CROSS REFERENCE TO RELATED APPLICATIONS )

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/303,741, filed March 4, 2016, which is incorporated herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. C A 172310 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

The chemical diversity of secondary metabolites reported to date has shown that marine cyanobacteria are an excellent resource for the discovery of new compounds.

Furthermore, marine cyanobacteria have been shown to be a source for secondary metabolites with interesting bioactivities and mechanisms of action.

Cocosolide (1) is a symmetrical dimer, which structurally resembles the sponge metabolites clavosolides A-D isolated from a Philippine collection of the sponge Myriastra clavosa (Erickson, K. L.; Gustafson, K. R.; Pannell, L. K.; Beutler, J. A.; Boyd, M. R. . Nat. Prod. 2002, 65, 1303-1306; Rao, M. R.; Faulkner, D. J. J. Nat. Prod. 2002, 65, 386-388) and cyanolide A obtained from a Papua New Guinea collection of Lyngbya bouillonii (FIG. 1). Pereira, A. R.; McCue, C. F.; Gerwick, W. H. J. Nat. Prod. 2010, 73, 217-220. Symmetrical dimeric secondary metabolites are rare in marine cyanobacteria. Two related symmetrical dimeric compounds tanikolide dimer (Gutierrez, M.; Andrianasolo, E. H.; Shin, W. K.;

Goeger, D. E.; Yokochi, A.; Schemies, J.; Jung, M.; France, D.; Cornell-Kennon, S.; Lee, E.; Gerwick, W. H. J. Org. Chem. 2009, 74, 5267-5275) and malyngolide dimer (Gutierrez, M.; Tidgewell, K.; Capson, T. L.; Engene, N.; Almanza, A.; Schemies, J.; Jung, M.; Gerwick, W. H. . Nat. Prod. 2010, 73, 709-711) were reported from Lyngbya majuscula collected from Malagasy and Panama, respectively.

An additional unusual structural feature in cocosolide (1) is the presence of cyclopropyl groups, previously encountered in only a few cyanobacterial metabolites. The details of isolation, structure elucidation, X-ray diffraction data of cocosolide (1), total synthesis and the biological activity studies of cocosolide (1), its anomer 26, monomeric analogues 2 and 3, and aglycon 28 (macrocyclic core) are described.

BRIEF SUMMARY OF THE INVENTION

The invention is directed towards compounds, compositions and methods of modulating disease and disease processes comprising compounds of the formulae herein.

The invention includes macrocyclic compounds of any of the formulae herein, compositions thereof, and methods of modulating disease, disorders, and symptoms thereof in a subject. The compounds and compositions are useful in methods of modulating immune response activity, and methods of treating immune disease and disorders in a subject.

In one embodiment, the invention rovides a compound according to Formula I:

Formula I

wherein:

each R is optionally substituted alkyl;

each R is optionally substituted alkyl;

independently H, optionally substituted alkyl, or

independently H, optionally substituted alkyl, or

and pharmaceutically acceptable salts, solvates, or hydrates thereof.

Another aspect is a compound of any of the formula herein (e.g., formula (I)) wherein, R ³ and R ⁴ are H;

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

1 2

wherein, R and R are ethyl;

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ¹ and R ² are ethyl, and R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ¹ and R ² are ethyl, and R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ¹ and R ² are ethyl, and R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ³ and R ⁴ are Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ³ and R ⁴ are

Another aspect is a compound of any of the formulae herein (e.g., formula (I))

wherein, R ³ and R ⁴ are

Other embodiments include a compound of any of the formulae herein, wherein the compound is any of Compounds 1, 2, 3, 26 or 28 herein.

In certain instances, the compounds of the invention are selected from the following of Formula (I) having the structure in Table A:

Table A

Formula I where SGI = SG2=

In other aspects, the compound of any of the formulae herein (e.g., formula I) is an isolated compound.

In another aspect, the invention provides a pharmaceutical composition comprising the compound of any of the formulae herein (e.g., formula I), and a pharmaceutically acceptable carrier.

In other aspects, the invention provides a method of treating a immune disease, disorder, or symptom thereof in a subject, comprising administering to the subject a compound of any of the formulae herein (e.g., formula I). In another aspect, the compound is administered in an amount and under conditions sufficient to ameliorate the immune disease, disorder, or symptom thereof in a subject.

In other aspects, the invention provides a method of modulating immune activity in a subject, comprising contacting the subject with a compound of any of the formulae herein (e.g., formula I), in an amount and under conditions sufficient to modulate immune disorder activity. In another aspect, the modulation is inhibition.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to a immune disorder or disease, wherein the subject has been identified as in need of treatment for a immune disorder or disease, comprising administering to said subject in need thereof, an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I), such that said subject is treated for said disorder.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to a immune disorder or disease, wherein the subject has been identified as in need of treatment for a immune -related disorder or disease, comprising administering to said subject in need thereof, an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I), such that immune response in said subject is modulated (e.g., down regulated).

In another aspect, the invention provides a method of treating diseases, disorders, or symptoms in a subject in need thereof comprising administering to said subject, an effective amount of a compound delineated herein (e.g., Formula I), or a pharmaceutically acceptable thereof. Such methods are useful for treating immune disorders described herein. In another aspect, the invention provides a method of treating immunomodultory disease, disorders, or symptoms thereof in a subject in need thereof comprising administering to said subject, an effective amount of a compound delineated herein (e.g., Formula I), or a pharmaceutically acceptable salt thereof. Such methods are useful for treating

immunomodultory disease, disorders, or symptoms thereof described herein.

Methods delineated herein include those wherein the subject is identified as in need of a particular stated treatment. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described below with reference to the following non-limiting examples and with reference to the following figures, in which:

FIG 1. depicts structures of cocosolide (1) from Symploca sp., clavosolide A from the sponge Myriastra clavosa and cyanolide A from the cyanobacterium Lyngbya bouillonii.

FIG 2. depicts a computer-generated perspective drawing of the X-ray model of cocosolide (1).

FIG 3. depicts biological effects of cocosolide (1), [a,a]-anomer 26, macrocyclic core 28 and monomer methyl ester 3 on T-cell systems.

DETAILED DESCRIPTION Definitions

In order that the invention may be more readily understood, certain terms are first defined here for convenience.

As used herein, the term "treating" a disorder encompasses preventing, ameliorating, mitigating and/or managing the disorder and/or conditions that may cause the disorder. The terms "treating" and "treatment" refer to a method of alleviating or abating a disease and/or its attendant symptoms. In accordance with the present invention "treating" includes preventing, blocking, inhibiting, attenuating, protecting against, modulating, reversing the effects of and reducing the occurrence of e.g., the harmful effects of a disorder. As used herein, "inhibiting" encompasses preventing, reducing and halting

progression.

The term "modulate" refers to increases or decreases in the activity of a cell in response to exposure to a compound of the invention.

The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid

chromatography. Particularly, in embodiments the compound is at least 85% pure, more preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

A "peptide" is a sequence of at least two amino acids. Peptides can consist of short as well as long amino acid sequences, including proteins.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phospho serine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

The term "protein" refers to series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I. The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 50 to 350 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and oc-helices.

"Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

The term "administration" or "administering" includes routes of introducing the compound(s) to a subject to perform their intended function. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), topical, oral, inhalation, rectal and transdermal.

The term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result. An effective amount of compound may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the elastase inhibitor compound are outweighed by the therapeutically beneficial effects. The phrases "systemic administration," "administered systemically", "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.

The term "therapeutically effective amount" refers to that amount of the compound being administered sufficient to prevent development of or alleviate to some extent one or more of the symptoms of the condition or disorder being treated.

A therapeutically effective amount of compound (i.e., an effective dosage) may range from about 0.005 g/kg to about 200 mg/kg, preferably about 0.1 mg/kg to about 200 mg/kg, more preferably about 10 mg/kg to about 100 mg/kg of body weight. In other embodiments, the therapeutically effect amount may range from about 1.0 pM to about 500nM. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.

Moreover, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a compound in the range of between about 0.005 g/kg to about 200 mg/kg of body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound used for treatment may increase or decrease over the course of a particular treatment.

The term "chiral" refers to molecules which have the property of non- superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.

The term "diastereomers" refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.

The term "enantiomers" refers to two stereoisomers of a compound which are non- superimposable mirror images of one another. An equimolar mixture of two enantiomers is called a "racemic mixture" or a "racemate."

The term "isomers" or "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space. The term "prodrug" includes compounds with moieties which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, {e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters {e.g., dimethylaminoethyl ester), acylamino lower alkyl esters {e.g., acetyloxymethyl ester), acyloxy lower alkyl esters {e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters {e.g., benzyl ester), substituted {e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di- lower alkyl amides, and hydroxy amides. Preferred prodrug moieties are propionoic acid esters and acyl esters. Prodrugs which are converted to active forms through other mechanisms in vivo are also included. In aspects, the compounds of the invention are prodrugs of any of the formulae herein.

The term "subject" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.

Furthermore the compounds of the invention include olefins having either geometry: "Z" refers to what is referred to as a "cis" (same side) conformation whereas "E" refers to what is referred to as a "trans" (opposite side) conformation. With respect to the

nomenclature of a chiral center, the terms "d" and "1" configuration are as defined by the IUPAC Recommendations. As to the use of the terms, diastereomer, racemate, epimer and enantiomer, these will be used in their normal context to describe the stereochemistry of preparations.

As used herein, the term "alkyl" refers to a straight-chained or branched hydrocarbon group containing 1 to 12 carbon atoms. The term "lower alkyl" refers to a C1-C6 alkyl chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-buty\, and n-pentyl. Alkyl groups may be optionally substituted with one or more substituents.

The term "alkenyl" refers to an unsaturated hydrocarbon chain that may be a straight chain or branched chain, containing 2 to 12 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may be optionally substituted with one or more substituents. The term "alkynyl" refers to an unsaturated hydrocarbon chain that may be a straight chain or branched chain, containing the 2 to 12 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups may be optionally substituted with one or more substituents.

The sp or sp carbons of an alkenyl group and an alkynyl group, respectively, may optionally be the point of attachment of the alkenyl or alkynyl groups.

The term "alkoxy" refers to an -O-alkyl radical.

As used herein, the term "halogen", "hal" or "halo" means -F, -CI, -Br or -I.

The term "haloalkoxy" refers to an -O-alkyl radical substitued by one or more halo.

The term "cycloalkyl" refers to a hydrocarbon 3-8 membered monocyclic or 7-14 membered bicyclic ring system having at least one saturated ring or having at least one non- aromatic ring, wherein the non-aromatic ring may have some degree of unsaturation.

Cycloalkyl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a cycloalkyl group may be substituted by a substituent. Representative examples of cycloalkyl group include cyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.

The term "aryl" refers to a hydrocarbon monocyclic, bicyclic or tricyclic aromatic ring system. Aryl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, 4, 5 or 6 atoms of each ring of an aryl group may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, and the like.

The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon (with appropriate hydrogen atoms unless otherwise indicated). Heteroaryl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a heteroaryl group may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furanyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,

isoquinolinyl, indazolyl, and the like.

The term "heterocycloalkyl" refers to a nonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic, or 10-14 membered tricyclic ring system comprising 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, B, P or Si, wherein the nonaromatic ring system is completely saturated. Heterocycloalkyl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a heterocycloalkyl group may be substituted by a substituent. Representative heterocycloalkyl groups include piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, 1,3-dioxolane, tetrahydrofuranyl, tetrahydrothienyl, thiirenyl, and the like.

The term "alkylamino" refers to an amino substituent which is further substituted with one or two alkyl groups. The term "aminoalkyl" refers to an alkyl substituent which is further substituted with one or more amino groups. The term "hydroxyalkyl" or

"hydroxylalkyl" refers to an alkyl substituent which is further substituted with one or more hydroxyl groups. The alkyl or aryl portion of alkylamino, aminoalkyl, mercaptoalkyl, hydroxyalkyl, mercaptoalkoxy, sulfonylalkyl, sulfonylaryl, alkylcarbonyl, and

alkylcarbonylalkyl may be optionally substituted with one or more substituents.

Acids and bases useful in the methods herein are known in the art. Acid catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic (e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium triflate) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g., triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.

Alkylating agents are any reagent that is capable of effecting the alkylation of the functional group at issue (e.g., oxygen atom of an alcohol, nitrogen atom of an amino group). Alkylating agents are known in the art, including in the references cited herein, and include alkyl halides (e.g., methyl iodide, benzyl bromide or chloride), alkyl sulfates (e.g., methyl sulfate), or other alkyl group-leaving group combinations known in the art. Leaving groups are any stable species that can detach from a molecule during a reaction (e.g., elimination reaction, substitution reaction) and are known in the art, including in the references cited herein, and include halides (e.g., I-, C1-, Br-, F-), hydroxy, alkoxy (e.g., -OMe, -O-t-Bu), acyloxy anions (e.g., -OAc, -OC(0)CF ₃ ), sulfonates (e.g., mesyl, tosyl), acetamides (e.g., - NHC(O)Me), carbamates (e.g., N(Me)C(0)Ot-Bu), phosphonates (e.g., -OP(0)(OEt) ₂ ), water or alcohols (protic conditions), and the like.

In certain embodiments, substituents on any group (such as, for example, alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, heterocycloalkyl) can be at any atom of that group, wherein any group that can be substituted (such as, for example, alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, heterocycloalkyl) can be optionally substituted with one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of suitable substituents include, but are not limited to alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halogen, haloalkyl, cyano, nitro, alkoxy, aryloxy, hydroxyl, hydroxylalkyl, oxo (i.e., carbonyl), carboxyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, aryloxycarbonyl, heteroaryloxy, heteroaryloxycarbonyl, thio, mercapto, mercaptoalkyl, arylsulfonyl, amino, aminoalkyl, dialkylamino, alkylcarbonylamino, alkylaminocarbonyl, alkoxycarbonylamino, alkylamino, arylamino, diarylamino,

alkylcarbonyl, or arylamino-substituted aryl; arylalkylamino, aralkylaminocarbonyl, amido, alkylaminosulfonyl, arylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, imino, carbamido, carbamyl, thioureido, thiocyanato, sulfoamido, sulfonylalkyl, sulfonylaryl, or mercaptoalkoxy.

Compounds of the Invention

Compounds of the invention can be made by means known in the art of organic synthesis. Methods for optimizing reaction conditions, if necessary minimizing competing by-products, are known in the art. Reaction optimization and scale-up may advantageously utilize high-speed parallel synthesis equipment and computer-controlled microreactors (e.g. Design And Optimization in Organic Synthesis, 2 ^nd Edition, Carlson R, Ed, 2005; Elsevier Science Ltd.; Jahnisch, K et al, Angew. Chem. Int. Ed. Engl. 2004 43: 406; and references therein). Additional reaction schemes and protocols may be determined by the skilled artesian by use of commercially available structure-searchable database software, for instance, SciFinder® (CAS division of the American Chemical Society) and CrossFire Beilstein® (Elsevier MDL), or by appropriate keyword searching using an internet search engine such as Google® or keyword databases such as the US Patent and Trademark Office text database.

The compounds herein may also contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis/trans and E/Z isomers are expressly included in the present invention. The compounds herein may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented. All such isomeric forms of such compounds herein are expressly included in the present invention. All crystal forms and polymorphs of the compounds described herein are expressly included in the present invention. Also embodied are extracts and fractions comprising compounds of the invention. The term isomers is intended to include

diastereoisomers, enantiomers, regioisomers, structural isomers, rotational isomers, tautomers, and the like. For compounds which contain one or more stereogenic centers, e.g., chiral compounds, the methods of the invention may be carried out with an enantiomerically enriched compound, a racemate, or a mixture of diastereomers.

Preferred enantiomerically enriched compounds have an enantiomeric excess of 50% or more, more preferably the compound has an enantiomeric excess of 60%, 70%, 80%, 90%, 95%, 98%, or 99% or more. In preferred embodiments, only one enantiomer or diastereomer of a chiral compound of the invention is administered to cells or a subject. In aspects, the compounds are isolated.

Methods of Treatment

The invention is directed towards macrocyclic compounds, and methods of treating disease and disorders using the compounds or compositions thereof delineated herein.

In other aspects, the invention provides a method of treating a subject suffering from or susceptible to a immune disorder or disease, wherein the subject has been identified as in need of treatment for a immune disorder or disease, comprising administering to said subject in need thereof, an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I), such that said subject is treated for said disorder.

Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

In one aspect, the invention provides a method of modulating the IL-2 activity in a subject, comprising contacting the subject with a compound of any of the formulae herein (e.g., formula I), in an amount and under conditions sufficient to modulate IL-2 activity.

In one embodiment, the modulation is inhibition.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to a IL-2 disorder or disease, comprising administering to the subject an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I). In other aspects, the invention provides a method of treating a subject suffering from or susceptible to a IL-2 disorder or disease, wherein the subject has been identified as in need of treatment for a 11-2 disorder or disease, comprising administering to said subject in need thereof, an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I), such that said subject is treated for said disorder.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to an anti-CD3-stimulated T-cell proliferation disorder or disease, comprising administering to the subject an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I).

In other aspects, the invention provides a method of treating a subject suffering from or susceptible to an anti-CD3-stimulated T-cell proliferation disorder or disease, wherein the subject has been identified as in need of treatment for an anti-CD3 -stimulated T-cell proliferation disorder or disease, comprising administering to said subject in need thereof, an effective amount of a compound or pharmaceutical composition of any of the formulae herein (e.g., formula I), such that said subject is treated for said disorder.

In certain embodiments, the invention provides a method as described above, wherein the compound of any of the formulae herein (e.g., formula I) is a compound of Table A..

In certain embodiments, the invention provides a method of treating a disorder, wherein the disorder is inflammatory skin diseases including psoriasis and dermatitis (e. g. atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis;

meningitis; encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes mellitus (e. g. Type I diabetes mellitus or insulin dependent diabetes mellitis); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener's disease; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia (including, but not limited to cryoglobinemia or Coombs positive anemia); myasthenia gravis; antigen- antibody complex mediated diseases; anti- glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis;

Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; vitiligo; Reiter's disease; stiff-man syndrome; Bechet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM

polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune

thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barre syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; polyarteritis nodosa; alopecia areata; idiopathic myxedema; graft versus host disease; or muscular dystrophy (Duchenne, Becker, Myotonic, Limb-girdle, Facioscapulohumeral, Congenital, Oculopharyngeal, Distal, Emery-Dreif u s s ) .

In another aspect, provides a method of treating a disorder, wherein the disorder is organ transplant rejection (e.g., liver, kidney, heart, lung, cornea, pancreas), bone marrow transplant rejection, rheumatoid arthritis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis).

In another aspect, the invention provides a method of inhibiting IL-2 in a subject in need thereof comprising administering to said subject, an effective amount of a compound delineated herein (e.g., formula I), and pharmaceutically acceptable salts thereof.

In another aspect, the invention provides a method of suppressing the proliferation of anti-CD3 -stimulated T-cells in a subject in need thereof comprising administering to said subject, an effective amount of a compound delineated herein (e.g., formula I), and pharmaceutically acceptable salts thereof.

In certain embodiments, the subject is a mammal, preferably a primate or human.

In another embodiment, the invention provides a method as described above, wherein the effective amount of the compound delineated herein ranges from about 0.005 g/kg to about 200 mg/kg. In certain embodiments, the effective amount of the compound of the formulae herein (e.g., formula I) ranges from about 0.1 mg/kg to about 200 mg/kg. In a further embodiment, the effective amount of compound delineated herein ranges from about 10 mg/kg to 100 mg/kg.

In other embodiments, the invention provides a method as described above wherein the effective amount of the compound delineated herein ranges from about 1.0 pM to about 500 nM. In certain embodiments, the effective amount ranges from about 10.0 pM to about 1000 M. In another embodiment, the effective amount ranges from about 1.0 nM to about 10 nM.

In another embodiment, the invention provides a method as described above, wherein the effective amount of the glucocorticoid compound ranges from about 0.005 g/kg to about 200 mg/kg. In certain embodiments, the effective amount of the compound delineated herein ranges from about 0.1 mg/kg to about 200 mg/kg. In a further embodiment, the effective amount of compound of formula I ranges from about 10 mg/kg to 100 mg/kg.

In another embodiment, the invention provides a method as described above, wherein the compound delineated herein is administered intravenously, intramuscularly,

subcutaneously, intracerebroventricularly, orally or topically.

In other embodiments, the invention provides a method as described above, wherein the compound delineated herein is administered alone or in combination with one or more other therapeutics. In a further embodiment, the additional therapeutic agent is an

immunosuppressive agent.

Another object of the present invention is the use of a compound as described herein (e.g., of any formulae herein) in the manufacture of a medicament for use in the treatment of an immune disorder or disease. Another object of the present invention is the use of a compound as described herein (e.g., of any formulae herein) for use in the treatment of an immune disorder or disease.

Pharmaceutical Compositions

In one aspect, the invention provides a pharmaceutical composition comprising the compound of any of the formulae herein (e.g., formula I), and a pharmaceutically acceptable carrier.

In one embodiment, the invention provides a pharmaceutical composition wherein the compound of any of the formulae herein (e.g., formula I) is a compound of Table A, and a pharmaceutically acceptable carrier.

In another embodiment, the invention provides a pharmaceutical composition further comprising an additional therapeutic agent. In a further embodiment, the additional therapeutic agent is an immunomodulatory agent.

In one aspect, the invention provides a kit comprising an effective amount of a compound of any of the formulae herein (e.g., formula I), in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to an immune disease or disorder, including any of those specifically listed herein.

In one aspect, the invention provides a kit comprising an effective amount of a compound of any of the formulae herein (e.g., formula I), in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a immune disease or disorder.

The term "pharmaceutically acceptable salts" or "pharmaceutically acceptable carrier" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,

dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et al., Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention.

The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

The invention also provides a pharmaceutical composition, comprising an effective amount a compound described herein and a pharmaceutically acceptable carrier. In an embodiment, compound is administered to the subject using a pharmaceutically-acceptable formulation, e.g., a pharmaceutically-acceptable formulation that provides sustained delivery of the compound to a subject for at least 12 hours, 24 hours, 36 hours, 48 hours, one week, two weeks, three weeks, or four weeks after the pharmaceutically-acceptable formulation is administered to the subject.

Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic (or unacceptably toxic) to the patient.

In use, at least one compound according to the present invention is administered in a pharmaceutically effective amount to a subject in need thereof in a pharmaceutical carrier by intravenous, intramuscular, subcutaneous, or intracerebroventricular injection or by oral administration or topical application. In accordance with the present invention, a compound of the invention may be administered alone or in conjunction with a second, different therapeutic. By "in conjunction with" is meant together, substantially simultaneously or sequentially. In one embodiment, a compound of the invention is administered acutely. The compound of the invention may therefore be administered for a short course of treatment, such as for about 1 day to about 1 week. In another embodiment, the compound of the invention may be administered over a longer period of time to ameliorate chronic disorders, such as, for example, for about one week to several months depending upon the condition to be treated.

By "pharmaceutically effective amount" as used herein is meant an amount of a compound of the invention, high enough to significantly positively modify the condition to be treated but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. A pharmaceutically effective amount of a compound of the invention will vary with the particular goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disease, the duration of treatment, the nature of concurrent therapy and the specific organozinc compound employed. For example, a therapeutically effective amount of a compound of the invention administered to a child or a neonate will be reduced proportionately in accordance with sound medical judgment. The effective amount of a compound of the invention will thus be the minimum amount which will provide the desired effect.

A decided practical advantage of the present invention is that the compound may be administered in a convenient manner such as by intravenous, intramuscular, subcutaneous, oral or intra-cerebroventricular injection routes or by topical application, such as in creams or gels. Depending on the route of administration, the active ingredients which comprise a compound of the invention may be required to be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. In order to administer a compound of the invention by other than parenteral administration, the compound can be coated by, or administered with, a material to prevent inactivation.

The compound may be administered parenterally or intraperitoneally. Dispersions can also be prepared, for example, in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage. The carrier can be a solvent or dispersion medium containing, for example, water, DMSO, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compound of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized compounds into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yields a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

For oral therapeutic administration, the compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains compound concentration sufficient to treat a disorder in a subject.

Some examples of substances which can serve as pharmaceutical carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate;

calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline; and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations such as Vitamin C, estrogen and echinacea, for example.

Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, tableting agents, stabilizers, anti-oxidants and preservatives, can also be present.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Examples

The present invention will now be demonstrated using specific examples that are not to be construed as limiting. Definitions of variables in the structures in schemes herein are commensurate with those of corresponding positions in the formulae delineated herein.

General Experimental Procedures.

Materials and Methods

Isolation and Structure Determination- The initial sample of the marine cyanobacterium Symploca sp., which appears as soft golden puffballs, was collected from a patch reef in Cocos Lagoon, Guam. The freeze-dried material was successively extracted with a mixture of CH ₂ Cl ₂ -MeOH (1: 1) and aqueous MeOH (1: 1). The combined extract was subsequently partitioned between EtOAc and H ₂ 0. The EtOAc-soluble portion was repeatedly fractionated by Si0 ₂ chromatography followed by reversed-phase C18 HPLC to give a new compound, cocosolide (1, 8.0 mg, 0.08 % extract wt). Crystallization of cocosolide (1) in 10% EtOAc - hexanes furnished colorless crystals, and 1 indicated negative specific rotation similar to the previously described clavosolides. The molecular formula of (-)-cocosolide (1) C ₄₆ H ₇₆ 0i ₆ was established from a high resolution ESEVIS measurement of the [M + Na]+ peak at m/z

907.5009. Since the 13 C spectrum (Table 1) showed only 23 signals, it was evident that 1 was a symmetrical dimer. The IR spectrum indicated the presence of alcohol, ester and ether functionalities by IR bands at 3462, 1741 and 1073 cm l, respectively. Following the interpretation of DQF COSY and edited HSQC experiments, the 1H and 13C NMR signals were assignable to three partial structures C-2 to C-3, C-5 to C-14 and C-17 to C-21. The presence of a high-field methylene group H2-14 (<5H 0.37, 0.23) that was coupled to two mutually coupled high-field methines H-10 (<5H 0.68) and H-l l (<5H 0.71) indicated the existence of a disubstituted cyclopropyl group in the C-5 to C-14 partial structure. In addition, the 1H NMR spectrum indicated two singlets corresponding to two OMe groups (<5, 3.49, 3.38) and another two singlets corresponding to two methyl groups (<5, 0.87, 0.79). The large geminal coupling constant (J = 17.2 Hz) for the H ₂ -2 methylene protons indicated that this methylene group is adjacent to the ester carbonyl. HMBC correlations (Table 1) from H ₂ -2 (<5H 2.38, 2.07) and H-3 (<5H 3.40) to C-l ester carbonyl (5C 171.7) and from H ₂ -2a to C-4 quaternary carbon (<5C 39.2) connected these two quaternary carbons to partial structure C-2 to C-3. The two methyl groups showed HMBC correlations to each other indicating the geminal arrangement of these methyl groups. Further HMBC correlations of these two methyls to C-3, C-4 and C-5 and HMBC correlation between H ₂ -6 and C-4 linked C-4 to C-5 and thus established the C-l to C-14 carbon skeleton. The partial structure C-17 to C-21was identified as a pyranose sugar through HMBC correlation of the oxymethylene H ₂ -21(<5H 3.90, 3.09) to the C-17 (<5C 106.4) anomeric carbon. The large diaxial coupling constant values (Table 1) observed for all oxymethines including the anomeric proton H-17 (J = 7.6 Hz) established a β-xylopyranose residue. HMBC correlations connected the two methoxy groups H ₃ -22 (53.49) and H ₃ -23 (<5 3.38) to C-19 (<5C 85.4) and C-20 (<5C 80.0), respectively. The free OH group at <5H 3.33 showed a COSY correlation to H-18 and HMBC correlation to C-17 and C-19, and therefore the position of the OH group was assigned to C-18. The oxymethine H-5 (<5H 3.34) showed HMBC correlation to the anomeric carbon C-17 of the sugar moiety indicating the position of connectivity. Absence of other hydroxyl groups in the molecule and the HMBC correlation seen from H-3 to C-7 showed that C-3 through C-7 formed the tetrahydropyran ring. HMBC correlation between oxymethine H-9 and carbonyl carbon C-l indicated the ester link at this position. These data established the symmetric 16- membered macrocyclic planar structure for cocosolide (1).

Table 1. NMR Spectroscopic Data for Cocosolide (1) in CD ₃ CN (600 MHz) position S mult. <¾ (J in Hz) COSY ^a HMBC NOESY ^fc

1, 171.7, C 2a, 2b, 3, 9

2a, 2a' 35.9, CH ₂ 2.38, dd (17.2, 2.0) 3 3 2b, 3, 15,

16

2b, 2b' 2.07, dd (17.2, 8.2) 3 2a, 3, 15,

16

3, 3' 80.5, CH 3.40, dd (8.2, 2.0) 2a, 2b 2a, 2b, 15, 16 2a, 2b, 15

4, 4' 39.2, C 2a, 5, 6a, 6b

15, 16

5, 5' 85.1, CH 3.34, dd (11.4, 4.8) 6a, 6b 3, 6, 7, 15, 16 15, 17

6a, 6a' 37.4, CH ₂ 1.80, m 5, 6b, 7 8a 5, 6b, 7

6b, 6b' 1.35, m 5, 6a, 7 5, 6a

7, 7' 76.3, CH 3.38, m 6a, 6b, 8a, 8b 3, 5, 6a, 6b, 9 8a, 8b, 9

8a, 8a' 41.4, CH ₂ 1.83, m 7, 8b, 9 6b, 7, 8, 9 8b

8b, 8b' 1.66, ddd (14.3, 4.1, 1.4) 7, 8a, 9 8a

9, 9' 77.6, CH 4.30, dt (8.2, 1.4) 8a, 8b, 10 11, 14a, 14b 7, 10, 14a

10, 10' 24.7, CH 0.68, m 9, 11, 14a, 14b 9, 12a, 12b 14b

14a, 14b

11, 11 ' 20.6, CH 0.71, m 10, 12a, 12b 9, 12a, 12b, 9, 12a, 12b

14a, 14b 13, 14b

12a, 12a' 27.2, CH ₂ 1.37, m 11, 12b, 13 10, 13, 14a, 14b 11, 12b, 13

12b, 12b' 0.90, m 11, 12a, 13 11, 12a, 13 13, 13' 13.8, CH ₃ 0.85, t (6.9) 12a, 12b 11, 12 12a, 12b

14a, 14a' 9.8, CH ₂ 0.37, dt (8.3, 4.9) 10, 11, 14b 9, 12a, 12b, 9, 11, 14b,

14b, 14b' 0.23, dt (8.3, 4.8) 10, 11, 14a 10, 14a

15, 15' 22.2, CH ₃ 0.87, s 3, 5, 16 2a, 2b, 3

16, 16' 13.5, CH ₃ 0.79, s 3, 5, 15 2a, 2b

17, 17' 106.4, CH 4.23, d (7.6) 18 5, 18, 18-OH 5, 21

19, 21a, 21b

18, 18' 74.2, CH 3.18, ddd (8.3, 7.6, 4.8) 17, 19 17, 18-OH, 19

18, 18'-OH 3.33, d (4.8)

19, 19' 85.4, CH 3.02, t (8.3) 18, 20 17, 18, 18-OH 22

20, 21a, 21b, 22

20, 20' 80.0, CH 3.17, ddd (9.6, 8.3, 4.8) 19, 21a, 21b 18, 19, 21a 21a, 23

21b, 23

21a, 21a' 63.4, CH ₂ 3.90, dd (11.6, 4.8) 20, 21b 17, 19, 20 20, 21b

21b, 21b' 3.09, dd (11.9, 9.6 20, 21a 21a

22, 22' 60.4, CH ₃ 3.49, s 19 19

23, 23' 58.5, CH ₃ 3.38, s 20 20

" ¹ H- ¹ H COSY and proton(s). "NOESY correlations are from proton(s) stated to the indicated protons.

The relative stereostructure of cocosolide (1) was determined by single crystal X-ray diffraction. Cocosolide (1) is structurally related to the sponge metabolites clavosolides A-D. The X-ray crystallography data of (-)-cocosolide (1) are in full agreement with the corresponding data reported for synthetic (-)-clavosolide A12 for all stereogenic centers (FIG. 2). Therefore, we established the relative configuration for all stereogenic centers of the structure of (-)-cocosolide (1) based on the absolute configuration of synthetic (-)- clavosolide A.

Base hydrolysis of cocosolide (1) furnished the monomer 2 (Scheme 1). Methylation of 2 with CH ₂ N ₂ gave the methyl ester 3 (Scheme 1). The structures of compounds 2 and 3 were determined by NMR studies and confirmed by HRMS studies. These two monomeric compounds 2 and 3 were prepared specifically for structure activity relationship studies, and subsequently the compound 3 was used to prepare the Mosher esters to establish the absolute stereochemistry of (-)-cocosolide (1). The two secondary hydroxy groups at C-9 and C-18 in compound 3 gave MTPA diesters. The MTPA ester of the sugar moiety (C-18) did not interfere with the stereochemical analysis given, and the Δδ values shown in Scheme 1 indicated the absolute configuration at C-9 was S. Applying this stereochemical information in the X-ray crystallography data established the absolute stereochemistry for all stereogenic centers in (-)-cocosolide (1).

Scheme 1

2 R = H

3 R = Me

Mosher diesters and analysis of 3

Total Synthesis of Cocosolide, Its [a,a]Anomer and Macrocyclic Core

A new dimeric macrolide xylopyranoside, cocosolide (1), was isolated from the lipophilic extracts of the marine cyanobacterium Symploca sp. from Cocos Lagoon and other reef flats around the island of Guam. The planar monomeric structure was determined based on NMR data and the symmetrical dimer was proposed based on HRMS data. The dimeric structure and its relative configuration were confirmed by X-ray diffraction studies and the absolute configuration established by Mosher' s analysis of the base hydrolysis product. Its carbon skeleton closely resembles that of clavosolides A-D isolated from the sponge

Myriastra clavosa and for which no bioactivity has been known. Cocosolide (1) differs from clavosolides by having a geminal dimethyl group in each tetrahydropyran ring and a terminal ethyl group at the end of the cyclopropyl-containing side chain. This is the first total synthesis of cocosolide (1) along with its [a,a]-anomer (26) and macrocyclic core (28), thus leading to the confirmation of the structure of natural 1. The convergent synthesis features Wadsworth- Emmons cyclopropanation, Sakurai annulation, Yamaguchi macrocyclization/dimerization reaction, α-selective glycosidation and ^-selective glycosidation. The synthesis also provided further quantities of the natural product, and structural derivatives for biological studies. Cocosolide (1) was non-cytotoxic to a range of cell types. Compounds 1 and 26 potently inhibit IL-2 production in immortalized T-cells in both a T-cell receptor dependent and independent manner. Full activity requires the presence of the sugar moiety as well as the intact dimeric configuration. Cocosolide (1) also suppressed the proliferation of anti-CD3- stimulated T-cells in a dose-dependent manner.

Cocosolide (1): Colorless crystals; mp 233-234 oC; [a] ²⁵ _D -65.3 (c 0.41, CH ₂ C1 ₂ ); UV (MeOH) max (log E) 210 (3.39) nm; IR (film) v _max 3462, 2957, 1741, 1464, 1252, 1165, 1096, 1073, 953 cm- 1 ; 1H NMR, 13C NMR, DQF COSY, HMBC and NOESY data, see Table 1 ; HRESI/APCIMS m/z 907.5009 [M + Na]+ (calcd for C ₄₆ H ₇ 60i6Na, 907.5026).

Retrosynthetic Analysis. The synthetic strategy to produce cocosolide involved cloasure of the macrocycle via the dimerization of the permethylated-D-xylose-containing monomer 21, which could be obtained from glycosylation of alcohol 19. The Sakurai reaction of allylsilane 9 and aldehyde 12 was anticipated to stereoselectively provide a 3,3-disubstituted 2,6-czs-tetrahydropyran precursor 17, which was readily converted into 19 by oxidative cleavage of double bond and subsequent reduction of the resulting ketone. The required allylsilane 9 could be derived from the known /^-hydroxy ester 4 (Reiff, E. a; Nair, S. K.; Henri, J. T.; Greiner, J. F.; Reddy, B. S.; Chakrasali, R.; David, S. a; Chiu, T.-L.; Amin, E. A.; Himes, R. H.; Vander Velde, D. G.; Georg, G. I. J. Org. Chem. 2010, 75, 86-94). The key intermediate 12 would be established through an asymmetric allylation of an appropriate aldehyde derived from the cyclopropane-containing acid 11 (Scheme 2).

Scheme 2. Retrosynthesis analysis of cocosolide (1). Synthesis of Allylsilane 9. The synthesis of fragment 9 started with the protection of the known chiral ester 4 as its triethylsilyl (TES) ether, followed by treatment of the methyl ester with trimethylsilylmethyllithium to give methyl ketone 6 in 83% yield over two steps (Scheme 3). Treatment of ketone 6 with potassium bis(trimethylsilyl)amide (KHMDS) and N-phenyltrifluoromethanesulfonimide afforded enol triflate 7 in 91% yield. Kumada coupling of enol triflate 7 with (trimethylsilyl)methyl Grignard reagent and selective desilylation of TES ether in the presence of a catalytic amount of 10-camphorsulfonic acid (CSA) allylsilane 9 in 61% overall ield.

Scheme 3. Synthesis of allylsilane 9.

Synthesis of Aldehyde 12. With the key intermediate 9 in hand, we next turned our attention to the synthesis of aldehyde 12, which contains a irans-disubstituted-cyclopropane moiety (Scheme 4). Thus, commercially available (S)-2-ethyloxirane 10 was subjected to optimized Wadsworth-Emmons cyclopropanation followed by an in situ saponification to furnish acid 11 in 82% yield. This acid was then converted into homoallylic alcohol 11a via a three- step sequence involving a LiAlH ₄ reduction to furnish the corresponding alcohol, a 2,2,6,6-Tetramethyl-l-piperidinyloxy (TEMPO) promoted oxidation to give rise to an aldehyde and Brown allylation to set the third stereocenter. Protection of the homoallylic alcohol of 11a as its benzyl ether, followed by oxidative cleavage of the terminal olefin with Os0 ₄ and NaI0 ₄ to produce the required aldehyde 12 in 26% overall yield from 11.

Scheme 4. Synthesis of aldehyde 12.

Synthesis of Thioglycoside 16. Thioglycosyl donor 16 was accessed from the known ortho ester 14 as shown in Scheme 5. Treatment of ortho ester 14 with thiophenol in the presence of BF ₃ "OEt ₂ afforded thioglycoside 15 in 36% yield. Hydrolysis of the acetate group in 15 and reprotection of the resulting secondary alcohol with ie/ -butyldimethylsilyl triflate afforded the desired thioglycosyl donor 16 in 86% yield.

15 ⁶

Scheme 5. Synthesis of thioglycoside 13.

Assembly of Subunits and Completion of the Synthesis of Cocosolide (1). With allylsilane 9, aldehyde 12 and thioglycoside 16 in hand, we were poised to complete the synthesis of cocosolide (1). The trimethylsilyl trifluoromethanesulfonate (TMSOTf)- promoted Sakurai annulation of allylsilane 9 with aldehyde 12, was found to occur rapidly at -78 °C to provide the 2,6-czs-tetrahydropyran (17) containing an ejco-methylene in the 4- position. Dihydroxylation of 17 using the Upjohn method (U.S. Patent 2,769,824, 1956; VanRheenen, V.; Kelly, R. C; Cha, D. Y. Tetrahedron Lett. 1976, 17, 1973-1976) and subsequent periodate cleavage afforded 18 in 85% yield. Reduction of the ketone with sodium borohydride gave rise to the secondary alcohol 19 in 92% yield as a single diastereomer. Treatment of thioglycoside 16 with one equivalent of N-bromosuccinimide (NBS) at -25 °C in dry acetonitrile, followed by addition of aglycone (19), the glycosidation proceeded with 5:3 ^-selectivity afforded the desired ?-anomer 20 in 60% isolated yield. Subsequent removal of the bis-benzyl ethers under hydrogenative conditions gave rise to diol 21. The primary alcohol in 21 was selectively converted into the corresponding acid with a one-step TEMPO-catalyzed oxidation protocol. Macrodiolide formation under Yamaguchi's conditions (Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn. 1979, 52, 1989-1993) provided macrocycle 22 in 43 % yield over two steps. Deprotection of 22 with tetra-n-butylammonium fluoride (TBAF) in THF then furnished cocosolide (1), which was identical to the natural product in all respects.

Scheme 6. Assembly of Subunits and Completion of the Synthesis of Cocosolide (1).

Synthesis of [«,«]-Anomer of Cocosolide (26). With the both 19 and 16 in hand, we prepared the [a,a]-anomer of cocosolide using the same strategy (Scheme 7). Wherein, a highly a-selective glycosidation of aglycone 19 with thioglycoside 16 was achieved under the promotion of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) in CH ₂ CI ₂ to furnish the desired a-anomer 23 in 71% isolated yield. Intermediate 23 was then elaborated to the [a,a]-anomer of cocosolide (26) in 19% yield by an identical strategy as described for 1, including removal of bis-benzyl ether, TEMPO-catalyzed oxidation, macrodiolide formation and desilylation. (Scheme 7).

Synthesis of Macrocyclic Core of Cocosolide (28). The macrocyclic core of cocosolide (28) was prepared from 19 by a similar strategy as described for 1. Thus, protection of the secondary alcohol as its ie/ -butyldimethylsilyl (TBS) ether, followed by removal of the bis- benzyl ethers under hydrogenative conditions to give rise to the corresponding diol, which was then selectively converted into the corresponding acid with a one-step TEMPO-catalyzed oxidation protocol. Direct macrocyclization using Yamaguchi' s conditions afforded the C2 symmetric diolide 27 in 19% yield over four steps. Deprotection of 27 with TBAF in THF then furnished 28 in 71% yield.

Scheme 8. Synthesis of macrocyclic core of cocosolide (28). Compounds of the invention can be made by means known in the art of organic synthesis. For example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof are representative and instructive. Methods for optimizing reaction conditions, if necessary minimizing competing by-products, are known in the art.

Synthesis Details.

Bn

4 5

To a solution of compound 4 (354 mg, 1.3 mmol) and 2,6-lutidine (0.35 mL, 4.1 mmol) in CH2CI2 (10 mL), TES-triflate (0.31 mL, 1.9 mmol) was added at 0 °C. The reaction mixture was stirred for an additional hour and then quenched by the addition of a saturated aqueous solution of NaHC0 ₃ (10 mL). The reaction mixture was extracted with EtOAc (2 x 30 mL); and the combined organic layers were washed successively with a saturated aqueous solution of NH ₄ C1 (10 mL), brine (10 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether = 95/5) to afford compound 5 (428 mg, 85%) as a colorless oil.

[a] _D ²⁰ = -10.2 (c 5.0, DCM); ¹ H NMR (400 MHz, CDC1 ₃ ) δ 7.38 - 7.26 (m, 5H), 4.48 (t, = 11.9 Hz, 2H), 4.08 (dd, = 8.6, 1.9 Hz, 1H), 3.63 (s, 3H), 3.53 - 3.46 (m, 2H), 1.81 - 1.67 (m, 1H), 1.67 - 1.55 (m, 1H), 1.16 (s, 3H), 1.08 (s, 3H), 0.92 (t, = 7.9 Hz, 9H), 0.55 (q, = 8.0 Hz, 6H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 177.53, 138.41, 128.34, 127.71, 127.53, 74.09, 72.88, 67.65, 51.64, 48.31, 33.55, 21.14, 20.48, 6.99, 5.41; HRMS (ESI) m/z calcd for C ₂ iH ₃₆ 0 ₄ NaSi [M + Na] ⁺ : 403.2275, found: 403.2273.

5 6

To a solution of compound 5 (5.3 g, 13.90 mmol) in pentane (100 mL), (trimethylsilyl)- methyllithium (27.5 mL, 27.50 mmol, 1.0 M in pentane) was cooled at 0 °C and stirred for 5 hours. MeOH (15 niL) was added dropwise to the reaction mixture and then stirred for 2 h. The reaction mixture was quenched with a saturated aqueous solution of NaHC0 ₃ (30 mL) and then extracted with EtOAc (2 x 60 mL). The combined organic layers were washed successively with a saturated aqueous solution of NH ₄ C1 (30 mL), brine (30 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether = 95/5) to give rise to compound 6 (5.0 g, 98%) as a colorless oil.

[a] _D ²⁰ = -10.4 (c 10.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.39 - 7.24 (m, 5H), 4.54 - 4.44 (dd, / = 11.83, 14.75 Hz, 2H), 4.08 (dd, = 8.5, 2.6 Hz, 1H), 3.51 (dd, 7 = 7.8, 5.4 Hz, 2H), 2.14 (s, 3H), 1.79 - 1.67 (m, 1H), 1.61 - 1.51 (m, 1H), 1.12 (s, 3H), 1.07 (s, 3H), 0.95 (t, / = 7.9 Hz, 9H), 0.60 (q, = 7.8 Hz, 6H). ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 213.46, 138.52, 128.42, 127.77, 127.62, 74.25, 73.00, 67.55, 53.30, 34.21, 26.95, 21.78, 20.15, 7.11, 5.54; HRMS (ESI) m/z calcd for C ₂ iH ₃₆ Na0 ₃ Si [M + Na] ⁺ : 387.2326, found: 387.2323.

To a solution of ketone 6 (2.6 g, 7.1 mmol) in THF (35 mL) at -78 °C, KHMDS (11.5 mL, 9.2 mmol, 0.8 M in THF) was added dropwise. After being stirred at -78 °C for 1 hour, a solution of N-phenyltrifluoromethanesulfonimide (2.9 g, 8.1 mmol) in THF (10 mL) was added dropwise to the reaction mixture and stirred for additional 2 h. The reaction mixture was quenched with a saturated aqueous solution of NH ₄ C1 (20 mL) and then extracted with hexanes (2 x 100 mL). The combined organic layers were washed successively with a saturated aqueous solution of NaHC0 ₃ (50 mL), brine (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether =96/4) to afford compound 7 (3.2 g, 91%) as a colorless oil. [a] _D ² °= -10.6 (c 2.0, DCM); 1H NMR (400 MHz, CDC1 ₃ ) δ 7.35 - 7.25 (m, 5H), 5.13 (d, = 4.3 Hz, 1H), 5.01 (d, = 4.3 Hz, 1H), 4.49 (dd, / = 11.90, 15.33 Hz, 2H), 3.83 (dd, = 8.8, 2.2 Hz, 1H), 3.56 - 3.44 (m, 2H), 1.88 (dtd, = 14.1, 8.0, 2.2 Hz, 1H), 1.63 - 1.51 (m, 2H), 1.57 (s, 1H), 1.15 (s, 3H), 1.09 (s, 3H), 0.92 (t, = 8.0 Hz, 9H), 0.56 (q, = 7.9 Hz, 6H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 162.09, 138.34, 128.33, 127.67, 127.54, 102.59, 72.92, 72.87, 67.55, 45.33, 33.31, 22.74, 20.40, 6.98, 5.35; HRMS (ESI) m/z calcd for C ₂ 2H ₃ 5F ₃ Na0 ₅ SSi[M + Na] ⁺ : 519.1819, found: 519.1820.

7 8

A dry flask was charged with LiCl (900 mg, 21.2 mmol) and the solid was flame-dried under reduced pressure and purged with argon. Et ₂ 0 (25 mL) was added, followed by a solution of triflate 7 (2.5 g, 5.1 mmol) in 15 mL of Et ₂ 0. The suspension was cooled to 0 °C and tetrakis(triphenylphosphine)-palladium(0) (471 mg, 0.4 mmol) was added, followed by (trimethylsilyl)methylmagnesium chloride (13.0 mL, 13.0 mmol, 1.0 M in Et ₂ 0). After being stirred for 5 hours, the yellow suspension was filtered over a pad of a celite, eluting with Et ₂ 0 (100 mL). The organic solution was poured over 50 mL of a saturated aqueous solution of NaHC0 ₃ and then extracted with Et ₂ 0 (2 x 50 mL). The combined organic layers were washed successively with a saturated aqueous solution of NaHC0 ₃ (50 mL), brine (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether =99/1) to afford compound 8 (1.8 g, 81%) as a colorless oil.

[a] _D ²⁰ = -0.96 (c 2.4, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.40 - 7.20 (m, 5H), 4.84 (s, 1H), 4.70 (s, 1H), 4.51 (dd, / = 12.09, 13.57 Hz, 2H), 3.85 (dd, 7 = 8.8, 1.9 Hz, 1H), 3.58 - 3.46 (m, 2H), 1.86 (dtd, = 14.3, 8.1, 2.0 Hz, 1H), 1.65 - 1.44 (m, 3H), 1.02 (s, 3H), 1.02 (s, 3H), 0.99 (t, J = 7.9 Hz, 9H), 0.63 (q, J = 7.6 Hz, 6H), 0.07 (s, 9H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 152.70, 138.83, 128.39, 127.69, 127.51, 109.21, 75.07, 72.88, 68.37, 45.16, 33.69, 25.85, 21.41, 20.50, 7.27, 5.71, -0.32; HRMS (ESI) m/z calcd for C ₂ 5H ₄₆ Na0 ₂ Si ₂ [M + Na] ⁺ :

457.2929, found: 457.2927.

8 9

To a solution of compound 8 (450 mg, 1.04 mmol) in MeOH (5 ml) was added a solution of camphorsulfonic acid (21 mg, 0.09 mmol) in 1.5 mL of MeOH. The reaction mixture was stirred for 2 hours at room temperature before being quenched by Et ₃ N (1 mL). The reaction mixture was poured over a saturated aqueous solution of NaHC0 ₃ (20 mL), and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed successively with a saturated aqueous solution of NH ₄ C1 (20 mL), brine (20 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc =6/1) to give rise to compound 9 (250 mg, 75%) as a colorless oil.

[a] _D ²⁰ = -7.3 (c 2.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.37 - 7.27 (m, 5H), 4.86 (d, = 1.0 Hz, 1H), 4.79 (d, = 0.7 Hz, 1H), 4.53 (dd, =11.88, 14.53 Hz, 2H), 3.75 - 3.64 (m, 3H), 2.46 (d, J = 2.1 Hz, 1H), 1.76 (ddd, = 11.6, 6.6, 1.4 Hz, 1H), 1.62 (s, 2H), 1.66 - 1.57 (m, 3H), 1.53 (s, 2H), 1.05 (s, 3H), 1.00 (s, 3H), 0.06 (s, 9H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 152.61, 138.38, 128.48, 127.76, 127.70, 109.85, 74.47, 73.37, 69.85, 44.33, 31.33, 22.19, 22.16, 21.22, -0.42; HRMS (ESI) m/z calcd for Ci ₉ H ₃₂ Na0 ₂ Si [M + Na] ⁺ : 343.2064, found: 343.2064.

13

To a suspension of sodium hydride (2.5 g, 60 mmol, 60% suspension in mineral oil) in toluene (100 mL) at 0 °C, triethylphosphonoacetate (13) (13 mL, 65 mmol) was added dropwise over 15 min. After being stirred for 10 min, epoxide 10 (4.2 mL, 50 mmol) was added dropwise over 35 min to the solution, and stirred for 6 hours. The reaction mixture was cooled to room temperature, water (50 mL) and 30% NaOH (50 mL) were added. The mixture was stirred under reflux for 2 hours. The layers were separated, and the organic phase is discarded. The pH value of the reaction mixture was adjusted to 2-3 by the addition of an aqueous solution of HC1. The mixture was then extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with 10% NaCl (3 x 50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 1/1) to yield compound 11 (4.7 g, 82%) as a light yellow oil.

[a] _D ²⁰ = -88.4 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 11.30 (b, 1H), 1.44-1.35 (m, 1H), 1.35-1.24 (m, 3H), 1.18 (dt, J = 8.7, 4.3 Hz, 1H), 0.95 (t, J = 7.3 Hz, 3H), 0.75 (ddd, 7 = 8.0, 6.4, 4.1 Hz, 1H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 181.43, 26.19, 25.72, 19.98, 16.21, 13.17; HRMS (ESI) m/z calcd for C ₆ H ₉ 0 ₂ [M - H] ^~ : 113.0608, found: 113.0608.

11 11a

A solution of acid 11 (1.5 g, 40 mmol) in 20 mL was added dropwise to a solution of LiAlH ₄ (3.5 g, 30 mmol) in diethyl ether (100 mL) at 0 °C. After being slowly warmed to room temperature and stirred for 2 hours, the reaction mixture was re-cooled to 0 °C and quenched by the addition of MeOH (10 mL), and followed by the addition of a saturated aqueous solution of Rochelle's salt (sodium potassium tartrate) (100 mL) and stirred for another 4 h. The mixture was extracted with ethyl ether (2 x 100 mL), and the combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (50 mL), brine (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure at low temperature to give a solution of alcohol 11a in ethyl ether, which was directly used in the next step. 1H

NMR (500 MHz, CDC1 ₃ ) δ 3.40 (dd, J = 7.1, 2.5 Hz, 2H), 1.64 (s, 1H), 1.27-1.19 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H), 0.84-0.75 (m, 1H), 0.59-0.50 (m, 1H), 0.35-0.29 (m, 1H), 0.29-0.24 (m, 1H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ = 67.25, 26.70, 21.08, 19.09, 13.74, 9.80.

1 ) TEMPO, TCCA

11a 12

To a solution of of alcohol 11a (30 mmol) in ethyl ether (100 mL) was added NaHC0 ₃ (7.6 g, 90 mmol), TEMPO (468 mg, 3.0 mmol). The reaction mixture was cooled to 0 °C and TCCA (7.7 g, 33 mmol) was added. After being stirred for an additional hour at 0 °C, the reaction mixture was filtered through a pad of silica gel. The filtrate was washed successively with saturated aqueous NaHC0 ₃ (30 mL), NH ₄ C1 (30 mL), brine (30 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure at low temperature to afford the crude aldehyde, which was used in the next step without further purification.

A solution of salt-free (-)-fi-Allyl bis(Isopinocampheyl)borane (35 mmol) in Et ₂ 0 (50 mL) was cooled to -82 °C. A solution of above fresh prepared aldehyde in Et ₂ 0 (30 mL) was cooled to -78 °C and added via cannula over 30 min to the allylboron reagent while maintaining the reaction temperature below -80 °C. After being stirred at -82 °C for 3 hours, the reaction was slowly warmed to room temperature over 2 h. A solution of 3N NaOH (40 mL) and H ₂ 0 ₂ (30%, 80 mL) was added dropwise over 30 min. The mixture was stirred for 18 hours at room temperature. The product was extracted with diethyl ether (2 x 200 ml). The combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (50 mL), NH ₄ C1 (50 mL), brine (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure at low temperature to give rise to a crude oil consisting of the desired allylation product and isopinocampheol. The product mixture was dissolved in THF (300 mL) and cooled to 0 °C. Sodium hydride (6.0 g, 150 mmol, 60% suspension in mineral oil) was added in portions to control the exothermic release of hydrogen. BnBr (16 mL, 135 mmol) and tetrabutylammonium iodide (3.5 g, 9.5 mmol) was then added and the mixture was gradually warmed to room temperature (the release of hydrogen was controlled by placing of the reaction vessel in an ice bath). The mixture was stirred at ambient temperature for 24 hours, then re-cooled to 0 °C and quenched by slow addition of MeOH (20 mL), followed by H ₂ 0 (50 mL). The mixture was extracted with Et ₂ 0 (2 x 300 mL). The combined organic layers were washed successively with saturated aqueous NH ₄ C1 (50 mL), brine (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether =95/5) to afford an inseparable mixture of desired benzyl ether and isopinocampheol-benzyl ether as a light yellow oil.

To a solution of above mixture in 60 mL of H ₂ 0 and 200 mL of acetone was added with 2,6- lutidine (7.0 mL, 60 mmol), osmium tetroxide (50 mL, 1 mmol, 0.02 M in i-BuOH) and sodium periodate (8.5 g, 40 mmol) at room temperature. After being stirred for 3 hours, the mixture was filtered, eluting with 50 mL of EtOAc and then concentrated. The slurry was then extracted with EtOAc (2 x 200 mL) and the combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (50 mL), brine (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether = 90/15) to yield compound 12 (1.84 g, 26 % for 5 steps) as a colorless oil.

[a] _D ²⁰ = -34.0 (c 2.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 9.81 (t, J = 2.4 Hz, 1H), 7.38 - 7.24 (m, 5H), 4.67 (dd, J = 126.1, 11.6 Hz, 2H), 3.29 (td, J = 8.5, 4.2 Hz, 1H), 2.77 (ddd, J = 15.9, 8.2, 2.8 Hz, 1H), 2.63 (ddd, J = 15.9, 4.1, 2.0 Hz, 1H), 1.45 - 1.33 (m, 1H), 1.33 - 1.22 (m, 1H), 1.03 (t, J = 7.4 Hz, 3H), 0.89 - 0.77 (m, 1H), 0.73 (ddd, J = 13.4, 8.9, 4.8 Hz, 1H), 0.34 - 0.21 (m, 2H). ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 201.61, 138.56, 128.47, 127.69, 127.67, 78.42, 70.90, 49.47, 26.71, 22.30, 21.43, 13.51, 8.03; HRMS (ESI) m/z calcd for Ci ₅ H ₂₀ O ₂ Na [M + Na] ⁺ : 255.1356, found: 255.1355.

17

To a -78 °C ether solution of aldehyde 12 (19 mg, 82 μηιοΐ) and flame-dried powdered 4A molecular sieves (30 mg), was added allylsilane 9 (20 mg, 62 μιηοΐ) in diethyl ether (2 mL). After being stirred for 30 min, TMSOTf (17 μί, 94 μιηοΐ) was added slowly and allowed to stir rapidly for an additional hour, then quenched with cooled saturated aqueous solution of NaHC0 ₃ (0.5 mL). The biphasic mixture was extracted with EtOAc (2 x 25 mL), and the combined organic layers were washed successively with saturated aqueous NH ₄ C1 (10 mL), brine (10 mL), dried over Na ₂ S0 ₄ , dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether =95/5) to yield compound 17 (22 mg, 76%) as a colorless oil.

[a] _D ²⁰ = -58.4 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ); δ 7.37 - 7.24 (m, 10H), 4.73 (d, = 11.7 Hz, 1H), 4.71 (s, 1H), 4.65 (s, 1H), 4.51 (d, / = 11.7 Hz, 1H), 4.47 (s, 2H), 3.59 - 3.53 (m, 1H), 3.53 - 3.46 (m, 2H), 3.12 (dd, J = 10.7, 1.7 Hz, 1H), 2.90 (dt, 7 = 8.5, 6.7 Hz, 1H), 2.24 - 2.16 (m, 1H), 2.08 - 2.00 (m, 2H), 1.85 (ddd, J = 9.5, 7.9, 3.9 Hz, 1H), 1.68 (ddd, = 9.1, 6.5, 2.1 Hz, 2H), 1.44 - 1.34 (m, 1H), 1.27 - 1.18 (m, 2H), 1.06 - 0.97 (m, 9H), 0.74 - 0.66 (m, 1H), 0.63 - 0.56 (m, 1H), 0.28 - 0.20 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 153.97, 139.32, 138.76, 128.41, 128.38, 127.73, 127.71, 127.53, 127.43, 106.09, 81.59, 79.50, 76.10, 73.10, 70.54, 68.33, 42.37, 39.68, 39.48, 30.23, 26.90, 22.58, 22.47, 20.59, 20.14, 13.65, 8.36; HRMS (ESI) m/z calcd for C ₃ iH ₄₂ 0 ₃ Na [M + Na] ⁺ : 485.3026, found: 485.3027.

17 18

To a solution of compound 17 (193 mg, 0.42 mmol) in 6.0 mL of H ₂ 0 and 20.0 mL of acetone was added Osmium tetroxide (0.5 mL, 0.05 mmol, 0.1M in i-BuOH) and N- methylmorpholine N-oxide (467 mg, 1.67 mmol). After being stirred for 24 h, a solution of sodium periodate (450 mg, 2.10 mmol) in 10 mL of H ₂ 0 was added, and the yellow solution was stirred for additional 3 h. The mixture was filtered, eluted with 50 mL of EtOAc and then concentrated. The slurry was extracted with EtOAc (2 x 50 mL) and the combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (30 mL), brine (30 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether = 85/15) to afford ketone 18 (164 mg, 85%) as a colorless oil.

[a] _D ²⁰ = -87.4 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.35 - 7.25 (m, 10H), 4.74 (d, 7 = 11.6 Hz, 1H), 4.45 (s, 2H), 4.45 (d, 2H), 3.86 - 3.75 (m, 1H), 3.61 - 3.47 (m, 2H), 3.39 (dd, 7 = 8.6, 3.8 Hz, 1H), 2.87 (dt, 7 = 8.6, 6.5 Hz, 1H), 2.47 (dd, 7 = 14.2, 11.9 Hz, 1H), 2.24 (dd, 7 = 14.2, 2.6 Hz, 1H), 2.10 (dt, 7 = 13.9, 7.0 Hz, 1H), 1.82 - 1.75 (m, 2H), 1.75 - 1.65 (m, 1H), 1.42 - 1.31 (m, 1H), 1.26 - 1.15 (m, 1H), 1.08 (s, 3H), 1.02 - 0.96 (m, 6H), 0.73 - 0.65 (m, 1H), 0.65 - 0.51 (m, 1H), 0.28 - 0.15 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 211.80, 139.04, 138.56, 128.46, 127.69, 127.66, 127.64, 127.56, 80.63, 79.28, 74.59, 73.12, 70.67, 67.58, 49.15, 44.70, 42.50, 29.87, 26.83, 22.35, 20.40, 19.40, 18.91, 13.62, 8.27; HRMS (ESI) m/z calcd for C ₃₀ H ₄₀ NaO ₄ [M + Na] ⁺ : 487.2819, found: 487.2815.

18 19

To a -40 °C solution of ketone 18 (41 mg, 88 μπιοΐ) in MeOH (1.5 mL) was added NaBH ₄ (10 mg, 0.26 mmol). The reaction was stirred at this temperature for 30 min. then quenched with saturated aqueous NH ₄ C1 (10 mL). The biphasic mixture was extracted with EtOAc (2 x 50 mL), and the combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 3/1) to yield compound 19 (39 mg, 92%) as a colorless oil.

[a] _D ²⁰ = -49.1 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.38 - 7.27 (m, 10H), 4.75 (d, 7 = 11.6 Hz, 1H), 4.46 (d, 7 = 11.5 Hz, 3H), 3.60 (dd, 7 = 11.2, 5.6 Hz, 1H), 3.58 - 3.53 (m, 1H), 3.53 - 3.46 (m, 1H), 3.39 (dd, 7 = 11.4, 4.3 Hz, 1H), 3.08 (d, 7 = 9.5 Hz, 1H), 2.88 (dd, 7 = 15.2, 6.7 Hz, 1H), 2.02 (dt, / = 14.1, 7.2 Hz, 1H), 1.87 - 1.78 (m, 1H), 1.68 - 1.63 (m, 2H), 1.44 - 1.34 (m, 2H), 1.32 (d, / = 12.0 Hz, 1H), 1.25 (dt, / = 14.0, 7.6 Hz, 2H), 1.01 (t, / = 7.4 Hz, 3H), 0.93 (s, 3H), 0.83 (s, 3H), 0.71 (dd, = 12.3, 5.7 Hz, 1H), 0.64 - 0.56 (m, 1H), 0.28 - 0.21 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 139.29, 138.74, 128.43, 128.39, 127.75, 127.70, 127.56, 127.46, 80.28, 79.54, 76.04, 73.08, 73.04, 70.62, 68.13, 42.04, 38.85, 37.13, 29.53, 26.90, 22.42, 20.24, 13.65, 12.55, 8.29; HRMS (ESI) m/z calcd for C ₃₀ H4 ₂ O ₄ Na [M + +: 489.2975, found: 489.2974.

The suspended solution of compound 16 (16 mg, 42 μιηοΐ) and flame-dried, powdered 4A molecular sieves (90 mg) in dry acetonitrile (1 mL) was stirred under nitrogen at room temperature for 1.5 h. To this solution at -25 °C, was added a solution of N- bromosuccinimide (7.5 mg, 42 μιηοΐ) in dry acetonitrile (0.5 mL). 10 min later a solution of alcohol 19 (10 mg, 21 μιηοΐ) in dry acetonitrile (0.5 mL) was added. The reaction mixture was stirred for 30 min at -25 °C before it was allowed to warm to room temperature over 3 h. The reaction was quenched with saturated aqueous NaHC0 ₃ (10 mL). The biphasic mixture was extracted with EtOAc (2 x 50 mL), and the combined organic layers were washed successively with saturated aqueous NH ₄ C1 (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to yield a crude product consisting a mixture of alpha:beta (3:5) isomers. The mixture was further purified by flash column chromatography on silica gel (hexanes/EtOAc = 3/1) to yield 9.4 mg of beta isomer (20) (60% yield) as a colorless oil. [a] _D ² °= -51.8 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.37 - 7.27 (m, 10H), 4.74 (d, = 11.5 Hz, 1H), 4.46 (s, 2H), 4.45 (d, = 11.6 Hz, 1H), 4.23 (d, = 7.3 Hz, 1H), 3.93 (dd, / = 11.5, 5.2 Hz, 1H), 3.63 - 3.57 (m, 2H), 3.55 (s, 3H), 3.52 (m, 1H), 3.48 (m, 2H), 3.44 (s, 3H), 3.41 - 3.34 (m, 1H), 3.30 - 3.22 (m, 2H), 3.09 (dd, = 21.0, 10.9 Hz, 2H), 3.00 (t, = 8.7 Hz, 1H), 2.92 - 2.86 (m, 1H), 2.05 - 1.98 (m, 1H), 1.83 (m, 1H), 1.78 (m, 1H), 1.64 (m, 1H), 1.42 - 1.33 (m, 1H), 1.25 - 1.17 (m, 1H), 1.01 (t, = 7.4 Hz, 3H), 0.95 (s, 3H), 0.92 (s, 9H), 0.89 (m, 5H), 0.68 (m, 1H), 0.59 (m, 1H), 0.28 - 0.22 (m, 2H), 0.13 (s, 6H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 139.23, 138.61, 128.32, 128.29, 127.61, 127.44, 127.34, 106.17, 86.26, 84.56, 80.68, 80.44, 79.96, 77.33, 77.01, 76.69, 74.31, 72.95, 70.71, 67.92, 63.13, 61.01, 58.34, 41.97, 38.97, 36.66, 29.69, 29.20, 26.82, 26.08, 22.32, 20.06, 18.11, 13.61, 13.49, 8.23, -3.70, -4.11; HRMS (ESI) m/z calcd for

C ₄₃ H ₆₈ 0 ₈ NaSi [(M + Na] ⁺ : 763.4576, found: 763.4579.

To a solution of Bn ether 20 (21 mg, 28 μηιοΐ) in MeOH (3 niL) was added 10% Pd/C (5 mg). The reaction flask was evacuated and purged with hydrogen three times. The reaction mixture was stirred under a ¾ atmosphere at ambient temperature for 2 h. The flask was then evacuated and purged with nitrogen three times and the catalyst was removed by filtration through Celite. The filtrate was concentrated and the crude product purified by flash column chromatography on silica gel (hexanes/EtOAc = 2/1) to yield compound 21 (13 mg, 83%) as a colorless oil. [a] _D ² °= -25.8 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 4.21 (d, = 7.2 Hz, 1H), 3.95 (dd, = 11.4, 5.2 Hz, 1H), 3.71 (dd, = 10.4, 4.4 Hz, 2H), 3.60 (dd, = 11.6, 8.8 Hz, 1H), 3.53 (s, 3H), 3.43 (s, 3H), 3.35 (dd, / = 8.7, 7.3 Hz, 1H), 3.24 (ddd, = 11.8, 8.2, 4.9 Hz, 2H), 3.18 - 3.13 (m, 1H), 3.10 (dd, = 11.4, 10.1 Hz, 2H), 2.98 (t, J = 8.7 Hz, 1H), 2.58 (s, 2H), 1.85 - 1.75 (m, 2H), 1.65 (dd, = 11.3, 6.4 Hz, 3H), 1.29 - 1.16 (m, 3H), 0.93 (dd, = 9.4, 5.1 Hz, 6H), 0.90 (d, J = 1.1 Hz, 12H), 0.65 (dd, J = 13.3, 8.1 Hz, 1H), 0.60 (dt, = 12.9, 4.2 Hz, 1H), 0.33 - 0.27 (m, 1H), 0.27 - 0.21 (m, 1H), 0.10 (s, 6H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 106.24, 86.27, 83.95, 80.81, 76.35, 75.42, 74.42, 63.22, 61.70, 60.96, 58.36, 42.78, 39.06, 37.06, 31.08, 26.80, 26.17, 25.60, 22.44, 18.88, 18.19, 13.74, 13.62, 9.32, 0.06, -3.65, -4.01; HRMS (ESI) m/z calcd for C ₂ 9H ₅₆ Na0 ₈ Si [M + Na] ⁺ : 583.3637, found: 583.3534.

To a solution of the diol 21 (13 mg, 23 μηιοΐ) in CH ₂ C1 ₂ (300 μί) was added TEMPO (0.2 mg, 1.2 μηιοΐ) and saturated NaHC0 ₃ (130 μί). KBr (50 μί, 2.5 μηιοΐ, 0.05 M aqueous solution) and Bu ₄ NCl (30 μί, 1.5 μηιοΐ, 0.05 M aqueous solution) were added subsequently and the mixture was cooled to 0 °C. To the vigorously stirring biphasic solution was added a stock solution of saturated NaHC0 ₃ (33 μί), brine (61 μί) and bleach (40 μί, 0.06 mmol, 1.5 M) dropwise over 45 min. The mixture was allowed to stir for an additional 30 min at 0 °C upon completion of the addition. The solution was then diluted with H ₂ 0 (2 mL) and CH ₂ C1 ₂ (2 mL). The diluted mixture was acidified with 10% citric acid (4-10 drops) to pH 3- 4. The biphasic mixture was extracted with EtOAc (2 x 50 mL), and the combined organic layers were washed with brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to give rise to the corresponding carboxylic acid, which was employed in the next step of reaction without further purification.

To the above acid in dry toluene (2 ml) was added DIPEA (N,N-Diisopropylethylamine) (30 μί, 0.18 mmol) and 2,4,6-trichlorobenzoyl chloride (20 μί, 0.13 mmol) dropwise. The reaction mixture was stirred at room temperature for 2 hours, then the mixture was added dropwise over 3 hours to a stirred solution of DMAP (4-dimethylaminopyridine) (35 mg, 0.29 mmol) in toluene (10 mL) at 80 °C, the reaction was allowed to stir for another 3 hours before it was quenched with saturated aqueous NH ₄ C1 (15 mL). The biphasic mixture was extracted with EtOAc (2 x 70 mL), and the combined organic layers were washed

successively with saturated aqueous NaHC0 ₃ (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 3/1) to yield compound 22 (5.5 mg, 43%) as a colorless oil.

[a] _D ²⁰ = -67.6 (c 0.2, DCM); 1H NMR (500 MHz, CD ₃ CN) δ 4.30 (t, = 8.4 Hz, 2H), 4.24 (d, = 7.2 Hz, 2H), 3.90 (dd, / = 11.3, 5.0 Hz, 2H), 3.47 (s, 6H), 3.40 - 3.38 (m, 2H), 3.37 (s, 6H), 3.34 (dd, / = 11.6, 4.8 Hz, 2H), 3.28 (dd, / = 8.6, 7.2 Hz, 2H), 3.22 - 3.16 (m, 2H), 3.11 (dd, 7 = 11.2, 9.7 Hz, 2H), 2.95 (t, 7 = 8.5 Hz, 2H), 2.66 (s, 2H), 2.37 (dd, 7 = 17.3, 1.7 Hz, 2H), 2.08 - 2.05 (m, 2H), 1.80 (dd, 7 = 4.9, 2.5 Hz, 2H), 1.79 - 1.75 (m, 2H), 1.66 (dd, 7 = 14.5, 2.9 Hz, 2H), 1.39 - 1.32 (m, 4H), 0.93 (d, 7 = 7.6 Hz, 2H), 0.90 (s, 18H), 0.87 (s, 6H), 0.85 (t, J = 1.0 Hz, 6H), 0.81 (s, 6H), 0.72 (dd, 7 = 11.5, 6.9 Hz, 2H), 0.69 - 0.64 (m, 2H), 0.40 - 0.32 (m, 2H), 0.26 - 0.18 (m, 2H), 0.10 (s, 6H), 0.09 (s, 6H); ¹³ C NMR (125 MHz, CD ₃ CN) δ 170.94, 108.71, 105.74, 89.27, 85.95, 84.05, 83.39, 81.51, 80.62, 80.27, 79.95, 77.24, 76.86, 76.60, 75.78, 74.56, 74.34, 66.23, 62.71, 61.46, 60.11, 57.62, 57.44, 56.42, 40.72, 39.14, 38.51, 38.15, 36.89, 36.08, 35.11, 27.12, 26.37, 26.27, 25.61, 24.40, 23.98, 21.95, 21.57, 20.41, 19.77, 18.81, 17.84, 14.61, 12.97, 9.29, 8.97, 8.27, -4.32, -4.63; HRMS (ESI) m/z calcd for C ₅₈ Hio ₄ NaOi6Si ₂ [M + Na] ⁺ : 1135.6755, found: 1135.6753.

To a stirred solution of silyl ether 22 (2.0 mg, 1.8 μιηοΐ) in THF (1.5 mL) at 0 °C was added TBAF (300 1 M in THF, 300 μιηοΐ), immediately producing a yellow solution. The mixture was stirred for 3 hours, then the reaction mixture was poured into a mixture of EtOAc (20 mL), H ₂ 0 (10 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (2 x 20 mL). The combined organic extracts were washed successively with saturated aqueous NaHC0 ₃ (15 mL), NH ₄ C1 (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was further purified by flash column chromatography on silica gel (hexanes/EtOAc = 1/1) to yield cocosolide (1) (1.3 mg, 82%) as a colorless oil. [a] _D ² °= -55.5 (c 0.4, DCM); 1H NMR (500 MHz, CD ₃ CN) δ 4.30 (t, 7 = 8.3 Hz, 2H), 4.23 (d, 7 = 7.3 Hz, 2H), 3.90 (dd, 7 = 11.3, 4.8 Hz, 2H), 3.50 (s, 6H), 3.40 (dd, 7 = 8.2, 1.5 Hz, 2H), 3.39 - 3.38 (m, 2H), 3.38 (s, 6H), 3.34 (dd, 7 = 11.6, 4.9 Hz, 6H), 3.32 (d, 7 = 4.7 Hz, 6H), 3.21 - 3.18 (m, 2H), 3.18 - 3.15 (m, 2H), 3.10 (dd, 7 = 11.2, 9.7 Hz, 2H), 3.02 (t, 7 = 8.5 Hz, 2H), 2.39 (dd, 7 = 17.2, 1.7 Hz, 2H), 2.07 (dd, 7 = 17.2, 8.3 Hz, 2H), 1.84 - 1.81 (m, 2H), 1.81 - 1.78 (m, 2H), 1.69 - 1.64 (m, 2H), 1.40 - 1.36 (m, 2H), 1.35 - 1.31 (m, 2H), 0.93 - 0.90 (m, 2H), 0.88 (s, 6H), 0.87 - 0.84 (m, 6H), 0.80 (s, 6H), 0.75 - 0.70 (m, 2H), 0.70 - 0.66 (m, 2H), 0.37 (dt, 7 = 8.7, 4.8 Hz, 2H), 0.24 (dt, J = 8.5, 5.0 Hz, 2H); ¹³ C NMR (125 MHz, CD ₃ CN) 5171.79, 106.37, 85.42, 85.21, 80.64, 80.00, 77.60, 76.36, 74.19, 63.36, 60.29, 58.44, 41.52, 39.26, 37.49, 35.98, 27.18, 24.73, 22.23, 20.60, 13.78, 13.48, 9.78; HRMS (ESI) m/z calcd for C ₄₆ H ₇₆ NaOi ₆ [M + Na] ⁺ : 907.5026, found: 907.5023.

14 15

To a -45 °C solution of compound 14 (1.3g, 5.2 mmol) in CH ₂ Cl ₂ (50 mL) was added dropwise BF ₃ Et ₂ 0 (980 μΐ,, 8 mmol), followed by the addition of PhSH (650 μΐ,, 6.3 mmol). The reaction mixture was allowed to stir for an additional 2 h before it was quenched by the addition of Et ₃ N (5 mL). The mixture was poured carefully into an Erlenmeyer flask and then partitioned between and then partitioned between H ₂ 0 (50 mL) and EtO Ac (150 mL). The layers were separated and the aqueous layer was extracted with EtO Ac (100 mL). The combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (30 mL), NH ₄ C1 (30 mL), brine (30 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 1/1) to yield compound 15 (650 mg, 36%) as a colorless oil.

[a] _D ²⁰ = -29.9 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.60 - 7.38 (m, 2H), 7.36 - 7.15 (m, 3H), 4.93 (t, J = 7.9 Hz, 1H), 4.76 (d, J = 8.1 Hz, 1H), 4.32 - 4.08 (m, 1H), 3.53 (s, 3H), 3.47 (s, 3H), 3.34 (ddd, = 24.0, 14.0, 5.7 Hz, 3H), 2.14 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 169.72, 133.78, 131.96, 129.01, 127.71, 86.75, 82.61, 78.30, 71.03, 65.69, 59.92, 58.61, 21.15; HRMS (ESI) m/z calcd. for C 19H32Na02Si(+)[(M+Na) +] : 343.2064, found: 343.2064; HRMS (ESI) m/z calcd for Ci ₉ H ₃₂ Na0 ₂ Si [M + Na] ⁺ : 335.0924, found: 335.0922.

15 16

To a solution of compound 15 (142 mg, 0.45 mmol) in MeOH (5 mL) was added a catalytic amount of sodium methoxide at room temperature. The reaction mixture was stirred for an hour, before it was concentrated under reduced pressure. The residue was dissolved in dry CH ₂ C1 ₂ (15 mL). To this solution at 0 °C was added 2,6-lutidine (0.18 mL, 1.5 mmol), followed by the addition of TBS-triflate (0.22 mL, 1.0 mmol). After being stirred for an additional hour, the reaction was quenched with saturated aqueous NaHC0 ₃ (10 mL), and the biphasic mixture was extracted with EtOAc (2 x 30 mL). The combined organic layers were washed successively with saturated aqueous NH ₄ C1 (10 mL), brine (10 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl ether = 95/5) to yield compound 16 (150 mg, 86%) as a colorless oil. [a] _D ² °= -42.7 (c 1.0, DCM); 1H NMR (400 MHz, CDC1 ₃ ) δ 7.56 - 7.38 (m, 2H), 7.36 - 7.15 (m, 3H), 4.57 (d, = 9.1 Hz, 1H), 4.13 - 4.04 (m, 1H), 3.58 (s, 2H), 3.51 - 3.47 (m, 1H), 3.45 (d, = 6.3 Hz, 3H), 3.35 - 3.28 (m, 1H), 3.17 (dd, = 11.4, 9.9 Hz, 1H), 3.08 (t, = 8.4 Hz, 1H), 0.95 (s, 9H), 0.19 (s, 3H), 0.14 (d, = 6.9 Hz, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 134.77, 131.24, 128.87, 127.16, 90.38, 87.12, 80.30, 73.20, 66.55, 60.93, 58.27, 26.10, 18.31, -3.89, -4.34; HRMS (ESI) m/z calcd for Ci ₉ H ₃₂ Na0 ₂ Si [M + Na] ⁺ : 407.1683, found: 407.1684.

The suspended solution of compound 16 (12 mg, 31 μιηοΐ) and flame-dried powdered 4 A molecular sieves (60 mg) in dry dichloromethane (1 mL) was stirred under nitrogen at room temperature for an hour and then cooled to- 78 °C. To this solution at -78 °C, was added a solution of N-iodosuccinimide (7 mg, 31 μιηοΐ) in dry dichlorometane (0.5 mL), followed by the addition of TfOH (0.30 μί, 3.4 μιηοΐ). 10 min later a solution of alcohol 19 (10 mg, 21 μιηοΐ) in dry dichloromethane (0.5 mL) was added. The reaction mixture was stirred for 0.5 hour at -78 °C before it was allowed to warm to room temperature over 2 hours. The reaction mixture was quenched by the addition of triethylamine (2 mL) and then partitioned between H ₂ 0 (15 mL) and EtOAc (50 mL). The layers were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (10 mL), NH ₄ C1 (10 mL), brine (10 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 10/1) to yield compound 23 (11 mg, 71%) as a colorless oil. [a] _D ² °= 3.3 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 7.37 - 7.22 (m, 10H), 4.73 (d, J = 11.7 Hz, 1H), 4.69 (d, 7 = 3.7 Hz, 1H), 4.48 (d, = 12.0 Hz, 1H), 4.47 (s, 2H), 3.68 (dd, J = 11.0, 5.7 Hz, 1H), 3.58 (s, 3H), 3.54 (ddd, J = 12.3, 8.5, 3.0 Hz, 3H), 3.49 (s, 3H), 3.48 - 3.42 (m, 1H), 3.36 (dd, J = 11.4, 4.4 Hz, 1H), 3.30 (t, J = 9.1 Hz, 1H), 3.22 (ddd, J = 10.6, 8.9, 5.8 Hz, 1H), 3.10 (d, J = 9.3 Hz, 1H), 2.85 (dd, J = 15.3, 6.7 Hz, 1H), 2.08 - 1.99 (m, 1H), 1.89 - 1.80 (m, 1H), 1.77 - 1.71 (m, 1H), 1.68 - 1.62 (m, 1H), 1.38 (dt, 7 = 21.2, 7.0 Hz, 1H), 1.21 (dd, / = 14.5, 9.2 Hz, 2H), 1.00 (dd, = 14.2, 6.8 Hz, 6H), 0.90 (d, J = 4.8 Hz, 3H), 0.89 - 0.85 (m, 9H), 0.72 - 0.64 (m, 1H), 0.61 - 0.54 (m, 1H), 0.24 (ddd, = 8.4, 6.6, 4.2 Hz, 2H), 0.08 (s, 3H), 0.00 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 139.28, 138.78, 128.41, 128.39, 127.73, 127.67, 127.52, 127.47, 94.90, 83.35, 80.55, 80.51, 79.46, 79.02, 73.16, 73.05, 72.73, 70.49, 68.15, 61.27, 60.28, 58.91, 42.41, 38.50, 32.61, 29.78, 29.50, 26.88, 25.92, 23.31, 22.35, 20.12, 18.08, 13.63, 13.43, 8.28, -4.57, -4.62;

HRMS (ESI) m/z calcd for C ₄₃ H ₆₈ 0 ₈ NaSi [M + Na] ⁺ : 763.4576, found: 763.4579.

To a solution of Bn ether 23 (72 mg, 97 μηιοΐ) in MeOH (6 niL) was added 10% Pd/C (10 mg). The reaction flask was evacuated and purged with hydrogen three times. The reaction mixture was stirred under a ¾ atmosphere at ambient temperature for 2 hours. The flask was then evacuated and purged with nitrogen three times and the catalyst was removed by filtration through Celite. The filtrate was concentrated and the crude product purified by flash column chromatography on silica gel (hexanes/EtOAc = 2/1) to yield compound 24 (35 mg, 64%) as a colorless oil. [a] _D ² °= 33.6 (c 1.0, DCM); 1H NMR (500 MHz, CDC1 ₃ ) δ 4.73 (d, = 3.5 Hz, 1H), 3.79 - 3.70 (m, 2H), 3.68 (dd, J = 11.0, 5.7 Hz, 1H), 3.58 (s, 3H), 3.54 - 3.47 (m, 2H), 3.49 (s, 3H), 3.37 (dd, 7 = 11.3, 4.3 Hz, 1H), 3.30 (t, J = 9.1 Hz, 1H), 3.25 - 3.16 (m, 2H), 3.15 - 3.05 (m, 1H), 1.88 - 1.80 (m, 2H), 1.74 - 1.64 (m, 4H), 1.60 (b, 3H), 1.39 - 1.31 (m, 2H), 1.24 - 1.19 (m, 1H), 0.98 (s, 3H), 0.97 - 0.93 (m, 6H), 0.91 (s, 9H), 0.73 - 0.65 (m, 1H), 0.65 - 0.58 (m, 1H), 0.35 - 0.31 (m, 1H), 0.31 - 0.23 (m, 1H), 0.10 (s, 3H), 0.05 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 95.04, 83.87, 83.33, 80.48, 75.91, 73.16, 61.72, 61.28, 60.35, 58.95, 42.93, 38.54, 32.81, 29.77, 26.79, 25.89, 25.58, 23.22, 18.89, 18.09, 13.72, 13.46, 9.34, -4.56, -4.62; HRMS (ESI) m/z calcd for [M + Na] ⁺ : 583.3637, found: 583.3534.

24

To a solution of the diol 24 (35 mg, 62 μηιοΐ) in CH ₂ C1 ₂ (0.7 niL) was added TEMPO (0.5 mg, 3.2 μιηοΐ) and saturated NaHC0 ₃ (380 μί). KBr (125 μΐ,, 6.2 μιηοΐ, 0.05 M aqueous solution) and Bu ₄ NCl (100 μί, 5.0 μιηοΐ, 0.05 M aqueous solution) were added subsequently and the mixture was cooled to 0 °C. To the vigorously stirring biphasic solution was added a stock solution of saturated NaHC0 ₃ (90 μί), brine (165 μί) and bleach (108 μί, 0.16 mmol, 1.5 M) dropwise over 70 min. The mixture was allowed to stir for an additional 30 min at 0 °C upon completion of the addition. The solution was then diluted with H ₂ 0 (2 mL) and CH ₂ C1 ₂ (2 mL). The diluted mixture was acidified with 10% citric acid (4-10 drops) to pH 3- 4. The biphasic mixture was extracted with EtOAc (2 x 50 mL), and the combined organic layers were washed with brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to give rise to the corresponding carboxylic acid (22 mg, 38 μιηοΐ), which was employed in the next step of reaction without further purification.

To the above acid (4 mg, 7 μιηοΐ) in dry toluene (2 mL) was added DIPEA (N,N- diisopropylethylamine) (15 μί, 90 μιηοΐ) and 2,4,6-trichlorobenzoyl chloride (10 μί, 64 μιηοΐ) dropwise. The reaction mixture was stirred at room temperature for 2 hours, then the mixture was added dropwise over 3 hours to a stirred solution of DMAP (4- dimethylaminopyridine) (17 mg, 0.14 mmol) in toluene (10 mL) at 80 °C, the reaction was allowed to stir for another 3 h before it was quenched with saturated aqueous NH ₄ C1 (15 mL). The biphasic mixture was extracted with EtOAc (2 x 70 mL), and the combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 3/1) to yield compound 25 (2.5 mg, 40%) as a colorless oil. [a] _D ² °= 29.4 (c 1.0, DCM); 1H NMR (500 MHz, CD ₃ CN) δ 4.74 (d, J = 3.7 Hz, 2H), 4.26 (t, / = 8.5 Hz, 2H), 3.66 (dd, / = 11.0, 5.3 Hz, 2H), 3.49 (d, = 4.6 Hz, 6H), 3.51 - 3.46 (m, 2H), 3.46 - 3.41 (m, 2H), 3.39 (s, 6H), 3.38 (d, = 7.5 Hz, 4H), 3.33 - 3.27 (m, 2H), 3.21 - 3.10 (m, 4H), 2.40 (dd, = 16.2, 1.4 Hz, 2H), 2.09 (s, 2H), 1.95 - 1.85 (m, 4H), 1.68 (dd, = 13.6, 5.3 Hz, 2H), 1.34 (dt, / = 20.0, 6.5 Hz, 2H), 1.20 - 1.12 (m, 2H), 1.00 - 0.93 (m, 2H), 0.91 (s, 6H), 0.90 (s, 18H), 0.87 (t, / = 7.2 Hz, 6H), 0.84 (s, 6H), 0.78 - 0.73 (m, 2H), 0.71 (dd, = 11.2, 6.1 Hz, 2H), 0.40 - 0.30 (m, 2H), 0.30 - 0.22 (m, 2H), 0.08 (s, 6H), 0.05 (s, 6H); ¹³ C NMR (125 MHz, CD ₃ CN) δ 171.36, 95.16, 83.25, 80.42, 80.29, 78.55, 76.44, 74.84, 73.04, 60.46, 60.04, 57.98, 41.18, 38.03, 35.41, 32.83, 26.36, 25.42, 23.68, 22.26, 19.85, 17.81, 12.98, 12.76, 9.06, -5.26, -5.34;

HRMS (ESI) m/z calcd for C ₅₈ Hio ₄ NaOi ₆ Si ₂ [M + Na] ⁺ : 1135.6755, found: 1135.6753.

To a stirred solution of silyl ether 25 (2.0 mg, 1.8 μιηοΐ) in THF (1 mL) at 0 °C was added TBAF (200 μί, 1 M in THF, 200 μιηοΐ), immediately producing a yellow solution. The mixture was stirred for 3 h, then the reaction mixture was poured into a mixture of EtOAc (20 mL), H ₂ 0 (10 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (2 x 20 mL). The combined organic extracts were washed successively with saturated aqueous NaHC0 ₃ (15 mL), NH ₄ C1 (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was further purified by flash column chromatography on silica gel (hexanes/EtOAc = 1/1) to yield compound 26 (1.2 mg, 75%) as a colorless oil. [a] _D ² °= 18.0 (c 1.0, DCM); 1H NMR (500 MHz, CD ₃ CN) δ 4.81 (d, = 3.8 Hz, 2H), 4.32 (t, = 8.1 Hz, 2H), 3.69 (dd, J = 11.0, 4.9 Hz, 2H), 3.54 (s, 6H), 3.50 - 3.42 (m, 6H), 3.41 (s, 6H), 3.39 - 3.32 (m, 4H), 3.24 - 3.12 (m, 4H), 2.70 (d, = 9.0 Hz, 2H), 2.70 (b, 2H), 2.44 (dd, = 16.8, 1.5 Hz, 2H), 1.92 - 1.84 (m, 4H), 1.73 (dd, = 14.8, 3.1 Hz, 2H), 1.38 (dd, = 14.9, 7.6 Hz, 4H), 1.00 - 0.94 (m, 2H), 0.93 (s, 6H), 0.91 - 0.85 (m, 12H), 0.75 (dd, = 10.7, 7.9 Hz, 4H), 0.44 - 0.34 (m, 2H), 0.27 (dd, = 13.5, 4.9 Hz, 2H); ¹³ C NMR (125 MHz, CD ₃ CN) δ 171.12, 95.36, 83.23, 80.15, 79.38, 79.34, 76.64, 75.25, 72.00, 60.21, 59.71, 57.87, 40.88, 37.89, 35.33, 32.80, 29.43, 26.38, 23.84, 22.23, 19.80, 12.98, 12.84, 9.01 ; HRMS (ESI) m/z calcd for C ₄₆ H ₇₆ NaOi ₆ [M + Na] ⁺ : 907.5026, found: 907.5023.

To a solution of compound 19 (37 mg, 0.08 mmol) and 2,6-lutidine (0.30 mL, 0.24 mmol) in CH ₂ C1 ₂ (1.5 mL), TBS-triflate (0.26 mL, 0.12 mmol) was added at 0 °C. The reaction mixture was stirred for one hour and then quenched by the addition of a saturated aqueous solution of NaHC0 ₃ (10 mL). The reaction mixture was extracted with EtOAc (2 x 30 mL); and the combined organic layers were washed successively with saturated aqueous solution of NH ₄ C1 (10 mL), brine (10 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (hexanes/ethyl ether = 85/15) to afford the corresponding TBS silyl ether (25 mg, 54%) as a light yellow oil.

To a solution of TBS silyl ether (25 mg, 0.043 mmol) in EtOAc (1 mL) was added 10% Pd/C (5 mg). The reaction flask was evacuated and purged with hydrogen three times. The reaction mixture was stirred under H ₂ atmosphere at ambient temperature for 2 h. The flask was then evacuated and purged with nitrogen three times and the catalyst was removed by filtration through a pad of Celite. The filtrate was concentrated and the crude product was purified by flash column chromatography on silica gel (hexanes/EtOAc = 3/1) to yield the corresponding diol (17 mg, 99%) as a colorless oil.

To a solution of the diol (17 mg, 0.043 μπιοΐ) in CH ₂ C1 ₂ (300 μί) was added TEMPO (0.3 mg, 2 μιηοΐ) and saturated NaHC0 ₃ (115 KBr (10 μί, 5 μιηοΐ, 0.05 M aqueous solution) and Bu ₄ NCl (40 μί, 3.2 μιηοΐ, 0.08 M aqueous solution) were added subsequently and the mixture was cooled to 0 °C. To the vigorously stirring biphasic solution was added a stock solution of saturated NaHC0 ₃ (31 μί), brine (60 iL and bleach (85 μί, 0.136 mmol, 1.6 M) dropwise over 60 min. The mixture was allowed to stir for an additional 30 min at 0 °C upon completion of the addition. The solution was then diluted with H ₂ 0 (2 mL) and CH ₂ C1 ₂ (2 mL). The diluted mixture was acidified with 10% citric acid (4-10 drops) to pH 3-4. The biphasic mixture was extracted with EtOAc (2 x 30 mL), and the combined organic layers were washed with brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to give rise to the corresponding carboxylic acid, which was employed in the next step of reaction without further purification.

To the above acid (8 mg, 0.02 mmol) in dry dichloromethane (0.6 ml) was added DIPEA

(N,N-diisopropylethylamine) (40 μί, 0.24 mmol) and 2,4,6-trichlorobenzoyl chloride (20 μί, 0.13 mmol) dropwise. The reaction mixture was stirred at room temperature for 2 hs, then the mixture was added dropwise over 3 hours to a stirred solution of DMAP (4- dimethylaminopyridine) (35 mg, 0.29 mmol) in dry dichloromethane (8 mL) at room temperature, the reaction was allowed to stir for another 10 hours before it was quenched with saturated aqueous NH ₄ C1 (15 mL). The biphasic mixture was extracted with EtOAc (2 x 40 mL), and the combined organic layers were washed successively with saturated aqueous NaHC0 ₃ (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 3/1) to yield compound 27 (2.8 mg, 36%) as a colorless oil.

[a] _D ²⁰ = -14.7 (c 0.4, DCM); 1H NMR (400 MHz, CDC1 ₃ ) δ 4.43 (t, J = 9.2 Hz, 2H), 3.44 (d, = 6.5 Hz, 4H), 3.39 (dd, J = 11.3, 4.9 Hz, 2H), 2.40 (dd, J = 17.4, 1.6 Hz, 1H), 2.24 (dd, J = 17.4, 8.2 Hz, 2H), 1.95 - 1.84 (m, 2H), 1.69 (dd, J = 14.9, 3.5 Hz, 2H), 1.55 (d, 7 = 5.0 Hz, 2H), 1.43 - 1.33 (m, 4H), 1.33 - 1.27 (m, 2H), 0.91 - 0.88 (m, 18H), 0.86 (s, 6H), 0.80 (s, 6H), 0.79 (s, 6H), 0.67 (ddd, J = 13.4, 9.0, 4.7 Hz, 2H), 0.41 - 0.31 (m, 2H), 0.26 (dt, J = 9.7, 4.8 Hz, 2H), 0.04 (s, 6H), 0.00 (s, 6H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 171.3, 79.6, 77.2, 75.7, 75.6, 41.0, 38.8, 38.1, 35.3, 29.7, 26.6, 25.8, 24.3, 22.7, 19.8, 18.0, 13.5, 12.9, 9.6, -3.9, -5.0; HRMS (ESI) m/z calcd for C ₄₄ H ₈ i0 ₈ Si ₂ [M + H] ⁺ : 793.5464, found: 793.5502

To a stirred solution of silyl ether 27 (2.0 mg, 2.5 μιηοΐ) in THF (1 mL) at 0 °C was added TBAF (1 mL, 0.5 mmol, 0.5 M in THF). The reaction mixture was stirred at room

temperature for 28 hours, then it was poured into a biphasic mixture solution of EtOAc (20 mL) and H ₂ 0 (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic extracts were washed successively with saturated aqueous NaHC0 ₃ (15 mL), NH ₄ C1 (15 mL), brine (15 mL), dried over anhydrous Na ₂ S0 ₄ , and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 2/1) to yield compound 28 (1.0 mg, 71%) as a colorless oil. [a] _D ² °= 5.0 (c 0.4, DCM); 1H NMR (300 MHz, CDC1 ₃ ) δ 4.41 (t, = 9.1 Hz, 2H), 3.52 - 3.37 (m, 6H), 2.45 - 2.24 (m, 4H), 2.02 - 1.88 (m, 2H), 1.83 - 1.69 (m, 4H), 1.41

- 1.30 (m, 4H), 1.00 - 0.93 (m, 2H), 0.90 (s, 6H), 0.87 (d, = 6.4 Hz, 6H), 0.82 (s, 6H), 0.74

- 0.63 (m, 2H), 0.40 - 0.31 (m, 2H), 0.31 - 0.22 (m, 2H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 171.3, 79.9, 77.2, 75.5, 75.3, 41.1, 38.4, 35.1, 26.6, 24.2, 22.2, 19.9, 13.5, 12.5, 9.6; HRMS (ESI) m/z calcd for C ₃₂ H ₅₃ 0 ₈ [M + H] ⁺ : 565.3735, found: 565.373

Biological studies

Methods

Biological Reagents and General Experimental Procedures. Phorbol myristate acetate (PMA) was purchased from Promega (Cat# VI 171; Madison, WI). Phytohemagglutinin (PHA) was from Sigma-Aldrich (Cat# L8902; St. Louis, MO) and ionomycin, calcium salt was purchased from EMD Chemicals, Inc (cat# 407952; Gibbstown, NJ). Human colon adenocarcinoma HCT116 cells and the human leukemic T-cell line Jurkat (Clone E6-1) were obtained from the American Type Culture Collection (Manassas, VA) and cultured either Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) for HCT116 cells and RPMI 1640 medium (Cellgro, Herndon, VA) for Jurkat cells, supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). Cells were maintained at 37 °C humidified air in 5% C0 ₂ .

Cell Viability Assays. HCT116, RAW 264.7 and Jurkat cells were seeded in

96-well plates and 24 h later treated with up to 50 μΜ cocosolide (1) for 24 h (RAW 264.7) or 48 h (HCT116, Jurkat). Cell viability was measured using MTT reagent according to the manufacturer's instructions (Promega, Madison, WI, USA).

Measurement of IL-2 Production (Jurkat Cells). Jurkat cells (1 x 10 ⁵ cells per well; 150 each well) were seeded in 96-well clear bottom plates and allowed to settle for one hour. Cells were then co-treated with compounds 1, 3, 26 and 28 (50 μΜ to 100 nM in EtOH) and TCR-dependent stimulants (80 nM PMA in DMSO and 10 μg/ml PHA in H ₂ 0) or TCR-independent stimulants (80 nM PMA in DMSO and 1 μΜ ionomycin in DMSO) along with EtOH/DMSO solvent controls. Cells were also treated with compounds alone to measure cell viability due PMA/PHA and PMA/ionomycin toxicities. DMSO concentrations were maintained at 0.21% to minimize toxicity for Jurkat cells. After 24 h incubation, 50 μί of culture supernatant were removed from each well into a separate plate and used for measuring IL-2 production using an alphaLISA kit (PerkinElmer, Waltham, MA). Briefly, acceptor bead and anti-IL-2 antibody were incubated with 5 μί of supernatant for 60 min and donor beads added and incubated for further 30 min following which, IL-2 was quantified using Envision-Reader (PerkinElmer). The cells were used to measure viability after 48 h. Proliferation Assay. Red blood cells (RBC) were removed from freshly isolated whole spleen cells using ammonium chloride potassium (ACK) lysis buffer for 2 min at room temperature then washed free of lysis buffer using PBS. RBC- depleted spleen cells were then cultured in RPMI 1640 supplemented with 10% FCS (Invitrogen Life Sciences), lx penicillin/ streptomycin/neomycin (Gibco), and 10 mM HEPES buffer (Gibco) at a concentration of 10 ⁶ cells/well in round-bottom 96-well plates at 37 °C. Anti-CD3e (0.05 μg/200 well) was added to stimulate cell proliferation in the presence of increasing concentrations of 1 (50 nM, 0.5 μΜ, 5 μΜ, and 50 μΜ). All treatment conditions were controlled to contain a final concentration of 1% DMSO solvent as used in drug preparation. At 72 h of culture, 1 μθι H- thymidine (Amersham Biosciences) in 50 μΐ _^ of media was added per well and allowed to incorporate for 12-16 h. Cells were harvested and washed using an automated cell harvester (Perkin Elmer), and radioactivity was analyzed using a liquid scintillation counter. Cell proliferation is measured as counts per minute (cpm).

NO Assay. RAW 264.7 cell assays to measure effects on LPS-induced NO production were performed as previously described (Ratnayake, R.; Liu, Y.; Paul, V. J.; Luesch, H. Cancer Prev. Res. 2013, 6, 989-999).

Antibacterial Assays. Activity against Bacillus cereus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis was assessed as we previously described (Montaser, R.; Abboud, K. A.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2011, 74, 109- 112; Montaser, R.; Paul, V. J.; Luesch, H. Phy to chemistry 2011, 72, 2068-2074).

Results

To study the effect of cocosolide (1) on biological function, we treated an array of cell types (HCT116 colorectal cancer cells, RAW macrophage cells, Jurkat T- cell lymphoma cells; IC ₅₀ > 50 μΜ) with cocosolide (1) and found no modulation of cell viability. Structural consideration, including the dimeric nature coupled with the glycosylation feature, hinted at the possibility of dimeric surface targets for geometric and recognition consideration, respectively. We tested the effects in immortalized T- cells (Jurkat) as a model to evaluate immunomodulatory activity (Schneider, U.; Schwenk, H.-U.; Bornkamm, G. Int. J. Cancer 1977, 19, 621-626). IL-2 production was induced via dual stimulation with phorbol 12-myristate 13-acetate (PMA, 80 nM) and phytohemagglutinin (PHA, 10 μg/mL), conditions for a T-cell receptor (TCR) dependent activation; or TCR-independent stimulants PMA (80 nM) and ionomycin (1 μΜ) (Fischer, B. S.; Qin, D.; Kim, K.; McDonald, T. V. J. Pharmacol. Exp. Ther. 2001, 299, 238-246). Cocosolide (1) and its [a,a]-anomer (26) equally and potently reduced IL-2 production without significantly affecting cell viability (Figures 3A and 3B). The susceptibility of the TCR-independent system was stronger, although both stimulations are abrogated in a dose-dependent manner. The macrocyclic core 28 and monomer ester 3 showed minimal effects in these assays compared with 1 and 26 (Figures 3C and D), indicating that the sugar and dimeric structure are important to the target recognition and engagement process. FIG 3. Biological effects of cocosolide (1), [a,a]-anomer 26, macrocyclic core 28 and monomer methyl ester 3 on T-cell systems. (A) Effect of 1 on IL-2 production by Jurkat cells and on cell viability in response to PMA/PHA and PMA/ionomycin stimulation. For comparison, cyclosporine A inhibited both activities at 1 μΜ by 90%. (B) Effect of cocosolide [a,a]-anomer 26 on IL-2 production by Jurkat cells and on cell viability in response to PMA/PHA and PMA/ionomycin stimulation. (C,D) Comparison of compounds 1 (natural product), 3 (monomer methyl ester), 26 ([α,α]- anomer) and 28 (aglycon) on their effect on IL-2 production in Jurkat cells (C) stimulated by PMA/PHA and (D) PMA/ionomycin. (E) Spleen cell proliferation assay. Anti-CD3 stimulated cells were cultured in triplicate wells in the presence of increasing concentrations of 1. Values shown represent the average spleen cell response from the mean triplicate values between two mice. * denotes p < 0.05 compared to DMSO.

To examine how cocosolide (1) may affect activated T cells, spleen cells were stimuklated with CD3 and cultured the cells under increasing concentrations of cocosolide (1) for 72 h. As shown in Figure 3E, cocosolide (1) inhibited anti-CD3- mediated T-cell proliferation in a dose-dependent manner. Cell viability was not affected compared with DMSO-treated controls, suggesting that the observed suppression of T cell expansion by 1 is not attributed to cell death.

Possible modulation of Toll-like receptor 4 mediated pathways was also investigated. Specifically, RAW264.7 macrophage cells were stimulated with lipopolysaccharides (LPS) and pretreatment with cocosolide (1) did not dampen the induction of NO production and also did not reduce the viability (up to 100 μΜ). Similarly, 1 did not exert antibacterial activity in assays {e.g., Bacillus cereus, Pseudomonas aeruginosa, Mycobacterium tuberculosis). Taken together, cocosolide' s effects appear to be fairly cell- and pathway-type specific.

The non-cytotoxic effects are consistent with data reported for the structurally related clavosolides, for which a bioactivity remained to be established. Clavosolides A and B were first reported from the sponge Myriastra clavosa but suggested to be of cyanobacterial origin as the structure did not relate to any known sponge metabolites and the presence of a high concentration of cyanobacterial cells in the sponge sample. These dimeric metabolites along with two other analogues, clavosolides C and D were concurrently reported from a cytotoxic extract of M. clavosa, displaying differential cytotoxicity in the NCI-60 cell panel (Erickson, K. L.; Gustafson, K. R.; Pannell, L. K.; Beutler, J. A.; Boyd, M. R. J. Nat. Prod. 2002, 65, 1303-1306). Evaluation of the purified compounds revealed that these are non-cytotoxic. The structurally related macrolide cyanolide A was reported from the cyanobacterium L. bouillonii with potent moUuscicidal activity against Biomphalaria glabrata, but non-cytotoxic effects against H-460 human lung adenocarcinoma and mouse neuroblastoma cells.

Incorporation by Reference

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended with be encompassed by the following claims.