TITLE OF THE INVENTION

HYBRID MOLECULES HAVING MIXED VITAMIN D RECEPTOR AGONISM AND HISTONE DEACETYLASE INHIBITORY PROPERTIES

FIELD OF THE INVENTION

[000 Ij The present invention relates to a seπes of new chemical agents that demonstrate antiproliferative and cytotoxic activity against cancer cells More particularly, but not exclusively, the present invention relates to hybrid molecules capable of mixed vitamin D receptor agonism and histone deacetylase inhibition The present invention also relates to methods of their synthesis

BACKGROUND OF THE INVENTION

[0002) 'alcitnol, 1), the biologically active metabolite of vitamin D ₃ (Calciferol), is a primary physiological regulator of calcium homeostasis, controlling intestinal calcium absorption, bone resorption and bone mineralization ' ³

[0003J The vitamin D receptor (VDR), a member of the nuclear receptor family of hgand-regulated transcription factors, plays a crucial role in calcitÏ€ol's signaling CalcitÏ€ol- bound VDR heterodimeÏ€zes with related retinoid X receptors and binds to specific DNA sequences called vitamin D response elements, located in the regulatory regions of target genes ²

[0004] Cancer progression has been associated with an acquired resistance to calcitriol. This resistance however, can be overcome by co-administration of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA, 2). HDAC inhibitors (a further example of which is provided by SAHA (3)) have emerged as a new and promising class of anticancer agents capable of regulating transcription and inhibiting cancer cell proliferation by induction of cell cycle arrest in either G0/G1 or G2/M, cell differentiation and/or apoptosis. ⁸ ' ⁹

[0005] Prior work has shown that it is possible to prepare hybrid molecules exhibiting VDR agonism and HDAC inhibitory activity (e.g. TSA-like hybrid 4 and SAHA-like hybrid 5).

[0006] Intensive efforts have been devoted to the development of calcitriol analogues that would combine therapeutic potential with lowered calcemic activity. While calcitriol regulates cell differentiation and proliferation, and has anti-cancer properties ² ' ³ , its calcemic activity has limited its use in the treatment of cancers due to hypercalcemia typically induced by the required supraphysiological levels used clincially. Structural changes and modifications in strategic locations throughout the calcitriol molecular backbone have led to the development of numerous analogues, many of which, including EB 1089 (6) have shown potent

antiproliferative and antidifferentiation activities, along with desired lowered calcemic activity. ^{4" 6} Several of these analogues have advanced to preclinical studies for the treatment of diverse human diseases, and some have become FDA-approved drugs. ⁷

[0007] Several currently investigated HDACi attempt to target various representatives of class I and II HDACs based on different structures, and as demonstrated in the following strui ; groups may be employed. MGCD-0103

(Methylgene) 7, a 2-aminophenylamide derivative is expected to be more selective for class I HDACs. Thiol-based SAHA derivatives were found to be weak HDAC inhibitors. Subsequently, it was tested whether their efficacy could be enhanced by designing disulfides and S-Acyl derivatives as prodrugs. While the disulfides still led to disappointing results, thioesters seemed more promising (8) and release a thiol upon thioester hydrolysis. Compounds 9 and 10 are examples of potent thioacetate based and alpha keto-amide HDAC inhibitors (Takeda). Trapoxin, (11) a cytotoxic cyclic tetrapeptide, is a natural product with irreversible HDAC inhibitory activity that shows an unusual epoxyketone zinc binding group. Fujisawa pursues FK228, also called depsipeptide or romidepsin, which is a cyclic peptide that inihibits class I HDAC. FK288 is again a prodrug which releases a thiol upon reduction of its disulfide group.

[0008] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0009] The present invention relates to hybrid molecules capable of mixed vitamin

D receptor agonism and histone deacetylase inhibition.

[0010] In an embodiment, the present invention relates to hybrid molecules comprising a vitamin D receptor agonist moiety and an HDAC inhibitor moiety.

[0011] In an embodiment, the present invention relates to hybrid molecules comprising a vitamin D receptor agonist moiety and an HDAC inhibitor moiety, wherein the vitamin D receptor agonist moiety is derived from vitamin D and wherein the HDAC inhibitor moiety is modelled after an HDAC inhibitor comprising a functionality including an N- hydroxyformate, a thioglycolate amide, a glycinamide, a bromoacetamide, a sulphonamide, a sulfamide, an α-keto ester or amide, or an ortho-aminoanilide.

[0012] In an embodiment, the present invention relates to hybrid molecules or pharmaceutically acceptable salts thereof selected from the group consisting of:

[0013] wherein:

[0014] Ri is sel H, OH and lower alkyl;

[0015] R ₂ is selected from the group consisting of H, lower alkyl and alkylene;

[0016] R ₃ is selected from the group consisting of H and OH;

[0017] R ₄ and R ₅ are independently selected from the group consisting of H, OH and lower alkyl;

[0018] R ₆ is selected from the group consisting of H and lower alkyl;

[0019] X is selected from the group consisting of H, NR ₄ R ₅ , F, Cl, Br, I, CF ₃ , OR ₆ , SR ₆ , CO ₂ R ₆ , S(C=S)SR ₆ , lower alkyl, phenyl and substituted phenyl;

[0020] Y is selected from the group consisting of CO, SO ₂ and NRj;

[0021] n is an integer ranging from 0 to 3; and

[0022] m is an integer ranging from 0 to 3.

[0023] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0024] wherein Ri and R ₆ are independently selected from the group consisting of H and Me; and n is an integer ranging from 1 to 2.

[0025] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0026] wherein n is an integer ranging from 1 to 2.

[0027] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0028] wherein n is an integer ranging from 1 to 3.

[0029J In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0030] wherein n is an integer ranging from 1 to 3.

[0031] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0032] wherein X is selected from the group consisting of CF ₃ , Et, Bu, Ph, 4-CN-Ph and OH; n is 2; and m is either 0 or 2.

[0033] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0034] wherein X is selected from the group consisting of SH, NMe ₂ , NH ₂ and Br; and n is either 1 or 2.

[0035] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0036] wherein n is either 1 or 2.

[0037] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0038] wherein X is selected from the group consisting of OMe and NHMe; and n is either 1 or

2.

[0039] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0040] wherein n is either 1 or 2.

[0041] In an embodiment, the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:

[0042] wherein n is either 1 or 2.

[0043] In an embodiment, the present invention relates to a method for the treatment of disorders or diseases wherein inhibition of HDAC and/or vitamin D agonism is beneficial, the method comprising administering to a subject in need thereof an affective amount of one or more hybrid molecules as disclosed herein.

[0044] In an embodiment, the present invention relates to a method of treating a patient afflicted with a condition selected from the group consisting of cancer, inflammation and auto-immune diseases, the method comprising administering to the patient a therapeutically effective amount of one or more of the hybrid molecules as disclosed herein.

[0045] In an embodiment, the present invention relates to a method of wound healing, the method comprising administering to a patient in need thereof a therapeutically effective amount of one or more of the hybrid molecules as disclosed herein.

[0046] In an embodiment, the present invention relates to a method of treating bacterial infections in a patient, the method comprising administering to the patient a therapeutically effective amount of one or more of the hybrid molecules as disclosed herein.

[0047] In an embodiment, the present invention relates to a method of reducing proliferation of/or inducing i method comprising contacting the neoplastic cells with one or more of the hybrid molecules as disclosed herein.

[0048] In an embodiment, the present invention relates to the use of one or more of the hybrid molecules as disclosed herein in the manufacture of a medicament for the treatment of a condition selected from the group consisting of cancer, inflammation and auto-immune diseases.

[0049] In an embodiment, the present invention relates to the use of one or more of the hybrid molecules as disclosed herein in the manufacture of a medicament for inducing wound healing.

[0050] In an embodiment, the present invention relates to the use of one or more of the hybrid molecules as disclosed herein in the manufacture of a medicament for treating bacterial infections.

[0051] In an embodiment, the present invention relates to a pharmaceutical composition comprising an effective amount of one or more of the hybrid molecules as disclosed herein in association with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0052] In an embodiment, the present invention relates to an admixture comprising an effective amount of one or more of the hybrid molecules as disclosed herein in association with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0053] The foregoing and other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of illustrative embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0055] FIG. 1 is an illustration of the VDR agonist activity of hybrid molecules 4, and 16-40. The capacity of the hybrid molecules to induce the expression of the 1,25D target gene encoding CYP24 was analyzed by RT/PCR using 1,25D (1) and hybrid molecule 4 as positive controls. Expression of the gene encoding GAPDH was used a negative control.

[0056] FIG. 2 is an illustration of the direct binding of 1 and hybrid molecules 5,

17-19, 21, 22, 25, 26, 28, 30, and, 32 to the VDR ligand binding domain as tested by fluorescence polarization (FP) competition assay. The apparent 1 ,25D-like affinity of 25 and 26 for the VDR was supportted by the results of dose-response curves analyzing induction of CYP24 expression by RT/PCR (bottom panel).

[0057] FIG. 3 is an illustration of the capacity of selected hybrid molecules of the present invention to inhibit HDAC activity. FIG. 3A illustrates the HDAC inhibitory activity of hybrid molecules 5 and 32 (using 2 as a positive control) as analyzed by colorimetric assay using

HeLa cell nuclear extracts as a source of HDAC activity. ¹⁰ Hybrid molecules were also tested using Western blotting for their capacity to induce hyperacetylation of tubulin (tub) or histones H3 and H4 (FIGs. 3B-E). As indicated, 2 or 4 were used as positive controls for induction of hyperacetylation. β-actin was used as control for protein loading.

[0058] FIG. 4 provides data from dose-response analyses of the capacity of hybrids to inhibit the activity of purified HDAC3 using the same colourimetric assay presented in FIG. 3A. IC50 values are presented. 3 was used as a positive control for HDAC inhibition.

[0059] Table. 1 provides a summary of the functional studies performed on compounds with 3-5 used as positive controls for analysis of HDAC inhibition and 1, 4 and 5 used as positive controls for VDR agonism. Data for CYP24 induction (CYP24) are taken in part from FIG. 1, and data for fluorescence polarization assays for VDR binding are taken from FIG. 2. Function of hybrids as HDAC inhibitors was tested using either a colorimetric assay or Western blottii ier tubulin (Tubulin) or histone (Histone) hyperacetylation using 2 and/or 4 as positive controls. HDAC inhibitory activity of hybrids 4, 5, 17-19, 21, 22, 25, 26, 28, 30, 32 and 38 was measured using an HDAC colorimetric assay with purified fractions of HDAC2, HDAC3 or HDAC6, as indicated. The IC ₅₀ values (μM) are presented for compounds tested. Mice fed a normal calcium diet were infused using minipumps with 1 (12 or 24 pmol/day) or hybrids 4, 17, 18, 21, 25, 26, and 32 (240 or 1,200 pmol/day). Unlike the animals treated with 1, which appeared unhealthy and were hypercalcemic (+++), all animals infused with hybrid compounds appeared healthy over the entire course of the experiment, and none developed hypercalcemia (-). Thus, all hybrids tested lacked the dose- limiting toxicity associated with treatment with 1.

[0060] FIG. 5 is an illustration of the capacity of hybrid molecules of the present invention to inhibit CYP24 enzymatic activity. The CYP24 enzyme, whose induction by 1 represents a physiological negative feedback loop, modifies 1 and inhibits its capacity to activate gene expression. A illustrates the comparison between the capacities of 31 and 39 to boost expression of the gene encoding CYP24 in the presence of limiting (1OnM) concentrations of 1. Cytochrome P450 inhibitor ketoconazole is used as a positive control for the effects of inhibition

of CYP24 enzymatic activity. B demonstrates that the effects of 31 and 39 are not linked to HDACi activity as 2 failed to boost CYP24 gene expression in the presence of limiting concentrations of 1.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0061] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

[0062] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a :

[0063] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0064] The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0065] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0066] Abbreviations: NMR: Nuclear Magnetic Resonance; MS: Mass Spectrometry; m.p.: melting point; HRMS: High Resolution Mass Spectrometry; EtOAc: Ethyl Acetate; CH ₂ Cl ₂ : Dichloromethane; CDCl ₃ : Chloroform-d; DMAP: 4-(N,N-dimethylamino)pyridine; TFA: Trifluoroacetic acid; TCDI: 1,1-thiocarbonyldiimidazole; AcOH: Acetic acid; TLC: Thin Layer Chromatography; FAB: Fast Atom Bombardment; FCC: Flash Column Chromatography.

[0067] As used herein, the term "alkyl" can be straight-chain or branched. This also applies if they carry substituents or occur as substituents on other residues, for example in alkoxy residues, alkoxycarbonyl residues or arylalkyl residues. Substituted alkyl residues can be substituted in any suitable position. Examples of alkyl residues containing from 1 to 18 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl and octadecyl, the ^-isomers of all these residues, isopropyl, isobutyl, isopentyl, neopentyl, isohexyl, isodecyl, 3-methylpentyl, 2,3,4-trimethylhexyl, sec-butyl, tert- butyl, or /erZ-pentyl. A specific group of alkyl residues is formed by the residues methyl, ethyl, ^-propyl, isopro ^' i terr-butyl.

[0068] As used herein, the term "lower alkyl" can be straight-chain or branched. This also applies if they carry substituents or occur as substituents on other residues, for example in alkoxy residues, alkoxycarbonyl residues or arylalkyl residues. Substituted alkyl residues can be substituted in any suitable position. Examples of lower alkyl residues containing from 1 to 6 carbon atoms are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-buty\, pentyl, isopentyl, neopentyl, and hexyl.

[0069] As used herein, the term "alkylene" can be a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms. Examples of alkylene residues are methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, and pentylene.

[0070] As used herein, the term "alkenyl" can be straight-chain or branched unsaturated alkyl residues that contain one or more, for example one, two or three double bonds which can be in any suitable position. Of course, an unsaturated alkyl residue has to contain at least two carbon

atoms. Examples of unsaturated alkyl residues are alkenyl residues such as vinyl, 1-propenyl, allyl, butenyl or 3-methyl-2-butenyl.

[0071] As used herein the term "alkynyl" can be straight-chain or branched unsaturated alkyl residues that contain one or more, for example one, two or three, triple bonds which can be in any suitable position. Of course, an unsaturated alkyl residue has to contain at least two carbon atoms. Examples of unsaturated alkyl residues are alkynyl residues such as ethynyl, 1-propynyl or propargyl.

[0072] As used herein, the term "cycloalkyl" can be monocyclic or polycyclic, for example monocyclic, bicyclic or tricyclic, i.e., they can for example be monocycloalkyl residues, bicycloalkyl residues and tricycloalkyl residues, provided they have a suitable number of carbon atoms and the parent hydrocarbon systems are stable. A bicyclic or tricyclic cycloalkyl residue has to contain at least 4 carbon atoms. In an embodiment, a bicyclic or tricyclic cycloalkyl residue contain further embodiment, a bicyclic or tricyclic cycloalkyl residue contains at least 6 carbon atoms and up to the number of carbon atoms specified in the respective definition. Cycloalkyl residues can be saturated or contain one or more double bonds within the ring system. In particular they can be saturated or contain one double bond within the ring system. In unsaturated cycloalkyl residues the double bonds can be present in any suitable positions. Monocycloalkyl residues are, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl or cyclotetradecyl, which can also be substituted, for example by Ci-C ₄ alkyl. Examples of substituted cycloalkyl residues are 4-methylcyclohexyl and 2,3-dimethylcyclopentyl. Examples of parent structures of bicyclic ring systems are norbornane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.1]octane.

[0073] As used herein, the term "aryl" means an aromatic substituent which is a single ring or multiple rings fused together. When formed of multiple rings, at least one of the constituent rings is aromatic. In an embodiment, aryl substituents include phenyl and naphthyl groups.

[0074] As used herein, the term "substituted phenyl" is understood as being phenyl having a substituent selected from the group consisting of amino, -NH(lower alkyl), and -N(lower alkyl) ₂ , as well as being mono-, di- and tri-substituted phenyl comprising substituents selected from the group consisting of lower alkyl, methoxy, methylthio, halo, cyanao, hydroxy, amino, NH(lower alkyl), and -N(lower alkyl) ₂ .

[0075] The invention contemplates that for any stereocenter or axis of chirality for which the stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures.

[0076] A novel class of chemical agents (i.e. novel hybrid molecules) having mixed vitamin D receptor agonism and histone deacetylase inhibitory properties are described herein.

[0077] A combinatory effect of calcitriol and TSA in prostate and breast cancer has been previously den showed a combinatory effect in low nM concentrations of TSA and calcitriol on the proliferation of calcitriol-resistant SCC4 HNSCC cells. Novel hybrid molecules combining both vitamin D receptor agonism and HDAC inhibition properties into a single molecular structure were developed.

[0078] Hybrid molecules have had considerable success in pharmacotherapy and offer several advantages over the use of the individual compounds (i.e. the compounds making-up the hybrid molecule) in combination therapy. ^12'14 Moreover, analyses of dose/toxicity relationships of hybrid molecules are simpler than those of combination therapies, and problems associated with differing pharmacokinetic profiles of individual components are eliminated.

[0079] The design of the hybrid molecules of the present invention is based on structure-activity relationship (SAR) and X-ray studies. A first generation of hybrid molecules, based on the structures of 1 and 2, has been disclosed in International Application PCT/CA2007/000885 published on November 22, 2007 under WO 2007/131364 Al . The crystal structure of calcitriol bound to the VDR-LBD reveals hydrogen bonding to all three hydroxyl functionalities (Ser237

and Arg274 for 1-OH; Ser278 and Tyrl43 for 3-OH; and His305 and His397 for 25-OH). ¹⁵ The remainder of the binding pocket is filled with hydrophobic residues which contact the triene and C/D-ring sections, as well as a portion of the side chain. The 25-OH group hydrogen bonds with His305 and His397, and this region is more tolerant of changes; with altered chain length and oxidation state, replacement of C26 and C27 with larger or cyclic structures, inversion of the C20 stereocenter and replacement of the 25-OH group with other hydrogen bonding groups all possible. The x-ray crystal structure of 2 bound to archaebacterial HDAC homolog HDLP, revealed 2 bound through bidentate coordination of the hydroxamic acid to the catalytic zinc ion, with the diene chain spanning the substrate-binding tube. The top of the tube ends at a surface groove which has several hydrophobic residues which contact the dimethylaniline group of 2. Most competitive HDACi consist of this general motif, a zinc-binding group (ZBG) connected by a saturated or unsaturated linker to a highly variable 'cap' group which binds at the protein surface. ¹ Hydrogen bonding to the hydroxyl functionalities of the A-ring of 1 is critical for binding, as deletion or alteration of the stereochemistry of the 1- or 3-OH group significantly decreases affinitv for the VDT? ¹⁶ Mn«t nntpnt analogs of calcitriol have hydroxyl moieties in the vicinity of C-2: , . __r exact location (e.g. in EB 1089) is tolerated.

The central C/D-ring is less critical, as it may be partially or fully excised in favor of a single 5- or 6-membered ring or a linear chain. ¹⁷ ' ¹⁸ 19-Nor and C-20 epi analogs are also well tolerated by the VDR. ¹⁶ ' ¹⁹

[0080] The crystal structure of 2 bound to an HDAC revealed a tube-like binding pocket possessing a zinc ion coordinated to two Asp residues and one His residue at a bottom portion of the tube-like binding pocket. ²⁰ The hydroxamic acid function of 2 forms a bidentate chelate with the zinc ion. There are two other Asp residues and two other His residues at the bottom portion of the tube-like binding pocket, the latter of which form hydrogen bonds with the NH and OH groups of the hydroxamic acid. The polyene chain of 2 spans the remainder of the tube-like binding pocket, consisting of hydrophobic residues. The top portion of the tube-like binding pocket terminates at a surface groove comprising several hydrophobic residues which come into contact with the dimethylamino group of 2.

[0081] Based on SAR studies, the γ-methyl dienylhydroxamic acid unit is required. However, the ketone and adjacent methyl substituted methyne may be excised, provided that the dimethylamino group is replaced with a larger unit such as an arylsulfonamide. ²¹ Thus, the dienyl chain in 2 seems to function as a tether, linking the zinc binding unit with a "cap" group which binds on the HDAC surface. Hydrogenation of the dienyl chain in analogs of 2 renders them inactive. However, straight chain analogs lacking the γ-methyl group (e.g. SAHA, 3) have been found to be potent HDAC inhibitors.

[0082] A first hybrid molecule (36) was designed in which the 25-hydroxyl moiety required for binding to the VDR was replaced by an N-hydroxyformate. It was hypothesized that the N- hydroxyformamide terminus of this polar side chain would allow hydrogen bond formation in the active site of the VDR, as well as permitting chelation to the zinc ion in the HDAC binding site. The backbone of the vitamin D core, including the A and C/D-ring systems were maintained along with the stereochemical relationships of the various substituents.

[0083] In an embodiment, the present invention relates to pharmaceutical compositions comprising a pharmaceutically effective amount of one or more hybrid molecules as defined herein or pharmaceutically acceptable salts thereof, in association with one or more pharmaceutically acceptable carriers, excipients and/or diluents. The term "pharmaceutically effective amount" is understood as being an amount of hybrid molecule required upon administration to a mammal in order to induce vitamin D receptor agonism and HDAC inhibition. Therapeutic methods comprise the step of treating patients in a pharmaceutically acceptable manner with one or more hybrid molecules or compositions comprising one or more hybrid molecules as disclosed herein. Such compositions may be in the form of tablets, capsules, caplets, powders, granules, lozenges, suppositories, reconstitutable powders, creams, lotions, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.

[0084] The therapeutic agents of the present invention (i.e. hybrid molecules) may be administered alone or in combination with pharmaceutically acceptable carriers. The proportion of each carrier is determined by the solubility and chemical nature of the agent(s), the route of administration, and standard pharmaceutical practice. In order to ensure consistency of

administration, in an embodiment of the present invention, the pharmaceutical composition is in the form of a unit dose. The unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients. Non-limiting examples of conventional excipients include binding agents such as acacia, gelatin, sorbitol, or polyvinylpyrolidone; fillers such as lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants such as magnesium stearate; disintegrants such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.

[0085] The hybrid molecules of the present invention may be injected parenterally; this being intramuscularly, intravenously, or subcutaneously. For parenteral administration, the hybrid molecules may be used in the form of sterile solutions containing solutes, for example sufficient saline or glucose to make the solution isotonic.

[0086] The hyi ;d orally in the form of tablets, capsules, or granules, containing suitable excipients such as starch, lactose, white sugar and the like. The hybrid molecules may be administered orally in the form of solutions which may contain coloring and/or flavoring agents. The hybrid molecules may also be administered sublingually in the form of tracheas or lozenges in which the active ingredient(s) is/are mixed with sugar or com syrups, flavoring agents and dyes, and then dehydrated sufficiently to make the mixture suitable for pressing into solid form.

[0087] The solid oral compositions may be prepared by conventional methods of blending, filling, tabletting, or the like. Repeated blending operations may be used to distribute the active agent(s) (i.e. hybrid molecules) throughout the compositions, employing large quantities of fillers. Such operations are, of course, conventional in the art. The tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

[0088] Oral liquid preparations may be in the form of emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or any other suitable vehicle before use.

Such liquid preparations may or may not contain conventional additives. Non limiting examples of conventional additives include suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifying agents such as sorbitan monooleate or acaci; non-aqueous vehicles (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters selected from the group consisting of glycerine, propylene glycol, ethylene glycol, and ethyl alcohol; preservatives such as for instance methyl parahydroxybenzoate, ethyl para-hydroxybenzoate, n- propyl para-hydroxybenzoate, «-butyl para-hydroxybenzoate and sorbic acid; and, if desired, conventional flavoring or coloring agents.

[0089] For parenteral administration, fluid unit dosage forms may be prepared by utilizing one or more hybrid molecules and a sterile vehicle, and, depending on the concentration employed, the hybrid molecule(s) may be either suspended or dissolved in the vehicle. Once in solution, the hybrid molecule(s) may be injected and filter sterilized before filling a suitable vial or ampoule followed by si ^{" "} - ^■ - ^• storage package. Adjuvants, such as a local anesthetic, a preservative or a tmrienng agent, may be dissolved in the vehicle prior to use. Stability of the pharmaceutical composition may be enhanced by freezing the composition after filling the vial and removing the water under vacuum, (e.g., freeze drying). Parenteral suspensions may be prepared in substantially the same manner, except that the hybrid molecule(s) should be suspended in the vehicle rather than being dissolved and, further, sterilization is not achievable by filtration. The hybrid molecule(s) may be sterilized, however, by exposing it to ethylene oxide before suspending it in the sterile vehicle. A surfactant or wetting solution may be advantageously included in the composition to facilitate uniform distribution of the hybrid molecule(s).

[0090] Topical administration can be used as the route of administration when local delivery of one or more hybrid molecules is desired at, or immediately adjacent to the point of application of the composition or formulation comprising one or more hybrid molecules.

[00911 The pharmaceutical compositions of the present invention comprise a pharmaceutically effective amount of one or more hybrid molecules as described herein and one or more

pharmaceutically acceptable carriers, excipients and/or diluents. In an embodiment of the present invention, the pharmaceutical compositions contain from about 0.1% to about 99% by weight of a hybrid molecule as disclosed herein. In a further embodiment of the present invention, the pharmaceutical compositions contain from about 10% to about 60% by weight of a hybrid molecule as disclosed herein, depending on which method of administration is employed. Physicians will determine the most-suitable dosage of the present therapeutic agents (i.e. hybrid molecules). Dosages may vary with the mode of administration and the particular hybrid molecule chosen. In addition, the dosage may vary with the particular patient under treatment. The dosage of the hybrid molecule used in the treatment may vary, depending on the condition, the weight of the patient, the relative efficacy of the compound and the judgment of the treating physician.

[0092] SYNTHESIS OF HYBRID MOLECULES

[0093] The C/ 'f all analogs, complete with stereochemical information, was conveniently prepared by oxidative degradation of vitamin D ₂ to afford diol 43. After installation of a secondary tert-butyldimethylsilyl protecting group and conversion of the primary alcohol to an iodide, the basic side-chain was installed by lithiation of iodide 45 and addition to acrolein diethylacetal. Subsequent oxidations of 46 afforded ketone 47, to which could be attached the A ring in a Horner coupling with previously reported phosphine oxide 41 [International Application PCT/CA2007/000885 published on November 22, 2007 under WO 2007/131364 Al] to afford ester 48.

[0094] To prepare precursors for forming a variety of hybrids, ester 48 could be reduced to aldehyde 49 (Scheme 1) for subsequent chain extension or hydrolyzed to its corresponding acid (Scheme 2). Curtius rearrangement of 50 afforded amine 51. Extended carboxylic acid 53 could be prepared by a methoxymethylene Wittig reaction on 49 followed by hydrolysis and oxidation of the olefÏŠnation product. One and two carbon homologues of amine 51 could be prepared by reductive amination from aldehydes 49 and 52. Acids 50 and 53 and amines 51, 54 and 55 are the main precursors for almost all hybrids described below.

imidazole r,, rreefπlu to rt 4 1 Ar uxx 3 o0υ m πnm

Scheme 1

toluene

D BnONH ₂ , toluene, reflux

1) BnONH ₂ , toluene, reflux 2) AILiH ₄ , Et ₂ O

2) AILlH ₄ , Et ₂ O 74%

80%

Scheme 2

SYNTHESIS OF HYBRID MOLECULES 5, 13, 14, 15 and 16

[0095] Hydroxamic acids 5 and 13 were synthesized by coupling acid chlorides derived from 53 and 50, respectively, with TBS protected hydroxylamine followed by deprotection (Scheme 3). Hydroxamic acid derivatives with methyl groups on either/both the nitrogen or/and oxygen could be prepared by a similar route using substituted hydroxylamines.

Scheme 3

[0096] SYNTHESIS OF HYBRID MOLECULES 17 and 18

[0097] Orthoaminoanilides 17 and 18 were prepared in a simple sequence from acids 50 and 53 by formation of an acid chloride and then coupling to with 1,2-phenylene diamine to form anilides. Deprotection with tetrabutylamonnium fluoride formed the final hybrids (Scheme 4)

o-Aminobenzamides

TBScy'

Scheme 4

[0098] SYNTHESIS OF HYBRID MOLECULES 19 to 30

[0099] Hybrid molecules 19 to 30 were prepared from amines 51, 54 and 55 through straightforward coupling with either sulfonyl chlorides, to form sulfonamides, or with N-(tert- Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)- 1 ,4-dihydropyridin- 1 -ylsulfonyl]azanide to form sulfamides (Scheme 5).

57% 61% 35% 54%

Scheme 5

[00100] SYNTHESIS OF HYBRID MOLECULES 31-35

[00101] Hybrids 31 to 35 were formed by coupling amines 51 and 54 with S-acetyl thioglycolic acid, glycine derivatives or bromoacetic acid under standard peptide coupling conditions (Scheme 6).

Scheme 6

[00102] SYNTHESIS OF HYBRID MOLECULE 36

Scheme 7

[00103] N-Hydroxyformate hybrid 36 was prepared from aldehyde 59 by partial reduction of the corresponding oxime followed by formylation and deprotection (Scheme 7).

[00104] SYNTHESIS OF HYBRID MOLECULES 37 and 38

71

70 Et ₃ N CHCI ₃

37

Scheme 8

[00105] α-Ketoester and amide hybrids 37 and 38 were prepared by Horner-Emmons olefination using a silyloxy substituted phosphonate ester. The resulting product, 69, could be deprotected directly to form 37 or partially desilylated and then aminated to form 38.

[00106] SYNTHESIS OF HYBRID MOLECULES 39 AND 40

N-hydroxyurea

Trith

Scheme 9

[00107] Preparation of N-hydroxyurea 39 was achieved by treating amine 54 with carbonyl diimidizole to form an intermediate which was directly treated with TBS protected hydroxylamine and then deprotected with HF. Trithiocarbonate 40 was prepared by addition of ethyl trithiocarbonate, formed from ethane thiol and carbon disulfide, to hybrid bromide 36.

[00108] BIOLOGICAL ACTIVITY

[00109] The degree of synthetic latitude available for development of further hybrids was investigated, given that hybrids must adapt to the binding pockets of two highly distinct targets. In addition, there is substantial interest in the pharmaceutical industry in development of isoform-specific HDACi. Therefore, nearly 30 secosteroidal hybrid compounds with a variety of ZBGs were synthesized and tested biochemically, including orthoaminoanilides, alpha- ketoamides and esters, thioglycolate amides, glycinamides, N-hydroxyureas, sulfonamides and sulfamides, some of which are effective in HDACi while others, most notably the latter two, have not been reported in HDACi's but function as inhibitors in other zinc metalloenzymes.

[00110] Bifunctionality of all compounds was tested with a series of assays. The

VDR agonist activity of the hybrid molecules of the present invention was tested by screening of induction of expression of the 1,25D target gene encoding CYP24 (FIG. 1), the enzyme responsible for initiation catabolic degradation of 1,25D, thus representing a physiological negative feedback loop. SCC4 cells were treated with hybrid molecules at either 10OnM or lμM, as indicated for 8h prior to harvesting RNA for reverse transcription/PCR analysis of CYP24 mRNA expression. 1 and/or 4 were used as positive controls for CYP24 mRNA induction. The results show that all hybrids, with the exceptions of compounds 33-35 and 39, showed substantial VDR agonist activity at 1 μM.

[00111] The capacity of hybrid molecules 5, 17-19, 21, 22, 25, 26, 28, 30, and 32 to bind directly to the VDR ligand binding domain (LBD) was tested using a fluorescence polarization (FP) competition assay. 1,25D was used a positive control. The assay was performed using a VDR competitor assay kit (Polarscreen, Invitrogen, Carlsbad, CA) set up using 0.5nM fix ires the decrease in FP accompanying loss of binding to the relatively high molecular weight VDR ligand binding domain of the fluorescent tracer due to the presence of a competitor. FP was measured using an Analyst HT fluorimeter (Molecular Devices) configured with absorption and emissions filters as recommended by the kit manufacturers. Dose response curves and IC ₅₀ determination were determined using XLfit (IDBS) Sigmoidal Dose-Response Model [fit = (A+((B-A)/(l+((C/x) ^λ D)))); inv = (C/((((B- A)/(y-A))-l) ^λ (l/D))); res = (y-fit)].

[00112] The capacity of the hybrid molecules to function as HDAC inhibitors was tested by Western blotting, testing for induction of either tubulin or histone hyperacetylation using TSA and/or triciferol as positive controls, or by colourimetric assay. Initial colourimetric experiments were performed using HeLa cell extracts as a source of HDAC activity (FIG 3A). An expanded series of compounds was tested by colourimetirc assay using fractions of purified HDACs 2, 3 or 6 (FIG 4). Compounds were generally more potent inhibitors of HDAC6 than HDAC3, consistent with the notion that the secosteroidal core confers higher affinity for HDAC6, and thus a degree of specificity in HDACi activity. HDACi activity of hybrids 5, 17- 19, 21, 22, 25, 26, 28, 30, 32 and 38 was measured using the HDAC colorimetric assay kit

following the supplier's protocol (Bio Vision, Mountain View, CA) using purified fractions of HDAC2, HDAC3 or HDAC6. The IC ₅₀ values (μM) for compounds tested are presented in Table 1. One notable compound was the thioglycolate (mercaptoamide) analogue 32, which was a potent VDR agonist, thus indicating that the thioglycolate moiety is an excellent substitute for the 25-OH group of 1. 32 functioned with low micromolar potency as an HDAC6 inhibitor; its HDACÏŒi activity was within a factor of 5 of that of 3 (Table 1). Consistent with data for other secosteroidal hybrids, 32 inhibited HDAC6 18.5- and >50-fold more potently than HDAC3 and HDAC2, respectively. Previous studies have suggested that thioglycolates (or mercaptoamides) are bidentate chelators of zinc ions, and that thioglycolate analogues of 3 could be developed with low micromolar HDACi activity when measured against HDACs present in HeLa cell extracts.

[00113] Hybrid molecules were tested for their capacity to induce acetyl ation of tubulin or histones H3 and H4 using specific acetyl-protein antibodies and Western blotting. 5 and 32 induced ^{' " "} . .. . tcetylation (FIG. 3B). Hybrid molecule 5, but not 32, also induced modest nistone nyperacetylation at 1 μM (FIGs. 3B, C). In contrast, while hybrid molecule 36 induced modest histone hyperacetylation, it had no effect on acetylation of tubulin (FIG. 3C). In other assays, hybrid molecules 21, 34 and 35 strongly induced hyperacetylation of histone H4, whereas hybrid molecule 20 induced moderate H4 hyperacetylation (FIG. 3D). Notably, unlike triciferol, hybrid molecules 20, 21, 34 and 35 had no effect on tubulin acetylation (FIG. 3D). The capacity of hybrid molecule 18 to induce protein acetylation was also tested by Western blotting (FIG. 3E). Remarkably, hybrid molecule 18 induced marked hyperacetylation of both tubulin and histone H3.

[00114] The results of testing hybrid molecules 5, 17-19, 21, 22, 25, 26, 28, 30, 32 and 38 for their capacity to induce acetylation of tubulin (Tubulin) or histones H3 or H4 (Histone) are summarized in Table 1, using 1 as a negative control and 3-5 as positive controls for induced protein acetylation. IC50 values inhibition of purified HDAC2, 3 or 6 are also presented in Table 1.

31/ 1 115] Table 1: Functional studies of hybrid molecules of the present invention.

Compound CYP24 FP Tubulin Kstone HDAC2 HDAC3 HDAC6 Hypercalcemia

1 +++ 13 - - +++

3 - - +++ +++ 0.21 0.15 0.35 ND

4 +++ 87 -H- + 10.4 13.3 0.58 -

5 ++ 248 -H- + 16.5 14.5 3 -

6 -H- - + ND

17 -H- 524 ++ ++ 460 330 -

18 -H- 185 -H- -H- 189 104 81.1 -

19 +++ 213 + + 730 71.3 ND

20 +++ ND

21 +++ 321 + ++ 718 191 -

22 ++ 14 ++ ++ 695 153 ND

23 ++ ND

24 -H- ND

25 +++ 29 - + 725 101 -

26 ++ 508 293 -

27 ND

28 -H- 280 + -H- 470 50.4 ND

29 +++ ND

30 + 196 -H- ++ 605 31.6 ND

31 + ND

32 ++ 36 + ++ 96.3 32.3 1.75 -

33 -/+ ND

34 -/+ ND

35 - ND

36 +++ ND

37 +++ ND

38 +++ + + 90.4 36 64 ND

39 -/+ ND

40 +++ ND

32

[00116] Some compounds induced cyp24 poorly, suggesting that they either bound the VDR weakly or were potential antagonists. Tests for antagonism (repression by lμM compound of cyp24 induced by 1OnM 1,25D), showed that cyp24 was superinduced more than additively by 1 combined with compound 31 or 39. Rather than VDR antagonism, this suggested that 31 or 39 were enhancing l,25D-induced gene expression by inhibition of CYP24 enzymatic activity; i.e. inhibiting the negative feedback loop that attenuates 1,25D signaling. As confirmation, two positive control experiments were performed; (i) cyp24 induction in the presence of a high (lμM) concentration of 1,25D, and (ii) cyp24 induced by 1OnM 1,25D in the presence of CYP24 inhibitor ketoconazole. Compound 31 or 39 boosted cyp24 induction by 1OnM 1,25D to levels comparable to those seen with lμM 1,25D or 1OnM l,25D+ketoconazole (FIG. 5A). 2 had no significant effect on cyp24 induced by 1OnM 1,25D (FIG. 5B), suggesting that the effects of 31 and 39 were not due to HDACi activity. As with other 1,25D analogs", 31 and 39 and other hybrids may thus be CYP24 inhibitors. This is intriguing and suggests that 31 and 39 are resistant to catabolism, and could boost signaling by endogenous 1,25D.

[00117] As hypercalcemia is the dose-limiting toxicity associated with 1,25D treatment, 4 and hybrids 5, 17, 18, 21, 25, 26, and 32 were compared with 1 for their capacity to induce hypercalcemia in mice. Compounds chosen had a range of affinities for the VDR as judged by FP assay (FIG. 2) and a variety of terminal zinc binding groups. Mice fed a normal calcium diet were infused using minipumps with 1 (12 or 24 pmol/day) or hybrids (240 or 1,200 pmol/day) over 6-7 days. Elevated concentrations of hybrids were chosen based on literature data that suggested induction of hypercalcemia with 1, and also to account for the lower affinity for the VDR of some hybrids. Some of the animals infused with 12pmol/day 1 were hypercalcemic after 6-7 days of treatment, and all animals treated with a 24pmol/day dose of 1 were hypercalcemic within 3 days (+++). In contrast, all animals infused with 240 or 1200pmol/day of hybrid compounds appeared healthy, and, none were hypercalcemic after 6-7 days (-). This includes animals infused with compounds 26 and 32, which have 1,25D- or near-l,25D-like affinities for the VDR as judged by FP assays and CYP24 induction (FIG. 1 and 2). Thus, all compounds tested lacked the dose-limiting toxicity associated with treatment with 1.

33

[00118] Experimental

[00119] General. MeCN, toluene and CH ₂ Cl ₂ were distilled from CaH ₂ under argon.

THF and Et ₂ O were distilled from sodium metal/benzophenone ketyl under argon. All other commercial solvents and reagents were used as received from the Aldrich Chemical Company, Fischer Scientific Ltd., EMD Chemicals Inc., Strem or BDH. All glassware was flame dried and allowed to cool under a stream of dry argon.

[00120] Silica gel (6θA, 230-400 mesh) used in flash column chromatography was obtained from Silicycle and was used as received. Analytical thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (Ultra Pure Silica Gel Plates purchased from Silicycle), visualized with a Spectroline UV2 ₅ 4 lamp, and stained with a 20% phosphomolybdic acid in ethanol solution, or a basic solution of KMnO ₄ . Solvent systems associated with R _f values and flash column chromatography are reported as percent by volume values.

[00121] ¹ H and ¹³ C NMR, recorded at 300 MHz and 75 MHz respectively, were performed on a Van an Mercury 300 spectrometer. ¹ H and ' ³ C NMR, recorded at 400 MHz and 100 MHz respectively, were performed on a Varian Mercury 400 spectrometer. Proton chemical shifts were internally referenced to the residual proton resonance in CDCI ₃ (δ 7.26 ppm), CD ₃ OD (δ 3.31 ppm), CD ₃ CN (δ 1.94 ppm), or αtø-DMSO (δ 2.50 ppm). Carbon chemical shifts were internally referenced to the deuterated solvent signals in CDCl ₃ (δ 77.2 ppm), CD ₃ OD (δ 49.0 ppm), CD ₃ CN (δ 118.3 ppm and 1.3 ppm) or d6-OMSO (δ 39.5 ppm). FT-IR spectra were recorded on a Nicolet Avatar 360 ESP spectrometer with samples loaded as neat films on NaCl plates. References following compound names indicate literature articles where ¹ H and ¹³ C NMR data have previously been reported.

34

[00122] Lythgoe-Inhoffen diol (43):

Chemical Formula: Ci ₃ H ₂₄ O ₂ Molecular Weight: 212,33

[00123] A flame-dried 100 mL three-necked flask was charged sequentially with 28 mg (0.33 mmol, 0.06 equiv) of NaHCO ₃ , 20 mL of anhydrous MeOH, 60 mL of anhydrous CH ₂ Cl ₂ , and 2.0 g (5.12 mmol, 1 equiv) of ergocalciferol. The solution was cooled to -78 ⁰ C and treated with O ₃ until a blue color appears. The solution was subsequently flushed with Ar for 10- 15 min until the blue color faded. Solid sodium borohydride (1.68 g, 44.55 mmol, 8.7 equiv) was added portionwise over a period of 10 min at -78 ⁰ C until complete disappearance of starting material was observed by TLC. The reaction mixture was warmed to 0 ⁰ C and stirred for 3 h.

After being stii ^{~ J r~ J J:} " ^' — ' ^" — ^:~ "* — >m temperature, the mixture was quenched with

1 N HCl (10 ^m L), dried (MgSO ₄ ), filtered, and concentrated in vacuo. Purification by silica gel chromatography (30% EtOAc in hexanes) afforded 670 mg (3.17 mmol) of Lythgoe-Inhoffen Diol in 62% yield as a white solid. Ry = 0.5 (50% EtOAc in hexanes); Mp 108-110 ⁰ C (lit. Mp 109-110 ⁰ C); ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.08 (IH, br s), 3.63 (IH, dd, J= 10.4, 2.8 Hz), 3.37 (IH, J= 10.0, 6.8 Hz), 1.98 (IH, d, J= 12.8 Hz ), 1.90-1.75 (3H, m), 1.60-1.40 (5H, m), 1.38-1.29 (4H, m), 1.22-1.13 (2H, m), 1.02 (3H, d, J= 6.8 Hz), 0.95 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 69.1, 67.7, 52.9, 52.3, 41.8, 40.2, 38.2, 33.5, 26.6, 22.5, 17.4, 16.6, 13.5; IR (KBr) D 3621, 3464, 3017, 2943 cm ^'1 .

35

[00124] (5)-2-((lR,3aR,4S,7aR)-4-(te/-r-butyldimethylsilyloxy)-7a- methyloctahydro-iH-inden-l-yl)propan-l-ol (44):

Chemical Formula: C ₁₉ H ₃₈ O ₂ Si Molecular Weight: 326,59

[00125] To a solution of Lythgoe-Inhoffen diol 43 (2.017 g, 9.5 mmol, 1 equiv) in 60 mL of dry DMF under argon atmosphere was added terÏŠ-butyldimethylsilylchloride (5.71 g, 38 mmol, 4 equiv) followed by NEt ₃ (5.54 mL, 42.7 mmol, 4.5 equiv) and sodium iodide (5.69 g, 38 mmol, 4 equiv). The reaction mixture was refluxed for 30 min and then cooled and quenched with H ₂ O (10 mL) and concentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and washed with F- ^{n n v} ^ ^{n mT} 1 ^ ^p ami ^p rms nnrtion was extracted with EtOAc (30 mL) and the combined orga. .„, _.„ _ (30 mL), dried (MgSO ₄ ), filtered, concentrated by rotary evaporation, and immediately purified by silica gel chromatography (10% EtOAc in hexanes) to afford 4.013 g (9.12 mmol) of bis-silylated diol intermediate in 96% yield. In a flame-dried round bottom flask under argon, 4.013 g (9.12 mmol) of the bis-silylated diol was dissolved in 80 mL of anhydrous THF and 4 mL of NEt ₃ . To this stirring solution was added TBAF (10.9 mL of a 1 M solution in THF, 10.9 mmol, 1.15 equiv). The resulting reaction mixture was stirred at room temperature for 3 h, concentrated in vacuo, and purified by silica gel column chromatography (20% EtOAc in hexanes) to afford 2.916 g (8.94 mmol) of the desired alcohol in 98% yield. R _/ = 0.5 (20% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.08 (IH, br s), 3.57 (IH, dd, J = 9.8, 3.0 Hz), 3.26 (IH, dd, J = 9.0, 7.8 Hz), 1.99 (IH, d, J = 13.6 Hz), 1.87-1.75 (3H, m), 1.58-1.40 (5H, m), 1.35-1.28 (3H, m), 1.40-1.09 (2H, m), 0.97 (3H, d, J = 6.8), 0.94 (3H, s), 0.89 (9H, s), 0.025 (6H, s) ; ¹³ C NMR (75 MHz, CDCl ₃ ) δ 69.3, 67.7, 53.2, 52.3, 41.8, 40.2, 38.5, 33.6, 26.6, 26.0, 22.6, 18.4, 17.4, 16.8, 13.6, -5.3, -5.4 ; IR (KBr) v 3300, 1470, 1250, 1160 cm ^"1 .

36

[00126] (5)-2-((li?,3aR,45,7ai?)-4-(fe^-butyldimethylsilyloxy)-7a- methyloctahydro-iH-inden-l-yl)-l-iodopropane (45):

Chemical Formula: C ₁₉ H ₃₇ IOSi Molecular Weight: 436,49

[00127] I ₂ (9.34 g, 36.8 mmol, 4.6 equiv) was added portionwise to an ice cooled solution of PPI1 ₃ (4.82 g, 18.4 mmol, 2.3 equiv) and imidazole (3.26 g, 48.0 mmol, 6 equiv) in 300 mL Of CH ₂ Cl ₂ . The cooled mixture, witch become heterogeneous after 5 min, was stirred for 35 min and treated with a solution of the primary alcohol 44 (2.61 g, 8 mmol, 1 equiv) in 100 mL of CH ₂ Cl ₂ during 30 min and then the mixture was stirred at room temperature for 4 h. The mixture was λiÏ€tVi a ? s% solution Of ISIa ₂ SO ₃ (30 mL). The organic layer was washed with H ₂ O (30 ,, _{v /7} _ __ _v U), concentrated in vacuo, and then purified by silica gel column chromatography (5% EtOAc in hexanes) to afford 3.14 g (6.96 mmol) of iodide 45 in 87% yield as a white solid. Ry = 0.6 (5% EtOAc in hexanes); Mp 40-41 ⁰ C (lit. Mascarenas, J. L. ;Mouinδ, A. ; Castedo, L. ; J. Org. Chem. 1986, 51, 1269-1272; Mp 41-42°C); ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.99 (IH, s), 3.32 (IH, d, J = 9.6 Hz), 3.17 (IH, dd, J = 9.6, 5.2 Hz), 1.90 (IH, d, J = 12.8 Hz), 1.84-1.76 (2H, m), 1.66 (IH, d, J= 13.6 Hz), 1.59-1.54 (IH, m), 1.40-1.08 (8H, m), 0.98 (3H, d, J = 5.2 Hz), 0.94 (3H, s), 0.88 (9H, s), -0.01 (6H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 69.3, 56.0, 52.7, 42.1 , 40.3, 36.4, 34.3, 26.6, 25.8, 22.9, 21.7, 20.7, 18.0, 17.6, 14.6, - 4.8, -5.2; IR (KBr), 2932, 2857, 1462, 1375, 1253, 1 160, 1084, 1032, 836, 774 cm ^"1 .

37

[00128] (^-S-CαR^aR^SJaRM-tte^butyldimethylsilyloxyHa- methyloctahydro-7H-inden-l-yl)-l-ethoyhex-l-ene (46):

Chemical Formula: C ₂₄ H ₄₆ O ₂ Si Molecular Weight: 394,71

[00129] In a flame-dried round bottom flask under argon atmosphere, 45 (678 mg, 1.5 mmol, 1 equiv) was dissolved in dry Et ₂ O (3 mL), the solution was cooled to -78 ⁰ C, then ^BuLi (1.42 mL of a 2.22 M solution in hexanes, 3.15 mmol, 2.1 equiv) was slowly added (ca. 1 h). The solution was stirred at -78 ⁰ C for 1 h, then warmed to 0 ⁰ C for 5 min and then recooled to -78 ⁰ C. A solution of acrolein acetal (251 μL, 1.65 mmol, 1.1 equiv) in dry Et ₂ O (2 mL) was slowly added via cannula tn thp rnrhaninn ^ointinn anH the mixture was warmed to room temperature.

StiÏ€ing was cc tion was quenched with sat. NH ₄ Cl (10 mL).

Extraction with Et ₂ O (3 x 20 mL) afforded an organic phase that was washed with H ₂ O (20 mL), brine (20 mL) and dried (MgSO ₄ ), concentrated in vacuo, and then purified by silica gel column chromatography (hexanes then 1% Et ₂ O in hexanes) to afford 444 mg (1.12 mmol) of the enol ether as a mixture 86/14 of £ and Z in 75% yield. R ₇ = 0.2 (hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ (46 E): 6.21 (IH, d, J = 12.4 Hz), 4.74 (IH, dt, J = 12.4, 6.2 Hz), 3.98 (IH, s), 3.69 (2H, q, J= 6.8 Hz), 1.95 (2H, br d, J= 12.4 Hz), 1.81-1.73 (3H, m), 1.65 (IH, br d, J= 13.2 Hz), 1.60-1.51 (IH, m), 1.42-1.29 (1OH, m), 1.27-1.00 (3H, m), 0.95-0.80 (15H, m), 0.00 (3H, s), - 0.01 (3H, m); (46 Z): 5.90 (IH, d, J = 9.2 Hz), 4.30 (IH, dd, J = 9.2, 7.5 Hz), 3.98 (IH, br s), 3.77 (2H, q, J= 7.5 Hz), 1.95 (2H, br d, J= 12.4 Hz), 1.81-1.73 (3H, m), 1.65 (IH, br d, J= 13.2 Hz), 1.60-1.51 (IH, m), 1.42-1.29 (1OH, m), 1.27-1.00 (3H, m), 0.95-0.80 (15H, m), 0.00 (3H, s), -0.01 (3H, m); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 145.6, 104.8, 69.5, 64.5, 56.7, 53.0, 42.1, 40.7, 37.0, 34.8, 34.4, 27.3, 25.8, 24.5, 23.0, 18.5, 18.0, 17.7, 14.8, 13.7, -4.8, -5.2; IR (KBr) v 2931, 2856, 1652, 1469, 1374, 1251, 1165, 1083, 1023, 977, 924, 836, 774 cm ⁴ ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 417.3165, found = 417.3155.

38

[00130] (φ-ethyl-S-tflλ^a/^aRHa-methyl^-oxo-octahydro-iH-inden-l- yl)hexanoate (47):

Chemical Formula: C18η30O3 Molecular Weight : 294,43

[00131] PCC (546 mg, 2.52 mmol, 2 eqÏ…iv) was added to a stirred solution of 46 (500 mg, 1.26 mmol) and celite (600 mg) in CH ₂ Cl ₂ (20 mL). The solution was stirred for 3 h, then the mixture was filtered on a silica pad. The precipitate was washed with CH ₂ Cl ₂ (3 x 20 mL) then the combined organic fractions were concentrated in vacuo to provide the crude ester as a colorless oil. The oil was diluted in CH ₂ Cl ₂ (10 mL) and CH ₃ CN (10 mL) and a 48% solution of HF (1 mL) was added. The solution was stirred for 24 h at room temperature. The reaction mixture was c on of a sat. solution of NaHCO ₃ until no effervescence was observed. The solution was extracted with CH ₂ Cl ₂ (3 x 20 mL), the combined organic layers were then washed with H ₂ O (20 mL) and brine (20 mL), then dried (MgSO ₄ ). The solution was concentrated in vacuo, then diluted in CH ₂ Cl ₂ (20 mL), celite was added (500 mg) followed by PCC (546 mg, 2.52 mmol, 2 equiv). After stirring for 3 h at room temperature, the reaction mixture was filtered on a silica pad, the precipitate was washed with CH ₂ Cl ₂ (3 x 20 mL) and the solution was concentrated in vacuo. The residual oil was purified by silica gel column chromatography (15% EtOAc in hexanes) to afford 363 mg (1.23 mmol) of 47 in 78% yield. Py= 0.2 (15% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.08 (IH, q, J= 7.2 Hz), 2.46 (IH, dd, J= 11.6, 7.6 Hz), 2.30-2.12 (4H, m), 2.07 (Ih, d, J= 13.6 Hz), 2.02-1.76 (4H, m), 1.75-1.60 (2H, m), 1.60-1.31 (3H, m), 1.21 (3H, t, J = 6.8 Hz), 0.93 (3H, d, J = 6.4 Hz), 0.84 (3H, t, J =6.8 Hz), 0.59 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 212.2, 173.9, 62.1, 60.4, 56.5, 50.1, 41.1, 39.1, 35.4, 31.8, 27.6, 25.5, 24.2, 22.8, 21.6, 19.2, 14.4, 12.6; IR (KBr) v 2957, 2874, 1710, 1704, 1463, 1377, 1239, 1 179, 1099, 1037 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 319.2093, found = 319.2086.

39

[00132] (R)-ethyl-5-((lR,3aS,7aR,E)-4-(2-((3R,5R)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-/H-inden-l- yl)hexanoate (48):

Chemical Formula: C ₃₈ H ₇₀ O ₄ Si ₂ Molecular Weight: 647,13

[00133] In a flame dried round bottom flask under argon atmosphere at -78 ⁰ C,

NaHMDS (514 μL of a 1 M solution in THF, 0.514 mmol, 1.05 equiv) was added to a solution of phosphine oxidi [uiv) in dry THF (6 mL). The reaction vessel was suspended above the ice bath for 5 min, then recooled to -78 ⁰ C. To this solution was added via cannula over a period of 5 min a solution of 47 (144 mg, 0.490 mmol, 1 equiv) in dry THF (2 mL). The reaction was left to stir at -78 ⁰ C for 2 h then warmed to room temperature for 1 h, and quenched with sat. NH ₄ Cl (10 mL). The layers were separated and the aqueous layer extracted with EtOAc (2 x 25mL). The organic layers were combined and extracted with sat. NH ₄ Cl (2 x 10 mL), H ₂ O (10 mL) and brine (10 mL), then dried (MgSO ₄ ), and concentrated in vacuo. Purified by silica gel column chromatography (20% EtOAc in hexanes) afforded 247 mg (0.382 mmol) of 48 in 78% yield. R _/ = 0.6 (20% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.16 (IH, d, J = 1 1.2 Hz), 5.80 (IH, d, J = 1 1.2 Hz), 4.20-4.00 (4H, m), 2.80 (IH, d, J = 11.6 Hz), 2.45-2.18 (4H, m), 2.09 (IH, dd, J = 12.8, 8.0 Hz), 1.98 (2H, d, J = 10.0 Hz), 1.80-1.25 (15H, m), 1.25 (3H, t, J= 6.8 Hz), 0.93 (3H, d, J= 6.4 Hz), 0.86 (9H, s), 0.85 (9H, s), 0.52 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.9, 140.7, 133.6, 121.7, 116.1, 68.1, 67.9, 68.1, 60.1, 56.2, 46.0, 45.6, 43.7, 40.6, 36.7, 35.9, 35.3, 34.7, 28.7, 27.7, 25.9, 25.8, 23.4, 22.2, 21.6, 18.7, 18.13, 18.09, 14.3, 12.0, -4.7, -4.76, -4.83, -4.9; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 669.4710, found = 669.4744.

40

[00134] (55)-5-((lR,3i?,7 _J E ^' ,17β)-l,3-bis[terf-butyl(diraethyl)siIyl-oxy]-9,10- secoestra-5,7-dien-17-yl)hexanal (49):

Chemical Formula: C ₃₆ H ₆₆ O ₃ Si ₂ Molecular Weight: 603,08

[00135] DIBALH (172 μL of a IM solution in toluene, 0.172 mmol, 1.1 equiv) was added dropwise to a stirred solution of ester 48 (101 mg, 0.157 mmol, 1 equiv) in dry CH ₂ Cl ₂ (1.5 mL) under Ar at -78°C. The solution was stirred at -78°C for Ih and warmed up to room temperature. Cr " '" " ^{Ï„ N} . ^t , . ., _;a ction mixture, then H ₂ O (0.16 mL) followed by 15% NaOH \v. iu UILJ men muic n ₂ w ^Ï….to mL). The solution was stirred for 30 min then MgSθ ₄ was added to the mixture. The solution was filtered, the precipitate washed with CH ₂ Cl ₂ (5 mL) and the combined organic fractions were concentrated in vacuo to provide the aldehyde 49 in quantitative yield without further purification (95 mg, 0.157 mmol). Ry= 0.6 (CH ₂ Cl ₂ ); ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.76 (IH, s), 6.16 (IH, d, J = 12.0 Hz), 5.81 (IH, d, J = 12.0 Hz), 4.40-4.43 (2H, m), 2.80 (IH, dd, J = 11.0, 3.1 Hz), 2.43-2.34 (4H, m), 2.24 (IH, br d, J = 13.6 Hz), 2.89 (IH, dd, J= 12.8, 8.4 Hz), 2.05-1.85 (2H, m), 1.85-1.20 (15H, m), 1.20-1.05 (IH, m), 0.94 (3H, d, J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s) ; ' ³ C NMR (75 MHz, CDCl ₃ ) δ 202.9, 140.6, 133.7, 121.7, 116.2, 68.1, 67.9, 56.22, 56.20, 46.0, 45.6, 44.3, 43.7, 40.5, 36.7, 36.0, 35.4, 28.7, 27.7, 25.87, 25.84, 23.4, 22.2, 18.7, 18.15, 18.10, 12.0, -4.6, - 4.7, -4.8, -4.9; IR (KBr) v 2950, 2883, 2856, 1728, 1468, 1377, 1361, 1253, 1087, 1025, 836, 775 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 625.4448, found = 625.4434.

41

[00136] (55)-5-((lR,3R,7 _J E,17β)-l,3-bis[tert-butyl(dimethyl)silyl-oxy]-9,10- secoestra-5,7-dien-17-yl)hexanoic acid (50):

Chemical Formula C ₃₆ H ₆₆ O ₄ Si ₂ Molecular Weight 619,08

[00137] LiOH H ₂ O was added to a stirred solution of ester 48 (128 mg, 0 197 mmol,

1 equiv) inn THF (2 5 mL), MeOH (0 75 mL) and H ₂ O (0 75 mmol) The solution was refluxed for 3h, then cooled to room temperature and diluted with EtOAc (20 mL), then quenched with a 1 M solution o1 was exctracted with EtOAc (2 x 10 mL) and then the combined organic fractions were washed with H ₂ O (10 mL), bÏ€ne (10 mL) then dÏ€ed (Na ₂ SO ₄ ) The solution was concentrated in vacuo to provide the acid 50 in 97% yield without further purification (1 18 mg, 0 191 mmol) R/ = 0 6 (40% EtOAc in hexanes), ¹ H NMR (400 MH/, CDCl ₃ ) δ 6 16 (IH, d, J = 10 8 Hz), 5 81 (IH, d, J = 10 8 Hz), 4 15-4 01 (2H, m), 2 81 (IH, br d, J = 1 1 6 Hz), 2 42-2 21 (5H, m), 2 12-1 05 (14H, m), 0 94 (3H, d, J = 7 2 Hz), 0 87 (9H, s), 0 86 (9H, s), 0 53 (3H, s), 0 04 (12H, s) , ¹³ C NMR (75 MHz, CDCl ₃ ) δ 180 0, 140 7, 133 7, 121 7, 1 16 1, 68 1, 68 0, 56 2, 46 0, 45 6, 43 7, 40 5, 36 7, 35 9, 35 3, 34 5, 28 7, 27 7, 25 9, 25 8, 23 4, 22 2, 21 3, 18 7, 18 2, 18 1, 12 0, -4 6, -4 7, -4 8, -4 9, IR (KBr) v 3435 (br), 2951 , 1637, 1459, 1247, 1086, 835, 780, 668 cm ¹ , HRMS (ESI) m/z calcd for [(M+Na) ⁺ ] = 641 4397, found = 641 4383

42

[00138] (R)-4-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-7H-inden-l- yl)pentan-l -amine (51):

Chemical Formula C ₃₅ H ₆₇ NO ₂ Si ₂ Molecular Weight. 590,08

[00139] To a solution of carboxylic acid 50 (102 mg, 0.165 mmol, 1 equiv) in 1 niL of toluene was added via syringue Et ₃ N (25 μL, 0.181 mmol, 1.1 equiv), followed by DPPA (35 μL, 0.165 mmol, 1 equiv). The solution was stirred at room temperature for 30 min then heated to reflux overnight and then concentrated under reduced pressure. The crude isocyanate was treated with 2 _% ιL) then stirred for 20 min at room temperature.

The resulting solution was extracted with CH ₂ Cl ₂ (2 x 10 mL), and the organic extract was washed with brine and then dried (MgSO ₄ ). The solution was concentrated in vacuo then purified by silica gel column chromatography (10/10/80, Et ₃ N/MeOH/EtOAc) to obtain the amine 51 in 68% yield (66 mg, 0.111 mmol). R ₇ = 0.35 (10/10/80, Et ₃ N/MeOH/EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.14 (IH, d, J= 11.2 Hz), 5.79 (IH, d, J= 11.2 Hz), 4.11-4.01 (2H, m), 2.79 (IH, d, J = 12.0 Hz), 2.71-2.52 (2H, m), 2.42-2.30 52H, m), 2.24 (IH, d, J= 12.4 Hz), 2.01-1.72 (5H, m), 1.71-0.98 (13H, m), 0.92 (3H, d, J= 8.0 Hz), 0.85 (9H, s), 0.84 (9H, s), 0.51 (3H, s), 0.04 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 140.7, 133.6, 121.7, 116.1, 68.1, 67.9, 56.4, 56.2, 46.0, 45.6, 43.7, 42.4, 40.6, 36.7, 36.0, 33.0, 29.7, 28.7, 27.7, 25.85, 25.83, 23.4, 22.2, 18.8, 18.13, 18.09, 12.0, -4.67, -4.77, -4.84, -4.9; IR (KBr) v 3350 (br), 2950, 2856, 1468, 1377, 1253, 1088, 836,

775 cm ^'1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 590.4789, found = 590.4792.

43

[00140] (65)-6-((lR,3i?,7 _J E',17β)-l,3-bis[fert-butyl(dimethyl)silyl-oxy]-9,10- secoestra-5,7-dien-17-yl)heptanal (52):

Chemical Formula: C ₃₇ H ₆₈ O ₃ Si ₂ Molecular Weight: 617,11

[00141] In a flame dried round bottom flask under argon atmosphere, NaHMDS (1.0 mL of a 1 M solution in THF, 1.0 mmol, 10 equiv) was added to a solution of (methoxymethyl)- triphenylphosphonium chloride (360 mg, 1.0 mmol, 10 equiv) in dry THF (10 mL) at -78°C. The reaction vessel i for 30 min, then recooled to -78 ⁰ C. To this solution was added via cannula over a period of 5 min a solution of 49 (60 mg, 0.100 mmol, 1 equiv) in dry THF (1 mL). The reaction was left to stir at -78 ⁰ C for 1 h then warmed to 0 ⁰ C for 4 h, and quenched with sat. NH ₄ Cl (25 mL). The layers were separated and the aqueous layer extracted with EtOAc (2 x 25mL). The organic layers were combined and extracted with sat. NH ₄ Cl (2 x 25 mL), H ₂ O (25 mL) and brine (25 mL), then dried (MgSO ₄ ), and concentrated in vacuo. The oil was then purified by silica gel column chromatography (5% EtOAc in hexanes) to provide 53 mg of a mixture 7/3 of E/Z enol ethers in 89% yield. R/= 0.6 (5% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.28 (0.6H, d, J= 12.8 Hz), 6.17 (IH, d, J= 11.0 Hz), 5.86 (0.3H, d, J = 6.4 Hz), 5.81 (IH, d, J = 11.0 Hz), 4.73 (0.7H, dt, J = 12.4, 7.2 Hz), 4.33 (0.3H, q, J = 6.8), 4.12-4.01 (2H, m), 3.58 (0.9H, s), 3.50 (2.1H, s), 2.81 (IH, d, J = 12.0 Hz), 2.43-2.33 (2H, m), 2.25 (IH, d, J = 13.6 Hz), 2.15-1.72 (6H, m), 1.70-1.17 (14H, m), 1.10-0.98 (IH, m), 0.91 (3H, d, J= 6.4), 8.87 (9H, s), 8.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s). Enol ether (40 mg, 0.063 mmol) was dissolved in a solution Of CHCl ₃ (1 mL), distilled H ₂ O (0.5 mL) and TFA (0.15 mL), and cooled to 0 ⁰ C. The reaction was stirred at 0 ⁰ C for approx. 30 min and monitored by TLC until complete consumption of the starting material, the reaction was quenched with sat.

44

NaHCO ₃ (5 mL). CH ₂ Cl ₂ (10 niL) was added to the mixture, the layers were separated and the aqueous layer extracted with CH ₂ Cl ₂ (2 x 1OmL). The organic layers were combined and washed with sat. NaHCO ₃ (2 x 25 mL), distilled H ₂ O (25 mL) and brine (25 mL), then dried (MgSO ₄ ), and concentrated in vacuo to give the crude product. 52 was isolated via FCC (10% EtOAc in hexanes) as a clear oil in 94% yield (51.8 mg, 0.084 mmol). R _/ = 0.2 (10% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) 59.77 (IH, t, J = 1.6 Hz), 6.16 (IH, d, J= 10.0 Hz), 5.81 (IH, d, J= 10.0 Hz), 4.12-4.12-4.02 (2H, m), 2.81 (IH, d, J= 11.6 Hz), 2.43 (2H, t, J= 7.2 Hz), 2.37 (2H, dd, J = 12.4, 5.2 Hz), 2.25 (IH, d, J= 11.6 Hz), 2.10 (IH, dd, J = 12.6, 8.2 Hz), 2.04- 1.93 (2H, m), 1.93-1.75 (2H, m), 1.70-1.45 (8H, m), 1.45-1.15 (7H, m), 1.15-1.00 (IH, m), 0.91 (3H, d, J= 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s) ; ¹³ C NMR (75 MHz, CDCl ₃ ) δ 203.3, 141.0, 133.9, 121.9, 116.4, 68.3, 68.2, 56.6, 56.5, 46.3, 45.9, 44.3, 43.9, 40.8, 37.0, 36.2, 35.8, 28.9, 27.9, 26.11, 26.08, 25.9, 23.6, 22.8, 22.5, 19.0, 18.4, 18.3, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 2949, 2884, 2856, 1728, 1468, 1252, 1052, 1025, 960, 920, 837, 776 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 639.4605, found = 639.4599.

[00142] (65)-6-((lR,3R,7 _J E,17β)-l,3-bis[terr-butyl(dimethyl)silyl-oxyl-9,10- secoestra-5,7-dien-17-yl)heptanoic acid (53):

Chemical Formula: C ₃₇ H ₆₈ O ₄ Si ₂ Molecular Weight: 633,1

[00143] A freshly prepared solution of NaClO ₂ (30 mg, 0.33 mmol, 3 equiv) and

NaH ₂ PO ₄ (76 mg, 0.55 mmol, 5 equiv) in H ₂ O (2 mL) was added to a stirred solution of the aldehyde 52 (69 mg, 0.1 1 mmol, 1 equiv) and 2-methyl-2-butene (0.5 mL) in ^BuOH (2 mL) and the mixture was stirred vigorously at room temperature for 1 h. H ₂ O (15 mL) was then added,

45

and the mixture was extracted with Et ₂ O (3 x 20 mL). The combined organic layers were washed with brine (10 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo then the oil was purified by silica gel column chromatography (19/1/80 EtOAc/AcOH/hexanes) to afford 65 mg (0.10 mmol) of acid 53 in 90% yield. R _/ = 0.6 (40% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.16 (IH, d, J = 11.0 Hz), 5.81 (IH, A, J = 11.0 Hz), 4.12-4.01 (2H, m), 2.80 (IH, d, J = 11.6 Hz), 2.44-2.31 (4H, m), 2.25 (IH, d, J = 13.6 Hz), 2.10 (IH, dd, J = 12.8, 8.4 Hz), 2.03- 1.94 (2H, m), 1.93-1.83 (IH, m), 1.83-1.75 (IH, m), 1.69-1.46 (9H, m), 1.45-1.34 (3H, m), 1.33- 1.18 (4H, m), 1.12-1.00 (IH, m), 0.91 (3H, d, J= 6.0 Hz), 0.87 (9H, s), 0.86 (9H, m), 0.53 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 179.9, 141.0, 133.9, 121.9, 116.4, 68.4, 68.2, 56.7, 56.5, 46.2, 45.9, 43.9, 40.8, 37.0, 36.2, 35.7, 34.3, 28.9, 27.9, 26.1 1, 26.08, 25.9, 25.4, 23.7, 22.5, 19.0, 18.4, 18.3, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 2950, 2857, 1710, 1468, 1253, 1088, 920, 836, 775 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M-Hy] = 631.4578, found = 631.4578.

[00144] (R)-5-((li?,3a5,7aλ^)-4-(2-((3R,5R)-3,5-bis(tert- butyldimethy ;ne)-7a-methyl-octahydro-7H-inden-l- yl)hexan-l-amine (54):

Chemical Formula: C ₃₆ H ₆₉ NO ₂ Si ₂ Molecular Weight: 604,11

[00145] A solution of aldehyde 49 (23 mg, 0.038 mmol, 1 equiv), O- benzylhydroxylamine hydrochloride (6.1 mg, 0.038 mmol, 1 equiv) and sodium acetate (3.1 mg, 0.03 mmol, 1 eq) in 3 mL of toluene was refluxed for 30 min. The mixture was then concentrated and loaded directly on to silica gel. The oximes were isolated by silica gel column chromatography (CH ₂ Cl ₂ ) as a mixture of E an Z compounds. In a flame dried round bottom flask under argon atmosphere, a solution of the oximes in Et ₂ O (1 mL) was added to a

46

suspension Of AlLiH ₄ (4 3 mg, 0 1 14 mmol, 3 equiv) m Et ₂ O (1 niL) at O ⁰ C The solution was stirred at room temperature for 4 h then cooled to 0 ⁰ C and quenched by addition of H ₂ O (5 μL), then 15% NaOH (5 μL) and then H ₂ O (15 μL) The mixture was diluted with Et ₂ O (10 mL), washed with water (5 mL), sat NaHCO ₃ (5 mL), brine (5 mL) and dÏ€ed (Na ₂ SO ₄ ) The solution was concentrated in vacuo then purified by silica gel column chromatography (10/10/80, Et ₃ N/MeOH/EtOAc) to provide 18 mg (0 030 mmol) of the amine 54 in 80 % yield Ry = 0 35 (10/10/80, Et ₃ N/MeOH/EtOAc), ¹ H NMR (400 MHz, CDCl ₃ ) δ 6 16 (IH, d, J = 11 1 Hz), 5 81 (IH, d, J = 11 1 Hz), 4 12-4 01 (2H, m), 2 80 (IH, d, J= 10 5 Hz), 2 75-2 62 (2H, m), 2 44-2 31 (2H, m), 2 25 (IH, d, J= 11 1 Hz), 2 10 (IH, dd, J= 12 6, 7 8 Hz), 2 04-1 92 (2H, m), 1 91-1 73 (2H, m), 1 70-0 98 (17H, m), 0 91 (3H, d, J = 6 3 Hz), 0 87 (9H, s), 0 86 (9H, s), 0 53 (3H, s), 0 05 (12H, s), ¹³ C NMR (75 MHz, CDCl ₃ ) δ 141 1, 133 9, 122 0, 116 3, 68 3, 68 2, 56 7, 56 5,

46 2, 45 9, 43 9, 40 8, 37 0, 36 3, 36 0, 28 9, 28 0, 27 9, 26 10, 26 08, 23 6, 22 5, 19 0, 18 4, 18 3, 12 3, -4 4, -4 5, -4 6, -4 7, IR (KBr) v 2930, 2857, 1469, 1377, 1362, 1253, 1085, 836, 775 cm ', HRMS (ESI) m/z calcd for [(M+H) ⁺ ] = 6044945, found = 604 4948

[00146] (R)-6-((lR,3a5,7a/? _r £)-4-(2-((3i?,5R)-3,5-bis(^^- butyldimethylsilyloxy)cyclohexylidene)ethyIidene)-7a-methyl- octahydro-/H-indeπ-l- yl)heptan-l-amine (55):

Chemical Formula C ₃₇ H _7I NO ₂ Si ₂ Molecular Weight 618,14

[00147] This was prepared from aldehyde 52 by following the same procedure descÏ€bed for 54 The reagents used were as follows 52 (30 mg, 0 048 mmol, 1 equiv), O- benzylhydroxylamme hydrochloÏ€de (7 7 mg, 0 048 mmol, 1 equiv), sodium acetate (4 9 mg, 0 03 mmol, 1 eq) and AlLiH ₄ (5 4 mg, 0 144 mmol, 3 equiv) This afforded 22 mg (0 035 mmol)

47

of amine 55 in 74% yield. R ₇ = 0.35 (10/10/80, Et ₃ N/MeOH/EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.16 (IH, d, J= 10.8 Hz), 5.81 (IH, d, J= 10.8 Hz), 4.15-4.01 (2H, m), 3.34 (IH, t, J = 7.2 Hz), 2.80 (IH, d, J = 12.0 Hz), 2.70 (IH, d, J= 7.2 Hz), 2.42-2.30 (2H, m), 2.25 (IH, d, J = 12.4 Hz), 2.10 (IH, dd, J= 12.4, 8.0 Hz), 2.02-1.71 (5H, m), 1.70-0.98 (17H, m), 0.91 (3H, d, J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 140.9, 133.6, 121.7, 116.1, 68.1, 67.9, 56.5, 56.3, 46.0, 45.6, 43.7, 40.6, 36.7, 36.1, 28.7, 27.7, 27.4, 25.9, 25.8, 23.4, 22.2, 18.8, 18.15, 18.10, 12.0, -4.7, -4.6, -4.8, -4.9; IR (KBr) v 2930, 2855, 1468, 1377, 1252, 1086, 836, 775 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 618.5102, found = 618.5089.

[00148] (R)-5-((lR,3aS,7aR£)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-i -inden-l-yl)-iV- hydroxyhexanamide (13):

Chemical Formula: C ₂₄ H ₃₉ NO ₄ Molecular Weight: 405,57

[00149] In a flame dried round bottom flask under argon atmosphere, oxalyl chloride

(12.9 μL, 0.153 mmol, 1.5 equiv) was added to a solution of the acid 50 (63 mg, 0.102 mmol, 1 equiv) in dry CH ₂ Cl ₂ (2 mL) and dry DMF (1 μL) at 0 ⁰ C then the solution was left to stir at room temperature for 1 h and recooled to 0 ⁰ C. A solution of DIPEA (53.3 μL, 0.306, 3 equiv) and <9-tert-butyldimethylsilylhydroxylamine (30 mg, 0.204 mmol, 2 equiv) in CH ₂ Cl ₂ (1 mL) was added to the reaction mixture. The solution was left to stir at 0 ⁰ C for 1 h then at room temperature for 1 h. A solution of citric acid (10 mL, 1 M) was then added, and the mixture was extracted with EtOAc (3 x 10 mL) and the combined organic layers were washed with H ₂ O (10 mL), brine (10 mL) and dried (MgSO ₄ ). The solution was concentrated in vacuo, then the crude

48

material was dissolved in CH ₂ Cl ₂ (1 mL) and CH ₃ CN (1 mL). A 48% solution of HF (70 μL, 2.04 mmol, 20 equiv) was added and the solution was stirred at room temperature for 2 h. The mixture was quenched cautionly by the addition of sat. NaHCO ₃ until no effervescence was observed then acidified with a 1 M aq. solution of citric acid (5 mL). The solution was extracted with CH ₂ Cl ₂ (3 x 5 mL) then the combined organic layers were washed with H ₂ O (5 mL), brine (5 mL), dried (MgSO ₄ ) and then concentrated in vacuo. The residual oil was purified by octadecyl-fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford the hydroxamic acid 13 in 68% yield. R _/ = 0.30 (88/10/2 CH ₂ Cl ₂ /MeOH/ CH ₃ COOH); ¹ H NMR (400 MHz, CD ₃ OD) δ 6.22 (IH, d, J= 11.0 Hz), 5.89 (IH, d, J= 11.0 Hz), 4.08-3.92 (2H, m), 2.83 (IH, br d, J= 12.4 Hz), 2.59 (IH, br d, J = 13.2 Hz), 2.52-1.04 (27H, m), 0.97 (3H, d, J = 3.2 Hz), 0.58 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 176.6, 140.9, 132.7, 122.3, 116.0, 66.8, 66.5, 56.6, 56.3, 45.6, 44.2, 41.5, 40.7, 36.5, 36.1, 35.3, 34.2, 28.7, 27.5, 23.4, 22.1, 21.6, 18.1, 11.3; IR (KBr) v 3390 (br), 2941, 2870, 1709, 1439, 1376, 1212, 1046 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 428.2777, found = 428.2769.

[00150] (R)-5-((lR,3aS,laRβ)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yI)-N- hydroxyheptanamide (5):

Chemical Formula: C ₂ SH ₄₁ NO ₄ Molecular Weight: 419,6

[00151] This was prepared from acid 53 by following the same procedure described for 13. The reagent used were as follows: acid 53 (30 mg, 0.047 mmol, 1 equiv), oxalylchloride (5.9 μL, 0.070 mmol, 1.5 equiv), DIPEA (24.5 μL, 0.141, 3 equiv) and O-tert- butyldimethylsilylhydroxylamine (14 mg, 0.094 mmol, 2 equiv). 5 was purified by octadecyl-

49

fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford 13 mg (0.032 mmol) of the hydroxamic acid 5 in 67% yield. R/ = 0.30 (88/10/2 CH ₂ Cl ₂ /Me0H/ CH ₃ COOH); ¹ H NMR (400 MHz, CD ₃ OD) δ 6.21 (IH, d, J = 1 1.0 Hz), 5.88 (IH, d, J = 1 1.0 Hz), 4.08-3.93 (2H, m), 2.83 (IH, d, J= 1 1.6), 2.59 (IH, dd, J= 13.2, 3.2 Hz), 2.41 (IH, dd, J = 13.2, 3.2 Hz), 2.18-2.12 (2H, m), 2.12-2.05 (2H, m), 2.05-1.98 (2H, m), 1.98-1.89 (IH, m), 1.89- 1.80 (IH, m), 1.80-1.71 (IH, m), 1.71-1.15 (17H, m), 1.15-1.03 (IH, m), 0.95 (3H, d, J = 6.0 Hz), 0.57 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 171.9, 140.9, 132.7, 122.2, 116.0, 66.8, 66.5, 56.7, 56.3, 45.6, 44.2, 41.5, 40.7, 36.4, 36.2, 35.6, 32.7, 28.6, 27.5, 26.1, 25.5, 23.3, 22.1 , 18.1, 1 1.2; IR (KBr) v 3370 (br), 2937, 2868, 1650, 1456, 1046 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 442.2933, found = 442.2925.

[00152] (R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)-λ ^r - methoxyheptanamide (14):

Chemical Formula: C ₂₆ H ₄₃ NO ₄ Molecular Weight: 433,62

[00153] This was prepared from acid 53 by following the same procedure described for 13. The reagent used were as follows: acid 53 (23 mg, 0.036 mmol, 1 equiv), oxalylchloride (4.5 μL, 0.054 mmol, 1.5 equiv), DIPEA (225.1 μL, 0.144, 4 equiv) and O- methylhydroxylamine hydrochloride (6 mg, 0.072 mmol, 2 equiv). 14 was purified by octadecyl-fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford 12.5 mg (0.029 mmol) of the hydroxamic acid 14 in 80% yield. R ₇ = 0.4 (1/9/90 AcOH/MeOH/CH ₂ Cl ₂ ); ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (IH, brs), 6.31 (IH, d, J= 11.2 Hz), 5.85 (IH, d, J = 11.2 Hz), 4.17-4.09 (IH, m), 4.09-4.01 (IH, m), 3.77 (3H, m), 2.79 (IH, d, J =

50

12.0), 2.74 (IH, dd, J = 13.6, 3.6 Hz), 2.41 (IH, dd, J= 13.2, 3.2 Hz), 2.48 (IH, d, J= 13.6 Hz), 2.28-2.17 (2H, m), 2.03-1.75 (5H, m), 1.73-1.15 (16H, m), 1.12-1.00 (IH, m), 0.91 (3H, d, J = 6.0 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 171.0, 143.1, 131.1, 123.8, 115.2, 67.4, 67.2, 60.4, 56.4, 56.3, 45.8, 44.6, 42.1, 40.4, 37.2, 35.9, 35.5, 28.9, 27.6, 25.7, 23.5, 22.3, 21.1, 18.8, 14.2, 12.1; IR (KBr) v 3342 (br), 2931, 2867, 1651, 1437, 1048, 975, 734 cm ^"1 ; HRMS (ESl): m/z calcd. for [(M+H) ⁺ ] = 434.3270, found = 434.3270.

[00154] (R)-6-((lR,3aS,7aRJS)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyloctahydro-iH-i nden-l-yl)-7V-methoxy-iV- methylheptanamide (15):

Chemical Formula: C _2Z H ₄₅ NO ₄ Molecular Weight: 447,65

[00155] This was prepared from acid 53 by following the same procedure described for 13. The reagent used were as follows: acid 53 (6.5 mg, 0.010 mmol, 1 equiv), oxalylchloride (1.3 μL, 0.015 mmol, 1.5 equiv), DIPEA (11 μL, 0.060, 6 equiv) and N ₁ O- dimethylhydroxylamine hydrochloride (4 mg, 0.040 mmol, 4 equiv). 15 was purified by silica gel column chromatography (50% EtOAc in hexanes) to afford 3.3 mg (0.0074 mmol) of the hydroxamic acid 15 in 74% yield. R ₇ = 0.3 (50% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J = 11.2 Hz), 5.84 (IH, d, J = 11.2 Hz), 4.1 1 (IH, brs), 4.04 (IH, brs), 3.68 (3H, m), 3.17 (3H, m), 2.84-2.79 (2H, m), 2.47 (IH, d, J = 13.2 Hz), 2.41 (2H, t, J = 7.2 Hz), 2.27-2.15 (2H, m), 2.04-1.84 (5H, m),1.83-1.74 (IH, m), 1.73-1.16 (14H, m), 1.12-1.02 (2H, m), 0.91 (3H, d, J = 6.0 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 175.2, 143.4, 131.3, 124.1, 115.5, 67.6, 67.5, 61.4, 56.7, 56.5, 46.0, 44.9, 42.4, 40.7, 37.4, 36.2, 35.8, 32.2, 29.2, 27.8, 26.2, 25.3, 23.8, 22.5, 19.0, 12.2; IR (KBr) v 3402 (br), 2938, 2876, 1645, 1440,

51

1380, 1049, 994, 732 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 470.3241 , found = 470.3241.

[00156] (R)-6-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)-7V-hydroxy-7V- methylheptanamide (16):

Chemical Formula: C ₂₆ H ₄₃ NO ₄ Molecular Weight: 433,62

[00157] nub wab μicμcucu Hum α^u J3 by following the same procedure described for 13. The reagent used were as follows: acid 53 (6.5 mg, 0.010 mmol, 1 equiv), oxalylchloride (1.2 μL, 0.014 mmol, 1.5 equiv), DIPEA (1.6 μL, 0.027, 3 equiv) and 0-tert-butyldimethylsilyl- yV-methyl hydroxylamine (2.26 μL, 0.018 mmol, 2 equiv). 16 was purified by octadecyl- fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford 2.2 mg (0.005 mmol) of the hydroxamic acid 16 in 51% yield. R/ = 0.40 (88/10/2 CH ₂ Cl ₂ /MeOH/CH ₃ COOH); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J = 1 1.2 Hz), 5.84 (IH, d, J = 1 1.2 Hz), 4.1 1 (IH, brs), 4.04 (IH, brs), 3.68 (3H, m), 3.17 (3H, m), 2.84-2.79 (2H, m), 2.47 (IH, d, J = 13.2 Hz), 2.41 (2H, t, J = 7.2 Hz), 2.27-2.15 (2H, m), 2.04-1.84 (5H, m),1.83- 1.74 (IH, m), 1.73-1.16 (14H, m), 1.12-1.02 (2H, m), 0.91 (3H, d, J = 6.0 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 175.2, 143.4, 131.3, 124.1, 115.5, 67.6, 67.5, 61.4, 56.7, 56.5, 46.0, 44.9, 42.4, 40.7, 37.4, 36.2, 35.8, 32.2, 29.2, 27.8, 26.2, 25.3, 23.8, 22.5, 19.0, 12.2; IR (KBr) D 3402 (br), 2938, 2876, 1645, 1440, 1380, 1049, 994, 732 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 470.3241, found = 470.3241.

52

[00158] N-((R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-2H-inden-l- yl)hexyl)hydroxylamine (60):

Chemical Formula: C ₃₆ H ₆ QNOsSi ₂ Molecular Weight: 620,11

[00159] A solution of aldehyde 59 (30 mg, 0.050 mmol, 1 equiv), hydroxylamine (9.2 μL of a 50% wt solution in water, 0.50 mmol, 10 equiv) in 4 mL of toluene was refluxed for 30 min. The mixtu ^" rude residue was disolved in AcOH:THF (2: 1,

2 mL) then cooled to U ^U C NaBH ₃ UJN (t> mg, U. lO mmol, 2 equiv) was added portionwise. The mixture was stirred at 0 ⁰ C for 1 h then 0.5 M NaOH was slowly added until alkaline pH. The solution was extracted with CH ₂ Cl ₂ (3 x 10 mL), the combined organic layers were washed with brine (10 mL) then dried (Na ₂ SO ₄ ). The solution was concentrated and the hydroxylamine 60 was obtained in quantitative yield without further purification (31 mg, 0.050 mmol). ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.16 (IH, d, J = 1 1.0 Hz), 5.81 (IH, d, J = 1 1.0 Hz), 4.13-4.01 (2H, m), 2.94 (2H, t, J= 6.8 Hz), 2.81 (IH, d, J= 1 1.6 Hz), 2.44-2.33 (2H, m), 2.25 (IH, d, J= 13.2 Hz), 2.10 (IH, dd, J= 12.4, 8.4 Hz), 2.04-1.94 (2H, m), 1.93-1.75 (2H, m), 1.70-1.15 (15H, m), 1.12- 0.98 (IH, m), 0.92 (3H, d, J= 6.4 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 141.0, 133.9, 122.0, 116.4, 68.3, 68.2, 56.7, 56.5, 54.2, 46.2, 45.9, 43.9, 40.8, 37.0, 36.3, 36.0, 28.9, 27.6, 26.10, 26.08, 23.9, 23.6, 22.5, 19.0, 18.4, 18.3, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 2931, 2857, 1468, 1253, 1087, 1052, 1026, 906, 835, 805, 775, 735 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 420.31 14, found = 420.3106.

53

[00160] N-((R)-5-((lR,3aS,7aRJE)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-iH- inden-l-yl)hexyl)-λ ^r - hydroxyformamide (36):

Chemical Formula: C ₂ SH ₄₁ NO ₄ Molecular Weight: 419,6

[00161] A solution of hydroxylamine 60 (30 mg, 0.048 mmol, 1 equiv) and trifluoroethylformate (7.2 mg of a 86% wt solution in formic acid, 0.48 mmol, 10 equiv) in THF (1 mL) was refli ;entrated in vacuo, then the crude material was dissolved in CH ₂ Cl ₂ (1 mL) and UH ₃ CJJN (1 mL). A 48% solution of HF (33 μL, 0.96 mmol, 20 equiv) was added and the solution was stirred at room temperature for 2 h. The mixture was quenched cautionly by the addition of a sat. NaHCO ₃ until no effervescence was observed. The solution was extracted with CH ₂ Cl ₂ (3 x 5 mL) then the combined organic layers were washed with H ₂ O (5 mL), brine (5 mL), dried (MgSO ₄ ) and then concentrated in vacuo. The oil was purified by octadecyl-fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford 8.2 mg (0.019 mmol) of 36 in 40% yield. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.16 (0.46H, s), 7.92 (0.54H,s), 6.21 (IH, d, J = 11.0 Hz), 5.88 (IH, d, J = 11.0 Hz), 4.09-3.93 (2H, m), 3.54 (0.92H, t, J = 6.8 Hz), 3.50 (1.08H, X, J= 6.8 Hz), 2.83 (IH, d, J= 11.6 Hz), 2.59 (IH, dd, J = 13.2, 3.2 Hz), 2.41 (IH, dd, J = 13.6, 3.2 Hz), 2.26-2.11 (2H, m), 2.08-1.98 (IH, m), 1.98-1.89 (IH, m), 1.89-1.80 (IH, m), 1.80-1.72 (IH, m), 1.72-1.27 (13H, m), 1.27-1.16 (IH, m), 1.16-1.04 (IH, m), 0.96 (3H, d, J = 6.4 Hz), 0.57 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 162.6, 158.1, 140.9, 132.7, 122.2, 116.0, 66.9, 66.5, 56.7, 56.3, 50.1, 46.3, 45.6, 41.5, 40.7, 36.4, 36.3, 35.4, 28.6, 27.6, 23.3, 22.9, 22.6, 22.1, 18.1, 11.2; IR (KBr) v 3342 (br), 2926, 2865, 1657, 1436, 1375, 1212, 1045, 975, 871, 810 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 620.4894, found = 620.4883.

54

[00162] (R)-N-(2-aminopheny\)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5-bis (tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methylo ctahydro-lH-inden-l- yl)hexanamide (61):

Chemical Formula: C ₄₂ H ₇₂ N ₂ O ₃ Si ₂ Molecular Weight: 709,2

[00163] HBTU (4.6 mg, 0.012 mmol, 1.1 equiv) was added to a solution of acid 50

(6.8 mg, 0.011 mmol, 1 equiv), 1 ,2-phenylenediamine (1.2 mg, 0.011 mmol, 1 equiv), HOBt (7.4 mg, 0.055 mmc 1.033 mmol, 3 equiv) in DMF (110 μL). The mixture was stirred at room temperature for 1 h, then diluted with EtOAc (10 mL). The organic solution was washed with sat. NaHCO ₃ (5 mL), water (5 mL), brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was filtered, concentrated, and the oil was purified by silica gel column chromatography (50/45/5 EtOAc/hexanes/Et ₃ N) to afford 7 mg (0.010 mmol) of 61 in 92% yield. Ry= 0.5 (50/45/5 EtOAc/hexanes/Et ₃ N); ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.16 (IH, d, J= 7.6 Hz), 7.11 (IH, s), 7.06 (2H, t, J = 7.6 Hz), 6.80 (IH, d, J = 8.0 Hz), 6.16 (IH, d, J = 11.0 Hz), 5.81 (IH, d, J = 1 1.0 Hz), 4.12-4.00 (2H, m), 3.87 (2H, brs), 2.80 (IH, dd, J = 11.6, 3.2 Hz), 2.59 (IH, q, J= 6.8 Hz), 2.45-2.30 (4H, m), 2.29-2.22 (IH, m), 2.10 (IH, dd, J= 12.4, 8.0 Hz), 2.03- 1.00 (19H, m), 0.97 (3H, d, J = 6.4 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.54 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 172.0, 140.9, 127.4, 125.4, 124.6, 121.9, 120.4, 119.8, 118.5, 116.4, 68.3, 68.2, 56.5, 46.2, 45.9, 43.9, 40.8, 37.8, 37.0, 36.2, 35.8, 30.3, 28.9, 28.0, 26.10, 26.08, 23.6, 22.7, 19.1, 18.4, 18.3, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 3269 (br), 2929, 2855, 1654, 1502, 1459, 1252, 1087, 835, 775, 740 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+H) ⁺ ] = 709.5160, found = 709.5155.

55

[00164] (R)-N-(2-aminophenyl)-6-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5-bis (tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-iH-inden-l- yl)heptanamide (62):

Chemical Formula C ₄₃ H ₇₄ N ₂ O ₃ Si ₂ Molecular Weight 723,23

[00165] This was prepared from acid 53 by following the same procedure described for 61 The reagent used were as follows acid 53 (19 mg, 0 030 mmol, 1 equiv), 1 ,2- phenylenediami ), HBTU (12 5 mg, 0 033 mmol, 1 1 equiv),

HOBt (20 2 mg, 0 150 mmol, 5 equiv) ana DIPEA (15 7 μL, 0 090 mmol, 3 equiv) oxalylchloÏ€de (1 2 μL, 0 014 mmol, 1 5 equiv), DIPEA (1 6 μL, 0 027, 3 equiv) and O-tert- butyldimethylsilyl-N-methyl hydroxylamine (2 26 μL, 0 018 mmol, 2 equiv) 62 was purified by silica gel column chromatography (50/45/5 EtOAc/hexanes/Et ₃ N) to afford 19 3 mg (0 027 mmol) of 62 in 89% yield R/= 0 5 (50/45/5 EtOAc/hexanes/Et ₃ N), ¹ H NMR (400 MHz, CDCl ₃ ) δ 7 17 (IH, d, J = 8 0 Hz), 7 12 (IH, s), 7 08 (2H, t, J = 7 6 Hz), 6 80 (IH, d, J = 8 0 Hz), 6 16 (IH, d, J= 11 0 Hz), 5 81 (IH, d, J= 11 0 Hz), 4 13-4 02 (2H, m), 3 87 (2H, brs), 2 81 (IH, dd, J = 1 1 6, 2 8 Hz), 2 61 (IH, q, J= 7 2 Hz), 2 45-2 33 (4H, m), 2 29-2 21 (IH, m), 2 10 (IH, dd, J = 12 8, 8 0 Hz), 2 03-1 00 (21H, m), 0 93 (3H, d, J = 6 4 Hz), 0 87 (9H, s), 0 86 (9H, s), 0 53 (3H, s), 0 05 (12H, s), ¹³ C NMR (75 MHz, CDCl ₃ ) δ 172 1 , 141 0, 133 9, 127 4, 125 4, 124 6, 121 9, 120 4, 119 8, 118 5, 116 3, 68 4, 68 2, 56 7, 56 5, 46 2, 45 9, 43 9, 40 8, 37 5, 37 0, 36 2, 35 8, 30 3, 28 9, 28 0, 26 6, 26 1 1, 26 08, 23 6, 22 5, 19 1 , 18 4, 18 3, 12 3, -4 4, -4 5, -4 6, -4 7, IR (KBr) v 3269 (br), 2930, 2856, 1655, 1502, 1459, 1252, 1087, 835, 774, 741 cm \ HRMS (ESI) m/z calcd For [(M+H) ⁺ ] = 723 5316, found = 723 5319

56

[00166] (R)-N-(2-aminophenyl)-5-((lR,3aS,7aR£)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-2H- inden-l-yl)hexanamide

(17):

Chemical Formula: C ₃₀ H ₄₄ N ₂ O ₃ Molecular Weight: 480,68

[00167] TBAF (28 μL of a 1 M solution in THF, 0.028 mmol, 4 equiv) and Et ₃ N (4 μL, 0.021 mmol, 3 equiv) were added to a stirred solution of 61 (5 mg, 0.007 mmol, 1 equiv) in THF (0.5 mL) v ^nAor <*™™ â„¢<* for 48 h. The solution was concentrated in vacuo and lc _^ . _^^ _„_, _o ... - . .vas purified by octadecyl-fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford 2.0 mg (0.004 mmol) of 17 in 60% yield. R ₇ = 0.33 (90/10 EtOAQEt ₃ N); ¹ H NMR (500 MHz, CD ₃ OD) δ 7.07 (IH, d, J = 8.0 Hz), 7.02 (IH, t, J= 7.6 Hz), 6.84 (IH, d, J= 8.0 Hz), 6.71 (IH, t, J= 7.5 Hz), 6.22 (IH, d, J = 11.0 Hz), 5.89 (IH, d, J = 11.0 Hz), 4.09-4.01 (IH, m), 4.00-3.96 (IH, m), 2.84 (IH, d, J = 12.0 Hz), 2.60 (IH, d, J = 13.5 Hz), 2.46-2.31 (3H, m), 2.29-2.11 (3H, m), 2.09-1.91 (4H, m), 1.88-1.73 (4H, m),1.72-1.12 (9H, m), 1.00 (3H, d, J= 7.0 Hz), 0.59 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 174.0, 142.1, 140.9, 132.7, 127.1, 126.0, 124.0, 122.2, 118.3, 117.4, 116.0, 66.8, 66.5, 56.6, 56.3, 45.6, 44.2, 41.5, 40.7, 36.4, 36.1, 35.4, 28.6, 27.5, 23.3, 22.5, 22.1, 18.1, 11.2; IR (KBr) v 3351 (br), 2943, 2872, 1653, 1503, 1454, 1376, 1306, 1047, 908, 732 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+H) ⁺ ] = 481.3426, found = 481.3430.

57

[00168] (R)-iV-(2-aminophenyl)-6-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-iH- inden-l-yl)heptanamide

Chemical Formula: C ₃₁ H ₄₆ N ₂ O ₃ Molecular Weight: 494,71

[00169] This was prepared from 62 by following the same procedure described for

17. The reagents used were as follows: 62 (18.8 mg, 0.026 mmol, 1 equiv), TBAF (78 μL of a 1 M solution in THF ω 078 mmnl 4 emiivÏŠ and Et ₃ N (15 μL, 0.078 mmol, 3 equiv). 18 was purified by oct; _, _^ ilumn chromatography (100% H ₂ O to 100%

MeOH) to afford 12.6 mg (0.025 mmol) of 18 in 95% yield. R ₇ = 0.33 (90/10 EtOACYEt ₃ N); ¹ H NMR (400 MHz, CD ₃ OD) δ 7.06 (IH, d, J = 8.0 Hz), 7.02 (IH, t, J = 7.6 Hz), 6.84 (IH, d, J = 8.0 Hz), 6.71 (IH, t, J= 7.5 Hz), 6.22 (IH, d, J= 11.0 Hz), 5.89 (IH, d, J = 11.0 Hz), 4.10-4.01 (IH, m), 4.00-3.93 (IH, m), 2.83 (IH, dd, J = 12.8, 4.0 Hz), 2.59 (IH, dd, J = 12.8, 3.6 Hz), 2.51-2.35 (3H, m), 2.30-2.10 (3H, m), 2.09-1.23 (17H, m), 1.22-1.08 (IH, m), 1.02 (IH, t, J = 7.2 Hz), 0.97 (3H, d, J = 6.4 Hz), 0.58 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 174.0, 140.9, 132.7, 127.1 , 125.9, 124.0, 122.3, 118.3, 117.4, 116.0, 66.8, 66.5, 56.7, 56.3, 45.6, 44.2, 41.4, 40.7, 36.5, 36.2, 36.1, 35.6, 28.6, 27.6, 26.4, 25.7, 23.6, 23.3, 22.1, 18.2, 11.2; IR (KBr) v 3350 (br), 2943, 2872, 1652, 1503, 1456, 1376, 1306, 1046, 909, 732 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+H) ⁺ ] = 495.3587, found = 495.3577.

58

[00170] S-2-((R)-4-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-7H-inden-l- yl)pentylamino)-2-oxoethyl ethanethioate (63):

Chemical Formula: 0 ₃₉ H ₇₁ NO ₄ SSi ₂ Molecular Weight: 706,22

[00171] EDCHCl (9.2 mg, 0.048 mmol, 1.2 equiv) was added to a stirred solution of the amine 51 (23 mg, 0.040 mmol, 1 equiv) and 2-(acethylthio)acetic acid (5.9 mg, 0.044 mmol, 1.1 equiv) in Cl - - - — ^{â–  â–}  ^ _wag g^g^ _{at room} temperature for 4 h then diluted with CH ₂ (Jl ₂ (15 mL), washed witn Ï….D M riCl (2 x 5 mL), with sat. NaHCO ₃ (2 x 5 mL), with brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo to give the product as an oil (27 mg, 0.046 mmol, 97% yield) and the product was carried forward without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.17 (IH, brs), 6.15 (IH, d, J = 1 1.2 Hz), 5.80 (IH, d, J = 1 1.2 Hz), 4.12-4.00 (2H, m), 3.51 (2H, s), 3.26-3.10 (2H, m), 2.80 (IH, d, J = 13.2 Hz), 2.42-2.30 (2H, m), 2.40 (3H, s), 2.25 (IH, d, J = 12.8 Hz), 2.14-2.05 (IH, m), 2.03- 1.73 (4H, m), 1.70-1.17 (13H, m), 0.91 (3H, d, J = 5.6 Hz), 0.86 (9H, s), 0.85 (9H, s), 0.51 (3H, s), 0.04 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) 5196.3, 168.3, 140.9, 134.0, 121.9, 116.4, 68.3, 68.2, 56.4, 46.2, 45.8, 43.9, 40.8, 40.5, 37.0, 36.0, 33.3, 33.1 , 30.5, 28.9, 27.9, 26.10, 26.07, 23.6, 22.4, 19.0, 18.38, 18.34, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 3310 (br), 2951, 2857, 1696, 1468, 1360, 1254, 1090, 836, 776, 733 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 728.4540, found = 728.4537.

59

[00172] S-2-((R)-5-((lR,3aS,laR£)-4-(2-((3R,SR)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-/H-inden-l- yI)hexyIamino)-2-oxoethylethanethioate (64):

Chemical Formula: C ₄₀ H ₇₃ NO ₄ SSi ₂ Molecular Weight: 720,25

[00173] This was prepared from amine 54 by following the same procedure described for 63. The reagents used were as follows: amine 54 (29 mg, 0.048 mmol, 1 equiv), 2- (acethylthio)act 1 equiv), EDCHCl (11 mg, 0.058 mmol, 1.2 equiv). 64 (31 mg, 0.043 mmol, 90% yield) was carried carried forward without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.17 (IH, brs), 6.16 (IH, d, J = 11.2 Hz), 5.81 (IH, d, J= 11.2 Hz), 4.11-4.02 (2H, m), 3.51 (2H, s), 3.21 (2H, q, J = 6.4 Hz), 2.80 (IH, d, J = 11.6 Hz), 2.42-2.33 (2H, m), 2.40 (3H, s), 2.25 (IH, d, J= 12.8 Hz), 2.09 (IH, dd, J= 12.8, 8.4 Hz), 2.02-1.94 (2H, m), 1.92-1.82 (IH, m), 1.82-1.74 (IH, m), 1.70-1.59 (3H, m), 1.58-0.98 (15H, m), 0.91 (3H, d, J = 6.4 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.52 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 196.3, 168.3, 141.0, 133.9, 122.0, 116.4, 68.3, 68.2, 56.6, 56.5, 46.2, 45.9, 43.9, 40.8, 40.1, 37.0, 36.3, 35.7, 33.3, 30.5, 30.0, 28.9, 28.0, 26.10, 26.08, 23.6, 23.5, 22.5, 19.0, 18.4, 18.3, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 3296 (br), 2930, 2856, 1696, 1653, 1540, 1468, 1252, 1087, 835, 775 cm ^'1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 742.4697, found = 742.4702.

60

[00174] N-((R)-4-((lR,3aS,laRJS)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-/H- inden-l-yl)pentyl)-2- mercaptoacetamide (31):

Chemical Formula: C ₂ SH ₄₁ NO ₃ S Molecular Weight: 435,66

100175] In a flame dried round bottom flask under argon atmosphere, a desoxygenated solution of MeONa (2 mg 0.036 mmol, 1 equiv) in MeOH was added by cannula to 63 (25 mg, C"' ' ¹ ----- ^' - ^λ ^ ¹ - ⁱ -^- _{n was} stirred at room temperature for 4 h then quenched by the αuumun ui .rivwn ^i uu _^ j. im_ solution was concentrated in vacuo then diluted in EtOAc (15 mL). The solution was washed with water (5 mL), brine (5 mL), dried (Na ₂ SO ₄ ) and concentrated to give the desacethylated compond witch was disolved in CH ₂ Cl ₂ (1 mL) and CH ₃ CN (1 mL). HF (50 μL) was added to the solution and the mixture was stirred at room temperature overnight. The reaction mixture was quenched with sat. NaHCO ₃ (2 mL) and exctrated with CH ₂ Cl ₂ (3 x 10 mL). The combined organic fractions were washed with brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo then purified by silica gel column chromatography (5% MeOH in CH ₂ Cl ₂ ) to give 31 in 71% yield. R _f = 0.5 (10% MeOH in CH ₂ Cl ₂ ); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.70 (IH, brs), 6.31 (IH, d, J= 11.6 Hz), 5.85 (IH, d, J = 10.8 Hz), 4.19-4.08 (IH, m), 4.08-4.01 (IH, m), 3.35-3.17 (3H, m), 3.80 (IH, d, J = 16.0 Hz), 2.74 (IH, d, J= 17.2 Hz), 2.48 (IH, d, J= 12.8 Hz), 2.26-2.15 (2H, m), 2.04-1.74 (6H, m), 1.73-1.00 (13H, m), 0.93 (3H, d, J= 6.4 Hz), 0.54 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 169.4, 143.3, 131.3, 124.1, 115.4, 67.6, 67.5, 56.60, 56.58, 46.3, 44.9, 42.4, 40.7, 40.3, 37.2, 36.0, 35.7, 29.9, 29.1, 28.6, 23.7, 2.5, 22.5, 19.0, 12.3 ; IR (KBr) v 3311 (br), 2925, 2869, 1651, 1558, 1540, 1457, 1261 , 1045, 802, 733 cm ^" '; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 458.2705, found = 458.2698.

61

[00176] N-((R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-raethyloctahydro-/H- inden-l-yl)hexyl)-2- mercaptoacetamide (32):

Chemical Formula: C ₂₆ H ₄₃ NO ₃ S Molecular Weight: 449,69

[00177] This was prepared from 64 by following the same procedure described for

31. The reagents used were as follows: 64 (30 mg, 0.041 mmol, 1 equiv), MeONa (2.2 mg 0.041 mmol, 1 equiv). 32 was purified by silica gel column chromatography (5% MeOH in CH ₂ Cl ₂ ) to give 13 mg (0.< = 0.5 (10% MeOH in CH ₂ Cl ₂ ); ¹ H NMR (400

MHz, CDCl ₃ ) 5 6.68 (IH, brs), 6.31 (IH, d, J = 11.4 Hz), 5.85 (IH, d, J = 11.4 Hz), 4.12 (IH, brs), 4.05 (IH, brs), 3.28 (2H, q, J= 6.4 Hz), 3.24 (2H, d, J= 8.8 Hz), 2.80 (IH, d, J= 12.4 Hz), 2.73 (IH, d, J= 13.2 Hz), 2.48 (IH, d, J= 11.2 Hz), 2.27-2.15 (2H, m), 2.05-1.74 (6H, m), 1.73- 1.16 (14H, m), 1.13-1.00 (IH, m), 0.92 (3H, d, J = 6.4 Hz), 0.54 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 169.2, 143.3, 131.4, 124.1, 115.5, 67.6, 67.4, 56.7, 56.5, 46.0, 44.9, 42.4, 40.7, 40.2, 37.4, 36.2, 35.7, 30.1, 29.2, 28.6, 27.9, 23.71, 23.66, 22.5, 19.0, 12.3; IR (KBr) v 3306 (br), 2935, 2869, 1650, 1550, 1439, 1048, 909, 732 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 450.3042, found = 450.3043.

62

[00178] N-((R)~5-((lR,3aS,7aR^)-4~(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)hexyl)-2- (dimethylamino)acetamide (34):

Chemical Formula: C ₂ SH ₄ SN ₂ O ₃ Molecular Weight: 460,69

[00179] EDCHCl (1.6 mg, 0.011 mmol, 1.1 equiv) was added to a stirred solution of amine 54 (6.3 mg, 0.010 mmol, 1 equiv) and dimethyl glycine (1.2 mg, 0.011 mmol, 1.1 equiv) in CH ₂ Cl ₂ (0.2 m ^{Ï„ λ} "* ^{A 0} ^ ^ ¹ " —' ^â– â–  *—- — ^~~4 _{at room} temperature for 4 h then diluted with CH ₂ Cl ₂ (10 mL,, vvdoiivu vvxtij ou... 1 ,UiX _^ Wj _v- - ,. 5 mL), with brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo to give the crude acetamide as a yellow oil. TBAF (4 μL of a 1 M solution in THF, 0.040 mmol, 4 equiv) and Et ₃ N (4 μL, 0.033 mmol, 3 equiv) were added to a stirred solution of the crude product in THF (0.5 mL) under argon and the mixture was stirred overnight. The solution was concentrated in vacuo and loaded directly on to silica gel column chromatography (80/10/10 EtOAc/MeOH/Et ₃ N) to obtain 34 as a clear oil in 58% yield (2.9 mg, 0.006 mmol). R/= 0.5 (80/10/10 EtOAc/MeOH/Et ₃ N); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J = 11.2 Hz), 5.85 (IH, d, J= 11.2 Hz), 3.32-3.20 (2H, m), 2.93 (IH, s), 2.79 (IH, dd, J = 13.2, 4.0 Hz), 2.74 (IH, dd, J = 14.0, 4.0 Hz), 2.48 (IH, d, J = 14,0 Hz), 2.28 (6H, s), 2.28-2.16 (2H, m), 2.06-1.02 (18H, m), 0.91 (3H, d, J= 6.0 Hz), 0.88-0.80 (2H, m), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 170.7, 143.3, 131.3, 124.1, 115.5, 67.7, 67.5, 63.4, 56.7, 56.5, 46.3, 46.0, 44.9, 42.4, 40.7, 39.2, 37.4, 36.2, 35.7, 30.4, 29.1, 27.9, 23.7, 22.5, 19.0, 12.3; IR (KBr) v 3332 (br), 2931, 2869, 1657, 1528, 1454, 1047, 732 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 461.3738, found = 431.3735.

63

[00180] tert-butyl-2-((R)-5-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5-bis(ter t- butyldimethylsilyloxy)cydohexylidene)ethylidene)-7a-methyloc tahydro-/H-inden-l- yl)hexylamino)-2-oxoethylcarbamate (65) :

Chemical Formula: C ₄₃ HSoNaO ₅ Si ₂ Molecular Weight: 761 ,28

[00181] DCC (2.2 mg, 0.0107 mmol, 1.05 equiv) then DMAP (catalytic amount) were added to a stirred solution of amine 54 (6.2 mg, 0.0102 mmol, 1 equiv) and N-Bocglycine

(1.9 mg, 0.01C ⁷ - ^{1 1 n<:} ~" "" ^{λ ;} " r ^{^} u ^. r ^λ. Ï€ ^Q μL) at 0 ⁰ C. The solution was then stirred at room temperatu.w .„, _. „ „, _^ Jl ₂ (10 mL), washed with sat. NaHCC>3 (2 x 5 raL), with brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo then purified by silica gel column chromatography (50% EtOAc in hexanes) to obtain 65 as a clear oil in 90% yield (7.3mg, 0.0096 mmol). R _/ = 0.3 (50% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.17 (IH, brs), 6.16 (IH, d, J = 10.8 Hz), 6.09 (IH, brs), 5.81 (IH, d, J = 10.8 Hz), 5.12 (IH, brs), 4.15-4.01 (2H, m), 3.76 (2H, d, J= 5.6 Hz), 3.26 (2H, q, J= 6.8 Hz), 2.81 (IH, d, J = 11.6 Hz), 2.43-2.33 (2H, m), 2.25 (IH, d, J = 11.2 Hz), 2.09 (IH, dd, J = 12.8, 8.0 Hz), 2.03-1.83 (5H, m), 1.82-1.75 (IH, m), 1.74-0.99 (14H, m), 1.45 (9H, s), 0.91 (3H, d, J= 6.4 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.52 (3H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 169.5, 156.9, 141.0, 133.9, 121.9, 116.3, 68.3, 68.2, 56.7, 56.5, 49.4, 46.2, 45.9, 43.9, 40.8, 39.8, 37.0, 36.3, 35.7, 34.2, 30.2, 28.9, 28.5, 28.0, 26.09, 26.07, 25.8, 25.2, 22.5, 19.0, 18.4, 18.3, 12.3, -4.4, _4.5 _; .4.6, -4.7; IR (KBr) v 3316 (br), 2929, 2854, 1635, 1558, 1470, 1365, 1252, 1 171, 1086, 835, 775 cm ^" '; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 761.5684, found = 716.5682.

64

[00182] 2-amino-iV-((λ)-5-((lR,3a5,7aR^)-4-(2-((3R,5 _J R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyloctahydro-/H-i nden-l-yl)hexyl)acetamide

(33):

Chemical Formula: C ₂ BH ₄₄ N ₂ O ₃ Molecular Weight: 432,64

[00183] HF (1 drop of a 48% solution) was added to a solution 65 (7.1 mg, 0.0096 mmol) in CH ₃ CN (lOOμL) and CH ₂ Cl ₂ (lOOμL) and the mixture was stirred at room temperature overnight. The * ^: — — ---* — — — ^â–  - — ^U ~ ^λ *vith sat. NaHCO ₃ (2 mL) and exctrated with

CH ₂ Cl ₂ (3 x IG Wi ₅ UiIiW iiuwJons were washed with brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo then purified by silica gel column chromatography (10/20/17 Et ₃ N/MeOH/EtOAc) to obtain 33 in 73% yield (3.3 mg, 0.0070 mmol). R/= 0.15 (10/20/17 Et ₃ N/MeOH/EtOAc); ¹ H NMR (400 MHz, CD ₃ OD) δ 6.21 (IH, d, J = 11.2 Hz), 5.80 (IH, d, J= 11.2 Hz), 4.09-4.02 (IH, m), 4.01-3.95 (IH, m), 3.25-3.15 (2H, m), 2.83 (IH, d, J = 11.2 Hz), 2.59 (IH, d, J = 10.0 Hz), 2.41 (IH, d, J = 10.8 Hz), 2.26-2.1 1 (2H, m), 2.09-1.80 (5H, m), 1.79-1.71 (IH, m), 1.70-1.02 (14H, m), 0.95 (3H, d, J = 6.0 Hz), 0.92- 0.84 (2H, m), 0.57 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 169.3, 140.8, 132.7, 122.2, 116.0, 66.8, 66.5, 56.7, 56.3, 45.6, 44.2, 43.0, 41.5, 40.7, 39.2, 36.4, 36.2, 35.6, 29.7, 29.6, 28.6, 27.6, 23.3, 22.1, 18.1, 11.2; IR (KBr) v 3301 (br), 2927, 2872, 1659, 1642, 1443, 1050, 978, 668 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+H) ⁺ ] = 433.3425, found = 433.3423.

65

[00184] N-((R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5-bis(tert- butyIdimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-iH-inden-l- yl)hexyl)-2-bromoacetamide (66)

Chemical Formula: C ₃₈ H ₇₀ BrNO ₃ Si ₂ Molecular Weight: 725,04

[00185] DCC (3.5 mg, 0.017 mmol, 1.05 equiv) then DMAP (catalytic amount) were added to a stirred solution of amine 54 (9.7 mg, 0.016 mmol, 1 equiv) and bromoacetic acid (2.33 mg, 0.017 ) μL) at 0 ⁰ C. The solution was then stirred at room temperature for 2 h then diluted with CH ₂ Cl ₂ (10 mL), washed with sat. NaHCO ₃ (2 x 5 mL), with brine (5 mL) and dried (Na ₂ SO ₄ ). The solution was concentrated in vacuo then purified by silica gel column chromatography (50% EtOAc in hexanes) to afford 10.1 mg (0.014 mmol) of 66 in 85% yield. R _/ = 0.8 (50% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.47 (IH, brs), 6.15 (IH, d, J= 10.8 Hz), 5.80 (IH, d, J= 10.8 Hz), 4.15-4.00 (2H, m), 3.88 (2H, s), 3.32-3.27 (2H, m), 3.18 (2H, brs), 2.79 (IH, d, J= 12.0 Hz), 2.42-2.30 (2H, m), 2.25 (IH, d, J = 13.6 Hz), 2.09 (IH, dd, J= 12.8, 7.6 Hz), 2.03-1.01 (17H, m), 1.45 (9H, s), 0.90 (3H, d, J= 6.0 Hz), 0.86 (9H, s), 0.85 (9H, s), 0.52 (3H, s), 0.04 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 165.3, 141.0, 133.9, 121.9, 116.4, 68.3, 68.2, 56.7, 56.5, 46.2, 45.9, 43.9, 40.8, 40.5, 37.0, 36.3, 35.7, 35.1, 30.0, 29.7, 28.9, 28.5, 28.0, 26.09, 26.08, 25.8, 24.9, 23.6, 22.4, 19.0, 18.38, 18.34, 12.3, - 4.4, -4.5, -4.6, -4.7. IR (KBr) v 2930, 2854, 2119, 1655, 1642, 1448, 1252, 1086, 835, 775 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 746.3970, found = 746.3970.

66

[00186] 2-bromo-N-((R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyloctahydro-iH-i nden-l-yl)hexyl)acetamide

(35):

Chemical Formula: C ₂₆ H ₄₂ BrNO ₃ Molecular Weight: 496,52

[00187] HF (1 drop of a 48% solution) was added to a solution 66 (4.8 mg, 0.0066 mmol) in CH3CN (100μL) and CH ₂ Cl ₂ (100μL) and the mixture was stirred at room temperature overnight. The reaction mixture was quenched with sat. NaHCO ₃ (2 mL) and exctrated with CH ₂ Cl ₂ (3 x 10 ons were washed with brine (5 mL) and dried

(Na ₂ SO ₄ ). The solution was concentrated in vacuo then purified by silica gel column chromatography (40% Acetone in hexanes) to afford 2.5 mg (0.0050 mmol) of 35 in 76% yield. R _/ = 0.15 (40% acetone in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.48 (IH, brs), 6.31 (IH, d, J = 11.4 Hz), 5.85 (IH, d, J= 11.4 Hz), 4.12 (IH, brs), 4.04 (IH, brs), 3.89 (2H, s), 3.29 (2H, q, J = 6.7 Hz), 2.79 (IH, dd, J = 12.2, 3.8 Hz), 2.74 (IH, dd, J = 13.6, 3.6 Hz), 2.26-2.14 (5H, m), 2.04-1.02 (19H, m), 0.92 (3H, d, J= 6.8 Hz), 0.54 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 165.4, 143.3, 131.3, 124.1, 1 15.5, 67.6, 67.5, 56.6, 56.5, 46.0, 44.9, 42.4, 40.5, 37.4, 36.2, 35.6, 30.0, 29.7, 29.5, 29.1, 27.9, 23.7, 22.5, 19.0, 12.3; IR (KBr) v 3305 (br), 2926, 2868, 1659, 1552,

1442, 1216, 1046, 977, 734 cm ^'1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 518.2240, found = 518.2242.

67

[00188] tert-butyl-N-((R)-4-((lR,3aS,7aRβ)-4-(2-((3R,5R)-3,5-bis(te rt- butyldimethyIsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-7H-inden-l- yl)pentyl)sulfamoylcarbamate (67):

Chemical Formula: C ₄₀ H ₇₆ N ₂ O ₆ SSi ₂ Molecular Weight: 769,28

[00189] iV-(ter?-Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)- 1 ,4-dihydropyridin- l-ylsulfonyl]azanide (8.6 mg, 0.028 mmol, 1.1 equiv; Winum, J. V.; Toupet, L.; Barragan, V.; Dewynter, G.; Montero, J. L. Org. Lett. 2001, 3, 2241-2243) was added to a stirred solution of amine 51 (15 n I ₂ (100 μh). The mixture was stirred at room temperature for 16 h, then concentrated and directly loaded on silica gel (20% EtOAc in hexanes) to give 17 mg (0.022 mmol) of the tert-butylsulfamoylcarbamate 67 in 85% yield. R/ = 0.3 (20% EtOAc in Hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.16 (IH, d, J = 10.8 Hz), 5.81 (IH, d, J = 11.2 Hz), 5.02 (IH, brs), 4.16-4.02 (2H, m), 3.10-2.96 (2H, m), 2.81 (IH, d, J = 12.0 Hz), 2.43-2.32 (2H, m), 2.25 (IH, d, J = 11.2 Hz), 2.10 (2H, dd, J = 12.8, 8.4 Hz), 2.05-1.95 (2H, m), 1.92-1.82 (IH, m), 1.81-1.75 (IH, m), 1.70-1.10 (13H, m), 1.50 (9H, m), 1.13-1.02 (IH, m), 0.93 (3H, d, J= 6.8 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.06 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 150.3, 140.8, 134.0, 121.9, 116.4, 84.0, 68.3, 68.2, 56.5, 56.4, 46.2, 45.8, 44.7, 43.9, 40.8, 40.0, 36.0, 33.0, 28.9, 28.2, 27.9, 26.10, 26.07, 23.6, 22.4, 18.9, 18.4, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 3275 (br), 2927, 2863, 1717, 1459, 1344, 1253, 1099, 1091, 830, 777 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 791.4860, found = 791.4865.

68

[00190] tert-butyl-N-((R)-5-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5 - bis (tert - butyldimethylsilyloxy ) cyclohexyl idene)ethylidene)-7a-methyI-octahydro-7H-inden-l- yl)hexyl)sulfamoylcarbamate (68) :

Chemical Formula: C ₄₁ H ₇₈ N ₂ O ₆ SSi ₂ Molecular Weight: 783,3

[00191] This was prepared from amine 54 by following the same procedure described for 67. The reagents used were as follows: 54 (7 mg, 0.01 1 mmol, 1 equiv) and N-(^r?- ButoxycarbonylVλ/-r4-Cflimethvla7aniÏ€mvlirl ^p nM-l ₅ 4-(iihydropyridin-l-ylsulfonyl]azanide (3.8 mg, 0.012 mmc , _x , - 3% yield (7.6 mg, 0.009 mmol). R/= 0.3 (20%

EtOAc in Hexanes); ¹ H NMR (400 MHz, CD ₃ OD) δ 6.15 (IH, d, J = 10.6 Hz), 5.82 (IH, d, J = 10.6 Hz), 4.17-4.05 (2H, m), 3.00 (2H, t, J= 6.4 Hz), 2.82 (IH, d, J= 12.4 Hz), 2.37 (2H, dd, J = 13.2, 4.4 Hz), 2.29 (IH, d, J= 12.0 Hz), 2.09 (IH, dd, J= 12.4, 4.4 Hz), 2.05-1.88 (3H, m), 1.84- 1.75 (IH, m), 1.71-1.20 (15H, m), 1.49 (9H, m), 1.13-1.02 (IH, m), 0.95 (3H, d, J = 6.0 Hz), 0.88 (9H, s), 0.87 (9H, s), 0.56 (3H, s), 0.06 (12H, s); ¹³ C NMR (75 MHz, CD ₃ OD) 6151.6, 140.3, 133.4, 121.7, 1 16.2, 81.9, 68.4, 68.2, 56.7, 56.3, 45.8, 45.6, 43.6, 43.3, 40.7, 36.5, 36.2, 35.5, 29.5, 28.5, 27.6, 27.2, 25.3, 25.2, 23.3, 23.2, 22.2, 18.1, 17.8, 1 1.5, -5.6, -5.7, -5.8, -5.9; IR (KBr) v 3278 (br), 2931, 2857, 1717, 1460, 1362, 1252, 1 147, 1086, 835, 775, 732 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 805.5011, found = 805.5015.

69

[001921 te^-butyl-7V-((λ)-6-((li?,3a5,7ai?^)-4-(2-((3i?,5/ϊ)-3,5-b is(te^- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyIo ctahydro-7H-inden-l- yl)heptyl)sulf amoylcarbamate (69) :

Chemical Formula: C ₄₂ H ₈₀ N ₂ O ₆ SSi ₂ Molecular Weight: 797,33

[00193] This was prepared from amine 55 by following the same procedure described for 67. The reagents used were as follows: 55 (10.3 mg, 0.016 mmol, 1 equiv) and N-(tert- Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)-l,4-dihydropyr idin-l-ylsulfonyl]azanide (5.5 mg, 0.018 mm. 85% yield (10.8 mg, 0.013 mmol). Ry = 0.3

(20% EtOAc in Hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.17 (IH, d, J= 11.2 Hz), 5.82 (IH, d, J = 1 1.2 Hz), 4.15-4.00 (2H, m), 3.06 (2H, q, J = 6.8 Hz), 2.81 (IH, d, J = 12.0 Hz), 2.45-2.32 (2H, m), 2.26 (IH, d, J = 13.6 Hz), 2.18-2.05 (2H, m), 2.01-1.92 (2H, m), 1.95-1.82 (IH, m), 1.81-1.75 (IH, m), 1.50 (9H,s), 1.74- 1.00 (18H, m), 0.91 (3H, d, J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.07 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) 5150.4, 141.2134.0, 122.0, 116.4, 84.1, 68.3, 68.2, 56.7, 56.5, 46.2, 45.9, 44.2, 43.9, 40.8, 37.0, 36.3, 36.0, 29.3, 28.9, 28.3, 28.0, 27.3, 26.1 1, 26.09, 25.9, 23.7, 22.5, 19.0, 18.4, 12.3, -4.4, -4.5, -4.6, -4.7; IR (KBr) v 3284 (br), 2930, 2856, 1720, 1459, 1362, 1252, 1142, 1087, 835, 775, 732 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 819.5173, found = 819.5173.

70

[00194] N-((R)-5-((lR,3aS,7aR&-4-(2-«3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl octahydro-iH-inden-1- yl)hexyl)methanesulfamide (20):

Chemical Formula: 0 ₂₃ H ₄₀ N ₂ O ₄ S Molecular Weight: 440,64

[00195] A 48% solution of HF (1 drop) was added to a stirred solution of 67 (17 mg,

0.022 mmol) in CH ₂ Cl ₂ (100 μL) and CH ₃ CN (100 μL) and the solution was stirred at room temperature overnight. The mixture was quenched cautionly by the addition of sat. NaHCO ₃ until no efferve 1 with a 1 M aq. solution of citric acid (5 mL).

The solution was extracted with CH ₂ Cl ₂ (3 x 5 mL) then the combined organic layers were washed with H ₂ O (5 mL), brine (5 mL), dried (MgSO ₄ ) and then concentrated in vacuo. The oil was purified by silica gel column chromatography (EtOAc) to afford 3.2 mg (0.007 mmol) of the sulfamide 20 in 32% yield. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J= 1 1.4 Hz), 5.86 (IH, d, J = 11.4 Hz), 4.46 (2H, s), 4.24-4.16 (IH, m), 4.10-4.02 (IH, m), 3.20-3.02 (2H, m), 2.80 (IH, dd, J = 12.0, 4.0 Hz), 2.74 (IH, dd, J= 13.6, 4.0 Hz), 2.49 (IH, dd, J= 13.2, 3.2 Hz), 2.28-2.17 (2H, m), 2.04-1.70 (5H, m), 1.74-1.24 (12H, m), 1.18-1.05 (IH, m), 0.94 (3H, d, J = 6.4 Hz), 0.55 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) 5143.1, 131.5, 124.1, 115.6, 67.7, 67.5, 56.51, 56.48, 46.0, 44.9, 44.4, 42.4, 40.7, 37.4, 36.0, 33.0, 29.1, 27.9, 26.4, 23.7, 22.5, 19.0, 12.3; IR (KBr) v 3290 (br), 2940, 2872, 1706, 1434, 1329, 1 156, 1045, 735, 668 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 463.2596, found = 463.2606.

71

[00196] N-((R)-5-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl octahydro-/H-inden-l- yl)hexyl)methanesulfamide (28):

Chemical Formula: C ₂₄ H ₄₂ N ₂ O ₄ S Molecular Weight: 454,67

[00197] This was prepared from 68 by following the same procedure described for

20. The reagents used were as follows: 68 (7 mg, 0.009 mmol, 1 equiv). 28 was obtained in 90% yield (3.6 mg, 0.008 mmol). R ₇ = 0.1 (EtOAc); ¹ H NMR (400 MHz, CD ₃ OD) δ 6.22 (IH, d, J = 11.2 Hz), 5.88 ( , m), 3.01 (2H, t, J= 6.6 Hz), 2.84 (IH, d; J =

11.6 Hz), 2.59 (IH, d, J= 10.8 Hz), 2.41 (IH, d, J= 12.0 Hz), 2.28-2.12 (3H, m), 2.08-1.99 (2H, m), 1.98-1.90 (IH, m), 1.89-1.80 (IH, m), 1.80-1.72 (IH, m), 1.71-1.20 (12H, m), 1.15-1.05 (IH, m), 0.96 (3H, d, J = 6.4 Hz), 0.94-0.85 (IH, m), 0.57 53H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δ 140.9, 132.7, 122.3, 116.0, 66.8, 66.5, 56.7, 56.3, 53.6, 45.6, 44.2, 43.1, 41.5, 40.7, 36.4, 36.2, 35.6, 29.9, 28.6, 27.6, 23.3, 22.1, 18.1, 1 1.2; IR (KBr) v 3302 (br), 2935, 2860, 1592, 1328, 1156, 1045, 669, 547 cm ^'1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = All.2151, found = 477.2760. 2606.

[00198] N-((R)-5-((lR,3aS,laRβ)-4-(2-((3R,5R)-3,5- dihydroxycycloheptylidene)ethylidene)-7a-methyloctahydro-7Hâ €” inden-1- yl)hexyl)methanesulfamide (30):

Chemical Formula: C ₂₅ H ₄₄ N ₂ O ₄ S Molecular Weight: 468,69

[00199] This was prepared from 69 by following the same procedure described for

20. The reagents used were as follows: 69 (5.4 mg, 0.007 mmol, 1 equiv). 30 was obtained in 81% yield (2.6 mg, 0.005 mmol). ¹ H NMR (400 MHz, CD ₃ OD) δ 6.22 (IH, d, J= 11.2 Hz), 5.88 (IH, d, J = 11.2 Hz), 4.07-4.01 (IH, m), 4.00-3.95 (IH, m), 3.00 (2H, t, J= 7.2 Hz), 2.83 (2H, d, J = 15.6 Hz), 2.59 (IH, dd, J = 13.2, 4.0 Hz), 2.44-2.35 (2H, m), 2.30-2.11 (3H, m), 2.05-1.00 (19H, m), 0.95 Ï€H H T = ^ή 4 H^ n S7 Ï€H «); ¹³ C NMR (75 MHz, CD ₃ OD) 5140.2134.4,

121.9, 1 16.5, 6'. . _^ , , , , .-._, ._. ., ...7, 37.4, 36.2, 36.0, 28.5, 27.7, 27.4, 25.8, 23.5,

22.3, 18.6, 11.7; IR (KBr) v 3298 (br), 2945, 2872, 1699, 1440, 1333, 1 145, 1044, 732, 667 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 491.2919, found = 491.2914.

[00200] N-((R)-4-((lR,3aS,7aRJE)-4-(2-((3R,5R)-3,5- dihydroxycyclohexyIidene)ethylidene)-7a-methyl-octahydro-/H- inden-l- yl)peπtyl)methanesulfonamide (19):

Chemical Formula: C ₂₄ H ₄₁ NO ₄ S Molecular Weight: 439,65

73

[00201] Methylsulfonyl chloride (2.3 μL, 0.031 mmol, 1.1 equiv) was added to a stirred solution of the amine 51 (16 mg, 0.028 mmol, 1 equiv) and Et ₃ N (11 μL, 0.084 mmol, 3eq) in CH ₂ Cl ₂ (1.5 niL) at 0 ⁰ C. The solution was stirred at 0 ⁰ C for 1 h then at room temperature for 1 h. The reaction mixture was then diluted with CH ₂ Cl ₂ (5 mL), washed with sat. NaHCO ₃ (2 x 5 mL), water (5 mL), brine (5 mL) then dried (Na ₂ SO ₄ ). The solution was filtered, concentrated and the residual oil was loaded on silical gel (35% EtOAc in hexanes) to give the sulfonamide as an oil. The oil was then dissolved into CH ₂ Cl ₂ (100 μL) and CH ₃ CN (100 μL) and then a 48% solution of HF was added (1 drop). The solution was stirred at room temperature overnight then quenched cautionly by the addition of a sat. NaHCO ₃ until no effervescence was observed. The solution was extracted with CH ₂ Cl ₂ (3 x 5 mL) then the combined organic layers were washed with H ₂ O (5 mL), brine (5 mL), dried (MgSO ₄ ) and then concentrated in vacuo. The oil was then purified by silica gel column chromatography (5% MeOH in EtOAc) to give 3.7 mg (0.0008 mmol) of the sulfonamide 19 in 30% yield. R ₇ = 0.7 (5% MeOH in EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J = 10.8 Hz), 5.85 (IH, d, J = 10.8 Hz), 4.19-4.00 (2H, m), 3.11 (2H, t ' - * ° ^u - ^{\ i n} * ^u ^ ⁱ en ^ H, d, J = 12.4 Hz), 2.74 (IH, d, J = 13.2 Hz), 2.48 (IH, d, J - _^ .u x^, t.. _^ - _* .. _{^ V} -, _" v, — J-1.02 (17H, m), 0.93 (3H, d, J= 6.4 Hz), 0.54 (3H, s) ¹³ C NMR (75 MHz, CDCl ₃ ) 6143.1 , 131.5, 124.0, 115.6, 67.6, 67.5, 56.53, 56.48, 46.0, 44.9, 44.1, 42.4, 40.7, 40.6, 37.4, 36.0, 33.0, 29.1, 27.9, 27.1 , 23.7, 22.5, 19.0, 12.3; IR (KBr) v 3303 (br), 2929, 2871, 1445, 1316, 1261, 1149, 1045, 974, 801 , 735 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 462.2624, found = 462.2650.

[00202] λ ^r -((λ)-5-((l/?,3a5',7a/?^>-4-(2-((3λ,5R)-3,5-dihydroxycy clohexylidene) ethylidene)-7a-methyloctahydro-/H-inden-l-yl)hexyI)methanesu lfonamide (21):

Chemical Formula: 0 ₂₅ H ₄₃ NO ₄ S Molecular Weight: 453,68

74

[00203] This was prepared from amine 54 by following the same procedure described for 19. The reagents used were as follows: amine 54 (5.5 mg, 0.009 mmol, 1 equiv), methylsulfonyl chloride (1 μL, 0.01 mmol, 1.1 equiv) and Et ₃ N (3 μL, 0.003 mmol, 3eq). 21 was purified by silica gel column chromatography (5% MeOH in EtOAc) to give 2 mg (0.004 mmol) the sulfonamide 21 in 48% yield. Ry= 0.7 (5% MeOH in EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J = 11.2 Hz), 5.85 (IH, d, J= 11.2 Hz), 4.20-4.10 (IH, m), 4.09-4.00 (IH, m), 3.34 (2H, q, J= 6.4 Hz), 2.96 (3H, s), 2.80 (IH, d, J= 12.0 Hz), 2.74 (IH, d, J= 13.6 Hz), 2.60 (IH, d, J = 13.6 Hz), 2.25-2.15 (2H, m), 2.08-0.85 (2OH, m), 0.92 (3H, d, J = 6.4 Hz), 0.54 (3H, s) ¹³ C NMR (75 MHz, CDCl ₃ ) δ 131.3, 124.1, 115.5, 100.0, 67.6, 67.5, 56.6, 56.5, 46.0, 44.9, 43.6, 42.4, 40.6, 37.4, 36.2, 35.6, 30.9, 29.9, 29.1, 27.9, 23.7, 23.4, 22.5, 19.0, 12.3; IR (KBr) v 3310 (br), 2939, 2857, 1750, 1347, 1169, 1030, 698 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 476.2816, found = 476.2804.

[00204] N-((R)-6-((lR,3aS,7aRβ)-4-(2-((3R,5R)-3,5- dihydroxycycl yl-octahydro-iH-inden-1- yl)heptyl)methanesulfonamide (29) :

Chemical Formula: C ₂ SH ₄₅ NO ₄ S Molecular Weight: 467,7

[00205] This was prepared from amine 55 by following the same procedure described for 19. The reagents used were as follows: amine 55 (10 mg, 0.016 mmol, 1 equiv), methylsulfonyl chloride (1.4 μL, 0.018 mmol, 1.1 equiv) and Et ₃ N (7 μL, 0.054 mmol, 3eq). 29 was purified by silica gel column chromatography (5% MeOH in EtOAc) to give 5.5 mg (0.012 mmol) the sulfonamide 29 in 73% yield. R ₇ = 0.7 (5% MeOH in EtOAc); ¹ H NMR (400 MHz,

75

CDCl ₃ ) δ 6.31 (IH, d, J= 1 1.2 Hz), 5.85 (IH, d, J= 11.2 Hz), 4.18-4.08 (IH, m), 4.07-4.00 (IH, m), 3.13 (2H, q, J = 6.8 Hz), 2.96 (3H, s), 2.80 (IH, d, J = 11.6 Hz), 2.74 (IH, d, J = 12.8 Hz), 2.62 (2H, brs), 2.48 (IH, d, J = 12.8 Hz), 2.26-2.16 (3H, m), 2.10-1.00 (19H, m), 0.91 (3H, d, J = 6.0 Hz), 0.54 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) 6143.4131.3, 124.1, 1 15.5, 67.6, 67.5, 56.7, 56.5, 46.0, 44.9, 43.6, 42.4, 40.6, 37.4, 36.2, 35.9, 30.5, 29.2, 27.9, 27.3, 25.9, 23.7, 22.5, 19.0, 12.3; IR (KBr) v 3294 (br), 2933, 2869, 1453, 1318, 1149, 1048, 975, 732, 523 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 490.2967, found = 490.2967.

[00206] N-((R)-5-((lR,3aS,laRβ)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l- yl)hexyl)trifluoromethanesulfonamide (22):

Chemical Formula: C ₂ SH ₄₀ F ₃ NO ₄ S Molecular Weight: 507,65

[00207] This was prepared from amine 54 by following the same procedure described for 19. The reagents used were as follows: amine 54 (20.9 mg, 0.035 mmol, 1 equiv), trifluoromethanesulfonyl chloride (A μL, 0.038 mmol, 1.1 equiv) and Et ₃ N (14 μL, 0.105 mmol, 3eq). 22 was purified by silica gel column chromatography (5% MeOH in EtOAc) to give 6.2 mg (0.012 mmol) the sulfonamide 22 in 35% yield. R ₇ = 0.4 (EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J= 11.4 Hz), 5.85 (IH, d, J= 1 1.4 Hz), 4.17-4.08 (IH, m), 4.08-4.00 (IH, m), 3.30 (2H, q, J= 7.2 Hz), 2.79 (IH, dd, J= 12.0, 4.0 Hz), 2.74 (IH, dd, J= 13.2, 4.0 Hz), 2.48 (IH, dd, J= 13.6, 3.2 Hz), 2.26-2.16 (2H, m), 2.04-1.75 (5H, m), 1.74-1.18 (14H, m), 1.13-1.02 (IH, m), 0.92 (3H, t, J = 6.4 Hz), 0.54 (3H, s); ' ³ C NMR (75 MHz, CDCl ₃ ) δ 143.2, 131.4, 124.1, 115.5, 67.7, 67.5, 56.6, 56.5, 46.0, 44.9, 44.8, 42.4, 40.7, 37.4, 36.2, 35.5, 31.0, 29.1, 27.9, 23.7, 23.1, 22.5, 18.9, 12.3; (KBr) v 3325 (br), 2940, 2871, 1435, 1371, 1221, 1188, 1148, 1045, 606 cm ^'1 ;

76

HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 530.2528, found = 530.2518.

[00208] N-((λ)-5-((lR,3a5,7aR^)-4-(2-((3λ,5λ)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)hexyl)butane-l- sulfonamide (25):

Chemical Formula: C ₂₈ H ₄₉ NO ₄ S Molecular Weight: 495,76

[00209] This was prepared from amine 54 by following the same procedure described for 19. The rs mine 54 (22.1 mg, 0.036 mmol, 1 equiv), butylsulfonyl chloride (5.2 μL, 0.040 mmol, 1.1 equiv) and Et ₃ N (15 μL, 0.108 mmol, 3eq). 25 was purified by silica gel column chromatography (EtOAc) to give 10.1 mg (0.020 mmol) the sulfonamide 25 in 57% yield. R _/ = 0.3 (EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.30 (IH, d, J = 11.4 Hz), 5.84 (IH, d, J = 11.4 Hz), 4.15-4.08 (IH, m), 4.07-3.98 (IH, m), 3.10 (2H, q, J = 6.8 Hz), 3.05-2.97 (2H, m), 2.79 (IH, dd, J = 12.0, 3.6 Hz), 2.74 (IH, dd, J = 13.6, 3.6 Hz), 2.47 (IH, dd, J= 13.6, 3.2 Hz), 2.27-2.16 (2H, m), 2.04-1.16 (23H, m), 1.13-1.01 (IH, m), 0.95 (3H, t, J= 7.6 Hz), 0.91 (3H, d, J= 6.8 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 143.2, 131.4, 124.0, 115.5, 67.6, 67.4, 56.6, 56.5, 52.6, 46.0, 44.9, 43.6, 42.4, 40.7, 37.4, 36.2, 35.6, 31.1, 29.1, 27.9, 25.9, 23.7, 23.4, 22.5, 21.8, 19.0, 13.8, 12.3; (KBr) v 3290 (br), 2937, 2871, 1449, 1320, 1141, 1047, 734 cm ^'1 ; HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 518.3280, found = 518.3267.

[00210] λH(/?)-5-((lλ,3a£,7afl,£)-4-(2-((3λ,5/?)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyI-octahydro-/H- inden-l- yl)hexyl)benzenesulfonamide (26):

77

Chemical Formula: C ₃₀ H ₄₅ NO ₄ S Molecular Weight: 515,75

[00211] This was prepared from amine 54 by following the same procedure described for 19. The reagents used were as follows: amine 54 (16.2 mg, 0.027 mmol, 1 equiv), benzenesulfonyl chloride (6.8 μL, 0.052 mmol, 2 equiv) and Et ₃ N (11 μL, 0.078 mmol, 3eq). 26 was purified by silica gel column chromatography (EtOAc) to give 5.1 mg (0.010 mmol) the sulfonamide 26 in 37% yield. R _/ = 0.4 (EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (2H, d, J = 7.2 Hz), 7.58 (l ^Ï„Ï„ ' ^Ï„ " - " ^ " ^« ^" - ^•■ - 7.2 Hz), 6.31 (IH, d, J = 11.6 Hz), 5.84 (IH, d, J= 11.6 Hz), A.Dj _U Ï€, i, j - Ï….u Ï€^, t.u-t.Ï…o _> JH, m), 4.08-3.99 (IH, m), 2.95 (2H, q, J= 6.4 Hz), 2.79 (IH, dd, J = 12.4, 4.0 Hz), 2.74 (IH, dd, J = 13.2, 3.6 Hz), 2.48 (IH, dd, J= 13.6, 2.8 Hz), 2.28-2.16 (2H, m), 2.04-1.91 (3H, m), 1.90-1.76 (2H, m), 1.65-1.59 (2H, m), 1.58-1.06 (1 IH, m), 1.05-0.92 (IH, m), 0.86 (3H, d, J= 6.0 Hz), 0.51 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 143.2, 140.2, 132.8, 131.4, 127.3, 124.1, 115.5, 67.6, 67.5, 56.5, 46.0, 44.9, 43.5, 42.4, 40.6, 37.4, 36.1, 35.5, 30.3, 29.1, 27.9, 23.2, 22.5, 18.9, 12.3; (KBr) v 3290 (br), 2940, 2870, 1446, 1323, 1159, 1046, 905, 732, 689, 586 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 538.2967, found = 538.2969.

[00212] 4-cyano-λK(φ-5-((li^3a£,7aR,£>4-(2-((3R,5/?)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-i// -inden-l- yl)hexyl)benzenesulfonamide (27):

78

Chemical Formula: C _3I H ₄₄ N ₂ O ₄ S Molecular Weight: 540,76

[00213] This was prepared from amine 54 by following the same procedure described for 19. The reagents used were as follows: amine 54 (14 mg, 0.024 mmol, 1 equiv), 4- cyanobenzenesulfonyl chloride (9.6 mg, 0.048 mmol, 2 equiv) and Et ₃ N (10 μL, 0.072 mmol, 3eq). 27 was purified by silica gel column chromatography (EtOAc) to give 8.0 mg (0.014 mmol) the sulfonamide 27 in 61% yield. R ₇ = 0.5 (EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.98 (2H, d, J= 8.0 1 IH, d, J= 11.6 Hz), 5.85 (IH, d, J= 11.6 Hz),

4.56 (IH, t, J = 6.0 Hz), 4.17-4.10 (IH, m), 4.10-4.01 (IH, m), 2.99 (2H, q, J = 6.7 Hz), 2.79 (IH, dd, J= 12.4, 4.0 Hz), 2.73 (IH, dd, J= 13.2, 4.0 Hz), 2.48 (IH, dd, J = 13.2, 3.2 Hz), 2.17- 2.29 (2H, m), 2.03-1.90 (3H, m), 1.76-1.88 (2H, m), 1.72-1.61 (2H, m), 1.61-1.08 (HH, m), 1.06-0.92 (IH, m), 0.87 (3H, d, J = 6.0 Hz), 0.51 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 144.7, 143.1 , 133.2, 131.5, 127.9, 124.0, 1 17.5, 1 16.6, 115.6, 67.6, 67.5, 56.53, 56.48, 45.9, 44.9, 43.6, 42.4, 40.6, 37.4, 36.1, 35.4, 30.3, 29.1, 27.9, 23.7, 23.2, 22.5, 18.9, 12.3; (KBr) v 3293 (br), 2939, 2869, 1434, 1332, 1 160, 1091, 1045, 837, 735, 634, 574, 517 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 563.2919, found = 563.2918.

[00214] N-((R)-5-((1R,3ΆS,1OR£)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)hexyl)-2- hydroxyethanesulfonamide (24):

79

Chemical Formula: C ₂ GH ₄₅ NO ₅ S Molecular Weight: 483,7

[00215] This was prepared from amine 54 by following the same procedure described for 19. The reagents used were as follows: amine 54 (14 mg, 0.023 mmol, 1 equiv), 2- hydroxyethanesulfonyl chloride (3.4 mg, 0.046 mmol, 2 equiv) and Et ₃ N (10 μL, 0.069 mmol, 3eq). 24 was purified by silica gel column chromatography (5% MeOH in EtOAc) to give 4 mg (0.008 mmol) the sulfonamide 24 in 35% yield. Ry= 0.2 (5% MeOH in EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ H, d, J = 11.8 Hz), 4.19-4.09 (IH, m), 4.09-

4.00 (IH, m), 4.07 (2H, t, J= 6.8 Hz), 3.17 (2H, t, J= 6.8 Hz), 3.03 (2H, t, J= 6.8 Hz), 2.79 (IH, d, J = 13.6 Hz), 2.75 (IH, d, J = 12.8 Hz), 2.48 (IH, d, J = 12.0 Hz), 2.17-2.25 (2H, m), 2.03- 1.15 (19H, m), 1.14-1.01 (IH, m), 0.92 (3H, d, J = 6.4 Hz), 0.54 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 143.3, 131.3, 124.1, 115.5, 67.7, 67.5, 57.4, 56.7, 56.5, 53.6, 47.3, 46.0, 44.9, 42.4, 40.7, 37.4, 36.2, 36.0, 29.1, 28.3, 27.9, 23.9, 23.7, 22.5, 19.0, 12.3; (KBr) v 3354 (br), 2934, 2867, 1724, 1316, 1201, 1170, 1143, 1046, 732 cm ^'1 ; HRMS (ESI): m/z calcd. For [(M+Na ⁺ ] = 506.2916, found = 506.2919.

[00216] N-((R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l- yl)hexyl)ethanesulfonamide (23):

80

Chemical Formula: 0 ₂₆ H ₄₅ NO ₄ S Molecular Weight: 467,7

[00217] This was prepared from amine 54 by following the same procedure described for 19. The reagents used were as follows: amine 54 (14 mg, 0.024 mmol, 1 equiv), ethanesulfonyl chloride (4.5 μL, 0.048 mmol, 2 equiv) and Et ₃ N (10 μL, 0.072 mmol, 3eq). 23 was purified by silica gel column chromatography (EtOAc) to give 6.0 mg (0.013 mmol) the sulfonamide 23 in 35% yield. R ₇ = 0.2 (EtOAc); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J = 1 1.4 Hz), 5.85 ( ^{" " " '} i, m), 4.08-4.01 (IH, m), 3.15-3.08 (2H, m),

3.04 (2H, q, J= 1.1 Hz), λ. /y (IH, dd, ./ = 1 1.0, 4.U Hz), 2.74 (IH, d, J= 13.2, 4.0 Hz), 2.48 (IH, dd, J= 13.6, 3.2 Hz), 2.27-2.16 (2H, m), 2.03-1.75 (5H, m), 1.73-1.16 (14H, m), 1.37 (3H, t, J = 7.2 Hz), 1.12-1.08 (IH, m), 0.92 (3H, d, J= 6.4 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 143.0, 131.1, 123.8, 115.3, 67.4, 67.2, 56.4, 56.3, 46.8, 45.8, 44.6, 43.4, 42.1, 40.4, 37.2, 36.0, 35.4, 30.9, 28.9, 27.7, 23.5, 23.1, 22.2, 18.8, 12.1, 8.4; (KBr) v 3291 (br), 2939, 2876, 1451, 1371, 1141, 1046, 910, 732 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 490.2967, found = 490.2966.

[00218] (R^-methyl7-((lR,3aS,7aR^-4-(2-((3λ,5R)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-iH-inden-l-yl)-2- (tert-butyldimethylsilyIoxy)oct-2-enoate (70):

81

Chemical Formula: C ₄₅ H ₈₄ O ₅ Si ₃ Molecular Weight: 789,4

[00219] In a flame dried round bottom flask under argon atmosphere at -78 ⁰ C,

NaHMDS (94 μL of a 1 M solution in THF, 0.095 mmol, 1.3 equiv) was added to a solution of methyl-2-(dimethoxyphosphoryl)-2-methoxyacetate (34 mg, 0.110 mmol, 1.5 equiv) in dry THF (1 mL). The solution was stirred for 15 min at -78 ⁰ C, then a solution of aldehyde 49 (44 mg, 0.073 mmol, 1 equiv) in dry THF (0.5 mL) was slowly added by cannula. The solution was then slowly warmed >vernight. The solution was quenched by the addition of aq. sat. NH ₄ Cl (2 mL) and exctrated with Et ₂ O (3 x 5 mL), the combined organic layers were washed with water (5 mL) and brine (5 mL) then dried (5 mL). The solution was concentrated in vacuo then purified by silica gel column chromatography (5% EtOAc in Hexanes) to obtain 70 in 71% yield (41 mg, 0.052 mmol). R ₇ = 0.7 (10% EtOAc in Hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.17 (IH, d, J= 10.0 Hz), 5.81 (IH, d, J= 10.0 Hz), 5.51 (IH, t, J = 8.0 Hz), 4.13-4.02 (2H, m), 3.75 (3H, s), 2.81 (IH, d, J= 12.0), 2.49-2.33 (4H, m), 2.25 (IH, d, J = 14.0 Hz), 2.10 (IH, dd, J = 12.2, 8.2 Hz), 2.04-1.94 (2H, m), 1.93-1.83 (IH, m), 1.83-1.73 (IH, m), 1.72-1.22 (14H, m), 1.14-1.01 (IH, m), 1.00-0.91 (12H, m), 0.87 (9H, m), 0.86 (9H, m), 0.53 (3H, s), 0.12 (6H, s), 0.05 (12H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 165.5, 141.0, 140.1, 133.9, 126.6, 122.0, 116.3, 68.3, 68.2, 56.7, 56.5, 51.6, 46.2, 45.8, 43.9, 40.8, 37.0, 36.2, 35.7, 29.9, 28.9, 27.9, 27.4, 26.7, 26.10, 26.07, 25.8, 23.6, 22.4, 19.0, 18.44, 18.38, 18.34, 12.3, -4.4, - 4.5, -4.6, -4.7 ; IR (KBr) v 2951, 2857, 1727, 1636, 1468, 1360, 1252, 1087, 837, 777 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 81 1.5524, found = 81 1.5509.

82

[00220] (R)-methyl-7-((lR,3aS,7aR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyloctahydro-/H-i nden-l-yl)-2-oxooctanoate

(71):

Chemical Formula: C27η42O5 Molecular Weight: 446,62

[00221] A 48% solution of HF (1 drop) was added to a solution of 70 (7 mg, 0.009 mmol) in CH ₂ Cl ₂ (0.3 raL) and CH ₃ CN (0.3 mL) and the solution was stirred at room temperature for 2 h. The mixture was concentrated in vacuo and the residue was loaded directly on to silica gel. ;OEt in hexanes) as a white solid in 85% yield

(3.9 mg, 0.007 mmol). ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J= 11.4 Hz), 5.85 (IH, d, J = 11.4 Hz), 4.18-4.08 (IH, m), 4.08-4.01 (IH, m), 3.87 (3H, s), 2.84 (2H, t, J= 7.2 Hz), 2.79 (IH, dd, J = 12.0, 3.6 Hz), 2.74 (IH, dd, J = 13.2, 3.6 Hz), 2.48 (IH, d, J = 13.6 Hz), 2.28-2.16 (2H, m), 2.06-1.74 (5H, m), 1.72-1.16 (14H, m), 1.14-1.02 (IH, m), 0.91 (3H, m, J = 6.0 Hz), 0.54 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 194.6, 161.8, 143.3, 131.3, 124.1, 115.5, 67.6, 67.5, 56.7, 53.1, 46.0, 44.9, 42.4, 40.7, 39.6, 37.4, 36.1, 35.7, 29.1, 27.9, 25.7, 23.7, 23.6, 22.5, 19.0, 12.3 ; IR (KBr) v 3380 (br), 2941, 2869, 1730, 1437, 1262, 1048, 733, 668 cm ^'1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 469.2930, found = 469.2920.

[00222] (R)-methy\-l-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5-bis(tert- butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl- octahydro-2H-inden-l-yl)-2- oxooctanoate (37):

83

Chemical Formula: 02 ₇ H ₄₂ O ₅ Molecular Weight: 446.62

[00223] To a solution of enol ether 70 (29 mg, 0.037 mmol, 1 equiv) and AcOH (11 μL, 0.185 mmol, 1 equiv) in 0.5 mL of MeCN and 0.5 mL Of CH ₂ Cl ₂ at 0 ⁰ C was added CsF (14 mg, 0.092 mmol, 2.5 equiv) in one portion. The mixture was allowed to warm to room temperature and then stirred for an additional 75 min. The reaction mixture then was diluted with 10 mL of EtOAc and 5 mL of sat. NaHCO ₃ . The layers were separated and the organic layer was washed with s? ^{f M} " ^urιπ - ^ â„¢ ^{Ï„ λ} * ^nA ^" ^« ° ^ mL), then dried (Na ₂ SO ₄ ) and concentrated.

Purification of . _o ~. _v _ ,„ ~ . .c in hexanes) gave 23 mg (0.034 mmol, 92%) of the a-keto ester 37. R _/ = 0.4 (5% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.16 (IH, d, J = 11.0 Hz), 5.81 (IH, d, J= 11.0 Hz), 4.13-4.01 (2H, m), 3.86 (3H, s), 2.84 (2H, t, J= 7.2), 2.44-2.32 (2H, m), 2.25 (IH, d, J= 12.4 Hz), 2.10 (IH, dd, J= 12.8, 8.4 Hz), 2.03-1.94 (2H, m), 1.93-1.82 (IH, m), 1.82-1.74 (IH, m), 1.71-1.16 (15H, m), 1.12-1.01 (IH, m), 0.91 (3H, d, J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (6H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 194.6, 166.3, 141.0, 133.9, 121.9, 116.4, 68.3, 68.2, 56.7, 56.5, 56.3, 53.1, 46.2, 45.9, 45.7, 43.9, 40.8, 39.6, 37.0, 36.2, 35.8, 28.9, 27.9, 26.10, 26.07, 25.7, 23.6, 22.4, 19.0, 18.4, 12.3, -4.4, -4.5, -4.6, -4.7 ; IR (KBr) v 2950, 2856, 1732, 1468, 1253, 1081, 1052, 836, 775 cm ^"1 ; HRMS (ESI): m/z calcd. for [(M+Na) ⁺ ] = 697.4659, found = 697.4647.

[00224] (R)-7-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)-λ ^L methyI-2- oxooctanamide (38):

84

Chemical Formula: C ₂₇ H ₄₃ NO ₄ Molecular Weight: 445,63

[00225] MeNH ₃ Cl (3.4 mg, 0.051 mmol, 1.5 equiv) was added to a stirred solution of keto ester 71 (23 mg, 0.034 mmol, 1 equiv) in NEt ₃ (1 mL) at 0 ⁰ C. The solution was warmed to room temperature and stirred for 2 days. The solution was concentrated and the residue disolved in EtOAc (10 mL). The organic layer was washed with sat. NaHCO ₃ (2 x 5 mL), H ₂ O (5 mL), brine (5 mL) and dried (Na ₂ SO ₄ ). Purification by silica gel column chromatography (5% to 10% EtOAc in hexa = 0.2 (10% EtOAc in hexanes)] witch was directly submited to the alcohols deprotection using the same procedure as for 37. Purification by silica gel column chromatography (50% EtOAc in hexanes) gave 38 in 53% yield (8.0 mg, 0.018 mmol). R _/ = 0.4 (50% EtOAc in hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.95 (IH, brs), 6.31 (IH, d, J= 10.8 Hz), 5.85 (IH, d, J= 10.8 Hz), 4.19-4.00 (2H, m), 2.97-2.85 (5H, m), 2.77 (2H, t, J= 14.1 Hz), 2.48 (IH, d, J= 12.0 Hz), 2.28-2.16 (2H, m), 2.07-1.15 (19H, m), 1.14-1.00 (IH, m), 0.91 (3H, d, J = 6.0 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 199.5, 161.1, 143.4, 131.3, 124.1, 115.5, 67.7, 67.5, 56.7, 56.5, 46.0, 44.9, 42.4, 40.7, 37.4, 37.0, 36.2, 35.8, 29.2, 27.9, 26.1, 25.8, 23.9, 23.7, 22.5, 19.0, 12.3 ; IR (KBr) v 3376 (br), 2928, 1717, 1673, 1538, 1453, 1047, 668 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 468.3096, found = 468.3082.

[00226] l-((R)-5-((l/?,3a£,7aR,£)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-/H- inden-l-yl)hexyl)-3- hydroxyurea (39):

85

Chemical Formula: C ₂ SH ₄₂ N ₂ O ₄ Molecular Weight: 434,61

[00227] Amine 54 (13.1 mg, 0.022 mmol, 1 equiv) in CH ₂ Cl ₂ (130 μL) was added dropwise to a stirred solution of 1 , 1 -carbonyldiimidazole (25.6 mg, 0.22 mmol, 10 equiv) in THF (50 μL) under argon cooled to 0 ⁰ C. The reaction mixture was stirred 16 h at room temperature, then O-terrt)utyldimetylsilylhydroxylamine (95 mg, 0.66 mmol, 30 equiv) was added to the mixture, the solution was then allowed to stir a further 16 h. The reaction mixture was diluted with EtOAc (5 mL), washed with 0.1 M HCl (2 x 5 mL), water (5mL), brine (5 mL) and then dried (Na ₂ SO ₄ ) :d in vacuo to provide an oil witch was purified by silica gel column chromatography (50% EtOAc in hexanes, R/ = 0.2) to give the O- tørλutyldimethylsilylhydroxyurea. The product was then directly submited to deprotection: the oil was dissolved in CH ₂ Cl ₂ (100 μL) and CH ₃ CN (100 μL) then a 48% solution of HF was added (2 drops) and the solution was stirred overnight. The mixture was quenched cautionly by the addition of sat. NaHCO ₃ until no effervescence was observed then acidified with a 1 M aq. solution of citric acid (5 mL). The solution was extracted with CH ₂ Cl ₂ (3 x 5 mL) then the combined organic layers were washed with H ₂ O (5 mL), brine (5 mL), dried (MgSO ₄ ) and then concentrated in vacuo. The oil was purified by octadecyl-fonctionalized silica gel column chromatography (100% H ₂ O to 100% MeOH) to afford the hydroxyurea in 44% yield (4.2 mg, 0.009 mmol). R ₇ = 0.2 (88/10/2 CH ₂ Cl ₂ MeOH/ CH ₃ COOH); ¹ H NMR (400 MHz, CD ₃ OD) δ 6.21 (IH, d, J = 11.4 Hz), 5.88 (IH, d, J = 1 1.4 Hz), 4.08-4.01 (IH, m), 4.00-3.93 (IH, m), 3.18 (2H, t, J = 6.8 Hz), 2.83 (IH, d, J = 9.6 Hz), 2.59 (IH, d, J= 6.0 Hz), 2.41 (IH, d, J = 10.4 Hz), 2.25-2.12 (2H, m), 2.06-1.98 (2H, m), 1.97-1.18 (17H, m), 1.15-1.04 (IH, m), 0.95 (3H, d, J = 6.0 Hz), 0.57 (3H, s); ¹³ C NMR (75 MHz, CD ₃ OD) δl70.9, 140.9, 132.7, 122.2, 1 15.9, 66.8, 66.5, 56.7, 56.3, 45.6, 44.2, 41.4, 40.7, 39.9, 36.4, 36.2, 35.6, 30.5, 28.6, 27.5, 23.3, 22.1, 18.1 ,

86

11.2; (KBr) v 3312 (br), 2937, 2841, 1651, 1457, 1049, 735 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+Na) ⁺ ] = 457.3042, found = 457.3042.

[00228] 2-((R)-5-((lR,3aS,laR^)-4-(2-((3R,5R)-3,5- dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-7H- inden-l-yl)hexylamino)-2- oxoethyl ethyl carbonotrithioate (40):

Chemical Formula: C29H47NO3S3 Molecular Weight: 553,88

[00229] NaHMDS (42 μL of a 1 M solution in THF, 0.042 mmol, 3 equiv) was added to a stirred solution of ethanethiol (3.1 μL, 0.042 mmol, 1 equiv) in dry THF (200 μL) cooled to 0 ⁰ C under argon. The solution was stirred at 0 ⁰ C for 10 min then CS ₂ (3.3 μL, 0.056 mmol, 4 equiv) was added dropwise and the reaction mixture was stirred at room temperature for 1 h before a solution of 35 (7 mg, 0.014 mmol, 1 equiv) in dry THF (150 μL) was added. After stirring for an additional 1 h at room temperature, the reaction mixture was quenched by addition of sat. NH ₄ CI (3 mL) and extracted with EtOAc (3 x 5 mL). The combined organic fraction were dried (Na ₂ SO ₄ ) and concentrated in vacuo, the oil was then purified by silica gel column chromatography (50% Acetone in Hexanes) to afford the trithiocarbonate 40 in 82% yield (6.4 mg, 0.011 mmol). R ₇ = 0.2 (50% Acetone in Hexanes); ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.31 (IH, d, J= 11.4 Hz), 6.29 (IH, brs), 5.85 (IH, d, J= 11.4 Hz), 4.15-4.09 (IH, m), 4.07-4.01 (IH, m), 4.06 (2H, s), 3.39 (2H, q, J= 7.6 Hz), 3.23 (2H, d, J= 7.6 Hz), 2.79 (IH, dd, J= 12.4, 3.6 Hz), 2.74 (IH, d, J = 13.2, 3.6 Hz), 2.47 (IH, dd, J = 13.2, 2.8 Hz), 2.25-2.16 (2H, m), 2.04-1.10 (19H, m), 1.37 (3H, t, J= 7.6 Hz), 1.09-0.99 (IH, m), 0.90 (3H, d, J= 6.4 Hz), 0.53 (3H, s); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 223.5, 166.8, 143.0, 131.3, 124.1, 115.5, 67.6, 67.5, 56.6, 56.5, 46.0,

87

44.9, 42.4, 40.7, 40.2, 39.4, 37.4, 36.2, 35.7, 32.3, 30.0, 29.1, 27.9, 23.7, 23.5, 23.5, 19.0, 13.5, 1122..33;; ((KKBBrr)) vv 33229944 ((bbrr)),, 22992299,, 22886699,, 11665533,, 11555511,, 11444466,, 113377f5, 1079, 1046, 811 cm ^"1 ; HRMS (ESI): m/z calcd. For [(M+H) ⁺ ] = 554.2796 , found = 554.2782.

88

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