95%).

Ether 206. Diisopropylethylamine (31 μl, 0.18 mmol) and MMTrCl (22 mg, 0.071 mmol) were added to a solution of alcohol 205 (28.4 mg, 0.0355 mmol) in CH ₂ Cl ₂ (4 ml) at 0° WITH. After 15 hours at room temperature, H ₂ O was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined extracts were washed with saline, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (40% EtOAc-hexanes) to obtain ester 206 as a ~1.5:1 mixture of C34 epimers (45 mg, quantitative yield), which contained a small amount of closely related impurities.

Alcohol 207.DDQ (40 mg, 0.18 mmol) was added to a solution of ether 206 (37 mg, 0.034 mmol) in CH ₂ Cl ₂ (4 ml) and a 1:10 mixture of tert-BuOH and phosphate buffer pH 7 (2 ml) at 0°C. The mixture was stirred vigorously in the dark for 15 minutes. Three additional portions of DDQ (40 mg, 0.18 mmol) were added at 10 minute intervals, then the reaction was diluted with saturated aqueous NaHCO ₃ and extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (30% EtOAc-hexanes) to give alcohol 207 (19.2 mg, 59%) as well as recovered ester 206 (9.7 mg , 26%).

Mesylates 208A and 208B. Et ₃ N (19 μl, 0.13 mmol) and Ms ₂ O (10 mg, 0.056 mmol) were successively added to a solution of alcohol 207 (21.3 mg, 0.022 mmol) in CH ₂ Cl ₂ (6 ml) at 0°C. After 30 minutes, saturated aqueous NaHCO ₃ was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined extracts were washed with saline, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (30% EtOAc-hexanes) to give mesylates 208A (11.7 mg 51%) and 208 B (6.5 mg, 28%) in in the form of individual C34 isomers.

B2039 and B2043. Similar to that described in Scheme 6 for the synthesis of B1794, both diastereomers 208A and 208B were independently converted to B2039 and B2043. HRMS (FAB): calc. for C ₄₅ H ₅₉ FO ₁₁ +Na 817.3939. Found: for B2039 817.3896, for B2043 817.3910.

Synthesis of B2086, B2088, B2091

Alcohol X-20. NaIO ₄ (1.16 g, 5.4 mmol) was added to a solution of diols 10 a, b (1.19 g, 3.0 mmol) in a MeOH-H ₂ O mixture (4:1, 75 ml) at 0° WITH. The reaction mixture was allowed to warm to room temperature. After stirring for 40 minutes, the mixture was diluted with EtOAc, filtered through celite, concentrated and partitioned between brine and CH ₂ Cl ₂ . The separated aqueous layer was extracted with CH ₂ Cl ₂ (2x). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to obtain the crude aldehyde intermediate.

NaBH ₄ (228 mg, 6.0 mmol) was added to a solution of the aldehyde in MeOH-Et ₂ O (1:1, 40 ml) at 0°C. The mixture was stirred for 30 minutes, carefully quenched with saturated aqueous NH ₄ Cl, stirred for 20 minutes at room temperature and extracted with CH ₂ Cl ₂ (3x). The combined extracts were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (4 0%-50% EtOAc-hexanes) to give X-20 alcohol (1.02 g, -93% for two steps).

Silyl ether. Imidazole (0.94 g, 13.9 mmol) and TBSC1 (0.59 g, 3.89 mmol) were sequentially added to a solution of alcohol X-20 (1.02 g, 2.78 mmol) in DMF (10 ml) at room temperature. After 14 hours, the reaction mixture was diluted with saturated aqueous NH ₄ Cl and extracted with EtOAc (3x). The combined organic extracts were washed with H ₂ O, brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (5%-15% EtOAc-hexanes) to give silyl ether 21 (1.3 g, 98%).

Alcohol 22. A mixture of Pd(OH) ₂ (20%, 0.8 g), silyl ether 21 (1.3 g, 2.70 mmol) and EtOAc (30 ml) was stirred for 1 hour under H ₂ (1 atm) at room temperature, filtered through celite, concentrated and purified by flash chromatography (20%-40% EtOAc-hexanes) to give alcohol 22 (0.96 g, 91%).

Alcohol 25. 4-methylmorpholine N-oxide (980 mg, 8.4 mmol) and TPAP (131 mg, 3.26 mmol) were successively added to a solution of alcohol 22 (1.78 g, 4.6 mmol) in CH ₂ Cl ₂ (45 ml) at room temperature. A cold bath was necessary to regulate this exothermic reaction. After 20 minutes, the reaction mixture was diluted with hexanes, filtered through a short column of SiO ₂ (15% EtOAc-hexanes) and concentrated to give the crude ketone.

Tebbe's reagent (14.9 mL, 9.0 mmol) was added over 10 minutes to a solution of the crude ketone in THF (60 mL) at 0°C. After 20 minutes, the reaction mixture was poured into Et ₂ O (100 ml), which was pre-cooled to -78°C, quenched by slow addition of H ₂ O (30 ml), warmed to room temperature, stirred for 30 minutes and extracted with Et ₂ O (4x). The combined extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (10% EtOAc-hexanes) to give the title heme-dimethyl product contaminated olefin (1.07 g). This mixture was used directly in the next step.

9-BBN (0.5 M in THF, 11.5 mL, 5.8 mmol) was added to the olefin solution in THF (15 mL) at 0°C. The reaction mixture was allowed to warm to room temperature, stirred for 5 hours and then re-cooled to 0°C. H ₂ O (60 ml), THF (60 ml) and NaBO ₃ ⋅ 4H ₂ O (5.7 g) were added. After stirring for 5 hours at room temperature, THF was removed under reduced pressure and the aqueous residue was extracted with EtOAc (4x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (20%-40% EtOAc-hexanes) to give alcohol 25 (605 mg, 18% for three steps).

Alcohol 26 Using the previously described procedure, alcohol 25 (604 mg, 1.49 mmol) was successively oxidized, isomerized, and reduced. Purification by flash chromatography (20%-40% EtOAc-hexanes) gave alcohol 26 (550 mg, 91% for three steps).

MRM ester 27. BF ₃ ⋅OEt ₂ (0.05 M in CH ₂ Cl ₂ , 270 μl, 0.013 mmol) was added to a solution of alcohol 26 (545 mg, 1.35 mmol) and MRM trichlorimidate (1.14 g , 4.0 mmol) in CH ₂ Cl ₂ (40 ml) at 0°C. After 1 hour, the reaction was quenched with saturated aqueous NaHCO ₃ , extracted with CH ₂ Cl ₂ , dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (10%-15% EtOAc-hexanes) to give MPM ester 27 (580 mg , 82%).

Alcohol 28. LAH (1 M in THF, 1.9 ml, 1.9 mmol) was added to a solution of MPM ester 27 (580 mg, 1.11 mmol) in Et ₂ O (100 ml) at 0°C. After 30 minutes, the reaction was carefully quenched with H ₂ O (0.5 ml) and 1 N. aqueous NaOH (0.5 ml), stirred for 1 hour at room temperature, filtered through celite, concentrated and purified by flash chromatography (30%-50% EtOAc-hexanes) to give alcohol 28 (460 mg, 95%).

Olefin 29. DMSO (441 µl, 6.23 mmol) was added to a solution of oxalyl chloride (272 µl, 3.12 mmol) in CH ₂ Cl ₂ (30 ml) at -7 8°C. After 15 minutes, a solution of alcohol 28 (458 mg, 1.04 mmol) in CH ₂ Cl ₂ (15 ml) was added to this reaction mixture. After stirring for 1 hour at -78°C, Et ₃ N (1.3 ml, 9.35 mmol) was added. The reaction mixture was heated to 0°C, stirred for 10 minutes, diluted with saturated aqueous NH ₄ Cl and extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and filtered through a short column of SiO ₂ (20%-30% EtOAc-hexanes) to obtain the crude aldehyde.

n-BuOH (1.63 M, 1.4 ml, 2.28 mmol) was added dropwise to a solution of CH ₃ PPh ₃ Br (815 mg, 2.28 mmol), THF (20 ml) and DMSO (7.5 ml) at 0°C. After 1 hour, a solution of aldehyde in THF (10 ml) was added. The reaction mixture was warmed to room temperature and stirred for 3 hours. Saturated aqueous NH ₄ Cl solution was added and the mixture was extracted with EtOAc (4x). The combined organic extracts were washed with H ₂ O, brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (10%-15% EtOAc-hexanes) to give olefin 29 (380 mg, 95% yield over two steps).

Compound 31. 9-BBN (0.5 M in THF, 6 mL, 3 mmol) was added to a solution of olefin 29 (370 mg, 0.85 mmol) in THF (7 mL) at 0°C. The mixture was allowed to warm to room temperature and stirred for 1 hour. After cooling again to 0°C, H ₂ O (30 ml), THF (20 ml) and NaBO ₃ ⋅ 4H ₂ O (2.8 g) were added. After stirring for 3 hours at room temperature, THF was removed under reduced pressure. The aqueous residue was extracted with EtOAc(4x), dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (25%-50% EtOAc-hexanes) to give alcohol 30, which was used directly in the next step.

Pivaloyl chloride (157 μl, 1.27 mmol) was added to a solution of alcohol 30 in a mixture of CH ₂ Cl ₂ -pyridine (1:1, 10 ml) at room temperature. After 18 hours, additional pivaloyl chloride (100 μl, 0.81 mmol) was added. After 1 hour, the reaction mixture was cooled to 0°C, quenched with MeOH (0.5 ml), concentrated, diluted with brine and extracted with CH ₂ Cl ₂ (4x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (10%-15% EtOAc-hexanes) to give compound 31 (410 mg, 90% for two steps).

Alcohol 32. TBAF (1 M d THF, 1.14 mL, 1.14 mmol) was added to a solution of 31 (410 mg, 0.761 mmol) in THF (5 mL) at room temperature. After 1.5 hours, the reaction mixture was concentrated and purified by flash chromatography (40% EtOAc-hexanes - 100% EtOAc) to give alcohol 32 (320 mg, 100%).

Alcohol 33a and 33b. Dess-Martin periodinane (925 mg, 2.18 mmol) was added to a solution of alcohol 32 (309 mg, 0.727 mmol) in CH ₂ Cl ₂ (19 ml) at room temperature. After 1 hour the reaction was diluted with Et ₂ O and filtered through celite. The filtrate was washed successively with a mixture of saturated aqueous NaHCO ₃ -Na ₂ S ₂ O ₃ 1:9 and brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (20%-30% EtOAc-hexanes) to obtain the target aldehyde , which was immediately used in the next stage.

BF ₃ ⋅OEt ₂ (135 μl, 1.1 mmol) was added to a solution of crude aldehyde, tri-n-butylallyltin (337 μl, 1.08 mmol) and CH ₂ Cl ₂ (16 ml) at -78°C. After 1 hour, the reaction was quenched with saturated aqueous NaHCO ₃ and extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and purified by medium pressure liquid chromatography (25%-30% EtOAc-hexanes) to give the major, more polar alcohol 33a (165 mg, 4 9% for two stages) and the minor, less polar product 33b (90 mg, 27% for two steps).

Compound 34 TBSOTf (163 μl, 0.710 mmol) was added to a solution of alcohol 33a (165 mg, 0.355 mmol), Et ₃ N (247 μl, 1.78 mmol) and CH ₂ Cl ₂ (5 ml) at 0°C. After 25 minutes, the reaction was quenched with saturated aqueous NaHCO ₃ , extracted with CH ₂ Cl ₂ (3x), dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (15%-20% EtOAc-hexanes) to give compound 34 (200 mg , 98%).

Diols 35a and 35b. OsO ₄ (0.1 M solution in toluene, 32 μl, 3.2 μmol) was added to a solution of K ₂ CO ₃ (168 mg, 1.22 mmol), K ₃ Fe(CN) ₆ (400 mg, 1.22 mmol), (DHQ) ₂ PYR (11 mg, 12 µmol), H ₂ O (3.2 ml), and tert-BuOH (2.2 ml) at 0°C. A solution of olefin 34 (200 mg, 0.345 mmol) in t-BuOH (1 mL) was then added to the reaction mixture. After 5 hours at 0°C, Na ₂ S ₂ O ₃ ⋅5H ₂ O (200 mg) was added. The reaction mixture was warmed to room temperature, stirred for 30 minutes and extracted with CH ₂ Cl ₂ (5x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (70% EtOAc-hexanes) to give the major, less polar diol 35a (118 mg, 56%) and the minor, more polar diastereomeric product 35b ( 74 mg, 35%). Individual diastereoisomers were then used separately.

Compound 36. TBSOTf (177 μl, 0.77 mmol) was added to a solution of diol 35a (118 mg, 0.192 mmol), Et ₃ N (267 μl, 1.92 mmol) and CH ₂ Cl ₂ (5 ml) at 0° WITH. After 25 minutes, the reaction was quenched with saturated aqueous NaHCO ₃ , extracted with CH ₂ Cl ₂ (3x), dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (10%-15% EtOAc-hexanes) to give compound 36 (161 mg , 100%).

Alcohol 37 Using the same procedure as previously described for alcohol 28, alcohol 37 (135 mg, 93%) was obtained from 36 (161 mg, 0.192 mmol) after purification by flash chromatography (20%-40% EtOAc-hexanes).

Aldehyde 38. Dess-Martin periodinane (227 mg, 0.535 mmol) was added to a solution of alcohol 37 (135 mg, 0.178 mmol) in CH ₂ Cl ₂ (5 ml) at room temperature. After 1 hour, the reaction mixture was diluted with Et ₂ O and filtered through celite. The filtrate was washed successively with a mixture of saturated aqueous NaHCO ₃ -Na ₂ S ₂ O ₃ 1:9 and brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (10%-20% EtOAc-hexanes) to obtain aldehyde 38 (127 mg, 95%).

B2086, B2102. Each of the diastereomers obtained above was separately brought to the final product similarly to the procedure described in Scheme 6 for B1794. Diastereomer 35a gave B2086. Diastereomer 35b gave B2102.

B2088. NaIO ₄ was added to a solution of B2086 (1 mg, 1.29 µmol) in MeOH-H ₂ O (4:1, 1 ml) at room temperature. After 30 minutes, the reaction mixture was diluted with H ₂ O, extracted with CH ₂ Cl ₂ (6x), dried over Na ₂ SO ₄ and concentrated to give B2088 (1.2 mg).

B2091. NaBH ₄ (0.013 M in EtOH, 20 μl, 0.27 μmol) was added to a solution of B2088 (1 mg, 1.29 μmol) in MeOH-CH ₂ Cl ₂ (4:1, 0.5 ml) at -78° WITH. Additional NaBEU was added periodically, with the reaction carefully monitored by TLC (a total of 220 μl of NaBH ₄ solution was required). The reaction mixture was quenched at 0°C with a saturated aqueous solution of NH ₄ Cl, stirred for 20 minutes at room temperature and extracted with CH ₂ Cl ₂ (6x). The combined extracts were dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (7% MeOH-EtOAc) to give B2091 (0.40 mg, 50%). Synthesis B1933:

Alcohol 303.9-BBN (0.5 M in THF, 23 ml, 0.012 mol) was added dropwise over 30 minutes to a solution of alkene 302 (1.51 g, 0.0038 6 mol) in THF (40 ml) at 0°C. After stirring at room temperature for 80 minutes, the mixture was cooled to 0°C and H ₂ O (80 ml) was added carefully, followed by NaBO ₃ ⋅ 4 _{H 2} O (4.2 g, 0.027 mol). The mixture was stirred vigorously at room temperature for 2.3 hours, then extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (50% EtOAc-hexanes) to give alcohol 303 (1.37 g, 87%).

Aldehyde 304 Oxalyl chloride (88 µl, 1.00 mmol) was added dropwise to a solution of DMSO (142 µl, 2.00 mmol) in CH ₂ Cl ₂ (20 ml) at -78°C. After 30 minutes, a solution of alcohol 303 (137 mg, 0.335 mmol) in CH ₂ Cl ₂ (5 ml) was added and stirred at -78°C for 1 hour. Et ₃ N (420 μl, 3.01 mmol) was added and after 10 minutes the reaction was stirred for 10 minutes at 0° C. at which point saturated aqueous NH ₄ Cl was added and the resulting mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (50% EtOAc-hexanes) to give intermediate aldehyde 304 (0.114 g, 84%), which was immediately used in the next step.

Alcohol 305. TBAF (1 M in THF, 5 µL, 0.005 mmol) was added to a solution of aldehyde 304 (0.114 g, 0.27 mmol) in CF3TMS (0.5 M in THF, 1.1 mL, 0.54 mmol) at 0°C. After 20 minutes, a second portion of TBAF (1 M in THF, 100 μl, 0.1 mmol) was added and the mixture was stirred for 10 minutes at which point excess TBAF (1 M in THF, 270 μl, 0.27 mmol) was added dropwise ) to cleave the silyl ether intermediate. After 30 minutes the mixture was diluted with H ₂ O and extracted with EtOAc (3x). Organic extracts were washed with H ₂ O, brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (50% EtOAc-hexanes) to give alcohol 305 (123 mg, 95%) as an unresolved 1:1 mixture of isomers.

Silyl ether 306. TBSOTf (265 µl, 1.16 mmol) was added to a solution of alcohol 305 (123 mg, 0.257 mmol) and Et ₃ N (430 µl, 3.08 mmol) in CH ₂ Cl ₂ (8 ml) at 0 °C. After stirring at room temperature for 20 hours, saturated aqueous NaHCO ₃ was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give silyl ether 306 (148 mg, 97%).

Alcohol 307.LAH (1 M in THF, 220 µl, 0.22 mmol) was added dropwise to a solution of silyl ether 306 (131 mg, 0.22 mmol) in Et ₂ O (5 ml) at 0°C. After 20 minutes, H ₂ O and 1 M NaOH were carefully added. The mixture was stirred at room temperature for 30 minutes, filtered through glass wool, concentrated and purified by column chromatography (50% EtOAc-hexanes) to give alcohol 307 (112 mg, quantitative yield).

Alkene 309. Oxalyl chloride (58 µl, 0.66 mmol) was added dropwise to a solution of DMSO (94 µl, 1.3 mmol) in CH ₂ Cl ₂ (10 ml) at -78°C. After 30 minutes, a solution of alcohol 307 (112 mg, 0.22 mmol) in CH ₂ Cl ₂ (3 ml) was added. After 1 hour, Et ₃ N (276 μl, 1.98 mmol) was added and after 10 minutes at -78°C, the reaction was stirred at 0°C for 10 minutes. Saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (50% EtOAc-hexanes) to give aldehyde 308 (101 mg, 91%), which was immediately used in the next step.

nBuLi (1.63 M in THF, 200 µl, 0.33 mmol) was added dropwise to a solution of CH ₃ PPh ₃ Br (118 mg, 0.33 mmol) in THF (3 ml) and DMSO (1.2 ml) at 0°C. After 70 minutes, a solution of aldehyde 308 (101 mg, 0.20 mmol) in THF (3 ml) was added and after 10 minutes at 0° C., the reaction was stirred at room temperature for 1 hour. The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give alkene 309 (90.9 mg, 90%).

Alcohol 310. 9-BBN (0.5 M in THF, 17 mL, 8.45 mmol) was added dropwise to a solution of alkene 309 (1.06 g, 2.11 mmol) in THF (30 mL) at 0°C . After stirring for 2/5 hours at room temperature, the reaction was cooled to 0° C. and H ₂ O (60 ml) was carefully added, followed by NaBO ₃ ⋅ 4 _{H 2} O (3.25 g, 21.1 mmol). The mixture was stirred vigorously at room temperature for 2 hours, then diluted with H ₂ O and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes) to give alcohol 310 (0.920 g, 84%).

Pivaloate 311. A mixture of alcohol 310 (65.8 mg, 0.012 6 mmol), pyridine (61 µl, 0.76 mmol) and PvCl (23 µl, 0.189 mmol) in CH ₂ Cl ₂ (3 ml) was stirred at room temperature in within 5 hours. A second reaction using alcohol 310 (0.92 g, 1.76 mmol) was carried out under the same conditions and both reactions were combined during the workup: saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give pivaloate 311 (1.08 g, quantitative yield).

Alcohol 312. A mixture of ester 311 (0.811 g, 1.33 mmol), DDQ (6.1 g, 27 mmol) and a 10:1 mixture of tert-BuOH phosphate buffer pH 7 (42 ml) in CH ₂ Cl ₂ ( 84 ml) was stirred vigorously in the dark at room temperature for 1.5 hours at which point additional DDQ (1.0 g, 4.4 mmol) was added. After 1 hour, saturated aqueous NaHCO ₃ was added and the mixture was extracted with CH ₂ Cl ₂ (4x). The combined organic extracts were washed successively with saturated aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ / concentrated and purified by column chromatography (20% EtOAc-hexanes) to give alcohol 312 (0.56 g, 87%), and with isolating starting material 311 (97 mg, 12%).

Ketone 313. Oxalyl chloride (21 µl, 0.12 mmol) was added dropwise to a solution of DMSO (34 µl, 0.48 mmol) in CH ₂ Cl ₂ (3 ml) at -78°C. After 1 hour, a solution of alcohol 312 (39.4 mg, 0.081 mmol) in CH ₂ Cl ₂ (1.5 ml) was added and the mixture was stirred for 1.5 hours. Et ₃ N (100 μl, 0.73 mmol) was added and after 10 minutes the mixture was heated to 0°C. A saturated aqueous solution of NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (30% EtOAc-hexanes) to give ketone 313 (36.6 mg, 93%), which was used directly in the next step.

Alkene 314. Tebbe's reagent (~0.65 M in toluene, 720 μL, 0.47 mmol) was added dropwise to a solution of ketone 313 (151 mg, 0.31 mmol) in THF (5 mL) at 0°C. After 5 minutes, H ₂ O was added carefully and the mixture was extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10% EtOAc-hexanes) to give alkene 314 (139 mg, 93%).

Alcohol 315. 9-BBN (0.5 M in THF, 6.0 mL, 2.9 mmol) was added dropwise to a solution of alkene 314 (468 mg, 0.97 mmol) in THF (10 mL) at 0°C. . The mixture was stirred at room temperature for 2 hours at which point additional 9-BBN (0.5 M in THF, 500 μL, 0.25 mmol) was added. After 2.5 hours the mixture was cooled to 0°C and H ₂ O (10 ml) was carefully added followed by NaBO ₃ 4H ₂ O (1.5 g, 9.7 mmol). The mixture was stirred vigorously at room temperature for 5 hours, diluted with H ₂ O and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes gradient) to give alcohol 315 (0.47 g, 97%).

Alcohol 316. Oxalyl chloride (246 µl, 2.82 mmol) was added dropwise to a solution of DMSO (400 µl, 5.64 mmol) in CH ₂ Cl ₂ (40 ml) at -78°C. After 1 hour, a solution of alcohol 315 (0.47 g, 0.94 mmol) in CH ₂ Cl ₂ (10 ml) was added and the mixture was stirred for 1 hour. Et ₃ N (1.2 ml, 8.5 mmol) was added and after 10 minutes the mixture was heated to 0° C. and stirred for 10 minutes. A saturated aqueous solution of NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ and concentrated. The crude aldehyde was stirred in CH ₂ Cl ₂ (20 ml) and Et ₃ N (2 ml) at room temperature overnight. Saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (30% EtOAc-hexanes) to give the epimerized aldehyde, which was immediately dissolved in 1:1 Et ₂ O:EtOH (10 ml) and cooled to 0°C. NaBH ₄ (35 mg, 0.94 mmol) was added and after 10 minutes the reaction was quenched with saturated aqueous NH ₄ Cl. The mixture was extracted with EtOAc (3x) and the combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (30% EtOAc-hexanes) to give alcohol 316 (0.410 g, 87% yield for 3 steps).

Ether 317. Alcohol 316 (60.7 mg, 0.12 mmol) and MPMOTCl (0.10 g, 0.36 mmol) were combined, azeotroped from toluene (3x) and dried under high vacuum overnight. CH ₂ Cl ₂ (3 ml) was added and the mixture was cooled to 0°C. BF ₃ ⋅OEt ₂ (approximately 1 µl, 0.01 mmol) was added and after stirring for 10 minutes the reaction was quenched with saturated aqueous NH ₄ Cl. The mixture was extracted with CH ₂ Cl ₂ (3x) and the combined extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (30% EtOAc-hexanes) to give ester 317 (55.4 mg, 74%).

Alcohol 318. LAH (1 M in THF, 104 µl, 0.104 mmol) was added dropwise to a solution of ether 317 (54 mg, 0.087 mmol) in Et ₂ O (5 ml) at 0°C. After 30 minutes, H ₂ O and 1 M NaOH were carefully added. The mixture was stirred at room temperature for 10 minutes, filtered through glass wool, concentrated and purified by column chromatography (30%-50% EtOAc-hexanes) to give alcohol 318 (45.5 mg, 98%).

Aldehyde 319. Oxalyl chloride (11 µl, 0.13 mmol) was added dropwise to a solution of DMSO (18 µl, 0.25 mmol) in CH ₂ Cl ₂ (2 ml) at -78°C. After 1.8 hours, a solution of alcohol 318 (22.6 mg, 0.042 mmol) in CH ₂ Cl ₂ (1 ml) was added and the mixture was stirred for 1 hour. Et ₃ N (53 μl, 0.38 mmol) was added and after 10 minutes the reaction was heated to 0°C and stirred for 10 minutes. Saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (20% EtOAc-hexanes) to give aldehyde 319 (21.7 mg, 97%).

B1933. Similar to Scheme 6 for the synthesis of B1794, intermediate 319 was converted to B1933. HRMS (FAB): calc. for C ₄₁ H ₅₇ F ₃ O ₁₁ + Na 783.3931. Found: 783, 3940.

B1942. A mixture of B1897 (2 mg, 2.73 µmol), NaIO ₄ (35 mg, 0.16 mmol), MeOH (0.8 ml) and H ₂ O (0.2 ml) was stirred at room temperature for 30 minutes. The reaction mixture was then diluted with H ₂ O (3 ml) and extracted with CH ₂ Cl ₂ (6x) and EtOAc (2x). The combined organic phases were dried over Na ₂ SO ₄ and purified by column chromatography (5% Me0H-CH ₂ Cl ₂ ) to obtain the target aldehyde.

This material was dissolved in THF (0.1 ml), cooled to 0°C and treated with 0.5 C CF ₃ TMS in THF (30 μl, 15 mmol) followed by the addition of 0.05 M TBAF in THF (5 ml, 0.025 mmol). After stirring for 30 minutes, the reaction mixture was diluted with saturated aqueous NaHCO ₃ (2 ml) and H ₂ O (1 ml), extracted with EtOAc (6x), dried over Na ₂ SO ₄ , filtered and concentrated to obtain crude bis-TMS- ether.

This material was dissolved in THF (0.5 mL) and treated with 1 M TBAF in THF containing 0.5 M imidazole hydrochloride (8 μL, 8 μmol) at room temperature for 30 minutes. The reaction mixture was eluted through a SiO ₂ column (50% EtOAc-hexanes - EtOAc) to give the diol as an intermediate.

A mixture of this product and Dess-Martin periodinane (10 mg, 24 mmol) in CH ₂ Cl ₂ (0.5 ml) was stirred at room temperature for 1 hour, diluted with Et ₂ O (5 ml) and filtered through celite. The filtrate was concentrated and purified by preparative TLC (50% EtOAc-hexanes) to give B1942 (1.5 mg, 72% for 5 steps). HRMS (FAB): calc. for C ₄₀ H ₅₅ F ₃ O ₁₁ +Na 767, 3516. Found: 767, 3542.

Synthesis of B2070/B2073:

Alcohol 401. A mixture of NaIO ₄ (375 mg, 1.74 mmol), X400 (674 mg, 1.58 mmol), MeOH (16 ml) and H ₂ O (4 ml) was stirred at room temperature for 1 hour. After dilution with _H2O , the mixture was extracted _{with CH2Cl2} (4x) and the combined organic extracts _were dried over _Na2SO4 , concentrated and purified by flash chromatography (30% EtOAc-hexanes) to give the aldehyde as an intermediate (570 mg), which was immediately dissolved in DMF (15 ml). Indium (275 mg, 2.4 mmol) and 3-bromo-3,3-difluoropropene (240 µl, 2.4 mmol) were added and after stirring at room temperature for 17 hours, H ₂ O and 0.1 M HCl were added . The mixture was extracted with EtOAc (3x) and the combined organic extracts were washed successively with _H2O and _brine , dried over _Na2SO4 , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes) to give alcohol 401 as mixture 1: 1 C34 isomers (605 mg, 81% for two stages).

Diol 402. Mixture of OsO ₄ (1 crystal), alcohol 401 (605 mg, 1.28 mmol), 4-methylmorpholine N-oxide (0.45 g, 3.84 mmol), acetone (30 ml) and H ₂ O (6 ml) was stirred at room temperature for 29 hours. Additional OsO ₄ (3 crystals) and 4-methylmorpholine N-oxide (0.1 g, 0.8 mmol) were added and saturated aqueous Na ₂ S ₂ O ₃ was added after 2 days. The mixture was extracted with CH ₂ Cl ₂ (6x) and the combined organic extracts were dried over Na ₂ SO ₄ and concentrated. The crude triol intermediate was immediately dissolved in 4:1:MeOH:H ₂ O (25 mL) and NaIO ₄ (0.41 g, 1.9 mmol) was added. After stirring vigorously at room temperature for 2 hours, the mixture was diluted with H ₂ O, extracted with CH ₂ Cl ₂ (3x) and the combined organic extracts dried over Na ₂ SO ₄ and concentrated to give the aldehyde as an intermediate, which was immediately dissolved in the mixture 1:1 EtOH-Et ₂ O (30 ml) and cooled to 0°C. NaBH ₄ (48 mg, 1.3 mmol) was added and after 20 minutes the reaction was quenched with H ₂ O and extracted with CH ₂ Cl ₂ (4x). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (50% EtOAc-hexanes) to give diol 402 (485 mg, 80% for 3 steps).

Silyl ether 403. TBSOTf (2.3 ml, 10 mmol) was added dropwise to a mixture of diol 402 (485 mg, 1.0 mmol), Et ₃ N (2.8 ml, 20 mmol) and CH ₂ Cl ₂ (30 ml) at 0°C. After stirring for 1 hour at room temperature, saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give silyl ether 403 (668 mg, 95%).

Alcohol 404. LAH (1 M in THF, 2.8 µl, 2.8 mmol) was added dropwise to a solution of silyl ether 403 (668 mg, 0.948 mmol) in Et ₂ O (60 ml) at 0°C. After 15 minutes, H ₂ O and 1 M NaOH were carefully added. The mixture was stirred at room temperature for 20 minutes, filtered through glass wool, concentrated and purified by column chromatography (30% EtOAc-hexanes) to give alcohol 404 (500 mg, 85%).

Aldehyde 405. Oxalyl chloride (210 µl, 2.42 mmol) was added dropwise to a solution of DMSO (345 µl, 4.84 mmol) in CH ₂ Cl ₂ (30 ml) at -78°C. After 1 hour, a solution of alcohol 404 (500 mg, 0.806 mmol) in CH ₂ Cl ₂ (10 ml) was added. After 40 minutes, Et ₃ N (1.0 ml, 7.2 mmol) was added. After stirring at -78°C for 10 minutes, the reaction mixture was warmed to 0°C and stirred for another 10 minutes. Saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed successively with H ₂ O, brine, dried over Na ₂ SO ₄ and concentrated. Purification by flash chromatography (30% EtOAc-hexanes) gave aldehyde 405 (486 mg, 98%), which was immediately used in the next step.

Alkene 406. nBuLi (1.63 M, 860 µl, 1.4 mmol) was added dropwise to a solution of CH ₃ PPh ₃ Br (500 mg, 1.4 mmol) in THF (15 ml) and DMSO (6 ml) at 0°C. After 1 hour, a solution of aldehyde 405 (486 mg) in THF (15 ml) was added. The reaction mixture was warmed to room temperature and stirred for 30 minutes. Saturated aqueous NH ₄ Cl was added, the mixture was extracted with EtOAc (3x) and the combined organic extracts were washed successively with H ₂ O and brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give alkene 406 ( 450 mg, 93%).

Ester 407.9-BBN (0.5 M in THF, 9.0 mL, 4.5 mmol) was added dropwise to a solution of alkene 406 (0.460 g, 0.746 mmol) in THF (10 mL) at 0° WITH. After warming: The mixture was stirred to room temperature for 3 hours and additional portions of 9-BBN (0.5 M in THF, 3.0 ml, 1.5 mmol) were added at 30 minute intervals. The reaction mixture was again cooled to 0°C, after which THF (10 ml), H ₂ O (10 ml) and NaBO ₃ ⋅ 4H ₂ O (1.72 g, 11.2 mmol) were carefully added. The mixture was stirred vigorously at room temperature for 1.5 hours and additional NaBO ₃ ⋅ 4H ₂ O (1.0 g, 6.5 mmol) was added. After 2 hours the mixture was diluted with H ₂ O and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes) to give an alcohol intermediate (509 mg), which was immediately dissolved in CH ₂ Cl ₂ ( 10 ml) and treated with pyridine (600 µl, 7.5 mmol) and PvCl (275 µl, 2.2 mmol). After 6 hours, a saturated aqueous solution of NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes) to give ester 407 (423 mg, 79% for 2 steps).

Alcohol 408. A mixture of ester 407 (11 mg, 0.015 mmol) and Pd(OH) ₂ /C (10 mg) in EtOAc (500 μl) was stirred vigorously under H ₂ at room temperature for 6 hours. The mixture was filtered through celite, concentrated and purified by column chromatography (30% EtOAc-hexanes) to give alcohol 408 (9.4 mg, quantitative yield).

Alkene 409. Oxalyl chloride (7 µl, 0.075 mmol) was added dropwise to a solution of DMSO (11 µl, 0.15 mmol) in CH ₂ Cl ₂ (2 ml) at -78°C under nitrogen atmosphere. After 40 minutes, a solution of alcohol 408 (15.2 mg, 0.025 mmol) in CH ₂ Cl ₂ (1 ml) was added and the reaction was stirred at -78°C for 1 hour. Et ₃ N (31 μl, 0.22 mmol) was added and after stirring for 10 minutes the mixture was warmed to 0°C. After 10 minutes, the reaction mixture was quenched with saturated aqueous NH ₄ Cl and extracted with CH ₂ Cl ₂ (3x). The combined extracts were washed successively with H ₂ O and brine, dried over Na ₂ SO ₄ and concentrated. After flash chromatography (30% EtOAc-hexanes), the ketone intermediate (13 mg) was immediately dissolved in THF (500 μL) and treated with Tebbe's reagent (~0.65 M in toluene, 62 μL, 0.040 mmol) at 0°C. After 1.5 hours, additional Tebbe's reagent was added (~0.65 M in toluene, 62 μl, 0.040 mmol) and after 10 minutes, H ₂ O was carefully added followed by saline. The mixture was extracted with EtOAc (3x) and the combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (10% EtOAc-hexanes) to give alkene 409 (11.9 mg, 80% for 2 steps).

Alcohol 410. 9-BBN (0.5 M in THF, 1.5 ml, 0.72 mmol) was added dropwise to a solution of alkene 409 (0.144 g, 0.242 mmol) in THF (2 ml) at 0°C . After warming to room temperature, the mixture was stirred for 3 hours. The reaction mixture was re-cooled to 0°C, after which THF (2 ml), H ₂ O (2 ml) and NaBO ₃ ⋅ 4H ₂ O (0.38 g, 2.4 mmol) were carefully added. The mixture was stirred vigorously at room temperature for 4 hours, diluted with H ₂ O and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20% EtOAc-hexanes) to give alcohol 410 (0.140 g, 94%).

Alcohol 411. Oxalyl chloride (26 µl, 0.30 ml) was added dropwise to a solution of DMSO (43 µl, 0.60 mmol) in CH ₂ Cl ₂ (4 ml) at -78°C. After 1 hour, a solution of alcohol 410 (57 mg, 0.093 mmol) in CH ₂ Cl ₂ (2 ml) was added. After 45 minutes, Et ₃ N (125 μl, 0.90 mmol) was added. After stirring at -78°C for 10 minutes, the reaction mixture was warmed to 0°C and stirred for another 10 minutes. Saturated aqueous NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ and concentrated. The crude product was dissolved in CH ₂ Cl ₂ (4 ml), treated with Et ₃ N (400 μl) and stirred at room temperature for 15 hours. A saturated aqueous solution of NH ₄ Cl was added and the mixture was extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (30% EtOAc-hexanes) to give the aldehyde as an intermediate (48 mg), which was immediately dissolved in a 1:1 mixture of Et ₂ O/EtOH (4 ml), cooled to 0°C and treated with solid NaBH ₄ (~4 mg, 0.09 mmol). After stirring for 15 minutes, saturated aqueous NH ₄ Cl solution was carefully added and the mixture was extracted with EtOAc (3x). The combined extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by column chromatography (20%-30% EtOAc-hexanes) to give alcohol 411 (45.6 mg, 80% for 3 steps).

412A and 412B. Alcohol 411 (120 mg, 0.196 mmol) and MPMOTC1 (0.17 g, 0.59 mmol) were combined, azeotroped from toluene (3x) and dried under high vacuum for 1 hour. CH ₂ Cl ₂ (9 ml) was added and the mixture was cooled to 0°C. BF ₃ ⋅OEt ₂ (0.016 M in CH ₂ Cl ₂ , 125 µl, 0.002 mmol) was added dropwise and after stirring for 20 minutes the reaction was quenched with saturated aqueous NH ₄ Cl. The mixture was extracted with CH ₂ Cl ₂ (3x) and the combined extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by preparative TLC (20% EtOAc-hexanes) to give an MPM ester as an intermediate which contained some coming impurities. This material was immediately dissolved in Et ₂ O (10 ml) and treated with LAH (1 M in THF, 300 μl, 0.300 mmol) at 0°C. After 10 minutes, H ₂ O and 1 M NaOH were added and after stirring for 10 minutes at room temperature, the mixture was filtered through celite, concentrated and purified by preparative TLC (35% EtOAc-hexanes) to give 412A (49 mg, 39% for 2 steps ) as the single C34 isomer and 412 B (46 mg, 3 6% for 2 stages) as a -9:1 mixture of C34 isomers.

B2070 and B2073. Similar to those described in Schemes 4 and 6 for the synthesis of B1794, intermediates 412A and 412B were converted to B2070 and B2073, respectively. For B2070: HRMS (FAB): calc. for C ₄₁ H ₅₈ F ₂ O ₁₂ +Na 803, 3794. Found: 803, 3801. For B2073: HRMS (FAB): calc. for C ₄₁ H ₅₈ F ₂ O ₁₂ + Na 803.3793. Found: 803.3781.

Synthesis B1963:

Diol 501 (64). Saturated aqueous NaHCO ₃ (21 ml) and KBr (89 mg, 0.75 mmol) were added to a solution of diol 10 (1.35 g, 3.4 mmol) in CH ₂ Cl ₂ (34 ml). The mixture was cooled to 0°C and 4-methoxy-2,2,6,6-tetramethyl-1-piperidinyloxy (0.05 M in CH ₂ Cl ₂ , 7.45 ml, 0.37 mmol) and NaOCl ( 0.07 M in H ₂ O, 5.6 ml, 0.39 mmol). After 1 hour, the reaction mixture was quenched with saturated aqueous Na ₂ S ₂ O ₃ , diluted with saturated aqueous NaHCO ₃ and extracted with CH ₂ Cl ₂ (3x). The combined extracts were dried over Na ₂ SO ₄ , concentrated and dissolved in THF (21 ml).

After cooling to 0°C, CF3TMS (1.5 g, 10.5 mmol) and TBAF (0.1 M in THF, 680 μL, 0.068 mmol) were added sequentially. After stirring for 40 minutes, additional TBAF (1 M in THF, 8.3 mL, 8.3 mmol) was added. After 30 minutes the reaction was quenched with H ₂ O and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by flash chromatography (30%, 40%, 50% EtOAc-hexanes then EtOAc) to give a 2:1 mixture of diols (553 mg, 35%) . Separation by medium pressure liquid chromatography (1.5% MeOH-CH ₂ Cl ₂ ) gave the major, more polar isomer 501 (64) (340 mg, 22%) and an additional, less polar isomer (152 mg, 10%).

B1963. Similar to Schemes 4 and 6 for the synthesis of B1794, intermediate 501 was converted to B1963.

Synthesis of B2320 and related analogues

These compounds are prepared by treating B2294 with a suitable amine in a solvent such as methanol over a period of several hours to several days. The progress of the reaction can be monitored using thin layer chromatography. A standard work-up procedure well known to those skilled in the art provides the target compounds. The procedure for obtaining ER803868 is described below; however, this procedure is general and can be used to obtain any target analogue.

Synthesis of ER803868

To a solution of B2294, 1.2 mg, in methanol, 0.5 ml, was added morpholine, 0.012 ml. The mixture was stirred for 10 days with the addition of additional amounts of morpholine, 0.012 ml, on days 1, 2, 3, 4 and 8. The mixture was then chromatographed to obtain 1.4 mg of the title compound.

D. Pharmacological activity

Many of the individually described drugs have been tested for in vitro and in vivo activity (see Table 1 below). Screening methods included a standard in vitro cell growth inhibition test using DLD-1 human colon cancer cells (ATCC accession number CCL 221) in a 96-well microtiter plate format (Finlay, G.J. et al., Analytical Biochemistry 139:272- 277, 1984), a mitotic block reversibility test using U937 (ATCC accession number CRL 1593) (described below), and in some cases, an in vivo growth inhibition test using a LOX human melanoma tumor xenograft (see Table 1). Chemical stability to degradation by esterase was also tested.

U937 Mitotic Blockage Reversibility Test Human U937 histiocytic lymphoma cells were added to 75 cm ² tissue culture flasks at 2.5 x 10 ⁶ cells in 22.5 ml of RPMI 1640 medium containing 10% fetal calf serum. The cells were allowed to adapt to the culture for 36 hours of incubation at 37°C in a humidified atmosphere containing 5% CO ₂ . Each test drug was then added to the flask in an amount of 2.5 ml of the 10x final concentration. The final concentrations obtained were 0.1-1000 nM, in semi-log increments for 10 concentration steps in total, including a drug-free control flask to which 2.5 ml of medium was added. The cells were incubated with the drug for a 12 hour pretreatment period at 37°C in a humidified atmosphere containing 5% CO ₂ .

The contents were removed from each flask and centrifuged at 300×g for 10 minutes at room temperature, after which the drug-containing medium was removed from the cell pellet. Cells were resuspended in 25 ml of warm drug-free medium and centrifuged at 300 × g for 10 minutes at room temperature. After removing medium from the cell pellet, cells were resuspended in 35 ml of warm drug-free medium, transferred to fresh flasks, and a 10 ml sample of cells was taken immediately from each flask, immediately processed as described below, and stored for later cell cycle analysis ( 0 hours after drug washout).

Incubation of the remaining 25 ml of cells was continued in drug-free medium for the next 10 hours. A sample of 10 ml of cells was taken from each flask, processed immediately and stored for later cell cycle analysis (10 hours after drug washout), and fresh replacement medium was added to each incubation flask. Incubation of cells in drug-free medium was continued for 5 days. On day 2, 20 ml of medium and cells were removed from each flask and replaced with 20 ml of fresh medium. Cell viability was quantified after 5 days according to the trypan blue exclusion method using cell counting with a hemocytometer.

Cells were processed for cell cycle analysis using modifications of the method published in Becton Dickinson Immunocytometry Systems source book section 1.11 (Preparation of Alcohol-Fixed Whole Cells From Suspensions For DNA Analysis). Briefly, each sample of 10 ml of cells taken from flasks at time points 0 and 10 hours after drug washout was separately centrifuged at 300 × g for 10 minutes. After removing the medium from the cell pellet, the cells were resuspended in 3 ml of cold saline. Seven milliliters of cold 100% ethanol was slowly added while vigorously vortexing. Ethanol-treated cell samples from the 0-hour and 10-hour periods were incubated overnight at 4°C after compound washout. Ethanol-treated cells were centrifuged at 300×g for 10 minutes, the ethanol was removed and then the cells were washed in 10 ml of phosphate buffered saline (PBS). Cells were resuspended in 0.5 ml of 0.2 mg/ml ribonuclease A (Sigma No. R-5503) in PBS and incubated in a 37°C water bath for 30 minutes.

Cells were transferred to suitable flow cytometry tubes and 0.5 ml of 10 mg/ml propidium iodide (PI) (Sigma No. P4170) in PBS was added to each tube. Cells were incubated with PI at room temperature in the dark for at least 15 minutes before analysis using a flow cytometer (Becton Dickinson FACScan flow cytometer or equivalent). Cells should be assayed within an hour and stored in the dark at 4°C until use. Cell cycle analysis was performed on 0 h and 10 h cells using flow cytometric measurement of cellular fluorescence intensity. Propidium iodide fluorescence intensity for each cell was measured on a linear gain scale, with doublet events being ignored by doublet discrimination. Results obtained from analysis of 15,000 cells are presented as a histogram with increasing fluorescence intensity on the × axis and the number of cells at a particular intensity level on the y axis.

The intensity of PI staining depends on the amount of DNA in the cell, so that it is possible to identify cells in different phases of the cell cycle, such as cells that have not yet synthesized DNA since the last mitosis (G ₁ phase), cells that are in intermediate stages of DNA synthesis (S-phase), and cells that have doubled their DNA complement and are ready to divide (G ₂ -phase). Cells that are blocked in the mitosis phase of the cell cycle also have double the amount of DNA compared to G ₁ phase cells. If all cells are blocked in mitosis, then there are no G ₁ phase cells, but if the block is removed by removing the junction, the cells complete mitosis and reappear in G ₁ phase. Thus, the number of cells that reappear in this way in G ₁ or S phase is a measure of the number of cells that have recently completed mitosis. For each sample at 0 and 10 hours after compound removal, the percentage of cells completing mitosis was quantified (as the number of cells re-entering G ₁ phase) and plotted as a function of the initial compound concentration applied during the 12-hour pretreatment period. The percentage of cells still viable 5 days after drug washout was plotted on the same graph. It was possible to determine the relationship between the concentration of compound required to completely block all cells in mitosis at 0 hours and the concentration required to maintain this block 10 hours after removal of the compound. This ratio was taken as a measure of the reversibility of the compound, with ratios close to or equal to one indicating compounds likely to be potent in vivo antitumor agents (see Table 1, columns 4-6).

The present invention also describes a method for identifying an agent that induces prolonged mitotic arrest in a cell after transient exposure of the cell to that agent. This invention describes the determination of the relative reversibility of test compounds by correlating the measurement of stage (d) and the measurement of stage (f), as described below. This definition could be, for example, a ratio or an arithmetic difference. In one aspect, this method provides:

(a) incubating a first sample of cells with a given concentration of test compound for a time interval between an interval sufficient to deplete the G ₁ population and an interval that is equivalent to one cell cycle (for example, typically 8-16 hours or about 12 hours );

(b) substantially completely separating the test compound from said first sample of cells (eg, by washing or replacing the medium);

(c) incubating said first sample of cells in medium not containing the test compound for a period of time sufficient to allow at least 80% (e.g., 85%, 90%, and preferably 95%, 98%, or 99%) cells freed from highly reversible mitotic inhibitor-induced mitotic block complete mitosis and return to G ₁ phase (eg, typically 6-14 hours or about 10 hours after separation stage (b); and

(d) measuring the percentage of transient cells from stage (c) that have completed mitosis and returned to G ₁ phase (eg, by measuring a cell cycle marker such as DNA _-3 dependent PI fluorescence).

In one aspect, this screening method includes the additional steps of:

(e) incubating a second sample of cells with a concentration of test compound less than or equal to the concentration used in step (a) for a time interval between an interval sufficient to deplete the G ₁ population and an interval that is equivalent to one cell cycle;

(f) measuring the percentage of cells from stage (e) that have completed mitosis and returned to G ₁ phase; And

(g) determination of the reversibility coefficient of the test compound.

In one embodiment of this method, the first and second cell samples are suspension culture cells selected from, for example, human leukemia, human lymphoma, murine leukemia and murine lymphoma cells. The first and second cell samples can be incubated simultaneously {steps (a) and (e)) or in separate portions. Other options further include, prior to step (a), the step of (i) determining a desired time interval for incubating said first sample of cells with a reversible mitotic blocking agent (or, alternatively, said test compound) to ensure a satisfactory majority of the cells collected from the mitotic blocking; wherein the incubation according to step (a) is carried out for the time interval determined in step (i). Another embodiment of this method further includes, prior to step (c), the step (ii) of determining a desired time interval for incubation without the test compound according to step (c), wherein said step (ii) involves determining a time interval after which at least 80% cells pretreated with a highly reversible antimitotic agent complete mitosis and re-enter G ₁ phase; wherein the incubation according to step (c) is carried out for the time interval determined in step (ii). Another embodiment of this method uses cells from a non-suspension culture, for example, from substrate-attached human or mouse cancer cells, collected by any suitable means to separate them from the culture flasks, structural pathways that prevent prolonged exposure. The effectiveness of relatively reversible antimitotic agents may depend on the period of maintained exposure.

Due to the high cost of developing pharmaceutical substances, the economic benefits of determining the reversibility factors described above are significant. The above methods can be used, for example, to predict whether a test compound with good in vitro activity will be effective in vivo, for example, in a clinical trial. This is shown, for example, by comparing data for two known compounds, the relatively irreversible antimitotic agent vincristine and the highly reversible antimitotic agent vinblastine.

Analyzes of the antimitotic drugs vinblastine and vincristine in the U937 mitotic block reversibility assay indicate that, despite identical efficacy in inducing initial mitotic block (0 hour values), the ability of these two drugs to induce mitotic block was maintained 10 hours after drug washout. agents (values for 10 hours) are quite different: vincristine induces an irreversible mitotic block, whereas the block induced by vinblastine is highly reversible.

In vivo assays of the anticancer activity of the antimitotic drugs vinblastine and vincristine against COLO 205 human colon cancer grafts grown subcutaneously in immunocompromised (nude) mice show that at equivalent doses of 1 mg/kg, vincristine exhibits significant tumor growth inhibitory activity. whereas vinblastine is inactive. At the lower dose of 0.3 mg/kg, vincristine still produces moderate growth inhibition, while vinblastine is again inactive. The higher in vivo activity of vincristine correlates with its irreversibility compared to the high reversibility of vinblastine.

E. Application

The described compounds have pharmacological activities, including anticancer and antimitotic activities, as shown in section D above. Examples of tumors include melanoma, fibrosarcoma, monocytic leukemia, colon cancer, ovarian cancer, breast cancer, osteosarcoma, prostate cancer, lung cancer and ras-transformed fibroblasts.

This invention describes pharmaceutical compositions that include a compound of formula (I) and a pharmaceutically acceptable carrier. The compositions may also contain a combination of the described compounds or a combination of one or more of the described compounds and other pharmaceutically active agents, such as an antitumor agent, an immunostimulating agent, an interferon, a cytokine, an anti-MDR agent or an angiogenesis inhibitory agent. The compositions can be prepared for oral, topical, parenteral, intravenous or intramuscular administration, administration by injection or inhalation. The compositions may also be formulated for controlled release, including in the form of transdermal patches.

A method of inhibiting the growth of tumors in a patient includes the step of administering to the patient an effective antitumor amount of a disclosed compound or composition. The present invention also contemplates combination therapies, including methods of co-administration of a compound of formula (I) before, during or after administration of another pharmaceutically active agent. The routes of administration may be the same or different. Inhibition of tumor growth involves growth of a cell or tissue exposed to a test compound that is at least 20% and preferably 30%, 50% or 75% less than that of a control (in the absence of a known inhibitor or test compound) .

Other Embodiments The basic features of the present invention can be readily understood from the above description and the appended claims. Based on the entire description, variants of the described compounds and methods can be developed and adapted to specific conditions without departing from the essence and scope of the claims and description. The references and publications described herein are hereby included in their entirety.

Claims (19)

1. Macrocyclic compound having the formula where A denotes a C ₃ saturated hydrocarbon skeleton, and the specified skeleton has 2 substituents independently selected from Q ₁ ; each Q ₁ is independently selected from OR ₁ and NR ₂ R ₁ ; R ₁ and R ₂ are each H; each of D and D' is independently selected from R ₃ and OR ₃ , where R ₃ is H or C ₁ alkyl; n is 0; E is missing; G stands for O; J and J' taken together represent =CH ₂ ; Q is C ₁ alkyl; T stands for ethylene; U and U' taken together denote =CH ₂ ; X is H; Y and Y' are each H; Z and Z' taken together denote =O; or a pharmaceutically acceptable salt thereof. 2. The connection according to claim 1 of the following structure: and pharmaceutically acceptable salts thereof.