WO2006060885A1 - 7-(2-cyclohexylidene-ethylidene)-spiro[5.5]undecanes useful for making pharmaceutical compositions - Google Patents

Abstract

The invention relates to novel analogues of the hormonally active metabolite of vitamin D3, and more specifically provides 7- (2-cyclohexylidene-ethylidene) -spiro [5.5] undecanes and methods for making them, as well as pharmaceutical compositions which comprise said 7- (2-cyclohexylidene-ethylidene) -spiro [5.5] undecanes, and methods for treating and preventing diseases, illnesses and disorders in humans, in particular conditions related to cell proliferation and bone disorders such a osteoporosis, by the administration of effective amounts of said compounds.

Description

7-(2-CYCLOHEXYLIDENE-ETHYLIDENE)-SPIRO[S-S]UNDECANES USEFUL FOR MAKING PHARMACEUTICAL COMPOSITIONS

FIELD OF THE INVENTION The present invention relates to novel analogues of the hormonally active metabolite of vitamin D₃. It relates more specifically to 7-(2-cyclohexylidene-ethylidene)- spiro[5.5]undecanes and methods for making them, as well as compositions, in particular pharmaceutical compositions which comprise said 7-(2-cyclohexylidene- ethylidene)-spiro[5.5] undecanes, and methods for treating and preventing diseases, illnesses and disorders in humans by the administration of effective amounts of said 7- (2-cyclohexylidene-ethylidene)-spiro[5.5]undecanes.

BACKGROUND OF THE INVENTION

Over the last decade there has been a continuous interest in the development of analogues of 1α, 25(OH)₂D₃ (calcitriol), the hormonally active metabolite of vitamin D₃ (cholecalciferol), with the purpose of separating calcemic and prodifferentiating and/or antiproliferative activities. Various structural modifications have been introduced in the parent compound, such as the 22-oxa modification in the side chain leading to a first analogue (OCT) that showed the desired dissociation in activities. Epimerisation at C-20 (MC1288) may also generate derivatives with enhanced biological activity, whereas removal of C-19 is usually accompanied by a reduction in calcemic activity. Whereas the above modifications are located in the flexible parts of the molecule, i.e. the side chain or A-ring, very few publications focused on structural changes within the central CD-ring skeleton of the molecule, the part that is, from a synthetic point of view, the least accessible one.

Although a few useful analogues of 1 α,25(OH)₂D₃ are already known via various structural modifications of the molecule, there is a continuous need in the art for the design of new such analogues being able to show a specific profile of biological activities and which may therefore be used in the manufacture of novel medicaments.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that 7-[2-(substituted or unsubstituted cyclohexylidene)-ethylidene]-spiro[5.5]undecanes can be made in good yield via a convenient synthetic route and that some of them show useful biological activities which make them suitable for the manufacture of pharmaceutical compositions for the prevention or treatment of bone disorders, immune disorders, inflammatory diseases, hyperproliferative disorders and cancer in humans and other mammals.

DEFINITIONS

As used herein, the term complex, or coordination compound, refers to the result of a donor-acceptor mechanism or Lewis acid-base reaction between a metal (the acceptor) and several neutral molecules or ionic compounds called ligands, each containing a non-metallic atom or ion (the donor).

As used herein, the term " monometallic " refers to a complex in which there is a single metal center.

As used herein, the term " heterobimetallic " refers to a complex in which there are two different metal centers. As used herein, the term " homobimetallic " refers to a complex having two identical metal centers, which however need not have identical ligands or coordination number.

As used herein with respect to a substituting group, the term " C_1-7 alkyl " means straight and branched chain saturated acyclic hydrocarbon monovalent groups or radicals having from 1 to 7 carbon atoms such as, for example, methyl, ethyl, propyl, n- butyl, 1 -methylethyl (isopropyl), 2-methylpropyl (isobutyl), 1 ,1-dimethylethyl (ter-butyl),

2-methylbutyl, n-pentyl, dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, n-heptyl and the like; optionally the carbon chain length of such group may be extended to 20 carbon atoms, such as n-octyl, n-decyl, n-dodecyl and the like; optionally the said C_1-7 alkyl or C_1-2O alkyl group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a substituting group, and unless otherwise stated, the term " acyl " broadly refers to a carbonyl (oxo) group adjacent to a C_1-7 alkyl or C_1-20 alkyl radical, a C_3-10 cycloalkyl radical, an aryl radical, an arylalkyl radical or a heterocyclic radical, all of them being such as herein defined; representative but non- limliting examples thereof include acetyl, benzoyl, naphthoyl and the like.

As used herein with respect to a linking group, the term " C_1-7 alkylene " means the divalent hydrocarbon radical corresponding to the above defined C_1-7 alkyl, such as methylene, bismethylene, trismethylene, tetramethylene, hexamethylene and the like; optionally the said C_1-7 alkylene linking group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a substituting radical or group, the term " C_3-10 cycloalkyl " means a mono- or polycyclic saturated hydrocarbon monovalent radical having from 3 to 10 carbon atoms as the only atoms in the ring(s), such as for instance cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl and the like, or a C_7-Io polycyclic or fused saturated hydrocarbon monovalent radical having from 7 to 10 carbon atoms as the only atoms in the ring(s) such as, for instance, octahydro- pentalenyl (C₈), octahydro-1H-indenyl (C₉), 3a,4,5,6,7,7a-hexahydro-3AV-inden-4-yl (C₉), decahydroazulenyl (C₁₀); bicyclo[6.2.0]decanyl (C₁₀), 1,2,3,4,4a,5,8,8a-octahydro- naphthalenyl (C₁₀), bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, 1 ,3-dimethyl[2.2.1]heptan-2-yl, bicyclo[2.2.2]octanyl norbornyl, fenchyl, trimethyltricycloheptyl or adamantyl; optionally the said C_3-I0 cycloalkyl group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a substituting radical, and unless otherwise stated, the term " C_3-10 cycloalkylalkyl " refers to an aliphatic saturated hydrocarbon monovalent radical (preferably a C_1-7 alkyl such as defined above) to which a C_3-10 cycloalkyl (such as defined above) is already linked such as, but not limited to, cyclohexylmethyl, cyclopentylmethyl and the like.

As used herein with respect to a linking group, and unless otherwise stated, the term " C_3-I₀ cycloalkylene " means the divalent hydrocarbon radical corresponding to the above defined C_3-10 cycloalkyl such as, but not limited to, 1 ,2-cyclohexylene and 1 ,4- cyclohexylene; optionally the said C_3-10 cycloalkylene linking group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a substituting radical or group, and unless otherwise stated, the term " aryl " designates any mono- or polycyclic aromatic monovalent hydrocarbon radical having from 6 up to 30 carbon atoms such as but not limited to phenyl, naphthyl, anthracenyl, phenantracyl, fluoranthenyl, chrysenyl, pyrenyl, biphenylyl, terphenyl, picenyl, indenyl, biphenyl, indacenyl, benzocyclobutenyl, benzocyclooctenyl and the like, including fused benzo-C_4-8 cycloalkyl radicals (the latter being as defined above) such as, for instance, indanyl, tetrahydronaphtyl, fluorenyl and the like, all of the said radicals being optionally substituted with one or more substituents being inter alia selected from the group consisting of halogen, amino, hydroxy, cyano, sulfhydryl, dimethylamino, diethylamino, methoxy, trifluoromethyl and nitro, such as for instance 4-fluorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 2,6-diisopropyl-4- bromophenyl, pentafluorophenyl, 4-cyanophenyl, 2,6-dichlorophenyl, 2-fluorophenyl, 3- chloro-phenyl, 3,5-dichlorophenyl and the like.

As used herein with respect to a linking group, and unless otherwise stated, the term " arylene " means the divalent hydrocarbon radical corresponding to the above defined aryl such as, but not limited to, phenylene, naphtylene, biphenylene and the like; optionally the said arylene linking group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a combination of two substituents, and unless otherwise stated, the term " homocyclic " means a mono- or polycyclic, saturated or mono-unsaturated or polyunsaturated hydrocarbon group or divalent radical having from 4 up to 15 carbon atoms but including no heteroatom in the said ring(s); for instance the said combination may form a C_2-6 alkylene radical, such as but not limited to tetramethylene, which cyclizes with the two preferably adjacent carbon atoms onto which the said two combined substituents are attached.

As used herein with respect to a substituting atom, the term " halogen " means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

As used herein with respect to a substituting radical or group, and unless otherwise stated, the term " halo C_1-7 alkyl " means a C_1-7 alkyl radical (such as above defined, i.e. optionally the carbon chain length of such group may be extended to 20 carbon atoms) in which one or more hydrogen atoms are independently replaced by one or more halogens (preferably fluorine, chlorine or bromine) such as, but not limited to, difluoromethyl, trifluoromethyl, trifluoroethyl, octafluoropentyl, dodecafluoroheptyl, dichloromethyl and the like.

As used herein with respect to a substituting radical or group, and unless otherwise stated, the term " C_2-7 alkenyl " means a straight or branched acyclic hydrocarbon monovalent radical having one or more ethylenic unsaturations and having from 2 to 7 carbon atoms such as, for example, vinyl, 1-propenyl, 2-propenyl (allyl), 1- butenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, 3-hexenyl, 2-hexenyl, 2- heptenyl, 1 ,3-butadienyl, pentadienyl, hexadienyl, heptadienyl, heptatrienyl and the like, including all possible isomers thereof; optionally the carbon chain length of such group may be extended to 20 carbon atoms, and optionally the said C_2-7 alkenyl group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below; for instance this definition includes 2-chlorovinyl, 4-hydroxybuten-1- yl and the like.

As used herein with respect to a substituting radical or group, and unless otherwise stated, the term " C_3-10 cycloalkenyl " means a monocyclic mono- or polyunsaturated hydrocarbon monovalent radical having from 3 to 8 carbon atoms, such as for instance cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, 1 ,3,5,7-cyclooctatetraenyl and the like, or a C_7-10 polycyclic mono- or polyunsaturated hydrocarbon monovalent radical having from 7 to 10 carbon atoms such as dicyclopentadienyl, fenchenyl (including all isomers thereof, such as α-pinolenyl), bicyclo[2.2.1]hept-2-enyl (norbornenyl), bicyclo[2.2.1] hepta-2,5-dienyl (norbornadienyl), cyclofenchenyl and the like; optionally the said C_3-10 cycloalkenyl group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a substituting radical or group, the term " C_2-7 alkynyl " defines straight and branched chain hydrocarbon radicals containing one or more triple bonds (i.e. acetylenic unsaturation) and optionally at least one double bond and having from 2 to 7 carbon atoms such as, for example, acetylenyl, 1-propynyl, 2- propynyl, 1-butynyl, 2-butynyl, 2-pentynyl, 1-pentynyl, 3-methyl-2-butynyl, 3-hexynyl, 2- hexynyl, 1-penten-4-ynyl, 3-penten-1-ynyl, 1,3-hexadien-1-ynyl and the like, including all possible isomers thereof; optionally the carbon chain length of such group may be extended to 20 carbon atoms, and optionally the said C_2-7 alkynyl group may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

As used herein with respect to a substituting radical or group, and unless otherwise stated, the terms " arylalkyl " and " arylalkenyl " refer to an aliphatic saturated or ethylenically unsaturated hydrocarbon monovalent radical (preferably a C_1-7 alkyl or C_2-7 alkenyl radical such as defined above, i.e. optionally the carbon chain length of such group may be extended to 20 carbon atoms) onto which an aryl radical (such as defined above) is already bonded, and wherein the said aliphatic radical and/or the said aryl radical may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, hydroxyl, sulfhydryl, Ci_-7 alkyl, trifluoromethyl and nitro such as, but not limited to, benzyl, 4-chlorobenzyl, A- fluorobenzyl, 2-fluorobenzyl, 3,4-dichlorobenzyl, 2,6-dichlorobenzyl, 3-methylbenzyl, A- methylbenzyl, 4-ter-butylbenzyl, phenylpropyl, 1-naphthylmethyl, phenylethyl, 1-amino-2- phenylethyl, 1-amino-2-[4-hydroxyphenyl]ethyl and styryl.

As used herein, and unless otherwise stated, the terms " alkylcycloalkyl ", " alkenylaryl " and " alkylaryl " respectively refer to an aryl or C_3-10 cycloalkyl radical (such as defined above) onto which are already bonded one or more aliphatic saturated or unsaturated hydrocarbon monovalent radicals, preferably one or more C-_I-7 alkyl, C_2-7 alkenyl or C_3-10 cycloalkyl radicals as defined above, such as, but not limited to, o-toluyl, m-toluyl, p-toluyl, 2,3-xylyl, 2,4-xylyl, 3,4-xylyl, o-cumenyl, m-cumenyl, p-cumenyl, o- cymenyl, m-cymenyl, p-cymenyl, mesityl and ter-butylphenyl; optionally the said aliphatic monovalent radical may be substituted with one or more functional heteroatoms or functional groups of atoms such as described below.

The term " substituted " is used throughout the specification and the above definitions. The term " substituted " as defined herein refers to " a hydrogen atom being replaced by a substituent or unit " as defined herein below. Some units or substituents are capable of replacing two or more hydrogen atoms of one or more carbon atoms at a time, e.g. on two adjacent carbons. For example, a substituted unit that requires a single hydrogen atom replacement includes halogen, hydroxyl, sulfhyfryl, nitro, amino and the like. A substituted unit that requires twin hydrogen atoms replacement on the same carbon atom includes carbonyl, oximino, and the like. A substituted unit that requires two hydrogen atoms replacement from adjacent carbon atoms includes epoxy, thiirane and the like. A substituted unit that requires three hydrogen atoms replacement includes cyano and the like. When a moiety, radical or group is described as " substituted ", this means that any number of the hydrogen atoms available for substitution may be replaced, and that such replacement may be effected independently from the replacement of other hydrogen atoms on the same carbon atom or another adjacent or non-adjacent carbon atom.

The following are non-limiting examples of categories of units (usually functional heteroatoms or functional groups of atoms) which can suitably and independently substitute for hydrogen atoms in the compounds of the present invention. In the following, each R³⁰ may be independently selected from the group consisting of hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl and aryl (each of the latter being as defined above). i) -NHCOR³⁰; for example, -NHCOCH₃, -NHCOCH₂CH₃, -NHCOC₆H₅; ii) -COR³⁰; for example, -COCH₃, -COCH₂CH₃, -COCH₂CH₂CH₃; iii) -CO₂R³⁰; for example, -CO₂CH₃, -CO₂CH₂CH₃, -CO₂CH₂CH₂CH₃; iv) -OCOR³⁰; for example, -OCOCH₃, -OCOCH₂CH₃, -OCOCH₂CH₂CH₃; v) -C(=NH)NH₂; vi) -NHC(=NH)NH₂; vii) -N(R³⁰)₂; for example, -NH₂, -NHCH₃, -N(CH₃)₂, -NH(CH₂CH₃); viii) -NHC₆H₅; ix) =CHC₆H₅; x) -CON(R³⁰)₂; for example, -CONH₂, -CONHCH₃, -CON(CH₃)₂; xi) -CONHNH₂; xii) -NHCN; xiii) -OCN; xiv) -CN; xv) halogen; xvi) -NHN(R³⁰)₂; for example, -NHNH₂, -NHNHCH₃, -NHN(CH₃)₂; xvii) -OR³⁰; for example, -OH, -OCH₃, -OCH₂CH₃, -OCH₂CH₂CH₃; xviii) -NO₂; xix) -CH₁₇₁X_n; wherein X is halogen, the index m can be selected from O, 1 and 2, the index n can be selected from O, 1 , 2 and 3, and m+n = 3; for example, -CF₃, -CCI₃, -CBr₃; xx) -SO₂R³⁰; for example, -SO₂CH₃, -SO₂CH₂CH₃, -SO₂C₆H₅; xxi) -OSO₂R³⁰; for example, -OSO₂CH₃, -OSO₂CH₂CH₃, -OSO₂C₆H₅; xxii) -OSO₃R³⁰; for example, -OSO₃CH₃, -OSO₃C₆H₅; xxiii) -SO₂N(R³⁰)₂; for example, -SO₂NH₂; -SO₂NHCH₃; -SO₂NHC₆H₅; xxiv) =0; xxv) =NR³⁰; for example, =NH, =NCH₃₎ =NCH₂CH₃, xxvi) two hydrogen atoms on adjacent carbon atoms are substituted by a single oxygen or sulfur atom, thereby forming an epoxy or thiirane unit; and xxvii) two hydrogen atoms on non-adjacent carbon atoms are substituted to form a homocyclic group.

As used herein and unless otherwise stated, the term " stereoisomer " refers to all possible different isomeric as well as conformational forms which the compounds of the invention may possess, in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.

As used herein and unless otherwise stated, the term " enantiomer " means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e. at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to 7-[2-(substituted or unsubstituted cyclohexylidenej-ethylidenej-spirofδ.δjundecanes, the core scaffolds thereof having two isomeric orientations with the following formulae:

wherein the rings of said core scaffolds are designated " A ", " C ", and " F " as indicated and referred to as such herein. The compounds of the present invention utilize the following conventional core scaffold numbering system for vitamin D compounds, referring to the individual core scaffold carbon atoms and side chain substitutents as shown herein below, as a representative example, for 5-{2-[2-(4-hydroxy-4- methylpentyO-spiro^.δjdec-Z-ylidene^cyclohexane-I .S-diol having the formula:

All possible stereoisomers, therefore all enantiomers and diasteriomers, are encompassed within the present invention and as such the above example should clraely be understood as non-limiting in the depiction of the relative stereochemistry at, for example, carbon atoms 1 , 3, 13, and 21. For the purpose of the present invention units with formulae having bonds depicted as:

encompass both orientations about the double bond, for example, both the formulae:

Therefore for the sake of brevity in the following definitions only one orientation, the 7£-isomer orientation, will be depicted. However, in the depiction of the following compound categories, the absolute orientation (7E-isomer or 7Z-isomer) will be defined. The same consideration also applies to the substituted cyclohexylidene Q units, wherein the following orientations apply to the various isomers:

COMPOUNDS

The compounds of the present invention comprise: i) a substituted spiro[5.5]undecane unit (the said unit including both rings C and F such as indicated herein above) having the following general formula:

wherein R^Oa, R^ob, R^1a, R^1b, R^2a, and R^2b are each independently selected from the group consisting of hydrogen and oxa-hydrocarbyl chains, wherein the oxygen atom of said oxa-hydrocarbyl chains is directly attached to the F ring of said spiro[5.5]undecane unit, and wherein the hydrocarbyl group of said oxa-hydrocarbyl chains is selected from the group consisting of substituted and unsubstituted Ci_-2o alkyl, substituted and unsubstituted C_3-10 cycloalkyl, substituted and unsubstituted C_2-20 alkenyl, and substituted and unsubstituted C_2-20 alkynyl; and ii) a substituted cyclohexylidene unit Q having the general formula:

wherein R^3a and R^3b are each hydrogen or R^3a and R^3b are taken together to form an exocyclic methylene unit; R^4a and R^4b are each hydrogen or R^4a and R^4b are taken together to form an exocyclic methylene unit; R^5a, R^5b, R^6a and R^6b are each independently selected from the group consisting of hydrogen, hydroxyl and OP wherein P is a hydroxyl-protecting group preferably selected from the group consisting of C_1-20 alkyl, C_3-10 cycloalkyl, acyl and the like. In addition, carbon 2 of the said compounds of the invention (i.e. the carbon atom located between the carbon atom bearing R^5a and R^5b and the carbon atom bearing R^6a and R^6b, according to conventional numbering system for vitamin D compounds, see representative example above) may further be optionally substituted with one or two substituent(s) R and/or R' (not shown in the above formula), wherein said substituent(s) R and R' are each independently selected from the group consisting of substituted and unsubstituted C_1-7 alkyl, preferably methyl or ethyl.

The critical feature of this invention is that one or more substituents of the substituted spiro[5.5]undecane unit is a (are) hydrocarbyl chain(s) directly linked to said spiro[5.5]undecane unit via an oxygen atom. In a preferred set of compounds of this invention, only one such oxa-hydrocarbyl chain is present, whatever its position on the F- ring of the vitamin D scaffold. Optionally the said oxa-hydrocarbyl chain substituent(s) may further contain one or more heteroatoms such as, but not limited to, oxygen, for instance in the form of a hydroxyl or epoxide function, said one or more heteroatom preferably being in the form of a terminal hydroxyl or epoxide function. A first embodiment of this invention relates to compounds wherein R^Oa is an oxa- hydrocarbyl chain containing a substituted or unsubstituted C_6-io alkyl group and R^ob is hydrogen. Preferably in this embodiment, each of R^1a, R^1b, R^2a, and R^2b is hydrogen. Non-limiting examples of R^Oa within this embodiment of the invention include the following: i) 4-hydroxy-4-methylpent-1 -yloxy; ii) 5-hydroxy-5-methylhex-2-yloxy; iii) 6-hydroxy-6-methylhept-2-yloxy; iv) 5-hydroxy-6-methylhept-2-yloxy; and v) 1-hydroxy-5,5-dimethylhex-1 -yloxy.

Another embodiment of this invention relates to compounds wherein R^ob is an oxa-hydrocarbyl chain containing a substituted or unsubstituted C_6-io alkyl group and R^Oa is hydrogen. Preferably in this embodiment, each of R^1a, R^1b, R^2a, and R^2b is hydrogen. Non-limiting examples of R^ob within this embodiment of the invention include the following: i) 4-hydroxy-4-methylpentyloxy; ii) 5-hydroxy-5-methylhex-2-yloxy; iii) 6-hydroxy-6-methylhept-2-yloxy; iv) 5-hydroxy-6-methylhept-2-yloxy; and v) 1-hydroxy-5,5-dimethylhexyloxy.

A further embodiment of this invention relates to compounds wherein R^Oa and R^ob are each independently selected from the group consisting of hydrogen and oxa- hydrocarbyl chains containing a substituted and unsubstituted C_3-10 alkenyl group. Preferably in this embodiment, each of R^1a, R^1b, R^2a, and R^2b is hydrogen. Non-limiting examples of substituted C₃-C₁₀ alkenyl groups included in such oxa-hydrocarbyl chains according to this embodiment of the present invention include the following: i) 7-hydroxy-7-methyloct-4-en-2-yl:

ii) 7-hydroxy-7-ethylnon-4-en-2-yl:

iii) 7-hydroxy-7-methyloct-3,5-dien-2-yl:

iv) 7-hydroxy-7-ethylnon-3,5-dien-2-yl:

Within these examples of R^Oa and R^ob are encompassed all enantiomers thereof, including, for example, those comprising: i) Z-(2S)-7-hydroxy-7-methyloct-4-en-2-yl:

as well as: ii) Z-(2f?)-7-hydroxy-7-methyloct-4-en-2-yl:

however, for a particular example only the substitution pattern across the double bond (E-isomer or Z-isomer), as depicted, is considered unless a generic structure is provided, for example, the formula:

A further embodiment of this invention relates to compounds wherein R^Oa and R^ob are each independently selected from the group consisting of hydrogen and oxa- hydrocarbyl chains containing a substituted and unsubstituted C_4-10 alkynyl group. ,1a

Preferably in this embodiment, each of R'^a, R^1b, R^2a, and R^2b is hydrogen. Non-limiting examples of substituted C_4-10 alkynyl groups included in such oxa-hydrocarbyl chains according to this embodiment of the present invention include the following: i) 5-hydroxy-5-methylhex-3-ynyl:

ii) 4-(2-methyl-oxyranyl)-but-3-ynyl:

iii) 6-hydroxy-6-methylhept-3-yn-2-yl:

iv) 5-(2-methyl-oxyranyl)-pent-4-yn-2-yl:

v) 5-hydroxy-5-ethylhept-3-ynyl:

vi) 6-hydroxy-6-ethyloctyn-4-yn-2-yl:

A further embodiment of this invention relates to compounds wherein R^Oa and R^ob are each independently selected from the group consisting of oxa-hydrocarbyl chains containing a substituted or unsubstituted C1-₁0 alkyl group or a substituted or unsubstituted C_3-10 cycloalkyl group. Preferably in this embodiment, each of R^1a, R^1b, R^2a, W

15 and R^2b is hydrogen. One representative example of such embodiment has R^Oa including a C₆-io alkyl group and R^ob including a methyl group. Another representative example of such embodiment relates to compounds wherein R^ob includes a C_6-10 alkyl group and R^Oa includes a methyl group. A more specific but non-limiting example of this embodiment has R^ob including a methyl group and R^Oa including a substituted or unsubstituted hexyl group.

Another embodiment of this invention relates to compounds wherein R^1a is an oxa-hydrocarbyl chain containing a substituted or unsubstituted C_6-10 alkyl group and R^1b is hydrogen. Preferably in this embodiment, each of R^Oa, R^ob, R^2a, and R^2b is hydrogen. Non-limiting examples of R^1a within this embodiment of the invention include the following: i) 4-hydroxy-4-methylpent-1 -yloxy; ii) 5-hydroxy-5-methylhex-2-yloxy; iii) 6-hydroxy-6-methylhept-2-yloxy; iv) 5-hydroxy-6-methylhept-2-yloxy; and v) 1 -hydroxy-5,5-dimethylhex-1 -yloxy.

Another embodiment of this invention relates to compounds wherein R^1b is an oxa-hydrocarbyl chain containing a substituted or unsubstituted C_6-10 alkyl group and R^1a is hydrogen. Preferably in this embodiment, each of R^Oa, R^ob, R^2a, and R^2b is hydrogen. Non-limiting examples of R^1b within this embodiment of the invention include the following: i) 4-hydroxy-4-methylpentyloxy; ii) 5-hydroxy-5-methylhex-2-yloxy; iii) 6-hydroxy-6-methylhept-2-yloxy; iv) 5-hydroxy-6-methylhept-2-yloxy; and v) 1-hydroxy-5,5-dimethylhexyloxy.

A further embodiment of this invention relates to compounds wherein R^1a and R^1b are each independently selected from the group consisting of hydrogen and oxa- hydrocarbyl chains containing a substituted and unsubstituted C_3--_I0 alkenyl group. Preferably in this embodiment, each of R^Oa, R^ob, R^2a, and R^2b is hydrogen. Non-limiting examples of substituted C₃-C₁₀ alkenyl groups included in such oxa-hydrocarbyl chains according to this embodiment of the present invention include the following: i) 7-hydroxy-7-methyloct-4-en-2-yl:

ii) 7-hydroxy-7-ethylnon-4-en-2-yl:

iii) 7-hydroxy-7-methyloct-3,5-dien-2-yl:

iv) 7-hydroxy-7-ethylnon-3,5-dien-2-yl:

Within these examples of R^1a and R^1b are encompassed all enantiomers thereof, including: i) Z-(2S)-7-hydroxy-7-methyloct-4-en-2-yl:

as well as: ii) Z-(2f?)-7-hydroxy-7-methyloct-4-en-2-yl:

however, for a particular example only the substitution pattern across the double bond (E-isomer or Z-isomer), as depicted, is considered unless a generic structure is provided, for example, the formula: W

17

A further embodiment of this invention relates to compounds wherein R^1a and R^1b are each independently selected from the group consisting of hydrogen and oxa- hydrocarbyl chains including a substituted and unsubstituted C₄-₁0 alkynyl group. Preferably in this embodiment, each of R^Oa, R^ob, R^2a, and R^2b is hydrogen. Non-limiting examples of substituted C_4-10 alkynyl groups included in this embodiment of the present invention include the following: i) 5-hydroxy-5-methylhex-3-ynyl:

ii) 4-(2-methyl-oxyranyl)-but-3-ynyl:

iii) 6-hydroxy-6-methylhept-3-yn-2-yl:

iv) 5-(2-methyl-oxyranyl)-pent-4-yn-2-yl:

v) 5-hydroxy-5-ethylhept-3-ynyl:

vi) 6-hydroxy-6-ethyloctyn-4-yn-2-yl:

A further embodiment of this invention relates to compounds wherein R^1a and R^1b are each independently selected from the group consisting of oxa-hydrocarbyl chains containing a substituted or unsubstituted Ci_-10 alkyl group or a substituted or unsubstituted C_3--_I0 cycloalkyl group. Preferably in this embodiment, each of R^Oa, R^ob, R^2a, and R^2b is hydrogen. One representative example of such embodiment includes R^1a including a C_6-io alkyl and R^1b including methyl. Another representative example of such embodiment has R^1b including a C_6-I0 alkyl group and R^1a including a methyl group. A more specific but non-limiting example of this embodiment has R^1b including a methyl group and R^1a including a substituted or unsubstituted hexyl group.

Another embodiment of this invention relates to compounds wherein R^2a is an oxa-hydrocarbyl chain including a substituted or unsubstituted C₆-_I0 alkyl group and R^2b is hydrogen. Preferably in this embodiment, each of R^Oa, R^ob, R^1a, and R^1b is hydrogen. Non-limiting examples of R^2a within this embodiment of the invention include the following: i) 4-hydroxy-4-methylpent-1 -yloxy;

H) 5-hydroxy-5-methylhex-2-yloxy; iii) 6-hydroxy-6-methylhept-2-yloxy; iv) 5-hydroxy-6-methylhept-2-yloxy; and v) 1 -hydroxy-5,5-dimethylhex-1 -yloxy.

Another embodiment of this invention relates to compounds wherein R^2b is an oxa-hydrocarbyl chain including a substituted or unsubstituted C_6-10 alkyl group and wherein R^2a is hydrogen. Preferably in this embodiment, each of R^Oa, R^ob, R^1a, and R^1b is hydrogen. Non-limiting examples of R^2b within this embodiment of the invention include the following: i) 4-hydroxy-4-methylpentyloxy; ii) 5-hydroxy-5-methylhex-2-yloxy; iii) 6-hydroxy-6-methylhept-2-yloxy; iv) 5-hydroxy-6-methylhept-2-yloxy; and v) 1 -hydroxy-5,5-dimethylhexyloxy. W

19

A further embodiment of this invention relates to compounds wherein R^2a and R^2b are each independently selected from the group consisting of hydrogen and oxa- hydrocarbyl chains including a substituted and unsubstituted C_3-10 alkenyl group. Preferably in this embodiment, each of R^Oa, R^ob, R^1a, and R^1b is hydrogen. Non-limiting examples of substituted C₃-Ci₀ alkenyl groups included within this embodiment of the present invention include the following: i) 7-hydroxy-7-methyloct-4-en-2-yl:

ϋ) 7-hydroxy-7-ethylnon-4-en-2-yl:

7-hydroxy-7-methylocta-3,5-dien-2-yl:

iv) 7-hyd roxy-7-ethyl nona-3 , 5-d ien-2-yl :

Within these examples of R^2a and R^2b are encompassed all enantiomers thereof, including: i) Z-(2S)-7-hydroxy-7-methyloct-4-en-2-yl:

as well as: i) Z-(2f?)-7-hydroxy-7-methyloct-4-en-2-yl:

however, only the substitution across the double bond (E-isomer or Z-isomer) as depicted in each example is considered.

A further embodiment of this invention relates to compounds wherein R^2a and R^2b are each independently selected from the group consisting of hydrogen and oxa- hydrocarbyl chains including a substituted and unsubstituted C_4-10 alkynyl group. Preferably in this embodiment, each of R^Oa, R^ob, R^1a, and R^1b is hydrogen. Non-limiting examples of substituted C_4--_I0 alkynyl groups included within this embodiment of the present invention include: i) 5-hydroxy-5-methylhex-3-ynyl:

ii) 4-(2-methyl-oxyranyl)-but-3-ynyl:

iii) 6-hydroxy-6-methylhept-3-yn-2-yl:

iv) 5-(2-methyl-oxyranyl)-pent-4-yn-2-yl:

v) 5-hydroxy-5-ethylhept-3-ynyl:

vi) 6-hydroxy-6-ethyloctyn-4-yn-2-yl

A further embodiment of this invention relates to compounds wherein R ^a and R are each independently selected from the group consisting of oxa-hydrocarbyl chains including a substituted or unsubstituted Ci_-10 alkyl group, or a substituted or unsubstituted C_3-10 cycloalkyl group. Preferably in this embodiment, each of R^Oa, R^Ob, R^1a, and R^1b is hydrogen. One representative example of such embodiment has R^2a including a C_6-I0 alkyl group and R^2b including a methyl group. Another representative example of such embodiment relates to compounds wherein R^2b includes a C_6-10 alkyl group and R^2a includes a methyl group.

The following are non-limiting categories of the substituted cyclohexylidene units Q which are comprised in the compounds of the present invention, as shown by the following structural formula:

A first category of such Q units have the following formula:

i.e. wherein R^3a, R^3b, R^4a, and R^4b are each hydrogen; and wherein R^5a, R^5b, R^6a and R^6b are each independently selected from the group consisting of hydrogen, hydroxyl and OP wherein P is a hydroxyl-protecting group such as defined herein. Preferably in this first category, at least two of R^5a, R^5b, R^6a and R^6b are hydrogen. In addition, carbon 2 of the said units (i.e. the carbon atom located between the carbon atom bearing R^5a and R^5b and the carbon atom bearing R^6a and R^6b, according to conventional numbering system for vitamin D compounds, see representative example above) may further be optionally substituted with one or two substituent(s) R and/or R' (not shown in the above formula), wherein said substituent(s) R and R' are each independently selected from the group consisting of substituted and unsubstituted Ci_-7 alkyl, preferably methyl or ethyl. A first aspect of the first category of Q units have the following formula:

i.e. wherein R^5b and R^6a are each hydroxyl and R^5a and R^6b are each hydrogen.

A second aspect of the first category of Q units have the following formula:

i.e. wherein R^5a and R^6b are each hydroxyl and R^5b and R^6a are each hydrogen.

A third aspect of the first category of Q units have the following formula:

i.e. wherein

o R6b a „_re_ e _a„c„h!, h u,y ,d_j_ro—x ...y ,ιl and R r->5a a __n,dj R o6a ^a are each hydrogen. A fourth aspect of the first category of Q units have the following formula:

i.e. wherein R^5a and R^6a are each hydroxyl, and wherein R^5b and R^6b are each hydrogen. A second category of Q units have the following formula: W 2

23

i.e. wherein R^3a and R^3b are taken together to form an exocyclic methylene unit; wherein R^4a and R^4b are each hydrogen; and wherein R^5a, R^5b, R^6a and R^6b are each independently selected from the group consisting of hydrogen, hydroxyl and OP wherein P is a hydroxyl-protecting group such as defined herein. Preferably in this second category, at least two of R^5a, R^5b, R^6a and R^6b are hydrogen. In addition, carbon 2 of the said units (i.e. the carbon atom located between the carbon atom bearing R^5a and R^5b and the carbon atom bearing R^6a and R^6b, according to conventional numbering system for vitamin D compounds, see representative example above) may further be optionally substituted with one or two substituent(s) R and/or R' (not shown in the above formula), wherein said substituent(s) R and R' are each independently selected from the group consisting of substituted and unsubstituted C_1-7 alkyl, preferably methyl or ethyl. A first aspect of the second category of Q units have the following formula:

i.e. wherein R^5b and R^6a are each hydroxyl, and wherein R^5a and R^6b are each hydrogen. A second aspect of the second category of Q units have the following formula:

i.e. wherein R^5a and R^6b are each hydroxyl, and wherein R^5b and R^6a are each hydrogen. A third aspect of the second category of Q units have the following formula:

i.e. wherein R^5b and R^6b are each hydroxyl, and wherein R^5a and R^6a are each hydrogen. A fourth aspect of the second category of Q units are rings having the formula:

i.e. wherein R^5a and R^6a are each hydroxyl, and wherein R^5b and R^6b are each hydrogen.

A third category of Q units have the following formula:

i.e. wherein R^3a, R^3b, R^4a and R^4b are each hydrogen; wherein one of R^5a, R^5b, R^6a and R^6b is hydroxyl or a hydroxyl-protecting group such as defined herein, and wherein the balance of R^5a, R^5b, R^6a and R^6b are hydrogen. In addition, carbon 2 of the said units (i.e. the carbon atom located between the carbon atom bearing R^5a and R^5b and the carbon atom bearing R^6a and R^6b, according to conventional numbering system for vitamin D compounds, see representative example above) may further be optionally substituted with one or two substituent(s) R and/or R' (not shown in the above formula), wherein said substituent(s) R and R' are each independently selected from the group consisting of substituted and unsubstituted C_1-7 alkyl, preferably methyl or ethyl.

Non-limiting aspects of this third category of Q units includes units having one of the following formulae:

A fourth category of Q units have the following formula:

wherein: a) an exocyclic methylene is present in the Q unit such that: i) R^3a and R^3b are taken together to form an exocyclic methylene unit, and R^4a and R^4b are each hydrogen; or ii) R^4a and R^4b are taken together to form an exocyclic methylene unit, and R^3a and R^3b are each hydrogen; and b) one of R^5a, R^5b, R^6a or R^6b is hydroxyl or a hydroxyl-protecting group such as defined herein, and the balance of R^5a, R^5b, R^6a and R^6b are hydrogen. Non-limiting aspects of this fourth category of Q units includes units having one of the following formulae:

A fifth category (not shown in any of the previous formulae) of Q units which may be comprised in the compounds of this invention includes, on carbon 2 of said compound (according to the conventional scaffold numbering system referred to herein above), one or two substituents R and/or R', wherein R and R' are each independently selected from the group consisting of substituted and unsubstituted Ci_-7 alkyl groups. Preferably within this fifth category, R and/or R' is methyl or ethyl, and R and/or R' is optionally substituted with one or more functional atoms or groups selected from the group consisting of fluoro, chloro, hydroxy, sulfhydryl and amino. More preferably carbon 2 is mono-substituted, i.e. only has one substituent R being most preferably methyl or ethyl. Specific examples of this fifth category are not illustrated hereunder, however their synthesis is similar to that of compounds of the four other categories, starting from precursors having the suitable substituents R and/or R', which can be made according to the teachings of prior art such as but not limited to De Luca and co-workers.

For a purpose of convenience, the compounds of the present invention are arranged hereinafter into twelve different categories in order to assist the skilled person in applying a rational synthetic strategy for the preparation of other compounds which are not expressly exampled herein. This arrangement into categories does not imply any ranking or hierarchy among the corresponding compounds, such as an increased or decreased medicinal importance or efficacy for any of the pharmaceutical compositions including such compounds described herein and/or for any of the methods of preventing or treating disorders described herein.

The present invention also relates to a number of intermediates, most of them having a specific stereochemistry, and which are useful in the preparation of the final vitamin D analogues of the various different categories stated herein above. The preparation and the detailed formulae of a number of these intermediates are shown in the following scheme I comprising a sequence of eleven process steps for making the pure (+) and (-) isomers of 1 ,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene, as well as in the following schemes hereinafter. Each step will now be illustrated in details, based on specific starting compounds, reactive agents, catalysts, solvents, temperature ranges and the like, but the skilled person understands that the specific materials and conditions disclosed herein may be replaced with similar or equivalent materials and conditions without significantly altering the resulting product of the relevant step, except perhaps for the reaction yield or, in the case of a stereoselective reaction, the enantiomeric excess of the product shown.

Scheme I

Synthesis of the pure (+) and (-) isomers of 1 ,4-dioxa-dispiro[4.1.5.3] pentadec- 8-ene starts, as a first step (a), with the halogenation, preferably the bromination, of 3- ethoxy-2-cyclohexenone, e.g. according to the procedure disclosed by Hara et al. in J. Am. Chem. Soc. (1999) 121, 3072-3082:

1

(preferred reagents and conditions for step (a) include: N-bromosuccinimide as a reagent, CH₂CI₂ as a solvent; temperature range from about O⁰C to about 40⁰C).

In a second step (b), the 2-bromo-3-ethoxy-cyclohex-2-enone resulting from step

(a) is alkylated, preferably n-butylated, for instance, but without limitation, by means of 1- chloro-4-methoxybutane (the latter may be prepared according to the procedures disclosed by Hara et al. in J. Org. Chem, (1975) 40, 2786-2791 and/or by De Buyck et al. in Bull. Soc. Chim. BeIg. (1992) 101, 807-815) in the presence of an effective amount of an organometallic derivative such as a Grignard reagent. In a more specific embodiment in order to improve butylation yield, said step may additionally be performed in the presence of a stoechiometric amount of cerium trichloride. This specific embodiment is believed to result in the formation of an organocerium active species as shown in the scheme below, although said presumed active species was not isolated:

(preferred reagents and conditions for step (b) include : THF as a solvent; temperature range from about O⁰C to about 4O⁰C after refluxing to create the Grignard reagent; magnesium, 1-chloro-4-methoxybutane and cerium trichloride as active reagents). In a third step (c), the 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enone resulting from step (b) is stereoselective^ reduced by means of a suitable stereoselective reducing agent in the presence of a catalytic amount of a suitable reduction catalyst. Suitable reducing agents for this reduction step include, but are not limited to, borane, catecholborane and borohydride reagents. Suitable stereoselective reduction catalyst for this reduction step include, but are not limited to, substantially pure methyloxazaborolidinone isomers and metal complexes, either monometallic, homobimetallic or heterobimetallic, such as lithium aluminum hydrides or transition metal complexes, having one or more chiral ligand. The skilled person understands that this step is very important for the present invention since it determines the success of the whole synthetic route up to the desired following stereoisomeric intermediates and vitamin D analogues. Therefore the skilled person, based on the general knowledge relating to reducing catalysts and agents, will make a careful selection of the reaction conditions in view of various considerations such as reaction yield, productivity and, mainly, enantiomeric excess of one stereoisomer of the resulting product with respect to the other stereoisomer.

2 3

(preferred reagents and conditions for step (c) as shown above include: (S)- methyloxazaborolidinone as a catalyst, catecholborane as a reducing agent, toluene as a solvent; temperature range from about -95⁰C to about 25°C, more preferably from - 78⁰C to about 0⁰C).

In a fourth step (d), the 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enol stereoisomer resulting from step (c) is submitted to a 3,3-sigmatropic rearrangement by means of a dimethylamino-dimethylacetal (preferably in an at least stoechiometric amount), preferably in the presence of a suitable solvent:

(preferred reagents and conditions for step (d) include: (CHs)₂N[CCH₃(OC!-^ as a reagent, toluene as a solvent; temperature: reflux of the solvent).

In a fifth step (e), the 2-[2-bromo-1-(4-methoxybutyl)-cyclohex-2-enyl]-/V,/V- dimethylacetamide stereoisomer resulting from step (d) is reduced by means of a reducing agent such as a metal hydride in the presence of a suitable solvent and optionally in the presence of a free radical initiator:

4 5

(preferred reagents and conditions for step (e) include: Bu₃SnH, azobis-isobutyronitrile as a reducing agent; THF a solvent; temperature: reflux of the solvent).

In a sixth step (f), the 2-[1-(4-methoxybutyl)-cyclohex-2-enyl]-Λ/,Λ/- dimethylacetamide stereoisomer resulting from step (e) is submitted to ether cleavage, preferably in the presence of a Lewis acid reagent, a nucleophile reagent and an effective amount of a suitable catalyst such as a crown ether.

5 Q

(preferred reagents and conditions for step (f) include: BBr₃ as a Lewis acid, NaI as a nucleophilic reagent, and 15-crown-5 as a catalyst, CH₂CI₂ as a solvent; temperature range from about -4O⁰C to about -2O⁰C).

In a seventh step (g), the 2-[1-(4-hydroxybutyl)-cyclohex-2-enyl]-/V,/V- dimethylacetamide stereoisomer resulting from step (f) is oxidized in the presence of a suitable oxidation reagent, preferably a chromium (Vl) compound, preferably at moderate temperature and in the presence of a suitable solvent:

(preferred reagents and conditions for step (g) include: pyridinium dichromate as an oxidation reagent, DMF as a solvent; temperature range from about 10°C to about 4O⁰C).

In an eighth step (h), the 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid stereoisomer resulting from step (g) is esterified, preferably in two sub-steps including first a strong base at elevated temperatures, and secondly the presence of an aliphatic alcohol optionally in the presence of an effective amount of an esterification catalyst:

(preferred reagents and conditions for step (h) include: first (i) KOH, dioxane / water mixture as a solvent, temperature range from about 16O⁰C to about 24O⁰C; then (ii) diazomethane CH₂N₂, MeOH).

In a ninth step (i), the 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid methyl ester stereoisomer resulting from step (h) is submitted to a Dieckmann condensation into a β-keto ester preferably in the presence of a strong base such as a sodium alkoxide or a lithium amide, and optionally in the presence of a suitable solvent:

(preferred reagents and conditions for step (i) include: lithium diisopropylamide as a base, THF as a solvent; temperature range from about -8O⁰C to about 4O⁰C).

In a tenth step (j), the 2-hydroxy-spiro[5.5]undecan-2,7-diene-3-carboxylic acid methyl ester stereoisomer resulting from step (i) is submitted to oxidative decarboxylation, preferably in the presence of a suitable oxidative agent:

(preferred reagents and conditions for step G) include: NaCI, water / DMSO as a solvent mixture; temperature range from about 14O⁰C to about 18O⁰C).

In an eleventh step (k), the spiro[5.5]undec-7-en-2-one stereoisomer resulting from step (i) is submitted to ketalization by means of an aliphatic diol, optionally in the presence of a suitable acidic catalyst:

40 Ai

(preferred reagents and conditions for step (k) include: HOCH₂CH₂OH as a reagent, p- toluenesulfonic acid as a catalyst, toluene as a solvent; temperature at reflux of the solvent).

The skilled person understands that, alternatively in step (k), thioacetalization may be performed by means of HSCH₂CH₂SH as a reagent and that the resulting 1 ,4- dithia-dispiro[4.1.5.3]pentadec-8-ene stereoisomers and their derivatives, although not specifically shown in the above and following formulae, also form part of the present invention.

For the ease of understanding and the completion of the disclosure of other stereoisomers which are also available by this methodology, scheme I' hereunder provides a full depiction of a similar synthetic route starting from 2-bromo-3-(4- methoxybutyl)-cyclohex-2-enone but wherein the stereoselective catalyst is (R)- methyloxazaborolidinone. Scheme I¹

EXAMPLE 1 - preparation of 1 ,4-dioxa-dispiror4.1.5.3lpentadec-8-ene stereoisomers (11) and (11') and 1 ,4-dithia-dispiror4.1.5.3]pentadec-8-ene stereoisomers

The preparation of 1 ,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene stereoisomers was effected via the principles of the eleven steps synthetic route described above, and more specifically as outlined hereunder.

a) preparation of 2-bromo-3-ethoxy-cvclohex-2-enone (1 )

To a solution of 3-ethoxy-2-cyclohexenone (70 g, 499 mmol) in CI(CH₂)₂CI (300 ml_) as a solvent at 0°C under argon atmosphere, /V-bromosuccinimide (91 g, 511 mmol) was added in portions over a period of 1 hour. The suspension was stirred at 0° C for 1 hour and then at room temperature for 2 hours. The solvent was removed in vacuo and CH₂CI₂ added. The solution was washed twice with a cold saturated NaHCO₃ solution and with cold H₂O, then dried over anhydrous MgSO₄, the solvent removed in vacuo and the resulting solid residue was triturated several times with Et₂O, the resulting solid dried in vacuo to afford 101 g (92% yield) of the desired product as white crystals which were characterized as follows:

- R_f (Et₂O) 0.33; - Ultraviolet (UV) absorption (MeOH) at λ_max 273 nm;

- Infrared (IR) main absorption bands (KBr) at v 2966, 2939, 1657, 1568, 1430, 1397, 1371 , 1348, 1324, 1295, 1260, 1246, 1195, 1150, 1107, 1076, 1030, 959, 922, 850, 798, 614 and 470 cm^"1;

- proton nuclear magnetic resonance (¹H NMR) (500 MHz, CDCI₃): δ 4.21 (2 H, q, J = 7.0 Hz), 2.69 (3 H, t, J = 6.2 Hz), 2.55-2.52 (2 H, m), 2.07-2.02 (2 H, m), and 1.43

(2 H, t, J = 7.0 Hz) ppm;

- carbon nuclear magnetic resonance (¹³C NMR) (50 MHz, CDCI₃): δ 191.0 (C), 172.8 (C), 103.0 (C), 65.2 (CH₂), 36.7 (CH₂), 27.2 (CH₂), 20.6 (CH₂) and 15.1 (CH₃) ppm; and - mass spectrum (MS) m/z (%): 220/218 (M⁺, 16/20), 192/190 (25/25), 164/162 (75/100), 149 (12), 111 (15), 67 (21) and 55 (51).

b) preparation of 2-bromo-3-(4-methoxy-butyl)-cyclohex-2-enone (2)

To magnesium turnings (9.84 g, 405 mmol) in dry THF (42 ml.) as a solvent were added a few crystals of iodine and a few drops of CH₂Br₂. A solution of 1-chloro-4- methoxybutane (46.8 g, 382 mmol) in dry THF (90 mL) was added dropwise at a rate sufficient to maintain gentle reflux. The reaction mixture was then refluxed for 1 hour and then cooled to room temperature. The resulting Grignard reagent was then added dropwise to a suspension of CeCI₃ (94.2 g, 382 mmol) in dry THF (750 mL) at 0° C. After stirring at room temperature for 2 hours, a solution of 2-bromo-3-ethoxy-2- cyclohexenone (1) (60 g, 274 mmol) in dry THF (300 mL) was added dropwise at 0° C. The reaction mixture was stirred an additional 2 hours at room temperature and then quenched by adding a saturated NH₄CI solution (1000 mL). The mixture was further acidified to approximately pH 1 by the addition of a 5% HCI solution and stirring was continued for an additional 2 hours. The organic layer was separated, the aqueous layer extracted with Et₂O and the combined organic layers were dried over anhydrous MgSO₄. The solvent was removed in vacuo and the residue purified over silica (using a isooctane/EtOAc 7:3 mixture as an eluent) to afford 65.5 g (92% yield) of the desired product as a light-yellow oil which was characterized as follows:

- R_f (isooctane/EtOAc, 7:3) 0.30;

- UV (MeOH) λ_max 254 nm; - IR (KBrfilm)v2929, 2865, 2828, 1682, 1595, 1455, 1427, 1385, 1337, 1313, 1272, 1175, 1117, 982and795cm^"1;

- ¹H NMR(500 MHz, CDCI₃): δ3.38 (2 H, t, J= 5.9 Hz), 3.31 (3 H, s), 2.56-2.53 (2 H, m), 2.50-2.47(4H, m), 1.99-1.94(2H, m),and 1.65-1.56(4H, m)ppm;

- ¹³C NMR (50 MHz, CDCI₃): δ 191.2 (C), 163.7 (C)₁ 122.6 (C), 72.1 (CH₂), 58.6 (CH₃), 38.9 (CH₂), 37.8 (CH₂), 32.3 (CH₂), 29.4 (CH₂), 23.5 (CH₂) and 21.9 (CH₂) ppm; and

- MS m/z (%): 230/228 (M⁺ - MeOH, <1/1), 200/188 (4/4), 181 (3), 149 (20), 131 (7), 121 (12), 107 (16), 93 (29), 91 (24), 79 (60), 77 (40), 65 (20), 51 (29) and 45 (100).

c) preparation of 2-bromo-3-(4-methoxybutyl)-cvclohex-2-enol stereoisomers (3) and (3') To a solution of 2-bromo-3-(4-methoxy-butyl)-cyclohex-2-enone (2) (32.0 g, 123 mmol) in dry toluene (480 ml_) at -95 ⁰C was added (S)-methyl-oxazaborolidinone (for making isomer 3) or(R)-methyl-oxazaborolidinone (for making isomer 3') (1 M solution in toluene; 25 ml_, 25 mmol). Catecholborane (1 M solution in toluene; 160 mL, 160 mmol) was added dropwise at -95°C over a period of 10 hours and the reaction mixture stirred at -78°C overnight. A 1 M NaOH solution (500 mL) was added and the temperature allowed to rise to room temperature. The organic layer was separated and the aqueous layer extracted with Et₂O. The combined organic layers were washed with a 1 M NaOH solution and dried over anhydrous MgSO₄. The solvent was removed in vacuo, and the resulting residue purified over silica (isooctane/EtOAc, 8:2) to afford 31.5 g (97% yield) of the desired product as a colorless oil which was characterized as follows:

- R_f (π-pentane/Et₂O, 7:3) 0.33;

- optical rotation at room temperature: [α]_D ^rt +71.6 (c = 1.53, CHCI₃) for isomer (3); [α]_D ^rt -70.0 (c = 1.13, CHCI₃) for isomer (3');

- IR (KBr film) v 3418, 2935, 2864, 2828, 1645, 1455, 1387, 1337, 1264, 1165, 1118, 1078, 990, 970, 939, 813 and 733 cnrf¹;

- ¹H NMR (500 MHz, CDCI₃): δ 3.38 (2 H, t, J = 6.4 Hz), 3.32 (3 H, s), 2.38 (1 H, d, J = 3.8 Hz), 2.20 (2 H, dd, J = 7.8, 7.8 Hz), 2.14 (2 H, ABt, J = 17.3, 5.0 Hz), 2.07 (1 H, ABdd, J = 17.3, 8.3, 5.6 Hz), 1.87-1.83 (2 H, m), 1.79-1.71 (1 H, m), 1.65-1.55 (3 H, m) and 1.51-1.45 (2 H, m) ppm;

- ¹³C NMR (50 MHz₁ CDCI₃): δ 140.5 (C), 122.9 (C), 72.5 (CH₂), 71.0 (CH), 58.5 (CH₃), 36.9 (CH₂), 32.0 (CH₂), 31.3 (CH₂), 29.3 (CH₂), 23.6 (CH₂), 18.3 (CH₂) ppm; - MS m/z (%) 246/244 (M⁺ - H₂O, <1/1), 214/212 (3/3), 165 (10), 133 (42), 123 (19), 105 (25), 91 (77), 79 (38), 67 (26), 55 (28), 53 (26), 45 (100) and 41 (33); and

- elemental analysis: calculated for CnH₁₉BrO₂: C, 50.20; H, 7.28; found: C, 50.17; H, 7.17.

d) preparation of 2-r2-bromo-1 -(4-methoxybutyl)-cvclohexy-2-enyll-A/,Λ/-dimethyl- acetamide stereoisomers (4) and (4')

To a solution of the relevant 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enol isomer (3) or (3') (24.5 g, 93.1 mmol) in dry toluene (400 mL) was added N,N- dimethylacetamide dimethyl acetal (27.2 mL, 186.2 mmol). The reaction mixture was brought to reflux for 8 hours removing the generated MeOH by azeotropic distillation. The solvent was removed in vacuo and the residue purified over silica (using cyclohexane/EtOAc 1:1 as the eluent) to afford 29.4 g (95% yield) of the desired product as a light-yellow oil which was characterized as follows:

- R_f (isooctane/EtOAc, 1 :1) 0.28; - optical rotation [α]_D ^rt -41.4 (c = 1.67, CHCI₃) for isomer (4); [α]_D ^rt +38.2 (c = 0.96, CHCI₃) for isomer (4');

- IR (KBrfilm): v 2934, 2865, 1644, 1490, 1456, 1393, 1258, 1179, 1149, 1118, 968, 891, 861, 813and761 cm^"1;

- ¹H NMR(500 MHz, CDCI₃):δ6.13(1 H, dd,J=4.2,4.2 Hz), 3.38 (1 H,ABt, J= 9.3, 6.5 Hz), 3.36 (1 H, ABt, J= 9.3, 6.6 Hz), 3.31 (3 H, s), 3.06 (3 H, s), 2.92 (3 H, s),

2.59 (1 H,AB, J= 14.9 Hz), 2.44 (1 H,AB, J= 14.9 Hz), 2.32 (1 H, m), 2.09-1.98 (2 H , m), 1.77-1.71 (1 H, m), 1.67-1.52(6 H, m)and 1.39-1.26(2H, m)ppm;

- ¹³C NMR (125 MHz, CDCI₃): δ 170.7 (C), 133.0 (C), 132.0 (CH), 72.6 (CH₂), 58.4 (CH₃), 43.8 (C), 39.7 (CH₂), 38.2 (CH₂), 38.0 (CH₃), 35.4 (CH₃), 30.7 (CH₂), 30.0 (CH₂), 27.8 (CH₂), 20.5 (CH₂), 18.6 (CH₂) ppm; MS m/z (%) 252 (M⁺ - Br, 3), 91 (8),

87 (3), 72 (14) and 45 (100); and

- elemental analysis: calculated for Ci₅H₂₆BrNO₂: C, 54.22; H, 7.89; N, 4.22; found: C, 54.04; H, 8.05; N, 4.22. e) preparation of 2-H -(4-methoxybutyl)-cvclohex-2-enyll-A/.Λ/-dimethyl-acetamide stereoisomers (5) and (5')

To a solution of the relevant 2-[2-bromo-1-(4-methoxybutyl)-cyclohexy-2-enyl]- Λ/,Λ/-dimethyl-acetamide isomer (4) or (4') (37.0 g, 111 mmol) in THF (600 mL) was added tributyltin hydride (H-Bu₃SnH) (35.3 mL, 133 mmol) followed by azobisisobutyronitrile (1.8 g, 11.1 mmol). The reaction mixture was brought to reflux for

4 hours and the solvent removed in vacuo. The resulting residue was purified over silica

(gradient elution: cyclohexane/EtOAc, 6:4 to EtOAc 100%) to afford 25.9 g (92% yield) of the desired product as a light-yellow oil which was characterized as follows:

- R_f (isooctane/EtOAc, 1 : 1 ) 0.24;

- optical rotation : [α]_D ^rt -24.0 (c = 1.59, CHCI₃) for isomer (5); [α]_D ^rt +22.9 (c = 1.07, CHCI₃) for isomer (5');

- IR (KBr film): v 3013, 2931 , 2864, 1637, 1490, 1449, 1384, 1261 , 1114, 1061 , 955 and 732 cm^"1;

- ¹H NMR (500 MHz, CDCI₃) δ 5.62 (1 H, ABt, J = 10.2, 3.7 Hz), 5.50 (1 H, AB, J = 10.2 Hz), 3.32 (2 H, t, J = 6.6 Hz), 3.26 (3 H, s), 2.96 (3 H, s), 2.87 (3 H, s), 2.40 (1 H, AB, J = 14.2 Hz), 2.19 (1 H, AB, J = 14.2 Hz), 1.93-1.84 (2 H, m), 1.63-1.43 (8 H, m) and 1.32-1.20 (2 H, m) ppm; - ¹³C NMR (50 MHz, CDCI₃) δ 171.3 (C), 134.3 (CH), 126.3 (CH), 72.6 (CH₂), 58.2 (CH₃), 40.9 (CH₂), 39.5 (CH₂), 38.1 (CH₃), 37.1 (C), 35.1 (CH₃), 32.5 (CH₂), 30.0 (CH₂), 24.7 (CH₂), 20.3 (CH₂) and 18.8 (CH₂) ppm;

- MS m/z (%): 253 (M⁺, 5), 238 (10), 210 (2), 166 (12), 87 (59), 72 (47) and 45 (100); and - elemental analysis: calculated for C₁₅H₂₇NO₂: C, 71.10; H, 10.74; N, 5.53; found: C, 70.97; H, 10.91 ; N, 5.45.

f) preparation of 2-H -(4-hvdroxybutyl)-cvclohex-2-envn-N,N-dimethyl-acetamide stereoisomers (6) and (6') To a solution of the relevant 2-[1-(4-methoxybutyl)-cyclohex-2-enyl]-Λ/,Λ/- dimethyl-acetamide isomer (5) or (5') (51.0 g, 201 mmol), NaI (60 g, 400 mmol) and 15- crown-5 (55 g, 250 mmol) in CH₂CI₂ (1000 mL) at -30 ⁰C was added dropwise BBr₃ (1 M solution in CH₂CI₂; 241 mL, 241 mmol). The reaction mixture was stirred at -30 ⁰C for 1 hour then a saturated NaHCO₃ solution (1000 ml_) was added. The organic layer was separated and the aqueous layer extracted with CH₂CI₂. The combined organic layers were washed with a saturated Na₂SO₃ solution and dried over anhydrous MgSO₄. The solvent was removed in vacuo and the residue purified over silica (gradient elution: Et₂O 100% to Et₂O/MeOH, 95:5) to afford 39.9 g (83% yield) of the desired product as a viscous oil which was characterized as follows:

- R_f (EtOAc) 0.25;

- optical rotation at room temperature [α]_D ^rt -21.4 (c = 1.03, CHCI₃) for isomer (6); [α]_D ^rt +20.2 (c = 0.99, CHCI₃) for isomer (6'); - IR (KBrfilm): v3409, 3012, 2932, 2863, 1626, 1498, 1457, 1398, 1261, 1146, 1060, 923and730cm^"1;

- ¹H NMR (500 MHz, CDCI₃): δ 5.65 (1 H, ABt, J= 10.2, 3.7 Hz), 5.48 (1 H, AB, J= 10.2 Hz), 3.68-3.59 (2 H, m), 3.00 (3 H, s), 2.91 (3 H, s), 2.79 (1 H, brs), 2.47 (1 H, AB, J= 13.9 Hz), 2.24 (1 H,AB, J= 13.9 Hz), 1.98-1.89 (2 H, m)and 1.67-1.31 (10 H, m) ppm;

- ¹³C NMR (50 MHz, CDCI₃) δ 171.8 (C), 134.6 (CH), 126.8 (CH), 61.5 (CH₂), 40.3 (CH₂), 38.5 (CH₃), 38.3 (CH₂), 37.4 (C), 35.5 (CH₃), 35.4 (CH₂), 32.4 (CH₂), 25.0 (CH₂), 19.2 (CH₂) and 18.9 (CH₂) ppm;

- MS m/z (%): 239 (M⁺, 3), 196 (2), 166 (12), 121 (2), 87 (100), 79 (35), 72 (90) and 45 (25);

- elemental analysis: calculated for C₁₄H₂₅NO₂: C, 70.25; H, 10.53; N, 5.85; found: C₁ 70.10; H, 10.49; N, 5.71.

g) preparation of 4-(1-dimethylcarbamoylmethyl-cvclohex-2-enyl)-butyric acid stereoisomers (7) and (7')

To a solution of the relevant 2-[1-(4-hydroxybutyl)-cyclohex-2-enyl]-N,N-dimethyl- acetamide isomer (6) or (6') (13.7 g, 57.1 mmol) in DMF (200 mL) was added pyridinium dichromate (65.3 g, 241.0 mmol) and the mixture stirred at room temperature for 24 hours. The reaction mixture was poured into H₂O (200 mL) and extracted with EtOAc (5 x). The combined organic layers were washed with a saturated NaCI solution and dried over anhydrous MgSO₄. The solvent was removed in vacuo, the residual DMF was removed by vacuum distillation (Kugelrohr-type) to afford 13.3 g of the desired product which was used without further purification. h) preparation of 4-(1-methoxycarbonylmethyl-cvclohex-2-enyl)-butyric acid methyl ester stereoisomers (8) and (8')

The 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid isomer (7) or (7') from the previous step (13.3 g, 52.0 mmol; viscous oil) was dissolved in 1 ,4-dioxane (75 ml_), KOH (1 M solution in H₂O; 225 mL) was added and the reaction mixture was heated at 200 ⁰C (autoclave) for 4 hours. The reaction mixture was allowed to cool and then acidified using a 37% HCI solution. The aqueous layer was then repeatedly extracted with CH₂CI₂. The combined organic layers were dried over anhydrous MgSO₄ and the solvent removed in vacuo. The resulting residue was dissolved in MeOH (200 mL) and a solution of CH₂N₂ in Et₂O added at 0 ⁰C with vigorous stirring until a yellow color persists. Excess CH₂N₂ was destroyed by the addition of silica gel, the mixture filtered, and the filtrate dried over anhydrous MgSO₄. The solvent was removed in vacuo and the resulting residue purified over silica (isooctane/EtOAc, 9:1) to afford 11.3 g (77% yield over 2 steps) of the desired product as a colorless oil which was characterized as follows:

- R_f (n-pentane/Et₂O, 8:2) 0.42;

- optical rotation at room temperature [α]_D ^rt : -1.7 (c = 1.25, CHCI₃) for isomer (8); [α]_D ^rt +1.4 (c = 1.4, CHCI₃) for isomer (8'); - IR(KBrfilm): v3011, 2933, 2867, 1737, 1436, 1356, 1256, 1194, 1163, 1096, 1014, 882and732cm^"1;

- ¹H NMR (500 MHz, CDCI₃): δ 5.70 (1 H₁ ABt, J= 10.2, 3.7 Hz), 5.49 (1 H, ABt, J= 10.2, 1.9 Hz), 3.66(3H, s), 3.64 (3 H, s),2.34(1 H,AB,J= 13.6 Hz), 2.31 (1 H,AB, J= 13.6 Hz), 2.28 (2 H, t, J= 7.5 Hz), 2.01-1.88 (2 H, m) and 1.65-1.38 (8 H, m) ppm;

- ¹³C NMR (50 MHz, CDCI₃): δ 174.1 (C), 172.3 (C), 133.4 (CH), 127.5 (CH), 51.5 (CH₃), 51.3 (CH₃), 43.9 (CH₂), 39.2 (CH₂), 36.8 (C), 34.5 (CH₂), 32.3 (CH₂), 24.8 (CH₂), 19.5 (CH₂)and 18.8(CH₂)ppm;

- MSm/z(%): 254(M⁺, <1), 223 (5), 222(9), 190 (5), 181 (26), 180(42), 153 (37), 149 (44), 121 (43), 107(44), 93(97), 79(100), 59(64)and42(35); and

- elemental analysis: calculated for C₁₄H₂₂O₄: C, 66.12; H, 8.72; found: C, 66.24; H, 8.79. (i) preparation of 2-hydroxy-spiror5.51undecan-2.7-diene-3-carboxylic acid methyl ester stereoisomers (9) and (9')

To a solution of the relevant 4-(1-methoxycarbonylmethyl-cyclohex-2-enyl)- butyric acid methyl ester isomer (8) or (8') (31.0 g, 122 mmol) in dry THF (580 ml_) at - 78 ⁰C was added dropwise /-Pr₂NLi (2 M solution in n-heptane; 122 ml_, 244 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. A saturated NH₄CI solution (700 mL) was added and the aqueous layer repeatedly extracted with Et₂O. The combined organic layers were dried over anhydrous MgSO₄ and the solvents removed in vacuo. The resulting residue was purified over silica (n- pentane/Et₂O, 95:5) to afford 24.7 g (91% yield) of the desired product as white crystals which were characterized as follows: melting point 46 °C;

- R_f (A7-pentane/Et₂O, 95:5) 0.50;

- optical rotation at room temperature [α]_D ^rt : -19.1 (c = 1.05, CHCI₃) for isomer (9); [α]_D ^rt +17.4 (c = 1.15, CHCI₃) for isomer (9');

- UV (MeOH) λ_max 255 nm;

- IR (KBr): v 3061, 2994, 2949, 2924, 2856, 1665, 1623, 1444, 1414, 1380, 1333, 1294, 1282, 1261, 1212, 1179, 1132, 1087, 1048, 1024, 986, 960, 926, 905, 813and 733cm^"1; - ¹H NMR (500 MHz, CDCI₃): δ 12.1 (1 H, s), 5.68 (1 H, dt, J= 10.1, 3.7 Hz), 5.45 (1 H, dt, J= 10.1, 2.0 Hz), 3.76 (3 H, s), 2.32-2.22 (2 H, m), 2.16 (1 H,ABm, J= 18.3 Hz),2.12(1 H,ABm,J= 18.3Hz),2.00-1.94(2H, m), 1.73-1.50(4H, m)and 1.44- 1.38(2H, m)ppm;

- ¹³C NMR (75 MHz, CDCI₃): δ 172.8 (C), 170.8 (C), 134.0 (CH), 127.1 (CH), 96.5 (CH), 51.4 (CH₃), 40.8 (CH₂), 34.1 (CH₂), 33.4 (CH₂), 25.4 (CH₂), 19.3 (CH₂) and

18.7 (CH₂) ppm;

- MSm/z(%): 222(M⁺,4), 194(14), 128 (15), 107 (14), 94 (68), 79(100), 77 (36), 65 (20), 55(36)and41 (45);and

- elemental analysis: calculated for C₁₃H₁₈O₃: C, 70.24; H₁ 8.16; found: C, 70.15; H, 8.18.

j) preparation of spiror5.51undec-7-en-2-one stereoisomers (10) and (10')

To a solution of the relevant 2-hydroxy-spiro[5.5]undecan-2,7-diene-3-carboxylic acid methyl ester isomer (9) or (9^!) (19.0 g, 85.0 mmol) in DMSO (66 ml.) was added NaCI (5.5 g, 93.5 mmol) and H₂O (4.6 ml_, 255 mmol). The reaction mixture was heated at 160 ⁰C for 6 hours then cooled and poured into H₂O (100 ml_). The aqueous layer was extracted with Et₂O (5 x) and the combined organic layers dried over anhydrous MgSO₄. The solvent was removed in vacuo and the resulting residue purified over silica (/7-pentane/Et₂O, 95:5) to afford 13.3 g (95% yield) of the desired product as a colorless oil which was characterized as follows:

- R_f (n-pentane/Et₂O, 9: 1 ) 0.36;

- optical rotation at room temperature [α]_D ^rt -67.0 (c = 1.49, CHCI₃) for isomer (10); [α]_D ^rt +68.9 (c = 1.02, CHCI₃) for isomer (10');

- IR(KBrfilm): v 3014, 2932, 2873, 1713, 1446, 1422, 1345, 1312, 1287, 1227, 1204, 1064, 1009, 923, 887, 730and 529cm^"1;

- ¹H NMR(500 MHz, CDCI₃)δ5.64 (1 H,ABt,J= 10.1, 3.7 Hz), 5.42 (1 H,AB(feJ), J= 10.1 Hz), 2.32-2.26 (2 H, m), 2.21 (1 H, AB, J= 13.9 Hz), 2.21 (1 H, AB, J= 13.9 Hz), 1.95-1.82 (4 H, m), 1.72 (1 H, m), 1.63-1.52 (3 H, m) and 1.51-1.40 (2 H, m) ppm;

- ¹³C NMR (50 MHz, CDCI₃) δ 211.6 (C), 133.7 (CH), 126.9 (CH), 52.8 (CH₂), 40.9 (CH₂), 39.4 (C), 36.9 (CH₂), 33.4(CH₂), 24.9(CH₂),21.6 (CH₂)and 18.3(CH₂)ppm;

- MSm/z(%) 164 (M⁺, 61), 146 (8), 136 (11), 131 (17), 121 (39), 107 (89), 91 (41), 79 (100), 67(17), 55 (18)and42(33);and

- elemental analysis: calculated for C₁₁H₁₆O: C, 80.44; H, 9.82; found: C, 80.33; H, 9.95.

k) preparation of 1 ,4-dioxa-dispiror4.1.5.3lpentadec-8-ene stereoisomers (11) and (11') To a solution of the relevant spiro[5.5]undec-7-en-2-one isomer (10) or (10') (10 g, 61 mmol) in toluene (150 ml_) was added HO(CH₂)₂OH (10 mL, 179 mmol) and p- toluenesulfonic acid- H₂O (0.57 g, 3 mmol). The reaction mixture was brought to reflux and the H₂O generated was removed by azeotropic distillation (using a Dean-Stark separator). The reaction mixture was then cooled and washed with a saturated NaHCO₃ solution. The aqueous phase was extracted with Et₂O and the combined organic layers dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the resulting residue purified over silica (isooctane/EtOAc, 95:5) to afford 12.1 g (95% yield) of the desired product which was characterized as follows: R_f (isooctane/EtOAc, 95:5) 0.34; optical rotation at room temperature [α]_D ^rt -25.8 (c = 1.41 , CHCI₃) for isomer (11);

[α]_D ^rt +25.2 (c = 1.03, CHCI₃) for isomer (11 ');

IR (KBr film) v 2933, 2871 , 2837, 1643, 1147, 1360, 1317, 1283, 1241 , 1219, 1178,

1156, 1074, 1051 , 1013, 947, 926, 888, 854, 821 and 732 cm^"1;

¹H NMR (500 MHz, CDCI₃): δ 5.67 (1 H, AB, J = 10.2 Hz), 5.59 (1 H, ABt, J = 10.2,

3.6 Hz), 3.94-3.89 (4 H, m), 1.96-1.92 (2 H, m), 1.68-1.50 (10 H, m) and 1.40-1.30

(2 H, m) ppm;

¹³C NMR (50 MHz, CDCI₃): δ 136.2 (CH), 125.6 (CH), 109.3 (C), 64.1 (CH₂), 63.9

(CH₂), 45.4 (CH₂), 37.3 (CH₂), 35.8 (C), 35.0 (CH₂), 25.5 (CH₂), 19.5 (CH₂) and 18.9

(CH₂) ppm; and

MS m/z(%): 208 (M⁺, 12), 193 (14), 165 (70), 125 (17), 99 (100), 86 (62), 79 (38) and41 (32).

As mentioned herein above, the corresponding 1 ,4-dithia-dispiro[4.1.5.3]pentadec-8- ene stereoisomers are also accessible in step (k) while using HS(CH₂)₂SH as an alternative reactant.

Compounds which are comprised in a first category of the present invention include, but are not limited to, the E-isomers of 5-[2-(3-R^0b-substituted-spiro[5.5]undec-7- ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following structural formula:

wherein the R^ob, R^5a, R^5b, R^6a and R^6b substituents are outlined herein below in Table 1 and wherein the first column attributes a number to each combination of substituents. TABLE 1

The skilled person understands that each compound of this first category of the present invention is usually obtained from the corresponding hydroxyl-protected intermediate having the same structural formula but wherein each substituent selected from the group consisting of R^5a, R^5b, R^6a and R^6b is, wherever applicable, a hydroxyl protected group OP (with P being as already defined herein) instead of hydroxyl.

The following schemes below outlines a sequence of process steps which may be used for the preparation of intermediates and precursors containing the R^Oa, R^Ob, R^1a, R^1b, R^2a and/or R^2b substituents of the F ring which are comprised in the compounds of the various categories of the present invention. Each step of these schemes will now be illustrated in details, based on specific starting compounds, reactive agents, catalysts, solvents, temperature ranges and the like, but the skilled person will understand that the specific materials and conditions disclosed herein may be replaced with similar or equivalent materials and conditions without significantly altering the resulting product of the relevant step. In particular the steps of the following scheme are illustrated below starting from 1 ,4-dioxa-dispiro intermediates, but the skilled person understands that a similar sequence of reactions can be performed successfully while starting from the corresponding 1 ,4-dithia-dispiro intermediates.

In the following step, the relevant 1 ,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene stereoisomer (referred as (11) in scheme (I), hereunder shown as compound 30) or 1 ,4- dithia-dispiro[4.1.5.3]pentadec-8-ene stereoisomer obtained in the eleventh and last step of the above scheme (I) is submitted, according to scheme II, to an allylic oxidation reaction by any suitable method, for instance by reaction with one or more molar equivalents of /--BuOOH, preferably in a suitable solvent (such as, but not limited to, acetonitrile, benzene and toluene) and in the presence of an effective amount of a suitable catalyst such as but not limited to copper iodide, hexacarbonyl chromium or pyridinium dichromate, at a temperature ranging from about 40⁰C to about 100⁰C (depending upon the solvent used).

Scheme Il

The above scheme combined with the following table show that, in addition to the desired intermediate 88, a certain amount of a by-product 89 resulting from undesired deacetalization and optionally a residual amount of the starting material 30 may still be found in the reaction mixture after 24 hours. However the third set of conditions provides an efficient way for making the desired intermediate 88.

(+) 88 (+) 89 (±) ³⁰ Reaction conditions (%) (^%) (%) f-BuOOH (7 eq.), CuI (0.01 eq.), CH₃CN, 5O⁰C

37 9 0 f-BuOOH (4 eq.), Cr(CO)₆ (0.5 eq.), CH₃CN, reflux

27 39 0 WBuOOH (4 eq.), pyridimium chromate (4 eq.),

64 7 10 Celite, benzene, room temperature

Intermediate 88 was characterized as follows:

- R_f (isooctane / ethyl acetate: 7/3) = 0.28; melting point: 46°C; - UV (CH₃OH): λ_max = 227 nm;

- IR(film): v 2935(C-H, s), 2887 (C-H, m), 1670 (C=O, s), 1605 (w), 1458 (w), 1426 (W)₁1392 (m), 1363 (m), 1326 (w), 1285 (w), 1252 (m), 1252 (w), 1185 (w), 1152 (m), 1108 (m), 1074 (m), 1053 (w), 1028 (w), 954 (m), 924 (w), 888 (w), 861 (w), 827(w)and797(w)cm^"1; - MS(m/z, %): 222(M^+", 14), 179 (50), 166(3), 151 (4), 135 (8), 113 (13), 99 (100), 86 (97), 65(9), 55 (26)and42(21) ;

- ¹H-NMR (500 MHz, CDCI₃): δ 7.05 (1H, d, J= 10.3 Hz), 5.86 (1H, d, J= 10.3 Hz), 3.95-3.87 (4H₁ m), 2.42 (2H, t, J= 6.8 Hz), 2.01 (1H, ABt, J= 13.4, 6.6 Hz), 1.89 (1H₁ABt, J= 13.5, 6.9Hz), 1.74(1H₁AB₁J= 13.9 Hz), 1.61 (1H₁AB₁J= 13.8 Hz), 1.77- 1.57(5H, m)and 1.44(1H, m) ppm ;

- ¹³C-NMR (50 MHz, CDCI₃): δ 199.7 (C), 158.1 (CH), 127.2 (CH), 108.4 (C), 64.3 (CH₂), 64.2(CH₂),43.4 (CH₂), 37.0 (C), 35.6 (CH₂), 34.8 (CH₂), 33.9 (CH₂)and 19.6 (CH₂)ppm;and

- elemental analysis: calculated: C = 70.24, H = 8.16; found: C = 70.17, H = 8.33.

According to scheme III, intermediate 88 is then hydrogenated, preferably in the presence of a suitable catalyst, into a mixture of stereoisomers 90a and 90b, preferably at a temperature ranging from about 1O⁰C to about 40⁰C. Scheme

(±)-88 90a (52%) 90b (45%)

Intermediate 90a was isolated as a colorless oil, whereas intermediate 90b was obtained as crystalline product and characterized as follows:

- R_f (isooctane / ethyl acetate: 3/7) = 0.43; melting point: 41 ⁰C;

- IR (film): v 3386 (O-H, s), 2930 (C-H, s), 2876 (C-H₁ s), 1446 (m), 1364 (m), 1313 (W), 1281 (W), 1242 (w), 1218 (w), 1173 (m), 1128 (m), 1084 (s), 1058 (s), 1021 (w),

983 (w), 964 (m), 940 (m), 921 (w), 854 (w) and 821 (w) cm^"1;

- MS (m/z): 226 (M^+', 3), 183 (70), 165 (15), 121 (6), 113 (11), 99 (100), 86 (34), 79 (10), 67 (9), 55 (22) and 41 (15) ;

- ¹H-NMR (500 MHz, CDCI₃): δ 3.95 - 3.89 (4H, m), 3.63 (1 H, m), 1.80 - 1.73 (4H, m), 1.63 - 1.56 (6H, m), 1.47 - 1.40 (3H, m), 1.26 (2H, m) and 1.14 (2H, m) ppm ;

- ¹³C-NMR (50 MHz, CDCI₃): δ 109.4 (C), 70.7 (CH), 64.1 (CH₂), 40.9 (CH₂), 38.4 (CH₂), 35.5 (CH₂), 33.7 (C), 30.6 (CH₂), and 19.6 (CH₂) ppm; and

- elemental analysis: calculated C = 68.99, H = 9.80; found C = 68.98, H = 9.84.

According to scheme IV, intermediate 90a is then submitted to a Williamson ether synthesis, preferably in two sub-steps, and transformed (yield: 83%) into intermediate 92a, preferably in the presence of a suitable solvent such as DMF, also preferably at a temperature ranging from about 0⁰C to about 30°C. The corresponding transformation of 90b into the corresponding intermediate stereoisomer 92b (not shown in the scheme below) proceeds similarly but with a yield of 71%. As indicated below, the first sub-step proceeds in the presence of sodium hydride (preferably a molar excess thereof with respect to the relevant intermediate 90), whereas the second sub-step proceeds via the addition of 4-bromo-2-methyl-2-butene in the presence of a catalytic amount of tetrabutylammonium iodide. Scheme IV

Intermediates 92a and 92b were isolated as colorless oils, and characterized as follows:

- R_f (isooctane / ethyl acetate: 9/1 ) = 0.30;

- IR (film): v 2932 (C-H, s), 2873 (C-H, s), 1673 (C=C, vw), 1447 (m), 1367 (w), 1241 (w), 1170 (w), 1121 (w), 1085 (s), 941 (w), 852 (w) and 823 (w) cm^"1 - MS (m/z): 294 (M^+", 3), 251 (8), 225 (19), 209 (49), 183 (15), 167 (10), 165 (12), 147 (11), 113 (24), 99 (100), 86 (25), 69 (27), 55 (32) and 41 (55) ;

- ¹H-NMR (500 MHz, CDCI₃): δ 5.35 (1H, t(fs), J = 6.9 Hz), 3.96 (2H, d, J = 6.9 Hz), 3.91 (4H, s), 3.24 (1H, tt, J = 9.5, 3.9 Hz), 1.77 - 1.71 (4H, m), 1.73 (3H, s), 1.66 (3H, s), 1.58 - 1.55 (4H, m), 1.47 - 1.40 (6H, m) and 1.18 (2H, m) ppm; - ¹³C-NMR (50 MHz, CDCI₃): δ 136.10 (C), 121.76 (CH), 109.37 (C), 76.79 (CH), 64.19 (CH₂), 63.97 (CH₂), 46.47 (CH₂), 35.30 (CH₂), 34.66 (CH₂), 34.12 (C), 32.78 (CH₂), 27.29 (CH₂), 25.78 (CH₃), 19.33 (CH₂) and 17.92 (CH₃) ppm; and

- elemental analysis: calculated C = 73.43, H = 10.27; found C = 73.26, H = 10.48.

According to scheme V, intermediate 92a is then transformed (yield: 92%), preferably in two sub-steps, into the hydroxyacetal 93a, preferably in the presence of a suitable solvent such as THF, also preferably at a temperature ranging from about 10⁰C to about 40°C. The corresponding transformation of 92b into the corresponding intermediate stereoisomer 93b (not shown in the scheme below) proceeds similarly but with a yield of 90%. As indicated below, the first sub-step proceeds in the presence of mercury acetate (preferably a molar excess thereof with respect to the relevant intermediate 92), whereas the second sub-step proceeds via the addition of suitable amounts of sodium borohydride and a sodium hydroxide solution. Scheme V

92a 93a

Intermediates 93a and 93b were isolated as colorless oils, and characterized as follows:

- R_f (isooctane / ethyl acetate: 7/3) = 0.25;

- IR (film): v 3462 (O-H, s), 2966 (C-H, m), 2931 (C-H, s), 2868 (C-H, s), 1448 (m), 1369 (m), 1281 (w), 1242 (w), 1222 (w), 1169 (m), 1149 (m), 1119 (w), 1081 (s), 1077 (s), 1042 (m), 1026 (w), 943 (m), 883 (w) and 852 (w) cm^"1

- MS (m/z, %): 312 (M⁺', 2), 297 (M⁺'- CH₃", 2), 269 (25), 225 (3), 209 (25), 165 (36), 113 (15), 99 (100) and 43 (33);

- ¹H-NMR (500 MHz, CDCI₃): δ 3.92 (4H, s), 3.88 (1 H, s(bή), 3.70 (2H, t, J = 5.7 Hz), 3.27 (1 H, tt, J = 8.6, 3.6 Hz), 1.74 (2H, t, J = 5.7 Hz), 1.73 - 1.64 (4H, m), 1.58 - 1.54 (4H, m), 1.49 - 1.42 (4H, m), 1.36 (2H, m(bή), 1.24 (2H, m) and 1.23 (6H, s) ppm;

- ¹³C-NMR (50 MHz, CDCI₃): δ 109.4 (C), 77.7 (CH), 70.6 (C), 65.3 (CH₂), 64.0 (CH₂), 45.5 (CH₂), 41.3 (CH₂), 35.3 (CH₂), 34.1 (CH₂), 33.8 (C), 29.2 (CH₃), 26.9 (CH₂) and 19.3 (CH₂) ppm; and

- elemental analysis: calculated C = 69.19, H = 10.32; found C = 69.35, H = 10.32.

According to scheme Vl, intermediate 90b is then transformed (yield: 70%), preferably in two sub-steps, into the desired intermediate 95b, preferably in the presence of a suitable solvent such as DMF. The corresponding transformation of 90a into the corresponding intermediate stereoisomer 95a (not shown in the scheme below) proceeds similarly but with a yield of 72%. As indicated below, the first sub-step proceeds in the presence of sodium hydride (preferably an equivalent amount thereof with respect to the relevant intermediate 90) preferably at a temperature ranging from about 1O⁰C to about 30⁰C, whereas the second sub-step proceeds via the addition of isobutene oxide and increasing the temperature up to at least 70⁰C, preferably from 80⁰C to about 100⁰C.

Scheme Vl

90b 95b 96b

The above scheme shows that, in addition to the desired intermediate 95, a certain amount of a by-product 96 may be formed. Optionally, a residual amount of the starting material 90 may still be found in the reaction mixture after 24 hours. However the above set of reaction conditions provides an efficient way for making the desired intermediate stereoisomers 95.

Intermediate 95b was isolated as a colorless oil, whereas the corresponding intermediate stereoisomer 95a was obtained as a crystalline product and characterized as follows: - Rf (isooctane / ethyl acetate: 8/2) = 0.17;

- melting point: 46 ⁰C;

- IR(film): v 3554 (O-H, s), 3416 (O-H, brs), 2967 (C-H, m), 2991 (C-H, s), 2887 (C- H, m), 1464 (w), 1448 (m), 1366 (m), 1340 (w), 1318 (w), 1282 (w), 1242 (w), 1172 (m), 1116 (s), 1076 (s), 1032 (m), 1014 (m), 943(m), 924 (m), 851 (w), 827 (w), 776 (w),704(w)and884(w)cm^"1

- MS (m/z, %): 298 (M^+*, <1), 255 (15), 225 (3), 209 (12), 165 (10), 131 (9), 99 (100), 86 (27), 79 (17), 59 (62), 55 (29), 43 (20) and 41 (22) ;

- ¹H-NMR (500 MHz, CDCI₃): δ 3.90 (4H, s), 3.26 (1 H₁ m), 3.23 (2H, s), 2.44 (1 H, s), 1.73 - 1.64 (4H, m), 1.57 - 1.56 (4H, m), 1.48 - 1.44 (4H, m), 1.38 (2H, m), 1.21 (2H, m) and 1.18 (6H, s) ppm ; and

- ¹³C-NMR (50 MHz, CDCI₃): δ 109.4 (C), 77.7 (CH), 76.1 (CH₂), 70.0 (C), 64.0 (CH₂), 45.6 (CH₂), 35.3 (CH₂), 34.2 (CH₂), 34.0 (CH₂), 33.7 (C), 27.0 (CH₂), 26.1 (CH₃), 19.3 (CH₂) ppm. The characterizing data of the by-product 96b are as follows:

- Rf (isooctane / ethyl acetate: 6/4) = 0.40;

- IR (film): v 3482 (O-H, br s), 2969 (C-H₁ m), 2929 (C-H, s), 2872 (C-H, m), 1496 (s), 1474 (W), 1485 (w), 1368 (w), 1251 (m), 1208 (m), 1172 (w), 1150 (w), 1102 (s), 1082 (m), 964 (w), 942 (w), 915 (s), 854 (w), 820 (w), 796 (w), 744 (m) and 734 (m) cm^"1

- MS (m/z, %): 370 (M⁺' , <1), 327 (2), 240 (4), 209 (11), 165 (15), 131 (55), 99 (46), 73 (44), 59 (C₃H₇O⁺, 100) and 43 (15);

- ¹H-NMR (500 MHz, CDCI₃): δ 3.93 - 3.87 (4H, m), 3.29 (2H, s), 3.22 (1 H, m), 3.21 (2H, s), 3.01 (1 H, s(bή), 1.75 - 1.68 (4H, m), 1.59 - 1.55 (6H, m), 1.42 (2H, m), 1.24

(2H, m), 1.16 (6H, s), 1.15 (6H, s) and 1.10 (2H, m) ppm; and

- ¹³C-NMR (50 MHz, CDCI₃): δ 109.4 (C), 78.2 (CH), 74.8 (CH₂), 73.2 (C), 70.1 (CH₂), 69.7 (C), 64.1 (2 X CH₂), 41.5 (CH₂), 37.9 (CH₂), 35.5 (CH₂), 34.6 (CH₂), 33.9 (C), 27.1 (CH₂), 26.1 (2 x CH₃), 23.3 (CH₃) and 19.6 (CH₂) ppm.

According to scheme VII, intermediate 93a is then submitted to a deacetalization step (yield: 85%), preferably by means of acid hydrolysis at a temperature ranging from about 10⁰C to about 4O⁰C, into the desired hydroxyketone intermediate 97a, preferably in the presence of a suitable solvent such as a ketone, desirably acetone. The corresponding transformation of 93b into the corresponding intermediate stereoisomer 97b (not shown in the scheme below) proceeds similarly but with a yield of 83%. As indicated below, this step proceeds for example in the presence of a catalytic amount of p-toluenesulfonic acid monohydrate at room temperature.

Scheme VII

93a 97a

The intermediate hydroxyketone 97a was characterized as follows: - R_f (isooctane / ethyl acetate: 1/1 ) = 0.29;

- IR (film): v 3457 (O-H, s), 2933 (C-H₁ s), 2866 (C-H, m), 1707 (C=O, s), 1448 (m), 1363(W), 1309(w), 1224(w), 1145(m), 1091 (s), 940(w)and876(w)cm^'1

- MS (m/z, %): 253 (M^+"- CH₃ ^', 3), 250 (M^+'- H₂O, 9), 181 (26), 165 (100), 147 (39), 125(28), 107(33), 87(37),79(38),69(69), 55(70), 59(77)and41 (62);

- ¹H-NMR (500 MHz, CDCI₃): δ3.69 (2H, t, J= 5.8 Hz), 3.62 (1H, s), 3.33 (1H, tt, J= 7.9, 3.9 Hz), 2.29 (2H, t, J=6.8 Hz), 2.17 (2H, s), 1.84 (2H, m), 1.75 (2H, t, J= 5.6 Hz), 1.74 (2H, m), 1.68 (2H, dd, J= 6.1, 6.1 Hz), 1.58 (1H, s), 1.58 - 1.49 (4H, m), 1.24(6H,s)and 1.22(2H, m)ppm; - ¹³C-NMR (50 MHz, CDCI₃): δ 211.9 (C), 76.7 (CH), 70.6 (C), 65.5 (CH₂), 53.0 (CH₂), 41.3 (CH₂), 41.2 (CH₂), 38.1 (C), 33.8 (CH₂), 33.0 (CH₂), 29.2 (CH₃), 26.4 (CH₂) and 21.5 (CH₂) ppm; and

- elemental analysis: calculated C = 71.60, H = 10.52; found: C = 71.74, H = 10.49.

Any of the previous intermediates 95a, 95b, 96a and 96b may be submitted to the above deacetalization procedure, under conditions similar to these of scheme VII, before being coupled in a next step to a suitable precursor of the A ring of the vitamin D molecule as described herein after.

According to scheme VIII, intermediate 97a is submitted to a Horner-Wittig (also named Lythgoe coupling) reaction involving a phosphine oxide (the latter being suitably selected in view of the desired final vitamin D compound), optionally in the presence of a catalyst. As indicated in the scheme below, this step proceeds for example in the presence of a 5-[2-(diphenyl-phosphinoyl)-ethylidene]-bis-(te/t-butyl-dimethyl-silanyloxy)- cyclohexane 28 as a phosphine oxide reagent (preferably a molar excess amount thereof with respect to the relevant intermediate 97), n-butyl lithium as a catalyst, THF as a solvent, at a temperature ranging from about -80⁰C to about 40⁰C, preferably with a temperature initially set at -78⁰C and progressively increasing as long as reaction proceeds. Scheme VIII

As indicated in the scheme above, this reaction produces a mixture of silyl ethers comprising about 85% of the E-isomer 98 and about 15% of the Z-isomer 99 which was characterized as follows:

- R_f (pentane / diethylether: 8/2) = 0.25; - IR (film): v 3496 (O-H, s), 2929 (C-H, s), 2887 (C-H, m), 2856 (C-H, m), 1654 (C=C, vw), 1636 (C=C, VW), 1473 (w), 1462 (w), 1448 (w), 1362 (w), 1253 (m), 1145 (w), 1086 (s), 1006 (w), 990 (w), 908 (m), 895 (w), 885 (w), 834 (s), 775 (m) and 734 (w) cm^"1

- UV (CH₃OH): λ_max = 212 and 260 nm; - ¹H-NMR (500 MHz, CDCI₃): δ 6.17 (1 H of Z-isomer, AB, J = 11.3 Hz), 6.15 (1 H of £-isomer, AB, J = 11.1 Hz), 6.09 (1 H of E-isomer, AB, J = 11.3 Hz), 6.02 (1 H of E-isomer, AB, J = 11.2 Hz), 5.20 (1H of Z-isomer, m), 5.19 (1 H of E-isomer, m), 4.84 (1H, m), 4.38 (1H, t, J = 5.4 Hz), 4.18 (1H, m), 3.75 (1H, s(bή), 3.69 (2H, t, J = 5.7 Hz), 3.30 (1H, m), 2.41 (1H, ABd, J = 13.1 , 3.2 Hz), 2.28 (1H,m), 2.19 (1H, ABd, J = 13.1 , 6.6 Hz), 2.20 (1H, m), 1.89 (2H, s), 1.81 (2H, t, J = 5.2 Hz), 1.74 (2H, t, J = 5.7

Hz), 1.69 (2H, m), 1.52 - 1.40 (8H, m), 1.23 (6H, s), 1.11 - 1.09 (2H, m), 0.88 (9H, s), 0.86 (9H, s), 0.07 (3H, s), 0.06 (3H, s), 0.05 (3H, s) and 0.04 (3H, s) ppm; and

- ¹³C-NMR (50 MHz, CDCI₃): (E-isomer) δ 148.4 (C)₁ 139.0 (C), 135.2 (C), 123.2 (CH), 120.6 (CH), 110.8 (CH₂), 77.8 (CH), 71.7 (CH), 70.6 (C), 67.5 (CH), 65.3 (CH₂), 49.1 (CH₂), 45.9 (CH₂), 44.7 (CH₂), 41.3 (CH₂), 35.2 (C), 33.2 (CH₂), 32.7 (CH₂), 29.2

(CH₃), 29.1 (CH₂), 26.6 (CH₂), 25.8 (CH₃), 22.4 (CH₂), 18.2 (C), 18.1 (C),-4.8 (CH₃) and -5.1 (CH₃) ppm. According to scheme IX, the mixture of intermediates 98 and 99 is then submitted, in a last step, to oxygen deprotection under standard conditions for this kind of reaction:

Scheme IX

Preferred reagents and conditions include, for instance, tetrabutylammonium fluoride as a reagent; THF as a solvent, and a temperature ranging from about 10⁰C to about 40⁰C. The resulting triol 100 was characterized as follows:

- R_f (ethyl acetate / isooctane: 9/1 ) = 0.33;

- IR (film): v 3340 (O-H, s), 2929 (C-H, s), 2865 (C-H, m), 1447 (s), 1082 (m), 1058 (s), 955 (w), 908 (m), 865 (w), 792 (w) and 733 (s) cm^"1

- UV (CH₃OH): λ_max = 211 and 262 nm; - ¹H-NMR (500 MHz, CDCI₃): δ 6.31 (1H, AB, J = 11.3 Hz), 6.05 (1 H, AB, J = 11.2 Hz), 5.32 (1H, s), 4.98 (1H, s), 4.43 (1H, s(bή), 4.23 - 4.19 (1 H, m(bή), 3.80 (1H, s(bή), 3.69 (2H, t, J = 5.7 Hz), 3.29 (1 H, tt, J = 7.9, 3.9 Hz), 2.60 (1 H, dd, J = 13.1 , 3.6 Hz), 2.31 - 2.22 (3H, m), 1.96 (2H, t, J = 5.6 Hz), 1.91 (2H, s), 1.74 (2H, t, J = 5.7 Hz), 1.71 - 1.69 (2H, m), 1.59 (2H, s(bή), 1.54 - 1.43 (8H, m), 1.24 (6H, s) and 1.13 - 1.08 (2H, m) ppm; and

- ¹³C-NMR (50 MHz, CD₂CI₂): δ 148.4, 141.4, 134.3, 125.0, 120.5, 112.3, 78.3, 71.5, 70.9, 67.2, 66.0, 49.7, 45.9, 43.5, 42.0, 35.8, 35.5, 33.9, 33.5, 29.7, 27.3 and 23.0 ppm.

According to scheme X, the stability of intermediate 100 was investigated and quantatively determined by the following procedure. A small amount of the pure intermediate 100 was dissolved in deuteroform, then brought into a NMR tube and shielded from light. Its transformation, at room temperature, into the pre-vitamin compound 102 was followed by ¹H-NMR spectroscopy. After three months, equilibrium was completed and resulted in about 75% of the pre-vitamin form, as shown by the scheme:

Scheme X

100

The pre-vitamin form 102 was characterized as follows:

- R_f (ethyl acetate / isooctane: 9/1 ) = 0.20;

- IR (film): v 3372 (O-H, s), 2965 (C-H, s), 2927 (C-H, s), 2867 (C-H, m), 1444 (m), 1367 (m), 1217 (w), 1153 (w), 1087 (s), 1046 (s), 956 (w), 907 (w), 842 (w) and 731 (m) cm^"1; and - ¹H-NMR (500 MHz, CDCI₃): δ 5.87 (1 H, AB, J = 12.4 Hz), 5.70 (1H, AB, J = 12.4 Hz), 5.64 (1 H, s(bή), 4.19 (1H, s(bή), 4.14 - 4.09 (1 H, m), 3.79 (1 H, s(bή), 3.70 (2H, t, J = 5.7 Hz), 3.32 (1 H, tt, J = 8.0, 4.0 Hz), 2.44 (1 H, dd, J = 16.7, 4.7 Hz), 2.08 - 2.03 (3H, m), 1.86 (1 H₁ AB₁ J = 16.9 Hz), 1.82 (1 H, AB, J = 16.9 Hz), 1.72 (3H₁ s), 1.76 - 1.41 (14H, m), 1.24 (6H₁ s) and 1.14 - 0.90 (2H, m) ppm.

In a manner similar to the procedures shown in schemes VIII and IX, the Horner- Wittig reaction (Lythgoe coupling) step was carried out onto intermediate 97a but starting from a different phosphine oxide reagent 29 (shown below) and then followed by a standard oxygen deprotection step as previously described. The following scheme Xl shows that this combined sequence of two steps results, with a 38% yield, in a mixture comprising about 80% of the E-isomer 103a and about 20% of the Z-isomer 104a. Scheme Xl

103a 104a

The resulting mixture was characterized as follows: R_f (ethyl acetate / isooctane: 95/5) = 0.19;

IR (film): v 3384 (O-H, s), 2924 (C-H, s), 2868 (C-H, m), 2842 (C-H, w), 1443 (w), 1368 (w), 1298 (w), 1218 (w), 1156 (w), 1086 (m), 1051 (m), 978 (w), 954 (w), 939 (w), 909 (w), 861 (w), 815 (w) and 711 (w) cm^"1; UV (CH₃OH): λ_max = 241 , 249 and 259 nm;

MS (m/z): 392 (M^+', 14), 374 (M^+'- H₂O, 9), 359 (1), 306 (3), 288 (11 ), 270 (6), 215 (3), 194 (5), 147 (18), 145 (21), 107 (73), 91 (50), 84 (81), 79 (53), 59 (46), 49 (100) and 43 (69);

¹H-NMR (500 MHz, CD₂CI₂): δ 6.22 (1 H of E-isomer, AB, J = 11.3 Hz), 6.20 (1 H of Z- isomer, AB, J = 11.3 Hz), 6.10 (1 H of Z-isomer, AB, J = 11.3 Hz), 5.95 (1 H E-isomer, AB, J = 11.3 Hz), 4.03 (2H, s(bή), 3.68 (2H, t, J = 5.8 Hz), 3.54 (1H, s), 3.30 (1 H, tt, J = 8.3, 4.1 Hz), 2.60 (1 H, dd, J = 13.3, 3.8 Hz), 2.45 (1 H, dd, J = 13.3, 3.6 Hz), 2.29 - 2.21 (2H, m), 2.17 - 2.12 (2H, m), 1.96 (2H, s), 1.87 - 1.70 (4H, m), 1.71 (2H, t, J = 5.8 Hz), 1.61 (2H, s(ό/-)), 1.55 - 1.40 (8H, m), 1.19 (6H, s) and 1.15 - 1.10 (2H₁ m) ppm; and

¹³C-NMR (50 MHz, CD₂CI₂): (£-isomer) δ 140.6 (C), 132.5 (C), 123.5 (CH), 118.7 (CH), 78.2 (CH), 70.7 (C), 67.7 (CH), 67.4 (CH), 65.7 (CH₂), 49.7 (CH₂), 45.2 (CH₂), 42.6 (CH₂), 41.8 (CH₂), 37.2 (CH₂), 35.4 (C), 35.0 (CH₂), 34.0 (CH₂), 33.7 (CH₂), 33.5 (CH₂), 29.4 (CH₃), 29.3 (CH₂), 27.1 (CH₂) and 22.6 (CH₂) ppm. In a similar manner, the following scheme XII shows the preparation, with a 59% yield, of a mixture comprising about 80% of the E-isomer 103b and about 20% of the Z- isomer 104b.

Scheme XII

After reaction, both isomers were separated by means of high performance liquid chromatography (HPLC). Isomer 103b was characterized as follows:

R_f (ethyl acetate / isooctane: 95/5) = 0.16;

- IR (film): v 3384 (O-H, s), 2924 (C-H, s), 2868 (C-H, m), 2842 (C-H, w), 1443 (w), 1368 (w), 1298 (w), 1218 (w), 1156 (w), 1086 (m), 1051 (m), 978 (w), 954 (w), 939 (w), 909 (W), 861 (w), 815 (w) and 711 (w) cm^"1; - UV (CH₃OH): λ_max = 241 , 249 and 259 nm;

- MS (m/z, %): 392 (M^+', 1 ), 374 (M^+'- H₂O, 7), 356 (10), 306 (4), 288 (23), 270 (17), 215 (14), 194 (10), 147 (26), 145 (19), 107 (51), 91 (75), 79 (76), 59 (68) and 43 (69);

- ¹H-NMR (500 MHz, CDCI₃): δ 6.24 (1 H, AB, J = 11.3 Hz), 5.94 (1 H, AB, J = 11.3 Hz), 4.12 - 4.07 (2H, m), 3.79 (1 H, s(bή), 3.72 (2H, t, J = 5.7 Hz), 3.27 (1 H, tt, J = 8.9, 3.9

Hz), 2.63 (1 H, dd, J = 13.4, 3.6 Hz), 2.49 (1 H, dd, J = 13.2, 3.6 Hz), 2.32 - 2.16 (4H, m), 2.06 (1 H, AB, J = 13.3 Hz), 2.02 (1 H, AB, J = 13.3 Hz), 1.92 - 1.82 (2H, m), 1.76 (2H, t, J = 5.7 Hz), 1.75 -1.72 (1OH, m), 1.58 - 1.42 (2H, m), 1.25 (6H, s) and 1.11 - 1.07 (2H, m) ppm; - ¹³C-NMR (75 MHz, CDCI₃): δ 140.2 (C), 131.8 (C), 123.7 (CH), 118.7 (CH), 78.1 (CH), 70.7 (C), 67.5 (CH), 67.2 (CH), 65.4 (CH₂), 45.9 (CH₂), 44.9 (CH₂), 42.0 (CH₂), 41.4 (CH₂), 38.5 (CH₂), 37.1 (CH₂), 35.1 (C), 33.6 (CH₂), 33.1 (CH₂), 29.3 (CH₃), 29.0 (CH₂), 26.8 (CH₂), 26.7 (CH₂) and 22.6 (CH₂) ppm.

Isomer 104b was characterized as follows:

- R_f (ethyl acetate / isooctane: 95/5) = 0.16;

- IR (film): v 3382 (O-H, s), 2966 (C-H, m), 2928 (C-H, s), 2872 (C-H, m), 2841 (C-H, w), 1616 (C=C, vw), 1443 (w), 1367 (w), 1351 (w), 1302 (w), 1214 (w), 1155 (w), 1084 (m), 1049 (m), 975 (w), 949 (w), 873 (w), 813 (w) and 701 (w) cm^"1;

- UV (CH₃OH): λ_max = 241 , 249 and 259 nm;

- ¹H-NMR (500 MHz, CD₂CI₂): δ 6.23 (1 H, AB, J = 11.2 Hz), 6.11 (1H, AB, J = 11.2 Hz), 4.05 - 4.01 (2H, m), 3.70 (2H, t, J = 5.8 Hz), 3.60 (1 H, s(bή), 3.26 (1 H, tt, J = 9.1 , 4.1 Hz), 2.59 (1H, dd, J = 13.3, 3.8 Hz), 2.46 (1H, dd, J = 13.1 , 3.6 Hz), 2.26 (1 H, dd, J = 13.3, 7.4 Hz)₁ 2.23 (1 H, AB, J = 13.2 Hz), 2.18 (1 H, AB, J = 13.2 Hz), 2.16 (1H₁ dd, J = 13.2, 7.1 Hz), 2.14 - 2.11 (2H, m), 1.86 - 1.73 (4H, m), 1.73 (2H, t, J = 5.8 Hz), 1.58 -1.54 (5H, m), 1.50 - 1.42 (3H, m), 1.40 - 1.38 (2H, m), 1.21 (6H, s) and 1.16 - 1.09 (2H, m) ppm; and

- ¹³C-NMR (50 MHz, CD₂CI₂): δ 140.5, 132.9, 123.5, 119.3, 78.5, 70.8, 67.6, 67.5, 65.9, 45.5, 42.6, 41.9, 39.3, 37.9, 37.4, 37.3, 35.4, 33.9, 33.8, 29.5, 27.7, 27.5 and 24.0 ppm.

In a manner similar to the procedure shown in scheme VII, the deacetalization reaction was carried out onto intermediate 95a but proceeds with a 82% yield as shown in the following scheme XIII:

Scheme XIII

95a 105a

The intermediate hydroxyketone 105a was characterized as follows: Rf (isooctane / ethyl acetate: 6/4) = 0.23; IR (KBr, film): v 3448 (O-H, s), 2931 (C-H, s), 2872 (C-H, m), 1702 (C=O, s), 1461

(W), 1449 (W), 1420 (w), 1373 (w), 1360 (w), 1307 (w), 1290 (w), 1237 (w), 1167 (m),

1102 (S), 1044 (w), 1020 (w), 997 (w), 914 (s), 773 (w) and 732 (s) cm^"1;

MS (m/z, %): 254 (M^+", <1), 239 (M^+'- CH₃ ^', <1), 196 (21), 165 (17), 147 (11), 123

(32), 110 (20), 67 (22), 59 (100), 55 (49)and41(44) ;

¹H-NMR (500 MHz, CDCI₃): δ 3.33 (1H, tt, J= 7.9, 3.9 Hz), 3.24 (2H ,s), 2.37 (1H, s),

2.30 (2H, t, J = 6.8 Hz), 2.18 (2H, s), 1.84 (2H, m), 1.75 - 1.69 (4H, m), 1.60 - 1.51

(4H, m), 1.21 (2H, m) and 1.19 (6H, s) ppm ; and

¹³C-NMR (125 MHz, CDCI₃): δ211.8 (C), 76.8 (CH), 76.3 (CH₂), 70.0 (C), 53.0 (CH₂),

41.1 (CH₂), 38.1 (C), 33.6 (CH₂), 33.0 (CH₂), 26.5 (CH₂), 26.0 (CH₃) and 21.4 (CH₂) ppm.

In a manner similar to the procedure shown in scheme VII, the deacetalization reaction was carried out onto intermediate 95b but proceeds with a 90% yield as shown in the following scheme XIV:

Scheme XIV

95b 105b

The intermediate hydroxyketone 105b was characterized as follows:

- Rf (isooctane / ethyl acetate: 6/4) = 0.23;

- melting point: 51 °C;

- IR (KBr, film): v 3430 (O-H, s), 2972 ( C-H, m), 2942 (C-H, s), 2912 (C-H, s), 2873 (C-H, m), 2849 (C-H, s), 1700 (C=O, s), 1466 (w), 1446 (m), 1425 (w), 1375 (m), 1356 (m), 1312 (w), 1296 (w), 1239 (m), 1189 (w), 1157 (m), 1115 (s), 1036 (w), 1000 (W), 952 (W), 935 (w)and 918 (w) cm^"1;

- MS (m/z, %): 239 (M^+'- CH₃ ^', <1), 196 (16), 165 (10), 147 (9), 123 (100), 110 (27), 73 (25), 59 (57)and 55 (42) ; - ¹H-NMR (500 MHz, CDCI₃): δ 3.31 (1H, tt, J = 8.2, 3.8 Hz), 3.23 (2H ,s), 2.37 (1 H, s), 2.30 (2H, t, J = 6.9 Hz), 2.27 (2H, s), 1.86 (2H, m), 1.73 (2H, m), 1.62 (2H₁ dd, J = 6.1 , 6.1 Hz), 1.60 - 1.47 (4H, m), 1.23 (2H, ddd, J = 13.4, 9.8, 3.7 Hz) and 1.18 (6H, s) ppm; - ¹³C-NMR (50 MHz, CDCI₃): δ 211.7 (C), 76.8 (CH), 76.1 (CH₂), 70.0 (C), 51.1 (CH₂), 41.2 (CH₂), 38.1 (C), 36.0 (CH₂), 33.3 (CH₂), 26.5 (CH₂), 26.0 (CH₃) and 21.8 (CH₂) ppm; and

- elemental analysis: calculated C = 70.83, H = 10.30; found C = 70.76, H = 10.31.

In a manner similar to the procedures shown in schemes VIII and IX, the Horner-

Wittig reaction (Lythgoe coupling) step was carried out onto intermediate 105a but starting from the phosphine oxide reagent 29 and then followed by a standard oxygen deprotection step. The following scheme XV shows that this combined sequence of two steps results, with a 70% yield, in a mixture comprising about 80% of the E-isomer 106a and about 20% of the Z-isomer 107a.

Scheme XV

106a 107a

The resulting mixture was characterized as follows:

- R_f (ethyl acetate / isooctane: 95/5) = 0.28;

- IR (film): v 3366 (O-H, s), 2929 (C-H, s), 2872 (C-H, m), 1613 (C=C, w), 1443 (m), 1374 (w), 1361 (w), 1173 (w), 1096 (m), 1044 (m), 973 (w), 955 (w), 920 (w), 861 (w), 808 (w) and 714 (w) cm^"1; - UV (CH₃OH): λ_max = 241 , 249 and 259 nm; MS (m/z, %): 378 (M^+", 27), 360 (M^+'- H₂O, 3), 320 (1), 288 (6), 265 (5), 253 (3), 215 (4), 163 (15), 145 (29), 107 (100), 91 (65), 79 (70) and 59 (94); ¹H-NMR (500 MHz, CD₂CI₂): δ 6.23 (1 H of £-isomer, AB, J = 11.2 Hz), 6.21 (1 H of Z- isomer, AB, J = 11.0 Hz), 6.10 (1 H of Z-isomer, AB, J = 11.0 Hz), 5.96 (1 H of E- isomer, AB, J = 11.2 Hz), 4.07 - 4.01 (2H, m), 3.34 - 3.29 (1 H, m), 3.23 (2H₁ s), 2.61 (1 H, dd, J = 13.3, 4.1 Hz), 2.46 (1 H, dd, J = 13.2, 3.7 Hz), 2.32 (1H, s(bή), 2.30 - 2.22 (2H, m), 2.17 - 2.10 (2H, m), 1.97 (2H₁ s), 1.87 - 1.77 (2H, m), 1.74 - 1.69 (2H, m), 1.59 - 1.43 (10H, m), 1.16 (6H, s) and 1.14 - 1.10 (2H, m) ppm; and ¹³C-NMR (75 MHz, CDCI₃): δ (E-isomer) δ 140.8 (C), 131.5 (C), 123.8 (CH), 118.3 (CH), 77.8 (CH), 76.2 (CH₂), 70.1 (C), 67.5 (CH), 67.2 (CH), 49.5 (CH₂), 44.8 (CH₂), 42.2 (CH₂), 36.9 (CH₂), 35.2 (C), 34.8 (CH₂), 33.4 (CH₂), 33.2 (CH₂), 29.0 (CH₃), 26.9 (CH₂), 26.1 (CH₂) and 22.3 (CH₂) ppm.

In a manner similar to the procedures shown in schemes VIII and IX, the Horner- Wittig reaction (Lythgoe coupling) step was carried out onto intermediate 105b but starting from the phosphine oxide reagent 29 and then followed by a standard oxygen deprotection step. The following scheme XVI shows that this combined sequence of two steps results, with a 52% yield, in a mixture comprising about 85% of the E-isomer 106b and about 15% of the Z-isomer 107b.

Scheme XVI

106b 107b

After reaction, both isomers were separated by means of high performance liquid chromatography (HPLC). Isomer 106b was characterized as follows:

- R_f (ethyl acetate / isooctane: 95/5) = 0.28;

- IR (film): v 3366 (O-H, s), 2929 (C-H, s), 2872 (C-H, m), 1613 (C=C, w), 1443 (m), 1374 (w), 1361 (w), 1173 (w), 1096 (m), 1044 (m), 973 (w), 955 (w), 920 (w), 861 (w), 808 (w) and 714 (w) cm^"1;

- UV (CH₃OH): λ_max = 241 , 249 and 259 nm;

- MS (m/z, %): 378 (M^+", 3), 360 (M^+"- H₂O, 6), 288 (14), 270 (12), 215 (7), 191 (6), 173 (10), 147 (15), 107 (24), 91 (42), 79 (39), 67 (24), 59 (100) and 43 (40);

- ¹H-NMR (500 MHz, CD₂CI₂): δ 6.22 (1 H, AB, J = 11.3 Hz), 5.99 (1 H, AB, J = 11.3 Hz), 4.07 - 4.00 (2H, m), 3.27 (1 H, tt, J = 9.1 , 4.2 Hz), 3.25 (2H, s), 2.61 (1 H, ABd, J

= 13.3, 3.8 Hz), 2.45 (1 H, ABd, J = 13.2, 3.7 Hz)₁ 2.40 (1 H, s(bή), 2.31 - 2.20 (3H, m), 2.15 (1 H, ABd, J = 13.2, 6.7 Hz), 2.09 (1 H, AB, J = 13.2 Hz), 2.06 (1 H, AB, J = 13.1 Hz), 1.98 - 1.71 (6H, m), 1.57 - 1.51 (4H, m), 1.49 - 1.42 (2H, m), 1.39 - 1.37 (2H, m), 1.17 (6H, s) and 1.13 - 1.06 (2H, m) ppm; and - ¹³C-NMR (50 MHz, CDCI₃): δ 140.3 (C), 132.7(C), 123.7(CH), 119.0 (CH), 78.6 (CH), 76.6 (CH₂), 70.2 (C), 67.6 (CH), 67.3 (CH), 46.0 (CH₂), 45.1 (CH₂), 42.6 (CH₂), 39.0 (CH₂), 37.2 (CH₂), 35.4 (C), 34.0 (CH₂), 33.6 (CH₂), 29.2 (CH₂), 27.3 (CH₂), 26.8 (CH₂), 26.3 (CH₃), and 23.0 (CH₂) ppm.

Isomer 107b was characterized as follows:

R_f (ethyl acetate / isooctane: 95/5) = 0.21 ;

- IR (film): v 3365 (O-H, s), 2928 (C-H, s), 2872 (C-H, m), 1615 (C=C, vw), 1444 (m), 1374 (w), 1356 (m), 1173 (w), 1093 (m), 1049 (m), 979 (w), 937 (w), 914 (w), 867 (w), 808 (w) and 726 (w) cm^'1; - UV (CH₃OH): λ_max = 242, 249 and 259 nm;

- ¹H-NMR (500 MHz, CD₂CI₂): δ 6.25 (1H, AB, J = 11.3 Hz), 6.12 (1H, AB, J = 11.3 Hz), 4.07 - 4.00 (2H, m), 3.27 (1 H, tt, J = 9.0, 4.1 Hz), 3.24 (2H, s), 2.60 (1 H, dd, J =

13.3, 3.8 Hz), 2.48 (1 H, dd, J = 13.1 , 3.7 Hz), 2.37 (1 H, s(bή), 2.27 (1 H, dd, J =

13.4, 7.5 Hz), 2.22 (1 H, AB, J = 13.5 Hz), 2.18 (1 H₁ AB, J = 13.4 Hz), 2.17 (1 H, m), 2.14 - 2.12 (2H, m), 1.86 - 1.79 (4H, m), 1.60 - 1.55 (6H, m), 1.51 - 1.44 (2H, m),

1.41 - 1.38 (2H, m), 1.17 (6H, s) and 1.15 - 1.10 (2H, m) ppm; and - ¹³C-NMR (50 MHz, CD₂CI₂): δ 140.6, 132.8, 123.5, 119.3, 78.5, 76.6, 70.2, 67.7, 67.5, 45.3, 42.5, 39.1 , 38.0, 37.6, 37.2, 35.4, 34.0, 33.7, 27.6, 27.5, 26.3 and 24.0 ppm.

In order to access vitamin D compounds from other categories of the present invention, the following schemes provide a series of suitable intermediates and precursors wherein the oxa-substituent substituent of the F ring is located at another position.

According to scheme XVII, the relevant 1 ,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene stereoisomer (referred as (11) in scheme (I), hereunder shown as 30), or the corresponding 1 ,4-dithia-dispiro[4.1.5.3]pentadec-8-ene stereoisomer, obtained in the eleventh and last step of scheme (I) is submitted to hydroboration. Reaction proceeds in two sub-steps, first in the presence of a suitable borating agent such as, but not limited to, 9-borabicyclo[3.3.1]nonane (preferably in a molar excess with respect to the starting material 30), and preferably in the presence of a suitable solvent such as, but not limited to, tetrahydrofuran (THF), and preferably within a temperature range from about 20⁰C to about 80⁰C. In the second sub-step, a sodium hydroxide solution and a hydrogen peroxide aqueous solution (with a concentration preferably ranging from about 10% to about 55% by volume) are added to the reaction mixture.

Scheme XVII

As shown in scheme XVII, this procedure does not result only in the desired intermediates but also in by-products resulting from undesired deacetalization.

Optionally a residual amount of the starting material 30 may still be present in the reaction mixture even after a substantial reaction time. Under the above specific conditions, running the first sub-step for 12 hours and the second sub-step for 3 hours resulted in 28% of a 1:1 mixture of isomers 108a and 108b, 10% of the regioisomer 109 shown hereunder, and 62% of a mixture of the hydroxyketones 110a and 110b. The latter may be converted, through acetalization under well known Dean-Stark conditions, into the desired acetal isomers 108a and 108b, if necessary.

109

The acetal isomer 108a was characterized as follows: - R_f (isooctane / ethyl acetate: 2/8) = 0.50;

- melting point: 84°C;

- optical rotation at room temperature [α]^kt _D: +4.9° (c 1.07, CHCI₃);

- IR (film): v3475 (O-H, s), 3414 (O-H, s), 2949 (C-H, s), 2926 (C-H, s), 2901 (C-H, m), 2845 (C-H, m), 1637 (w), 1617 (m), 1478 (w), 1456 (w), 1446 (m), 1427 (w), 1373 (m), 1348 (w), 1322 (w), 1288 (w), 1256 (w), 1204 (w), 1179 (w), 1146 (m), 1096 (S), 1049 (S), 1018(w), 961 (m), 937(m), 856 (w)and 826 (w)cm^"1

- MS(m/z, %): 150 (17), 123 (63), 110(38), 95 (33), 94 (38), 82 (32), 79 (59), 71 (35), 67(25), 55(25), 53 (39),45 (100)and41 (35) ;

- ¹H-NMR(500 MHz, CDCI₃): δ3.92-3.86 (4H, m), 3.74(1H,tt, J= 11.0,4.2Hz), 2.09 (1H, d(fe), J= 12.2 Hz), 1.95 (1H, d(fe), J= 12.0 Hz), 1.71 (1H, d(fe), J= 13.3 Hz),

1.63 - 1.48 (8H, m), 1.41 (1H, qt, J= 13.5, 3.6 Hz), 1.27 (2H₁ dd, J- 5.3, 5.3 Hz), 1.10 (1H, q(fs), J= 12.2 Hz), 0.95 (1H,td, J= 13.2, 4.0 Hz) and 0.90 (1H, t, J= 11.6 Hz) ppm; and

- ¹³C-NMR (50 MHz, CDCI₃): δ 109.3 (C), 67.2 (CH), 64.1 (CH₂), 64.0 (CH₂), 46.4 (CH₂), 40.7 (CH₂), 40.4 (CH₂), 36.7 (CH₂), 36.4 (C), 35.9 (CH₂), 35.1 (CH₂), 20.1

(CH₂)and 19.2 (CH₂) ppm.

The acetal isomer 108b was characterized as follows:

- R_f (isooctane / ethyl acetate: 2/8) = 0.52; - IR (KBr, film): v 3390 (O-H, s), 2930 (C-H, s), 2867 (C-H, m), 1450 (m), 1364 (m), 1338 (W), 1270 (w), 1241 (w), 1209 (w), 1175 (w), 1127 (m), 1090 (s), 1048 (s), 1030 (m), 1008 (W), 975 (w), 964 (w), 930 (w), 886 (w), and 842 (w) cm^"1

- MS (m/z, %): 226 (M^+', 3), 183 (63), 165 (17), 139 (7), 121 (7), 111 (92), 99 (100), 86 (63), 79 (18), 67 (19), 55 (40) and 41 (34) ; - ¹H-NMR (500 MHz, CDCI₃): 5 3.94 - 3.89 (4H, m), 3.77 (1 H, ttd, J = 10.1 , 4.3, 4.2 Hz), 1.94 - 1.87 (2H₁ m), 1.64 - 1.54 (7H, m), 1.44 - 1.30 (5H, m), 1.21 - 1.12 (2H, m) and 1.05 (1 H, m) ppm ; and

- ¹³C-NMR (50 MHz, CDCI₃): 5 109.2 (C), 67.2 (CH), 64.1 (CH₂), 64.0 (CH₂), 47.9 (CH₂), 46.6 (CH₂), 36.8 (CH₂), 36.3 (C), 35.4 (CH₂), 35.2 (CH₂), 33.2 (CH₂), 19.7

(CH₂) and 19.4 (CH₂) ppm.

The acetal regioisomer 109 was characterized as follows: R_f (isooctane / ethyl acetate: 1/1 ) = 0.49; - IR (film): v 3444 (O-H, s), 2931 (C-H, s), 2963 (C-H, s), 1449 (m), 1365 (w), 1243 (w), 1174 (w), 1082 (m), 1050 (w), 1005 (w), 947 (w), 915 (w), 840 (w), 814 (w) and 742 (w) cm^"1

- MS (m/z, %): 226 (M^+', 9), 183 (31), 167 (15), 141 (26), 126 (9), 112 (21), 99 (100), 86 (39), 79 (13), 67 (14), 55 (34) and 41 (26) ; - ¹H-NMR (500 MHz, CDCI₃): 5 3.99 - 3.89 (4H+4H, m), 3.51 - 3.47 (1 H, m), 3.43 - 3.41 (1 H, m), 3.03 (1 H, sφή), 2.23 (1 H, d, J = 6.1 Hz) and 1.81 - 1.02 (32H, m) ppm; and

- ¹³C-NMR (75 MHz, CDCI₃): δ 109.8 (C), 109.6 (C), 77.0 (CH), 75.1 (CH), 64.3 (CH₂), 64.1 (CH₂), 45.3 (CH₂), 39.7 (C), 39.4 (C), 38.1 (CH₂), 36.7 (CH₂), 35.2 (CH₂), 35.1 (CH₂), 34.9 (CH₂), 34.7 (CH₂), 30.0 (CH₂), 29.8 (CH₂), 28.4 (CH₂), 23.9 (CH₂), 23.1

(CH₂), 21.1 (CH₂), 21.0 (CH₂), 20.0 (CH₂) and 19.7 (CH₂) ppm.

Each of the acetal isomers 108a, 108b and 109 can then be transformed into a suitable compound of the invention by making use of the sequence of methodologies that were applied to the corresponding intermediates 90a and 90b, respectively, and were described herein before, in particular:

- a Williamson ether synthesis similar to that illustrated in scheme IV,

- a two-steps transformation similar to that illustrated in scheme V, a two-steps transformation similar to that illustrated in scheme Vl, - a deacetalization step similar to that illustrated in schemes VII, XIII and XIV,

- a Horner-Wittig reaction similar to that illustrated in schemes VIII, Xl, XII, XV and XVI, and - an oxygen deprotection step similar to that illustrated in scheme IX or included in the second sub-step of schemes XII, XV and XVI.

By making use of these methodologies, vitamin D compounds included in each of the various above categories of the invention can suitably be made. However, for the sake of concision, the detailed set of steps from the above intermediates to the compounds of each such category was not repeated.

Compounds which are comprised in category II of the present invention include, but are not limited to, the Z-isomers of 5-[2-(3-R^0b-substituted-spiro[5.5]undec-7- ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R^ob, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 1 hereinabove.

Compounds which are comprised in category III of the present invention include, but are not limited to, the E-isomers of 5-[2-(3-R^0a-substituted-spiro[5.5]undec-7- ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R^Oa, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 2 below and wherein the first column attributes a number to each combination of substituents.

TABLE 2

The skilled person understands that each compound of this third category of the present invention is usually obtained from the corresponding hydroxyl-protected intermediate having the same structural formula but wherein each substituent selected from the group consisting of R^5a, R^5b, R^6a and R^6b is, wherever applicable, a hydroxyl protected group OP (with P being as already defined herein) instead of hydroxyl.

Compounds which are comprised in category IV of the present invention include, but are not limited to, the Z-isomers of 5-[2-(3-R^0a-substituted-spiro[5.5]undec-7- ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R^Oa, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 2 herein before.

Compounds which are comprised in category V of the present invention include, but are not limited to, the E-isomers of 5-[2-(2-R^1b-substituted-spiro[5.5]undec-7-ylidene)- ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R^1b, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 3 below and wherein the first column attributes a number to each combination of substituents. TABLE 3

The skilled person understands that each compound of this fifth category of the present invention is usually obtained from the corresponding hydroxyl-protected intermediate having the same structural formula but wherein each substituent selected from the group consisting of R^5a, R^5b, R^6a and R^6b is, wherever applicable, a hydroxyl protected group OP (with P being as already defined herein) instead of hydroxyl.

Compounds which are comprised in category Vl of the present invention include, but are not limited to, the Z-isomers of 5-[2-(2-R^1b-substituted-spiro[5.5]undec-7- ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following structural formula:

wherein the R^1b, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 3 herein before.

Compounds which are comprised in category VII of the present invention include, but are not limited to, the E-isomers of 5-[2-(2-R^1a-substituted- spiro[5.5]undec-7-ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following structural formula:

wherein the R ,1a , _D R5a , F R->5b , ι R-,6a and R -,6b substituents are as outlined in Table 4 below and wherein the first column attributes a number to each combination of substituents.

TABLE 4

The skilled person understands that each compound of this seventh category of the present invention is usually obtained from the corresponding hydroxyl- protected intermediate having the same structural formula but wherein each substituent selected from the group consisting of R^5a, R^5b, R^6a and R^6b is, wherever applicable, a hydroxyl protected group OP (with P being as already defined herein) instead of hydroxyl.

Compounds which are comprised in category VIII of the present invention include, but are not limited to, the Z-isomers of 5-[2-(2-R^1a-substituted- spiro[5.5]undec-7-ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R^1a, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 4 herein before.

Compounds which are comprised in category IX of the present invention include, but are not limited to, the E-isomers of 5-[2-(4-R^2b-substituted- spiro[5.5]undec-7-ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R , R , R , R^6a and R^6b substituents are as outlined in Table 5 below and wherein the first column attributes a number to each combination of substituents.

TABLE 5

The skilled person understands that each compound of this ninth category of the present invention is usually obtained from the corresponding hydroxyl-protected intermediate having the same structural formula but wherein each substituent selected from the group consisting of R^5a, R^5b, R^6a and R^6b is, wherever applicable, a hydroxyl protected group OP (with P being as already defined herein) instead of hydroxyl.

Compounds which are comprised in category X of the present invention include, but are not limited to, the Z-isomers of 5-[2-(2-R^2b-substituted- spiro[5.5]undec-7-ylidene)-ethylidene]-cyclohexane-1,3-diols having the following general formula:

wherein the R^2b, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 5 herein before. Compounds which are comprised in category Xl of the present invention include, but are not limited to, the E-isomers of 5-[2-(4-R^2a-substituted- spiro[5.5]undec-7-ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following general formula:

wherein the R^2a, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 6 below and wherein the first column attributes a number to each combination of substituents. TABLE 6

The skilled person understands that each compound of this first category of the present invention is usually obtained from the corresponding hydroxyl-protected intermediate having the same structural formula but wherein each substituent selected from the group consisting of R^5a, R^5b, R^6a and R^6b is, wherever applicable, a hydroxyl protected group OP (with P being as already defined herein) instead of hydroxyl. Compounds which are comprised in category XII of the present invention include, but are not limited to, the Z-isomers of 5-[2-(2-R^2a-substituted- spiro[5.5]undec-7-ylidene)-ethylidene]-cyclohexane-1 ,3-diols having the following structural formula:

wherein the R^2a, R^5a, R^5b, R^6a and R^6b substituents are as outlined in Table 6 herein before. As mentioned herein above, the compounds of the present invention may further include, at carbon 2 thereof (according the scaffold numbering system previously described), i.e. on the cyclohexylidene unit Q, one or two substituents R and/or R', wherein R and R' are each independently selected from the group consisting of substituted and unsubstituted Ci_-7 alkyl. Preferably R and R' are each independently selected from the group consisting of methyl and ethyl. More preferably, substitution at carbon 2 involves only one such substituent R or R'. Methods for introducing one or two such substituents R and/or R' at carbon 2 of the molecule are well known to those skilled in the art, as illustrated for instance in WO 2004/080922.

Compounds listed and described herein above have been found in many instances to exhibit useful biological activity (such as an IC₅₀ in the cell based assay described herein below or in one or more other assays which are referenced herein) at a level below about 1 mM, more particularly below about 1 micromolar (μM). Consequently such compounds are useful as active ingredients in pharmaceutical _M

83

formulations and are useful in the manu- facture of medicaments for the prevention and/or treatment of one or more of the disorders and diseases listed herein below.

Each of the disease states or conditions which may be desirable to treat or prevent may require differing levels or amounts of the compounds described herein in order to obtain a therapeutic level. The formulator can determine this therapeutic amount by any of the known testing procedures known to the artisan. It is well known that the effective amount of a certain compound may be predicated on various parameters which are germane to delivery of an effective active ingredient, inter alia, in vivo activity, bioavailability, metabolism, ease of formulation, stability of compound formulation and the like. The artisan of ordinary skill will recognize that a compound having superior properties in vitro may, when introduced into humans or higher mammals, not succeed as the best compound of a category defined herein above.

Diseases to be prevented or treated by the Compounds of the Invention The compounds described herein have a selective activity on cell function.

One key selective action relates to the inhibition of cell proliferation. Non-limiting examples of cells which are a target of cell proliferation inhibition include both non- malignant cells, inter alia, keratinocytes and malignant cells, inter alia, breast carcinoma cells, leukemia cells. A second selective activity on cell function relates to induction of cell differentiation, for example, leukemia cells.

However, unlike current compounds used against diseases relating to cell proliferation, inter alia, classical vitamin D compounds, the compounds of the present invention have a strikingly lower effect on calcium and bone homeostasis. Indeed, as described herein below, 1 α,25-dihydroxyvitamin D₃ [hereinafter 1α,25(OH)₂D₃], a Vitamin D metabolite], was used as a control when evaluating the compounds of the present invention. One test utilizing vitamin D repleted normal mice, demonstrated that the compounds of the present invention exhibited a lower toxic effect on calcium and bone homeostatsis when measured at key end points, inter alia, calcium levels in serum, urine and the femur; and osteocalcin levels in serum; and against body weight.

The following diseases or disease states may be affected by successfully controlling, modulating, or inhibiting cell proliferation.

A) Calcium and Phosphate Disorders: The compounds of the present invention are suitable for use in diseases and conditions which are related to calcium and phosphate metabolism. Vitamin D and metabolites have potent effects on calcium and phosphate metabolism, and therefore they can be used for prevention and therapy of Vitamin D deficiency and other disorders of plasma and bone mineral homeostasis, inter alia, osteoporosis, especially low bone turnover osteoporosis, steroid induced osteoporosis, senile osteoporosis or postmenauposal osteoporosis, as well as osteomalacia, renal osteodystrophy, Paget's disease and vitamin D resistant rickets and other diseases whereby bone growth is desired, and disorders of the parathyroid function. It has also been found that Vitamin D and metabolites affect intercellular calcium concentration.

B) Immune Disorders: i) For the purposes of the present invention, type (i) immune disorders are auto-immune diseases, inter alia, diabetes mellitus type 1 , multiple sclerosis, lupus and lupus like disorders, asthma, glomerulonephritis, and auto-immune throiditis. ii) For the purposes of the present invention, type (ii) immune disorders are selective dysfunctions of the immune system, inter alia, Acquired Immune Deficiency Syndrome (AIDS). iii) For the purposes of the present invention, type (iii) immune disorders are medically induced immune disorders, inter alia, the body's rejection of foreign tissue due to tissue grafts (e.g. kidney, heart, bone marrow, liver, islets or whole pancreas, and skin). iv) For the purposes of the present invention, type (iv) immune disorders are autoimmune and other inflammatory diseases, inter alia, rheumatoid arthritis. v) For the purposes of the present invention, type (v) immune disorders are skin disorders. These disorders can be due to any immune system imbalance, inter alia, those characterized by hyperproliferation, inflammation, (auto)immune reactions. Non-limiting examples include psoriasis, dyskeratosis, and acne.

Because of the advantages exhibited by the compounds of the present invention, inter alia, a lower toxic effect on calcium and bone homeostasis, the present invention also relates to the use of the compounds described herein in combination with one or more other immuno-modulating or immuno-suppressing drugs known to affect the immune system, inter alia cyclosporin A, tacrolimus (FK 506), rapamycin, leflunomide, mofetil, methylxanthine derivatives, glucocorticoids, monoclonal antibodies, cytokines and growth factors, for the manufacture of pharmaceutical preparations for the treatment or prevention of the above immune disorders.

C) Cell Proliferation Disorders:

The compounds of the present invention are also suitable for use in the treatment or prevention of diseases, disease states, and disorders which are due to abnormal cell proliferation in humans such as in cancer or tumor formation. Among these conditions are breast cancer, leukemia, myelo-dysplastic syndromes and lymphomas, squamous cell carcinomas and gastrointestinal cancers, melanomas, and osteosarcoma.

Another advantage related to the cell differentiation capacity of the compounds of the present invention relates to the treatment or prevention of alopecia. The present invention therefore relates to methods of treating or preventing alopecia, especially when induced by chemotherapy or irradiation, in humans.

The present invention further relates to certain forms of the present compounds, which under normal human or higher mammalian physiological conditions, release the compounds described herein. One first embodiment of this aspect of the invention includes the pharmaceutically acceptable salts of the compounds described herein.

The term " pharmaceutically acceptable salts " as used herein means the therapeutically active non-toxic salt forms which some of the compounds of the above formulae, including all stereoisomers and embodiments thereof, are able to form. Therefore, the compounds of this invention optionally comprise salts of the compounds herein, especially pharmaceutically acceptable non-toxic salts containing, for example, cations such as, but not limited to, Na⁺, Li⁺, K⁺, Ca²⁺ and Mg²⁺. Such salts may include those derived from the combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium or quaternary ammonium ions with an acid, typically a carboxylic acid, anion moiety. The compounds of the invention may bear multiple positive or negative charges. The net charge of the compounds of the invention may be either positive or negative. Any associated counter ions are typically dictated by the synthesis and/or isolation methods by which the compounds are obtained. Typical counter ions include, but are not limited to ammonium, sodium, potassium, lithium, halides, acetate, trifluoroacetate, etc., and mixtures thereof. It will be understood that the identity of any associated counter ion is not a critical feature of the invention, and that the invention encompasses the compounds in association with any type of counter ion. Included within this definition is any therapeutically active non-toxic addition salt which the compounds of the present invention are able to form with a salt- forming agent. Such addition salts may conveniently be obtained by treating the compounds of the invention with an appropriate salt-forming acid or base. For instance, compounds having basic properties may be converted into the corresponding therapeutically active, non-toxic acid addition salt form by treating the free base form with a suitable amount of an appropiate acid following conventional procedures. Examples of such appropriate salt-forming acids include, for instance, inorganic acids resulting in forming salts such as but not limited to hydrohalides (e.g. hydrochloride and hydrobromide), sulfate, nitrate, phosphate, diphosphate, carbonate, bicarbonate, and the like; and organic monocarboxylic or dicarboxylic acids resulting in forming salts such as, for example, acetate, propanoate, hydroxyacetate, 2-hydroxypropanoate, 2-oxopropanoate, lactate, pyruvate, oxalate, malonate, succinate, maleate, fumarate, malate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzoate, 2-hydroxybenzoate, 4-amino-2-hydroxybenzoate, benzene-sulfonate, p-toluenesulfonate, salicylate, p-aminosalicylate, pamoate, bitartrate, camphorsulfonate, edetate, 1 ,2-ethanedisulfonate, fumarate, glucoheptonate, gluconate, glutamate, hexylresorcinate, hydroxynaphtoate, hydroxyethanesulfonate, mandelate, methylsulfate, pantothenate, stearate, as well as salts derived from ethanedioic, propanedioic, butanedioic, (Z)-2-butenedioic, (E)2- butenedioic, 2-hydroxybutanedioic, 2,3-dihydroxy-butanedioic, 2-hydroxy-1 ,2,3- propanetricarboxylic and cyclohexanesulfamic acids and the like.

Compounds having acidic properties may be converted in a similar manner into the corresponding therapeutically active, non-toxic base addition salt form. Examples of appropriate salt-forming bases include, for instance, inorganic bases like metallic hydroxides such as but not limited to those of alkali and alkaline-earth metals like calcium, lithium, magnesium, potassium and sodium, or zinc, resulting in the corresponding metal salt; organic bases such as but not limited to ammonia, alkylamines, benzathine, hydrabamine, arginine, lysine, N₁N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylene-diamine, N-methylglucamine, procaine and the like. _Q7

Reaction conditions for treating the compounds of this invention with an appropriate salt-forming acid or base are similar to standard conditions involving the same acid or base but different organic compounds with basic or acidic properties, respectively. Preferably, in view of its use in a pharmaceutical composition or in the manufacture of medicament for treating specific diseases, the pharmaceutically acceptable salt will be designed, i.e. the salt-forming acid or base will be selected so as to impart greater water-solubility, lower toxicity, greater stability and/or slower dissolution rate to the pteridine derivative of this invention.

Moreover, as the compounds of the invention may exist in a variety of different forms, the invention is intended to encompass not only forms of these compounds that are in association with counter ions (e.g., dry salts), but also forms that are not in association with counter ions (e.g., aqueous or organic solutions). Furthermore, this term also includes the solvates which the compounds of the above formulae, including all specific stereoisomers and embodiments thereof, as well as their salts (such as above defined), are able to form, such as for example hydrates, alcoholates, solvates with solvents such as acetonitrile, esters, hydrocarbons and the like. Finally, it should be understood that the invention also includes such compounds in their non-ionized, as well as zwitterionic form, and combinations thereof with stoichiometric amounts of water as in hydrates. Also included within the scope of this invention are the salts that certain compounds are able to form with one or more amino acids, especially the naturally-occurring amino acids found as protein components. Suitable amino acids for this purpose typically have a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine. The compounds of the invention also include physiologically acceptable salts thereof. Examples of physiologically acceptable salts of the compounds of the invention include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth metal (for example, magnesium), ammonium and NX⁴⁺ (wherein X preferably is hydrogen or C_1-7 alkyl). Physiologically acceptable salts of a compound containing a hydroxy group include the anion of said compound in combination with a suitable cation such as Na⁺ and NX⁴⁺ (wherein X is as above defined). However, salts of acids or bases which are not physiologically acceptable may also find use, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived form a physiologically acceptable acid or base, are within the scope of the present invention. The skilled artisan, for the purposes of compatibility with delivery mode, bioavailability, excipients, and the like, can select one salt form of the present compounds over another since the compounds themselves are the active species which mitigate the disease processes described herein.

Pro-drug Forms

Another embodiment of this aspect of the invention are the various precursor or "pro-drug" forms of the compounds of the present invention. It may be desirable to formulate the compounds of the present invention in the form of a chemical species which itself is not an agonist of a vitamin D receptor, but instead are forms of the present compounds which when delivered to the body of a human or higher mammal will undergo a chemical reaction catalyzed by the normal function of the body, inter alia, enzymes present in the stomach or in blood serum, said chemical reaction having the effect of releasing a compound as defined herein. The term "pro- drug " thus relates to these species which are converted in vivo to the active pharmaceutical ingredient.

The pro-drugs of the present invention can have any form suitable to the formulator, for example, esters are common pro-drug forms. In the present case, however, the pro-drug may necessarily exist in a form wherein a covalent bond is cleaved by the action of an enzyme present at the target situs. For example, a C-C covalent bond may be selectively cleaved by one or more enzymes at said target situs and, therefore, a pro-drug in a form other than an easily hydrolysable precursor, inter alia, esters, amides, and the like, may be utilized.

For the purposes of the present invention the term " therapeutically suitable pro-drug " is defined herein as " a vitamin D receptor agonist modified in such a way as to be transformed in vivo to the therapeutically active form, whether by way of a single or by multiple biological transformations, when in contact with the tissues of humans or mammals to which the pro-drug has been administered, and without undue toxicity, irritation, or allergic response, and achieving the intended therapeutic outcome ".

FORMULATIONS

The present invention also relates to compositions or formulations which comprise the compounds according to the present invention. In general, the compositions of the present invention comprise an effective amount of one or more _..

7-(2-cyclohexylidene-ethylidene)-spiro[5.5]undecanes and derivatives thereof according to the present invention which are effective for controlling cell proliferation. A further category of formulations relates to pharmaceutical compositions, said compositions comprising: a) an effective amount of one or more 7-(2-cyclohexylidene-ethylidene)- spiro[5.5]undecanes and derivatives thereof according to the present invention which are effective for controlling cell proliferation; and b) one or more pharmaceutically acceptable excipients or carriers. For the purposes of the present invention the term " excipient " and " carrier " are used interchangeably throughout the description of the present invention and said terms are defined herein as " ingredients which are used in the practice of formulating a safe and effective pharmaceutical composition ".

The formulator will understand that excipients are used primarily to serve in delivering a safe, stable, and functional pharmaceutical, serving not only as part of the overall vehicle for delivery but also as a means for achieving effective absorption by the recipient of the active ingredient. An excipient may fill a role as simple and direct as being an inert filler, or an excipient as used herein may be part of a pH stabilizing system or coating to insure delivery of the ingredients safely to the stomach. The formulator can also take advantage of the fact the compounds of the present invention have improved cellular potency, pharmacokinetic properties, as well as improved oral bioavailability.

Another category of formulation according to the present invention relates to compositions or formulations which comprise a precursor or " pro-drug " form of the 7-(2-cyclohexylidene-ethylidene)-spiro[5.5]undecanes according to the present invention. For the purposes of the present invention, as it relates to the subject of chemical entities which are converted in vivo to 7-(2-cyclohexylidene-ethylidene)- spiro[5.5]undecanes, the terms "pro-drug" and " precursor " are considered to be interchangeable and represent the same concept. In general, these precursor- comprising compositions of the present invention comprise: a) an effective amount of one or more derivatives or pro-drug of 7-(2- cyclohexyl-idene-ethylidene)-spiro[5.5]undecanes according to the present invention which act to release in vivo the corresponding analog which is effective for effective for controlling cell proliferation; and b) one or more pharmaceutically acceptable excipients. The terms "effective amount " and " therapeutic amount " as discussed herein above, is typically determined because of a range of factors. For the purposes of the present invention a first aspect of " therapeutic amount " relates to compositions which deliver a compound according to the present invention wherein the plasma level of said compound is from about 0.001 pg/mL, more specifically from 1 pg/mL, yet more specifically from 1 ng/mL, to about 100 mg/mL in humans or higher mammals. Another category of pharmaceutical compositions according to this invention relates to compositions wherein said plasma level of said compound is from about 10 ng/mL to about 25 mg/mL in humans or higher mammals. Administration of the compositions of the present invention can achieve the desired therapeutic amounts in vivo as measured by the plasma level in various ways. It is not necessary to provide the therapeutic amount of compound in a single dose, for example, a single pill. Therefore, the formulator can vary the size of the dosage, and therefore the amount of compound in the compositions. Non-limiting examples of compositions according to the present invention include: a) from about 1 pg to about 1000 mg of one or more 7-(2-cyclohexyl- idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and b) one or more (preferably pharmaceutically acceptable) excipients. Another embodiment according to the present invention relates to the following compositions: a) from about 1 ng to about 500 mg of one or more 7-(2-cyclohexyl- idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and b) one or more (preferably pharmaceutically acceptable) excipients.

Another embodiment according to the present invention relates to the following compositions: a) from about 1 mg to about 500 mg of one or more 7-(2-cyclohexyl- idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and b) one or more (preferably pharmaceutically acceptable) excipients. Another embodiment according to the present invention relates to the following compositions: a) from about 100 mg to about 500 mg of one or more 7-(2-cyclohexyl- idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and _QΛ y i

b) one or more (preferably pharmaceutically acceptable) excipients. Another embodiment according to the present invention relates to the following compositions: a) from about 0.01 mg to about 20 mg (e.g. up to 5 mg) of one or more 7-(2-cyclohexyl-idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and b) one or more pharmaceutical excipients.

Another embodiment according to the present invention relates to the following compositions: a) from about 0.01 mg to about 5 mg of one or more 7-(2-cyclohexyl- idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and b) one or more (preferably pharmaceutically acceptable) excipients. A further embodiment according to the present invention relates to the following compositions: a) from about 0.1 mg to about 1 mg of one or more 7-(2-cyclohexyl- idene-ethylidene)-spiro[5.5]undecanes according to the present invention; and b) one or more pharmaceutical excipient. In addition, the compositions of the present invention can be administered as frequently as necessary to achieve a therapeutic amount.

The formulator will understand that excipients are used primarily to serve in delivering a safe, stable, and functional pharmaceutical, serving not only as part of the overall vehicle for delivery but also as a means for achieving effective absorption by the recipient of the active ingredient. An excipient may fill a role as simple and direct as being an inert filler, or an excipient as used herein may be part of a pH stabilizing system or coating to insure delivery of the ingredients safely to the stomach.

The compounds of the invention may be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. Formulations optionally contain excipients such as those set forth in the "Handbook of Pharmaceutical Excipients" (1986) and include ascorbic acid and other antioxidants, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The term " pharmaceutically acceptable carrier " as used herein also means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders. Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 μm, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients.

Suitable surface-active agents, also known as emulgent or emulsifier, to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water- soluble synthetic surface-active agents. Suitable soaps are alkaline or alkaline-earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (Ci₀- C₂₂). e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable from coconut oil or tallow oil. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulphonates and sulphates; sulphonated benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or sulphates are usually in the form of alkaline or alkaline-earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a mixture of fatty alcohol sulphates obtained from natural fatty acids, alkaline or alkaline-earth metal salts of sulphuric or sulphonic acid esters (such as sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are the sodium, calcium or alcanolamine salts of dodecylbenzene sulphonic acid or dibutyl- naphtalenesulphonic acid or a naphtalene-sulphonic acid/formaldehyde condensation product. Also suitable are the corresponding phosphates, e.g. salts of phosphoric acid ester and an adduct of p-nonylphenol with ethylene and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl -choline and their mixtures.

Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulphonates and dialkylsulphosuccinates, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with poylypropylene glycol, ethylenediaminopolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups. Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit. Representative examples of non-ionic surfactants are nonylphenol -polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable non-ionic surfactants. Suitable cationic surfactants include quaternary ammonium salts, particularly halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C₈-C₂₂ alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for this purpose may be found for instance in "McCutcheon's Detergents and Emulsifiers Annual" (MC

Publishing Crop., Ridgewood, New Jersey, 1981 ), "Tensid-Taschenbucw¹, 2 d ed. (Hanser Verlag, Vienna, 1981) and "Encyclopaedia of Surfactants, (Chemical

Publishing Co., New York, 1981).

Compounds of the invention and their physiologically acceptable salts (hereafter collectively referred to as the active ingredients) may be administered by any route appropriate to the condition to be treated, suitable routes including, oral, rectal, nasal, topical (including transdermal^, ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intra-arterially, intradermal, intrathecal and epidural). The preferred route of administration may vary, for example with the condition of the recipient.

While it is possible for the active ingredients to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations, both for veterinary and for human use, of the present invention comprise at least one active ingredient, as above described, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic ingredients. The carrier(s) optimally are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet comprising a compound of this invention may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. The formulations of the invention are optionally applied as a topical ointment or cream. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30 weight-% of a polyhydric alcohol, i.e. an alcohol having two or more hydroxy! groups such as propylene glycol, butane 1 ,3- diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Optionally, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations could be quite high, looking at Vitamin D. Thus the cream should optionally be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di- isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range of about 20 to 500 microns, which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Compounds of the invention can be used to provide controlled release pharmaceutical formulations containing as active ingredient one or more compounds of the invention (" controlled release formulations ") in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given compound. Controlled release formulations adapted for oral administration in which discrete units comprising one or more compounds of the invention can be prepared according to conventional methods.

Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxymethylcellulose, polyniethyl methacrylate and the other above-described polymers. Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on. Depending on the route of administration, the pharmaceutical composition may require protective coatings. Pharmaceutical forms suitable for injectionable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol and the like and mixtures thereof.

METHODS OF USE

The present invention also relates to a method for controlling cell proliferation and diseases which may be related to cell proliferation. The present invention thus also relates to a method for preventing or treating cell proliferation and diseases which are related to cell proliferation. The present method comprises the step of administering to a human or higher mammal an effective amount of a composition comprising one or more 7-(2-cyclohexylidene-ethylidene)-spiro[5.5]undecanes according to the present invention.

Because the 7-(2-cyclohexyl-idene-ethylidene)-spiro[5.5]undecanes according to the present invention can be delivered in a manner wherein more than one site of control can be achieved, more than one disease state can be affectively treated at the same time.

A first aspect of the methods for controlling cellular proliferation relate to calcium and phosphate metabolism: plasma and bone mineral homeostasis, inter alia, osteomalacia, osteoporosis, renal osteodystrophy, and disorders of the parathyroid function.

A second aspect of the methods for controlling cellular proliferation relate to immune disorders, non-limiting examples of which include: i) auto-immune diseases, inter alia, diabetes mellitus type 1 , multiple sclerosis, lupus and lupus like disorders, asthma, glomerulonephritis, and auto-immune thyroiditis; ii) selective dysfunctions of the immune system, inter alia, Acquired

Immune Deficiency Syndrome (AIDS). iii) medically induced immune disorders, inter alia, the body's rejection of foreign tissue due to tissue grafts (e.g. kidney, heart, bone marrow, liver, islets or whole pancreas, and skin); iv) skin disorders, inter alia, those characterized by hyperproliferation, inflammation, (auto)immune reactions, examples of which include psoriasis, dyskeratosis, and acne. 00181

99

A third aspect of the methods for controlling cellular proliferation relate to cell differentiation disorders, non-limiting examples of which include breast cancer, leukemia, myelo-dysplastic syndromes and lymphomas, squamous cell carcinomas and gastrointestinal cancers, melanomas, and osteosarcoma. The present invention comprises a method for providing a therapeutic level of a 7-(2-cyclohexyl-idene-ethylidene)-spiro[5.5]undecane according to the present invention, said method comprising administering to a human or higher mammal one or more 7-(2-cyclohexyl-idene-ethylidene)-spiro[5.5]undecanes described herein.

For the purposes of the present invention the term " effective amount " as it relates to the amount of one or more 7-(2-cyclohexyl-idene-ethylidene)- spiro[5.5]undecanes delivered to a patient in need of treatment, is defined herein as an amount of a pharmaceutically active compound which produces the alleviation of symptoms or the suppression of cellular proliferation as measured directly, for example, by a laboratory test or procedure which provides a measure of the active ingredient in plasma, or indirectly, for example, by the ability of the patient not to experience undesirable disease or disease state symptoms. Said symptoms are necessarily dependent upon one or more factors, inter alia, level of cellular proliferation or differentiation activity, age of the patient, degree of disease involvement, other diseases or disease states present, desired outcome (complete cure as in a chronic illness or temporary relief as in an acute illness condition). It is recognized that the compositions of the present invention can be delivered in various dosages and therefore, the effective amount can be determined on a patient by patient basis if necessary.

PROCEDURES

For the purposes of testing compounds according to the present invention 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], a Vitamin D metabolite, is used as the control.

BINDING AFFINITY EVALUATION Affinity for Vitamin D Receptor (herein after referred to as VDR)

Selected compounds of the present invention were evaluated for their affinity to bind to a VDR, versus the active metabolite 1α,25(OH)₂D₃. The method employed and described herein are used for determining steroid hormone and steroid hormone mimetic binding. Utilizing the tritium labeled analog, [³H]I α,25(OH)₂D₃, the said compounds were evaluated for their binding to a high speed supernatant from porcine intestinal mucosa homogenates.

Incubation was performed at 4 ⁰C for 20 hours and the phase separation was obtained by adding dextran-coated charcoal. The relative affinity of the subject compound was then calculated by determining the concentration needed to displace 50 % of the [³H]I α,25(OH)₂D₃ from the VRD as compared to the amount of [³H]I α,25(OH)₂D₃ displaced by non-tritium labeled 1α,25(OH)₂D₃, which was assigned the relative value of 100%.

Affinity for Human Vitamin D Binding Protein (hDBP) Selected compounds of the present invention were evaluated for their affinity to bind to a human VDR, versus the active metabolite 1α,25(OH)₂D₃. The methods employed and described herein are used for determining steroid hormone and steroid hormone mimetic binding. Utilizing the tritium labeled analogue, [³H]I α,25(OH)₂D₃, the compounds were evaluated for their binding to a high speed supernatant from porcine intestinal mucosa homogenates.

A 5 μl_ solution of [³H]I α,25(OH)₂D₃ together with the compound to be tested, or with 1α,25(OH)₂D₃ in the case of the control experiment, dissolved in ethanol were added to glass tubes and incubated with hDBP (0.18 μM) made up to a final volume of 1 mL with a solution containing 0.01 M Tris-HCI buffer and 0.154 M NaCI at pH 7.4. The solution was incubated for 3 hours at 4 ⁰C. Final phase separation was obtained by adding 0.5 mL of cold destran-coated charcoal. The relative affinity of the subject compound was then calculated by determining the concentration needed to displace 50 % of the [³H]I α,25(OH)₂D₃ from the VDR as compared to the amount of [³H]I α,25(OH)₂D₃ displaced by non-tritium labeled 1α,25(OH)₂D₃, which was assigned the relative value of 100%.

MEASUREMENT OF CELL PROLIFERATION AND DIFFERENTIATION Breast Carcinoma Cells: MCF-7

Selected compounds of the present invention were evaluated for their effect on cell proliferation. Malignant MCF-7 cells were cultured in DMEM nutrient mix F12

(HAM) medium which was supplemented with 10 % heat inactivated FCS, glutamine

(2 mM), penicillin (100 units/mL) and streptomycin (0.1 mg/mL). The cultures were maintained at 37 ⁰C in an atmosphere of humidified air containing 5% CO₂. The MCF-7 cells were seeded at approximately 5 x 10³ cells/well in the DMEM modified medium in a 96-well microtiter plate. Each plate had wells made up to a final volume of 0.2 ml_. After 24 hours incubation, the control 1α,25(OH)₂D₃ or the compound to be tested was added in the appropriate concentration for an incubation period of 72 hours. Finally, 1 μCi of [³H]thymidine was added to each well and the cells were harvested after a further 4 hour incubation period. The cells were harvested with a Parckard Harvester and the cell count measured by a Packard Topcount System. Results are usually expressed as percentage activity (at 50 % of the dose response) in comparison with 1α,25(OH)₂D₃ as a control (having 100 % activity)

Promyelocyte Leukemia Cells (HL-60)

Selected compounds of the present invention were evaluated for their effect promyelocytic leukemia on cell proliferation. Falcon tissue chambers were seeded with 4 x 104 cells/cm² of HL-60 cells using RPMI 1640 medium supplemented with 20e% FCS and gentamycin (50 μg/mL) in a final cell volume of 5 mL. The cultures were maintained at 37 ⁰C in an atmosphere of humidified air containing 5% CO₂. After 24 hours incubation, the control 1α,25(OH)₂D₃ and the compound to be tested were each dissolved in ethanol and added to the cell culture with the final concentration being less than 0.2 %. After 4 days, the dishes were shaken to lose adherent cells. The cells were then washed twice in RPMI medium, counted, and then assayed for differentiation markers (NBT reduction assay). Superoxide production was measured as NBT reducing activity as described by Ostrem et al. in Proc. Natl. Acad. ScL USA, (1987) 84: 2610-2614. HL-60 cells at 1 x 10⁶/mL were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13- acetate (200 ng/mL) and NBT (2 μg/mL) and incubated for 30 minutes at 37 ⁰C. The percentage of cells containing black formazan deposits was determined using a hemacytometer. Results are usually expressed as.percentage activity (at 50 % of the dose response) in comparison with 1α,25(OH)₂D₃ as a control (having 100 % activity)

IN VIVO DETERMINATION OF CALCIUM LEVELS Eight weeks old, male NMRI mice were obtained from the Animal Center of

Leuven (Belgium) and fed a vitamin D-replete diet (0.2 % calcium, 1 % phosphate, 2000 U vitamin D/kg, available from Hope Farms, Woerden, The Netherlands). Groups of six mice were intraperitoneously injected daily during 7 consecutive days with different doses of 1α,25(OH)₂D₃ (0.2 and 0.4 μg/kg/day) or the vitamin D analog identified hereinabove as the E-isomer 103b (5 μg/kg/day). The control group was injected with vehicle (arachis oil). The average weight of each group of 6 mice was determined at the beginning and at the end of the experiment. The following parameters were evaluated: serum calcium, femur calcium. Serum calcium was measured by a microcolorimetric assay (Sigma, St. Louis, MO). Femurs were removed and femur calcium content was measured in HCI-dissolved bone ash (obtained by heating for 24hours in an oven at 100 ⁰C), using the same technique as for serum calcium.

Results of these experiments are presented in table 7. Several biological profiles could be demonstrated in this first screening assay: 1 ,25(OH)₂Vit D3 was found to have increased serum calcium levels together with decreased femur calcium levels compared to the vehicle treated animals (referred to as the " vitamin D toxic effect ". On the contrary, the compound of the invention exhibits a desired and highly advantageous selectivity profile with low serum calcium levels (equal or lower versus vehicle treated animals) together with increased calcium content in bone (> 10 % compared to vehicle treated animals). The preferential activity of this compound on bone without major calcemic side effects allows a safe in vivo administration of this compound for the treatment of metabolic bone diseases where bone loss is a major concern. TABLE 7

MEASUREMENTOFPREVENTIONOFOSTEOPOROSIS

A) Primary Prevention of Osteoporosis by Compound of the Present Invention. 12 Week old C3H female mice are subjected to bilateral ovariectomy or sham surgery. Animals are treated with a test compound or vehicle by oral gavage or intraperitoneally. Dosing is started 3 days after surgery and continued for 8-9 weeks. Prior to the first treatment, in vivo measurements are performed to determine bone mineral density (BMD), bone mineral content (BMC) of total body and spine by dual-energy X-ray absorptiometry (DXA). Urine and serum is collected to measure calcium levels together with collagen cross-links in urine and osteocalcin in serum. Animals are weighed regularly during the experimental period for determination of their general healthness. After 4 weeks treatment urine and serum is again collected and biochemical parameters are determined. At the end of the experiment (8-9 weeks) urine is collected and DXA measurement is performed in vivo to determine BMD and BMC. After sacrificing the animals, the tibiae and femora are dissected. The following biochemical parameters are investigated: serum calcium, serum osteocalcin, urine calcium, urine collagen cross-links, femur calcium.

Tibiae are used for histomorphometric analysis and femurs for measurement of cortical and trabecular volumetric density and geometry by peripheral quantitative computed tomography (pQCT) ex vivo.

B) Secondary Prevention of Osteoporosis.

12 Week old C3H female mice are subjected to bilateral ovariectomy or sham surgery. The animals are treated with the test compound or vehicle by oral gavage or intraperitoneally. Dosing was started 4 weeks after surgery and continued for 4 to 10 weeks. Prior to the first treatment in vivo measurements are performed to determine bone mineral density (BMD), bone mineral content (BMC) of total body and spine by dual-energy X-ray absorptiometry (DXA). Urine and serum is collected to measure calcium levels together with collagen cross-links in urine and osteocalcin in serum. The animals are weighed regularly during the experimental period. After 4 weeks treatment urine and serum is again collected and biochemical parameters were determined and urine was collected and DXA measurement is performed in vivo to determine BMD and BMC. After sacrificing the animals tibiae and femora are dissected. The following biochemical parameters are investigated: serum calcium, serum osteocalcin, urine calcium, urine collagen cross-links, and femur calcium. Tibiae are used for histomorphometric analysis and femurs for measurement of cortical and trabecular volumetric density and geometry by peripheral quantitative computed tomography (pQCT) ex vivo.

While particular embodiments of the present invention have been illustrated and described, various modifications can be made without departing from the scope of the invention. It is therefore intended to cover in the appended claims all such modifications that are within the scope of this invention.

Claims

CLAIMS 1. A compound, including all enantiomeric and diasteriomeric forms and salts thereof, said compound comprising: a substituted spiro[5.5]undecane unit having the following structural formula:

,1b ,2a ,2b wherein R^Oa, R^ob, R^1a, R^1D, R^, and R^z° are each independently selected from the group consisting of hydrogen and oxa-hydrocarbyl chains, wherein the oxygen atom of said oxa-hydrocarbyl chains is directly attached to the F ring of said spiro[5.5]undecane unit, and wherein the hydrocarbyl group of said oxa-hydrocarbyl chains is selected from the group consisting of substituted and unsubstituted C_1-2O alkyl, substituted and unsubstituted C_3-10 cycloalkyl, substituted and unsubstituted C_{2- 20} alkenyl, and substituted and unsubstituted C_2-20 alkynyl; and ii) a substituted cyclohexylidene unit Q having the structural formula:

wherein:

R^3a and R^3b are each hydrogen or R^3a and R -,^ύ3°b are taken together to form an exocyclic methylene unit;

R^4a and R^4b are each hydrogen or R^4a and R^4b are taken together to form an exocyclic methylene unit;

R^5a, R^5b, R^6a and R^6b are each independently selected from the group consisting of hydrogen, hydroxyl and OP wherein P is a hydroxyl-protecting group.

2. A compound according to claim 1 , wherein P is selected from the group consisting of C_1-20 alkyl, C_3-10 cycloalkyl and acyl.

3. A compound according to claim 1 or claim 2, wherein carbon 2 of said compound is further substituted with one or two substituents R and/or R', wherein R and R' are each independently selected from the group consisting of substituted and unsubstituted C_1-7 alkyl.

4. A compound according to any of claims 1 to 3, wherein Q has a formula selected from the group consisting of: i)

5. A compound according to any of claims 1 to 3, wherein Q has a formula selected from the group consisting of: i)

6. A composition comprising a compound according to any of claims 1 to 5.

7. A composition according to claim 6, further comprising one or more pharmaceutically acceptable carriers.

8. A composition according to claim 6 or claim 7, in the form of a pharmaceutical composition comprising a therapeutically effective amount of said compound.

9. A composition according to any of claims 6 to 8, in the form of a pharmaceutical composition comprising from 1 pg to 1000 mg of said compound.

10. A method of treating or preventing cell proliferation, comprising the administration to a human of a therapeutically effective amount of a compound according to any of claims 1 to 5.

11. A method of treating or preventing a bone disorder, comprising the administration to a human of a therapeutically effective amount of a compound according to any of claims 1 to 5.

12. A method according to claim 11 , wherein said bone disorder is osteoporosis.